Abstract
Multipotent adipose-derived stromal/stem cells (ASCs) are candidates for use in cellular therapies for the treatment of a variety of conditions/diseases. Ex vivo expansion of freshly isolated ASCs may be necessary prior to clinical application to ensure that clinically relevant cell numbers are administered during treatment. In addition, cryopreserving cells at early passages allows for storage of freshly isolated cells for extended periods of time before expanding these cells for clinical usage. There are however several concerns that these laboratory-based procedures may alter the characteristics of the cells and in so doing decrease their regenerative potential. In this study we report on the impact of early rounds of cryopreservation (P0) and ex vivo expansion (P0 to P5) on the phenotypic characteristics and adipogenic differentiation potential of ASCs. Our results show that ASCs that upregulate CD36 expression during adipogenic differentiation gradually decrease with increasing expansion rounds. The consequent decrease in adipogenic differentiation capacity was evident in both gene expression and flow cytometry-based phenotypic studies. Successive rounds of expansion did not however alter cell surface marker expression of the cells. We also show that early cryopreservation of ASCs (at P0) does not affect the adipogenic differentiation potential of the cells.
Highlights
Most clinical trials currently underway make use of SVF, with only a few studies using ex vivo expanded ASCs11–14
In order for cells to be characterized as MSCs they need to be plastic adherent, express a pre-defined set of cell surface proteins and have the ability to differentiate into adipocytes, chondrocytes and osteoblasts[31]
The main phenotypic difference between undifferentiated and differentiated adipose-derived stromal/stem cells (ASCs) was the upregulation of CD36 expression during adipocyte differentiation (Fig. 1), confirming the findings reported by several other groups that CD36 is associated with adipocyte differentiation[30,34,35,36]
Summary
Most clinical trials currently underway make use of SVF, with only a few studies using ex vivo expanded ASCs11–14. ASCs, undergo fundamental changes during ex vivo expansion[16,20,21] These ex vivo-associated changes include reduced proliferation potential, increased cell senescence, impaired multipotent differentiation capabilities and genomic instability[22]. CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) are main regulators[26,27,28], with PPARγ being an essential master regulator of the adipogenic differentiation process[27]. Upon activation, these transcription factors induce the upregulation of enzymes responsible for fatty acid biosynthesis, transport and incorporation into triglycerides, the main component of intracellular lipid droplet cores[28]. Cryopreservation at P0, did not affect the adipogenic differentiation potential of ASCs
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