Embryonic stem cells (ESCs) are a unique cell population that can differentiate into all three embryonic germ layers (endoderm, mesoderm, and ectoderm), rendering them an invaluable cell source for studying the molecular mechanisms of embryogenesis. Signaling molecules that direct tissue patterning during embryonic development are secreted by ESC aggregates, known as embryoid bodies (EBs). As many of these signaling proteins interact with the extracellular matrix (ECM), manipulation of the ESC extracellular environment provides a means to direct differentiation. ECM components, such as glycosaminoglycans (GAGs), play crucial roles in cell signaling and regulation of morphogen gradients during early development through binding and concentration of secreted growth factors. Thus, engineered biomaterials fabricated from highly sulfated GAGs, such as heparin, provide matrices for manipulation and efficient capture of ESC morphogens via reversible electrostatic and affinity interactions. Ultimately, biomaterials designed to efficiently capture and retain morphogenic factors offer an attractive platform to enhance the differentiation of ESCs toward defined cell types. The overall objective of this work was to examine the ability of microparticles synthesized from both synthetic and naturally-derived materials to enhance the local presentation of morphogens to direct ESC differentiation. The overall hypothesis was that microparticles that mimic the ECM can modulate ESC differentiation through sequestration of endogenous morphogens present within the EB microenvironment.

The cardiovascular system is the first functional organ to develop during embryogenesis. As the embryo reaches above a certain size, passive diffusion of gases and nutrients is no longer compatible with efficient growth. During embryogenesis, endothelial progenitor cells (angioblasts) are recruited from the primitive streak mesoderm and instructed to express vascular endothelial growth factor receptor-2 (VEGFR-2). This thesis examines the roles played by VEGFR-2 in the events through which a subpopulation of embryonic stem (ES) cells differentiate into endothelial cells and form the vasculature. We show that ES cells gene targeted for VEGFR-2 (flk1-/-) develop immature endothelial cells (ECs), precursors, when differentiated in vitro as embryoid bodies (EBs). The flk1-/- ECs are unresponsive to VEGF-stimulation and consistently fail to form vessels. However, when co-cultured with wild type ES cells in chimeric EBs, flk1-/- endothelial precursors are guided by wild type ECs to form transient, chimeric vascular structures. Use of lentivirus in an add-back approach allowed reconstitution of VEGFR-2 expression in flk1-/- ES cells, and rescue of vasculogenesis and sprouting angiogenesis. We propose that recruitment to the endothelial lineage is not dependent on VEGFR-2, although this receptor tyrosine kinase appears indispensible for EC integrity, survival and for differentation of endothelial precursors into mature ECs formating a stable vasculature. Neuropilin-1 (NRP1) and heparan sulfate proteoglycans (HSPGs) function as co-receptors for VEGFs. The co-receptors influence, qualitatively and quantitatively, the intracellular signal relayed by VEGFR-2 but it is unclear how. We examined the contribution of NRP1 to VEGFR-2 signaling in EB cultures, in zebrafish and in mice. Only NRP1-binding VEGFs were able to promote sprouting angiogenesis and formation of properly branched vascular tubes, supported by pericytes. Downstream of VEGFR-2/NRP1 activation, we identified recruitment of p38MAPK in signal transduction regulating sprouting angiogenesis.

Embryonic stem cells (ESCs) hold great promise for treatment of degenerative disorders such as Parkinson's and Alzheimer's disease, diabetes, and cardiovascular disease. The ability of ESCs to differentiate to all somatic cell types suggests that they may serve as a robust cell source for production of differentiated cells for regenerative medicine and other cell-based therapeutics. In order for ESCs to be used effectively in clinical settings, efficient and reproducible differentiation to targeted cell types must be demonstrated. The overall objective of this project was to engineer microenvironmental control over differentiating ESCs through the formation of embryoid bodies (EBs) uniform in size and shape, and through the incorporation of morphogen-containing polymer microspheres within the interior of EBs. The central hypothesis was that morphogen delivery through incorporated polymer microspheres within a uniform population of EBs will induce controlled and uniform differentiation of ESCs. Rotary suspension culture was developed in order to efficiently produce uniform EBs in high yield. Compared to static suspension culture, rotary suspension significantly improved the production of differentiating cells and EBs over the course of 7 days, while simultaneously improving the homogeneity of EB size and shape compared to both hanging drop and static EBs. The diffusive transport properties of EBs formed via rotary suspension were investigated using a fluorescent, cell permeable dye to model the movement of small morphogenic molecules within EBs. Confocal microscopy, cryosections and EB dissociation all demonstrated that the dye was not able to fully penetrate EB, and that the larger EBs at later time points (7 days) retarded dye movement to a greater extent than earlier EBs (days 2 and 4). Polymer microspheres capable of encapsulating morphogenic factors were incorporated into EBs in order to overcome the diffusional limitations of traditional soluble delivery. The size of microspheres, microsphere coating, microsphere to cell ratio, and rotary mixing speed were all observed to influence incorporation within EBs. The use of microsphere-mediated delivery within EBs to direct cell differentiation was examined. Microsphere-mediated delivery of retinoic acid (RA) induced formation of uniquely cystic spheroids with a visceral endoderm layer enveloping a pseudo-stratified columnar epithelium, and with spatial localization of transcriptional profiles similar to the early primitive streak stage of mouse development. Continued differentiation of RA MS EBs in defined media conditions was assessed. Gene expression demonstrated that regular serum enhanced endoderm induction, serum-free media supported ectoderm differentiation, while mesoderm was most prominent in untreated EBs in full serum. In summary, this work has realized a unique approach for stem cell differentiation through modification of the internal microenvironment of ESC spheroids. This novel inside-out method toward engineering EBs demonstrated that the mode of morphogen delivery significantly affected the course of differentiation. These studies provide the basis for ongoing work, which will utilize the choice of microsphere material, coating, and morphogen in order to uniquely study mechanisms of ESC differentiation and achieve unparalleled engineering of the EB microenvironment.

Stem cell differentiation is regulated by the complex interplay of multiple parameters, including adhesive intercellular interactions, cytoskeletal and extracellular matrix remodeling, and gradients of agonists and antagonists that individually and collectively vary as a function of spatial locale and temporal stages of development. Directed differentiation approaches have traditionally focused on the delivery of soluble morphogens and/or the manipulation of culture substrates in two-dimensional, monolayer cultures, with the objective of achieving large yields of homogeneously differentiated cells. However, a more complete understanding of stem cell niche complexity motivates tissue engineering approaches to inform the development of physiologically relevant, biomimetic models of stem cell differentiation. The capacity of pluripotent stem cells to simultaneously differentiate toward multiple tissue-specific cell lineages has prompted the development of new strategies to guide complex, three-dimensional morphogenesis of functional tissue structures. The objective of this project was to characterize the spatiotemporal dynamics of stem cell biophysical characteristics and morphogenesis, to inform the development of ESC culture technologies to present defined and tunable cues within the three-dimensional spheroid microenvironment. The hypothesis was that the biophysical and biochemical cues present within the 3D microenvironment are altered in conjunction with morphogenesis as a function of stem cell differentiation stage. Understanding biochemical and physical tissue morphogenesis, including the relationships between remodeling of cytoskeletal elements and intercellular adhesions, associated developmentally relevant signaling pathways, and the physical properties of the EB structure together elucidate fundamental cellular interactions governing embryonic morphogenesis and cell specification. Together, this project has established a foundation for controlling, characterizing, and systematically perturbing aspects of stem cell microenvironments in order to guide the development of complex, functional tissue structures for regenerative therapies.

The objective of the proposed research is to design an experimental setup to assess the ability of impedance measurements to characterize mouse embryonic stem cell (ESC) and embryoid body (EB) growth and differentiation. Existing quality assurance measurements used to stage the growth and differentiation of embryoid bodies are labor intensive and most often destructive to the cells, thus present methods are typically valid for a single time point. Bioimpedance measurements are non-invasive and non-destructive, presenting an alternative approach to this challenge. These measurements can be done continuously for real-time measurements on the changes in embryoid body growth and differentiation. A system capable of making bioimpedance measurements of ESC and EB suspensions was designed along with a biocompatible test device to hold the cells and Ag-AgCl electrodes. The system uses a lock-in amplifier to record the magnitude and phase changes of the ESC and EB suspensions when a 1 Vpp signal sweeping frequencies from 100 Hz to 100 kHz is applied. The system performance was validated with a test case of 1 mL of 0.1 M KCl. Then experiments with cell culture media, ESCs, and EBs were performed, with varying concentrations of cells and EBs. Experimental results for single ESC suspensions showed promise in detecting a difference in cell concentration between 2 million and 4 million cells in 0.5 mL of media. Results for four day old EBs were ambiguous, and we conclude that a different experimental set up is required due to EB settling during experimentation.