I have isolated MSC from the placental warton's jelly and wanted to differentiate into fibroblasts. Can someone suggest protocols for the same.
Also select the markers for the fibroblasts.
Regards,
Tarun
Few important MSC markers are (CD44, CD73 and CD105) and fibroblastic markers a are i.e. collagen I, HSP47 (heat shock protein 47), vimentin, FSP (fibroblast surface protein) and αSMA (α smooth muscle actin)], .Use negative markers as endothelial cell marker CD31 and haemopoietic lineage markers (CD14 and CD45). Further, stem cell-associated gene expressions by performing real-time PCR may found different expression between these two cells types. The expression of embryonic stem cell markers [OCT4, KLF4, NANOG, LIN28, FGF4 (fibroblast growth factor 4) and REST] in hASCs and hSDFs differ more than 2.5-fold as compared with WI38. Commercially available fibroblast preparations from skin (hSDFs) consist of a significant number of cells with differentiation potential apart from terminally differentiated fibroblasts; (ii) colony-forming capacity and differentiation potential are specific important properties that discriminate MSCs from fibroblasts (WI38), while conventional stem cell properties such as plastic adherence and the expression of CD44, CD90 and CD105 are unspecific for stem cells.
This is not at all straightforward. Though some helpful positive and negative biomarkers have been used to help define hMSC, the biomarkers mentioned above for hMSC were not considered to be exclusively specific to stem cells. Rather, they serve as useful qualifying indicators that combined with functional assays demonstrating multi-potent differentiation potential provided a multi parametric consensus to help define the cell population as being hMSC. Like hMSC, fibroblasts can also be heterogenous with regard to phenotype expression and strict definitions as to what constitutes a fibroblast can vary: the clearest discrimination coming more from the tissue source and how the cells were derived rather than studies that differentiate hMSC into fibroblasts. Additional complexity comes from "phenotypic drift" as the hMSC cultures age with passage and show susceptibility to stress induced senescence from the culture conditions that can alter their phenotype. I'm sorry I don't have a more positive easy answer for you, I'd be very keen to learn if anyone does. Some suggest that multivariate parameters focused on small cell diameter, cell stiffness and high nuclear membrane fluctuations may contribute to functional signatures of multi-potency (Lee et al., 2014), but if in your model you define a differentiated fibroblast as a cell that has lost multi-potency there are still challenges. It may be more than semantics to ask how does one distinguish an hMSC that has differentiated to a fibroblast from an hMSC that has lost multi potency as a result of senescence.
In my experience I cultured MSC in D.MEM Low glucose medium and fibrobolast in high gluocse medium. MSC do not grow well in high glucose medium, it could help you?
From my experience two cytokines that will induce fibrogenesis in mesodermal stem cells are basic-FGF and TGF-beta. You will see the results after 1-2 passages of your cultures [see attached articles]. These two cytokines are actually the bane of most people doing cell culture. Most think that cytokines have a single activity, when actually they have several activities based on the identity of the cells they are working with. And of course that brings up another point, are you working with a 100% purified clonal population of MSCs are are you using a mixture of cells? The last attached article lists techniques to identify various cell types differentiated from 100% purified clonal populations of precursor stem cells and progenitor cells, fibroblasts being one of the differentiated cell types identified.
- Badges
- Science topic