Hypoxia promotes differentiation of human induced pluripotent stem cells into embryoid bodies in vitro / 南方医科大学学报

OBJECTIVE@#To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms.@*METHODS@#EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment.@*RESULTS@#The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, β-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05).@*CONCLUSION@#Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.

CRISPR/Cas9-Mediated Suds3 Knockout Inhibited Mouse Embryonic Stem Cell Proliferation and Mesoderm/Endoderm Differentiation / 中国生物化学与分子生物学报

Embryonic stem cells (ESCs) have the ability to differentiate into various adult cells, and their fate in the process of development and differentiation is determined by the comprehensive regulation of multiple factors such as gene expression, epigenetics, and extracellular signals. Epigenetic regulation, such as DNA methylation, histone acetylation, and methylation, plays an important role in the maintenance of pluripotency and differentiation of ESCs. Suds3 (Sin3 histone deacetylase corepressor complex component SDS3) is one of the important components of Sin3 histone deacetylase complex. It played an important role in embryonic development, cell proliferation, chromosome separation and other biological processes. However, the functions of Suds3 in ESCs, such as its influence on the proliferation, maintenance of pluripotency and differentiation of ESCs, were rarely reported. In this study, we used CRISPR/Cas9 gene editing technology to construct a Suds3 knockout mouse embryonic stem cell line, and combined cell culture, in vitro embryoid body (EB) formation and in vivo teratoma formation, CCK-8 and cell counting experiments to study the function of Suds3 in ESCs. Western blotting results showed that the SUDS3 protein was not expressed, and the Suds3 gene was successfully knocked out. Through the observation of cell morphology and fluorescence quantitative PCR (QRT-PCR) to detect the expression of pluripotency genes, we found that the knockout of Suds3 had no significant effect on the maintenance of pluripotency of ESCs. Embryoid body (EB) formation experiments revealed that on the fourth and sixth days of EB formation, the pluripotency gene expression was not down-regulated as quickly as WT cells but increased in Suds3

Directing Human Embryonic Stem Cells towards Functional Endothelial Cells Easily and without Purification

Hemangioblasts or blood islands only arise in early development thereby the sources to obtain these bi-potential cells are limited. While previous studies have isolated both lineages in vitro through the hemangioblast, derivation efficiency was rather low due to cellular damage attributed by enzyme usage and fluorescent activated cell sorting (FACS). This study focused on avoiding the use of damaging factors in the derivation of endothelial cells (ECs). Single cell H9-human embryonic stem cells (hESCs) were obtained by using a mild dissociation protocol then human embryoid body (hEB) formation was performed under hemangioblast differentiation conditions. The hEBs were subjected to a two-stage cytokine treatment procedure. Subsequent culture of the adhesive cells in day 4 hEBs gave arise to a seemingly pure population of ECs. The hESC-derived ECs were characterized by identifying signature endothelial gene and protein markers as well as testing for in vitro functionality. Furthermore, in vivo functionality was also confirmed by transplanting the cells in hindlimb ischemic murine models. We demonstrate that the genetic change required for EC derivation precedes blast colony formation. Furthermore, cell damage was prevented by abating enzyme usage and FACS, resulting in a high yield of ECs upon adhesion. Under this method, confluent cultures of ECs were obtainable 4 days after hEB formation which is significantly faster than previous protocols.

Assessment of Developmental Toxicants using Human Embryonic Stem Cells

Embryonic stem (ES) cells have potential for use in evaluation of developmental toxicity because they are generated in large numbers and differentiate into three germ layers following formation of embryoid bodies (EBs). In earlier study, embryonic stem cell test (EST) was established for assessment of the embryotoxic potential of compounds. Using EBs indicating the onset of differentiation of mouse ES cells, many toxicologists have refined the developmental toxicity of a variety of compounds. However, due to some limitation of the EST method resulting from species-specific differences between humans and mouse, it is an incomplete approach. In this regard, we examined the effects of several developmental toxic chemicals on formation of EBs using human ES cells. Although human ES cells are fastidious in culture and differentiation, we concluded that the relevancy of our experimental method is more accurate than that of EST using mouse ES cells. These types of studies could extend our understanding of how human ES cells could be used for monitoring developmental toxicity and its relevance in relation to its differentiation progress. In addition, this concept will be used as a model system for screening for developmental toxicity of various chemicals. This article might update new information about the usage of embryonic stem cells in the context of their possible ability in the toxicological fields.

Residual undifferentiated embryonic stem cells in embryoid bodies / 上海第二医科大学学报

Objective To explore the residual undifferentiated mouse embryonic stem cells (ESCs) in embryoid bodies. Methods Mouse R1 and Oct-4-GFP transgenic ESCs were firstly cultured in suspension to form embryoid bodies (EBs). Twenty days later, EBs were digested into single cells and then re-plated in standard ESC culture condition. The morphology of residual undifferentiated cells in EBs was observed, and surface makers and in vitro redifferentiation potency of residual cells were examined by flow cytometry and immunofluoreseent staining. The residual cells were expanded and subcutaneously injected into nude mice, and the specimens were harvested from the injection site for histological analysis 6 weeks after injection. Results There were residual undifferentiated ESCs in EBs differentiated for 20 days, which displayed clonal morphology and expressed undifferentiated cell markers of ESCs, including SSEA1, CD31, CD9 and Oct-4. The cells could be differentiated to form EBs again, and could be re-expanded from secondary EBs. The residual cells were able to form teratoma at the injection site, and mature endoderm, mesoderm and ectoderm tissues could be found in teratoma tissues. Conclusion There are residual undifferentiated ESCs after differentiation of ESCs into EBs. The residual ESCs can differentiate again in vitro and in vivo, and can residue again in the in vitro differentiation.

Two conditional media promoting the differentiation murine embryonic stem cells into hematopoietic stem cells / 中南大学学报(医学版)

Objective To derive hematopoietic stem cells with functional properties of hematopoietic reconstitution from murine embryonic stem (ES) cells. Methods ES-D3 cells by formation of the day-4 embryoid bodies (4dEBs) were induced into hematopoietic stem cells by co-culture with murine bone marrow endothelial cell-conditional medium (mBMEC-CM) and the fetal liver stromal cell-conditional medium (FLSC-CM). This experiment was designed to 4 groups (mBMEC-CM + FLSC-CM group, mBMEC-CM group, FLSC-CM group, and the control group). Results The total cell numbers, CD34 + cell numbers, and colony numbers formed in the mBMEC-CM + FLSC-CM group were the highest among the 4 groups. The cells in the mBMEC-CM + FLSC-CM group resumed the hematopoietic system of the mice after being transplanted with the inducing cells. Conclusion The culture condition combing mBMEC-CM with FLSC-CM can promote murine ES cells differentiating into hematopoietic stem cells with functional properties of hematopoietic reconstitution.

Stirred Bioreactor Cultivation Enhances the Efficiency of Embryoid Bodies Formation and Differentiation into Cardiomyocytes / 中国生物工程杂志

Objective:To determine the optimal condition for mouse embryonic stem cells (mESC) culture with stirred bioreactor,and to develop a method for mass production of embryoid bodies (EB). Methods:The different initial cell concentrations of mESC and the initial stirring speed of bioreactor were investigated to determine the optimal condition for EB formation. Induced by ascorbic acid,the differentiation of EBs formed in stirred bioreactor into cardiomyocytes was compared with EBs formed in Petri dish. Immunofluorescence staining and RT-PCR were used to identify the cardiomyocytes derived from mESC. Results:The formation of a large number of uniform relatively EBs was achieved in stirred bioreactor when mESC were seeded initially with 1?105~3?105 cells/ml and stirring speed was set to 15~30r/min. Most of cells in the EBs formed in bioreactor were viable. EBs produced in bioreactor differentiated into cardiomyocytes more efficiently compared with EBs from Petri dish. The cardiac specific genes were expressed in ESC-derived cardiomyocytes. Conclusions:Stirred bioreactor culture could enhance the efficiency of EB formation and differentiation into cardiomyocytes,which may be a more ideal culture system for EB formation.

Bridge PCR,An Easy Way for Concatemerizing DNA Tags / 中国生物工程杂志

In MAST (mRNA accessible site tagging),the DNA tags from synthesized library were employed for identifying mRNA accessible sites. A large number of tags were amplified and subcloned for sequencing to verify mRNA binding profiles. A PCR was designed by using one primer which bridges over the tag terminal sequences. In PCR reaction DNA tag fragments were concatemerized by a bridge primer in reaction cycles. The concatemerized tag fragments were subcloned and sequenced. Dozens of the concatemerized sequences contained thousands tags. The PCR was a simple,effective way which for sequencing tags in a high through put manner.

Detection of clock genes in embryoid body derived single cells with nested RT-PCR / 基础医学与临床

Objective To establish methods for examining expression of clock genes including BMAL1,CLOCK,CRY1and CRY2,and to figure out expression profile of these genes in single cell derived from embryoid body(EB).Methods Total RNA isolated from EB was subjected to reverse transcription and amplification with clock genes specific primers to determine thermo-cycle condition,which was used consequently to examine the expression profiles of these clock genes in single cells isolated with patch clamp from embryoid bodies.Results Parameters in amplification and detection were determined.No unspecific band was amplified.At least 2 molecules could be detected with established systems.In the differentiating EB cells,co-expression of these clock genes was rare.Conclusion Theses systems are sensitive enough to detect expression of clock genes in single cells.Transcription and translation loop among clock genes are not intact in differentiating EB cells,and the clock genes may play a role inthe early development and differentiation.

A novel method for preparing embryoid bodies with attached embryonic stem cells / 中国实用内科杂志

Objective To improve disadvantages such as asynchrony during embryoid bodies(EBs)preparation,and to establish a novel method to prepare EBs by using attached mouse embryonic stem Cells.Methods Mouse R1 embryonic stem(ES)cells were trypsinized to a single cell suspension when reaching a sub-confluent state of 70 %～80 %,then 1?106 ES cells were plated into 100 mm tissue culture dishes.After being cultured in medium containing low concentration leukemia inhibitory factor(LIF,1 ?g/L)for 3 days,EBs were collected and suspended in EBs culture medium for further development.Morphology studies were performed on suspended EBs and their cryosections,then immunofluorescent staining of ?-fetoprotein(AFP),platelet endothelial cell adhesion molecule-1(PECAM-1),and neurofilament 68 KD(NF-68)were performed on suspended and attached EBs.Results EBs obtained by this method were homogeneous in size,synchronous in developmental stage;typical in structure and could well display the developmental process from simple EBs to mature cystic EBs.Immunofluorescent staining showed that AFP,PECAM-1 and NF-68 were positive.Conclusion Compared with present methods of preparing EBs,this novel method have advantages of simple manipulation,high efficiency of EBs formation and obtaining EBs at synchronous developmental stage,furthermore,these EBs have typical structures and the potential to differentiate into derivatives of all three embryonic germ layers.Thus this method can be used as an ideal tool for studies on early embryonal development,ES cell differentiation and so on.

Mouse Embryonic Stem Cells Derived Feeder Cells Support the Growth of Themselves / 上海第二医科大学学报

Objective To test whether feeder cells derived from mouse embryonic stem cells(mESCs) could support the growth of mESCs themselves. Methods mESCs were induced to form mouse embryoid bodies(EB),and then fibroblast-like cells were derived from further differentiated mEB(mEB-dF),which served as feeder cells.The undifferentiation of mESCs grown on mEB-dF was confirmed by morphological analysis,colony efficiency and cell differentiation rate of mESCs,immunocytochemistry,alkaline phosphatase staining and RT-PCR.The pluripotency of mESCs grown on mEB-dF was examined by RT-PCR,inducing their differentiation in vivo and in vitro. Results(Forty-eight) fibroblast-like cells lines were derived from the same EB at three periods(d 10,d 15 and d 20),and five of them,mostly derived from d15 EB,were able to maintain mESCs in undifferentiated status and pluripotential ability over 10 passages.mESCs cultured on these feeder cell lines expressed alkaline phosphatase and specific mESCs markers,including SSEA-1,OCT-4,NANOG,and formed EB in vitro and teratomas in vivo.However,the majority of mEB-dF lines(43/48) has no such ability. Conclusion This study not only provides a novel feeder system for mESCs culture,avoiding lot of disadvantages of mouse embryo fibroblasts used as the feeder,but also indicates that fibroblast-like cells derived from mESCs take on different functions.The molecular mechanism of different function of these fibroblast cells is worthy of further investigations.

Establishment of Embryonic Stem Cell Line from Isolated Blastomeres from Mouse Preimplantation Embryos / 대한불임학회지

OBJECTIVE: The aim of this study was to investigate whether embryonic stem (ES) cells can be established from isolated blastomeres of mouse embryos. METHODS: Blastomeres were separated from mouse (C57Bl/6J) 2- or 4-cell embryos. Isolated blastomeres or whole 4-cell embryos were co-cultured with mitosis-arrested STO feeder cells in DMEM supplemented with recombinant murine leukemia inhibitory factor and ES-qualified fetal bovine serum. After the tentative ES cell lines were maintained from isolated blastomeres or whole embryos, some of them were frozen and the others were sub-cultured continually. Characteristics of tentative ES cell lines as were evaluated for specific gene expressions with immunocytochemistry and RT-PCR. RESULTS: One ES cell line (3.0%) was established from isolated blastomere of 2-cell embryo and one cell line (4.0%) from isolated two blastomeres of 4-cell embryo. And five cell lines (16.7%) were established from whole 4-cell embryos. Both cell lines from isolated blastomere and whole embryo expressed mouse ES cells specific markers such as SSEA-1, Oct-4 and alkaline phosphatase. Marker genes of three germ layers were expressed from embryoid bodies of both cell lines. CONCLUSION: This study suggests that mouse ES cells could be established from isolated blastomeres, although the efficiency is lower than whole embryos. This animal model could be applied to establishment of autologous human ES cells from biopsied blastomeres of preimplantation embryos in human IVF-ET program.

Hematopoietic Differentiation of Embryoid Bodies Derived from the Human Embryonic Stem Cell Line SNUhES3 in Co-culture with Human Bone Marrow Stromal Cells

Human embryonic stem (ES) cells can be induced to differentiate into hematopoietic precursor cells via two methods: the formation of embryoid bodies (EBs) and co-culture with mouse bone marrow (BM) stromal cells. In this study, the above two methods have been combined by co-culture of human ES-cell-derived EBs with human BM stromal cells. The efficacy of this method was compared with that using EB formation alone. The undifferentiated human ES cell line SNUhES3 was allowed to form EBs for two days, then EBs were induced to differentiate in the presence of a different serum concentration (EB and EB/high FBS group), or co- cultured with human BM stromal cells (EB/BM co-culture group). Flow cytometry and hematopoietic colony-forming assays were used to assess hematopoietic differentiation in the three groups. While no significant increase of CD34+/CD45- or CD34+/CD38- cells was noted in the three groups on days 3 and 5, the percentage of CD34+/CD45- cells and CD34+/ CD38- cells was significantly higher in the EB/BM co-culture group than in the EB and EB/high FBS groups on day 10. The number of colony-forming cells (CFCs) was increased in the EB/BM co-culture group on days 7 and 10, implying a possible role for human BM stromal cells in supporting hematopoietic differentiation from human ES cell-derived EBs. These results demonstrate that co-culture of human ES-cell-derived EBs with human BM stromal cells might lead to more efficient hematopoietic differentiation from human ES cells cultured alone. Further study is warranted to evaluate the underlying mechanism.

Efficient gene delivery in differentiated human embryonic stem cells

Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.

Human embryonic stem cells forming embryoid bodies comprising multiple types of cells / 北京大学学报(医学版)

Objective:To investigate the pluripotency of human embryonic stem cells in vitro.Meth-ods:Human embryonic stem cells were cultured in suspension without feeder layers and bFGF in medi-um.mRNAs of different types of cells were analyzed by RT-PCR.Results:When Human embryonicstem cells were cultured in suspension without feeder layers and bFGF in medium,they formed floatingaggregates termed embryoid bodies(EBs),in which many precursors and functional cells were detectedby RT-PCR,such as neural and islet precursors,neurons,insulin-secreting cells,glucagon-secretingcells and liver cells et al.Conclusion:Human embryonic stem cells are able to form embryoid bodiesnaturally in vitro which express specific markers of many types of precursors and mature functional cells.

MHC Expression in Human Embryonic Stem Cells and Embryoid Bodies / 대한이식학회지

PURPOSE: Human embryonic stem (ES) cell is pluripotent cell derived from a group of cells called the inner cell mass and has the ability to reproduce itself for long periods and give rise to types of cells that develop from the three germ layers. Due to its pluripotency, ES cell holds the promise of being able to replace cells that are damaged or destroyed by many devastating diseases. However, the potential for the recipient of an ES cell transplant to reject this cell as foreign is very high. Thus, it is essential to determine whether human ES cells express MHC antigens. The purpose of this study is to characterize the stem cell properties of our cell line (SNUhES1) and the expression profile of MHC antigens on the surface of these cells and their differentiated derivatives, embryoid bodies (EBs). METHODS: The ES cells were grown on STO fibroblast in DMEM-F12. The EBs were grown in the same medium with exception that it lacked LIF and bFGF. The expression of self-renewal-associated genes and three germ layer cell-specific genes in ES cells and EBs were measured by RT-PCR at varying time point of incubation (1, 7, 14 and 28 day). The expression of MHC molecules were measured by RT-PCR and FACS analysis. RESULTS: The SNUhES1 cells expressed all self-renewal- associated genes (Fgf4, FoxD3, Oct4, Sox2 and TERT) we tested. During the differentiation three germ layer cell-specific genes in EBs were expressed as following order: ecto-, meso- and endodermal cell-specific genes. MHC class I proteins (HLA-ABC and beta2m) on the surfaces of ES cells and EBs were expressed in very low levels. MHC class II proteins (HLA-DP, -DQ and -DR) and HLA-G were not expressed on the surface of these cells. However, the expression of MHC class II proteins were detected in 1% more or less cells of 28-day-old EBs which were hardly detected in the population of 1-day-old EBs. CONCLUSION: These data imply that SNUhES1 cells and EBs have stem cell properties. Although they express very low MHC antigens, further investigation determining whether the MHC expression in the ES cells and EBs may alter under inflammatory condition which can be occurred in damaged tissue or through surgical process.

Vascular endothelial growth factor promotes hematopoietic differentiation from mouse embryonic stem cells / 中国病理生理杂志

AIM: To study the effect of vascular endothelial growth factor(VEGF) on hematopoietic differentiation from mouse embryonic stem cells(ESC) in vitro.METHODS: ES-D3 was allowed to grow on mouse fetal fibroblast feeder layer,and then was developed into embryoid bodies(EB).EB cells were transferred into medium supplemented with different concentration of VEGF and VEGF+SCF for 1 week.Six groups,including.VEGF 5 ?g/L,VEGF 10 ?g/L,VEGF 20 ?g/L, VEGF 5 ?g/L+SCF,VEGF 10 ?g/L+SCF and VEGF 20 ?g/L+SCF,were designed.The group of spontaneous differentiation without cytokine(s) was used as control.Hematopoietic transcription factor GATA-2 and early hematopoietic differentiation genes(c-kit and ?-H1) were detected by RT-PCR.The content of CD34~+ cells in each group were measured by flow cytometry.The cells derived from ESC were incubated in semisolid methycellulose cultures.The numbers of total colony-forming units in culture(CFU-C) were counted by reverse microscope.RESULTS: ES-D3 grew and formed EB at day 4.VEGF had a stimulatory effect as a single factor on the expression of genes associated with early hematopoietic differentiation(GATA-2,c-kit and ?-H1),the generation of CD34~+ cells and CFU-C in EB.The effects of VEGF+SCF were the most potent in the experimental groups according to the percent of CD34~+ cells and the numbers of hematopoietic colonies.The most highest inducing efficacy was achieved in VEGF 20 ?g/L or VEGF 10 ?g/L combined with SCF.CONCLUSION: VEGF promotes the differentiation of ESC into hematopoietic cells in vitro.The strongest effect was achieved when VEGF was combined with SCF.

THE RESEARCH ON THE PROLIFERATION AND DIFFERENTIATION OF MOUSE PRIMORDIAL GERM CELLS IN VITRO / 解剖学报

Objective To research on the proliferation and differentiation in vitro of primordial germ cells. Methods We got plenty of primordial germ cells(PGCs) by co-culture of PGCs with sertoli cells(SCs).The primary cells formed embryoid bodies(EBs) by suspension culture.furthermore,these EBs were plated on gelatin-coated culture plates for spontaneous differentiation. Results We got plenty of PGCs by coculture.Primary PGCs and passaged PGCs for histochemistry staining with AKP and immunohistochemistry staining for SSEA-1 display positive character.The EBs by suspension culture differentiated into epithelial-like,fibroblast-like and neuronal-like cells in vitro. Conclusion Primordial germ cells can proliferate and keep the character of stem cells by co-culture with SCs.PGCs can form EBs by suspension culture,most of these EBs can differentiate into neuronal-like cells,they also can differentiate into epithelial-like and fibroblast-like cells in vitro.