Yield and characterization of subcutaneous human adipose-derived stem cells by flow cytometric and adipogenic mRNA analyzes

Abstract

Background aims. Adipose-derived stromal/stem cells (ASC) capable of multipotential differentiation can be isolated with high yields from human subcutaneous lipoaspirates. This study reports our recent experience of isolating and immunophenotypically characterizing ASC from >60 human patients with a mean age of 43.6 and body mass index (BMI) of 27. Methods. We examined the ASC yield per unit volume of lipoaspirate tissue, the surface antigen profile based on flow cytometry, histochemical differentiation potential along the adipogenic and osteogenic pathways, and expression of adipogenic mRNA by transcriptomic microarray and reverse transcription (RT)–polymerase chain reaction (PCR). Results. The population (n = 64) of predominantly Caucasian (84.3%) female (90.6%) donors had a mean age of 43.6 ± 11.1 years and a mean BMI of 27.0 ± 3.8. A yield of 375 ± 142 × 10³ ASC was obtained per milliliter of lipoaspirate within a 4.1 ± 0.7-day culture period (n = 62). The ASC population was uniformly CD29⁺ CD34⁺ CD44^lo CD45^lo CD73⁺ CD90⁺ CD105⁺ and capable of undergoing both adipogenesis and osteogenesis in vitro based on Oil Red O and Alizarin Red staining, respectively. Adipogenic differentiation was associated with a significant induction of multiple mRNA associated with lipid storage and synthesis based on microarray analysis of n = 3 donors. During an adipogenic differentiation time–course, representative mRNA (adiponectin, C/EBPα, leptin and LPL) displayed increases of several orders of magnitude. Conclusions. These findings demonstrate the reproducibility of subcutaneous lipoaspirates as a consistent and abundant source of functional ASC from donors across a spectrum of ages and BMI. These results have relevance for regenerative medical applications exploiting autologous and allogeneic ASC for soft and hard tissue engineering.

Key Words::

• adipogenesis
• adipose-derived stem cells
• cell yield
• differentiation
• flow cytometry
• human
• transcriptome

Acknowledgments

The investigators express their appreciation to Elizabeth Clubb MD, James Wade MD, and their office staff; patients for providing the human lipoaspirate tissue used in this study; and Laura Dallam for administrative assistant supporting this work.

This work was partially supported by a CNRU Center Grant, number 1P30 DK072476, entitled ‘Nutritional programming: environmental and molecular interactions’ sponsored by NIDDK.

Disclosure of interest: JMG has been a consultant with companies pursuing the use of ASC for therapeutic purposes, including Cognate Bioservices, Toucan Capital, Vesta Therapeutics, VetStem and Zen-Bio.