P-1019: Mesenchymal stem cells from adipose tissue can be a novel feeder cell source for human embryonic stem cell culture

Abstract

Nearly all published human embryonic stem cell (hESC) lines are derived on mouse embryonic fibroblast (MEF) feeder cells. Although MEF is a convenient culture system for research purposes, therapeutic use of hESCs will likely require either feeder-free or an effective human feeder system to be developed. Several feeder systems of human origin have recently been reported, however each source has its own advantages/problems such as difficulty in sampling, culture conditions and hESC support. Recent results indicate that adipose tissue can be a novel and abundant feeder cell source for hESC culture. Here, we report the isolation, establishment and characterization of a human adipose derived mesenchymal stem (hADMS) cell lines and their potential as hESC feeder cells. Laboratory data. Adipose tissue was obtained from liposuction or caesarian section procedures. After collection, tissue pieces were extensively washed with PBS and processed for enzymatic digestion. Final cell suspensions were seeded on tissue culture plates containing Ko-DMEM with %10 FBS. Cellular morphology and antigenic properties were assessed by phase-contrast microscopy and flow cytometry against antibodies, CD29, CD90, CD45, CD34 and CD44. One of our previously characterized hESC lines, NS-4 was cultured on feeder system derived from mitomycin treated ADMS cells. Feeder systems derived from MEF, human foreskin fibroblasts (HFF) and human endometrial stromal (HES) cells were also used as controls. After extended culture, characterization procedures involving karyotyping, gene expression analysis by RT-PCR, immunocytochemistry analysis of surface antigens (SSEA-4, TRA-1-60, TRA-1-81, OCT-4), and alkaline phosphatase expression were performed. Feeder system prepared from ADMS cells supported the continuous proliferation of NS-4 line for at least 12 passages. NS-4 cells grown on HFF, HES and ADMS showed very similar morphological characteristics and growth behavior. They all displayed a well-defined colony boundary and feeder polarity-dependent angular shape. However, hESC colonies grown on HES feeder were found to be slightly thinner and flatter than HFF and ADMS counterparts. hESCs were round and small, contained prominent nucleoli, had high nucleus to cytoplasm ratio and typical space was seen between cells. Due to slightly extended doubling rates, passaging interval was approximately 8-9 days in all human feeder cultured hESCs compared to hESC grown on MEF. Comparative evaluation of surface markers SSEA-4, TRA-1-60 and TRA-1-81 as well as Oct-4 expression pattern of hESCs grown on human feeders exhibited that HFF, HES or ADMS feeders did not negatively affect the expression of the Oct-4 gene. Human embryonic stem cells, besides current ethical and technical limitations in many countries, are likely to be one of the major cell sources for both research and therapy in the near future. Therefore, isolation, culture and characterization of new human embryonic cell lines under a variety of in vitro conditions are of key importance in order to expand our knowledge. Our results and experience on hESC culture on various human feeders could extend our knowledge on the required growth conditions and can lead to the development of novel culture protocols.