RESUMEN
Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors1. Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2V617F (JAK2VF) mutation, which increases JAK-STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease1,2. Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2VF selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2VF lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2VF lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1ß reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2VF macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1ß or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk.
Asunto(s)
Aterosclerosis/patología , Hematopoyesis Clonal , Proteínas de Unión al ADN/metabolismo , Inflamasomas/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/inmunología , Médula Ósea/metabolismo , Caspasa 1/metabolismo , Caspasas Iniciadoras/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/metabolismo , Inflamación/patología , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Interleucina-1beta/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión a Fosfato/metabolismo , Piroptosis , RNA-Seq , Análisis de la Célula IndividualRESUMEN
Although strategies for directed differentiation of human pluripotent stem cells (hPSCs) into lung and airway have been established, terminal maturation of the cells remains a vexing problem. We show here that in collagen I 3D cultures in the absence of glycogen synthase kinase 3 (GSK3) inhibition, hPSC-derived lung progenitors (LPs) undergo multilineage maturation into proximal cells, type I alveolar epithelial cells and morphologically mature type II cells. Enhanced cell cycling, one of the signaling outputs of GSK3 inhibition, plays a role in the maturation-inhibiting effect of GSK3 inhibition. Using this model, we show NOTCH signaling induced a distal cell fate at the expense of a proximal and ciliated cell fate, whereas WNT signaling promoted a proximal club cell fate, thus implicating both signaling pathways in proximodistal specification in human lung development. These findings establish an approach to achieve multilineage maturation of lung and airway cells from hPSCs, demonstrate a pivotal role of GSK3 in the maturation of lung progenitors and provide novel insight into proximodistal specification during human lung development.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Linaje de la Célula , Glucógeno Sintasa Quinasa 3/metabolismo , Células Madre Pluripotentes Inducidas/citología , Pulmón/citología , Piridinas/farmacología , Animales , Tipificación del Cuerpo/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Colágeno Tipo I/metabolismo , Genoma Humano , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/ultraestructura , Ratones , Receptores Notch/metabolismo , Reproducibilidad de los Resultados , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Pluripotent and post-natal, tissue-specific stem cells share functional features such as the capacity to differentiate into multiple lineages and to self-renew, and are endowed with specific cell maintenance mechanism as well as transcriptional and epigenetic signatures that determine stem cell identity and distinguish them from their progeny. Calcium is a highly versatile and ubiquitous second messenger that regulates a wide variety of cellular functions. Specific roles of calcium in stem cell niches and stem cell maintenance mechanisms are only beginning to be explored, however. In this review, I discuss stem cell-specific regulation and roles of calcium, focusing on its potential involvement in the intertwined metabolic and epigenetic regulation of stem cells.
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Calcio , Epigénesis Genética , Diferenciación Celular , Células MadreRESUMEN
Haematopoietic stem cells (HSCs), which sustain production of all blood cell lineages, rely on glycolysis for ATP production, yet little attention has been paid to the role of mitochondria. Here we show in mice that the short isoform of a critical regulator of HSCs, Prdm16 (refs 4, 5), induces mitofusin 2 (Mfn2), a protein involved in mitochondrial fusion and in tethering of mitochondria to the endoplasmic reticulum. Overexpression and deletion studies, including single-cell transplantation assays, revealed that Mfn2 is specifically required for the maintenance of HSCs with extensive lymphoid potential, but not, or less so, for the maintenance of myeloid-dominant HSCs. Mfn2 increased buffering of intracellular Ca(2+), an effect mediated through its endoplasmic reticulum-mitochondria tethering activity, thereby negatively regulating nuclear translocation and transcriptional activity of nuclear factor of activated T cells (Nfat). Nfat inhibition rescued the effects of Mfn2 deletion in HSCs, demonstrating that negative regulation of Nfat is the prime downstream mechanism of Mfn2 in the maintenance of HSCs with extensive lymphoid potential. Mitochondria therefore have an important role in HSCs. These findings provide a mechanism underlying clonal heterogeneity among HSCs and may lead to the design of approaches to bias HSC differentiation into desired lineages after transplantation.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Linfocitos/citología , Transporte Activo de Núcleo Celular , Animales , Calcio/metabolismo , Señalización del Calcio , Diferenciación Celular , Linaje de la Célula , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Femenino , Fibroblastos , Linfocitos/metabolismo , Masculino , Ratones , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Células Mieloides/citología , Factores de Transcripción NFATC/antagonistas & inhibidores , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Directed differentiation of human pluripotent stem cells (hPSCs) into mature cells, tissues and organs holds major promise for the development of novel approaches in regenerative medicine, and provides a unique tool for disease modeling and drug discovery. Sometimes underappreciated is the fact that directed differentiation of hPSCs also provides a unique model for human development, with a number of important advantages over model organisms. Here, I discuss the importance of using human stem cell models for understanding human lung development and disease.
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Enfermedades Pulmonares/embriología , Pulmón/embriología , Pulmón/patología , Modelos Biológicos , Células Madre Pluripotentes/patología , HumanosRESUMEN
The B-myb (MYBL2) gene is a member of the MYB family of transcription factors and is involved in cell cycle regulation, DNA replication, and maintenance of genomic integrity. However, its function during adult development and hematopoiesis is unknown. We show here that conditional inactivation of B-myb in vivo results in depletion of the hematopoietic stem cell (HSC) pool, leading to profound reductions in mature lymphoid, erythroid, and myeloid cells. This defect is autonomous to the bone marrow and is first evident in stem cells, which accumulate in the S and G2/M phases. B-myb inactivation also causes defects in the myeloid progenitor compartment, consisting of depletion of common myeloid progenitors but relative sparing of granulocyte-macrophage progenitors. Microarray studies indicate that B-myb-null LSK(+) cells differentially express genes that direct myeloid lineage development and commitment, suggesting that B-myb is a key player in controlling cell fate. Collectively, these studies demonstrate that B-myb is essential for HSC and progenitor maintenance and survival during hematopoiesis.
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Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/fisiología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/fisiología , Células Progenitoras Mieloides/fisiología , Transactivadores/metabolismo , Animales , Trasplante de Médula Ósea , Cruzamientos Genéticos , Cartilla de ADN/genética , Citometría de Flujo , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We review recent progress in the stem cell biology of the respiratory system, and discuss its scientific and translational ramifications. Several studies have defined novel stem cells in postnatal lung and airways and implicated their roles in tissue homeostasis and repair. In addition, significant advances in the generation of respiratory epithelium from pluripotent stem cells (PSCs) now provide a novel and powerful platform for understanding lung development, modeling pulmonary diseases, and implementing drug screening. Finally, breakthroughs have been made in the generation of decellularized lung matrices that can serve as a scaffold for repopulation with respiratory cells derived from either postnatal or PSCs. These studies are a critical step forward towards the still distant goal of stem cell-based regenerative medicine for diseases of lung and airways.
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Diferenciación Celular , Células Epiteliales/citología , Epitelio/crecimiento & desarrollo , Sistema Respiratorio/citología , Células Madre/citología , Animales , HumanosRESUMEN
PURPOSE OF REVIEW: Aging of the hematopoietic system is associated with myeloid malignancies, anemia and immune dysfunction. As hematopoietic stem cells (HSCs) generate all cells of the hematopoietic system, age-associated changes in HSCs may underlie many features of the aged hematopoietic system. Recent findings on age-associated changes in HSCs are reviewed here. RECENT FINDINGS: Aged HSCs are myeloid biased, have acquired DNA damage and are functionally compromised. However, overall function of the HSC compartment is well maintained through age-associated expansion of HSCs. Many age-related changes in the hematopoietic system, in particular the clonal myeloid bias of HSCs and the decrease in B and T-cell development, in fact begin during development. Furthermore, HSCs possess specific protective mechanisms aimed at maintaining their number, even at the expense of accumulating damaged cells. SUMMARY: We argue that age-related changes in HSCs and in the hematopoietic system may not entirely be due to a degenerative aging process, but are the result of developmental and stem cell-protective mechanisms aimed at maximizing fitness during reproductive life. These mechanisms may be disadvantageous later in life as damaged HSCs accumulate and establishment of responses to neoantigens becomes compromised because of the reduced generation of naive T and B cells.
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Envejecimiento/fisiología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/fisiología , Recuento de Células , Diferenciación Celular/fisiología , Proliferación Celular , Senescencia Celular/fisiología , HumanosRESUMEN
In contrast to most hematopoietic lineages, megakaryocytes (MKs) can derive rapidly and directly from hematopoietic stem cells (HSCs). The underlying mechanism is unclear, however. Here, we show that DNA damage induces MK markers in HSCs and that G2 arrest, an integral part of the DNA damage response, suffices for MK priming followed by irreversible MK differentiation in HSCs, but not in progenitors. We also show that replication stress causes DNA damage in HSCs and is at least in part due to uracil misincorporation in vitro and in vivo. Consistent with this notion, thymidine attenuated DNA damage, improved HSC maintenance, and reduced the generation of CD41+ MK-committed HSCs. Replication stress and concomitant MK differentiation is therefore one of the barriers to HSC maintenance. DNA damage-induced MK priming may allow rapid generation of a lineage essential to immediate organismal survival, while also removing damaged cells from the HSC pool.
Asunto(s)
Diferenciación Celular , Daño del ADN , Células Madre Hematopoyéticas , Megacariocitos , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Animales , Ratones , Megacariocitos/metabolismo , Megacariocitos/citología , Trombopoyesis , Puntos de Control de la Fase G2 del Ciclo Celular , Ratones Endogámicos C57BL , HumanosRESUMEN
Hematopoiesis is the process whereby BM HSCs renew to maintain their number or to differentiate into committed progenitors to generate all blood cells. One approach to gain mechanistic insight into this complex process is the investigation of quantitative genetic variation in hematopoietic function among inbred mouse strains. We previously showed that TGF-ß2 is a genetically determined positive regulator of hematopoiesis. In the presence of unknown nonprotein serum factors TGF-ß2, but not TGF-ß1 or -ß3, enhances progenitor proliferation in vitro, an effect that is subject to mouse strain-dependent variation mapping to a locus on chr.4, Tb2r1. TGF-ß2-deficient mice show hematopoietic defects, demonstrating the physiologic role of this cytokine. Here, we show that TGF-ß2 specifically and predominantly cell autonomously enhances signaling by FLT3 in vitro and in vivo. A coding polymorphism in Prdm16 (PR-domain-containing 16) underlies Tb2r1 and differentially regulates transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ), identifying lipid PPAR ligands as the serum factors required for regulation of FLT3 signaling by TGF-ß2. We furthermore show that PPARγ agonists play a FLT3-dependent role in stress responses of progenitor cells. These observations identify a novel regulatory axis that includes PPARs, Prdm16, and TGF-ß2 in hematopoiesis.
Asunto(s)
Proteínas de Unión al ADN/genética , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/fisiología , PPAR gamma/genética , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta2/genética , Tirosina Quinasa 3 Similar a fms/genética , Animales , Células COS , Diferenciación Celular/fisiología , División Celular/fisiología , Chlorocebus aethiops , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/fisiología , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , PPAR gamma/agonistas , PPAR gamma/metabolismo , Polimorfismo Genético/fisiología , Sitios de Carácter Cuantitativo/fisiología , Estrés Fisiológico/fisiología , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Fetal liver and adult bone marrow hematopoietic stem cells (HSCs) renew or differentiate into committed progenitors to generate all blood cells. PRDM16 is involved in human leukemic translocations and is expressed highly in some karyotypically normal acute myeloblastic leukemias. As many genes involved in leukemogenic fusions play a role in normal hematopoiesis, we analyzed the role of Prdm16 in the biology of HSCs using Prdm16-deficient mice. We show here that, within the hematopoietic system, Prdm16 is expressed very selectively in the earliest stem and progenitor compartments, and, consistent with this expression pattern, is critical for the establishment and maintenance of the HSC pool during development and after transplantation. Prdm16 deletion enhances apoptosis and cycling of HSCs. Expression analysis revealed that Prdm16 regulates a remarkable number of genes that, based on knockout models, both enhance and suppress HSC function, and affect quiescence, cell cycling, renewal, differentiation, and apoptosis to various extents. These data suggest that Prdm16 may be a critical node in a network that contains negative and positive feedback loops and integrates HSC renewal, quiescence, apoptosis, and differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Factores de Transcripción/metabolismo , Animales , Apoptosis/fisiología , Separación Celular , Proteínas de Unión al ADN/genética , Citometría de Flujo , Expresión Génica , Perfilación de la Expresión Génica , Genotipo , Células Madre Hematopoyéticas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
Human lungs contain unique cell populations in distal respiratory airways (RAs). These populations accumulate in patients with lung injury, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Their lineage potentials and roles are unknown, however. As they are absent in rodents, deeper understanding of these cells requires a human in vitro model. Here we report the generation from human pluripotent stem cells (hPSCs) of expandable spheres (induced respiratory airway progenitors (iRAPs)) consisting of all RA-associated cell types. iRAPs could differentiate into type 1 (AT1) and type 2 alveolar (AT2) epithelial cells in defined conditions, showing that alveolar cells can be derived from RAs. iRAPs with deletion of HPS1, which causes pulmonary fibrosis in humans, display defects that are hallmarks of IPF, indicating involvement of intrinsic dysfunction of RA-associated cells in IPF. iRAPs thus provide a model to gain insight into human lung regeneration and into pathogenesis of IPF.
RESUMEN
Hematopoietic stem cells (HSCs) reside in the bone marrow (BM), can self-renew, and generate all cells of the hematopoietic system. 1 Most hematopoietic lineages arise through successive, increasingly lineage-committed progenitors. In contrast, megakaryocytes (MKs), hyperploid cells that generate platelets essential to hemostasis, can derive rapidly and directly from HSCs. 2 The underlying mechanism is unknown however. Here we show that DNA damage and subsequent arrest in the G2 phase of the cell cycle rapidly induce MK commitment specifically in HSCs, but not in progenitors, through an initially predominantly post-transcriptional mechanism. Cycling HSCs show extensive replication-induced DNA damage associated with uracil misincorporation in vivo and in vitro . Consistent with this notion, thymidine attenuated DNA damage, rescued HSC maintenance and reduced the generation of CD41 + MK-committed HSCs in vitro . Similarly, overexpression of the dUTP-scavenging enzyme, dUTPase, enhanced in vitro maintenance of HSCs. We conclude that a DNA damage response drives direct megakaryopoiesis and that replication stress-induced direct megakaryopoiesis, at least in part caused by uracil misincorporation, is a barrier to HSC maintenance in vitro . DNA damage-induced direct megakaryopoiesis may allow rapid generation of a lineage essential to immediate organismal survival, while simultaneously removing damaged HSCs and potentially avoiding malignant transformation of self-renewing stem cells.
RESUMEN
Organoids have been an exciting advancement in stem cell research. Here we describe a strategy for directed differentiation of human pluripotent stem cells into distal lung organoids. This protocol recapitulates lung development by sequentially specifying human pluripotent stem cells to definitive endoderm, anterior foregut endoderm, ventral anterior foregut endoderm, lung bud organoids and finally lung organoids. The organoids take ~40 d to generate and can be maintained more than 180 d, while progressively maturing up to a stage consistent with the second trimester of human gestation. They are unique because of their branching morphology, the near absence of non-lung endodermal lineages, presence of mesenchyme and capacity to recapitulate interstitial lung diseases. This protocol can be performed by anyone familiar with cell culture techniques, is conducted in serum-free conditions and does not require lineage-specific reporters or enrichment steps. We also provide a protocol for the generation of single-cell suspensions for single-cell RNA sequencing.
Asunto(s)
Enfermedades Pulmonares Intersticiales , Células Madre Pluripotentes , Virosis , Humanos , Pulmón , Organoides , Diferenciación CelularRESUMEN
Although lung disease is a major cause of mortality, the mechanisms involved in human lung regeneration are unclear because of the lack of experimental models. Here we report a novel model where human pluripotent stem cell-derived expandable cell lines sharing features of airway secretory and basal cells engraft in the distal rat lung after conditioning by locoregional de-epithelialization followed by irradiation and immunosuppression. The engrafting cells, which we named distal lung epithelial progenitors (DLEPs), contributed to alveolar epithelial cells and generated 'KRT5-pods', structures involved in distal lung repair after severe injury, but only rarely to distal airways. Most strikingly, however, injury induced by the conditioning regimen was largely prevented by the engrafting DLEPs. The approach described here provides a model to study mechanisms involved in human lung regeneration, and potentially lays the foundation for the preclinical development of cell therapy to treat lung injury and disease.
RESUMEN
B cells, the Ab-producing cells of the immune system, develop from hematopoietic stem cells (HSCs) through well-defined stages during which Ig genes are rearranged to generate a clonal BCR. Signaling through the BCR plays a role in the subsequent cell fate decisions leading to the generation of three distinct types of B cells: B1, marginal zone, and follicular B cells. Common lymphoid progenitors (CLPs) are descended from HSCs, and although recent observations suggest that CLPs may not be physiological T cell precursors, it is generally accepted that CLPs are obligate progenitors for B cells. In addition, a CLP-like progenitor of unknown significance that lacks expression of c-kit (kit(-)CLP) was recently identified in the mouse model. In this study, we show that CLPs, kit(-)CLPs and a population within the lin(-)Sca1(+)kit(+)flt3(-) HSC compartment generate mature B cell types in different proportions: CLPs and kit(-)CLPs show a stronger marginal zone/follicular ratio than lin(-)Sca1(+)kit(+)flt3(-) cells, whereas kit(-)CLPs show a stronger B1 bias than any other progenitor population. Furthermore, expression of Sca1 on B cells depends on their progenitor origin as B cells derived from CLPs and kit(-)CLPs express more Sca1 than those derived from lin(-)Sca1(+)kit(+)flt3(-) cells. These observations indicate a role for progenitor origin in B cell fate choices and suggest the existence of CLP-independent B cell development.
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Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/inmunología , Animales , Líquido Ascítico/citología , Líquido Ascítico/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-kit/metabolismo , Bazo/citología , Bazo/inmunologíaAsunto(s)
Señales (Psicología) , Queratinocitos , Diferenciación Celular , Células Cultivadas , HumanosRESUMEN
Pluripotent stem cells have the capacity to generate all cell lineages, and substantial progress has been made in realizing this potential. One fascinating but as yet unrealized possibility is the differentiation of pluripotent stem cells into thymic epithelial cells. The thymus is a primary lymphoid organ essential for naïve T-cell generation. T cells play an important role in adaptive immunity, and their loss or dysfunction underlies in a wide range of autoimmune and infectious diseases. T cells are generated and selected through interaction with thymic epithelial cells, the functionally essential element of thymus. The ability to generate functional thymic epithelial cells from pluripotent stem cells would have applications in modeling human immune responses in mice, in tissue transplantation, and in modulating autoimmune and infectious disease.
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Células Madre Pluripotentes/trasplante , Linfocitos T/inmunología , Timo/fisiología , Inmunidad Adaptativa , Animales , Enfermedades Transmisibles/inmunología , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here, we describe a strategy for directed differentiation of hPSCs into mature lung and airway epithelial cells obtained through maturation of NKX2.1+ hPSC-derived lung progenitors in a 3D matrix of collagen I in the absence of glycogen synthase kinase 3 inhibition. This protocol is an extension of our previously published protocol on the directed differentiation of lung and airway epithelium from hPSCs that modifies the technique and offers additional applications. This protocol is conducted in defined media conditions, has a duration of 50-80 d, does not require reporter lines and results in cultures containing mature alveolar type II and I cells as well as airway basal, ciliated, club and neuroendocrine cells. We also present a flow cytometry strategy to assess maturation in the cultures. Several of these populations, including mature NGFR+ basal cells, can be prospectively isolated by cell sorting and expanded for further investigation.
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Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Linaje de la Célula , Imagenología Tridimensional , Pulmón/citología , Células Madre Pluripotentes/citología , Animales , Biomarcadores/metabolismo , Células Cultivadas , Endodermo/citología , Células Madre Embrionarias Humanas/citología , Humanos , Ratones , Virus de la Parainfluenza 3 Humana/fisiologíaRESUMEN
Elucidation of pathways involved in mouse strain-dependent variation in the hematopoietic stem cell (HSC) compartment may reveal novel mechanisms relevant in vivo. Here, we demonstrate genetically determined variation in the proliferation of lin-Sca1++kit+ (LSK) primitive hematopoietic progenitor cells in response to transforming growth factor-beta (TGF-beta) 2, the dose response of which was biphasic with a stimulatory effect at low concentrations. In contrast, the dose responses of TGF-beta1 or -beta3 were inhibitory and did not show mouse strain-dependent variation. A quantitative trait locus (QTL) for the effect of TGF-beta2 was identified on chromosome 4 overlapping with a QTL regulating the frequency of LSK cells. These overlapping QTL were corroborated by the observation that the frequency of LSK cells is lower in adult Tgfb2+/- mice than in wild-type littermates, indicating that TGF-beta2 is a genetically determined positive regulator LSK number in vivo. Furthermore, adult Tgfb2+/- mice have a defect in competitive repopulation potential that becomes more pronounced upon serial transplantation. In fetal TGF-beta2-deficient HSCs, a defect only appears after serial reconstitution. These data suggest that TGF-beta2 can act cell autonomously and is important for HSCs that have undergone replicative stress. Thus, TGF-beta2 is a novel, genetically determined positive regulator of adult HSCs.