carbocyanines and pyrazolanthrone

Dedifferentiation of cells to multipotential cells is of interest since they have a potential regenerative capacity. Our purpose was to de- and redifferentiate murine RAW 264.7 cells, a committed macrophage cell line of hematopoietic origin, into mesenchymal-like cells such as osteoblasts. RAW 264.7 cells in culture were treated with 5 μM reversine, a purine analog that was shown to dedifferentiate myoblasts in osteoblasts. Treatment with reversine resulted in a significant increase in the expression of the STRO-1 antigen, a marker of mesenchymal stem/progenitor cells: from 0.6%±0.5% cells in untreated RAW cells to 19.0%±8.6% in treated cells, but there was no increase in the expression of SH-2 (CD105), an earlier marker of mesenchymal stem cells. The effects of reversine were significantly curtailed by 67% when cultures were pretreated with the c-Jun N-terminal kinase pathway blocker SP600125. These STRO-1+ cells retained a multipotential status and were capable of redifferentiating into cells with osteogenic and lipogenic characteristics under inductive conditions. We showed that STRO-1+ cells in an osteogenic medium significantly increased expression of the osteoblast marker osteocalcin, and formed mineralized nodules. When seeded on a demineralized scaffold of human bone in vitro, these cells deposited a calcium matrix. Under adipogenic conditions, expression of the adipocyte marker peroxisome proliferator-activated receptor gamma 2 on STRO-1+ cells was elevated, and cultures stained positive with Oil red O. Our results demonstrated that treating a committed hematopoietic cell line with a purine analog can alter cell development and result in cellular reverse transformation into stage-limited multipotential cells. These cells could subsequently be redifferentiated into cells with characteristics of the mesenchymal lineage, such as those of an osteoblast and/or adipocyte, under inductive conditions.

Topics: Adipogenesis; Animals; Anthracenes; Antigens, Surface; Bone and Bones; Carbocyanines; Cell Dedifferentiation; Cell Line; Endoglin; Flow Cytometry; Humans; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Macrophages; Mesenchymal Stem Cells; Mice; Microscopy, Fluorescence; Morpholines; Protein Kinase Inhibitors; Purines; Rats; Reverse Transcriptase Polymerase Chain Reaction; Staining and Labeling; Tissue Scaffolds; X-Ray Microtomography