Cixian Bai

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.

Structure of human spindlin1. Tandem tudor-like domains for cell cycle regulation

Spindlin1, a meiotic spindle-binding protein that is highly expressed in ovarian cancer cells, was first identified as a gene involved in gametogenesis. It appeared to be a target for cell cycle-dependent phosphorylation and was demonstrated to disturb the cell cycle. Here we report the crystal structure of human spindlin1 to 2.2Å of resolution, representing the first three-dimensional structure from the spin/ssty (Y-linked spermiogenesis-specific transcript) gene family. The refined structure, containing three repeats of five/four anti-parallel β-strands, exhibits a novel arrangement of tandem Tudor-like domains. Two phosphate ions, chelated by Thr-95 and other residues, appear to stabilize the long loop between domains I and II, which might mediate the cell cycle regulation activity of spindlin1. Flow cytometry experiments indicate that cells expressing spindlin1 display a different cell cycle distribution in mitosis, whereas those expressing a T95A mutant, which had a great decrease in phosphorous content, have little effect on the cell cycle. We further identified associations of spindlin1 with nucleic acid to provide a biochemical basis for its cell cycle regulation and other functions.

Self‐renewal and pluripotency is maintained in human embryonic stem cells by co‐culture with human fetal liver stromal cells expressing hypoxia inducible factor 1α

Human embryonic stem (hES) cells are typically maintained on mouse embryonic fibroblast (MEF) feeders or with MEF‐conditioned medium. However, these xenosupport systems greatly limit the therapeutic applications of hES cells because of the risk of cross‐transfer of animal pathogens. The stem cell niche is a unique tissue microenvironment that regulates the self‐renewal and differentiation of stem cells. Recent evidence suggests that stem cells are localized in the microenvironment of low oxygen. We hypothesized that hypoxia could maintain the undifferentiated phenotype of embryonic stem cells. We have co‐cultured a human embryonic cell line with human fetal liver stromal cells (hFLSCs) feeder cells stably expressing hypoxia‐inducible factor‐1 alpha (HIF‐1α), which is known as the key transcription factor in hypoxia. The results suggested HIF‐1α was critical for preventing differentiation of hES cells in culture. Consistent with this observation, hypoxia upregulated the expression of Nanog and Oct‐4, the key factors expressed in undifferentiated stem cells. We further demonstrated that HIF‐1α could upregulate the expression of some soluble factors including bFGF and SDF‐1α, which are released into the microenvironment to maintain the undifferentiated status of hES cells. This suggests that the targets of HIF‐1α are secreted soluble factors rather than a cell–cell contact mechanism, and defines an important mechanism for the inhibition of hESCs differentiation by hypoxia. Our findings developed a transgene feeder co‐culture system and will provide a more reliable alternative for future therapeutic applications of hES cells. J. Cell. Physiol. 221: 54–66, 2009.

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.

Overexpression of spindlin1 induces metaphase arrest and chromosomal instability

Spin/Ssty gene family is high conserved and very abundant transcript involved in gametogenesis, which was repeatedly detected in early embryo. Nevertheless, the biologic roles of the members are still largely unknown. Previously we have identified human gene spindlin1 as a homologue of the family from ovarian cancer cells, and reported that stable overexpression of spindlin1 could transform NIH3T3 cells and induce tumorigenesis in nude mouse. Here, we showed that spindlin1, as a nuclear protein, was relocated during mitosis. A fraction of spindlin1 proteins was dynamic distributed along mitotic spindle tubulin and enriched at midzone following anaphase entering. We also showed that transient overexpression of spindlin1 induced cell cycle delay in metaphase, caused mitotic spindle defects, and resulted in chromosome instability, micronucleus and multinuclear giant cells formation. Moreover, time‐lapse microscopy revealed that these cells arrested at metaphase for more than 3 h with chromosome nondisjunction or missegregation. Furthermore, Mad2 up‐regulation in these cells suggested that overexpression of spindlin1 may affect the bipolar spindle correctly attachment to chromosomes and activate spindle checkpoint. Taken together, these data demonstrated that excess spindlin1 protein may be detrimental for spindle microtubule organization, chromosomal stability and can potentially contribute to the development of cancer. J. Cell. Physiol. 217: 400–408, 2008.

Lineage Restriction and Differentiation of Human Embryonic Stem Cells into Hepatic Progenitors and Zone 1 Hepatocytes

Human embryonic stem (hES) cells can self-renew, which enables them to have considerable expansion potential, and are pluripotent. If their differentiation can be controlled, they can offer promise for clinical programs in cell therapies. A novel strategy has been developed to derive early hepatocytic lineage stages from hES cells using four sequential inducing steps lasting 16 days. First, embryoid bodies (EBs) were generated by growing hES cells in suspension for 2 days; second, EBs were lineage restricted to definitive endoderm with 3 days of treatment with human activin A; third, cells were differentiated further by coculturing for 5 days with human fetal liver stromal cells (hFLSCs) made transgenic to stably release basic fibroblast growth factor (bFGF); fourth, treating them for 6 days with soluble signals comprised of hFLSC-derived bFGF, hepatocyte growth factor, oncostatin M, and dexamethasone. Induced cells displayed morphological, immunohistochemical, and biochemical characteristics of hepatocytic committed progenitors and of early lineage stage hepatocytes found in zone 1 of the liver acinus. They expressed alpha-fetoprotein, albumin, cytokeratin 18, glycogen, a fetal P450 isoform, and CYP1B1, and demonstrated indocyanine green uptake and excretion. In conclusion, we have developed a novel method to lineage restrict hES cells into early lineage stages of hepatocytic fates.

Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells

To explore the feasibility of repairing clinical cutaneous deficiency, autogenic bone marrow mesenchymal stem cells (BMSCs) were isolated and differentiated into epidermal cells and fibroblasts in vitro supplemented with different inducing factors and biomaterials to construct functional tissueengineered skin. The results showed that after 72 h induction, BMSCs displayed morphologic changes such as typical epidermal cell arrangement, from spindle shape to round or oval; tonofibrils, melanosomes and keratohyaline granules were observed under a transmission electronic microscope. The differentiated cells expressed epidermal stem cell surface marker CK19 (59.66% ± 4.2%) and epidermal cells differentiation marker CK10. In addition, the induced epidermal cells acquired the anti-radiation capacity featured by lowered apoptosis following exposure to UVB. On the other hand, the collagen microfibrils deposition was noticed under a transmission electronic microscope after differentiating into dermis fibroblasts; RT-PCR identified collagen type I mRNA expression in differentiated cells; radioimmunoassay detected the secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) (up to 115.06 pg/mL and 0.84 ng/mL, respectively). Further in vivo implanting BMSCs with scaffold material shortened skin wound repair significantly. In one word, autogenic BMSCs have the potential to differentiate into epidermal cells and fibroblasts in vitro, and show clinical feasibility acting as epidermis-like and dermis-like seed cells in skin engineering.

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.

Cells Extract from Fetal Liver Promotes the Hematopoietic Differentiation of Human Embryonic Stem Cells

Here, we have now developed a new inducing system to promote the differentiation of human stem cells (hESCs) toward hematopoietic lineages by the treatment with cells extract of human fetal liver tissue (hFLT). The embryoid bodies (EBs) obtained from human H1 embryonic stem cells were exposed to buffer, hFLT cells extract, heated hFLT cell extract, and cell extract of human liver cells lines-LO2. Then, the feature of EBs in different groups was characterized by real-time RT-PCR and colony-forming assays. The results showed the treatment by hFLT cells extract could activate the hematopoietic genes expression and improve the capacity for hematopoietic progenitor development of hEBs. After that, we cocultured hFLT extract treated hEBs on the hFLSCs (human fetal liver stromal cells) feeder to differentiate them into hematopoietic cells. As a control, untreated hEBs were cocultured on hFLSCs feeder with cytokines. The feature of induced cells from hEBs was characterized by flow cytometry, Wright-Giemsa staining, and colony-forming assays. The results demonstrated that hFLT cells extract was capable of inducing hEBs into hematopoietic cells and combing it with hFLSCs feeder could largely promote hematopoietic differentiation of hESCs. This method may supply a new way to substitute the cytokines required in hematopoietic induction of hESCs.

Legume Lectin FRIL Preserves Neural Progenitor Cells in Suspension Culture In Vitro

In vitro maintenance of stem cells is crucial for many clinical applications. Stem cell preservation factor FRIL (Flt3 receptor-interacting lectin) is a plant lectin extracted from Dolichos Lablab and has been found preserve hematopoietic stem cells in vitro for a month in our previous studies. To investigate whether FRIL can preserve neural progenitor cells (NPCs), it was supplemented into serum-free suspension culture media. FRIL made NPC grow slowly, induced cell adhesion, and delayed neurospheres formation. However, FRIL did not initiate NPC differentiation according to immunofluorescence and semiquantitive RT-PCR results. In conclusion, FRIL could also preserve neural progenitor cells in vitro by inhibiting both cell proliferation and differentiation.