Ontogeny of human umbilical cord perivascular cells: molecular and fate potential changes during gestation.

Human umbilical cord-derived perivascular cells (PVCs) are a recently characterized source of mesenchymal stromal cells that has gained much interest in the field of cellular therapeutics. However, very little is known about the changes in fate potential and restrictions that these cells undergo during gestational development. This study is the first to examine the phenotypic, molecular, and functional properties of first trimester (FTM)-derived PVCs, outlining properties that are unique to this population when compared to term (TERM) counterparts. FTM- and TERM-PVCs displayed analogous mesenchymal, perivascular, and immunological immunophenotypes. Both PVCs could be maintained in culture without alteration to these phenotypes or mesenchymal lineage differentiation potential. Some unique features of FTM-PVCs were uncovered in this study: (1) while the gene signatures of FTM- and TERM-PVCs were similar, key differences were observed, namely, that the Oct4A and Sox17 proteins were detected in FTM-PVCs, but not in TERM counterparts; (2) FTM-PVCs exhibited a greater proliferative potential; and (3) FTM-PVCs were more efficient in their in vitro differentiation toward selective mesenchymal cell types, including the chondrogenic and adipogenic lineages, as well as toward neuronal- and hepatocyte-like lineages, when compared to TERM-PVCs. Both PVCs were able to generate osteocytes and cardiomyocyte-like cells with similar efficiencies in vitro. Overall, FTM-PVCs show more plasticity than TERM-PVCs with regard to fate acquisition, suggesting that a restriction in multipotentiality is imposed on PVCs as gestation progresses. Taken together, our findings support the idea that PVCs from earlier in gestation may be better than later sources of multipotent stromal cells (MSCs) for some regenerative medicine applications.

Withaferin A induces proteasome inhibition, endoplasmic reticulum stress, the heat shock response and acquisition of thermotolerance.

In the present study, withaferin A (WA), a steroidal lactone with anti-inflammatory and anti-tumor properties, inhibited proteasome activity and induced endoplasmic reticulum (ER) and cytoplasmic HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Proteasomal inhibition by WA was indicated by an accumulation of ubiquitinated protein and a decrease in chymotrypsin-like activity. Additionally, immunoblot analysis revealed that treatment of cells with WA induced the accumulation of HSPs including ER chaperones, BiP and GRP94, as well as cytoplasmic/nuclear HSPs, HSP70 and HSP30. Furthermore, WA-induced an increase in the relative levels of the protein kinase, Akt, while the levels of actin were unchanged compared to control. Northern blot experiments determined that WA induced an accumulation in bip, hsp70 and hsp30 mRNA but not eIF-1α mRNA. Interestingly, WA acted synergistically with mild heat shock to enhance HSP70 and HSP30 accumulation to a greater extent than the sum of both stressors individually. This latter phenomenon was not observed with BiP or GRP94. Immunocytochemical analysis indicated that WA-induced BiP accumulation occurred mainly in the perinuclear region in a punctate pattern, while HSP30 accumulation occurred primarily in a granular pattern in the cytoplasm with some staining in the nucleus. Prolonged exposure to WA resulted in disorganization of the F-actin cytoskeleton as well as the production of relatively large HSP30 staining structures that co-localized with F-actin. Finally, prior exposure of cells to WA treatment, which induced the accumulation of HSPs conferred a state of thermal protection since it protected the F-actin cytoskeleton against a subsequent cytotoxic thermal challenge.