<p>Hematopoietic stem cell (HSC) self-renewal, proliferation and differentiation are regulated by signaling through protein tyrosine kinases (PTK) such as c-kit, Flt-3 and Tie2. PTKs work in concert with receptor protein tyrosine phosphatases (PTPs) to maintain cellular equilibrium. The functions of PTPs in counterbalancing PTK signaling in HSCs however remain incompletely understood. Our laboratory has demonstrated that a heparin binding growth factor, Pleiotrophin (PTN), promotes the expansion of murine long-term (LT)-HSCs via binding to a PTP, protein tyrosine phosphatase receptor type Z (PTPRZ). The addition of PTN to murine PTPRZ-/- c-Kit+Sca-1+Lineage- (KSL) cells caused no expansion of HSCs in culture, suggesting that PTPRZ mediates PTN effects on HSC growth. We subsequently screened for the expression of other receptor PTPs in murine HSCs. Among 21 different receptor PTPs, we found that protein tyrosine phosphatase receptor type S (PTPRS) was significantly overexpressed in mouse and human HSCs compared to more mature hematopoietic cells. Ptprs-/- mice displayed no difference in mature blood counts or phenotypic HSC frequency compared to Ptprs+/+ mice. However, competitive transplantation of bone marrow (BM) cells from Ptprs-/- mice resulted in more than 8-fold increased multilineage hematopoietic repopulation in primary and secondary recipient mice compared to mice transplanted with BM cells from Ptprs+/+ mice. While Ptprs-/- mice displayed no differences in cell cycle status, HSC survival or homing capability compared to Ptprs+/+ mice, PTPRS-/- BM cells expressed significantly increased levels of activated Rac1, a RhoGTPase which regulates HSC engraftment capacity, compared to PTPRS+/+ BM cells. PTPRS-/- BM cells displayed significantly increased transendothelial migration capacity and cobblestone area forming cells (CAFC), consistent with increased Rac1 activation. Furthermore, inhibition of Rac1 abrogated the increased transendothelial migration capacity of PTPRS-/- BM cells, suggesting that the augmented engraftment capacity of PTPRS-/- BM cells was mediated via Rac1. Translationally, we demonstrated that negative selection of human cord blood Lin-CD34+CD38-CD45RA- cells for PTPRS expression yielded a 15-fold enrichment for human long term HSCs compared to Lin-CD34+CD38-CD45RA- cells or Lin-CD34+CD38-CD45RA- PTPRS+ cells. These data suggest that PTPRS regulates HSC repopulating capacity via inhibition of Rac1 and selection of human PTPRS - negative HSCs is a translatable strategy to significantly enrich human cord blood HSCs for transplantation.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/9810 |
| Date | January 2015 |
| Creators | Quarmyne, Mamle |
| Contributors | Chute, John P |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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