Porcine adipose tissue-derived mesenchymal stem cells retain their proliferative characteristics, senescence, karyotype and plasticity after long-term cryopreservation

Abstract

We and others have provided evidence that adipose tissue-derived mesenchymal stem cells (ASCs) can mitigate rat cardiac functional deterioration after myocardial ischemia, even though the mechanism of action or the relevance of these findings to human conditions remains elusive. In this regard, the porcine model is a key translational step, because it displays heart anatomic-physiological features that are similar to those found in the human heart. Towards this end, we wanted to establish the cultural characteristics of porcine ASCs (pASCs) with or without long-term cryostorage, considering that allogeneic transplantation may also be a future option. Compared to fresh pASCs, thawed cells displayed 90-95% viability and no changes in morphological characteristics or in the expression of surface markers (being pASCs characterized by positive markers CD29(+); CD90(+); CD44(+); CD140b(+); CD105(+); and negative markers CD31(-); CD34(-); CD45(-) and SLA-DR(-); n = 3). Mean population doubling time was also comparable (64.26±15.11 hours to thawed cells vs. 62.74±18.07 hours to fresh cells) and cumulative population doubling increased constantly until Passage 10 (P10) in the entire cell population, with a small and gradual increase in senescence (P5, 3.25%±0.26 vs. 3.47%±0.32 and P10, 9.6%±0.29 vs. 10.67%±1.25, thawed vs. fresh; SA-β-Gal staining). Chromosomal aberrations were not observed. In addition, under both conditions pASCs responded to adipogenic and osteogenic chemical cues in vitro. In conclusion, we have demonstrated the growth characteristics, senescence, and the capacity of pASCs to respond to chemical cues in vitro and have provided evidence that these properties are not influenced by cryostorage in 10% DMSO solution.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. pASC morphology and viability are maintained after long-term cryostorage.

Morphological profiles for pASCs A) after passage 3 (before freezing) and passages 5, 10, and 15 (after thawing). Note similar fibroblast–like morphology in all represented passages. B) Quantification of viability of pASCs by Tripan blue dye assay in Passage 5 after thawing displayed a small but significant reduction versus fresh cells (P6–P10; *p<0.001; n = 3). C) Representative FACS plot showing the viability of P5 pASC by Annexin/PI assay (mean%±SEM; n = 3). D) Quantification of cell death by type (*p<0.0001 fresh late apoptotic vs. thawed late apoptotic cells; n = 3). Correlation curve of percentage of viability between Tripan blue dye assay×Annexin/PI assay.

Figure 2. Cryostorage does not influence the expression of mesenchymal surface markers.

A) FACS histogram representations for each analyzed surface marker in pASCs. B) Quantification of surface markers (means expressed as percentages; n = 3). C) Representative semi quantitative RT-PCR comparing fresh and thawed cells by mRNA expression for positive and negative phenotypic markers (2% agarose gel). Numbers 1, 2 and 3 are relative to different animals used to extract the pASCs.

Figure 3. Growing kinetics are similar for thawed and fresh pASC.

A) There were no significant differences in doubling times between passages in different conditions (64.26±15.11 hours to thawed cells vs. 62.74±18.07 hours to fresh cells; p>0.05; n = 4). B) pASCs cumulative population doubling (CPD) indicates a relatively constant population doubling rate between thawed and fresh cells from Passage 5 to 10. (thawed cells CPD mean = 1.27 vs. fresh cells CPD mean = 1.20; p>0.05. n = 4).

Figure 4. Senescence rate in pASC is not influenced by cryostorage.

A) pASCs senescence assay results for passages 5, 10 and pASCs passage 5 positive control (400 µM H2O2) respectively. Note the elevation in green-labeled cells in passage 10 vs. 5 (white head arrows); yellow arrows indicate senescent cells. B) Senescence quantification for passages 5 vs. P7 * = p<0.05; P5 vs. P8 ** = p<0.01; P5 vs. P9. 10 and positive controls *** = p<0.001. P5 vs. P6 p>0.05. No significant differences were observed between fresh and thawed cells in each analyzed passage (p>0.05). C) Linear regression showing that the mean number between thawed and fresh senescent cells increased gradually during Passages 5–10 (Pearson’s r: fresh = 0.9694. thawed = 0.9895; R squared: fresh = 0.9397. thawed = 0.9792; p<0.001; linear regression - p slope>0.5). D) Representative P5 and P10 karyotypes and metaphases. No chromosomal aberrations were observed in pASC after thawing (P5) or after long-term cultivation (P10).

Figure 5. Cryostorage does not influence the plasticity of pASCs.

A) pASCs adipogenesis induced by supplemented medium during 3 weeks in culture. B) pASCs osteogenesis induced by supplemented medium after 3 weeks in culture after Alizarin Red S staining.