Direct Reprogramming of Mouse Fibroblasts into Functional Osteoblasts

Abstract

Although induced pluripotent stem cells hold promise as a potential source of osteoblasts for skeletal regeneration, the induction of pluripotency followed by directed differentiation into osteoblasts is time consuming and low yield. In contrast, direct lineage reprogramming without an intervening stem/progenitor cell stage would be a more efficient approach to generate osteoblasts. We screened combinations of osteogenic transcription factors and identified four factors, Runx2, Osx, Dlx5, and ATF4, that rapidly and efficiently reprogram mouse fibroblasts derived from 2.3 kb type I collagen promoter‐driven green fluorescent protein (Col2.3GFP) transgenic mice into induced osteoblast cells (iOBs). iOBs exhibit osteoblast morphology, form mineralized nodules, and express Col2.3GFP and gene markers of osteoblast differentiation. The global transcriptome profiles validated that iOBs resemble primary osteoblasts. Genomewide DNA methylation analysis demonstrates that within differentially methylated loci, the methylation status of iOBs more closely resembles primary osteoblasts than mouse fibroblasts. We further demonstrate that Col2.3GFP+ iOBs have transcriptome profiles similar to GFP+ cells harvested from Col2.3GFP mouse bone chips. Functionally, Col2.3GFP+ iOBs form mineralized bone structures after subcutaneous implantation in immunodeficient mice and contribute to bone healing in a tibia bone fracture model. These findings provide an approach to derive and study osteoblasts for skeletal regeneration. © 2019 American Society for Bone and Mineral Research.

Collagen Niches Affect Direct Transcriptional Conversion toward Human Nucleus Pulposus Cells via Actomyosin Contractility

“…[ 29 ] The results presented that the expression of CD24, GD2, and TIE2 was no significantly expression in iNPLCs, compared with hNPPCs (Figure 1i). Meanwhile, GOCircle, a gene ontology analysis to reveal the functional pathway change between different cells, [ 30 ] also showed that Wnt pathway, Shh pathway, and transforming growth factor β pathway of iNPLCs were significantly increased than ones of hDFs, which were closely related with hNPCs lineage development (Figure 1j). These findings revealed that hDFs were directly converted into iNPLCs without the state of progenitor cells.…”

Direct Reprogramming of Fibroblasts to Osteoblasts: Techniques and Methodologies

“…Transcription factors play a crucial role in guiding a source cell toward a desired target phenotype, and selecting them necessitates a comprehensive understanding of the factors significantly involved in the developmental lineage of the target cell. 10 , 23 By overexpressing transcription factors, it becomes possible to reconfigure gene regulating networks, thereby modifying the identity of the cells ( Fig. 2 ).…”

“…2 ). 23 Dlx5 enhances Runx2 expression and regulates osteogenic genes, while ATF4 regulates osteocalcin and osteopontin, crucial in osteoblast differentiation and bone formation. 23 …”

“…1B ). 10 , 22 , 23 DR eliminates the potential hazard of introducing random DNA mutations into the targeted cell type which is a potential issue when starting with iPSCs. Cells that have been directly reprogrammed ex vivo can then be transplanted in a scaffold to kickstart the ossification process using the body’s own cells 22 ( Fig.…”

Direct cellular reprogramming and transdifferentiation of fibroblasts on wound healing—Fantasy or reality?

“…Therefore, an alternative source of cells that can be used as osteoblasts is desired. The ability of fibroblasts to undergo differentiation into osteoblasts has been demonstrated by some researchers 22–26 . Rheumatoid arthritis synovial fibroblasts travel from cartilage destruction sites to new locations where they reinitiate the destructive processes at distant articular cartilage surfaces and affect the function of joints 27 .…”