Background and ObjectivesAdipose-derived mesenchymal stem cells (ADSCs) are promising candidates in regenerative medicine. The need for in vitro propagation to obtain therapeutic quantities of the cells imposes a risk of impaired functionality due to cellular senescence. The aim of the study was to analyze in vitro senescence of previously cryopreserved human ADSCs subjected to serial passages in cell culture.Methods and ResultsADSC cultures from 8 donors were cultivated until proliferation arrest was reached. A gradual decline of ADSC fitness was observed by altered cell morphology, loss of proliferative, clonogenic and differentiation abilities and increased β-galactosidase expression all of which occurred in a donor-specific manner. Relative telomere length (RTL) analysis revealed that only three tested cultures encountered replicative senescence. The presence of two ADSC subsets with significantly different RTL and cell size was discovered. The heterogeneity of ADSC cultures was supported by the intermittent nature of aging seen in tested samples.ConclusionsWe conclude that the onset of in vitro senescence of ADSCs is a strongly donor-specific process which is complicated by the intricate dynamics of cell subsets present in ADSC population. This complexity needs to be carefully considered when elaborating protocols for personalized cellular therapy.

Background and Objectives:Recent findings suggest that therapeutic potential of mesenchymal stem cells (MSCs) could be increased through aggregation into three-dimensional (3D) bodies, and different culture methods have been employed to obtain 3D spheroids of MSCs. In the current study we report accidentally encountered spontaneous formation of adipose-derived stem cell (ASC) bodies in standard ASC culture of a single donor.Methods and Results:Human ASCs from passages 1 to 3, cultured in a medium containing 5% autologous serum (AS), spontaneously clustered and formed floating 3D bodies. After a transfer of floating ASC bodies onto new adherent plastic dish, they attached to the surface and gradual migration of spindle-shaped ASCs out of the bodies was detected. A substitution of AS with allogeneic sera did not hinder this ability, but commercial medium containing fetal bovine serum delayed the process. Substantial part of ASCs surrounding transferred ASC bodies showed alkaline phosphatase (AP) activity, while ASC aggregates were AP negative. Similar 3D bodies formed when ASCs were grown on an uncoated glass surface. These ASC aggregates as well as clusters of ASCs, where formation of the 3D bodies is initiated, expressed pluripotency marker NANOG, but the expression of OCT4A was not detected.Conclusions:Obtained results suggest that spontaneously formed ASC aggregates may represent a more primitive cell subpopulation within the individual ASC culture. The ability to form 3D aggregates, the expression of NANOG, and the lack of the AP activity may be used to enrich ASC cultures with potentially more primitive cells serving as an excellent basis for therapeutic applications.

Adipose-derived stem cells cultured in autologous serum maintain the characteristics of mesenchymal stem cells Human adipose tissue is known to be an attractive and readily available source of mesenchymal stem cells (MSC), which are becoming increasingly popular for application in regenerative medicine. Most of the protocols currently used for in vitro expansion of MSC include fetal bovine serum (FBS) supplementation. When MSC are cultured in such a way for clinical applications this rises concerns about immunogenicity of FBS proteins. A possible solution to this problem is the use of autologous serum (AS) instead of FBS. In this study we investigated whether adipose-derived stem cells (ADSC), cultivated in medium containing AS, maintain characteristics of MSC. The results show that the obtained ADSC were plastic adherent, rapidly dividing (doubling time 40 ± 4 hours), spindle-shaped cells with fibroblastoid morphology and exhibited normal karyotype. No less than 95% of the obtained cells displayed MSC surface markers, including CD73, CD90 and CD105, but showed no expression of the hematopoietic markers CD34 and CD45. ADSC cultured in the presence of AS underwent in vitro differentiation into adipocytes, osteoblasts and chondroblasts, confirmed by Oil Red O, Alizarin Red S and Alcian Blue stains, respectively. These findings suggest that ADSC may be expanded in the AS without the loss of characteristics of MSC.

The study characterises canine adipose-derived stem cells (cASCs) in comparison to human ASCs (hASCs) and tests their safety in a canine model after intravenous administration. cASCs from two dogs were cultured under hypoxic conditions in a medium supplemented with autologous serum. They were plastic adherent, spindle-shaped cells that expressed CD73, CD90, and CD44 but lacked CD45, CD14, HLA-DR, and CD34. cASCs differentiated toward adipogenic, osteogenic, and chondrogenic lineages, although adipogenic differentiation capacity was low. Blast transformation reaction demonstrated that these cells significantly suppress T-cell proliferation, and this ability is dose-dependent. Intravenous administration of a cell freezing medium, therapeutic dose of cASCs (2 × 106 live cells/kg), and five times higher dose of cASCs showed no significant side effects in two dogs. Microscopic tissue lesions were limited to only mild, non-specific changes. There were no signs of malignancy. The results of the study indicate that cASCs are similar to hASCs and are safe for therapeutic applications in a canine model. The proposed methodology for ASC preparation on a non-routine basis, which includes individually optimised cell culture conditions and offers risk-adapted treatment, could be used for future personalised off-the-shelf therapies, for example, in myocardial infarction or stroke.

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