Gene expression profile in human induced pluripotent stem cells: Chondrogenic differentiation in vitro, part B

Abstract

The development of human induced pluripotent stem cells (hiPSCs) is considered a turning point in tissue engineering. However, more data are required to improve understanding of key aspects of the cell differentiation process, including how specific chondrogenic processes affect the gene expression profile of chondrocyte-like cells and the relative value of cell differentiation markers. The main aims of the present study were as follows: To determine the gene expression profile of chondrogenic-like cells derived from hiPSCs cultured in mediums conditioned with HC-402-05a cells or supplemented with transforming growth factor β3 (TGF-β3), and to assess the relative utility of the most commonly-used chondrogenic markers as indicators of cell differentiation. These issues are relevant with regard to the use of human fibroblasts in the reprogramming process to obtain hiPSCs. Human fibroblasts are derived from mesoderm and thus share a wide range of properties with chondrocytes, which originate from the mesenchyme. The hiPSCs were obtained from human primary dermal fibroblasts during a reprogramming process. Two methods, both involving embryoid bodies (EB), were used to obtain chondrocytes from the hiPSCs: EBs formed in the presence of a chondrogenic medium with TGF-β3 (10 ng/ml) and EBs formed in a medium conditioned with growth factors from HC-402-05a cells. Based on reverse transcription-quantitative polymerase chain reaction analysis, the results demonstrated that hiPSCs are capable of effective chondrogenic differentiation, with the cells obtained in the HC-402-05a medium presenting with morphological features and markers characteristic of mature human chondrocytes. In contrast, cells differentiated in the presence of TGF-β3 presented with certain undesirable hypertrophic characteristics. Several genes, most notably runt-related transcription factor 2, transforming growth factor β2 and transforming growth factor β3, were good markers of advanced and late hiPSC chondrogenic differentiation, whereas transforming growth factor β3I, II, III receptors and bone morphogenetic protein-2, bone morphogenetic protein-4 and growth differentiation factor 5 were less valuable. These findings provide valuable data on the use of stem cells in cartilage tissue regeneration.

Gene expression profile in human induced pluripotent stem cells: Chondrogenic differentiation in vitro, part A

“…This phenomenon is associated with the transition from chondrogenic-like cells into hypertrophic chondrogenic-like cells, rather than cells from early chondrogenesis. This observation is confirmed by the high expression of RUNX2 ( 16 ), which is associated with IGF-1 via the P13K/Akt signaling pathway.…”

“…7 ). In addition, these cells did not express hypertrophic markers, which include RUNX2 ( 16 ). In contrast, the cells differentiated in the TGF-β3 medium expressed low levels of SOX9 and high levels of RUNX2 , an observation that suggests these cells underwent hypertrophy.…”

“…PAX9 is required to induce the chondrogenic process, and PAX9 expression was demonstrated to be associated with IHH expression. The presence of PAX9 mRNA was visible in cells differentiated in the conditioned medium, and these cells also exhibited a high level of IHH expression ( 16 ). In cells differentiated in the TGF-β3 medium, expression of IHH was lower ( 16 ) and PAX9 expression was lower compared with cells cultured in the conditioned medium.…”

“…The presence of PAX9 mRNA was visible in cells differentiated in the conditioned medium, and these cells also exhibited a high level of IHH expression ( 16 ). In cells differentiated in the TGF-β3 medium, expression of IHH was lower ( 16 ) and PAX9 expression was lower compared with cells cultured in the conditioned medium. This observation indicated that these cells originated from late stages of chondrogenesis, during which PAX9 expression is downregulated ( Fig.…”

“…Part A, presented here, describes the markers that are characteristic for pluripotency state and early-stage chondrogenesis ( Table I ). The second part of the study ( 16 ) focused on markers that are characteristic of late stage chondrogenesis, hypertrophy and ossification.…”