Date of Award

1-1-2015

Embargo Period

1-1-2020

Document Type

Thesis

Degree Name

Master of Biomedical Science

Department

Pathology and Laboratory Medicine

College

College of Graduate Studies

First Advisor

Steven L. Carroll

Second Advisor

Amanda C. LaRue

Third Advisor

Meenal Mehrotra

Fourth Advisor

Victoria J. Findlay

Fifth Advisor

Jeffrey A. Jones

Sixth Advisor

Lee R. Leddy

Abstract

Cartilage is a complex tissue that has a very low regenerative capacity, which significantly hinders its ability to repair itself. Stem cell therapy for the purpose of cartilage regeneration has gathered much attention. Bone marrow consists of two types of stem cell populations, the mesenchymal stromal cell (MSC) and the hematopoietic stem cell (HSC). While MSCs have been demonstrated to have the capacity of differentiating into osteoblasts, chondrocytes, and adipoctyes, recent studies are beginning to delve into the possibility of hematopoietic stem cells also having this differentiation capability. Our laboratory has demonstrated an HSC origin for cells such as osteoblasts, cancer-associated fibroblasts, and immature adipocytes, revealing the ability of HSCs to give rise to cell types not typically associated with this lineage. Of particular relevance to this study are our in vivo studies using a clonal cell transplantation model that demonstrated that HSCs can give rise to hypertrophic chondrogenic cells during non-stabilized fracture repair. This work has led to the hypothesis that chondrocytes differentiate through an HSC lineage. To address this hypothesis, in vitro studies first elucidated culture conditions for HSC-derived chondroprogenitors. Using the defined cell types of articular cartilage chondrocytes and the chondrogenic cell line ATDC5 morphology, and in vitro chondrogenic potential was examined. The ability of TGF-β1, TGF-β3, BMP-2, and BMP-7 to promote chondrogenesis in the ATDC5 cell line was determined. The potency of these factors on chondrogenic differentiation was demonstrated with Alcian Blue staining and gene expression profiling by qRT-PCR analysis. Culture conditions and endpoints as defined using this cell line then served as a basis for culture of HSC enriched cells. Novel HSC origins for chondroprogenitors were examined through chondrogenic induction of two HSC-derived cell sources: non-adherent bone marrow fraction (the fraction enriched for HSCs) and a monocytic precursor lineage. Chondrogenic differentiation and maturation was confirmed by comparing morphology and Alcian Blue staining to those derived from articular cartilage chondrocytes and ATDC5 cells and by the presence of immunofluorescent staining for cartilage specific markers, Collagen II and Aggrecan. Together, these data demonstrate the potential of the HSC to give rise to chondrocytes via the monocyte lineage. Future studies directed at determining the mechanisms regulating the HSC contribution to chondrogenic lineages and the associated process of differentiation/maturation has the potential to enhance stem cell therapies for cartilage repair.

Rights

All rights reserved. Copyright is held by the author.

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