Altered properties of feline adipose-derived mesenchymal stem cells during continuous <i>in vitro</i> cultivation

Abstract

Cytotherapy with mesenchymal stem cells (MSCs) has been studied in many species, and often requires in vitro cell expansion to obtain therapeutic doses of stem cells. Because the characteristics of MSCs, such as self-renewal and multi-lineage differentiation, can be altered by long-term culture, it is important to maintain stemness during cultivation. This study assessed the changes in the characteristics of feline adipose tissue-derived (fAT)-MSCs during in vitro passaging. Stem cells isolated from the adipose tissue of donor cats were cultured for seven sub-passages. Proliferation capacity was analyzed by calculating the cell doubling time and by colorimetric assay. Expression of stem cell-specific markers was evaluated by quantitative reverse transcription (qRT)-PCR and immunophenotyping. Expression of adipogenic and osteogenic differentiation markers was also measured by qRT-PCR. Histochemical staining and measurement of β-galactosidase activity were conducted to detect cellular senescence. The cell proliferation rate decreased significantly at passage 5 (P5). Gene expression levels of pluripotency markers (Sox2, Nanog and Klf4) and stem cell surface markers (CD9, CD44, CD90 and CD105) decreased during continuous culture; in most assays, statistically significant changes were observed at P5. The ability of cells to undergo adipogenic or osteogenic differentiation was inversely proportional to the number of passages. The proportion of senescent cells increased with the number of passages. These results suggest that repeated passages alter the proliferation and multipotency of fAT-MSCs. In clinical trials, early-passage cells should be used to achieve the maximum therapeutic effect.

Viability, Yield and Expansion Capability of Feline MSCs Obtained from Subcutaneous and Reproductive Organ Adipose Depots

“…These samples also had the slowest P2 doubling time. The range of doubling times reported here are consistent with some previous publications of feline abdominal adipose tissue [28][29][30] but slower than those reported by others [20,31].…”

“…Some have reported the inability to detect Sox2 or the resulting protein in feline abdominal or subcutaneous adipose MSCs [28,29]. In contrast, our results are consistent with those of Lee et al, showing detection of Sox2 in the abdominal adipose tissue [31]. Although not very likely, we point out that our choice of PCR primers for Sox2 might also result in some differences compared to earlier studies.…”

“…Because extensive work has already been published on the general MSC markers found in feline adipose tissue from numerous sources [19,28,30,31], we opted to study biomarkers of pluripotency and lineage assignment to further characterize the tissue. While the expression levels of some of the pluripotent biomarkers reached statistical significance, the differences were small and likely not biologically relevant.…”

Prostaglandin E2 secreted from feline adipose tissue-derived mesenchymal stem cells alleviate DSS-induced colitis by increasing regulatory T cells in mice

“…Cells were characterized by flow cytometry using antibodies against the following proteins: CD9, CD44 (GeneTex, CA, USA), CD34-phycoerythrin (PE), and CD45-fluorescein isothiocyanate (FITC; eBiosciences, San Diego, CA, USA). For CD9 and CD44, indirect immunofluorescence was performed using goat anti-mouse IgG-FITC and goat anti-rat IgG-PE (Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively [ 43 , 59 ]. Characterization was conducted using FlowJo 7.6.5 software (TreeStar, Inc., Ashland, OR, USA).…”

Hypoxic Preconditioning as a Strategy to Maintain the Regenerative Potential of Mesenchymal Stem Cells

“…Human Wharton's jelly-derived mesenchymal stem cells showed significant decrease in growth kinetics and differentiation when cultured for longer time as compared to the cells in initial passages [22]. Feline adipose tissue derived MSC showed a progressive decrease in pluripotency and proliferation over continuous passaging [23].…”