Design Approaches for Generating Organ Constructs

Cell Stem Cell

Review Design Approaches for Generating Organ Constructs Yun Xia1,* and Juan Carlos Izpisua Belmonte2,* 1Lee

Kong Chian School of Medicine, Nanyang Technological University, 11 Mandalay Road, Singapore 308232, Singapore Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA *Correspondence: [email protected] (Y.X.), [email protected] (J.C.I.B.) https://doi.org/10.1016/j.stem.2019.05.016 2Gene

Organ constructs are organ-like structures grown in vitro or in vivo that harbor the components, architecture, and function of in vivo organs, in part or in toto. The convergence of stem cell biology, bioengineering, and gene editing tools have substantially broadened our ability to generate various types of organ constructs for regenerative medicine as well as to address pressing biomedical questions. In this Review, we highlight prevailing approaches for generating organ constructs, from organoids to chimeric organ engineering. We also discuss design principles of different approaches, their utility and limitations, and propose strategies to resolve existing hurdles. Introduction Over the last two decades, rapid expansion of stem cell research has substantially deepened our understanding of cell fate plasticity, leading to various approaches toward manipulating cell fate determination. Among these, development of methods that use a patient’s own cells to form specific cell types and even organ-like miniatures has significantly extended our capability to study normal and abnormal biological processes (Table S1; Clevers, 2016; Lancaster and Knoblich, 2014; Takahashi and Yamanaka, 2006). Meanwhile, progress in design, fabrication, and printing of biomaterials has markedly increased our control over the generation of desired cell products in vitro (Lutolf et al., 2009; Murphy and Atala, 2014). Thanks to the advances in multiple biotechnologies, such as gene editing and micromanipulation, our ability to generate desired cell types has been extended in vivo (Cong et al., 2013; Doudna and Charpentier, 2014; Jinek et al., 2012; Ran et al., 2013). In this Review, we propose the concept of ‘‘organ construct,’’ which is an entity, constructed by cellular and non-cellular elements in vitro or in vivo, that present in part or in toto, the components, architecture, and function of in vivo organs. A growing repertoire of organ constructs are being produced and utilized to address a number of important biological needs, from studying development, modeling disease initiation and progression, and generating tissue biobanks to regenerative medicine. In this article, we will broadly categorize existing approaches for generating human organ constructs (Figure 1). We will first focus our discussion on organoid approaches that, in principle, are established upon a framework centered on self-organization (Figure 2A), followed by a comparison with bioengineering and organs-on-a-chip approaches, which engage higher levels of extrinsic manipulations. Next, we will switch gears to elaborate on ‘‘in situ’’ approaches, with a focus on interspecies organogenesis. We will also briefly highlight how to generate organ constructs in situ via lineage reprogramming and gene editing. Finally, we will collectively consider the utility and limitations of these approaches for addressing important biological questions and speculate on how future approaches can combine each other’s strengths.

In Vitro Approaches Organoids Gradual understanding of embryonic development has allowed us to partially and/or temporally reproduce this process in vitro. Since the establishment of the first human embryonic stem cell (ESC) lines in vitro (Thomson et al., 1998), tremendous efforts have been dedicated to coax human pluripotent stem cells (PSCs) into desired lineages, with the hope of generating transplantable cell resources. Despite progress in generating many cell types in 2D (two dimensions) via direct differentiation, their inherent lack of cell-cell and cell-matrix interactions precludes the possibility to study phenotypes usually seen in 3D (three-dimensional) organs/tissues in vivo. To distinguish organoids from their ‘‘close relatives,’’ bioengineered tissues/organs and organs-on-a-chip, we define them as 3D structures, derived from pluripotent or adult stem cells (ASCs) through self-organization. Traditionally, organoids require minimal external manipulation compared with the amount of external input required to generate bioengineered tissues/organs and organs-on-a-chip, which makes organoid approaches technically amenable and reproducible. As expected from a self-organized process, much of organoid mechanistic assembly can recapitulate in vivo development and assembly processes, which are still not well understood (Figure 2A). Organoid advantages include experimental accessibility, phenotype presentation at a tissue/organ level, and preservation of pathological and genetic abnormalities in a patient-specific manner. Over the last decade, organoids have been extensively used for studying development and disease, developing personalized medicine (Schwank et al., 2013), and regenerative medicine. Alignment of human organoid models with genetic animal models can highlight developmental similarity (McCracken et al., 2014, 2017) and divergence (Eiraku et al., 2011; Nakano et al., 2012) between human and model organisms. The recent establishment of various early stage embryoids from human and mouse PSCs provides a new avenue for studying mechanisms underpinning self-organization (Harrison et al., 2017; Martyn et al., 2018; Rivron et al., 2018; Sozen et al., 2018). Facilitated by gene editing technologies, organoids have already been widely used for modeling genetic diseases and host-pathogen interactions Cell Stem Cell 24, June 6, 2019 ª 2019 Elsevier Inc. 877

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Figure 1. Categorization of Approaches for Generating Organ Constructs Organ construct generation approaches can be mainly categorized into in vitro and in situ. In vitro approaches include organoid, bioengineering, and organs-ona-chip, while in situ approaches include interspecies chimeras, direct lineage reprogramming, and gene editing. Abbreviations: PSC, pluripotent stem cells; ASC, adult stem cells; CRISPR, clustered regularly interspaced short palindromic repeats; AAV, adeno-associated virus.

(Bartfeld et al., 2015; Co et al., 2019; McCracken et al., 2014; Qian et al., 2016). Furthermore, organoids represent a highly accessible and amenable platform for validating genetic lesions that are associated with various human diseases (Klaus et al., 2019) and tumor formation (Bian et al., 2018). Although using organoids for regenerative medicine may require considerably more time and efforts to realize, promising achievements have been made over the last few years (Haller et al., 2019; Sampaziotis et al., 2017; Sharma et al., 2019; Sugimoto et al., 2018). Progress in developing this myriad of organoid technologies have been extensively documented in several excellent reviews (Clevers, 2016; Drost and Clevers, 2018; Li and Izpisua Belmonte, 2019; Smith and Tabar, 2019). 878 Cell Stem Cell 24, June 6, 2019

Complex Organoids Require Stage-Specific Self-Assembly. Based on our understanding of embryonic development, we can typically figure out what growth signaling molecules should be employed to devise a specific organoid. For instance, a precise modulation of WNT/FGF or Activin signaling often acts as the starting point for defining a specific embryonic region (Lancaster et al., 2017; Sharon et al., 2019; Takasato et al., 2014, 2016). It is noteworthy that embryonic, neuroectoderm, and mesoderm organoids are mainly derived from PSCs via differentiation, whereas most endoderm organoids can be derived from PSCs, as well as ASCs and dissociated tissue (healthy or pathological) (Table S1). Several mesoderm organoids fall into the ‘‘grey zone,’’ such as organotypic culture formed from

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Review B

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t – Ɵming for self-assembly Fetal stem cells

PSC

Gastruloids

Adult stem cells Adult Ɵssues Pathological Ɵssues

Neural tube Kidney organoids GI organoids

Brain OpƟc cup organoids Lung organoids

Liver organoids Prostate organoids GI organoids PancreaƟc organoids

SpaƟal Temporal

Lung organoids

Tubuloids Liver organoids

Tumoroids Clonal liver organoids

Figure 2. Multicellular Self-organization during Organoid Formation (A) Multicellular self-organization mainly includes three basic processes: cell sorting (self-assembly), self-patterning, and morphogenesis (Sasai, 2013). Cell sorting usually cooperates with self-patterning to establish a pattern from a cluster of cells that have no pre-pattern through spatiotemporal control of cell status, total cell number, and relative location, driven by intrinsic as well as extrinsic cues. A symmetry-breaking event usually triggers the initiation of self-patterning. Morphogenesis employs the intrinsic mechanic constraints and physical properties to establish a higher-level tissue/organ architecture. (B) For different types of organoids, the optimal timing for self-assembly correlates with the intrinsic developmental program of the cells. (Fetal stem cells refer to organ-specific stem cells derived from either PSCs or fetal tissues. GI indicates gastrointestinal. Clonal liver organoids refer to liver organoids derived from damaged postnatal liver tissue.)

aggregated mouse embryonic kidney or gonad cells, as well as adult kidney tubuloids that were recently described (Schutgens et al., 2019), because these organoids/organotypic culture do not possess unlimited capability for in vitro culture. PSCs may represent the optimal starting point for generating various early stage embryonic structures, such as gastruloids (Etoc et al., 2016; Martyn et al., 2018) and neural cysts (Figure 2B; Meinhardt et al., 2014; Ranga et al., 2016). Following this line of logic, PSC aggregates can be efficiently steered into eye cups and various brain organoids, a remarkable feat as these structures emerge without a recognizable reference coordinating system (Figure 2B; Eiraku et al., 2008, 2011; Lancaster et al., 2013; Nakano et al., 2012). One possible explanation for this observation may lie in the intrinsic tendency of PSCs to develop into anterior neural fates. In comparison, endoderm and mesoderm organoid formation engages self-assembly at a stage when organ-specific patterning has already initiated (Figure 2B). For example, differentiation of intestinal and colonic organoids from human PSCs both involve a stage when human gut tube (organ primordium) spontaneously detaches from 2D monolayer culture (McCracken et al., 2011; Mu´nera et al., 2017; Spence et al., 2011). Likewise, at the anterior foregut endoderm stage, cells self-assemble and protrude out from 2D monolayer, finally detach to establish human lung bud (Chen et al., 2017; McCauley et al., 2017; Miller et al., 2019). Similarly, human PSC-derived pancreatic progenitors constitute the start of self-assembly, followed by guided patterning toward polarized pancreatic ducts (Huang et al., 2015). Kidney organoid formation can employ either spontaneous (protrusion from 2D monolayer) or facilitated self-assembly at the onset of nephrogenesis to realize organoid formation (Morizane et al., 2015; Takasato et al., 2016). Although some kidney organoid

differentiation protocols employ self-assembly at the pluripotency stage, these protocols often require more delicate and exhaustive extrinsic instructions during differentiation (Taguchi et al., 2014; Taguchi and Nishinakamura, 2017). This general principle may be easier to appreciate with ASC-derived organoids, when adult stem cells constitute the only or main cellular component for organoid generation (Figure 2B). Adult tissuederived organoids, especially those derived from damaged organs (non-genetic lesion), may self-organize in a manner that can hardly be distinguished from ASC-derived organoids (Figure 2B). Notably, tumor organoids (tumoroids) represent a special form of self-organization, in which cell sorting acts as the major player to preserve both architectural and genetic heterogeneity that resembles the original tumor (Broutier et al., 2017; van de Wetering et al., 2015). When we try to align the timing of self-assembly employed in organoid generation with developmental and pathophysiological stages, spontaneous or facilitated self-assembly is usually engaged at the point when the targeted lineage is about to segregate from the initial system (Figure 2B). Thus, different organoid generation approaches engage self-organization at specific differentiation stages, depending on whether sufficient organ-specific intrinsic guidance is garnered at the point of self-assembly. Multicellular Self-Assembly. For most organoids, organ-specific cell types are better represented, such as dopaminergic neurons in brain organoids, retinal pigment epithelium (RPE) in optic cups, hepatocytes in liver organoids, and podocytes in kidney organoids. In contrast, generic cell types that are ubiquitously existing in multiple organs are usually under-represented in organoids, such as vascular endothelial cells, immune cells, neurons, and stromal cells. Cell Stem Cell 24, June 6, 2019 879

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Review As far as cellular composition is concerned, self-organization can generously tolerate the addition of cell types that share different origins (Figure 2A). Based on existing evidence, such tolerance may be dependent upon two factors: (1) a defined lineage propensity within the ‘‘guest’’ cells and (2) a close-to-native ratio between ‘‘host’’ and ‘‘guest’’ cells. This is not a completely new concept, as mouse embryonic rudimentary aggregates have been utilized to validate the lineage properties of exogenous (guest) cells (Garreta et al., 2019; Mae et al., 2013; Xia et al., 2013). Similarly, human PSC-derived germ cells require co-culture with neonatal testes (Hayashi et al., 2011) or E12.5 ovaries (Hayashi et al., 2012; Hikabe et al., 2016; Yamashiro et al., 2018) to reach full potency. Self-organization has already been exploited to introduce missing cell types during organoid formation. Takebe and colleagues introduced extrinsic endothelial cells and mesenchyme stem cells (MSCs) into liver organ bud, which anastomosed with and was perfused by the host circulation system quickly after implantation (Takebe et al., 2013). Such an approach was later generalized to establish multiple organ buds using mouse embryonic tissues (Takebe et al., 2015). The recent establishment of blood vessel organoids from human PSCs may offer an alternative source of endothelial cells (Wimmer et al., 2019). Co-culture or co-transplantation of blood vessel organoids with other types of avascular organoids may lead to inter-organoid vessel migration. Employing a similar additive approach (Gilmont et al., 2014; Raghavan et al., 2011), human PSC-derived neural crest cells were successfully assembled into intestinal organoids derived from the same parental PSC line to establish organoids with a higher order of structural sophistication (Workman et al., 2017). Such an intestinal organoid platform enabled the modeling of Hirschsprung disease caused by mutations in PHOX2B, which underpins aganglionosis of human bowel (Workman et al., 2017). In a recent study, liver organoids self-assembled from human PSC-derived hepatic stellate cells and hepatocytes offer a new avenue for studying liver fibrosis and examining liver toxicity (Coll et al., 2018). Organoids have recently been used as ‘‘incubators’’ for the generation and maturation of immune cells. Artificial thymus organoids facilitated T cell differentiation from human PSC-derived hematopoietic cells (Montel-Hagen et al., 2019), while tumoroids switch on tumor reactivity in patient-derived circulating T cells (Dijkstra et al., 2018). Challenges and Limitations of Current Organoid Systems. Within self-organization system, lineage specification is determined by the combinatory effects of two factors, the intrinsic cell characteristics (genetic and non-genetic) and the local interactions with the surroundings (chemical and physical). Chance constitutes a nontrivial component in cell fate determination under the framework of self-organization. Only when one factor is dominant, would the system stably and reproducibly acquire a specific pattern. This may explain why ASC-derived organoids display lower variability, because the starting cells have already acquired the intrinsic characteristics that will later dominate selforganization. Self-organization can only bring us so far. Although the organoid research field is moving forward fast, we may need to spend the next decade figuring out how to bridge the gap in between organoids and native organs. For organoid approaches, the most ‘‘notorious’’ limitation is the high variability that lies in 880 Cell Stem Cell 24, June 6, 2019

various aspects of their formation, such as the relative ratio of different cellular components and the dimension of the organoid. The inter-organoid variability is hampering many applications, as it is extremely difficult to conclude whether an observed phenotype is a real one or a mere consequence of intrinsic variability. As a result, there is an accumulating effort toward comprehensively evaluating the levels of variability in organoids (Capowski et al., 2019; Phipson et al., 2019; Wu et al., 2018; Yoon et al., 2019). Another ‘‘lethal’’ limitation of organoids is the low level of maturation, particularly those derived from human PSCs (Camp et al., 2017; Takasato et al., 2016). There is a consensus that the majority of human PSC derivatives more resemble their fetal instead of post-natal counterparts. There have already been increasing arguments on whether human PSC-derived organoids are suitable for modeling adult human tissue biology. In this regard, human PSC-derived organoids are considered suitable for disease modeling only if the disease-related cellular apparatus are precisely represented within the organoids, in parallel with rigorous controls. Furthermore, we need to interpret experiment results with extra caution, preferentially in combination with additional disease modeling systems. The recent explosion of single-cell sequencing tools has enabled in-depth analysis of the cellular composition of human PSC-derived organoids (Camp et al., 2017; Czerniecki et al., 2018; Kumar et al., 2019; Phipson et al., 2019; Quadrato et al., 2017; Wu et al., 2018). In addition to the confirmation of the overarching fetal-like status, single-cell transcriptomics identified missing components as well as off-target cells (Phipson et al., 2019; Wu et al., 2018). Furthermore, single-cell transcriptomics managed to unveil causative signaling pathways of some off-target cells, leading to the refinement of organoid differentiation protocols (Wu et al., 2018). Organoid dimension remains another concern, as most existing organoids are micrometers in size, limiting their potential for transplantation. Various approaches and methodologies are being developed to overcome the limitations of organoids, and bioengineering-based approaches have already brought forward promising outcomes. Bioengineering Bioengineering refers to the ‘‘marriage’’ between biological principles and engineering tools to generate highly useful and tangible products. Organoid generation usually employs selforganization, thus requiring minimum extrinsic cues. This convenience is obtained at the expense of high intrinsic variabilities seen in various types of organoids. When additional instructions are given, the ‘‘working mode’’ of organoid formation will shift from permissive to instructive, while self-organization gradually gives way to engineering. As a result, these organ constructs will adopt more consistent cellular composition and structural organization, as well as organ-scale dimensions. It requires multifaceted considerations to select suitable engineering approaches for intervening in self-organization with high accuracy. ‘‘Deconstructing’’ Self-Organization: Cells and Microenvironment. To steer self-organization toward the desired direction, we need to thoroughly deconstruct self-organization to gain a fine-grained understanding on how microenvironment instructs cellular behaviors, and vice versa. The input signals are processed by the receiving cells and decision made in cooperation with cells’ intrinsic properties, which are mainly comprised of

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Review Figure 3. Cell-Environment Interactions Underpin Derivation of Organ Constructs The principle cellular behaviors that are usually implicated in self-organization include viability, division, contact, movement, and production. The reciprocal interaction between cells and their microenvironment leads to constant change of both the cellular behaviors and the properties of microenvironment, constituting the foundation for organ construct generation.

transcription factor expression and epigenetic regulation. The self-organization framework includes five distinctive and interactive cellular behaviors: viability, division, contact, movement, and production (Figure 3). Among these, viability constitutes the foundation of all the other cellular behaviors. The balance between symmetric and asymmetric cell division largely determines the bifurcation between self-renewal and differentiation. Different modes of cell movement are critical for cell sorting and morphogenesis, and they are essentially influenced by the type and number of contacts established between the cells and their surroundings (cell-cell or cell-ECM). Cells are not only receivers, but also producers of a large variety of bioactive molecules. Thus, cells have every capability to actively re-write the environment that they are living in, as they progress from one statute to the other (Figure 3). The extrinsic cues provided by local microenvironment can be primarily categorized into chemical and physical cues (Figure 3). Historically, the control of stem cell fate has been predominantly attributed to chemical factors such as chemokines, growth factors, and hormones, as strongly indicated by embryology studies. The choice and timely delivery of chemical factors largely determines the outcome of in vitro lineage specification. In addition to these classic chemical cues, metabolites are being recognized as active players in determining cellular status. The preferential use of glycolysis as the main energy metabolic pathway has been identified in PSCs and many organ-specific stem cells (Ito and Suda, 2014; Shyh-Chang and Ng, 2017). Although metabolites alone could hardly steer cell fate determination, accumulating evidence indicates that metabolites may be an ideal cell fate facilitator with neglectable side effects (Gonzalez et al., 2018; Ryall et al., 2015; Wu et al., 2016). Compared with other chemical cues, extracellular matrix (ECM) is the most complex, because it provides both chemical cues (bound growth factors and integrin binding proteins) and physical instructions. New discoveries continue to mount on the influence of physical cues on stem cell behaviors. In fact, most known physical cues can be ascribed to various aspects of ECM, including stiffness, topography, viscosity, and various mechanical stimuli. Cell-ECM interaction is highly context dependent, according to the tissue type as well as the pathophysiological status. To establish efficient cell-matrix interaction, cells need to express correlative type of integrin heterodimers that can bind to their respective ECM proteins (Watt and Huck, 2013). The binding between cells and ECM is critical for the establishment of a

mechanical link between them. The effects of ECM stiffness on cell fate determination were most well studied using MSCs (Engler et al., 2006), as well as some other stem cell types (Holst et al., 2010; Przybyla et al., 2016; Saha et al., 2008). Cells also have the ability to sense micro- and nano-topographic cues (surface topography, fiber diameter, etc.) from the environment (Deglincerti et al., 2016; Martyn et al., 2018, 2019). The synergism between 3D substrate topography and matrix proteins (laminin) facilitates neuronal differentiation and neurite alignment (Recknor et al., 2006). In addition to these static physical cues, cells inside our body are constantly facing a variety of dynamic mechanical stimuli, such as stretch (tension, cyclic mechanic strain), compression, and shear stresses. Although the correlation between mechanical stimuli and cell fate determination is less clear, mechanical stimuli exert remarkable influences on the maturation of multiple lineages, such as endothelial cells and cardiomyocytes (Mihic et al., 2014; Yamamoto et al., 2003). Engineering ECM. ECM represents the most appealing engineerable environmental element, due to its essential contribution to both physical and chemical properties of the microenvironment. Matrigel, a natural ECM purified from Engelbreth-HolmSwarm mouse sarcoma, is highly effective for both human PSC maintenance and generation of various organoids. Matrigel is a reservoir of a large variety of ECM components (laminin, collagen, heparin sulfate proteoglycans, etc.) and growth factors. Despite its high potency in promoting cell proliferation and differentiation, it is not well defined, and displays high variability. Furthermore, Matrigel contains animal-derived products, making it ill qualified for clinical application. Accumulative efforts have been dedicated to synthesizing chemically defined hydrogels that display physical and chemical properties reminiscent of the natural stem cell microenvironment. Poly (ethylene glycol) (PEG)-based hydrogels have been most successfully devised for replacing Matrigel in both primary (Gjorevski et al., 2016) and PSC-derived intestinal organoid (Capeling et al., 2019; CruzAcun˜a et al., 2017) culture. PEG-based hydrogels are also amenable for the growth of neural tube-like structures (Meinhardt et al., 2014; Ranga et al., 2016). Brain organoids have been generated in hyaluronan-based hydrogels (Lindborg et al., 2016), and microfilament scaffolds have improved their cortical development in vitro (Lancaster et al., 2017). By controlling matrix stiffness and 3D topography, human PSCs can be steered toward amnion-like structures through self-organization (Shao et al., 2017). Cell Stem Cell 24, June 6, 2019 881

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Review It is also critical to enhance the bioactivity of synthetic matrixes, seeing that biomolecular signaling plays a key role in directing stem cell fate. Traditionally, gradients of signaling molecules can be established by local diffusion from microspheres (Carpenedo et al., 2009). It is becoming standard practice to confer engineered ECM with cell-instructive and -responsive properties, including cell-adhesive domains from collagen (RGD, DGEA), fibronectin (RGDS, REDV), and laminin (RGD, IKVAV, YIGSR), as well as growth factor binding domains and MMP (matrix metalloproteinase) recognition sites (Caliari and Burdick, 2016; Patterson and Hubbell, 2010). Spatially controlled immobilization and patterning of multiple growth factors is also achieved in 3D hydrogels (Wylie et al., 2011). In real life, native microenvironment is spatiotemporally dynamic. The chemical and physical properties of microenvironment continue being modified by the resident cells as well as the systemic environment. The development of biomimetic ECM will allow us to incorporate spatiotemporal and thermal (Roberts et al., 2018) mechanisms that are critical for tissue engineering. The last decade has witnessed the establishment of enzyme- and light-mediated approaches to manipulate ECM stiffness and bioactive factor presentation (Bonnans et al., 2014; DeForest and Tirrell, 2015; Kloxin et al., 2009). For example, matrix stiffening is very often observed during aging and pathogenesis when ECM deposition increases. Multiple approaches are available for recapitulating this in vivo environmental change (Baker et al., 2015; Guvendiren and Burdick, 2012; Ondeck et al., 2019). Hydrogels with tunable stress relaxation and stress stiffening have been developed to control stem cell fate (Chaudhuri et al., 2016; Das et al., 2016). However, most of these pioneering studies were conducted using human MSCs that are amenable for in vitro culture and have a limited lineage potency. Dynamically tunable hydrogels have only been used in determining whether intestinal stem cells should proliferate or differentiate (Gjorevski et al., 2016). Substantial improvement and synergism of existing approaches may be required to synthesize biomimetic ECMs that are highly biocompatible, and thus could be broadly utilized for generating human organ constructs. 3D Bioprinting. Bioprinting is a 3D fabrication technology that can precisely dispense cell-laden biomaterials to generate complex organ constructs. Different from ECM engineering, 3D bioprinting can position multiple types of cells and their respective ECMs in the desired conformation and dimension with equivalent resolution as the native organ. There are three bioprinting approaches: biomimicry, autonomous self-organization, and mini-tissue generation (Murphy and Atala, 2014). Biomimicry requires the utmost accuracy in recapitulating various aspects of the native organ (Ingber et al., 2006). Autonomous self-organization employs a cell-driven organization and patterning of the generated organ constructs, similar with organoid approaches (Derby, 2012). Mini-tissue generation synergizes biomimicry and self-organization to multiplex the smallest structural and functional unit of a tissue/organ, such as islet and nephron (Mironov et al., 2009; Tan et al., 2014). For detailed explanation and comparison of bioprinting approaches, please refer to an excellent review by Murphy and Atala (2014). In a more recent study, Tuszynski and colleagues developed a microscale continuous projection printing method to produce central nervous system (CNS) constructs (hydrogel and neural progenitor cells) that 882 Cell Stem Cell 24, June 6, 2019

can be tailored to rodent spinal cord dimensions (Koffler et al., 2019). The selection of printing materials and cells, as well as the printing strategy, substantially influences the outcome of bioprinting. In many aspects, we have similar expectations of printing materials as what we have in engineered ECM, such as inclusion of bioactive factors, tailorability for presenting tissue/organ-specific topography, and the desired mechanical properties. Furthermore, printing material selection requires more deliberation, including printability, crosslinkability, biocompatibility, degradation dynamics, and by-products. The swelling characteristics of printing materials also need to be taken into consideration if the printed organ constructs will be transplanted (Gladman et al., 2016). The selection of printing material is also dependent upon the bioprinting strategy employed. Cell types for bioprinting mainly include (1) functional primary cells with supporting cells and (2) stem cells or progenitors that can further differentiate. Bioprinted organ constructs usually contain multiple cell types presenting their respective biological functions. Thus, one of the major challenges related to primary cell-based bioprinting lies in the requirement for preparing a large variety of mixtures comprised of different combinations of cells and their respective printing material. Based on the capability of existing bioprinting approaches, the initial cell density can hardly reach the levels equivalent with native organs. This requires the printed cells to have a high proliferation rate during the initial stage to sufficiently populate the organ constructs. In this regard, primary cells have difficulty realizing the desired proliferation rate. Using stem cells or progenitors for bioprinting requires starting cells but a more rigorous control over cell fate determination. A combination of organ-specific stem cells (isolated from patients or differentiated from human PSCs) and generic primary supporting cells may be able to address some of the existing concerns regarding cell selection. Native ECM Scaffold. Organ-specific ECM scaffolds have been a very valuable resource for us to understand the composition and organization of native ECM, as well as their function as a natural reservoir of various growth factors, such as EGF, FGF, and VEGF (Song and Ott, 2011). Tremendous efforts have been dedicated to deriving organ scaffolds that possess both structural and functional integrity, followed by recellularization of the acellular scaffolds. There is encouraging evidence that human PSC-derived cardiomyocytes, endothelial cells, and intestinal epithelial cells can repopulate heart (Lu et al., 2013), lung (Ren et al., 2015), and intestinal (Kitano et al., 2017) ECM scaffolds, respectively. In addition to using native ECM scaffolds to guide tissue engineering, decellularized ECM scaffolds have recently been solubilized into bioinks for bioprinting (Pati et al., 2014; Yu et al., 2019a). Such bioinks usually mirror the protein composition of organ-specific ECM, whereas the organizational properties are not preserved in a comparable manner. To this point, Chen and colleagues have successfully developed digital light processing (DLP)-based 3D bioprinting that is capable of patterning a large variety of functional elements within the biomimetic liver organ construct, including the geometry and the mechanical properties (Ma et al., 2016). It is possible that native ECM gel and synthetic polymers can be leveraged to generate ‘‘hybrid’’ printing materials.

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Review Challenges Shared by ECM Engineering and 3D Bioprinting. Although ECM engineering and 3D bioprinting have unique advantages, they are facing similar challenges as far as transplantation is concerned. The biosafety of both cellular and noncellular components within human organ constructs need to be rigorously evaluated. Using autologous cells can largely avoid immune attack by recipients upon transplantation. However, it is not always possible to obtain a sufficient number of primary autologous cells. To tackle this issue, HLA-matched iPSC biobanks may offer cell resources for generating the desired cell types for transplantation with a minimal requirement of immunosuppression (Taylor et al., 2012). Establishing universal donor cells represents an alternative approach to avoid the tedious work related to establishing patient-specific iPSCs (Deuse et al., 2019; Gornalusse et al., 2017; Han et al., 2019; Xu et al., 2019). Meanwhile, we should also carefully evaluate whether non-cellular components, such as hydrogels, are going to induce foreign body responses in vivo (Vegas et al., 2016). The optimal survival of transplanted organ constructs is primarily determined by a quick and efficient establishment of vascular networks and perfusion within the transplant. Multiple approaches have been developed to facilitate both in vitro and in vivo vascularization of organ constructs. It has been proven that native lung ECM scaffolds serve as a promising framework to support pulmonary vascularization itself (Ren et al., 2015) and the vascularization of islet engraftment (Citro et al., 2019). A dynamic modification (activation of caged RGD peptide) of synthetic hydrogels (PEGDA) greatly facilitated vascularization after implantation (Lee et al., 2015). Recent progress in 3D bioprinting using biocompatible and photopolymerizable hydrogels (GM-HA and GelMA) have enabled in vitro generation of pre-vascularized tissue constructs that demonstrate successful establishment of endothelial networks upon implantation (BenShaul et al., 2019; Jia et al., 2016; Zhu et al., 2017). In a recent study, pre-vascularization of bioengineered esophagus was realized via omentum transplantation (Urbani et al., 2018). Another hurdle is how to generate organ constructs of human organ scales. Although 3D bioprinting seems to be an enabling strategy, limited success has been achieved (Kang et al., 2016). Since it is much less challenging to print smaller tissue/organ unit with higher accuracy, it may be advisable to multiplex a large number of small organ constructs in the presence of a scaffold-like backbone. To this point, a 3D bioprinting approach that is capable of printing organoids or small tissue/organ units alongside an engineered biocompatible scaffold may provide unprecedented opportunities. Organs-on-a-Chip Organs-on-a-chip, situated between reductionist cell culture and holistic animal models, represent a branch of bioengineering. Organ-on-a-chip approaches synergize microfluidics with 3D cell culture to simulate multifaceted intrinsic and extrinsic properties of an in vivo organ/tissue (Ronaldson-Bouchard and Vunjak-Novakovic, 2018). Compared with all the other in vitro approaches, organs-on-a-chip are the most purposeful, as they aim to emulate only very defined functional features of the target organ/tissue instead of constructing the entire organ/tissue. These properties make organs-on-a-chip highly suitable for drug evaluation, which can hardly be realized using organoids despite their higher levels of structural and compositional

sophistication. Nonetheless, the design principles, especially the dimensional limit, largely preclude organs-on-a-chip from being used for replacement therapy. Design Principles. The design of organs-on-a-chip mainly concerns the selection of human cell types and the construction of a biomimetic environment within a microfluidic device. The selected cells are expected to recapitulate multidimensional phenotypes of the target organ/tissue, from genetic and molecular characterists to functionality. Additional issues also need to be considered, including cell number, viability, and sustainability. Organs-on-a-chip are less scrupulous toward the accuracy of 3D structural organization, as long as the functional features can be correctly recapitulated. For example, glomerular filtration barrier is flattened in a 2D format within a chip instead of adopting its usual 3D format (Musah et al., 2017). Such simplification is crucial for increasing the throughput that is required for drug screening. Furthermore, organs-on-a-chip approaches are amenable for incorporating dynamic physical, mechanical (stretch, pressure, sheer stress) (Mihic et al., 2014; Yamamoto et al., 2003), and electrical (Rorsman and Ashcroft, 2018) forces, which are critical for determining the functional maturation of native organs. This feature has recently been exploited to enhance the structural and functional maturation of human PSC-derived tissue (heart) (Ronaldson-Bouchard et al., 2018) and organoids (kidney) (Homan et al., 2019). Applications and Limitations. If organoids are more suitable for evaluating qualitative phenotypes, organs-on-a-chip are designed for quantitative phenotyping. A large variety of organs-on-a-chip have been developed for evaluating drug response and toxicity. Drug development very often fails at the clinical trial stage due to toxicity and off-target effects. Animal models may not be able to correctly predict drug toxicity due to inter-species differences. Furthermore, drug toxicity should not be simply concluded by a drastic effect on cell viability or proliferation. The beauty of organs-on-a-chip is the capability to reflect highly specific function of a small organ/tissue unit. There is a continuous interest in developing readout approaches that do not require culture termination, such as measurements of cellular production and changes in live cell behaviors. The possibility of collecting online readouts allows researchers to dynamically track the longitudinal drug response. Drugs usually pass through multiple organs before reaching the desired target organ, depending on the delivery route. This may dramatically change the amount and form of the drug, leading to a completely unexpected outcome. To address this issue, multi-organs-on-a-chip may offer a new avenue for comprehensively analyzing drug response at an organismal level. Another similar problem shared by various organ constructs is the absence of generic cell types, such as immune cells and microbiota, that impart significant effects on organ/tissue physiology and drug responsiveness. The cooperation between material science and bioengineering should further improve the capability of organs-on-a-chip to more accurately recapitulate human pathophysiology. In Situ Approaches Interspecies Chimeras—Promises and Challenges Nature has evolved highly sophisticated and faithful systems to generate functional organs in the right place and at the correct Cell Stem Cell 24, June 6, 2019 883

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Review time during embryonic development. Historically, chimeric assays have been employed to investigate the developmental potency or differentiation potential of various types of stem cells. During the last decade, interspecies chimeras have been regaining popularity in regenerative medicine, due to advances in gene editing technology and stem cell biology. Compared with traditional chimeras that are highly random, chimeric complementation is a strategy that can selectively enrich chimerism in the target organ/tissue (Kobayashi et al., 2010; Suchy et al., 2018; Wu and Izpisua Belmonte, 2015). It is now highly feasible to delete specific types of cells via conditionally deleting essential genes or direct ablation of specific cells to create a niche for the organ/tissue of interest (Masaki and Nakauchi, 2017; Suchy and Nakauchi, 2017; Wu and Izpisua Belmonte, 2015). Hypothetically, introduction of PSCs into the defective embryo could fill up the created niche by differentiating into the deleted cells. One landmark study by Nakauchi and colleagues demonstrated that both mouse and rat PSCs are competent to form pancreas within Pdx1 / mice through chimeric complementation (Kobayashi et al., 2010). Is it possible to employ similar approaches for generating human organs within large animals to fulfill the unmet demand for transplantable organs? What Determines the Efficiency of Interspecies Chimeras? The success of interspecies chimeras is dependent upon the selection of donor stem cells (cells) and host embryo (environment). Despite a lack of systemic understanding of low efficiency commonly seen in interspecies chimeras, recent studies have proposed a few possible explanations. Synchronization of the developmental timing between donor stem cells and the host embryo is essential for chimera formation. This critical attribute had not been realized until stem cell biologists found out that stem cells derived from post-implantation mouse epiblast (EpiSCs) were not able to integrate into pre-implantation blastocyst ICM (inner cell mass) efficiently (Rossant and Tam, 2017). Interestingly, the chimeric competency of EpiSCs is much more robust following their grafting into post-implantation mouse epiblast (Huang et al., 2012), as well as human ESCs/ iPSCs (Wu et al., 2015; Mascetti et al., 2016). The different signaling requirements of human versus mouse ESC culture strongly hinted at intrinsic differences in the developmental timing, in addition to inter-species differences. Besides naive (pre-implantation) and primed (post-implantation) pluripotent states, it is now possible to capture various intermediate pluripotent states in a spatiotemporally confined manner (Wu and Izpisua Belmonte, 2015). Although some studies demonstrated that human naive-like PSCs formed interspecies chimeras upon injection into mouse pre-implantation embryos, the efficiency of chimerism was far below that seen in mouse-rat interspecies chimeras (Gafni et al., 2013; Yang et al., 2017). Mice are evolutionarily more distant from human than many other large animals including pig and non-human primates. It is thus not unexpected that interspecies chimeras have been successfully generated between more closely related species, such as mouse-rat (Kobayashi et al., 2010), sheep-goat (Fehilly et al., 1984), and cow-zebu (Williams et al., 1990). Furthermore, mouse embryos form a cupshaped egg cylinder after blastocyst formation, whereas other mammalian embryos form a simple flattened embryonic disc. This particular feature of mouse embryos might make it 884 Cell Stem Cell 24, June 6, 2019

unsuitable for accommodating donor stem cells from other species. In support of these notions, Wu et al. (2017) reported on the establishment of the first human-pig chimera by injecting naive or intermediate human PSCs into pig blastocysts, albeit with low efficiency. What is the destiny of injected PSCs if they fail to be integrated into the host blastocysts? Masaki et al. provided strong evidence that mouse EpiSCs failed to contribute to chimeras upon injection into mouse pre-implantation embryos due to apoptotic cell death (Huang et al., 2018; Masaki et al., 2016). Forced BCL2 expression in primed ESCs made them apoptosis resistant and competent to form chimeras upon injection into pre-implantation embryos (Masaki et al., 2016). Later on, a similar approach was employed to increase the competence of human primed ESCs in contributing to both embryonic and extraembryonic lineages upon injection into mouse pre-implantation embryos (Wang et al., 2018). As far as evolutionary distance and developmental timing are concerned, incompatibility between donor stem cells and the host embryo may cause apoptosis of the ‘‘intruder,’’ leading to a lack of contribution to the host embryo. However, Cohen et al. 2018) recently reported that inhibition of apoptosis did not help neural crest cells (NCCs) integrate into mouse preimplantation embryos, nor did it facilitate the integration of Sox17+ endoderm cells. It is plausible that different experimental systems lead to discrepant observations. We also cannot rule out the possibility that donor stem cells from different germ layers differ greatly in the way that they behave upon injection into the same type of host embryos. Major Hurdles to Be Overcome. Notwithstanding the difficulty and ethical concerns, there is continuous enthusiasm in perfecting interspecies organogenesis (Sawai et al., 2019). One of the major advantages of employing interspecies chimeric complementation for generating transplantable organs is the immune compatibility with the organ recipient. Nakauchi and colleagues have provided the first proof-of-principle of the therapeutic potential of PSC-derived islets generated through interspecies chimeric complementation (Yamaguchi et al., 2017). However, we are facing numerous hurdles in realizing human organogenesis via interspecies chimeric complementation. As far as host embryos are considered, accurate creation of the prospective niche lays the foundation for successful chimeric complementation. There is a consensus that removal of organ-specific determinants, such as PDX1 or NKX2.5, will be sufficient to vacant the desired niche (Wu et al., 2017). However, the developmental program of many organs is dependent upon a highly intricate regulatory network involving multiple critical genes. Furthermore, studies in mouse-rat interspecies chimeras showed that generic supporting cells such as endothelial and stromal cells are mostly comprised of host-derived cells (Goto et al., 2019; Yamaguchi et al., 2017). With recent advances in CRISPR-Cas9 gene editing tools, it is now highly feasible to eliminate multiple unwanted genes for creating organ-specific niches that can accommodate both organ-specific and generic cell types of a same donor origin. It is equally important to obtain matching donor stem cells for the host embryos to realize optimal chimerism. Although groundstate ESCs are hypothetically more potent in differentiating into all lineages in vivo, intermediate-state ESCs or organ-specific stem cells may be borne with higher propensity toward specific

Cell Stem Cell

Review lineages. Recent studies have employed NCCs or Sox17+ endodermal progenitors to complement post-implantation mouse embryos (Cohen et al., 2018). More importantly, utilization of region- or organ-specific progenitor cells may facilitate the generation of organ-specific chimeras. The major ethical concern in relation to interspecies organogenesis is the possible human contribution to neural lineages and gametes. Employing regionor organ-specific stem cells for interspecies chimeras may circumvent these potentially troubling possibilities, while increasing the specificity and efficiency of chimerism. Genetically engineered donor stem cells have already displayed beneficial effects on avoiding apoptosis upon injection into a suboptimal host embryo. However, such genetic manipulation may add on another layer of ethical concerns when it comes to human PSCs. Case-Specific Design and In Vitro Test Run. Different organs have their distinctive developmental timeline. In this regard, it is necessary to design interspecies chimeric complementation in a case-specific manner. For example, if an organ is defined at a later time point during development, performing complementation using ground-state ESCs and pre-implantation embryos is more likely to result in random (instead of organ-specific) contribution, due to the longer developmental journey that donor stem cells need to travel. Therefore, establishing stem cells that can accurately fall in the created niche in a spatiotemporally confined manner will result in a better outcome. For example, the current approach of using mouse ESCs to generate kidney in anephric Sall1 / rats leads to high mouse ESC contribution to metanephric mesenchyme (MM)-derived lineages, whereas there is low contribution to ureteric bud (UB)-derived collecting ducts (Goto et al., 2019). The specific capture of anterior and posterior T+ primitive streak cells might facilitate the derivation of different mesodermal organs, because UB and MM originate from anterior and posterior intermediate mesoderm, respectively (Takasato et al., 2016). It would be extremely beneficial to generate a 4-dimensional repertoire of embryonic stem cell lines that could accurately reflect the spatiotemporal location of their in vivo counterparts. Embryonic development can be viewed as a paradigm of self-organization. The successful integration of donor stem cells into the host embryo may similarly employ a self-organization framework (Figure 2A). The last few years have witnessed the realization of in vitro culture of early mammalian embryos, as well as the establishment of ‘‘synthetic embryos’’ in vitro. Zernicka-Goetz and colleagues successfully re-constructed early mammalian embryo-like structures by selfassembling embryonic stem cells in the absence (Bedzhov and Zernicka-Goetz, 2014) or presence of extra-embryonic stem cell types (trophectoderm stem cells and extraembryonic endoderm stem cells) (Harrison et al., 2017; Rivron et al., 2018; Sozen et al., 2018). Such synthetic early embryo-like structures displayed remarkable potential to initiate implantation in vivo (Zhang et al., 2019). An exciting recent study demonstrated successful induction of three pre-implantation blastocyst cell types from fibroblasts (Benchetrit et al., 2019). The possibility to coax stem cells into epiblast-like structures offers a promising in vitro platform to test the integration potential of donor stem cells (Figure 4). Compared with in vivo evaluation approaches, such an in vitro platform

is more amenable and cost effective. The in vitro ‘‘test run’’ may be performed in two different ways: (1) introducing donor stem cells via self-assembly or (2) injecting donor stem cells into the assembled early embryo-like structures (Figure 4). The first approach may be more informative about the compatibility between donor stem cells and the host embryonic stem cells. However, direct self-assembly of both donor and host stem cells may not reflect the situation in real life, which should be better represented by the second approach. Last but not the least, implementation and innovation of existing methodologies for in vitro embryo culture and manipulation will be crucial for the establishment of a versatile platform for interspecies organogenesis. In Vivo Lineage Reprogramming Most if not all the organs inside our body have considerable abilities to replenish the lost cells for the restoration of physiological function. These observations strongly indicated that cell fate plasticity is much broader than what was endowed by the stereotypical development program. It is becoming increasingly clear that cell fate plasticity is the central player of injury response in a myriad of mammalian organs. However, natural cell fate plasticity does not permit cell fate transition in between very distantly related lineages. The goal of in vivo lineage reprogramming is to force resident supporting cells to the desired cell types in situ for the regeneration of lost or damaged cells. In vitro lineage reprogramming has already been realized in a spectrum of lineages (Srivastava and DeWitt, 2016), such as cardiac, neuronal, hepatic, and pancreatic lineages, as well as renal (Kaminski et al., 2016), intestinal (Miura and Suzuki, 2017), and hematopoietic (Sugimura et al., 2017) lineages that were achieved more recently. The first step toward realizing in vitro lineage reprogramming is selection of an optimal combination of lineage determining factors that usually include transcription factors and microRNAs (Rackham et al., 2016). Interestingly, loss of specific genes may represent an alternative approach to realize lineage conversion between cell types that are developmentally related (Chakravarthy et al., 2017). It was not until recently that small molecules come onto the stage of in vitro lineage reprogramming. Human fibroblasts can be successfully converted into beating cardiomyocytes by a combination of chemicals (Cao et al., 2016). Similar chemical approaches have been employed to generate a plastic progenitor/multipotent cell status, followed by refinement into specific lineages (Kurian et al., 2013; Lalit et al., 2016; Li et al., 2017; Zhang et al., 2016). Some studies argued against such a plastic cell status by providing evidence that Yamanaka factor-mediated lineage conversion involves a transient detour via an iPSC stage (Bar-Nur et al., 2015; Maza et al., 2015). Furthermore, such approaches usually lead to the generation of immature cells with a fetus-like status. The efficiency and quality of in vitro lineage reprogramming is generally low. Nonetheless, in vivo lineage reprogramming is very often more efficient and higher quality, possibly owing to unknown factors from the native environment. It is worth noting that some organs are amenable to direct in vivo lineage conversion due to the higher plasticity that exists between closely related cell types. The most representative example is the conversion between pancreatic a cells and b cells, which takes place naturally in adult mice after extreme b cell loss (Thorel et al., 2010). Cell Stem Cell 24, June 6, 2019 885

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Review A

B

Figure 4. In vitro ‘‘Test Run’’ of Interspecies Chimeric Complementation using ‘‘Synthetic Embryos’’ (A) Schematic of early mouse embryonic development. (B) Time-matched synthetic embryos can be employed for validating the integration potential of donor stem cells.

Successful a-b cell conversion led to the normalization of diabetic phenotypes caused by b cell lesion in mouse (Cigliola et al., 2018; Furuyama et al., 2019; Xiao et al., 2018). It was quite unexpected that b-like cells can also be successfully derived from liver cells (Banga et al., 2012) and stomach cells (Ariyachet et al., 2016) via in vivo reprogramming to mitigate diabetic phenotypes in mouse, possibly attributed to the common endodermal origin of these cells. Besides, a stress paradigm is beneficial for cellular reprogramming. For example, IL-6 promotes both direct lineage reprogramming (Chiche et al., 2017; Mosteiro et al., 2016) and iPSC reprogramming (Brady et al., 2013). A similar strategy was employed to promote Ngn3-driven in vivo b cell regeneration to rescue mice from both type I and type II diabetes (Cheng et al., 2017). In two groundbreaking studies, tissue-resident cells (hepatic myofibroblasts and wound-resident mesenchyme cells) were converted to the lost functional cells (hepatocytes and keratinocytes) via direct in vivo reprogramming to attenuate the pathogenic phenotypes (Kurita et al., 2018; Song et al., 2016). For more information on cardiac and neuronal in vivo reprogramming, please refer to some excellent recent reviews (Gasco´n et al., 2017; Sadahiro et al., 2015). Major obstacles need to be addressed before clinical realization of in vivo 886 Cell Stem Cell 24, June 6, 2019

lineage reprogramming. The screening for the optimal minimum combination of reprogramming factors is laborious and time consuming. Because most of the target lineages are terminally differentiated cells that have already exited mitosis, it is challenging to achieve the desired cell number. A better understanding of the mechanisms underpinning direct lineage reprogramming will allow us to develop novel approaches to increase efficiency (Biddy et al., 2018; Gasco´n et al., 2016; Zhou et al., 2016). Delivery approaches greatly influence the efficacy and safety of in vivo lineage reprogramming. The possibility of including chemicals as reprogramming factors and the advances in nanotechnology will facilitate organ-specific delivery, while reducing safety concerns that are commonly associated with virus-based delivery approaches. In Vivo Gene Editing For many genetic diseases, pathological phenotypes are usually presented within specific organs/tissues, such as Duchenne muscular dystrophy and retinitis pigmentosa. Given such situations, it may be the least harmful to perform in vivo gene editing in an organ/tissue-specific manner. This approach can potentially preclude the necessity of organ/tissue transplantation that typically involves surgical and immune

Cell Stem Cell

Review Figure 5. Balance the ‘‘Pros’’ and ‘‘Cons’’ across Various Organ Construct Approaches Color shade correlates with the levels of advantage.

been utilized to ameliorate systemic aging phenotypes in progeria mice (Beyret et al., 2019; Santiago-Ferna´ndez et al., 2019).

complications. Traditionally, site-specific transgene integration is achieved via homology-dependent repair pathway, which can be realized only in dividing cells. Unfortunately, most somatic cells are post-mitotic, laying the biggest challenge for in vivo gene editing. Over the last few years, CRISPR-Cas9-based gene editing tools have been tailored to perform gene correction with higher efficiency and precision, such as the recent establishment of high-fidelity SpCas9 (Kleinstiver et al., 2016) and the development of homology-independent targeted integration (HITI) strategy (Suzuki et al., 2016). Besides, the utilization of adeno-associated virus (AAV) has greatly lessened our concerns of undesired gene integration that may cause tumor formation later in life (Samulski and Muzyczka, 2014). Several landmark studies reported on successful postnatal in vivo gene editing of mutations in the DMD gene (encoding Dystrophin) of animal models, leading to partial recovery of muscle functional deficiencies (Amoasii et al., 2018; Long et al., 2016; Nelson et al., 2016, 2019; Tabebordbar et al., 2016). Suzuki et al. (2016) utilized an innovative CRISPR/Cas9-mediated HITI integration strategy to realize in vivo gene editing in mouse neurons via either in utero electroporation or an AAV-based delivery method. In utero editing of metabolic gene Pcsk9 via AAV-mediated delivery successfully rescued the lethal phenotype of hereditary tyrosinemia type 1 (Rossidis et al., 2018). Likewise, in utero gene editing of Sftpc in a mouse model improved lung morphology and increased survival (Alapati et al., 2019). To alleviate potential off-target effects of gene editing, Liao et al. established a novel approach to activate target genes through CRISPR-Cas9-mediated trans-epigenetic modulation in vivo. This represents a highly versatile platform that can potentially allow us to treat a wider range of human diseases and injuries (Liao et al., 2017). It would be more beneficial if such epigenetic modulations are subjected to rigorous temporal control to reduce undesirable over-dosing effect. It is also highly encouraging that in vivo gene editing has recently

Conclusion Over the last decade, advances in stem cell technology, material science, bioengineering, and gene editing have enabled us to develop a wide selection of approaches for generating human organ constructs that can recapitulate organ architecture, functionality, and pathophysiology. Different approaches employ distinct design principles, each bearing advantages and shortcomings (Figure 5). Nevertheless, each approach is established upon the reciprocal interaction between two elementary components, the intrinsic property and the extrinsic instruction. The selection of an approach to generate a specific type of organ construct is primarily dependent on the biological question that needs to be addressed. Generally speaking, in vitro approaches require more instructions due to the lack of constraints that are incurred by the surrounding organs/tissues as in vivo. Among in vitro approaches, organoid approaches most heavily involve self-organization, the mechanism of which is only beginning to be unveiled (Serra et al., 2019). The low levels of extrinsic instructions enable organoids to preserve the innate molecular and cellular program, thus making organoid approaches ‘‘user friendly’’ for studying development and modeling diseases. The explosion of single-cell sequencing methods (Ku et al., 2019; Palii et al., 2019) and analysis tools (Becht et al., 2018; Deng et al., 2019; Lin et al., 2019; Setty et al., 2019) offer unprecedented opportunities to thoroughly profile existing organoids. The lack of maturity is a common feature of most human PSC-derived organoids, which can be largely attributed to a lack of understanding of late gestational as well as neonatal development. Single-cell sequencing tools have been extensively exploited to fill this knowledge gap (Cao et al., 2019; Pijuan-Sala et al., 2019; Taylor et al., 2019; Yu et al., 2019b). On the other hand, recent implementation of novel culture approaches greatly increased the throughput of organoid generation (Czerniecki et al., 2018; Kumar et al., 2019; Przepiorski et al., 2018), expanding their utility for drug screening and regenerative medicine, in spite of inter-organoid variability associated with self-organization. Compared with organoid approaches, bioengineering approaches involve a higher degree of extrinsic instructions for generating organ constructs with improved precision and adjustable dimensions, especially by engaging bio-fabrication and 3D bioprinting. Bioengineering approaches are usually employed for regenerative medicine purposes. Due to more rigorous design Cell Stem Cell 24, June 6, 2019 887

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Review principles, bioengineered organs/tissues are also suitable for drug evaluation and disease modeling. Several recent studies forecast the upcoming revolution in using bioengineering approaches for both organ and tissue bioprinting (Kelly et al., 2019) and for mechanistic study of synthetic biology (Gobbo et al., 2018). Organs-on-a-chip approaches mostly heavily engage extrinsic instructions, making it suitable for performing accurate phenotyping and large-scale drug screening. Although initially designed for emulating highly specific pathophysiological phenotypes, organs-on-a-chip approaches have been proven highly useful in providing dynamic physical cues for maturating human PSC derivatives (Homan et al., 2019; Ronaldson-Bouchard et al., 2018), as well as for the direct generation of naive iPSCs (Giulitti et al., 2019). The capability of organs-on-a-chip in the mechanical training of immature muscle cells has been recently extended to developing mechanoresponsive selfgrowing hydrogels (Matsuda et al., 2019), which may serve as an adaptive biomaterial for tissue engineering. There is an increasing interest in synergizing multiple organs-on-a-chip to recapitulate systemic pathophysiology. In parallel, recent advances in organoid approaches have led to the establishment of systemic biological interfaces, including thalamus-cortical (Xiang et al., 2019), microglia-cerebral (Ormel et al., 2018), and immune cells-tumor microenvironment (Neal et al., 2018; Schnalzger et al., 2019). Multi-organs-on-a-chip and organoids harboring systemic biological interfaces may complement each other to present highly complex phenotypes at an organ or system level. Using in vitro derived organ constructs for regenerative medicine typically encounter several major challenges, including structural sophistication, dimension, functionality, immune compatibility, and sustainability. Compared with in vitro approaches, in situ approaches offer better opportunities to circumvent several of these major hurdles, despite numerous technical difficulties. Most importantly, organ constructs generated in situ usually present higher resemblance with organs in vivo. Currently, interspecies chimeric complementation still sounds like science fiction as far as human organogenesis is concerned. Successful chimeric complementation requires a perfect match between donor stem cells and the host embryo. We are still trying to better understand the regulatory mechanisms of different types of embryonic stem cells, and thus how to establish in vitro culture approaches that can preserve their spatiotemporal properties (Nakanishi et al., 2019). Besides, we are far from figuring out how to overcome the evolutionary barriers between different species. The technical difficulties and ethical concerns may preclude researchers from studying interspecies organogenesis. As discussed earlier, prior to executing in vivo experiments, the latest advances in generating synthetic embryos could serve as a complementary in vitro platform for validating the primary design approaches for generating chimeric organs. It might take decades before full organ generation by interspecies chimeric complementation can be realized. Nevertheless, it should be less challenging to generate functional human organ/tissue units by interspecies organogenesis, for example, hepatocytes and b cells. Given such situations, bioengineering approaches could be used to facilitate the assembly and multiplexing of organ/tissue units. The development of a wide spectrum of gene editing tools, which require minimal surgical interventions, has expedited the 888 Cell Stem Cell 24, June 6, 2019

progress of in vivo lineage reprogramming and gene editing approaches. Furthermore, these two approaches aim to exert minimal interference to the whole organism, by providing highly specific genetic manipulation of the target cells. In vivo lineage reprogramming approaches are predominantly restricted by the developmental lineage distance as well as the geographic distance between the starting and the targeting cells. Additionally, when compared with in vivo gene editing, the tissue microenvironment has greater influence on in vivo lineage reprogramming. All these advantages together have led to several clinical and pre-clinical studies with promising outcomes throughout the last decade of in vivo gene editing interventions (Figure S1). This has been more successful when it comes to the pathological phenotypes that are highly restricted to specific organs and cell types, such as eye, muscle, and pancreas. Although systemic gene editing has also been successful, a case in point is the extension of lifespan of progeria mice, systemic gene editing via AAV-mediated delivery approaches remain to be further investigated. More needs to be learned before we could translate these proof-of-concept studies into standard clinical practice in a cost-effective manner. These versatile approaches for generating organ constructs complement each another to satisfy the demands of both basic and translational biomedical research. The multidisciplinary nature of these approaches requires collective efforts from different research fields. Future work that garners continued innovation in stem cell biology and bioengineering, as well as material science and artificial intelligence, will foster successful establishment of translatable strategies. SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. stem.2019.05.016. ACKNOWLEDGMENTS We would like to thank M. Schwarz and P. Schwarz for administrative help. Work in the laboratory of Y.X. was supported by Nanyang Assistant Professorship Grant from Lee Kong Chian School of Medicine, Nanyang Technological University, and Singapore National Medical Research Council (NMRC/OFIRG/ 0076/2018 and NMRC/OFLCG/001/2017). Work in the laboratory of J.C.I.B. was supported by The G. Harold and Leila Y. Mathers Charitable Foundation, Universidad Cato´lica San Antonio de Murcia, The Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002), The Moxie Foundation, and the NIH (R21 AG055938). REFERENCES Alapati, D., Zacharias, W.J., Hartman, H.A., Rossidis, A.C., Stratigis, J.D., Ahn, N.J., Coons, B., Zhou, S., Li, H., Singh, K., et al. (2019). In utero gene editing for monogenic lung disease. Sci. Transl. Med. 11, 11. Amoasii, L., Hildyard, J.C.W., Li, H., Sanchez-Ortiz, E., Mireault, A., Caballero, D., Harron, R., Stathopoulou, T.R., Massey, C., Shelton, J.M., et al. (2018). Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science 362, 86–91. Ariyachet, C., Tovaglieri, A., Xiang, G., Lu, J., Shah, M.S., Richmond, C.A., Verbeke, C., Melton, D.A., Stanger, B.Z., Mooney, D., et al. (2016). Reprogrammed Stomach Tissue as a Renewable Source of Functional b Cells for Blood Glucose Regulation. Cell Stem Cell 18, 410–421. Baker, B.M., Trappmann, B., Wang, W.Y., Sakar, M.S., Kim, I.L., Shenoy, V.B., Burdick, J.A., and Chen, C.S. (2015). Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments. Nat. Mater. 14, 1262–1268.

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