Repairing Effect of Mesenchymal Stem Cells on Lead Acetate-Induced Testicular Injury in Mice.

Lead acetate can cause testicular damage in males. In this study, we assessed the repairing effects of human umbilical cord mesenchymal stem cells (MSCs) on testicular injury caused by lead acetate in mice. MSCs were injected into mice with testicular injury by intraperitoneal injection, and the organ coefficient of reproductive organs, sperm motility, hormone level and antioxidant index of mice were tested. Compared with the normal group, the coefficient of reproductive organs and sperm motility were reduced in the model group, and histopathology showed obvious testicular injury, proving successful modeling. Compared with the model group, the reproductive organ coefficient and sperm motility were improved in the experimental group, and histopathology showed that the testicular injury could be significantly improved. Sex hormone secretion tends to be normal, and the antioxidant index increased. Sequencing results showed that there were 485 upregulated genes and 172 downregulated genes between the model group and the control group, and 210 upregulated genes and 482 downregulated genes between the experimental group and the model group. Differentially expressed genes are mainly concentrated in AMP-activated protein kinase (AMPK) signaling pathway, apoptosis signaling pathway, and arginine biosynthesis signaling pathway. Overall, MSCs can significantly improve the degree of damages to mice testis caused by lead acetate and have a certain repairing effect.

Mesenchymal stem cells from human umbilical cord regulate the expression of major histocompatibility complex in human neural stem cells and their lineages.

hNSCs (human neural stem cells) derived from embryonic tissue and aborted fetal brains are considered to be the most promising candidates for neurodegenerative and other CNS(central nervous system) diseases. However, the most common problem, which limited successful use of these allogeneic hNSC therapy, is immune rejection. Mesenchymal stem cells (MSCs) from human umbilical cord (hUC-MSCs) are receiving increasing attention for their immune-modulatory properties. In the current studies, we firstly investigated the immunogenecity of hNSCs as well as their lineages in cultures with the presence or absence of interferon gamma (IFNγ), a pro-inflammatory factors. Our data revealed that the majority of hNSCs and astrocytes expressed MHCI (major histocompatibility complex class I) while neurons hardly expressed MHCI (<5%) in the absence of IFNγ. In addition, neither hNSCs nor neurons expressed MHCII while a subpopulation (about 18 %) of astrocytes expressed MHCII without IFNγ stimulation. However, the addition of IFNγ in cultures significantly increased the expressions of MHCII on hNSCs and astrocytes. However, IFNγ did not affect the expression of MHCI on hNSCs and astrocytes. We then investigated whether hUC-MSCs had the capacity of regulating the immunogenecity of hNSCs as well as their lineages in a co-culture system. We found that hUC-MSCs did not affect the expression of MHCI on hNSCs and their lineages, however, these cells were able to significantly inhibit the IFNγ-induced up-regulation of MHCII on hNSCs and astrocytes (p < 0.001). Thus, our results suggest that hUC-MSCs may serve as potentially useful modulators to reduce the immunogenicity of allogeneic hNSCs in clinical application.

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat's weight (260-330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa's score, a larger infarct volume and a greater model success ratio were screened using the Longa's score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

[Isolation, Culture and Biological Characteristics of Endothelial Progenitor Cells from Cryopreserved Umbilical Cord Blood].

OBJECTIVE: To establish a novel method to isolate endothelial progenitor cells（EPC） from cryopreserved umbilical cord blood (cryoUCB), to investigate the biological characteristics of EPC and to improve the rate of EPC obtained from cryoUCB. METHODS: Twelve cryoUCB samples during 2000 to 2001 years were collected from allogeneic cord blood bank, cryoUCB was thawed rapidly in a water bath at 37 ℃, total nucleated cells (TNCs) were washed by phosphate-buffered saline (PBS). TNCs were seeded onto fibronectin-coated dishes to isolate EPC. Flow cytometry and immunofluorescence were used to identify EPC. The function of EPC was identified in vitro, such as the incorporation of Dil-Ac-LDL and FITC-UEA-I, the formation of capillary-like structure in matrigel, and the release of VEGF by ELISA. RESULTS: One to five cluster of cobble stone-like cells appeared at 2-3 weeks after seeding. Flow cytometric analysis showed that positive rates of CD31, CD34, CD144, and VEGFR (CD309) were（92.91±5.20）%, （30.0±23.27）%, （88.55±3.83）% and （67.21±12.12）% in passage 1 to passage 3 of EPC. EPC could uptake Dil-Ac-LDL and FITC-UEA-I, form capillary-like network on Matriget and release VEGF. CONCLUSION: EPC had been successfully isolated from cryopreserved umbilical cord blood by this method with high stability and reproducibility. EPC can be obtained in 85% frozen umbilical cord blood. This method may lay a foundation to supply abundant EPC for clinical application.

Dopaminergic Neuronal Differentiation from the Forebrain-Derived Human Neural Stem Cells Induced in Cultures by Using a Combination of BMP-7 and Pramipexole with Growth Factors.

Transplantation of dopaminergic (DA) neurons is considered to be the most promising therapeutic strategy for replacing degenerated dopamine cells in the midbrain of Parkinson's disease (PD), thereby restoring normal neural circuit function and slow clinical progression of the disease. Human neural stem cells (hNSCs) derived from fetal forebrain are thought to be the important cell sources for producing DA neurons because of their multipotency for differentiation and long-term expansion property in cultures. However, low DA differentiation of the forebrain-derived hNSCs limited their therapeutic potential in PD. In the current study, we explored a combined application of Pramipexole (PRX), bone morphogenetic proteins 7 (BMP-7), and growth factors, including acidic fibroblast factor (aFGF), forskolin, and phorbol-12-myristae-13-acetate (TPA), to induce differentiation of forebrain-derived hNSCs toward DA neurons in cultures. We found that DA neuron-associated genes, including Nurr1, Neurogenin2 (Ngn2), and tyrosine hydroxylase (TH) were significantly increased after 24 h of differentiation by RT-PCR analysis (p < 0.01). Fluorescent examination showed that about 25% of cells became TH-positive neurons at 24 h, about 5% of cells became VMAT2 (vascular monoamine transporter 2)-positive neurons, and less than 5% of cells became DAT (dopamine transporter)-positive neurons at 72 h following differentiation in cultures. Importantly, these TH-, VMAT2-, and DAT-expressing neurons were able to release dopamine into cultures under both of the basal and evoked conditions. Dopamine levels released by DA neurons produced using our protocol were significantly higher compared to the control groups (P < 0.01), as examined by ELISA. Our results demonstrated that the combination of PRX, BMP-7, and growth factors was able to greatly promote differentiation of the forebrain-derived hNSCs into DA-releasing neurons.

A new method to effectively and rapidly generate neurons from SH-SY5Y cells.

It is well known that neurons differentiated from SH-SY5Y cells can serve as cell models for neuroscience research; i.e., neurotoxicity and tolerance to morphine in vitro. To differentiate SH-SY5Y cells into neurons, RA (retinoic acid) is commonly used to produce the inductive effect. However, the percentage of neuronal cells produced from SH-SY5Y cells is low, either from the use of RA treatment alone or from the combined application of RA and other chemicals. In the current study, we used CM-hNSCs (conditioned medium of human neural stem cells) as the combinational inducer with RA to prompt neuronal differentiation of SH-SY5Y cells. We found that neuronal differentiation was improved and that neurons were greatly increased in the differentiated SH-SY5Y cells using a combined treatment of CM-hNSCs and RA compared to RA treatment alone. The neuronal percentage was higher than 80% (about 88%) on the 3rd day and about 91% on the 7th day examined after a combined treatment with CM-hNSCs and RA. Cell maturation and neurite growth of these neuronal cells were also improved. In addition, the use of CM-hNSCs inhibited the apoptosis of RA-treated SH-SY5Y cells in culture. We are the first to report the use of CM-hNSCs in combination with RA to induce neuronal differentiation of RA-treated SH-SY5Y cells. Our method can rapidly and effectively promote the neuronal production of SH-SY5Y cells in culture conditions.

[Expansion and function of MHC restricted killer T cells derived from umbilical cord blood].

OBJECTIVE: This study was to expand the cytotoxic T lymphocytes (CTL) through inducing the differentiation of umbilical blood monomuclear cells (UBMNC) by using various combination of cytokines, and to investigate the functions of expanded CTL. METHODS: The MNC were isolated by ficoll density gradient centrifugation. Then, the PHA-P, IFN-γ combined with IL-2, IL-15 and other cytokines were used for induction and expansion of the cord blood-derived CTL. The biological function of CTL was examined by phenotype analysis, cytotoxic tests and real-time fluorescence quantitative PCR. RESULTS: After expansion for 15 days, the cell number increased by 1522% ± 137%. The content of CD3(-)CD8(-) cells in uncultured cord blood MNC was 95%, and the CD3(+)CD8(+) CTL cells reached 82.77% in cultured cord blood MNC after expansion for 15 days. The expanded CTL cell showed the cytotoxic activity against K562 and HeLa cell line. The killing rate of MNC was 61.88 ± 1.08%. After expansion, the killing rate could reach to 90% with the average value of 90.33 ± 2.02%. The expanded CTL cells highly expressed some key cytokines, such as granzyme A, granzyme B, GM-CSF, granulysin, IFN-γ, TGF-ß, TNF-α and perforin. Compared with the control group, the expression of IFN-γ and TGF-ß significantly increased (P < 0.05), and the other factors dramatically increased (P < 0.01). CONCLUSION: The cord blood-derived CTL can be expanded by different combinations of cytokines. These protocols may provide alternative choices for CTL cell expansion in tumor adoptive immunotherapy.

[Gene expression profile changes induced upon umbilical cord mesenchymal cell infusion therapy in a rat model of hepatic cirrhosis].

OBJECTIVE: To investigate changes in gene expression that occur upon treatment with human umbilical cord mesenchymal stem cells (UC-MSCs) for hepatic cirrhosis using a rat model system. METHODS: Hepatic cirrhosis was induced in Sprague-Dawley rats by subcutaneous injection of carbon tetrachloride and oral administration of alcohol.UC-MSCs were isolated from human umbilical cord and the cells' immunophenotype and differentiation towards osteogenic and adipogenic lineages were confirmed.The UC-MSC sample or vehicle alone (phosphate buffered saline, PBS) was transplanted by intravenous injection.Histopathological staining and serological testing were used to compare the liver morphology and function among the different groups.The gene expression in the PBS group and UC-MSC group were detected by gene microarray and differences between the groups were statistically analyzed by t-test. RESULTS: Transplantation of the UC-MSCs improved liver function in the hepatic cirrhosis rats.Comparison of the gene expression profiles of the PBS group and the UC-MSC group showed that the latter had up-regulation of the genes related to the complement and coagulation cascades and down-regulation of the genes related to cell proliferation, cell cycle, and collagen synthesis. CONCLUSION: UC-MSC therapy might improve liver function in cirrhosis by increasing the expression of genes related to the complement and coagulation cascades and by decreasing genes involved in cell proliferation and collagen deposition.

[IFN-γ stimulation enhances immunosuppressive capability of human umbilical cord mesenchymal stem cells].

This study was objective to explore the effect of IFN-γ on immunosuppressive capability of mesenchymal stem cells (MSC) derived from umbilical cord. The immunomodulating capability of MSC was changed by stimulating cell surface receptors like Toll-like receptors (TLR). The inhibition of T-lymphocyte proliferation by MSC was tested via cell co-cultures. Further RT-PCR and ELISA were performed to examine the expression changes in gene and protein level. The results showed that the IFN-γ could promote the immunosuppressive effect of umbilical cord derived MSC. IFN-γ-stimulated MSC could suppress the proliferation of T cells more effectively. IFN-γ stimulation up-regulated the expression of immunosuppressive genes like IDO1, COX2, HLA-G, and soluble suppressive proteins such as HLA-G, KYN, IL10, PGE2 of MSC. And the immuno suppression capability of IFN-γ-stimulated MSC was 2-7 folds higher than control in MSC and lymphocyte co-culture tests. It is concluded that IFN-γ can effectively enhance the immunosuppressive capability of MSC.