Proteomic and phosphorylated proteomic landscape of injured lung in juvenile septic rats with therapeutic application of umbilical cord mesenchymal stem cells.

Acute lung injury (ALI) is the most common complication of sepsis. Intravenous injection of HUMSCs can regulate the level of circulating endothelial cytokines and alleviate lung injury in juvenile septic rats. In this study, we performed proteomic and phosphorylated proteomic analysis of lung tissue of juvenile septic rats after Human Umbilical Cord Mesenchymal Stem Cells (HUMSCs) intervention for the first time, and screened the potential proteins and pathways of HUMSCs for therapeutic effect. The 4D proteome quantitative technique was used to quantitatively analyze the lung tissues of septic rats 24 hours (3 biological samples) and 24 hours after HUMSCs intervention (3 biological samples). A total of 213 proteins were identified as differentially expressed proteins, and 971 phosphorylation sites changed significantly. Based on the public database, we analyzed the functional enrichment of these proteins and phosphorylated proteins. In addition, Tenascin-C may be the key differential protein and ECM receptor interaction pathway may be the main signal pathway by using various algorithms to analyze the protein-protein interaction network. Phosphorylation analysis showed that tight junction pathway was closely related to immune inflammatory reaction, and EGFR interacted most, which may be the key differential phosphorylated protein. Finally, 123 conserved motifs of serine phosphorylation site (pS) and 17 conserved motifs of threonine (pT) phosphorylation sites were identified by motif analysis of phosphorylation sites. Results from proteomics and phosphorylated proteomics, the potential new therapeutic targets of HUMSCs in alleviating lung injury in juvenile septic rats were revealed.

Single-cell diploid Hi-C reveals the role of spatial aggregations in complex rearrangements and KMT2A fusions in leukemia.

BACKGROUND: Simple translocations and complex rearrangements are formed through illegitimate ligations of double-strand breaks of fusion partners and lead to generation of oncogenic fusion genes that affect cellular function. The contact first hypothesis states that fusion partners tend to colocalize prior to fusion in normal cells. Here we test this hypothesis at the single-cell level and explore the underlying mechanism. RESULTS: By analyzing published single-cell diploid Hi-C datasets, we find partner genes fused in leukemia exhibit smaller spatial distances than those fused in solid tumor and control gene pairs. Intriguingly, multiple partners tend to colocalize with KMT2A in the same cell. 3D genome architecture has little association with lineage decision of KMT2A fusion types in leukemia. Besides simple translocations, complex rearrangement-related KMT2A fusion genes (CRGs) also show closer proximity and belong to a genome-wide mutual proximity network. We find CRGs are co-expressed, co-localized, and enriched in the targets of the transcriptional factor RUNX1, suggesting they may be involved in RUNX1-mediated transcription factories. Knockdown of RUNX1 leads to significantly fewer contacts among CRGs. We also find CRGs are enriched in active transcriptional regions and loop anchors, and exhibit high levels of TOP2-mediated DNA breakages. Inhibition of transcription leads to reduced DNA breakages of CRGs. CONCLUSIONS: Our results demonstrate KMT2A partners and CRGs may form dynamic and multipartite spatial clusters in individual cells that may be involved in RUNX1-mediated transcription factories, wherein massive DNA damages and illegitimate ligations of genes may occur, leading to complex rearrangements and KMT2A fusions in leukemia.

Single-cell RNA sequencing reveals the potential mechanism of heterogeneity of immunomodulatory properties of foreskin and umbilical cord mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) are heterogeneous populations. Heterogeneity exists within the same tissue and between different tissues. Some studies have found enormous heterogeneity in immunomodulatory function among MSCs derived from different tissues. Moreover, the underlying mechanism of heterogeneity in immunomodulatory abilities is still unclear. METHODS: Foreskin mesenchymal stromal cells (FSMSCs) and human umbilical cord mesenchymal stromal cells (HuMSCs) were isolated and cultured until the third passage. According to the International Association for Cell Therapy standard, we confirmed the cell type. Then, FSMSCs and HuMSCs were cocultured with human peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide (LPS) in vitro. Furthermore, the supernatant was sampled for an enzyme-linked immunosorbent assay to investigate the secretion of IL-1ß, IL-6, IL-10, TNF-α, and TGF-ß1. Finally, we performed single-cell RNA sequencing (scRNA-seq) of FSMSCs and HuMSCs. RESULTS: We successfully identified FSMSCs and HuMSCs as MSCs. When cocultured with LPS pretreated PBMCs, FSMSCs and HuMSCs could effectively reduced the secretion of IL-1ß and TNF-α. However, FSMSCs stimulated the PBMCs to secrete more IL-10, TGF-ß1, and IL-6. Furthermore, 4 cell subsets were identified from integrated scRNA-seq data, including proliferative MSCs (MKI67+, CD146low+, NG2+, PDGFRB-), pericytes (CD146high+, PDGFRB+, MKI67-, CD31-, CD45-, CD34-), immune MSCs (CXCL12high+, PTGIShigh+, PDGFRB+, CD146-, MKI67-) and progenitor proliferative MSCs (CXCL12low+, PTGISlow+, PDGFRB+, CD146-, MKI67-). Among them, we found that immune MSCs with strengthened transcriptional activity were similar to pericytes with regard to the degree of differentiated. Various of immune-related genes, gene sets, and regulons were also enriched in immune MSCs. Moreover, immune MSCs were determined to be close to other cell subsets in cell-cell communication analysis. Finally, we found that the proportion of immune MSCs in foreskin tissue was highest when comparing the subset compositions of MSCs derived from different tissues. CONCLUSIONS: FSMSCs show better immunomodulatory capacity than HuMSCs in vitro. Moreover, immune MSCs may play a vital role in the heterogeneity of immunoregulatory properties. This study provides new insights suggesting that immune MSCs can be isolated to exert stable immunoregulatory functions without being limited by the heterogeneity of MSCs derived from different tissues.

Targeting metabolism: A potential strategy for hematological cancer therapy.

Most hematological cancer-related relapses and deaths are caused by metastasis; thus, the importance of this process as a target of therapy should be considered. Hematological cancer is a type of cancer in which metabolism plays an essential role in progression. Therefore, we are required to block fundamental metastatic processes and develop specific preclinical and clinical strategies against those biomarkers involved in the metabolic regulation of hematological cancer cells, which do not rely on primary tumor responses. To understand progress in this field, we provide a summary of recent developments in the understanding of metabolism in hematological cancer and a general understanding of biomarkers currently used and under investigation for clinical and preclinical applications involving drug development. The signaling pathways involved in cancer cell metabolism are highlighted and shed light on how we could identify novel biomarkers involved in cancer development and treatment. This review provides new insights into biomolecular carriers that could be targeted as anticancer biomarkers.

Single-Cell Transcriptome Integration Analysis Reveals the Correlation Between Mesenchymal Stromal Cells and Fibroblasts.

Background: Mesenchymal stromal cells (MSCs) and fibroblasts show similar morphology, surface marker expression, and proliferation, differentiation, and immunomodulatory capacities. These similarities not only blur their cell identities but also limit their application. Methods: We performed single-cell transcriptome sequencing of the human umbilical cord and foreskin MSCs (HuMSCs and FSMSCs) and extracted the single-cell transcriptome data of the bone marrow and adipose MSCs (BMSCs and ADMSCs) from the Gene Expression Omnibus (GEO) database. Then, we performed quality control, batch effect correction, integration, and clustering analysis of the integrated single-cell transcriptome data from the HuMSCs, FMSCs, BMSCs, and ADMSCs. The cell subsets were annotated based on the surface marker phenotypes for the MSCs (CD105 + , CD90 +, CD73 +, CD45 -, CD34 -, CD19 -, HLA-DRA -, and CD11b -), fibroblasts (VIM +, PECAM1 -, CD34 -, CD45 -, EPCAM -, and MYH11 -), and pericytes (CD146 +, PDGFRB +, PECAM1 -, CD34 -, and CD45 -). The expression levels of common fibroblast markers (ACTA2, FAP, PDGFRA, PDGFRB, S100A4, FN1, COL1A1, POSTN, DCN, COL1A2, FBLN2, COL1A2, DES, and CDH11) were also analyzed in all cell subsets. Finally, the gene expression profiles, differentiation status, and the enrichment status of various gene sets and regulons were compared between the cell subsets. Results: We demonstrated 15 distinct cell subsets in the integrated single-cell transcriptome sequencing data. Surface marker annotation demonstrated the MSC phenotype in 12 of the 15 cell subsets. C10 and C14 subsets demonstrated both the MSC and pericyte phenotypes. All 15 cell subsets demonstrated the fibroblast phenotype. C8, C12, and C13 subsets exclusively demonstrated the fibroblast phenotype. We identified 3,275 differentially expressed genes, 305 enriched gene sets, and 34 enriched regulons between the 15 cell subsets. The cell subsets that exclusively demonstrated the fibroblast phenotype represented less primitive and more differentiated cell types. Conclusion: Cell subsets with the MSC phenotype also demonstrated the fibroblast phenotype, but cell subsets with the fibroblast phenotype did not necessarily demonstrate the MSC phenotype, suggesting that MSCs represented a subclass of fibroblasts. We also demonstrated that the MSCs and fibroblasts represented highly heterogeneous populations with distinct cell subsets, which could be identified based on the differentially enriched gene sets and regulons that specify proliferating, differentiating, metabolic, and/or immunomodulatory functions.

Chimeric antigen receptor T cells targeting CD7 in a child with high-risk T-cell acute lymphoblastic leukemia.

Effective systemic treatments for relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) are limited. Recent clinical application of chimeric antigen receptor (CAR) immunotherapy has demonstrated successful control of B-cell malignancies by CAR-T cells; however, designing CARs for T-ALL remains a challenge. CD7 overexpression in T-cell malignancies may be an attractive target for immunotherapy in T-ALL. This study aimed to describe the safe and effective use of autologous CD7-CAR T cells (4SCAR7) for the treatment of T-ALL with induction failure in an 11-year-old patient. Based on The Chinese Children's Cancer Group-ALL (CCCG-ALL) study protocol, minimal residual disease (MRD) by flow cytometry (FC) analysis was detected on days 19 and 46 of remission induction. At the end of remission-induction chemotherapy, the patient achieved morphologic complete remission, though with MRD 16.13% and RT-PCR of KMT2A-MLLT1 fusion positive, which indicated induction failure. The cerebrospinal fluid (CSF) was negative for blasts at diagnosed. CAR-T therapy and allogeneic transplant were recommended as the next treatment options. CD3+ lymphocytes were collected from the patient 18 days after the high-dose MTX chemotherapy through leukapheresis. The 4SCAR7 CD7-targeting CAR-T cells were generated thereafter. The patient received lymphodepleting chemotherapy prior to 4SCAR7 infusion. Oral administration of itraconazole and sulfamethoxazole was performed from day 0 after CAR-T cell infusion. The patient did not have hypotension, hypoxia, or serious biochemical change or abnormality, but had fever on day 9. Although grade 1 cytokine-release syndrome (CRS) was diagnosed, it was successfully treated with ibuprofen. Anti-CD7 CAR transgene copy numbers in peripheral blood were determined by qPCR, which showed effective expansion initially, then dropped quickly, and persisted at a low level. Although experienced cytopenia from days 14 to 21, the patient achieved remission on day 17. After complete remission, the patient received hematopoietic stem cell transplantation (HSCT) and has recovered well to thisdate. Overall, this report suggested that 4SCAR7 could be a safe and effective strategy for the treatment of pediatric patients with high-risk T-cell malignancies.

Identification of a De Novoc.1000delA ANK1 mutation associated to hereditary spherocytosis in a neonate with Coombs-negative hemolytic jaundice-case reports and review of the literature.

BACKGROUND: To strengthen the understanding of Hereditary Spherocytosis (HS) and determine the disease-causing mutation present with neonatal jaundice. HS is a hemolytic condition resulting from various erythrocyte membrane defects. Many different mutations result in HS, including mutations in ANK1. CASE PRESENTATION: A term neonate presented at ten hours with severe jaundice requiring exchange transfusion. At two months he was hospitalized due to repeated pallor and anemia requiring blood transfusions. Using next-generation sequencing, we discovered the responsible mutation in the proband but not in his parents; a heterozygous nucleotide variation of c.1000delA (p.1334Sfs*6) in ANK1. Thus hereditary spherocytosis was diagnosed. CONCLUSIONS: Genetic detection is an important means of discovering the cause of hemolytic anemia in neonates and infants where routine diagnostic tests are unrevealing. We found a novel de novo mutation, c.1000delA (p.1334Sfs*6) in ANK1 that might account for other cases of HS in the Chinese population.

Successful outcome of disseminated Candida tropicalis osteomyelitis on remission induction for childhood Philadelphia chromosome-positive acute lymphoblastic leukaemia-case report.

BACKGROUND: Invasive fungal infection (IFI) is one of the most challenging complications in children undergoing acute lymphoblastic leukaemia (ALL) treatment, but acute fungal osteomyelitis (OM) is rarely encountered. CASE PRESENTATION: Here, we describe a case of Candida tropicalis osteomyelitis in a 10-year-old patient with Philadelphia chromosome (Ph)-positive ALL. He was on remission induction therapy at the time of neutropenia, and an abscess developed in his right arm. The blood and bone cultures were positive for C. tropicalis. Antibiotics and antifungals were administered. Magnetic resonance imaging of the arm revealed an intraosseous abscess, suggestive of OM. Surgical irrigation and debridement of the bone were performed immediately. The patient was effectively treated with antifungal therapy and ALL treatment. He has fully recovered into complete clinical remission but with visible sequelae on magnetic resonance imaging (MRI). He took oral posaconazole for consolidation until disappearance of the lesion shadows on MRI and received subsequent cycles of chemotherapy in parallel. CONCLUSIONS: In the successful management of Ph-positive ALL, dasatinib, a second-generation Abl-tyrosine kinase inhibitor, is crucial. The recommended treatment for Candida osteomyelitis in Ph-positive ALL patients is a fungicidal agent combined with surgery and modification chemotherapy with dasatinib. The use of combined modalities of treatment seems to be crucial in the successful management of Ph-positive ALL.

Intranasal administration of Cytoglobin modifies human umbilical cord­derived mesenchymal stem cells and improves hypoxic­ischemia brain damage in neonatal rats by modulating p38 MAPK signaling­mediated apoptosis.

Neonatal hypoxic­ischemic brain damage (HIBD) is a common clinical syndrome in newborns. Hypothermia is the only approved therapy for the clinical treatment; however, the therapeutic window of hypothermia is confined to 6 h after birth and even then, >40% of the infants either die or survive with various impairments, including cerebral palsy, seizure disorder and intellectual disability following hypothermic treatment. The aim of the present study was to determine whether nasal transplantation of Cytoglobin (CYGB) genetically modified human umbilical cord­derived mesenchymal stem cells (CYGB­HuMSCs) exhibited protective effects in neonatal rats with HIBD compared with those treated without genetically modified CYGB. A total of 120 neonatal Sprague­Dawley rats (postnatal day 7) were assigned to either a Sham, HIBD, HuMSCs or CYGB­HuMSCs group (n = 30 rats/group). For HIBD modeling, rats underwent left carotid artery ligation and were exposed to 8% oxygen for 2.5 h. A total of 30 min after HI, HuMSCs (or CYGB­HuMSCs) labeled with enhanced­green fluorescent protein (eGFP) were intranasally administered. After modeling for 3, 14 and 29 days, five randomly selected rats were sacrificed in each group, and the expression levels of CYGB, ERK, JNK and p38 in brain tissues were determined. Nissl staining of the cortex and hippocampal Cornu Ammonis 1 area of rats in each group were compared after 3 days of modeling. TUNEL assay and immunofluorescence were performed 3 days after modeling. Long term memory in rats was assessed using a Morris­water maze 29 days after modeling. The HIBD group demonstrated significant deficiencies compared with the Sham group based on Nissl staining, TUNEL assay and the Morris­water maze test. HuMSC treated rats exhibited improvement on in all the tests, and CYGB­HuMSCs treatment resulted in further improvements. PCR and western blotting results indicated that the CYGB mRNA and protein levels were increased from day 3 to day 29 after transplantation of CYGB­HuMSCs. Furthermore, it was identified that CYGB­HuMSC transplantation suppressed p38 signaling at all experimental time points. Immunofluorescence indicated the scattered presence of HuMSCs or CYGB­HuMSCs in damaged brain tissue. No eGFP and glial fibrillary acidic protein or eGFP and neuron­specific enolase double­stained positive cells were found in the brain tissues. Therefore, CYGB­HuMSCs may serve as a gene transporter, as well as exert a neuroprotective and antiapoptotic effect in HIBD, potentially via the p38 mitogen­activated protein kinase signaling pathway.

Human umbilical cord mesenchymal stem cells alleviate interstitial fibrosis and cardiac dysfunction in a dilated cardiomyopathy rat model by inhibiting TNF­α and TGF­ß1/ERK1/2 signaling pathways.

Dilated cardiomyopathy (DCM) is a disease of the heart characterized by pathological remodeling, including patchy interstitial fibrosis and degeneration of cardiomyocytes. In the present study, the beneficial role of human umbilical cord­derived mesenchymal stem cells (HuMSCs) derived from Wharton's jelly was evaluated in the myosin­induced rat model of DCM. Male Lewis rats (aged 8­weeks) were injected with porcine myosin to induce DCM. Cultured HuMSCs (1x106 cells/rat) were intravenously injected 28 days after myosin injection and the effects on myocardial fibrosis and the underlying signaling pathways were investigated and compared with vehicle­injected and negative control rats. Myosin injections in rats (vehicle group and experimental group) for 28 days led to severe fibrosis and significant deterioration of cardiac function indicative of DCM. HuMSC treatment reduced fibrosis as determined by Masson's staining of collagen deposits, as well as quantification of molecular markers of myocardial fibrosis such as collagen I/III, profibrotic factors transforming growth factor­ß1 (TGF­ß1), tumor necrosis factor­α (TNF­α), and connective tissue growth factor (CTGF). HuMSC treatment restored cardiac function as observed using echocardiography. In addition, western blot analysis indicated that HuMSC injections in DCM rats inhibited the expression of TNF­α, extracellular­signal regulated kinase 1/2 (ERK1/2) and TGF­ß1, which is a master switch for inducing myocardial fibrosis. These findings suggested that HuMSC injections attenuated myocardial fibrosis and dysfunction in a rat model of DCM, likely by inhibiting TNF­α and the TGF­ß1/ERK1/2 fibrosis pathways. Therefore, HuMSC treatment may represent a potential therapeutic method for treatment of DCM.

Exome sequencing identifies a novel mutation of the GDI1 gene in a Chinese non-syndromic X-linked intellectual disability family.

X-linked intellectual disability (XLID) has been associated with various genes. Diagnosis of XLID, especially for non-syndromic ones (NS-XLID), is often hampered by the heterogeneity of this disease. Here we report the case of a Chinese family in which three males suffer from intellectual disability (ID). The three patients shared the same phenotype: no typical clinical manifestation other than IQ score ≤ 70. For a genetic diagnosis for this family we carried out whole exome sequencing on the proband, and validated 16 variants of interest in the genomic DNA of all the family members. A missense mutation (c.710G > T), which mapped to exon 6 of the Rab GDP-Dissociation Inhibitor 1 (GDI1) gene, was found segregating with the ID phenotype, and this mutation changes the 237th position in the guanosine diphosphate dissociation inhibitor (GDI) protein from glycine to valine (p. Gly237Val). Through molecular dynamics simulations we found that this substitution results in a conformational change of GDI, possibly affecting the Rab-binding capacity of this protein. In conclusion, our study identified a novel GDI1 mutation that is possibly NS-XLID causative, and showed that whole exome sequencing provides advantages for detecting novel ID-associated variants and can greatly facilitate the genetic diagnosis of the disease.

Exome sequencing identifies a novel mutation of the GDI1 gene in a Chinese non-syndromic X-linked intellectual disability family

Abstract X-linked intellectual disability (XLID) has been associated with various genes. Diagnosis of XLID, especially for non-syndromic ones (NS-XLID), is often hampered by the heterogeneity of this disease. Here we report the case of a Chinese family in which three males suffer from intellectual disability (ID). The three patients shared the same phenotype: no typical clinical manifestation other than IQ score ≤ 70. For a genetic diagnosis for this family we carried out whole exome sequencing on the proband, and validated 16 variants of interest in the genomic DNA of all the family members. A missense mutation (c.710G > T), which mapped to exon 6 of the Rab GDP-Dissociation Inhibitor 1 (GDI1) gene, was found segregating with the ID phenotype, and this mutation changes the 237th position in the guanosine diphosphate dissociation inhibitor (GDI) protein from glycine to valine (p. Gly237Val). Through molecular dynamics simulations we found that this substitution results in a conformational change of GDI, possibly affecting the Rab-binding capacity of this protein. In conclusion, our study identified a novel GDI1 mutation that is possibly NS-XLID causative, and showed that whole exome sequencing provides advantages for detecting novel ID-associated variants and can greatly facilitate the genetic diagnosis of the disease.

Human umbilical cord mesenchymal stem cells alleviate acute myocarditis by modulating endoplasmic reticulum stress and extracellular signal regulated 1/2-mediated apoptosis.

Acute myocarditis is a non-ischemic inflammatory disease of the myocardium, and there is currently no standard treatment. Mesenchymal stem cells (MSCs) can alleviate myosin­induced myocarditis; however, the mechanism has not been clearly elucidated. In the present study, the authors investigated the ability of human umbilical cordMSCs (HuMSCs) to attenuate myocardial injury and dysfunction during the acute phase of experimental myocarditis. Male Lewis rats (aged 8 weeks) were injected with porcine myosin to induce myocarditis. Cultured HuMSCs (1x106 cells/rat) were intravenously injected 10 days following myosin injection. A total of 3 weeks following injection, this resulted in severe inflammation and significant deterioration of cardiac function. HuMSC transplantation attenuated infiltration of inflammatory cells and adverse cardiac remodeling, as well as reduced cardiomyocyte apoptosis. Furthermore, it was identified that HuMSC transplantation suppressed endoplasmic reticulum stress and extracellular signal­regulated kinase (ERK)1/2 signaling in experimental autoimmune myocarditis (EAM). The reduced number of TUNEL­positive apoptotic cells in myocardial sections from HuMSC­treated EAM rats compared with control demonstrates HuMSCs' anti­apoptotic function. Based on these data, the author suggested that treatment with HuMSCs inhibits myocardial apoptosis in EAM rats, ultimately protecting them from myocardial damage. The conclusion demonstrated that HuMSC transplantation attenuates myocardial injury and dysfunction in a rat model of acute myocarditis, potentially via regulation of ER stress, ERK1/2 signaling and induction of cardiomyocyte apoptosis.

Differentiation of human umbilical cord mesenchymal stem cells into germ-like cells in mouse seminiferous tubules.

Our previous study demonstrated that human umbilical cord mesenchymal stem cells (HUMSCs) were capable of differentiation into germ cells in vitro. To assess this potential in vivo, HUMSCs were microinjected into the lumen of seminiferous tubules of immunocompetent mice, which were treated with busulfan to destroy endogenous spermatogenesis. Bromodeoxyuridine labeling studies demonstrated that HUMSCs survived in the tubule for at least 120 days, exhibited a round cell shape typical of proliferating or differentiating germ cells, migrated to the basement of the tubule, where proliferating spermatogonia reside and returned to the luminal compartment, where differentiating spermatids and spermatozoa reside. The migration pattern resembled that of germ cell development in vivo. Immunohistochemical and colocalization studies revealed that transplanted HUMSCs expressed the germ cell markers octamer-binding transcription factor 4, α6 integrin, C-kit and VASA, confirming the germ cell differentiation. In addition, it was observed that tubules transplanted with HUMSCs exhibited marked improvement in the histological features damaged by the chemotherapeutic busulfan, as judged by morphology and quantitative histology. Taken together, these data demonstrated the capacity of HUMSCs to form germ cells in the testes and to repair testicular tissue. These findings suggest a potential utility of HUMSCs to treat the infertility and testicular insufficiency caused by cancer therapeutics.

Sertoli cell-mediated differentiation of male germ cell-like cells from human umbilical cord Wharton's jelly-derived mesenchymal stem cells in an in vitro co-culture system.

BACKGROUND: Microenvironment signals play a critical role in directing the differentiation of stem cells. Sertoli cells (SCs) provide a unique microenvironment that is essential for germ cell differentiation. METHODS: Our previous study has demonstrated that human umbilical cord Wharton's jelly-derived mesenchymal stem cells (HUMSCs) could differentiate towards male germ cells in vitro, but HUMSC-derived germ-like cells expressed only few germ cell markers. The aim of this study was to investigate the effect of SCs on the differentiation of HUMSCs towards male germ cells using a co-culture system that mimicked the in vivo male germ cell microenvironment. RESULTS: HUMSCs formed clump-like features on SC monolayers after seeding for 3 weeks. Differentiated cells formed round colonies that share the morphological features of spermatogonial colonies. RT-PCR, immunofluorescence, confocal microscopy, and Western blot analyses revealed the expression of early germ cell markers STELLA and VASA and male germ cell-specific marker DAZL in differentiated HUMSCs, confirming the presence of cells with characteristics of male germ cells. CONCLUSION: The HUMSC-SC co-culture system mimics a native microenvironment for germ cell colonization without any in vitro artificial manipulation and can be used to explore the mechanisms controlling the differentiation of male germ cells from HUMSCs. Male germ cells derived from HUMSCs may be used in the therapy for male infertility.

Immunological characteristics of human umbilical cord mesenchymal stem cells and the therapeutic effects of their transplantion on hyperglycemia in diabetic rats.

Islet transplantation involves the transplantation of pancreatic islets from the pancreas of a donor to another individual. It has proven to be an effective method for the treatment of type 1 diabetes. However, islet transplantation is hampered by immune rejection, as well as the shortage of donor islets. Human umbilical cord Wharton's jelly-derived mesenchymal stem cells (HUMSCs) are an ideal cell source for use in transplantation due to their biological characteristics and their use does not provoke any ethical issues. In this study, we investigated the immunological characteristics of HUMSCs and their effects on lymphocyte proliferation and the secretion of interferon (IFN)-γ, and explored whether direct cell-to-cell interactions and soluble factors, such as IFN-γ were important for balancing HUMSC-mediated immune regulation. We transplanted HUMSCs into diabetic rats to investigate whether these cells can colonize in vivo and differentiate into pancreatic ß-cells, and whether the hyperglycemia of diabetic rats can be improved by transplantation. Our results revealed that HUMSCs did not stimulate the proliferation of lymphocytes and did not induce allogeneic or xenogeneic immune cell responses. qRT-PCR demonstrated that the HUMSCs produced an immunosuppressive isoform of human leukocyte antigen (HLA-I) and did not express HLA-DR. Flow cytometry revealed that the HUMSCs did not express immune response-related surface antigens such as, CD40, CD40L, CD80 and CD86. IFN-γ secretion by human peripheral blood lymphocytes was reduced when the cells were co-cultured with HUMSCs. These results suggest that HUMSCs are tolerated by the host in an allogeneic transplant. We transplanted HUMSCs into diabetic rats, and the cells survived in the liver and pancreas. Hyperglycemia of the diabetic rats was improved and the destruction of pancreatic cells was partly repaired by HUMSC transplantation. Hyperglycemic improvement may be related to the immunomodulatory effects of HUMSCs. However, the exact mechanisms involved remain to be further clarified.

[Prokaryote diversity in water environment of land-ocean ecotone of Zhuhai City].

By constructing 16S rDNA clone library with PCR-RFLP, the prokaryote diversity in the seawater and groundwater of land-ocean ecotone of Zhuhai City was investigated, and the similarity and cluster analyses were implemented with the database of the sequences in Genbank. In the seawater, Proteobacteria was dominant, followed by Archaeon, Gemmatimonadetes, Candidate division OP3 and OP8, and Planctomycetes, etc.; while in the groundwater, Archaeon was dominant, followed by Proteobacteria, Sphingobacteria, Candidate division OP3, Actinobacterium, and Pseudomonas. The dominant taxa in the groundwater had high similarity to the unculturable groups of marine microorganisms. Large amount of bacteria capable of degrading organic matter and purifying water body existed in the groundwater, suggesting that after long-term evolution, the land-ocean ecotone of Zhuhai City had the characteristics of both land and ocean.

[Spatial characteristics of microbial groups associated with the groundwater flow in a small watershed].

Few reports of microbial groups associated with the groundwater flow system are available in China. 16S rRNA gene library was constructed by the cultured-independent approach to investigate gene sequences of microorganism in groundwater samples from the recharge (R), intermediate (M) and discharge (D) zones of an experimental watershed at Zhuhai campus of Sun Yat-sen University. Proteobacterium, Candidate division OPx, uncultured archaeon (uncultured Crenarchaeote and Euryarchaeote) and Actinobacterium are found predominant in all these three zones with the Proteobacterium accounting for 23.21%, 36.21%, and 28.84% in R, M, D zone respectively. The other predominant microbial groups were identified for varied zones, e. g. Eubacterium and Nitrospira in the R wells, Eubacterium and Acidobacterium in the M wells, and Bacteroidetes bacterium in the D wells. Linkages and potential evolution of microbial groups among three zones were examined by using the genetic neighbor-joining tree. Environmental adaptation along the groundwater flow contributes to the similarity and discrepancy of microorganism in term of the genetic tree, and the ecological functions of the microbial groups need further assessment.