gms | German Medical Science

Questionnaire: Tissue engeneering and regenerative medicine approaches are promising approaches to regenerate tissues damaged by trauma or diseases. Several cell types were evaluated based on their „biologic potential“, of which cells from adipose tissue, easily harvested subcutaneously. The stromal vascular fraction (SVF) is a heterogeneous mixture of cells obtained by enzymatic digestion of human adipose tissue and depletion of mature adipocytes by centrifugation. It contains multipotent mesenchymal cells, referred to as adipose-derived stem/stromal cells (ASC), in higher numbers of clonogenic cells than the standardly-used bone marrow-derived cells, as well as hematopoietic and vascular cells. The sialomucin CD34 is a type-I transmembrane glycoprotein, the function of which is still largely unknown, broadly expressed by cells in SVF but rapidly lost by CD90⁺/CD34⁺ human ASC expanded in monolayer culture. We thus aimed at defining culture conditions allowing the maintenance of CD34 in ASC to investigate this role.

Methods: Long-term cultures of SVF cells in Petri dishes were initiated, avoiding the standard replating into new dishes at cell confluence, in order to possibly favor niche-establishment and interactions between cells.

Results and Conclusions: In this setup, a subpopulation of around 20% of CD34⁺ cells was maintained from day 21 to day 56, e.g. 22.3±18% (n=7) of CD34⁺ cells at day 28. The cells proliferated until day 56 as demonstrated by cell counting and by positivity for Ki67. 95% of them were positive for mesenchymal markers, such as CD90 or CD73. SVF cells cultured with serial, weekly replating generated ASC but with minimal expression of CD34. CD34⁺ ASC exhibited a spread, rounded morphology and expressed PODXL, another sialomucin, as well as stem cell markers like SSEA-1 or POU5F1 (Oct3-4). To study the potential role of CD34, ASC were sorted at day 28 according to CD34.Preliminary data suggest that CD34⁺ ASC are more clonogenic (CD34⁺: 31%, CD 34^-: 8%) and more proliferative than CD34^- ASC, as demonstrated by colony forming efficiency assays. CD34⁺ ASC also displayed increased activity of aldehyde dehydrogenase (ALDH), an enzyme associated with clonogenic stem cells, as compared to CD34^- ASC. A first series of differentiation assays showed that CD34⁺ ASC are likely more osteogenic than CD34^- ones, which instead have a higher capacity to differentiate into the adipogenic lineage.

This study demonstrates that CD34 expression is maintained by human, mesenchymal ASC under specific culture conditions, possibly by establishing niche-like structures in long-time cultures. Clearly, more work is required to investigate the parameters responsible for the maintenance of such phenotypes of ASC and the precise impact of the expression of CD34, and other sialomucins such as PODXL, on the biology of ASC. ASC cultured without replating could represent a cell source of choice for bone tissue engineering applications and beyond.