Scaffold Materials for hES Cell Culture and Differentiation

5.508.

Scaffold Materials for hES Cell Culture and Differentiation

S Willerth and D Schaffer, University of California, Berkeley, CA, USA ã 2011 Elsevier Ltd. All rights reserved.

5.508.1. 5.508.1.1. 5.508.1.2. 5.508.1.3. 5.508.1.4. 5.508.1.4.1. 5.508.1.4.2. 5.508.1.5. 5.508.1.6. 5.508.2. 5.508.2.1. 5.508.2.1.1. 5.508.2.1.2. 5.508.2.2. 5.508.3. 5.508.3.1. 5.508.3.1.1. 5.508.3.1.2. 5.508.3.1.3. 5.508.3.1.4. 5.508.3.1.5. 5.508.3.2. 5.508.3.2.1. 5.508.3.2.2. 5.508.3.2.3. 5.508.4. 5.508.4.1. 5.508.4.2. 5.508.4.2.1. 5.508.4.2.2. 5.508.5. References

Introduction Defining Stem Cells and Potency Derivation of hESCs Standard Culture Methods for hESCs Techniques for Evaluating the Differentiation State of hESCs Markers for observing the differentiation state of hESCs Experimental techniques for observing the differentiation state of hESCs Using Biomaterials for the Culture and Differentiation of hESCs Comparison of Natural Versus Synthetic Biomaterials Biomaterials for Undifferentiated hESC Culture Natural Biomaterials for Undifferentiated hESC Culture Heterogeneous biomaterial scaffolds Homogeneous biomaterial scaffolds Synthetic Biomaterials for Undifferentiated hESC Culture Biomaterials for hESC Differentiation Natural Biomaterials for hESC Differentiation Alginate Dextran Collagen/laminin/matrigel Fibronectin Other naturally derived materials Synthetic Biomaterials for hESC Differentiation Poly(lactic-co-glycolic acid) Poly(ethylene glycol) Other synthetic materials Conclusions and Future Work Reflections on the Current State of Biomaterial Scaffolds for hESC Culture and Differentiation Future Directions Issues with hESC culture to be addressed Potential new materials for use in combination with hESCs Summary

Glossary Biomaterial A material that can be either natural or synthetic and that comprises either the whole or a part of a living structure or biomedical device that performs, supplements, or replaces a natural function. Conditioned media Media taken from cell culture that contains growth factors and other molecules that have been secreted by the cells. Ectoderm One of the three germ layers formed during embryogenesis that gives rise to the nervous system and the epidermis. Embryoid body Aggregates of embryonic stem cells usually grown in suspension culture. Embryonic stem cell Pluripotent cells derived from embryos or fetal tissue with the capacity for self-renewal.

96 96 96 97 98 98 98 99 100 100 100 100 102 103 104 104 104 105 105 106 106 107 107 109 110 111 111 111 111 111 112 112

Endoderm One of the three germ layers formed during embryogenesis that gives rise to the gastrointestinal and respiratory tracts along with the organs of the endocrine system. Extracellular matrix The matrix that surrounds cells and consists of a complex mixture of proteins, proteoglycans, and other molecules. Feeder layer A layer of cells that secrete specific factors into the media to allow for the culture of the desired cell line in coculture. Hydrogel A network of polymer chains that are water insoluble but possess a high water content. Karotype Characterization of the chromosomal content of a eukaryotic cell that can be used to evaluate if a gross genomic aberrations or abnormalities are present in a cell line.

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Matrigel A commercially available mixture of basement membrane proteins, including laminin and collagen, and proteoglycans that are secreted by Engelbreth–Holm–Swarm (EHS) mouse sarcoma cells as their extracellular matrix. Mesoderm One of the three germ layers formed during embryogenesis that gives rise to muscle, bone, connective tissues, and the dermis layer of skin. Niche Term used to describe the microenvironment that encompasses stem cells, composed of matrix, growth factors, small molecules, and adjacent cells.

Passage The process of maintaining a cell culture that has neared or reached confluence by diluting the number of cells present and replating to allow for continuous culture. Self-assembled monolayer A monolayer that is formed by self-organizing amphipilic molecules that possess a polar head group and nonpolar tail where the polar head group shows a specific affinity for the substrate for monolayer formation. Vasculature The network of blood vessels found in tissues.

Abbreviations

PDMS PEG pHEMA PLGA PLLA PTFE qPCR RGD SAMs SCID sIPNs SSEA-3 SSEA-4 TGF-b TRA-1-60 TRA-1-81 VEGF 2D 3D

AT bFGF Cbfa1 EBs ECM EHS ELISAs GABA HA hESC HGF IKVAV IPSCs LIF MEF NGF PCR PDL

5.508.1.

Alkanethiols Basic fibroblast growth factor Core binding factor alpha1 Embryoid bodies Extracellular matrix Engelbreth-Holm-Swarm Enzyme linked immunosorption assays g-aminobutyric acid Hyaluronic acid Human embryonic stem cell Hepatocyte growth factor Isoleucine–lysine–valine–alanine–valine Induced pluripotent stem cells Leukemia inhibitory factor Mouse embryonic fibroblast Nerve growth factor Polymerase chain reaction Poly-(D-lysine)

Introduction

Polydimethylsiloxane Poly-(ethylene glycol) Poly-(hydroxyethyl methacrylate) Poly-(lactic-co-glycolic acid) Poly-(L-lactic acid) Poly-tetrafluroethylene Quantitative polymerase chain reaction Arginine–glycine–aspartic acid Self-assembled monolayers Severe combined immune deficiency Semi-interpenetrating polymer networks Stage-specific antigen 3 Stage-specific antigen 4 Transforming growth factor b Tumor recognition antigen 1-60 Tumor recognition antigen 1-81 Vascular endothelial growth factor Two dimensional Three dimensional

This chapter will discuss the use of biomaterial scaffolds for the culture and differentiation of human embryonic stem cells (hESCs). The first section will provide definitions of stem cells and biomaterials and associated terminology, as well as describe the experimental techniques needed to study these cells. Section 5.508.2 will describe the research that has been conducted using biomaterials for culturing undifferentiated hESCs, while Section 5.508.3 will discuss the use of such materials for promoting hESC differentiation into specific phenotypes. Finally, Section 5.508.4 summarizes this field along with a discussion of future trends and directions.

the ability to differentiate into all of the cell types found in an organism along with the associated extraembryonic structures, such as the placenta, are referred to as totipotent. Pluripotent cells can differentiate into any cells in the different germ layers of an organism, and thereby give rise to all cell types in an adult organism, but lack the initial ability to generate the extraembryonic structures. Other classes of stem cells include unipotent (possessing the abilities to self-renew, but only to differentiate into one cell type) and multipotent (the ability to give rise to a subset of cell types). These final two typically include tissue-specific stem cells, such as muscle stem cells and neural stem cells, that typically differentiate into cells associated with that tissue.

5.508.1.1. Defining Stem Cells and Potency

5.508.1.2. Derivation of hESCs

Stem cells possess two defining characteristics: (1) the ability to self-renew, or proliferate and maintain themselves in an undifferentiated state, and (2) the ability to differentiate into one or more cell phenotypes. A variety of different stem cell types exist – including embryonic, fetal, and adult – and each has different properties such as varying degrees of potency, that is, the ability to differentiate into multiple cell types. Cells with

hESCs are pluripotent cells derived from human embryos. The first hESC lines were generated in 1998 from the inner cell mass of human blastocysts (Figure 1),1 which were obtained by culturing cleavage stage human embryos generated via in vitro fertilization. To confirm that viable and stable hESC lines are produced, the cells were continuously cultured for 4–5 months to demonstrate their ability to continuously proliferate,

Scaffold Materials for hES Cell Culture and Differentiation

(a)

(b)

(c)

(d)

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Figure 1 Derivation of the H9 cell line. (a) Inner cell mass-derived cells attached to a mouse embryonic fibroblast feeder layer after 8 days of culture, 24 h before their first dissociation. Scale bar, 100 mm. (b) H9 colony. Scale bar, 100 mm. (c) H9 cells at higher magnification. Scale bar, 50 mm. (d) Differentiated H9 cells, cultured for 5 days in the absence of mouse embryonic fibroblasts, but in the presence of human leukemia inhibitory factor (LIF) (20 ng ml
1; Sigma). Scale bar, 100 mm. Reprinted from Thomson, J. A.; Itskovitz-Eldor, J.; Shapiro, S. S.; et al. Science 1998, 282, 1145–1147, with permission from The American Association for the Advancement of Science.

differentiate into cells from all three germ layers (endoderm, ectoderm, and mesoderm), and maintain a stable karyotype. Additionally, undifferentiated hESCs express a number of markers – including stage-specific antigens 3 and 4 (SSEA-3 and SSEA-4) and the transcription factors Oct4, Sox2, and Nanog – that can be used to confirm the successful generation of a new hESC line and confirm the maintenance of a hESC state for existing lines. Further studies have confirmed the ability of hESCs to differentiate into numerous cell types from various tissues, including neuronal, glial, endothelial, cardiomyocytes, beta cells, muscle cells, and osteogenic lineages.2–5 Their pluripotency makes hESCs highly desirable for tissue engineering applications, as this single cell type provides the means to produce any cell present in the adult body, and their ability to self-renew yields a way to continually generate cells for these therapeutic purposes.

5.508.1.3. Standard Culture Methods for hESCs The first hESC lines were isolated and cultured on feeder layers of irradiated mouse embryonic fibroblasts.1,6 In general, feeder layers are a cellular monolayer that is cocultured with the desired cell type (in this case hESCs) and provides nutrients and growth factors necessary for the latter’s survival and proliferation. A variety of cell types have since been used as feeder layers to support hESC culture, including human fetal, foreskin, and adult fibroblasts as well as adult human marrow cells.7–9 In the interest of avoiding the use of feeder layers and thereby moving toward more defined and reproducible culture systems, hESC were then propagated on the substrate Matrigel in the presence of conditioned media derived from

mouse embryonic fibroblast cultures.10 Matrigel consists primarily of a mixture of basement membrane proteins, including laminin and collagen as well as proteoglycans that are secreted by the Engelbreth–Holm–Swarm (EHS) mouse sarcoma cell line as their extracellular matrix.11 The highly complex and heterogeneous nature of Matrigel12 means that its exact composition varies from batch to batch, making it less than ideal for hESC culture. Furthermore, the animal proteins present in Matrigel as well as from feeder layers could potentially transfer immunogens to hESCs, which could elicit a downstream immune response against transplanted stem cells.13 To generate hESC lines for clinical applications, the cells should ideally be cultured in fully defined conditions. To this end, many groups have worked toward developing defined hESC culture conditions, a challenging problem since the factors known to support mouse ESC culture did not directly translate to hESC culture. In 2006, Ludwig et al.14 described the derivation of new hESC lines in defined media that did not require the use of a feeder layer. Their methodology involved using chemically defined media referred to as TeSR1 that contained basic fibroblast growth factor (bFGF), lithium chloride, g-aminobutyric acid (GABA), pipecolic acid, and transforming growth factor-b (TGF-b), though the system still utilized Matrigel as a substrate for hESC adhesion. The Geron Corporation, a leading company advancing human stem cell therapies, has developed an alternative system composed of X-VIVO 10 medium (Cambrex Corporation) supplemented with bFGF and TGF-b1 for culturing their hESC lines on Matrigel.15 More recent work has suggested that the use of Noggin in combination with the Matrigel culture system can help hESCs remain undifferentiated.16 However, each of these

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systems utilized Matrigel as a substrate, and there are considerable efforts underway to replace Matrigel with a defined substrate.

5.508.1.4. Techniques for Evaluating the Differentiation State of hESCs Some of the most important considerations when working with hESCs are how to evaluate and characterize their differentiation potential and state. This section will discuss the different experimental techniques that are used for such purposes, including both analysis of marker expression and functional validation of pluripotency. It is important to have markers that can be used to identify hESCs to validate that such a line has been produced and confirm that the cells have remained undifferentiated after extended periods of culture. Similarly, it is necessary to identify specific markers of differentiation states when trying to generate specific cellular phenotypes from hESCs or confirm the pluripotency of a hESC line. Finally, these marker expression analyses should be combined with functional assays to confirm cell potency. See Chapter 3.314, Materials to Control and Measure Cell Function for further reading on analyzing cell behavior.

5.508.1.4.1. of hESCs

Markers for observing the differentiation state

To take advantage of the potential of hESCs for therapeutic applications, it is important to analyze biological markers that can be used to assess their differentiation state. Previously identified markers that could be used for studying mouse ESCs do not always directly translate to hESCs, and this section will discuss the specific markers that can be used to study hESCs (summarized in Table 1). Numerous markers are specifically or selectively expressed by undifferentiated hESCs,25 including the glycolipids SSEA-3 and SSEA-417,26 that can be detected with antigen-specific antibodies. Another set of hESC markers includes the tumor rejection or tumor recognition antigens, TRA-1-60 and TRA-1-81, which are keratan sulfate derivatives.1 Other very commonly used ones are transcription factors that are critical in maintaining hESCs pluripotency: Oct4, Sox2, and Nanog.18–20 In addition to analyzing self-renewal markers, the capacity for pluripotent differentiation, which can be characterized by Table 1

List of biological markers indicating differentiation state

Differentiation state

Marker

References

Undifferentiated hESCs

SSEA-3 SSEA-4 TRA-1-60 TRA-1-81 Nanog Oct4 Sox2

[1] [17] [1] [1] [18] [19] [20]

Nestin b-tubulin a-fetoprotein Smooth muscle actin

[21] [22] [23] [24]

Differentiated hESCs Ectoderm Endoderm Mesoderm

the ability to differentiate into cells of the three germ layers found during human embryogenesis, must often be assessed. Markers used to identify the ectoderm lineage include nestin and bIII-tubulin.21,22 Smooth muscle actin is often employed to detect cells from the mesoderm,24 and a-fetoprotein is frequently utilized to identify the endodermal germ layer cells.23 In addition to these commonly used markers, there are many alternatives used to detect cells from these germ layers. Finally, numerous downstream lineage- and cell-specific markers are utilized to monitor the process of hESC differentiation into a final desired differentiated phenotype.

5.508.1.4.2. Experimental techniques for observing the differentiation state of hESCs Numerous experimental methods exist to confirm the expression of the aforementioned markers, and this section will discuss the most frequently used techniques along with their relative advantages and disadvantages. Furthermore, in addition to marker expression, it is always important and often critical to utilize functional assays to confirm differentiation into specific cell phenotypes. While a variety of methods are employed to validate specific cellular phenotypes (such as electrophysiology for neuronal or cardiac differentiation, or enzyme and albumin synthesis for hepatocytes),27,28 the commonly used functional assays for confirming hESC identity will be discussed in Section 5.508.1.4.2.3. 5.508.1.4.2.1. Immunostaining Immunostaining is a standard technique that employs antibodies to detect and quantify antigen levels. This process involves using an antibody targeted against a specific molecule, referred to as the primary antibody, to detect its presence. A secondary antibody conjugated with either a fluorescent or enzymatic tag is used to bind to the primary antibody, and the presence of the marker can thereby be visualized and/or quantified through the use of a fluorescence microscope or by the addition of a colored substrate. While this process is extremely useful in allowing observation of proteins, it is important to perform proper controls to ensure that the desired antigen as opposed to background staining is being observed. Additionally, the quality of the staining is dependent on the specificity and quality of the primary antibody, and thus, different primary antibodies directed against the same target may yield different staining results. Nonspecific binding can often be reduced through enhancing blocking steps, often requiring the use of serum or other proteins, and through the use of wash steps that remove nonspecifically bound antibody. Immunohistochemistry is a variant of immunostaining where the cells or tissue to be stained is preserved through fixation prior to the staining process. This method has the advantage of showing the various structures formed by cells in culture and in tissue. Other ways of performing immunostaining include the use of flow cytometry where the resulting mixture from the cells stained in solution is analyzed quantitatively using a flow cytometer instrument, allowing for the determination of the fraction of cells expressing a specific marker and at what levels. In addition to measuring fluorescence, the flow cytometer characterizes cells based on their size and granularity, which allows for the exclusion of dead cellular

Scaffold Materials for hES Cell Culture and Differentiation

matter as well as for the analysis of the composition of a specific cellular population based on size. Another commonly used immunostaining method is Western blotting. In this process, the cells to be analyzed are lysed, and the resulting protein mixture is electrophoresed on a gel to separate the proteins by size. The separated proteins are then transferred to a membrane that can be immunostained. The separation of proteins by size allows for a second confirmation of the antibody specificity by confirming that the protein being identified is the correct weight. Western blotting can also be used to compare the expression levels of target proteins in a semiquantitative manner. A more effective method for quantitatively comparing protein expression levels using immunostaining is enzymelinked immunosorption assays (ELISAs). In this assay, the protein mixture to be analyzed is adsorped to a plate either through nonspecific binding or through the use of a capture antibody. A detection antibody conjugated with an enzyme is then used to detect the presence of the protein and the levels of protein are detected based on the enzymatic activity using either a colorimetric or chemiluminescence based substrate. One issue is the presence of background activity, which can be reduced through the use of blocking steps and washing. Overall, a variety of immunostaining techniques exist, and specific techniques should be chosen based on the desired information, such as protein location versus protein expression levels. 5.508.1.4.2.2. Polymerase chain reaction Another means to assess the expression of the various differentiation markers is polymerase chain reaction (PCR). The process of PCR involves amplifying small quantities of DNA to generate detectable levels of DNA using a preset number of cycles that heat and cool the reaction containing the target DNA, gene-specific primers (short specific DNA sequences that bind to the target gene) and a thermostable polymerase, the enzyme that synthesizes new copies of the target gene. Thus, this method can be used to study the mRNA expression levels of target genes by first performing a reverse transcription reaction that converts mRNA isolated from hESCs into DNA. The PCR reaction can then be performed and the resulting products electrophoresed on an agarose gel to confirm that the target genes were expressed. While examining these products on a gel will allow for semiquantitative comparison of gene expression, numerous methods for quantifying DNA amplified in a PCR reaction, that is, quantitative PCR (qPCR), have been developed. One uses a fluorescently labeled dye that binds to double-stranded DNA, such that the amount of PCR product at each cycle can be quantified in real time using a qPCR cycler. Other methods involve the use of gene-specific DNA probes that fluoresce when they bind to a complimentary piece of DNA, or upon degradation of the probe by the polymerase during the amplification. These fluorescence-based detection methods require the use of a thermocycler (the instrument that cycles the temperatures during the PCR reactions) that is specially equipped to detect fluorescence. The additional use of a standard curve of known concentrations of the target gene can be used to calculate the number of copies of mRNA present in a sample. It is also possible to examine relative gene expression between two samples by normalizing the data for the samples obtained from a housekeeping gene such as b-actin that is constitutively

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expressed by the cells, though caution should be applied since the expression of housekeeping genes can sometimes vary with cell state. Additional caveats with using qPCR to analyze differentiation are that mRNA levels do not necessarily translate directly to protein expression, and that glycoproteins and polysaccharides cannot be detected by this method. 5.508.1.4.2.3. Functional assays for confirming hESC identity In addition to these methods for analyzing the expression of characteristic markers by hESCs and their differentiated progeny, there are several standard assays conducted to confirm the integrity and functionality of hESC cells. These assays should also be performed periodically to confirm that an established hESC line is maintaining its properties. One of these methods is karotyping, a method used for characterizing the chromosomal content of a eukaryotic cell and evaluating whether aberrations or abnormalities such as chromosomal duplications or translocations are present in a cell line.29 This process is important to ensure that cells with no gross genomic abnormalities are used for basic biological studies or downstream translational efforts, though modern methods such as next generation sequencing may aid in higher resolution analysis of genomic integrity. Another important assay for confirming hESC identity is to validate that the cells can undergo self-renewal – or be continuously cultured while maintaining pluripotency marker expression and normal karyotype – for an extended period of time, such as between 4 and 6 months. Another key assay is teratoma analysis, the injection of hESCs into immunodeficient mice to determine if they have the capacity to form teratomas, benign tumors that express tissues similar to those found in the three embryonic germ layers.30 To confirm the presence of all three layers (ectoderm, endoderm, and mesoderm), histology followed by immunostaining for the aforementioned germ layer markers is performed.

5.508.1.5. Using Biomaterials for the Culture and Differentiation of hESCs As described in Section 5.508.1.3, the use of chemically defined media in combination with Matrigel has been shown to support undifferentiated hESC culture. Matrigel is considered a biomaterial, and its success as a substrate for hESC culture suggests that other, more defined and controlled biomaterial surfaces could be used for this application. In general, a biomaterial can be defined as a material, either natural or synthetic, that comprises either the whole or a part of a living structure or biomedical device that performs, supplements, or replaces a natural function. In addition to coating two-dimensional (2D) surfaces for the culture and differentiation of hESCs, biomaterials can also be used as 3D scaffolds for these applications. This chapter will detail the various studies that have used biomaterials in combination with hESCs in both 2D and 3D culture, along with the relative advantages and disadvantages of such scaffolds. The first part of this chapter focuses on biomaterials used to culture and expand undifferentiated hESCs, and the latter discusses the use of scaffolds to direct hESC differentiation into specific phenotypes. These parts are further subdivided into natural materials, obtained from animals or plants, and synthetic biomaterials, of artificial origins.

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5.508.1.6. Comparison of Natural Versus Synthetic Biomaterials It is important to first consider the advantages and disadvantages of both natural and synthetic biomaterials. Natural materials have been developed from the different components that make up the extracellular matrix, such as proteins and polysaccharides, including, for example, Matrigel.31 These components perform many roles in vivo, making the resulting materials attractive for tissue engineering applications since they typically contain sites for cellular adhesion and are biocompatible, meaning that the material does not typically induce an immune response upon implantation. However, some disadvantages include potentially large variability of the material depending on the source and the method of isolation, the need for robust purification of the protein or polysaccharide to both reduce variability and avoid triggering an immune response in vivo, potential challenges with supply in the case of human materials, and concerns with the transfer of pathogens from the animal or human materials into stem cells. Furthermore, as discussed below, scaffolds made from natural biomaterials tend to possess a limited range of mechanical properties compared to synthetic biomaterials, and it is very difficult to independently tune mechanics and the presentation of biochemical signals. Synthetic biomaterials offer a number of advantages compared to natural biomaterials, including reproducibility due to their defined chemical composition and the ability to control the mechanical properties, degradation rate, and shape independently.31 The mechanical properties of a scaffold have been shown to play an important role in influencing stem cell differentiation.32,33 Synthetic scaffolds can also be shaped, allowing for production of scaffolds that conform to specifications of an injury site. Engineering synthetic scaffolds with specific degradation rates can regulate the lifespan of the scaffolds in an in vivo setting as well as offer the capacity for controlled release of drugs incorporated into such scaffolds. However, a disadvantage for synthetic biomaterials is their inherent lack of bioactivity, such as sites for cell adhesion. These scaffolds are thus often biofunctionalized to introduce adhesion ligands, growth factors, and other activities. Other challenges include the need to carefully tune the biocompatibility of the material and its potential for inducing an immune response. See Chapter 4.410, Sterilization of Biomaterials of Synthetic and Biological Origin for further reading on preparation of scaffolds for implantation.

5.508.2. Culture

Biomaterials for Undifferentiated hESC

As mentioned in Section 5.508.1.3, the current methods for culturing and expanding undifferentiated hESCs suffer from numerous limitations. The current methods for passaging hESCs are laborious and time consuming. Additionally, the highly complex and undefined nature of Matrigel may result in considerable batch to batch variability in its constituent proteins and polysaccharides.12 This variability could, for example, introduce challenges in consistently maintaining pluripotency over time. Thus, an increasing focus has been placed on identifying the specific cues necessary to culture

Table 2

List of biomaterials used for hESC culture

Type of material

Material

References

Natural

Alginate Hyaluronic acid Matrigel Serum Acrylates Self-assembling monolayers Semi-interpenetrating polymers

[36,37] [38] [10,16,39–41] [41,42] [43] [44] [34]

Synthetic

undifferentiated hESCs and designing biomaterial surfaces with defined chemical compositions to eliminate the variability encountered when using Matrigel.34,35 A summary of the studies discussed in this Section is given in Table 2.

5.508.2.1. Natural Biomaterials for Undifferentiated hESC Culture This Section will detail the use of natural biomaterials for hESC culture and expansion. Natural biomaterials have been developed from the various proteins and polysaccharides that compose the extracellular matrix. These materials can be classified as heterogeneous biomaterials scaffolds, such as Matrigel and adsorbed human serum, and homogenous scaffolds, which have undergone more rigorous purification processes. See Chapter 2.207, Extracellular Matrix: Inspired Biomaterials; Chapter 2.220, Extracellular Matrix as Biomimetic Biomaterial: Biological Matrices for Tissue Regeneration; and Chapter 2.221, Decellularized Scaffolds for further reading on this subject.

5.508.2.1.1.

Heterogeneous biomaterial scaffolds

Despite the limitations discussed in Section 5.508.1.3, the combination of defined media and Matrigel coated onto a 2D surface remains the current standard for culturing hESCs.14,15,45 However, even when hESCs are cultured under these specific conditions, some random differentiation occurs due to local variations in the microenvironment. Patterning of Matrigel onto surfaces has been used as a method to help dissect such microenvironmental factors and properties that regulate self-renewal and differentiation. As a key example, Zandstra and colleagues used Matrigel micropatterning on 2D substrates to show that hESC colony size and composition regulated hESC differentiation by controlling local cell–cell signaling.46 As mentioned in Section 5.508.1.3, the signaling activity of TGF-b helps hESCs remain undifferentiated by inhibiting the activity of Smad1. The effect of colony size on both Oct4 (expressed by undifferentiated hESCs) and Smad1 activity (as indicated by phosphoSmad activity) are shown in Figure 2. As colony size increases, a corresponding increase in Oct4 staining and decrease in pSmad staining are observed. Additionally they used small interfering RNA to knock down Smad1, which prevented differentiation. This study suggests that a critical hESC colony size is needed to antagonize the effects of Smad1 and thereby prevent hESCs from differentiating when seeded on Matrigel, thereby illustrating the principle that controlling cell–cell interactions may be necessary for maintaining hESC self-renewal.

Scaffold Materials for hES Cell Culture and Differentiation

101

D = 200 mm

20´

D = 400 mm

20´

D = 800 mm

10´

pSmad1 (Oct4+)

%Oct4+

60 40

*

20 T=0

Gene expression (day 2)

0.8 0.6

*

*

*

*

0.4 0.2 0

D = 200 mm D = 400 mm D = 800 mm

D = 200 mm

D = 400 mm

5 4

BMP 2 Follistatin GDF3 LeftyB Smad6 Smad7

*

3

* **

2 1 0

D = 200 mm

**

**

**

D = 400 mm

D = 800 mm

Pattern type

3 2 1

**

**

**

0

D = 800 mm

(ii) 6

D = 200 mm D = 400 mm D = 800 mm

7 6 5 4

Oct4

Pattern type

(i)

(c)

6h Day 2

Pattern type

(b)

Merged (iii) 8

1.0

0h 6h Day 2

80

0

pSmad1

(ii)

100

Fold change over BMP-2

(i)

Oct4

Gene expression (day 2)

Hoechst

Nanog

Sox2

(iii) (Diameter, pitch) * 4

3

D = 200 mm D = 400 mm D = 800 mm

* **

*

2 1 0

Follistatin

GDF3

LeftyB

GDF3 gene expression normalized to Oct4%

(a)

7 6 5 4 3 2 1 0

D = 200 mm

D = 400 mm

D = 800 mm

Pattern type

Figure 2 Micropatterning can be used to manipulate the human embryonic stem cell (hESC) microenvironment and control hESC fate. To demonstrate that the hESC microenvironment, and in particular local cell–cell interaction, directly controls hESC fate, H9 hESCs were plated on patterned tissue–culture substrates with Matrigel using microcontact printing. Colony diameters (D) ranged from 200 to 800 mm and distance between colonies, pitch (P), was fixed at 500 or 1000 mm. (a) Quantitative fluorescent microscopy of patterned H9 cultures in XV media after withdrawal of all growth factors for 48 h and stained for Hoechst, Oct4, and pSmad1. Scale bar is 200 mm. (b) Analysis of micropatterned colonies after 48 h. The percentage of Oct4 cells as a function of colony size (b-i). pSmad1 in the Oct4þ population as a function of colony size (b-ii). Gene expression for Oct4, Sox2, Nanog normalized to b-actin as a function of colony size (b-iii). (c) To elucidate the molecular mechanism for the changes in pSmad1 levels, gene expression was quantified as a function of colony size for Smad1 agonist, BMP2, and antagonists, Follistatin, GDF3 and LeftyB and I-Smads, Smad6, and Smad7. Gene expression after normalization to b-actin and relative to the 200 mm data set (c-i). Ratios of the transcripts of Smad1 antagonists, Follistatin, GDF3 and LeftyB, to the transcripts of Smad1 agonist, BMP2, as a function of colony size (c-ii). GDF3 gene expression as a function of colony size normalized to the percentage of Oct4þ cells present (c-iii). For (c-i), statistical comparisons were made between gene expression for each cytokine in 400 and 800 mm colonies against the expression at 200 mm colonies. For (c-ii), statistical comparisons were made between the ratio of the levels of transcripts of the antagonist to BMP2 between the 400 and 800 mm colonies against the ratio present in the 200 mm. Reprinted from Peerani, R.; Rao, B. M.; Bauwens, C.; et al. Embo J. 2007, 26, 4744–4755, with permission from the Nature Publishing Group.

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Tissue Engineering – Fundamentals

Molded PU substrate Gold coating EG3 terminated SAM Matrigel ECM hESCs Figure 3 Schematic of microwell fabrication depicting spatial localization of physical and chemical constraints to human embryonic stem cell (hESC) attachment and propagation. Microwells are formed by cross-linking polyurethane (PU) prepolymer. Gold is evaporated on the surfaces surrounding the wells and the sides of the wells at an oblique angle, but the bottoms of the wells are shielded from gold. A triethylene glycol-terminated alkanethiol self-assembled monolayer (EG terminated SAM) is then assembled on the gold surface to repel extracellular matrix (ECM) proteins and cells. The wells are then treated with Matrigel, which adsorbs to the bare PU at the bottoms of the wells. Cells are then cultured in the Matrigel-coated wells. Structures are not to scale. Reprinted from Mohr, J. C.; De Pablo, J. J.; Palecek, S. P. Biomaterials 2006, 27, 6032–6042, with permission from Elsevier.

hESCs tend to aggregate, thereby forming 3D structures in 2D culture, and another group developed a 3D hESC culture that also involved Matrigel patterning (Figure 3).39 To first generate the microwell structure, photolithography was used to pattern polydimethylsiloxane (PDMS) masters, which were used to mold polyurethane into the desired structures. The resulting microwells were stabilized by UV-induced cross-linking. The resulting microwells were then functionalized with self-assembled monolayers (SAMs) – a monolayer that is formed by selforganizing amphiphilic molecules that possess a polar head group and nonpolar tail, where the polar head group has a specific affinity for the substrate to yield monolayer coverage – which were then coated with Matrigel. The study showed that hESCs could be cultured as colonies inside of these 3D Matrigelcoated scaffolds while maintaining their expression of Oct4. The resulting hESCs maintained their ability to proliferate even when transferred back to traditional 2D Matrigel culture. This system demonstrated that there are beneficial effects to using 3D culture for maintaining the undifferentiated state of hESCs without passaging the cells, and as an additional benefit, it allows for generation of uniform hESC embryoid bodies. Another group investigated human serum-coated substrates as a 2D biomaterial matrix for the culture of undifferentiated hESCs, as a potential alternative to Matrigel.42 The use of human serum would avoid the xenogenic pathogens associated with the use of Matrigel, which is derived from a mouse tumor. Tissue culture plates were coated with human serum, and hESCs were then cultured on this biomaterial surface in the presence of conditioned media obtained from human fibroblast-like cells derived from hESCs. This combination of medium and surface maintained the undifferentiated, pluripotent state of the hESCs as well as a normal karotype after 21 passages, suggesting human serum could potentially be used as a Matrigel replacement. That said, although the

use of human serum does avoid xenogenic components, it is even more heterogeneous and undefined than Matrigel. A subsequent study directly compared a variety of biomaterials and their suitability for culturing five different hESC lines.41 Of the materials analyzed, interestingly only surfaces coated with human serum, fetal bovine serum, and Matrigel supported undifferentiated hESC culture for multiple passages. Of these options, Matrigel was the only surface that supported long term (>30 passages) culture of hESCs. On the whole, the materials that could support multiple passages all consisted of heterogeneous, undefined mixtures of extracellular matrix or serum, whereas a fully chemically defined biomaterial surface and medium that can support undifferentiated hESC culture would be ideal.

5.508.2.1.2.

Homogeneous biomaterial scaffolds

This section will focus on scaffolds comprising a single natural biomaterial in contrast to the heterogeneous scaffold materials discussed previously in Section 5.508.2.1.1. One of the first studies to use a single 3D biomaterial scaffold for hESC culture involved using alginate scaffolds to promote the formation of embryoid bodies.36 Alginate is a linear polysaccharide derived from algae that can be cross-linked with divalent cations such as calcium to form 3D scaffolds.47 The alginate scaffolds were produced by cross-linking an alginate solution with calcium, followed by freezing the scaffold and a lyophilization process. The resulting porous scaffold was seeded with hESCs at a low or high density (Figure 4(b)). The microenvironments offered by these scaffolds promoted formation of round embryoid bodies and supported hESC proliferation, suggesting a potential method for hESC culture. Such scaffolds were later examined as a method of promoting the differentiation of hESCs after embryoid body formation. Another study also showed the use of alginate hydrogels for extended culture of undifferentiated hESCs.37 Hydrogels consist of a network of polymer chains that are water insoluble but possess high water content.48 Siti-Ismail et al. used alginate hydrogels to encapsulate the hESCs, which could then be cultured for up to 260 days in chemically defined media while maintaining their undifferentiated state provided the media was changed every 3–4 days. Although the number of hESCs produced was limited by the size of the alginate capsules, this process avoided the use of both animal products and a process of embryoid formation, suggesting the strong potential of hydrogels for hESC culture. Another study investigated the use of hyaluronic acid (HA) hydrogels for the culture of hESCs.38 HA is a linear glycosaminoglycan expressed in the extracellular matrix of hESCs as well as in the brain. In this work, the HA was chemically crosslinked to form stable hydrogels for hESC culture. When encapsulated inside of 3D HA scaffolds in the presence of conditioned media obtained from mouse embryonic fibroblasts, hESCs maintained their pluripotent state, whereas hESCs cultured on 2D HA surfaces did not. All of these studies indicate the potential for homogenous natural biomaterial scaffolds to be developed for maintenance of hESC culture. However, more work and rigorous testing will have to be performed before these systems can be coupled with chemically defined media and replace Matrigel as the standard for hESC culture.

Scaffold Materials for hES Cell Culture and Differentiation

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Figure 4 Scanning electron micrographs of human embryonic stem cell (hESC)-seeded LF120 alginate scaffolds after 1 month of cultivation. (a) A construct made with low hESC seeding concentration, demonstrating the porous structure of alginate scaffold. (b) Low-magnification image of a scaffold seeded with a high hESC concentration, demonstrating homogeneous cell distribution throughout the scaffold. Reprinted from Gerecht-Nir, S.; Cohen, S.; Ziskind, A.; Itskovitz-Eldor, J. Biotechnol. Bioeng. 2004, 88, 313–320, with permission of John Wiley & Sons, Inc.

5.508.2.2. Synthetic Biomaterials for Undifferentiated hESC Culture While natural systems such as Matrigel supply the substrate signals needed to support hESC attachment and maintenance, synthetic materials offer the potential for a defined and reproducible system if they can be engineered with the necessary bioactivity. See Chapter 2.209, Materials as Artificial Stem Cell Microenvironments for further reading on this subject. To identify numerous synthetic biomaterials to support hESC culture, Anderson et al. developed combinatorial microarray to screen potential substrates.43 Their microarray consisted of 576 combinations of acrylate, diacrylate, dimethacrylate, and triacrylate monomers presented on 2D surfaces consisting of poly (hydroxyethyl methacrylate) (pHEMA) using the mircroarray shown in Figure 5. For this study, they cultured the hESCs in knock-out replacement medium containing serum

103

and other defined supplements. This high-throughput screening of potential biomaterials surfaces for hESC culture allowed them to identify several candidate materials, and additional characterization found that several could effectively support hESC differentiation induced by retinoic acid. Future work may analyze whether the materials themselves were bioactive or selectively adsorbed different factors from serum. Regardless, additional characterization may reveal valuable new signals that can regulate cell differentiation. An analogous approach invovled a SAM array for screening potential peptide substrates for 2D hESC culture.44 To generate the array, one layer of SAMs consisting of alkanethiols (ATs) was photopatterned onto the surface followed by the spot application of various peptide sequences conjugated to ATs to generate the desired array. These peptide sequences were derived from the sequence of laminin, a significant component of Matrigel. The authors hypothesized that peptide fragments based on laminin would be able to support hESC culture in a manner similar to Matrigel. hESCs were then cultured in mouse embryonic fibroblast (MEF)-conditioned media on the microarray for 6 days and evaluated for their ability to support hESC growth and maintain their undifferentiated state. Their results showed that hESCs prefer to be grown on peptides containing the arginine–glycine–asparatic acid (RGD) sequence when presented at high concentrations. Earlier work from the Healy Lab investigated the use of semi-interpenetrating polymer networks (sIPNs, Figure 6) for the culture of undifferentiated hESCs.34 For this study, the sIPN was designed to contain cross-links that could be degraded by proteases, to simulate ‘natural’ remodeling of tissues by cells, and functionalized with an RGD sequence derived from an integrin-binding site of bone sialoprotein to allow for hESC adhesion to the network. These networks were capable of supporting hESC culture for short time periods ( 5 days) when used in combination with chemically defined media, representing for the first time a completely synthetic microenvironment used for short-term hESC maintenance. More recent work from the Healy and Schaffer Labs investigated the integrin ‘fingerprint’ necessary to support hESC attachment and to allow for culture and maintenance of undifferentiated hESC culture in the presence of chemically defined media.35 They determined that the aVb3, a6b1, and a2b1 integrins mediated hESC adhesion to Matrigel-coated surfaces. Based on these results, they synthesized peptides previously reported to bind these integrins and found that a combination of two integrin-engaging peptides along with an additional peptide designed to bind the heparan sulfate proteoglycan syndecan could support hESC adhesion and propagation for three passages, though the cells eventually lost expression of pluripotency markers. However, this study illustrates the concept that emulating the pattern of adhesion receptor engagement mediated by a natural biomaterial can extend the duration of hESC maintenance in a fully chemically defined culture system, a promising starting point for further work in developing synthetic scaffolds for hESC culture. Overall, these studies highlight the promise and advantages of using synthetic scaffolds for hESCs. However, significant challenges remain in investigating the mechanisms of interaction between hESCs and natural substrates such as Matrigel, the downstream signaling mechanisms that support hESC

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Figure 5 Biomaterial microarray design. (a) Monomers used for microarray synthesis. (b) Monomers were mixed at a 70:30 ratio pairwise in all possible combinations with the exception of monomer 17, which was substituted with * to increase polymer hydrophilicity. To facilitate analysis, all 24 polymers composed of 70% of a particular monomer were printed as a 6  4 group on the array, as highlighted by the red and yellow boxes. Three blocks of 576 polymers were printed on each slide, with a center to center spacing of 740 mm. (c) Printed polymer array imaged by an Arrayworx reader. Blocks composed of 70% monomer 1 and 70% monomer 6 are highlighted in red and yellow, respectively. (d) Differential interference contrast light microscopy of typical polymer spot overlaid with a few fluorescent cells (red). Reprinted from Anderson, D. G.; Levenberg, S.; Langer, R. Nat. Biotechnol. 2004, 22, 863–866, with permission from the Nature Publishing Group.

self-renewal, and synthetic ligands that can emulate these engagements. The first two studies described43,44 demonstrate how such synthetic materials can be screened in a highthroughput manner, allowing for the identification of potential scaffold materials and ligands, and the final study initiates the process of dissecting Matrigel for key signals that activate particular adhesion receptors. As many of the signals that promote hESC survival and proliferation remain unidentified, additional searches aided by high-throughput approaches could potentially be used for identifying novel molecules and peptide sequences for these applications.

5.508.3.

Biomaterials for hESC Differentiation

A considerable body of prior work has explored the use of biomaterial scaffolds for engineering various types of tissues (neural, dental, bone, vasculature),47,49–51 and it is natural to use these biomaterial-based approaches as starting places when investigating methods to promote the differentiation of hESCs into specific lineages for therapeutic applications. As discussed in Section 5.508.2, maintaining hESCs in an undifferentiated state requires a complex combination of signals. Likewise, directing hESC differentiation requires specific cues, such as growth factors and morphogens, combined with biomaterial

scaffolds. This section will discuss the current use of both natural and synthetic biomaterial scaffolds for promoting hESC differentiation into specific cell types. When selecting a material for promoting hESC differentiation, scaffold materials should meet a number of criteria: to be biocompatible, to contain sites for cell adhesion, to promote the desired differentiation, and – if included as part of an implantable graft – to not induce an immune response upon implantation. Furthermore, the degradation rate of the scaffold should also be considered, especially in comparison to the time course required for the desired differentiation. If differentiation-promoting cues are incorporated into the scaffold, this degradation rate can also influence the release rate of these cues. Finally, as previously mentioned in Section 5.508.1.6, it is also important to consider the fact that the mechanical properties of the scaffolds can also influence the resulting differentiation.32,33 Table 3 summarizes the efforts to engineer materials that investigate a number of these design criteria.

5.508.3.1. Natural Biomaterials for hESC Differentiation 5.508.3.1.1.

Alginate

As mentioned in Section 5.508.2.1.1, the biomaterial alginate has been shown to support the hESC cell culture, and in the same study, Gerecht-Nir et al.36 showed that embryoid bodies

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Figure 6 Semi-interpenetrating polymer networks (sIPN) synthesis: poly(N-isopropylacrylamide-co-acrylic acid) [p(NIPAAm-co-AAc)], interpenetrated by polyacrylic acid-graft-Arg-Gly-Asp [p(AAc)-g-RGD] linear polymer chains; (a) polymerization scheme and (b) schematic representation of the polymerized sIPN network. Reprinted from Li, Y. J.; Chung, E. H.; Rodriguez, R. T.; Firpo, M. T.; Healy, K. E. J. Biomed. Mater. Res. A 2006, 79, 1–5, with permission of John Wiley & Sons, Inc. Table 3

List of biomaterials used for hESC differentiation

Type of material

Material

References

Natural

Alginate Dextran Fibronectin Laminin Matrigel Carbon nanotubes Poly-D-lysine Poly(ethylene glycol) Poly(lactic-co-glycolic acid) Poly(tetrafluroethylene)

[36,52] [53] [40,54] [40] [40,55] [56] [40] [57] [58–62] [63]

Synthetic

made from the hESCs could differentiate into vasculature. The authors hypothesized that the porous nature of their alginate scaffolds helped enhance the differentiation process compared to traditional 2D culture. Another group also investigated that use of alginate to encapsulate hESCs, followed by implantation into mice, and characterized the resulting phenotypes to determine the effects of encapsulation on differentiation.52 They found that the encapsulated, transplanted cells differentiated primarily into an endoderm lineage. In addition, although it is unlikely that undifferentiated hESCs would be implanted for therapeutic application, this study found that the cells did not form teratomas, benign tumors that can be formed by immature transplanted hESCs, which could

enhance the safety of a graft. They also hypothesized that the encapsulation process may help protect the hESCs from immune rejection.

5.508.3.1.2.

Dextran

The use of a functionalized dextran scaffold for directing the differentiation of hESCs into vasculature was also investigated.53 Derived from bacteria, dextran is a complex polysaccharide consisting of glucose subunits, and it resists both cell and protein adsorption.47 To engineer the capacity for specific interactions with cells, the authors functionalized their dextran hydrogel scaffolds with RGD peptides. Additionally, they incorporated poly(lactic-co-glycolic acid) (PLGA, a synthetic polymer) microspheres (Figure 7(a)) that released vascular endothelial growth factor (VEGF) over a 10-day period (Figure 7(b)). Figure 7(c) shows the entire system including the dextran scaffold, the VEGF releasing PLGA microspheres, and hESCs. The hESCs cultured inside the hydrogels exhibited an increase in markers characteristic of differentiation into vasculature, including the VEGF receptor KDF/Flk-1. The authors emphasized that this biomaterial scaffold could potentially generate large numbers of endothelial cells for tissue engineering applications.

5.508.3.1.3.

Collagen/laminin/matrigel

Another group explored a wide variety of 2D biomaterial surfaces for directing hESCs into neural phenotypes, that is, neurons, oligodendrocytes, and astrocytes – including Matrigel, type 1 collagen, laminin/poly-D-lysine (PDL) mixture,

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Figure 7 Distribution and viability of human embryonic stem cell (hESC) aggregates encapsulated within bioactive dextran-based hydrogels: (a) scanning electron micrographs of 7 mm (a1, a2) and 20 mm (a3, a4) poly(lactic-co-glycolic acid) (PLGA) microparticles at day 0 (a1, a3) and 10 (a2, a4), during the release of vascular endothelial growth factor (VEGF)165. (b) Release profile of VEGF165 from 7 mm (□) and 20 mm (○) microparticle formulations. (c) Distribution of hESC aggregates on dextran-based hydrogels with 0.5 mm Acr-PEG-RGD and 5 mg ml
1 VEGF-loaded 20-mm microparticles, at day 0 (c1, c2) and day 10 (c3, c4). Top (c1, c3) and side (c2, c4) views. Arrows in c3 and c4 indicate a clump of cells proliferating outside the hydrogel. Inset in c3 shows the incorporation of microparticles (arrow) within the hESC aggregates. Scale bar corresponds to 200 mm. (D) Mitochondrial metabolic activity (average  SD, n ¼ 6) of hESC aggregates encapsulated in dextran-based hydrogels with no Acr-PEG-RGD (1), 0.5 mm Acr-PEG-RGD (2), 5 mm Acr-PEG-RGD (3), 0.5 mm Acr-PEG-RGD with 5 mg ml
1 of blank microparticles (4), and 0.5 mm Acr-PEG-RGD with 5 mg ml
1 of VEGF165 loaded 20 mm (5), or 7 mm (6) microparticles. The absorbance at 540 nm was measured after 1 day (black bars) and 10 days (white bars). Both absorbances were normalized to the day 1 absorbance. Reprinted from Ferreira, L. S.; Gerecht, S.; Fuller, J.; et al. Biomaterials 2007, 28, 2706–2717, with permission from Elsevier.

fibronectin/PDL mixture, and PDL alone (whose results will be discussed below in Section 5.508.3.2.40 This study found that Matrigel and the laminin/PDL mixture promoted an increase in neural progenitor generation as well as subsequent neuronal generation compared to the other substrates (the neuronal subtypes were not analyzed). They also noted a laminin dosedependency in these responses and thus concluded that laminin is a key molecule for promoting the generation of neural progenitors from hESCs. Other researchers used a 2D micropatterning approach, with Matrigel in combination with growth factors, to direct hESC differentiation in mesoderm and endoderm lineages.55 As discussed in Section 5.508.2, the local cellular microenvironment plays an important role in promoting hESC differentiation, and this study restricted the size of hESC colonies using patterns of adsorbed Matrigel. They showed that small, 200 mm colonies promoted hESC differentiation into endoderm, whereas larger, 1200 mm spots promoted mesodermal formation. This study thus illustrates how limiting the size of hESCs cultures can influence the resulting differentiation, which suggest the need to control the uniformity of embryoid body (EB) sizes to tune cell differentiation.

5.508.3.1.4.

Fibronectin

Another interesting approach to promoting hESC differentiation involved coating gold nanoparticles with a layer-by-layer deposition of the extracellular matrix (ECM) protein fibronectin to enable cell attachment.54 Gold is electrically conductive and enables an investigation of the effects of electrical stimulation on hESCs differentiation (Figure 8). They found that electrical stimulation promoted differentiation into osteogenic lineages, as indicated by the expression of collagen type 1 and the transcription factor core-binding factor alpha1 (Cbfa1), while a dramatic decrease in the expression of Oct4 was observed, suggesting that the majority of the hESCs had differentiated. The same effects were not observed in unstimulated cells.

5.508.3.1.5.

Other naturally derived materials

Another potential application of hESCs involves periodontal tissue engineering.64 Inanc et al. examined the capacity of tooth root slices, which consist of the soft tissue found in the root canal, as a natural ‘microenvironment’ to promote the differentiation of hESCs into tooth structures. They examined three different sets of culture conditions for hESCs, including

Scaffold Materials for hES Cell Culture and Differentiation

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Figure 8 Schematic diagram of a fibronectin-coated gold nanoparticle surface for human embryonic stem cell (hESC) differentiation by alternating electrical stimulation. Reprinted from Woo, D. G.; Shim, M. S.; Park, J. S.; Yang, H. N.; Lee, D. R.; Park, K. H. Biomaterials 2009, 30, 5631–5638, with permission from Elsevier.

seeding them next to these root slices, seeding them on a feeder layer of periodontal fibroblastic cells with the root slices present in the presence of osteogenic media, and allowing the hESCs to spontaneously differentiate. They found that the presence of the tooth root slices and a feeder layer along with the osteogenic media promoted differentiation into bone tissue, suggesting a potential mechanism for engineering periodontal tissue. These examples show the variety of natural biomaterials and approaches that can be used for directing hESC differentiation. The cell adhesion ligands naturally present in many of these materials, in conjunction with specific cues such as growth factors or small molecules, can aid in guiding cell differentiation; however, natural materials again suffer from some shortcomings, including reproducibility of composition and properties as well as potential transfer of immunogens or pathogens from the material to the cells.

5.508.3.2. Synthetic Biomaterials for hESC Differentiation A number of synthetic biomaterial classes have been evaluated for directing hESC differentiation into numerous lineages, and this section will review the current state of the field.

5.508.3.2.1.

Poly(lactic-co-glycolic acid)

A key early study examined how to control the differentiation of hESCs seeded inside a 3D scaffold consisting of a 50/50% blend of PLGA and poly(L-lactic acid) (PLLA).58 hESCs were first mixed with Matrigel to aid survival upon seeding into the

scaffolds. To direct hESCs into specific lineages, the following cues were used: retinoic acid (neural), TGF-b (osteogenic), and insulin-like growth factor (vasculature). They observed the ability of these factors to direct hESC differentiation into neural, cartilage, and liver tissue, respectively, through the use of immunohistochemistry, in vitro. Additionally, they observed the ability of hESCs to form blood vessels. Based on these promising results, they transplanted these scaffold-containing hESCs after 2 weeks of in vitro culture into severe combined immune deficiency (SCID) mice, such that implantation of such scaffolds containing hESCs would not trigger an immune response. Staining results indicate that the hESCs could survive and maintain these individual phenotypes in an in vivo setting (Figure 9). This key study demonstrated the potential of synthetic scaffold for promoting hESC differentiation by providing a 3D environment. Many other studies have used PLGA as a scaffold to promote hESC differentiation. One group encapsulated hESCs in PLGA scaffolds that were coated with laminin, implanted these scaffolds into the livers of SCID mice, and characterized the resulting differentiation.59 The cells differentiated into all three germ layers, and the scaffolds became highly vascularized. One issue encountered was the formation of teratomas inside these scaffolds due to the presence of undifferentiated hESCs. This issue would have to be addressed before such a strategy could be considered for clinical translation by ensuring that only differentiated hESCs are transplanted. Another group used scaffolds that consisted of PLGA-mixed hydroxyapatite to promote the differentiation of hESCs into bone tissue.60 To achieve this goal, they first induced the hESCs to differentiate

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Figure 9 Transplantation of human embryonic stem cell (hESC)–scaffold constructs into severe combined immune deficiency (SCID) mice. Two-week-old constructs (treated with retinoic acid (RA) or control medium) were implanted subcutaneously in the dorsal region of SCID mice. Fourteen-day-old implants were stained with hematoxylin and eosin (H&E) or with antibodies against human CD31, cytokeratin, AFP, or bIII-tubulin. Human-specific CD31 staining demonstrated the presence of both immunoreactive (construct-origin, arrows) and nonimmunoreactive (host origin, arrowhead) vessels. In certain instances there appeared to be continued differentiation and organization of constructs postimplantation. After continued construct maturation in vivo, RA-conditioned constructs exhibited larger and better organized neural structures than those seen in vitro (or with control medium in vitro or in vivo) including ductular structures lined by tall columnar epithelium invested with long cilia resembling ependymal cells, and rosettes with abundant melanin granules (brown/black in H&E section; confirmed by bleaching with potassium permanganate, data not shown). bIII-tubulin antibodies stained neuroectodermal structures within the implant as well as murine peripheral nerve fibers in surrounding connective tissue (asterisk). Scale bar, 50 mm. Reprinted from Levenberg, S.; Huang, N. F.; Lavik, E.; Rogers, A. B.; Itskovitz-Eldor, J.; Langer, R. Proc. Natl. Acad. Sci. USA 2003, 100, 12741–12746, with permission from the National Academy of Sciences.

into osteogenic lineages through coculture with human bonederived cells, since the latter secrete the factors necessary for promoting differentiation as previously reported.65 They seeded the resulting hESC-derived osteogenic progenitor cells inside the aforementioned 3D scaffolds and implanted them subcutaneously in SCID mice. These cell–material scaffolds promoted the regeneration of bone (Figure 10), whereas

control scaffolds seeded with dermal fibroblasts did not, suggesting that it was the hESC-derived cells that were responsible for the observed effects. This study illustrates the utility of hESC-derived cells for bone tissue engineering applications. To engineer neural tissue, 3D PLGA scaffolds combined with hESCs were treated with a combination of retinoic acid and nerve growth factor (NGF).61 The researchers made the scaffold

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Figure 10 Histochemical evaluation of osteogenic induction 4 and 8 weeks after in vivo implantation. In vivo bone formations of osteogenic cells derived from human embryonic stem cells (OC-hESCs) were evaluated and compared in four treatment groups: scaffold only, fibroblasts in the scaffold, OC-hESCs in the scaffold, and OC-hESCs in the scaffold with BMP2: (a) H&E staining (B, C, and S represent bone tissue, connective tissue, and scaffold, respectively); (b) von Kossa staining; (c) an enlarged image of (b); and (d) osteocalcin immunostaining (100). Reprinted from Kim, S.; Kim, S. S.; Lee, S. H.; et al. Biomaterials 2008, 29, 1043–1053, with permission from Elsevier.

porous to better enable cell proliferation and migration as well as the diffusion of the retinoic acid and NGF. They found that using 3D culture enhanced the expression of nestin and troponin (a skeletal muscle marker) compared to culturing the hESCs in 2D monolayer culture. The authors suggested that it may be beneficial to study hESCs in 3D to better emulate and understand behavior in an in vivo setting. A final study used hESCs and 3D PLGA scaffold to generate islet-like cells as a potential treatment for diabetes.62 In this work, a five-step protocol for generating the islet-like cells in vitro that could secrete insulin was adapted. They then encapsulated these isletlike cells inside PLGA scaffolds and transplanted them into diabetic SCID mice. The mice receiving this treatment maintained the presence of the islet-like cells in vivo and exhibited a decrease in glucose levels compared to levels in control mice (Figure 11). These studies collectively illustrate the utility of

PLGA scaffolds for promoting hESC differentiation for a variety of tissue engineering applications.

5.508.3.2.2.

Poly(ethylene glycol)

An early study investigated the use of 3D poly(ethylene glycol) (PEG) hydrogels as a potential scaffold material for hESC differentiation into chondrocytes, resident cells of cartilage.57 This important work by Elisseeff and colleagues used PEG diacrylate hydrogels chemically modified to present different sites that allow hESCs to bind to the scaffolds. Specifically, they compared the ability of PEG scaffolds conjugated with HA, type I collagen, and the RGD peptide to promote hESC adhesion and found that only RGD allowed hESCs to differentiate into neocartilage, as indicated by both cellular markers and the deposition of basophilic extracellular matrix indicated by glycosaminoglycan content.

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Random blood glucose levels 6 h fasted blood glucose levels

#

30

#

D Blood glucose (mmol l –1)

25

*

D

*

20 15 10 5

(a)

0

N Normal

C K S Pretransplant

Kidney bearing the grafts

C

K S Posttransplant

Cell–scaffold complex

C-peptide

(b)

C-peptide

(c)

Cell–scaffold complex

Cell–scaffold complex Granules

CD31

N

g (d)

(e)

Figure 11 (a) Random-fed and 6 h fasted blood glucose levels in the mice transplanted under the kidney capsules and embedded subcutaneous cell–scaffold complexes. N, normal mice; C, sham-transplanted mice; K, cell transplanted under kidney capsule mice; S, cell–scaffold complex transplanted mice. * p < 0.05 versus sham-transplanted mice; D p < 0.05 versus random blood glucose levels; # p < 0.05 versus pretransplant 6 h fasted blood glucose. (b–e): identification of islet-like cells during cell transplantation process. (b) c-peptideþ cells seeded in kidney capsule. c-peptide is a peptide derived from proinsulin. (c) c-peptideþ cells seeded in scaffold graft. (d) Immunohistochemical staining of rabbit anti-mouse CD31 in blood vessels within scaffold. (e) Transmission electron microscopic examination of secretory granules (g) in islet-like cells seeded in scaffold. The cells show a round nucleus (N). Magnification (b and c) 400, (d) 100, (e) 40 000. Reprinted from Mao, G. H.; Chen, G. A.; Bai, H. Y.; Song, T. R.; Wang, Y. X. Biomaterials 2009, 30, 1706–1714, with permission from Elsevier.

5.508.3.2.3.

Other synthetic materials

Other synthetic polymers have been studied for their suitability as substrates for hESC differentiation. 2D polytetrafluroethylene (PTFE) surfaces coated with poly-amino-urethane were seeded with hESCs, and a variant of hepatocyte growth factor

(HGF) as well as basic fibroblast growth factor (bFGF) were added to the media to induce differentiation into hepatocytes.63 The resulting cells secreted both albumin and urea, indicating that the hESCs were becoming functional hepatocytes. A study discussed in Section 5.508.3.1,

Scaffold Materials for hES Cell Culture and Differentiation

investigated a number of natural and synthetic materials for the ability to support neural differentiation.40 The study found that PDL – a synthetic polymer that is often used to enhance the ability of cells to attach to surfaces via ionic interactions between the amine groups and the negatively charged cellular glycocalyx66 – could modestly support hESC differentiation into neural phenotypes, though the ECM proteins were more effective. A recent study investigated the use of thin films of carbon nanotubes as a 2D substrate for promoting the differentiation of hESCs into neurons,56 and the nanotube-based surfaces allowed the cells to adhere and differentiate without cytotoxic effects. These are examples of other polymers that could potentially be used as substrates for hESC adhesion and differentiation.

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5.508.4.2. Future Directions

must be made in identifying biochemical and mechanical cues in the stem cell microenvironment, including both substrate and matrix, to enable the development of defined biomimetic materials and liquid culture systems that can fully substitute for undefined components and support cell function. To address this issue, proteomic analysis of conditioned media was conducted to determine what components are necessary for maintaining undifferentiated hESCs. One hundred seventy-five proteins were found in the media, and a key subset of these factors must aid in maintaining undifferentiated hESCs.67 Likewise, a recent proteomic study identified hundreds of proteins in Matrigel,12 which renders the development of a synthetic matrix quite challenging. See Chapter 4.409, Surfaces and Cell Behavior and Chapter 4.427, CellDemanded Release of Growth Factors for further reading on presentation of cues that influence cell behavior. Bioactive materials along with hESC-derived progeny may aid in overcoming challenges with engraftment, such as low cell viability. In addition, immune responses against the graft may be a challenge. hESCs cultured on Matrigel, in conditioned medium, in serum, or in the presence of animal proteins in general likely internalize and incorporate murine antigens,13 which could trigger an immune reaction when implanted in vivo. This issue would be mitigated by the use of a defined culture system prior to implantation, and the coimplantation of nonimmunogenic biomaterials may to an extent shield cells from immune responses. Cells may well also face immune responses due to challenges in the histocompatibility of donor cells and patient recipients. However, patient-specific pluripotent stem cell lines are theoretically possible with the advent of induced pluripotent stem cells (iPSCs). iPSCs are generated from adult somatic cells, such as fibroblasts, by inducing the expression of specific genes that restore pluripotency.68 Human iPS cells are similar to ESCs in their selfrenewal, pluripotency, and teratoma formation,69 and it is likely that biomaterials development studies will increasingly involve human iPSC lines. An additional consideration when developing hESC-based therapies is avoiding teratoma formation in vivo. To achieve clinically relevant therapies, it is important to ensure that the cells are sufficiently and uniformly differentiated so as to avoid this hazardous possibility, and Section 5.508.3, details potential scaffold materials that can be used to control and potentially provide uniform hESC differentiation. Furthermore, cell encapsulation inside materials, in some cases, may physically contain cells and thereby inhibit tumor formation.

This section discusses challenges with current approaches, as well as suggests potential novel biomaterial scaffolds for the culture and differentiation of hESCs.

5.508.4.2.2. with hESCs

5.508.4.

Conclusions and Future Work

Overall, this chapter summarizes the current state of biomaterial scaffolds for the culture and differentiation of hESCs. The use of hESCs in conjunction with bioactive materials holds enormous therapeutic potential that these studies begin to highlight and help harness. This final section will discuss the challenges in current efforts and offer directions for the future of the field.

5.508.4.1. Reflections on the Current State of Biomaterial Scaffolds for hESC Culture and Differentiation This chapter has discussed the current state of the field, and many valuable studies have shown that hESCs can differentiate into a wide variety of cells types for therapeutic applications, including neurons, bone, and islet-like cells. This work has also demonstrated that hESC behavior is heavily influenced by the culture system, and cues ranging from biochemical signals from matrix, small molecules, and growth factors can support and regulate hESC self-renewal and differentiation. Furthermore, ‘geometric’ cues such as local cell density and composition, as well as 2D versus 3D culture systems, can profoundly influence cell function. Collectively, this work has highlighted the promise and potential of hESC and biomaterial scaffolds for serving as a culture systems so as to aid cell expansion and differentiation in vitro, as well as for supporting the implantation of cells in vivo.

5.508.4.2.1.

Issues with hESC culture to be addressed

As discussed in Section 5.508.1.3, progress must still be made in the development of in vitro culture systems for hESCs. A major limitation of current hESC culture methods is the use of Matrigel or other poorly defined components such as serum, conditioned medium, or other ECM proteins. The biomaterials described in this chapter have the potential to provide fully chemically defined conditions for cell expansion and differentiation, which would enhance safety, reproducibility, and scaleability. However, considerable progress

Potential new materials for use in combination

A number of materials have been successfully investigated for hESC culture and differentiation, though a number of additional materials offer other key advantages that may be explored in future work. 5.508.4.2.2.1. Additional natural biomaterials Many other natural biomaterials have been used in combination with mouse ESC culture, and these could also potentially be applied to hESCs. For example, fibrin and collagen are two potential protein-based materials that are found in the extracellular matrix, and these materials have been extensively

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studied in biomaterial scaffolds.70,71 Fibrin is a polymer that consists of fibrinogen monomers that are found in blood, which coagulate to form fibrin clots in response to vascular injury. Several groups have shown the feasibility of using fibrin scaffolds for both mouse and mesenchymal stem cell culture.72–76 Another commonly used biomaterial for scaffolds is collagen, one of the major components in Matrigel, but little work has been conducted using pure collagen scaffolds for the culture and differentiation of hESCs. Previous work has shown that mouse neural stem cells seeded inside collagen scaffolds can form functional neuronal circuits.77 The other major group of natural biomaterials that could be investigated is polysaccharides. As mentioned in Section 5.508.3.1, dextran and alginate scaffolds have been successfully used for hESC culture and differentiation, suggesting that other polysaccharide-based biomaterial scaffolds could be used in a similar manner. Dextran is resistant to cell and protein adsorption, but it can be chemically modified to contain sites for cell adhesion, making it a potentially promising substrate for hESC culture. Agarose, a readily available polysaccharide isolated from seaweed, has been used as a scaffold material for differentiating primate ESC lines into cardiomyocytes.78 The thermoresponsive properties of agarose can be used to regulate scaffold formation, suggesting that such scaffolds could be formulated to produce an injectable version. Another potential scaffold material is chitosan, which consists of glucosamine subunits. Chitosan scaffolds have been used to culture stem cells derived from human cord blood as well as mesenchymal stem cells.79,80 It can fabricated using a variety of methods, such as encapsulation, spray drying, and gelation, to provide different methods of drug delivery.81 It would be interesting to study hESC behavior inside scaffolds made from these polysaccharide, based on their suitability for the culture and differentiation of these other stem cell lines. 5.508.4.2.2.2. Additional synthetic biomaterials Numerous synthetic polymers have been characterized for use as scaffolds in combination with stem cells other than hESCs, including pHEMA and polypyrrole. Scaffolds made from pHEMA do not degrade, and thus, maintain their initial shape after implantation. Such pHEMA scaffolds have been successfully fabricated into hydrogels that support mesenchymal stem culture.82 Another attractive synthetic material for investigation is polypyrrole, a polymer consisting of connected pyrrole ring structures. It is highly conductive, a potentially interesting property as electrical stimulation has been shown to influence hESC behavior.54 It has also been used to study mesenchymal stem cell behavior on 2D surfaces, where it was determined that the concentration of polypyrrole used influenced the resulting stem cell differentiation. These studies suggest that both of these synthetic polymers have the potential for use with hESCs. Recently, several groups have developed novel selfassembling scaffolds based on short synthetic peptide sequences for stem cell culture and differentiation. Stupp and colleagues showed self-assembling scaffolds based on the peptide sequence IKVAV (isoleucine–lysine–valine–alanine– valine) derived from laminin promote the differentiation of fetal neural cells into neurons.83 More recent studies have examined the use of similar peptide-based self-assembling

scaffolds in combination with mesenchymal stem cells for osteogenic differentiation.4,84,85 Likewise, Zhang and colleagues developed a novel self-assembling material based on a peptide, RADA-16-I, and used scaffolds derived from this peptide and related variants for promoting the differentiation of mesenchymal and neural stem cells.86,87 These studies illustrate the utility of such novel peptide-based scaffolds, and such a strategy could be applied for hESC culture and differentiation, though precisely controlling the 3D assembly and mechanical properties of these short oligomers can be challenging. Furthermore, successfully implementing such a strategy may require the use of multiple peptide sequences due to the complex requirements of hESC culture.

5.508.5.

Summary

This chapter has detailed the current state of the field, including the use of both natural and synthetic scaffolds for hESC culture and differentiation, along with discussing potential trends for future work to help maximize the therapeutic potential of hESCs. In general, hESCs have great therapeutic potential due to their ability to self-renew, which in principle can provide limitless quantities of cells, and their pluripotency, such that a single cell type can be harnessed to address numerous diseases. Many different biomaterial scaffolds have been investigated for hESC culture and differentiation due to their ability to provide the signals and cues, and thereby recreate cellular microenvironments, necessary to regulate and control self-renewal in vitro and differentiation in vitro and in vivo. Therefore, the integrated use of hESCs and engineered biomaterials hold great promise for greatly enhancing the therapeutic potential of tissue engineering and regenerative medicine.

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Relevant Websites http://www.stembook.org/ – StemBook is a comprehensive, open-access collection of original, peer-reviewed chapters covering topics related to Stem Cell Biology. http://www.cirm.ca.gov/ – The official website of the California Institute of Regenerative Medicine. http://stemcells.nih.gov/research/nihresearch/scunit/ – The official website of the NIH Stem Cell Unit. http://www.biomaterials.org/ – The official website of the Society of Biomaterials. http://stemcells.wisc.edu/ – The official website of the University of Wisconsin–Madison Stem Cell and Regenerative Medicine Center.