RESUMO
While the cholesterol biosynthesis pathway has been extensively studied, recent work has forged new links between inhibition of specific sterol pathway enzymes, accumulation of their unique sterol substrates, and biological areas as diverse as cancer, immunology, and neurodegenerative disease. We recently reported that dozens of small molecules enhance formation of oligodendrocytes, a glial cell type lost in multiple sclerosis, by inhibiting CYP51, Sterol 14-reductase, or EBP and inducing cellular accumulation of their 8,9-unsaturated sterol substrates. Several adjacent pathway enzymes also have 8,9-unsaturated sterol substrates but have not yet been evaluated as potential targets for oligodendrocyte formation or in many other biological contexts, in part due to a lack of available small-molecule probes. Here, we show that genetic suppression of SC4MOL or HSD17B7 increases the formation of oligodendrocytes. Additionally, we have identified and optimized multiple potent new series of SC4MOL and HSD17B7 inhibitors and shown that these small molecules enhance oligodendrocyte formation. SC4MOL inhibitor CW4142 induced accumulation of SC4MOL's sterol substrates in mouse brain and represents an in vivo probe of SC4MOL activity. Mechanistically, the cellular accumulation of these 8,9-unsaturated sterols represents a central driver of enhanced oligodendrocyte formation, as exogenous addition of purified SC4MOL and HSD17B7 substrates but not their 8,9-saturated analogs promotes OPC differentiation. Our work validates SC4MOL and HSD17B7 as novel targets for promoting oligodendrocyte formation, underlines a broad role for 8,9-unsaturated sterols as enhancers of oligodendrocyte formation, and establishes the first high-quality small molecules targeting SC4MOL and HSD17B7 as novel tools for probing diverse areas of biology.
RESUMO
Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months. Despite substantial efforts to define druggable targets, there are no therapeutic options that notably extend the lifespan of patients with glioblastoma. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology for use in orthotopic patient-derived xenograft models, creating a high-throughput negative-selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators needed for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies, and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, suggesting that targeting transcription elongation machinery may be an effective therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of 'cancer dependencies' not identified by previous in vitro approaches, and could supply new opportunities for therapeutic intervention.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Terapia de Alvo Molecular/tendências , Fatores de Elongação da Transcrição/antagonistas & inibidores , Fatores de Elongação da Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Humanos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Camundongos , Interferência de RNA , Transcrição Gênica , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Theiler's murine encephalomyelitis virus (TMEV) infects the central nervous system of mice and causes a demyelinating disease that is a model for multiple sclerosis. During the chronic phase of the disease, TMEV persists in oligodendrocytes and macrophages. Lack of remyelination has been attributed to insufficient proliferation and differentiation of oligodendrocyte progenitor cells (OPCs), but the molecular mechanisms remain unknown. Here, we employed pluripotent stem cell technologies to generate pure populations of mouse OPCs to study the temporal and molecular effects of TMEV infection. Global transcriptome analysis of RNA sequencing data revealed that TMEV infection of OPCs caused significant up-regulation of 1926 genes, whereas 1853 genes were significantly down-regulated compared to uninfected cells. Pathway analysis revealed that TMEV disrupted many genes required for OPC growth and maturation. Down-regulation of Olig2, a transcription factor necessary for OPC proliferation, was confirmed by real-time PCR, immunofluorescence microscopy, and western blot analysis. Depletion of Olig2 was not found to be specific to viral strain and did not require expression of the leader (L) protein, which is a multifunctional protein important for persistence, modulation of gene expression, and cell death. These data suggest that direct infection of OPCs by TMEV may inhibit remyelination during the chronic phase of TMEV-induced demyelinating disease.
Assuntos
Doenças Desmielinizantes/virologia , Interações Hospedeiro-Patógeno , Células Precursoras de Oligodendrócitos/virologia , Fator de Transcrição 2 de Oligodendrócitos/genética , Células-Tronco Pluripotentes/virologia , Theilovirus/genética , Animais , Diferenciação Celular , Linhagem Celular , Cricetinae , Doenças Desmielinizantes/patologia , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Anotação de Sequência Molecular , Células Precursoras de Oligodendrócitos/metabolismo , Fator de Transcrição 2 de Oligodendrócitos/deficiência , Células-Tronco Pluripotentes/metabolismo , Cultura Primária de Células , Theilovirus/metabolismo , TranscriptomaRESUMO
Glioblastomas display hierarchies with self-renewing cancer stem-like cells (CSCs). RNA sequencing and enhancer mapping revealed regulatory programs unique to CSCs causing upregulation of the iron transporter transferrin, the top differentially expressed gene compared with tissue-specific progenitors. Direct interrogation of iron uptake demonstrated that CSCs potently extract iron from the microenvironment more effectively than other tumor cells. Systematic interrogation of iron flux determined that CSCs preferentially require transferrin receptor and ferritin, two core iron regulators, to propagate and form tumors in vivo. Depleting ferritin disrupted CSC mitotic progression, through the STAT3-FoxM1 regulatory axis, revealing an iron-regulated CSC pathway. Iron is a unique, primordial metal fundamental for earliest life forms, on which CSCs have an epigenetically programmed, targetable dependence.
Assuntos
Neoplasias Encefálicas/patologia , Ferritinas/metabolismo , Glioblastoma/patologia , Ferro/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptores da Transferrina/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Células Cultivadas , Células-Tronco Embrionárias , Epigênese Genética , Ferritinas/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Receptores da Transferrina/genética , Análise de Sequência de RNA , Transdução de Sinais , Transferrina/metabolismoRESUMO
The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.
Assuntos
Células-Tronco Embrionárias/citologia , Animais , Linhagem da Célula , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Transgenes , Inativação do Cromossomo XRESUMO
Neural induction is the first fundamental step in nervous system formation. During development, a tightly regulated niche modulates transient extracellular signals to influence neural lineage commitment. To date, however, the cascade of molecular events that sustain these signals in humans is not well understood. Here we show that NPTX1, a secreted protein, is rapidly upregulated during neural induction from human pluripotent stem cells (hPSCs). By manipulating its expression, we were able to reduce or initiate neural lineage commitment. A time-course transcriptome analysis and functional assays show that NPTX1 acts in part by binding the Nodal receptor cofactor TDGF1, reducing both Nodal and BMP signaling. Our findings identify one of the earliest genes expressed upon neural induction and provide insight into human neural lineage specification.
Assuntos
Proteína C-Reativa/metabolismo , Linhagem da Célula , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Proteína C-Reativa/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Neurogênese , Ligação Proteica , Transcriptoma , Regulação para CimaRESUMO
Differentiated retinal pigmented epithelial (RPE) cells have been obtained from human induced pluripotent stem (hiPS) cells. However, the visual (retinoid) cycle in hiPS-RPE cells has not been adequately examined. Here we determined the expression of functional visual cycle enzymes in hiPS-RPE cells compared with that of isolated wild-type mouse primary RPE (mpRPE) cells in vitro and in vivo. hiPS-RPE cells appeared morphologically similar to mpRPE cells. Notably, expression of certain visual cycle proteins was maintained during cell culture of hiPS-RPE cells, whereas expression of these same molecules rapidly decreased in mpRPE cells. Production of the visual chromophore, 11-cis-retinal, and retinosome formation also were documented in hiPS-RPE cells in vitro. When mpRPE cells with luciferase activity were transplanted into the subretinal space of mice, bioluminance intensity was preserved for >3 months. Additionally, transplantation of mpRPE into blind Lrat(-/-) and Rpe65(-/-) mice resulted in the recovery of visual function, including increased electrographic signaling and endogenous 11-cis-retinal production. Finally, when hiPS-RPE cells were transplanted into the subretinal space of Lrat(-/-) and Rpe65(-/-) mice, their vision improved as well. Moreover, histological analyses of these eyes displayed replacement of dysfunctional RPE cells by hiPS-RPE cells. Together, our results show that hiPS-RPE cells can exhibit a functional visual cycle in vitro and in vivo. These cells could provide potential treatment options for certain blinding retinal degenerative diseases.
Assuntos
Células-Tronco Pluripotentes Induzidas/transplante , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Epitélio Pigmentado da Retina/transplante , cis-trans-Isomerases/genética , Animais , Diferenciação Celular , Células Cultivadas , Regulação Enzimológica da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/enzimologia , Camundongos , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/enzimologia , Retinaldeído/biossíntese , Retinaldeído/genética , Visão Ocular/genética , Visão Ocular/fisiologia , cis-trans-Isomerases/deficiênciaRESUMO
BACKGROUND: Certain mutations in the Deadend1 (Dnd1) gene are the most potent modifiers of testicular germ cell tumor (TGCT) susceptibility in mice and rats. In the 129 family of mice, the Dnd1Ter mutation significantly increases occurrence of TGCT-affected males. To test the hypothesis that he Dnd1Ter allele is a loss-of-function mutation; we characterized the consequences of a genetically-engineered loss-of-function mutation in mice, and compared these results with those for Dnd1Ter. RESULTS: We found that intercrossing Dnd1+/KO heterozygotes to generate a complete loss-of-function led to absence of Dnd1KO/KO homozygotes and significantly reduced numbers of Dnd1+/KO heterozygotes. Further crosses showed that Dnd1Ter partially rescues loss of Dnd1KO mice. We also found that loss of a single copy of Dnd1 in Dnd1KO/+ heterozygotes did not affect baseline occurrence of TGCT-affected males and that Dnd1Ter increased TGCT risk regardless whether the alternative allele was loss-of-function (Dnd1KO) or wild-type (Dnd1âº). Finally, we found that the action of Dnd1Ter was not limited to testicular cancer, but also significantly increased polyp number and burden in the Apc+/Min model of intestinal polyposis. CONCLUSION: These results show that Dnd1 is essential for normal allelic inheritance and that Dnd1Ter has a novel combination of functions that significantly increase risk for both testicular and intestinal cancer.
Assuntos
Alelos , Polipose Intestinal/genética , Mutação , Proteínas de Neoplasias/genética , Neoplasias Testiculares/genética , Animais , Heterozigoto , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/genéticaRESUMO
Cell-based therapies for myelin disorders, such as multiple sclerosis and leukodystrophies, require technologies to generate functional oligodendrocyte progenitor cells. Here we describe direct conversion of mouse embryonic and lung fibroblasts to induced oligodendrocyte progenitor cells (iOPCs) using sets of either eight or three defined transcription factors. iOPCs exhibit a bipolar morphology and global gene expression profile consistent with bona fide OPCs. They can be expanded in vitro for at least five passages while retaining the ability to differentiate into multiprocessed oligodendrocytes. When transplanted to hypomyelinated mice, iOPCs are capable of ensheathing host axons and generating compact myelin. Lineage conversion of somatic cells to expandable iOPCs provides a strategy to study the molecular control of oligodendrocyte lineage identity and may facilitate neurological disease modeling and autologous remyelinating therapies.
Assuntos
Fibroblastos/citologia , Bainha de Mielina/metabolismo , Oligodendroglia/citologia , Oligodendroglia/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Fibroblastos/fisiologia , Melhoramento Genético/métodos , Camundongos , Transplante de Células-Tronco/métodosRESUMO
Environmental agents and genetic variants can induce heritable epigenetic changes that affect phenotypic variation and disease risk in many species. These transgenerational effects challenge conventional understanding about the modes and mechanisms of inheritance, but their molecular basis is poorly understood. The Deadend1 (Dnd1) gene enhances susceptibility to testicular germ cell tumors (TGCTs) in mice, in part by interacting epigenetically with other TGCT modifier genes in previous generations. Sequence homology to A1cf, the RNA-binding subunit of the ApoB editing complex, raises the possibility that the function of Dnd1 is related to Apobec1 activity as a cytidine deaminase. We conducted a series of experiments with a genetically engineered deficiency of Apobec1 on the TGCT-susceptible 129/Sv inbred background to determine whether dosage of Apobec1 modifies susceptibility, either alone or in combination with Dnd1, and either in a conventional or a transgenerational manner. In the paternal germ-lineage, Apobec1 deficiency significantly increased susceptibility among heterozygous but not wild-type male offspring, without subsequent transgenerational effects, showing that increased TGCT risk resulting from partial loss of Apobec1 function is inherited in a conventional manner. By contrast, partial deficiency in the maternal germ-lineage led to suppression of TGCTs in both partially and fully deficient males and significantly reduced TGCT risk in a transgenerational manner among wild-type offspring. These heritable epigenetic changes persisted for multiple generations and were fully reversed after consecutive crosses through the alternative germ-lineage. These results suggest that Apobec1 plays a central role in controlling TGCT susceptibility in both a conventional and a transgenerational manner.
Assuntos
Citidina Desaminase/genética , Embrião de Mamíferos/metabolismo , Predisposição Genética para Doença , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Testiculares/genética , Desaminase APOBEC-1 , Animais , Distribuição de Qui-Quadrado , Citidina Desaminase/deficiência , Embrião de Mamíferos/embriologia , Epigenômica , Feminino , Frequência do Gene , Genótipo , Padrões de Herança , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Mutação , Proteínas de Neoplasias/genética , Neoplasias Embrionárias de Células Germinativas/enzimologia , Fatores Sexuais , Neoplasias Testiculares/enzimologiaRESUMO
Cancer is characterized by gene expression aberrations. Studies have largely focused on coding sequences and promoters, even though distal regulatory elements play a central role in controlling transcription patterns. We used the histone mark H3K4me1 to analyze gain and loss of enhancer activity genome-wide in primary colon cancer lines relative to normal colon crypts. We identified thousands of variant enhancer loci (VELs) that comprise a signature that is robustly predictive of the in vivo colon cancer transcriptome. Furthermore, VELs are enriched in haplotype blocks containing colon cancer genetic risk variants, implicating these genomic regions in colon cancer pathogenesis. We propose that reproducible changes in the epigenome at enhancer elements drive a specific transcriptional program to promote colon carcinogenesis.
Assuntos
Neoplasias do Colo/genética , Elementos Facilitadores Genéticos , Epigênese Genética , Histonas/metabolismo , Transcriptoma , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Colo/metabolismo , Neoplasias do Colo/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Loci Gênicos , Humanos , Mucosa Intestinal/metabolismo , Metilação , Polimorfismo de Nucleotídeo Único , Regiões Promotoras GenéticasRESUMO
Pluripotency manifests during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Rodent pluripotent stem cells can be considered as two distinct states: naïve and primed. Naïve pluripotent stem cell lines are distinguished from primed cells by self-renewal in response to LIF signaling and MEK/GSK3 inhibition (LIF/2i conditions) and two active X chromosomes in female cells. In rodent cells, the naïve pluripotent state may be accessed through at least three routes: explantation of the inner cell mass, somatic cell reprogramming by ectopic Oct4, Sox2, Klf4, and C-myc, and direct reversion of primed post-implantation-associated epiblast stem cells (EpiSCs). In contrast to their rodent counterparts, human embryonic stem cells and induced pluripotent stem cells more closely resemble rodent primed EpiSCs. A critical question is whether naïve human pluripotent stem cells with bona fide features of both a pluripotent state and naïve-specific features can be obtained. In this review, we outline current understanding of the differences between these pluripotent states in mice, new perspectives on the origins of naïve pluripotency in rodents, and recent attempts to apply the rodent paradigm to capture naïve pluripotency in human cells. Unraveling how to stably induce naïve pluripotency in human cells will influence the full realization of human pluripotent stem cell biology and medicine.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Quimera/genética , Técnicas de Cultura Embrionária , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Células-Tronco Embrionárias/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Sistema de Sinalização das MAP Quinases , Camundongos , Proteína Homeobox Nanog , Ratos , Especificidade da Espécie , Teratoma/patologiaRESUMO
BACKGROUND & AIMS: Many studies of embryonic stem cells have investigated direct cell replacement of damaged tissues, but little is known about how donor cell-derived signals affect host tissue regeneration. We investigated the direct and indirect roles of human embryonic stem cell-derived cells in liver repair in mice. METHODS: To promote the initial differentiation of human embryonic stem cells into mesendoderm, we activated the ß-catenin signaling pathway with lithium; cells were then further differentiated into hepatocyte-like cells. The differentiated cells were purified by indocyanine green staining and laser microdissection and characterized by immunostaining, polymerase chain reaction, biochemical function, electron microscopy, and transplantation analyses. To investigate indirect effects of these cells, secreted proteins (secretomes) were analyzed by a label-free quantitative mass spectrometry. Carbon tetrachloride was used to induce acute liver injury in mice; cells or secreted proteins were administered by intrasplenic or intraperitoneal injection, respectively. RESULTS: The differentiated hepatocyte-like cells had multiple features of normal hepatocytes, engrafted efficiently into mice, and continued to have hepatic features; they promoted proliferation of host hepatocytes and revascularization of injured host liver tissues. Proteomic analysis identified proteins secreted from these cells that might promote host tissue repair. Injection of the secreted proteins into injured livers of mice promoted significant amounts of tissue regeneration without cell grafts. CONCLUSIONS: Hepatocyte-like cells derived from human embryonic stem cells contribute to recovery of injured liver tissues in mice, not only by cell replacement but also by delivering trophic factors that support endogenous liver regeneration.
Assuntos
Diferenciação Celular , Proliferação de Células , Doença Hepática Induzida por Substâncias e Drogas/cirurgia , Células-Tronco Embrionárias/transplante , Hepatócitos/transplante , Células-Tronco Pluripotentes Induzidas/transplante , Regeneração Hepática , Fígado/patologia , Animais , Biomarcadores/metabolismo , Tetracloreto de Carbono , Diferenciação Celular/efeitos dos fármacos , Separação Celular/métodos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Técnicas de Cocultura , Modelos Animais de Doenças , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microdissecção e Captura a Laser , Cloreto de Lítio/farmacologia , Fígado/irrigação sanguínea , Fígado/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Eletrônica , Neovascularização Fisiológica , Reação em Cadeia da Polimerase , Proteômica/métodos , Fatores de Tempo , CicatrizaçãoRESUMO
Despite strong heritability, little is known about the genetic control of susceptibility to testicular germ cell tumors (TGCT) in humans or mice. Although the mouse model of spontaneous TGCTs has been extensively studied, conventional linkage analysis has failed to locate the factors that control teratocarcinogenesis in the susceptible 129 family of inbred strains. As an alternative approach, we used both chromosome substitution strains (CSS) to identify individual chromosomes that harbor susceptibility genes and a panel of congenic strains derived from a selected CSS to determine the number and location of susceptibility variants on the substituted chromosome. We showed that 129-Chr 18(MOLF) males are resistant to spontaneous TGCTs and that at least four genetic variants control susceptibility in males with this substituted chromosome. In addition, early embryonic cells from this strain fail to establish embryonic stem cell lines as efficiently as those from the parental 129/Sv strain. For the first time, 129-derived genetic variants that control TGCT susceptibility and fundamental aspects of embryonic stem cell biology have been localized in a genetic context in which the genes can be identified and functionally characterized.