Human Umbilical Cord Mesenchymal Cells Research Articles

Plasma Redox Balance in Advanced-Maternal-Age Pregnant Women and Effects of Plasma on Umbilical Cord Mesenchymal Stem Cells.

Pregnancy at advanced maternal age (AMA) is a condition of potential risk for the development of maternal-fetal complications with possible repercussions even in the long term. Here, we analyzed the changes in plasma redox balance and the effects of plasma on human umbilical cord mesenchymal cells (hUMSCs) in AMA pregnant women (patients) at various timings of pregnancy. One hundred patients and twenty pregnant women younger than 40 years (controls) were recruited and evaluated at various timings during pregnancy until after delivery. Plasma samples were used to measure the thiobarbituric acid reactive substances (TBARS), glutathione and nitric oxide (NO). In addition, plasma was used to stimulate the hUMSCs, which were tested for cell viability, reactive oxygen species (ROS) and NO release. The obtained results showed that, throughout pregnancy until after delivery in patients, the levels of plasma glutathione and NO were lower than those of controls, while those of TBARS were higher. Moreover, plasma of patients reduced cell viability and NO release, and increased ROS release in hUMSCs. Our results highlighted alterations in the redox balance and the presence of potentially harmful circulating factors in plasma of patients. They could have clinical relevance for the prevention of complications related to AMA pregnancy.

Multiomics reveal human umbilical cord mesenchymal stem cells improving acute lung injury via the lung-gut axis.

Acute lung injury (ALI) and its final severe stage, acute respiratory distress syndrome, are associated with high morbidity and mortality rates in patients due to the lack of effective specific treatments. Gut microbiota homeostasis, including that in ALI, is important for human health. Evidence suggests that the gut microbiota improves lung injury through the lung-gut axis. Human umbilical cord mesenchymal cells (HUC-MSCs) have attractive prospects for ALI treatment. This study hypothesized that HUC-MSCs improve ALI via the lung-gut microflora. To explore the effects of HUC-MSCs on lipopolysaccharide (LPS)-induced ALI in mice and the involvement of the lung-gut axis in this process. C57BL/6 mice were randomly divided into four groups (18 rats per group): Sham, sham + HUC-MSCs, LPS, and LPS + HUC-MSCs. ALI was induced in mice by intraperitoneal injections of LPS (10 mg/kg). After 6 h, mice were intervened with 0.5 mL phosphate buffered saline (PBS) containing 1 × 106 HUC-MSCs by intraperitoneal injections. For the negative control, 100 mL 0.9% NaCl and 0.5 mL PBS were used. Bronchoalveolar lavage fluid (BALF) was obtained from anesthetized mice, and their blood, lungs, ileum, and feces were obtained by an aseptic technique following CO2 euthanasia. Wright's staining, enzyme-linked immunosorbent assay, hematoxylin-eosin staining, Evans blue dye leakage assay, immunohistochemistry, fluorescence in situ hybridization, western blot, 16S rDNA sequencing, and non-targeted metabolomics were used to observe the effect of HUC-MSCs on ALI mice, and the involvement of the lung-gut axis in this process was explored. One-way analysis of variance with post-hoc Tukey's test, independent-sample Student's t-test, Wilcoxon rank-sum test, and Pearson correlation analysis were used for statistical analyses. HUC-MSCs were observed to improve pulmonary edema and lung and ileal injury, and decrease mononuclear cell and neutrophil counts, protein concentrations in BALF and inflammatory cytokine levels in the serum, lung, and ileum of ALI mice. Especially, HUC-MSCs decreased Evans blue concentration and Toll-like receptor 4, myeloid differentiation factor 88, p-nuclear factor kappa-B (NF-κB)/NF-κB, and p-inhibitor α of NF-κB (p-IκBα)/IκBα expression levels in the lung, and raised the pulmonary vascular endothelial-cadherin, zonula occludens-1 (ZO-1), and occludin levels and ileal ZO-1, claudin-1, and occludin expression levels. HUC-MSCs improved gut and BALF microbial homeostases. The number of pathogenic bacteria decreased in the BALF of ALI mice treated with HUC-MSCs. Concurrently, the abundances of Oscillospira and Coprococcus in the feces of HUS-MSC-treated ALI mice were significantly increased. In addition, Lactobacillus, Bacteroides, and unidentified_Rikenellaceae genera appeared in both feces and BALF. Moreover, this study performed metabolomic analysis on the lung tissue and identified five upregulated metabolites and 11 downregulated metabolites in the LPS + MSC group compared to the LPS group, which were related to the purine metabolism and the taste transduction signaling pathways. Therefore, an intrinsic link between lung metabolite levels and BALF flora homeostasis was established. This study suggests that HUM-MSCs attenuate ALI by redefining the gut and lung microbiota.

Recent Advances in InVitro Maturation (IVM): A Systematic Literature Review

In Vitro Maturation (IVM) is one of the Assisted Reproductive Technologies (ART) that involves the retrieval of immature oocytes from antral follicles in the ovaries, either in a non-stimulated or minimally stimulated state. IVM has drawn the attention of fertility specialists due to its reliability, cost-effectiveness, low risk of Ovarian Hyperstimulation Syndrome (OHSS), and acceptable clinical pregnancy rates Objective: Understanding the Process and Factors Influencing In Vitro Maturation of Human Oocytes (IVM) Method: The research methodology employed in this study is the Systematic Literature Review (SLR) method. Data was collected by documenting all articles based on predetermined inclusion and exclusion criteria. A total of 10 national and international journal articles were utilized for this study, obtained from databases such as Google Scholar, PubMed, and Science Direct, using specific keywords including "In Vitro Maturation" and "human oocyte maturation". The literature search and study selection process followed the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Results: There were no instances of moderate-severe OHSS (ovarian hyperstimulation syndrome) in the IVM group. The fertilization rate in the group receiving growth hormone (GH) was 12.8% higher compared to the control group. Additionally, the blastocyst rate in the GH group was 9.5% higher than that of the control group. Treatment with a concentration of 50 µmol/L ALA (alpha-lipoic acid) significantly accelerated oocyte maturation. It resulted in a significantly higher mitochondrial DNA (mtDNA) copy number in mature oocytes compared to the control group (0 µmol/L ALA). The highest maturation rates were observed in human umbilical cord mesenchymal cells (hUCM) in the in vitro maturation (fIVM) of germinal vesicle (GV) oocytes, while the lowest maturation rates were observed using alpha-Minimum Essential Medium (α-MEM) in the in vitro maturation (vIVM) of GV oocytes. When AMH (anti-Müllerian hormone) was added to the IVM medium, a maturation rate of 100% was achieved in mature oocytes. The inclusion of 50 mmol/L CoQ10 in IVM media was found to reduce the level of oocyte aneuploidy by nearly 50%. The formation rate of metaphase II (MII) stage oocytes was higher in culture medium containing 1.0 mm resveratrol compared to the control group. Subsequently, a total of eight cleavage-stage embryos were frozen after intracytoplasmic sperm injection (ICSI). One year later, when the patient returned for the transfer of cryopreserved embryos, two embryos were thawed and transferred into the uterus. The patient successfully became pregnant, and the twin pregnancy progressed without complications, delivering two healthy full-term baby boys. Conclusion: The occurrence of OHSS was not observed in any of the IVM intervention groups. Supplementation with growth hormone (GH), alpha-lipoic acid (ALA), umbilical cord-derived mesenchymal stem cells (CM-MSC), anti-Müllerian hormone (AMH), and resveratrol in the culture media each demonstrated a significant improvement in human oocyte maturation. Furthermore, resveratrol and coenzyme Q10 were effective in addressing abnormal spindle morphology, irregular chromosome arrangement, and high postmeiotic aneuploidy. It is worth noting that IVM is not exclusively limited to patients with polycystic ovary syndrome (PCOS), as it can yield satisfactory outcomes in patients with autoimmune premature ovarian insufficiency (POI).

Exosome Derived from Human Umbilical Cord Mesenchymal Cell Exerts Immunomodulatory Effects on B Cells from SLE Patients.

Excessive proliferation and activation of B cells, resulting in the production of various autoantibodies, is a crucial link and significant feature of the pathogenesis of systemic lupus erythematosus (SLE), as well as the pathological basis of systemic multiorgan damage. However, whether exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-Exo) are involved in the immune regulation of SLE has not been clarified. Therefore, our study aimed to investigate the efficacy of hucMSCs-Exo for treating SLE. hucMSCs-Exo and peripheral blood mononuclear cells (PBMCs) of SLE patients were cocultured in vitro, and B cell apoptosis, activation, proliferation, and inflammation levels were detected by flow cytometry. Subsequently, the expression level of miR-155 in B lymphocytes of SLE patients was detected by qRT-PCR, and the target gene relationship between miR-155 and SHIP-1 was found through bioinformatics and dual luciferase activity experiments, which verified the inhibition of miR-155 in B lymphocytes of SLE patients to regulate immunity. We found that hucMSCs-Exo promoted B cell apoptosis, prevented B cell overactivation, and reduced inflammation. MicroRNA-155 (miR-155) has a powerful regulatory function in B cells. It was demonstrated that hucMSCs-Exo acts synergistically with miR-155 inhibitors to target SHIP-1 to B cells more effectively than exosomes alone. Our results provide insight into how hucMSCs-Exo regulates autoimmunity in patients with lupus and suggest targeting miR-155 for autoimmunity while protecting immunity.

Inhibitory effect of exosomes secreted by human umbilical cord mesenchymal stem cells on apoptosis of oxygen-glucose deprived reoxygenation model of venous endothelial cells

Objective To explore the inhibitory effect of exosomes secreted by human umbilical cord mesenchymal stem cells(HUCMSC) on apoptosis of human umbilical vein endothelial cells(HUVEC) after model group(oxygen-glucose deprivation reoxygenation), and to clarify its possible mechanism. Methods Human umbilical cord mesenchymal stem cells were cultured. The collected cell supernatant was stored in a centrifugal tube. The exosomes secreted by human umbilical cord mesenchymal stem cells were extracted by ultracentrifugation and identified. Human umbilical vein endothelial cells were randomly divided into control group, model group and different concentrations of HUCMSC-EXO(20 μg/ml, 40 μg/ml, 60 μg/ml) treatment groups(adding HUCMSC-EXO into the model group) . The morphological changes of HUVEC cells in each group were observed by inverted phase contrast microscope, and the proliferation inhibition rate of HUVEC in each group was measured by CCK-8 reagent. Western blot was used to detect the expression of apoptosis-related proteins Caspase-3, Bax, Bcl-2 and hypoxia-associated protein hypoxia inducible factor 1α(HIF-1α). Inhibitor(HIF-1α inhibitor) + model group and HUCMSC-EXO + inhibitor + model group were added on the basis of the above experiments. Western blot analysis was performed to observe the effects of HUCMSC-EXO, inhibitor and both of them on HIF-1α and Bax expressions in HUVEC. Results HUCMSC-EXO was successfully extracted and identified. Compared with the control group, the volume of HUVEC in the model group and the HUCMSC-EXO group with different concentrations decreased, became round, connected and evacuated, and the growth state was poor under the inverted phase contrast microscope.CCK-8 detection showed that the cell viability in the HUCMSC-EXO group was significantly higher than that in the model group, the difference was statistically significant (t=9.23, P 0.05)and Bax protein ((0.363±0.069), (0.370±0.064); t=0.18, P>0.05). But both of them were down-regulated compared with the model group (HIF-1α protein (0.919±0.064), Bax protein (0.902±0.071)), the differences were significant( t=13.56, t=13.03, both P<0.05). Conclusion HUCMSC-EXO has a protective effect on OGD/R model of HUVEC, and its mechanism may be related to the down-regulation of HIF-1α expression. Key words: Human umbilical mesenchymal stem cells; Exosomes; Oxygen-glucose deprivation reoxygenation; Human umbilical venous endothelial cells; Apoptosis

The impact of inflammatory factors and related cell growth factors of human umbilical cord mesenchymal stem cells for model rats with diabetic foot ulcer

Objective To study effect of inflammatory factors and related cell growth factors of human umbilical cord mesenchymal stem cells (HUCMSCs) for model rats with diabetic foot ulcer (DFU), and analyzed the possible action mechanism of HUCMSCs therapy DFU. Methods 50 specific pathogen free (SPF)-level male rats were fed with high-fat beverage for 4 weeks. The DFU models were established by means of intraperitoneal injection of 1% streptozotocin (40 mg/kg) and steam fumigation. 30 successful model rats were divided into model group, high-dose HUCMSCs group (high-dose group: femoral artery injection with HUCMSCs 2.0×106/0.2 ml) and low-dose HUCMSCs group (low-dose group: femoral artery injection with HUCMSCs 1.0×106/0.2 ml), 10 rats/each group. Another 10 SPF-level male rats were selected and set as blank control group (blank group). Then wound healing rate, serum inflammatory factors, related cell growth factors, granulation tissue fibroblasts and newly born capillaries were compared between the groups. Results (1) Wound healing rate: The model group, low-dose group, high-dose group respectively were (26.31±4.32)%, (66.92±7.24)%, (74.74±8.32)%. low-dose group and high-dose group wound healing rate were significantly higher than model group (t=8.424, 11.201, P<0.01), high-dose group wound healing rate were significantly higher than low-dose group (t=3.148, P<0.05). (2) Serum inflammatory factors: model group, low-dose group and high-dose group interleukin (IL)-1, tumor necrosis factor-α (TNF-α), hs-CRP respectively were (6.12±0.25), (4.54±0.36), (4.20±0.33) ng/L; (29.45±3.12), (21.13±3.24), (18.20±2.65) ng/L; (0.61±0.08), (0.45±0.07), (0.37±0.06) ng/L. low-dose group and high-dose group IL-1, TNF-α, hs-CRP were significantly lower than model group (t=11.400, 5.849, 5.111, 14.666, 8.691, 8.222, P<0.05, P<0.01), high-dose group were significantly lower low-dose group (t=2.202, 2.214, 2.744, P<0.05); (3) Related cell growth factors: model group, low-dose group and high-dose group basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) respectively were (46.42±6.36), (61.35±7.20), (68.24±8.12) ng/L; (546.32±30.24), (722.36±32.17), (772.45±34.26) μg/L; (2.24±0.32), (3.65±0.41), (4.26±0.55) μg/L. Low-dose group and high-dose group bFGF, EGF, VEGF were significantly higher than model group (t=4.915, 12.609, 8.573, 6.690, 15.648, 10.039, P<0.05, P<0.01). high-dose group bFGF, EGF and VEGF were significantly higher than low-dose group (t=2.031, 3.740, 2.812, P<0.05); (4) Granulation tissue fibroblasts and newly born capillaries: model group, low-dose group and high-dose group granulation tissue fibroblasts and newly born capillaries were respectively (155.24±8.45), (278.65±9.12), (305.12±10.25) cells/visual field; (4.36±0.45), (6.45±0.62), (7.24±0.60)×10/μm2. The low-dose group and high-dose group were significantly higher than model group (t=31.389, 8.627, 35.679, 12.143, P<0.01), high-dose group were significantly higher than low-dose group (t=6.101, 2.896, P<0.05). Conclusion HUMSCs help to promote the ulcerative healing of DFU rats. Key words: Diabetic foot ulcer; Human umbilical cord mesenchymal stem cells; Inflammatory factor; Growth factor

Neural like cells and acetyl-salicylic acid alter rat brain structure and function following transient middle cerebral artery occlusion.

Introduction Transient cerebral ischemia is a pandemic neurological disorder and the main aim of medical intervention is to reduce complications. Human umbilical cord mesenchymal cells (hUCMs) are capable of differentiating into neural-like cells (NLC) in vitro, therefore we investigated the neuroprotective potential of these cells in comparison to aspirin and in combination (NLC-Aspirin) on spatial memory and neural morphologic changes in male rats submitted to transient cerebral ischemia. Methods Ten days after the intervention, the improvement in learning and memory were assessed in the animals by Morris Water Maze. Thence, the animals were examined for the presence of PKH26 labeled cells in the ischemic area of the brain, the infarct volume and neural changes in the brain tissue. Results Significant spatial memory deficits in the ischemic animals were detected compared with the control animals. The learning and memory were significantly improved (p ≤ 0.05) in the aspirin and NLC groups compared with the ischemic animals. Co-treatment of aspirin and NLCs did not improve the outcome. Moreover, infarction volume and neural changes were significantly altered when aspirin or NLCs were administered. Conclusions Our data suggest the significant neuroprotective potential of aspirin and neural-like cells derived from hUCM cells in the treatment of brain ischemic stroke. Further studies are required to evaluate possible underlying mechanisms, and to evaluate the possible interactions between aspirin and stem cells in a joint treatment aimed at the recovery of cognitive impairments.

Human umbilical cord mesenchymal stem cell′s transplantation for the treatment of neonatal hypoxic-ischemic encephalopathy

Neonatal hypoxic-ischemic encephalopathy(HIE)is a common disease in neonatal nervous system injury, which often treated by the traditional measures such as three support′s and three symptomatic treatments.Moreover, there is no specific treatment nowadays.The human umbilical cord mesenchymal stem cells(HUC-MSC)are mixture of multifunctional stem cells from neonatal umbilical cord tissues, which has strong abilities of self-renewal and multilineage differentiation potential.HUC-MSC are easy to harvest and have more sources, low immunogenicity and without ethical problems.It can differentiate into neural cells under certain conditions, secrete neurotrophic factors, repair the damaged brain and promote the recovery of cerebral function.Transplantation of HUC-MSC provides a new way for the treatment of HIE. Key words: Human umbilical cord mesenchymal stem cell; Newborn; Hypoxic-ischemic encephalopathy; Transplantation

Therapeutic effect and mechanism of human umbilical cord mesenchymal stem cell transplantation for Sjögren syndrome in mice

Background The incidence of dry eye is gradually increased, and researches showed that inflammation participated in the pathogenesis and development of dry eye.The current therapy for dry eye can only relieve symptom but not achieve final cure.Stem cell therapy has been used in the treatment of limbal stem cell deficiency.However, whether it is feasible for the stem cell treating dry eye is still unclear. Objective This study attempted to investigate a new approach to treat dry eye syndrom by using human umbilical cord mesenchymal stem cells (HUCMSCs). Methods The use and care of experimental animals complied with the Tsinghua University School of Medicine Laboratory Animal Care Details.HUCMSCs were cultured and cell suspension was prepared with the cell density of 5×105/ml.Twenty 24-week-old male NOD/Ltj mice were randomized into 4 groups.0.1 ml PBS-HUCMSCs suspension was injected via tail vein or lacrimal respectively in the caudal vein injection group and lacrimal injection group, and 10 μl PBS-HUCMSCs suspension was topically administed in the eye drops group.The NOD/Ltj mice without any treatment served as the model group.Five male ICR mice were used as the normal control group.Tear secretion was quantitatively detected with phenol red cotton thread in 1, 2, 3 weeks after injection, and corneal epithelial defect was scored by fluorescein staining.The serum levels of interleukin (IL)-6, IL-17a, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were assayed by ELISA.The relative expression levels of p65, Stat3, Stat5 and extracellular signal-regulated kinases (Erk)-1 in lacrimal gland were detected by Western blot. Results The tear secretion amount was significantly different among the model group, caudal vein injection group, lacrimal injection group and eye drops group in various time points (1 week: F=3.700, P=0.040; 2 weeks: F=5.150, P=0.008; 3 weeks: F=10.130, P 0.05). The fluorescein staining score was 3.00±0.63, 9.40±1.62, 5.20±1.17, 4.20±1.17 and 7.20±0.98 in the ICR mouse control group, model group, caudal vein injection group, lacrimal injection group and eye drops group 1 week after injection respectively, and the scores were significantly lower in the caudal vein injection group, lacrimal injection group and eye drops group than those in the model group (P=0.001, 0.000, 0.033). The serum levels of IL-6, IL-17a and TNF-α in the caudal vein injection group were evidently lower than those in the model group (t=4.70, 3.46, 11.0, all at P<0.01), but no significant difference was displayed in the serum IFN-γ level among the five groups (F=1.740, P=0.170). The expressions of STAT5 were significantly decreased in the mice treated with tail vein injection and lacrimal injection compared with mice without treatment (both at P<0.05). Conclusions Administration of HUCMSCs via intravenous and lacrimal injection can alleviate the inflammatory response during progression of dry eye syndrome by down-regulating the serum level and expression of inflammation-related factors in NOD/Ltj mice.The topical administration of HUCMSCs eye drops can attenuate the symptom of dry eyes by lubricating the cornea and suppling nutrition. Key words: Umbilical cord/cytology; Mesenchymal stem cell/cytology; Stem cell transplantation/methods; Dry eye syndromes/therapy; Inflammation/immunology; Cytokines; Mice, inbred NOD; Lacrimal apparatus/pathology

Comparison between isolation protocols highlights intrinsic variability of human umbilical cord mesenchymal cells

Mesenchymal stem cells (MSCs) though multipotent exhibit limited lifespan in vitro, with progressive reduction in capacity for self-renewal leading to irreversible arrest of cell division, which limits their use for therapeutic purposes. Human umbilical cord wall MSCs are easy to process and proliferate rapidly in culture, but variability of individual samples and impact upon in vitro expansion and aging processes is unknown. We compared isolation protocols to determine which one yields the highest number of viable cells with the best proliferation capacity. Three different protocols were tested: two were enzymatic procedures and one explant method. Isolated cells were evaluated in terms of proliferation, differentiation capacity, and phenotype. All samples were processed using one or more protocols. After passage 2 adherent cells displayed standard phenotypic and differentiation characteristics of MSCs, but our results show that isolating cells directly from Wharton's jelly is more advantageous. Cells obtained from explants presented similar characteristics to those from enzymatic protocols, but always reached proliferation arrest earlier, irrespective of initial population doubling times. From the same sample, cells obtained with enzymatic protocol ii reached later passages while exhibiting shorter doubling times in culture than cells from other protocols, that is, took longer to reach senescence. More important, each individual MSC sample exhibited different population doubling rates and reached senescence at different passages, irrespective of protocol. Thus, even when in strict conformity with procedures and quality control, each cord sample shows a unique behavior, a finding that should be taken into account when planning for therapeutic approaches.

Evaluation of neurogenic potential of human umbilical cord mesenchymal cells; a time- and concentration-dependent manner.

Background:Retinoic acid as one of the most important regulators for cell differentiation was examined in this study for differentiation of human umbilical mesenchymal cells (hUCM). Methods: After isolation, hUCM were evaluated for mesenchymal stem cell properties by flow cytometry and alkaline phosphatase assay. Also, doubling time of the cells and their differentiation potential into adipogenic and osteogenic cells were tested. hUCM were then cultured with different concentrations of retinoic acid, and on days 1, 7, and 12, the percentage of differentiated cells was determined by immunostaining for nestin, anti-microtubule associated protein 2 (MAP2), glutamic acid decarboxylase (GAD), and gamma-aminobutyric acid (GABA) markers. Results:The isolated cells were negative for the hematopoietic markers and positive for the mesenchymal markers. They showed the population doubling time 60 ± 3 hours and differentiated into osteogenic and adipogenic cells. A descending trend in nestin and an ascending trend in MAP2, GAD, and GABA expression were observed from the first day until the last day between different concentrations of retinoic acid. Conclusion: hUCM cells may have the potential to differentiate into neural cells in the presence of different incubation period and concentration of retinoic acid.

PO-0370 Germ Cells Induced From Human Umbilical Cord Mesenchymal Cell-derived Induced Pluripotent Stem Cells By Bmp4

<h3>Objective</h3> To reprogramme the induced pluripotent stem (iPS) cells from Human umbilical cord mesenchymal cells (HuMSCs) and induce the iPS cells into germ cells by BMP4. <h3>Methods</h3> OCT4, SOX2, Klf4,c-myc, Nanog, Lin28 were transfected into HuMSCs with lentivirus to reprogram HuMSCs into iPS cells. Morphological observation, Alkaline Phosphatase staining, karyotype analysis, RT-PCR, immunofluorescence staining, tumour formation <i><i>in vivo</i></i> and embryniod body formation <i><i>in vitro</i></i> were performed to examine the pluripotency of the iPS cell lines. Then we induced one of the iPS cells lines into germ cells by BMP4. Gene expression was measured by qRT-PCR at days 0, 3, 7, 10 and 14. Early-stage germ specific protein VASA and meiosis specific protein SYCP3 were assesssed by immunofluorescence staining. <h3>Result</h3> We obtained two iPS cell lines completely reprogrammed, HuMSC-iPS1 and HuMSC-iPS2. HuMSC-iPS1 expresses germ cell markers at undifferentiated state. BMP4 can upregulate germ cell markers at different time points highly while the spontaneous differentiation just upregulate DPPA3, DAZL and VASA modestly at day 3. However, all of these genes were downregulated at day 14. VASA and SYCP3 immunofluorescence staining indicates there is a high VASA expression in BMP4 induced group in contrast to low expression in the spontaneous group at day 7. Meanwhile, there is a modest SYCP3 fluorescence in BMP4 induced group in contrast to no immunofluoresence in the spontaneous group. <h3>Conclusion</h3> This system can reprogram HuMSCs into iPS cells effectively. The MSC- iPS1 can differentiate into early germ cells spontaneously while the germ cells induced by BMP4 can enter meiosis.

Induced pluripotent stem cells can be reprogrammed from human umbilical cord mesenchymal cells by six transcription factors

Objective To establish and identify the induced pluripotent stem cell(iPSC) line reprogrammed from human umbilical cord mesenchymal cells(HuMSCs). Methods HuMSCs were cultured by adhesion method, and OCT4, SOX2, KLF4, c-Myc, NANOG, LIN-28 were transfected into HuMSCs with lentiviral victor to reprogramme HuMSCs into iPSC.Morphological observation, pluripotency genes(SOX2, TDGF1, THY-1, OCT4, REX1 and TERF1) expression, alkaline phosphatase detection, karyotype analysis, embryonic stem cells(ESC) specific proteins(NANOG, OCT4, SSEA-4, TRA-1-81) immunofluorescence staining, differentiated into teratomas in vivo(inject the iPSC into SCID mice) and embryniod bodies in vitro were performed to exam the pluripotency of the iPSC. Results Four days after being infected by lentivirus, the HuMSCs became round-shape; 10 days after infection, some embryonic stem(ESC)-like colonies appeared.Fourteen days after infection, picked up the regularly shaped colonies and cultured several passages.About 1.25% HuMSCs were reprogrammed into iPSC.The iPSC presented clone-like growth like ESC.All the cells were positive to alkaline phosphatase staining and expressed the pluripotency genes.The iPSC also expressed the ESC specific proteins, and karyotype analysis showed normal chromosome caryotype(46, XY). Furthermore, the iPSC could form embryoid bodies in vitro, expressed alpha fetoprotein(AFP), smooth muscle actin(SMA) and β-tubulin.The iPSC could alsoform teratomas in vivo. Conclusion OCT4, SOX2, KLF4, c-Myc, NANOG, LIN-28 can reprogram HuMSCs into iPSC efficiently. Key words: Umbilical cord mesenchymal stem cells; Induced pluripotent stem cells; Transcription factor; Lentiviral vector

[Effect of interleukin-18 gene modified human umbilical cord mesenchymal stem cells on proliferation of breast cancer cell].

To establish human umbilical cord mesenchymal stem cells (HUMSCs) strain transfected with interleukin-18 (IL-18) gene and examine its effects on the proliferation of breast cancer cell (MCF-7). HUMSCs were isolated and cultured. And the lentivirus-IL-18 vector containing human IL-18 gene was constructed and transfected into HUMSCs. The expressions of IL-18 gene mRNA and protein were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. After co-culturing for 1, 3, 5 days, Transwell and cell counting kit-8 (CCK-8) assays were performed on MCF-7 to plot the cell growth curve. IL-18-HUMSCs could stably express of IL-18 gene and inhibit the proliferation of breast cancer cells. The IL-18 mRNA relative expression amounts were 1.40 ± 0.21 for experimental group (IL-18-HUMSCs), 0.59 ± 0.09 for negative control group (NV-HUMSCs) and 0.71 ± 0.05 for blank control group (HUMSCs). As compared with control group, the difference was statistically significant (F = 31.81, P = 0.001). The relative expressions of IL-18 protein were 1.54 ± 0.27 for experimental group (IL-18-HUMSCs), 0.57 ± 0.04 for negative control group (NV-HUMSCs) and 0.59 ± 0.23 for blank control group (HUMSCs). As compared with control group, the difference was statistically significant (F = 22.32, P = 0.002). After co-culturing for 5 days, the cellular proliferation was significantly inhibited. IL-18 gene has been successfully transfected into HUMSCs and has a stable expression. And IL-18-HUMSCs can effectively inhibit the proliferation of breast cancer cells in vitro.

Distinct adipogenic differentiation phenotypes of human umbilical cord mesenchymal cells dependent on adipogenic conditions.

The umbilical cord (UC) matrix is a source of multipotent mesenchymal stem cells (MSCs) that have adipogenic potential and thus can be a model to study adipogenesis. However, existing variability in adipocytic differentiation outcomes may be due to discrepancies in methods utilized for adipogenic differentiation. Additionally, functional characterization of UCMSCs as adipocytes has not been described. We tested the potential of three well-established adipogenic cocktails containing IBMX, dexamethasone, and insulin (MDI) plus indomethacin (MDI-I) or rosiglitazone (MDI-R) to stimulate adipocyte differentiation in UCMSCs. MDI, MDI-I, and MDI-R treatment significantly increased peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT-enhancer binding protein alpha (C/EBPα) mRNA and induced lipid droplet formation. However, MDI-I had the greatest impact on mRNA expression of PPARγ, C/EBPα, FABP4, GPD1, PLIN1, PLIN2, and ADIPOQ and lipid accumulation, whereas MDI showed the least. Interestingly, there were no treatment group differences in the amount of PPARγ protein. However, MDI-I treated cells had significantly more C/EBPα protein compared to MDI or MDI-R, suggesting that indomethacin-dependent increased C/EBPα may contribute to the adipogenesis-inducing potency of MDI-I. Additionally, bone morphogenetic protein 4 (BMP4) treatment of UCMSCs did not enhance responsiveness to MDI-induced differentiation. Finally to characterize adipocyte function, differentiated UCMSCs were stimulated with insulin and downstream signaling was assessed. Differentiated UCMSCs were responsive to insulin at two weeks but showed decreased sensitivity by five weeks following differentiation, suggesting that long-term differentiation may induce insulin resistance. Together, these data indicate that UCMSCs undergo adipogenesis when differentiated in MDI, MDI-I, and MDI-R, however the presence of indomethacin greatly enhances their adipogenic potential beyond that of rosiglitazone. Furthermore, our results suggest that insulin signaling pathways of differentiated UCMSCs are functionally similar to adipocytes.

Low Immunogenicity of Neural Progenitor Cells Differentiated from Induced Pluripotent Stem Cells Derived from Less Immunogenic Somatic Cells

The groundbreaking discovery of induced pluripotent stem cells (iPS cells) provides a new source for cell therapy. However, whether the iPS derived functional lineages from different cell origins have different immunogenicity remains unknown. It had been known that the cells isolated from extra-embryonic tissues, such as umbilical cord mesenchymal cells (UMCs), are less immunogenic than other adult lineages such as skin fibroblasts (SFs). In this report, we differentiated iPS cells from human UMCs and SFs into neural progenitor cells (NPCs) and analyzed their immunogenicity. Through co-culture with allologous peripheral blood mononuclear cells (PBMCs), we showed that UMCs were indeed less immunogenic than skin cells to simulate proliferation of PBMCs. Surprisingly, we found that the NPCs differentiated from UMC-iPS cells retained low immunogenicity as the parental UMCs based on the PBMC proliferation assay. In cytotoxic expression assay, reactions in most kinds of immune effector cells showed more perforin and granzyme B expression with SF-NPCs stimulation than that with UMC-NPCs stimulation in PBMC co-culture system, in T cell co-culture system as well. Furthermore, through whole genome expression microarray analysis, we showed that over 70 immune genes, including all members of HLA-I, were expressed at lower levels in NPCs derived from UMC-iPS cells than that from SF-iPS cells. Our results demonstrated a phenomenon that the low immunogenicity of the less immunogenic cells could be retained after cell reprogramming and further differentiation, thus provide a new concept to generate functional lineages with lower immunogenicity for regenerative medicine.

Effect of EGF and FGF on the expansion properties of human umbilical cord mesenchymal cells

Mesenchymal stem cells have been increasingly introduced to have great potential in regenerative medicine, immunotherapy, and gene therapy due to their unique properties of self-renewal and differentiation into multiple cell lineages. Studies have shown that these properties may be limited and changed by senescence-associated growth arrest under different culture conditions. This study aimed to present the ability of some growth factors on human umbilical cord mesenchymal (hUCM) cells expansion and telomerase activity. To optimize hUCM cell growth, epidermal growth factor (EGF) and fibroblast growth factor (FGF) were utilized in culture media, and the ability of these growth factors on the expression of the telomerase reverse transcriptase (TERT) gene and cell cycle phases was investigated. TERT mRNA expression increased in the hUCM cells treated by EGF and FGF. So, the untreated hUCM cells expressed 30.49 ± 7.15% of TERT, while EGF-treated cells expressed 51.82 ± 12.96% and FGF-treated cells expressed 33.77 ± 11.55% of TERT. Exposure of hUCM cells to EGF or FGF also promoted the progression of cells from G1 to S phase of the cell cycle and induced them to decrease the number of cells entering the G2/M phase. Our study showed that EGF and, to a lesser extent, FGF amplify the proliferation and expansion of hUCM cells.

Improvement in Cardiac Function following Transplantation of Human Umbilical Cord Matrix-Derived Mesenchymal Cells

Objectives: Human umbilical cord mesenchymal cells (hUCM) can be easily obtained and processed in a laboratory. These cells may be considered as a suitable source in the repair of heart failure diseases. We, therefore, examined whether these cells may contribute to heart regeneration following an acute experimental myocardial infarction (MI). Methods: MI-induced animals received 5 × 10⁶ hUCM cells, 5 × 10⁶ 5-azacytidine-treated cells (dhUCM), or PBS alone, subepicardially. A group of animals with MI and no other former intervention served as controls. dhUCM cells were assessed for F-actin, myogenin and troponin-I expression. Results: dhUCM cells appeared as binucleated cells with extensive cytoplasmic processes. These differentiated cells were F-actin and myogenin positive. Thirty days after LAD ligation, left ventricular ejection fraction and the percentage of fractional shortening improved significantly in cell-receiving animals. In addition, the amount of scar tissue was significantly reduced in hUCM and dhUCM groups compared to MI group (p < 0.05). These parameters were comparable between hUCM and dhUCM groups. Histopathological evaluations revealed that some engrafted cells adjacent to and remote from the MI area expressed troponin-I, F-actin and connexin43. Conclusion: These findings demonstrated the potential therapeutic use of either differentiated or undifferentiated hUCM cells in treatment of heart failure conditions.

Antioxidants rescue stressed embryos at a rate comparable with co-culturing of embryos with human umbilical cord mesenchymal cells

During laboratory manipulations, oocytes and embryos are inevitably exposed to suboptimal conditions that interfere with the normal development of embryos. In this study, we examined the effects of antioxidants, feeder cells and a conditioned medium on embryo development and cleavage rate following exposure of the embryos to suboptimal conditions. We exposed mouse two-cell embryos to visible light and divided them into four groups: control (E-ctr), co-culture (Co-c), conditioned medium (Cndm) and antioxidant-plus medium (Aopm). We used human umbilical cord matrix-derived mesenchymal cells for co-culture. A group of embryos was not exposed to visible light and served as the non-exposed control (NE-ctr) group. The developmental rate was higher in NE-ctr embryos than in the E-ctr group. Exposed embryos in the various groups showed a comparable developmental rate at different stages. Blastomere number significantly increased (P < 0.05) in the Co-c and Aopm groups compared with the E-ctr and Cndm groups. No significant difference was observed between the Co-c and Aopm groups. Our data indicate that in suboptimal conditions, antioxidants could improve the embryo cleavage rate in the same way as feeder cells. Antioxidants probably improve embryo quality through their ability to scavenge reactive oxygen species.

Effect of human umbilical cord mesenchymal stem cell-secretion on proliferation and apoptosis in hepatocytes

Objective To investigate the effect of human umbilical cord mesenchymal stem cell paracrine substance on proliferation and apoptosis of liver cells in vitro. Methods Mesenchymal stem cells (MSC)were separated from human umbilical cord with type Ⅳ collagenase and trypsogen digestion method and cultured in vitro. The human umbilical cord mesenchymal stem cells-conditioned medium(MSC-CM) which contain paracrine substance of human umbilical cord mesenchymal stem cells (HUCMSC) was prepared. Hepatocytes were isolated from SD rats by low concentration collagenase perfusion procedure. There were three groups in the experiment, control group, 2% MSC-CM group and 8% MSC-CM group. The proliferation of normal hepatocytes were assayed with MTT method. We detected the urea and albumin level in culture supernatant to assay the hepatocyte function under different concentration MSC-CM. Hepatocytes were induced for apoptosis by Actinomycin D and tumor necrosis factor alpha (TNF-α),and the apoptosis effect of different concentration MSC-CM was assayed with LIVE/DEAD Viability/Cytotoxicity Kit. Results The MTT assay showed that the absorbance of 2% MSC-CM group was significantly increased (P＜0. 01), and the urea and albumin levels of 2 % MSC-CM group were also significantly increased when compared with control group(P＜0. 01).LIVE/DEAD Viability/Cytotoxicity Kit revealed that hepatocyte survival rate of 2 % MSC-CM group was increased when compared with control group(P＜0. 05), there were no significant differences in above-mentioned experiments when 8% MSC-CM group compared with control group. Conclusion The low concentration MSC-CM could stimulate normal hepatocyte proliferation, inhibit impaired hepatocyte apoptosis and improve hepatocyte function. Key words: Umbilical cord mesenchymal stem cell; Paracrine; Hepatocyte; Regeneration; Apoptosis