Generation of induced pluripotent stem cells (iPSCs) from adult camelid cells followed by differentiation into a muscle cell line

Abstract

Representatives of the Camelidae family of suborder Tylopoda, such as Alpacas, Guanacos, Vicunas, Llamas, Camels, Dromedaries, among others, have a strong presence in four (America, Asia, Africa, and Oceania) of the seven continents and are of great economic and social importance. due to the ability to adapt to adverse conditions, typical of the arid regions they inhabit, given their ability to submit to numerous tasks, such as transport, agricultural work, leisure, production of wool, meat, milk and their derivatives.The main objective of this study is to generate greater knowledge about the biology of development and new technologies in these animals, and in particular, aiming to contribute to possibilities in the future of species preservation, in view of the threat of extinction of some camelids in the wild, such as vicuñas (almost decimated by poachers) and camels. To this end, in view of the role of stem cells in promoting greater reproductive efficiency and the possibility of innovative therapies, such as regenerative and reproductive ones, in this study we sought, in an unprecedented way, the production and characterization of induced pluripotency stem cells (iPSC) , that is, reprogramming adult cells in vitro to a pluripotent state, similar to the embryo, in camelids and using part of these cells for in vitro differentiation into myocytes, an initiative that is also a pioneer for the species that brings numerous benefits to regenerative therapies for tissue replacement or repair , avoid immunological rejection, envisioning greater success of transplants, in addition to the possibility of testing new drugs and their toxicity, improvement of genetic bioengineering and greater understanding of the embryogenesis process.For this, we will use commercial fibroblasts of the species Camelus dromedarius, which will be isolated, cultured and, later, transduced with polycistronic lentiviral vectors OCT4, SOX2, KLF4 and c-MYC or murine (hOSKM or mOSKM). Positive strains for the expression of green fluorescent protein (GFP+) will also be generated, in order to obtain cells with long-lasting tracking, helping their tracking in later studies.The generated strains are expected to be positive for: alkaline phosphatase detection and immunocytochemistry for pluripotency proteins OCT4, SOX2 and NANOG, formation of embryoid bodies and in vitro differentiation into myocytes, followed by polymerase chain reaction (PCR) analysis for quantification of gene expression of pluripotency or differentiation targets and, finally, teratoma assay.The present proposal proves to be relevant both as a model for the study of diseases, development of new drugs and countless therapeutic actions and innovations, as well as for promoting advanced reproductive technologies, such as the possibility of generating embryos and gametes in vitro, representing an important role in the species preservation.