Endothelial Progenitor Cells Derived from mESCs Were Directed to Differentiate into Vascular Endothelial Cells and Promote Wound Healing / 中国生物化学与分子生物学报

Ischemic dysfunction is an important global health problem. Vascular endothelial cells(VECs) play a key role in angiogenesis, and insufficient vascular remodeling may lead to chronic nonhealing wounds. Therefore, effective VEC generation strategies of exploration help improve angiogenesisin damaged tissues. Embryonic stem cells (ESCs) are widely used in the study of tissueendothelialization, and endothelial progenitor cells (EPCs) are indispensable parts of the development ofVECs. The aims of this study were to find a rapid, easily screened and reproducible method for thederivation of EPCs from mouse embryonic stem cells (mESCs), and obtain VECs with high survival ratesand strong functions from the directed differentiation of the EPCs. The results showed that mESCs weredifferentiated into " stepping stone" -like progenitor cells with active proliferative ability by 10 ng / mLVEGF and 5 ng / mL bFGF. At the same time, the method of differential adherence was helpful for theselection of EPCs, and EPCs induced high expression of CD133 and CD34 (The relative expressionlevels were 0. 88 ± 0. 04 and 2. 12 ± 0. 02, respectively) for 3 days. Then EPCs were digested withacctuse enzymes, and induced to differentiate into vascular endothelial-like cells by 50 ng / mL VEGF and25 ng / mL bFGF for 7 days. The endothelial cells not only expressed endothelial marker genes (CD31, CD144, LAMA5, Tek, KDR and vWF),and marker proteins CD31, CD144 and LAMA5 (The relativeexpression levels were 1. 07 ± 0. 03, 0. 60 ± 0. 02 and 0. 70 ± 0. 02, respectively), but also had thegood ability of migration, tubulogenesis and formation of W-P bodies. Moreover, PBS, EPC and VECwere used to treat wounds of the same size. Both EPC and VEC could accelerate the degree of tissuehealing (The relative healing rates were 78. 93 ± 75. 35%, 95. 57 ± 83. 73% and 100. 00 ± 0. 00%, respectively), and VEC significantly enhanced the ability of wound angiogenesis and inflammatoryresponses. In consequence, this study preliminarily confirmed that mESC-derived EPCs coulddifferentiate into VECs after directional induction for 7 days, which had good function of tissue repair. The physiological pathway on stem cells by stimulating angiogenesis is expected to become a new target fortissue remodeling.

Effects of Concave and Convex Interface on Pluripotency of Mouse Embryonic Stem Cells / 医用生物力学

Objective To explore the effect of concave and convex interface on in vitro culture of mouse embryonic stem cells. Methods Mouse embryonic stem cells were cultured on substrate with concave and convex interface. The biological morphology of cell colony was observed. The pluripotency of embryonic stem cells was detected by immunofluorescence and alkaline phosphatase (ALP) staining. Results Embryonic stem cells on concave substrates and convex substrates had higher stereo degree and circularity than those on flat substrates, but it was more obvious on concave substrates. Besides, the expression level of Oct4-GFP and the staining intensity of ALP in embryonic stem cells which were cultured on concave substrates and convex substrates were significantly higher than those on flat basement, especially on concave substrates. Conclusion sCompared with flat substrates, concave substrates and convex substrates had positive effects on the pluripotency maintenance of embryonic stem cells, which could help to maintain pluripotency, but concave substrates had better effects. Changing the substrate curvature could help to maintain pluripotency of embryonic stem cells cultured in vitro. The research findings are of great significance to the study and clinical application of embryonic stem cells.

Splitomicin, a SIRT1 Inhibitor, Enhances Hematopoietic Differentiation of Mouse Embryonic Stem Cells

BACKGROUND AND OBJECTIVES: Embryonic stem (ES) cells have pluripotent ability to differentiate into multiple tissue lineages. SIRT1 is a class III histone deacetylase which modulates chromatin remodeling, gene silencing, cell survival, metabolism, and development. In this study, we examined the effects of SIRT1 inhibitors on the hematopoietic differentiation of mouse ES cells. METHODS AND RESULTS: Treatment with the SIRT1 inhibitors, nicotinamide and splitomicin, during the hematopoietic differentiation of ES cells enhanced the production of hematopoietic progenitors and slightly up-regulated erythroid and myeloid specific gene expression. Furthermore, treatment with splitomicin increased the percentage of erythroid and myeloid lineage cells. CONCLUSIONS: Application of the SIRT1 inhibitor splitomicin during ES cell differentiation to hematopoietic cells enhanced the yield of specific hematopoietic lineage cells from ES cells. This result suggests that SIRT1 is involved in the regulation of hematopoietic differentiation of specific lineages and that the modulation of the SIRT1 activity can be a strategy to enhance the efficiency of hematopoietic differentiation.

Determining Osteogenic Differentiation Efficacy of Pluripotent Stem Cells by Telomerase Activity

BACKGROUND: Bone tissue engineering based on pluripotent stem cells (PSCs) is a new approach to deal with bone defects. Protocols have been developed to generate osteoblasts from PSCs. However, the low efficiency of this process is still an important issue that needs to be resolved. Many studies have aimed to improve efficiency, but developing accurate methods to determine efficacy is also critical. Studies using pluripotency to estimate efficacy are rare. Telomerase is highly associated with pluripotency. METHODS: We have described a quantitative method to measure telomerase activity, telomeric repeat elongation assay based on quartz crystal microbalance (QCM). To investigate whether this method could be used to determine the efficiency of in vitro osteogenic differentiation based on pluripotency, we measured the pluripotency pattern of cultures through stemness gene expression, proliferation ability and telomerase activity, measured by QCM. RESULTS: We showed that the pluripotency pattern determined by QCM was similar to the patterns of proliferation ability and gene expression, which showed a slight upregulation at the late stages, within the context of the general downregulation tendency during differentiation. Additionally, a comprehensive gene expression pattern covering nearly every stage of differentiation was identified. CONCLUSION: Therefore, this assay may be powerful tools for determining the efficiency of differentiation systems based on pluripotency. In this study, we not only introduce a new method for determining efficiency based on pluripotency, but also provide more information about the characteristics of osteogenic differentiation which help facilitate future development of more efficient protocols.

Effects of CHIR99021 and Wnt3 a on cardiac differentiation of mouse embryonic stem cells / 安徽医科大学学报

Objective To investigate the effects of CHIR99021 and Wnt3a, Wnt/β-catenin signaling pathway acti-vators, on cardiac differentiation of mouse embryonic stem cells ( mESCs ) . Methods The embryonic bodies ( EBs) were formed through suspension culture method, CHIR99021 or Wnt3a was added into differentiated medi-um from day 2 to 5, named CHIR99021 group or Wnt3a group, respectively. In addition, there was a control group in which EBs were automatically differentiated. The expression levels of Brachyury, the mesoderm specific target gene, and Nkx2. 5, cardiac-precursor marker, as well as the transcripts of cardiomyocyte markers,α-myosin heavychain (α-MHC ) , cardiac troponin T ( cTnT ) and connexin-43 ( Cx43 ) were analyzed through quantitative RT-PCR. Besides, the cardiac-specific proteins including α-MHC, cTNT and CX43 were detected by immunofluores-cence and Western blot. Results The mESCs in every group did differentiate into cardiomyocytes. The expression of Brachyury was substantially augmented by treatment with CHIR99021 and Wnt3a, showing a peak of expression at day 7. Similarly, CHIR99021 and Wnt3a dramatically increased the expression levels of Nkx2. 5,α-MHC, cT-nT and Cx43 with the time of differentiation, with the expression of target genes in CHIR99021 group and Wnt3a group was greater than that in the control group and CHIR99021 group was higher than Wnt3 a group at day 15 ( P<0. 05, P < 0. 01 ). Western blot analysis suggested that the expressions of α-MHC, cTNT and CX43 in CHIR99021 group and Wnt3a group were greater than those in the control group, and CHIR99021 group was higher than Wnt3 a group at day 15 . Conclusion Both CHIR99021 and Wnt3 a could improve cardiogenesis from mESCs through activate Wnt/β-catenin signaling pathway at the early stage of differentiation while the former is better than the latter.

Role of circadian gene Clock during differentiation of mouse pluripotent stem cells

Biological rhythms controlled by the circadian clock are absent in embryonic stem cells (ESCs). However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells (iPSCs). These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any difference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before (DeBruyne et al., 2007b). After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.

Impact of different mouse blastocyst donors on the efficiency of germline chimera production from C57BL/6 embryonic stem cells / 中国实验动物学报

Objective To improve the gene targeting efficiency with C57BL/6 embryonic stem ( ES) cells.Meth-ods Three different genetically modified C57BL/6 ES cell lines, named TLX3, Ai3K and SL, were microinjected into ICR, B6( Cg)-Tyrc-2J and BALB/c mouse blastocysts, respectively.The efficiency was statistically evaluated according to three aspects:blastocyst collection, chimera production and germline transmission.Results None of the three ES cell lines was germline transmitted with B6(Cg)-Tyrc-2J mice as blastocyst donors, while it was achieved with both BALB/c and ICR mouse blastocysts.Compared in the aspect of blastocysts collection, ICR mouse was much better than BALB/c mouse (P<0.05), and the chimera production efficiency of ICR mouse was comparable to that of BALB/c mouse (P =0.115). As to the germline transmission efficiency, that of BALB/c mice is significantly higher than that of the ICR mice ( P<0.01).Conclusions The germline transmission efficiency of BALB/c mouse is highest among these three mouse strains. However, it has the disadvantages of blastocyst collection, developmental delay and zona pellucida fragility, compared with ICR mouse.Therefore, ICR mouse is also a good candidate as blastocyst donor for embryonic stem cell microinjection.

Promoting effects of activated Nodal signal pathway on definitive endo-derm induction from mouse embryonic stem cells / 中国病理生理杂志

[ ABSTRACT] AIM:To study the process of promoting mouse embryonic stem cells ( ESC) to specify to definitive endoderm by up-regulating of Nodal signal pathway in order to find the best cultivated systems of differentiation of mouse ESC to definitive endoderm cells.METHODS:The cells were divided into different groups based on the culture medium:ESC group ( serum-free medium +LIF) , natural differentiation group ( serum-free medium) and activin A group ( serum-free medium +50μg/L activin A).The cells and the sterilized coverslips with cells were collected at 1, 3, 5 and 7 d of the cultivation.The proportion of CXCR4 +cells was detected by flow cytometry.The expression of CXCR4 was determined by immunocytochemical method, and the protein expression of OCT4 and CXCR4 was detected by Western blot.RE-SULTS:The proportion of CXCR4 +cells showed no dramatic change in ESC group along with the extending of cultivation day, while there were gradually increased in natural differentiation group and activin A group and the highest level was ob-served at 5 d.Among the 3 groups, the proportion of CXCR4 +cells at 5 d was the highest in activin A group.The brown or tan staining in the cells observed under microscope was considered as positive CXCR4 by immunocytochemistry.The pro-tein levels of OCT4 and CXCR4 in ESC group along with the extending of cultivation days was observed.The expression levels of OCT4 were gradually decreased in the cells in natural differentiation group and activin A group, while those of CX-CR4 were gradually increased with the highest level at 5 d.It was highest in the cells in activin A group.CONCLUSION:The proportion of definitive endoderm was the highest at 5 d of the induction during in vitro mouse ESC differentiation.Up-regulation of Nodal signal pathway by adding activin A at the early stage of mouse ESC differentiation promotes mouse ESC to specify to definitive endoderm with CXCR4 molecular marker.

Expression and function of calcium-sensing receptor in mouse embryonic stem cells / 中国病理生理杂志

AIM:To observe the functional expression of calcium-sensing receptor (CaSR) in the mouse em-bryonic stem cells (mESCs).METHODS:The expression and distribution of CaSR were detected by Western blotting and immunofluorescence observation in 129 mouse ES-D3 cells.The intracellular concentration of free calcium ([Ca2+]i) was determined by confocal laser scanning microscopy .The cell viability was analyzed by MTT assay and flow cytometry .RE-SULTS:CaSR protein was expressed in mESCs .Extracellular calcium or neomycin significantly increased the expression of CaSR and [Ca2+]i.Neomycin increased the cell viability , up-regulated the protein expression of p-ERK2.These effects of neomycin were inhibited by NPS2390.CONCLUSION:CaSR is expressed in mESCs .The activation of CaSR is involved in the proliferation of mESCs .

MiR-3099 is Overexpressed in Differentiating 46c Mouse Embryonic Stem Cells upon Neural Induction

Background: MicroRNAs (miRNAs) have a crucial role in gene expression regulation and protein synthesis, especially in the central nervous system. In developing mouse embryos a novel miRNA, miR-3099, is highly expressed, particularly in the central nervous system. This study aims to determine the expression of miR-3099 during cellular differentiation of 46C mouse embryonic stem cells after neural induction with N2/B27 medium. Methods: 46C mouse embryonic stem cells were subjected to neural induction with N2/B27 medium. At 0, 3, 7, 11, 17, and 22 days after neural induction, the cells were screened for various pluripotent, progenitor, and differentiating/differentiated cells markers by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR). Stem-loop pulse RT-PCR was performed to determine the expression of miR-3099 at all selected time points after neural induction. Results: Our findings showed that after induction, mouse embryonic stem cells differentiated into heterogeneous pools of cells containing neurons, astrocytes, and oligodendrocytes. Mouse embryonic stem cells and neural progenitor/precursor cells were also present in culture up to day 22 as indicated by RT-PCR analysis. Elucidation of miR-3099 expression during in vitro neural induction revealed that this miRNA was expressed throughout the differentiation process of 46C mouse embryonic stem cells. miR-3099 was expressed at higher levels on day 11, 17, and 22 as compared to day 0, 3 and 7 after neural induction. Conclusion: The level of miR-3099 expression was higher in differentiated mouse embryonic stem cells after neural induction. This finding suggested that miR-3099 might play a role in regulating neural stem cell differentiation. However, further characterisation of miR-3099 in a better characterised or optimised differentiated neural stem cell culture would provide increased understanding of the cellular function and molecular targets of miR-3099, especially in neuron development.

La lipofección incrementa la eficiencia de mutagénesis dirigida en células troncoembrionarias de ratón E14 TG2a / Lipofection improves gene targeting efficiency in E14 TG2a mouse embryonic stem cells

Electroporation has been the method of election for transfection of murine embryonic stem cells for over 15 years; however, it is a time consuming protocol because it requires large amounts of DNA and cells, as well as expensive and delicate equipment. Lipofection is a transfection method that requires lower amounts of cells and DNA than electroporation, and has proven to be efficient in a large number of cell lines. It has been shown that after lipofection, mouse embryonic stem cells remain pluripotent, capable of forming germ line chimeras and can be transfected with greater efficiency than with electroporation; however, gene targeting of mouse embryonic stem cells by lipofection has not been reported. The objective of this work was to find out if lipofection can be used as efficiently as electroporation for regular gene targeting protocols. This context compares gene targeting efficiency between these techniques in mouse embryonic stem cells E14TG2a, using a gene replacement type vector. No differences were found in gene targeting efficiency between groups; however, lipofection was three times more efficient than electroporation in transfection efficiency, which makes lipofection a less expensive alternative method to produce gene targeting in mouse embryonic stem cells.

Durante los últimos 15 años se ha demostrado que la electroporación representa el método ideal para la transfección de células troncoembrionarias de ratón; sin embargo, demanda grandes cantidades de ADN y células, así como equipo caro y delicado, ello hace que este proceso sea costoso y laborioso. La lipofección es un método de transfección que requiere menos de células y ADN que la electroporación; asimismo, ha probado ser eficiente en gran número de líneas celulares. Se ha demostrado que después de lipofectar células troncoembrionarias de ratón, éstas mantienen su pluripotencia y son capaces de formar quimeras de línea germinal y se transfectan con mayor eficiencia que con electroporación, pero no se ha notificado la mutagénesis dirigida mediante la lipofección de células troncoembrionarias de ratón. El objetivo del presente trabajo fue saber si la lipofección puede ser utilizada con la misma o mayor eficiencia que la electroporación para los protocolos regulares de mutagénesis dirigida; en este contexto, se compara la eficiencia en mutagénesis dirigida entre estas técnicas en células troncoembrionarias de ratón E14TG2a, utilizando un vector de reemplazo. Entre las células transfectadas no se hallan diferencias en la eficiencia en mutagénesis dirigida entre grupos; sin embargo, los resultados que aquí se ofrecen muestran que la lipofección es tres veces más eficiente en la transfección, lo cual indica que la lipofección es un método alternativo menos costoso para obtener mutagénesis dirigida en células troncoembrionarias de ratón.

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.

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.

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.

Establishment of Mouse Embryonic Stem Cell-like Cells from In Vitro Fertilized Embryos / 대한불임학회지

OBJECTIVE: In order to acquire the technique for the establishment of human embryonic stem cells (ESC) derived from the human frozen-thawed embryos produced in IVF-ET program, this study was performed to establish mouse ESC derived from the in vitro fertilized embryos. MATERIALS AND METHODS: After F1 hybrid (C57BL x CBA) female mice were superovulated with PMSG and hCG treatment, their oocytes were retrieved and inseminated, and the fertilized embryos were cultured for 96-120 hours until the expected stages of blastocysts were obtained. To isolate the inner cell mass (ICM), either the blastocysts were treated with immunosurgery, or the whole embryos were cultured for 4 days. Isolated ICMs were then cultured onto STO feeder cell layer, and the resultant ICM colonies were subcultured with trypsin-EDTA treatment. During the subculture process, ESC-like cell colonies were observed with phase contrast microscopy. To identify ESC in the subcultured ESC-like cell colonies, alkaline phosphatase activity and Oct-4 (octamer-binding transcription factor-4) expression were examined by immunohistochemistry and RT-PCR, respectively. To examine the spontaneous differentiation, ESC-like cell colonies were cultured without STO feeder cell layer and leukemia inhibitory factor (LIF). RESULTS: Seven ESC-like cell lines were established from ICMs isolated from the in vitro fertilized embryos. According to the developmental stage, the growth of ICMs isolated from the expanded blastocysts was significantly better than that of ICMs isolated from the hatched blastocysts (80.3% vs. 58.7%, p<0.05). ESC-like cell colonies were only obtained from ICMs of expanded blastocysts. However, the ICMs isolated from the embryos treated with immunosurgery were poorly grown and frequently differentiated during the culture process. The established ESC-like cell colonies were positively stained with alkaline phosphatase and expressed Oct-4, and their morphology resembled that observed in the previously reported mouse ESC. In addition, following the extended in vitro culture process, they maintained their expression of cell surface markers characteristic of the pluripotent stem cells such as alkaline phosphatase and Oct-4. When cultured without STO feeder cell layer and LIF, they were spontaneously differentiated into the various types of cells. CONCLUSION: The findings of this study suggest that the establishment of mouse ESC can be successfully derived from the in vitro fertilized embryos. The established ESC-like cells expressed the cell surface markers characteristic of the pluripotent stem cells and spontaneously differentiated into the various types of cells.