Gadd45g诱导小鼠胚胎干细胞向中内胚层分化

为探究生长阻滞和DNA损伤诱导蛋白45γ(Growth arrest and DNA-damage-inducible protein GADD45 gamma,Gadd45g)对小鼠胚胎干细胞(mouse embryonic stem cells,m ESCs)在体外培养条件下自我更新状态的影响,通过设计并构建含有Gadd45g基因的重组质粒,将其导入m ESCs内,过表达目标基因;在含有白血病抑制因子(LIF)的血清培养条件下,通过细胞计数、碱性磷酸酶染色、qRT-PCR以及免疫荧光等实验手段检测m ESCs的生长情况。结果显示,与对照组相比,过表达Gadd45g基因后,m ESCs的生长速度减缓,碱性磷酸酶活性降低,且中内胚层标志基因的表达水平显著上升。进一步研究发现,在添加LIF的有血清或2i无血清培养体系中,过表达Gadd45g均可以降低细胞内STAT3蛋白的磷酸化水平,由此推断上调Gadd45g的表达会抑制STAT3的活性,从而推动m ESCs向中内胚层分化。研究结果扩大了目前人们对于ESCs分化机制的理解,有利于胚胎干细胞未来的基础研究与安全应用。     

BMP Induces Cochlin Expression to Facilitate Self-renewal and Suppress Neural Differentiation of Mouse Embryonic Stem Cells

BMP4 maintains self-renewal of mouse embryonic stem cells (ESCs) in collaboration with LIF. Here, we report the identification of a novel key BMP target gene, cochlin (Coch) in mouse ESCs. Coch can be significantly up-regulated by BMP4 specifically in ESCs but not in somatic differentiated cells, and this up-regulation is dependent on the BMP signaling mediators Smad1/5 and Smad4. Overexpression of Coch can partially substitute BMP4 to promote self-renewal of mouse ESCs together with LIF, whereas knockdown of Coch impairs self-renewal marker gene expression even in the presence of both BMP4 and LIF. Further studies showed that COCH could mimic BMP4 in repressing neural differentiation of mouse ESCs upon LIF withdrawal and the inhibitory effect of BMP4 on neural differentiation is compromised by Coch knockdown. Taken together, our data suggest that COCH is a part of the downstream target network of BMP signaling and serves as another important effector to fine-tune mouse ESC fates.     

Figla基因过表达促进小鼠胚胎干细胞向雌性生殖细胞分化

生殖系a因子(Figla）是最早表达的生殖细胞特异性转录因子之一,对卵泡的发育、Zp基因的表达和透明带的形成具有调节作用. Figla基因异常会引起卵巢早衰的发生. 本研究通过 PCR自小鼠基因组中扩增出Figla基因,将其克隆到真核报告载体pDsRed1 N1,构建了携带609 bp 的Figla重组载体pDsRed1 N1 Figla. 用该载体转染小鼠胚胎干细胞（mESCs）系J1、小鼠成纤维细胞系NIH 3T3、小鼠畸胎瘤细胞P19和小鼠精原细胞系GC1,在荧光显微镜下观察红色荧光蛋白(RFP)在细胞中的表达,同时检测转染细胞中Figla基因及其它生殖细胞特异性基因的表达. 结果显示,转染2 d,mESCs内Figla总表达量明显增加,且内源性表达量亦有所提高,即转入的外源性Figla基因可以促进内源性Figla的启动和表达. 免疫荧光染色显示,表达RFP 的细胞同时表达生殖特异性基因Vasa,减数分裂特异性基因Stra8、Scp3及卵母细胞标志基因Zp3. 通过QRT PCR检测发现,在转染3 d的细胞中,Vasa、Scp3和Zp1的表达较对照组均有明显上调,而Oct4和Stra8的表达量下降. 研究表明,Figla基因对生殖特异性基因的表达具有调控作用,可以激活雌性生殖基因表达,为更清楚地了解Figla基因在生殖细胞生长发育过程中的调控机制,以及发现该基因在生殖细胞中的新功能奠定了基础.     

Characterization of TLX Expression in Neural Stem Cells and Progenitor Cells in Adult Brains

TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression.Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells.     

Analysis of Epigenetic Factors in Mouse Embryonic Neural Stem Cells Exposed to Hyperglycemia

Background

Maternal diabetes alters gene expression leading to neural tube defects (NTDs) in the developing brain. The mechanistic pathways that deregulate the gene expression remain unknown. It is hypothesized that exposure of neural stem cells (NSCs) to high glucose/hyperglycemia results in activation of epigenetic mechanisms which alter gene expression and cell fate during brain development.

Methods and Findings

NSCs were isolated from normal pregnancy and streptozotocin induced-diabetic pregnancy and cultured in physiological glucose. In order to examine hyperglycemia induced epigenetic changes in NSCs, chromatin reorganization, global histone status at lysine 9 residue of histone H3 (acetylation and trimethylation) and global DNA methylation were examined and found to be altered by hyperglycemia. In NSCs, hyperglycemia increased the expression of Dcx (Doublecortin) and Pafah1b1 (Platelet activating factor acetyl hydrolase, isoform 1b, subunit 1) proteins concomitant with decreased expression of four microRNAs (mmu-miR-200a, mmu-miR-200b, mmu-miR-466a-3p and mmu-miR-466 d-3p) predicted to target these genes. Knockdown of specific microRNAs in NSCs resulted in increased expression of Dcx and Pafah1b1 proteins confirming target prediction and altered NSC fate by increasing the expression of neuronal and glial lineage markers.

Conclusion/Interpretation

This study revealed that hyperglycemia alters the epigenetic mechanisms in NSCs, resulting in altered expression of some development control genes which may form the basis for the NTDs. Since epigenetic changes are reversible, they may be valuable therapeutic targets in order to improve fetal outcomes in diabetic pregnancy.     

Noggin Over-Expressing Mouse Embryonic Fibroblasts and MS5 Stromal Cells Enhance Directed Differentiation of Dopaminergic Neurons from Human Embryonic Stem Cells

Directed methods for differentiating human embryonic stem cells (hESCs) into dopaminergic (DA) precursor cells using stromal cells co-culture systems are already well established. However, not all of the hESCs differentiate into DA precursors using these methods. HSF6, H1, H7, and H9 cells differentiate well into DA precursors, but CHA13 and CHA15 cells hardly differentiate. To overcome this problem, we modified the differentiation system to include a co-culturing step that exposes the cells to noggin early in the differentiation process. This was done using γ-irradiated noggin-overexpressing CF1-mouse embryonic fibroblasts (MEF-noggin) and MS5 stromal cells (MS5-noggin and MS5-sonic hedgehog). After directed differentiation, RT-PCR analyses revealed that engrailed-1 (En-1), Lmx1b, and Nurr1, which are midbrain DA markers, were expressed regardless of differentiation stage. Moreover, tyrosine hydroxylase (Th) and an A9 midbrain-specific DA marker (Girk2) were expressed during differentiation, whereas levels of Oct3/4, an undifferentiated marker, decreased. Immunocytochemical analyses revealed that protein levels of the neuronal markers TH and TuJ1 increased during the final differentiation stage. These results demonstrate that early noggin exposure may play a specific role in the directed differentiation of DA cells from human embryonic stem cells.     

Self-Contained Induction of Neurons from Human Embryonic Stem Cells

Background

Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES) cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM). However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins.

Methodology/Principal Findings

We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES) cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM) could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation.

Conclusion/Significance

By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.     

The Tight-Junction Protein Claudin-6 Induces Epithelial Differentiation from Mouse F9 and Embryonic Stem Cells

During epithelialization, cell adhesions and polarity must be established to maintain tissue assemblies and separate the biological compartments in the body. However, the molecular basis of epithelial morphogenesis, in particular, a role of cell adhesion molecules in epithelial differentiation from stem cells, remains unclear. Here, we show that the stable and conditional expression of a tight-junction protein, claudin-6 (Cldn6), triggers epithelial morphogenesis in mouse F9 stem cells. We also demonstrate that Cldn6 induces the expression of other tight-junction and microvillus molecules including Cldn7, occludin, ZO-1α+, and ezrin/radixin/moesin-binding phosphoprotein50. These events were inhibited by attenuation of Cldn6 using RNA interference or the C-terminal half of Clostridium Perfringens enterotoxin. Furthermore, similar results were obtained in mouse embryonic stem cells. Thus, we have uncovered that the Cldn6 functions as a novel cue to induce epithelial differentiation.     

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.     

In Vitro Differentiation of Mouse Embryonic Stem Cells into Neurons of the Dorsal Forebrain

Pluripotent embryonic stem cells (ESCs) are able to differentiate into all cell types in the organism including cortical neurons. To follow the dynamic generation of progenitors of the dorsal forebrain in vitro, we generated ESCs from D6-GFP mice in which GFP marks neocortical progenitors and neurons after embryonic day (E) 10.5. We used several cell culture protocols for differentiation of ESCs into progenitors and neurons of the dorsal forebrain. In cell culture, GFP-positive cells were induced under differentiation conditions in quickly formed embryoid bodies (qEBs) after 10–12 day incubation. Activation of Wnt signaling during ESC differentiation further stimulated generation of D6-GFP-positive cortical cells. In contrast, differentiation protocols using normal embryoid bodies (nEBs) yielded only a few D6-GFP-positive cells. Gene expression analysis revealed that multiple components of the canonical Wnt signaling pathway were expressed during the development of embryoid bodies. As shown by immunohistochemistry and quantitative qRT-PCR, D6-GFP-positive cells from qEBs expressed genes that are characteristic for the dorsal forebrain such as Pax6, Dach1, Tbr1, Tbr2, or Sox5. qEBs culture allowed the formation of a D6-GFP positive pseudo-polarized neuroepithelium with the characteristic presence of N-cadherin at the apical pole resembling the structure of the developing neocortex.     

pINC－lacZ逆转录病毒载体的构建及其在小鼠胚胎干细胞中的表达

小鼠胚胎干细胞系（ＥＳ）是从囊胚的内细胞团中建立起来的多潜能胚胎干细胞系。ＥＳ细胞系目前正广泛用于将基因打靶后的突变和其它遗传变化导入小鼠的种系中。其中,嵌合鼠的获得是非常必要的一步。这就需要用一个可以广泛表达的基因对ＥＳ细胞进行标记。大肠杆菌β－半乳糖苷酶（β－ｇａｌ）基因的表达在细胞水平就能很容易地观察到,而且对哺乳动物细胞没有任何毒害作用,因而是一个被广泛地用于各种细胞基因表达研究中的报导基因。猿类巨细胞病毒早期（ＳｉＣＭＶＩＥ）启动子是一个广泛用于转基因小鼠研究中的强启动子。然而,该启动子在ＥＳ细胞方面应用的报道尚未见到。本研究用ＢａｍＨＩ和ＨｉｎｄⅢ双酶切,从ｐｓｖ－β－ｇａｌａｃｔｏｓｉｄａｓｅ中得到３．７Ｋｂ的ｌａｃＺ基因片断,将其插入到ｐＩＮＣ载体上（Ｆｉｇ．１）,得到ｐＩＮＣ－ｌａｃＺ（Ｆｉｇ．２）。用ＮｏｔⅠ线性化ｐＩＮＣ－ｌａｃＺ后,电击导入ＭＥＳＰＵ－１３细胞中。ＭＥＳＰＵ－１３为本实验室从１２９／Ｔｅｒ品系小鼠的囊胚中建立的一个ＥＳ细胞系。转化细胞在含有２５０μｇ／ｍｌＧ４１８的培养基上培养两周,进行ＭＥＳＰＵ－１３细胞稳定转化子的筛选。在一次转化实验中,从１ｘ１０７个转化的ＭＥＳ     

CREG基因敲除小鼠胚胎干细胞的筛选及鉴定

目的:为阐明E1A激活基因阻遏子(Cellular repressor of E1A-stimulated genes,CREG)在发育过程中的作用,本研究拟通过高浓度药物筛选获得基因敲除的小鼠胚胎干细胞(Embryonic stem cell,ESC)。方法:用0.5 mg/ml、1.0 mg/ml、1.5 mg/ml、2.0mg/ml、2.5 mg/ml及3.0 mg/ml 6个浓度的G418培养CREG杂合型(CREG其中一个等位基因被新霉素抗性neo基因替代)小鼠ESC 2周,确定最佳的G418筛选浓度。挑取该浓度下存活的ESC克隆进行扩增。将每个ESC克隆一半冻存,另一半贴壁培养。待ESC生长至80%融合后分别提取基因组DNA和蛋白。PCR方法扩增CREG基因明确基因组中是否存在CREG基因,WesternBlot方法鉴定是否有CREG蛋白表达。结果:确定2.0 mg/ml G418为最佳的筛选浓度。在该浓度下,共获得存活的克隆10个,PCR证实C2及C7克隆基因组中没有CREG基因,Western Blot证实C2及C7无CREG蛋白表达。结论:成功获得CREG基因敲除的小鼠ESC 2株,为深入研究CREG功能奠定了基础。     

敲除Pofut1不影响胚胎干细胞向神经元分化

蛋白O-连接岩藻糖基转移酶1 (Pofut1)基因缺失可导致Notch分子无法与配体结合并启动信号传递. 为研究Pofut1基因对哺乳动物胚胎干细胞（ESC）向神经分化的影响,利用Pofut1基因敲除的胚胎干细胞与野生型胚胎干细胞,经体外培养诱导拟胚体（EB）分化为神经细胞,计数分化为神经细胞的比例,采用细胞免疫组化染色和real-time PCR等方法,分析神经细胞特异性标志分子的表达. 结果显示,Pofut1基因缺失后,对EBC生长没有明显影响,分化过程中形成的拟胚体数量明显增多,分化的神经样细胞以及神经标志物分子的表达也明显多于对照组;Notch信号缺失对小鼠胚胎干细胞生长无明显影响,但可以促进ES细胞向神经细胞分化.     

Pancreatic Insulin-Producing Cells Differentiated from Human Embryonic Stem Cells Correct Hyperglycemia in SCID/NOD Mice,an Animal Model of Diabetes

Background

Human pancreatic islet transplantation is a prospective curative treatment for diabetes. However, the lack of donor pancreases greatly limits this approach. One approach to overcome the limited supply of donor pancreases is to generate functional islets from human embryonic stem cells (hESCs), a cell line with unlimited proliferative capacity, through rapid directed differentiation. This study investigated whether pancreatic insulin-producing cells (IPCs) differentiated from hESCs could correct hyperglycemia in severe combined immunodeficient (SCID)/non-obese diabetic (NOD) mice, an animal model of diabetes.

Methods

We generated pancreatic IPCs from two hESC lines, YT1 and YT2, using an optimized four-stage differentiation protocol in a chemically defined culture system. Then, about 5–7×10⁶ differentiated cells were transplanted into the epididymal fat pad of SCID/NOD mice (n = 20). The control group were transplanted with undifferentiated hESCs (n = 6). Graft survival and function were assessed using immunohistochemistry, and measuring serum human C-peptide and blood glucose levels.

Results

The pancreatic IPCs were generated by the four-stage differentiation protocol using hESCs. About 17.1% of differentiated cells expressed insulin, as determined by flow cytometry. These cells secreted insulin/C-peptide following glucose stimulation, similarly to adult human islets. Most of these IPCs co-expressed mature β cell-specific markers, including human C-peptide, GLUT2, PDX1, insulin, and glucagon. After implantation into the epididymal fat pad of SCID/NOD mice, the hESC-derived pancreatic IPCs corrected hyperglycemia for ≥8 weeks. None of the animals transplanted with pancreatic IPCs developed tumors during the time. The mean survival of recipients was increased by implanted IPCs as compared to implanted undifferentiated hESCs (P<0.0001).

Conclusions

The results of this study confirmed that human terminally differentiated pancreatic IPCs derived from hESCs can correct hyperglycemia in SCID/NOD mice for ≥8 weeks.