In traditional 2D monolayer cultures, exogenous soluble factors or cell-secreted endogenous factors diffuse freely throughout the medium, and thereby reach an equilibrium in which all cells are exposed to similar biochemical environments.5 In contrast, in 3D aggregate Tenofovir (Viread) cultures, a concentration gradient is established between the surrounding culture environment and the interior of the spheroids.165 The distinct cellular dynamics in 2D and 3D stem cell culture136,170 as well as the heterogeneity within individual EBs most likely arise, at least in part, due to the aforementioned disparity in mass transport between the culture systems128 The mass transport within EBs has been measured experimentally128 and modeled mathematically165 as Rabbit Polyclonal to 14-3-3 gamma a function of the EB size (radius), extracellular matrix composition, cell packing density and molecular uptake rate. bioprocessing, and regenerative therapies. INTRODUCTION The balance between stem cell proliferation and differentiation is tightly controlled by local cues present in the stem cell niche microenvironment.111,137 In response to chemical or physical perturbations, cells exit the niche and undergo differentiation processes,102 often to mediate regeneration or repair in pathological contexts such as hemogenic repopulation92 or wound Tenofovir (Viread) healing.156 One particularly dynamic example of stem cell microenvironment regulation occurs within the blastocyst-stage embryo, whereby a compact cluster of cells, known as the inner cell mass (ICM), develop into all somatic tissues and organs.61 During the early stages of pre-implantation development, the cells of the ICM undergo sequential specification, through Tenofovir (Viread) which cells commit along the three germ lineages C endoderm, ectoderm, and mesoderm C and continue to make cell fate decisions in a spatially and temporally controlled manner, thereby providing a robust model by which to study cell plasticity and tissue formation. The patterning of cell fates is mediated by physical processes, such as proliferation62 and migration,56 which occur concomitant with biochemical gradients,47 thereby highlighting the need for novel technologies to recapitulate the multiparametric stimuli present within the tissue microenvironment. For example, during gastrulation, the prospective mesoderm cells undergo a dynamic epithelial-to-mesenchymal transition (EMT) and migrate through the primitive streak.18,31 Similarly, collective cell migration of epithelial sheets has been implicated in processes such as branching morphogenesis.50 Biophysical signals mediating the spatiotemporal dynamics of cell migration mediate the formation of functionally and structurally distinct, yet adjacent, tissue structures, such as heart, lungs and kidney, each of which is defined by precisely controlled, heterotypic multicellular organization. The precise presentation of biochemical and biophysical cues motivates the development of engineering approaches that recapitulate the stem cell niche in order to create functional heterotypic multicellular structures which are amenable to the replacement of damaged or diseased tissue through scalable bioprocessing and tissue engineering approaches, and offer new cellular platforms for high-throughput pharmaceutical screening and drug development. In order to emulate tissue-scale morphogenic processes, platforms have been developed to present chemical and physical cues in three-dimensional configurations, analogous to the multicellular structure of native tissues. Early studies of pluripotent embryonal carcinoma cells created high-density cellular environments organoid model of intestinal structure and function.149 Another model exhibiting self-formation of complex cerebral structures97 was developed to study the pathogenesis of human microcephaly using iPS cells. Moreover, similar approaches have yielded functional anterior pituitary,151 thyroid,4 and hepatic,154 structures which exhibit secretory functions when transplanted recapitulates aspects of EMT,25 including alterations in ECM composition and cellular organization as a function of differentiation. For example, GAGs such as hyaluronan and versican are increasingly synthesized with EB differentiation and co-localize within mesenchymal regions of the EBs.143 GAGs are known to sequester and bind growth factors within the extracellular matrix to facilitate the local presentation to cells,180 which reflects the ability of ECM to regulate biochemical signals in addition to providing physical cues. In addition to GAGs, other fibrillar ECM molecules such as collagen I and IV, fibronectin, and laminin are observed throughout EBs;63,113,128 while generally in lower abundance within pluripotent aggregates compared to mature tissues ECM synthesis and deposition may play an important role in early stem cell morphogenesis. While three-dimensional culture of PSCs recapitulates many early developmental events, the specific role of extracellular matrix in PSC morphogenic processes remains largely unknown due to the limited techniques for achieving spatial and temporal precision similar to developmental processes, as well as the complexity associated with studying such multivariate processes in.
