Generation of Directly Converted Human Osteoblasts That Are Free of Exogenous Gene and Xenogenic Protein

Abstract

Generation of osteoblasts from human somatic cells may be applicable in an effective transplantation therapy against bone diseases. Recently we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some transcription factor genes via retroviral vectors. However, retroviral vector-mediated transduction may potentially cause tumor formation from the infected cells, thus a non-viral gene transfection method may be more preferable for preparation of osteoblasts to be used for transplantation therapy. Here, we constructed a plasmid vector encoding Oct4, Osterix, and L-Myc that were an appropriate combination of transcription factors for this purpose. Osteoblast-like phenotypes including high alkaline phosphatase (ALP) activity, bone matrix production and osteoblast-specific gene expression were induced in normal human fibroblasts that were transfected with the plasmid followed by culturing in osteogenic medium. The plasmid-driven directly converted osteoblasts (p-dOBs) were obtained even in the absence of a xenogenic protein. The plasmid vector sequence had fallen out of the p-dOBs. The cells formed deposition of calcified bodies in situ after transplantation into mice. These results strongly suggest that p-dOBs can be put into practical use for a novel cell-based therapy against bone diseases. J. Cell. Biochem. 117: 2538-2545, 2016. © 2016 Wiley Periodicals, Inc.

Nanogel tectonic porous 3D scaffold for direct reprogramming fibroblasts into osteoblasts and bone regeneration

“…Direct reprogramming technologies enable phenotypic conversion of a somatic cell type into another without passing through an intermediate pluripotent state 10 – 13 . Recently we succeeded in reprogramming human fibroblasts into osteoblasts (directly converted osteoblasts; dOBs) by transducing four genes each encoding Runx2 (R), Osterix (X), Oct3/4 (O), and L-Myc (L), or three genes (X, O and L) 14 – 16 . As fibroblasts can be easily obtained from a small piece of biopsy sample of a patient without any invasive procedure 17 and expanded into an enough large number 18 , 19 , our procedure may be quite adequate for preparation of a large number of autologous osteoblasts with a high bone forming ability.…”

“…Therefore, we tried to combine the above two technologies, the nanogel tectonic material and dOBs, to generate a feasible tissue engineering procedure for a novel regeneration therapy of bone diseases. In the present study, we newly developed NanoCliP-FD gel from the NanoCliP gel that we previously reported 8 , and examined its potential as a scaffold for bone tissue engineering using the dOBs induced by our previously reported procedure 14 – 16 .…”

Reprogramming of human fibroblasts into osteoblasts by insulin-like growth factor-binding protein 7

“…Genetic approaches have successfully converted dermal fibroblasts into osteoblasts in several studies . For example, overexpression of the muscle master transcription factor MyoD in myoblasts can increase their sensitivity to bone morphogenetic protein (BMP‐2), thus enhancing BMP signaling to drive trans‐differentiation into OB.…”

“…Genetic approaches have successfully converted dermal fibroblasts into osteoblasts in several studies. [36][37][38][39] For example, overexpression of the muscle master transcription factor MyoD in F I G U R E 7 IGFBP7-induced osteoblastic reprogramming is dependent on Il-6. Fibroblasts were treated with IGFBP7 (1000 ng/mL) together with IgG control antibody (50 μg/mL) or IL-6 neutralizing antibody (5 μg/mL).…”

Direct Reprogramming of Mouse Fibroblasts into Functional Osteoblasts

“…Forced expression of Runx2, a master regulator of osteoblast differentiation, in non‐osteoblastic fibroblast cells and osteoblast‐like cells can increase osteogenic gene expression and mineralized nodule deposition . Recently, it was reported that forced expression of four transcription factors, Runt‐related transcription factor 2 (Runx2), Osterix (Osx), Octamer‐binding transcription factor 3/4 (Oct4), and L‐Myc, can directly convert human fibroblasts to functional osteoblasts as assayed by gene expression and mineralization . Other combinations, including Oct6, Oct9, or N‐myc, can also convert human fibroblasts to osteoblasts .…”