222 GENERATION OF INDUCED PLURIPOTENT STEM CELL-LIKE CELLS FROM RAT EAR SKIN CELLS

Abstract

Generation of induced pluripotent stem cells (iPSC) present not only an opportunity to use the autologous cells clinically but also as cellular models for studying molecular mechanisms of various diseases and screening of drugs. The objective of the present study was to generate iPSC-like cells from rat ear skin cells by evaluating the expression of alkaline phosphatase (AP) activity, detection of markers specific to embryonic stem cells (ESC) and in vitro differentiation into selected cell lineages. Ear skin cells were isolated from 6-week-old s.d. rats and cultured in advanced-DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO₂ in air. Cells were reprogrammed with retroviral factors, namely OCT4, SOX2, KLF4 and c-Myc, the combination commonly used to generate mouse and human iPSCs. These cells were cultured in media specific to ESC on mitomycin treated mouse embryonic fibroblasts at 37°C in a humidified atmosphere of 5% CO₂ in air. AP activity was assessed with Western Blue^® kit (Promega, Madison, WI, USA). Expressions of ESC specific markers, such as OCT4, NANOG, SSEA-1, SSEA-4 and REX1 were evaluated by reverse transcriptase-PCR (RT-PCR). Reprogrammed iPSC-like cells were differentiated into adipogenic, osteogenic and neurogenic lineages by following previously described protocols. Cytochemical staining was performed to confirm the adipogenesis and osteogenesis. Further, the lineage specific marker genes in adipocytes (PPARγ2, adiponectin and aP2), osteocytes (osteocalcin, osteonectin, osteopontin and Runx2) and neuronal cells (nestin, neurogenin-1, β-tubulin and nerve growth factor) were evaluated by RT-PCR. Following transduction, ear skin cells were successfully reprogrammed into iPSC-like cells. Generated iPSC-like cells were positive for AP activity and clearly expressed the markers of ESC, including OCT4, NANOG, SSEA-1, SSEA-4 and REX1. After induction, iPSC-like cells were capable of differentiating in vitro into adipocytes as indicated by Oil red O staining and expressed the specific markers, such as PPARγ2, adiponectin and aP2. Differentiation of iPSC-like cells into osteocytes was evidenced by von Kossa staining and the detection of marker genes, osteocalcin, osteonectin, osteopontin and Runx2. Furthermore, upon neuronal specific induction, iPSC-like cells were able to form neuronal cells as demonstrated by the expression of neuronal specific markers, such as nestin, neurogenin-1, β-tubulin and nerve growth factor. In conclusion, our results showed the successful generation of iPSC-like cells from rat ear skin cells by exhibiting similar features of ESCs and demonstrated their ability to differentiate in vitro into adipocytes, osteocytes and neuronal cells. Further studies are being carried out to characterise and demonstrate the pluripotency capacity of generated iPS-like cells in a rat model.

This work was supported by Grant No. 2007031034040 from Bio-organ and Grant No. 200908FHT010204005 from Biogreen21.