Knockdown of IKK1/2 Promotes Differentiation of Mouse Embryonic Stem Cells into Neuroectoderm at the Expense of Mesoderm

Activation of nuclear factor kappa B (NF-??B) is accomplished by a specific kinase complex (IKK-complex), phosphorylating inhibitors of NF-??B (I??B). In embryonic stem cells (ESCs), NF-??B signaling causes loss of pluripotency and promotes differentiation towards a mesodermal phenotype. Here we show that NF-??B signaling is involved in cell fate determination during retinoic acid (RA) mediated differentiation of ESCs. Knockdown of IKK1 and IKK2 promotes differentiation of ESCs into neuroectoderm at the expense of neural crest derived myofibroblasts. Our data indicate that RA is not only able to induce neuronal differentiation in vitro but also drives ESCs into a neural crest cell lineage represented by differentiation towards peripheral neurons and myofibroblasts. The NC is a transiently existing, highly multipotent embryonic cell population generating a wide range of different cell types. During embryonic development the NC gives rise to distinct precursor lineages along the anterior-posterior axis determining differentiation towards specific derivates. Retinoic acid (RA) signaling provides essential instructive cues for patterning the neuroectoderm along the anterior-posterior axis. The demonstration of RA as a sufficient instructive signal for the differentiation of pluripotent cells towards NC and the involvement of NF-??B during this process provides useful information for the generation of specific NC-lineages, which are valuable for studying NC development or disease modeling.     

星形胶质细胞条件培养液促进胚胎干细胞分化为神经元样细胞的实验研究

目的:探讨星形胶质细胞条件培养液在体外能否促进胚胎干细胞（ESCs）向神经元方向分化。 方法:在Bain等建立的4-/4+方案基础上，分成两组：单用ATRA组和ATRA联合胎脑星形胶质细胞条件培养液组，分3阶段诱导ESCs定向生成神经元样细胞。形态学观察及畸胎瘤形成试验对ESCs的全能性进行鉴定；免疫组化染色法检测nestin、GFAP、NSE和NF-200的表达对诱导过程进行动态监测。 结果:（1）体外培养的小鼠ESC-D3细胞在胚胎成纤维饲养层细胞上连续传代培养，仍保持向3胚层分化的能力。（2）在诱导分化阶段，联合诱导组分化细胞NF-200和NSE的表达阳性率高于单用ATRA组。（3） 联合诱导组最终诱导ESCs生成纯度高达73.5%神经元样细胞。 结论:采用胚胎脑星形胶质细胞条件培养液可较高产率及纯度地促进ESCs生成神经元样细胞，值得推广应用。     

Defining a developmental path to neural fate by global expression profiling of mouse embryonic stem cells and adult neural stem/progenitor cells

To understand global features of gene expression changes during in vitro neural differentiation, we carried out the microarray analysis of embryonic stem cells (ESCs), embryonal carcinoma cells, and adult neural stem/progenitor (NS) cells. Expression profiling of ESCs during differentiation in monolayer culture revealed three distinct phases: undifferentiated ESCs, primitive ectoderm-like cells, and neural progenitor cells. Principal component (PC) analysis revealed that these cells were aligned on PC1 over the course of 6 days. This PC1 represents approximately 4,000 genes, the expression of which increased with neural commitment/differentiation. Furthermore, NS cells derived from adult brain and their differentiated cells were positioned along this PC axis further away from undifferentiated ESCs than embryonic stem-derived neural progenitors. We suggest that this PC1 defines a path to neural fate, providing a scale for the degree of commitment/differentiation.     

Influence of scaffold physical properties and stromal cell coculture on hematopoietic differentiation of mouse embryonic stem cells

Recent studies have suggested that three-dimensional (3D) biomaterial-based scaffolds and dynamic culture conditions could provide significant enhancement in the differentiation efficiency of embryonic stem cells (ESCs). Here we report that scaffold physical properties, like pore size, polymer concentration and compression modulus as well as specific culture conditions, e.g. cell seeding density and coculture with stromal cells can significantly influence hematopoietic differentiation of ESCs. PLLA scaffolds of various polymer concentrations (7.5%, 10% and 20% w/v) and pore size distributions (<150 μm, 150–425 μm, >425 μm) were fabricated using a standard solvent casting-salt leaching method. Mouse R1 ESCs were allowed to differentiate on these scaffolds either alone or in coculture with OP9 cells, a bone-marrow derived murine stromal cell line. Following one week of culture, cells were detached and analyzed using flow cytometry to evaluate the frequency of hematopoietic progenitor cells (HPC). Our results indicate that decreasing scaffold pore size increases hematopoietic differentiation of ESCs. In addition, increasing polymer concentration which resulted in increased scaffold compression modulus also provided significantly enhanced hematopoiesis. Furthermore, higher cell seeding densities as well as coculture with marrow-derived stromal cells increased HPC generation. Collectively, these results indicate that physical and mechanical properties of the 3D microenvironment as well as cell–cell and cell–stromal interactions might play a significant role in ESC differentiation and therefore warrants further investigation to elucidate the molecular mechanisms.     

Neural stem cells directly differentiated from partially reprogrammed fibroblasts rapidly acquire gliogenic competency

Neural stem cells (NSCs) were directly induced from mouse fibroblasts using four reprogramming factors (Oct4, Sox2, Klf4, and cMyc) without the clonal isolation of induced pluripotent stem cells (iPSCs). These NSCs gave rise to both neurons and glial cells even at early passages, while early NSCs derived from clonal embryonic stem cells (ESCs)/iPSCs differentiated mainly into neurons. Epidermal growth factor-dependent neurosphere cultivation efficiently propagated these gliogenic NSCs and eliminated residual pluripotent cells that could form teratomas in vivo. We concluded that these directly induced NSCs were derived from partially reprogrammed cells, because dissociated ESCs/iPSCs did not form neurospheres in this culture condition. These NSCs differentiated into both neurons and glial cells in vivo after being transplanted intracranially into mouse striatum. NSCs could also be directly induced from adult human fibroblasts. The direct differentiation of partially reprogrammed cells may be useful for rapidly preparing NSCs with a strongly reduced propensity for tumorigenesis.     

Fluorescence-activated cell sorting-based purification of embryonic stem cell-derived neural precursors averts tumor formation after transplantation

The differentiation of dopaminergic (DA) neurons from mouse embryonic stem cells (ESCs) can be efficiently induced, making these neurons a potential source for transplantation as a treatment for Parkinson's disease, a condition characterized by the gradual loss of midbrain DA neurons. One of the major persistent obstacles to the successful implementation of therapeutic ESC transplantation is the propensity of ESC-derived grafts to form tumors in vivo. To address this problem, we used fluorescence-activated cell sorting to purify mouse ESC-derived neural precursors expressing the neural precursor marker Sox1. ESC-derived, Sox1+ cells began to express neuronal cell markers and differentiated into DA neurons upon transplantation into mouse brains but did not generate tumors in this site. In contrast, Sox1- cells that expressed ESC markers frequently formed tumors in vivo. These results indicate that Sox1-based cell sorting of neural precursors prevents graft-derived tumor formation after transplantation, providing a promising strategy for cell transplantation therapy of neurodegenerative disorders.     

大鼠胚胎神经干细胞与永生化神经干细胞系C17.2定向分化为神经元潜能的差异

目的 对比大鼠早期胚胎神经干细胞( NSCs)与永生化神经干细胞系C17.2(C17.2-NSC)定向分化为神经元潜能的差异,确立适用于诱导NSCs定向分化为神经元高内涵药物筛选的NSCs筛选模型. 方法 C17.2-NSC、17d胚胎海马NSCs(E17-NSC)及11d胚胎大脑皮层NSCs(E11-NSC)均设定对照组和实验组,对照组与实验组的细胞在含有2％ (v/v) B27的DMEM/F12培养液中,分别经0μmol/L和1μmol/L维甲酸(RA)在37℃、5％CO2常规培养条件下诱导分化5d,通过免疫组织化学技术检测对照组与实验组中NSCs或前体细胞特异性标志蛋白巢蛋白(Nestin)及神经元特异性标志蛋白βⅢ微管蛋白(Tuj1)表达量的差异. 结果 与对照组相比,C17.2-NSC经1μμmol/L RA诱导后未定向分化为神经元,而E17-NSC及E11-NSC经1μmol/L RA诱导后可有效定向分化为神经元. 结论 相对于C17.2-NSC,大鼠早期胚胎NSCs定向分化为神经元的潜能更强,适用于诱导NSCs定向分化为神经元的高内涵药物筛选.     

Cell extract-derived differentiation of embryonic stem cells

Various means have been used to encourage the differentiation of embryonic stem cells (ESCs) toward specific lineages, including growth factor administration, genetic modification, and coculture with relevant cells/tissues. Cell extract-based reprogramming has recently been used to derive mature cells from nonrelated phenotypes. In this communication, we tested whether this in vitro reprogramming approach can be used to direct ESC differentiation. Permeabilized murine ESCs exposed to extracts of murine type II pneumocytes showed increased expression of surfactant protein C and its corresponding mRNA, reflecting enhanced differentiation of pneumocytes. Subsequent differentiation to a type I phenotype was demonstrated by expression of aquaporin 5. Pneumocyte formation occurred quicker than with growth factor-induced differentiation. Our findings establish that ESCs can be differentiated in vitro using cellular extracts. This model provides a tool for analysis of the key factors involved in the differentiation of ESCs to type II pneumocytes.     

Retinoic acid induced the differentiation of neural stem cells from embryonic spinal cord into functional neurons in vitro

Retinoic acid is an important molecular taking part in the development and homeostasis of nervous system. Neural stem cells (NSCs) are pluripotent cells that can differentiate into three main neural cells including neuron, astrocyte and oligodendrocyte. However, whether retinoic acid can induce NSCs derived from embryonic spinal cord differentiating into functional neurons and its efficiency are not clear. In this experiment, NSCs were isolated from embryonic 14 d spinal cord of rats. The growth and neuronal differentiation of NSCs induced by 500 nM RA was examined in vitro. It was indicated that compared with the control group, there were more differentiated cells with longer cytodendrites in the medium treated with RA at different time. And more, there were more neuronal marker positive cells in 500 nM RA group than the control group seven days after differentiation. At the same time, the expression of β-tublin III protein in RA group was higher than those in control group, which was contrary to the expression of astrocyte marker GFAP protein at seven days after differentiation. However the differentiated neurons, whether treated with RA or not both exhibited biological electrical reactivity after stimulated by glutamine. Therefore, these findings indicated that RA could promote growth of cellular dendrites and neuronal differentiation of NSCs, which also induce functional maturation of differentiated neurons finally.     

序贯培养法诱导小鼠胚胎干细胞向神经细胞分化的研究

目的诱导小鼠胚胎干细胞向神经细胞分化。方法通过"序贯培养法"诱导小鼠胚胎干细胞分化为神经细胞,并应用RT-PCR、免疫荧光、端粒酶活性及基因芯片等对诱导结果进行检测。结果分化细胞表达神经元特异性烯醇化酶(NSE)、低分子量神经微丝蛋白(NF-L)、神经细胞粘附分子1(NCAM1)、微管相关蛋白Tau等多种神经元标志物,免疫荧光显示NSE阳性率为(81±9.2)%,而神经胶质细胞原纤维酸性蛋白(GFAP)几乎不表达。基因芯片分析结果显示,在3张芯片中有8179个基因共表达,另有998个基因的表达完全不同,且有多种神经元基因的标志物表达明显升高。结论通过"序贯培养法"能有效地诱导小鼠胚胎干细胞向神经元分化。     

Transplantable neural progenitor populations derived from rhesus monkey embryonic stem cells

Cell-based therapies using embryonic stem cells (ESCs) in the treatment of neural disease will require the generation of homogenous donor neural progenitor (NP) populations. Here we describe an efficient culture system containing hepatocyte growth factor (HGF) and G5 supplement for the production of highly enriched (88.3%+/-8.1%) populations of NPs from rhesus monkey ESCs. Additional purification resulted in NP preparations that were 98% nestin positive. Moreover, NPs, as monolayers or neurospheres, could be maintained for prolonged periods of time in media containing HGF+G5 or G5 alone. In vitro differentiation and in vivo transplantation assays showed that NPs could differentiate into neurons, astrocytes, and oligodendrocytes. The kinds and quantities of differentiated cells derived from NPs were closely correlated with their niches in vivo. Glial differentiation was predominant in periventricular areas, whereas cells migrating into the cortex were mostly neurons. Cell counts showed that 2 months after transplantation, approximately 25% of transplanted NPs survived and 65%-80% of the surviving transplanted cells migrated along the ventricular wall or in a radial fashion. Subcloning demonstrated that several clonal lines derived from NPs expressed nestin and differentiated into three neural lineages in vitro and in rat brains in vivo. In contrast, some subcloned lines showed restricted differentiation both in vitro and in vivo in rat brains. These observations set the stage for obtaining highly enriched NPs and evaluating the efficacy of NP-based transplantation therapy in the nonhuman primate and will provide a platform for probing the molecular mechanisms that control neural induction.     

p38alpha MAP kinase-deficient mouse embryonic stem cells can differentiate to endothelial cells, smooth muscle cells, and neurons.

p38 MAP kinase alpha (p38alpha) regulates various cellular processes in adult cells, but little is known about its function in stem cells. We investigated the potential of wild type and p38alpha deficient mouse embryonic stem cells (ESCs) to differentiate into endothelial cells (ECs), smooth muscle cells (SMCs), and neurons. Our differentiation methods allowed simultaneous development of all these cell types. ECs formed monolayers similar to mature ECs and could assemble into vessel-like structures. SMCs had well-organized actin filaments with morphology similar to adult SMCs. Neurons exhibited well-developed cell bodies and elongated axons. Deletion of the p38alpha gene did not significantly compromise ESC differentiation since p38alpha-/- cells could express cell-specific markers and displayed similar overall morphology to the cells differentiated from p38alpha+/+ ESCs. Although p38alpha regulates various cellular activities of adult SMCs, ECs, and neurons, our data demonstrate that p38alpha is not essential for ESC differentiation to these cell types.     

A focused microarray to assess dopaminergic and glial cell differentiation from fetal tissue or embryonic stem cells

We designed oligonucleotide gene-specific probes to develop a focused array that can be used to discriminate between neural phenotypes, identify biomarkers, and provide an overview of the process of dopaminergic neuron and glial differentiation. We have arrayed approximately 100 genes expressed in dopaminergic neurons, oligodendrocytes, and astrocytes, an additional 200 known cytokines, chemokines, and their respective receptors, as well as markers for pluripotent and progenitor cells. The gene-specific 60-mer 3' biased oligonucleotides for these 281 genes were arrayed in a 25 x 12 format based on function. Using human adult brain substantia nigra, human embryonic stem cells (ESCs), and the differentiated progeny of pluripotent cells, we showed that this array was capable of distinguishing dopaminergic neurons, glial cells, and pluripotent cells by their gene expression profiles in a concentration-dependent manner. Using linear correlation coefficients of input RNA with output intensity, we identified a list of genes that can serve as reporting genes for detecting dopaminergic neurons, glial cells, and contaminating ESCs and progenitors. Finally, we monitored NTera2 differentiation toward dopaminergic neurons and have shown the ability of this array to distinguish stages of differentiation and provide important clues to factors regulating differentiation, the degree of contaminating populations, and stage of cell maturity. We suggest that this focused array will serve as a useful complement to other large-scale arrays in routine assessment of cell properties prior to their therapeutic use.     

Tenascin-R promotes neuronal differentiation of embryonic stem cells and recruitment of host-derived neural precursor cells after excitotoxic lesion of the mouse striatum

Loss of GABAergic projection neurons under excitotoxic conditions in the striatum is associated with a disturbance of motor and cognitive functions as seen, for instance, in Huntington's disease. Since current treatments cannot replace degenerated neurons, research on alternative therapeutic approaches needs to be pursued. In this context, the transplantation of genetically modified stem cells into lesioned brain areas of patients is a possible alternative. In this study, green fluorescent protein-labeled murine embryonic stem cells (ESCs) were stably transfected to overexpress the extracellular matrix molecule tenascin-R (TNR), which is expressed by striatal GABAergic neurons. TNR-overexpressing ESCs were analyzed in comparison with their parental cells regarding neural differentiation and migration in vitro, and after transplantation into the striatum of quinolinic acid-treated mice, which serve as a model for Huntington's disease. In comparison with sham-transfected control cells, TNR-overexpressing ESCs showed enhanced differentiation into neurons in vitro, reduced migration in vitro and in vivo, and increased generation of GABAergic neurons and decreased numbers of astrocytes 1 month and 2 months after transplantation, but without significant effects on locomotor functions. Interestingly, TNR-overexpressing ESCs transplanted into the striatum attracted host-derived neuroblasts from the rostral migratory stream and promoted stem cell-mediated recruitment of host-derived newborn neurons within the grafted area. Thus, we show for the first time that overexpression of an extracellular matrix molecule by in vitro predifferentiated ESCs exerts beneficial effects on tissue regeneration in a mouse model of neurodegenerative disease. Disclosure of potential conflicts of interest is found at the end of this article.