Umbilical Cord Matrix-derived Stem Cells Research Articles

Hepatogenic Potential and Liver Regeneration Effect of Human Liver-derived Mesenchymal-Like Stem Cells.

Human liver-derived stem cells (hLD-SCs) have been proposed as a possible resource for stem cell therapy in patients with irreversible liver diseases. However, it is not known whether liver resident hLD-SCs can differentiate toward a hepatic fate better than mesenchymal stem cells (MSCs) obtained from other origins. In this study, we compared the differentiation ability and regeneration potency of hLD-SCs with those of human umbilical cord matrix-derived stem cells (hUC-MSCs) by inducing hepatic differentiation. Undifferentiated hLD-SCs expressed relatively high levels of endoderm-related markers (GATA4 and FOXA1). During directed hepatic differentiation supported by two small molecules (Fasudil and 5-azacytidine), hLD-SCs presented more advanced mitochondrial respiration compared to hUC-MSCs. Moreover, hLD-SCs featured higher numbers of hepatic progenitor cell markers on day 14 of differentiation (CPM and CD133) and matured into hepatocyte-like cells by day 7 through 21 with increased hepatocyte markers (ALB, HNF4A, and AFP). During in vivo cell transplantation, hLD-SCs migrated into the liver of ischemia-reperfusion injury-induced mice within 2 h and relieved liver injury. In the thioacetamide (TAA)-induced liver injury mouse model, transplanted hLD-SCs trafficked into the liver and spontaneously matured into hepatocyte-like cells within 14 days. These results collectively suggest that hLD-SCs hold greater hepatogenic potential, and hepatic differentiation-induced hLD-SCs may be a promising source of stem cells for liver regeneration.

Fibrin scaffold enhances function of insulin producing cells differentiated from human umbilical cord matrix-derived stem cells.

Tissue engineering is a new strategy which proposed to treat numerous human diseases nowadays. Three dimensional (3D) scaffolds fill the gap between two dimensional cell culture (2D) and animal tissues through mimicking the environmental behaviors surrounding the cells. In this study, hUCMs into insulin producing cells in fibrin scaffold were differentiated compare to conventional culture condition. Differentiation rate was estimated by real time PCR, immunocytochemistry (ICC) and the chemiluminesence (CLIA) and enzyme immunoassay (EIA). Real time PCR's results showed an increasing expression in NKX2.2, PDX1 and INS (producing the hormone insulin) genes in fibrin scaffold. Furthermore ICC analysis exhibited that insulin and pro-insulin proteins were more in fibrin scaffolds. CLIA and EIA on insulin and C peptide secretion indicated that both of groups were sensitive to the glucose challenge test but significant higher response was observed in fibrin scaffold (6.5 fold in 3D, 1.8 fold in 2D culture). It could be concluded that differentiation of hUCM cells into insulin producing cells in fibrin scaffold 3D culture system is much more efficient than 2D conventional culture system.

Human umbilical cord matrix-derived stem cells expressing interferon-β gene inhibit breast cancer cells via apoptosis.

Human umbilical cord mesenchymal stem cells (hUCMSCs) derived from the umbilical cord matrix have been reported to be used as anti-tumor gene carrier for attenuation of tumor growth, which extends the half-life and lowers the unexpected cytotoxicity of the gene in vivo. Interferon-β (IFNβ) is known to possess robust antitumor effects on different types of cancer cell lines in vitro. The present study was aimed to investigate the anti-tumor effect of IFNβ gene-transfected hUCMSCs (IFNβ-hUCMSCs) on breast cancer cells with emphasis on triple negative breast carcinoma. Our findings revealed that the co-culture of IFNβ-hUCMSCs with the human triple negative breast carcinoma cell lines MDA-MB-231 or Hs578T significantly inhibited growth of both carcinoma cells. In addition, the culture medium conditioned by these cells also significantly suppressed the growth and induced apoptosis of both carcinoma cells. Further investigation showed that the suppressed growth and the apoptosis induced by co-culture of IFNβ-hUCMSCs or conditioned medium were abolished by pretreating anti-IFNβ neutralizing antibody. These findings indicate that IFNβ-hUCMSCs triggered cell death of breast carcinoma cells through IFN-β production, thereby induced apoptosis and suppressed tumor cell growth. In conclusion, we demonstrated that IFNβ-hUCMSCs inhibited the growth of breast cancer cells through apoptosis. with potent anti-cancer activity, it represents as an anti-cancer cytotherapeutic modality against breast cancer.

Human umbilical cord matrix-derived stem cells exert trophic effects on β-cell survival in diabetic rats and isolated islets

ABSTRACTHuman umbilical cord matrix-derived stem cells (uMSCs), owing to their cellular and procurement advantages compared with mesenchymal stem cells derived from other tissue sources, are in clinical trials to treat type 1 (T1D) and type 2 diabetes (T2D). However, the therapeutic basis remains to be fully understood. The immunomodulatory property of uMSCs could explain the use in treating T1D; however, the mere immune modulation might not be sufficient to support the use in T2D. We thus tested whether uMSCs could exert direct trophic effects on β-cells. Infusion of uMSCs into chemically induced diabetic rats prevented hyperglycemic progression with a parallel preservation of islet size and cellularity, demonstrating the protective effect of uMSCs on β-cells. Mechanistic analyses revealed that uMSCs engrafted long-term in the injured pancreas and the engraftment markedly activated the pancreatic PI3K pathway and its downstream anti-apoptotic machinery. The pro-survival pathway activation was associated with the expression and secretion of β-cell growth factors by uMSCs, among which insulin-like growth factor 1 (IGF1) was highly abundant. To establish the causal relationship between the uMSC-secreted factors and β-cell survival, isolated rat islets were co-cultured with uMSCs in the transwell system. Co-culturing improved the islet viability and insulin secretion. Furthermore, reduction of uMSC-secreted IGF1 via siRNA knockdown diminished the protective effects on islets in the co-culture. Thus, our data support a model whereby uMSCs exert trophic effects on islets by secreting β-cell growth factors such as IGF1. The study reveals a novel therapeutic role of uMSCs and suggests that multiple mechanisms are employed by uMSCs to treat diabetes.

A Systemic Approach to Cancer Treatment: Tumor Cell Reprogramming Focused on Endocrine-Related Cancers

The term "cancer cell reprogramming" is used to define any kind of intervention aimed at transforming cancer cells into terminally differentiated cells. Using this approach, new technologies have been applied with different methods for a more systemic approach to cancer treatment. This review reports on advances of these technologies, including our personal contributions, mainly carried out on endocrine-related cancers. Some of the interventions, aimed at reverting cancer cells into a normal phenotype, are based on the evidence that tumor development is suppressed by the embryonic microenvironment. On the basis of this rationale, experiments have been conducted using stem cell differentiation stage factors (SCDSFs) taken at different stages of development of Zebrafish embryos, oocyte extracts, or naïve human umbilical cord matrix derived stem cells (UMDSCs). SCDSFs induce significant growth inhibition on different tumor cell lines in vitro, likely because of increases in cell cycle regulatory molecules, such as p53 and pRb. Treatment with these factors activates apoptosis and differentiation related to caspase-3. This is achieved via p73 apoptotic-dependent pathway activation with a concurrent normalization of the E-cadherin and beta-catenin ratio. Extracts from prophase amphibian oocytes could reprogram relevant epigenetic alterations in MCF-7 and HCC1954 breast cancer cell lines, while un-engineered (naïve) human UMDSCs attenuated growth of MDA-231 human breast carcinoma cells. A product prepared for human treatments, containing SCDSFs at very low doses, yielded favorable results in breast cancer and in intermediate-advanced hepatocellular carcinoma. Other reprogramming interventions used in the models of breast, prostate and ovarian cancer cell lines are described. Finally, current and future perspectives of this novel technology are discussed and a new hallmark of cancer is suggested: the loss of differentiation of cancer cells.

Abstract 220: Human umbilical cord matrix-derived stem cells control tumor growth by tumor suppressor gene expression.

Abstract Umbilical cord matrix derived stem cells (UCMSC) have the potential to treat various diseases including cancer. We have shown that naïve human and rat UCMSC significantly attenuate proliferation of multiple cancer cells (Ganta et al., Cancer Res., 2009. Ayuzawa et al., Cancer Lett., 2009). However, the growth attenuation ability of rat UCMSC is stronger than that of human UCMSC. To clarify their different tumoricidal abilities, differential gene expression profiles of these two cells were studied by micro-array analysis using Illumina HumanRef-8 V2 for human and RatRef-12 BeadChip for rat UCMSC. The gene expression profiles were compared between UCMSC untreated and co-cultured with either human (MDA-231) or rat breast carcinoma cells (Mat B III). Selection criteria used for the screening of candidate genes associated with UCMSC-dependent tumoricidal ability are as follows; 1) gene expression difference should be at least a 1.5 fold between naive UCMSC and those co-cultured with mammary tumor cells; 2) they must encode secretory proteins; and 3) they must encode cell growth regulation-related proteins. These analyses screened 16 common genes from both human and rat UCMSC. The comparison between two sets of gene expression profiles identified that two genes, adipose-differentiation related protein (ADRP) and follistatin (FST), were specifically up-regulated in rat UCMSC, whereas they were down-regulated in human UCMSC when they were co-cultured with the corresponding species’ breast cancer cells. The suppression of either protein by adding specific neutralizing antibody in culture of rat UCMSC significantly abrogated their ability to attenuate cell growth. Over-expression of both genes by adenovirus vector in human UCMSC enhanced their ability to suppress the growth of MDA-231 cells. FST over-expression in MDA 231 cells also decreased their growth significantly. In the experimental lung metastasis model with MDA 231 cells in immunodeficient mice, the treatment with FST-over-expressing human UCMSC was administered three times, at one week intervals, via tail vein injection, starting 6 days after tumor cell inoculation. Examination 4 weeks after tumor inoculation showed a significant decrease in the number of tumor nodules in the lungs. Both ADRP and FST are considered to be tumor suppressor genes. These results suggest that both ADRP and FST may play important roles in exhibiting a stronger tumoricidal ability in rat UCMSC than human UCMSC. These results imply that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression. This work was supported by the Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU College of Veterinary Medicine Dean's Fund, NIH RR017686, RR15563, CA135599, Kansas Bioscience Authority research grant. Citation Format: Naomi Ohta, Susumu Ishiguro, Atsushi Kawabata, Deepthi Uppalapati, Marla Pyle, Deryl Troyer, Supriyo De, Yongqing Zhang, Kevin G. Becker, Masaaki Tamura. Human umbilical cord matrix-derived stem cells control tumor growth by tumor suppressor gene expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 220. doi:10.1158/1538-7445.AM2013-220

Stem cell-based photodynamic therapy

We have transfected murine neural stem cells (NSCs) and rat umbilical cord matrix-derived stem cells (RUCMSCs) with a plasmid expressing gaussia luciferase (gLuc). These cells are engineered to secrete the luciferase. We have used gLuc containing supernatant from culturing the NSCs to perform in vitro photodynamic therapy of murine melanoma cells (B16F10), and RUCMSCs to perform in vivo PDT of lung melanomas in C57BL/6 mice. The treatment system was comprised of aminolevulic acid as a prodrug for the synthesis of the photosensitizer protoporphyrin IX, gaussia luciferase, and its' substrate coelenterazine. A significant reduction of the number of live melanoma cells in vitro and a borderline significant retardation of tumour growth in vivo was observed after coelenterazine-mediated PDT.

Abstract B46: Umbilical cord matrix-derived stem cells control tumor growth by their tumor suppressor and promoter gene expression

Abstract Umbilical cord matrix-derived stem cells (UCMSC) have the potential to treat various diseases including cancer. We have shown that naïve human and rat UCMSC significantly attenuate proliferation of multiple cancer cells. However, our previous study showed that the growth attenuation ability of rat UCMSC is stronger than that of human UCMSC. To clarify their different tumoricidal abilities, differential gene expression profiles were studied by microarray analysis using IIIumina HumanRef-8 V2 BeadChip for human and RatRef-12 BeadChip for rat UCMSC. The differential gene expression profile between untreated human UCMSC and those co-cultured with MDA-MB 231 human breast carcinoma cells was compared with that between untreated rat UCMSC and those co-cultured with Mat B III rat mammary gland carcinoma cells. Strict screening criteria used to identify putative genes associated with UCMSC-dependent tumoricidal activity were as follows: gene expression should (1) be over than 1.5 fold different, (2) encode secretory proteins, and (3) be associated with cell growth regulation. Seventeen genes were identified as being associated with either human or rat UCMSC-dependent tumor growth regulation. Among these genes, eight were up-regulated in both human and rat UCMSC (two being known tumor suppressor and six being putative tumor promoter genes). Seven out of seventeen genes were up-regulated in human UCMSC but not in rat UCMSC (three were identified to be tumor suppressor and four were tumor promoter genes). Two out of the seventeen genes, adipose-differentiation related protein (ADRP) and follistatin (FST), which are known tumor suppressor genes, were specifically up-regulated in rat UCMSC, whereas they were down-regulated in human UCMSC when they were co-cultured with carcinoma cells. These results strongly suggest that the balance of the up-regulation of tumor suppressor genes and down-regulation of tumor promoter genes in UCMSC appear to control tumor growth. Since both ADRP and FST are considered to be tumor suppressor genes and were specifically up-regulated in only rat UCMSC, these two genes expression may play central role in strong tumoricidal activity by rat UCMSC. In support of this hypothesis, suppression of ADRP and FST protein by adding a neutralizing antibody (4 µg/ml) in culture medium of rat UCMSC significantly abrogated their ability to attenuate DNA synthesis. Over-expression of ADRP and FST genes by adenoviral vector (100 MOI) in human UCMSC promoted their ability to suppress the DNA synthesis of MDA-MB 231 cells in [3H]-thymidine uptake assay. Interestingly, ADRP expression in human UCMSC stimulated differentiation to adipose type cell morphology (cell enlargement and oil droplet accumulations in cytoplasm). This result suggests that ADRP may stimulate adiponectin production in vivo thereby attenuating tumor growth. Taken together, these results suggest that both ADRP and FST may be key genes that exhibit stronger tumoricidal ability in rat UCMSC than human UCMSC. This work was supported by the Kansas State University (KSU) Terry C. Johnson Center for Basic Cancer Research, KSU College of Veterinary Medicine Dean's Fund, NIH RR017686, RR15563, CA135599, Kansas Bioscience Authority Collaborative Cancer Research grant and by the Intramural Research Program of the NIH, National Institute on Aging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B46.

Human umbilical cord matrix-derived stem cells expressing interferon-β gene significantly attenuate bronchioloalveolar carcinoma xenografts in SCID mice

Mesenchymal stem cells derived from the human umbilical cord matrix (hUCMSCs) have great potential for therapeutic use for multiple diseases. The strategy that uses therapeutic gene-transfected hUCMSCs as cellular vehicles for targeted biologic agent delivery has solved the problem of short half-life or excessive toxicity of biological agent(s) in vivo. Interferon-β (IFN-β) has demonstrated a potent antitumor effect on many types of cancer cell lines in vivo. The aim of this study was to determine the anti-cancer effect of IFN-β gene-transfected hUCMSCs (IFN-β-hUCMSCs) on cells derived from bronchioloalveolar carcinoma, a subset of lung adenocarcinoma that is difficult to treat. The co-culture of a small number of IFN-β-hUCMSCs with the human bronchioloalveolar carcinoma cell lines H358 or SW1573 significantly inhibited growth of both types of carcinoma cell lines. The culture medium conditioned by these cells also significantly attenuated the growth of both carcinoma cells, but this attenuation was abolished by adding anti-IFN-β antibody. Finally, systemic administration of IFN-β-hUCMSCs through the tail vein markedly attenuated growth of orthotopic H358 bronchioloalveolar carcinoma xenografts in SCID mice by increasing apoptosis. These results clearly indicate that IFN-β-hUCMSCs caused cell death of bronchioloalveolar carcinoma cells through IFN-β production, thereby attenuating tumor growth in vivo. These results indicate that IFN-β-hUCMSCs are a powerful anti-cancer cytotherapeutic tool for bronchioloalveolar carcinoma.

Cytotherapy with naive rat umbilical cord matrix stem cells significantly attenuates growth of murine pancreatic cancer cells and increases survival in syngeneic mice

Pancreatic cancer, sometimes called a 'silent killer', is one of the most aggressive human malignancies, with a very poor prognosis. It is the fourth leading cause of cancer-related morbidity and mortality in the USA. A mouse peritoneal model was used to test the ability of unengineered rat umbilical cord matrix-derived stem cells (UCMSC) to control growth of pancreatic cancer. In vivo results were supported by various in vitro assays, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), direct cell count, [3H]thymidine uptake and soft agar colony assays. Co-culture of rat UCMSC with PAN02 murine pancreatic carcinoma cells (UCMSC:PAN02, 1:6 and 1:3) caused G0/G1 arrest and significantly attenuated the proliferation of PAN02 tumor cells, as monitored by MTT assay, direct cell counts and [3H]thymidine uptake assay. Rat UCMSC also significantly reduced PAN02 colony size and number, as measured by soft agar colony assay. The in vivo mouse studies showed that rat UCMSC treatment significantly decreased the peritoneal PAN02 tumor burden 3 weeks after tumor transplantation and increased mouse survival time. Histologic study revealed that intraperitoneally administered rat UCMSC survived for at least 3 weeks, and the majority were found near or inside the tumor. These results indicate that naive rat UCMSC alone remarkably attenuate the growth of pancreatic carcinoma cells in vitro and in a mouse peritoneal model. This implies that UCMSC could be a potential tool for targeted cytotherapy for pancreatic cancer.

Abstracts

Pancreatic cancer, sometimes called a ‘silent killer’, is one of the most aggressive human malignancies, with a very poor prognosis. It is the fourth leading cause of cancer-related morbidity and mortality in the USA.A mouse peritoneal model was used to test the ability of unengineered rat umbilical cord matrix-derived stem cells (UCMSC) to control growth of pancreatic cancer. In vivo results were supported by various in vitro assays, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), direct cell count, [3H]thymidine uptake and soft agar colony assays.Co-culture of rat UCMSC with PAN02 murine pancreatic carcinoma cells (UCMSC:PAN02, 1:6 and 1:3) caused G0/G1 arrest and significantly attenuated the proliferation of PAN02 tumor cells, as monitored by MTT assay, direct cell counts and [3H]thymidine uptake assay. Rat UCMSC also significantly reduced PAN02 colony size and number, as measured by soft agar colony assay. The in vivo mouse studies showed that rat UCMSC treatment significantly decreased the peritoneal PAN02 tumor burden 3 weeks after tumor transplantation and increased mouse survival time. Histologic study revealed that intraperitoneally administered rat UCMSC survived for at least 3 weeks, and the majority were found near or inside the tumor.These results indicate that naive rat UCMSC alone remarkably attenuate the growth of pancreatic carcinoma cells in vitro and in a mouse peritoneal model. This implies that UCMSC could be a potential tool for targeted cytotherapy for pancreatic cancer.