Work

Derivation of Equine Mesenchymal Stem Cells (msc) From Induced Pluripotentstem Cells (ipsc): a Promising Step for Veterinary and Translational Regenerative Medicine

Year: 2024

Type: article

Abstract: Horses frequently suffer from similar diseases to humans, like osteoarthritis or asthma, constituting an important veterinary patient and a relevant model owed to the physiopathological analogies between both species. Mesenchymal stem cells (MSCs) are very promising to treat several disorders, but enter senescence after several subculture steps, requiring repeated tissue harvesting that increases product heterogeneity. Using induced pluripotent stem cells (iPSCs) to derive MSCs (iMSCs) can increase their availability, reduce donor-associated variability and diminish the need for invasive and repeated tissue harvesting. While human iMSCs are being intensively studied, only two equine studies have reported the generation of iMSCs to date. To extend iMSC advantages to the veterinary field, we aimed at obtaining equine iMSCs (eiMSCs) and comparing their main characterization criteria over primary equine MSCs. Three protocols were tested on already established eiPSC lines: the two only protocols described in horses, both based on spontaneous differentiation, and a directed differentiation protocol via neural crest pathway adapted from human iPSCs. The eiMSCs obtained were characterised over equine bone marrow (BM)-MSCs by immunophenotype, trilineage differentiation and gene expression. The two spontaneous protocols previously described did not work in our eiPSCs: morphology changed but cells did not attain the MSC typical spindle-shape but a polygonal shape, with poor plastic adherence. Only the directed protocol was successful and 3 lines of eiMSCs, from 3 different eiPSC lines, were obtained, all presenting the morphology and plastic adherence typical of MSCs. We observed immunophenotype differences between eiMSCs and BM-MSCs, but inter-donor variability was also observed in the latter. Moreover, some differences were noted for the trilineage differentiation: while adipogenesis was complete and similar in both MSC types, osteogenic and chondrogeneic potentials were lower for eiMSCs. Gene expression analysis supported these observations with few differences, and confirmed the downregulation of pluripotency genes over eiPSCs. In conclusion, we demonstrated that eiMSCs can be derived from eiPSCs, constituting an interesting alternative to primary MSCs. More studies are needed to further characterize eiMSC and to examine their function over primary MSCs, especially their immunomodulatory and immunogenic profiles, and to determine their safety and efficacy in vivo.

Source: Zenodo (CERN European Organization for Nuclear Research)

DOI: 10.5281/zenodo.13753053