Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Bioengineering

First Advisor

Scott L. Diamond

Abstract

Human embryonic stem cells (hESCs) offer the potential to develop in vitro protocols for the generation of any human somatic cell. In order for protocols to allow for both comprehension of underlying developmental mechanics and future clinical application, they will need to rely upon efficient differentiation of cells without the reliance upon animal-derived components. This thesis presents the development of a 96-well plate culture system that allows 4-color, flow cytometry based high throughput screening of defined, serum-free hESC differentiation conditions. In the first portion, broad applicability is proven by demonstrating highly efficient differentiation toward the three primary germ layers. Using four separate biomarkers, we were able to distinguish between ectoderm, endoderm, mesoderm and pluripotent hESCs. We demonstrated the ability to perform both cytokine screens and siRNA-mediated knockdown in this assay. In the second portion, we establish conditions to apply this assay to study hematopoietic differentiation. We performed numerous cytokine and inhibitor screens to develop a stepwise protocol that generates high yields of primitive megakaryocyte-erythromyeloid progenitors and megakaryocytes after 8 and 11 days of embryoid body differentiation, respectively. This work provides a novel tool to streamline the development of hESC differentiation protocols and advances the hematopoietic field towards future hESC-derived therapies.

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