The generation of programmable cells of monocytic origin involves partial repression of monocyte/macrophage markers and reactivation of pluripotency genes.
ABSTRACT We have recently demonstrated that peripheral blood monocytes can be differentiated in vitro into hepatocyte-like cells using appropriate differentiation media. Phenotype conversion required prior in vitro culture in the presence of M-CSF, IL-3, and human serum, during which the cells acquired a state of plasticity, so were termed "programmable cells of monocytic origin" (PCMO). Here, we have further characterized the process of PCMO generation with respect to markers of monocyte-to-macrophage transition and pluripotency. During a 6-day culture period, various monocyte/macrophage differentiation markers were down-regulated being indicative of a process of partial dedifferentiation. Dedifferentiation and hepatic redifferentiation also proceeded in highly purified monocyte preparations, albeit with different kinetics, suggesting that the presence of nonmonocytes, or soluble factors derived from them, is not essential in order for monocytes to acquire a multipotent state. PCMOs expressed various markers of human embryonic stem cells with early induction of NANOG and OCT4. Expression of the pluripotency-associated OCT4A isoform was paralleled by a global rise in histone H3 methylation on Lys-4, a marker of active chromatin, and coincided with peak sensitivity to tissue-specific differentiation. These results show that peripheral blood monocytes can be induced in vitro to transiently acquire stem cell-like properties and concomitantly a state of increased differentiation potential toward the hepatocytic phenotype.
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ABSTRACT: Hepatocyte-like cells (NeoHepatocytes) generated from a peripheral blood monocyte-derived stem cell-like cell (the PCMO) are a promising alternative for primary hepatocytes in cell transplantation studies to cure liver diseases. However, to be therapeutically effective NeoHepatocytes are needed in large quantities. It was the aim of the present study to investigate i) whether the proportion of actively proliferating NeoHepatocytes can be enhanced by supplementing the PCMO differentiation medium (containing M-CSF, IL-3, and human serum) with either EGF or HB-EGF and ii) which signaling pathway underlies the promitotic effect. EGF and HB-EGF enhanced cell proliferation of PCMOs as demonstrated by increased expression of cycle control genes (ABL, ANAPC2, CDC2, CDK4, CDK6), phosphorylation of the retinoblastoma protein, and increased PCMO cell numbers after stimulation with EGF or HB-EGF. EGF also raised the number of monocytes expressing the proliferation marker Ki67. PCMOs expressed the EGF receptors EGFR (ERBB1) and ERBB3, and expression of both increased during PCMO generation. Phosphoimmunoblotting of PCMOs indicated that both EGF and HB-EGF activated MEK-1/2 and ERK1/2 in a concentration-dependent fashion with the effect of EGF being more prominent. EGF treatment further decreased expression of p47phox and increased that of Nanog indicating enhanced dedifferentiation and pluripotency, respectively. Treatment with both EGF and HB-EGF resulted in NeoHepatocytes with improved functional parameters. The results suggested that the addition of EGF or HB-EGF to PCMO differentiation medium superactivates MEK/ERK signaling which then increases both PCMO proliferation, number, and functional differentiation of PCMO-derived NeoHepatocytes.Cell Communication and Signaling 08/2012; 10(1):23. · 5.09 Impact Factor
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ABSTRACT: 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.Tissue Engineering Part A 04/2012; 18(17-18):1890-901. · 4.64 Impact Factor
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ABSTRACT: Objective: The vision of potential autologous cell therapy for the cure of diabetes encourages ongoing research. According to a previously published protocol for the generation of insulin-producing cells from human monocytes, we analyzed whether the addition of growth factors could increase insulin production. This protocol was then transferred to a non-human primate model by using either blood- or spleen-derived monocytes. Methods: Human monocytes were treated to dedifferentiate into programmable cells of monocytic origin (PCMO). In addition to the published protocol, PCMOs were then treated with either activin A, betacellulin, exendin 3 or 4. Cells were characterized by protein expression of insulin, Pdx-1, C-peptide and Glut-2. After identifying the optimal protocol, monocytes from baboon blood were isolated and the procedure was repeated. Spleen monocytes following splenectomy of a live baboon were differentiated and analyzed in the same manner and calculated in number and volume. Results: Insulin content of human cells was highest when cells were treated with activin A and their insulin content was 13 000 µU/1 million cells. Insulin-producing cells form primate monocytes could successfully be generated despite using human growth factors and serum. Expression of insulin, Pdx-1, C-peptide and Glut-2 was comparable to that of human neo-islets. Total insulin content of activin A-treated baboon monocytes was 16 000 µU/1 million cells. Conclusion: We were able to show that insulin-producing cells can be generated from baboon monocytes with human growth factors. The amount generated from one spleen could be enough to cure a baboon from experimentally induced diabetes in an autologous cell transplant setting.Journal of Clinical Research in Pediatric Endocrinology 06/2014; 6(2):93-9.