Xue Nan

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.

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.

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)

A critical challenge: dosage-related efficacy and acute complication intracoronary injection of autologous bone marrow mesenchymal stem cells in acute myocardial infarction.

BACKGROUND Previous studies showed improvement in heart function by injecting bone marrow mesenchymal stem cells (BMSCs) after AMI. Emerging evidence suggested that both the number and function of BMSCs decline with ageing. We designed a randomized, controlled trial to further investigate the safety and efficacy of this treatment. METHODS Patients with ST-elevation AMI undergoing successful reperfusion treatment within 12 hours were randomly assigned to receive an intracoronary infusion of BMSCs (n=21) or standard medical treatment (n=22) (the numbers of patients were limited because of the complication of coronary artery obstruction). RESULTS There is a closely positive correlation of the number and function of BMSCs vs. the cardiac function reflected by LVEF at baseline (r=0.679, P=0.001) and at 12-month follow-up (r=0.477, P=0.039). Six months after cell administration, myocardial viability within the infarct area by 18-FDG SPECT was improved in both groups compared with baseline, but no significant difference in the BMSCs compared with control groups (4.0±0.4% 95%CI 3.1-4.9 vs. 3.2±0.5% 95%CI 2.1-4.3, P=0.237). 99mTc-sestamibi SPECT demonstrated that myocardial perfusion within the infarct area in the BMSCs did not differ from the control group (4.4±0.5% 95%CI 3.2-5.5 vs. 3.9±0.6% 95%CI 2.6-5.2, P=0.594). Similarly, LVEF after 12 and 24 months follow-up did not show any difference between the two groups. In the BMSCs group, one patient suffered a serious complication of coronary artery occlusion during the BMSCs injection procedure. CONCLUSIONS The clinical benefits of intracoronary injection of autologous BMSCs in acute STEMI patients need further investigation and reevaluation.

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.

Superior osteogenic capacity of different mesenchymal stem cells for bone tissue engineering

OBJECTIVE We evaluated the effect of human bone marrow stromal cells (hBMSCs), human adipose tissue-derived mesenchymal stem cells (hAD-MSCs), and umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in bone tissue engineering and identified a reliable cell source. STUDY DESIGN Alkaline phosphatase (ALP) activity and quantitative polymerase chain reaction were used to evaluate osteogenic in vitro, X-ray and histologic analysis in vivo. RESULTS hBMSCs exhibited strongest ALP staining, followed by hAD-MSCs and hUC-MSCs. At 7 days, hUC-MSCs and hAD-MSCs had higher expression of collagen type I and Runt-related transcription factor 2 than hBMSCs, and hUC-MSCs showed higher osteopontin expression. Bone structure was observed in the hUC-MSC group. Defects showed good healing in the hBMSC and hAD-MSC groups. Enhanced green fluorescent protein and osteopontin were detected in newly formed bone at 8 weeks. CONCLUSIONS Our results suggested that hUC-MSCs and hAD-MSCs could be used for bone tissue engineering effectively; hUC-MSCs could serve as a new alternative cell source.

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.