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Background Aims: The immunomodulating capacity of multipotent mesenchymal stromal cells (MSCs) qualifies them as a therapeutic tool in several diseases. However, repeated transplantation with products of reproducible characteristics may be required. This could be achieved with cryopreserved aliquots of Good Manufacturing Practice (GMP)-grade MSCs. However, the impact of cryopreservation on the characteristics of GMP-MSCs is ill defined., Methods: We produced fresh and cryopreserved MSCs from human donors with a xenogen-free GMP protocol. Immunogenicity and immunomodulating capacity were tested in co-culture with putative recipient-specific peripheral blood mononuclear cells (PBMCs). Risk of malignant transformation was assessed in vitro and in vivo., Results: Cryopreservation had no impact on viability and consensus criteria of MSCs. In co-culture with PBMCs, MSCs showed low immunogenicity and suppressed mitogen-stimulated proliferation of PBMC irrespective of cryopreservation. Cytogenetic aberrations were not observed consistently in fresh and cryopreserved products, and no signs of malignant transformation occurred in functional assays. MSC products from an elderly pretreated donor showed reduced functional quality, but imminent failure of functional criteria could be detected by an increased population doubling time in early passages., Discussion: This study is the first systematic analysis on cryopreservation of xenogen-free human bone marrow-derived GMP-MSCs. The data support that cryopreservation does not alter the characteristics of the cells and thus may allow the generation of products for serial transplantation. In addition, the protocol allowed early detection of MSC products with low functional capacity., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Tumor-integrating multipotent mesenchymal stromal cells (MSC) expressing transgenes with anti-tumor activity may serve as vehicles for tumor therapy. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) represents such a factor; however, TRAIL-resistant tumor cells exist. Based on our previous work, here we investigated whether MSC with lentiviral TRAIL expression (TRAIL-MSC) inhibit the growth of TRAIL-resistant colorectal carcinoma (CRC) cells. Our data show that TRAIL-MSC induce apoptosis in selected TRAIL-resistant CRC cell lines and effectively inhibit the growth of TRAIL-resistant HCT8 cells. This sensitization to TRAIL-induced apoptosis required the presence of MSC-expressed TRAIL. However, for the first time we show that selected CRC cells are resistant to TRAIL-MSC. In the cell line HT29, this resistance could be overcome by concomitant subapoptotic genotoxic damage in vitro. However, such sensitization was not achieved in vivo as treatment of mixed HT29/TRAIL-MSC xenografts with 5-FU rather resulted in enhanced growth. Taken together, our data prove that TRAIL-MSC overcome TRAIL resistance in selected CRC cells through direct intercellular interaction and may, therefore, represent a clinical tool to overcome TRAIL resistance. However, such potential clinical use requires further preclinical studies as our data also prove that TRAIL-MSC-resistant CRC cells exist. Our data add to the notion that TRAIL resistance of CRC cells is conferred by different mechanisms.

Colorectal carcinoma (CRC) constitutes a common malignancy with limited therapeutic options in metastasized stages. Mesenchymal stem cells (MSC) home to tumours and may therefore serve as a novel therapeutic tool for intratumoral delivery of antineoplastic factors. Tumour necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) which promises apoptosis induction preferentially in tumour cells represents such a factor. We generated TRAIL-MSC by transduction of human MSC with a third generation lentiviral vector system and analysed their characteristics and capacity to inhibit CRC growth. (1) TRAIL-MSC showed stable transgene expression with neither changes in the defining MSC characteristics nor signs of malignant transformation. (2) Upon direct in vitro coculture TRAIL-MSC induced apoptosis in TRAIL-sensitive CRC-cell lines (DLD-1 and HCT-15) but also in CRC-cell lines resistant to soluble TRAIL (HCT-8 and SW480). (3) In mixed subcutaneous (s.c.) xenografts TRAIL-MSC inhibited CRC-tumour growth presumably by apoptosis induction but a substantial proportion of TRAIL-MSC within the total tumour cell number was needed to yield such anti-tumour effect. (4) Systemic application of TRAIL-MSC had no effect on the growth of s.c. DLD-1 xenografts which appeared to be due to a pulmonary entrapment and low rate of tumour integration of TRAIL-MSC. Systemic TRAIL-MSC caused no toxicity in this model. (5) Wild-type MSC seemed to exert a tumour growth-supporting effect in mixed s.c. DLD-1 xenografts. These novel results support the idea that lentiviral TRAIL-transgenic human MSC may serve as vehicles for clinical tumour therapy but also highlight the need for further investigations to improve tumour integration of transgenic MSC and to clarify a potential tumour-supporting effect by MSC., (© 2009 The Authors Journal compilation © 2010 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Differentiation and malignant transformation of stem cells are regulated by epigenetic mechanisms. We analyzed promoter methylation and expression of the stem cell determining genes Brachyury, DPPA5, FGF4, FOXD3, LIN28, NESTIN and ZFP42 depending on the differentiation state in human mesenchymal stem cells (MSC), human embryonal carcinoma cells (ECC) and somatic tumor cells. Differentiation of MSC into osteoblasts and adipocytes was accompanied with a loss of expression of the Brachyury gene and downregulation of LIN28. Inactivation of Brachyury was associated with progressive methylation of its CpG island promoter. In ECC promoter methylation of stem cell markers was more frequent in the differentiated subgroup (71%) compared to undifferentiated ECC (29%) and this was associated with downregulation of Brachyury, DPPA5, FGF4, FOXD3, LIN28 and ZFP42. DPPA5 was methylated and NESTIN was unmethylated in most tumor cells. In somatic tumor cells, methylation of stem cell markers (Brachyury, DPPA5, FGF4, FOXD3, LIN28 and ZFP42) was frequently observed (85%). Treatment of cell lines with an inhibitor of DNA methyltransferase reactivated the expression of DPPA5, FGF4, FOXD3, LIN28 and ZFP42, indicating that aberrant promoter methylation is a crucial event that results in their silencing. Our results suggest that epigenetic inactivation of stem cell associated genes is mediated by promoter methylation and that this may represent a fundamental mechanism during normal differentiation processes.

OCT4 is considered a main regulator of embryonic stem cell pluripotency and self renewal capacity. It was shown that relevant OCT4 expression only occurs in cells of embryonic pluripotent nature. However, several recent publications claimed to have demonstrated OCT4 expression in human somatic tumor cells, human adult stem or progenitor cells and differentiated cells.We analysed 42 human tumor cell lines from 13 entities and human bone marrow-derived mesenchymal stem cells (MSC). To validate OCT4 expression we used germ cell tumor (GCT) cell lines, derived xenografts and GCT samples. Analysis by RT-PCR, western blotting, immunocytochemistry and immunohistochemistry was performed. With exception of typical embryonal carcinoma cells, we did not observe reliable OCT4 expression in somatic tumor cell lines and MSC. We suggest that a high level of expression of the OCT4 protein together with its nuclear localization still remains a reliable and definitive feature of cells with embryonic pluripotent nature.

Aims: At present, clinical success of hepatocyte transplantation as an alternative to whole liver transplantation is hampered by the limited availability of suitable donor organs for the isolation of transplantable hepatocytes. Hence, novel cell sources are required to deliver hepatocytes of adequate quality for clinical use. Mesenchymal stem cells (MSCs) from human bone marrow may have the potential to differentiate into hepatocytes in vitro and in vivo., Methods: Isolated MSCs were selected by density gradient centrifugation and plastic adherence, differentiated in the presence of human hepatocyte growth medium and transplanted in immunodeficient Pfp/Rag2 mice., Results: Here, we demonstrate that human MSCs gain in vitro the characteristic morphology and function of hepatocytes in response to specified growth factors. Specifically, preconditioned MSCs store glycogen, synthesise urea and feature the active hepatocyte-specific gene promoter of phosphoenolpyruvate carboxykinase (PCK1). After transplantation into livers of immunodeficient mice, preconditioned MSCs engraft predominantly in the periportal portion of the liver lobule. In situ, the cells continue to store glycogen and express PCK1, connexin32, albumin and the human hepatocyte-specific antigen HepPar1, indicating that the transplanted cells retain prominent qualities of hepatocytes after their regional integration., Conclusion: MSCs derived from human bone marrow may serve as a novel source for the propagation of hepatocyte-like cells suitable for cell therapy in liver diseases.

For various potential clinical applications, the use of autologous human MSCs (hMSCs) would be favorable. In vitro observations suggested that hMSCs are resistant for chemotherapeutic substances; however, no data exist on the characteristics of hMSCs from bone marrow (BM) of chemotherapeutically treated patients. Here, we analyzed the character of hMSCs derived from chemotherapy-exposed BM and the in vitro response of hMSCs to chemotherapeutic substances. Colony-forming units-fibroblast (CFU-Fs) were isolated from BM aspirates of patients after high-dose or standard chemotherapy and of donors with unaffected BM. CFU-Fs from chemotherapy-exposed and unaffected BM contained hMSCs with similar phenotype, proliferation capacity, and differentiation potential. No obvious influence of age, sex, or time since chemotherapy exposure on the presence and characteristics of hMSCs was observed. In vitro, hMSCs showed a significant resistance for cisplatin, vincristine, and etoposide compared with sensitive tumor cell lines, particularly at apoptosis-inducing doses. The phenotype and differentiation potential of hMSCs was not altered by genotoxic treatment under clinically relevant conditions in vitro. hMSCs showed an elevated threshold for cisplatin-induced apoptosis, which was characterized by a lack of caspase-9 activity in apoptotic cells. In vitro exposure of hMSCs to cisplatin, vincristine, and etoposide resulted in an increased p53 expression, independent of apoptosis induction. We conclude that hMSCs can be isolated from chemotherapy-exposed BM in sufficient number and quality for potential clinical applications in chemotherapeutically treated patients. Our data suggest that an elevated apoptotic threshold contributes not only to the persistence of hMSCs in the BM after chemotherapy but also to their lifelong presence in the adult BM.