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Tissue Eng Part C Methods 2012 Nov;18(11):877-89

Quantitative approaches to detect donor and passage differences in adipogenic potential and clonogenicity in human bone marrow-derived multipotent stromal cells.

Lo Surdo JL, Bauer SR

Abstract

Bone marrow-derived multipotent stromal cells (MSCs), also known as mesenchymal stem cells, have great promise due to their capacity for tri-lineage differentiation and immunosuppressive properties, which allows for their allogeneic use and ultimately may allow for treatment of many diseases. MSCs will require extensive expansion and passaging to obtain cells in sufficient numbers necessary for cell therapies. MSCs from many donors could potentially be used. Because of this, there is a need to understand the role of passaging and donor differences on differentiation capacity using quantitative approaches. Here, we evaluated MSCs from 2 donors (noted as PCBM1632 and PCBM1641 by the manufacturer) at tissue culture passages 3, 5 and 7. We used a colony forming unit (CFU) assay and limiting dilution to quantify clonogenicity and precursor frequency during adipogenesis, as well as qRT-PCR for adipogenic markers to evaluate changes on a gene expression level. Further, we observed changes in cell size, and sorted small and large populations to evaluate size-related adipogenic potential. While the adipogenic precursor frequency of approximately 1 in 76 cells remained similar through passages for cells from PCBM1641, we found a large decrease in the adipogenic potential of MSCs from PCBM1632, with 1 in 2,035 cells being capable of differentiating into an adipocyte at passage 7. MSCs from both donors showed an increase in cell diameter with increasing passage, which correlates with a decrease in clonogenicity by CFU analysis. We also measured adipose lineage gene expression following induction of adipocyte differentiation. Expression of these genes decreased with passage number for MSCs from PCBM1632 and correlated with the decrease in adipogenic potential by passage 7. In contrast, MSCs from PCBM1641 showed increased expression of these genes with increasing passage. We have shown that several quantitative assays can detect differences in MSC differentiation capacity, clonogenicity and cell size between donors and passages. These quantitative methods are useful to assess the quality of MSCs.


Category: Journal Article
PubMed ID: #22563812 DOI: 10.1089/ten.TEC.2011.0736
PubMed Central ID: #PMC3483050
Includes FDA Authors from Scientific Area(s): Biologics
Entry Created: 2012-02-15 Entry Last Modified: 2013-09-22
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