ORCID

http://orcid.org/0000-0002-5147-3866

Date of Award

Summer 8-15-2020

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The current standard of treatment for a variety of hematopoietic malignancies and genetic disorders is allogenic bone marrow transplantation, where donor hematopoietic stem cells (HSCs) engraft within the host and give rise to all of them hematopoietic lineages necessary for homeostasis. In many cases, finding a compatible human leukocyte antigen (HLA) matching donor is not possible, due to the large amount of genetic variation at those loci, but with the advent of induced pluripotent stem cells (iPSCs), unlimited sources of patient matched cells can be derived. Hematopoietic differentiations of human pluripotent stem cells (hPSCs) have been shown to recapitulate the early development of the embryo, producing known progenitors such as the hemangioblast, but current efforts have been unable to produce an HSC without the use of transgene expression. This is partly due to the complex nature of human developmental hematopoiesis, which is known to contain multiple hematopoietic programs of varying potential.These programs or ‘waves’ can be generally fit into two categories, the first being extraembryonic hematopoiesis that occurs within the yolk sac earliest in development and produces mainly transient, primitive progenitors that support the developing embryo. The second category is intraembryonic hematopoiesis, which occurs within the embryo proper, producing more mature progenitors, including the HSC, which arises from hemogenic endothelium (HE) in the dorsal aorta. Our lab has developed an hPSCs differentiation model that can identify and specify WNT independent, extraembryonic, primitive hematopoietic progenitors, and WNT dependent, intraembryonic, definitive hematopoietic progenitors through stage specific modulation of WNT signaling during the mesodermal stage of differentiation. I have shown that mesodermal expression of CDX4, a caudal-like homeobox transcription factor, is an important regulator of the specification of definitive HE, by utilizing a doxycycline inducible CDX4 hPSC line and a CDX4y/- KO hPSC line. In this work, I have demonstrated that CDX4 acts to induce canonical gene targets, such as medial HOXA genes, in different subsets of hemogenic mesoderm, likely impacting the specification of definitive HE. Surprisingly, TBX20, a cardiomyocyte transcription factor, was found to be a negatively regulated CDX4 target, suggesting that CDX4 might also play a role in regulating cardiac specification. I performed cardiomyocyte differentiations and demonstrated that CDX4y/- KO lead to a significant expansion of cardiomyocytes over WT and that mesodermal CDX4 expression abrogated this expansion. Additionally, single cell transcriptomics revealed that CDX4+ mesoderm also expresses CD1D, and when functionally characterized, definitive CD1D+ hemogenic mesoderm contained nearly all lymphoid, erythroid,

and myeloid potential.

These results will impact the field of hematopoietic differentiation, having extensively characterized the role of CDX4 in hemogenic mesoderm, it’s gene targets, and correlative markers. Further studies will be able to leverage CDX4 and its downstream targets, such as HOXA genes, to improve hematopoietic differentiations, and perhaps improve cardiomyocyte differentiations by reducing the expression of CDX4.

Language

English (en)

Chair and Committee

Christopher Sturgeon

Committee Members

Kyunghee Choi, Kory Lavine, Grant Challen, Samantha Morris,

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