Germline-competent Chimeras Research Articles

Oncostatin M Maintains Naïve Pluripotency of mESCs by Tetraploid Embryo Complementation (TEC) Assay.

It has been well established that leukemia inhibitory factor (LIF) is essential for maintaining naïve pluripotency of embryonic stem cells (ESCs). Oncostatin M (OSM) is a member of the IL-6 family of cytokines which share gp130 as a receptor subunit, and the OSM-gp130 complex can recruit either LIF receptor β or OSM receptor β. Here we show that OSM can completely replace LIF to maintain naïve pluripotency of ESCs. Mouse ESCs (mESCs) cultured in the presence of LIF or OSM not only express pluripotency genes at similar levels but also exhibit the same developmental pluripotency as evidenced by the generation of germline competent chimeras, supporting previous findings. Moreover, we demonstrate by tetraploid embryo complementation assay, the most stringent functional test of authentic pluripotency that mESCs cultured in OSM produce viable all-ESC pups. Furthermore, telomere length and telomerase activity, which are also crucial for unlimited self-renewal and genomic stability of mESCs, do not differ in mESCs cultured under OSM or LIF. The transcriptome of mESCs cultured in OSM overall is very similar to that of LIF, and OSM activates Stat3 signaling pathway, like LIF. Additionally, OSM upregulates pentose and glucuronate interconversion, ascorbate and aldarate metabolism, and steroid and retinol metabolic pathways. Although the significance of these pathways remains to be determined, our data shows that OSM can maintain naïve pluripotent stem cells in the absence of LIF.

Pluripotent Stem Cells for Transgenesis in the Rabbit: A Utopia?

Pluripotent stem cells (PSCs) possess the following two main properties: self-renewal and pluripotency. Self-renewal is defined as the ability to proliferate in an undifferentiated state and pluripotency as the capacity to differentiate into cells of the three germ layers, i.e., ectoderm, mesoderm, and endoderm. PSCs are derived from early embryos as embryonic stem cells (ESCs) or are produced by reprogramming somatic cells into induced pluripotent stem cells (iPSCs). In mice, PSCs can be stabilized into two states of pluripotency, namely naive and primed. Naive and primed PSCs notably differ by their ability to colonize a host blastocyst to produce germline-competent chimeras; hence, naive PSCs are valuable for transgenesis, whereas primed PSCs are not. Thanks to its physiological and developmental peculiarities similar to those of primates, the rabbit is an interesting animal model for studying human diseases and early embryonic development. Both ESCs and iPSCs have been described in rabbits. They self-renew in the primed state of pluripotency and, therefore, cannot be used for transgenesis. This review presents the available data on the pluripotent state and the chimeric ability of these rabbit PSCs. It also examines the potential barriers that compromise their intended use as producers of germline-competent chimeras and proposes possible alternatives to exploit them for transgenesis.

MLL1 Inhibition Reprograms Epiblast Stem Cells to Naive Pluripotency

The interconversion between naive and primed pluripotent states is accompanied by drastic epigenetic rearrangements. However, it is unclear whether intrinsic epigenetic events can drive reprogramming to naive pluripotency or if distinct chromatin states are instead simply a reflection of discrete pluripotent states. Here, we show that blocking histone H3K4 methyltransferase MLL1 activity with the small-molecule inhibitor MM-401 reprograms mouse epiblast stem cells (EpiSCs) to naive pluripotency. This reversion is highly efficient and synchronized, with more than 50% of treated EpiSCs exhibiting features of naive embryonic stem cells (ESCs) within 3 days. Reverted ESCs reactivate the silenced X chromosome and contribute to embryos following blastocyst injection, generating germline-competent chimeras. Importantly, blocking MLL1 leads to global redistribution of H3K4me1 at enhancers and represses lineage determinant factors and EpiSC markers, which indirectly regulate ESC transcription circuitry. These findings show that discrete perturbation of H3K4 methylation is sufficient to drive reprogramming to naive pluripotency.

Mouse embryos' fusion for the tetraploid complementation assay.

Production of the germline-competent chimeras using genetically modified ES cell lines is an essential step in the establishment of novel mouse models. In addition chimeras provide a powerful tool to study the cell lineage and to analyze complex phenotypes of mutant mice. Mouse chimeras with tetraploid embryos are used to rescue extraembryonic defects, to analyze an impact of gene function on specific lineage, to study the interaction between embryonic and extraembryonic tissues, and to produce mutant embryos and mice for the phenotype analysis. Tetraploid embryos are generated by the fusion of two blastomeres of the mouse embryo. The applications of tetraploid complementation assay and the protocol are described below.

Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system

One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo.

Rapid target gene validation in complex cancer mouse models using re‐derived embryonic stem cells

Human cancers modeled in Genetically Engineered Mouse Models (GEMMs) can provide important mechanistic insights into the molecular basis of tumor development and enable testing of new intervention strategies. The inherent complexity of these models, with often multiple modified tumor suppressor genes and oncogenes, has hampered their use as preclinical models for validating cancer genes and drug targets. In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle, as exemplified for three GEMMs; two lung cancer models and one mesothelioma model. Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs, (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls, the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation. As proof-of-principle, we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer.

Do all roads lead to Oct4? The emerging concepts of induced pluripotency

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

202 EFFECT OF LEUKEMIA INHIBITORY FACTOR AND FORSKOLIN ON ESTABLISHMENT OF RAT EMBRYONIC STEM CELL LINES

Rat embryonic stem (ES) cell lines can be established in culture medium containing inhibitors for glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK). We confirmed reproducibility of the 2i culture system in establishing rat ES cell lines (Hirabayashi et al. 2010 Mol. Reprod. Dev. 77, 94) and the likelihood of successful germline transmission (genuine) of rat ES cell lines established in leukemia inhibitory factor (LIF)- and forskolin (FK)-supplemented 2i medium (Hirabayashi et al. 2013 Transgenic Res. 22, 411–416). This study was designed to investigate whether LIF and/or FK supplemented to the 2i medium support establishment of germline-competent rat ES cell lines. E4.5 blastocysts were recovered from BLK rat females, and zona-free embryos were plated on mitomycin-treated mouse embryonic fibroblasts in N2B27 medium containing 1 mM MEK inhibitor PD0325901 and 3 mM GSK3 inhibitor CHIR99021, with rat 1000 U mL–1 LIF and/or 10 μM FK. Outgrowth rate of the blastocysts after 1 wk culture and establishment efficiency of ES cell lines after third passage were analyzed by Fisher's exact probability test. Arcsin-transformed percentage data on full-term development of ES cell-injected blastocysts, chimeric rat production, and germline-competent chimeras were analyzed by Fisher's least significant difference test after one-way ANOVA. Because of the higher outgrowth rates of blastocysts, supplementation of rat LIF, FK, or both contributed to the higher (P < 0.05) establishment efficiency of ES cell lines in BLK rat strain (76% to 92% v. 50% in LIF/FK-free 2i medium). Neither efficiency of producing chimeric rats (14% to 39% of blastocysts injected) nor germline transmission competency of the chimeric rats (67% to 100% of cell lines analyzed) was influenced by the pre-treatment of ES cell lines. When the LIF/FK-supplemented 2i medium was used, rat strain for blastocyst donor such as F344 or WI was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, however, all of them (9/9 in overall) were found to be germline-competent by progeny test of chimeric rats. In conclusion, both LIF and FK are not essential, but can play a beneficial role, for the establishment of genuine rat ES cell lines. This work was supported by a grant-in-aid for basic research from Japan Society for the Promotion of Science (No. 25290037; to M.H.).

Stem cell potency and the ability to contribute to chimeric organisms

Mouse embryonic chimeras are a well-established tool for studying cell lineage commitment and pluripotency. Experimental chimeras were successfully produced by combining two or more preimplantation embryos or by introducing into host embryo cultured pluripotent embryonic stem cells (ESCs). Chimera production using genetically modified ESCs became the method of choice for the generation of knockout or knockin mice. Although the derivation of ESCs or ESC-like cells has been reported for other species, only mouse and rat pluripotent stem cells have been shown to contribute to germline-competent chimeras, which is the defining feature of ESCs. Herein, we describe different approaches employed for the generation of embryonic chimeras, define chimera-competent cell types, and describe cases of spontaneous chimerism in humans. We also review the current state of derivation of pluripotent stem cells in several species and discuss outcomes of various chimera studies when such cells are used.

Generation of gene-targeted mice using embryonic stem cells derived from a transgenic mouse model of Alzheimer’s disease

Gene-targeting technology using mouse embryonic stem (ES) cells has become the "gold standard" for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer's disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409-421, 2003) harboring 3 mutated genes (APPswe, TauP301L, and PS1M146V) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F1 mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background.

Optimization of Protocols for Derivation of Mouse Embryonic Stem Cell Lines from Refractory Strains, Including the Non Obese Diabetic Mouse

The derivation of pluripotent embryonic stem cells (ESCs) from a variety of genetic backgrounds remains a desirable objective in the generation of mice functionally deficient in genes of interest and the modeling of human disease. Nevertheless, disparity in the ease with which different strains of mice yield ESC lines has long been acknowledged. Indeed, the generation of bona fide ESCs from the non obese diabetic (NOD) mouse, a well-characterized model of human type I diabetes, has historically proved especially difficult to achieve. Here, we report the development of protocols for the derivation of novel ESC lines from C57Bl/6 mice based on the combined use of high concentrations of leukemia inhibitory factor and serum-replacement, which is equally applicable to fresh and cryo-preserved embryos. Further, we demonstrate the success of this approach using Balb/K and CBA/Ca mice, widely considered to be refractory strains. CBA/Ca ESCs contributed to the somatic germ layers of chimeras and displayed a very high competence at germline transmission. Importantly, we were able to use the same protocol for the derivation of ESC lines from nonpermissive NOD mice. These ESCs displayed a normal karyotype that was robustly stable during long-term culture, were capable of forming teratomas in vivo and germline competent chimeras after injection into recipient blastocysts. Further, these novel ESC lines efficiently formed embryoid bodies in vitro and could be directed in their differentiation along the dendritic cell lineage, thus illustrating their potential application to the generation of cell types of relevance to the pathogenesis of type I diabetes.

Loss of Dnd1 facilitates the cultivation of genital ridge-derived rat embryonic germ cells

Pluripotent cells referred to as embryonic germ cells (EGCs) can be derived from the embryonic precursors of the mature gametes: the primordial germ cells (PGCs). A homozygous mutation ( ter) of the dead-end homolog 1 gene ( Dnd1) in the rat causes gonadal teratocarcinogenesis and sterility due to neoplastic transformation and loss of germ cells. We mated heterozygous ter/+ WKY- Dnd1 ter /Ztm rats and were able to cultivate the first genital ridge-derived EGCs of the rat embryo at day 14.5 post coitum (pc). Genotyping revealed that ten EGC lines were Dnd1 deficient, while only one wild type cell line had survived in culture. This suggests that the inactivation of the putative tumor suppressor gene Dnd1 facilitates the immortalization of late EGCs in vitro. Injection of the wild type EGCs into blastocysts resulted in the first germ-line competent chimeras. These new pluripotent rat EGCs offer an innovative approach for studies on germ cell tumor development as well as a new tool for genetic manipulations in rats.

Gene-manipulated embryonic stem cells for rat transgenesis.

Embryonic stem cells (ESCs) are derived from blastocysts and are capable of differentiating into whole tissues and organs. Transplantation of ESCs into recipient blastocysts leads to the generation of germline-competent chimeras in mice. Transgenic, knockin, and knockout gene manipulations are available in mouse ESCs, enabling the production of genetically modified animals. Rats have important advantages over mice as an experimental system for physiological and pharmacological investigations. However, in contrast to mouse ESCs, rat ESCs were not established until 2008 because of the difficulty of maintaining pluripotency. Although the use of signaling inhibitors has allowed the generation of rat ESCs, the production of genetically modified rats has been difficult due to problems in rat ESCs after gene introduction. In this review, we will focus on some well-documented examples of gene manipulation in rat ESCs.

Association of telomere length with authentic pluripotency of ES/iPS cells

Telomerase and telomeres are important for indefinite replication of stem cells. Recently, telomeres of somatic cells were found to be reprogrammed to elongate in induced pluripotent stem cells (iPSCs). The role of telomeres in developmental pluripotency in vivo of embryonic stem cells (ESCs) or iPSCs, however, has not been directly addressed. We show that ESCs with long telomeres exhibit authentic developmental pluripotency, as evidenced by generation of complete ESC pups as well as germline-competent chimeras, the most stringent tests available in rodents. ESCs with short telomeres show reduced teratoma formation and chimera production, and fail to generate complete ESC pups. Telomere lengths are highly correlated (r > 0.8) with the developmental pluripotency of ESCs. Short telomeres decrease the proliferative rate or capacity of ESCs, alter the expression of genes related to telomere epigenetics, down-regulate genes important for embryogenesis and disrupt germ cell differentiation. Moreover, iPSCs with longer telomeres generate chimeras with higher efficiency than those with short telomeres. Our data show that functional telomeres are essential for the developmental pluripotency of ESCs/iPSCs and suggest that telomere length may provide a valuable marker to evaluate stem cell pluripotency, particularly when the stringent tests are not feasible.

Generation of Parthenogenetic Induced Pluripotent Stem Cells from Parthenogenetic Neural Stem Cells

Somatic cells can achieve a pluripotent cell state in a process called pluripotential reprogramming. Multipotent stem cells can differentiate into cells of only one lineage, but pluripotent stem cells can give rise to cells of all three germ layers of an organism. In this study, we generated induced pluripotent stem (iPS) cells from bimaternal (uniparental) parthenogenetic neural stem cells (pNSCs) by transduction with either four (4F: Oct4, Klf4, Sox2, and c-Myc) or two (2F: Oct4 and Klf4) transcription factors. The resultant maternal iPS cells, which were reprogrammed directly from pNSCs, were capable of generating germ line-competent chimeras. Interestingly, analysis of global gene expression and imprinting status revealed that parthenogenetic iPS cells clustered closer to parthenogenetic ESCs than to female ESCs, with patterns that were clearly distinct from those of pNSCs.

Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts

Direct reprogramming of somatic cells provides an opportunity to generate patient- or disease-specific pluripotent stem cells. Such induced pluripotent stem (iPS) cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. Mouse iPS cells are indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras. Reactivation of the c-Myc retrovirus, however, increases tumorigenicity in the chimeras and progeny mice, hindering clinical applications. Here we describe a modified protocol for the generation of iPS cells that does not require the Myc retrovirus. With this protocol, we obtained significantly fewer non-iPS background cells, and the iPS cells generated were consistently of high quality. Mice derived from Myc(-) iPS cells did not develop tumors during the study period. The protocol also enabled efficient isolation of iPS cells without drug selection. Furthermore, we generated human iPS cells from adult dermal fibroblasts without MYC.

Easy assessment of ES cell clone potency for chimeric development and germ-line competency by an optimized aggregation method

Production of germ-line competent chimeric mice from embryonic stem (ES) cells is an inevitable step in establishing gene-manipulated mouse lineages. A common method used for creating chimeric mice is the injection of ES cells into the blastocoelic cavity (blastocyst injection). The aggregation method is an alternative way to introduce ES cells to the host embryo which is less difficult than blastocyst injection. Here we re-examined the condition of embryo-ES cell coculture on the aggregation method and found improvement of germ-line competent chimeric production by a simple modification of the coculture medium. Moreover, R1 ES cell and its 10 gene-manipulated subclones were tested by this method. Although all ES cell clones showed good morphology and a normal karyotype, the efficiency of chimeric development and germ-line transmission varied among clones and were classified into three grades according to germ-line competency. In the first group (class A), both the incidence of chimera with high ES cell contribution and the rate of germ-line transmission were fairly high. Germ-line competent chimeras were obtained but with rather low efficiency in the second group (class B), while another group (class C) showed an absence of high ES cell-contributed chimeras and no germ-line transmission. These results suggest the usefulness of this modified aggregation method to predict the potency of ES cell clones for germ-line competency.