Xuetao Pei

Generation of insulin-producing cells from PDX-1 gene-modified human mesenchymal stem cells

Islet cell replacement is considered as the optimal treatment for type I diabetes. However, the availability of human pancreatic islets for transplantation is limited. Here, we show that human bone marrow‐derived mesenchymal stem cells (hMSCs) could be induced to differentiate into functional insulin‐producing cells by introduction of the pancreatic duodenal homeobox‐1 (PDX‐1). Recombinant adenoviral vector was used to deliver PDX‐1 gene into hMSCs. After being infected with Ad‐PDX‐1, hMSCs were successfully induced to differentiate into insulin‐secreting cells. The differentiated PDX‐1+ hMSCs expressed multiple islet‐cell genes including neurogenin3 (Ngn3), insulin, GK, Glut2, and glucagon, produced and released insulin/C‐peptide in a weak glucose‐regulated manner. After the differentiated PDX‐1+ hMSCs were transplanted into STZ‐induced diabetic mice, euglycemia can be obtained within 2 weeks and maintained for at least 42 days. These findings validate the hMSCs model system as a potential basis for enrichment of human beta cells or their precursors, and a possible source for cell replacement therapy in diabetes. J. Cell. Physiol. 211: 36–44, 2007.

Sonic Hedgehog Pathway Is Essential for Maintenance of Cancer Stem-Like Cells in Human Gastric Cancer

Abnormal activation of the Sonic hedgehog (SHH) pathway has been described in a wide variety of human cancers and in cancer stem cells (CSCs), however, the role of SHH pathway in gastric CSCs has not been reported. In this study, we investigated the possibility that abnormal activation of the SHH pathway maintained the characteristics of gastric CSCs. First, we identified cancer stem-like cells (CSLCs) from human gastric cancer cell lines (HGC-27, MGC-803 and MKN-45) using tumorsphere culture. Compared with adherent cells, the floating tumorsphere cells had more self-renewing capacity and chemoresistance. The cells expressing CSCs markers (CD44, CD24 and CD133) were also significantly more in tumorsphere cells than in adherent cells. More importantly, in vivo xenograft studies showed that tumors could be generated with 2×104 tumorsphere cells, which was 100-fold less than those required for tumors seeding by adherent cells. Next, RT-PCR and Western blot showed that the expression levels of Ptch and Gli1 (SHH pathway target genes) were significantly higher in tumorsphere cells than in adherent cells. The results of quantitative real-time PCR were similar to those of RT-PCR and Western blot. Further analysis revealed that SHH pathway blocked by cyclopamine or 5E1 caused a higher reduction in self-renewing capacity of HGC-27 tumorsphere cells than that of adherent cells. We also found that SHH pathway blocking strongly enhanced the efficacy of chemotherapeutic drugs in HGC-27 tumorsphere cells in vitro and in vivo but had no significant effect in adherent cells. Finally, we isolated the tumorspheres from gastric cancer specimen, these cells also had chemoresistance and tumorigenic capacity, and SHH pathway maintained the gastric CSLCs characteristics of tumorsphere cells from primary tumor samples. In conclusion, our data suggested that SHH pathway was essential for maintenance of CSLCs in human gastric cancer.

Hepatocellular carcinoma‐associated mesenchymal stem cells promote hepatocarcinoma progression: Role of the S100A4‐miR155‐SOCS1‐MMP9 axis

Cancer‐associated mesenchymal stem cells (MSCs) play a pivotal role in modulating tumor progression. However, the interactions between liver cancer‐associated MSCs (LC‐MSCs) and hepatocellular carcinoma (HCC) remain unreported. Here, we identified the presence of MSCs in HCC tissues. We also showed that LC‐MSCs significantly enhanced tumor growth in vivo and promoted tumor sphere formation in vitro. LC‐MSCs also promoted HCC metastasis in an orthotopic liver transplantation model. Complementary DNA (cDNA) microarray analysis showed that S100A4 expression was significantly higher in LC‐MSCs compared with liver normal MSCs (LN‐MSCs) from adjacent cancer‐free tissues. Importantly, the inhibition of S100A4 led to a reduction of proliferation and invasion of HCC cells, while exogenous S100A4 expression in HCC cells resulted in heavier tumors and more metastasis sites. Our results indicate that S100A4 secreted from LC‐MSCs can promote HCC cell proliferation and invasion. We then found the expression of oncogenic microRNA (miR)‐155 in HCC cells was significantly up‐regulated by coculture with LC‐MSCs and by S100A4 ectopic overexpression. The invasion‐promoting effects of S100A4 were significantly attenuated by a miR‐155 inhibitor. These results suggest that S100A4 exerts its effects through the regulation of miR‐155 expression in HCC cells. We demonstrate that S100A4 secreted from LC‐MSCs promotes the expression of miR‐155, which mediates the down‐regulation of suppressor of cytokine signaling 1, leading to the subsequent activation of STAT3 signaling. This promotes the expression of matrix metalloproteinases 9, which results in increased tumor invasiveness. Conclusion: S100A4 secreted from LC‐MSCs is involved in the modulation of HCC progression, and may be a potential therapeutic target. (HEPATOLOGY 2013)

Mesenchymal stem cells from primary breast cancer tissue promote cancer proliferation and enhance mammosphere formation partially via EGF/EGFR/Akt pathway

Mesenchymal stem cells (MSCs) play a critical role in promoting cancer progression. However, it is not clear whether MSCs are located in breast cancer tissues and correlated with tumor proliferation. The aim of this study was to investigate the presence of MSCs in breast cancer tissues and evaluate their interactions with cancer cells. We successfully isolated and identified MSCs from primary breast cancer tissues. Breast cancer-associated MSCs (BC-MSCs) showed homogenous immunophenotype, and possessed tri-lineage differentiation potential (osteoblast, adipocyte, and chondrocyte). When co-transplanted with cancer cells in a xenograft model in vivo, BC-MSCs significantly increased the volume and weight of tumors. We observed that BC-MSCs stimulated mammosphere formation in the transwell co-culture system in vitro. This effect was significantly suppressed by the EGF receptor inhibitor. We verified that BC-MSCs could secrete EGF and activate cancer cell’s EGF receptors. Furthermore, our data showed that EGF derived from BC-MSCs could promote mammosphere formation via the PI3K/Akt signaling pathway. Our results confirmed the presence of MSC in primary breast cancer tissues, and they could provide a favorable microenvironment for tumor cell growth in vivo, partially enhance mammosphere formation via the EGF/EGFR/Akt pathway.

Gingiva-Derived Mesenchymal Stem Cell-Mediated Therapeutic Approach for Bone Tissue Regeneration

Up to now, the gingiva-derived mesenchymal stem cells (GMSCs) as a new postnatal stem cells have been isolated and characterized with multipotential differentiation capabilities in vitro. However, the in vivo efficacy of utilizing the GMSCs in bone regeneration remains obscure. First of all, we identified canonical MSCs in human gingival tissue, which possessed homogenous immunophenotype (CD34(-)CD45(-)CD29(+)CD105(+)CD90(+) STRO-1(+)) and had tri-lineage differentiation potential (osteoblasts, adipocytes, and chondrocytes). Next, we examined the efficacy of utilizing these stem cells in bone tissue regeneration; the enhanced green fluorescent protein-labeled GMSCs seeded on type I collagen gel were implanted into the mandibular defects as well as the critical-sized calvarial defects in Sprague Dawley rats. We first demonstrated that GMSCs could repair the mandibular wounds and calvarial defects at 2 months in rats postsurgical reconstruction. Histomorphological analysis and image of fluorescence microscope certified that new bone in the defect areas was derived from the transplanted GMSCs. Immunohistochemical analysis of green fluorescent protein, human collagen I, and osteopontin further confirmed our conclusion. The above results implied that mesenchymal stem cells derived from gingival tissue could be a novel source for stem cell-based therapy in bone reconstruction in clinical applications.

SIRT1 is required for long-term growth of human mesenchymal stem cells

Human mesenchymal stem cells (MSCs) have therapeutic potential because of their ability to self-renew and differentiate into multiple tissues. However, senescence often occurs in MSCs when they are cultured in vitro and the molecular mechanisms underlying this effect remain unclear. In this study, we found that NAD-dependent protein deacetylase SIRT1 is differentially expressed in both human bone marrow-derived MSCs (B-MSCs) and adipose tissue-derived MSCs after increasing passages of cell culture. Using lentiviral shRNA we demonstrated that selective knockdown of SIRT1 in human MSCs at early passage slows down cell growth and accelerates cellular senescence. Conversely, overexpression of SIRT1 delays senescence in B-MSCs that have undergone prolonged in vitro culturing and the cells do not lose adipogenic and osteogenic potential. In addition, we found that the delayed accumulation of the protein p16 is involved in the effect of SIRT1. However, resveratrol, which has been used as an activator of SIRT1 deacetylase activity, only transiently promotes proliferation of B-MSCs. Our findings will help us understand the role of SIRT1 in the aging of normal diploid cells and may contribute to the prevention of human MSCs senescence thus benefiting MSCs-based tissue engineering and therapies.

Modification of the brain-derived neurotrophic factor gene: a portal to transform mesenchymal stem cells into advantageous engineering cells for neuroregeneration and neuroprotection

Multipotential mesenchymal stem cells (MSCs) are ideal seed cells for recruiting the loss of neural cells due to their strong proliferative capacity, easy acquisition, and considerable tolerance of genetic modifications. After transduction of brain-derived neurotrophic factor (BDNF) gene via recombinant retroviral vectors into the human MSCs, nearly 100% of cells expressed BDNF (which were therefore transformed into BNDF-MSCs) as detected by immunocytochemistry, and the quantity of BDNF in the culture medium was increased by approximately 20,000-fold. In spite of the genomic integration of an exogenous gene, BDNF-MSCs did not present any structural aberration in the chromosomes. All-trans-retinoic acid (RA) induction caused the BDNF-MSCs to differentiate into neural cells with significantly increased expressions of such neural-specific proteins as nestin, NeuN, O4, and glial fibrillary acidic protein (GFAP). The voltage-dependent K+/Ca2+ currents were recorded from the induced BDNF-MSCs using patch-clamp technique. Compared with the MSCs induced by both RA and BDNF, BDNF-MSCs survived in significantly greater number in the induction medium, and also more cells were induced into neuron-like cells (NeuN, P < 0.01) and oligodendrocyte-like cells (O4, P < 0.05). We suppose that, once engrafted into human central nervous system, the BDNF-MSCs would not only recruit the neuronal losses, but also provide, by way of paracrine, large quantities of BDNF that effectively perform the functions of neuroprotection and neuroregeneration, promoting the activation of endogenous neural stem/progenitor cells and their chemotactic migration. On the other hand, the BDNF-MSCs that can survive in the host environment and differentiate subsequently into functional mature cells may also serve as specifically targeting vectors for ex vivo gene therapy.

MicroRNA-125b attenuates epithelial-mesenchymal transitions and targets stem-like liver cancer cells through small mothers against decapentaplegic 2 and 4

Emerging evidence suggests that epithelial‐mesenchymal transitions (EMTs) play important roles in tumor metastasis and recurrence. Understanding molecular mechanisms that regulate the EMT process is crucial for improving treatment of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) play important roles in HCC; however, the mechanisms by which miRNAs target the EMT and their therapeutic potential remains largely unknown. To better explore the roles of miRNAs in the EMT process, we established an EMT model in HCC cells by transforming growth factor beta 1 treatment and found that several tumor‐related miRNAs were significantly decreased. Among these miRNAs, miR‐125b expression was most strongly suppressed. We also found down‐regulation of miR‐125b in most HCC cells and clinical specimens, which correlated with cellular differentiation in HCC patients. We then demonstrated that miR‐125b overexpression attenuated EMT phenotype in HCC cancer cells, whereas knockdown of miR‐125b promoted the EMT phenotype in vitro and in vivo. Moreover, we found that miR‐125b attenuated EMT‐associated traits, including chemoresistance, migration, and stemness in HCC cells, and negatively correlated with EMT and cancer stem cell (CSC) marker expressions in HCC specimens. miR‐125b overexpression could inhibit CSC generation and decrease tumor incidence in the mouse xenograft model. Mechanistically, our data revealed that miR‐125b suppressed EMT and EMT‐associated traits of HCC cells by targeting small mothers against decapentaplegic (SMAD)2 and 4. Most important, the therapeutic delivery of synthetic miR‐125b mimics decreased the target molecule of CSC and inhibited metastasis in the mice model. These findings suggest a potential therapeutic treatment of miR‐125b for liver cancer. Conclusion: miR‐125b exerts inhibitory effects on EMT and EMT‐associated traits in HCC by SMAD2 and 4. Ectopic expression of miR‐125b provides a promising strategy to treat HCC. (Hepatology 2015;62:801–815)

Epimorphin promotes human hepatocellular carcinoma invasion and metastasis through activation of focal adhesion kinase/extracellular signal‐regulated kinase/matrix metalloproteinase‐9 axis

The high incidence rate of hepatocellular carcinoma (HCC) is mainly the result of frequent metastasis and tumor recurrence. Unfortunately, the underlying molecular mechanisms driving HCC metastasis are still not fully understood. It has been demonstrated that tumor stroma cells contribute to primary tumor growth and metastasis. Within the HCC environment, activated hepatic stellate cells (HSCs) can release a number of molecules and enhance cancer cell proliferation and invasiveness in a paracrine manner. Here, for the first time, we demonstrate that epimorphin (EPM; also called syntaxin‐2), an extracellular protein, is strongly elevated in activated HSCs within tumor stroma. We show that knockdown of EPM expression in HSCs substantially abolishes their effects on cancer cell invasion and metastasis. Ectopic expression of EPM in HCC cancer cells enhances their invasiveness; we demonstrate that the cells expressing EPM have markedly increased metastasis potential. Furthermore, EPM‐mediated invasion and metastasis of cancer cells is found to require up‐regulation of matrix metalloproteinase‐9 (MMP‐9) through the activation of focal adhesion kinase (FAK)/extracellular signal‐regulated kinase (ERK) axis. Conclusion: Our results show that EPM, secreted by activated HSCs within HCC stroma, promotes invasion and metastasis of cancer cells by activating MMP‐9 expression through the FAK‐ERK pathway. (HEPATOLOGY 2011;)

NRSF silencing induces neuronal differentiation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent cells that have the potential to differentiate into the neuronal cell lineage. Here, we describe the highly efficient and specific induction of cells with neuronal characteristics, without glial differentiation, from human bone marrow-derived mesenchymal stem cells by NRSF silencing. Cells that have the characteristics of MSCs were obtained from human bone marrow. Lentiviral vectors were used to deliver small interference NRSF RNA (siNRSF) into MSCs. After being infected with lentivirus containing siNRSF, MSCs were successfully induced to differentiate into neuronal cells, which exhibited neuron-like morphology and formed nissl bodies. These differentiated cells expressed multiple neuron-specific genes including brain-derived neurotrophic factor (BDNF), neurogenin 1 (NGN1), neuron-specific enolase (NSE), synaptophysin (SYP), and neuron-specific growth-associated protein (SCG10), as well as expressing mature neuronal marker proteins, such as beta-tubulin III, NSE, microtubule-associated protein type 2 (MAP-2), and neurofilament-200 (NF-200), yet did not express the glial markers glial fibrillary acidic protein (GFAP) and oligodendrocyte transcription factor 2 (Olig2), as verified by immunofluorescence staining. The whole cell patch-clamp technique recorded TTX-sensitive Na(+) currents and action potential from these differentiated cells. Thus, our results demonstrate that NRSF silencing can activate some neuronal genes and induce neuronal differentiation of mesenchymal stem cells.