BACKGROUND Premature ovarian failure(POF)affects many adult women less than 40 years of age and leads to infertility.According to previous reports,various tissue-specific stem cells can restore ovarian function and fo...BACKGROUND Premature ovarian failure(POF)affects many adult women less than 40 years of age and leads to infertility.According to previous reports,various tissue-specific stem cells can restore ovarian function and folliculogenesis in mice with chemotherapy-induced POF.Human embryonic stem cells(ES)provide an alternative source for mesenchymal stem cells(MSCs)because of their similarities in phenotype and immunomodulatory and anti-inflammatory characteristics.Embryonic stem cell-derived mesenchymal stem cells(ES-MSCs)are attractive candidates for regenerative medicine because of their high proliferation and lack of barriers for harvesting tissue-specific MSCs.However,possible therapeutic effects and underlying mechanisms of transplanted ES-MSCs on cyclophosphamide and busulfan-induced mouse ovarian damage have not been evaluated.AIM To evaluate ES-MSCs vs bone marrow-derived mesenchymal stem cells(BMMSCs)in restoring ovarian function in a mouse model of chemotherapy-induced premature ovarian failure.METHODS Female mice received intraperitoneal injections of different doses of cyclophosphamide and busulfan to induce POF.Either human ES-MSCs or BMMSCs were transplanted into these mice.Ten days after the mice were injected with cyclophosphamide and busulfan and 4 wk after transplantation of the ESMSCs and/or BM-MSCs,we evaluated body weight,estrous cyclicity,folliclestimulating hormone and estradiol hormone concentrations and follicle count were used to evaluate the POF model and cell transplantation.Moreover,terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling,real-time PCR,Western blot analysis and immunohistochemistry and mating was used to evaluate cell transplantation.Enzyme-linked immunosorbent assay was used to analyze vascular endothelial growth factor,insulin-like growth factor 2 and hepatocyte growth factor levels in ES-MSC condition medium in order to investigate the mechanisms that underlie their function.RESULTS The human ES-MSCs significantly restored hormone secretion,survival rate and reproductive function in POF mice,which was similar to the results obtained with BM-MSCs.Gene expression analysis and the terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling assay results indicated that the ES-MSCs and/or BM-MSCs reduced apoptosis in the follicles.Notably,the transplanted mice generated new offspring.The results of different analyses showed increases in antiapoptotic and trophic proteins and genes.CONCLUSION These results suggested that transplantation of human ES-MSCs were similar to BM-MSCs in that they could restore the structure of the injured ovarian tissue and its function in chemotherapy-induced damaged POF mice and rescue fertility.The possible mechanisms of human ES-MSC were related to promotion of follicular development,ovarian secretion,fertility via a paracrine effect and ovarian cell survival.展开更多
The difference between Noggin and basic fibroblast growth factor for the neural precursor differen- tiation from human embryonic stem cells has not been studied. In this study, 100 tJg/L Noggin or 20 IJg/L basic fibro...The difference between Noggin and basic fibroblast growth factor for the neural precursor differen- tiation from human embryonic stem cells has not been studied. In this study, 100 tJg/L Noggin or 20 IJg/L basic fibroblast growth factor in serum-free neural induction medium was used to differen- tiate human embryonic stem cells H14 into neural precursors using monolayer differentiation. Two weeks after induction, significantly higher numbers of neural rosettes formed in the Noggin-induced group than the basic fibroblast growth factor-induced group, as detected by phase contrast micro- scope. Immunofluorescence staining revealed expression levels of Nestin, [3-111 Tubulin and Sox-1 were higher in the induced cells and reverse-transcription PCR showed induced cells expressed Nestin, Sox-1 and Neurofilament mRNA. Protein and mRNA expression in the Noggin-induced group was increased compared with the basic fibroblast growth factor-induced group. Noggin has a greater effect than basic fibroblast growth factor on the induction of human embryonic stem cell differentiation into neural precursors by monolayer differentiation, as Noggin accelerates and in- creases the differentiation of neural precursors.展开更多
This study aimed to compare epithelial cells derived from human embryonic stem cells(hESCs) to human ameloblast-lineage cells (ALCs),as a way to determine their potential use as a cell source for ameloblast regenerati...This study aimed to compare epithelial cells derived from human embryonic stem cells(hESCs) to human ameloblast-lineage cells (ALCs),as a way to determine their potential use as a cell source for ameloblast regeneration.Induced by various concentrations of bone morphogenetic protein 4(BMP4),retinoic acid(RA) and lithium chloride(LiCI) for 7 days,hESCs adopted cobble-stone epithelial phenotype(hESC-derived epithelial cells(ES-ECs)) and expressed cytokeratin 14.Compared with ALCs and oral epithelial cells(OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs.ES-ECs were compared with human fetal skin epithelium,human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well.ALCs had relatively high expression levels of cytokeratin 76,which was also found to be upregulated in ES-ECs.Based on the present study,with the similarity of gene expression with ALCs,ES-ECs are a promising potential cell source for regeneration,which are not available in erupted human teeth for regeneration of enamel.展开更多
The effects of vascular endothelial growth factor (VEGF) on neural differentiation of human embryonic stem cells (hESCs) in vitro and the possible mechanism were observed. The hESCs lines, TJMU1 and TJMU2, were es...The effects of vascular endothelial growth factor (VEGF) on neural differentiation of human embryonic stem cells (hESCs) in vitro and the possible mechanism were observed. The hESCs lines, TJMU1 and TJMU2, were established and stored by our laboratory, hESCs differentiated into neuronal cells through embryonic body formation. In this induction process, hESCs were divided into three groups: group A, routine induction; group B, routine induction+10 ng/mL VEGF; group C, routine in- duction+10 ng/mL VEGF+10 ng/mL VEGFR2/Fc. OCT4, Nestin and GFAP in each group were de- tected by RT-PCR, and the cells expressing Nestin and GFAP were counted by immunofluorescence. The percentage of Nestin positive cells in group B was significantly higher than in groups A and C, while the percentage of GFAP positive cells in group B was significantly lower than in groups A and C (P〈0.01). There was no significant difference between groups A and C (P〉0.05). It was concluded that VEGF, via VEGFR2, stimulated the neural differentiation of hESCs in vitro.展开更多
BACKGROUND: Induced differentiation strategies and cytochemical properties of human embryonic stem ceils (hESCs) have been investigated. However, the electrophysiological functions of tyrosine hydroxylase (TH)-po...BACKGROUND: Induced differentiation strategies and cytochemical properties of human embryonic stem ceils (hESCs) have been investigated. However, the electrophysiological functions of tyrosine hydroxylase (TH)-positive cells dedved from hESCs remain unclear. OBJECTIVE: To investigate the differentiation efficiency of TH-positive cells from hESCs in vitro using modified four-step culture methods, including embryoid body formation, and to examine the functional characteristics of the differentiated TH-positive cells using electrophysiological techniques. DESIGN, TIME AND SETTING: Neuroelectrophysiology was performed at the Reproductive Medicine Center and Stem Cell Research Center, Peking University Third Hospital, and the Neuroscience Research Institute and Department of Neurobiology, Peking University, from September 2004 to August 2008. MATERIALS: The hESC line, PKU-1.1, a monoclonal cell line derived from a pre-implantation human blastocyst in the Reproductive Medical Center of Peking University Third Hospital. The patch clamp recording system was provided by the Neuroscience Research Institute and Department of Neurobiology, Peking University. METHODS: The hESC line was induced to differentiate into TH-positive cells in vitro using a modified four-step culture method, including the formation of embryoid body, as well as the presence of sonic hedgehog and fibroblast growth factor 8. The cell karyotype was assessed by G-banding karyotype analysis techniques and specific markers were detected immunocytochemically. Whole-cell configuration was obtained after obtaining a tight seal of over 1 GΩ. Ionic currents were detected by holding the cells at -70 mV and stepping to test voltages between -80 and 40 mV in 10-mV increments in voltage-clamp configuration. MAIN OUTCOME MEASURES: We measured the cell karyotype, specific cell markers, and the electrophysiological properties of the voltage-gated ion channels on the cell membrane of TH-positive dopaminergic cells differentiated from our hESCs line in vitro. RESULTS: The differentiated cells had a consistent appearance, and the majority of cells (〉 90%) expressed TH and β-tubulion, as well as the neural progenitor marker, nestino Cell karyotype analysis demonstrated that all of the hESCs had a stable and normal karyotype (46, XX) after differentiation. In addition, patch clamp recording showed that the 10 recorded TH-positive cells exhibited a fast inward current when the test voltage depolarized to -30 mV, and a delayed outward current when the test voltage depolarized to -10 mV. The peak of inward current was obtained at voltage between 10 mV and 0 mV, while the peak of outward current was obtained at 40 mV. The average peak of inward current density was ( -50.05 ± 15.50) pA/pF, and the average peak of outward current density was (41.98 ± 13.55) pA/pE CONCLUSION: More than 90% of the differentiated hESC-derived cells induced by the modified four-step culture method exhibit dopaminergic neuronal properties, including general electrophysiological functional properties, such as functional potassium and sodium channels.展开更多
Overexpression of receptor-interacting protein 140(RIP140) promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2(ERK1/2) signaling.However,involvement of RIP140 in human neural dif...Overexpression of receptor-interacting protein 140(RIP140) promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2(ERK1/2) signaling.However,involvement of RIP140 in human neural differentiation remains unclear.We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells.Moreover,RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation,and positively correlated with the neural stem cell marker Nestin during later stages.Thus,ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.展开更多
This experiment is the first report on N, N'-dini-trosopiperazine (DNF)-induced neoplastic transformation of human embryonic nasopharyngeal (HENPE) cells. The transformed cells showed a prolonged life span, anchor...This experiment is the first report on N, N'-dini-trosopiperazine (DNF)-induced neoplastic transformation of human embryonic nasopharyngeal (HENPE) cells. The transformed cells showed a prolonged life span, anchorage independent growth, chromosome aberration, tumorigenicity and an altered cell morphological appearance. The results demonstrated that DNP was able to induce not only nasopharyngeal carcinoma (NPC) of rats in vivo, but also neoplastic transformation of HENPE cells in vitro.展开更多
The developmental origins of health and diseases(DOHaD)is a concept stating that adverse intrauterine environments contribute to the health risks of offspring.Since the theory emerged more than 30 years ago,many epide...The developmental origins of health and diseases(DOHaD)is a concept stating that adverse intrauterine environments contribute to the health risks of offspring.Since the theory emerged more than 30 years ago,many epidemiological and animal studies have confirmed that in utero exposure to environmental insults,including hyperglycemia and chemicals,increased the risk of developing noncommunicable diseases(NCDs).These NCDs include metabolic syndrome,type 2 diabetes,and complications such as diabetic cardiomyopathy.Studying the effects of different environmental insults on early embryo development would aid in understanding the underlying mechanisms by which these insults promote NCD development.Embryonic stem cells(ESCs)have also been utilized by researchers to study the DOHaD.ESCs have pluripotent characteristics and can be differentiated into almost every cell lineage;therefore,they are excellent in vitro models for studying early developmental events.More importantly,human ESCs(hESCs)are the best alternative to human embryos for research because of ethical concerns.In this review,we will discuss different maternal conditions associated with DOHaD,focusing on the complications of maternal diabetes.Next,we will review the differentiation protocols developed to generate different cell lineages from hESCs.Additionally,we will review how hESCs are utilized as a model for research into the DOHaD.The effects of environmental insults on hESC differentiation and the possible involvement of epigenetic regulation will be discussed.展开更多
Normal mouse pluripotent stem cells were originally derived from the inner cell mass(ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryoni...Normal mouse pluripotent stem cells were originally derived from the inner cell mass(ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryonic stem cells(ESCs). More than a decade later, pluripotent cells were isolated from the ICM of human blastocysts. Despite being called human ESCs, these cells differ significantly from mouse ESCs, including different morphology and mechanisms of control of pluripotency, suggesting distinct embryonic origins of ESCs from the two species. Subsequently, mouse pluripotent stem cells were established from the ICMderived epiblast of post-implantation embryos. These mouse epiblast stem cells(Epi SCs) are morphological and epigenetically more similar to human ESCs. This raised the question of whether cells from the human ICM are in a more advanced differentiation stage than their murine counterpart, or whether the available culture conditions were not adequate to maintain those human cells in their in vivo state, leading to a transition into Epi SC-like cells in vitro. More recently, novel culture conditions allowed the conversion of human ESCs into mouse ESC-like cells called nave(or ground state) human ESCs, and the derivation of nave human ESCs from blastocysts. Here we will review the characteristics of each type of pluripotent stem cells, how(and whether) these relate to different stages of embryonic development, and discuss the potential implications of nave human ESCs in research and therapy.展开更多
In recent years,neonicotinoids(NEOs)and organophosphate esters(OPEs)have been widely used as substitutes for traditional pesticides and brominated fame-retardants,respectively.Previous studies have shown that those co...In recent years,neonicotinoids(NEOs)and organophosphate esters(OPEs)have been widely used as substitutes for traditional pesticides and brominated fame-retardants,respectively.Previous studies have shown that those compounds can be frequently detected in environmental and human samples,are able to penetrate the placental barrier,and are toxic to animals.Thus,it is reasonable to speculate that NEOs and OPEs may have potential adverse effects in humans,especially during development.We employed a human embryonic stem cell differentiation-and liver S9 fraction metabolism-based fast screening model to assess the potential embryonic toxicity of those two types of chemicals.We show that four NEO and five OPE prototypes targeted mostly ectoderm specification,as neural ectoderm and neural crest genes were down-regulated,and surface ectoderm and placode markers up-regulated.Human liver S9 fraction's treatment could generally reduce the effects of the chemicals,except in a few specific instances,indicating the liver may detoxify NEOs and OPEs.Our findings suggest that NEOs and OPEs interfere with human early embryonic development.展开更多
AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenit...AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.展开更多
Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor (p75NTR) expression in rat models of cauda equi...Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor (p75NTR) expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA (shRNA) for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments (shRNA) were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector (carrying shRNA) and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells (PC12). Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.展开更多
AIM:To explore the temporal mitochondrial characteristics of retinal pigment epithelium(RPE)cells obtained from human embryonic stem cells(hESC)-derived retinal organoids(hEROs-RPE),to verify the optimal period for us...AIM:To explore the temporal mitochondrial characteristics of retinal pigment epithelium(RPE)cells obtained from human embryonic stem cells(hESC)-derived retinal organoids(hEROs-RPE),to verify the optimal period for using hEROs-RPE as donor cells from the aspect of mitochondria and to optimize RPE cell-based therapeutic strategies for age-related macular degeneration(AMD).METHODS:RPE cells were obtained from hEROs and from spontaneous differentiation(SD-RPE).The mitochondrial characteristics were analyzed every 20 d from day 60 to 160.Mitochondrial quantity was measured by MitoTracker Green staining.Transmission electron microscopy(TEM)was adopted to assess the morphological features of the mitochondria,including their distribution,length,and cristae.Mitochondrial membrane potentials(MMPs)were determined by JC-1 staining and evaluated by flow cytometry,reactive oxygen species(ROS)levels were evaluated by flow cytometry,and adenosine triphosphate(ATP)levels were measured by a luminometer.Differences between two groups were analyzed by the independentsamples t-test,and comparisons among multiple groups were made using one-way ANOVA or Kruskal-Wallis H test when equal variance was not assumed.RESULTS:hEROs-RPE and SD-RPE cells from day 60 to 160 were successfully differentiated from hESCs and expressed RPE markers(Pax6,MITF,Bestrophin-1,RPE65,Cralbp).RPE features,including a cobblestonelike morphology with tight junctions(ZO-1),pigments and microvilli,were also observed in both hEROs-RPE and SDRPE cells.The mitochondrial quantities of hEROs-RPE and SD-RPE cells both peaked at day 80.However,the cristae of hEROs-RPE mitochondria were less mature and abundant than those of SD-RPE mitochondria at day 80,with hEROsRPE mitochondria becoming mature at day 100.Both hEROs-RPE and SD-RPE cells showed low ROS levels from day 100 to 140 and maintained a normal MMP during this period.However,hEROs-RPE mitochondria maintained a longer time to produce high levels of ATP(from day 120 to 140)than SD-RPE cells(only day 120).CONCLUSION:hEROs-RPE mitochondria develop more slowly and maintain a longer time to supply high-level energy than SD-RPE mitochondria.From the mitochondrial perspective,hEROs-RPE cells from day 100 to 140 are an optimal cell source for treating AMD.展开更多
Background Mono-(2-ethylhexyl) phthalate (MEHP), the metabolite of di-(2-ethylhexyl) phthalate (DEHP), was suspected to be toxic to human embryos. This study contributes to investigating its toxic effects by a...Background Mono-(2-ethylhexyl) phthalate (MEHP), the metabolite of di-(2-ethylhexyl) phthalate (DEHP), was suspected to be toxic to human embryos. This study contributes to investigating its toxic effects by an embryonic stem cell test (EST) based on two human embryonic stem cell (hESCs) lines. Methods CH1 established in our own lab and H1, a federally registered cell line were two human embryonic stem cell lines used in this test. Four endpoint measurements were performed consisting of cell viability, proliferation ability, apoptosis as well as changes of gene expression patterns after spontaneous differentiation were determined. For measuring effects on the first three endpoints, the cells were treated with various concentrations of MEHP dissolved in dimethyl sulfoxide (DMSO) and only with DMSO which served as control and harvested after 5 days. For measuring effects during spontaneous differentiation, the RNA of embryoid bodies (EBs) formed after 8 days' MEHP exposure was collected and changes in differentiation specific gene expression patterns were analyzed by quantitative real time RT-PCR. Results As a result the viability and proliferation ability of both cell lines decreased significantly at 1000 μmol/L MEHP, while there was no effect on apoptosis or cell morphology. In addition MEHP also changed the gene expression pattern in the EBs of both cell lines. Conclusion MEHP in a high dose was cytotoxic and affected the development of hESCs, which indicates its embryo toxicitv in human embrvos.展开更多
In order to develop a novel method of visualizing possible Ca^(2+) signaling during the early differentiation of h ESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent rep...In order to develop a novel method of visualizing possible Ca^(2+) signaling during the early differentiation of h ESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca^(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca^(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca^(2+) transients(generated by release from intracellular stores) were detected in 1–12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or Ca Cl_2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor(IP_3R) agonist, small Ca^(2+) transients were generated from day 1 onward. That ATP was inducing Ca^(2+) release from functional IP_3 Rs was demonstrated by treatment with 2-APB, a known IP_3 R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor(Ry R) agonist, a minimal Ca^(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike Ry Rs, IP_3 Rs are present and continually functional at these early stages of cardiomyocyte differentiation.展开更多
Background The efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evalua...Background The efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evaluate the efficiency of cryopreservation of human ES cells using vitrification method.Methods Human ES cells clumped from an identical cell line were randomly allocated to be cryopreserved by vitrification or by slow freezing. The recovery rates, the growth and differentiation potential of thawed human ES cells were compared between these two groups. The pluripotency of human ES cells after thawing was identified.Results Eighty-one point nine percent (59/72) of human ES cell clumps were recovered after vitrification, while only 22.8% (16/70) were recovered after slow freezing (P<0.01). The colonies after vitrification manifested have not only faster growth but also a lower level of differentiation when compared to colonies subjected to the slow freezing protocol. However, the rates of growth and differentiation in undifferentiated colonies from both groups were identical to the rates in those of non-cryopreserved stem cells after a prolonged culture period. Passage 6 of vitrified human ES cells retained the properties of pluripotent cells, a normal karyotype and expressed the transcription factor OCT-4, stage specific expressed antigen-4 (SSEA-4) and SSEA-3. Teratoma growth of these cells demonstrated the ability to develop into all three germ layers.Conclusions Vitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation.展开更多
Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for d...Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embrvonic stem cell lines with clarified directly differentiation ability展开更多
Background:Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body.Ets variant 2(ETV2)is transiently expressed in both zebrafish and mice and is n...Background:Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body.Ets variant 2(ETV2)is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification.Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells.Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.Findings:We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells(ESCs)to determine when the peak of ETV2 expression occurs.We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.Conclusions:Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification.This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.展开更多
OBJECTIVE:To investigate whether Zichong granules(资冲颗粒,ZCKL),a very effective herbal formula for treating infertility,have an impact on the differentiation of ovarian granulosa cells from human embryonic stem cell...OBJECTIVE:To investigate whether Zichong granules(资冲颗粒,ZCKL),a very effective herbal formula for treating infertility,have an impact on the differentiation of ovarian granulosa cells from human embryonic stem cells(hESCs)in vitro,and to explore the cellular mechanisms of its clinical effects.METHODS:Serum from ZCKL-medicated rats was prepared and used to treat mesoderm cells derived from hESCs for 6 d.Normal rat serum and a set of growth factors were used as negative and positive controls,respectively.RESULTS:ZCKL-medicated rat serum,but not normal rat serum,induced hESCs-derived mesoderm cells to differentiate into functional ovarian granulosa-like cells(OGLCs)in a similar manner to defined growth factors.The induced OGLCs resembled the morphology of native human granulosa cells,expressed granulosa cell-specific markers at both the mRNA and protein levels,produced high levels of estradiol and strongly responded to follicle-stimulating hormone stimulation.Furthermore,mRNA levels of follistatin,mothers against decapentaplegic homolog 8 and bone morphogenetic protein 6 were dynamically changed during the process.CONCLUSION:In the ZCKL treatment of infertility,one mechanism by which ZCKL may act is by influencing ovarian granulosa cell differentiation and development,possibly through the follistatin and BMP/SMAD signaling pathways.展开更多
文摘BACKGROUND Premature ovarian failure(POF)affects many adult women less than 40 years of age and leads to infertility.According to previous reports,various tissue-specific stem cells can restore ovarian function and folliculogenesis in mice with chemotherapy-induced POF.Human embryonic stem cells(ES)provide an alternative source for mesenchymal stem cells(MSCs)because of their similarities in phenotype and immunomodulatory and anti-inflammatory characteristics.Embryonic stem cell-derived mesenchymal stem cells(ES-MSCs)are attractive candidates for regenerative medicine because of their high proliferation and lack of barriers for harvesting tissue-specific MSCs.However,possible therapeutic effects and underlying mechanisms of transplanted ES-MSCs on cyclophosphamide and busulfan-induced mouse ovarian damage have not been evaluated.AIM To evaluate ES-MSCs vs bone marrow-derived mesenchymal stem cells(BMMSCs)in restoring ovarian function in a mouse model of chemotherapy-induced premature ovarian failure.METHODS Female mice received intraperitoneal injections of different doses of cyclophosphamide and busulfan to induce POF.Either human ES-MSCs or BMMSCs were transplanted into these mice.Ten days after the mice were injected with cyclophosphamide and busulfan and 4 wk after transplantation of the ESMSCs and/or BM-MSCs,we evaluated body weight,estrous cyclicity,folliclestimulating hormone and estradiol hormone concentrations and follicle count were used to evaluate the POF model and cell transplantation.Moreover,terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling,real-time PCR,Western blot analysis and immunohistochemistry and mating was used to evaluate cell transplantation.Enzyme-linked immunosorbent assay was used to analyze vascular endothelial growth factor,insulin-like growth factor 2 and hepatocyte growth factor levels in ES-MSC condition medium in order to investigate the mechanisms that underlie their function.RESULTS The human ES-MSCs significantly restored hormone secretion,survival rate and reproductive function in POF mice,which was similar to the results obtained with BM-MSCs.Gene expression analysis and the terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling assay results indicated that the ES-MSCs and/or BM-MSCs reduced apoptosis in the follicles.Notably,the transplanted mice generated new offspring.The results of different analyses showed increases in antiapoptotic and trophic proteins and genes.CONCLUSION These results suggested that transplantation of human ES-MSCs were similar to BM-MSCs in that they could restore the structure of the injured ovarian tissue and its function in chemotherapy-induced damaged POF mice and rescue fertility.The possible mechanisms of human ES-MSC were related to promotion of follicular development,ovarian secretion,fertility via a paracrine effect and ovarian cell survival.
基金sponsored by Shanghai Key Projects of Basic Research,No.08JC1413900
文摘The difference between Noggin and basic fibroblast growth factor for the neural precursor differen- tiation from human embryonic stem cells has not been studied. In this study, 100 tJg/L Noggin or 20 IJg/L basic fibroblast growth factor in serum-free neural induction medium was used to differen- tiate human embryonic stem cells H14 into neural precursors using monolayer differentiation. Two weeks after induction, significantly higher numbers of neural rosettes formed in the Noggin-induced group than the basic fibroblast growth factor-induced group, as detected by phase contrast micro- scope. Immunofluorescence staining revealed expression levels of Nestin, [3-111 Tubulin and Sox-1 were higher in the induced cells and reverse-transcription PCR showed induced cells expressed Nestin, Sox-1 and Neurofilament mRNA. Protein and mRNA expression in the Noggin-induced group was increased compared with the basic fibroblast growth factor-induced group. Noggin has a greater effect than basic fibroblast growth factor on the induction of human embryonic stem cell differentiation into neural precursors by monolayer differentiation, as Noggin accelerates and in- creases the differentiation of neural precursors.
基金supported by NIH/NIDCR grants R03 DE019507-02 to Yan Zhang,R21 D E0 18633 to Pamela K DenBesten and 2011SCU 11999-3/ NSFC81200760to Li-Wei Zheng
文摘This study aimed to compare epithelial cells derived from human embryonic stem cells(hESCs) to human ameloblast-lineage cells (ALCs),as a way to determine their potential use as a cell source for ameloblast regeneration.Induced by various concentrations of bone morphogenetic protein 4(BMP4),retinoic acid(RA) and lithium chloride(LiCI) for 7 days,hESCs adopted cobble-stone epithelial phenotype(hESC-derived epithelial cells(ES-ECs)) and expressed cytokeratin 14.Compared with ALCs and oral epithelial cells(OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs.ES-ECs were compared with human fetal skin epithelium,human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well.ALCs had relatively high expression levels of cytokeratin 76,which was also found to be upregulated in ES-ECs.Based on the present study,with the similarity of gene expression with ALCs,ES-ECs are a promising potential cell source for regeneration,which are not available in erupted human teeth for regeneration of enamel.
文摘The effects of vascular endothelial growth factor (VEGF) on neural differentiation of human embryonic stem cells (hESCs) in vitro and the possible mechanism were observed. The hESCs lines, TJMU1 and TJMU2, were established and stored by our laboratory, hESCs differentiated into neuronal cells through embryonic body formation. In this induction process, hESCs were divided into three groups: group A, routine induction; group B, routine induction+10 ng/mL VEGF; group C, routine in- duction+10 ng/mL VEGF+10 ng/mL VEGFR2/Fc. OCT4, Nestin and GFAP in each group were de- tected by RT-PCR, and the cells expressing Nestin and GFAP were counted by immunofluorescence. The percentage of Nestin positive cells in group B was significantly higher than in groups A and C, while the percentage of GFAP positive cells in group B was significantly lower than in groups A and C (P〈0.01). There was no significant difference between groups A and C (P〉0.05). It was concluded that VEGF, via VEGFR2, stimulated the neural differentiation of hESCs in vitro.
基金the National Natural Science Foundation of China, No. 30672239
文摘BACKGROUND: Induced differentiation strategies and cytochemical properties of human embryonic stem ceils (hESCs) have been investigated. However, the electrophysiological functions of tyrosine hydroxylase (TH)-positive cells dedved from hESCs remain unclear. OBJECTIVE: To investigate the differentiation efficiency of TH-positive cells from hESCs in vitro using modified four-step culture methods, including embryoid body formation, and to examine the functional characteristics of the differentiated TH-positive cells using electrophysiological techniques. DESIGN, TIME AND SETTING: Neuroelectrophysiology was performed at the Reproductive Medicine Center and Stem Cell Research Center, Peking University Third Hospital, and the Neuroscience Research Institute and Department of Neurobiology, Peking University, from September 2004 to August 2008. MATERIALS: The hESC line, PKU-1.1, a monoclonal cell line derived from a pre-implantation human blastocyst in the Reproductive Medical Center of Peking University Third Hospital. The patch clamp recording system was provided by the Neuroscience Research Institute and Department of Neurobiology, Peking University. METHODS: The hESC line was induced to differentiate into TH-positive cells in vitro using a modified four-step culture method, including the formation of embryoid body, as well as the presence of sonic hedgehog and fibroblast growth factor 8. The cell karyotype was assessed by G-banding karyotype analysis techniques and specific markers were detected immunocytochemically. Whole-cell configuration was obtained after obtaining a tight seal of over 1 GΩ. Ionic currents were detected by holding the cells at -70 mV and stepping to test voltages between -80 and 40 mV in 10-mV increments in voltage-clamp configuration. MAIN OUTCOME MEASURES: We measured the cell karyotype, specific cell markers, and the electrophysiological properties of the voltage-gated ion channels on the cell membrane of TH-positive dopaminergic cells differentiated from our hESCs line in vitro. RESULTS: The differentiated cells had a consistent appearance, and the majority of cells (〉 90%) expressed TH and β-tubulion, as well as the neural progenitor marker, nestino Cell karyotype analysis demonstrated that all of the hESCs had a stable and normal karyotype (46, XX) after differentiation. In addition, patch clamp recording showed that the 10 recorded TH-positive cells exhibited a fast inward current when the test voltage depolarized to -30 mV, and a delayed outward current when the test voltage depolarized to -10 mV. The peak of inward current was obtained at voltage between 10 mV and 0 mV, while the peak of outward current was obtained at 40 mV. The average peak of inward current density was ( -50.05 ± 15.50) pA/pF, and the average peak of outward current density was (41.98 ± 13.55) pA/pE CONCLUSION: More than 90% of the differentiated hESC-derived cells induced by the modified four-step culture method exhibit dopaminergic neuronal properties, including general electrophysiological functional properties, such as functional potassium and sodium channels.
基金supported by the National Natural Science Foundation of China,No.31340024
文摘Overexpression of receptor-interacting protein 140(RIP140) promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2(ERK1/2) signaling.However,involvement of RIP140 in human neural differentiation remains unclear.We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells.Moreover,RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation,and positively correlated with the neural stem cell marker Nestin during later stages.Thus,ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.
文摘This experiment is the first report on N, N'-dini-trosopiperazine (DNF)-induced neoplastic transformation of human embryonic nasopharyngeal (HENPE) cells. The transformed cells showed a prolonged life span, anchorage independent growth, chromosome aberration, tumorigenicity and an altered cell morphological appearance. The results demonstrated that DNP was able to induce not only nasopharyngeal carcinoma (NPC) of rats in vivo, but also neoplastic transformation of HENPE cells in vitro.
文摘The developmental origins of health and diseases(DOHaD)is a concept stating that adverse intrauterine environments contribute to the health risks of offspring.Since the theory emerged more than 30 years ago,many epidemiological and animal studies have confirmed that in utero exposure to environmental insults,including hyperglycemia and chemicals,increased the risk of developing noncommunicable diseases(NCDs).These NCDs include metabolic syndrome,type 2 diabetes,and complications such as diabetic cardiomyopathy.Studying the effects of different environmental insults on early embryo development would aid in understanding the underlying mechanisms by which these insults promote NCD development.Embryonic stem cells(ESCs)have also been utilized by researchers to study the DOHaD.ESCs have pluripotent characteristics and can be differentiated into almost every cell lineage;therefore,they are excellent in vitro models for studying early developmental events.More importantly,human ESCs(hESCs)are the best alternative to human embryos for research because of ethical concerns.In this review,we will discuss different maternal conditions associated with DOHaD,focusing on the complications of maternal diabetes.Next,we will review the differentiation protocols developed to generate different cell lineages from hESCs.Additionally,we will review how hESCs are utilized as a model for research into the DOHaD.The effects of environmental insults on hESC differentiation and the possible involvement of epigenetic regulation will be discussed.
基金Conselho Nacional de Desenvolvimento Cientifico e Tecnologico/Departamento de Ciencia e Tecnologia do Ministerio da Saude(CNPq/MS/DECIT)Banco Nacional de Desenvolvimento Economico e Social(BNDES)+2 种基金Financiadora de Estudos e Projetos(FINEP)the fellowship from CNPq(Costas RM)a fellowship from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior(Fonseca SAS)
文摘Normal mouse pluripotent stem cells were originally derived from the inner cell mass(ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryonic stem cells(ESCs). More than a decade later, pluripotent cells were isolated from the ICM of human blastocysts. Despite being called human ESCs, these cells differ significantly from mouse ESCs, including different morphology and mechanisms of control of pluripotency, suggesting distinct embryonic origins of ESCs from the two species. Subsequently, mouse pluripotent stem cells were established from the ICMderived epiblast of post-implantation embryos. These mouse epiblast stem cells(Epi SCs) are morphological and epigenetically more similar to human ESCs. This raised the question of whether cells from the human ICM are in a more advanced differentiation stage than their murine counterpart, or whether the available culture conditions were not adequate to maintain those human cells in their in vivo state, leading to a transition into Epi SC-like cells in vitro. More recently, novel culture conditions allowed the conversion of human ESCs into mouse ESC-like cells called nave(or ground state) human ESCs, and the derivation of nave human ESCs from blastocysts. Here we will review the characteristics of each type of pluripotent stem cells, how(and whether) these relate to different stages of embryonic development, and discuss the potential implications of nave human ESCs in research and therapy.
基金supported by the Ministry of Science and Technology of the People’s Republic of China (No.2020YFA0907500)the National Natural Science Foundation of China (Nos.22150710514,22021003,and 22106174)+1 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences (No.XDPB200202)the Postdoc Science Foundation of China (No.2021M693322)。
文摘In recent years,neonicotinoids(NEOs)and organophosphate esters(OPEs)have been widely used as substitutes for traditional pesticides and brominated fame-retardants,respectively.Previous studies have shown that those compounds can be frequently detected in environmental and human samples,are able to penetrate the placental barrier,and are toxic to animals.Thus,it is reasonable to speculate that NEOs and OPEs may have potential adverse effects in humans,especially during development.We employed a human embryonic stem cell differentiation-and liver S9 fraction metabolism-based fast screening model to assess the potential embryonic toxicity of those two types of chemicals.We show that four NEO and five OPE prototypes targeted mostly ectoderm specification,as neural ectoderm and neural crest genes were down-regulated,and surface ectoderm and placode markers up-regulated.Human liver S9 fraction's treatment could generally reduce the effects of the chemicals,except in a few specific instances,indicating the liver may detoxify NEOs and OPEs.Our findings suggest that NEOs and OPEs interfere with human early embryonic development.
基金Supported by The German Federal Ministry for Education and Research(BMBF),No.01GN0818 and No.01GN0819the Max-Planck Society,and initially by the Dr.Helmut Storz Stiftung
文摘AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.
基金the National Natural Science Foundation of China, No. 30672136
文摘Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor (p75NTR) expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA (shRNA) for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments (shRNA) were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector (carrying shRNA) and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells (PC12). Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.
基金Supported by the National Key Research and Development Program of China(No.2018YFA01017302)the National Natural Science Foundation of China(No.82000945)。
文摘AIM:To explore the temporal mitochondrial characteristics of retinal pigment epithelium(RPE)cells obtained from human embryonic stem cells(hESC)-derived retinal organoids(hEROs-RPE),to verify the optimal period for using hEROs-RPE as donor cells from the aspect of mitochondria and to optimize RPE cell-based therapeutic strategies for age-related macular degeneration(AMD).METHODS:RPE cells were obtained from hEROs and from spontaneous differentiation(SD-RPE).The mitochondrial characteristics were analyzed every 20 d from day 60 to 160.Mitochondrial quantity was measured by MitoTracker Green staining.Transmission electron microscopy(TEM)was adopted to assess the morphological features of the mitochondria,including their distribution,length,and cristae.Mitochondrial membrane potentials(MMPs)were determined by JC-1 staining and evaluated by flow cytometry,reactive oxygen species(ROS)levels were evaluated by flow cytometry,and adenosine triphosphate(ATP)levels were measured by a luminometer.Differences between two groups were analyzed by the independentsamples t-test,and comparisons among multiple groups were made using one-way ANOVA or Kruskal-Wallis H test when equal variance was not assumed.RESULTS:hEROs-RPE and SD-RPE cells from day 60 to 160 were successfully differentiated from hESCs and expressed RPE markers(Pax6,MITF,Bestrophin-1,RPE65,Cralbp).RPE features,including a cobblestonelike morphology with tight junctions(ZO-1),pigments and microvilli,were also observed in both hEROs-RPE and SDRPE cells.The mitochondrial quantities of hEROs-RPE and SD-RPE cells both peaked at day 80.However,the cristae of hEROs-RPE mitochondria were less mature and abundant than those of SD-RPE mitochondria at day 80,with hEROsRPE mitochondria becoming mature at day 100.Both hEROs-RPE and SD-RPE cells showed low ROS levels from day 100 to 140 and maintained a normal MMP during this period.However,hEROs-RPE mitochondria maintained a longer time to produce high levels of ATP(from day 120 to 140)than SD-RPE cells(only day 120).CONCLUSION:hEROs-RPE mitochondria develop more slowly and maintain a longer time to supply high-level energy than SD-RPE mitochondria.From the mitochondrial perspective,hEROs-RPE cells from day 100 to 140 are an optimal cell source for treating AMD.
基金This study was supported by Beijing Natural Science Foundation (No. 7112141).
文摘Background Mono-(2-ethylhexyl) phthalate (MEHP), the metabolite of di-(2-ethylhexyl) phthalate (DEHP), was suspected to be toxic to human embryos. This study contributes to investigating its toxic effects by an embryonic stem cell test (EST) based on two human embryonic stem cell (hESCs) lines. Methods CH1 established in our own lab and H1, a federally registered cell line were two human embryonic stem cell lines used in this test. Four endpoint measurements were performed consisting of cell viability, proliferation ability, apoptosis as well as changes of gene expression patterns after spontaneous differentiation were determined. For measuring effects on the first three endpoints, the cells were treated with various concentrations of MEHP dissolved in dimethyl sulfoxide (DMSO) and only with DMSO which served as control and harvested after 5 days. For measuring effects during spontaneous differentiation, the RNA of embryoid bodies (EBs) formed after 8 days' MEHP exposure was collected and changes in differentiation specific gene expression patterns were analyzed by quantitative real time RT-PCR. Results As a result the viability and proliferation ability of both cell lines decreased significantly at 1000 μmol/L MEHP, while there was no effect on apoptosis or cell morphology. In addition MEHP also changed the gene expression pattern in the EBs of both cell lines. Conclusion MEHP in a high dose was cytotoxic and affected the development of hESCs, which indicates its embryo toxicitv in human embrvos.
基金supported by the Hong Kong Theme-based Research Scheme award(T13-706/11-1)the Hong Kong Research Grants Council(RGC)General Research Fund awards(662113,16101714,16100115)+2 种基金the ANR/RGC joint research scheme award(A-HKUST601/13)the Innovation and Technology Commission(ITCPD/17-9)supported by a Hong Kong University Grants Council post-graduate studentship(T13-706/11-11PG)
文摘In order to develop a novel method of visualizing possible Ca^(2+) signaling during the early differentiation of h ESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca^(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca^(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca^(2+) transients(generated by release from intracellular stores) were detected in 1–12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or Ca Cl_2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor(IP_3R) agonist, small Ca^(2+) transients were generated from day 1 onward. That ATP was inducing Ca^(2+) release from functional IP_3 Rs was demonstrated by treatment with 2-APB, a known IP_3 R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor(Ry R) agonist, a minimal Ca^(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike Ry Rs, IP_3 Rs are present and continually functional at these early stages of cardiomyocyte differentiation.
文摘Background The efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evaluate the efficiency of cryopreservation of human ES cells using vitrification method.Methods Human ES cells clumped from an identical cell line were randomly allocated to be cryopreserved by vitrification or by slow freezing. The recovery rates, the growth and differentiation potential of thawed human ES cells were compared between these two groups. The pluripotency of human ES cells after thawing was identified.Results Eighty-one point nine percent (59/72) of human ES cell clumps were recovered after vitrification, while only 22.8% (16/70) were recovered after slow freezing (P<0.01). The colonies after vitrification manifested have not only faster growth but also a lower level of differentiation when compared to colonies subjected to the slow freezing protocol. However, the rates of growth and differentiation in undifferentiated colonies from both groups were identical to the rates in those of non-cryopreserved stem cells after a prolonged culture period. Passage 6 of vitrified human ES cells retained the properties of pluripotent cells, a normal karyotype and expressed the transcription factor OCT-4, stage specific expressed antigen-4 (SSEA-4) and SSEA-3. Teratoma growth of these cells demonstrated the ability to develop into all three germ layers.Conclusions Vitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation.
文摘Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embrvonic stem cell lines with clarified directly differentiation ability
基金The UCLA vector core is supported by JCCC/P30 CA016042 and CURE/P30 DK041301The cells were supplied through the UCLA BSCRC stem cell core laboratoryThis work was supported by funds from the California Institute for Regenerative Medicine(CIRM RB3-02165).
文摘Background:Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body.Ets variant 2(ETV2)is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification.Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells.Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.Findings:We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells(ESCs)to determine when the peak of ETV2 expression occurs.We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.Conclusions:Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification.This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.
基金Supported by The National Basic Research Program of China:Study on the theory of"kidney governs reproduction"from the perspective of infertility(973 Plan,Grant Nos.2010CB530403 and 2010CB530400)。
文摘OBJECTIVE:To investigate whether Zichong granules(资冲颗粒,ZCKL),a very effective herbal formula for treating infertility,have an impact on the differentiation of ovarian granulosa cells from human embryonic stem cells(hESCs)in vitro,and to explore the cellular mechanisms of its clinical effects.METHODS:Serum from ZCKL-medicated rats was prepared and used to treat mesoderm cells derived from hESCs for 6 d.Normal rat serum and a set of growth factors were used as negative and positive controls,respectively.RESULTS:ZCKL-medicated rat serum,but not normal rat serum,induced hESCs-derived mesoderm cells to differentiate into functional ovarian granulosa-like cells(OGLCs)in a similar manner to defined growth factors.The induced OGLCs resembled the morphology of native human granulosa cells,expressed granulosa cell-specific markers at both the mRNA and protein levels,produced high levels of estradiol and strongly responded to follicle-stimulating hormone stimulation.Furthermore,mRNA levels of follistatin,mothers against decapentaplegic homolog 8 and bone morphogenetic protein 6 were dynamically changed during the process.CONCLUSION:In the ZCKL treatment of infertility,one mechanism by which ZCKL may act is by influencing ovarian granulosa cell differentiation and development,possibly through the follistatin and BMP/SMAD signaling pathways.