Gene-Specific Reporters as Potential Tools for the Enrichment of ESC-Derived Osteogenic Progenitor Cells
Open Access
- Author:
- DeMarco, Jean E.
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 29, 2009
- Committee Members:
- Sarah Bronson, Dissertation Advisor/Co-Advisor
Sarah Bronson, Committee Chair/Co-Chair
Rebecca C Craven, Committee Member
Edward Joseph Gunther, Committee Member
Christopher Niyibizi, Committee Member
Timothy M Ritty, Committee Member - Keywords:
- stem cell therapy
GFP
osteogenesis
bone
embryonic stem cells
Twist2 - Abstract:
- To utilize ESC-derived progenitors for clinical transplantation, methods to identify and enrich for specific lineages from heterogeneous differentiation cultures must be developed, and stages of differentiation harboring therapeutic capacity must be determined. To this end, we have engineered murine ESCs to contain fluorescent reporters, permitting enrichment of progenitors from osteogenic differentiation cultures using FACS. The overall hypothesis is that we can identify specific genes with expression characteristic of various stages in osteogenic differentiation, generate transcriptional reporters in ESCs, and use reporter expression to identify osteogenic intermediates. These cell populations will be tested by in vitro assays and ultimately these cells could be tested in vivo in the context of a therapeutic paradigm. Three bacterial artificial chromosome (BAC) based reporters have been engineered to place GFP under the transcriptional control of specific genes expressed at various times during osteogenic differentiation (Twist2, Sox9, or Osterix). Subsequently, these BAC-based reporters were targeted in a single copy to a chosen site in an ESC. An additional reporter, Nestin-GFP, was targeted to the same genomic site. A fifth reporter, Brachyury-GFP, was also tested for enrichment ability. A constitutively expressed YFP transgene has been integrated and tested for ubiquitous expression to allow for tracking of the cells at all times upon transplantation in a murine host model. The Twist2-EGFP BAC-based reporter demonstrated the most promising results: (i) Twist2-EGFP fluorescent expression paralleled the endogenous Twist2 mRNA expression pattern, (ii) transgenic Twist2-EGFP mRNA expression recapitulated endogenous Twist2 mRNA expression, (iii) QRT-PCR analysis of mRNA from Twist2-EGFP positive cells sorted from the osteogenic cultures showed higher expression of osteogenic progenitor markers as compared to the EGFP negative cells, demonstrating enrichment for osteogenic progenitors, and (iv) sorted populations of Twist2-EGFP cells survived and were able to undergo osteogenic differentiation. This thesis provides proof of concept for FACS-based osteogenic enrichment of ESC-derived differentiation cultures, while simultaneously revealing which stages of the differentiation culture are most accessible, and the possible limitations of interrupting the differentiation process. Ultimately this approach has the potential to enrich for therapeutic osteogenic cells for a variety of applications that can improve and inform in vivo therapeutic modalities.