A retrospective analysis of germline competence in rat embryonic stem cell lines

The factors responsible for conferring germline competence in embryonic stem (ES) cell lines remain unidentified. In the present study, rat ES cell lines (n = 17) were established with 3i medium (SU5402, PD0325901, CHIR99021), 2i medium (PD0325901, CHIR99021) or 2iF medium (PD0325901, CHIR99021, forskolin), and their potential for germline transmission to the G1 generation was examined. Rat strains were divided into an albino group (F344, Wistar or CAG/Venus transgenic rats with the Wistar background) or a colored coat group (Brown-Norway, Dark-Agouti, or BLK rats selected from >F3 generations of Wistar × Dark-Agouti rats based on their black coat color). Successful germline transmission was observed in 57 % (4/7), 40 % (2/5) and 100 % (5/5) of the ES cells established with 3i, 2i and 2iF media, respectively. ES cell lines from the homozygous CAG/Venus transgenic rats were established in all three media, but only the lines established with the 2iF medium were germline-competent. Neither coat-color (albino: 64 %, 7/11; colored: 67 %, 4/6) nor gender of the ES cell lines (XX: 67 %, 2/3; XY: 64 %, 9/14) were likely to affect germline transmission.     

A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing

Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.     

Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions

Embryonic stem cells and induced pluripotent stem (iPS) cells are usually maintained on feeder cells derived from mouse embryonic fibroblasts (MEFs). In recent years, the cell culture of iPS cells under serum- and feeder-free conditions is gaining attention in overcoming the biosafety issues for clinical applications. In this study, we report on the use of multiple small-molecular inhibitors (i.e., CHIR99021, PD0325901, and Thiazovivin) to efficiently cultivate mouse iPS cells without feeder cells in a chemically-defined and serum-free condition. In this condition, we showed that mouse iPS cells are expressing the Nanog, Oct3/4, and SSEA-1 pluripotent markers, indicating that the culture condition is optimized to maintain the pluripotent status of iPS cells. Without these small-molecular inhibitors, mouse iPS cells required the adaptation period to start the stable cell proliferation. The application of these inhibitors enabled us the shortcut culture method for the cellular adaptation. This study will be useful to efficiently establish mouse iPS cell lines without MEF-derived feeder cells.     

The Roles of Notch3 on the Cell Proliferation and Apoptosis Induced by CHIR99021 in NSCLC Cell Lines: A Functional Link between Wnt and Notch Signaling Pathways

Wnt and Notch signaling pathways both play essential roles and interact closely in development and carcinogenesis, but their interaction in non-small-cell lung cancer (NSCLC) is poorly unknown. Here we investigated the effects of CHIR99021, a Wnt signaling agonist, or Notch3-shRNA, or the combined application of CHIR99021 and Notch3-shRNA on cell proliferation and apoptosis, as well as the expressions of Notch3, its downstream genes, cyclinA and caspase-3. Our results showed that CHIR99021 up-regulated the expression of Notch3 protein and HES1 and HEYL mRNA. CHIR99021 promoted cell proliferation and the expression of cyclinA, which were inhibited by Notch3-shRNA in these three cell lines. Moreover, Notch3-shRNA significantly attenuated the positive effects of CHIR99021 on cell proliferation and cyclinA in H460 and H157. As for apoptosis, Notch3-shRNA induced cell apoptosis and increased the expression of caspase-3, whereas CHIR99021 showed the different effects in these three cell lines. The inhibitory effect of CHIR99021 on apoptosis was significantly weakened by Notch3-shRNA only in H460. Overall, although the effects of CHIR99021 and the combined application of CHIR99021 and Notch3-shRNA on the cell proliferation and apoptosis aren’t completely similar in the three cell lines, our findings still indicate that Notch3 signaling can be activated by canonical Wnt signaling and a functional link between Wnt and Notch signaling pathways exists in NSCLC, at least, which partially is associated with their regulations on the expressions of cyclinA and caspase-3.     

两种GSK-3β抑制剂对间充质干细胞神经分化的影响

目的:观察两种GSK-3β抑制剂(氯化锂和CHIR99021)对间充质干细胞向神经元样细胞分化的影响作用。方法:体外分离、扩增人胎盘间充质干细胞,分别用低浓度氯化锂和CHIR99021对细胞进行刺激,检测其对增值活力及Wnt通路中β连环蛋白调节作用的影响,并在加药条件下对细胞进行神经诱导。结果:在细胞活力相近的状况下,CHIR99021更能促进细胞分裂增殖和向神经元样细胞分化的能力,而免疫荧光结果显示,CHIR99021对Wnt通路的起效时间比氯化锂更为迟缓。结论:GSK-3β受抑制可促进间充质干细胞向神经细胞分化。在低浓度条件下,CHIR99021的抑制作用起效比氯化锂缓慢,但作用延续时间比氯化锂更长,因而对神经分化的长期促进作用也更加明显。     

A Modified EpiSC Culture Condition Containing a GSK3 Inhibitor Can Support Germline-Competent Pluripotency in Mice

Embryonic stem cells (ESCs) can contribute to the tissues of chimeric animals, including the germline. By contrast, epiblast stem cells (EpiSCs) barely contribute to chimeras. These two types of cells are established and maintained under different culture conditions. Here, we show that a modified EpiSC culture condition containing the GSK3 inhibitor CHIR99021 can support a germline-competent pluripotent state that is intermediate between ESCs and EpiSCs. When ESCs were cultured under a modified condition containing bFGF, Activin A, and CHIR99021, they converted to intermediate pluripotent stem cells (INTPSCs). These INTPSCs were able to form teratomas in vivo and contribute to chimeras by blastocyst injection. We also induced formation of INTPSCs (iINTPSCs) from mouse embryonic fibroblasts by exogenous expression of four reprogramming factors, Oct3/4, Sox2, Klf4, and c-Myc, under the INTPSC culture condition. These iINTPSCs contributed efficiently to chimeras, including the germline, by blastocyst injection. The INTPSCs exhibited several characteristic properties of both ESCs and EpiSCs. Our results suggest that the modified EpiSC culture condition can support growth of cells that meet the most stringent criteria for pluripotency, and that germline-competent pluripotency may depend on the activation state of Wnt signaling.     

Wnt signaling associated small molecules improve the viability of pPSCs in a PI3K/Akt pathway dependent way

Although we have obtained porcine pluripotent stem cell lines (pPSCs) from blastocysts, the cells exhibit flat clonal morphology and do not support single-cell passage. There is massive cell death after cell dissociation, and the efficiency of single-cell colony is generally ≤10%. In a recent study, we got a new pPSCs using two Wnt signaling pathway regulators CHIR99021 and XAV939. This cell had strong biological viability, small-domed morphology, and its cloning efficiency after dissociation was 80–90%. The CH/XAV-treated cells expressed elevated levels of pluripotent genes, and possessed differentiation abilities both in vitro and in vivo, proven by the formation of embryonic bodies and teratomas with three germ layers. Furthermore, we found that the combinative use of CHIR99021 and XAV939 resulted in β-catenin-maintained expression in the cytoplasm but not translocation to the nuclei for WNT/TCF activation. In the meanwhile, E-cadherin located on the cell membrane, thereby activated the PI3K/Akt signaling pathway to enhance the pluripotency of the cells. Our study obtained new pPSCs, which were even closer to the naïve state with only two small molecule inhibitors, and the improved pluripotency of pPSCs could facilitate transgenic manipulation and regenerative medicine research. Besides, our study casted a light on the understanding of pPSCs and the derivation of authentic porcine embryonic stem cells.     

CHIR-99021与FGF8协同诱导人表皮干细胞牙向分化

人表皮干细胞(human keratinocyte stem cells, hKSCs)可作为上皮源性的成体干细胞应用于牙齿再生,但是其诱导效率较低.本研究利用小分子化合物CHIR-99021提高hKSCs的Wnt/β-catenin信号活性,再与具有诱导成牙潜能的小鼠牙胚间充质重组,构建嵌合体,并移植裸鼠肾囊膜下培养20 d. 将嵌合体组织切片,并利用组织染色和免疫组化等方法鉴定牙齿结构. 结果显示,经FGF8诱导处理的hKSCs与小鼠牙胚间充质构成的嵌合体的成牙率为27.80%,其中成釉率仅为40.00%;经CHIR 99021诱导处理的hKSCs与小鼠牙胚间充质构成的嵌合体的成牙率仅为18.20%,其中成釉率高达100%;而CHIR 99021与FGF8协同作用,则进一步提高嵌合体成牙率至40.00%,其中成釉率也达75.00%. 进一步的研究发现,经CHIR-99021处理后,hKSCs的Wnt/β-catenin信号活性明显提高,同时FGF8的表达水平也显著上调. 以上结果表明,CHIR-99021可通过上调Wnt/β-catenin信号活性水平,同时促进FGF8表达,与FGF8协同,高效诱导hKSCs分化为具有分泌釉质功能的成釉质细胞. 研究结果对利用hKSCs作为上皮来源的成体细胞应用于人类牙齿再生的研究具有重要意义.     

大鼠心脏细胞条件培养基对小鼠ES细胞特性的维持

以C19-2和MESPU-13为供试细胞,用克隆测试、传代培养等方法对17种细胞的条件培养基进行了筛选,结果表明,大鼠心脏细胞的条件培养基（RH-CM）具有显著抑制小鼠ES细胞分化、维持其二倍体核型、促进ES细胞贴壁生长的作用。经RH-CM培养10代和20代的小鼠ES细胞在体内外分化能力上仍保留了原ES细胞的多方向分化潜能和特征;RH-CM也可作为小鼠ES细胞培养基的添加物,用含70%RH-CM的ES细胞培养基和小鼠胚胎原代成纤维细胞饲养层（PMEF）培养ES细胞,可长期有效地维持其未分化状态和二倍体核型。RT-PCR检测到大鼠心脏细胞有LIF mRNA表达。     

Mouse ES cells maintained in different pluripotency-promoting conditions differ in their neural differentiation propensity

Prior to differentiation, embryonic stem (ES) cells in culture are maintained in a so-called “undifferentiated” state, allowing derivation of multiple downstream cell lineages when induced in a directed manner, which in turn grants these cells their “pluripotent” state. The current work is based on a simple observation that the initial culture condition for maintaining mouse ES cells in an “undifferentiated” state does impact on the differentiation propensity of these cells, in this case to a neuronal fate. We point out the importance in judging the “pluripotency” of a given stem cell culture, as this clearly demonstrated that the “undifferentiated” state of these cells is not necessarily a “pluripotent” state, even for a widely used mouse ES cell line. We partly attribute this difference in the initial value of ES cells to the naïve-to-primed status of pluripotency, which in turn may affect early events of the differentiation in vitro.     

小鼠胚胎干细胞的培养

ＥＳ细胞的培养应满足促进细胞增殖和抑制细胞分化而保持其高度未分化潜能。我们用胚胎成纤维细胞作为饲养层,培养基ＤＭＥＭ中加白血病抑制因子的方法来培养ＥＳ细胞。培养的ＥＳ细胞呈集落状生长,细胞排列紧密,呈未分化状态。胚胎成纤维细胞作为饲养层是分离培养ＥＳ细胞最普遍使用而且有效的方法。本文还围绕ＥＳ细胞培养中的促进增殖和抑制分化这两个方面的影响因素进行了讨论。     

Chromosomal instability of mouse pluripotent cells cultured in vitro

The perspectives of using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSs) in clinics makes the karyological analysis of these cells an important issue. In the present study, using methods of classical and molecular cytogenetics of chromosome, we carried out a karyological study of two mouse ES and two iPS cell lines derived de novo. We obsererved the X chromosome monosomy in all studied ES and iPS cell lines, which makes the modal number of chromosomes in these cell lines equal to 39. The chromosomal instability (aneuploidy) was revealed in both studied iPS cell lines. Moreover, we have detected chromosomal rearrangements and chromosomal fragments in one of studied iPS. Our findings stress the importance of the careful cytogenetic evaluation of a pluripotent cell line, especially iPS cell lines, which should be carried out prior to any clinical use of these cells.     

Cardiac differentiation at an initial low density of human-induced pluripotent stem cells

A high density of human-induced pluripotent stem cells (hiPSCs) improves the efficiency of cardiac differentiation, suggesting the existence of indispensable cell-cell interaction signals. The complexity of interactions among cells at high density hinders the understanding of the roles of cell signals. In this study, we determined the minimum cell density that can initiate differentiation to facilitate cell-cell interaction studies. First, we co-induced cardiac differentiation in the presence of the glycogen synthase kinase-3β inhibitor CHIR99021 and activin A at various cell densities. At an initial low density, cells died within a few days in RPMI-based medium. We then investigated the culture conditions required to maintain cell viability. We used a basal medium excluding important components for the maintenance of hiPSC pluripotency, including activin A, basic fibroblast growth factor, and insulin. Supplementation of the basal medium with Rho-associated protein kinase inhibitor and insulin improved cell viability. Interestingly, addition of basic fibroblast growth factor enabled the expression of cardiac markers at the mRNA level but not the protein level. After further modification of the culture conditions, 10% of the cells expressed the cardiac troponin T protein, which is associated with cell contraction. The novel protocol for cardiac differentiation at an initial low cell density can also be used to evaluate high cell density conditions. The findings will facilitate the identification of cell signals required for cardiomyocyte formation.     

Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods

The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM), an embryonic germ layer that gives rise to kidneys. In order to achieve this goal, establishing an efficient, stable and low-cost method for differentiating IM cells using small molecules is required. In this study, we identified two retinoids, AM580 and TTNPB, as potent IM inducers by high-throughput chemical screening, and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator, CHIR99021, combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step, directly inducing IM cells by activating Wnt, retinoic acid (RA), and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy.     

Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.

Human embryonic stem (ES) cells are pluripotent cell lines that have been derived from the inner cell mass (ICM) of blastocyst stage embryos [1--3]. They are characterized by their ability to be propagated indefinitely in culture as undifferentiated cells with a normal karyotype and can be induced to differentiate in vitro into various cell types [1, 2, 4-- 6]. Thus, human ES cells promise to serve as an unlimited cell source for transplantation. However, these unique cell lines tend to spontaneously differentiate in culture and therefore are difficult to maintain. Furthermore, colonies may contain several cell types and may be composed of cells other than pluripotent cells [1, 2, 6]. In order to overcome these difficulties and establish lines of cells with an undifferentiated phenotype, we have introduced a reporter gene that is regulated by a promoter of an ES cell-enriched gene into the cells. For the introduction of DNA into human ES cells, we have established a specific transfection protocol that is different from the one used for murine ES cells. Human ES cells were transfected with enhanced green fluorescence protein (EGFP), under the control of murine Rex1 promoter. The transfected cells show high levels of GFP expression when in an undifferentiated state. As the cells differentiate, this expression is dramatically reduced in monolayer cultures as well as in the primitive endoderm of early stage (simple) embryoid bodies (EBs) and in mature EBs. The undifferentiated cells expressing GFP can be analyzed and sorted by using a Fluorescence Activated Cell Sorter (FACS). Thus, we have established lines of human ES cells in which only undifferentiated cells are fluorescent, and these cells can be followed and selected for in culture. We also propose that the pluripotent nature of the culture is made evident by the ability of the homogeneous cell population to form EBs. The ability to efficiently transfect human ES cells will provide the means to study and manipulate these cells for the purpose of basic and applied research.     

Natural and artificial routes to pluripotency

Pluripotent cells of the blastocyst inner cell mass (ICM) and their in vitro derivatives, embryonic stem (ES) cells, contain genomes in an epigenetic state that are poised for subsequent differentiation. Their chromatin is hyperdynamic in nature and relatively uncondensed. In addition, a large number of genes are expressed at low levels in both ICM and ES cells. Also, the chromatin of naturally pluripotent cells contains specialized histone modification patterns such as bivalent domains, which mark genes destined for later developmentally-regulated expression states. Female pluripotent cells contain X chromosomes that have yet to undergo the process of X chromosome inactivation. Collectively, these features of very early embyronic chromatin are required for the successful specification and production of differentiated cell lineages. Artificial reprogramming methods such as somatic nuclear transfer (SCNT), ES cell fusion-mediated reprogramming (FMR), and induced pluripotency (iPS) yield pluripotent cells that recapitulate many features of naturally pluripotent cells, including many of their epigenetic features. However, the route to pluripotent epigenomic states in artificial pluripotent cells differs drastically from that of their natural counterparts. Here, we compare and contrast the differing routes to pluripotency under natural and artificial conditions. In addition, we discuss the intrinsically metastable nature of the pluripotent epigenome and consider epigenetic aspects of reprogramming that may lead to incomplete or inaccurate reprogrammed states. Artificial methods of reprogramming hold immense promise for the development of autologous cell graft sources and for the development of cell culture models for human genetic disorders. However, the utility of artificially reprogrammed cells is highly dependent on the fidelity of the reprogramming process and it is therefore critically important to assess the epigenetic similarities between embryonic and induced pluripotent stem cells.     

Microbioreactor Arrays for Full Factorial Screening of Exogenous and Paracrine Factors in Human Embryonic Stem Cell Differentiation

Timed exposure of pluripotent stem cell cultures to exogenous molecules is widely used to drive differentiation towards desired cell lineages. However, screening differentiation conditions in conventional static cultures can become impractical in large parameter spaces, and is intrinsically limited by poor spatiotemporal control of the microenvironment that also makes it impossible to determine whether exogenous factors act directly or through paracrine-dependent mechanisms. We detail here the development of a continuous flow microbioreactor array platform that combines full-factorial multiplexing of input factors with progressive accumulation of paracrine factors through serially-connected culture chambers, and further, the use of this system to explore the combinatorial parameter space of both exogenous and paracrine factors involved in human embryonic stem cell (hESC) differentiation to a MIXL1-GFP⁺ primitive streak-like population. We show that well known inducers of primitive streak (BMP, Activin and Wnt signals) do not simply act directly on hESC to induce MIXL1 expression, but that this requires accumulation of surplus, endogenous factors; and, that conditioned medium or FGF-2 supplementation is able to offset this. Our approach further reveals the presence of a paracrine, negative feedback loop to the MIXL1-GFP⁺ population, which can be overcome with GSK-3β inhibitors (BIO or CHIR99021), implicating secreted Wnt inhibitory signals such as DKKs and sFRPs as candidate effectors. Importantly, modulating paracrine effects identified in microbioreactor arrays by supplementing FGF-2 and CHIR in conventional static culture vessels resulted in improved differentiation outcomes. We therefore demonstrate that this microbioreactor array platform uniquely enables the identification and decoding of complex soluble factor signalling hierarchies, and that this not only challenges prevailing strategies for extrinsic control of hESC differentiation, but also is translatable to conventional culture systems.     

Retinoic acid-treated pluripotent stem cells undergoing neurogenesis present increased aneuploidy and micronuclei formation

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.