Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a p...Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.展开更多
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
BACKGROUND: Induced differentiation strategies and cytochemical properties of human embryonic stem cells (hESCs) have been investigated. However, the electrophysiological functions of tyrosine hydroxylase (TH)-positiv...BACKGROUND: Induced differentiation strategies and cytochemical properties of human embryonic stem cells (hESCs) have been investigated. However, the electrophysiological functions of tyrosine hydroxylase (TH)-positive cells derived 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, nestin. Cell karyotype analysis demonstrated that all of the hESCs had a stable and normal karyotype (46, XX) after dif-ferentiation. 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/pF. 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
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
AIM: To investigate the epigenetic states and expres- sion of imprinted genes in five human embryonic stem cell (hESC) lines derived in Taiwan. METHODS: The heterozygous alleles of single nucleo- tide polymorphisms (S...AIM: To investigate the epigenetic states and expres- sion of imprinted genes in five human embryonic stem cell (hESC) lines derived in Taiwan. METHODS: The heterozygous alleles of single nucleo- tide polymorphisms (SNPs) at imprinted genes were analyzed by sequencing genomic DNAs of hESC lines and the monoallelic expression of the imprinted genes were confirmed by sequencing the cDNAs. The expres- sion profiles of 32 known imprinted genes of five hESC lines were determined using Affymetrix human genome U133 plus 2.0 DNA microarray. RESULTS: The heterozygous alleles of SNPs at seven imprinted genes, IPW , PEG10 , NESP55 , KCNQ1 , ATP10A ,TCEB3C and IGF2 , were identified and the monoallelic expression of these imprinted genes except IGF2 were confirmed. The IGF2 gene was found to be imprinted in hESC line T2 but partially imprinted in line T3 and not imprinted in line T4 embryoid bodies. Ten imprinted genes, namely GRB10 , PEG10 , SGCE, MEST , SDHD , SN- RPN , SNURF , NDN , IPW and NESP55 , were found to be highly expressed in the undifferentiated hESC lines and down-regulated in differentiated derivatives. The UBE3A gene abundantly expressed in undifferentiated hESC lines and further up-regulated in differentiated tissues. The expression levels of other 21 imprinted genes were relatively low in undifferentiated hESC lines and five of these genes (TP73 , COPG2 , OSBPL5 , IGF2 and ATP10A ) were found to be up-regulated in differentiated tissues. CONCLUSION: The epigenetic states and expression of imprinted genes in hESC lines should be thoroughly studied after extended culture and upon differentiation in order to understand epigenetic stability in hESC lines before their clinical applications.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 rege...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 ,vas 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
AIM:To evaluate the safety and efficacy of human embryonic stem cells(h ESCs)for the management of type 2 diabetes mellitus(T2DM).METHODS:Patients with a previous history of diabetes and its associated complications w...AIM:To evaluate the safety and efficacy of human embryonic stem cells(h ESCs)for the management of type 2 diabetes mellitus(T2DM).METHODS:Patients with a previous history of diabetes and its associated complications were enrolled and injected with hE SC lines as per the defined protocol.The patients were assessed using Nutech functional score(NFS),a numeric scoring scale to evaluate the patients for 11 diagnostic parameters.Patients were evaluated at baseline and at the end of treatment period 1(T1).All the parameters were graded on the NFS scale from 1to 5.Highest possible grade(HPG)of 5 was considered as the grade of best improvement.RESULTS:Overall,94.8%of the patients showed improvement by at least one grade of NFS at the end of T1.For all the 11 parameters evaluated,54%of patients achieved HPG after treatment.The four essential parameters(improvement in glycated hemoglobin(HbA 1c)and insulin level,and fall in number of other oral hypoglycemic drugs with and without insulin)are presented in detail.For Hb A1c,72.6%of patients at the end of T1 met the World Health Organization cut off value,i.e.,6.5%of HbA 1c.For insulin level,65.9%of patients at the end of T1 were able to achieve HPG.After treatment,the improvement was seen in 16.3%of patients who required no more than two medications along with insulin.Similarly,21.5%of patients were improved as their dosage regimen for using oral drugs was reduced to 1-2 from 5.CONCLUSION:hE SC therapy is beneficial in patients with diabetes and helps in reducing their dependence on insulin and other medicines.展开更多
AIM:The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein. METHODS:Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter d...AIM:The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein. METHODS:Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter driven mCherry transgene was performed in MEL2 hESC. Red fluore-scence was assessed by immunofluorescence and flow cytometry. Pluripotency of stably transduced hESC was determined by immunofluorescent pluripotency marker expression, flow cytometry, teratoma assays andembryoid body-based differentiation followed by reverse transcriptase-polymerase chain reaction. Quantification of cell motility and survival was performed with time lapse microscopy. RESULTS:Constitutively fluorescently-labeled hESCs are useful tools for facile in vitro and in vivo tracking of survival, motility and cell spreading on various surfaces before and after differentiation. Here we describe the generation and characterization of a hESC line (MEL2) stably expressing red fluorescent protein, mCherry. This line was generated by random integration of a fluorescent protein-expressing cassette, driven by the ubiquitously-expressed human EF-1α promoter. Stably transfected MEL2-mCherry hESC were shown to express pluripo-tency markers in the nucleus (POU5F1/OCT4, NANOG and SOX2) and on the cell surface (SSEA4, TRA1-60 and TG30/CD9) and were shown to maintain a normal karyotype in long-term (for at least 35 passages) culture. MEL2-mCherry hESC further readily differentiated into representative cell types of the three germ layers in embryoid body and teratoma based assays and, importantly, maintained robust mCherry expression throughout differentiation. The cell line was next adapted to singlecell passaging, rendering it compatible with numerous bioengineering applications such as measurement of cell motility and cell spreading on various protein modified surfaces, quantification of cell attachment to nanoparticles and rapid estimation of cell survival. CONCLUSION:The MEL2-mCherry hESC line conforms to the criteria of bona fide pluripotent stem cells and maintains red fluorescence throughout differentiation, making it a useful tool for bioengineering and in vivo tracking experiments.展开更多
Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, ...Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.展开更多
Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to ...Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to determine the optimal source of MSCs. We investigated if this biological heterogeneity in MSCs from different sources results in different mechanisms for their differentiation. In this study, we compared the gene expression patterns of phenotypically defined MSCs derived from three ontogenically different sources: Embryonic stem cells (hES-MSCs), Fetal limb (Flb-MSCs) and Bone Marrow (BM-MSCs). Differentially expressed genes between differentiated cells and undifferentiated controls were compared across the three MSC sources. We found minimal overlap (5% - 16%) in differentially expressed gene sets among the three sources. Flb-MSCs were similar to BM-MSCs based on differential gene expression patterns. Pathway analysis of the differentially expressed genes using Ingenuity Pathway Analysis (IPA) revealed a large variation in the canonical pathways leading to MSC differentiation. The similar canonical pathways among the three sources were lineage specific. The Flb-MSCs showed maximum overlap of canonical pathways with the BM-MSCs, indicating that the Flb-MSCs are an intermediate source between the less specialised hES-MSC source and the more specialised BM-MSC source. The source specific pathways prove that MSCs from the three ontogenically different sources use different biological pathways to obtain similar differentiation outcomes. Thus our study advocates the understanding of biological pathways to obtain optimal sources of MSCs for various clinical applications.展开更多
We established a QT interval assessment system that uses human embryonic stem cell-derived cardiomyocyte clusters (hES-CMCs) in which the field potential duration (FPD) or corrected FPD (FPDc) was measured as an indic...We established a QT interval assessment system that uses human embryonic stem cell-derived cardiomyocyte clusters (hES-CMCs) in which the field potential duration (FPD) or corrected FPD (FPDc) was measured as an indicator of drug-induced QT interval prolongation. To investigate the applicability of the hES-CMC system to drug safety assessment, we investigated short-term variability in FPDc (STVFPDc) (beat rate rhythmicity) as a marker of torsadogenic risk. We investigated the FPDc and STVFPDc of hES-CMCs treated with hERG channel blockers (E-4031 or cisapride) or with our proprietary compounds X, Y, and Z. We also evaluated the electrocardiograms and hemodynamics of dogs treated with compound X, Y, or Z. The torsadogenic hERG channel blockers increased STVFPDc and prolonged FPDc. Compounds X, Y, and Z had hERG inhibitory activity. Compound X prolonged FPDc with increased STVFPDc, whereas compounds Y and Z tended to shorten FPDc in the hES-CMC system. In the in vivo canine study, compound X prolonged corrected QT (QTc), and compounds Y and Z tended to shorten QTc, showing a good correlation with the results in hES-CMCs. These findings suggest that combined assessment of FPDc and STVFPDc in the hES-CMC system increases the predictability of torsadogenic risk.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 μg/L Noggin or 20 μg/L basic fibrob...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 μg/L Noggin or 20 μg/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, β-III 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
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 differe...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
A 32bp deletion in the chemokine receptor 5 (CCR5) gene (CMKBR5) was shown to be linked to HIV resistance. Bone marrow transplantation from the homozygous CCR5-del32 donor to a CDC Stage 2 HIV-positive recipient was d...A 32bp deletion in the chemokine receptor 5 (CCR5) gene (CMKBR5) was shown to be linked to HIV resistance. Bone marrow transplantation from the homozygous CCR5-del32 donor to a CDC Stage 2 HIV-positive recipient was demonstrated to confer a HIV resistance, resulting in discontinuation of antiretroviral therapy. In search for an unlimited source of CCR5-del32 cells for transplantation purposes, we tested 137 human embryonic stem cell (hESC) lines from the Reproductive Genetics Institute’s hESC lines collection, and report here the finding of 12 hESC lines with the CCR5-del32 allele, one of which represents a unique partenogenetic ESC line containing two copies of this deletion and may be studied for utility in stem cell transplantation treatment of HIV.展开更多
基金supported by the National Key Research and Development Program of China,Nos.2017YFE0122900(to BH),2019YFA0110800(to WL),2019YFA0903802(to YW),2021YFA1101604(to LW),2018YFA0108502(to LF),and 2020YFA0804003(to JW)the National Natural Science Foundation of China,Nos.31621004(to WL,BH)and 31970821(to YW)+1 种基金CAS Project for Young Scientists in Basic Research,No.YSBR-041(to YW)Joint Funds of the National Natural Science Foundation of China,No.U21A20396(to BH)。
文摘Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.
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the National Natural Science Foundation of China, No. 30672239
文摘BACKGROUND: Induced differentiation strategies and cytochemical properties of human embryonic stem cells (hESCs) have been investigated. However, the electrophysiological functions of tyrosine hydroxylase (TH)-positive cells derived 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, nestin. Cell karyotype analysis demonstrated that all of the hESCs had a stable and normal karyotype (46, XX) after dif-ferentiation. 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/pF. 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.
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This research was supported by the Ministry of Science and Technology Grant （2001CB510106）；Science and Technology Plan of Beijing Municipal Government （H020220050290）；National Natural Science Foundation of China Awards for 0utstanding Young Scientists （30125022）；for Creative Research Groups （30421004）；Bill ＆ Melinda Gates Foundation Grant （37871） to H Deng.
文摘为人的胚胎的茎(ES ) 的自强和区别的能力细胞为对待类型 Idiabetes mellitus 为胰腺的贝它细胞的产生使他们成为潜在的来源。这里，我们报导一最新发展了并且有效方法，在aserum免费的系统执行了，区分进生产胰岛素的 cells.Activin A 的导致的人的 ES 房间它在起始的阶段被使用从人的 EScells 导致权威的内胚叶区别，是由权威的内胚叶标记 Sox17 和 Brachyury.Further 的表示检测了， all-trans retinoic 酸( RA )被用来支持胰腺的区别，由早胰腺的抄写因素 pdx1 和 hlxb9 的表示显示了。在成熟 inDMEM/F12 以后有 bFGF 和菸碱的没有浆液的媒介，区分的房间表示了小岛特定的标记象 C 肽，胰岛素，胰高血糖素和 glut2 那样。百分比 ofC-peptide-positive 房间超过了 15% 。由这些房间的胰岛素和 C 肽的分泌物在葡萄糖层次对应于变化。当移植了进肾的囊时， ofStreptozotocin (STZ ) 对待裸体老鼠，这些区分的人的 ES 房间熬过并且维持贝它房间标记基因的表示包括 C 肽， pdx1， glucokinase， nkx6.1， IAPP， pax6and Tcf1。百分之三十只移植裸体老鼠展出了 stableeuglycemia 的明显的恢复；并且改正的显型被支撑超过六个星期。我们的新方法为学习人的胰开发的机制提供一个有希望的试管内区别模特儿并且说明为类型 Idiabetes mellitus 的处理使用人的 ES 房间的潜力。
基金Supported by National Program for Genomic Medicine GrantsNSC95/96/97-3112-B-037-002 of National Science Council inTaiwan (to Li SS)a Chair Professorship of The Medical Educationand Development Foundation of Kaohsiung Medical University (to Li SS)
文摘AIM: To investigate the epigenetic states and expres- sion of imprinted genes in five human embryonic stem cell (hESC) lines derived in Taiwan. METHODS: The heterozygous alleles of single nucleo- tide polymorphisms (SNPs) at imprinted genes were analyzed by sequencing genomic DNAs of hESC lines and the monoallelic expression of the imprinted genes were confirmed by sequencing the cDNAs. The expres- sion profiles of 32 known imprinted genes of five hESC lines were determined using Affymetrix human genome U133 plus 2.0 DNA microarray. RESULTS: The heterozygous alleles of SNPs at seven imprinted genes, IPW , PEG10 , NESP55 , KCNQ1 , ATP10A ,TCEB3C and IGF2 , were identified and the monoallelic expression of these imprinted genes except IGF2 were confirmed. The IGF2 gene was found to be imprinted in hESC line T2 but partially imprinted in line T3 and not imprinted in line T4 embryoid bodies. Ten imprinted genes, namely GRB10 , PEG10 , SGCE, MEST , SDHD , SN- RPN , SNURF , NDN , IPW and NESP55 , were found to be highly expressed in the undifferentiated hESC lines and down-regulated in differentiated derivatives. The UBE3A gene abundantly expressed in undifferentiated hESC lines and further up-regulated in differentiated tissues. The expression levels of other 21 imprinted genes were relatively low in undifferentiated hESC lines and five of these genes (TP73 , COPG2 , OSBPL5 , IGF2 and ATP10A ) were found to be up-regulated in differentiated tissues. CONCLUSION: The epigenetic states and expression of imprinted genes in hESC lines should be thoroughly studied after extended culture and upon differentiation in order to understand epigenetic stability in hESC lines before their clinical applications.
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 ,vas 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AIM:To evaluate the safety and efficacy of human embryonic stem cells(h ESCs)for the management of type 2 diabetes mellitus(T2DM).METHODS:Patients with a previous history of diabetes and its associated complications were enrolled and injected with hE SC lines as per the defined protocol.The patients were assessed using Nutech functional score(NFS),a numeric scoring scale to evaluate the patients for 11 diagnostic parameters.Patients were evaluated at baseline and at the end of treatment period 1(T1).All the parameters were graded on the NFS scale from 1to 5.Highest possible grade(HPG)of 5 was considered as the grade of best improvement.RESULTS:Overall,94.8%of the patients showed improvement by at least one grade of NFS at the end of T1.For all the 11 parameters evaluated,54%of patients achieved HPG after treatment.The four essential parameters(improvement in glycated hemoglobin(HbA 1c)and insulin level,and fall in number of other oral hypoglycemic drugs with and without insulin)are presented in detail.For Hb A1c,72.6%of patients at the end of T1 met the World Health Organization cut off value,i.e.,6.5%of HbA 1c.For insulin level,65.9%of patients at the end of T1 were able to achieve HPG.After treatment,the improvement was seen in 16.3%of patients who required no more than two medications along with insulin.Similarly,21.5%of patients were improved as their dosage regimen for using oral drugs was reduced to 1-2 from 5.CONCLUSION:hE SC therapy is beneficial in patients with diabetes and helps in reducing their dependence on insulin and other medicines.
基金the Australian Stem CellCentre (ASCC), UQ AIBN Challenge Grant Scheme for financial supportthe ASCC’s StemCore pluripotent stem cell core facility for the provision of hES cell lines and logistical support
文摘AIM:The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein. METHODS:Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter driven mCherry transgene was performed in MEL2 hESC. Red fluore-scence was assessed by immunofluorescence and flow cytometry. Pluripotency of stably transduced hESC was determined by immunofluorescent pluripotency marker expression, flow cytometry, teratoma assays andembryoid body-based differentiation followed by reverse transcriptase-polymerase chain reaction. Quantification of cell motility and survival was performed with time lapse microscopy. RESULTS:Constitutively fluorescently-labeled hESCs are useful tools for facile in vitro and in vivo tracking of survival, motility and cell spreading on various surfaces before and after differentiation. Here we describe the generation and characterization of a hESC line (MEL2) stably expressing red fluorescent protein, mCherry. This line was generated by random integration of a fluorescent protein-expressing cassette, driven by the ubiquitously-expressed human EF-1α promoter. Stably transfected MEL2-mCherry hESC were shown to express pluripo-tency markers in the nucleus (POU5F1/OCT4, NANOG and SOX2) and on the cell surface (SSEA4, TRA1-60 and TG30/CD9) and were shown to maintain a normal karyotype in long-term (for at least 35 passages) culture. MEL2-mCherry hESC further readily differentiated into representative cell types of the three germ layers in embryoid body and teratoma based assays and, importantly, maintained robust mCherry expression throughout differentiation. The cell line was next adapted to singlecell passaging, rendering it compatible with numerous bioengineering applications such as measurement of cell motility and cell spreading on various protein modified surfaces, quantification of cell attachment to nanoparticles and rapid estimation of cell survival. CONCLUSION:The MEL2-mCherry hESC line conforms to the criteria of bona fide pluripotent stem cells and maintains red fluorescence throughout differentiation, making it a useful tool for bioengineering and in vivo tracking experiments.
基金This work was supported by grants from National Ba-sic Research Program of China(973 Program)(No:001CB509903,001CB509904)Hi-Tech Research and Development Program of China(973 Program)(No:2001A A216121.2004AA205010)+3 种基金National Natural Science Foun-dation of China(No:30040003)Science and Technology Committee of Shanghai Municipality(No:99DJ 14002,00DJI 4033,0IDJ 14003.03DJ 14017)Chinese Academy of Science(No:KSCX-2-3-08)Shanghai Municipal Edu-cation Commission and Shanghai Second Medical University.
文摘Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.
基金Acknowledgments We are grateful to Dr DA Melton （Harvard University） for shar- ing his human ES cells with us. The study was supported by grants from the National High Technology Research and Development Program of China （2006CB943900）, the National Natural Science Foundation of China （General Program, 30500088）, the Shang- hai Jiao Tong University School of Medicine, and the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The study was also
supported by the Shanghai Leading Academic Deciline Project （S30201）.
文摘Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to determine the optimal source of MSCs. We investigated if this biological heterogeneity in MSCs from different sources results in different mechanisms for their differentiation. In this study, we compared the gene expression patterns of phenotypically defined MSCs derived from three ontogenically different sources: Embryonic stem cells (hES-MSCs), Fetal limb (Flb-MSCs) and Bone Marrow (BM-MSCs). Differentially expressed genes between differentiated cells and undifferentiated controls were compared across the three MSC sources. We found minimal overlap (5% - 16%) in differentially expressed gene sets among the three sources. Flb-MSCs were similar to BM-MSCs based on differential gene expression patterns. Pathway analysis of the differentially expressed genes using Ingenuity Pathway Analysis (IPA) revealed a large variation in the canonical pathways leading to MSC differentiation. The similar canonical pathways among the three sources were lineage specific. The Flb-MSCs showed maximum overlap of canonical pathways with the BM-MSCs, indicating that the Flb-MSCs are an intermediate source between the less specialised hES-MSC source and the more specialised BM-MSC source. The source specific pathways prove that MSCs from the three ontogenically different sources use different biological pathways to obtain similar differentiation outcomes. Thus our study advocates the understanding of biological pathways to obtain optimal sources of MSCs for various clinical applications.
文摘We established a QT interval assessment system that uses human embryonic stem cell-derived cardiomyocyte clusters (hES-CMCs) in which the field potential duration (FPD) or corrected FPD (FPDc) was measured as an indicator of drug-induced QT interval prolongation. To investigate the applicability of the hES-CMC system to drug safety assessment, we investigated short-term variability in FPDc (STVFPDc) (beat rate rhythmicity) as a marker of torsadogenic risk. We investigated the FPDc and STVFPDc of hES-CMCs treated with hERG channel blockers (E-4031 or cisapride) or with our proprietary compounds X, Y, and Z. We also evaluated the electrocardiograms and hemodynamics of dogs treated with compound X, Y, or Z. The torsadogenic hERG channel blockers increased STVFPDc and prolonged FPDc. Compounds X, Y, and Z had hERG inhibitory activity. Compound X prolonged FPDc with increased STVFPDc, whereas compounds Y and Z tended to shorten FPDc in the hES-CMC system. In the in vivo canine study, compound X prolonged corrected QT (QTc), and compounds Y and Z tended to shorten QTc, showing a good correlation with the results in hES-CMCs. These findings suggest that combined assessment of FPDc and STVFPDc in the hES-CMC system increases the predictability of torsadogenic risk.
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 μg/L Noggin or 20 μg/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, β-III 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 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A 32bp deletion in the chemokine receptor 5 (CCR5) gene (CMKBR5) was shown to be linked to HIV resistance. Bone marrow transplantation from the homozygous CCR5-del32 donor to a CDC Stage 2 HIV-positive recipient was demonstrated to confer a HIV resistance, resulting in discontinuation of antiretroviral therapy. In search for an unlimited source of CCR5-del32 cells for transplantation purposes, we tested 137 human embryonic stem cell (hESC) lines from the Reproductive Genetics Institute’s hESC lines collection, and report here the finding of 12 hESC lines with the CCR5-del32 allele, one of which represents a unique partenogenetic ESC line containing two copies of this deletion and may be studied for utility in stem cell transplantation treatment of HIV.
