Pluripotency potential of equine mesenchymal cells obtained from different sources subjected to genetically induced reprogramming (iPS cells)

Author(s)

Laís Vicari de Figueiredo Pessôa

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2016-12-20

Examining board members

Flavio Vieira Meirelles; Carlos Eduardo Ambrosio; Virgínia Picanço e Castro; Heidge Fukumasu; Tathiane Maistro Malta Pereira

• Advisor: Flavio Vieira Meirelles; Fabiana Fernandes Bressan

Abstract

The insertion of known transcription factors into somatic cells is capable of reprogramming them into a pluripotency stage, generating induced pluripotent stem cells (iPS). The influence of the origin of the somatic cell used in the generation of iPS is currently discussed, and the use of these cells in regenerative medicine has great potential in both human and veterinary medicine. Domestic mammals, such as the equine are great models for the study of human and veterinary medicine, highlighting cell therapy using mesenchymal cells in orthopedic and articular injuries. Hypothesizing that equine cells derived from different tissues show variable pluripotency induction potentials and in vitro differentiation capacity, depending on the source of derivation; this proposal aims to generate equine iPS cells obtained by viral transduction of human or murine OSKM transcription factors in adult fibroblasts (eFibro) and mesenchymal cells derived from adipose tissue (eAdMSC), bone marrow(eMO), umbilical cord tissue (etCU). Four umbilical cord tissue cell lines, three equine fibroblast cells lines, three equine adipose tissue mesenchymal cell lines and 2 equine bone marrow cell lines were isolated and cultured. These cells had their doubling time calculated in hours (eMO 61.3 ± 15, etCU 53.8 ± 5, eFIBRO 27 ± 2, and ADMSC 18.2 ± 4.5) and were characterized by chondrogenic, osteogenic and adipogenic and by immunophenotyping. From these cells, 85 equine iPS (eiPS) cell lines were produced (30 fibroblast-derived cell lines, 33 cell lines derived from adipose tissue cells, 21 cell lines derived from umbilical cord tissue cells, with human factors and 1 cell line derived from eAdMSC using murine factors). Alkaline phosphatase and OCT4 detection tests were performed on eiPS cells obtained from each cell line and eAdMSC and eFibro derived iPS cells were positive for the detection of NANOG,TRA1-60, TRA1-81 and eiPS derived from eAdMSC were positive for SSEA-1 detection. Embryonic bodies, which were later dissociated for spontaneous differentiation test in vitro were derived from each cell type. Results show that equine cells can be more easily reprogrammed with human factors when compared to murine factors (P<0.05). While murine factors produced only one eiPS cell line derived from eAdMSC, human reprogramming factors were able to produce multiple eiPS cell lines, and colony formation was more efficient for cells derived from eAdMSC (322 P <0.01) than for cells derived from eFibro (65) and etCU (58), which did not differ (P = 0.95) and It has not yet been possible to produce iPS cells from the eMO cell lines. Using the induction system standardized in our laboratory, the use of human factors in direct reprogramming results in greater efficiency in the production of equine iPS cells when compared to murine factors. To our knowledge, this is the first report of eiPS cells produced solely dependent on bFGF, without the need for addition of LIF in the culture medium. (AU)

• FAPESP's process: 13/17955-3 - Study of the pluripotency potential of equine stem cells derived from adult tissue and umbilical cord, submitted to genetically induced reprogramming (IPS cells)
• Grantee: Laís Vicari de Figueiredo Pessôa
• Support Opportunities: Scholarships in Brazil - Doctorate