Date of Award

9-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Shannon McKinney-Freeman, PhD

Committee

John V. Cox, PhD Eric J. Enemark, PhD Gerard C. Grosveld, PhD Susan A. Miranda, PhD Martine F. Roussel, PhD Mitchell J. Weiss, PhD

Keywords

Bone Marrow, Cell Biology, Development, Hematopoiesis, Mouse Models, Stem Cells

Abstract

Hematopoietic stem cells (HSCs) and their downstream progenitors are a heterogeneous population of cells that are indispensable for lifelong hematopoiesis and are often utilized in the clinic for the treatment of hematologic maladies via hematopoietic stem cell transplantation (HSCT). Over several decades, it has been discovered that HSCs arise in the dorsal aorta of the developing embryo, migrate to the fetal liver (FL), and undergo a large expansion before reaching their final resting place in the bone marrow (BM). Many resources have been invested in understanding the roles of the different niches HSCs encounter along their journey. A greater understanding of HSC niche regulation could provide clues for HSC maintenance and expansion in vitro. One critical niche during HSC ontogeny that has been greatly overlooked is the fetal BM (FBM), of which the hematopoietic and HSC niche compartments are poorly understood. For this reason, we meticulously characterized the hematopoietic progenitor compartment of the whole skeleton FBM from its colonization until after birth via competitive transplantation, immunophenotypic analysis of the hematopoietic stem and progenitor cell (HSPC) compartment, functional assessment of specific progenitor populations, and single-cell RNA-sequencing (scRNA-Seq) of the hematopoietic and stromal FBM environment. Here, we provide the first report of the presence of bona fide HSCs within the E15.5 FBM. We also found that HSCs were present in the all sources of BM, including the forelimbs, hindlimbs, and trunk of E15.5 embryos. We are also the first to assess the BM immunophenotypic HSPC compartment from initial seeding to adulthood and found that specific multipotent progenitor (MPP) cells (MPP2s) are the predominant HSPC population in the FBM, appearing to have the ability to migrate to and seed the FBM directly from the FL. Interestingly, immunophenotypic MPP2s are not functional in vitro or in vivo until birth (E18.5-P0), and display reduced repopulating capacity compared to adult BM and time-matched FL MPP2s. Also, the frequencies of the different FBM HSPCs shift around birth from an MPP2-dominant phenotype to the MPP3/MPP4-dominant phenotype seen in adult BM. To identify the intrinsic and extrinsic mechanisms controlling MPP2 functional maturation, we isolated stromal and hematopoietic progenitor (HP) populations from E16.5, E18.5, P0, and adult BM, and constructed the first known scRNA-Seq dataset spanning the HP and stromal compartment across BM ontogeny. Preliminary analysis of our scRNA-Seq datasets show that the FBM stroma and HP compartments are compositionally distinct from the adult BM compartments, and this disparity in composition is even more drastic at E16.5, suggesting that the reduced function of FBM MPP2s may be due to a semi-incompatible FBM niche. Our future studies will focus on identifying intrinsic differences between immunophenotypic HSPC populations across FBM ontogeny in our HP scRNA-Seq dataset, as well as defining putative niches for HSPCs in the FBM. We hope that these analyses will identify novel hematopoietic factors in the FBM niche that can be therapeutically exploited to enhance HSC expansion/function/differentiation in the clinic.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0003-0197-4328

DOI

10.21007/etd.cghs.2020.0517

Additional Files
2020-023-Hall-DOA.pdf (322 kB)
Declaration of Authorship
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