Microsphere-mediated control of embryoid body microenvironments

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. The overall objective of this project was to engineer microenvironmental control over differentiating ESCs through the formation of 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.;The diffusive transport properties of EBs formed via rotary suspension were investigated. The diffusion of a fluorescent, cell permeable dye was used to model the movement of small morphogenic molecules within EBs. Confocal microscopy 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). Dye diffusion was further assessed by imaging cryosections of labeled EBs, which confirmed that dye is preferentially sequestered by cells at the periphery of EBs. Analysis of the fluorescence of dissociated EBs fortified the observed trend that later EBs (7 day) were labeled less efficiently than earlier EBs (2 or 4 days). Calculation of the percentage of cells at a given depth in EBs as a function of spheroid radius was performed, and matched well with experimental data.;Polymer microspheres capable of encapsulating morphogenic factors were incorporated into EBs in order to overcome the diffusional limitations of traditional soluble delivery. First, control over microsphere integration within EBs was demonstrated. The size of poly (lactic-co-glycolic acid) (PLGA) microspheres was observed to have an impact on incorporation, with smaller microspheres promoting greater incorporation. The coating of microspheres with an adhesive protein also promoted incorporation, while coating with poly(lysine) did not encourage microsphere incorporation. Additionally, microsphere to cell ratio and rotary mixing speed were observed to impact integration of microspheres, with higher microsphere ratios and slower rotary speeds resulting in greater numbers of incorporated particles. Analysis of EB morphology as well as cell viability and differentiation demonstrated that the presence of microspheres did not significantly affect cells within EBs.;The use of microsphere-mediated delivery within EBs to direct cell differentiation was examined. Retinoic acid (RA), a small molecule morphogen commonly employed in ESC differentiation, was encapsulated in PLGA microspheres. The morphology of EBs containing RA microspheres (RA MS EBs) was assessed and compared to untreated EBs, EBs containing unloaded microspheres, and soluble RA treated EBs. RA MS EBs formed uniquely cystic spheroids with a visceral endoderm layer enveloping a pseudo-stratified columnar epithelium. Transcriptional analysis using microarrays indicated that RA MS EBs cultured for 10 days were in a differentiation state similar to the early primitive streak stage of mouse development. The organization of epiblast and endoderm cells was examined using immunostaining, and RA MS EB ultrastructure was analyzed with scanning electron microscopy; both of these analyses confirmed the primitive streak-like differentiation state of the EBs.;Finally, the continued differentiation of RA MS EBs in defined media conditions was assessed. RA MS EBs were transferred to regular serum, low serum, or serum-free media after 10 days of differentiation in regular serum media. The choice of media appeared to influence the germ lineage differentiation of the epiblast-like cells in RA MS EBs. 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. (Abstract shortened by UMI.)...