In Vitro Microscale Models for Embryogenesis - Wiley Online Library

Review Embryogenesis

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In Vitro Microscale Models for Embryogenesis Jennifer Rico-Varela, Dominic Ho, and Leo Q. Wan* For instance, the gradient of Nodal across the ventral node is widely considered to determinate the asymmetry between left and right sides, resulting in the lateralization of tissues and organs.[3] Cell–cell and cell–niche interactions further facilitate stem cell fate determination and tissue morphogenesis. In particular, cells respond to the mechanics, microstructure, and chemical motifs of the extracellular matrix (ECM) with changes in cell functions, and remodel cellular environment by ECM degradation, secretion, and reorganization. In addition, the diverse cellular mechanical forces transmitted between cells and between cells and ECM are critical for the structural and functional coherence of the developing embryos.[4,5] All these factors lead to the coordinated establishment of different polarity and axes at cell, tissue, and organ levels, and determines the positioning and morphology of tissues and organs within the body.[1,6,7] Deficiencies or flaws in embryogenesis result in birth defects, which are regarded as the leading cause of deaths and disorders among human fetuses and infants. In the United States, an estimated 3–5% of children exhibit birth defects.[8] Teratogens account for 2–3% of all malformations.[9] Teratogens are chemicals, drugs, infections, and other factors that interfere with normal embryonic developmental process while showing little or no toxicity in adults.[10] Teratogens lead to physiological abnormalities and birth defects in fetuses and infants. Despite these devastating effects, hurdles still remain in the assessment of embryonic toxicity. Due to restrictions in direct manipulation of human embryos, traditional methods for teratogen screening involve either epidemiological studies of human populations or controlled exposure of various animal models.[11] For instance, many animal studies have demonstrated that disruption of the Nodal/Lefty signal has been associated to a spectrum of malformations (e.g., heterotaxy, left isomerism, and right isomerism).[12] The labor intensive and costly nature of animal studies and the animal–human discrepancies have led to interests in the development of new approaches especially with human pluripotent stem cells (hPSCs). The hPSCs including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) offer great opportunities of studying human embryogenesis with in vitro models in a highly efficient manner. In order to build a physiologically meaningful model with these cells, the native cellular microenvironment, both biochemical and biomechanical, has to be recapitulated. Microtechnologies such as micropatterning, microwells, and microfluidic systems can

Embryogenesis is a highly regulated developmental process requiring complex mechanical and biochemical microenvironments to give rise to a fully developed and functional embryo. Significant efforts are taken to recapitulate specific features of embryogenesis by presenting the cells with developmentally relevant signals. The outcomes, however, are limited partly due to the complexity of this biological process. Microtechnologies such as micropatterned and microfluidic systems, along with new emerging embryonic stem cell-based models, can potentially serve as powerful tools to study embryogenesis. The aim of this article is to review major studies involving the culturing of pluripotent stem cells using different geometrical patterns, microfluidic platforms, and embryo/embryoid body-on-a-chip modalities. Indeed, new research opportunities have emerged for establishing in vitro culture for studying human embryogenesis and for high-throughput pharmacological testing platforms and disease models to prevent defects in early stages of human development.

1. Introduction During embryogenesis, biological tissues undergo drastic morphological changes accompanied by spatiotemporal patterns of stem cell proliferation, differentiation, and orchestrated cell migration.[1,2] During this process, multiple factors are involved including morphogen gradients, cell–cell, and cell–substrate interactions, and biomechanical forces. Morphogen gradients are widely considered as a central player of early embryonic patterning such as the generation of the three germ layers (ectoderm, mesoderm, and endoderm) during gastrulation. In this process, a single-cell epithelial layer differentiation turns into a trilaminar structure, and the embryo sets up the basic axes (i.e., anterior–posterior, dorsal–ventral, and left–right) of the body. J. Rico-Varela, Dr. D. Ho, Dr. L. Q. Wan Department of Biomedical Engineering Rensselaer Polytechnic Institute 110 8th Street, Troy, NY 12180, USA E-mail: [email protected] Dr. L. Q. Wan Center for Biotechnology and Interdisciplinary Studies Rensselaer Polytechnic Institute 110 8th Street, Troy, NY 12180, USA Dr. L. Q. Wan Center for Modeling Simulation and Imaging in Medicine Rensselaer Polytechnic Institute 110 8th Street, Troy, NY 12180, USA The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adbi.201700235.

DOI: 10.1002/adbi.201700235

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provide precise control over the cells at the cellular, multicellular, and even subcellular levels. In addition, these systems can be used to mimic spatiotemporal organizations and chemical signals associated with stem cell niche, physiological fluid flow, and other natural tissue-like properties.[13] The high-throughput nature of microtechnologies allows for efficient teratogen screening and full characterization of teratoxicity. Recent advances in synthetic human models with embryolike features (SHEEFs) allow us to better recapitulate the in vivo spatial organization of tissues that is typically only found in the embryo.[14] Several research groups have successfully cultured human embryos in vitro within the stipulated 14-day culture rule limit.[15–17] This rule stipulates restrictions on human embryo research beyond 14 days of development since it involves the primitive streak formation and neural tube development.[18] For instance, Deglincerti et al. revealed that human blastocysts self-organized and recapitulated lineage differentiation and separation in culture dish, equivalently to in vivo human embryonic development.[15] Likewise, Shahbazi et al. presented an in vitro culture of human embryos that allows observation of key events during pregrastrulation stages.[17] Although inevitable moral concerns are raised and debated, these findings promote further investigations with synthetic human models to better recapitulate embryogenesis. In this review, an overview of studies on in vitro culture of hESCs and main microtechnologies as tools to assess cellular toxicity will be described in great detail (Figure 1). Particularly, in vitro control of stem cell colony size can be detailed into several groundbreaking studies that describe different methods of size control and various geometrical patterns used to manipulate stem cell pluripotency, germ layer differentiation, and terminal differentiation. We will then highlight studies on microfluidics-based systems that define stem cell micro environment, offer reliable chemical gradients, and accurately manipulate embryos and embryoid bodies (EBs). Furthermore, we briefly report current research being carried out on prolonged in vitro cultures of synthetic human models and their potential applications for the study of embryogenesis.

2. The Use of Human Embryonic Stem Cell Assays Human ESCs can be differentiated into the three germ layers to further become the different cell types that comprise an organism.[19] It is therefore believed that their use allows for a more accurate representation of human developmental processes. Mouse embryonic stem cells (mESCs) were the first used for teratogen screening.[20] Similar to mESCs, hESCs were utilized in various forms such as monolayers and EBs, treated with chemical compounds, and assessed by their morphology and changes in gene expression related to their differentiation. Chemical compounds can be classified into three classes “nonembryotoxic,” “weakly embryotoxic,” and “strongly embryotoxic,” based on the effects on cell viability, proliferation, and differentiation.[21] In addition, these techniques have been further improved by employing additional cell types or molecular and genomic analysis.[22]

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Jennifer Rico-Varela is currently a Ph.D. student in Dr. Leo Q. Wan’s group at the Department of Biomedical Engineering, Rensselaer Polytechnic Institute. She received her bachelor’s and master’s degrees from The City College of New York in 2013 and 2015, respectively before joining Dr. Wan’s lab for cell chirality research. Her current research focuses on understanding physical and biochemical mechanisms of embryogenesis and tissue morphogenesis using principles from developmental biology and tissue engineering. Dominic Ho received his bachelor’s and Ph.D. degrees in the field of nanotechnology from the University of Western Australia. He then undertook his postdoctoral training at the Rensselaer Polytechnic Institute in the Laboratory for Tissue Engineering and Morphogenesis under the guidance of Dr. Leo Q. Wan. Following this, he left academia to join the field of medical communications where he is now a medical writer for MediTech Media at their APAC headquarters in Singapore. Leo Q. Wan is an associate professor in the Department of Biomedical Engineering at the Rensselaer Polytechnic Institute. He received his bachelor’s and master’s degrees from the University of Science and Technology of China, and his Ph.D. degree in Biomedical Engineering at Columbia University under the guidance of Prof. Van C. Mow. He was a postdoctoral scientist working with Prof. Gordana Vunjak-Novakovic. His research interests focus on understanding physical biology in tissue development and regeneration, developing novel strategies for tissue repair, and establishing innovative organon-a-chip devices for disease diagnosis and drug screening.

The use of hESCs, instead of mESCs, in toxicity assays can potentially improve the predictive accuracy of embryotoxicity.[22] There are currently significant concerns regarding correlating animal–human teratogenicity responses. At a molecular and

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Figure 1. Schematic of technologies for mimicking embryogenesis. I) 2D culture of embryonic stem cells and 3D culture of embryoid bodies have been utilized to study stem cell renewal and differentiation during embryogenesis. II) Micropatterning techniques have been used to control embryonic stem cell colony size and shape. III) Microfluidics-based systems control autocrine and paracrine cell signaling and generate defined biochemical gradients. IV) Emerging areas such as in vitro humanized models involve the creation of embryo-like structures in vitro (e.g., amniotic cavity).

genomic level, studies comparing expression patterns between murine and human embryonic stem cell lines have indicated differences in their survival and self-renewal pathways despite certain fundamental developmental mechanisms being conserved.[23] Genome-wide profiling has also demonstrated that most human miRNAs are not expressed in murine embryonic stem cells.[24] The significant disparities between human and animal physiologies were also demonstrated with the Thalidomide tragedy of the 1950s.[25] Prior to its release into the market, its teratogenic potential was assessed in various animal models using traditional clinical and histopathological measurements. While congenital defects were found in rabbits, moderate to no fetal changes were observed in rats and mice respectively.[26] Its subsequent use for the treatment of morning sickness among pregnant women was linked to an increase in limb defects among newborns with an estimated 10,000 cases of birth defects worldwide.[27] As such, there remains a demand for hESC based teratogenic testing systems, allowing for improved prediction of teratogenicity. A recent example using hESCs for a teratogenic assay utilized 3-day directed differentiation of hESC monolayers with a readout of nuclear translocation of Sox17.[28] The directed mesoendoderm differentiation allowed for a threefold reduction in assay time, when compared to 10 days for the spontaneous differentiation of mESCs in a previously report.[29] Subsequent testing with 71 drugs with known teratogenic effects and 15 environmental toxicants in a 96-well plate format yielded accuracies of 94% and 87%, respectively. Lastly, as proof of its feasibility as a high-throughput screening (HTS) platform, 300 kinase inhibitors were screened to identify pathways whose disruption led to teratogenic outcomes that disrupt the mesoendoderm lineage differentiation. From this library, five compounds were identified as potentially teratogenic. Though

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this approach is promising, studies in this area are limited and additional work is required to ascertain their suitability. In this regard, the screening using 2D geometric patterns, 3D embryonic bodies, and 3D organoids with developmental features can provide more reliable approaches for defining effects of teratogens on different embryonic events and correlating those to alterations during embryogenesis. Indeed, the use of hESCs with new engineering approaches such as microfabrication and biomaterials opened new opportunities to study embryonic processes and birth defects.

3. Microtechniques to Control Stem Cell Function by Colony Size Various engineering approaches have been utilized to regulate stem cell function. Among them, biomaterial-based 3D approaches have been successful to promote stem cell renewal and lineage-specific differentiation. For instance, functionali zed hydrogels with RGD sequences were used to encapsu late hESCs, together with vascular endothelial growth factor microparticles to promote vascular differentiation.[30] Similarly, encapsulation hESC-derived mesenchymal stem cells in alginate microbeads promoted osteogenic differentiation.[31] In addition, 3D microfibrous scaffolds such as alginate and watersoluble chitin blend fibers aided in stem cell self-renewal.[32] Likewise, 3D nanofibrous matrices prepared by electrospinning are shown to benefit neuronal differentiation and proliferation, depending on their physical properties (e.g., size, porosity, interconnectivity, and aspect ratios).[33] Despite of these successes, these systems do not necessarily recapitulate developmental processes, especially the highly regulated multicellular morphogenesis observed in embryonic development.

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Therefore, though important for tissue engineering and regenerative medicine, they may not be insightful models for studying embryogenesis. Microfabrication technologies are highly efficient approaches to emulate, control, and reproduce complex and dynamic in vitro stem cell microenvironments or stem cell niches to further enhance our understanding of cell differentiation and embryogenesis.[1,34,35] A number of methods have been used to generate stem cell patterns, including microcontact printing, microwells, and direct cell printing. These different approaches may produce similar but distinct outcomes in stem cell function, particularly due to the nature and quality of cell–cell and cell–substrate interactions. Microcontact printing typically involves the fabrication of a stamp of polydimethylsiloxane (PDMS), poly(ethylene glycol) (PEG), or poly(lactide-co-glycolide) substrate configurations at the micrometer scale (1–500 µm) with a resolution at ≈1 µm, using soft lithography techniques.[6,34,36,37] The elastomeric nature of the stamps allows for protein printing and molding, thus mimicking important aspects of the cellular microenvironment (i.e., cell–cell and cell–ECM interactions, colony size, and distance between colonies).[5,38–40] Microwells, on the other hand, minimize the cell–substrate adhesion. Cells adhere to each other and form a cell cluster when they are confined into a narrow space. The microwell technique is therefore an effective way to control the size of EBs and regulate stem cell renewal and differentiation. Finally, the increasingly popular direct cell printing is a bottom-up approach that places cells onto a 2D surface or into 3D hydrogel in a predefined pattern.[41] The cellular self-assembly process drives the formation of the final geometric patterns. This approach allows for the study of multicellular interactions and cell–protein interactions that may better mimic the complexity in embryonic development. Direct printing may suffer in throughput compared to other two methods, and therefore has not been widely used for studying embryogenesis or screening drugs. Confining stem cells onto well-defined geometric patterns with these techniques has allowed for the study of dynamic processes such as cell fate determination and gastrulation.[34,42,43] The geometric confinement of cells onto different geometries have been demonstrated to change the local mechanical (through multicellular interaction) and biochemical (through paracrine signaling) environment, and therefore generate spatiotemporal patterns of stem cell differentiation, depending on the size and shape of the geometric confinement.[35] Numerous studies have been reported in this field, but only a few will be briefly introduced with an emphasis on recent studies on maintaining stem cell pluripotency, recapitulating early stages of gastrulation, and guiding stem cell differentiation within geometrically varying micropatterns.

3.1. Maintaining Stem Cell Pluripotency Pluripotency is the ability of ESCs to maintain the status that allows the cells to later choose a specific path for differentiation into adult cells. Even though the pluripotent state during development exists for a short period of time, its maintenance requires key extrinsic biological signals to maintain a network

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of transcription factors (e.g., Oct4, Nanog, Sox2) in the early embryo and in culture.[44] Changes in gene expression due to alterations in cellular microenvironment or exposure to extrinsic biological signaling result in loss of pluripotency. While the ability of ESCs to become any cell type is crucial to understand human development, it is as important to study the mechanisms that allow ESCs to maintain their pluripotent state. Geometric control of stem cell colonies is an important consideration for stem cell pluripotency. An early study exploring geometric constraints controls stem cell colony size by using microwells.[45] Common EB formation practice often produces EBs with different sizes and significantly affects the efficiency of subsequent differentiation. Using microwells fabricated with PDMS, the hESCs are more uniform, resulting in increased viability and a stronger expression of Oct4, a transcription marker indicating the stem cell pluripotency. Similarly, a recent study patterned H9 cells into Matrigel-coated 384-well plates at high densities, and exposed them to 2800 compounds. The results demonstrated that treatment with theanine, sinomenine, gatifloxacin, and flurbiprofen significantly promoted hESCs self-renewal in a dose-dependent manner.[46] The restricted spontaneous differentiation of hESCs has also been achieved by controlling the size of cell clusters through microcontact printing. Indeed, stem cell fate highly depends on the size of micropattens.[47] The pluripotency of embryonic stem cells can be maintained better on larger colonies when their diameter varies from 200 to 800 µm. This colony size-dependent stem cell renewal is related to the level of an SMAD1 antagonist, growth differentiation factor 3, which is increased with the colony size (Figure 2A). Rapid and versatile micropatterning techniques have also been introduced for large-scale manufacturing. For instance, micropatterns were generated through aerosol deposition of Matrigel onto PDMS patterns at the macro-scale (area > 1 cm2), utilizing an airbrushing technique.[48] The surface was further treated with plasma polymerized allylamine to enhance cell attachment. Results showed that ECM aerosol deposition allowed for production of a defined cell–substrate interface without differentiating hESCs and hiPSCs cultures.[48]

3.2. Recapitulating Gastrulation The use of microtechnologies such as microprinting and microwells has been shown to not only maintain the pluripotent state of different embryonic stem cell lines, but also to recreate highly regulated and short morphogenetic processes such as the gastrulation. These assays may potentially be utilized for a range of applications including drug toxicity testing for earlystage embryonic development. During development, the proper positioning of the three germ layers and body axes specification occurs during gastrulation. This highly regulated process is mediated by a group of signaling pathways (i.e., Nodal, BMP, FGF, Wnt) which are essential for stem cell differentiation and functioning.[44] Recently, hPSCs on micropatterned surfaces have been shown to recapitulate in vivo self-organization programs at early

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Figure 2. Microtechnology-based systems control embryonic stem cell renewal and differentiation by controlling colony size and shape. A) H9 hESCs colonies patterned onto single islands with 400 µm in diameter expressed higher pluripotency markers such as Oct4 and lower differentiation markers such as pSmad1, than those patterned onto smaller islands (200 µm). Scale bar is 200 µm (in white). Adapted with permission.[47] Copyright 2007, Wiley. B) RUES2 hESCs patterned onto circular islands displaying radial patterning of the three germ layers (endoderm, mesoderm, ectoderm) as well as the trophectoderm-like tissue (TE) as indicated by different gastrulation markers (EOMES, SOX17, CDX2, BRA, NANOG) when treated with BMP4. Adapted with permission.[53] Copyright 2014, Nature Publishing Group. C) hiPSCs patterned onto circular islands with smaller diameters ranging from 80 to 140 µm displayed a large number of endothelial cells expressing VE-cadherin with significant sprouting compared to those patterned onto islands with larger diameters (225–500 µm). Adapted with permission.[60] Copyright 2015, Wiley.

developmental stages and to mimic the gastrulation and the germ layer formation.[43,49] Several studies have demonstrated the impact of manipulating the colony size of hESCs and media components within geometrical patterns to induce their differentiation and enhance their lineage commitment.[47] For example, Lee et al. micropatterned hESC colonies onto Matrigel circular islands to control the mesoderm and endoderm differentiation of primitive streak transcription factors (Brachyury and Mix 1 homeobox-like 1) via BMP2 and activin A in a serum-free environment.[50] This study demonstrated that large circular colonies (1200 µm) preferred mesoderm differentiation into hematopoietic progenitor cells, while small circular colonies (200 µm) favored the primitive gut endoderm differentiation, both under prolonged treatments and high doses of the above-mentioned inductive factors.[50] Despite their significant findings, the authors proposed the use of exogenous factors to specify lineage differentiation more efficiently while reducing heterogeneity in cell responses that arise from endogenous ligands and ECM proteins. Thus, the same group introduced an HTS method using microcontact printing to analyze early hPSCs cell fate responses to exogenous factors and reduced heterogeneity in differentiation bias.[51] Nazareth et al. directly printed a fibronectin and gelatin mixture into 96-well plates in circular patterns using different exogenous testing factors (i.e., FGF2, SB431542, BMP4, and Activin A). Their study

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concluded that the HTS achieved a rapid and robust characterization of cell-fate across the different exogenous factors tested, and reported that Oct4 and Sox2 can discriminate colonies undergoing endoderm and primitive-streak differentiation.[51] Similar platforms of geometric confinements have also been used for teratogen detection. For example, Xing et al. generated hPSC colonies on circular Matrigel islands (1 mm in diameter) patterned with a PDMS stencil, and treated with induction factors (i.e., BMP4, Activin A, and FGF2) to promote mesendoderm differentiation.[52] Known teratogens (such as Thalidomide) and nonteratogens (e.g., d-penicillamine) were classified based on their dose-dependent characteristics of developmentally relevant cellular behavior such as collective cell migration and spatial mesendoderm lineage differentiation. Recently Warmflash et al. observed the radial patterning of the three germ layers on micropatterns (Figure 2B).[53] They noticed the presence of trophectoderm-like embryonic-tissue in the outer layer when hESCs colonies were confined in different circular PDMS microwells (200, 500, and 1000 µm in diameter), and treated with bone morphogenic protein 4 (BMP4). This was one of the first studies that showed that with prolonged treatment of BMP4, hESCs can exhibit spatial patterning phenotypes on middle-sized circular patterns which fully reproduced the beginning of gastrulation. One of the potential applications of the Warmflash et al. study would be using their patterning

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technique as a promising tool to investigate symmetry breaking and body axis formation within an in vitro system. A recent study utilized 1000 µm circular hPSCs colonies to induce peri-gastrulation-like biological cell fate patterning.[54] By supplementing patterned colonies of hPSCs with BMP4 on circular islands, Tewary et al. observed high phosphorylated SMAD1 (pSMAD1) activity at the periphery of the differentiating hPSC colonies, and their self-organization in a radial gradient via BMP4-Noggin reaction–diffusion (RD). In addition, the fate acquisition was consistent with the positional information (PI) model by the strength of the pSMAD1 activity at the center and periphery of the colonies as well as the induction time of BMP signaling gradients. For several decades which mechanism was debated, RD or PI, influences tissue patterning as well as morphogenesis.[54,55] This study provided evidence that RD and PI mechanisms work interdependently to induce human perigastrulation-like events as a two-step process for cell fate patterning which might take place in the developing embryo.

3.3. Controlling Stem Cell Terminal Differentiation In addition to conserving stem cell pluripotency and recapitulating gastrulation events, many other studies have demonstrated that manipulating the colony size results in differences in terminal differentiation of ESCs. Several efforts in the field have demonstrated that cardiomyocyte and endothelial differentiation require an optimal micropattern size to influence their respective differentiation trajectories. For instance, Bauwens et al. proposed a multistage differentiation technique to control EB size to maximize cardiac induction and mesoderm differentiation.[56] The authors patterned Matrigel using microcontact printing with PDMS stamps containing circular features of 200–800 µm in dia meter. The quantitative evaluation of differentiation indicated that small EB colonies (200 µm in diameter) had endodermenriched induction due to low expression levels of BMP-2, while large-diameter EBs (800 µm in diameter) underwent neural-enriched induction with high BMP-2 levels. In addition, for endoderm-biased hESCs, a larger EB size (800 µm) promoted cardiac induction, while for neural-biased stem cells, a smaller size facilitated cardiac differentiation. From this study, the authors revealed that control of hESC colony and EB size influences stem cell differentiation trajectories. In MP-nt, CD31, and SMA, form this study, the authors proposed that by controlling the size of EBs with microwells independently from ECM substrate, researchers can enhance the homogeneity of EBs to direct mesoderm lineage differentiation. The abovementioned studies summarized the effect of micropatterned sizes along with EBs colonies’ sizes with and without morphogenic signaling treatments. Hwang et al. utilized a PEG hydrogel microwell array to direct the differentiation of mouse EBs to endothelial and cardiac lineages. In this study, mESCs aggregate to form different sizes (150, 300, and 450 µm) of EBs under prolonged treatment of Wnt5a and Wnt11 due to their known role in controlling cardiogenesis and vasculogenesis during tissue development.[57] The spontaneous cardiogenic differentiation of ESCs was aided by a larger size of EBs (450 µm) with higher expression levels of sarco-

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meric α-actinin, GATA4, and NKx2.5 and a higher number of beating colonies.[58] On the other hand, the endothelial cell differentiation was enhanced by smaller EBs (150 µm) with more significant vessel sprouting activity and higher expression of Wnt5a, CD31 and smooth muscle actin. From this study, the authors proposed that by controlling the size of EBs with microwells, EBs can be uniformly directed into mesoderm lineage differentiation. The abovementioned studies summarized the effect of micropatterned sizes and EB sizes with and without morphogenic signaling treatments. Similarly, Ma et al. used PEG-patterned substrates to geometrically confine hiPSCs colonies onto Matrigel-coated circular islands. With the modulation of Wnt/β-catenin signaling and the presence of the biophysical forces from geometrical confinement, the cells differentiated and formed 3D cardiac microchambers.[37] Likewise, other recent studies have confirmed cardiac, endothelial, and neural differentiation from micropattern size-dependent studies. For instance, Salick et al. wanted to understand the role of micropattern widths and aspects ratios of rectangular micropatterns on the alignment, polarization, and structure of sarcomeres (necessary for cardiomyocyte maturation). Cardiomyocytes derived from H9 cells were patterned onto rectangles of different aspect ratios and line widths (15–400 µm). Authors reported that high levels of cardiomyocyte alignment occurred on rectangles between widths of 30 and 80 µm promoting sarcomere structures, which can emulate important physiological features during cardiomyocyte development.[59] Another study by Kusuma et al., proposed to pattern hiPSCs onto islands of different diameter to control endothelial differentiation. In this study, patterning hiPSCs onto single circular fibronectin islands of smaller diameters (80 and 140 µm) led to a higher percentage of endothelial cells with positive VE-Cadherin staining and an increased spouting frequency, compared to the cells on larger islands (e.g., 500 µm), where no sprouting was observed (Figure 2C).[60] Joshi et al. controlled stem cell colony size with an aqueous two-phase microtechnology system for stromal cells-mediated neural differentiation. The differentiation of mESCs on stromal PA6 cells depended on the colony size. Large colonies significantly promoted neural differentiation as demonstrated by high neurite protein expression levels (e.g., TuJ, Nestin, and GFAP).[61] A follow-up study evaluated the self-regulatory factors of embryonic stem cells in coculture with stromal cells on neural differentiation of a single mESC colony.[62] The abovedescribed studies demonstrate the importance of pattern geo metries on the induction of specific lineage differentiation. Micropatterning along with other assays should be further examined to enhance our understanding of stem cell biology in the areas of pluripotency of stem cells, gastrulation events, and lineage differentiation. Overall, these techniques lack the ability to control defined and controlled concentration gradients of chemical signaling factors on the patterned cells and tissues. Such a more controlled microenvironment can be achieved by microfluidic-based systems.

4. Microfluidic Devices to Mimic Stem Cell Microenvironment Biochemical signals, morphogen gradients, and fluidinduced shear stress are crucial elements of stem cell

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microenvironments. Microfluidic devices provide dynamic mechanical and biochemical properties which not only support tissue differentiation and recapitulation of cell–cell and cell–ECM interactions, but also reproduce key aspects of native microenvironments in microengineered organ and tissue models.[38,63,64] These miniaturized devices usually consist of PDMS, polycarbonate, and off stoichiometry thiolene–epoxy (OSTE(+)). Their physical and chemical properties can be finetuned during fabrication using soft lithography or photolithography techniques.[39,65] These devices are composed of small channels on the micrometer and microliter scale, achieving high spatiotemporal resolution and control over the stem cell microenvironment. Stem cell culture can be enhanced with microfluidics by utilizing important characters of generated biophysical environment such as defining biochemical environment, generating varying levels of cytokines, and sorting of cell clusters or embryos.

4.1. Controlling Stem Cell Response via Defined Biochemical Environment Fluid flow within microfluidic devices exposes cells to welldefined chemical gradients of soluble or insoluble factors as well as to laminar shear stresses. The dynamical stresses experienced by cells are responsible for washing away their autocrine and paracrine signals, thus affecting cell attachment, differentiation, and proliferation.[64,66] These systems deliver chemical factors while removing cell-secreted signaling factors, allowing for precise regulation of cellular microenvironments, gene expression profiling, and cell fate. For instance, Villa-Diaz et al. utilized a PDMS microfluidic system to culture hESCs colonies without affecting nutrient delivery, differentiation, and proliferation potential.[67] Another study focused on whether FGF4 autocrine signaling is sufficient for neuroectodermal differentiation of mESCs (Figure 3A).[68] Using a carefully defined multiplex microfluidic platform, the autocrine cytokines could be rinsed away without generating undesired side effects and the cells were only exposed to exogenous cytokines provided in the fluid flow. They found that cellsecreted factors were required for the differentiation, but exogenous supplementation of FGF4 could not fully recover the differentiation, indicating that FGF4 independent autocrine/ paracrine signaling is necessary for the neuroectodermal differentiation. Yoshimitsu et al. employed a microchamber array chip to culture hiPSCs which enhanced proliferation, in comparison to static cultures in the same microchamber assay.[69] Other applications of these systems involve the clonal population analysis and gene expression profiling of hESCs colonies to examine their growth rate and differentiation patterns. For instance, Matsumura et al. combined a microfluidic system with microwells to compare the cellular properties of single cells and its progeny through single cell clonal analysis. The study reported that hiPSCs did not show any signs of chromosomal abnormalities and maintained genetic characteristics of the parental lineage.[70] The abovementioned studies demonstrated that flow-based systems facilitate tight control over cellular environment, permitting mechanistic studies of stem cell renewal and differentiation.

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4.2. Studying Dose-Dependent Responses with Microfluidics Biochemical cues, particularly gradients of morphogens and soluble factors, play an important role on cell patterning, differentiation, and guide the body plan during development. Various devices have quantitatively assessed the cell microenvironment features such as lineage specification. Many studies have utilized gradient generating microfluidic systems to study dosedependent stem cell differentiation under continuous flow. For instance, Chung et al. utilized a gradient-generator device capable of mixing gradients of different growth factors (EGF, FGF, and PDGF) to optimize neuronal differentiation conditions.[71] This Christmas tree-shaped microfluidic platform allowed for the combination of multiple gradients of growth factors simultaneously while minimizing the autocrine and paracrine signals under constant fluid flow. Similarly, another group developed an osmotic pump-based microfluidic system that produces two laminar flows with gradients of signaling molecules such as sonic hedgehog (Shh), fibroblast growth factor 8 (FGF8), and BMP4, to study proliferation and differentiation of neuronal progenitor cells.[72] The device provided stable concentration gradients for over a week, under controlled liquid fluid flow. In addition, a proof-of-concept microfluidic device was used to examine cell growth, pluripotency status, and differentiation potential of hESC colonies in real time.[73] The integrated PDMS-based microfluidic platform contains multiple hydraulic valves and nanoliter-level peristaltic pumps, allowing for selection and manipulation of hESCs colonies in a semiautomated fashion, and the culture and analysis of the pluripotency of hESCs on the chip. The abovementioned systems were shown to control the spatiotemporal gradients of diffusible biological molecules important for cell fate specification. However, such platforms expose cells to fluid flow and shear stresses that might alter intracellular signaling cascades, and in many cases make it difficult to quantitatively determine what gradients individual cells are experiencing.[74] Cimetta et al. proposed a microbioreactor platform capable of generating stable spatiotemporal morphogen gradients of Wnt3a, Activin A, and BMP4 over EBs of hESCs and iPSCs in a shear-free microenvironment (Figure 3B).[75] This system trapped individual EBs into conical microwells positioned in independent rows connected to lateral flow channels. They found that stem cell differentiation was activated by local environment in response to stable morphogen gradients, confirming previous cardiac differentiation studies.[76]

4.3. Microfluidic Embryo/EB-on-a-Chip To better emulate key features during embryonic development, embryo-on-a-chip and EB-on-a-chip microfluidic devices have been presented as powerful platforms to recapitulate the anatomy and physiology within one system. These devices have been utilized not only to study developmental processes, but also to introduce high-throughput drug screening platforms to predict the toxicity of drugs on embryos.[77] Zebrafish embryos have been widely used to evaluate toxicity of drugs during development and to determine the effect of flow rate over full-term development. Yang et al. designed a three-layer

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Figure 3. Microfluidics-based systems to manipulate stem cell colonies and microenvironment. A) A microfluidic perfusion-based device dissects autocrine and paracrine signaling for stem cell differentiation. Microfluidics can take away cell-secreted signals while exogenously supplying autocrine cytokines such as FGF4 for neuroectodermal lineage specification. Adapted with permission.[68] Copyright 2011, Public Library of Science. B) A microfluidic bioreactor device utilized to cultivate EBs under controlled and defined concentration gradients of Wnt3a/ActivinA/BMP4 with minimal fluid shear. Adapted with permission.[75] Copyright 2013, Royal Society of Chemistry. C) A high-throughput fusion-actuated microfluidic device used to trap mouse EBs under different gradients of BMP4 to study the formation of the primitive streak. Adapted with permission.[83] Copyright 2013, Royal Society of Chemistry.

microfluidic system capable of generating controlled concentration gradients of cancer drugs such as doxorubicin, cisplatin, and fluorouracil (5-FU), over trapped zebrafish embryos in culture chambers.[78] The study demonstrated the feasibility of monitoring of embryos in real-time, and the characterization of embryotoxicity in a dose-dependent manner. In addition, embryo-on-chip devices at the optimal mechanical stimulations provide precise control over embryo growth and development in different animal models. Similarly, Wielhouwer et al. demo nstrated that zebrafish embryos could develop in biochips for more than 5 days under the proper pressure and fluid flow using a perfusion system.[79] Despite the challenging task of manipulating embryos within in vitro conditions, as aforementioned, this modality should be further explored to provide data that may be extrapolated to humans. Several studies have utilized similar flow-based microfluidic devices to manipulate ESCs or iPSCs to regulate their renewal, differentiation and even polarization.[80] Agarwal et al. introduced the first droplet microfluidic device to encapsulate mouse EBs with an alginate hydrogel shell in one-step to enhance their pluripotency.[81] After a week, higher levels of pluripotent markers such as Sox2, Nanog, and Klf2 were expressed within these 3D cultures as opposed to 2D conventional studies.[81] Khoury et al. reported a microfluidic trap system to support prolonged cultures of mouse EBs, within a controlled

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environment where an oxygen and medium mixture facilitated their differentiation.[82] The fluid flow rate of nutrients within the system was controlled, enabling appropriate EB growth conditions and thus stimulated the expression of ectoderm, mesoderm, and endoderm markers. This mechanical microtrap system has the potential to be used as a culturing method to mimic innate cell polarization during embryogenesis.[82] Suri et al. designed a high-throughput in vitro multicellular EB fusion device to investigate the potential of introducing a primitive streak formation-like pattern within microenvironments of BMP4 (linked to mesoderm differentiation) (Figure 3C).[83] The authors built a reliable trapping microchip with high efficiency and reproducibility. Within cross-flow serpentine channels, two mouse EBs were trapped into fusion microarrays guided by fluid flow, and Brachyury-T-GFP expression (an early developmental marker associated with the primitive streak formation) in cells were monitored over time.[83] Although the trapping systems were complex microengineered models, the integration flow and biochemical signals allow for the study of specific stages of development. Despite the easy fabrication, low cost, and high reproducibility of stable and controlled gradients of soluble factors, several limitations exist with the use of microfluidic devices. The PDMS has a strong affinity for hydrophobic molecules, including drugs and soluble factors diluted in the cell culture

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medium, which can be absorbed over time.[84] In addition, several studies have shown that cell metabolism and proliferation are affected during prolonged culture.[85] Regardless of the clinical potential of embryo/EB -on-a-chip devices to emulate complex architectural microenvironments, transform the development of drug testing models, and perhaps help to understand embryo development from animal models, these systems face several challenges. For instance, performing measurements on 3D complex cultures at high resolutions at the microscale level would become more difficult, and therefore further improvements in imaging techniques are urgently needed.[86] Crucially, embryo/EBon-a-chip devices should be rigorously investigated to fully recapitulate embryogenesis.

5. Emerging Areas of Humanized In Vitro Models In addition to the field of microtechnologies, several bioengineering and developmental biology approaches have contributed to important advances in modeling early human developmental events using hESCs and embryos, despite some ethical debates. In the recent years, the fields of tissue engineering and embryology have shown Figure 4. In vitro humanized platforms for the development of embryo-like tissue structures. A) Illustration of an artificial or ETS-embryo formation from two populations of stem cells different strategies to either mimic the (embryonic and trophoblast stem cells). The ETS-embryo appears to accurately undergo the architecture of tissues and embryos or cul- same self-assembly as observed with the natural embryo cultured on a dish. Adapted with ture embryos on a dish. For instance, there permission.[88] Copyright 2017, American Association for the Advancement of Science (AAAS). are a large variety of organs and tissues that B) Representation of hPSCs utilized to create amnion/amnioblast-like structures on 2D and 3D have been modeled as 3D organoids in vitro glass and gel-based culturing conditions. In order to achieve the development of the amniotic to understand their function and determine cavity, the presence of a gel bed (in cyan) and 3D ECM overlay (in pink) is necessary to form squamous cysts with E-cadherin (ECAD) positive protrusions. Scale bar: 50 µm. Adapted with pathogenesis and malformations. However, permission.[89] Copyright 2017, Nature Publishing Group. organoids have several limitations including their inability to fully recapitulate the in vivo microenvironment in which, for instance, embryos selfhESC-based model for peri-implantation of human amnion assemble and organize, grow, and develop.[87] development. The above studies have significantly broadened our potential to directly think about research with synthetic In addition to synthetic human models with embryohuman models. Their clinical relevance and potential applilike features mentioned in the Introduction, the study of cations in development, birth defects modeling, and drug extraembryonic tissues, trophoblast, and amniotic cavity, was screening and discovery, make these models desirable, but not considered before due to serious ethical concerns. Haronly if the appropriate ethical guidelines are followed.[15,17] rison et al. revealed the potential of artificial embryos using a mouse model (Figure 4A).[88] This study recapitulated key architectural components of the embryo from two different populations of stem cells (embryonic and trophoblast stem 6. Future Directions cells) along with a 3D-scaffold and a cocktail of signaling molecules. This study was the first in vitro self-organized While various approaches have been, and continue to be develmouse artificial embryo with structures notably similar to natoped to guide initial cell organization in vitro, very few efforts ural embryos, revealing the localized expression of the germ incorporate multiple microscale approaches into the design of layers more accurately than embryoid bodies.[88] In addition, engineered stem cell niches to ensure in vitro long-term spatiotemporal and architectural control of tissue patterning and Shao et al. mimicked the microenvironment at which human organization. Embryo/EB-on-a-chip devices, in combination with amnion-like tissue (amniotic cavity) self-organized in the microfluidic gradients in multiple axes, could be integrated to recabsence of biochemical cues from maternal sources by using reate in vitro microenvironments for guiding proper polarization hESCs (Figure 4B).[89] The study was the first to establish an

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during embryonic axis development. Furthermore, despite the moral and ethical limitations of using human embryo-like tissues, novel systems could be still developed and used within this period to promote efficient drug screening for embryotoxicity to improve our understanding of human development as well as birth malformations from teratogenic factors. Finally, the potential of using 3D printing to build complex tissues with multiple types of cells and proteins to study embryogenesis can really expand the horizons in the field. Overall, the field of microtechnology, the use of synthetic embryos, and 3D printing perhaps could be foreseen as important future directions in developing tissue-engineering products with better-controlled tissue organization and arrangement, and robust systems to study embryogenesis as well as birth defects.

7. Concluding Remarks The stem cell microenvironment plays a crucial role on embryogenesis, and many techniques have been utilized to mimic tissue and organ formation. The main objective of this review paper is to emphasize that the recapitulation of embryogenesis, an orchestrated, multistep, complex, and highly regulated process, requires multimodal approaches. Understanding the underlying in vivo tissue patterning domains and signaling-mediated mechanisms is essential for the design, model, and production of biological platforms that would mimic early stages of this developmental process. Microfabrication platforms can provide reliable control of spatiotemporal cellular biochemical and biomechanical environment, but have their shortcomings in term of recapitulating complex embryogenesis. The humanized in vitro models, despite strict ethical regulations, have promises to transform the field of research. New advances in developmental biology might allow us to understand embryonic development and explore disease models and reconstruct a reliable in vitro model to improve the current research state for embryogenesis and pathogenesis.

Acknowledgements The authors would like to thank Amanda Chin and Kathryn Worley for editing this manuscript. This work was supported by the National Institutes of Health (OD/NICHD DP2HD083961 and 3DP2HD083961-01S1), the National Science Foundation (CAREER CMMI-1254656), and American Heart Association (13SDG17230047). L.Q.W. is a Pew Scholar in Biomedical Sciences (PEW 00026185), supported by the Pew Charitable Trusts.

Conflict of Interest The authors declare no conflict of interest.

Keywords cell microenvironment, development, embryogenesis, embryo-on-a-chip, microfluidics, micropatterning, teratogens Received: November 30, 2017 Revised: March 1, 2018 Published online: May 7, 2018

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