An efficient method for the derivation of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) represent a unique tool for many researchers; however, the process of ESC derivation is often very inefficient and requires high specialization, training, and expertise. To circumvent these limitations, we aimed to develop a simple and efficient protocol based on the use of commercially available products. Here, we present an optimized protocol that we successfully applied to derive ESCs from several knockout mouse strains (Wnt-1, Wnt-5a, Lrp6, and parkin) with 50%-75% efficiency. The methodology is based on the use of mouse embryonic fibroblast feeders, knockout serum replacement (SR), and minimal handling of the blastocyst. In this protocol, all centrifugation steps (as well as the use of trypsin inhibitor) were avoided and replaced by an ESC medium containing fetal calf serum (FCS) after the trypsinizations. We define the potential advantages and disadvantages of using SR and FCS in individual steps of the protocol. We also characterize the ESCs for the expression of ESC markers by immunohistochemistry, Western blot, and a stem cell focused microarray. In summary, we provide a simplified and improved protocol to derive mESCs that can be useful for laboratories aiming to isolate transgenic mESCs for the first time.     

Simple and efficient derivation of mouse embryonic stem cell lines using differentiation inhibitors or proliferation stimulators

The inhibition of endogenous differentiation-inducing signaling or the enhancement of growth capacity and viability of preimplantation embryos, via 2i (PD0325901 and CHIR99021), dramatically improves the establishment of mouse embryonic stem cells (mESCs). Using adrenocorticotropic hormone fragments 1-24 (ACTH 1-24), which enhances survival and/or proliferation of mESCs, also increases the derivation of mESCs from single blastomeres significantly. The CHIR99021 pathway and the proposed ACTH pathway are likely different. Therefore, this study aimed to assess the synergetic effects of 2i and ACTH 1-24 on derivation of mESCs. Results in the present study demonstrate that germline-transmitted mESCs could be efficiently derived from ICR and C57BL/6J at 0.5-4.5 days postcoitum denuded zygotes to blastocysts or isolated blastomeres of 2-8-cell embryos and cultured in 10 μL droplets with human foreskin fibroblast (Hs68) or STO (a mouse embryonic fibroblast line) feeders and in knockout serum replacement (KSR) ESC medium containing 2i or ACTH 1-24. The overall success rates for C57BL/6J and ICR were 56.2% when cultured in 2i+ACTH 1-24, 26.6% in 2i, 6.7% in ACTH 1-24, and 4.8% in KSR ESC medium. These results imply that CHIR99021 and ACTH 1-24 are synergistically enhancing the establishment of mESCs. The proposed protocol also demonstrates a highly efficient and reproducible method, has a simple layout, is easy to apply, and could be used as an alternative method for routinely establishing mESC lines.     

Characterization of MSH2 variants by endogenous gene modification in mouse embryonic stem cells

Mutations in the mismatch repair gene MSH2 underlie hereditary nonpolyposis colorectal cancer (Lynch syndrome). Whereas disruptive mutations are overtly pathogenic, the implications of missense mutations found in sporadic colorectal cancer patients or in suspected Lynch syndrome families are often unknown. Adequate genetic counseling of mutation carriers requires phenotypic characterization of the variant allele. We present a novel approach to functionally characterize MSH2 missense mutations. Our approach involves introduction of the mutation into the endogenous gene of murine embryonic stem cells (ESC) by oligonucleotide-directed gene modification, a technique we recently developed in our lab. Subsequently, the mismatch repair capacity of mutant ESC is determined using a set of validated functional assays. We have evaluated four clinically relevant MSH2 variants and found one to completely lack mismatch repair capacity while three behaved as wild-type MSH2 and can therefore be considered as polymorphisms. Our approach contributes to an adequate risk assessment of mismatch repair missense mutations. We have also shown that oligonucleotide-directed gene modification provides a straightforward approach to recreate allelic variants in the endogenous gene in murine ESC. This approach can be extended to other hereditary conditions.     

小鼠孤雌胚胎及孤雌胚胎干细胞中印记基因的表达

小鼠孤雌胚胎体内发育最多不能超过10.5d,发育失败的主要原因是胚外组织发育缺陷和印记基因表达的异常。随着小鼠孤雌二倍体和单倍体胚胎干细胞的建系成功,不仅能作为研究印记基因的理想模型,还能够作为种子细胞应用于细胞治疗,拓宽了小鼠孤雌生殖的研究领域和再生医学应用范围。孤雌胚胎聚合作为一种简便易行的技术手段,能够显著提高孤雌胚胎干细胞的建系效率,还能促使异质胚胎间的印记基因相互补偿从而更加趋近于正常受精的胚胎干细胞。我们在文中主要阐释了小鼠孤雌胚胎、孤雌聚合胚胎、孤雌二倍体胚胎干细胞、孤雌单倍体胚胎、孤雌聚合胚胎干细胞中印记基因的表达。     

Mouse hematopoietic stem cells, unlike human and mouse embryonic stem cells, exhibit checkpoint-apoptosis coupling

Previously, we reported that the spindle assembly checkpoint (SAC), which is coupled in somatic cells, is uncoupled from apoptosis-initiation in mouse and human embryonic stem cells (ESCs). This condition allows ESCs to tolerate and proliferate as polyploidy/aneuploid cells. Proper function of the SAC is vital to prevent polyploidy/aneuploidy during ex vivo hematopoietic stem cell (HSC) expansion. Here we address, for the first time, whether HSCs are more like ESCs or somatic cells with respect to SAC-apoptosis coupling. Using multiparametric permeablized cell flow-cytometric analysis to identify and analyze the mouse sca 1(+)/c-kit(+)/lin(-) (LSK) population, we found the mitotic spindle checkpoint to be functional in primary murine LSK cells, a population enriched in primitive hematopoietic stem/progenitor cells, after prolonged activation of the SAC by microtubule-depolymerizing agents such as nocodazole. HSCs can efficiently initiate apoptosis after activation of the SAC in LSK cells as indicated by increased hypodiploidy and increased levels of activated caspase 3, suggesting that HSCs behave more like somatic cells instead of ESCs with respect to this important cell cycle checkpoint. We conclude that mouse HSCs are not subject to the same kinds of chromosomal instability as are ESCs, knowledge that might aid in optimizing in vitro culture and expansion of human bone marrow or cord blood HSC for clinical applications.     

Simvastatin suppresses self-renewal of mouse embryonic stem cells by inhibiting RhoA geranylgeranylation

Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, were originally developed to lower cholesterol. Their pleiotropic (or cholesterol-independent) effects at the cellular and molecular levels are highly related to numerous cellular functions, such as proliferation and differentiation. However, they are hardly studied in embryonic stem cells. In this study, we evaluated the effects of statins on mouse ESCs (J1, D3, and RW.4) to enhance our understanding of the molecular basis of ESC self-renewal. Treatment of ESCs with simvastatin, mevastatin, atorvastatin, or pravastatin induced morphological change and decreased cell proliferation. We observed that the use of simvastatin was most effective in all three ESCs. Loss of ESC self-renewal by simvastatin was determined by marked downregulation of ESC markers alkaline phosphatase, Oct4, Nanog, Rex-1, and SSEA-1. Simvastatin effects were selectively reversed by either mevalonate or its metabolite geranylgeranyl pyrophosphate (GGPP) but not by cholesterol or farnesyl pyrophosphate. These results suggest that simvastatin effects were mainly derived from depletion of intracellular pools of GGPP, the substrate required for the geranylgeranylation. Using this approach, we found that GGPP, a derivative of the mevalonate pathway, is critical for ESC self-renewal. Furthermore, we identified that simvastatin selectively blocked cytosol-to-membrane translocalization of RhoA small guanosine triphosphate-binding protein, known to be the major target for geranylgeranylation, and lowered the levels of Rho-kinase (ROCK)2 protein in ESCs. In addition, simvastatin downregulated the ROCK activity, and this effect was reversed by addition of GGPP. Our data suggest that simvastatin, independently of its cholesterol-lowering properties, impairs the ESC self-renewal by modulating RhoA/ROCK-dependent cell-signaling.     

Feeder-free culture of human embryonic stem cells for scalable expansion in a reproducible manner

Human embryonic stem (hES) cells have the potential as starting materials for a wide variety of applications in cell therapy, drug discovery and development. However, the challenge is to produce large numbers of well-characterized hES cells that are pluripotent and of high quality. This is needed to be capable of producing future cell therapies that are safe, effective, and affordable for use in routine clinical practice. A major bottleneck is the present requirement for complex culturing regimes that are very labor intensive and unscalable. hES cells have traditionally been grown on feeder layers made from inactivated mouse or human embryonic fibroblasts, in medium containing serum and other nondefined factors. This makes conditions difficult to reproduce over multiple passages. With a view to simplifying culture conditions we have tested a novel proprietary good manufacturing practice-based system that circumvents the use of feeders completely. The system consists of a matrix and a formulated medium that, in combination, demonstrate a reliable and reproducible way to culture hES cells without the use of feeders. We have been able to grow hES cells (Shef3 and Shef6) for over 20 passages, in this system, without loss of pluripotency, capacity to differentiate, or acquisition of karyotypic abnormalities. Furthermore, we have demonstrated the feasibility of propagating hES cells at clonal dilutions from single cells using this system.     

Cardiomyocyte differentiation of mouse and human embryonic stem cells*

Ischaemic heart disease is the leading cause of morbidity and mortality in the western world. Cardiac ischaemia caused by oxygen deprivation and subsequent oxygen reperfusion initiates irreversible cell damage, eventually leading to widespread cell death and loss of function. Strategies to regenerate damaged cardiac tissue by cardiomyocyte transplantation may prevent or limit post‐infarction cardiac failure. We are searching for methods for inducing pluripotent stem cells to differentiate into transplantable cardiomyocytes. We have already shown that an endoderm‐like cell line induced the differentiation of embryonal carcinoma cells into immature cardiomyoctyes. Preliminary results show that human and mouse embryonic stem cells respond in a similar manner. This study presents initial characterization of these cardiomyocytes and the mouse myocardial infarction model in which we will test their ability to restore cardiac function.     

Migration of dorsal aorta mesenchymal stem cells induced by mouse embryonic circulation

Mesenchymal stem cells (MSCs) represent powerful tools for regenerative medicine for their differentiation and migration capacity. However, ontogeny and migration of MSCs in mammalian mid‐gestation conceptus is poorly understood. We identified canonical MSCs in the mouse embryonic day (E) 11.5 dorsal aorta (DA). They possessed homogenous immunophenotype (CD45^?CD31^?Flk‐1^?CD44⁺CD29⁺), expressed perivascular markers (α‐SMA⁺NG2⁺PDGFRβ⁺PDGFRα⁺), and had tri‐lineage differentiation potential (osteoblasts, adipocytes, and chondrocytes). Of interest, MSCs were also detected in E12.5–E13.5 embryonic circulation, 24 hr later than in DA, suggesting migration like hematopoietic stem cells. Functionally, E12.5 embryonic blood could trigger efficient migration of DA‐MSCs through platelet‐derived growth factor (PDGF) receptor‐, transforming growth factor‐beta receptor‐, but not basic fibroblast growth factor receptor‐mediated signaling. Moreover, downstream JNK and AKT signaling pathway played important roles in embryonic blood‐ or PDGF‐mediated migration of DA‐derived MSCs. Taken together, these results revealed that clonal MSCs developed in the mouse DA. More importantly, the embryonic circulation, in addition to its conventional transporting roles, could modulate migration of MSC during early embryogenesis. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

Large-scale gene trapping in C57BL/6N mouse embryonic stem cells

We report the construction and analysis of a mouse gene trap mutant resource created in the C57BL/6N genetic background containing more than 350,000 sequence-tagged embryonic stem (ES) cell clones. We also demonstrate the ability of these ES cell clones to contribute to the germline and produce knockout mice. Each mutant clone is identified by a genomic sequence tag representing the exact insertion location, allowing accurate prediction of mutagenicity and enabling direct genotyping of mutant alleles. Mutations have been identified in more than 10,000 genes and show a bias toward the first intron. The trapped ES cell lines, which can be requested from the Texas A&M Institute for Genomic Medicine, are readily available to the scientific community.     

Immunosuppression by embryonic stem cells

Embryonic stem cells or their progeny inevitably differ genetically from those who might receive the cells as transplants. We tested the barriers to engraftment of embryonic stem cells and the mechanisms that determine those barriers. Using formation of teratomas as a measure of engraftment, we found that semiallogeneic and fully allogeneic embryonic stem cells engraft successfully in mice, provided a sufficient number of cells are delivered. Successfully engrafted cells did not generate immunological memory; unsuccessfully engrafted cells did. Embryonic stem cells reversibly, and in a dose-dependent manner, inhibited T-cell proliferation to various stimuli and the maturation of antigen-presenting cells induced by lipopolysaccharide. Inhibition of both was owed at least in part to production of transforming growth factor-beta by the embryonic stem cells. Thus, murine embryonic stem cells exert "immunosuppression" locally, enabling engraftment across allogeneic barriers.     

Uni-directional differentiation of mouse embryonic stem cells into neurons by the neural stem sphere method

We previously showed that our neural stem sphere (NSS) method promotes the neuronal differentiation of mouse, monkey and human embryonic stem (ES) cells. Here we analyzed changes in expression of marker genes and proteins during neuronal differentiation. When cultured in astrocyte-conditioned medium (ACM) under free-floating conditions, colonies of ES cells formed floating cell spheres, which, within 4 days, gave rise to NSSs. In the spheres, the expression of ES cell marker genes was consistently down-regulated, while expression of an epiblast marker was transiently up-regulated, beginning on day 2, and the expression of neuroectoderm, neural stem cell and neuron markers was up-regulated, beginning on days 3, 4 and 6, respectively. The expression of the marker genes was consistent with that of marker proteins. The time course of expression of these markers in the spheres resembled that of neuronal differentiation from the inner cell mass (ICM) cells of blastula. In contrast, the expression of endoderm, mesoderm, epidermis, astrocyte and oligodendrocyte markers was low and not up-regulated during differentiation. Only a small number of apoptotic cells were present in the spheres. These results suggest that mouse ES cells uni-directionally differentiate into neurons via epiblast cells, neuroectodermal cells and neural stem cells.     

胚胎干细胞与四倍体互补产生转基因小鼠

目的 将四倍体胚胎补偿技术与转基因相结合，构建基因打靶载体转化的胚胎干细胞(ESCs)，获得小概率的同源重组事件，进而直接得到所要的突变品系。 方法 通过电穿孔的方法将增强型绿色荧光蛋白（EGFP）表达质粒转入ESCs，用G418筛选获得EGFP-ESCs。另外，通过电融合获得四倍体囊胚细胞，显微注射法将19～21个EGFP-ESCs注入每个四倍体囊胚腔，分别移植到假孕2.5d雌鼠子宫或假孕0.5d的输卵管内。 结果 获得稳定表达的EGFP-ESCs，染色体数目正常 (2n＝40条)；四倍体细胞融合率为95.07%，囊胚发育率为95%；共获得410个重构囊胚；移植后未得到出生小鼠，但共观察到了151个着床位点，子宫移植的着床率为29.41%，输卵管移植的着床率为64.37%，获得的胚胎中EGFP蛋白有散在的表达。 结论 稳定表达的EGFP-ESCs参与到了转基因胎鼠的发育中；本实验中输卵管移植的着床率高于子宫移植的着床率。     

昆明小鼠胚胎干细胞向心肌细胞的分化

目的饲养层制作及复苏培养昆明小鼠胚胎干细胞,探索体外诱导胚胎干细胞向心肌细胞分化。方法饲养层制作,复苏及体外培养胚胎干细胞,采用一步法消化贴壁胚胎干细胞,悬浮培养5d,形成拟胚体(EB),待EB贴壁后,加入淫羊藿苷(Icraiin,ICA)诱导液对其诱导并每天观察,免疫荧光检测心肌细胞特异性肌钙蛋白T(cTnT)和心室肌球蛋白轻链(MLC-2v)的表达。结果胚胎干细胞悬浮聚集5d,形成类似球状的拟胚体,将拟胚体贴壁诱导8d,发现拟胚体中出现跳动,诱导后10d拟胚体跳动率达65%,显著高于空白对照组和阴性对照组,一个分化拟胚体中一般出现1-3个跳动点,节律约为50-80times/min,在诱导10d时跳动的合胞体,免疫荧光表明cTnT和MLC-2v表达为阳性。结论胚胎干细胞经悬浮聚集培养5d后经10-7mol/L淫羊藿苷诱导,得到了可以跳动的心肌细胞团。     

BMP-4 increases activin A gene expression during osteogenic differentiation of mouse embryonic stem cells

Abstract

Activin A is a growth factor released by mature osteoblasts that has a critical effect on bone formation. We investigated the effect of bone morphogenetic protein (BMP)-4 on activin A gene expression during in vitro osteogenic differentiation of mouse embryonic stem (ES) cells. Embryoid bodies were cultured in retinoic acid (RA) for three days and then without RA for two days. Seeded cells received osteogenic medium with β-glycerophosphate, L-ascorbic acid 2-phosphate and dexamethasone during 19 days, with or without BMP-4. Six independent experiments were carried out. Real-time PCR was used to detect gene expression of activin A, Oct-4, Nanog, osteocalcin, RUNX2 and bone alkaline phosphatase. Immunofluorescence was used to co-localize activin A with the undifferentiation marker stage-specific embryonic antigen 1. Cells treated with BMP-4 had an increased gene expression of activin A, osteocalcin and bone alkaline phosphatase (p?<?0.05). In conclusion, BMP-4 increases activin A gene expression during mouse ES cell differentiation into bone precursors.     

小鼠pdx-1基因真核表达载体的构建及其在胚胎干细胞中的表达

目的： 克隆小鼠pdx-1基因，构建其真核表达载体，并在小鼠胚胎干细胞中表达，为糖尿病的细胞移植治疗奠定基础。方法： PCR扩增小鼠胰腺pdx-1基因 cDNA，酶切后和携带绿色荧光蛋白报告基因的真核表达载体pEGFP-N1重组，将pdx-1基因 cDNA片段连接到pEGFP-N1载体的多克隆位点，形成重组载体pEGFP/pdx-1，转化大肠杆菌DH5α菌株，构建成pdx-1基因真核表达载体质粒。扩增DH5α后抽提质粒DNA，Hind Ⅲ 和BamHⅠ酶切，电泳，DNA测序鉴定。鉴定正确的质粒DNA用脂质体包裹后转染小鼠胚胎干细胞MESPU13。结果： 从小鼠胰腺cDNA扩增出876 bp的DNA片段并成功重组到pEGFP-N1载体中。经酶切和DNA测序验证，插入载体的DNA片段为pdx-1基因，插入方向正确。重组质粒经脂质体转染胚胎干细胞MESPU13，24 h 后观察到绿色荧光蛋白报告基因和目的基因的pdx-1表达。结论： 小鼠pdx-1基因的克隆和真核表达载体构建获得成功，为进一步研究其功能奠定了基础。