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Characterization of pluripotent states, in which cells can both self-renew or differentiate, with the irreversible loss of pluripotency, are important research areas in developmental biology. Although microRNAs (miRNAs) have been shown to play a relevant role in cellular differentiation, the role of miRNAs integrated into gene regulatory networks and its dynamic changes during these early stages of embryonic stem cell (ESC) differentiation remain elusive. Here we describe the dynamic transcriptional regulatory circuitry of stem cells that incorporate protein-coding and miRNA genes based on miRNA array expression and quantitative sequencing of short transcripts upon the downregulation of the Estrogen Related Receptor Beta (Esrrb). The data reveals how Esrrb, a key stem cell transcription factor, regulates a specific stem cell miRNA expression program and integrates dynamic changes of feed-forward loops contributing to the early stages of cell differentiation upon its downregulation. Together these findings provide new insights on the architecture of the combined transcriptional post-transcriptional regulatory network in embryonic stem cells.
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Cell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory "dimensions" coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator. Comparative analyses of expression changes subsequent to depletion of Esrrb or Nanog, indicated that a system of interlocked feed-forward loops involving both factors, plays a central part in regulating the timing of mESC fate decisions. Taken together, our meta-analyses support a hierarchical model in which pluripotency is maintained by an Oct4-Sox2 regulatory module, while the timing of differentiation is regulated by a Nanog-Esrrb module.
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Osteosarcoma is one of the most frequent common primary malignant tumors in childhood and adolescence. Long non-coding RNAs (lncRNAs) have been reported to regulate the initiation and progression of tumors. However, the exact molecular mechanisms involving lncRNA in osteosarcomagenesis remain largely unknown. Li-Fraumeni syndrome (LFS) is a familial cancer syndrome caused by germline p53 mutation. We investigated the tumor suppressor function of lncRNA H19 in LFS-associated osteosarcoma. Analyzing H19-induced transcriptome alterations in LFS induced pluripotent stem cell (iPSC)-derived osteoblasts, we unexpectedly discovered a large group of snoRNAs whose expression was significantly affected by H19. We identified SNORA7A among the H19-suppressed snoRNAs. SNORA7A restoration impairs H19-mediated osteogenesis and tumor suppression, indicating an oncogenic role of SNORA7A. TCGA analysis indicated that SNORA7A expression is associated with activation of oncogenic signaling and poor survival in cancer patients. Using an optimized streptavidin-binding RNA aptamer designed from H19 lncRNA, we revealed that H19-tethered protein complexes include proteins critical for DNA damage response and repair, confirming H19's tumor suppressor role. In summary, our findings demonstrate a critical role of H19-modulated SNORA7A expression in LFS-associated osteosarcomas.
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Transcription factor (TF)-based reprogramming of somatic tissues holds great promise for regenerative medicine. Previously, we demonstrated that the TFs GATA2, GFI1B, and FOS convert mouse and human fibroblasts to hemogenic endothelial-like precursors that generate hematopoietic stem progenitor (HSPC)-like cells over time. This conversion is lacking in robustness both in yield and biological function. Herein, we show that inclusion of GFI1 to the reprogramming cocktail significantly expands the HSPC-like population. AFT024 coculture imparts functional potential to these cells and allows quantification of stem cell frequency. Altogether, we demonstrate an improved human hemogenic induction protocol that could provide a valuable human in vitro model of hematopoiesis for disease modeling and a platform for cell-based therapeutics. DATABASE: Gene expression data are available in the Gene Expression Omnibus (GEO) database under the accession number GSE130361.
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Diferenciación Celular/genética , Reprogramación Celular/genética , Hemangioblastos/citología , Células Madre Hematopoyéticas/citología , Animales , Técnicas de Cocultivo/métodos , Fibroblastos/citología , Fibroblastos/metabolismo , Factor de Transcripción GATA2/genética , Regulación del Desarrollo de la Expresión Génica/genética , Hemangioblastos/metabolismo , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Represoras/genética , Factores de Transcripción/genéticaRESUMEN
A multitude of signals are coordinated to maintain self-renewal in embryonic stem cells (ESCs). To unravel the essential internal and external signals required for sustaining the ESC state, we expand upon a set of ESC pluripotency-associated phosphoregulators (PRs) identified previously by short hairpin RNA (shRNA) screening. In addition to the previously described Aurka, we identify 4 additional PRs (Bub1b, Chek1, Ppm1g, and Ppp2r1b) whose depletion compromises self-renewal and leads to consequent differentiation. Global gene expression profiling and computational analyses reveal that knockdown of the 5 PRs leads to DNA damage/genome instability, activating p53 and culminating in ESC differentiation. Similarly, depletion of genome integrity-associated genes involved in DNA replication and checkpoint, mRNA processing, and Charcot-Marie-Tooth disease lead to compromise of ESC self-renewal via an increase in p53 activity. Our studies demonstrate an essential link between genomic integrity and developmental cell fate regulation in ESCs.
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Diferenciación Celular/genética , Células Madre Embrionarias/fisiología , Inestabilidad Genómica , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Línea Celular , Daño del ADN , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Ratones , Fosfoproteínas/genética , Fosfoproteínas/fisiología , ARN Interferente Pequeño , Transducción de Señal , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
During development, hematopoietic stem and progenitor cells (HSPCs) arise from specialized endothelial cells by a process termed endothelial-to-hematopoietic transition (EHT). The genetic program driving human HSPC emergence remains largely unknown. We previously reported that the generation of hemogenic precursor cells from mouse fibroblasts recapitulates developmental hematopoiesis. Here, we demonstrate that human fibroblasts can be reprogrammed into hemogenic cells by the same transcription factors. Induced cells display dynamic EHT transcriptional programs, generate hematopoietic progeny, possess HSPC cell surface phenotype, and repopulate immunodeficient mice for 3 months. Mechanistically, GATA2 and GFI1B interact and co-occupy a cohort of targets. This cooperative binding is reflected by engagement of open enhancers and promoters, initiating silencing of fibroblast genes and activating the hemogenic program. However, GATA2 displays dominant and independent targeting activity during the early phases of reprogramming. These findings shed light on the processes controlling human HSC specification and support generation of reprogrammed HSCs for clinical applications.
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Reprogramación Celular , Hemangioblastos/citología , Hemangioblastos/metabolismo , Factores de Transcripción/metabolismo , Adulto , Secuencia de Bases , Elementos de Facilitación Genéticos/genética , Fibroblastos/metabolismo , Factor de Transcripción GATA2/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Recién Nacido , Fenotipo , Regiones Promotoras Genéticas/genética , Unión ProteicaRESUMEN
Osteosarcoma (OS), the most common primary bone tumor, is highly metastatic with high chemotherapeutic resistance and poor survival rates. Using induced pluripotent stem cells (iPSCs) generated from Li-Fraumeni syndrome (LFS) patients, we investigate an oncogenic role of secreted frizzled-related protein 2 (SFRP2) in p53 mutation-associated OS development. Interestingly, we find that high SFRP2 expression in OS patient samples correlates with poor survival. Systems-level analyses identified that expression of SFRP2 increases during LFS OS development and can induce angiogenesis. Ectopic SFRP2 overexpression in normal osteoblast precursors is sufficient to suppress normal osteoblast differentiation and to promote OS phenotypes through induction of oncogenic molecules such as FOXM1 and CYR61 in a ß-catenin-independent manner. Conversely, inhibition of SFRP2, FOXM1, or CYR61 represses the tumorigenic potential. In summary, these findings demonstrate the oncogenic role of SFRP2 in the development of p53 mutation-associated OS and that inhibition of SFRP2 is a potential therapeutic strategy.
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Neoplasias Óseas/genética , Carcinogénesis/genética , Síndrome de Li-Fraumeni/patología , Proteínas de la Membrana/genética , Osteosarcoma/genética , Proteína p53 Supresora de Tumor/genética , Animales , Neoplasias Óseas/patología , Línea Celular Tumoral , Proteína 61 Rica en Cisteína/antagonistas & inhibidores , Proteína 61 Rica en Cisteína/genética , Proteína Forkhead Box M1/antagonistas & inhibidores , Proteína Forkhead Box M1/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Síndrome de Li-Fraumeni/genética , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Desnudos , Neovascularización Patológica/genética , Osteoblastos/citología , Osteosarcoma/patologíaRESUMEN
Research in photobiology is currently limited by a lack of devices capable of delivering precise and tunable irradiation to cells in a high-throughput format. This limits researchers to using expensive commercially available or custom-built light sources which make it difficult to replicate, standardize, optimize, and scale experiments. Here we present an open-source Microplate Photoirradiation System (MPS) developed to enable high-throughput light experiments in standard 96 and 24-well microplates for a variety of applications in photobiology research. This open-source system features 96 independently controlled LEDs (4 LEDs per well in 24-well), Wi-Fi connected control and programmable graphical user interface (GUI) for control and programming, automated calibration GUI, and modular control and LED boards for maximum flexibility. A web-based GUI generates light program files containing irradiation parameters for groups of LEDs. These parameters are then uploaded wirelessly, stored and used on the MPS to run photoirradiation experiments inside any incubator. A rapid and semi-quantitative porphyrin metabolism assay was also developed to validate the system in wild-type fibroblasts. Protoporphyrin IX (PpIX) fluorescence accumulation was induced by incubation with 5-aminolevulinic acid (ALA), a photosensitization method leveraged clinically to destroy malignant cell types in a process termed photodynamic therapy (PDT), and cells were irradiated with 405nm light with varying irradiance, duration and pulsation parameters. Immediately after light treatment with the MPS, subsequent photobleaching was measured in live, adherent cells in both 96-well and a 24-well microplates using a microplate reader. Results demonstrate the utility and reliability of the Microplate Photoirradiation System to irradiate cells with precise irradiance and timing parameters in order to measure PpIx photobleaching kinetics in live adherent cells and perform comparable experiments with both 24 and 96 well microplate formats. The high-throughput capability of the MPS enabled measurement of enough irradiance conditions in a single microplate to fit PpIX fluorescence to a bioexponential decay model of photobleaching, as well as reveal a dependency of photobleaching on duty-cycle-but not frequency-in a pulsed irradiance regimen.
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Fotobiología/métodos , Fotoquimioterapia/métodos , Trastornos por Fotosensibilidad , Protoporfirinas/química , Ácido Aminolevulínico/química , Ácido Aminolevulínico/efectos de la radiación , Gráficos por Computador , Humanos , Luz , Fotoblanqueo , Protoporfirinas/efectos de la radiación , Radiación , Tecnología InalámbricaRESUMEN
Embryonic stem cells (ESCs) can proliferate and self-renew, maintaining their pluripotency status in vitro for a long period of time. Pluripotent states of ESCs in vitro are supported by a network of signaling, transcriptional and epigenetic regulatory interactions known as the pluripotency gene regulatory network (PGRN). Despite extensive investigation of the network, the exact order of regulatory links and many structural features of the network are still missing. Analysis of published data and literature reveals numerous PGRN components regulating each other in a mutual fashion, thus creating multiple regulatory feedback control circuits. Here we consider possible organizational features of PGRN and describe examples representing known feedback control loops in the context of mouse ESCs. We discuss how the feedback control interactions can contribute to learning behavior and dynamic responses of pluripotency gene network to changing environments.
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Epigénesis Genética/fisiología , Redes Reguladoras de Genes/fisiología , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Animales , Células Madre Embrionarias Humanas/citología , Humanos , Ratones , Células Madre Embrionarias de Ratones/citologíaRESUMEN
Notch signaling plays a role in specifying a cardiac fate but the downstream effectors remain unknown. In this study we implicate the Notch downstream effector HES5 in cardiogenesis. We show transient Hes5 expression in early mesoderm of gastrulating embryos and demonstrate, by loss and gain-of-function experiments in mouse embryonic stem cells, that HES5 favors cardiac over primitive erythroid fate. Hes5 overexpression promotes upregulation of the cardiac gene Isl1, while the hematopoietic regulator Scl is downregulated. Moreover, whereas a pulse of Hes5 instructs cardiac commitment, sustained expression after lineage specification impairs progression of differentiation to contracting cardiomyocytes. These findings establish a role for HES5 in cardiogenesis and provide insights into the early cardiac molecular network.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Mesodermo/citología , Células Madre Embrionarias de Ratones/citología , Miocitos Cardíacos/citología , Proteínas Represoras/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Proliferación Celular , Eritropoyesis , Gastrulación , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Represoras/genética , Transducción de SeñalRESUMEN
Variability in induced pluripotent stem cell (iPSC) lines remains a concern for disease modeling and regenerative medicine. We have used RNA-sequencing analysis and linear mixed models to examine the sources of gene expression variability in 317 human iPSC lines from 101 individuals. We found that â¼50% of genome-wide expression variability is explained by variation across individuals and identified a set of expression quantitative trait loci that contribute to this variation. These analyses coupled with allele-specific expression show that iPSCs retain a donor-specific gene expression pattern. Network, pathway, and key driver analyses showed that Polycomb targets contribute significantly to the non-genetic variability seen within and across individuals, highlighting this chromatin regulator as a likely source of reprogramming-based variability. Our findings therefore shed light on variation between iPSC lines and illustrate the potential for our dataset and other similar large-scale analyses to identify underlying drivers relevant to iPSC applications.
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Heterogeneidad Genética , Células Madre Pluripotentes Inducidas/metabolismo , Transcripción Genética , Alelos , Teorema de Bayes , Diferenciación Celular/genética , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Estudios de Asociación Genética , Humanos , Proteínas del Grupo Polycomb/metabolismo , Sitios de Carácter Cuantitativo/genética , Reproducibilidad de los ResultadosRESUMEN
The elucidation of cancer pathogenesis has been hindered by limited access to patient samples, tumor heterogeneity and the lack of reliable model organisms. Characterized by their ability to self-renew indefinitely and differentiate into all cell lineages of an organism, pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful and unlimited source to generate differentiated cells that can be used to study disease biology, facilitate drug discovery and development, and provide key insights for developing personalized therapies. This article reviews the recent developments and technologies converting PSCs into clinically relevant model systems for cancer research.
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Modelos Biológicos , Neoplasias , Células Madre Pluripotentes , Animales , Diferenciación Celular , Humanos , OrganoidesRESUMEN
The maintenance of hematopoietic stem cells (HSCs) during ex vivo culture is an important prerequisite for their therapeutic manipulation. However, despite intense research, culture conditions for robust maintenance of HSCs are still missing. Cultured HSCs are quickly lost, preventing their improved analysis and manipulation. Identification of novel factors supporting HSC ex vivo maintenance is therefore necessary. Coculture with the AFT024 stroma cell line is capable of maintaining HSCs ex vivo long-term, but the responsible molecular players remain unknown. Here, we use continuous long-term single-cell observation to identify the HSC behavioral signature under supportive or nonsupportive stroma cocultures. We report early HSC survival as a major characteristic of HSC-maintaining conditions. Behavioral screening after manipulation of candidate molecules revealed that the extracellular matrix protein dermatopontin (Dpt) is involved in HSC maintenance. DPT knockdown in supportive stroma impaired HSC survival, whereas ectopic expression of the Dpt gene or protein in nonsupportive conditions restored HSC survival. Supplementing defined stroma- and serum-free culture conditions with recombinant DPT protein improved HSC clonogenicity. These findings illustrate a previously uncharacterized role of Dpt in maintaining HSCs ex vivo.
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Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Células Madre Hematopoyéticas/metabolismo , Animales , Técnicas de Cultivo de Célula , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteoglicanos Tipo Condroitín Sulfato/farmacología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/farmacología , Células Madre Hematopoyéticas/citología , Masculino , Ratones , Ratones Transgénicos , Células del Estroma/citología , Células del Estroma/metabolismo , Factores de TiempoRESUMEN
Loss-of-function studies are fundamental for dissecting gene function. Yet, methods to rapidly and effectively perturb genes in mammalian cells, and particularly in stem cells, are scarce. Here we present a system for simultaneous conditional regulation of two different proteins in the same mammalian cell. This system harnesses the plant auxin and jasmonate hormone-induced degradation pathways, and is deliverable with only two lentiviral vectors. It combines RNAi-mediated silencing of two endogenous proteins with the expression of two exogenous proteins whose degradation is induced by external ligands in a rapid, reversible, titratable and independent manner. By engineering molecular tuners for NANOG, CHK1, p53 and NOTCH1 in mammalian stem cells, we have validated the applicability of the system and demonstrated its potential to unravel complex biological processes.
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Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Interferencia de ARN , Animales , Línea Celular , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Células HCT116 , Células HEK293 , Humanos , Lentivirus/genética , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Definitive hematopoiesis emerges via an endothelial-to-hematopoietic transition in the embryo and placenta; however, the precursor cells to hemogenic endothelium are not defined phenotypically. We previously demonstrated that the induction of hematopoietic progenitors from fibroblasts progresses through hemogenic precursors that are Prom1(+)Sca1(+)CD34(+)CD45(-) (PS34CD45(-)). Guided by these studies, we analyzed mouse placentas and identified a population with this phenotype. These cells express endothelial markers, are heterogeneous for early hematopoietic markers, and localize to the vascular labyrinth. Remarkably, global gene expression profiles of PS34CD45(-) cells correlate with reprogrammed precursors and establish a hemogenic precursor cell molecular signature. PS34CD45(-) cells are also present in intra-embryonic hemogenic sites. After stromal co-culture, PS34CD45(-) cells give rise to all blood lineages and engraft primary and secondary immunodeficient mice. In summary, we show that reprogramming reveals a phenotype for in vivo precursors to hemogenic endothelium, establishing that direct in vitro conversion informs developmental processes in vivo.
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Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Reprogramación Celular , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Embrionarias de Ratones/citología , Animales , Células Cultivadas , Endotelio/metabolismo , Femenino , Fibroblastos/citología , Ratones , Ratones Endogámicos C57BL , EmbarazoRESUMEN
Modern stem cell research, as well as other fields of contemporary biology involves quantitative sciences in many ways. Identifying candidates for key differentiation or reprogramming factors, tracing global transcriptome changes, or finding drugs is now broadly involves bioinformatics and biostatistics. However, the next key step, understanding the underlying reasons and establishing causal links leading to differentiation or reprogramming requires qualitative and quantitative biological models describing complex biological systems. Currently, quantitative modeling is a challenging science, capable to deliver rather modest results or predictions. What model types are the most popular and what features of stem cell behavior they are capturing? What new insights do we expect from the computational modeling of stem cells in the foreseeable future? Current review attempts to approach these essential questions by considering published quantitative models and solutions emerging in the area of stem cell research.
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Diferenciación Celular , Reprogramación Celular , Células Madre Embrionarias/citología , Modelos Biológicos , Investigación con Células Madre , Animales , HumanosRESUMEN
Even though all paradigms of stem cell therapy and regenerative medicine emerged from the study of hematopoietic stem cells (HSCs), the inability to generate these cells de novo or expand them in vitro persists. Initial efforts to obtain these cells began with the use of embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) technologies, but these strategies have yet to yield fully functional cells. Subsequently, more recent approaches involve transcription factor (TF) overexpression to reprogram PSCs and various somatic cells. The induction of pluripotency with just four TFs by Yamanaka informs our ability to convert cell fates and demonstrates the feasibility of utilizing terminally differentiated cells to generate cells with multilineage potential. In this review, we discuss the recent efforts undertaken using TF-based reprogramming strategies to convert several cell types into HSCs.
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Linaje de la Célula/fisiología , Células Madre Hematopoyéticas/metabolismo , Trasplante de Células Madre/métodos , Factores de Transcripción/metabolismo , Animales , Células Madre Hematopoyéticas/citología , Humanos , Medicina RegenerativaRESUMEN
This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.