RESUMEN
The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD+) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative single-cell analysis with pre- and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Diferenciación Celular , Linaje de la Célula , Desarrollo Embrionario , Células Madre Embrionarias/metabolismo , Endodermo/metabolismo , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Análisis de la Célula Individual , Vía de Señalización WntRESUMEN
DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn(2+)-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired nicking of newly replicated DNA and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair.
Asunto(s)
Adenosina Trifosfatasas/química , Bacillus subtilis/enzimología , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Reparación de la Incompatibilidad de ADN , Endonucleasas/química , Activación Enzimática , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Zinc/metabolismoRESUMEN
Histone variants (HVs) are a subfamily of epigenetic regulators implicated in embryonic development, but their role in human stem cell fate remains unclear. Here, we reveal that the phosphorylation state of the HV H2A.X (γH2A.X) regulates self-renewal and differentiation of human pluripotent stem cells (hPSCs) and leukemic progenitors. As demonstrated by CRISPR-Cas deletion, H2A.X is essential in maintaining normal hPSC behavior. However, reduced levels of γH2A.X enhances hPSC differentiation toward the hematopoietic lineage with concomitant inhibition of neural development. In contrast, activation and sustained levels of phosphorylated H2A.X enhance hPSC neural fate while suppressing hematopoiesis. This controlled lineage bias correlates to occupancy of γH2A.X at genomic loci associated with ectoderm versus mesoderm specification. Finally, drug modulation of H2A.X phosphorylation overcomes differentiation block of patient-derived leukemic progenitors. Our study demonstrates HVs may serve to regulate pluripotent cell fate and that this biology could be extended to somatic cancer stem cell control.
Asunto(s)
Autorrenovación de las Células/fisiología , Histonas/metabolismo , Células Madre Neoplásicas/citología , Células Madre Pluripotentes/citología , Sistemas CRISPR-Cas/genética , Diferenciación Celular , Linaje de la Célula , Ectodermo/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Histonas/deficiencia , Histonas/genética , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Mesodermo/metabolismo , Células Madre Neoplásicas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Nucleosomas/metabolismo , Fosforilación , Células Madre Pluripotentes/metabolismoRESUMEN
The aberrant expression of dopamine receptors (DRDs) in acute myeloid leukemia (AML) cells has encouraged the repurposing of DRD antagonists such as thioridazine (TDZ) as anti-leukemic agents. Here, we access patient cells from a Phase I dose escalation trial to resolve the cellular and molecular bases of response to TDZ, and we extend these findings to an additional independent cohort of AML patient samples tested preclinically. We reveal that in DRD2+ AML patients, DRD signaling in leukemic progenitors provides leukemia-exclusive networks of sensitivity that spare healthy hematopoiesis. AML progenitor cell suppression can be increased by the isolation of the positive enantiomer from the racemic TDZ mixture (TDZ+), and this is accompanied by reduced cardiac liability. Our study indicates that the development of DRD-directed therapies provides a targeting strategy for a subset of AML patients and potentially other cancers that acquire DRD expression upon transformation from healthy tissue.
Asunto(s)
Hematopoyesis/fisiología , Leucemia Mieloide Aguda/tratamiento farmacológico , Células Madre Neoplásicas/metabolismo , Receptores Dopaminérgicos/metabolismo , Tioridazina/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Natural products (NPs) encompass a rich source of bioactive chemical entities. Here, we used human cancer stem cells (CSCs) in a chemical genomics campaign with NP chemical space to interrogate extracts from diverse strains of actinomycete for anti-cancer properties. We identified a compound (McM25044) capable of selectively inhibiting human CSC function versus normal stem cell counterparts. Biochemical and molecular studies revealed that McM025044 exerts inhibition on human CSCs through the small ubiquitin-like modifier (SUMO) cascade, found to be hyperactive in a variety of human cancers. McM025044 impedes the SUMOylation pathway via direct targeting of the SAE1/2 complex. Treatment of patient-derived CSCs resulted in reduced levels of SUMOylated proteins and suppression of progenitor and stem cell capacity measured in vitro and in vivo. Our study overcomes a barrier in chemically inhibiting oncogenic SUMOylation activity and uncovers a unique role for SAE2 in the biology of human cancers.
Asunto(s)
Células Madre Neoplásicas/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Sitios de Unión , Productos Biológicos/química , Productos Biológicos/metabolismo , Productos Biológicos/farmacología , Productos Biológicos/uso terapéutico , Línea Celular Tumoral , Autorrenovación de las Células , Supervivencia Celular/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Ratones , Simulación del Acoplamiento Molecular , Células Madre Neoplásicas/citología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Sumoilación/efectos de los fármacos , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/genéticaRESUMEN
Human pluripotent stem cells (hPSCs) have been reported in naive and primed states. However, the ability to generate mature cell types remains the imperative property for utility of hPSCs. Here, we reveal that the naive state enhances self-renewal while restricting lineage differentiation in vitro to neural default fate. Molecular analyses indicate expression of multiple lineage-associated transcripts in naive hPSCs that failed to predict biased functional differentiation capacity. Naive hPSCs can be converted to primed state over long-term serial passage that permits recovery of multi-germ layer differentiation. Suppression of OCT4 but not NANOG allows immediate recovery directly from naive state. To this end, we identified chemical inhibitors of OCT4 that restore naive hPSC differentiation. Our study reveals unique cell-fate restrictions in human pluripotent states and provides an approach to overcome these barriers that harness both efficient naive hPSC growth while maintaining in vitro differentiation essential for hPSC applications.
Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , Reprogramación Celular/genética , Estratos Germinativos/citología , Células Madre Pluripotentes/citología , Animales , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Hepatocitos/metabolismo , Humanos , Ratones , Miocitos Cardíacos/metabolismo , Proteína Homeótica Nanog/metabolismo , Nistatina/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , ARN/genética , Teratoma/metabolismoRESUMEN
Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/ß-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/ß-catenin signaling within CSCs. Disruption of CBP-ß-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Neoplásicas/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Sialoglicoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Anciano , Animales , Apoptosis/efectos de los fármacos , Compuestos de Azabiciclo/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Ratones Endogámicos NOD , Persona de Mediana Edad , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/trasplante , Organofosfatos/farmacología , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Pirimidinonas/farmacología , Interferencia de ARN , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Sialoglicoproteínas/antagonistas & inhibidores , Sialoglicoproteínas/genética , Sumoilación/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismoRESUMEN
The combination of OCT4 expression and short-term exposure to reprogramming media induces a state of transcriptional plasticity in human fibroblasts, capable of responding to changes in the extracellular environment that facilitate direct cell fate conversion toward lineage specific progenitors. Here we reveal that continued exposure of OCT4-induced plastic human fibroblasts to reprogramming media (RM) is sufficient to induce pluripotency. OCT4-derived induced pluripotent stem cell (iPSC(OCT4)) colonies emerged after prolonged culture in RM, and formed independently of lineage specific progenitors. Human iPSC(OCT4) are morphologically indistinguishable from conventionally derived iPSCs and express core proteins involved in maintenance of pluripotency. iPSC(OCT4) display in vivo functional pluripotency as measured by teratoma formation consisting of the three germ layers, and are capable of targeted in vitro differentiation. Our study indicates that acquisition of pluripotency is one of multiple cell fate choices that can be facilitated through environmental stimulation of OCT4-induced plasticity, and suggests the role of other reprogramming factors to induce pluripotency can be substituted by prolonged culture of plastic fibroblasts.
Asunto(s)
Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Factor 3 de Transcripción de Unión a Octámeros/farmacología , Adulto , Animales , Linaje de la Célula/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Humanos , Inmunofenotipificación , Ratones Endogámicos NOD , Ratones SCID , Modelos BiológicosRESUMEN
The combination of OCT4 expression and short-term exposure to reprogramming media induces a state of transcriptional plasticity in human fibroblasts, capable of responding to changes in the extracellular environment. Here we provide characterization of iPSCs established through continued culture of OCT4-induced plastic human fibroblasts in pluripotent-supportive reprogramming media. Human iPSC(OCT4) are morphologically indistinguishable from conventionally derived iPSCs and express core proteins involved in maintenance of pluripotency. iPSC(OCT4) display bona fide functional pluripotency as measured by in vivo teratoma formation consisting of the three germ layers.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Factor 3 de Transcripción de Unión a Octámeros/farmacología , Adulto , Animales , Reprogramación Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
The clinical applicability of direct cell fate conversion depends on obtaining tissue from patients that is easy to harvest, store, and manipulate for reprogramming. Here, we generate induced neural progenitor cells (iNPCs) from neonatal and adult peripheral blood using single-factor OCT4 reprogramming. Unlike fibroblasts that share molecular hallmarks of neural crest, OCT4 reprogramming of blood was facilitated by SMAD+GSK-3 inhibition to overcome restrictions on neural fate conversion. Blood-derived (BD) iNPCs differentiate in vivo and respond to guided differentiation in vitro, producing glia (astrocytes and oligodendrocytes) and multiple neuronal subtypes, including dopaminergic (CNS related) and nociceptive neurons (peripheral nervous system [PNS]). Furthermore, nociceptive neurons phenocopy chemotherapy-induced neurotoxicity in a system suitable for high-throughput drug screening. Our findings provide an easily accessible approach for generating human NPCs that harbor extensive developmental potential, enabling the study of clinically relevant neural diseases directly from patient cohorts.
Asunto(s)
Técnicas de Reprogramación Celular/métodos , Células-Madre Neurales/citología , Diferenciación Celular/fisiología , Humanos , Factor 3 de Transcripción de Unión a Octámeros/genéticaRESUMEN
Human-induced pluripotent stem cells (hiPSCs) provide an invaluable source for regenerative medicine, but are limited by proficient lineage-specific differentiation. Here we reveal that hiPSCs derived from human fibroblasts (Fibs) versus human cord blood (CB) exhibit indistinguishable pluripotency, but harbour biased propensities for differentiation. Genes associated with germ layer specification were identical in Fib- or CB-derived iPSCs, whereas lineage-specific marks emerge upon differentiation induction of hiPSCs that were correlated to the cell of origin. Differentiation propensities come at the expense of other lineages and cannot be overcome with stimuli for alternative cell fates. Although incomplete DNA methylation and distinct histone modifications of lineage-specific loci correlate to lineage-specific transcriptome priming, transitioning hiPSCs into naive state of pluripotency removes iPSC-memorized transcriptome. Upon re-entry to the primed state, transcriptome memory is restored, indicating a human-specific phenomenon whereby lineage gated developmental potential is not permanently erased, but can be modulated by the pluripotent state.
Asunto(s)
Linaje de la Célula/genética , Sangre Fetal/citología , Fibroblastos/citología , Células Madre Pluripotentes Inducidas/metabolismo , Transcriptoma/genética , Diferenciación Celular/genética , Metilación de ADN , Epigénesis Genética , Histonas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citologíaRESUMEN
Several transcription factors and methods have been used to convert fibroblasts directly to neural fate and have provided insights into molecular mechanisms as to how each of these required factors orchestrate neural fate conversion. Here, we provide evidence and detailed characterization of the direct conversion process of primary adult human fibroblasts (hFib) to neural progenitor cells (NPC) using OCT4 alone. Factors previously associated with neural cell fate conversion were induced during hFib-NPC(OCT-4) generation, where OCT-4 alone was sufficient to induce neural fate conversion without the use of promiscuous small-molecule manipulation. Human Fib-NPC(OCT-4) proliferate, express neural stem/progenitor markers, and possess developmental potential that gives rise to all three major subtypes of neural cells: astrocytes, oligodendrocytes, and neurons with functional capacity. We propose a de-convoluted reprogramming approach for neural fate conversion in which OCT4 is sufficient for inducing neural conversion from hFib for disease modeling as well as the fundamental study of early neural fate induction.