人脐带间充质干细胞移植对小鼠干燥综合征的治疗作用及其机制

背景干眼的发病率有逐渐升高的趋势，炎症反应参与干眼的发生和进展过程，而目前的治疗方法通常仅能缓解症状，而不能达到彻底治愈的目的。已有研究将干细胞移植用于眼表损伤引起的角膜缘干细胞缺乏症，但干细胞是否可用于干眼及其作用机制的研究鲜见报道。目的探讨人脐带间充质干细胞（HUCMSCs）移植治疗干燥综合征的可行性及治疗途径。方法采用完全随机方法将20只24周龄雄性NOD／Ltj小鼠分为尾静脉注射组、泪腺注射组、点眼组和模型组，分别采用HUCMSCs悬液尾静脉注射法、泪腺注射法和点眼法进行HUCMSCs移植，模型组小鼠不移植HUCMSCs，采用ICR雄性小鼠5只作为正常对照。分别于注射后1、2和3周采用酚红棉线法测量各组小鼠的泪液分泌量；采用角膜荧光素钠染色法对角膜上皮的缺损情况进行评分；采用ELISA法检测各组小鼠血清白细胞介素（IL）-6、IL-17a、肿瘤坏死因子-α（TNF—α）和γ干扰素（IFN-γ）质量浓度；采用Western blot法检测各组小鼠泪腺组织中p65、Stat3、Stat5和细胞外信号调节激酶-1（Erk-1）的相对表达量。对各组小鼠上述指标的测定结果进行比较。结果模型组、尾静脉注射组、泪腺注射组和点眼组NOD／Ltj小鼠干预后1、2和3周时组间泪液分泌量的总体比较差异均有统计学意义（第1周：F=3．700，P=0．040；第2周：F=5．150，P=0．008；第3周：F=10．130，P〈0．001），其中注射后第1、2和3周尾静脉注射组、泪腺注射组小鼠的泪液分泌量均明显多于模型组，差异均有统计学意义（均P〈0．05），但各时间点点眼组小鼠的泪液分泌量与模型组比较差异均无统计学意义（均P〉0．05）。注射后1周ICR小鼠对照组、模型组、尾静脉注射组、泪腺注射组和点眼组角膜荧光素钠染色评分分别为3．00±0．63、9．40±1．62、5．20±1．17、4．20±1．17和7．20±0．98，其中尾静脉注射组、泪腺注射组和点眼组小鼠角膜荧光素染色评分值明显低于模型组，差异均有统计学意义（P=0．001、0．000、0．033）。注射HUCMSCs后尾静脉注射组小鼠血清IL-6、IL-17a和TNF-α质量浓度均明显低于模型组，差异均有统计学意义（t=4．70、3．46、11．00，均P〈0．01），而各组间小鼠血清IFN-γ质量浓度总体比较差异无统计学意义（F=1．740，P=0．170）。尾静脉注射组和泪腺注射组小鼠眶外泪腺中Stat5蛋白相对表达量低于模型对照组，差异均有统计学意义（均P〈0．05）。结论NOD／Ltj干燥综合征模型小鼠全身和泪腺局部注射HUCMSCs能够分别抑制血清和泪腺中炎性相关因子的表达，减轻炎症反应，从而治疗干眼。HUCMSCs点眼法能够发挥润滑和营养角膜的作用，从而缓解干眼症状。

Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes

AIM： To investigate the differentiation of human umbilical cord blood （HUCB）-derived mesenchymal stem cells （MSCs） into hepatocytes by induction of fibroblast growth factor-4 （FGF-4） and hepatocyte growth factor （HGF）, and to find a new source of cell types for therapies of hepatic diseases. METHODS： MSCs were isolated by combining gradient density centrifugation with plastic adherence. When HUCB-derived MSCs reached 70% confluence, they were cultured in Iscove modified Dulbecco medium （IMDM） supplemented with 10 mL/L FBS, 20 ng/mL HGF and 10 ng/mL FGF-4. The medium was changed every 4 d and stored for albumin, alpha-fetoprotein （AFP） and urea assay. Expression of CK-18 was detected by immunocytochemistry. Glycogen storage in hepatocytes was determined by PAS staining. RESULTS： By combining gradient density centrifugation with plastic adherence, we could isolate MSCs from 25.6% of human umbilical cord blood. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on d 28 by morphology. Compared with the control, the level of AFP increased significantly from d 12 to 18.20：1=1.16 μg/L （t = 2.884, P〈0.05） in MSCs cultured with FGF-4 and HGF, and was higher （54.28±3.11 μg/L） on d 28 （t = 13.493, P〈0.01）. Albumin increased significantly on d 16 （t = 6.68, P〈0.01） to 1.02±0.15 μg/mL, and to 3.63±0.30 μg/mL on d 28 （t = 11.748, P〈0.01）. Urea （4.72±1.03 μmol/L） was detected on d 20 （t = 4.272, P〈0.01）, and continued to increase to 10.28±1.06 μmol/L on d 28 （t = 9.276, P〈0.01）. Cells expressed CK-18 on d 16. Glycogen storage was observed on d 24. CONCLUSION： HUCB-derived MSCs can differentiate into hepatocytes by induction of F-GF-4 and HGF. HUCB derived MSCs are a new source of cell types for cell transplantation therapy of hepatic diseases.

Differentiation of human umbilical cord blood stem cells into hepatocytes in vivo and in vitro

AIM： To study the condition and potentiality of human umbilical cord blood stem cells （HUCBSC） to differentiate into hepatocytes in vivo or in vitro. METHODS： In a cell culture study of human umbilical cord blood stem cell （HUCBSC） differentiation, human umbilical cord blood mononuclear cells （HUCBMNC） were separated by density gradient centrifugation. Fibroblast growth factor （FGF） and hepatocyte growth factor （HGF） and the supernatant of fetal liver were added in the inducing groups. Only FGF was added in the control group. The expansion and differentiation of HUCBMNC in each group were observed. Human alpha fetoprotein （AFP） and albumin （ALB） were detected by immunohistochemistry. In the animal experiments, the survival SD rats with acute hepatic injury after carbon tetrachloride （CCL4） injection 48 h were randomly divided into three groups. The rats in group A were treated with human umbilical cord blood serum. The rats in group B were treated with HUCBMNC transplantation. The rats in group C were treated with HUCBMNC transplantation followed by intraperitoneal cyclophosphamide for 7 d. The rats were killed at different time points after the treatment and the liver tissue was histopathologically studied and human AFP and ALB detected by immunohistochemistry. The human X inactive-specific transcript gene fragment in the liver tissue was amplified by PCR to find human DNA. RESULTS： The results of cell culture showed that adherent cells were stained negative for AFP or ALB in control group. However, the adherent cells in the inducing groups stained positive for AFP or ALB. The result of animal experiment showed that no human AFP or ALB positive cells present in the liver tissue of group A （control group）. However, many human AFP or ALB positive cells were scattered around sinus hepaUcus and the central veins of hepatic Iobules and in the portal area in group B and group C after one month. The fragment of human X chromagene could be detected in the liver tissue of groups B and C, but not in group A.CONCLUSION： Under certain conditions HUCBSC can differentiate into liver cells in vivo and in vitro.

Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocyte proliferation

Human placenta-derived mononuclear cells （MNC） were isolated by a Percoll density gradient and cultured in mesenchymal stem cell （MSC） maintenance medium. The homogenous layer of adherent cells exhibited a typical fibroblastlike morphology, a large expansive potential, and cell cycle characteristics including a subset of quiescent cells. In vitro differentiation assays showed the tripotential differentiation capacity of these cells toward adipogenic, osteogenic and chondrogenic lineages. Flow cytometry analyses and immunocytochemistry stain showed that placental MSC was a homogeneous cell population devoid of hematopoietic cells, which uniformly expressed CD29, CD44, CD73, CD105, CD166, laminin, fibronectin and vimentin while being negative for expression of CD31, CD34, CD45 and m-smooth muscle actin. Most importantly, immuno-phenotypic analyses demonstrated that these cells expressed class Ⅰ major histocompatibility complex （MHC-I）, but they did not express MHC-Ⅱ molecules. Additionally these cells could suppress umbilical cord blood （UCB） lymphocytes proliferation induced by cellular or nonspecific mitogenic stimuli. This strongly implies that they may have potential application in allograft transplantation. Since placenta and UCB are homogeneous, the MSC derived from human placenta can be transplanted combined with hematopoietic stem cells （HSC） from UCB to reduce the potential graft-versus-host disease （GVHD） in recipients.

Umbilical cord mesenchymal stem cell transplantation for the treatment of Duchenne muscular dystrophy

Due to their relative abundance,stable biological properties and excellent reproductive activity,umbilical cord mesenchymal stem cells have previously been utilized for the treatment of Duchenne muscular dystrophy,which is a muscular atrophy disease.Three patients who were clinically and pathologically diagnosed with Duchenne muscular dystrophy were transplanted with umbilical cord mesenchymal stem cells by intravenous infusion,in combination with multi-point intramuscular injection.They were followed up for 12 months after cell transplantation.Results showed that clinical symptoms significantly improved,daily living activity and muscle strength were enhanced,the sero-enzyme,electromyogram,and MRI scans showed improvement,and dystrophin was expressed in the muscle cell membrane.Hematoxylin-eosin staining of a muscle biopsy revealed that muscle fibers were well arranged,fibrous degeneration was alleviated,and fat infiltration was improved.These pieces of evidence suggest that umbilical cord mesenchymal stem cell transplantation can be considered as a new regimen for Duchenne muscular dystrophy.

Transplantation of microencapsulated umbilical-cord-blood-derived hepatic-like cells for treatment of hepatic failure

AIM: To investigate intraperitoneal transplantation of microencapsulated hepatic-like cells from human umbilical cord blood for treatment of hepatic failure in rats. METHODS: CD34+ cells in umbilical cord blood cells were isolated by magnetic cell sorting. In the in vitro experiment, sorted CD34+ cells were amplified and induced into hepatic-like cells by culturing with a combination of fibroblast growth factor 4 and hepatocyte growth factor. Cultures without growth factor addition served as controls. mRNA and protein levels for hepatic-like cells were analyzed by reverse transcription-polymerase chain reaction, immunohistochemistry and immunofluorescence. In the in vivo experiment, the hepatic-like cells were encapsulated and transplanted into the abdominal cavity of acute hepatic failure (AHF) rats at 48 h after D-galactosamine induction of acute hepatic failure. Transplantation with PBS and unencapsulated hepatic-like cells served as controls. The mortality rate, hepatic pathological changes and serum biochemical indexes were determined. The morphology and structure of microcapsules in the greater omentum were observed. RESULTS: Human albumin, alpha-fetoprotein and GATA-4 mRNA and albumin protein positive cells were found among cultured cells after 16 d. Albumin level in culture medium was significantly increased after culturing with growth factors in comparison with culturing without growth factor addition (P < 0.01). Compared with the unencapsulated group, the mortality rate of the encapsulated hepatic-like cell-transplanted group was significantly lower (P < 0.05). Serum biochemical parameters, alanine aminotransferase, aspartate aminotransferase and total bilirubin in the encapsulated group were significantly improvement compared with the PBS control group (P < 0.01). Pathological staining further supported these findings. At 1-2 wk post-transplantation, free microcapsules with a round clear structure and a smooth surface were observed in peritoneal lavage fluid, surviving cells inside microcapsules were found by trypan blue staining, but some fibrous tissue around microcapsules was also detected in the greater omentum of encapsulated group by hematoxylin and eosin staining. CONCLUSION: Transplantation of microencapsulated hepatic-like cells derived from umbilical cord blood cells could preliminarily alleviate the symptoms of AHF rats.

Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen

Objective: To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen. Methods:Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination.Results: The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor,hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-α (tumor necrosis factor-α), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found. Conclusion: Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.

Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells

AIM： To study the selective killing of human umbilical vein endothelial cells （HUVECs） by a double suicide gene under the regulation of a kinase domain insert containing receptor （KDR） promoter and mediated by an adenoviral gene vector. METHODS： Human KDR promoter was cloned by polymerase chain reaction （PCR）, and two recombinant adenoviral plasmids pAdKDR-CdgIyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses （AdKDR-CDglyTK and AdCMV-CDglyTK） respectively, and the infection rates were estimated by detection of green fluorescent protein （GFP） expression. Infected cells were cultured in culture media containing different concentrations of 5-fiuoroo/tosine （5-FC） and ganciclovir （GCV）, and the killing effects were measured. RESULTS： The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs： HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMVo CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK （P 〈 0.001）. CONCLUSION： Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

Cocultivation of umbilical cord blood CD34^+ cells with retro-transduced hMSCs leads to effective amplification of long-term culture-initiating cells

AIM： To establish a novel coculture system for ex vivo expansion of umbilical cord blood（UCB） hematopoietic progenitors using thrombopoietin （TPO）/FIt-3 ligand （FL）-transduced human marrow-derived mesenchymal stem cells （tfhMSCs） as feeder. METHODS： UCB CD34^＋ cells were isolated and cultured using four culture systems in serum-containing or serumfree medium. Suitable aliquots of cultured cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture （LTC-IC） output. Finally, the severe-combined immunodeficient （SCID） mouse-repopulating cell （SRC） assay was performed to confirm ability of the cultured cells to reconstitute longterm hematopoiesis. RESULTS： There were no significant differences in the number of total nudeated cells among different culture systems in serum-containing medium during 21-d culture. However, on d 14, the outputs of CD34^＋ cells, CFU-C and CFU-GEMM in tfhMSCs coculture system were significantly enhanced. LTC-IC assay demonstrated that the tfhMSCs coculture system had the most powerful activity. The severe-combined immunodeficient （SCID） mouse repopulating cell （SRC） assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. Furthermore, PCR analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of NOD/SCID mice.CONCLUSION： The TPO/FL-transduced hMSCs, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro and the tfhMSCs coculture system may be a suitable system for ex vivo manipulation of primitive progenitor cells under contact culture conditions.

Effects of static magnetic field on human umbilical vessel endothelial cell

Objective: To investigate the effects of static magnetic field(SMF) on the viability, adhesion molecule expression of human umbilical vessel endothelial cell. Methods: Magnetic flux intensity was 0. 1 mT, 1 mT, 10 mT. Cell viability and proliferation were measured with 3H-TdR and MTT methods; and apoptosis of human umbilical vein endothelial cell (HUVEC) was studied by flow cytometry and transmission electric microscopy. ELISA was used to measure the expression of ICAM-1 and VCAM-1 on endothelium. Results: 0. 1 mT SMF had no effects on the growth of HUVEC. however,SMF of 1 mT, 10 mT attenuated growth of HUVEC. 10 mT static magnetic field could induce apoptosis and necrosis of HUVEC. 10 mT SMF enhanced the expression of ICAM-1 and VCAM-1 on endothelium. Conclusion: The effect of SMF depends on the intensity of SMF. 10 mT SMF has adverse effects on human umbilical vessel endothelial cell.

Conversion of mononuclear cells from human umbilical cord blood into hepatocyte-like cells

Objective： To evaluate the dltterentlatlon ot human umbilical cord blood ceils into hepatocyte-like cells. Methods： Mononuclear cells （MNCs） derived from human umbilical cord blood were isolated using Ficoll. The experiment was derived into 3 categories： （1） MNCs co-cultured with 50 mg minced liver tissue separated by a trans-well membrane and then collected at 0 h, 24 h, 48 h and 72 h; （2） MNCs cultured along supplemented with 100 ml/L FBS, 100 μ/ml penicillin, 100 μg/ml streptomycin, 4.7 μg/ml linoleic acid, 1×ITS, 10^-4 mol/L L-Ascorbic acid 2-P and a combination of FGF4 （100 ng/ml） and HGF （20 ng/mL）. Cells were then collected at 0 d and 16 d to examine the expression profile of hepatocyte correlating markers; （3） 0.2-0.3 ml of MNCs with a cell density of 2×10^7/ml were transplanted into prepared recipient mice [n=12, injected with 0.4 ml/kg （20%） CCl4 and 150 ng/kg 5-fluorouracil （5-Fu） prior the transplant 24 h and 48 h, respectively] via injection through tail vein. Mice were sacrificed 4 weeks after transplantation. The hepatocyte correlating mRNAs and proteins were determined by RT-PCR, immunohistochemical analysis and immunoflurence technique. Results： （1） After 72 h, a number of glycogen positive stained cells were observed with MNCs co-cultured with damaged mouse liver tissues. The expression of hepatocyte markers, human albumin （ALB）, α-fetal protein （AFP） and human GATA4 mRNA and proteins were detected by RT-PCR and immunohistochemistry as well. For the confirmation, the DNA sequencing of PCR products was performed. In control groups, MNCs co-cuhured with normal mouse hepatocytes or MNCs cultured alone, all markers remained negative. （2） In growth factor supplemented culture system, MNCs developed into larger volume with richer cytoplasm and binucleation after 16 d. Positive expression of ALB, AFP, CK18 and CK19 mRNA were detected with RT-PCR, and ALB positive staining was observed by immunocytochemistry as well. In contrast, MNCs cultured without exogenous growth factors scarcely attached to the culture dish and ALB mRNA was not detected. （3） In transplantation experiment, both of ALB and AFP mRNA were detected by RT-PCR and HSA, PCNA and ALB positive staining were observed in the livers of recipient mice by immunocytochemistry. Conclusion： MNCs from human umbilical cord blood could convert into hepatocyte-like ceils in 3 different ways, indicating their potential use in the clinic applications for the treatment of human liver diseases.

Expression of Caspase－3 in Cord Blood CD34^＋ Cells during Culture in vitro

Objective: To investigate the expression and significance of caspase-3 protein in CD34^+ cells from cord blood (CB) during culture in vitro with different growth factors. Methods: RT-PCR, Western blot and flow cytometry techniques were used to detect the expression of caspase-3 in CD34^+ CB cells during culture in vitro. Results: Caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34^+ cells. The expression of caspase-3 mRNA and protein was upregulated when these cellswere first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3 proenzyme was detected in the freshly isolated CD34^+ cells as well as during the first 3 days expansion with cytokines. With longer culture time in vitro, especially in the presence of the combination of IL-3, IL-6 and GM-CSF, caspase-3 was activated and a cleavage product of 20 kDa became detectable.Conclusion: Caspase-3 is involved in apoptosis of primitive CB CD34^+ cells during expansion in vitro.

Generation of human/rat xenograft animal model for the study of human donor stem cell behaviors in vivo

AIM： TO accurately and realistically elucidate human stem cell behaviors in vivo and the fundamental mechanisms controlling human stem cell fates in vivo, which is urgently required in regenerative medicine and treatments for some human diseases, a surrogate human-rat chimera model was developed. METHODS： Human-rat chimeras were achieved by in utero transplanting low-density mononuclear cells from human umbilical cord blood into the fetal rats at 9-11 d of gestation, and subsequently, a variety of methods, including flow cytometry, PCR as well as immunohistochemical assay, were used to test the human donor contribution in the recipients. RESULTS： Of 29 live-born recipients, 19 had the presence of human CD45^＋ cells in peripheral blood （PB） detected by flow cytometry, while PCR analysis on genomic DNA from 11 different adult tissues showed that 14 selected from flow cytometry-positive 19 animals possessed of donor-derived human cell engraftment in multiple tissues （i.e. liver, spleen, thymus, heart, kidney, blood, lung, muscle, gut and skin） examined at the time of tissue collection, as confirmed by detecting human 132- microglobulin expression using immunohistochemistry. Tn this xenogeneic system, the engrafted donor-derived human cells persisted in multiple tissues for at least 6 mo after birth. Moreover, transplanted human donor cells underwent site-specific differentiation into CK18-positive human cells in chimeric liver and CEHS-positive human cells in chimeric spleen and thymus of recipients. CONCLUSION： Taken together, these findings suggest that we successfully developed human-rat chimeras, in which xenogeneic human cells exist up to 6 mo later. This humanized small animal model, which offers an in vivo environment more closely resembling to the situations in human, provides an invaluable and effective approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future. The potential for new advances in our better understanding the living biological systems in human provided by investigators in humanized animals will remain promising.

Comparison of human amniotic fluid-derived and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells： Characterization and myocardial differentiation capacity

Objective To compare the characterization and myocardial differentiation capacity of arnniotic fluid-derived mesenchymal stromal cells （AF MSCs） and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells （WJ MSCs）. Methods The human AF MSCs were cultured from amniotic fluid samples obtained by amniocentesis. The umbilical cord WJ MSCs were obtained from Wharton＇s Jelly of umbilical cords of infants delivered full-term by normal labor. The morphology, growth curves, and analyses by flow cytometry of cell surface markers were compared between the two types of cells. Myocardial genes （GATA-4, c-TnT, a-actin, and Cx43） were detected by real-time PCR and the corresponding protein expressions were detected by Western blot analysis after myocardial induced in AF MSCs and WJ MSCs. Results Our findings revealed AF MSCs and WJ MSCs shared similar morphological characteristics of the fibroblastoid shape. The AF MSCs were easily obtained than the WJ MSCs and had a shorter time to reach adherence of 2.7 ± 1.6 days to WJ MSCs of 6.5 ± 1.8 days. The growth curves by MTT cytotoxic assay showed the AF MSCs had a similar proliferative capacity at passage 5 and passage 10. However, the proliferative capacities ofWJ MSCs were decreased at 5 passage relative to 10 passage. Both AF stem cells and WJ stem cells had the characteristics of mesenchymal stromal cells with some characteristics of embryonic stem cells. They express CD29 and CD105, but not CD34. They were positive for Class I major histocompatibility （MHC I） antigens （HLA-ABC）, and were negative, or mildly positive, for MHC Class II （HLA-DR） antigen. Oct-4 was positive in all the two cells types. Both AF MSCs and WJ MSCs could differentiate along myocardium. The differentiation capacities were detected by the expression of GATA-4, c-TnT, a-actin, Cx43 after myocardial induction. Conclusions Both AF MSCs and WJ MSCs have the potential clinical application for myogenesis in cardiac regenerative therapy.

Effect of IBD sera on expression of inducible and endothelial nitric oxide synthase in human umbilical vein endothelial cells

AIM： To study the expression of endothelial and inducible nitric oxide synthases （eNOS and iNOS） and their role in inflammatory bowel disease （IBD）. METHODS： We examined the effect of sera obtained from patients with active Crohn＇s disease （CD） and ulcerative colitis （UC） on the function and viability of human umbilical vein endothelial cells （HUVEC）. HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofluorescence, and quantified by the densitometry of Western blots. Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10^-4 mol/L L-NAME. Apoptosis and necrosis was examined by the annexin-V-biotin method and by propidium iodide staining, respectively. RESULTS： In HUVEC immediately after exposure to UC, serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control （18%±16% vs 23%± 15% P〈0.01）. UC or CD serum caused a significant increase in iNOS compared to control （UC： 300%±21%; CD： 275% ± 27% vs 108% ± 14%, P〈0.01）. Apoptosis/necrosis characteristics did not differ significantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum. CONCLUSION： IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced antiinflammatory protection of endothelial cells.

Ex vivo expansion of CD34^+ cells and immunocytes from umbilical cord blood

Objective： Umbilical cord blood stem cell transplantation （CBSCT） has approached significant success in leukemia treatment, but it is associated with higher rates of delayed or failed engraftment and relapse. This may be caused by immature immune calls of umbilical cord blood. We try to expand stem/progenitor calls and T, NK, DC immunocytes from umbilical cord blood for transplantation and immunotherapy. Methods： CB MNCs were cultured and analyzed for progenitor/stem calls, immunocytes at day 0, 3, 7 and 14 by using flowcytometry. Results： The combinations of SCF, IL-3, IL-6, plus IL-2 or/and IL-4 showed significantly expanded results both for UCB MNCs and CD34^＋ cells. CD34^＋ percentage went up from fresh CB 1.6% to the highest group E （SCF＋IL-3, 6, 2, 4） 11.1%. The average expansion multiples of CD34^＋ calls at 7th culture days were from 10 to 50 （SCF＋IL-3, 6, 2, 4）. The CD3^＋ T cells was （18.7±4.3）% in fresh cord blood, and decreased sharply in the medium without cytokine, while markedly increased in groups with cytokines combination, in group B, E, G and F, their level were about 2 times of fresh control. The fresh UCB contained （3.6±1.9）% CD56^＋ calls, NK cells only were expanded in groups with IL-2. DCs markers CDla, CD80, CD83 and CD86 expressed a lower level at day 3 in all test groups, and then increased sharply in groups E, F and G with IL-4 cytokin at 7th culture days. Conclusion： T calls, NK cells and DCs as well as stem/progenitor calls could be expanded in the same medium from CB MNCs with the combinations of cytokines. The combination of SCF, IL-2, IL-3, IL-6 and IL-4 showed a balancaable expansion result of both CD34^＋ cells and immunocytes at 7th culture days.

Mechanism Study of Endothelial Protection and Inhibits Platelet Activation of Low Molecular Weight Fucoidan from Laminaria japonica

Several studies have indicated that fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica could inhibit the activation of platelets directly by reducing the platelet aggregation. To explore the direct effect of LMW fucoidan on the platelet system furthermore and examine the possible mechanism, the endothelial protection and inhibits platelet activation effects of two LMW fucoidan were investigated. In the present study, Endothelial injury model of rats was made by injection of adrenaline(0.4 mg kg-1) and human umbilical vein endothelial cells were cultured. v WF level was be investigated in vivo and in vitro as an important index of endothelial injury. LMW fucoidan could significantly reduce v WF level in vascular endothelial injury rats and also significantly reduce v WF level in vitro. The number of EMPs was be detected as another important index of endothelial injury. The results showed that LMW fucoidan reduced EMPs stimulated by tumor necrosis factor. In this study, it was found that by inhibiting platelet adhesion, LMW fucoidan played a role in anti-thrombosis and the specific mechanism of action is to inhibit the flow of extracellular Ca2+. All in a word, LMW fucoidan could inhibit the activation of platelets indirectly by reducing the concentration of EMPs and v WF, at the same time; LMW fucoidan inhibited the activation of platelets directly by inhibiting the flow of extracellular Ca2+.

Ex vivo expansion of hematopoietic stem cells

Ex vivo expansion of hematopoietic stem cells(HSCs) would benefit clinical applications in several aspects, to improve patient survival, utilize cord blood stem cells for adult applications, and selectively propagate stem cell populations after genetic manipulation. In this review we summarize and discuss recent advances in the culture systems of mouse and human HSCs, which include stroma/HSC co-culture, continuous perfusion and fed-batch cultures, and those supplemented with extrinsic ligands, membrane transportable transcription factors, complement components, protein modification enzymes, metabolites, or small molecule chemicals. Some of the expansion systems have been tested in clinical trials. The optimal condition for ex vivo expansion of the primitive and functional human HSCs is still under development. An improved understanding of the mechanisms for HSC cell fate determination and the HSC culture characteristics will guide development of new strategies to overcome difficulties. In the future, development of a combination treatment regimen with agents that enhance self-renewal, block differentiation, and improve homing will be critical. Methods to enhance yields and lower cost during collection and processing should be employed. The employment of an efficient system for ex vivo expansion of HSCs will facilitate the further development of novel strategies for cell and gene therapies including genome editing.

CAPE promotes the expansion of human umbilical cord blood-derived hematopoietic stem and progenitor cells in vitro

Due to the low number of collectable stem cells from single umbilical cord blood(UCB)unit,their initial uses were limited to pediatric therapies.Clinical applications of UCB hematopoietic stem and progenitor cells(HSPCs)would become feasible if there were a culture method that can effectively expand HSPCs while maintaining their self-renewal capacity.In recent years,numerous attempts have been made to expand human UCB HSPCs in vitro.In this study,we report that caffeic acid phenethyl ester(CAPE),a small molecule from honeybee extract,can promote in vitro expansion of HSPCs.Treatment with CAPE increased the percentage of HSPCs in cultured mononuclear cells.Importantly,culture of CD34+HSPCs with CAPE resulted in a significant increase in total colony-forming units and high proliferative potential colony-forming units.Burst-forming unit-erythroid was the mostly affected colony type,which increased more than 3.7-fold in 1μg mL 1CAPE treatment group when compared to the controls.CAPE appears to induce HSPC expansion by upregulating the expression of SCF and HIF1-α.Our data suggest that CAPE may become a potent medium supplement for in vitro HSPC expansion.

Distinguish on the viability of human umbilical cord mesenchymal stem cells using delayed luminescence

In this paper, we report the discrimination of the viability of human umbilical cord mesenchymal stem cells(hUC-MSCs) with photo-induced delayed luminescence(DL). We measure the DL decay kinetics of hUC-MSCs using an ultraweak luminescence detection system, and find the significant difference in the weight distributions of the decay rate for hUC-MSCs with high and low viabilities. Spectral discrimination of hUC-MSCs with high and low viabilities is thus carried out by comparing the DL kinetics parameters, including the initial intensity, the peak decay rate and the peak weight value. Our results show that the novel optical method for the viability diagnosis of hUC-MSCs has a promising prospect.