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Dissertation
The potential of human bone marrow stromal cells as vehicles for ex vivo gene therapy applications
Doctor of Philosophy (Ph.D.), Medical College of Pennsylvania and Hahnemann University
Jun 1999
DOI:
https://doi.org/10.17918/00008132
Abstract
The bone marrow stroma, is postulated to contain multipotential, mesenchymal progenitors capable of differentiation into osteoblasts, adipocytes, chondrocytes, and myoblasts. These properties make marrow stromal cells (MSCs) attractive candidates as vehicles for gene therapy of various diseases. However, much of these data characterizing MSCs are derived from animal models, with knowledge of the human marrow stromal system slowly emerging. In our efforts to characterize human bone marrow stromal cells (hMSCs), we analyzed the growth and differentiation properties of hMSCs obtained from iliac crest aspirates of normal human volunteers. We show that hMSCs are heterogeneous with respect to morphology, differentiation capacity, and growth dynamics. We also identified a subpopulation that is enriched in highly proliferative, multipotential mesenchymal progenitors based on a simple colony forming unit assay. Analysis of this population revealed that the proliferative potential of these cells increases dramatically at low density relative to higher plating density. Furthermore, we demonstrate that low density propagation enriches hMSC cultures for colony forming cells. We conclude that the mesenchymal progenitors reside within the colony forming population of adherent cultures and that progentior cells exist at different stages of differentiation. When MSCs are propagated in vitro, inhibitory factors that limit lifespan and colony forming potential appear to be present at high density, but not evident at lower density. These findings have significant implications for the biological phenomena occurring under various conditions. Furthermore, exploiting the proliferative capacity of hMSCs by low density propagation is significant for ex vivo expansion to obtain adequate numbers for genetic manipulation and therapeutic use. Finally, we developed an ex-vivo gene therapy strategy for osteogenesis imperfecta (OI) utilizing hMSCs genetically engineered with the wild type [alpha]1 chain of type I procollagen (COL1Al). We designed a COL1A1 cDNA construct that expresses an appropriate transcript in transfected HT1080 cells. We also devised a strategy to assay expression of COLlA1 in hMSCs. These studies are the first step in determining the feasibility of using modified hMSCs in future clinical trials for OI.
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Details
- Title
- The potential of human bone marrow stromal cells as vehicles for ex vivo gene therapy applications
- Creators
- Carla Marie DiGirolamo
- Contributors
- Darwin J. Prockop (Advisor) - Drexel University, Medical College of Pennsylvania and Hahnemann University (1993-1996, 1998-2002)
- Awarding Institution
- Medical College of Pennsylvania and Hahnemann University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Medical College of Pennsylvania and Hahnemann University; Philadelphia, Pennsylvania
- Number of pages
- x, 127 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Medical College of Pennsylvania and Hahnemann University (1993-1996, 1998-2002)
- Other Identifier
- 991021888720504721