Generation of Mesenchymal Progenitors From Human Gingiva-derived iPS Cells

Objectives: Regenerative therapy, using mesenchymal progenitors (MPs)-derived embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, is an effective therapeutic approach for the treatment of diseases and lost tissues. Our first approach involves the establishment of iPS cells derived from human gingiva tissues, which is discarded during general dental treatments. Therefore, the objectives of the study were to demonstrate an efficient method for generating MPs from iPS cells, and to characterize its differentiation into osteoblasts, chondrocytes, and adipocytes.
Methods: Human gingiva fibroblasts were transfected with episomal vectors by electroporation. The established iPS cells were characterized by immunocytochemistry, gene analysis, and embryoid body formation. These iPS cells were differentiated and inducted without feeder; the cells were passaged three times, and sorted with CD24- CD73+ CD105+ for MP generation. Furthermore, the generated MPs were differentiated into osteoblasts, chondrocytes and adipocytes.
Results: The established iPS cells exhibited ES cell-like morphology and proliferated similar to ES cells. By immunostaining and quantitative reverse transcription polymerase chain reaction (qRT-PCR), iPS cells were observed as expression similar to the genes and proteins of ES cell-specific markers. Embryoid bodies were formed in order to investigate the three germ layers differentiation capacity of iPS cells in vitro. The Karyotest on established cells showed normal results. The generated MPs were able to passage for 15 times and were observed as the expression of mesenchymal stem cells (MSC) markers using flow cytometric analysis. MPs were confirmed for differentiation capacity of osteoblasts, chondrocytes and adipocytes by qRT-PCR, alizarin red staining, oil red staining, and toluidine blue staining.
Conclusions: From the above data, we established iPS cells using episomal vectors from human gingival fibroblasts. The MPs generated from iPS cells were capable of differentiation, such as MSC, and proliferating at a relatively higher rate than that of MSCs. MPs may be efficient cell sources for regenerative therapy.