UVM Theses and Dissertations
Format:
Print
Author:
Bakondi, Benjamn
Dept./Program:
Cell and Molecular Biology Program
Year:
2011
Degree:
Ph. D.
Abstract:
The reparative properties of adult human bone marrow-derived multipotent stromal cells (BMSCs) have been attributed in part to the paracrine action of secreted factors that act to reduce cell death, inflammation, and promote healing of tissues in several models of disease and injury. However, direct evidence for trophic support of injured tissues is lacking and it is not clear whether BMSC sub-populations possess different reparative characteristics.
In chapter 2, it is demonstrated that conditioned medium (CdM) from BMSCs reduced cerebral infarct size after stroke when injected into immunodeficient mice. Furthermore, the neuroprotection conferred by CdM from BMSCs isolated by magnetic activated cell sorting against CD133 (CD133-derived BMSCs, CD133BMSCs) was superior to that of heterogeneous unfractionated BMSCs as well as epitope-isolated P75-derived BMSCs and fibroblast control cells. These BMSC populations exhibited differences in the secretion levels of various growth factors and cytokines by ELISA. Results indicated that the CD133BMSC population may be better suited to produce CdM for neuroprotection and prompted further analysis of CD133BMSC CdM components.
In chapter 3, the transcriptional responses in BMSCs for the secreted factors analyzed in chapter 2 were investigated after BMSC injection into the ischemic brains of immunocompetent mice. Stromal derived factor 1alpha(SDP-I) was upregulated 80 fold in CD133BMSCs 48 hours after injection compared with expression in CD133BMSCs transplanted into the brain parenchyma of uninjured animals. The involvement ofSDP-l in neural cell protection during hypoxia exposure was confinned through the use of CdM from cDl33BMSCs that could no longer express SDP-1. It was also determined that SDP-1 transduced survival signaling through an SDF-1 receptor previously unidentified on neural cells, CXCR7.
SDF-1 is important for hematopoietic cell migration and hematopoiesis. In chapter 4, the stem-like multipotency of the CDl33BMSC population was validated by the most stringent criteria known to date. CD133BMSCs were differentiated in vivo and formed niches of active hematopoiesis at ectopic sites in immunodeficient mice. Mesenchymal cells were donor derived, whereas hematopoietic cells were mouse derived. This indicated that CD133BMSCs retained the primitive instructions for de novo reconstruction of compartments phenotypically similar to native bone marrow. The ability to prospectively isolate mesenchymal stem cells by CD 133 indicates that native human bone marrow stem cells express CD133 in vivo.
In summary, my data show that the prospective isolation of adult human bone marrow stem/progenitor cells by CD133 generates cells that provide enhanced neuroprotection through the action of trophic factors. Despite identification of SDF-l as one of these factors, many BMSC-secreted soluble mediators of neuroprotection remain unidentified. Future discovery of the full spectrum of secreted factors may yield medicines that promote recovery from devastating injuries such as stroke.
In chapter 2, it is demonstrated that conditioned medium (CdM) from BMSCs reduced cerebral infarct size after stroke when injected into immunodeficient mice. Furthermore, the neuroprotection conferred by CdM from BMSCs isolated by magnetic activated cell sorting against CD133 (CD133-derived BMSCs, CD133BMSCs) was superior to that of heterogeneous unfractionated BMSCs as well as epitope-isolated P75-derived BMSCs and fibroblast control cells. These BMSC populations exhibited differences in the secretion levels of various growth factors and cytokines by ELISA. Results indicated that the CD133BMSC population may be better suited to produce CdM for neuroprotection and prompted further analysis of CD133BMSC CdM components.
In chapter 3, the transcriptional responses in BMSCs for the secreted factors analyzed in chapter 2 were investigated after BMSC injection into the ischemic brains of immunocompetent mice. Stromal derived factor 1alpha(SDP-I) was upregulated 80 fold in CD133BMSCs 48 hours after injection compared with expression in CD133BMSCs transplanted into the brain parenchyma of uninjured animals. The involvement ofSDP-l in neural cell protection during hypoxia exposure was confinned through the use of CdM from cDl33BMSCs that could no longer express SDP-1. It was also determined that SDP-1 transduced survival signaling through an SDF-1 receptor previously unidentified on neural cells, CXCR7.
SDF-1 is important for hematopoietic cell migration and hematopoiesis. In chapter 4, the stem-like multipotency of the CDl33BMSC population was validated by the most stringent criteria known to date. CD133BMSCs were differentiated in vivo and formed niches of active hematopoiesis at ectopic sites in immunodeficient mice. Mesenchymal cells were donor derived, whereas hematopoietic cells were mouse derived. This indicated that CD133BMSCs retained the primitive instructions for de novo reconstruction of compartments phenotypically similar to native bone marrow. The ability to prospectively isolate mesenchymal stem cells by CD 133 indicates that native human bone marrow stem cells express CD133 in vivo.
In summary, my data show that the prospective isolation of adult human bone marrow stem/progenitor cells by CD133 generates cells that provide enhanced neuroprotection through the action of trophic factors. Despite identification of SDF-l as one of these factors, many BMSC-secreted soluble mediators of neuroprotection remain unidentified. Future discovery of the full spectrum of secreted factors may yield medicines that promote recovery from devastating injuries such as stroke.