Mesenchymal stem cells (MSCs) are best characterized by their capacity to differentiate into connective tissue cell lineages. Many aspects of their basic biology including the molecular mechanisms that regulate lineage commitment are poorly described. We developed a method to enrich MSCs from mouse bone marrow and reported that FGF2 exposure induced proliferation but reversibly inhibited their multi-lineage differentiation. Herein, kinetic studies performed via real-time PCR revealed that FGF2 down regulated expression of soluble frizzled-related receptors 1 and 2 (sfrp1, 2), but induced expression of twist 2 and sprouty 4 (spry4), respectively. In contrast, Wnt3a exposure up-regulated expression of twist1 and had no effect on spry4 expression. Consistent with these differential effects on gene expression, FGF2 was shown to inhibit osteogenic differentiation of MSCs, while Wnt3a modestly inhibited osteogenesis but promoted chondrogenic differentiation.
Efforts to localize Twist and Spry4 in cells revealed that the proteins were expressed in a small percentage (∼7 %) of MSCs but both proteins were co-expressed in the majority (∼80 %) of these cells. Exposure to FGF2 afforded a significant enrichment of Twist/Spry4 double positive cells, which represent a novel progenitor population in bone marrow. To study the nature of this subpopulation, we developed a Twist-promoter GFP-reporter expression vector and showed that it afforded significant enrichment of Twist-expressing Neuro2A cells in vitro. An HIV-1 based expression vector was constructed to ectopically express Twist1 in human MSCs, which completely blocked the capacity of cells to differentiate into osteoblasts.
FGF2 was shown to promote growth of MSCs, but even exposure cells to FGF2 failed to prevent their growth arrest after prolonged culture in vitro. Culture of MSCs in hypoxia (5% O2) increased their proliferative capacity and had no effect on FGF2 or Wnt3a induced cell signaling, indicating that effects on growth were not related to Twist regulation. However, MSCs cultured in normoxia (21% O2) exhibited rapid increasing p53 levels, which were down regulated after cells were switched to low oxygen condition. These data indicates that the poor growth of MSCs in room air (21% O2) is related to oxygen-induced toxicity that results in cell cycle arrest due to p53 induction.
|Advisor:||Phinney, Donald G.|
|Commitee:||Betancourt, Aline M., Sullivan, Deborah, Wang, Guoshun|
|Department:||Microbiology & Immunology|
|School Location:||United States -- Louisiana|
|Source:||DAI-B 70/07, Dissertation Abstracts International|
|Subjects:||Genetics, Cellular biology|
|Keywords:||FGF2, Mesenchymal stem cells, Self-renewal, Sprouty4, Twist, Wnt signaling|
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