The role of Pref-1 in in vitro adipogenic differenctiation of mesenchymal stromal/stem cells

Abstract

Obesity is characterized by an excessive amount of body fat which can negatively affect health as it is a risk factor for various non-communicable diseases such as cardiovascular disease and diabetes, amongst others. A better understanding of the process of fat cell formation, a process known as adipogenesis, would aid in finding sustainable solutions to this growing global health challenge. The ability of human derived mesenchymal stromal/stem cells (hMSCs) to differentiate into adipocytes holds great promise to serve as an in vitro model to study human adipogenesis. It is known from the literature that hMSCs from different sources within the body may differ in their differentiation potential. It is well reported that human Wharton’s jelly derived MSCs (hWJSCs) have reduced adipogenic differentiation capacity when compared to hMSCs isolated from adipose tissue. hMSCs isolated from adipose tissue are referred to as adipose-derived mesenchymal stromal/stem cells (hASCs). The poor adipogenic differentiation potential of hWJSCs has also been observed in our group. We also observed elevated pre-adipocyte factor 1 (Pref-1) Mrna levels in hWJSCs. The observed elevated Pref-1 mRNA levels in hWJSCs led to the hypothesis that this may be responsible for hWJSCs poor adipogenic differentiation capacity. Pref-1 is a transmembrane protein capable of being cleaved to give rise to a 50 kDa soluble protein which is thought to be an important adipogenic inhibitor by preventing cells from maturing into adipocytes thereby keeping them in a pre-adipocyte state. It was therefore important to confirm whether the elevated Pref-1 gene expression observed translated into higher levels of Pref-1 protein in hWJSCs when compared to hASCs. The aim of this study was to further investigate a previous observation in our group of elevated Pref-1 gene expression levels in hWJSCs undergoing adipogenesis. This will be done by examining the association between Pref-1 protein expression levels in hWJSCs and hASCs and their respective in vitro adipogenic differentiation potential. The cells were induced to undergo adipogenesis for a period of 21 days and monitored on days 0, 1, 3, 7, 14 and 21. During this time period, the adipogenic differentiation potential of the cells was assessed by means of flow cytometry (quantitative) and microscopy (qualitative). The level of Pref-1 protein expression was investigated using various techniques which included flow cytometry, ELISA and western blot assays. Gene expression was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). As previously determined, hWJSCs displayed poor adipogenic differentiation despite the upregulation of peroxisome proliferator activated receptor gamma (PPARγ). Flow cytometry data revealed hASCs to have greater levels of Pref-1 protein expression on the cell surface. We were unable to detect any Pref-1 protein in the cell culture supernatant collected at each time point using ELISA, but western blot data suggested that there may be more soluble Pref-1 protein in the cell culture supernatant obtained from hWJSCs. Gene expression data also revealed higher levels of Pref-1 mRNA in hWJSCs. However, the amount of Pref-1 protein detected was very low and no statistical significance could be determined between Pref-1 expression in hASCs versus hWJSCs in any of the assays. The in vivo data obtained by immunohistochemistry shows that both adipose tissue and umbilical cord express Pref-1 around blood vessels. The umbilical cord does however have additional Pref-1 staining that is not associated with blood vessels. The stromal cells in the Wharton’s jelly displayed positive Pref-1 staining. How this finding correlates with the in vitro results obtained is unclear. Comparing the adipogenic differentiation potential of hWJSCs to hASCs did however allow for an interesting observation. HASCs were able to upregulate CD36 (a fatty acid translocase) during the adipogenic differentiation period, while this was not observed for hWJSCs. The low levels of Pref-1 protein expression combined with the upregulation of PPARγ in hWJSCs thus raises the question as to whether other regulatory elements, currently unknown, downstream of PPARγ, suppress the upregulation of endstage adipogenic genes and consequently prevent the induced hWJSCs from acquiring the phenotypic characteristics associated with mature adipocytes.