Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites

Abstract

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating wide-spread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their “stem cell” characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73, and CD105 and negative for CD14, CD31, and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes, and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2, and NANOG when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.

NANOG Plays a Hierarchical Role in the Transcription Network Regulating the Pluripotency and Plasticity of Adipose Tissue-Derived Stem Cells (ASCs)

“…Our results indicate that the stemness of ASCs should be defined by their ability to differentiate into multiple lineages coupled with their expression of the pluripotent stem cell-related genes Oct-4, SOX2, and NANOG in order to functionally distinguish ASCs as more stem cell-like [20]. We found that the embryonic stem cell marker NANOG is over-expressed in MSCs derived from adipose tissue, confirming recent studies that have demonstrated a central role of NANOG in embryonic stem cells [21].…”

Multipotency and secretome: the mechanisms behind the regenerative potential of adipose-derived stem cells

“…[15] This might lead the researcher to acquire inaccurate results if senescent cells of latter passages are used. For the reliability and standardization of the experiments, it is also advised that the ASCs' multilineage (i.e.…”

Modeling early stage bone regeneration with biomimetic electrospun fibrinogen nanofibers and adipose-derived mesenchymal stem cells

“…ASCs were procured from donors as we described previously [34,35] and in accordance with Virginia Commonwealth University's Institutional Review Board. Briefly, lipoaspirate was obtained as surgical waste from patients undergoing elective, cosmetic surgery.…”

“…The cells were cultured in low glucose DMEM with 10% FBS, and 1% ABAM, known as basic ASC growth media. Adherent cells were expanded and then confirmed by flow cytometry to exhibit the MSC immunophenotype and by histology and qRT-PCR to display tri-lineage mesenchymal differentiation potential, as previously described [34,35].…”

“…An ABI PRISM 7900 (Life Technologies) was used for gene expression (qRT-PCR) analysis from quintuplicate samples. Primers for 18S (ABI standard sequences [35]), osteocalcin (Fwd: 5'-AGCAAAGGTGCAGCCTTTGT-3'; Rev: 5'-GCGCCTGGGTCTCT TCACT-3'; TaqMan probe: 5'-CCTCGCTGCCCTCCTGCTTGG-3'), RUNX2 (Fwd: 5'-CCCGTGGCCTTCAAGGT-3'; Rev: 5'-CGTTACCCGCCATGACAGTA-3'; TaqMan probe: 5'-CCACAGTCCCATCTGGTACCTCTCCG-3'), and alkaline phosphatase (Fwd: 5'-GCACTCCCACTTCATCTGGAA-3'; Rev: 5'-GAAACCCAATAGGTAGTCCACATTG-3'; TaqMan probe: 5'-CGCACGGAACTCCTGACCCTTGAC-3') were used. Relative expression levels for the lineage specific genes were calculated using standard curves generated from the triplicate dilution series of the cDNA with normalization to the 18S housekeeping gene.…”