Yanhua Li

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)

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.

Overexpression of SPINDLIN1 induces cellular senescence, multinucleation and apoptosis.

Human or mouse Spindlin1 is expressed in various tissues and cells, but its biological functions are poorly understood. In this study, we show that human SPINDLIN1 is localized to interphase nucleus and mitotic chromosomes, and its expression in HeLa cells is not regulated in a cell cycle-dependent manner. When SPINDLIN1 is stably overexpressed in HeLa cells, it results in multinucleation of cells, and these multinucleated cells exhibits characteristic features of senescence and apoptosis shown by growth and morphological alterations, beta-galactosidase activity, and Annexin V/7-Aminoactinomycin D staining. Mouse Spindlin1 is highly homologous with human Spindlin1, when overexpressed in NIH3T3 cells, it also induces multinucleation, senescence and apoptosis in murine cells. Our results demonstrate that SPINDLIN1 is an important gene for mammalian mitotic chromosome functions, and disrupted regulation results in abnormal cell division, a mechanism that may be involved in tumorigenesis.

Induction of umbilical cord blood–derived β2m−c-Met+ cells into hepatocyte-like cells by coculture with CFSC/HGF cells

Several studies have indicated that adult stem cells derived from bone marrow (BM) and cord blood (CB) can differentiate into hepatocyte‐like cells. This ability is important for the treatment of hepatic diseases with BM or CB as a potential approach. However, methods are still being developed for the efficient induction of stem cell differentiation and expansion to get enough cells to be useful. In the present study, we enriched a subset of umbilical cord blood β2m−c‐Met+ cells (UCBCCs) and investigated the combination effect of liver nonparenchymal cells (cirrhotic fat‐storing cells [CFSCs]) and hepatocyte growth factor (HGF) on the induction of UCBCCs into hepatocyte‐like cells. UCBCCs were cocultured with CFSC/HGF feeder layers either directly or separately using insert wells. Flow cytometric analysis showed that most UCBCCs were CD34+/−CD90+/−CD49f+CD29+Alb+AFP+. After cocultured with transgenic feeder layers for 7 days, UCBCCs displayed some morphologic characteristics of hepatocytes. Reverse‐transcription polymerase chain reaction (RT‐PCR) and immunofluorescence cell staining proved that the induced UCBCCs expressed several hepatocyte specific genes including AFP, Alb, CYP1B1 and cytokeratins CK18 and CK19. Furthermore, the induced cells displayed liver specific functions of indocyanine green (ICG) uptake, ammonium metabolism and albumin secretion. Hence, our data have demonstrated that UCBCCs might represent a novel subpopulation of CB‐derived stem/progenitor cells capable of successful differentiation into hepatocyte‐like cells when incubated with CFSC/HGF cells. In conclusion, not only HGF but also CFSCs and/or the secreted extracellular matrix (ECM) have been shown to be able to serve as essential microenvironment for hepatocyte differentiation. (Liver Transpl 2005;11:635–643.)

Small molecule Me6TREN mobilizes hematopoietic stem/progenitor cells by activating MMP-9 expression and disrupting SDF-1/CXCR4 axis

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into the blood circulation has been widely used for hematopoietic transplantation. However, the current methods of cytokine- or small-molecule-stimulated HSPC mobilization are far from satisfactory. New mobilizing agents are needed to increase the number of stem cells in peripheral blood for effective reconstitution of hematopoiesis. Here, we report that the molecule Me6TREN (Me6) can induce rapid mobilization of hematopoietic progenitor cells and that Me6 exhibits more significant effects than granulocyte colony-stimulating factor (G-CSF) or AMD3100. Me6 also mobilizes long-term repopulating cells, which successfully engraft and expand in a multilineage fashion in primary and secondary transplant recipients. Mechanistically, Me6 inhibits both the SDF-1α-induced migration and VLA-4-mediated adhesion of mouse and human hematopoietic cells. Me6 appears to mobilize HSPCs by activating MMP-9 expression and disrupting the SDF-1α/CXCR4 axis. Therefore, Me6 may become a new potent and efficacious mobilizing agent of HSPCs.

Infusion of Megakaryocytic Progenitor Products Generated from Cord Blood Hematopoietic Stem/Progenitor Cells: Results of the Phase 1 Study

Background Currently, a constant shortage in the supply of platelets has become an important medical and society challenge, especially in developing country, and the in vitro production of megakaryocytic progenitor cells (MPs) from cord blood could represent an effective platelet substitute. In the present study, our objective was to determine the safety and feasibility of ex vivo generated MPs in patients. Methods and Findings MPs were produced and characterized from cord blood mononuclear cells under a serum free medium with cytokines. We investigated the feasibility of expansion and infusion of cord blood-derived MPs in 24 patients with advanced hematological malignancyes. The primary end point was the safety and tolerability of the infusion of cord blood-derived MPs. No adverse effects were observed in patients who received ex vivo-generated cells at concentrations of up to a median value of 5.45×106cells/kg of body weight. With one year follow-up, acute and chronic GVHD had not been observed among patients who received MPs infusion, even without ABO blood group and HLA typing matching. Conclusions These initial results in patients are very encouraging. They suggest that infusion of cord blood-derived MPs appears safe and feasible for treatment of thrombocytopenia. Trial Registration www.chictr.org ChiCTR-TCH-09000333.

Neuronal restrictive silencing factor silencing induces human amniotic fluid-derived stem cells differentiation into insulin-producing cells.

Islet cell replacement represents the most promising approach for the treatment of type I diabetes. However, it is limited by a shortage of pancreas donors. Here, we report that human amniotic fluid-derived stem cells (hAFSCs) can be induced to differentiate into functional insulin-producing cells by knocking down neuronal restrictive silencing factor (NRSF). In this study, lentiviral vectors were used to deliver small interference NRSF (siNRSF) RNA into hAFSCs. After infection with lentivirus containing siNRSF, hAFSCs were successfully induced to differentiate into insulin-producing cells. The differentiated siNRSF-hAFSCs expressed genes specific for islet cells, such as Pdx1, Hnf4α, Isl-1, Nkx6.1, Insulin, and Glut2. These cells also produced and released C-peptide in a glucose-responsive manner. These findings indicated that hAFSCs could be induced to differentiate into insulin-producing β-like cells by NRSF silencing.

Regulatory role of neuron-restrictive silencing factor in the specific expression of cocaine- and amphetamine-regulated transcript gene

Cocaine‐ and amphetamine‐regulated transcript (CART) peptide is an endogenous peptide which is widely expressed in the CNS and PNS as well as in endocrine cells. Despite the functional knowledge about CART, the mechanisms that regulate CART gene transcription are poorly characterized. Here, we showed that neuron‐restrictive silencer factor (NRSF) functions as a negative regulator of CART gene expression in neuroendocrine cells. A putative neuron‐restrictive silencer element (NRSE) conserved between the rodent and human CART promoter was identified and demonstrated to bind to NRSF in sequence‐specific manner by the electrophoretic mobility shift and chromatin immunoprecipitation assays. Ectopic expression of NRSF in pheochromocytoma cells (PC12) and insulin‐secreting cells (INS‐1) induced a marked reduction in the level of CART mRNA and the activity of CART promoter or NRSE reporter. The CART promoter showed very low activity in endogenous NRSF‐expressing HeLa cells. When expression of NRSF was down‐regulated in HeLa cells using a RNA interfering technique, the transcriptional activity of the CART promoter or a NRSE reporter was significantly increased. Taken together, our data suggested that CART gene expression in neuroendocrine cells is strictly controlled by NRSF, via a mechanism dependent upon the CART NRSE.

In Vitro Large Scale Production of Human Mature Red Blood Cells from Hematopoietic Stem Cells by Coculturing with Human Fetal Liver Stromal Cells

In vitro models of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 109-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitive β-globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.