235 Isolation and in vitro induction into pluripotency of adult camelid (Lama glama) fibroblasts

Camelids are ruminants with a wide social and economic application due to their adaptive capacity to adverse conditions and the variety of tasks to which they are submitted. The recent generation of induced pluripotent stem cells (iPSCs), through cellular in vitro reprogramming, has revolutionised animal science due to its contribution to regenerative medicine, the conservation and propagation of genetic material, and animal and food production by enabling the formation of specialised tissues such as cultured gametes; however, in vitro-induced cellular reprogramming is still not reported in camelids. In this study, adult Lama glama fibroblasts were isolated, characterised, and in vitro reprogrammed for further use in reproductive or regenerative biotechnologies. A skin biopsy was submitted to either explantation or enzymatic incubation and fibroblasts were obtained and maintained in vitro or cryopreserved until use. The cellular doubling time was analysed (72.5 h), and cells were maintained in culture (DMEM, 10% FBS, 1% pen/strep) or cryopreserved until use. The reprogramming process consisted of the lentiviral transduction overnight with human or murine OCT4, SOX2, KLF4 and c-MYC pluripotency-related transcription factors (hOSKM or mOSKM, respectively). Six days after the transduction, the cells were replated onto mitomycin-treated MEFs and cultured in iPSC media (DMEM/F12 knockout, 20% KSR, 1% GlutaMAX, 1% NEAA, 1% pen/strep, 3.85 uM β-mercaptoethanol supplemented with 10 ng/mL bFGF, replaced at every two days. Cellular aggregation resembling colony formation was observed ∼30 days after transduction. RT-qPCR was performed to evaluate the abundance of endogenous (OCT4, SOX2, and NANOG) and exogenous pluripotency factors (hOSKM or mOSKM groups) on Day 6 after the transduction, using GAPDH as a housekeeping gene, and non-transduced cells were used as controls. In both groups, the endogenous factors OCT4, SOX2, and NANOG were present and increased when compared to fibroblasts (P < 0.05), as well as presented expression of the exogenous factors, as expected. The colonies did not maintain typical morphology after passaging, suggesting that the reprogramming protocol still requires further optimisation in this species. These initial results show, for the first time, the endogenous re-activation of pluripotency genes in vitro in Lama glama after induced reprogramming, contributing to the improvement of the knowledge in pluripotency acquisition and maintenance in economically important, however non-trivial, farm animals, also opening up further possibilities for regenerative medicine and animal reproduction.

This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (#2015/26818-5, #2020/07921-8), CNPq, and Coordination for the Improvement of Higher Education Personnel (CAPES).