Human umbilical cord mesenchymal stem cells derived from Wharton's jelly differentiate into insulin-producing cells in vitro / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Islet transplantation is an effective way of reversing type I diabetes. However, islet transplantation is hampered by issues such as immune rejection and shortage of donor islets. Mesenchymal stem cells can differentiate into insulin-producing cells. However, the potential of human umbilical cord mesenchymal stem cells (huMSCs) to become insulin-producing cells remains undetermined.</p><p><b>METHODS</b>We isolated and induced cultured huMSCs under islet cell culture conditions. The response of huMSCs were monitored under an inverted phase contrast microscope. Immunocytochemical and immunofluorescence staining methods were used to measure insulin and glucagon protein levels. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect gene expression of human insulin and PDX-1. Dithizone-staining was employed to determine the zinc contents in huMSCs. Insulin secretion was also evaluated through radioimmunoassay.</p><p><b>RESULTS</b>HuMSCs induced by nicotinamide and β-mercaptoethanol or by neurogenic differentiation 1 gene (NeuroD1) transfection gradually changed morphology from typically elongated fibroblast-shaped cells to round cells. They had a tendency to form clusters. Immunocytochemical studies showed positive expression of human insulin and glucagon in these cells in response to induction. RT-PCR experiments found that huMSCs expressed insulin and PDX-1 genes following induction and dithizone stained the cytoplasm of huMSCs a brownish red color after induction. Insulin secretion in induced huMSCs was significantly elevated compared with the control group (t = 6.183, P < 0.05).</p><p><b>CONCLUSIONS</b>HuMSCs are able to differentiate into insulin-producing cells in vitro. The potential use of huMSCs in β cell replacement therapy of diabetes needs to be studied further.</p>

Key sperm membrane proteins in sperm-egg fusion / 中华男科学杂志

Fertilization is a complex process involving multiple steps, of which sperm-egg fusion is most important. This article presents a detailed review of some of the key sperm membrane proteins closely related with fertilization, such as the Izumo, the ADAMs gene family and the Crisp gene family proteins, which is of practical significance for deeper insights into the mechanisms of sperm-egg fusion, as well as for the improvement of clinical diagnosis of male infertility and development of novel contraceptive drugs.

Effects of diethylstilbestrol on cultured testis gubernacular cells in mice / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish a primary culture of the testis gubernacular cells of Kunming mice, observe the morphological characteristics of the cells, and explore the effects of exogenous estrogens (EEs) on the development of the testis gubernacula in vitro.</p><p><b>METHODS</b>We removed the gubernacula from 3-day-old mice with the surgical magnifier and cultured the gubernacular cells. Then we detected the cell viability by trypan blue and cell morphology by HE staining. The subcultured cells were randomly divided into a blank control, a DMSO (0.1%, v/v) control, and 4 experimental groups (given 0.01, 0.10, 1.00 and 10.00 micdrog/ml of diethylstilbestrol [DES] dissolved in DMSO, respectively). After treated for 12, 24 and 48 hours, the gubernacular cells were observed for morphological changes and proliferation inhibition by CCK-8.</p><p><b>RESULTS</b>Most of the cultured gubernacular cells were fibroblasts, and a few were epithelioids. The primary cells showed a viability of 85%-90%. Dose- and time-dependent inhibition of cell proliferation was found in the four experimental groups at three different times, with statistically significant differences (P < 0.01).</p><p><b>CONCLUSION</b>Gubernacular cells can be cultured in vitro. EEs inhibit the proliferation of gubernacular cells in a dose- and time-dependent manner. An in- sight into the effects EES on cultured gubernacular cells is an effective approach to the study of their influence on the development of the reproductive system.</p>

Biological characteristics of human umbilical cord-derived mesenchymal stem cells and their differentiation into neurocyte-like cells / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the isolation and expansion of mesenchymal stem cells (MSCs) from human umbilical cord Wharton's jelly and their biological identities, and explore the possibility of inducing human umbilical cord-derived MSCs to differentiate into neurocyte-like cells.</p><p><b>METHODS</b>The growth and proliferative abilities of human umbilical cord-derived MSCs were observed, and their immunophenotypes were determined by flow cytometry. Salvia miltiorrhiza and beta-sulfhydryl alcohol were adopted to induce the cells to differentiate. The differentiated and undifferentiated cells were identified with immunocytochemistry. The pleiotrophin and nestin genes were measured by RT-PCR.</p><p><b>RESULTS</b>A population of human umbilical cord-derived MSCs were isolated from human umbilical Wharton's jelly; they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 10 passages, indicating their proliferative capacity. The human umbilical cord-derived MSCs were positive for CD(29), CD(44), CD(59), CD(105), but negative or weakly expressed the markers of hematopoietic cells such as CD(14), CD(33), CD(34), CD(28), CD(45) and CD(117). The important GVHD correlation markers were negative or weakly expressed, including CD(80) (B7-1), CD(86) (B7-2), CD(40) and CD(40L). Salvia miltiorrhiza beta-sulfhydryl alcohol could induce the MSCs to express nestin, a marker of neuronal precursor stem cells at early stage of differentiation. Later, they exhibited neural phenotypes, expressing beta-tubulin III and neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that the MSCs could express pleiotrophin either before or after the induction of salvia miltiorrhiza, furthermore, after the induction the expression was markedly enhanced and the nestin gene was also expressed.</p><p><b>CONCLUSION</b>The human MSCs could be isolated from human umbilical cord Wharton's jelly, and it was easy to propagate these MSCs. The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier, which may suggest potential clinical significance. The MSCs are capable of differentiating into neurocyte-like cells and they may represent an alternative stem cell source for CNS cells transplantation.</p>

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To detect the expression of hepatitis B virus (HBV) genes (HB S and C genes) in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization (IVF) technique.</p><p><b>METHODS</b>Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction (PCR) was used to detect HB S and pre-Core/Core (pre-C/C) coding genes both in one- and two-cell embryos. Reverse transcription-PCR (RT-PCR) analysis was used to study the expression of the two genes. Fluorescence in situ hybridization (FISH) analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome.</p><p><b>RESULTS</b>Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences.</p><p><b>CONCLUSION</b>Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.</p>

Bone marrow stromal cells express neural phenotypes in vitro and migrate in brain after transplantation in vivo / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the differentiation of bone marrow stromal cells (BMSC) into neuron-like cells and to explore their potential use for neural transplantation.</p><p><b>METHODS</b>BMSC from rats and adult humans were cultured in serum-containing media. Salvia miltiorrhiza was used to induce human BMSC (hBMSC) to differentiate. BMSC were identified with immunocytochemistry. Semi-quantitative RT-PCR was used to examine mRNA expression of neurofilamentl (NF1), nestin and neuron-specific enolase (NSE) in rat BMSC (rBMSC). Rat BMSC labelled by Hoschst33258 were transplanted into striatum of rats to trace migration and distribution.</p><p><b>RESULTS</b>rBMSC expressed NSE, NF1 and nestin mRNA, and NF1 mRNA and expression was increased with induction of Salvia miltiorrhiza. A small number of hBMSC were stained by anti-nestin, anti-GFAP and anti-S100. Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. Some differentiated neuron-like cells, that expressed NSE, beta-tubulin and NF-200, showed typical neuron morphology, but some neuron-like cells also expressed alpha smooth muscle protein, making their neuron identification complicated. rBMSC could migrate and adapted in the host brains after being transplanted.</p><p><b>CONCLUSION</b>Bone marrow stromal cells could express phenotypes of neurons, and Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. If BMSC could be converted into neurons instead of mesenchymal derivatives, they would be an abundant and accessible cellular source to treat a variety of neurological diseases.</p>

Study on Transplantation of Human Umbilical Cord Mesenchymal Stem Cells into Infertile Mouse Testis / 实用儿科临床杂志

Objective To observe clone ability of human umbilical cord mesenchymal stem cells (MSCs) into infertile mouse seminife-rous tubules and the effects of MSCs on reproductive function.Methods Busulfan was used to destroy endogenous spermatogenesis of the recipient mice.To isolate,culture and purify MSCs with adherent method before marked with Brdu and Hoechst 33258 respectively,and then transplanted into the seminiferous tubules by microinjection.The survival of MSCs in recipient testes were evaluated by immunohistochemistry stained for Brdu and Fluorescent microscopy for Hoechst 33258 observation at different times.The diameter of seminiferous tubules was detected with HMIAS-2000 high-definition colored analyzing system for medical pictures.SPSS 13.0 software was used to analyze the data.Results The dosage of Busulfan resulted in 15% death in the mice,the testis of survived mice showed only basilar membrane in seminiferous tubules after 4 weeks.A lot of purified MSCs were obtained at the third generation and transplantation them into mouse seminiferous tubules survive for at least 4 months and appear to migration.The average diameter in experimental groups were higher than those in controls not only on 26 days but also on 120 days(P

Human Umbilical Cord Blood Mesenchymal Stem Cells Colonizing in Hypoxic-Ischemic Encephalopathy Rats Brain / 实用儿科临床杂志

Objective To explore human umbilical cord blood mesenchymal stem cells(MSCs)colonization in hypoxic-ischemic encephalopathy(HIE)rats brain.Methods Models of 7-day-old newborn rats with HIE brain injury were established.Meanwhile,on the same day,MSCs were transplanted with Hoechst 33258 for 24 hours into rats models marked by flurescent nucleotide dye injected through caudal vein or with stereotactic instrument.After 15-30 days,then MSCs were detected with fluorescene microscope.Results With the improved rice methods,HIE animal model was successfully attained.Majority of MSCs were distributed in the cortex,hippocampus of the lesioned hemisphere,especially in the forehead.And abtained a good fusion with HIE rats brain tissue.Conclusion Human umbilical cord blood MSCs can be cultured,when transplanted into the HIE rats model,they can move into intracranial,and integer with rats brain tissue.

Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The two most basic properties of mesenchymal stem cells (MSCs) are the capacities to self-renew indefinitely and differentiate into multiple cells and tissue types. The cells from human umbilical cord Wharton's Jelly have properties of MSCs and represent a rich source of primitive cells. This study was conducted to explore the possibility of inducing human umbilical cord Wharton's Jelly-derived MSCs to differentiate into nerve-like cells.</p><p><b>METHODS</b>MSCs were cultured from the Wharton's Jelly taken from human umbilical cord of babies delivered after full-term normal labor. Salvia miltiorrhiza and beta-mercaptoethanol were used to induce the human umbilical cord-derived MSCs to differentiate. The expression of neural protein markers was shown by immunocytochemistry. The induction process was monitored by phase contrast microscopy, electron microscopy (EM), and laser scanning confocal microscopy (LSCM). The pleiotrophin and nestin genes were measured by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>MSCs in the Wharton's Jelly were easily attainable and could be maintained and expanded in culture. They were positive for markers of MSCs, but negative for markers of hematopoietic cells and graft-versus-host disease (GVHD)-related cells. Treatment with Salvia miltiorrhiza caused Wharton's Jelly cells to undergo profound morphological changes. The induced MSCs developed rounded cell bodies with multiple neurite-like extensions. Eventually they developed processes that formed networks reminiscent of primary cultures of neurons. Salvia miltiorrhiza and beta-mercaptoethanol also induced MSCs to express nestin, beta-tubulinIII, neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that MSCs could express pleiotrophin both before and after induction by Salvia miltiorrhiza. The expression was markedly enhanced after induction and the nestin gene was also expressed.</p><p><b>CONCLUSIONS</b>MSCs could be isolated from human umbilical cord Wharton's Jelly. They were capable of differentiating into nerve-like cells using Salvia miltiorrhiza or beta-mercaptoethanol. The induced MSCs not only underwent morphologic changes, but also expressed the neuron-related genes and neuronal cell markers. They may represent an alternative source of stem cells for central nervous system cell transplantation.</p>

Effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 mRNA level / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To evaluate the effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 transcription level.</p><p><b>METHODS</b>Immature mouse oocytes were cultured in vitro with a known dynein ATPase activity inhibitor-sodium orthovanadate (SOV) to detect the changes of maturation rate, and semi-quantitative RT-PCR and single cell RT-PCR were performed to detect the changes of cyclin B1 mRNA level.</p><p><b>RESULTS</b>In dose-dependent experiments, the maturation rates of oocytes were significantly different between 5 micromol/L SOV and control groups (P < 0.05), and decreased with SOV increasing doses. However, the elevation of cyclin B1 mRNA level of immatured oocytes cultured for 12 h depended on SOV concentrations ranging from 50 to 500 micromol/L. In incontinuity exposed SOV experiments, the maturation rates of oocytes markedly reduced after the first incubation with 400 micromol/L SOV at least for 1 h and were first cultured in SOV-free medium for 4 h or 8 h before exposure to SOV (P < 0.05). In time-course experiment, the opposite changes of cyclin B1 mRNA level in oocytes between SOV and control groups were observed.</p><p><b>CONCLUSION</b>Dynein inhibitor might delay oocytes meiosis process, and cause ectopic expression of cyclin B1 in oocytes. Most Oocytes incubated with SOV blocked at germinal vesicles (GV) stage or M I to anaphase transition due to dynein dysfunction and ectopic transcription level of cyclin B1.</p>

Effect of prenatal exposure to diethylstilbestrol on gubernacular development in fetal male mice / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To study the effect of prenatal exposure to diethylstilbestrol (DES) and the role of actin and proliferating cell nuclear antigen (PCNA) on testicular gubernaculum development in fetal male Kunming mice.</p><p><b>METHODS</b>Pregnant mice were randomly assigned to 6 groups and injected with DES subcutaneously from gestational day 9 (E9) to day 17 (E17) at doses of 0, 25, 50, 100, 200 microg.kg-1.d-1 in 0.2 mL dimethyl sulfoxide (DMSO). On E17 they were sacrificed and fetuses quickly removed for fixation. Male fetuses were sliced on serial coronal plane. Histological changes were observed under the light microscope (LM) and ultrastructural changes with the scanning and transmission electron microscopes (SEM and TEM). The expression intensity of actin and PCNA in the gubernacula was quantitated by immunohistochemistry.</p><p><b>RESULTS</b>The mortality of the fetuses was higher in the DES-treated groups than that in the DMSO and saline controls (P<0.05). Under LM the gubernacula were seen to be poorly developed with smaller bulbs. On SEM the bulbs lose the clear demarcation between the mesenchymal inner core and the muscular outer layer and looked like a small cone instead of the normal cylindrical appearance. On TEM there were some smaller disordered myofibrils and sparse cytoplasmic organelles in the gubernacular muscular cells of the treated groups. The expression intensity of actin and PCNA in the gubernacula was significantly weaker in the treated groups than that in the DMSO and saline controls (P<0.05).</p><p><b>CONCLUSION</b>DES induces underdevelopment of the gubernacula in a dose-dependent manner in fetal male mice and down regulates the actin and PCNA expression.</p>

Studies on the integration of hepatitis B virus DNA sequence in human sperm chromosomes / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To study the integration of hepatitis B virus (HBV) DNA into sperm chromosomes in hepatitis B patients and the features of its integration.</p><p><b>METHODS</b>Sperm chromosomes of 14 subjects (5 healthy controls and 9 HB patients, including 1 acute hepatitis B, 2 chronic active hepatitis B, 4 chronic persistent hepatitis B, 2 HBsAg chronic carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free hamster oocytes and human spermatozoa. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes.</p><p><b>RESULTS</b>Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis B. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots and the others 2 to 4 signals. The fluorescence intensity showed significant difference among the signal spots. The distribution of signal sites among chromosomes seems to be random.</p><p><b>CONCLUSION</b>HBV could integrate into human sperm chromosomes. Results suggest that the possibility of vertical transmission of HBV via the germ line to the next generation is present.</p>

Biological Characteristics of Mesenchymal Stem Cells Cultured in Conditions for Spermatogonia in Vitro / 实用儿科临床杂志

Objective To investigate the possibility of inducing mesenchymal stem cells(MSCs)from human umbilical cord Wharton's Jelly to differentiate into spermatogonia.Methods To isolate,culture and purify MSCs with adherent method,the growth and proliferation of human umbilical cord-derived MSCs were observed,and their immunophenotypes were determined by flow cytometry;MSCs of the third generation were divided into 2 groups to be induced and cultured,MSCs of the control group were cultured in basal medium,while those of the experimental group with conditional medium.The morphologic and ultrastructure changes of control group and experimental group cells were compared with phase contrast microscopy,electron microscopy(EM)and transmission electron microscope(TEM)respectively ;the spermatogonial cells differentiated were then evaluated by immunohistochemistry stained for CD117and CD49f ;the method of Western-blot was used to test if the cells induced could express CD49f.Results A population of MSCs were isolated from human umbilical Wharton's Jelly;they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling;After induction,the shape of MSCs changed greatly from the fibroblast to the round,even familiar to the tadpole;expressed the known molecular markers of spermatogonial cells,such as CD49f,CD117.Conclusion The induced MSCs not only undergo spfermatogonial-cell like morphologic changes,ultramicrostructure mature with increasing cell organs,but also express the spermatogonial cell markers,which show that human umbilical cord derived MSCs are capable of differentiating into spermatogonial cell.