Abstract
In this study, we developed a new method to stimulate osteogenic differentiation in tissue-nonspecific alkaline phosphatase (TNAP)-positive cells liberated from human induced pluripotent stem cells (hiPSCs)-derived embryoid bodies (EBs) with 14 days long TGF-β/IGF-1/FGF-2 treatment. TNAP is a marker protein of osteolineage cells. We analyzed and isolated TNAP-positive and E-cadherin-negative nonepithelial cells by fluorescence-activated cell sorting. Treating the cells with a combination of transforming growth factor (TGF)-β, insulin-like growth factor (IGF)-1, and fibroblast growth factor (FGF)-2 for 14 days greatly enhanced TNAP expression and maximized expression frequency up to 77.3%. The isolated cells expressed high levels of osterix, which is an exclusive osteogenic marker. Culturing these TNAP-positive cells in osteoblast differentiation medium (OBM) led to the expression of runt-related transcription factor 2, type I collagen, bone sialoprotein, and osteocalcin (OCN). These cells responded to treatment with activated vitamin D3 by upregulating OCN. Furthermore, in OBM they were capable of generating many mineralized nodules with strong expression of receptor activator of NF-kappaB ligand and sclerostin (SOST). Real-time RT-PCR showed a significant increase in the expression of osteocyte marker genes, including SOST, neuropeptide Y, and reelin. Scanning electron microscopy showed dendritic morphology. Examination of semi-thin toluidine blue-stained sections showed many interconnected dendrites. Thus, TNAP-positive cells cultured in OBM may eventually become terminally differentiated osteocyte-like cells. In conclusion, treating hiPSCs-derived cells with a combination of TGF-β, IGF-1, and FGF-2 generated TNAP-positive cells at high frequency. These TNAP-positive cells had a high osteogenic potential and could terminally differentiate into osteocyte-like cells. The method described here may reveal new pathways of osteogenesis and provide a novel tool for regenerative medicine and drug development.
Highlights
The development of new treatment strategies for osteoporosis and other skeletal tissue diseases has become increasingly important considering the growing population of elderly people
tissue-nonspecific alkaline phosphatase (TNAP)-positive cells derived from human Induced pluripotent stem cells (iPSCs) We induced embryoid bodies (EBs) from hiPSCs and the trypsinized EBs
Parental hiPSCs and Alkaline phosphatase (ALP)-positive cells derived from hiPSCs by EB formation expressed the TNAP isoenzyme, whereas ALP-negative cells did not express any ALP isoenzyme (Fig. 2c)
Summary
The development of new treatment strategies for osteoporosis and other skeletal tissue diseases has become increasingly important considering the growing population of elderly people. Utilization of hMSCs for drug development is difficult because of their limited proliferative ability and the poor reproducibility of the method. These problems could be resolved using human iPSCs (hiPSCs). MSCs or MSC-like cells can be obtained from human ESCs by methods, such as fluorescence-activated cell sorting (FACS) after embryoid body (EB) formation. These protocols require prolonged serial passages or multiple cell sorting steps and are laborintensive, time-consuming, and generally inefficient [3]
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