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.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/33948 |
| Date | 05 April 2010 |
| Creators | Carpenedo, Richard L. |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Detected Language | English |
| Type | Dissertation |
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