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: 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. DOI:10.4274/Jcrpe.1284
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ABSTRACT: Previous studies have shown that peripheral blood monocytes can be converted in vitro to a stem cell-like cell termed PCMO as evidenced by the re-expression of pluripotency-associated genes, transient proliferation, and the ability to adopt the phenotype of hepatocytes and insulin-producing cells upon tissue-specific differentiation. However, the regulatory interactions between cultured cells governing pluripotency and mitotic activity have remained elusive. Here we asked whether activin(s) and TGF-β(s), are involved in PCMO generation. De novo proliferation of PCMO was higher under adherent vs. suspended culture conditions as revealed by the appearance of a subset of Ki67-positive monocytes and correlated with down-regulation of p21WAF1 beyond day 2 of culture. Realtime-PCR analysis showed that PCMO express ActRIIA, ALK4, TβRII, ALK5 as well as TGF-β1 and the βA subunit of activin. Interestingly, expression of ActRIIA and ALK4, and activin A levels in the culture supernatants increased until day 4 of culture, while levels of total and active TGF-β1 strongly declined. PCMO responded to both growth factors in an autocrine fashion with intracellular signaling as evidenced by a rise in the levels of phospho-Smad2 and a drop in those of phospho-Smad3. Stimulation of PCMO with recombinant activins (A, B, AB) and TGF-β1 induced phosphorylation of Smad2 but not Smad3. Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation. Inhibition of autocrine TGF-β signaling by either SB431542 or anti-TGF-β antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF-β antibody moderately enhanced Oct4A/Nanog expression. Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-β/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-β/Smad3, and to a lesser extent, activin/Smad2 signaling.PLoS ONE 02/2015; 10(2):e0118097. DOI:10.1371/journal.pone.0118097 · 3.53 Impact Factor
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ABSTRACT: Impaired bone healing can occur with numerous pathologic conditions like trauma, osteoporosis, and infection. Therefore tissue-engineering strategies that aim to enhance osteogenic differentiation of stem cells in order to accelerate bone healing are a major goal of contemporary regenerative research. In this study we cultivated mesenchymal stem cells (MSC) together with the recently patented programmable cells of monocytic origin (PCMO) to test whether co-cultures promote an osteogenic differentiation process. PCMO have recently been shown to have pluripotent characteristics and do support the regeneration processes of liver and heart diseases. Quantitative real time PCR expression profiles of osteogenic marker genes such as alkaline phosphatase in co-cultures of PCMO and MSC showed that MSC differentiated into osteoblast-like cells more rapidly as compared to mono-cultures. Alkaline phosphatase expression and enzyme activity levels were highly increased in co-cultures compared to mono-cultures of MSC. Tests for mineralized matrix formation also indicated that PCMO have a positive effect on co-cultured MSC under osteogenic culture conditions. However, analysis of collagen 1A did not show enhanced expression. In summary, PCMO obviously have the ability to promote osteogenic differentiation of MSC in vitro while their own pluripotent potential is not sufficient to develop osteoblast-like characteristics themselves. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop ResJournal of Orthopaedic Research 10/2014; 32(10). DOI:10.1002/jor.22663 · 2.97 Impact Factor