PLoS One. 2014 Jun 9;9(6):e99534.

A novel strategy for enrichment and isolation of osteoprogenitor cells from induced pluripotent stem cells based on surface marker combination.

Hiromi Ochiai-Shino1, Toshifumi Azuma1

1Department of Biochemistry, Tokyo Dental College, Tokyo, Japan

 

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 transforming growth factor (TGF)-β/insulin-like growth factor (IGF)-1/fibroblast growth factor (FGF)-2 treatment for 14 days. Treating the cells with these factors greatly enhanced TNAP expression and maximized expression frequency up to 77.3%. TNAP is a marker protein of osteolineage cells. We isolated TNAP-positive and E-cadherin-negative nonepithelial cells by fluorescence-activated cell sorting. 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 (RUNX2), type I collagen, bone sialoprotein (BSP), and osteocalcin (OCN). These cells responded to treatment with activated vitamin D3 by upregulating OCN. Furthermore, they were capable of generating many mineralized nodules with strong expression of many osteocyte markers, such as receptor activator of NF-kappaB ligand (RANKL), sclerostin (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 induced 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.

PMID: 24911063

 

Supplement

iPSCs are powerful tools in many fields of basic scientific research. Several reports have shown that osteogenic cells can be generated from iPSCs. However, the osteogenic differentiation of hiPSCs presents numerous problems, including time-consuming methods, poor reproducibility, and low efficiency. In this study, we developed a new strategy to purify osteoprogenitors from EB-derived cells by isolating tissue-nonspecific alkaline phosphatase (TNAP)-positive cells using fluorescence-activated cell sorting (FACS).

Alkaline phosphatase (ALP) as well as type I collagen is the most essential protein for mineralization by osteolineage cells. Human have four ALP genes encoding intestinal, placental, placenta-like, and liver/bone/kidney (i.e., TNAP) gene products. TNAP in osteolineage cells is expressed relatively early during differentiation and is abundantly expressed on the membrane surface. Apart from osteolineage cells, epithelial cells also express TNAP. Therefore, purification of osteolineage cells among TNAP-positive cells will probably require elimination of epithelial cells. In addition to isolate TNAP-positive cells, we isolated E-cadherin-negative and CD90-positive cells. These cells were not epithelial cells and morphologically fibroblastic and spindle shaped rather than cuboidal or epithelial shaped.

WBF Figure 2

Figure: Protocol for differentiation of hiPSCs into osteoblast-like cells and FACS analysis of the differentiated hiPSCs.

 

Next, We investigated methods of maximizing TNAP expression. We attempted several combinations of cytokines and found that activins, retinoic acid, and BMPs did not effectively induce TNAP expression. However, treating the cells with combination of TGF-β, IGF-1, and FGF-2 greatly enhanced TNAP expression. Furthermore, TNAP-positive cells began to express high levels of osterix (OSX), which is an exclusive osteogenic marker. The cells initially expressed low levels of RUNX2, and continuous culture induced high levels of RUNX2, BSP, type I collagen, and eventually, OCN. To the best of our knowledge, these are the first observations of osteoprogenitors expressing high levels of TNAP and OSX but low levels of RUNX2 and type I collagen. Because epigenetic conditions during embryonic development are quite different from those during ESC/iPSC differentiation, the transcription factors required for TNAP expression may be different. In embryos, Runx2 is required for the differentiation of prechondrogenic mesenchymal cells into osteoblasts, whereas Osx is believed to induce subsequent maturation of osteoblasts and inhibit chondrogenic differentiation. In Osx-null embryos, cartilage forms normally but the embryos completely lack bone. OSX, which is specifically and exclusively expressed in all osteoblasts, showed markedly high expression in TNAP-positive cells, although TNAP-positive and -negative cells expressed almost similar levels of RUNX2. These findings indicated that iPSCs may not require the prechondrogenic process and may induce OSX without a RUNX2 surge.

We found that TNAP-positive cells derived from hiPSCs formed several bone nodules that contained intensely anti-RANKL, and anti-SOST-immunopositive cells. The expression of other osteocyte marker genes were increased significantly. SEM showed that TNAP-positive cells were flattened with multiple dendritic morphologies after long cultivation in OBM. These findings suggested that they could terminally differentiate into osteocyte-like cells.

In conclusion, we found that human iPSCs have distinct signaling pathway to differentiate into osteolineage cells. We believe that single step preparation of TNAP-positive and E-cadherin-negative cells is a powerful tool for studying developmental process of bone tissues.

 

 

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann