ORCID

http://orcid.org/0000-0002-2701-8847

Date of Award

Summer 8-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Immunology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Hematopoietic stem cells (HSCs) have the unique ability to self-renew for life, to differentiate into mature blood lineages, and to readily engraft upon intravenous transplantation. As such, they are the only types of stem cells in routine clinical use. Understanding HSCs and hematopoietic development can provide many lessons for other types of stem cells as they near clinical utility. Through bone marrow transplantation, it was discovered that cells exist with regenerative potential. This led to the search to purify these cells and to determine the function of other hematopoietic cells. By isolating and transplanting cells expressing different combinations of surface markers, scientists were able to identify hematopoietic stem and progenitor cells and the blood lineages they regenerate. Additionally, the transplantation of lineage-committed cells led to the identification of their functions including their ability to protect recipients from various infections. This interwoven history demonstrates the importance of understanding the basic biology of hematopoiesis to improve clinical transplantations. Reciprocally, transplant studies have informed our basic understanding of hematopoiesis. While the fields of hematopoiesis and regenerative medicine have come a long way, there is still much to learn about hematopoiesis, and barriers such as immunological rejection remain to successful transplantation.Due to the rarity of HSCs, their differentiation into more differentiated progenitors and mature cells must occur with proliferation, leading to an expansion of each downstream step. This process, called transit amplification, is required to produce sufficient numbers of mature cells. This output must be balanced with the turnover rates of mature lineages to maintain homeostasis and prevent hematological malignancies. How transit amplification and subsequent homeostasis is controlled may involve metabolic switches. Hematopoietic expansion requires a switch from glycolysis to oxidative phosphorylation, but the signals that induce this switch and the specific carbon sources that fuel it are unknown. To address these questions, we employed mice conditionally deficient in mitochondrial pyruvate import, fatty acid oxidation, or glutamine hydrolysis and examined the requirements of these pathways in hematopoietic homeostasis. Ablation of carnitine palmitoyltransferase 2 (Cpt2) or glutaminase (Gls) had no effect on most blood lineages, suggesting fatty acid oxidation and glutaminolysis are not required for the production or survival of most hematopoietic cells. In contrast, upon deletion of mitochondrial pyruvate carrier 2 (Mpc2), peripheral myeloid cell numbers declined dramatically. Over time after Mpc2 ablation, a recovery of myeloid cells was observed. This recovery was associated with a myeloid progenitor-intrinsic switch to glutaminolysis. This switch was accompanied by a transient increase in proliferation to regenerate themselves and their mature myeloid progeny. These data suggest that hematopoietic progenitors are capable of intrinsically sensing metabolic perturbations and adjusting accordingly to maintain homeostasis. As our understanding of hematopoiesis has increased over the last few decades, interest in regenerative medicine and transplantation using stem cells has rapidly gained traction and proceeded into clinical trials. Transplantations in the 1950s and 1960s, were largely unsuccessful unless they were allogeneic transplants into immunodeficient patients, highlighting immune rejection as a major barrier to transplantation. This rejection is prominently due to T cells, natural killer (NK) cells, complement deposition, and phagocytes. Currently many transplant recipients undergo immunosuppressive therapies to help prevent rejection, but there are still concerns with long-term transplant efficacy, and patients are at a significantly higher risk of infection. To overcome these issues, we have generated a line of human embryonic stem cells (hESCs) that have been genetically modified to evade recognition by T cells, NK cells, and phagocytes, as well as to prevent complement deposition. These cells are devoid of HLA-I and -II to prevent recognition of T cells. They also lack expression of MICA and B, which are activating ligands of NK cells. Additionally, they express mouse inhibitory proteins for NK cells (Kb and Qa1), complement (Crry, CD55, and CD59), and phagocytes (CD47). When transplanted into immunocompetent mice, these modified hESCs persisted, demonstrating their ability to cross a xenogeneic barrier and evade immune rejection. In preliminary studies, these hESCs have been differentiated into pancreatic b cells, which also persist when transplanted into immunocompetent mice, and modified hESCs that express the human versions of the immune evasion proteins have also evaded rejection in nonhuman primates. These data demonstrate the ability to genetically engineer hESCs to prevent immunological rejection and provide a framework that could be applied to various types of stem cell therapies and transplants for the treatment of many diseases. This work focuses on both the basic biology of hematopoiesis, as well as the application of stem cells in transplantation. Specifically, these data show that hematopoietic progenitors can maintain homeostasis by intrinsically sensing and responding to metabolic changes. Additionally, based on the current understanding of immune rejection obtained from past transplantation studies, we describe a strategy to genetically modify human pluripotent stem cells that allows successful transplantation into immunocompetent recipients, even across xenogeneic barriers. Overall, the future of the field lies in continuing to understand the basic biology of hematopoiesis, for instance the role of metabolism, and applying this knowledge to pluripotent stem cells to advance clinical transplantation.

Language

English (en)

Chair and Committee

Deepta Bhattacharya Gwendalyn Randolph

Committee Members

John P. Atkinson, Todd A. Fehniger, Daniel C. Link, Gary J. Patti,

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