Biophysical Regulation of Human Mesenchymal Stem Cell Proliferation

Open Access
Riddle, Ryan Christopher
Graduate Program:
Cell and Molecular Biology
Doctor of Philosophy
Document Type:
Date of Defense:
June 13, 2007
Committee Members:
  • Henry Joseph Donahue, Committee Chair
  • Sarah Bronson, Committee Member
  • Douglas Cavener, Committee Member
  • Christopher J Lynch, Committee Member
  • Timothy Ritty, Committee Member
  • Osteoporosis
  • Mesenchymal stem cell
  • Orthopaedics
Osteoporosis, a disease characterized by low bone mass and increased fracture susceptibility, affects more than 10 million Americans and an additional 34 million are at risk of developing this disease. As our population continues to age these numbers are likely to grow significantly. Estimates of the economic burden of treating this disease in 2005 are on the order of $17 billion and are expected to increase by as much as 50% by 2025. As a result, the development of new therapeutic techniques designed to enhance bone formation and strength is a necessity. One way to achieve such goals is to harness the proliferation and differentiation potential of mesenchymal stem cells (MSCs) derived from skeletal tissue. MSCs provide a source of new bone-forming osteoblasts important for skeletal homeostasis, but the factors that regulate the proliferation and differentiation of MSCs are not completely understood. Mechanical signals are widely accepted regulators of skeletal homeostasis, such that the addition of exogenous mechanical load enhances osteoblastic bone formation while inhibiting osteoclastic bone resoption. Current hypotheses suggest that the deformation of bone in response to an applied load generate substrate strains that drive the movement of interstitial fluid and bone cells sense this fluid movement in the form of chemotransport or a shear stress across cell bodies and processes. Indeed, numerous in vitro studies suggest that both osteoblastic and osteocytic cells respond to the predicted physiologic levels of fluid shear stress by releasing paracrine factors necessary for the anabolic response of bone to mechanical loads. It is likely that fluid flow also regulates the behavior of MSCs, but there is little experimental evidence for this and the signaling cascades activated in MSCs in response to fluid flow have not been examined. Thus the goals of this thesis are three-fold: 1) to examine the effect of oscillatory fluid flow on human mesenchymal stem cell (hMSC) proliferation and to identify candidate signaling cascades involved in this response; 2) to identify the factor that initiates the activation of these signaling cascades; and, 3) to identify the biophysical signal that hMSCs perceive. One hour of fluid flow exposure induced a significant increase in cellular proliferation over static controls, indicating that like osteoblasts and osteocytes, hMSCs are responsive to fluid flow. Since intracellular calcium is a vital mediator of the processes by which extracellular signals are conveyed to the cell’s interior, we next examined whether calcium signaling pathways contribute to the effect of fluid flow on hMSC proliferation. Fluid flow exposure triggered a robust, but transient increase in intracellular calcium concentration that was partially mediated by the activation of phospholipase C. Increased intracellular calcium concentration stimulated the activation of the protein phosphatase calcineurin and the nuclear translocation of its target transcription factor, nuclear factor of activated T cells (NFAT). Fluid flow also stimulated the activating, phosphorylation of the MAP kinases, ERK-1 and -2. Pharmacological inhibition of calcineurin activation or ERK1/2 phosphorylation blocked the effect of fluid flow on hMSC proliferation. Having determined that fluid flow stimulates hMSC proliferation, we attempted to identify the factor responsible for this response. As previous studies from our laboratory and others suggested a role for extracellular ATP in osteoblastic and osteocytic mechanotransduction, we hypothesized that the release of ATP may also account for the mechanosensitivity of hMSCs. hMSCs actively release ATP in response to fluid flow stimulation and express a number of purinergic receptors necessary to respond to extracellular nucleotides. Treating hMSCs with ATP, but not other nucleotides, induced cellular proliferation at a level similar to fluid flow. Further, enzymatically degrading extracellular nucleotides with apyrase abolished the effect of fluid flow on hMSC proliferation. Degrading extracellular nucleotides also abrogated the effect of fluid flow on intracellular calcium signaling, the activation of calcineurin, and the nuclear translocation of NFAT. These data strongly suggest that ATP is the factor that mediates the induction of hMSC proliferation in response to fluid flow. Finally, we examined the contribution of chemotransport and fluid shear stress, two biophysical signals induced by interstitial fluid flow, to the effect of fluid flow on hMSC proliferation. Alterations in chemotransport, but not fluid shear stress, altered the sensitivity of hMSCs to fluid flow. We found that decreasing chemotransport inhibited hMSC proliferation as well as intracellular calcium signaling and ATP release in response to fluid flow. Incrementally increasing fluid shear stress did not alter any of these parameters. These data suggest the clearance of cellular metabolites and replacement of nutrient levels are a prerequisite for hMSC mechanotransduction. In summary, these studies provide new evidence that mechanical signals regulate the behavior of mesenchymal stem cells and outline for the first time the molecular mechanisms by which fluid flow affects these cells. The similarities between the signaling cascades activated by fluid flow in more mature osteoblastic cells and in hMSCs imply that a common pathway exists by which mechanical signals are translated to cellular responses. These data could be used in the development of new therapeutic techniques designed to enhance the recruitment of mesenchymal stem cells and promote there proliferation and subsequent differentiation into bone-forming osteoblasts. Such techniques would be beneficial in the treatment of diseases in which decreased bone formation compromises the integrity of skeletal tissue. Additionally, given the interest in hMSCs in tissue engineering protocols, these data could be applied in such a way as to control the phenotype of stem cells in vitro and drive their proliferation and differentiation towards specific lineages.