A Novel Strategy for Enrichment and Isolation of Osteoprogenitor Cells from Induced Pluripotent Stem Cells Based on Surface Marker Combination

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.

Fibromodulin reprogrammed cells: A novel cell source for bone regeneration

“…Fibroblast growth factor (FGF)2 ( aka . basic fibroblast growth factor) also plays an essential role in promoting the conversion of uncommitted pluripotent/multipotent cells to osteochondroprogenitors and the subsequent osteogenic differentiation via multiple pathways [28, 39–45]. Transcription of FGF2 was markedly reduced in week 2 when BJ-fibroblasts were cultured in the osteogenic medium (Supplementary Fig.…”

AML-induced osteogenic differentiation in mesenchymal stromal cells supports leukemia growth

“…Flow cytometry revealed that TNAP (clone W8B2), known to be expressed on osteoprogenitor cells (20), mature osteoblasts, and naive MSCs (21), was significantly upregulated in AML-MSCs compared with N-MSCs ( Figure 1A). In the cohort of primary MSC samples isolated from AML patients with different disease status (newly diagnosed or in remission or relapsed; n = 29), the average mean fluorescence intensity (MFI) of TNAP was approximately 10-fold higher than that in N-MSCs (n = 11; Figure 1B, P < 0.01).…”

Induced Pluripotent Stem Cells in Dental and Nondental Tissue Regeneration: A Review of an Unexploited Potential

“…In order to attain iPSC osteogenic commitment, various inductive factors were applied including chemical inducers, biomolecules [101][102][103], growth factors [100], gene modification [104], two-dimensional culture environment [105], and modified three-dimensional scaffolds [100,101,[106][107][108]. Tissue-nonspecific alkaline phosphatase (TNAP) was demonstrated to influence the osteogenic differentiation potential of iPSCs, where TNAP-positive cells isolated from human EBs derived from iPSCs and cultured in osteogenic media expressed high levels of OSX, RUNX2, COL1A1, BSP, and OCN as well as generated mineralized nodules and revealed a significant expression of osteocyte marker genes, including sclerostin, neuropeptide Y, and reelin [109]. Similarly, extremely low-frequency electromagnetic field (ELF-EMF) (50 Hz and 1.5 mT) also significantly improved the osteogenic potential of iPSCs [110].…”