Characterisation of adipose-derived stromal cell heterogeneity

Abstract

Human adipose-derived stromal cells (hASCs) have gained increasing attention in the past decade as a potential cell therapeutic product. hASCs are classified as multipotent, fibroblast-like, plastic-adherent cells that can easily be expanded in vitro and has the ability to differentiate into multiple cell lineages. A distinct advantage of hASCs is that large numbers of cells can be extracted with minor donor site morbidity. This has sparked the worldwide growth of a new research field and industry. There is however, still much to learn about these cells before they can be used with confidence in a clinical setting.

An inherent characteristic of hASCs that is not well understood is their heterogeneity. Freshly isolated hASCs consists of various sub-populations of cells. Whether this heterogeneity is beneficial or detrimental with respect to the potential therapeutic effect of hASCs is unknown. Thus, a great deal of effort is currently being made to understand the heterogeneous nature of hASCs. In the attempt to characterise the heterogeneity of hASCs in this study, two different approaches were used. The SP assay was used in an attempt to identify a sub-population of cells with greater dye efflux capability than the rest of the hASC population. We also examined levels of ABC transporter gene and protein expression (known to cause the SP phenotype). Results revealed a sub-population of cells with efflux ability present in hASCs. Results also provided novel insight into the expression of ABC transporter genes and the expression pattern of ABC transporter proteins in hASCs. The second approach used was the first study, to the author’s knowledge, that attempted to characterize the heterogeneity of hASCs at P2 using whole transcriptome analysis at the single-cell level. Results revealed the presence of a minimum of two and a maximum of five sub-populations in hASCs at P2. In addition, one of the sub-populations identified is believed to be contractile cells such as pericytes and/or vascular smooth muscle cells (vSMCs).

Another aspect that needs to be taken into consideration in order to use hASCs in a clinical setting, is the proliferation and maintenance of the isolated cells ex vivo. Many research groups are moving away from foetal bovine serum (FBS) as the standard serum supplement in order to make the isolation and expansion of cells compliant to good manufacturing practice. Suggested replacements for FBS are human blood alternatives which can create a culture environment that more accurately resembles the human environment. The human alternatives suggested include human serum (HS), platelet-rich plasma (PRP), platelet-poor plasma (PPP), freshly frozen plasma (FFP) and platelet lysate (PL). This was, to the author’s knowledge, the first head to head comparison done between FBS and all the listed human alternatives simultaneously. Results revealed that all the different human alternatives were able to sustain hASCs in vitro with no effect on the viability or immunophenotypic profiles of the cells. Results also revealed PL and PRP to cause a significant increase in the proliferation rate of hASC.