Investigating the Stage-Specific Roles of NOTCH Signaling during Hemato-endothelial Development from Human Pluripotent Stem Cells
Başlık:
Investigating the Stage-Specific Roles of NOTCH Signaling during Hemato-endothelial Development from Human Pluripotent Stem Cells
Yazar:
Uenishi, Gene Ichiro, author.
ISBN:
9780438024106
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (197 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Advisors: Igor I. Slukvin Committee members: Fotis Asimakopoulos; Emery Bresnick; William J. Burlingham; Jing Zhang.
Özet:
The technology surrounding human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) have opened the opportunity to generate blood cells in vitro for research and therapeutic applications. However, the effectiveness of hPSC-derived hematopoietic stem cells (HSCs) and their progeny depends on their long-term engraftment potential, which has thus far been unobtainable. The ability to generate long-term engrafting definitive HSCs (LT-dHSCs) in vitro depends on our ability to recapitulate in vivo development. The current understanding of mammalian hematopoietic development is that there are two waves: The transient extra-embryonic primitive wave that starts in blood islands of the yolk sac, and the life-long intra-embryonic definitive wave that emerge from hemogenic endothelium (HE) in the dorsal aorta by the process of endothelial-to-hematopoietic transition (EHT). Molecular profiling analyses comparing hPSC-derived HPs to de novo LT-dHSCs have identified aberrant NOTCH signaling activation, a critical signaling pathway found to be required for definitive hematopoiesis in animal models, though exactly what role NOTCH signaling plays during HE development, EHT, and LT-dHSC specification have been contended. While specific stages analogous to in vivo murine hematopoietic development were previously identified during hPSC differentiation by co-culturing hPSCs on the OP9 mouse stromal cell line, the undefined and variable conditions of this method impedes the ability to identify, isolate, and investigate individual signaling pathways during hemato-endothelial differentiation. Thus, we have created a directed chemically defined two-dimensional differentiation platform that reproduces all of the developmental stages of hPSC hemato-endothelial differentiation. In addition, we have identified that Tenascin C is critical for definitive hematopoiesis from hPSCs. Next, we adapted our defined directed differentiation platform in order to manipulate NOTCH signaling by using an immobilized DLL1-Fc ligand for increased activation of NOTCH, and the small molecule gamma-secretase inhibitor, DAPT, to inhibit NOTCH. We found that hPSC-derived D4 CD144 +CD43-CD73-DLL4- HEP in secondary culture conditions with DLL1-Fc increases EHT and HP expansion, while adding DAPT decreases hematopoietic activity. Kinetic analysis of EHT following culture of HE with NOTCH signaling modulators revealed that NOTCH activation increases a unique population of CD144+CD43 -CD73- arterial-like HE that are DLL4+Notch hi and only undergoes EHT with increased NOTCH activation. Continued NOTCH activation post-EHT increased CD34+CD43+ HP expansion while maintaining CFC-GEMM potential, increased T-cell potential, and alpha- and beta-globin expressing erythrocyte potential. Overall, the methods developed in this work have created a defined platform on which to study the isolated effects of the NOTCH signaling pathway. We have identified that NOTCH activation increases arterial HE specification, is necessary for subsequent EHT, and facilitates definitive-type HP specification and expansion. In conclusion, we are one step closer to generating human HSCs from hPSCs with long-term engraftment and multilineage potential.
Notlar:
School code: 0262
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(677915.1) | 677915-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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