Human peripheral blood stem cell differentiation, transduction, and transplantation into the nude rat brain | Posted on:2005-03-27 | Degree:Ph.D | Type:Thesis | University:State University of New York at Buffalo | Candidate:Morrow, Jay Toby | Full Text:PDF | GTID:2454390008978090 | Subject:Biology | Abstract/Summary: | | Hematopoietic stem cells (HSCs) exhibit transdifferentiation into cells indigenous to the central nervous system (CNS). Human peripheral blood stem cells (PBSCs) are a renewable source of autologous HSCs obtained with minimal invasiveness. Retrovirally transduced PBSCs, therefore, are an ideal ex vivo gene therapy vehicle to the CNS.; The major hypothesis was that human PBSCs could serve as an ex vivo gene transfer vehicle to the CNS. The first specific aim is to acquire, expand, and differentiate human CD 34+ PBSCs to a monocyte/microglial progenitor cell (M/MPC) in vitro. The second specific aim is to clone CNS therapeutic genes, and to develop an efficient retroviral transduction system for CD 34+ PBSCs. The final specific aim is to establish transduced M/MPCs as a viable CNS graft for gene transfer into the nude rat brain.; PBSCs obtained from mobilized peripheral blood were antibody-conjugated column enriched for CD 34 expression. PBSC culture in granulocyte-macrophage colony-stimulating factor (CSF), macrophage-CSF, stem cell factor, and interleukin-3 induced cell cycling, and differentiation down the myelo-monocytic lineage.; Ciliary neurotrophic factor (CNTF) and osteopontin (OP) were cloned into a retroviral vector containing the enhanced green fluorescent protein (EGFP) reporter gene, and high titer replication incompetent retrovirus was made. Use of a human packaging cell line, RetroNectin(TM), and spinoculation resulted in 20% of PBSCs transduced after one round of infection. PBSCs enriched for EGFP by flow sorting yielded a 95% stably transduced population. CNTF or OP transduced PBSCs effectively secreted the transgenic proteins.; EGFP + PBSCs stereotactically implanted into the nude rat caudate/putamen (CPu) were detected in the brain 6 months post-implantation. The majority of PBSCs migrated out of the brain via the vasculature after 7 days. To circumvent this problem, PBSCs transplanted in a basement membrane matrix stayed in the CPu for at least 2 months. Some transplanted cells assumed microglial immunophenotype and morphology. PBSCs implanted into an excitotoxic lesion or brain tumor maintained significant engraftment.; In conclusion, human PBSCs are efficiently transduced, and engraft upon implantation to the CPu. Vast potential exists for transduced PBSCs to serve as an ex vivo gene therapy vehicle to the CNS. | Keywords/Search Tags: | CNS, Into the nude rat, Peripheral blood, Human, Pbscs, Cell, Stem, Ex vivo | | Related items |
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