title: Pluripotency gene expression and growth control in cultures of peripheral blood monocytes during their conversion into programmable cells of monocytic origin (PCMO): evidence for a regulatory role of autocrine activin and TGF-β.
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.
authors.affiliation: Clinic for Applied Cellular Medicine, UKSH, Kiel, Germany.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Ungefroren
authors.affiliation: Clinic for Applied Cellular Medicine, UKSH, Kiel, Germany.
authors.forename: Ayman
authors.initials: A
authors.lastname: Hyder
authors.affiliation: Clinic for Applied Cellular Medicine, UKSH, Kiel, Germany.
authors.forename: Hebke
authors.initials: H
authors.lastname: Hinz
authors.affiliation: Clinic for Applied Cellular Medicine, UKSH, Kiel, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Groth
authors.affiliation: Clinic for Applied Cellular Medicine, UKSH, Kiel, Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Lange
authors.affiliation: Clinic for Conservative Dentistry and Periodontology, School of Dental Medicine, Kiel, Germany.
authors.forename: Karim M Fawzy
authors.initials: KM
authors.lastname: El-Sayed
authors.affiliation: Siegfried Weller Institute for Trauma Research, BG Trauma Center, Eberhard Karls University Tübingen, Tübingen, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Ehnert
authors.affiliation: Siegfried Weller Institute for Trauma Research, BG Trauma Center, Eberhard Karls University Tübingen, Tübingen, Germany.
authors.forename: Andreas K
authors.initials: AK
authors.lastname: Nüssler
authors.affiliation: Clinic for Applied Cellular Medicine, UKSH, Kiel, Germany.
authors.forename: Fred
authors.initials: F
authors.lastname: Fändrich
authors.affiliation: First Department of Medicine, UKSH, Lübeck, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Gieseler
mesh_terms: D030201:Activin Receptors, Type I / Q000378:metabolism; D030301:Activin Receptors, Type II / Q000378:metabolism; D028341:Activins / Q000378:metabolism*; D019898:Autocrine Communication / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D015536:Down-Regulation / Q000502:physiology; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D009000:Monocytes / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D015854:Up-Regulation / Q000502:physiology
chemical_list: D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; C422405:activin A; D028341:Activins; D017346:Protein Serine-Threonine Kinases; C498932:ACVR1B protein, human; D030201:Activin Receptors, Type I; D030301:Activin Receptors, Type II; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; C000625841:TGFBR1 protein, human; C425585:activin receptor type II-A