Investigations into the in vitro developmental plasticity of adult mesenchymal stem cells

Abstract

Bone marrow (BM) derived stem cells contribute to the regeneration of diverse adult tissues including heart, liver and brain following BM transplantation. Trans- differentiation is a mechanism proposed to explain how tissue specific stem cells could generate cells of other organs, thus supporting the emerging concept of enhanced adult stem cell plasticity. New studies have demonstrated that spontaneous cell fusion rather than trans-differentiation is the cause of unexpected cell fate changes in vivo. In contrast, several authors have reported that trans-differentiation can occur in vitro in the absence of cell fusion, including the generation of neural derivatives from non-neural tissues. These findings have profound implications for stem cell biology and cell replacement therapy, and as a result require extensive validation. Mesenchymal stem cells (MSCs) nave been isolated from the postnatal BM and more recently many other sites including adipose tissue, skin and placental cord blood. As such these cells have attracted interest as candidates for cell replacement therapies. This interest follows recent observations both in vitro and in transplant studies that these cells are capable of broader differentiation potential beyond those cell lineages associated with the organ in which they reside. The aim of the present thesis was to examine the developmental plasticity of MSCs in vitro including the capacity of these cells to cross lineage boundaries by differentiating into neuro-ectodermal cell derivatives. There are no universally accepted procedures for the prospective isolation of these cells. In the present thesis, procedures for the isolation of MSCs from rat BM and optimal conditions for the propagation of these cells in culture without loss of multipotent differentiation potential and proliferative capacity are first described. Secondly, the response of cultured MSCs with a consistent immunophenotype to defined culture conditions, previously reported to induce neuronal differentiation of MSCs are evaluated. Thirdly, evidence is presented that suggests that previous claims of trans-differentiation and apparent changes in cell phenotype have been incorrectly interpreted. Evidence is provided that MSCs respond to neural cues in vitro with a stress response, which is characterized by aberrant changes in the expression of constitutive neural proteins, an event previously interpreted as trans-differentiation. MSCs do not have the attributes of early or mature neural derivatives and therefore such changes in protein expression do not equate to true neural differentiation. Finally, evidence is presented that demonstrates that MSCs cultured under defined culture conditions release soluble factors that instruct a neurogenic cell fate decision on neural stem cells (NSCs). In addition, these soluble factors also increase neurite outgrowth of Tuj-1+ differentiating cell progeny. These effects may in part explain the therapeutic benefit of MSG transplantation in animal models of CNS lesions.