RESUMEN

OBJECTIVE: This article describes an interdisciplinary treatment that helped a patient with displaced upper anterior teeth. A gingivectomy, root canal therapies, digital smile design, digital wax-up, and guided tooth preparations were applied. CLINICAL CONSIDERATIONS: A patient with pathologically migrated teeth asked for treatment without orthodontic involvement due to a primary failed orthodontic treatment history. A smile photo was taken and superimposed with the dentition in a CAD software to accomplish a digital smile design. The jaw movements were recorded with two different methods, a mechanical articulator and an intraoral scanner with Patient-Specific-Motion function. The occlusal contacts during protrusive and lateral movements were compared and the digital wax-up was designed according to the later occlusal data. An aesthetic crown lengthening and pre-op root canal treatment were carried out in advance accordingly. After guided tooth preparation with a silicone index, the final fixed restorations were manufactured and cemented. A 2-year follow-up showed that our prosthesis functions well. CONCLUSIONS: This clinical report revealed that an intraoral scanner with Patient-Specific-Motion function can effectively record individual dynamic occlusal patterns and these data can be integrated into the CAD/CAM process to enhance the fulfillment of clinical requirements. CLINICAL SIGNIFICANCE: This clinical procedure with a 2-year follow-up demonstrated that a prosthodontic-based interdisciplinary treatment of pathologically migrated teeth using dynamic occlusal recording with an intraoral scanner could achieve satisfactory esthetics in a relatively short treatment period. The Patient Specific Motion module may be used to record a personalized functional movement and the data can be integrated into the design process of the final restorations.

RESUMEN

Bronchiolitis obliterans syndrome (BOS) is a common and potentially devastating noninfectious pulmonary complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Currently, predictive tools for BOS are not available. We aimed to identify the clinical risk factors and establish a prognostic model for BOS in patients who undergo allo-HSCT. We retrospectively identified a cohort comprising 195 BOS patients from 6100 consecutive patients who were allografted between 2008 and 2022. The entire cohort was divided into a derivation cohort and a validation cohort based on the time of transplantation. Via multivariable Cox regression methods, declining forced expiratory volume at 1 s (FEV1) to <40%, pneumonia, cGVHD except lung, and respiratory failure were found to be independent risk factors for the 3-year mortality of BOS. A risk score called FACT was constructed based on the regression coefficients. The FACT model had an AUC of 0.863 (95% CI: 0.797-0.928) in internal validation and 0.749 (95% CI: 0.621-0.876) in external validation. The calibration curves showed good agreement between the FACT-predicted probabilities and actual observations. The FACT risk score will help to identify patients at high risk and facilitate future research on developing novel, effective interventions to personalize treatment.

Asunto(s)

Síndrome de Bronquiolitis Obliterante , Bronquiolitis Obliterante , Trasplante de Células Madre Hematopoyéticas , Humanos , Bronquiolitis Obliterante/terapia , Pronóstico , Estudios Retrospectivos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Factores de Riesgo

RESUMEN

Septic shock remains a potentially life-threatening complication among allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients. There is a paucity of information on the clinical characteristics, outcome and prognostic factors of septic shock patients after allo-HSCT. We aimed to describe the clinical characteristics of septic shock after allo-HSCT and its associated health outcomes and to evaluate the role of patient demographics, transplantation-related laboratory and clinical variables associated with the short-term mortality of septic shock after allo-HSCT. We retrospectively studied 242 septic shock patients from 6105 consecutive patients allografted between 2007 and 2021. We assessed 29 risk factors as candidate predictors and used multivariable logistic regression to establish clinical model. The primary outcome was 28-day mortality. The median age of the subjects was 34 (IQR 24 to 45) years. A total of 148 patients (61.2%) had positive blood cultures. Gram-negative bacilli accounted for 61.5% of the positive isolates, gram-positive cocci accounted for 12.2%, and fungi accounted for 6.1%. Coinfections were found in 30 (20.3%) patients. Escherichia coli was the dominant isolated pathogen (31.1%), followed by Pseudomonas spp. (12.8%) and Klebsiella pneumoniae (10.1%). With a median follow-up of 34 (IQR: 2 to 528) days, a total of 142 (58.7%) patients died, of whom 118 (48.8%) died within the first 28 days after septic shock diagnosis, 131 (54.1%) died within 90 days, and 141 (58.3%) died within 1 year. A large majority of deaths (83.1% [118/142]) occurred within 28 days of septic shock diagnosis. Finally, 6 independent predictive variables of 28-day mortality were identified by multivariable logistic regression: time of septic shock, albumin, bilirubin, PaO2/FiO2, lactate, and sepsis-induced coagulopathy. Patients with late onset shock had higher 28-day mortality rates (64.6% versus 25.5%, P < .001) and more ICU admission (32.6% versus 7.1%, P < .001) than those with early onset shock. We highlight the poor survival outcomes in patients who develop septic shock, emphasizing the need for increasing awareness regarding septic shock after allo-HSCT. The information from the current study may help to assist clinicians in identifying high-risk patients.

Asunto(s)

Trasplante de Células Madre Hematopoyéticas , Choque Séptico , Humanos , Adulto Joven , Adulto , Persona de Mediana Edad , Pronóstico , Choque Séptico/etiología , Estudios Retrospectivos , Trasplante Homólogo/efectos adversos , Trasplante de Células Madre Hematopoyéticas/efectos adversos

RESUMEN

BACKGROUND: Pulmonary arteriovenous malformation (PAVM) is an abnormal communication between pulmonary vasculatures and has an unclear boundary with surrounding lung tissues. At present, surgeons can only determine its location by preoperative imaging and intraoperative palpation, despite its soft texture. Indocyanine green(ICG), a near-infrared fluorophore, has been demonstrated useful in the accurate identification of vascular tissue. Therefore, we explored its application in PAVM cases. CASE PRESENTATION: We present two PAVM cases using near-infrared fluorescence (NIF) with 25 mg ICG at 5 mg/ml to achieve intraoperative visualization of the lesion in video-assisted thoracoscopic surgery (VATS). Under the NIF mode, ICG systemic injection led to successive signaling of the anomaly and normal tissues in merely 10 s, which helped us distinguish them efficiently and precisely. A peak signal-to-background ratio of 2.2 confirmed the significant fluorescence difference and excluded interference from carbon dust. CONCLUSIONS: We are the first to report the use of such an approach in delineating the margin of vascular malformation with high contrast, and this new finding may help minimize the damage to lung function in PAVM treatment. Further exploration and validation are needed to determine its role.

Asunto(s)

Fístula Arteriovenosa , Verde de Indocianina , Humanos , Cirugía Torácica Asistida por Video/métodos , Fluorescencia , Pulmón

RESUMEN

The hematopoietic niche is a supportive microenvironment composed of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses in zebrafish, we define a conserved gene expression signature and cis-regulatory landscape that are unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code that involves members of the Ets, Sox, and nuclear hormone receptor families and is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance, and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche.

Asunto(s)

Nicho de Células Madre , Factores de Transcripción , Animales , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Endoteliales/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Regulación de la Expresión Génica

RESUMEN

The National Heart, Lung, and Blood Institute Progenitor Cell Translational Consortium Blood Progenitor Meeting was hosted virtually on November 5, 2020, with 93 attendees across 20 research groups. The purpose of this meeting was to exchange recent findings, discuss current efforts, and identify prospective opportunities in the field of hematopoietic stem and progenitor cell research and therapeutic discovery.

Asunto(s)

Células Sanguíneas/citología , Conducta Cooperativa , Células Madre/citología , Microambiente Celular , Hematopoyesis , Humanos , Nicho de Células Madre

RESUMEN

Strontium loaded titania nanotube arrays (NTSr), as well as titania nanotube arrays (NT), have been regarded as effective coatings to promote bone regeneration on titanium implants, but an understanding of the full extent of early processes affected by such surface modifications is absent. To address this limitation, we performed RNA sequencing (RNA-seq) of Sprague-Dawley rat bone marrow mesenchymal stem cells (rBMMSCs) cultured on unmodified titanium sheets (Con), NT and NTSr specimens. By pairwise comparisons we found that NT and NTSr shared a majority of differentially expressed genes. The Gene Ontology (GO) analysis revealed that NT and NTSr up-regulated a bunch of genes that are annotated to the cytoskeleton. The results were supported by immunofluorescent, transmission electron microscopy (TEM) and western blotting assays. By inhibiting the cytoskeleton through pharmacological agents, the activities of alkaline phosphatase (ALP) on NT and NTSr were also suppressed. Informed by these results, we concluded that NT and NTSr specimens reorganized the cytoskeleton of cultured cells that may play a crucial role in osteogenic lineage commitment.

Asunto(s)

Células Madre Mesenquimatosas , Nanotubos , Animales , Diferenciación Celular , Células Cultivadas , Citoesqueleto , Osteogénesis , Ratas , Ratas Sprague-Dawley , Análisis de Secuencia de ARN , Estroncio/farmacología , Titanio/farmacología

RESUMEN

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) exhibit a fetal phenotype that limits in vitro and therapeutic applications. Strategies to promote cardiomyocyte maturation have focused interventions on differentiated hPSC-CMs, but this study tests priming of early cardiac progenitor cells (CPCs) with polyinosinic-polycytidylic acid (pIC) to accelerate cardiomyocyte maturation. CPCs were differentiated from hPSCs using a monolayer differentiation protocol with defined small molecule Wnt temporal modulation, and pIC was added during the formation of early CPCs. pIC priming did not alter the expression of cell surface markers for CPCs (>80% KDR+/PDGFRα+), expression of common cardiac transcription factors, or final purity of differentiated hPSC-CMs (∼90%). However, CPC differentiation in basal medium revealed that pIC priming resulted in hPSC-CMs with enhanced maturity manifested by increased cell size, greater contractility, faster electrical upstrokes, increased oxidative metabolism, and more mature sarcomeric structure and composition. To investigate the mechanisms of CPC priming, RNAseq revealed that cardiac progenitor-stage pIC modulated early Notch signaling and cardiomyogenic transcriptional programs. Chromatin immunoprecipitation of CPCs showed that pIC treatment increased deposition of the H3K9ac activating epigenetic mark at core promoters of cardiac myofilament genes and the Notch ligand, JAG1. Inhibition of Notch signaling blocked the effects of pIC on differentiation and cardiomyocyte maturation. Furthermore, primed CPCs showed more robust formation of hPSC-CMs grafts when transplanted to the NSGW mouse kidney capsule. Overall, epigenetic modulation of CPCs with pIC accelerates cardiomyocyte maturation enabling basic research applications and potential therapeutic uses. Stem Cells 2019;37:910-923.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Epigénesis Genética , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Poli I-C/farmacología , Receptores Notch/genética , Animales , Tamaño de la Célula , Histonas/genética , Histonas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Riñón , Ratones , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Fosforilación Oxidativa , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores Notch/metabolismo , Sarcómeros/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal , Trasplante de Células Madre/métodos , Trasplante Heterotópico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Adult stem cells are responsible for life-long tissue maintenance. They reside in and interact with specialized tissue microenvironments (niches). Using murine hair follicle as a model, we show that when junctional perturbations in the niche disrupt barrier function, adjacent stem cells dramatically change their transcriptome independent of bacterial invasion and become capable of directly signaling to and recruiting immune cells. Additionally, these stem cells elevate cell cycle transcripts which reduce their quiescence threshold, enabling them to selectively proliferate within this microenvironment of immune distress cues. However, rather than mobilizing to fuel new tissue regeneration, these ectopically proliferative stem cells remain within their niche to contain the breach. Together, our findings expose a potential communication relay system that operates from the niche to the stem cells to the immune system and back. The repurposing of proliferation by these stem cells patch the breached barrier, stoke the immune response and restore niche integrity.

Asunto(s)

Proliferación Celular/genética , Perfilación de la Expresión Génica/métodos , Folículo Piloso/metabolismo , Nicho de Células Madre , Células Madre/metabolismo , Animales , Comunicación Celular/genética , Ciclo Celular/genética , Células Cultivadas , Folículo Piloso/citología , Folículo Piloso/ultraestructura , Homeostasis/genética , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Células Madre/citología , Células Madre/ultraestructura

RESUMEN

During heart development, epicardial progenitors contribute various cardiac lineages including smooth muscle cells, cardiac fibroblasts, and endothelial cells. However, their specific contribution to the human endothelium has not yet been resolved, at least in part due to the inability to expand and maintain human primary or pluripotent stem cell (hPSC)-derived epicardial cells. Here we first generated CDH5-2A-eGFP knock-in hPSC lines and differentiated them into self-renewing WT1+ epicardial cells, which gave rise to endothelial cells upon VEGF treatment in vitro. In addition, we found that the percentage of endothelial cells correlated with WT1 expression in a WT1-2A-eGFP reporter line. The resulting endothelial cells displayed many endocardium-like endothelial cell properties, including high expression levels of endocardial-specific markers, nutrient transporters and well-organized tight junctions. These findings suggest that human epicardial progenitors may have the capacity to form endocardial endothelium during development and have implications for heart regeneration and cardiac tissue engineering.

RESUMEN

Here, we describe how to efficiently direct human pluripotent stem cells (hPSCs) differentiation into self-renewing epicardial cells in a completely defined, xeno-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate differentiation-stage-specific application of Gsk3 inhibitor, Wnt inhibitor, and Gsk3 inhibitor (GiWiGi) is sufficient to produce cells expressing epicardial markers and exhibiting epicardial phenotypes with a high yield and purity from multiple hPSC lines in 16 d. Characterization of differentiated cells is performed via flow cytometry and immunostaining to assess quantitative expression and localization of epicardial cell-specific proteins. In vitro differentiation into fibroblasts and smooth muscle cells (SMCs) is also described. In addition, culture in the presence of transforming growth factor (TGF)-ß inhibitors allows long-term expansion of hPSC-derived epicardial cells (for at least 25 population doublings). Functional human epicardial cells differentiated via this protocol may constitute a potential cell source for heart disease modeling, drug screening, and cell-based therapeutic applications.

Asunto(s)

Diferenciación Celular/fisiología , Ingeniería Celular/métodos , Pericardio/citología , Células Madre Pluripotentes/citología , Técnicas de Cultivo de Célula , Humanos , Vía de Señalización Wnt/fisiología

RESUMEN

The topography of biomaterials can significantly influence the osteogenic differentiation of cells. Understanding topographical signal transduction is critical for developing biofunctional surfaces, but the current knowledge is insufficient. Recently, numerous reports have suggested that the unfolded protein response (UPR) and osteogenic differentiation are inter-linked. Therefore, we hypothesize that the UPR pathway may be involved in the topography-induced osteogenesis. In the present study, different surface topographies were fabricated on pure titanium foils and the endoplasmic reticulum (ER) stress and UPR pathway were systematically investigated. We found that ER stress and the PERK-eIF2α-ATF4 pathway were activated in a time- and topography-dependent manner. Additionally, the activation of the PERK-eIF2α-ATF4 pathway by different topographies was in line with their osteogenic induction capability. More specifically, the osteogenic differentiation could be enhanced or weakened when the PERK-eIF2α-ATF4 pathway was promoted or inhibited, respectively. Furthermore, tuning of the degree of ER stress with different concentrations of thapsigargin revealed that mild ER stress promotes osteogenic differentiation, whereas excessive ER stress inhibits osteogenic differentiation and causes apoptosis. Taken together, our findings suggest that the UPR may play a critical role in topography-induced osteogenic differentiation, which may help to provide new insights into topographical signal transduction. STATEMENT OF SIGNIFICANCE: Suitable implant surface topography can effectively improve bioactivity and eventual bone affinity. However, the mechanism of topographical signaling transduction is unclear and criteria for designation of an appropriate implant surface topography is lacking. This study shows that the ER stress and PERK-eIF2α-ATF4 pathway were activated by micro- and micro/nano-topographies, which is corresponding to the osteogenic induction abilities of these topographies. Furthermore, we have found that mild ER stress improves osteogenic differentiation, whereas excessive ER stress inhibits osteogenic differentiation and causes apoptosis. Our findings demonstrate that the UPR plays a critical role in the topography induced osteogenic differentiation, which may help to provide new insights into the topographical signaling transduction.

Asunto(s)

Células de la Médula Ósea/metabolismo , Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Transducción de Señal , Respuesta de Proteína Desplegada , Animales , Células de la Médula Ósea/citología , Estrés del Retículo Endoplásmico , Células Madre Mesenquimatosas/citología , Ratas , Ratas Sprague-Dawley

RESUMEN

While much progress has been made in the resolution of the cellular hierarchy underlying cardiogenesis, our understanding of chamber-specific myocardium differentiation remains incomplete. To better understand ventricular myocardium differentiation, we targeted the ventricle-specific gene, Irx4, in mouse embryonic stem cells to generate a reporter cell line. Using an antibiotic-selection approach, we purified Irx4+ cells in vitro from differentiating embryoid bodies. The isolated Irx4+ cells proved to be highly proliferative and presented Cxcr4, Pdgfr-alpha, Flk1, and Flt1 on the cell surface. Single Irx4+ ventricular progenitor cells (VPCs) exhibited cardiovascular potency, generating endothelial cells, smooth muscle cells, and ventricular myocytes in vitro. The ventricular specificity of the Irx4+ population was further demonstrated in vivo as VPCs injected into the cardiac crescent subsequently produced Mlc2v+ myocytes that exclusively contributed to the nascent ventricle at E9.5. These findings support the existence of a newly identified ventricular myocardial progenitor. This is the first report of a multipotent cardiac progenitor that contributes progeny specific to the ventricular myocardium. Stem Cells 2016;34:2875-2888.

Asunto(s)

Ventrículos Cardíacos/citología , Proteínas de Homeodominio/metabolismo , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Animales , Biomarcadores/metabolismo , Línea Celular , Membrana Celular/metabolismo , Proliferación Celular , Separación Celular , Células Clonales , Desarrollo Embrionario , Células Endoteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos del Músculo Liso/citología , Especificidad de Órganos , Análisis de la Célula Individual , Factores de Tiempo

RESUMEN

Novel biomedical titanium (Ti) implants with high osteogenic ability for fast and good osseointegration under normal as well as osteoporotic conditions are urgently needed. Expanding on our previous in vitro results, we hypothesized that nanotubular, strontium-loaded (NT-Sr) structures on Ti implants would have favorable osteogenic effects and evaluated the in vivo osseointegration of these implants in rats. The structures with nanotubes of different diameters were fabricated by electrochemical anodization at 10 and 40 V, and the amounts of Sr loaded were adjusted by using two hydrothermal treatment times of 1 and 3 hours. Qualitative microcomputed tomography in two and three dimensions showed that the NT-Sr formed with an anodization voltage of 10 V and hydrothermal treatment time of 3 hours best supported bone growth in vivo. Histomorphometric examination of osseointegration also showed that more newly formed bone was found at its surface. The bone-implant contact percentage was highest (92.48%±0.76%) at 12 weeks. In conclusion, the NT-Sr formed with an anodization voltage of 10 V and hydrothermal treatment time of 3 hours showed excellent osteogenic properties, making it an attractive option for Ti surface modification with considerable clinical potential.

Asunto(s)

Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Nanotubos/química , Oseointegración/efectos de los fármacos , Prótesis e Implantes , Estroncio/química , Titanio/química , Animales , Electroquímica , Femenino , Ratas , Microtomografía por Rayos X

RESUMEN

The present work assesses the potential of three-dimensional (3D) porous titanium (pore size of 188-390 µm and porosity of 70%) fabricated by vacuum diffusion bonding of titanium meshes for applications in bone engineering. Rat bone marrow mesenchymal stem cells were used to investigate the proliferation and differentiation of cells on titanium scaffolds with different pore sizes at day 7, day 14 and day 21 based on DNA contents, alkaline phosphatase (ALP) activity, collagen (COL) secretion and osteogenic gene expressions including ALP, COL-1, bone morphogenetic protein-2 (BMP-2), osteopontin (OPN), runt-related transcription factor 2 (RUNX2), using smooth solid titanium plate as reference material. The rabbit models with distal femoral condyles defect were used to investigate the bone ingrowth into the porous titanium. All samples were subjected to Micro-CT and histological analysis after 4 and 12 weeks of healing. A one-way ANOVA followed by Tukey post hoc tests was used to analyze the data. It was found that the differentiation stage of cells on the porous titanium delayed compared with the smooth solid titanium plate and Ti 188 was more inclined to promote cell differentiation at the initial stage (day 14) while cell proliferation (day 1, 4, 7, 10, 14 and 21) and bone ingrowth (4 and 12 weeks) were biased to Ti 313 and Ti 390. The study indicates that the hybrid porous implant design which combines the advantages of different pore sizes may be meaningful and promising for bone defect restoration. STATEMENT OF SIGNIFICANCE: One of the significant challenges in bone defect restoration is the integration of biomaterials and surrounding bone tissue. Porous titanium may be a promising choice for bone ingrowth and mineralization with appropriate mechanical and biological properties. In this study, based on porous titanium fabricated by vacuum diffusion bonding of titanium meshes, we have evaluated the influence of various pore sizes on rat bone marrow mesenchymal stem cells (rBMMSCs) penetration in vitro and bone ingrowth in vivo. It was interesting that we found the proliferation and differentiation abilities of rBMMSCs, as well as bone ingrowth were related to different pore sizes of such porous scaffolds. The results may provide guidance for porous titanium design for bone defect restoration.

Asunto(s)

Desarrollo Óseo/efectos de los fármacos , Huesos/efectos de los fármacos , Huesos/fisiología , Células Madre Mesenquimatosas/citología , Titanio/farmacología , Vacio , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN/metabolismo , Difusión , Colorantes Fluorescentes/metabolismo , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Tamaño de la Partícula , Porosidad , Implantación de Prótesis , Conejos , Ratas Sprague-Dawley , Regeneración/efectos de los fármacos , Agua/química , Microtomografía por Rayos X

RESUMEN

The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair, underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here, we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines, including a WT1-2A-eGFP knock-in reporter line, under chemically-defined, xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-ß)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells, resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo, as determined by morphological and functional assays, including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies.

RESUMEN

Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors are important for vascular research and therapeutic revascularization. Here, we report a completely defined endothelial progenitor differentiation platform that uses a minimalistic medium consisting of Dulbecco's modified eagle medium and ascorbic acid, lacking of albumin and growth factors. Following hPSC treatment with a GSK-3ß inhibitor and culture in this medium, this protocol generates more than 30% multipotent CD34+ CD31+ endothelial progenitors that can be purified to >95% CD34+ cells via magnetic activated cell sorting (MACS). These CD34+ progenitors are capable of differentiating into endothelial cells in serum-free inductive media. These hPSC-derived endothelial cells express key endothelial markers including CD31, VE-cadherin, and von Willebrand factor (vWF), exhibit endothelial-specific phenotypes and functions including tube formation and acetylated low-density lipoprotein (Ac-LDL) uptake. This fully defined platform should facilitate production of proliferative, xeno-free endothelial progenitor cells for both research and clinical applications.

Asunto(s)

Albúminas/química , Diferenciación Celular , Células Progenitoras Endoteliales/citología , Células Madre Pluripotentes/citología , Antígenos CD/metabolismo , Diferenciación Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Células Progenitoras Endoteliales/efectos de los fármacos , Humanos , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos