Biomaterials control of pluripotent stem cell fate for regenerative therapy

Progress in Materials Science 82 (2016) 234–293

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Progress in Materials Science journal homepage: www.elsevier.com/locate/pmatsci

Biomaterials control of pluripotent stem cell fate for regenerative therapy Roman A. Perez a,b, Seong-Jun Choi a,b, Cheol-Min Han b,c,d, Jung-Ju Kim a,b, Hosup Shim a,b, Kam W. Leong b,e, Hae-Won Kim a,b,c,e,⇑ a

Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 330-714, Republic of Korea Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 330-714, Republic of Korea c Department of Biomaterials Science, College of Dentistry, Dankook University, Cheonan 330-714, Republic of Korea d Division of Orthodontics, College of Dentistry, The Ohio State University, Columbus, OH 43210, USA e Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA b

a r t i c l e

i n f o

Article history: Received 29 January 2016 Received in revised form 18 May 2016 Accepted 21 May 2016 Available online 24 May 2016 Keywords: Pluripotent stem cells Self-renewal Lineage differentiation Biomaterials Microenvironment

a b s t r a c t Pluripotent stem cells (PSCs) derived from either the embryo or reprogramming processes have the capacity to self-renew and differentiate into various cells in the body, thereby offering a valuable cell source for regenerative therapy of intractable disease and serious tissue damage. Traditionally, methods to expand and differentiate PSCs have been confined to 2D culture through the use of biochemical signals; the use of biomaterials beyond the commercially available culture dish has not been widespread. Nevertheless, biomaterials with tailored physical, chemical, and geometrical cues can mimic the native stem cell niche to tune the microenvironmental conditions for PSCs to preserve their self-renewal capacity or to switch their phenotype, a status ultimately needed to gain regenerative functions ex vivo and in vivo. Recently efforts to explore biomaterials to regulate PSC behavior have accelerated. The biomaterials properties investigated include surface chemistry, immobilized ligand, nano-/micro-topography, matrix stiffness, geometrical complexity, 3D configuration, and combinations thereof. This review aims to cover the current advances of biomaterials-based control over PSCs, particularly for the preservation of self-renewal capacity as well as for their differentiation into target cells. Furthermore, it aims to suggest future research directions that would facilitate the eventual translation of these advances. Ó 2016 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Establishment of PSCs: A brief summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Niche and microenvironments of PSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomaterials for the self-renewal and proliferation of PSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Surface functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Chemical groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. Biomolecular tethering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

⇑ Corresponding author at: Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 330-714, Republic of Korea. E-mail address: [email protected] (H.-W. Kim). http://dx.doi.org/10.1016/j.pmatsci.2016.05.003 0079-6425/Ó 2016 Elsevier Ltd. All rights reserved.

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4.2.

5.

6.

Physical modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Nano-/micro-topography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Matrix stiffness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Three dimensional environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. 3D gel encapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2. 3D geometrical scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomaterials for lineage differentiation of PSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Surface tailoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Physical modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1. Nano-/micro-topography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.2. Matrix stiffness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Three dimensional environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.1. 3D gel culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.2. 3D geometrical scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Patients suffering from various degenerative diseases create a high demand for effective treatments. To satisfy the therapeutic needs of these degenerative diseases, regenerative medicine offers an exciting promise [1–3]. Regenerative medicine seeks to utilize the therapeutic capacity of stem cells because most of tissues in the body have their own endogenous stem cells to regenerate upon injury [4]. When the damage exceeds the capacity of the injured tissues to regenerate, transplantation with exogenous stem cells is required [5]. Stem cells have a capacity to self-renew and differentiate [6–8]. While adult stem cells are multipotent or unipotent, embryonic stem cells (ESCs), embryonic germ cells (EGCs), and embryonic carcinoma cells (ECCs) are pluripotent [9–14]. Induced pluripotent stem cells (iPSCs), recently developed by epigenetically reprogramming somatic cells, are also pluripotent [15]. PSCs have several advantages over adult stem cells: (i) they can proliferate indefinitely in vitro under proper conditions, whereas adult stem cells have a limited proliferation capacity, (ii) they are capable of differentiating into most cell types in the body, while adult stem cells are only able to differentiate into cells of lineages from which they originate, and (iii) they are permissive to genetic modification, while adult stem cells are largely resistant to genetic engineering [9–11]. The establishment of human ESCs and EGCs is thus a milestone in the medical applications of stem cells, including modeling embryonic development and disease progression in human, drug screening using ESC-derived somatic cells in vitro, and regenerative therapy using ESC-derived somatic cells as donor cells [16–19]. The shortage of donor tissues and organs has always been a concern in tackling organ failure and intractable diseases [20]. In fact, the treatments of injured tissues and organs have been carried out by transplantation of physiologically functional cells, tissues or organs derived from stem cells. Although adult stem cells, such as hematopoietic stem cells (HSCs), have been used for the treatment of leukemia [21], their use in organ failure or degenerative diseases is still far from being practical due to their limited proliferation capacity in vitro [22,23]. PSCs in contrast can provide an inexhaustible cell source. Some of the life-threatening diseases and injuries, like hematopoietic disorders, liver damage and spinal cord injuries, have started to utilize the PSCs-based regenerative approaches [24]. Above all, for the successful clinical applications of the PSCs, two major assets of PSCs – unlimited self-renewal and differentiation into all types of cells – should be defined and controlled well. Securing a large population of cells is a prerequisite to achieve therapeutic capacities in stem cell based regenerative medicine. Maintaining the characteristics and phenotype of PSCs over prolonged passages is not easy – often the cells loose the pluripotency with a heterogeneous population of unwanted or poorly-defined differentiated cells. Directing the secured large number of PSCs toward a targeted lineage is a challenging issue for safe and potential use of them in clinical settings. In fact, human PSCs were potentially tumorigenic upon their transplantation into the body [25,26], limiting their clinical uses. Therefore, strategies and technologies to target tissue differentiation in vivo as well as the control of the differentiation level in vitro should be explored. Substantial effort has been given to this. For example, antibody-based strategy was used to remove the undifferentiated cells before transplantation [27–29]; however, the lack of specificity of the targeted PSC markers limits their clinical availability. Some cells originated from PSCs have also been shown to spontaneously dedifferentiate into a pluripotent state after transplantation, leading to a teratoma formation [30]. A method to inhibit the pathways in the generation of pluripotency and teratoma was thus used; NANOG, known as a key gene for the pluripotency, was suppressed to reduce the teratoma formation [31–33]. Apart from the biological strategies that have employed 2D culture plates and soluble molecules, other culture parameters like 3D environments and substrate physico-chemical properties can be considered to overcome those limitations of PSCs – how to multiply cell population without losing a pluripotency and to differentiate to specific cell types in vitro and in vivo – ultimately to satisfy safety and therapeutic efficacy. A substantial body of literatures has recently witnessed the

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Fig. 1.1. Schematic illustration of the stream of self-renewal of PSCs (ESCs and iPSCs) and their differentiation down to three-germ layers and further to specified tissue cells that hold therapeutic promise for curing diseases and regenerating dysfunctional tissues; while well-defined soluble biochemical factors are mostly used, the ‘biomaterials’ can play significant roles in those biological processes.

important role of biomaterials and matrices in controlling those PSCs behaviors. The culture of PSCs over the biomaterials with tailored surface chemistry, specific ligands, and physical topology and stiffness, and the culture under 3D environments either with mechanical cues or with the supply of soluble factors, opens the possibility to tackle those self-renewal and differentiation issues raised in PSCs. Another issue often faced in the applications of differentiated PSCs is the immune response; a question might be raised if the differentiated PSCs express a high level of major histocompatibility complex molecules that cause immune reaction. To address this, patient’s immune system is possibly suppressed although a long term immunosuppression may incur serious side effects [34–36]. Here the role of biomaterials and matrices can also be found. Biomaterials have been designed to suppress and modulate the immune responses of cells, altering the phenotype of surrounding macrophages from an inflammatory phase to a regenerative stage. It is recommended to refer to a recent review which details this topic [37]. Thus this Review will discuss on using biomaterials to culture PSCs aiming (i) to preserve the pluripotency, and/or (ii) to provide differentiation cues to specific lineage cells. While the culture of adult stem cells like MSCs with biomaterials has already been extensively studied and reviewed [38,39], the current direction of biomaterials interactions with PSCs to control their self-renewal capacity and differentiation yet need to be discussed. Although most of the studies so far have been empirical, they have provided valuable insights to define the merits of PSC-based therapy. There is ample room for design and production of novel biomaterials to further advance PSC-based therapy. Fig. 1.1 schematically illustrates the stream of self-renewal and differentiation (three-germ layers then down to specific lineage cells) of PSCs and the possible regulation of these processes through the biomaterials. 2. Establishment of PSCs: A brief summary PSCs developed thus far are categorized as ESCs and iPSCs. This section briefly summarizes these two types of PSCs, including the historical establishment, their potential, and hurdles in clinical application. First, the ESCs, as derived from pre-implantation embryos, are capable of maintaining an undifferentiated state even after cell division and differentiating into all cell lineages of three embryonic germ layers. Since ESCs have these properties, called self-renewal and pluripotency, they offer the possibility to repair or replace damaged tissues and to treat degenerative diseases such as Parkinson’s and Alzheimer’s disease. For this reason, ESCs are considered to have strong potential for regenerative medicine [40]. ESCs originate in a blastocyst, the embryo that is developed five days after fertilization and consists of inner cell mass (ICM) and enclosing trophoblast cells. To obtain the ICM from a blastocyst, the immunosurgical method developed by Solter and Knowles in 1975 is currently used in laboratories. In immunosurgery, blastocysts are incubated with an antibody and

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then exposed to guinea pig complement for lysis of the outer trophoblast cells, resulting in isolation of ICM cells. Since the isolated ICM cells are not able to maintain self-renewal and pluripotent properties in vitro, the cells are co-cultured on the feeder cells which are mitotically-inactivated fibroblasts, which results in the establishment of mouse and human ESC lines [12,41]. After the establishment of an ESC line, characterization is carried out to ascertain its pluripotency. In ESCs, the maintenance of self-renewal and pluripotency is achieved by three core transcription factors, Oct4 (octamer-binding transcription factor 4), Sox2 (sex determining region), and Nanog. Oct4, a POU domain-containing transcription factor, is highly expressed in ESCs and its expression is dependent on the status of ESCs; overexpression of Oct4 in ESCs causes their differentiation into primitive endoderm and mesoderm, whereas its repression induces differentiation to trophectoderm [42]. Sox2 is an HMG (high mobility group)-family protein and one of the targets of Oct4. Moreover, Sox2 interacts and works together with Oct4 to maintain pluripotency of ESCs [43]. Similarly, Nanog, a homeodomain-containing protein, is also specifically expressed in pluripotent cells and modified expression can change the phenotype of the ESCs [31,32]. These three proteins work together to activate a set of genes needed for the maintenance of stemness of ESCs, and they also activate their own genes, therefore constructing the interlinked auto-regulatory loop [44]. Together with progressive understanding of the gene regulation in human ESCs, traditional methods for generating ESCs have been improved for clinical applications, including using ‘‘dead” embryos which present arrested cell division [45], creating ESCs from single cells taken from pre-implantation embryos [46,47], and using parthenogenetically-activated oocyte instead of a fertilized oocyte [48]. Society, even so, has yet major concerns about using human ESCs in clinical settings, thus requiring more technological advances that enable clinical availability. For generation of human ESCs, animal-derived materials such as fetal bovine serum and mouse feeder cells are indispensable in conventional systems. To overcome this limitation, xeno-free culture systems have been developed for human ESCs [49,50]. Another problem is the immune incompatibility of human ESCs in clinical applications. Because ESCs cannot always be derived from the patient, their therapeutic applications naturally imply risk of immune rejection. These immune concerns can be solved by reprogramming the somatic cell nucleus in an enucleated oocyte [51,52]. Recently, human ESCs have been derived from embryos transferred with somatic cell nucleus [53,54]. One of the major limitations is an ethical issue caused by the destruction of a fertilized embryo. Although the methodologies newly developed for human ESCs are able to solve some of the limitations, they are still not quite free of the ethical controversy because some of their sources still involve human embryonic elements. These barriers can be overcome by the use of a novel cell type, iPSCs, first reported by Yamanaka’s group, demonstrating ES-like characteristics, such as self-renewal and differentiation capacities [15]. Since iPSCs are created from differentiated somatic cells not from embryonic cells, they do not raise the ethical controversy associated with ESC research. Furthermore, iPSCs can be established from patient to avoid the immune rejection afflicting the ESC counterpart. These merits currently make iPSC technologies a major research area in regenerative medicine. Reprogramming is a key feature to change somatic cells into pluripotent cells in iPSC generation. Yamanaka’s group achieved mouse fibroblast reprogramming by retroviral infection, leading to ectopic expression. Twenty-four genes were selected as the ones considered to play a key role in pluripotency induction. The combination of these 24 genes gave rise to the discovery of 4 specific genes that were able to derive iPSCs with similar growth and morphology to ESCs. These genes were, Oct4, Sox2, Klf4 (Kruppel-like factor 4), and c-Myc, which were implemented through the use of retroviruses [15,55] but the efficiency of the process was shown to be around 0.1%. In a similar way, two sets of transcription factors were shown to generate iPSCs from the somatic cells in human. The combinations with other factors like Nanog and Lin28 were effective as well in the reprogramming process [56,57]. A few years later, adult human fibroblasts were genetically reprogrammed into human iPSCs, although the reprogramming efficiency decreased to a value of 0.01% [58]. Furthermore, c-Myc, a wellknown oncogene, could be omitted, in order to avoid tumor generation, even though the transformation efficiency further decreased to as low as 0.001% [58]. When only two transcription factors, Oct4 and Sox2, were used for reprogramming fibroblasts, the induction of the PSCs failed [59], although iPSCs were formed with one factor (Oct4) when the host cells were neural stem cells [60]. The combination of the two factors and valproic acid, a histone deacetylase inhibitor, allowed the generation of human iPSCs from fibroblasts [61], showing that the conversion could also take place through epigenetic modification [61]. This study shows that chemicals can replace some genetic factors for reprogramming cells, suggesting the potential use of small molecules for cell reprogramming that can address safety issues raised by using viral vectors to carry genetic materials into cells. Besides retroviruses, other strategies have also been used for the production of iPSCs. For instance, lentiviruses have been shown to generate human iPSCs by overexpressing Oct4, Sox4, Nanog and Lin28 [57], obtaining similar efficiencies to those obtained by Takahashi. A much higher efficiency, up to 10%, was achieved by combining Oct4, Sox2, c-Myc, Klf4, Nanog, and LIN28 [62]. However, the genes introduced through lentiviruses, unlike the use of retroviruses, were not shut down after iPSC formation, making it inappropriate for clinical application. Transfection with plasmids has also appeared as an alternative strategy. When embryonic fibroblasts were transfected with two different plasmids, one with Oct3/4, Sox2, and Klf4 and another with c-Myc, human iPSCs were produced, but with very low efficiency (0.0015%), showing a decreased potential for clinical application [63]. Nevertheless, a year later, much higher efficiencies, up to 0.1%, were obtained when three plasmids containing 6 transcriptional factors (Lin28, Klf4, Nanog, c-Myc, Sox2, and Oct4) were used to generate human iPSCs. Another method which is safer, but has lower efficiency as well is the incorporation of recombinant proteins into cells. Four reprograming proteins (Oct4, Sox2, Klf4, and c-Myc) fused with a cell penetrating peptide were delivered to human fibroblasts to obtain iPSCs, although the reprogramming efficiency was too low to be practical in clinical applications [64]. Recent

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Refs.

Somatic cell nuclear transfer in frog Isolation of mouse embryonic stem cells Somatic cell nuclear transfer in sheep Isolation of human embryonic stem cells Isolation of mouse induced pluripotent stem cells Isolation of human induced pluripotent stem cells Cell therapy using mouse induced pluripotent stem cells Direct cell lineage conversion Isolation of human nuclear transfer embryonic stem cells Isolation of mouse induced pluripotent stem cells using small molecules

[67] [41,68] [69] [12] [15] [56,57] [70] [71] [53] [72]

Table 2 Comparison of the two types of PSCs, ESCs and iPSCs, in terms of their safety, efficacy, and clinical availability. Category

ESCs

iPSCs

Origin Source availability Establishment

Somatic cells Unlimited Reprogramming by transcription factors

Efficiency of establishment

Inner cell mass cells of blastocyst-stage embryo Rare Culture of ICM under the condition of inhibiting differentiation Low

Risk of immune rejection Probability of mutation

High in allograft Low

Remaining epigenetic memory of somatic cells Differentiation potential

None

Ethical concern

Serious

Pluripotent

Vary depending on reprogramming efficiencies Low if patient-specific Vary depending on reprogramming procedures Exists depending on cell sources Skewed depending on epigenetic signatures None

advances showed that the introduction of modified RNA or microRNA (miRNA) into somatic cells was relatively efficient, demonstrating their potential use in clinical practices [65,66]. Table 1 summarizes the history of ESCs and iPSCs that have been established thus far. As noted, a prominent difference between PSCs and adult stem cells is their capacities of differentiation. PSCs can differentiate into all cell types comprising the body, whereas adult stem cells may differentiate into limited cell types, commonly those of their tissues of origin. Once established, PSCs can be maintained in culture almost indefinitely, thus providing an unlimited source for cell therapy. In contrast, adult stem cells are rare in mature tissues, so that isolation of pure population in large numbers is often challenging. Expansion in culture may be required to obtain sufficient number of cells for cell therapy. However, proliferation of adult stem cells in culture is limited since they may become aged progressively. Overall, PSCs have advantages in their accessibility, purity, expansion, and potency over adult stem cells. Among the PSCs, the application of iPSCs in regenerative medicine appears to be close to being practical, given that some of the limitations have been overcome. On the other hand, because of the long history of study, ESCs are well-characterized, including their self-renewal and differentiation properties. Development of human ESCs has increased hope for the treatment of degenerative diseases. There was a clinical application for spinal cord injury led by Geron, USA, initially using ESCs. Subsequent trial involves iPSCs that are free of ethical issues, a prime asset relative to ESCs. However, some limitations of iPSCs are yet to be solved. The major hurdle of human iPSCs is safety and the low efficiency of generation. However, various methodologies are being rapidly developed to enhance the efficiency of iPSC generation. The safety concern of iPSCs in their generation because of the use of viruses has also been a primary hurdle, as the viral vectors can cause potential pathological conflicts, such as tumor formation; however, some of the efforts using viral-free systems made of chemically derived carriers have begun to show promise. Another safety concern of iPSCs is tumor development, since the iPSCs are pluripotent. This problem can be solved by priming the iPSCs application in vivo. Table 2 summarizes the pros and cons of the two types PSCs, ESCs and iPSCs, considering their safety, efficacy, and clinical availability. 3. Niche and microenvironments of PSCs The ability of the PSCs to maintain their pluripotency or to differentiate into specific lineages depends on the stimuli given by their local microenvironment, called niche [73]. The complexity and dynamics of the extracellular matrices (with immobilized molecules) and the soluble molecules created by cells define the niche [74]. In the niche the stem cells are quiescent, but switch to a proliferative phenotype in response to injury for repair of the tissue. Since stem cell fate is closely

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Fig. 3.1. Schematic illustration showing the niche of PSCs and the possible strategies to regulate the maintenance or differentiation of PSCs using microenvironmental factors, including surface chemistry, ECM molecules tethered, signaling molecules delivered, nano/microtopology, matrix stiffness, scaffold geometry, co-cultures (and the paracrine signals), 3D cultures, mechanical force and oxygen level.

influenced by interaction with the niche, many studies have focused on engineering the microenvironment to regulate stem cell behavior. A well-engineered niche requires the integration of physical and biochemical cues such as cell–cell contact, cell–matrix interaction, tethered biomolecules, oxygen tension, and external mechanical forces as schematically presented in Fig. 3.1. In this section, the basic strategies for regulating the maintenance or differentiation of PSCs using these microenvironmental cues are outlined. The first step for the interaction of PSCs with the surrounding is adhesion, which occurs between cells or between cells and a matrix. Both ESCs and iPSCs present specific receptors that lead to adhesion to the molecules on neighboring cells or to the matrix to maintain their colony status. Cell adhesion molecules (CAMs) are proteins located on mammalian cell surfaces. When cells interact with neighboring cells or extracellular matrices (ECMs), CAMs such as cadherins, integrins, selectins, and immunoglobulins are involved in the interaction [75]. Cadherins are cell surface glycoproteins responsible for Ca2+-mediated cell–cell interaction. Among the cadherin subfamily, epithelial (E-), neural (N-), and vascular endothelial (VE-) cadherins have been widely studied. Undifferentiated ESCs express E-cadherin, but not N- and VE-cadherins [76,77], suggesting the important role of E-cadherin in maintaining the pluripotent state of ESCs. The E-cadherins establish homophilic interactions between cells through bonds between their long extracellular domain and p120- and b-catenins at the end of cytoplasmic domain. Recently, several molecular mechanisms describing E-cadherin in regulation of human ESC fate have been described. When human ESCs form colonies, non-muscle myosin IIA (NMM IIA) promotes the localized accumulation of E-cadherin at intercellular junctions by stabilizing p120-catenin. This NMM IIA-mediated regulatory signaling help maintain the Oct4-Nanog-Sox2 circuitry [78]. These properties of E-cadherin need to be considered in designing the desired niche. Several studies have been carried out on E-cadherin-coated substrates to provide ESCs with an environment similar to cell–cell interactions, resulting in the maintenance of pluripotency in ESCs for several passages without feeder cells [79,80]. On the other hand, if the appropriate external cues are given, N-cadherin coated substrates can be helpful to differentiate cells into specific lineages, such as neurons. The main rationale is that the substrate dependent activation of Ncadherin reduces Rho/ROCK signal and b-catenin expression which in turn can enhance the neurite outgrowth [81]. Several ECM proteins have been shown to play key roles in self-renewal capacity as well as differentiation. ECM molecules such as collagen I, laminin, vitronectin, and fibronectin offer excellent adhesion sites for ESCs, allowing the maintenance of their pluripotency [82–87]. For instance, fibronectin plays a key role in mouse ESC attachment and self-renewal, although excessive amounts of fibronectin would give rise to spreading and differentiation [88]. Both ESCs and iPSCs express integrins a3, a5, a6 and b1. The integrins a2 and a11 are also expressed by ESCs and a7, aV, and b5 by iPSCs [84,89–91]. Laminin has also been shown to allow self-renewal, although not all of the different forms of laminin are effective in supporting self-renewal. Antibody-blocking studies show that adhesion of ESCs to laminin requires a6b1, but not other integrins [92]. On the other hand, iPSCs maintain initial attachment and self-renewal capacity in the presence of aVb5 integrin when vitronectin is used as a substrate, while they significantly lose the adhesion capacity upon the inhibition of aVb5 and b1 [90]. Specific peptide sequences have been analyzed for the adhesion of human ESCs. When the substrate contains the RGD domains of bone sialoprotein and vitronectin, ESCs are able to adhere and proliferate, whereas the RGD sequencecontaining fibronectin and laminin do not allow their adhesion and proliferation, proving that the RGD sequence alone is not sufficient for the adhesion of human ESCs to substrates [93]. In a similar way, glycosaminoglycans (GAGs), such as hyaluronic acid (HA), may possess unique properties for the self-renewal of ESCs as well. Low molecular weight HA is particularly suitable for long-term culture of ESCs in an undifferentiated state [94,95]. Self-renewal is an important characteristic that allows stem cells to arrive in an undifferentiated state to the site of defect. The differentiation of PSCs is initiated with the formation of an embryo body in which cells are held together through cell– cell contact and gap junctions. During culture, the embryo body is capable of differentiating into various cell types following reseeding on proper ECMs. The chemical composition and the physical and mechanical properties of the surrounding matri-

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ces are the key factors that determine differentiation. Cells are able to change their phenotype according to the substrate stiffness [96]. When ESCs are cultured on fibronectin substrates, they tend to differentiate into vascular endothelial cells, whereas they have a tendency to differentiate into cardiomyocytes on laminin [97]. Specific types of integrins guide cell differentiation. For instance, a5b1 and a6b1 drive the differentiation of ESCs into mesodermal lineages [98]. Surface chemistry also affects the differentiation of ESCs. When ESCs are cultured independently on type I or type III collagen, they are not able to differentiate even in the cardiac differentiation medium. However, when both collagen types are mixed, the substrate can guide the differentiation of ESCs into the cardiac lineage [99]. Type I collagen has also shown to allow the differentiation of iPSCs to hepatocytes in 3D culture conditions when combined with 3T3 cell sheets [100]. Soluble biochemical factors are potent cues that affect the maintenance and differentiation of PSCs. The soluble factors secreted from cells are signaling molecules for indirect communication between stem cells and surrounding cells in the niche. These factors regulate cell fate and self-renewal by directly binding to surface receptors on stem cells [101]. Flaim et al. evaluated the effect of four soluble factors, wnt3a, activin A, bone morphogenetic protein 4 (BMP4), and fibroblast growth factor 4 (FGF4) on the differentiation of mouse ESCs using combinatorial methods. The results showed that the addition of wnt3a inhibited the differentiation of ESCs, whereas the presence of activin A and BMP4 activated differentiation into cardiomyocytes [99]. Wnt signaling plays an important role in maintenance of pluripotency in ESCs by inhibition of glycogen synthase kinase 3, which in turn stabilizes b-catenin. In the nucleus, b-catenin forms a complex with T-cell factor 3 (Tcf3), which inhibits cell differentiation in multiple tissues [102,103]. However, several studies have reported that wnt signaling promotes mesodermal differentiation of mouse ESCs [104–106]. Different results from the same signaling pathway can be explained by the phenomenon that the wnt signaling pathway functions differently at different times during embryonic development. In a similar way, this phenomenon occurs during the embryonic development in zebrafish, in which wnt signaling at early stages promotes mesodermal development, while the treatment at the later stage rather inhibits the mesodermal development [107]. In addition, wnt signaling not only supports short-term self-renewal of human ESCs [108,109], but also promotes their mesodermal differentiation [110]. Basic fibroblast growth factor is another soluble factor that promotes the self-renewal of human ESCs [111,112], while BMP4 promotes the differentiation of ESCs into various lineages of cells. When BMP4 is added with other soluble factors, such as leukemia inhibitory factor (LIF), it synergistically supports selfrenewal of mouse ESCs [113], whereas alone it stimulates the differentiation of human ESCs into trophoblast [114] and hematopoietic progenitors [115]. Furthermore, addition of BMP4 with activin A promotes the differentiation of mouse and human ESCs into cardiomyocytes [99,116]. Beside the use of different types of factors, the concentration of soluble factors is also important in stem cell behavior regulation. For example, BMP4 drives hepatic differentiation of mouse ESCs only at an optimal concentration 50 ng/ml, while the differentiation is barely observed with <10 ng/ml [117]. The introduction of soluble factors is a simple and effective way to regulate stem cell fate. A fertile direction of bioengineering research is to develop controlled release systems to delivery these biochemical cues in a local and sustained manner, which would be particularly useful for in vivo tissue regeneration. In addition to soluble factors, the partial pressure of oxygen, or oxygen tension, is an important microenvironment factor that can modulate the stem cell fate. The oxygen tension ranges between 2% and 5% in mammalian tissues [118], which is much lower than oxygen concentration in air (21%). The exposure of ESCs to low oxygen tension in vitro has been shown to enhance their clonal recovery without altering the status of pluripotency and to reduce the differentiation [119–122]. The beneficial effects of hypoxic conditions on the maintenance of human PSCs is in part explained by Ezashi et al., who showed that pre-implantation embryos could be exposed to an oxygen pressure more naturally to produce more inner cell mass [119]. When cells are grown in hypoxic conditions, they initiate oxygen-dependent processes regulated by hypoxia inducible factors (HIFs). HIFs consist of three oxygen-dependent a-subunits (HIF1a, HIF2a and HIF3a) and a constitutively expressed b subunit (HIF1b). Under normal oxygen tension, HIF1b is degraded via hydroxylation by prolyl hydroxylase proteins, while the hydroxylation reaction is inhibited under hypoxic conditions [121]. Gustafsson et al. found that notch signaling is required to support the undifferentiated state of stem and precursor cells. The intracellular domain of notch protein interacts with and stabilizes HIF1a, leading to maintenance of undifferentiated states of ESCs [123]. However, HIF1a plays a role in maintaining the undifferentiated state only for short-term culture (1–2 days) [121,122]. In long-term culture under hypoxic conditions, HIF3a expression, instead of HIF1a, is upregulated which also positively regulates HIF2a. HIF2a plays a role in maintaining the undifferentiated state of ESCs by regulating the expression of Sox2 and Nanog [121]. External mechanical forces have also been shown to play a role during embryonic development in drosophila [124]. The external forces can be referred to contractile forces that are applied outside of the cells, such as shear, tensile or compressive loads. Each mode of external forces can be applied by fluidic flow, stretching or compressing the substrate [125]. Several studies have shown the relationship between external mechanical forces and ESC fate. Chowdhury et al. applied the local cyclic stress to mouse ESCs to observe their behavior. A sinusoidal magnetic field was applied to ESCs incubated with integrin receptor-coated ferromagnetic microbeads to generate the local cyclic stress. The results showed that the small cyclic stress (17.5 Pa at 0.3 Hz) applied to adherent ESCs allowed the cells to present a soft morphology. Undifferentiated ESCs are more sensitive to the stresses than the differentiated counterparts. Moreover, the expression of oct3/4 is down-regulated upon small cyclic stress, suggesting that the differentiation of ESCs can be driven by small external mechanical stress [126]. The effect of external mechanical forces on human ESC fate has also been evaluated by Saha et al. The cyclic external mechanical force (10% strain, 10 cycles/min) was applied to the ESCs using a Flexcell culture system. Interestingly, the external mechanical force promotes the maintenance of undifferentiated state of ESCs, resulting in the higher fraction of undifferentiated ESCs compared to the static control. However, the differentiation of human ESCs is enhanced by mechanical

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stress under the differentiation culture conditions. To regulate the fate of ESCs, therefore, mechanical forces should be considered in conjunction with biochemical signals [127]. The detailed mechanisms that ESCs respond to external mechanical forces are not fully clarified, but the deformation of focal adhesion proteins and other structural proteins is considered the molecular mechanism at least partially responsible for strain sensing of the cells [126]. Taken together, the general consensus is that a microenvironment that has similar physical properties and chemical compositions as the native niche conditions would be beneficial for controlling the self-renewal and differentiation of PSCs. Providing a reproducible cell source that can be applied in clinical applications requires proper cell expansion protocols, which are currently based on the expansion of mouse embryonic fibroblasts or Matrigel as feeder layers. Despite their efficiency to support the self-renewal, major concerns on the possible transmission of pathogens and contaminants still remain. For this reason, there is a great need of biomaterials that offer proper substrates to replace the feeder layer and mimic the natural environment of PSCs. Natural substrates may often lack reproducibility, making outcomes inconsistent in research on PSCs and thus reducing the potential of PSCs for clinical applications. Synthetic substrates, on the other hand, have poor biological activity and matrix signals to control PSC fate. For this reason, combinations of natural and synthetic substrates that may control and regulate the PSC microenvironments has become of interest. Further to this, maintaining the desired phenotype is a key prerequisite for successful cell therapies in clinical applications; this needs well-controlled physico-chemical properties of a substrate as well as the surrounding 3D matrix cues. In the following sections, we thus discuss the control over the self-renewal and differentiation of PSCs based on biomaterials parameters, including chemical groups and immobilized ligands, physical surface topology and mechanical stiffness, and 3D geometrical and culture environments.

4. Biomaterials for the self-renewal and proliferation of PSCs Research on PSCs is aimed to study strategies that allow maintaining the self-renewal and proliferation ability of PSCs without changing their phenotype, as well as on controlling their differentiation behaviors into specific lineages. For this purpose, PSCs have been conventionally cultured on 2D substrates that contained defined ingredients and signaling molecules. However, the use of novel biomaterials with specific 3D environments and optimized physico-chemical properties for the culture of PSCs has been shown to be more efficient in their ability to maintain the proliferation capacity or to differentiate into the desired phenotypes. This part review the biomaterials developed for the self-renewal and proliferation of PSCs. First, the surface properties of biomaterials are an important parameter that controls cell behaviors. The surface of biomaterials is the first ligand that cells are able to recognize and the one that will dictate the behavior of cells in the subsequent steps. In order to find the optimum substrate for self-renewal of PSCs, the combination of synthetic substrates with different surface chemistry, topography, and elasticity are investigated. Furthermore, the use of 3D hydrogels and scaffolds that provide great geometrical similarities with the native microenvironment of PSCs for their self-renewal is currently an area of extensive research.

4.1. Surface functionality 4.1.1. Chemical groups The slight modification of surface chemistry has been shown to significantly alter the pluripotency of PSCs. Substrates lacking of biological signaling, must regulate self-renewal through their physico-chemical properties which need to support colony formation to maintain the pluripotency. For instance, varying the concentration of negatively charged carboxylic groups is an example of how surface chemistry plays a key role [128]. Modifying the surface of glass with different amounts of hydrophobic (e.g. octadiene) and hydrophilic (e.g. acrylic acid) molecules allows obtaining a gradient concentration of carboxylic acid groups. In the low carboxylic acid concentration zone, mESCs failed to attach, whereas in the high carboxylic acid concentration zone, cells formed mono-layered colonies with substantial differentiation. In the intermediate carboxylic acid concentration zone, cells were able to form compact and multilayered colonies with little sign of differentiation. The intermediate concentration of carboxylic acid is thought to provide spatial arrangement of surface chemistry, consequently restricting the cell spreading properly for the self-renewal process (Fig. 4.1) [128]. Since the carboxylic acid is negatively charged, high concentrations lead to more hydrophilic surfaces, allowing cells to attach and spread better compared to a hydrophobic surface [129]. Other chemical groups than the carboxylic group can also be studied to support the PSC maintenance because the cell behaviors (not PSCs) mainly the cell spreading have been shown to significantly change by the substrates with different chemical groups, like amine, carboxylic, hydroxyl, methyl or mercaptol [129–133]. An important message is that the limited spreading of PSCs induced by the tailored simple surface chemistry is indicative of the self-renewal ability. Specific molecules, such as leukemia inhibitory factor (LIF) or Rho-associated protein kinase (ROCK) inhibitor are known to contribute to reducing the PSCs spreading, and consequently the pluripotency [134], and the methods to regulate those specific signaling molecules may also need other functional biochemical moieties, which remains further studies. Conclusively, limiting the spreading of PSCs by a simple surface chemistry control is considered a facile strategy to preserve the pluripotency of cells.

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Fig. 4.1. Effect of carboxylic concentration of substrates on the adhesion of mouse ESCs. (A) Variation of %C in carboxylic group with distance along the coverslip, where arrows indicate the regions 1–5 labelled in the coverslip. (B) Phase contrast micrographs of mouse ESCs cultured for 7 days on the gradient coverslips. Scale bar 100 lm. Modified with permission from Ref. [128].

4.1.2. Biomolecular tethering Not just a simple chemical group, but the ECM molecules are considered more potential to change the surface biochemical properties. Those molecules are mostly adhesive proteins (either full sequenced or the derivative peptides) and their combination with proteoglycans to mimic native ECMs. The ECM mimic molecules can dictate the response of surface receptors of PSCs and signal the intracellular processes involved in the self-renewal. As described in the previous part, it is essential that the interaction of PSCs with their niche matrices takes place via integrins for phenotype maintenance and self-renewal. For instance, vitronectin is a protein abundantly present in serum, and its interaction with specific integrins, such as avb5 is well-known to support PSC self-renewal [84,135]. One study found that human ESCs were shown to form colonies on substrates when coated with vitronectin through the binding of integrins avb3 and avb5, which are vitronectin receptors present in human ESCs (Fig. 4.2) [136]. The blocking experiment of those receptors significantly reduced the adhesion of human ESCs. The combination of ECM molecules is known to be more effective than single molecules to support PSC self-renewal. While fibronectin-coated plates induced the differentiation of human PSCs only after 5 passages, the combination of fibronectin with heparin sulfate proteoglycan (HSPG) allowed maintaining the human PSCs in an undifferentiated state for longer passages [137]. In a similar way, the combination of vitronectin peptides immobilized by polydopamine facilitated the long-term maintenance of human PSCs through enhanced focal adhesion, cell– cell interaction and biophysical signals provided by the designed platform [138,139]. Gelatin has also been shown to be important for the self-renewal of mouse PSCs. Gelatin-coated hydroxyapatite was efficient in self-renewal of ESCs, enabling the expression of Oct-4 and Nanog factors and maintained the ability to differentiate into three germ layers [140]. Meanwhile, other than the biological proteins, synthetic molecular surfaces were also developed for long-term culture of PSCs. Human ESCs cultured on aminopropylmethacrylamide (APMAAm) surface showed pluripotency for more than 20 passages, and the result was similar to the cells cultured on Matrigel-coated substrate, evidencing the effectiveness of synthetic substrates for the self-renewal of the human ESCs without the need for biological coatings (Fig. 4.3) [141]. In the study, the bovine serum albumin (BSA) in the mTeSRTM1 media was found to be critical for cell adhesion on APMAAm surfaces, signifying the involvement of biological molecules in the cell–synthetic substrate interfaces. Interestingly, the cell adhesion on the surface with the soluble growth factors either bFGF or TGF-b was lower than the case when used only with BSA; moreover, the quartz crystal microbalance analysis of the protein adsorption study revealed a dominant adsorption of BSA over other proteins in the medium. The results indicate that the protein layer adsorbed to APMAAm surface is primarily BSA and the altered interface supports the long-term growth of human ESCs. Although the authors used specific aminated surface of APMAAm and observed BSA as the dominating protein layer, the finding highlights an important biological event of the competitive adsorption of proteins in monolayer that generally occurs at the engineered biomaterial interface and its significance in the biomaterial-based culture of PSCs.

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Fig. 4.2. Fraction of adhered human ESCs after 24 h of culture in mTeSR1 media on hit polymer arrays coated with either human serum (HuSerum) or human vitronectin (HuVitronectin) and with the specified integrin blocking antibody. b1 blocking had minimal effect either alone or in combination with avb5 and avb3 blocking, whereas both avb5 and avb3 blocking reduced adhesion. Modified with permission from Ref. [136].

As discussed, the surface modification of biomatrices with either chemical groups or ECM molecules has enabled longterm cultures of PSCs without losing the pluripotency. One of the key considerations here is that the cellular interaction with the underlying substrate is through the protein molecules adsorbed which can ultimately mediate the surface receptors of PSCs to recognize the substrate – this offers a rationale on the proper selection of chemical groups and molecules, and helps interpreting the PSC self-renewal phenomena in relation with the protein adsorption, which remains as future study.

4.2. Physical modifications 4.2.1. Nano-/micro-topography Surface topologies tailored at the micro/nanoscale have been shown to play key roles in regulating the cellular interactions with matrices. This also applies to the pluripotency behavior of PSCs. The rationale is that the adhesive molecules and integrins of PSCs recognize this nano/micro-scale topology, and the receptor–matrix interactions contribute to governing cell spreading, cell–cell interactions, and the pluripotency phenotype expressions. In order to obtain the topological features, several strategies have been used, such as coating with different sized nanoparticles (such as carbon nanotubes; CNTs) or the use of photolithography (to generate nanogrooves or nanopillars in random or ordered). CNTs have often been used to produce the surface with nanotubular structures. Mouse iPSCs cultured on collagen coated multi-wall CNT culture dish formed hemi-round colonies and strongly expressed early undifferentiation markers after 5 days. On the other hand, when the cells were cultured in the absence of the CNT, cells had the tendency to spread and weakly expressed undifferentiated markers, implying that the nanotopological CNTs devoted to maintain the pluripotency of mouse iPSCs [142]. Another strategy is to change the surface nanotopography by sonication of CNTs during different periods of time to achieve different roughness values between 465 nm and 75 nm. The human ESCs were shown to

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Fig. 4.3. Pluripotency of ESCs maintained on the engineered APMAAm substrate over 22 passages. (A) Representative confocal images at passage 10 on APMAAm and Matrigel stained for (c and f) Dapi, (d and g) Oct-4 and (e and h) SEEA-4. (B) Quantitative immunostaining of H1 and H9-hOct4-pGZs for pluripotent markers and RT-PCR results (RQ) of the markers. (C) H9-hOct4-pGZ attachment to APMAAm surfaces after 24 h in complete mTeSRTM1 and incomplete mTeSRTM1 supplemented with BSA, TGF-b, or bFGF. Presence of BSA allowed four times higher cell attachment compared to bFGF or TGF-b. Modified with permission from Ref. [141].

prefer rough surfaces compared to smoother surfaces (Fig. 4.4) [143]. However, the CNTs are electrically conductive, which might complicate the nanotopological effects on the pluripotency maintenance of cells and the mechanisms behind. While the CNTs provide nanotopological surfaces, the structure is rather limited – nanotubular and random – thus other nanotechnological processings are more widely used to generate different structured nanotopologies, such as nanogrooves and nanopillars. Nanolithography can generate nanopillar patterns with gradient pillar diameter ranging from 120 to 360 nm with fixed distance of 440 nm, offering an ordered nanotopology platform for examining the effect of nanotopography on the maintenance of human ESC. The human ESCs formed compact colonies expressing high levels of undifferentiated markers on the small diameter (120–170 nm) pillars. On this small region, the cell colonies made very limited adhesion with the nanopillars. The colony morphology was spherical with fine peripheral outgrowths of filopodia and limited protrusions of lamellopodia, which was contrasted to the flat and broad lamellopodia observed in mid (170–290 nm) and high (290– 360 nm) regions (Fig. 4.5) [144]. The limited cell–substrate interaction on the small pillars hindered cell spreading, but forced the cells to rely more on cell–cell interaction mediated by E-cadherin, a phenomenon closely related to pluripotency of ESCs. Not only the size, but also the shape of the topographical features showed an effect on the behavior of PSCs. While flattened colonies were primarily observed on groove and pillar surfaces of polyacrylamide gel, spheroid colonies were dominant on hexagonal surfaces [145]. Although the mechanism needs more in-dept study, it is expected that the cells are able to fill the pits on the hexagonal substrates with the expression of Oct-4 and Nanog highly regulated. This was a result of altered distribution of focal adhesion complexes as well as modulated cell tractions. An interesting study has recently been carried using dendrimers to culture human iPSCs. Depending on the amount of dendrimers on the surface which was enabled by the generation passage, i.e., 1st, 3rd, 5th generation, and presenting increasing amounts of dendrimers, the selective maintenance of stemness or differentiation was possible; the lower density (1st generation dendrimers) allowed the maintenance of the undifferentiated state of iPSC, whereas the higher density (5th generation) exhibited early commitment to differentiation toward an endodermal fate (Fig. 4.6) [146]. The 1st generation dendrimers enhanced the formation of tightly packed colonies by enhancing the cell–cell contact, whereas the 5th generation promoted the formation of an aggregated colony with a ring-like structure. This phenomenon was correlated with the amount of adsorbed fibronectin. The increased dendrimer generation was able to promote the initial adsorption of fibronectin; thus possibly enhanced the secretion of fibronectin from the feeder cells, which increasing the cell–substrate interactions while reducing the cell–cell contacts. The signaling mechanism was reasoned to be the

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Fig. 4.4. Mouse iPSCs cultured on CNT surfaces with different roughness levels. (A) (a–c) Phase contrast and (d–f) Hoechst nucleus staining of cells. High roughness surface showed extensive growth after 120 h, intermediate roughness showed clustered colonies with limited expansion after 120 h, and low roughness surfaces showed no adherence after 36 h. The graph represents the number of cells at 36 h for each condition. (B) Colony growth on low surface CNT surface vs. Matrigel or feeder cell layer (left), and the percentage of viable cells after 5 days plating (right). Modified with permission from Ref. [143].

endogenous Rho family GTPases Rac1 and E-cadherin, which expressed and distributed differently in the cell colonies depending on the generation of dendrimers. These above findings prove the possible role of nanotopological surfaces in maintaining undifferentiated PSCs and provide an effective technological platform for developing feeder free PSC culture systems for the purpose of regenerative medicine. While only a limited set of nanotopological platforms (including CNTs, dendrimers and nanogroove-/nanopillar-patterns) have been studied thus far, many other nano-structured surfaces that have been developed in the nanotechnology area may help in better understanding the mechanism of nanotopological effects on the pluripotency maintenance of PSCs. 4.2.2. Matrix stiffness Along with the surface topography, the stiffness of the matrix has been considered an important parameter of physical properties of biomaterials that determines the cellular behaviors, including the adhesion, proliferation, migration and differentiation of various cell types [147–149]. The biomaterials that match the stiffness level of the ECM of the cells in concern are preferred to maintain and drive them to express proper phenotypes [96,150]. For the preservation of PSC pluripotency the

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Fig. 4.5. Control of human ESC pluripotency on the nanopatterns tailored with different domain sizes. (A) Nanopillar patterns with different sizes of domain and spacing. Scale bar (SEM images) = 1 lm. (B) Expression of undifferentiated markers in human ESCs analyzed by qPCR. (C) SEM images of cells cultured on three different nanopatterns. Low nanopillar surface reveals colonies with fine filopodia and limited lamellipodia protrusions, whereas mid and high nanopillar surfaces exhibit flat and broad protrusions of lamellipodia (black and white arrows indicate filopodia (F) and lamellipodia (L), respectively). (D) Confocal immunocytochemistry of E-cadherin, phalloidin and b-catenin on the different substrates (top). Cell spreading images within human ESC colonies and the quantitative measuring of an average distance of the nucleus of each cell from the center of the colony (bottom). Modified with permission from Ref. [144].

stiffness of a matrix has a significant impact: the PSCs sense the elasticity of surfaces and the force exchange preserves selfrenew. Thus synthetic and natural polymeric materials have been developed to possess the stiffness level that the PSCs favor to self-renew without losing the pluripotency. In fact, there has been no given reference to this matrix stiffness of PSCs; however, when we consider the ECM stiffness values of different tissues (as provided in Fig. 4.7), the PSCs are considered to favor very low stiffness levels – a range of less than a few kPa (comparable to or lower than neural tissues, but possibly higher than the hematopoietic niche). Based on this rationale of stiffness, certain compositions of biomaterials that could satisfy the processing techniques were engineered, and the effects of stiffness values on the PSCs self-renewal behavior were investigated. Different stiffness levels could be obtained on the synthetic or natural biopolymers by changing the molecular weight or the crosslinking conditions. First, poly(L-lysine) (PLL) nanofilms that present different stiffness values (from 190 to 400 kPa) were prepared by changing the crosslinking degree. The pluripotency of mouse ESCs was maintained better on the noncrosslinked films (lowest stiffness) than on the crosslinked films (Fig. 4.8A). It was also hypothesized that the release of PLL due to poor crosslinking could create a charged environment that allowed ESCs to aggregate together. Therefore, the surface chemistry as well as the stiffness might play a combined role in modulating the self-renewal and fate of the cells [152]. In a similar approach, hyaluronic acid (HA) with different molecular weights was immobilized onto the coverslips to examine its effect on the pluripotency and proliferation of mouse ESCs (Fig. 4.8B) [95]. The interactions between the cells and the surface were shown to be highly dependent on the molecular weight of HA. The ESCs interacted with high molecular weight HA via CD44, whereas they interacted with low molecular weight HA mostly via CD168, which is indicative of pluripotency of ESCs [153]. The results concluded that the low molecular weight HA was more effective in maintaining the ESCs in a viable and undifferentiated state. Polyacrylamide (PAA) gel has often been a model substrate to study the stiffness effects on cellular behaviors, including MSC differentiation [154–156]. A recent work also compared the ability of different stiffness gels to maintain stemness of ESCs. Two stiffness values (soft 6.1 kPa and stiff 46.7 kPa) were compared. The stemness of ESCs was shown to be preserved better in the soft substrate, expressing higher Oct-4 and Nanog [145]. In a similar way, a much softer substrate (E = 0.6 kPa) that is considered to mimic the mESC niche, was compared with a stiffer substrate (E = 3.5–8 kPa) [157]. The softer matrix presented undifferentiated colonies for a series of different mouse ESC lines, even without exogenous LIF, exhibiting upregulation of Oct3/4 expression and alkaline phosphatase activity, up to at least 15 passages, which suggested the possibility of long-term cultures (Fig. 4.9) [157]. Combining those two results on PAA gels, the softer the matrix the better the PSCs were shown to preserve pluripotency; furthermore, when we consider the whole range of the stiffness examined in the two studies (softest E = 0.6 kPa; middle E = 3.5, 6.1 or 8 kPa; stiffest E = 46.7 kPa), it is envisaged that the softest stiffness value may provide the most favorable matrix conditions for PSCs to self-renew and preserve pluripotency, and this level (less than

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Fig. 4.6. Cell morphology and pluripotency of human iPSC cultured on dendrimer-like substrates with different generations (G1, G3 and G5). (A) Optical cell images for up to 5 days of culture. Apoptotic cells (red arrows) were also noticed. (B) Immunofluorescence staining of Oct3/4 (green) and DAPI (blue). Scale bar = 400 lm (top). Quantitative PCR assay showing undifferentiated cell markers (Oct3/4, Nanog, and Sox2) and the three germ layer markers (bottom). (C) Immunofluorescence staining of Rac1 and E-cadherin after 5 days. Scale bar = 400 lm. Modified with permission from Ref. [146].

Fig. 4.7. Stiffness range for the different cells and tissues. Reprint permission from Ref. [151].

1 kPa) is considered to mimic the native physical environments of PSCs to survive. Recently, a result seemingly opposite to this phenomenon on the stiffness effect was provided [158]. However a closer look at the results revealed the stiffness range was totally different; three different stiffness values (10.3, 25, and 30.4 kPa) were engineered using polyvinyl alcohol (PVA) gel substrates. From the report, an intermediate stiffness (25 kPa) showed higher pluripotency for 20 passages compared to stiffer (30.4 kPa) or lower (10.3 kPa) stiffness; although lower stiffness also showed enhanced maintenance compared to the 30.4 kPa substrate, the optimum was 25 kPa. Although more factors such as surface chemistry, composition or even cell type were reasoned to be involved in the sensing mechanism of cells in terms of stiffness [158], the stiffness range that the authors investigated are not considered optimal, i.e., much higher to drive PSCs to optimally preserve pluripotency. Taken together the findings on the stiffness effects of matrix surfaces on the pluripotency behaviors of PSCs, the lower stiffness matrix favors PSCs to self-renew. The low cell–matrix tractions and the consequent low stiffness of cell colonies

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Fig. 4.8. Effects of stiffness level on the self-renewal capacity of mouse ESCs. (A) Stiffness increased with increasing EDC crosslink concentration (10, 40, 70, and 100 mg/ml) in PLL-HA solutions; cells observed at day 4, showing better colony formation and self-renewal in the lower stiffness substrate. Scale bar 100 lm, 5 lm (inset images). (B) Stiffness varied with different HA molecular weight; high molecular weight (HMW) (IV–VI) and low molecular weight (LMW) HA immobilized on coverslips (VII–IX) compared with gelatin (I–III). Fluorescence images showing the expression of main pluripotent marker Oct3/ 4 (red) (I, IV, VI) with DAPI nuclei stain (blue) (II, V, VIII) and their combined image (III, VI, IX), showing higher Oct3/4 on the LMW substrate. Reprint permission from Refs. [95,152].

were largely noticed on softer substrates, envisaging that the PSC self-renewal and pluripotency could be maintained homogeneously on softer substrates via the biophysical mechanism of lowered cell–matrix tractions. Not the stiffness, but the surface hardness was also considered in the self-renewal behavior of PSCs. Hardness, defined as a resistance to a plastic deformation of a material, is a slightly different mechanical property from stiffness, which is a proportionality of a strain (normalized deformation) with respect to an applied stress (normalized force). While hardness is related more with impact force, stiffness is considered under tensile-, compressive- or shear-force conditions; thus both values cannot be directly converted each other, but are closely related (mostly proportional). Polyethylene terephthalate (PET) membranes with different surface hardness values (0.135–0.345 GPa) were used to observe the self-renewal ability of human ESCs [159]. Results showed that an intermediate range of hardness (0.291 GPa) supported optimal self-renewal and proliferation of human ESCs than the other hardness surfaces (Fig. 4.10). While the authors reported the effect of hardness parameter, the corresponding elasticity (stiffness) of the matrices is also considered to correlate similarly. One thing to note is that, the PET substrates used in that study were relatively hard (and also stiff), similar to the study of using PVA gel with a relatively higher stiffness range – thus observing that the intermediate range was optimal; however, matrices with much lower hardness values would be better to interpret the PSC self-renewal behaviors. Conclusively, the stiffness of matrices is an important physical parameter that determines the microenvironment to which PSCs sense and decide to preserve or change pluripotency, and the stiffness level tailored to that of native niche (lower than at least a few kPa) is believed to favor their maintenance of pluripotency. The study reporting the preservation of pluripotency of mouse ESCs only via matching the substrate stiffness, while nullifying the effect of differentiation biochemical signal LIF, reflects the importance of physical cues. Unfortunately, most studies have explored PSC behaviors on 2D matrices with varied stiffness values, which can limit full demonstration of the native 3D niche phenomena; therefore future works are required to design 3D culture models with tailoring of physical stiffness level.

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Fig. 4.9. Polyacrylamide gels with different stiffness levels used to maintain pluripotency of mouse ESCs. (A–D) Oct3/4 FACS plots of mouse ESCs cultured on soft (0.6 kPa) and rigid (>4 MPa) substrates with or w/o LIF. (E and F) Representative plots showing high Oct3/4:GFP expression of cells with or w/o LIF, showing a reduced number of Oct3/4:GFP+ cells when cultured on the rigid substrates w/o LIF. (G) Data summary of Oct3/4:GFP+ cells. Modified with permission from Ref. [157].

Fig. 4.10. Effects of matrix hardness on the self-renewal capacity of human ESCs. Ethylene-based substrate with intermediate hardness (0.2913 GPa) shows reduced Rho-associated kinase (ROCK-1) by RT-PCR, indicating lower cell differentiation compared to the other substrates. Controls are cells harvested with peripheral cells (Con/W) and without peripheral cells (Ex-Peri). Immunostaining of pluripotent marker (TRA1-60) shows higher expression on the 0.2913 GPa substrate. Modified with permission from Ref. [159].

4.3. Three dimensional environments Not just the surface (chemical or physical) conditions of 2D matrices, but the 3D environments have significant impact on the responses of various types of cells [160–164]. For instance, MSCs cultured on 3D microspheres presented higher

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expression of osteoblastic genes and mineralization when compared to those on monolayer cultures, which was reasoned from the altered traction forces of cells induced by the round surface [162]. Another example is the culture of MSCs on the 2D and 3D conditions of collagen type I, where the 3D system enhanced the differentiation of cells into different lineages, especially adipogenic lineage, although the mechanism is not clarified [164]. Recently, 3D graphene foam was used to

Fig. 4.11. 3D scaffolds (collagen, PLGA, and chitosan) for the pluripotency maintenance of mouse ESCs, compared to flat 2D surfaces. (A) SEM images of porous scaffold morphology. (B) qRT-PCR for pluripotent markers (Oct4 and Nanog) and differentiating marker (Sox2) of mouse ESCs cultured for 5 days in different scaffolds compared to those cultured on feeder cells (C2DmES) and to those differentiated 5 days from (EB 5 day). ⁄⁄ Indicates significant differences compared to C2DmES group. (C) Immunostaining of mouse ESCs cultured for 5 days. Scale bar 100 lm. Modified with permission from Ref. [165].

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differentiate neural stem cells (NSCs). Compared to 2D graphene substrates, 3D foam structures upregulated Ki67 expression and stimulated differentiation into astrocytes and neurons [163]. The 3D cultures were also shown to be influential on the long-term self-renewal of PSCs. Recently, the change from 2D to 3D culture matrices was shown to significantly alter the self-renewal ability of ESCs, suggesting the possibility to preserve pluripotency of cells using scaffolds with an extra 3-dimensionality (Fig. 4.11) [165]. The 3-dimensinality as well as the fibrillar pore structure enabled ESCs to conform less flat morphology than 2D culture condition. This cellular shape was then correlated with the membrane proteins differently distributed between 2D and 3D, which played a significant role in cell– cell contact and cell–matrix interaction; consequently contributing to maintenance of the stemness of ESCs with upregulation of Oct4 and concurrent down-regulation of Sox2. In fact, the methods to provide 3-dimensionality to cells are through either cell-encapsulating hydrogels or prefabricated scaffolds. The hydrogels incorporate cells initially before the gelation, thus allowing them to distribute homogeneously throughout and them to recognize the matrix. The prefabricated scaffolds, produced in the form of either porous foams, nanofibers, or microspheres, can provide 3-dimensionality at the time of cell seeding, which however, often limits uniform distribution of cells; thus the stage of cellular response to the 3D matrices is variable, i.e., initially anchored cells at the subsurface need to migrate to recognize the 3D network deep inside the scaffold. In the following, the 3D culture studies of PSCs to improve the self-renewal capacity are discussed in terms of those (i) encapsulating cells within 3D gel environments and (ii) seeding cells on the prefabricated 3D shaped scaffolds. 4.3.1. 3D gel encapsulation Hydrogels can be effectively used for PSC culture by encapsulating the cells within the geometry of materials. Because of their high water content and hence their soft nature while preserving a 3D-networked structure, they are considered to mimic the in vivo ECM environments that favor cells to properly adopt and conform proliferative and self-renewal status. Many natural and water-based synthetic polymers can be designed to encapsulate cells and then to form gels. Although chemically-modified and bioactive polymers have been progressively engineered for the encapsulation of tissue cells including MSCs, NSCs, chondrocytes, and cardiomyocytes [166–170], those studied for PSC self-renewal are relatively simple compositions. Among others, the alginate system (mostly in bead form) is relatively more widely-studied hydrogel to store PSCs. The human ESCs encapsulated in calcium alginate beads (2 mm) grew in basic maintenance medium for up to 260 days and showed pluripotency without showing the formation of three germ layers. At the ultrastructural level, the cells presented a closely packed morphology without cytoplasmic organelles, demonstrating a typical undifferentiated state (Fig. 4.12)

Fig. 4.12. Alginate hydrogels encapsulating human ESCs for the prolonged culture and self-renewal. (A) Human ESC aggregate in the hydrogel with no sign of differentiation or cyst formation. (B) Morphology of decapsulated human ESCs after 110 days of culture in the hydrogel. (C) Viable cell cultured for 100 days in the hydrogel. (D and E) Transmission electron micrographs showing ultrastructure of (D) human ESCs encapsulated in alginate hydrogels without signs of organelles after 110 days, indicating their pluripotency and (E) non-encapsulated human ESCs showing the presence of organelles indicating their differentiated status. Modified with permission from Ref. [87].

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[87]. Similarly, the alginate beads (400–500 lm) were shown to be a good candidate to effectively culture human ESCs in serum replacement medium or human fetal fibroblast-conditioned medium, with the treatment of a ROCK inhibitor Y-27632, resulting in enhanced cell survival, cell cluster formation and proliferation. This was eventually aimed to isolate human ESCs and to use them for differentiation into definitive endoderm because the alginate encapsulation system could allow the immune-isolation and prevention of teratoma formation of human ESCs during transplantation [171]. Due to the micron size and spherical morphology, the bead culture is a scalable approach to expand PSCs in bioreactor. For instance, alginate hydrogel microcapsules combined with PEG showed excellent ability to expand mouse iPSCs with limited differentiation into an endodermal lineage. Furthermore, the beads can allocate signaling cues, such as adhesive molecules or growth factors that can alter the cellular behaviors [172]. The alginate hydrogel was further engineered into 3D microfibers to support long-term self-renewal of human ESCs and iPSCs under feeder-free and chemically-defined conditions (Fig. 4.13) [173]. The microfibers were fabricated from interactions between cationic (e.g. chitin) and anionic (e.g. sodium alginate) polymer solutions, allowing cell encapsulation in the alginate. This system showed uniform and high cell loading with a capacity to maintain self-renewal of cells.

Fig. 4.13. Alginate-based microfibers (AWC) used to provide 3D environments for the pluripotency maintenance of human iPSC and human ESCs. Matrigel used as a control gel. Data showing similar doubling time and Oct4-positive cell fraction with increase in passage number. Modified with permission from Ref. [173].

Fig. 4.14. Engineered integrin signaling 3D hydrogels based on PEG to promote mouse ESC self-renewal. Pluripotency preservation observed by (A) Nanog protein expression and (B) Akt1 gene expression in 2D and 3D PEG-hydrogels containing various integrin sets. Significant differences noted. Modified with permission from Ref. [174].

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Furthermore, the cells could be stored under cryopreservation while maintaining their self-renewal phenotype and genetic integrity. Similar to the alginate beads, HA hydrogels were also used to encapsulate ESCs for the maintenance. In the synthetic HA hydrogel, human ESCs formed colonies and maintained undifferentiated states for up to 20 days in the presence of conditioned medium from mouse embryonic fibroblast feeder layers [94]. The functionalization of 3D hydrogel networks with specific molecules provides favorable physical and biochemical environments for PSC maintenance. Polyethylene glycol (PEG)-based hydrogels functionalized with different integrin subsets to mimic native adhesive receptors is an example. The transcriptional analysis of 24 integrin subunits followed by a confirmation at the translational and functional levels suggests that a5b1, avb5, a6b1 and a9b1 are effective in maintaining the undifferentiated state of mouse ESCs (Fig. 4.14) [174]. Although different compositions of hydrogels can be developed to provide 3D tissue-mimic environments for PSCs, the chemical modification or molecular tailoring of the hydrogel networks to control PSC behaviors has been limitedly carried out, which thus remains as an important unexplored research area. Furthermore, the modulation of stiffness of the gels and the investigation into PSC pluripotency under 3D culture environments also warrant future exploration. The biochemical and physical parameters studied on 2D surfaces should be re-investigated under the 3D matrix conditions that can optimally mimic the in vivo tissue. 4.3.2. 3D geometrical scaffolds Along with hydrogels, 3D-shaped polymeric scaffolds including porous foams, fibers, and microspheres have also been used to culture PSCs that maintain the ability to self-renew. Compared to hydrogels, the 3D-shaped preformed scaffolds can be more diverse in the composition because many synthetic polymers processed under organic solvent conditions are also candidates to culture and self-renew PSCs. However, the synthetic polymeric scaffolds developed with stable networks and solid thick frameworks generally possess physical stiffness values higher than those of natural polymers and mainly of hydrogel forms, thus the choice of 3D matrices for the maintenance of PSC pluripotency is also considered limited. As a first report, porous scaffolds of natural polymers composed of alginate and chitosan were prepared to support the self-renewal of human ESCs without feeder cells or conditioned medium [175] (Fig. 4.15). The results showed that the pluripotency of the self-renewed human ESCs was maintained in vitro and in vivo. Furthermore, the cells recovered by decomposing the scaffolds in mild conditions still maintained their pluripotency in further subcultures, offering an alternative to the hydrogel-based maintenance of cells. While the hydrogels need enzymes or sometimes toxic agents to isolate cells, the scaffold system applies relatively mild conditions to gather the cells. While many different forms of polymeric scaffolds can be developed, the electrospun nanofibrous scaffolds are considered to be one of the most attractive cell culture matrices [176–179]. The simplicity and versatility of electrospinning process enables a wide range of compositions and morphologies (alignment and fiber size) to be achieved [180–184]. More fascinating is the nanofibrous network that is considered to mimic the native ECM fibers [185–187]. However, the line-ofsight process creates a very thin membrane structure, which is thus considered ‘pseudo-3D’ (rather than ‘exactly 3D’) structure, holding some limitations in completely mimicking the native 3D architecture and in progressive cellular penetration [176–179,188]. Even so, the electrospun nanofibers have been potentially used to cultivate stem cells to provide ECM mimic matrix and to interpret the cellular interactions with the underlying ECM-like nanostructure [185,189,190]. Furthermore, owing to the ultrathin nanofibrous network, the electrospun scaffold can possess very low rigidity (stiffness) providing a sufficient flexibility for cells to sense the mechanical elasticity; albeit its preformed nature, this is different from the case of other scaffolds, like solid foamed or microfiber networked ones, offering a matrix platform suitable for the PSC culture. Collagen (type I)-grafted polyethersulfone (PES) nanofiber matrix was prepared to culture mouse ESCs [191]. Cells presented an undifferentiated morphology and enhanced proliferation upon the matrix, maintaining the stemness for 7 passages. Moreover, the number of colonies expressing pluripotency markers, including Oct-4 and SSEA-1 (stage-specific embryonic antigen 1), was significantly higher on collagen-grafted PES nanofiber compared with uncoated PES nanofiber. Interestingly, when the cells were cultured on a gelatin-coated nanofiber scaffold, the pluripotency was not well preserved. Thus the grafted collagen, as one of the major ECM components, was hypothesized to provide a biochemical cue to play a major role in self-renewal of mouse ESCs [191] (Fig. 4.16a and b). This is one of the rare representative studies that combine biomolecular tailoring with 3D matrix in the culture of PSCs. Upon the nanofibrous 3D environment, the integrin (a2b1) in mouse ESCs is considered to recognize the collagen type I ligand specifically in a way to stimulate the proliferation and expression of stemness markers. The significant role of electrospun nanofibrous structures played in maintaining the pluripotent state of PSCs has also been demonstrated in different compositions, such as poly(e-caprolactone) (PCL) containing calcium deficient hydroxyapatite crystals [192], and poly(lactic-co-glycolic acid) (PLGA) [193]. One of the recent studies systematically analyzed the effect of nanofiber size and gelatin surface density on the self-renewal of human ESCs using gelatin nanofibers. The nanofiber size ranged from 70 to 500 nm by altering the gelatin concentration while the gelatin density ranged from 0.1 to 5.8 lg/cm2 by changing the amount of gelatin incorporated. Results revealed that the nanofiber with intermediate size (240 nm) and high gelatin density (4.8 lg/cm2) was optimal in cultivating human ESCs (H9 and H1) for long-term periods without sacrificing a self-renewal capacity [194] (Fig. 4.16c and d). Such biochemical and nanotopological

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Fig. 4.15. Alginate porous scaffolds for the pluripotency maintenance of human ESCs. (A) SEM image of scaffold morphology and the pore size distribution measured by mercury intrusion porosimetry. (B) Proliferation and pluripotency (ALP activity) of cells compared to those cultured on human fibroblast feeder (hFF) cell layer. (C) mRNA levels of various genes expressed in human ESC cultured for 21 days in the alginate scaffolds (data normalized to those cultured on hFF cell layer). Modified with permission from Ref. [175].

conditions provided by the gelatin nanofibers are considered to offer anchorage sites for human ESCs optimally, i.e., a weak adhesive substrate for cells; in fact, a higher cell adhesion was observed on the low density nanofibers and 2D glass surfaces where the human ESCs made cell–matrix interaction more but cell–cell contact less, eventually losing the pluripotency markers. Polymeric microspheres have often been used to support and cultivate cells, multiplying them under dynamic conditions and consequently deliver into tissue defects [195–198]. Moreover, they can carry therapeutic molecules inside to provide signaling cues to cells. Some of the PSC studies have also utilized the polymeric microspheres. Polystyrene microspheres ranging from 200 nm to 2 lm were prepared and functionalized with amino groups to deliver molecules such as fluorescent dyes, proteins, and nucleic acids. The results showed that microspheres did not require a transfection reagent and could successfully deliver the molecules without toxicity or loss of pluripotency of the ESCs [199]. The ESCs

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Fig. 4.16. Nanofibrous scaffolds used to maintain the pluripotency of human ESCs. (A) SEM images of cell colonies cultured on collagen-grafted polyethersulfone (PES-COL) and polyethersulfone (PES). (B) Oct4 and Nanog gene expressions by qRT-PCR analysis. ⁄ Indicates significant difference. (C) FACS and (D) RT-PCR analysis of stage specific embryonic antigen (SEE4) cultured on the nanofibrous scaffolds, showing comparable levels to those cultured on Matrigel (positive control). Modified with permission from Refs. [191,194].

attached on microspherical carriers by a spinner flask culture system also maintained their pluripotency. Commercially available microspheres, Cytodex 3 and Cultispher S, were used as a carrier for mouse ESCs. A number of mouse ESCs can be expanded while maintaining pluripotency on the Cultispher S, a macroporous microspherical carrier, in serum-free conditions [200]. Meanwhile, trimethyl ammonium-coated polystyrene microspheres were tested as microspherical carriers for human ESCs and showed a prolonged self-renewal of 6 passage with a 14-fold increase in cell number [201]. As discussed, these microspheres can be useful to cultivate PSCs under the dynamic culture conditions like spinner flask cultures, and to apply them as an injectable delivery system into tissue defects, but the area needs further studies. Table 4.1 summarizes the representative biomaterials that have been used to culture PSCs that maintain the pluripotency and self-renewal capacity of the PSCs.

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Table 4.1 Summarizing representative biomaterials-based cultures of PSCs to preserve the pluripotency and self-renewal capacity. Culture type 2D culture

Cell type

Period

Note

Ref.

Chemical groups

COOH gradient surface

mESCs (E14, R1)

7 days

[128]

Biomolecular tethering

APMAAm coated surface Acrylate polymers + serums or proteins ECM protein/HSPG-coated culture plate Multiwalled CNT-coated culture plate

hESCs (H1, H9) hESCs (BG01v, WIBR3) hiPSs hESCs (WA09, BG01v)

20 passages 10 passages

MEF-derived iPSCs

Multiple passages 5 days

Self-renewal is maintained when the cell spreading is restricted to <120 lm2 Similar results between the APMAAm substrate and Matrigel High acrylate content and adsorbed vitronectin promote colony formation Fibronectin/HSPG coating was shown to support self-renewal

[142]

Nanopillar array

hESCs (H9)

4 days

Matrix stiffness

PLL/HA nanofilm PET membrane HA coated coverslip

mESCs (CGR8) hESCs (HSF6) mESCs (EB3)

Not specified 10 passages 3 days

iPSCs on CNT formed hemi-round colonies and expressed early undifferentiation markers Undifferentiated state of colonies maintained better on smaller pillar diameter region Pluripotency was better maintained on low stiffness film Pluripotency was better maintained on 0.291 GPa membrane Low molecular weight HA better supported self-renewal

Gel environment

Alginate beads

hESCs (H1)

260 days

Porous scaffold

Alginate microfiber

14 days 10 passages

HA scaffold

hESCs hESCs (HUES7, BG01V/ hOG) hiPSC (PD-iPS5, hFib2iPS4) hESCs (H1, H9, H13)

PEG/dicystein-containing peptides

mESCs (E14tg2a)

Not specified

Alginate/chitosan

hESCs (BG01v)

21 days

Collagen grafted PES scaffold

mESCs

7 passages

PCL/CDHA scaffold Cytodex 3, Cultispher S Trimethyl ammonium-coated PS microsphere

mESCs (R1) mESCs (46C) hESCs (ESI-017)

3 days 8 days 6 passages

3D culture

Electrospun scaffold

Microspheres

20 passages

[137]

[144] [152] [159] [145]

Cells encapsulated presented a packed morphology without cytoplasmic organelles, demonstrating a typical undifferentiated state Cell viability was enhanced by ROCK inhibitor treatment Long-term self-renewal of human ESCs and iPSCs under feederfree and chemically-defined conditions

[87]

ESCs formed colonies and maintained undifferentiated state in the presence of conditioned medium from mouse embryonic fibroblast feeder layers Integrins a5b1, aVb5, a6b1 and a9b1 play important roles in selfrenewal Pluripotency of the self-renewed human ESCs was maintained in vitro and in vivo Pluripotency markers were significantly higher on collagengrafted PES nanofiber compared with uncoated PES nanofiber ESCs were able to proliferate in a pluripotent state Porous microsphere was more useful for cell culturing Prolonged self-renewal of 6 passages with a 14-fold increase in cell number

[94]

[171] [173]

[174] [175] [191] [192] [200] [201]

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Nano/microtopography

[141] [136]

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5. Biomaterials for lineage differentiation of PSCs Regulating differentiation into a desired lineage is necessary to apply the pluripotent cells more safely and effectively to clinical settings. To achieve this, the cell culture platforms have been chemically and physically modified to provide differentiation cues, and engineered to endow 3D scaffolding conditions for the PSC-derived tissue constructs in regenerative medicine. While the parameters of biomaterials tailored to maintain the pluripotency of PSCs are largely limited with narrow stiffness values and defined adhesive motifs, those for the differentiation of PSCs can be more diverse; depending on the target cells the biochemical functionality and physical nanotopology and stiffness should be optimized, and the properties of 3D gel matrix and scaffolds need more targeted design. When the matrices are tailored to mimic the ECM properties of the target tissue, PSCs can be more likely to change their fate into the lineage cells. In many cases, the differentiation and maturation to functional cells require long-term periods; therefore, the cultures on the biomaterials should be robust without a breakdown (due to degradation) or eliciting toxic elements, and different cocktails of signaling molecules should be provided in a time-dependent manner. During long-term cellular processes, a homogeneous and well-developed population of differentiated cells is highly recommended for safe and effective clinical applications. In this part, we survey the literatures that investigated the lineage differentiation of PSCs using biomaterials tailored with different properties and designs.

5.1. Surface tailoring As mentioned earlier, surface chemistry is considered a first motif to tailor biomaterials in order to provide PSCs proper sites to adhere and grow. PSC differentiation down to a lineage in concern also requires surface dependent cellular recognition and process. Signaling molecules and proteins have been functionalized on the biomaterials surface to drive PSCs to differentiate into specific lineages. Compared to the surfaces used for PSC self-renewal, where simple chemical groups were found to be supportive, those for differentiation were tailored to be more functionalized with specific signaling molecules which have already been identified in other studies like primary cell cultures and differentiation of progenitor cells. Moreover, the physical and chemical tailoring was often explored on the 3D scaffolds to drive PSCs more effectively to lineage cells, benefiting from the 3D culture conditions as well as the functionalized surfaces. Galactose, considered as a promising molecule for the specific regulator for liver cells, was used for the hepatocyte differentiation of PSCs. The polystyrene well plate was co-immobilized with poly(N-p-vinylbenzyl-4-O-b-Dgalactopyranosyl-D-gluconamide) (PVLA) and E-cadherin-IgC Fc as a galactose carrying substratum. Mouse ESCs cultured on the plate were shown to maintain their proliferative ability initially and then differentiated directly into a hepatocyte lineage, expressing substantial level of hepatocyte marker, asialoglycoprotein receptor ASGPR [202] (Fig. 5.1). These cells were re-seeded onto the PVLA coated surface, which allowed the selective attachment of the ASGPR-expressing hepatocytes while eliminating the cells differentiated poorly or into other lineages. Although the pristine E-cadherin substrate allowed the adhesion of other endodermal lineages, such as pancreatic cells, the co-use of PVLA was selective for hepatocytes, priming a surface for mouse ESCs to recognize the galactose molecules. Adhesion ligands for PSCs have been designed and tethered to biomaterials to regulate the adhesion of cells and further differentiation processes. The polystyrene well plate was functionalized with peptide ligands for a5b1 and a6b1 integrins those present on human ESCs. In the presence of differentiation factor bone morphogenetic protein-4 (BMP-4), human ESCs cultured on the substrate functionalized with both ligands expressed early mesodermal markers, while the cells cultured on the substrates functionalized with ligand either for a5b1 or for a6b1 alone, did not [98]. The results suggest that the synthetic substrate engineered with proper adhesive molecules can support a well-controlled niche for human ESC differentiation; furthermore, the integrin combinations were important for proper lineage differentiation of human ESCs (Fig. 5.2) [98]. Of note is that the use of other soluble differentiation factors (BMP-4 in that study) along with the adhesive ligand seems to be important when one seeks to differentiate properly. In a similar way, more studies on the differentiation to other lineages (such as endodermal and ectodermal) using different soluble factors might help in confirming if the adhesive ligand combination is effective in general. While the above study performed the differentiation of human ESCs down to one of the three germ layers, a commitment of final staged cells (e.g., osteoblasts, myoblasts and chondrocytes) may need additional inductive matrices and/or long-term culture conditions with more soluble supplements. The immobilization of soluble signaling molecules (like growth factors) onto the matrix improves the target differentiation [203–206]. Vascular endothelial growth factor (VEGF) was immobilized onto the biomaterial surface to induce the ESC differentiation toward an endothelial lineage. VEGF-A was covalently immobilized onto type IV collagen by carbodiimide crosslinking. The mouse ESCs cultured on this surface differentiated into primarily endothelial cells, whereas those on the surface without VEGF differentiated into vascular smooth muscle cells [207]. The immobilized VEGF was considered to be more potent in the molecular signaling of mouse ESC differentiation down to endothelial cell lineage compared to those toward smooth muscle cells, which was sufficiently guided by the underlying type IV collagen substrate; in fact, other growth factors like platelet derived growth factor has been shown to drive the differentiation into this vascular smooth muscle lineage. Because of the degradable nature of substrate biopolymer used, it might be expected that the immobilized growth factor could be released and negate the differentiation effect on cells; however, the immobilization was sufficient to guide the cellular adhesion and differentiation. Likewise, the differentiation of mouse ESCs toward cardiomyocyte could be induced by immobilizing insulin like growth factor binding protein 4 (IGFBP4) onto the polystyrene plate through the

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Fig. 5.1. Differentiation and isolation of mouse ESC toward hepatocytes using galactose based substrates. (A) Schematic representation to prepare hybrid substratum containing PVLA and E-cadherin for the differentiation of mouse ESC into hepatocytes. PVLA contains the galactose residue which binds with the ASGPR from hepatocyte. (B) Transcription markers of final differentiating stage to hepatocytes when cultured on the substrates, as analyzed by RT-PCR. (C) Secretion of key hepatic molecules after 20 and 24 days of differentiation. Modified with permission from Ref. [202].

Fig. 5.2. Biomimetic substrate functionalized with peptide ligands for mesodermal differentiation of human ESC. (A) Molecular design of substrates functionalized with either La5b1, La6b1, or both of them, showing that substrates were immobilized through cysteine linking onto gold coated substrates. (B) Mesodermal gene expression of human ESCs cultured for 48 h in differentiation medium (vs. Matrigel control). Modified with permission from Ref. [98].

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fusion of elastin-like polypeptides [208]. In fact, IGFBP4, as the smallest IGFBPs, binds IGF-1 and IFG-II and inhibits their actions under almost all in vitro and in vivo conditions, including cancer, reproductive physiology, bone formation and renal pathophysiology [209]. In recent study, the immobilized IGFBP4 was shown to promote cardiomyocyte differentiation of mouse ESCs by strong and continuous inhibition of Wnt/b-catenin signaling in the late phase after embryo body formation (Fig. 5.3) [210]. The elastin-like polypeptides were able to effectively immobilize the IGFBP4. Compared to the soluble form, the immobilized IGFBP4 particularly at low concentration showed more continuous and stronger inhibition of the Wnt/bcatenin signaling. This study again confirmed the biological role of signaling molecules could be enhanced when immobilized onto the matrix. While the defined ECM components such as the adhesive proteins and growth factors have been used mostly, the whole ECM molecules have also been tethered onto the biomaterial surface. For this, native ECMs were formed on the surface of PLGA microspheres by culturing osteoblasts for 14 days and then decellularizing the cellular constructs. The remaining ECMs were used to allow the culture and osteogenic differentiation of mouse ESCs. During the culture of cells in an osteogenic medium for 21 days the mouse ESCs were developed into an osteogenic lineage. Although the uncoated microspheres also induced mouse ESCs toward an osteogenic lineage with the supply of differentiating soluble factors, the ECM-coated microspheres significantly enhanced the osteogenesis and maturation of cells with higher levels of osteocalcin expression and calcium deposition than the uncoated microspheres (Fig. 5.4) [211]. The ECM of osteoblasts, holding sufficient level of biochemical molecules and biophysical properties mimicking native bone tissue, may properly guide PSCs to change their fate into an osteogenic lineage. Of note was that the study applied the biochemical modification strategy to the 3D scaffold matrix, aiming to utilize the functionalized scaffolds and cellular constructs for tissue regeneration, specifically bone. Apart from the biomolecules, a unique class of materials, carbon nanomaterials, has also been studied in the culture of PSCs and their differentiation. Carbon nanomaterials, such as graphene (G), graphene oxide (GO), and carbon nanotubes (CNTs) are considered intriguing substrate platforms to modulate PSC differentiation. While G is electrically conductive, its oxidized form GO loses this electrical property, which is thus often treated to a reduced form, reduced GO (rGO), to regain the electrical conductivity. Moreover, GO contains lots of oxides defects which serve as the site for molecular interactions [212–215]. On the other hand, CNT is also electrically conductive and nanotubular in morphology which can provide nanotopography as well as electrical conductivity to the substrate. For these reasons, some types of cells that need electrical signals like neural cells have been shown to be stimulated upon the carbon nanomaterials, primarily CNTs and rGO, in their neurite outgrowth (for primary neurons) and neuronal differentiation (for NSCs) [216–220]. On the other hand, GO, even though its low electrical conductivity, has been shown to stimulate the differentiation of other types of progenitor/stem cells, such as the differentiation of MSCs into osteoblasts, MSCs into chondrocytes, and muscle precursor cells into myoblasts [221–225]. This phenomenon is mainly related with the strong adsorption of relevant biological molecules that can play key roles in the adhesion and differentiation of cells.

Fig. 5.3. Bimolecular modification of substrates to enable the differentiation of mouse ESC. IGFBP4 functionalized polystyrene to promote cardiomyocyte differentiation. Modified with permission from Refs. [208] and [210].

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Fig. 5.4. Differentiation of human ESCs on ECM containing osteomimetic microspheres for bone tissue engineering. Osteoblasts were cultured for 14 days under osteogenic conditions to allow production of osteogenic related ECM molecules. Cells were then removed, exposing only the ECM osteogenic molecules on the surface of the microspheres. Analysis of osteomimetic microspheres after 14 day culture of osteoblasts, revealing (A) collagen II (scale bar 50 lm) staining and (B) ALP staining (scale bar 500 lm). (C–F) Confocal images at day 35 of differentiated human ESCs culture on osteomimetic microspheres, showing (C) actin (red), (D) DAPI (blue), (E) osteocalcin (green) and (F) merged image. (G) Higher expression of osteogenic genes compared to TCPS control and gelatin coated microspheres (Geltrex-coated PLGA). Modified with permission from Ref. [211].

Therefore, the PSC responses to the carbon nanomaterials are of an intriguing issue. The GO-coated glass substrate was prepared via functionalization with an amine group, whereas its reduction produced rGO-coated substrate. The mouse iPSCs cultured on rGO-coated substrate exhibited similar cell adhesion and proliferation to that on the glass substrate, however the cells cultured on the GO-coated substrate showed a faster adhesion and proliferation (Fig. 5.5A–D) [226]. In terms of differentiation, both of the surfaces enabled the iPSCs to spontaneously differentiate into ectodermal and mesodermal lineages; interestingly but, G suppressed the iPSCs differentiation into endodermal lineage whereas the GO augmented this type of differentiation. The higher adhesion of iPSCs onto GO- than rGO-coated surface might be due to the larger number of exposed active surface chemical groups (primarily carboxyl and hydroxyl groups) and thus the stronger adsorption of bioactive molecules in serum medium, which has also been noticed in the culture of other types of cells [227–229]. A notable aspect is the differential three-germ layer lineage specification of iPSCs between GO and rGO, and this was considered to be related with the significantly different polarity and hydrophilicity of the two surfaces, which might affect the type of iPSC surface receptors that could recognize, and regulate signal transduction and differentiation behavior. However, the transduction pathways of PSCs are poorly understood and thus the exact mechanisms underlying this phenomenon need further study. In a similar way, CNTs also stimulated the differentiation of human ESCs into neurons with good cell viability (Fig. 5.5E) [230]. Acid-treated CNTs were grafted with poly(acrylic acid) (PAA) and then deposited onto a glass coverslip. Compared to a poly-L-ornithine surface, a conventional material used for neuron growth, the PAA-grafted CNT thin film showed comparable viability and even better adhesion and neuronal differentiation of human ESCs. This study concluded that the neuronal differentiation of human ESCs was affected by the nanoscale tubular morphology of the CNTs which enhanced both protein

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Fig. 5.5. Carbon-based substrates for the differentiation of pluripotent cells. (A–D) Pluripotent and lineage specific mRNA levels of mouse iPSCs cultured on graphene (G) and graphene oxide (GO), compared to glass slide. (A) Pluripotent markers (Nanog and Oct4) were reduced with time indicating differentiation into different lineages. (B) Endoderm markers (Gata 4 and Ihh) were greatly expressed on GO, whereas (C) mesoderm (T and BMP4) and (D) ectoderm markers (Fgf5 and Nestin) were greatly expressed on G. (E) Expression of b-tubulin III and fluorescence images of human ESCs cultured on poly(acrylic acid) substrates in the presence and absence of CNT, compared to poly-L-ornithine (PLO). Modified with permission from Refs. [226,230].

adsorption and cell adhesion. However, the electrical properties of CNTs were not considered in that study, which practically might not be easy to prove such effects in relation with the cellular responses; even so the electrical conductivity of the CNTs cannot be ignored because the neural cell behaviors (like primary neurons and NSCs) have often been shown to change closely related with the electrical conductivity of the underling substrates [231–234]. Conclusively, the carbon nanomaterials, due to their unique physico-chemical properties, such as high electrical conductivity (G, rGO and CNT), nanoscale topology (CNT), and high molecular binding capacity (rGO, CNT), hold great promise for use as the matrix for PSC culture and differentiation. However, the underlying mechanisms of their control over the PSC fate are yet to be clarified. 5.2. Physical modifications 5.2.1. Nano-/micro-topography Along with the chemical modifications, controlling PSC differentiation into a specific lineage is possible by physical tailoring. Creating well-defined micro- or nano-patterns provides nano-/micro-topological cues to control the fate of cells. In the previous part of PSC self-renewal, the nano-grooves and nano-pillars tailored at certain sizes have already been demonstrated to be effective in preserving the pluripotency, through the change in PSC integrin interactions with underlying nano-recognition sites, and consequently the altered cell–cell interactions and cellular aggregation [142–144,146]. For the PSC differentiation, not only the initial integrin-ligand interactions but the distribution of patterns of the nano-/ micro-topologies that can affect the middle- and long-term behaviors, such as polarization, migration, and cytoskeletal elongation is also important. Furthermore, the secreting molecules from cells and the sequestering effect of the nano-/ micro-topologies cannot be ignored, which may alter the surface conditions progressively during prolonged cultures. Designs of nano-/micro-topologies for the PSC differentiation studies were mostly found to be aligned patterns, grooves or gratings, those optimized for the neural cells that require axonal growth along the topologies. The composition of patterns, size (width) of grooves, and ordering (or randomness) of the patterns has been considered as the key parameters. Different compositions of micro-patterned arrays, made of ECM proteins, such as gelatin, collagen IV, and fibronectin, were prepared using direct writing technique. The mouse ESCs selectively attached to the micro-patterned surface, showing pluripotent phenotypes in growth medium containing LIF. The mouse ESCs were then able to successfully differentiate, in the presence of retinoic acid and BMP-4, to the ectodermal lineage (Fig. 5.6) [235]. Interestingly, the mouse ESCs cultured on gelatin substrates expressed higher levels of pan cytokeratin compared to those on collagen IV and fibronectin, indicating

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Fig. 5.6. ECM micropatterning for the ectodermal differentiation of mouse ESCs. (A) Immunofluorescence images showing pan cytokeratin (pCK) and Hoechst staining on different ECM substrates (gelatin, collagen IV, and fibronectin, and bovine serum albumin control), and (B) mean fluorescence intensity of pCK on the different substrates. ⁄Indicates significant difference. Scale bar 100 lm. Modified with permission from Ref. [235].

a preferential differentiation into an ectodermal lineage. This work utilized the combined cues of the underlying matrices, i.e., micro-topology and surface protein chemistry. Nanoscale ridge/groove pattern arrays gained more interest to modulate the differentiation of the human ESCs. UVcurable polyurethane acrylate (PUA) nanoscale patterns with a spacing of 350 nm and a height of 500 nm were prepared by capillary force lithography. The human ESCs cultured on the nano-patterned arrays differentiated into a neural lineage effectively and rapidly after 5 days without the addition of differentiation-inducing biochemical factors (Fig. 5.7) [236]. Cells expressed neuronal markers such as Tuj1 (neuron-specific class III beta-tubulin), HuC/D and MAP2 (microtubule-associated protein 2), and a fraction of cells was even positive for serotonergic and GABAergic type neurons at 5 and 10 days, while the differentiation into dopaminergic neurons was not possible. Moreover, the nano-patterned surface did not allow a differentiation into astrocytes. Although the mechanism was not clarified, the authors hypothesized that the nano-grooves forced cytoskeleton elongation and cell shape change, which in turn transferred a tensional force to the nucleus, eventually altering gene expression and signal transduction. Another study compared 350 nm nanograting with the micron-sized (2 and 5 lm) gratings [237]. The nanograting showed higher neural differentiation of human iPSC than those microgratings, with up-regulation of neural related markers such as NeuroD1 and NeuroG1, as well as with highly aligned and elongated cell morphology along the gratings (Fig. 5.8). The mechanism of this behavior still remains unclear, although it was postulated that in the smaller grooves, a capillary force might induce cell alignment, whereas the bigger grooves favored cell attachment on the edges, providing more efficient anchorage points. However, a previous study showed that 2 lm sized microgratings were more effective in allowing the elongation of PSCs (both human ESCs and iPSCs) than the nanogratings, although without presenting a direct relationship between the elongation and the neural differentiation behaviors [238]. It was thought that the 2 lm gratings could match well the average size of filopodia extensions, hence allowing strong guidance; on the other hand, the sensing mechanism of cells on the nanogratings was considered to be slightly different, i.e., cells might bend to bridge the grating gaps, not being able to extend along the grating axis. Although the grating size clearly affected the cell morphology, the effects on neural differentiation was not elucidated, thus more studies might give clarity. The nanograting pattern was also compared with other patterns (like nanopillars and nanowells). While the nanogratings provide one-dimensional anisotropic cue to the cells, the nanopillars and nanowells are considered two-dimensional isotropic topologies. Results showed that the nanogratings enabled the human ESCs to better differentiate into neurons, whereas the isotropic patterns drove them to differentiate more likely into glial cells (Fig. 5.9) [239]. The cells on the anisotropic

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Fig. 5.7. Neural differentiation of human ESCs on the nanoscale ridge/groove patterned arrays. (A) SEM image of acrylate based substrate with 350 nm spacing and 500 nm height, with cross-section image in inset. Black and white scale bar is 2 lm and 500 nm, respectively. (B) Early differentiation of human ESCs cultured on 350 nm compared with flat surfaces after 5 days. Undifferentiated human ESCs and differentiated embryoid bodies (hEBS) are also shown as controls. (C–H) Presence of neural differentiation markers (Tuj1, HuC/D, and MAP2) and absence of glial markers (GFAP) in human ESCs cultured on 350 nm ridge/grooved substrates after 5 and 10 days. Modified with permission from Ref. [236].

substrates were able to adopt the aligned nanotopology, elongating along the gratings and expressing high levels of immature (Tuj-1) and mature (MAP-2) neuronal markers; however, those grown on the isotropic substrates contacted the surroundings more randomly, with higher extensions in different directions and thus higher level of spreading, and the behaviors could be transduced to increase the GFAP marker expression. These series of studies on the effects of nanostructured surfaces on the neural differentiation of PSCs support the determinant roles of the nanotopological features in driving their lineage to neurons or other surrounding neural cells. Although the mechanism on the phenomenon still remains unclear, the nanotopology can alter the focal adhesion alignment and the intracellular signaling processes, which consequently changes the morphology and differentiation of PSCs. The intracellular machineries involved in the mechanotransduction physically and chemically connect the outside topological cue toward the inside cytoskeletons and even the nucleus, implying the possible impact of physical signals on the nuclear deformation and PSC differentiation control [147,240,241]. As discussed, most recent interest has been focused on the differentiation of PSCs into neural lineage cells; however, the impact of nanotopologies on other types of cells, those also sensitive to elongation behaviors under topological cues (e.g. musculoskeletal cells), might be of significant interest and hence still remains as a further important research area. 5.2.2. Matrix stiffness Matrix stiffness has been considered a key physical regulator of stem cell behaviors. As discussed in the previous section, the self-renewal and pluripotency could be maintained well on matrices with stiffness that matched the native ECM of cells. Therefore, a subtle change in matrix elasticity can result in the loss of the pluripotency of cells. Researchers have thus

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Fig. 5.8. Nano- and micro-grooved (350 nm, 2 lm, 5 lm, and flat) topographical effects on the differentiation of human iPSC toward neuronal lineage. (A) Atomic force microscopy images of the different substrates. (B) Immunostaining images of human iPSC cultured on different substrates for 7 days, showing beta-III-tubulin in green while the red arrows indicate direction of gratings. (C) mRNA levels of neuronal markers stimulated by the 350 nm nano-grooved topographical cue. Modified with permission from Ref. [237].

engineered the matrix stiffness to drive differentiation into intermediate stage (ecto-, endo, or meso-dermal lineage) or terminal stage (e.g., neurons, cardiomyocytes, and osteoblasts). Among the different biomaterials, PAA hydrogels have been the most widely studied for this purpose. Acrylamide gels with different stiffness levels (4–80 kPa) were used as substrates to explore cardiac differentiation of human ESCs. The cardiogenic differentiation was analyzed by the expression of the cardiogenic marker cTnT (Troponin complex) and was found to be higher for the hydrogels with stiffness tailored in the intermediate range (E = 18.4–49.4 kPa) at 15 and 30 days, suggesting that the stiffness guided the differentiation of ESCs into a mesoderm lineage (Fig. 5.10) [242]. However, a recent study using mouse iPSCs reported that the PAA gels tailored with different stiffness levels (0.6 kPa, 14 kPa, and 50 kPa) had similar effects on the expression of cardiogenic marker cTnT at 12 days of culture, although a slight difference was observed between collagen-I and fibronectin coating substrates. This may suggest different sensitivity between ESC and iPSC (or human and mouse) when seeded onto substrates with different stiffness, but still a more detailed study may be needed to confirm this. Nevertheless, a clear tendency to differentiate into neural lineage was observed in the lower stiffness (0.6 kPa)

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Fig. 5.9. Human ESC differentiation on the different nano-topographical (nanogratings, nanopillars, and nanowells) substrates. (A) SEM image of the gratings and patterned substrates. Patterns are identified with letters next to numbers; ‘‘S” refers to spacing between gratings, ‘‘H” refers to height of the topography, and ‘‘pr” refers to perpendicular to. Scale bar 100 lm. (B) Immunostaining quantification of human ESCs cultured for 7 days on the substrates; immature neuronal markers (Tuj1), mature neuronal maker (MAP2), and glial marker glial fibrillary acid protein (GFAP). Modified with permission from Ref. [239].

and the difference was more pronounced when fibronectin was used as coating material instead of collagen-I. The results implied that the differentiation was not only controlled by the stiffness but also modulated by the composition [243]. The acrylamide was also combined (not coated but mixed) with natural polymers to improve cell adhesion, such as collagen. The mouse ESCs were cultured on top of the collagen-acrylamide hydrogels tailored with elastic modulus ranging from 0.2 to 40 kPa. The results showed that cellular organization into the form of vascularized cardiac muscle sheet was maximal on the hydrogels with stiffness similarly tailored to that of mouse cardiac muscle (i.e. 6 kPa) (Fig. 5.11) [244]. The percentage of EBs with contractility was 7-fold higher on the 6 kPa substrate than on the other conditions, although the beating frequency of cells was similar for all groups. The mRNA levels of Actn2 and Tnnt2, which encode sarcomeric a-actinin and cardiac troponin T type 2, respectively, and are indicative of cardiomyogenic differentiation, were slightly enhanced on the 6 kPa substrate. Moreover, the protein level of sarcomeric a-actinin exhibited 2-fold increase on the 6 kPa substrate compared to other groups. The cardiomyogenic differentiation of EBs was highly dependent on the transmission and interaction between cellular adhesion molecules, and the stiffness close to cardiac muscle could stimulate this signaling and enhance cardiomyogenic differentiation. However, too soft or stiff substrates inhibited this signaling; soft substrates were unable to transmit the signaling, whereas high stiffness substrates reduced cell–cell contact, resulting in migration of cells from EBs and disrupting the spheroidal 3D environment. Polydimethylsiloxane (PDMS) gel substrates were also similarly used to tailor the stiffness of matrix by modifying the cross-linker concentration, and then covalently linking collagen on the surface. The stiffness was tailored at much higher levels, from 41 kPa to 2700 kPa, to target terminal differentiation into hard tissue forming cells, osteoblasts. Mouse ESCs were cultured over the gel matrices and the role of stiffness both in early and terminal ESC differentiation was investigated. The expression of mesendoderm-related genes (early differentiation) such as, Brachyury, Mixl1, and Eomes, was shown to increase with increasing stiffness (up to 2300 and 2700 kPa). Furthermore, their terminal differentiation into an osteogenic lineage was also enhanced on the stiffer matrix (2300 kPa) in comparison to the softer matrices (41 and 260 kPa) (Fig. 5.12) [245]. Not only the synthetic polymer gels, but also natural polymer gel matrices have been used. The fibrin gels with different stiffness levels from 4 kPa to 247 kPa could be produced by altering the fibrinogen concentration and the

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Fig. 5.10. Cardiomyocytes differentiation of human ESCs cultured on polyacrylamide gel substrates with different stiffness values (4, 20, 50, and 80 kPa). (A) % of cardiac troponin T-expressing (cTnT+) cardiomyocytes after 5 days culture. (B) Fluorescent images of cardiomyocytes at day 15, showing organized sarcomeres. Cardiac troponin I (in green), a-actinin (in red), and nuclei (in blue). Scale bar 10 lm. Modified with permission from Ref. [242].

fibrinogen-thrombin crosslinking ratio. Compared to the stiffer substrates (171–247 kPa), the softer ones (4–35 kPa) drove the mouse ESCs to proliferate more and then to express the endoderm-related genes (AFP, Sox17 and Hnf4) to higher levels [246]. The results share in common with those observed in synthetic hydrogels in a sense that the stiffness matching the native matrix of the cells in concern favors the PSCs to adopt and to differentiate down the lineage. While other natural polymers, such as collagen, alginate, hyaluronic acid, and silk can also be produced into gel matrices with varied stiffness levels, the studies on PSC differentiation on the gel matrices are still in infancy, suggesting more works need to follow to use the compositional merits. Compared to synthetic polymer gels, those with natural polymers can provide adhesive ligands to cells, favoring PSCs to recognize the surface and to adopt proper differentiation processes. As explained, some synthetic and natural polymers were effective in controlling the stiffness level through changing the material concentration and crosslinking degree, which have ultimately enabled the pioneering studies on matching stiffness conditions to be optimized for specific lineage differentiation of PSCs. However, as discussed for the self-renewal and proliferation, the differentiation behaviors of PSCs have been studied primarily on the 2D gel matrix conditions to see the

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Fig. 5.11. Matrix rigidity (0.2, 6, and 40 kPa) to modulate cardiovascular organoid formation from mouse ESCs. (A) Bright field images of EBs cultured on hydrogels with different stiffness levels after 15 days. (B) Increased percentage of contracting EBs after two weeks of culture on the 6 kPa substrate. (C) Enhanced immunohistochemical expression of sarcomeric a-actinin after 23 days on the 6 kPa substrate. (D) Cell cycle analysis showing fluorescent images of a-actinin positive cells (red) incorporating EdU (green) in the different substrates. Scale bar 20 lm. Modified with permission from Ref. [244].

stiffness effects; thus further exploration of works within the 3D gel matrix conditions with stiffness controlled is considered a more promising platform. 5.3. Three dimensional environments 5.3.1. 3D gel culture Like the maintenance of the pluripotency of cells, the differentiation of PSCs can be better implemented under 3D culture environments than 2D. Furthermore, the culture of PSCs within 3D hydrogels and scaffold matrices enables the use of PSCdifferentiated constructs directly in diseased and dysfunctional tissues. The encapsulation of PSCs within the hydrogels not only maintains the pluripotency of stem cells, but also directs cell differentiation. For example, encapsulated ESCs within the alginate hydrogel microcapsule can differentiate into desired cell lineages including endoderm, mesendoderm, and primitive gut tube under differentiation conditions by adding specific molecules [171]. Furthermore, the mouse ESCs encapsulated within alginate microbeads differentiate toward hepatocyte lineage without the addition of growth factors enabling the cells to form aggregates [247]. When the alginate microbeads were

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Fig. 5.12. Substrate stiffness directs osteogenic differentiation of mouse ESCs. Material stiffness was tuned by changing the cross-linker concentration of polydimethylsiloxane (PDMS). (A) Material characterization, showing reduced water contact angle in the collage coated substrates, and the increased Young modulus with increasing cross-linker concentration. (B) Relative gene expression of mouse ESCs at 6 days, showing stiffness-dependent increase of ectoderm (Fgf5) and primitive mesendoderm markers (Brachyury, Foxa2, Eomes, Mixl1), with low expression of anterior mesendoderm (Twist1), neuroepithelium (Sox1), primitive endoderm (Gata6) and cadherins 1 and 2 precursors (E- and N cadherin; Cdh1 and Cdh2). (C) Osteogenic related gene expression of mouse ESCs cultured for 28 days, showing stiffness-dependent increase of osteogenic genes and bone nodule formation. Modified with permission from Ref. [245].

cross-linked properly at 2.2% and were incorporated with soluble inducer (retinoic acid (RA)) into the permeable microcapsule system, the cell aggregation was reduced which in turn enhanced the neural differentiation. Even in the absence of RA, the differentiation was directed away from the hepatocyte and toward the neural lineage by physical cell–cell aggregation blocking, which offered insights into targeting cellular differentiation toward both endodermal and ectodermal cell lineages using the cell encapsulating gel systems (Fig. 5.13) [248]. Because the microgels that encapsulate cells differentiated to a proper lineage are implantable (even as an injectable system) into a wide range of tissues diseased or defected, the differentiation control of PSCs through the 3D microgel and their delivery to the target tissues are considered a practical approach. The alginate hydrogel with micro-cavities was recently employed to culture the mouse ESCs and iPSCs to form colonies/ EBs and continuously differentiate into hepatic cells, where the hydrogel system could enhance nutrient exchange and also permit greater living space for the encapsulated PSCs. After culturing the PSCs for 10 days in the encapsulating hydrogels, colonies/EBs were spontaneously formed, subsequently directed toward an endodermal lineage, followed by differentiation into hepatic lineage through the introduction of differentiation conditions. Compared to monolayer culture, the employed hydrogel system up-regulated endoderm and hepatic markers and enhanced the urea and albumin production significantly (Fig. 5.14) [249]. One notable merit of the alginate hydrogels is that mechanical stiffness can be easily tuned to direct stem cell fate into a desired lineage. For instance, alginate capsules incorporating human ESCs were crosslinked in different BaCl2 concentrations,

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Fig. 5.13. Neural lineage differentiation of mouse ESCs within alginate microbeads. (A) Representative 3D surface view of mouse ESC encapsulated. (B–D) Kinetic profile of neural markers of mouse ESCs obtained by blocking cell-to-cell contact with different blocking concentrations of E-cadherin (0.5 and 1 lg/ ml); (B) O1, (C) neurofilament M, and (D) GFAP. Modified with permission from Ref. [248].

to provide stiffness values from 4 to 80 kPa. The encapsulated human ESCs within an intermediate stiffness gel were stimulated to differentiate into intermediate definitive endoderm and early pancreatic lineage, through the activation of the TGFb signaling. On the other hand, the cells encapsulated in the higher stiffness capsules showed reduced proliferation and cell growth, possibly due to the higher doses of barium as well as to the stiffness of the substrates; furthermore, the pancreatic progenitor induction was significantly suppressed. It is known that a high level of TGFb is needed for endoderm differentiation whereas continuous activation of TGFb is detrimental to subsequent pancreatic progenitor induction. The pathway for pancreatic progenitor cell differentiation was then correlated with the retinoic acid signaling and sonic hedgehog inhibition. Conclusively, the stiffness levels modulated in the alginate capsules were proven to be a powerful tool to differentiate human ESCs down to an endodermal lineage and further to pancreatic progenitor cells [250]. The alginate gel system was also developed into an array of one-dimensional-like microstrands after coating with PLL to mimic the ‘liquid-like’ embryonic microenvironment. Mouse ESCs were encapsulated within the liquid-like alginate/PLL microstrands with different diameters ranging from 30 to 300 lm. It was shown that mouse ESCs exhibited self-assembly behaviors within the one-dimensional microstrands. When compared to the alginate gelled microstrands, the liquid-like microstrands showed the formation of bigger self-assembled micro-tissues with compact structure. Interestingly, the micro-tissue within the aqueous alginate/PLL microstrands tended to differentiate toward the endoderm and mesoderm lineages, whereas the cells recovered from alginate gelled microstrands differentiated into ectoderm and mesoderm lineages (Fig. 5.15) [251]. While the narrow one-dimensional-like geometrical cue drives mouse ESCs to self-assemble into aggregates, the inner matrix elasticity, e.g., either gel-like or fluid-like nature, appears to deliver the differentiation cue into different lineages. The 3D culture can be combined with dynamic cultures, like in a shaking bath, a spinner flask, or a rotating bioreactor, to provide better 3D dynamic environments for the differentiation of PSCs. Mouse ESCs were encapsulated within the alginate microbeads and then cultured in a rotating bioreactor to generate hard tissue forming cells [252]. Interestingly, a cell population with enhanced mesodermal differentiation capability was first generated by the treatment with 50% medium of hepatic cell line HepG2. Successively, these cells differentiated into an osteogenic lineage under osteogenic medium

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Fig. 5.14. Hepatogenesis of mouse iPSCs and mouse ESCs in 3D microcavitary hydrogel system. (A) Schematic representation of protocol for cell encapsulation in hydrogel construct; n alginate hydrogel incorporates gelatin microspheres which are removed by a temperature increase to allow the migration of pluripotent cells to form EB within the hydrogel microcavity. (B) Phase contrast images of cell laden hydrogel constructs at various stages of growth and differentiation. (C) Assessment of liver specific functionality of cells cultured in 3D hydrogel vs. in 2D; urea and albumin production by mouse ESCs and mouse iPSCs. Modified with permission from Ref. [249].

conditions. The cellular constructs displayed morphological, phenotypical, and molecular attributes of the osteogenic lineage, as well as calcium-phosphate mineral deposition, showing macroscopic 3D bone-like constructs (Fig. 5.16). Although it was not clarified whether the rotating culture of cells might have some influence on the osteogenic differentiation, the combinatory approach of hydrogel encapsulation, rotating culture, and the osteogenic medium was effective in achieving ex vivo calcified hard tissues from pluripotent cells.

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Fig. 5.15. Array of hydrogel microstrands used to self-assemble the mouse ESCs. (A) SEM images of porous microfluidic device (a; top view, b; cross-section view; scale bar 20 lm), scheme showing a drop of alginate/cell suspension soaked in calcium chloride solution to encapsulate cells (c), and SEM image showing the formed microstrands (d). (B) Fluorescent images showing the differentiation potential of mouse ESCs recovered from alginate/PLL microstrands (a–c) and alginate microstrands (d–f); expression of ectoderm marker nestin (a and d), mesoderm marker Myf-5 (b and e), and endoderm marker PDX-1 (c and f). Scale bar 50 lm. Modified with permission from Ref. [251].

While the alginate-based hydrogels have been the most widely studied, other hydrogel systems have also been used for cell encapsulation to regulate differentiation behaviors. For instance, human ESCs encapsulated within a dextran-based hydrogel scaffold containing regulatory factors, such as tethered RGD peptides and VEGF, tended to differentiate into vascular lineages. Compared to spontaneously differentiated embryoid bodies, the number of cells expressing KDR/Flk-1 (kinase insert domain receptor/fetal liver kinase 1), a vascular marker, increased up to 20-fold. Furthermore, when the cells were removed from the hydrogels and cultured on tissue culture polystyrene, they presented higher vascular differentiation than those from embryoid bodies (Fig. 5.17) [253]. In a similar way, agarose hydrogels functionalized with VEGF were synthesized in order to understand the effect of VEGF on the encapsulated ESCs. Under serum-free conditions, the ESCs within the VEGF-functionalized agarose hydrogel were induced to blood progenitors more efficiently than when the VEGF was used as a soluble factor. It was believed that three-dimensionally immobilized VEGF can play a similar role as the VEGF found in the native ECM in vivo [254]. Functionalized hydrogels made of PEG were also prepared to incorporate matrix metalloproteinase (MMP) and RGD peptide. The MMP acted as a cross-linker between the different PEG chains, while the RGD sequence was incorporated to increase cell

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Fig. 5.16. Alginate hydrogel encapsulation of mouse ESCs cultured in a rotatory microgravity bioreactor effective for bone applications. (A) Schematic representation of the design. (B) Live/dead assays of the cells encapsulated within alginate hydrogel after encapsulation (a), 11 days (b) and 16 days (c). (C) SEM and EDS analysis of mineralized constructs with differentiated mouse ESCs. (D) Force displacement curves of the tissue engineered 3D bone like structure, compared with alginate hydrogel and alginate hydrogels with undifferentiated mouse ESCs. Modified with permission from Ref. [252].

adhesion. Human iPSCs encapsulated in the gel matrix, as compared to those cultured on 2D dish, were able to develop vasculogenesis more profoundly through the upregulation of matrix remodeling and Notch signaling pathways, which was also found in the vasculature development in vivo [255]. Among the PEG hydrogels with different MMP cross-linking ratios, the intermediate MMP cross-linking showed an optimum vascularization, implying the importance of an appropriate matrix density and cell-induced degradation for vasculogenesis. The combination of collagen and Matrigel was shown to provide the necessary components of ECMs as well as physical supports for ESC development and differentiation. The mouse ESCs seeded within the 3D gel of collagen/Matrigel grew into aggregates and formed embryoid bodies, which eventually differentiated into three embryonic germ layers after 7 days of culture [256]. Moreover, the human ESCs differentiated into cardiomyocytes with the introduction of ascorbic acid and demonstrated the beating behavior at 19 days. Similarly, the PEG-fibrinogen based hydrogels were also shown to be successful to 3D culture iPSCs and then differentiate them into cardiomyocytes [257]. The hydrogels were able to culture both cellular clusters and single cells to create reproducible cardiac tissue. During cultures, a spontaneous contraction frequency increased steadily, which was related to the temporal development of cell–cell junctions. Compared to the single cell encapsulation, where the initial cell–cell contact was low, the cell cluster encapsulation showed the spontaneous contraction more quickly (6 days vs. 9–11 days), suggesting the need of cluster culture for cardiac tissue engineering. The iPSC clusters encapsulated within the 3D hydrogel were able to develop into functional cardiac tissues composed of self-aligned cardiomyocytes with structural maturation and mimicking heart development (Fig. 5.18). Overall, the current 3D hydrogels offer a significant

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Fig. 5.17. Bioactive hydrogels for the vascular differentiation of human ESCs. (A) Schematic presentation of the dextran based hydrogels showing RGD tethering on the monomer and the encapsulation of VEGF in PLGA microparticles placed within the hydrogel. The swelling ratio (white column) increased with lowering monomer concentration, whereas the elastic modulus decreased with increasing monomer concentrations (black column). (B) Expression of endothelial and undifferentiating cell markers of human ESC encapsulated in hydrogel analyzed by FACS. (C) Expression of endothelial and undifferentiated cell markers when removed from the hydrogel. Modified with permission from Ref. [253].

advantage over the conventional 2D cultures, enabling sufficient cell–cell junctions, differentiation of cardiomyocytes and their maturation, and long-term culture for cellular secretion of ECM molecules to create a continuous tissue, replicating the ontogenic cardiac tissue development. Fibrin hydrogels combined with mouse ESCs and certain growth factors, such as neurotrophin-3, platelet derived growth factor AA and sonic hedgehog, which are known to promote neural fiber sprouting, angiogenesis, and proliferation of neural progenitors, respectively, were prepared for the effective repair of spinal cord injury. The affinity-based controlled delivery of the growth factors promoted the differentiation of mouse ESCs seeded inside the gels into neural progenitors, neurons, and oligodendrocytes whereas it reduced differentiation of mouse ESCs into astrocytes [258]. The work demonstrated the importance of signaling molecules in the PSC differentiation when combined with 3D matrices and then released in a controlled manner. One recent study reported a micro-device to culture PSCs with a capacity to release signaling molecules at determined time points [259]. Soft lithography was introduced to produce PDMS molds with different sizes and shapes of

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Fig. 35 (continued)

Fig. 5.18. Hydrogels encapsulating pluripotent cells used to differentiate into specified lineage. (A) Human iPSCs encapsulated in PEG-fibrinogen either in clustered or single cellular form allows the differentiation and growth of engineered heart tissues. (B) Long-term culture of human ESCs showing highly aligned sarcomere and large and elongated cell nuclei, and (C) ultrastructural images at 124 days, showing characteristics of muscle cell with sarcomere structures, including Z-lines (Z), I-bands (I), H-zones (H), intercalated discs (ID), gap junctions (GJ) and mitochondria (m). Modified with permission from Ref. [257].

microscale features, which was designed to enhance the homogeneity of EB formation and the subsequent lineage commitment depending on the spheroid size. Importantly, the micro-device platform was embedded with microchannels for the precise control of biomolecular delivery. Neurogenic differentiation was tested by the spatiotemporal delivery of gradient retinoic acid solutions. Near the highest retinoic acid concentration the Sox-1 expression and thus neural induction was the strongest. Furthermore, the size of EB closer to retinoic acid was larger, presumably due to the differential cell proliferation. The micro-device therefore offers the possibility to control the spheroid formation and differentiation behavior of PSCs through the precise delivery of signaling molecules, which can be performed in a simple culture and rapid analysis platform. However, those micro-devices made of PDMS are not practical for the in vivo delivery of PSCs but mainly useful to interpret the cellular phenomena in interactions with biomolecules and substrates and to enable high throughput screening of those combinatorial sets. 5.3.2. 3D geometrical scaffolds Together with hydrogel matrices, 3D shaped polymeric scaffolds have been extensively used for the regulation of PSC differentiation. A recent attempt was made to systematically study the effects of matrix environments on pluripotent cell viability and neuronal differentiation with self-assembling peptides based scaffolds. Three different niche factors, including dimension, laminin biochemical signal, and mechanical stiffness, were varied and the P19 cell line was used as a pluripotent cell model. In particular, two-level, three-factor factorial experimental design was employed to investigate the effects on

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Fig. 5.19. Self-assembling peptides tuned (with or w/o IKVAV; tailored with hard or soft) to stimulate neural differentiation of human ESCs. Gene expression of neural markers, demonstrating significant role of IKVAV. Modified with permission from Ref. [260].

neuronal differentiation of cells [260]. Results showed that the differentiation of the mouse ESCs was promoted synergistically when cultured on the 3D geometrical substrate in the presence of Ile-Lys-Val-Ala-Val (IKVAV) (Fig. 5.19). However, the stiffness did not influence the differentiation, which might be due to the relatively low stiffness levels of all scaffolds (0.262– 0.672 kPa). While the study could show systematically the effects of geometrical, physical, and biochemical factors on the PSC behaviors, some limitations are the use of matrices in a narrow stiffness range and the interpretation of data using pluripotent cell line, thus further studies need to follow. Among other types of scaffolds, the nanofibrous form has been the most-widely studied to model the differentiation behaviors of PSCs. A recent elegant work demonstrated the effect of fiber scaffold stiffness on the embryonic mesenchymal progenitor cell differentiation [261]. In fact, the stiffness effects of 2D hydrogel coatings on stem cell behaviors, such as MSCs differentiation, have been relatively well studied; however, the stiffness tailoring of 3D scaffolds and the effects on PSC differentiation have been unexplored. In the study, pure PCL and core-shell PES/PCL fibrous scaffolds were created using electrospinning. Both fibrous scaffolds were similar in fiber diameter, inter-fiber distance, and tensile strength, however, the stiffness of the PCL/PES scaffold (30.6 MPa) was much higher than that of pure PCL scaffolds (7.1 MPa). The differentiation of cells was shown to be significantly influenced by the scaffold stiffness. The results demonstrated that the softer scaffolds provided adequate environments for chondrogenesis, whereas the stiffer scaffolds supported enhanced osteogenic differentiation (Fig. 5.20). The phenomenon is in good agreement with previous findings that other types of stem/progenitor cells preferentially differentiate when the matrix is tailored to match the native tissues. PCL/gelatin nanofibrous scaffolds were also used to allow the chondrogenesis of iPSCs using two-separated electrospinning process. The cells were able to express higher levels of chondrogenic markers compared to the control cells, and when the constructs were implanted in vivo, the results showed higher cartilage specific gene and protein expressions as well as subchondral bone regeneration [262]. A recent approach prepared silk matrices by self-assembly of recombinant silk, and demonstrated the ability of silk matrices to allow long-term culture and the differentiation of both human iPSCs and ESCs in 3D conditions, providing a new chemically-defined and xeno-free culture system [263]. The alignment of fibrous scaffolds also influences the differentiation capacity of PSCs. Random and uniaxial-aligned PCL fiber scaffolds were prepared by electrospinning using different types of collectors and the mouse embryo bodies containing neural progenitor cells and undifferentiated ESCs were seeded onto the scaffolds [264]. The results demonstrated that wellaligned fiber scaffolds could enhance the differentiation of embryo bodies into mature neural lineage cells. Moreover, the aligned PCL fiber scaffolds not only promoted differentiation of ESCs into neuronal lineage cells (except for astrocytes), but also directed neurite outgrowth (Fig. 5.21). The matrix guided elongation of cells has been considered a morphological indication of the possible phenotype change of cells, particularly those favoring directional growth like neural and muscle cells. The combined effects of 3D geometry with extracellular proteins were also studied using electrospun fibers. The electrospun PCL scaffolds were coated with different adhesive ECM molecules, such as collagen IV, fibronectin, laminin, and vitronectin, and the differentiation of mouse ESCs into cardiovascular cells was examined. The results showed that the number of cardiac progenitor cells found in the 3D electrospun scaffolds coated with laminin or vitronectin was higher when compared to the matrices coated with collagen IV (Fig. 5.22) [265], which has been shown to maintain the mouse ESCs in an

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Fig. 5.20. Nanofibers with tailored mechanical modulus by the PES core/PCL shell electrospinning design, to control the differentiation of mouse ESCs. (A) SEM image of the microstructure of pure poly(e-caprolactone) PCL (a) and co-axial poly(ether sulfone)-PCL (PES-PCL) (a and b). Illustration of co-axial microstructure of the core–shell fiber (c) and TEM image of core–shell PES-PCL fiber (d). (B) The different stiffness was able to guide direct cellular differentiation into different lineages depending on their modulus, showing enhanced chondrogenic differentiation (Sox9 and Acan expressions in a and b) for PCL nanofiber (E = 7.1 MPa) and enhanced osteogenic differentiation (alkaline phosphatase A/p and osteocalcin Oc expressions in c and d) for PES-PCL nanofiber (E = 30.6 MPa). ⁄Indicates significant difference. Modified with permission from Ref. [261].

undifferentiated state [191,266], signifying the importance of ECM adhesive molecules on the cardiac differentiation of mouse ESCs. Although the work shows that 3D culture is sufficient to induce a number of cardiovascular progenitor cells, the mechanisms to direct cell fate through the 3-dimensionality together with the ECM molecules, have not been clarified. Thermally induced phase separation (TIPS) is one of the 3D scaffold fabricating techniques which often generates porous or fibrous structure. The 3D poly(lactic-co-glycolic acid) (PLGA) scaffold was fabricated by TIPS and then coated with laminin to enhance cell adhesion. The results showed that human ESCs cultured on the 3D scaffolds directly differentiated into an endoderm lineage without the formation of embryo bodies [267]. In a similar way, nanofibrous and porous scaffolds of poly(L-lactic acid) (PLLA) could be prepared by the combination of 3D printing and TIPS [268,269]. Compared to 2D solid film of the same material, on the nanofibrous matrix, both mouse and human ESCs expressed osteogenic differentiation markers more rapidly. Furthermore, 3D structured nanofibrous surface showed higher levels of osteogenic differentiation markers and mineralization compared to non-nanofibrous 3D scaffolds (Fig. 5.23). These studies strongly support the beneficial effects of nanofibrous topology on the differentiation of PSCs (particularly to an osteogenic lineage) in the 3D geometrical conditions. The differentiation of PSCs to lineages other than the osteogenic upon the 3D nanofibrous network scaffolds will be an interesting area of research to follow. Another notable approach to directing differentiation of human ESCs within the 3D scaffolds is to combine gene delivery systems. Porous scaffolds of PLA/PLGA blend were prepared by salt-leaching and then combined with a complex of small interference RNA (siRNA) and lipid-like non-viral carrier material [270]. The scaffolds with the delivery of siRNA (with green fluorescent protein (GFP) tagging) showed a 90% knockdown efficiency of GFP expression in human ESCs. It was also observed that the silencing of KDR receptor gene resulted in down-regulation (60–90%) of genes related to endoderm layer and up-regulation (27–90-fold) of genes related to mesoderm layer. The results suggest that the methodology adopting gene delivery system with 3D scaffolds can be used to isolate endoderm layer from the mesoderm and ectoderm (Fig. 5.24). Therefore, similar to ECM proteins and peptides, the genetic molecules are also a possible biochemical cue that can synergize with the 3-dimensionality of scaffolds in the regulation of PSC differentiation; in this case however, the nanoparticulate carriers that deliver the genetic molecules into the intracellular compartments need to be designed carefully for efficient cellular uptake and transfection. Meanwhile, the differentiation of PSCs could be influenced not only by surrounding matrices, but also from those signals created within the cell aggregates. Microparticles made of different materials, including agarose, PLGA, and gelatin, were prepared and combined with mouse ESC aggregates by forced aggregation technique to examine the interaction between

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Fig. 5.21. Nanofiber substrates of aligned (PCL-A) and randomly oriented (PCL-R) used to differentiate mouse ESCs into neural lineage. (A) SEM images of PCL-A (a) and PCL-R (b). (B) Maximum neurite outgrowth and cell phenotype after 14 days in culture. Maximum neurite length was significantly higher on the aligned nanofiber. While undifferentiated markers (SSEA-1 and Nestin) were down-regulated the neural differentiation markers (Tuj1, O4, and GFAP) were stimulated on the nanofibers (vs. undifferentiated ESCs and differentiated embryoid bodies). Modified with permission from Ref. [264].

Fig. 5.22. Effect of ECM molecules coated either on 2D or 3D matrices on the differentiation of mouse ESCs. FACS analysis showing enhanced number of Flk1+ cells when cultured on the 2D collagen IV and 3D vitronectin substrates. With permission from Ref. [265].

the cell and the biomaterials within the aggregates [271]. The presence of the different materials modulated the gene and protein expressions differently without affecting cell viability. The distribution of cell differentiation was also observed. For example, a-sarcomeric actin, a mesoderm marker, was localized on the exterior of untreated cell aggregates and agarose microparticle-treated aggregates, while it was observed both on the exterior and interior of the low concentration gelatin microparticle-treated aggregates. This study suggests that the biomaterials incorporated within the stem cell aggregates can control the final phenotypic expression of the cells (Fig. 5.25). The intercalated biomaterials between cells appear to replace in part the cell–cell interactions with cell–matrix(material) interactions, influencing the aggregation behaviors of PSCs, the contact-induced microenvironments of cell aggregates, and the subsequent differentiation. This expands the

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Fig. 5.23. Nanofibrous form of PLGA scaffolds used for the osteogenic differentiation of human ESCs. (A) SEM images of human ESC after 48 h of culture on nanofibrous substrates compared to solid substrates. (B) Expression of osteogenic markers of differentiated human ESC on nanofibrous and solid walled scaffolds after 4 weeks in culture, showing significant differences. Modified with permission from Ref. [269].

possible role of intercalated biomaterials in the spatial control over lineage-specific differentiation of PSCs, which remains an interesting yet unexplored area. While extensive research has been carried out in vitro to direct PSC fate, recent work has started to apply those wellestablished concepts to in vivo models [272–275]. The major limitation is the poor survival rate as well as the teratoma formation of the implanted cells. In order to overcome this, the biomaterials to support and deliver cells in vivo need to be optimally designed which can provide microenvironments suitable for cellular survival and lineage differentiation. As an exemplar study, scaffolds were designed to induce in vivo osteoblastic differentiation of PSCs. For this, the bone mineral hydroxyapatite (HA) was covered over the PLGA scaffolds, which was then tethered with BMP2 to enable sustained release; thus the optimized scaffolds could act as an osteogenic niche to properly guide the in vivo differentiation of human ESCs and iPSCs [272] (Fig. 5.26). Both cell types did not show any signs of teratoma formation in the scaffolds tethered with BMP2, but did show in the scaffolds absent of BMP2, suggesting that the slow releasing BMP2 was critical in driving pluripotent cells to strongly commit an osteogenic lineage. This work was meaningful in that the in vivo osteogenic commitment was achieved without the predifferentiation of PSCs into any osteogenic progenitors, but through the direct differentiation with the engineered niche. The finding suggests that the local biomaterial-based microenvironments in vivo were able to cue the pluripotent cells constructed with the scaffolds to survive and differentiate properly, allowing the successful engraftment of the cells into regenerated tissue. In vivo PSC therapy was also confirmed in central nervous system, such as spinal cord injury and brain. Injectable gels made of hyaluronic acid and methylcellulose modified with RGD peptide and platelet derived growth factor A (PDGF-A) were used for in vivo delivery of human iPSC to spinal cord injury of rat [273]. When the cells were directly injected to the nerve defect they showed a sign of teratoma formation (in 100% animals) with poor survivability; however, when the cells were injected with the hydrogel they were mostly able to differentiate into a glial phenotype although the teratoma formation was still observed (in 50% animals) at long survival times (Fig. 5.27). The attenuated teratoma formation of iPSCs by the hydrogel delivery with respect to those delivered in media was due to the enhanced differentiation of cells in vivo, which was also correlated with the higher cell survival and migration ability attributed to the biochemically modified gels with RGD peptide and PDGF-A growth factor. In this way, the engineered gel niche was effective in differentiating iPSCs to oligodendrocyte progenitor cells (OPCs) at an early phase, however, the teratoma formation could not be completely prevented at

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Fig. 5.24. siRNA delivery to control human ESCs differentiation upon 3D polymeric scaffolds; (A) knockdown of GFP in GFP-human ESCs and GFP-human EBs scaffolds after 2 and 3 days, compared to untreated control. (B) 3D scaffolds containing siRNA(GFP) showing 90% knockdown efficiency (a), siRNA (GFP) delivery efficiency with lipidoid observed higher in 3D than in 2D culture in a siRNA-dose dependent manner (b), and target gene KDR siRNA delivery to show down-regulation of endoderm-related genes concomitantly with up-regulation of mesoderm-related genes (c). Modified with permission from Ref. [270].

long-term transplantation periods, and this was ascribed to the immature OPCs remained. Therefore, driving the immature population of cells toward a well-defined differentiation status is critical to tackle the teratoma formation of PSCs, which ultimately necessary for clinical uses in the future. Instead of using the PSCs, their differentiated lineages were used for the in vivo transplantation for nerve regeneration [274,275]. The iPSC-derived neural crest stem cells (NCSCs) were implanted together with nanofibrous nerve conduits to bridge rat sciatic nerve gap [274]. Nanofibrous matrices have already been demonstrated to stimulate neuronal growth of neural stem cells. All the expanded NCSC lines were capable of differentiating into mesodermal and ectodermal lineages, including neural cells. The nerve conduits constructed with NCSCs demonstrated an accelerated regeneration of sciatic nerve at 1 month with promoted axonal myelination. The NCSCs were shown to differentiate into Schwann cells and were integrated into the myelin sheath around axons. Furthermore, no teratoma formation was noticed for up to 1 year after the transplantation. This study used iPSC-derived NCSCs, instead of pluripotent cells, for the in vivo applications, and demonstrated the differentiated multipotent cells were safe and effective in peripheral nerve regeneration therapy. Recently, iPSC-derived neurons (iN) were also used for the in vivo transplantation with microfiber scaffolds in rat brain [275]. Two types of electrospun microfibers (thick fiber that allows cellular penetration, and thin fiber that limits this) were

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Fig. 5.25. Effects of polymeric microparticles (PLGA, agarose, and gelatin) incorporated within the aggregates of mouse ESCs. (A) SEM images of the PLGA, agarose and gelatin microparticles. (B) Incorporation measurement dependent on the microparticle dose quantified in number or fluorescence-stained in red (a), and different differentiation behaviors analyzed, showing mesoderm markers a-Sacromeric actin and AFP (b). Modified with permission from Ref. [271].

prepared, and the iN were differentiated on the fibers. Compared to thin fibers or 2D control, thick fibers presented more extensive neurite outgrowth and higher expression of bIII-tubulin, MAP2 and synaptophysin after 12 days. The microfiber architecture was shown to enhance the cell–cell contact and influence the functional and phenotypic maturity of the iN. In parallel, pluripotent markers including Ki67 and Oct-4 were higher in the 2D culture compared to the fibrous substrates, suggesting that the 3D architecture was able to selectively reduce the presence of residual pluripotent cells, which could potentially lead to reduced teratoma formation in the in vivo conditions. The iN-seeded microfibers injected into the rat brain showed significantly higher neurite outgrowth (831 lm) than the direct injection of iN (241 lm). Furthermore, the survival rate of cells was 38-fold higher when injected with the microfibers than when directly injected (Fig. 5.28).

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Fig. 5.26. In vivo differentiation of human iPSCs and ESCs combined with scaffolds (HA-PLGA with or without BGM-2) for skeletal defect regeneration. MicroCT images and quantification of bone regeneration during 8 weeks implantation in calvarium defects for the different treatment groups. Modified with permission from Ref. [272].

As noticed, the in vivo applications of PSCs for neural tissues still hold concerns mainly on the possible teratoma formation due to the poorly differentiated population of cells and their long-term survival in the in vivo conditions (over weeks to months in vivo vs. days to a few weeks in vitro); thus their differentiated lineages (such as neural progenitor cells or neurons) can be preferred cell sources for in vivo applications. Clearly, the use of scaffold and gel matrices is helpful for the in vivo implantation of those potent cells in lesions and injury sites, possibly improving the survivability, functional differentiation, and integration into native tissues. Although only a handful of in vivo studies have been carried out thus far, more works on different tissue models are considered to follow in the near future – a necessary preclinical step prior to applications. Collectively, the representative biomaterials that have been used to differentiate PSCs into specific lineages in vitro and in vivo are summarized in Table 5.1. 6. Concluding remarks PSCs including ESCs and iPSCs hold great promise in regenerative therapy of incurable diseases, irreparable damages, and dysfunctions of tissues and organs, due to two distinctive properties, unlimited self-renewal and capacity of pluripotent differentiation. However, control of these two phenotypes is challenging, particularly for clinical applications. Ideally one would like to expand the cells with stemness remained intact or to differentiate the cells into certain linage with specificity on demand. While the control over self-renewal and target differentiation of PSCs has been traditionally achieved by biological cues such as growth factors, hormones, and small molecules, the use of biomaterials can complement and synergize with the different biological signaling molecules. Engineered biomatrices can mimic the native stem cell niche and alter the microenvironmental conditions where pluripotent cells reside. PSCs can sense the physical and chemical properties of the matrix, the distribution of soluble factors, the mechanical profiles and oxygen levels to interactively adopt a proliferative or differentiated state to fulfill their mission. Recent advances have seen intense activity on engineering biomaterials that can regulate the microenvironmental effectors of PSCs. Innovations range from simple modification of functional groups to tether bioactive signals to design of recombinant biopolymers with built-in bioactivity and to advanced micro-/nano-engineering fabrication techniques to pattern biochemical cues to influence the adhesion machineries of PSCs for preservation of pluripotency. Nano- and microtopographical cues can also influence cellular adhesion confinement and the capacity to maintain pluripotency. Not only does the topographical dimension matter, but the arrangement (random or ordered patterns) can also lead to different PSC self-renewal stability. Matrix stiffness has been a key mechanical sensing effector on the self-renewal of PSCs. 3D cell culture matrices are more potent than the 2D counterparts in the regulation of PSCs fate. Hydrogels, including alginate, collagen, agarose, HA, and PEG, in combination with specific molecules, such as integrin adhesive ligands and growth factors, have shown promise in preserving PSC pluripotency. Furthermore, 3D geometrical scaffolds with complex shapes like porous

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Fig. 5.27. In vivo injection in spinal cord injury of the human iPSC encapsulated in hydrogels containing RGD and PDGF-A. (A) Cells transplanted within the hydrogel showed markers of glial lineage (MBP, red; SOX10, yellow), but not CD44 (red) or SOX2 (yellow). There were few cells with mesodermal marker expression (SMA, red) or endodermal (AFP, yellow). (B) On the other hand, cells transplanted in the absence of the hydrogel did not express the glial lineage markers, but expressed CD44, as well as mesodermal and endodermal markers. (C) Teratoma formation attenuated significantly when cells were injected with hydrogel, which was due to the higher OPC differentiation of pluripotent cells. Modified with permission from Ref. [273].

foams, nanofibers, and microspheres have also proved capable of culturing PSCs in an undifferentiated state for long-term periods. In most cases, the cells maintain their native pluripotency better when the cell aggregates are more compact and smaller over the biomaterial matrices with those tailored properties. The differentiation behaviors of PSCs stimulated by biomaterials in 2D or 3D environments have also been extensively and diversely studied. Surfaces immobilized with ligands for integrins, growth factors, and proteins promote the differentiation of PSCs into 3-germ layers or more highly specified cells. The fates of PSCs are influenced by the scale of surface patterns. For example, the micro-scale pattern of ECM proteins promotes the differentiation of PSCs into ectoderms, while nanoscale pattern would guide differentiation into neuronal cells, even in the absence of differentiation-inducing agents. Matrices with certain elastic properties can deliver mechanical cues to properly regulate lineage differentiation of PSCs. Furthermore, 3D hydrogels with specific signaling molecules are useful 3D culture platforms for PSC differentiation into various cell types, such as hepatocytes, neurons, bone cells, vascular cells or cardiomyocytes. Along with gel matrices, porous foams, nanofibrous meshes, and microparticles have also been studied for 3D culture of PSCs and their geometry-induced differentiation. Signaling molecules like growth factors and genetic molecules can be introduced to provide biochemical signals in concert with 3D matrix cues, which is likely to better mimic the native tissue microenvironment. Thus far, 2D studies involving change in chemical groups, surface modification with ECM molecules, patterning with micro/nanostructures, and tuning underlying matrix stiffness have shed light on the mechanisms that possibly govern the self-renewal and lineage differentiation of PSCs. Some of the 2D studies that combine the parameters together, such as linking adhesive ligands to gels with different elasticity values, patterning nanotopologies with tethered signaling molecules, and designing nanofibrous matrices with varied stiffness levels, provide substrate conditions better tuned for PSC selfrenewal or lineage differentiation. Moreover, ongoing 3D studies will likely gain more mechanistic insights to facilitate

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Fig. 5.28. In vivo outgrowth and survival of human iPSC-derived iNs in mouse striatum, supported by electrospun microfiber scaffolds. (A) iN-seeded scaffolds were injected into mouse striatum and compared with injected dissociated cells. GFP-expressing surviving iNs were found 3 weeks post transplantation, both for dissociated iNs (B) and with iN-seeded microscaffolds (C–E), located in the vicinity of microfiber scaffolds (indicated with dashed line). (F) Post-synaptic density protein 95 (PSD-95, blue), co-localized with synaptophysin (red), suggestive of synaptic integration with host tissue. (G) Quantification of surviving cells. ⁄P < 0.05 by one-way ANOVA. There was no significant difference in neurite length between the two groups (h). GFPexpressing NeuroD1 iNs and RFP-expressing dopamine neurons were preserved in close proximity when co-transplanted on microfiber scaffolds (indicated with dashed line) (j), while only some sparse dissociated and transplanted iNs survived (i) 1 week post transplantation. Modified with permission from Ref. [275].

Culture type 2D culture (in vitro)

Cell type

Period

Note

Ref.

PVLA and E-cadherin IgC

mESCs (EB3)

7 days

Differentiation into hepatocytes

[202]

Substrate functionalization with peptide ligands for integrins a5b1 and a6b1 VEGF immobilization onto collagen IV substrate Immobilization of insulin like growth factor binding protein 4 onto PS substrate Decellularized 14 days seeded osteoblast/PLGA MS Graphene (G) and graphene oxide (GO) coated substrates PAA grafted CNT

hESCs (H9)

7 days

Mesodermal differentiation in presence of both ligands No differentiation in either of the ligands alone

[98]

mESCs (D4T)

3 days

[207]

ESCs (P19CL6)

20 days

Differentiation into primary endothelial cells in the presence of VEGF Differentaition into vascular smooth muscle cells in the absence of VEGF Cardiomycytes differentiation by inhibition of Wntb/catenin signaling

hESCs (h9p38)

21 days

[211]

iPSCs (20D17)

9 days

hESCs (H9)

5 days

mESC (D3)

2 days

Enhanced calcium deposition and OCN expression of ESC on decellularized MS compared to pristine MS Both surfaces allowed ectodermal and mesodermal differentiation. G suppressed endodermal differentiation and GO enhanced it Nanoscale features allowed enhanced protein adsorption, enhancing cell adhesion allowing high levels of neuronal differentiation In presence of RA and BMP4, cells differentiated into ectodermal lineages

[235]

hESCs (H9)

10 days

Neural differentiation without the addition of biochemical factors

[236]

hESCs (H1, H9)

7 days

[239]

hESCs (H9)

5 days

mESCs (W4)

23 days

mESCs (ESD3)

5 days

Differentiation into neuron in the nanogrooves and into glial cells in the pillars Cardiac differentiation peak found at day 1 on intermediate stiffness hydrogels Cellular organization in the form of vascularized cardiac muscle was enhanced in the 6 kPa hydrogels Upregulation of endoderm related genes on soft substrates

Alginate beads

mESCs (D3)

24 days

Differentiation toward neural lineages by cell–cell aggregation blocking

[248]

Alginate and Alginate/PLL microstrands

mESCs (CCE)

5 days

[251]

Alginate hydrogel combined with microgravity bioreactor VEGF functionalized agarose

mESCs (E14/Tg2a)

24 days

mESCs (Brachyury/Tgreen fluorescent cell line)

7 days

Nanofibrous scaffolds

PCL and core shell PES/PCL fibers with different stiffness

2 days

Macroporous scaffold

PCL/gelatin scaffolds 2D and 3D PLLA scaffolds Salt leached PLA/PLGA scaffold combined with siRNA

Mouse embryonic mesenchymal progenitor cells (C3H10T1/2) miPSCs (S103F9) mESCs (D3) hESCs (H9 and GFP-ES, EF1a-EmGFP-BG01v)

Cells within the alginate/PLL strands differentiated into endoderm and mesoderm. Cells within alginate strands differentiated toward ectoderm and mesoderm Alginate microcapsules cultured in bioreactor allowed osteogenic differentiation, showing 3D macroscopic bone like constructs Differentiation into blood progenitor cells was induced more efficient when VEGF was immobilized on agarose surface than when used as soluble factor Softer scaffolds (PCL) allowed chondrogenesis whereas stiffer scaffolds (PES/PCL) allowed osteogenesis

Biomolecular modification

Nano/microtopography

Stiffness

Cell encapsulation within hydrogels

Micropatterned arrays of ECM proteins Polyurethane acrylate nanoscale ridge/grooves PDMS nanogrooves and nanopillars Polyacrylamide hydrogels. Stiffness range 4–80 kPa Acrylamide collagen hydrogels stiffness range 0.2–40 kPa Fibrinogen-thrombin hydrogel Stiffness range 4–247 Pa

21 days 14 days 3 days

Expression of higher chondrogenic factors than the control ESCs expressed faster osteogenic markers on 3D compared to 2D Down-regulation of endoderm germ layer and up-regulation of mesoderm germ layer

[208,210]

[226] [230]

[242] [244] [246]

[252] [254]

[261]

[262] [268] [270]

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Carbon-based materials

3D culture (in vitro)

284

Table 5.1 Summary of representative biomaterials-based cultures of PSCs to induce differentiation into specific lineage in vitro and in vivo.

Table 5.1 (continued) Culture type

3D culture (in vivo)

Cell type

Period

Note

Ref.

Microspheres

Agarose, PLGA and gelatin

mESCs (D3)

14 days

Biomaterials incorporated within the stem cell aggregates can control the final phenotypic expression of cells

[271]

Scaffold

PLGA scaffold covered with HA and BMP2

8 weeks

De novo bone formation without teratoma formation in the scaffolds implanted with PSC and in the presence of BMP2

[272]

Hydrogel

Hyaluronan and methylcellulose hydrogels containing RGD and PDF-A Poly(lactide-co-caprolactone)

hiPSC (reprogramming of patient adipose derived stromal cells) hESC (H9) hiPSC

9 weeks

Spinal cord injury regeneration. Cell differentiated more, and teratoma formation was attenuated when cells were incorporated within hydrogel compared to direct cell injection Sciatic nerve conduit with cells. NCSC derived from pluripotent cells differentiated into Schwan cells and facilitated myelination of axons, promoting nerve regeneration Microfiber based scaffolds showed optimum results in brain in vivo implantation, showing higher neurite outgrowth and survival rate compared to direct cell injection

[273]

Nanofibrous scaffold

Polycarbonate based microfibrous substrates

1 month

hiPSC (derived from human foreskin) and derived neurons

3 weeks

[274]

[275]

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Mrofibrous scaffold

hiPSC, hESC (H1,H9), and derived NCSC

285

286

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design of more functional cell delivery matrices relevant for in vivo applications. Especially, introducing the 2D parameters like the matrix stiffness, cell sensing signaling molecules, and nanopatterns to the 3D geometrical regime can provide optimized PSC-biomatrix constructs that are most relevant for clinical translation. Thanks to considerable research over the last decade, our knowledge of PSC biology and advances in materials science and technologies have converged to empower biomaterials control over the PSC fate. However, there are still many issues to consider and hurdles to overcome on the road to clinical translation. Expansion of PSCs without the loss of pluripotency under feeder cell- or xeno-free conditions still awaits the development of cost-effective engineered biomaterials. Particularly for iPSCs, most generation methods rely on viral vectors to achieve efficient transfection, raising long-term safety issue. Biomaterials-based non-viral vectors are safer but remain suboptimal. More efficient non-viral intracellular delivery technologies must be developed to overcome this deficiency. Scaling up the quantities of PSCs to a level available for regenerative therapies is a prerequisite for translation. This is an area where biomaterials can play an important role to advance the field. Specifically, biomaterials endowed with specified cues to drive the pluripotent cells to target tissues would be most welcome. At the same time, more relevant animal models should be developed to facilitate meaningful evaluation of regenerative therapies in pre-clinical studies. Acknowledgements Supports from research funds include Global Research Laboratory Program (No. 2015032163) and Priority Research Centers Program (No. 2009-0093829) through National Research Foundation, South Korea, and NIH (5R25EB013127), USA. References [1] Fisher MB, Mauck RL. 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