Role of wnt-5a in the determination of human mesenchymal stem cells to preadipocytes

Aims: Increasing adipocyte size as well as numbers is important in the development of obesity, with adipocytes being generated from mesenchymal precursor cells. This process comprises the determination from mesenchymal stem cells (MSC) into preadipocytes (PA) and the differentiation of PA into mature fat cells. While the process of differentiation is highly investigated, the determination from MSC to PA in humans is poorly understood. Therefore, in the present study we have examined the role of different wnt molecules in the process of determination of human MSC into PA.

Methods: Human MSC were prepared from umbilical cord blood. Human PA were isolated from fat biopsies of metabolically healthy men aged of 18 to 35 undergoing elective surgery. These two cell populations were analyzed by FACS and induced to differentiate into adipocytes and osteocytes. Expression of wnt-10b and wnt-5a in the two cell populations was compared by western-blot of whole cell lysates. Effect of wnt-5a in the process of determination was investigated by induction of adipogenesis and osteogenesis in the presence and absence of neutralizing wnt-5a antibodies in the culture medium. These antibody experiments were also used to investigate intracellular wnt-5a signalling in MSC.

Results: The two cell populations, MSC and PA, showed similar expression of the mesenchymal surface markers CD29, CD44 and CD73 and were both negative for the hematopoetic surface markers CD34 and CD45. However, while MSC were able to differentiate into adipocytes and osteocytes, PA were only able to undergo adipogenesis, indicating that PA lost their pluripotency during determination. Wnt-10b was found not to be expressed at significant levels in human MSC and PA. However, examination of wnt-5a expression in the two cell populations resulted in significant (p<0.01) higher levels in MSC compared to PA, suggesting wnt-5a down-regulation might be important in the determination of MSC to PA in humans. This was further supported by the fact that incubation of human MSC in medium containing neutralizing wnt-5a antibodies abolished the ability to undergo osteogenesis while adipogenesis was still possible. Finally, beta-Catenin levels were found to be similar in cells incubated with and without anti-wnt-5a antibodies, suggesting that wnt-5a signalling in MSC is mediated by the non-canonical pathway.

Conclusion: These data suggest that down-regulation of wnt-5a expression plays a major role in the determination of human MSC to PA.