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ABSTRACT
Mesenchymal Stem Cells (MSCs) are multipotent adult cells capable of differentiating both in vivo and in vitro, into cells of different mesenchymal tissues, including adipocytes, osteocytes and chondrocytes. MSCs were originally isolated from the bone marrow but are virtually present in all embryonic and adult tissues. Within the bone marrow, the MSCs, togheter with their progeny, in particular adipocytes and osteoblasts, constitute the hematopoietic niche, in which the Hematopoietic Stem Cell (HSC) reside, and contribute to regulating their function. MSCs are studied for their widespread clinical potential in regenerative medicine, given their easy accessibility, multipotency, immunomodulatory properties and the ability to create a regenerative microenvironment subsequent to tissue damage. However their optimal use in clinical settings requires an improved understanding of the molecular mechanisms regulating their proliferation and differentiation, especially during the early stages of their commitment toward a specific lineage. Given the important cross-talk occurring between MSCs and HSCs and since recent results obtained in our laboratory indicated that Prep1 (Pbx-regulating protein 1) is involved in MSCs regulation, my thesis project was aimed at investigating whether Prep1 may play a role in the commitment of MSCs towards the adipogenic lineage. Prep1 is a member of the TALE (Three Aminoacidic Loop Extension) family, which encodes homeodomain transcription factors, known for their important role in embryonic development, organogenesis, and the hematopoietic stem and progenitor cells. Our results have shown that Prep1 is expressed in cultured undifferentiated murine MSCs and that its expression changes during their in vitro differentiation toward the adipocytic and osteogenic lineages. In particular, Prep1 protein levels decrease significantly three days after adipogenic induction and, conversely, significantly increase during the osteogenic process. To understand the biological function that Prep1 plays in MSC-derived adipogenesis we analyzed the effects of its in vivo downregulation, since its complete inactivation is lethal in the early stages of embryonic development. Therefore we used transgenic Prep1 hypomorphic mice, that express lower levels of protein, 2-20% as compared to wild type, and set up mMSCs cultures derived from their bone marrows. We analyzed cultured MSCs from wt and Prep1i/i mice, both at the undifferentiated state and at different time points after adipogenic induction.
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higher efficiency in differentiating along the adipogenic lineage as compared to the wt cells, used as controls. In particular, Oil Red staining, which highlights lipidic formation in adipose cells, showed a significantly greater number of mature adipocytes in Prep1i/i cultures at intermediate differentiation times, as compared to the normal counterpart. Furthermore, Real Time qPCR assays showed that the expression of adipogenic markers are more highly expressed in hypomorphic cells, already at the undifferentiated state level, suggesting that the undifferentiated mMSCs derived from hypomorphic mice may be enriched for adipogenic-committed cells. Further experiments are currently underway to confirm this hypothesis. In addition, Prep1 in vitro down-regulation is associated to altered cytokine/chemokine production. The in vivo read-out for such phenomenon could be an altered signaling downstream of such chemical mediator/s, resulting in a defective hematopoietic niche that may in turn contribute to the altered hematopoiesis observed in the Prep1i/i mice. I also performed gain-of-function assays, by using lentiviral vectors to overexpress Prep1. Our preliminary data suggest that forced Prep1 expression affectes MSCs survival.
In conclusion, our results support the hypothesis that Prep1 acts as a major inhibitor along the adipogenic differentiation program, and that Prep1 dysregulation affects both MSCs survival and differentiation potential.
