Lijuan He

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)

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)

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.

Human Fetal Liver Stromal Cells That Overexpress bFGF Support Growth and Maintenance of Human Embryonic Stem Cells

In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs) we isolated human fetal liver stromal cells (hFLSCs) from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days). Basic fibroblast growth factor (bFGF) is known to play an important role in promoting self-renewal of human embryonic stem (hES) cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells — bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2), and transforming growth factor β (TGF-β), thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

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.

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.

Recombinant human thrombopoietin promotes hematopoietic reconstruction after severe whole body irradiation.

Recombinant human thrombopoietin (rHuTPO) is a drug that is used clinically to promote megakaryocyte and platelet generation. Here, we report the mitigative effect of rHuTPO (administered after exposure) against severe whole body irradiation in mice. Injection of rHuTPO for 14 consecutive days following exposure significantly improved the survival rate of lethally irradiated mice. RHuTPO treatment notably increased bone marrow cell density and LSK cell numbers in the mice after sub-lethal irradiation primarily by promoting residual HSC proliferation. In lethally irradiated mice with hematopoietic cell transplantation, rHuTPO treatment increased the survival rate and enhanced hematopoietic cell engraftment compared with the placebo treatment. Our observations indicate that recombinant human TPO might have a therapeutic role in promoting hematopoietic reconstitution and HSC engraftment.

A novel molecule Me6TREN promotes angiogenesis via enhancing endothelial progenitor cell mobilization and recruitment

Critical limb ischaemia is the most severe clinical manifestation of peripheral arterial disease. The circulating endothelial progenitor cells (EPCs) play important roles in angiogenesis and ischemic tissue repair. The increase of circulating EPC numbers by using mobilization agents is critical for obtaining a better therapeutic outcome in patients with ischemic disease. Here, we firstly report a novel small molecule, Me6TREN (Me6), can efficiently mobilize EPCs into the blood circulation. Single injection of Me6 induced a long-lasting increase in circulating Flk-1+ Sca-1+ EPC numbers. In a mouse hind limb ischemia (HLI) model, local intramuscular transplantation of these Me6-mobilized cells accelerated the blood flow restoration in the ischemic muscles. More importantly, systemic administration of Me6 notably increased the capillary density, arteriole density and regenerative muscle weight in the ischemic tissue of HLI. Mechanistically, we found Me6 reduced stromal cell-derived factor-1α level in bone marrow by up-regulation of matrix metallopeptidase-9 expression, which allowed the dissemination of EPCs into peripheral blood. These data indicate that Me6 may represent a potentially useful therapy for ischemic disease via enhancing autologous EPC recruitment and promote angiogenesis.

Differentiation of adipose-derived adult stem cells into epithelial-like stem cells

Adipose-derived adult stem (ADAS) cells can be easily obtained in large quantities. Previous studies have suggested that all-trans-retinoic acid (ATRA) plays an important role in the differentiation of mesenchymal stem cells toward an epithelial lineage. In order to verify that ADAS-cells can differentiate into an epithelial lineage retaining most of the characteristics of stem cells, ADAS-cells were isolated and cultured. They were induced to differentiate toward an epithelial lineage in vitro. Differentiated epithelial cells were assayed as to whether they retain characteristics of stem cells by RT-PCR and cell cycle stage analysis, and were further induced to differentiate toward an osteogenic lineage. RT-PCR analysis revealed that no CK5, CK10 or CK19 mRNA was detected in ADAS-cells, CK19 but not CK5 or CK10 mRNA was detected in differentiated cells at passage 1, CK10 and CK19 expression but not CK5 mRNA was detected in differentiated cells at passage 10. After induction, the expression of CK19 was observed by immunofluorescent staining. Positive staining with alkaline phosphatase (ALP) and Von Kossa staining verified that differentiated epithelial cells still had potential to further differentiate toward an osteogenic lineage. These experiments provide proof that ADAS-cells can differentiate into an epithelial lineage retaining most of the characteristics of stem cells.