RESUMEN
Stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the epigenetic events and molecular mechanisms occurring between consecutive cell divisions have been insufficiently studied due to technical limitations. Here, using the FUCCI reporter we developed a cell-cycle synchronised human pluripotent stem cell (hPSC) differentiation system for uncovering epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We observed that transcription of key differentiation markers occurs before cell division, while chromatin accessibility analyses revealed the early inhibition of alternative cell fates. We found that Activator protein-1 members controlled by p38/MAPK signalling are necessary for inducing endoderm while blocking cell fate shifting toward mesoderm, and that enhancers are rapidly established and decommissioned between different cell divisions. Our study has practical biomedical utility for producing hPSC-derived patient-specific cell types since p38/MAPK induction increased the differentiation efficiency of insulin-producing pancreatic beta-cells.
Asunto(s)
Células Madre Pluripotentes , Humanos , Diferenciación Celular/genética , Regulación de la Expresión Génica , Antígenos de Diferenciación/metabolismo , Epigénesis Genética , EndodermoRESUMEN
Basal cells are multipotent stem cells responsible for the repair and regeneration of all the epithelial cell types present in the proximal lung. In mice, the elusive origins of basal cells and their contribution to lung development were recently revealed by high-resolution, lineage tracing studies. It however remains unclear if human basal cells originate and participate in lung development in a similar fashion, particularly with mounting evidence for significant species-specific differences in this process. To address this outstanding question, in the last several years differentiation protocols incorporating human pluripotent stem cells (hPSC) have been developed to produce human basal cells in vitro with varying efficiencies. To facilitate this endeavour, we introduced tdTomato into the human TP63 gene, whose expression specifically labels basal cells, in the background of a previously described hPSC line harbouring an NKX2-1GFP reporter allele. The functionality and specificity of the NKX2-1GFP;TP63tdTomato hPSC line was validated by directed differentiation into lung progenitors as well as more specialised lung epithelial subtypes using an organoid platform. This dual fluorescent reporter hPSC line will be useful for tracking, isolating and expanding basal cells from heterogenous differentiation cultures for further study.
Asunto(s)
Proteínas Fluorescentes Verdes/análisis , Proteínas Luminiscentes/análisis , Pulmón/citología , Células Madre Pluripotentes/citología , Factor Nuclear Tiroideo 1/análisis , Factores de Transcripción/análisis , Proteínas Supresoras de Tumor/análisis , Línea Celular , Proteínas Fluorescentes Verdes/genética , Humanos , Proteínas Luminiscentes/genética , Pulmón/metabolismo , Organoides/citología , Organoides/metabolismo , Células Madre Pluripotentes/metabolismo , Factor Nuclear Tiroideo 1/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Proteína Fluorescente RojaRESUMEN
Werner Syndrome (WS) and Bloom Syndrome (BS) are disorders of DNA damage repair caused by biallelic disruption of the WRN or BLM DNA helicases respectively. Both are commonly associated with insulin resistant diabetes, usually accompanied by dyslipidemia and fatty liver, as seen in lipodystrophies. In keeping with this, progressive reduction of subcutaneous adipose tissue is commonly observed. To interrogate the underlying cause of adipose tissue dysfunction in these syndromes, CRISPR/Cas9 genome editing was used to generate human pluripotent stem cell (hPSC) lacking either functional WRN or BLM helicase. No deleterious effects were observed in WRN-/- or BLM-/- embryonic stem cells, however upon their differentiation into adipocyte precursors (AP), premature senescence emerged, impairing later stages of adipogenesis. The resulting adipocytes were also found to be senescent, with increased levels of senescent markers and senescence-associated secretory phenotype (SASP) components. SASP components initiate and reinforce senescence in adjacent cells, which is likely to create a positive feedback loop of cellular senescence within the adipocyte precursor compartment, as demonstrated in normal ageing. Such a scenario could progressively attenuate adipose mass and function, giving rise to "lipodystrophy-like" insulin resistance. Further assessment of pharmacological senolytic strategies are warranted to mitigate this component of Werner and Bloom syndromes.
Asunto(s)
Adipocitos/metabolismo , Síndrome de Bloom , Senescencia Celular , Células Madre Embrionarias Humanas/metabolismo , Modelos Biológicos , Síndrome de Werner , Adipocitos/patología , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Síndrome de Bloom/patología , Sistemas CRISPR-Cas , Línea Celular , Eliminación de Gen , Células Madre Embrionarias Humanas/patología , Humanos , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Síndrome de Werner/patologíaRESUMEN
The liver, lung, pancreas, and digestive tract all originate from the endoderm germ layer, and these vital organs are subject to many life-threatening diseases affecting millions of patients. However, primary cells from endodermal organs are often difficult to grow in vitro. Human pluripotent stem cells thus hold great promise for generating endoderm cells and their derivatives as tools for the development of new therapeutics against a variety of global healthcare challenges. Here we describe recent advances in methods for generating endodermal cell types from human pluripotent stem cells and their use for disease modeling and cell-based therapy.
Asunto(s)
Endodermo/citología , Endodermo/metabolismo , Modelos Biológicos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , HumanosRESUMEN
Adipose tissue is the primary tissue affected in most single gene forms of severe insulin resistance, and growing evidence has implicated it as a site at which many risk alleles for insulin resistance identified in population-wide studies might exert their effect. There is thus increasing need for human adipocyte models in which to interrogate the function of known and emerging genetic risk variants. However, primary adipocyte cultures, existing immortalised cell lines and stem-cell based models all have significant biological or practical limitations. In an attempt to widen the repertoire of human cell models in which to study adipocyte-autonomous effects of relevant human genetic variants, we have undertaken direct reprogramming of skin fibroblasts to adipocyte-like cells by employing an inducible recombinant lentivirus overexpressing the master adipogenic transcription factor PPARγ2. Doxycycline-driven expression of PPARγ2 and adipogenic culture conditions converted dermal fibroblasts into triglyceride-laden cells within days. The resulting cells recapitulated most of the crucial aspects of adipocyte biology in vivo, including the expression of mature adipocyte markers, secreted high levels of the adipokine adiponectin, and underwent lipolysis when treated with isoproterenol/3-isobutyl-1-methylxanthine (IBMX). They did not, however, exhibit insulin-inducible glucose uptake, and withdrawal of doxycycline produced rapid delipidation and loss of adipogenic markers. This protocol was applied successfully to a panel of skin cells from individuals with monogenic severe insulin resistance; however, surprisingly, even cell lines harbouring mutations causing severe, generalised lipodystrophy accumulated large lipid droplets and induced adipocyte-specific genes. The direct reprogramming protocol of human dermal fibroblasts to adipocyte-like cells we established is simple, fast and efficient, and has the potential to generate cells which can serve as a tool to address some, though not all, aspects of adipocyte function in the presence of endogenous disease-causing mutations.
Asunto(s)
Adipocitos/patología , Reprogramación Celular , Dermis/patología , Fibroblastos/patología , Enfermedades Metabólicas/patología , Modelos Biológicos , Humanos , Mutación/genética , PPAR gamma/genética , PPAR gamma/metabolismo , Células Madre/patologíaRESUMEN
DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for lagging-strand DNA synthesis during DNA replication. It carries out this synthesis with high fidelity owing to its intrinsic 3'- to 5'-exonuclease activity, which confers proofreading ability. Missense mutations affecting the exonuclease domain of POLD1 have recently been shown to predispose to colorectal and endometrial cancers. Here we report a recurring heterozygous single-codon deletion in POLD1 affecting the polymerase active site that abolishes DNA polymerase activity but only mildly impairs 3'- to 5'-exonuclease activity. This mutation causes a distinct multisystem disorder that includes subcutaneous lipodystrophy, deafness, mandibular hypoplasia and hypogonadism in males. This discovery suggests that perturbing the function of the ubiquitously expressed POLD1 polymerase has unexpectedly tissue-specific effects in humans and argues for an important role for POLD1 function in adipose tissue homeostasis.
Asunto(s)
Anomalías Múltiples/genética , Dominio Catalítico/genética , ADN Polimerasa III/genética , Lipodistrofia/genética , Sistemas de Lectura , Eliminación de Secuencia , Anomalías Múltiples/diagnóstico , Animales , Línea Celular , ADN Polimerasa III/química , Activación Enzimática/genética , Facies , Fibrosis , Humanos , Lipodistrofia/complicaciones , Imagen por Resonancia Magnética , Masculino , Ratones , Modelos Moleculares , Fenotipo , Conformación Proteica , Grasa Subcutánea Abdominal/patología , SíndromeRESUMEN
Embryonic stem cells (ESCs) depend on extensive regulatory networks to coordinate their self-renewal and differentiation. The polyamine pathway regulator AMD1 was recently implicated in ESC self-renewal and directed differentiation of ESCs to neural precursor cells (NPCs). The polyamines spermine and spermidine are essential for a wide range of biological processes, and their levels are tightly regulated. Here, we review the polyamine pathway and discuss how it can impact polyamine levels, cellular methylation and hypusinated EIF5A levels. We discuss how it could feed into regulation of ESC self-renewal and directed differentiation. We show that in addition to AMD1, a second rate-limiting enzyme in the polyamine pathway, ODC1, can also promote ESC self-renewal, and that both Amd1 and Odc1 can partially substitute for Myc during cellular reprogramming. We propose that both Amd1 and Odc1 are essential regulators of ESCs and function to ensure high polyamine levels to promote ESC self-renewal.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Poliaminas/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Humanos , Transducción de SeñalRESUMEN
Neural cell adhesion molecule (NCAM) has recently been found on adult stem cells, but its biological significance remains largely unknown. In this study, we used bone-marrow-derived mesenchymal stem cells (MSCs) from wild-type and NCAM knockout mice to investigate the role of NCAM in adipocyte differentiation. It was demonstrated that NCAM isoforms 180 and 140 but not NCAM-120 are expressed on almost all wild-type MSCs. Upon adipogenic induction, Ncam(-/-) MSCs exhibited a marked decrease in adipocyte differentiation compared with wild-type cells. The role of NCAM in adipocyte differentiation was also confirmed in NCAM-silenced preadipocyte 3T3-L1 cells, which also had a phenotype with reduced adipogenic potential. In addition, we found that Ncam(-/-) MSCs appeared to be insulin resistant, as shown by their impaired insulin signaling cascade, such as the activation of the insulin-IGF-1 receptor, PI3K-Akt and CREB pathways. The PI3K-Akt inhibitor, LY294002, completely blocked adipocyte differentiation of MSCs, unveiling that the reduced adipogenic potential of Ncam(-/-) MSCs is due to insulin resistance as a result of loss of NCAM function. Furthermore, insulin resistance of Ncam(-/-) MSCs was shown to be associated with induction of tumor necrosis factor α (TNF-α), a key mediator of insulin resistance. Finally, we demonstrated that re-expression of NCAM-180, but not NCAM-140, inhibits induction of TNF-α and thereby improves insulin resistance and adipogenic potential of Ncam(-/-) MSCs. Our results suggest a novel role of NCAM in promoting insulin signaling and adipocyte differentiation of adult stem cells. These findings raise the possibility of using NCAM intervention to improve insulin resistance.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Diferenciación Celular/fisiología , Insulina/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Células 3T3-L1 , Animales , Western Blotting , Diferenciación Celular/genética , Línea Celular , Citometría de Flujo , Inmunoprecipitación , Resistencia a la Insulina/genética , Resistencia a la Insulina/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Moléculas de Adhesión de Célula Nerviosa/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
DNA damage triggers a network of signaling events that leads to cell cycle arrest or apoptosis. This DNA damage response acts as a mechanism to prevent cancer development. It has been reported that fatty acids (FAs) synthesis is increased in many human tumors while inhibition of fatty acid synthase (FASN) could suppress tumor growth. Here we report that saturated fatty acids (SFAs) play a negative role in DNA damage response. Palmitic acid, as well as stearic acid and myristic acid, compromised the induction of p21 and Bax expression in response to double stranded breaks and ssDNA, while inhibition or knockdown of FASN enhanced these cellular events. SFAs appeared to regulate p21 and Bax expression via Atr-p53 dependent and independent pathways. These effects were only observed in primary mouse embryonic fibroblasts and osteoblasts, but not in immortalized murine NIH3T3, or transformed HCT116 and MCF-7 cell lines. Accordingly, SFAs showed some positive effects on proliferation of MEFs in response to DNA damage. These results suggest that SFAs, by negatively regulating the DNA damage response pathway, might promote cell transformation, and that increased synthesis of SFAs in precancer/cancer cells might contribute to tumor progression and drug resistance.