Single cell sequencing analysis identifies genetics-modulated ORMDL3+ cholangiocytes having higher metabolic effects on primary biliary cholangitis.

BACKGROUND: Primary biliary cholangitis (PBC) is a classical autoimmune disease, which is highly influenced by genetic determinants. Many genome-wide association studies (GWAS) have reported that numerous genetic loci were significantly associated with PBC susceptibility. However, the effects of genetic determinants on liver cells and its immune microenvironment for PBC remain unclear. RESULTS: We constructed a powerful computational framework to integrate GWAS summary statistics with scRNA-seq data to uncover genetics-modulated liver cell subpopulations for PBC. Based on our multi-omics integrative analysis, 29 risk genes including ORMDL3, GSNK2B, and DDAH2 were significantly associated with PBC susceptibility. By combining GWAS summary statistics with scRNA-seq data, we found that cholangiocytes exhibited a notable enrichment by PBC-related genetic association signals (Permuted P < 0.05). The risk gene of ORMDL3 showed the highest expression proportion in cholangiocytes than other liver cells (22.38%). The ORMDL3+ cholangiocytes have prominently higher metabolism activity score than ORMDL3- cholangiocytes (P = 1.38 × 10-15). Compared with ORMDL3- cholangiocytes, there were 77 significantly differentially expressed genes among ORMDL3+ cholangiocytes (FDR < 0.05), and these significant genes were associated with autoimmune diseases-related functional terms or pathways. The ORMDL3+ cholangiocytes exhibited relatively high communications with macrophage and monocyte. Compared with ORMDL3- cholangiocytes, the VEGF signaling pathway is specific for ORMDL3+ cholangiocytes to interact with other cell populations. CONCLUSIONS: To the best of our knowledge, this is the first study to integrate genetic information with single cell sequencing data for parsing genetics-influenced liver cells for PBC risk. We identified that ORMDL3+ cholangiocytes with higher metabolism activity play important immune-modulatory roles in the etiology of PBC.

Human menstrual blood-derived mesenchymal stem cells as a cellular vehicle for malignant glioma gene therapy.

Despite many advances in conventional treatment strategies, there is no effective treatment modality for malignant gliomas. Gene therapy may offer a promising option for gliomas and several gene therapy approaches have shown anti-tumor efficiency in previous studies. Mesenchymal stem cell-based gene therapies, in which stem cells are genetically engineered to express therapeutic molecules, have shown tremendous potential because of their innate homing ability. In this study, human menstrual blood-derived MSCs (MenSC), a novel type of multipotential MSCs displays tropism for human malignant glioma when used as a gene delivery vehicle for therapeutics. Secretable trimeric TRAIL (stTRAIL) contains the receptor-binding domain of TRAIL, a death ligand that induces apoptosis in tumor cells. To overexpress stTRAIL, MenSCs were infected with efficient adenoviral serotype 35 vectors that had no influence on its broad multipotency and low immunophenotype. The modified MenSCs served as an excellent local drug delivery system for tumor site-specific targeted delivery and demonstrated therapeutic efficacy in an animal xenografts tumor model of U-87 MG cells. The MenSC-stTRAIL cells induced antitumor effects in vitro by significantly increasing apoptosis (P < 0.05). It also significantly reduced tumor burden in vivo (P < 0.05). The results showed that the proliferation of tumor cells was significantly reduced (P < 0.05). The MenSC, as a cellular delivery vehicle has a wide potential therapeutic role, which includes the treatment of tumors.

Transplantation of Menstrual Blood-Derived Mesenchymal Stem Cells Promotes the Repair of LPS-Induced Acute Lung Injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality. Menstrual blood-derived stem cells (MenSCs) have been shown to be good therapeutic tools in diseases such as ovarian failure and cardiac fibrosis. However, relevant studies of MenSCs in ALI have not yet proceeded. We hypothesized that MenSC could attenuate the inflammation in lipopolysaccharide (LPS)-induced ALI and promote the repair of damaged lung. ALI model was induced by LPS in C57 mice, and saline or MenSCs were administered via tail vein after four hours. The MenSCs were subsequently detected in the lungs by a live imaging system. The MenSCs not only improved pulmonary microvascular permeability and attenuated histopathological damage, but also mediated the downregulation of IL-1ß and the upregulation of IL-10 in bronchoalveolar lavage fluid (BALF) and the damaged lung. Immunohistochemistry revealed the increased expression of proliferating cell nuclear antigen (PCNA) and the reduced expression of caspase-3 indicating the beneficial effect of MenSCs. Keratinocyte growth factor (KGF) was also upregulated after MenSCs administrated. As shown using transwell co-culture, the MenSCs also could improve the viability of BEAS-2B cells and inhibit LPS-induced apoptosis. These findings suggest that MenSC-based therapies could be promising strategies for treating ALI.

Human Menstrual Blood-Derived Stem Cells Ameliorate Liver Fibrosis in Mice by Targeting Hepatic Stellate Cells via Paracrine Mediators.

Mesenchymal stem cells (MSCs) may have potential applications in regenerative medicine for the treatment of chronic liver diseases (CLDs). Human menstrual blood is a novel source of MSCs, termed menstrual blood-derived stem cells (MenSCs). Compared with bone marrow MSCs, MenSCs exhibit a higher proliferation rate and they can be obtained through a simple, safe, painless procedure without ethical concerns. Although the therapeutic efficacy of MenSCs has been explored in some diseases, their effects on liver fibrosis are still unclear. In the present study, we investigated the therapeutic effects of MenSC transplantation in a carbon tetrachloride-induced mouse model of liver fibrosis. These results revealed that MenSCs markedly improved liver function, attenuated collagen deposition, and inhibited activated hepatic stellate cells up to 2 weeks after transplantation. Moreover, tracking of green fluorescent protein-expressing MenSCs demonstrated that transplanted cells migrated to the sites of injury, but few differentiated into functional hepatocyte-like cells. Transwell coculturing experiments also showed that MenSCs suppressed proliferation of LX-2 cells (an immortalized hepatic stellate cell line) through secretion of monocyte chemoattractant protein-1, interleukin-6, hepatocyte growth factor, growth-related oncogene, interleukin-8, and osteoprotegerin. Collectively, our results provided preliminary evidence for the antifibrotic capacity of MenSCs in liver fibrosis and suggested that these cells may be an alternative therapeutic approach for the treatment of CLDs. Stem Cells Translational Medicine 2017;6:272-284.

Exosomes derived from human menstrual blood-derived stem cells alleviate fulminant hepatic failure.

BACKGROUND: Human menstrual blood-derived stem cells (MenSCs) are a novel source of MSCs that provide the advantage of being easy to collect and isolate. Exosomes contain some mRNAs and adhesion molecules that can potentially impact cellular and animal physiology. This study aimed to investigate the therapeutic potential of MenSC-derived exosomes (MenSC-Ex) on AML12 cells (in vitro) and D-GalN/LPS-induced FHF mice (in vivo). METHODS: Transmission electron microscopy and Western blot were used to identify MenSC-Ex. Antibody array was used to examine cytokine levels on MenSC-Ex. MenSC-Ex were treated in D-GalN/LPS-induced AML12 in vitro. Cell proliferation and apoptosis were measured. MenSC-Ex were injected into the tail veins of mice 24 h before treatment with D-GalN/LPS. Blood and liver tissues served as physiological and biochemical indexes. The number of liver mononuclear cells (MNCs) and the amount of the active apoptotic protein caspase-3 were determined to elaborate the mechanism of hepatoprotective activity. RESULTS: Human menstrual blood-derived stem cell-derived exosomes (MenSC-Ex) are bi-lipid membrane vesicles that have a round, ball-like shape with a diameter of approximately 30-100 nm. Cytokine arrays have shown that MenSC-Ex expressed cytokines, including ICAM-1, angiopoietin-2, Axl, angiogenin, IGFBP-6, osteoprotegerin, IL-6, and IL-8. MenSC-Ex markedly improved liver function, enhanced survival rates, and inhibited liver cell apoptosis at 6 h after transplantation. MenSC-Ex migrated to sites of injury and to AML12 cells (a mouse hepatocyte cell line), respectively. Moreover, MenSC-Ex reduced the number of liver mononuclear cells (MNCs) and the amount of the active apoptotic protein caspase-3 in injured livers. CONCLUSIONS: In conclusion, our results provide preliminary evidence for the anti-apoptotic capacity of MenSC-Ex in FHF and suggest that MenSC-Ex may be an alternative therapeutic approach to treat FHF.

Stem cells as cellular vehicles for gene therapy against glioblastoma.

Glioblastoma (GBM) is the most common and deadliest primary tumor in adults, with current treatments having limited specific and efficient delivery of therapeutic drugs to tumor sites or cells. Therefore, the development of alternative treatment options is urgently needed. Stem cells are considered as ideal cellular vehicles for gene therapy against glioblastoma. In this paper, we reviewed the recent studies investigating the use of different types of stem cells as cellular vehicles and the gene of interests against the glioblastoma, as well as the future directions of the application of cellular vehicles mediated therapy for glioblastoma.

Transplantation of human menstrual blood progenitor cells improves hyperglycemia by promoting endogenous progenitor differentiation in type 1 diabetic mice.

Recently, a unique population of progenitor cells was isolated from human menstrual blood. The human menstrual blood progenitor cells (MBPCs) possess many advantages, such as the noninvasive acquisition procedure, broad multipotency, a higher proliferative rate, and low immunogenicity, and have attracted extensive attention in regenerative medicine. Preclinical studies to test the safety and efficacy of MBPCs have been underway in several animal models. However, relevant studies in type 1 diabetes mellitus (T1DM) have not yet been proceeded. Herein, we studied the therapeutic effect of MBPCs and the mechanism of ß-cell regeneration after MBPC transplantation in the T1DM model. Intravenous injection of MBPCs can reverse hyperglycemia and weight loss, prolong lifespan, and increase insulin production in diabetic mice. Histological and immunohistochemistry analyses indicated that T1DM mice with MBPC transplantation recovered islet structures and increased the ß-cell number. We further analyzed in vivo distribution of MBPCs and discovered that a majority of MBPCs migrated into damaged pancreas and located at the islet, duct, and exocrine tissue. MBPCs did not differentiate into insulin-producing cells, but enhanced neurogenin3 (ngn3) expression, which represented endocrine progenitors that were activated. Ngn3(+) cells were not only in the ductal epithelium, but also in the islet and exocrine tissue. We analyzed a series of genes associated with the embryonic mode of ß-cell development by real-time polymerase chain reaction and the results showed that the levels of those gene expressions all increased after cell transplantation. According to the results, we concluded that MBPCs stimulated ß-cell regeneration through promoting differentiation of endogenous progenitor cells.

Menstrual blood-derived mesenchymal stem cells differentiate into functional hepatocyte-like cells.

Orthotopic liver transplantation (OLT) is the only proven effective treatment for both end-stage and metabolic liver diseases. Hepatocyte transplantation is a promising alternative for OLT, but the lack of available donor livers has hampered its clinical application. Hepatocyte-like cells (HLCs) differentiated from many multi-potential stem cells can help repair damaged liver tissue. Yet almost suitable cells currently identified for human use are difficult to harvest and involve invasive procedures. Recently, a novel mesenchymal stem cell derived from human menstrual blood (MenSC) has been discovered and obtained easily and repeatedly. In this study, we examined whether the MenSCs are able to differentiate into functional HLCs in vitro. After three weeks of incubation in hepatogenic differentiation medium containing hepatocyte growth factor (HGF), fibroblast growth factor-4 (FGF-4), and oncostain M (OSM), cuboidal HLCs were observed, and cells also expressed hepatocyte-specific marker genes including albumin (ALB), α-fetoprotein (AFP), cytokeratin 18/19 (CK18/19), and cytochrome P450 1A1/3A4 (CYP1A1/3A4). Differentiated cells further demonstrated in vitro mature hepatocyte functions such as urea synthesis, glycogen storage, and indocyanine green (ICG) uptake. After intrasplenic transplantation into mice with 2/3 partial hepatectomy, the MenSC-derived HLCs were detected in recipient livers and expressed human ALB protein. We also showed that MenSC-derived HLC transplantation could restore the serum ALB level and significantly suppressed transaminase activity of liver injury animals. In conclusion, MenSCs may serve as an ideal, easily accessible source of material for tissue engineering and cell therapy of liver tissues.