Clinic for Applied Cellular Medicine

Kiel, Germany

Clinic for Applied Cellular Medicine

Kiel, Germany

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Ungefroren H.,Clinic for Applied Cellular Medicine | Groth S.,Clinic for Applied Cellular Medicine | Hyder A.,Clinic for Applied Cellular Medicine | Thomsen N.,Clinic for Applied Cellular Medicine | And 6 more authors.
Stem Cells and Development | Year: 2010

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. © Copyright 2010, Mary Ann Liebert, Inc.


Ungefroren H.,UKSH | Hyder A.,Clinic for Applied Cellular Medicine | Hyder A.,Damietta University | Schulze M.,University of Duisburg - Essen | And 5 more authors.
Stem Cells International | Year: 2016

Adult stem or programmable cells hold great promise in diseases in which damaged or nonfunctional cells need to be replaced. We have recently demonstrated that peripheral blood monocytes can be differentiated in vitro into cells resembling specialized cell types like hepatocytes and pancreatic beta cells. During phenotypic conversion, the monocytes downregulate monocyte/macrophage differentiation markers, being indicative of partial dedifferentiation, and are partially reprogrammed to acquire a state of plasticity along with expression of various markers of pluripotency and resumption of mitosis. Upregulation of stem cell markers and mitotic activity in the cultures was shown to be controlled by autocrine production/secretion of activin A and transforming growth factor-beta (TGF-β). These reprogrammed monocyte derivatives were termed "programmable cells of monocytic origin" (PCMO). Current efforts focus on establishing culture conditions that increase both the plasticity and proliferation potential of PCMO in order to be able to generate large amounts of blood-derived cells suitable for both autologous and allogeneic therapies. © 2016 Hendrik Ungefroren et al.


PubMed | University of Tübingen, UKSH, Clinic for Applied Cellular Medicine, Damietta University and 2 more.
Type: | Journal: Stem cells international | Year: 2016

Adult stem or programmable cells hold great promise in diseases in which damaged or nonfunctional cells need to be replaced. We have recently demonstrated that peripheral blood monocytes can be differentiated in vitro into cells resembling specialized cell types like hepatocytes and pancreatic beta cells. During phenotypic conversion, the monocytes downregulate monocyte/macrophage differentiation markers, being indicative of partial dedifferentiation, and are partially reprogrammed to acquire a state of plasticity along with expression of various markers of pluripotency and resumption of mitosis. Upregulation of stem cell markers and mitotic activity in the cultures was shown to be controlled by autocrine production/secretion of activin A and transforming growth factor-beta (TGF-). These reprogrammed monocyte derivatives were termed programmable cells of monocytic origin (PCMO). Current efforts focus on establishing culture conditions that increase both the plasticity and proliferation potential of PCMO in order to be able to generate large amounts of blood-derived cells suitable for both autologous and allogeneic therapies.


Ungefroren H.,Clinic for Applied Cellular Medicine | Sebens S.,University of Kiel | Sebens S.,Laboratory of Molecular Gastroenterology and Hepatology | Groth S.,Clinic for Applied Cellular Medicine | And 2 more authors.
Current Cancer Drug Targets | Year: 2011

Both the nonreceptor tyrosine kinase Src and the receptors for transforming growth factor (TGF)-β (TβRI, TβRII) play major roles during tumorigenesis by regulating cell growth, migration/invasion and metastasis. The common Src family kinase inhibitors PP2 and PP1 effectively block Src activity in vitro and in vivo, however, they may exert nonspecific effects on other kinases. In this study, we have evaluated PP2 and PP1 for their ability to inhibit TGFβ1-mediated responses in the TGF-β-responsive pancreatic adenocarcinoma cell line Panc1. We show that PP2 and PP1 but not the more specific Src inhibitor SU6656 effectively relieved TGF-β1-induced growth arrest and p21WAF1 induction, while basal growth was enhanced by PP2 and PP1, and suppressed by SU6656. PP2 and PP1 but not SU6656 also suppressed TGF-β1-induced epithelial-to-mesenchymal transition (EMT) as evidenced by their ability to inhibit downregulation of the epithelial marker E-cadherin, and upregulation of the EMT-associated transcription factor Slug. Likewise, PP2 and PP1 but not SU6656 effectively blocked TGF-β1-induced activation of Smad2 and p38 MAPK and partially suppressed Smad activation and transcriptional activity on TGF-β/Smad-responsive reporters of a kinase-active TβRI mutant ectopically expressed in Panc1 cells. Interestingly, PP2 and PP1 strongly inhibited recombinant TβRI in an in vitro kinase assay, with PP1 being more potent and PP2 being nearly as potent as the established TβRI inhibitor SB431542. PP2 but not PP1 also weakly inhibited the TβRII kinase. Together, these data provide evidence that PP2 and PP1 are powerful inhibitors of TβR function that can block TGF-β/Smad signaling in a Src-unrelated fashion. Both agents may be useful as dual TGF-β/Src inhibitors in experimental therapeutics of late stage metastatic disease. © 2011 Bentham Science Publishers Ltd.


Held-Feindt J.,University of Kiel | Hattermann K.,University of Kiel | Muerkoster S.S.,Laboratory of Molecular Gastroenterology and Hepatology | Wedderkopp H.,University of Kiel | And 4 more authors.
Experimental Cell Research | Year: 2010

The transmembrane chemokine CX3CL1 and its receptor CX3CR1 are thought to be involved in the trafficking of immune cells during an immune response and in the pathology of various human diseases including cancer. However, little is known about the expression and function of CX3CR1 in human glioma-infiltrating microglia/macrophages (GIMs), representing the major cellular stroma component of highly malignant gliomas. Here, we show that CX3CR1 is overexpressed at both the mRNA and protein level in solid human astrocytomas of different malignancy grades and in glioblastomas. CX3CR1 was localized in ionized calcium-binding adapter molecule 1 (Iba1) and CD11b/c positive GIMs in situ as shown by fluorescence microscopy. In accordance with this, freshly isolated human GIM-enriched fractions separated by CD11b MACS technology displayed high Iba1 and CX3CR1 mRNA expression levels in vitro. Moreover, cultured human GIMs responded to CX3CL1-triggered activation of CX3CR1 with adhesion and migration in vitro. Besides an increase in motility, CX3CL1 also enhanced expression of matrix metalloproteases 2, 9, and 14 in GIM fractions in vitro. These data indicate that the CX3CL1/CX3CR1 system has a crucial tumor-promoting role in human glioblastomas via its impact on glioma-infiltrating immune subsets. © 2010.


Ungefroren H.,Clinic for Applied Cellular Medicine | Hyder A.,Clinic for Applied Cellular Medicine | Hinz H.,Clinic for Applied Cellular Medicine | Groth S.,Clinic for Applied Cellular Medicine | And 7 more authors.
PLoS ONE | Year: 2015

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. © 2015 Ungefroren et al.


Ungefroren H.,Clinic for Applied Cellular Medicine | Sebens S.,University of Kiel | Sebens S.,C o Laboratory of Molecular Gastroenterology and Hepatology | Groth S.,Clinic for Applied Cellular Medicine | And 2 more authors.
International Journal of Oncology | Year: 2011

Both transforming growth factor (TGF)-β and the non-receptor tyrosine kinase Src play major roles during tumorigenesis by regulating cell growth, epithelial-to-mesenchymal transition (EMT), migration/invasion and metastasis, but little is known about the signaling crosstalk between them. To interfere with Src function in vitro and in vivo many studies have employed the pharmacologic Src inhibitors PP2 and PP1. Both agents have recently been shown to be powerful inhibitors of TGF-β receptor type I/ALK5 and type II. As this situation prohibited any definite conclusions with respect to the relative contribution of TGF-β vs. Src signaling, we decided to reappraise a potential role of Src in TGF-β1-mediated cellular responses using RNA and dominant-negative (dn) interference to block Src expression and function, respectively. In TGF-β-responsive pancreatic ductal adenocarcinoma (PDAC) cells, we show that Src is activated by TGF-β1 and that its specific inhibition strongly attenuated basal proliferation and enhanced TGF-β1-mediated growth arrest. However, Src inhibition was unable to impair TGF-β1-controlled EMT as evidenced by cell morphology and regulation of the epithelial marker E-cadherin. Despite its dispensibility for TGF-β-induced EMT, specific inhibition of Src dramatically reduced basal and TGF-β1-induced cell migration in Panc-1 cells as measured with a novel real-time migration assay (xCELLigence DP system). Biochemically, dnSrc inhibition failed to block TGF-β1/ALK5-induced activation of Smad2 and Smad3, but partially inhibited transcriptional activation of TGF-β/Smad-responsive reporter genes, and effectively blocked basal and TGF-β1-induced activation of p38 MAPK. Together, the data provide evidence for a role of Src in the regulation of basal proliferation as well as in basal and TGFβ1-mediated cell motility but not EMT in TGF-β-responsive pancreatic (tumor) cells.


Ungefroren H.,Clinic for Applied Cellular Medicine
Advances in Experimental Medicine and Biology | Year: 2010

Adult stem or programmable cells hold great promise in diseases in which damaged or non-functional cells need to be replaced, such as in type 1 diabetes. We have recently demonstrated that peripheral blood monocytes can be differentiated in vitro into pancreatic β-cell-like cells capable of synthesizing insulin. The two-step phenotypic conversion commences with growth factor-induced partial reprogramming during which the cells acquire a state of plasticity along with expression of various markers of pluripotency. These cells, termed "programmable cells of monocytic origin" (PCMOs), can then be induced with appropriate differentiation media to become insulin-producing cells (NeoIslet cells). Expression profiling of transcription factors known to determine endocrine and β-cell development in vivo indicated that NeoIslet cells resemble cells with an immature β-cell phenotype. Current efforts focus on establishing culture conditions that (i) increase the plasticity and proliferation potential of PCMOs by enhancing the reprogramming process and (ii) improve insulin production by mimicking in vivo lineage specification and normal pancreatic endocrine development. Combining these two strategies has great potential in generating large amounts of blood-derived cells suitable for both autologous and allogeneic therapy of type 1 diabetes. © Springer Science+Business Media B.V. 2010.


Hyder A.,Clinic for Applied Cellular Medicine | Ehnert S.,University of Tübingen | Hinz H.,Clinic for Applied Cellular Medicine | Nussler A.K.,University of Tübingen | And 2 more authors.
Cell Communication and Signaling | Year: 2012

Background: 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. Results: 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 p47 phoxand increased that of Nanog indicating enhanced dedifferentiation and pluripotency, respectively. Treatment with both EGF and HB-EGF resulted in NeoHepatocytes with improved functional parameters. Conclusions: 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. © 2012 Hyder et al.; licensee BioMed Central Ltd.


PubMed | Clinic for Applied Cellular Medicine, University of Tübingen, Clinic for Conservative Dentistry and Periodontology and UKSH
Type: Journal Article | Journal: PloS one | Year: 2015

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-(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, TRII, ALK5 as well as TGF-1 and the A 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-1 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-1 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- signaling by either SB431542 or anti-TGF- antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF- 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-/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-/Smad3, and to a lesser extent, activin/Smad2 signaling.

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