RESUMO
BACKGROUND: A non-invasive imaging technology that can monitor cell viability, retention, distribution, and interaction with host tissue after transplantation is needed for optimizing and translating stem cell-based therapies. Current cell imaging approaches are limited in sensitivity or specificity, or both, for in vivo cell tracking. The objective of this study was to apply a novel ferritin-based magnetic resonance imaging (MRI) platform to longitudinal tracking of human embryonic stem cells (hESCs) in vivo. METHODS: Human embryonic stem cells (hESCs) were genetically modified to stably overexpress ferritin using the CRISPR-Cas9 system. Cellular toxicity associated with ferritin overexpression and manganese (Mn) supplementation were assessed based on cell viability, proliferation, and metabolic activity. Ferritin-overexpressing hESCs were characterized based on stem cell pluripotency and cardiac-lineage differentiation capability. Cells were supplemented with Mn and imaged in vitro as cell pellets on a preclinical 3 T MR scanner. T1-weighted images and T1 relaxation times were analyzed to assess contrast. For in vivo study, three million cells were injected into the leg muscle of non-obese diabetic severe combined immunodeficiency (NOD SCID) mice. Mn was administrated subcutaneously. T1-weighted sequences and T1 mapping were used to image the animals for longitudinal in vivo cell tracking. Cell survival, proliferation, and teratoma formation were non-invasively monitored by MRI. Histological analysis was used to validate MRI results. RESULTS: Ferritin-overexpressing hESCs labeled with 0.1 mM MnCl2 provided significant T1-induced bright contrast on in vitro MRI, with no adverse effect on cell viability, proliferation, pluripotency, and differentiation into cardiomyocytes. Transplanted hESCs displayed significant bright contrast on MRI 24 h after Mn administration, with contrast persisting for 5 days. Bright contrast was recalled at 4-6 weeks with early teratoma outgrowth. CONCLUSIONS: The bright-ferritin platform provides the first demonstration of longitudinal cell tracking with signal recall, opening a window on the massive cell death that hESCs undergo in the weeks following transplantation before the surviving cell fraction proliferates to form teratomas.
Assuntos
Células-Tronco Embrionárias Humanas , Teratoma , Camundongos , Animais , Humanos , Células-Tronco Embrionárias Humanas/patologia , Ferritinas/genética , Camundongos SCID , Imageamento por Ressonância Magnética/métodos , Células-Tronco EmbrionáriasRESUMO
There are still no definite treatment modalities for interstitial cystitis (IC). Meanwhile, stem cell therapy is rising as potential alternative for various chronic diseases. This study aimed to investigate the safety of the clinical-grade mesenchymal stem cells (MSCs) derived from human embryonic stem cells (hESCs), code name MR-MC-01 (SNU42-MMSCs), in IC patients. Three female IC patients with (1) symptom duration >6 months, (2) visual pain analog scale (VAS) ≥4, and (3) one or two Hunner lesions <2 cm in-office cystoscopy within 1 month were included. Under general anesthesia, participants received cystoscopic submucosal injection of SNU42-MMSCs (2.0 × 107/5 mL) at the center or margin of Hunner lesions and other parts of the bladder wall except trigone with each injection volume of 1 mL. Follow-up was 1, 3, 6, 9, and 12 months postoperatively. Patients underwent scheduled follow-ups, and symptoms were evaluated with validated questionnaires at each visit. No SNU42-MMSCs-related adverse events including immune reaction and abnormalities on laboratory tests and image examinations were reported up to 12-month follow-up. VAS pain was temporarily improved in all subjects. No de novo Hunner lesions were observed and one lesion of the first subject was not identifiable on 12-month cystoscopy. This study reports the first clinical application of transurethral hESC-derived MSC injection in three patients with IC. hESC-based therapeutics was safe and proved to have potential therapeutic efficacy in IC patients. Stem cell therapy could be a potential therapeutic option for treating IC.
Assuntos
Cistite Intersticial , Células-Tronco Embrionárias Humanas , Células-Tronco Mesenquimais , Humanos , Feminino , Cistite Intersticial/terapia , Cistite Intersticial/diagnóstico , Cistite Intersticial/patologia , Células-Tronco Embrionárias Humanas/patologia , Bexiga Urinária , Dor , Células-Tronco Mesenquimais/patologiaRESUMO
BACKGROUND: Spinal cord injury (SCI) results in permanent impairment of motor and sensory functions at and below the lesion site. There is no therapeutic option to the functional recovery of SCI involving diverse injury responses of different cell types in the lesion that limit endogenous nerve regeneration. In this regard, cell replacement therapy utilizing stem cells or their derivatives has become a highly promising approach to promote locomotor recovery. For this reason, the demand for a safe and efficient multipotent cell source that can differentiate into various neural cells is increasing. In this study, we evaluated the efficacy and safety of human polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive neural precursor cells (hNPCsPSA-NCAM+) as a treatment for SCI. METHODS: One hundred thousand hNPCsPSA-NCAM+ isolated from human embryonic stem cell-derived NPCs were transplanted into the lesion site by microinjection 7 days after contusive SCI at the thoracic level. We examined the histological characteristics of the graft and behavioral improvement in the SCI rats 10 weeks after transplantation. RESULTS: Locomotor activity improvement was estimated by the Basso-Beattie-Bresnahan locomotor rating scale. Behavioral tests revealed that the transplantation of the hNPCsPSA-NCAM+ into the injured spinal cords of rats significantly improved locomotor function. Histological examination showed that hNPCsPSA-NCAM+ had differentiated into neural cells and successfully integrated into the host tissue with no evidence of tumor formation. We investigated cytokine expressions, which led to the early therapeutic effect of hNPCsPSA-NCAM+, and found that some undifferentiated NPCs still expressed midkine, a well-known neurotrophic factor involved in neural development and inflammatory responses, 10 weeks after transplantation. CONCLUSION: Our results demonstrate that hNPCsPSA-NCAM+ serve as a safe and efficient cell source which has the potential to improve impaired motor function following SCI.
Assuntos
Células-Tronco Embrionárias Humanas , Células-Tronco Neurais , Traumatismos da Medula Espinal , Ratos , Animais , Humanos , Moléculas de Adesão de Célula Nervosa/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Células-Tronco Embrionárias Humanas/transplante , Traumatismos da Medula Espinal/terapia , Modelos Animais de DoençasRESUMO
BACKGROUND: The slowly activated delayed rectifier potassium current (IKs) mediated by the KCNQ1 gene is one of the main currents involved in repolarization. KCNQ1 mutation can result in long-QT syndrome type 1 (LQT1). IKs does not participate in repolarization in mice; thus, no good model is currently available for research on the mechanism of and drug screening for LQT1. In this study, we established a KCNQ1-deficient human cardiomyocyte (CM) model and performed a series of microelectrode array (MEA) detection experiments on KCNQ1-mutant CMs constructed in other studies to explore the pathogenic mechanism of KCNQ1 deletion and mutation and perform drug screening. METHOD: KCNQ1 was knocked out in human embryonic stem cell (hESC) H9 line using the CRISPR/cas9 system. KCNQ1-deficient and KCNQ1-mutant hESCs were differentiated into CMs through a chemically defined differentiation protocol. Subsequently, high-throughput MEA analysis and drug intervention were performed to determine the electrophysiological characteristics of KCNQ1-deficient and KCNQ1-mutant CMs. RESULTS: During high-throughput MEA analysis, the electric field potential and action potential durations in KCNQ1-deficient CMs were significantly longer than those in wild-type CMs. KCNQ1-deficient CMs also showed an irregular rhythm. Furthermore, KCNQ1-deficient and KCNQ1-mutant CMs showed different responses to different drug treatments, which reflected the differences in their pathogenic mechanisms. CONCLUSION: We established a human CM model with KCNQ1 deficiency showing a prolonged QT interval and an irregular heart rhythm. Further, we used various drugs to treat KCNQ1-deficient and KCNQ1-mutant CMs, and the three models showed different responses to these drugs. These models can be used as important tools for studying the different pathogenic mechanisms of KCNQ1 mutation and the relationship between the genotype and phenotype of KCNQ1, thereby facilitating drug development.
Assuntos
Células-Tronco Embrionárias Humanas , Canal de Potássio KCNQ1 , Síndrome do QT Longo , Linhagem Celular , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Canal de Potássio KCNQ1/deficiência , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Síndrome do QT Longo/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologiaRESUMO
Cell-based therapies face challenges, including poor cell survival, immune rejection, and integration into pathologic tissue. We conducted an open-label phase 1/2a clinical trial to assess the safety and preliminary efficacy of a subretinal implant consisting of a polarized monolayer of allogeneic human embryonic stem cell-derived retinal pigmented epithelium (RPE) cells in subjects with geographic atrophy (GA) secondary to dry age-related macular degeneration. Postmortem histology from one subject with very advanced disease shows the presence of donor RPE cells 2 years after implantation by immunoreactivity for RPE65 and donor-specific human leukocyte antigen (HLA) class I molecules. Markers of RPE cell polarity and phagocytosis suggest donor RPE function. Further histologic examination demonstrated CD34+ structures beneath the implant and CD4+, CD68+, and FoxP3+ cells in the tissue. Despite significant donor-host HLA mismatch, no clinical signs of retinitis, vitreitis, vasculitis, choroiditis, or serologic immune response were detected in the deceased subject or any other subject in the study. Subretinally implanted, HLA-mismatched donor RPE cells survive, express functional markers, and do not elicit clinically detectable intraocular inflammation or serologic immune responses even without long-term immunosuppression.
Assuntos
Atrofia Geográfica , Degeneração Macular , Próteses e Implantes , Atrofia Geográfica/terapia , Células-Tronco Embrionárias Humanas/patologia , Humanos , Degeneração Macular/patologia , Degeneração Macular/terapia , Próteses e Implantes/efeitos adversos , Epitélio Pigmentado da Retina/patologiaRESUMO
Trichloroethylene (TCE) is a major environmental contaminant. Maternal exposure of TCE is linked to developmental defects, but the mechanisms remain to be elucidated. Along with a strategy of 3Rs principle, human embryonic stem cells (hESCs) are regarded as most promising in vitro models for developmental toxicity studies. TCE interfered with hESCs differentiation, but no report was available for TCE effects on hESCs proliferation. Here, we aimed to explore the toxic effects and mechanisms of TCE on hESCs proliferation. Treatment with TCE, did not affect the pluripotency genes expression. However, TCE enhanced hESCs proliferation, manifested by increased cell number, PCNA expression and EdU incorporation. Moreover, TCE exposure upregulated the protein expression levels of Cx43 and cyclin-dependent kinases. Knockdown of Cx43 attenuated the TCE-induced cell hyper-proliferation and CDK2 upregulation. Furthermore, TCE increased Akt phosphorylation, and the inhibition of Akt blocked the TCE-induced Cx43 overexpression and cell proliferation. In conclusion, TCE exposure resulted in upregulation of Cx43 via Akt phosphorylation, consequently stimulated CDK2 expression, contributing to hyper-proliferation in hESCs. Our study brings to light that TCE stimulated the proliferation of hESCs via Cx43, providing a new research avenue for the causes of TCE-induced developmental toxicity.
Assuntos
Proliferação de Células/efeitos dos fármacos , Conexina 43/metabolismo , Poluentes Ambientais/toxicidade , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Tricloroetileno/toxicidade , Linhagem Celular , Conexina 43/genética , Quinase 2 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
BACKGROUND: Neuroblastoma (NB) is a common childhood malignant tumor of neural crest (NC) origin with remarkable heterogeneity in outcomes. Amplification of the oncogene MYCN is strongly associated with highly malignant behaviour and poor prognosis. METHODS: This study aims to use a human embryonic stem cell (hESC)-derived NC model to identify novel downstream effectors of MYCN that can be potentially used as prognostic marker and/or therapeutic target. RESULTS: We show that MYCN-driven NB derived from human neural crest cells (hNCCs) recapitulate the pathological and molecular features of MYCN-amplified neuroblastoma (MNA-NB). By using this platform, we identify a group of 14 surface protein-encoding genes that are associated with MYCN expression level in MNA-NB. Among these genes, high CD55 expression is correlated with poor survival in MNA-NB but not in non-MNA-NB. Furthermore, CD55 promotes tumorigenesis, tumor growth, and cancer stemness in MNA-NB cell lines (MNA-NBL) through regulating the JNK pathway. Mechanistically, MYCN binds to both canonical and noncanonical E-boxes on the promoter of CD55 to regulate its transcriptional expression. Finally, neutralizing antibody targeting CD55 significantly attenuates cancer stemness, suppresses tumor growth, and improves survival exclusively in MNA-NBL-inoculated mice. CONCLUSION: MYCN shapes CD55 into a cancer stem cell regulator which represents a prognostic marker and therapeutic target of MNA-NB. The hESC-derived NC model serves as a valuable platform for investigating NB initiation and progression and developing potential therapeutic targets.
Assuntos
Células-Tronco Embrionárias Humanas , Neuroblastoma , Animais , Linhagem Celular Tumoral , Criança , Regulação Neoplásica da Expressão Gênica , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteína Proto-Oncogênica N-Myc/uso terapêutico , Células-Tronco Neoplásicas/metabolismo , Crista Neural/metabolismo , Crista Neural/patologia , Neuroblastoma/genética , Fatores de Transcrição/genéticaRESUMO
Approximately 45% of the human genome is composed of transposable elements (TEs). Expression of these elements is tightly regulated during normal development. TEs may be expressed at high levels in embryonic stem cells but are epigenetically silenced in terminally differentiated cells. As part of the global 'epigenetic dysregulation' that cells undergo during transformation from normal to cancer, TEs can lose epigenetic silencing and become transcribed, and, in some cases, active. Here, we summarize recent advances detailing the consequences of TE activation in cancer and describe how these understudied residents of our genome can both aid tumorigenesis and potentially be harnessed for anticancer therapies.
Assuntos
Metilação de DNA , Elementos de DNA Transponíveis , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Redes e Vias Metabólicas/genética , Neoplasias/genética , Antineoplásicos/uso terapêutico , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Diferenciação Celular , Inativação Gênica , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Elementos Nucleotídeos Longos e Dispersos , Redes e Vias Metabólicas/efeitos dos fármacos , Mutagênese Insercional , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Processamento de Proteína Pós-Traducional , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mature cardiomyocytes (CMs) obtained from human pluripotent stem cells (hPSCs) have been required for more accurate in vitro modeling of adult-onset cardiac disease and drug discovery. Here, we found that FGF4 and ascorbic acid (AA) induce differentiation of BG01 human embryonic stem cell-cardiogenic mesoderm cells (hESC-CMCs) into mature and ventricular CMs. Co-treatment of BG01 hESC-CMCs with FGF4+AA synergistically induced differentiation into mature and ventricular CMs. FGF4+AA-treated BG01 hESC-CMs robustly released acute myocardial infarction (AMI) biomarkers (cTnI, CK-MB, and myoglobin) into culture medium in response to hypoxic injury. Hypoxia-responsive genes and potential cardiac biomarkers proved in the diagnosis and prognosis of coronary artery diseases were induced in FGF4+AA-treated BG01 hESC-CMs in response to hypoxia based on transcriptome analyses. This study demonstrates that it is feasible to model hypoxic stress in vitro using hESC-CMs matured by soluble factors.
Assuntos
Ácido Ascórbico/farmacologia , Diferenciação Celular , Fator 4 de Crescimento de Fibroblastos/farmacologia , Células-Tronco Embrionárias Humanas/patologia , Modelos Biológicos , Miócitos Cardíacos/patologia , Estresse Fisiológico , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultura/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ventrículos do Coração/patologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Infarto do Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Chromosome 4q deletion is one of the most frequently detected genomic imbalance events in congenital heart disease (CHD) patients. However, a portion of CHD-associated 4q deletions without known CHD genes suggests unknown CHD genes within these intervals. Here, we have shown that knockdown of SORBS2, a 4q interval gene, disrupted sarcomeric integrity of cardiomyocytes and caused reduced cardiomyocyte number in human embryonic stem cell differentiation model. Molecular analyses revealed decreased expression of second heart field (SHF) marker genes and impaired NOTCH and SHH signaling in SORBS2-knockdown cells. Exogenous SHH rescued SORBS2 knockdown-induced cardiomyocyte differentiation defects. Sorbs2-/- mouse mutants had atrial septal hypoplasia/aplasia or double atrial septum (DAS) derived from impaired posterior SHF with a similar expression alteration. Rare SORBS2 variants were significantly enriched in a cohort of 300 CHD patients. Our findings indicate that SORBS2 is a regulator of SHF development and its variants contribute to CHD pathogenesis. The presence of DAS in Sorbs2-/- hearts reveals the first molecular etiology of this rare anomaly linked to paradoxical thromboembolism.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Diferenciação Celular , Transtornos Cromossômicos/genética , Cardiopatias Congênitas/genética , Células-Tronco Embrionárias Humanas/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Animais , Estudos de Casos e Controles , Criança , Pré-Escolar , Deleção Cromossômica , Transtornos Cromossômicos/diagnóstico , Cromossomos Humanos Par 4/genética , Bases de Dados Genéticas , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Células HEK293 , Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Miócitos Cardíacos/patologia , Fenótipo , Proteínas de Ligação a RNA/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: The therapeutic effects of human embryonic stem cell-derived multipotent mesenchymal stem cells (M-MSCs) were evaluated for detrusor underactivity (DUA) in a rat model with atherosclerosis-induced chronic bladder ischemia (CBI) and associated mechanisms. METHODS: Sixteen-week-old male Sprague-Dawley rats were divided into five groups (n = 10). The DUA groups underwent 30 bilateral repetitions of endothelial injury to the iliac arteries to induce CBI, while the sham control group underwent a sham operation. All rats used in this study received a 1.25% cholesterol diet for 8 weeks. M-MSCs at a density of 2.5, 5.0, or 10.0 × 105 cells (250 K, 500 K, or 1000 K; K = a thousand) were injected directly into the bladder 7 weeks post-injury, while the sham and DUA group were treated only with vehicle (phosphate buffer solution). One week after M-MSC injection, awake cystometry was performed on the rats. Then, the bladders were harvested, studied in an organ bath, and prepared for histological and gene expression analyses. RESULTS: CBI by iliac artery injury reproduced voiding defects characteristic of DUA with decreased micturition pressure, increased micturition interval, and a larger residual volume. The pathological DUA properties were improved by M-MSC treatment in a dose-dependent manner, with the 1000 K group producing the best efficacy. Histological analysis revealed that M-MSC therapy reduced CBI-induced injuries including bladder fibrosis, muscular loss, and apoptosis. Transplanted M-MSCs mainly engrafted as vimentin and NG2 positive pericytes rather than myocytes, leading to increased angiogenesis in the CBI bladder. Transcriptomes of the CBI-injured bladders were characterized by the complement system, inflammatory, and ion transport-related pathways, which were restored by M-MSC therapy. CONCLUSIONS: Single injection of M-MSCs directly into the bladder of a CBI-induced DUA rat model improved voiding profiles and repaired the bladder muscle atrophy in a dose-dependent manner.
Assuntos
Células-Tronco Embrionárias Humanas , Células-Tronco Mesenquimais , Bexiga Inativa , Animais , Modelos Animais de Doenças , Células-Tronco Embrionárias Humanas/patologia , Humanos , Isquemia/patologia , Isquemia/terapia , Masculino , Ratos , Ratos Sprague-Dawley , Bexiga Urinária/patologia , Bexiga Inativa/patologiaRESUMO
Telomere length control is critical for cellular lifespan and tumor suppression. Telomerase is transiently activated in the inner cell mass of the developing blastocyst to reset telomere reserves. Its silencing upon differentiation leads to gradual telomere shortening in somatic cells. Here, we report that transcriptional regulation through cis-regulatory elements only partially accounts for telomerase activation in pluripotent cells. Instead, developmental control of telomerase is primarily driven by an alternative splicing event, centered around hTERT exon 2. Skipping of exon 2 triggers hTERT mRNA decay in differentiated cells, and conversely, its retention promotes telomerase accumulation in pluripotent cells. We identify SON as a regulator of exon 2 alternative splicing and report a patient carrying a SON mutation and suffering from insufficient telomerase and short telomeres. In summary, our study highlights a critical role for hTERT alternative splicing in the developmental regulation of telomerase and implicates defective splicing in telomere biology disorders.
Assuntos
Processamento Alternativo , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Antígenos de Histocompatibilidade Menor/genética , Telomerase/genética , Homeostase do Telômero , Telômero/metabolismo , Blastocisto/metabolismo , Blastocisto/patologia , Diferenciação Celular , Pré-Escolar , Proteínas de Ligação a DNA/deficiência , Feminino , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Linhagem , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , Cultura Primária de Células , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/deficiência , Telômero/patologiaRESUMO
Familial adenomatous polyposis (FAP) is an inherited syndrome caused by a heterozygous adenomatous polyposis coli (APC) germline mutation, associated with a profound lifetime risk for colorectal cancer. While it is well accepted that tumorigenic transformation is initiated following acquisition of a second mutation and loss of function of the APC gene, the role of heterozygous APC mutation in this process is yet to be discovered. This work aimed to explore whether a heterozygous APC mutation induces molecular defects underlying tumorigenic transformation and how different APC germline mutations predict disease severity. Three FAP-human embryonic stem cell lines (FAP1/2/3-hESC lines) carrying germline mutations at different locations of the APC gene, and two control hESC lines free of the APC mutation, were differentiated into colon organoids and analyzed by immunohistochemistry and RNA sequencing. In addition, data regarding the genotype and clinical phenotype of the embryo donor parents were collected from medical records. FAP-hESCs carrying a complete loss-of-function of a single APC allele (FAP3) generated complex and molecularly mature colon organoids, which were similar to controls. In contrast, FAP-hESCs carrying APC truncation mutations (FAP1 and FAP2) generated only few cyst-like structures and cell aggregates of various shape, occasionally with luminal parts, which aligned with their failure to upregulate critical differentiation genes early in the process, as shown by RNA sequencing. Abnormal disease phenotype was shown also in non-pathological colon of FAP patients by the randomly distribution of proliferating cells throughout the crypts, compared to their focused localization in the lower part of the crypt in healthy/non-FAP patients. Genotype/phenotype analysis revealed correlations between the colon organoid maturation potential and FAP severity in the carrier parents. In conclusion, this study suggest that a single truncated APC allele is sufficient to initiate early molecular tumorigenic activity. In addition, the results hint that patient-specific hESC-derived colon organoids can probably predict disease severity among FAP patients.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , Predisposição Genética para Doença , Polipose Adenomatosa do Colo/patologia , Adulto , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/patologia , Feminino , Genótipo , Mutação em Linhagem Germinativa/genética , Heterozigoto , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
The Wnt/ß-catenin signaling pathway plays a crucial role in early embryonic development. Wnt/ß-catenin signaling is a major regulator of cell proliferation and keeps embryonic stem cells (ESCs) in the pluripotent state. Dysregulation of Wnt signaling in the early developmental stages causes several hereditary diseases that lead to embryonic abnormalities. Several other signaling molecules are directly or indirectly activated in response to Wnt/ß-catenin stimulation. The crosstalk of these signaling factors either synergizes or opposes the transcriptional activation of ß-catenin/Tcf4-mediated target gene expression. Recently, the crosstalk between the peroxisome proliferator-activated receptor delta (PPARδ), which belongs to the steroid superfamily, and Wnt/ß-catenin signaling has been reported to take place during several aspects of embryonic development. However, numerous questions need to be answered regarding the function and regulation of PPARδ in coordination with the Wnt/ß-catenin pathway. Here, we have summarized the functional activation of the PPARδ in co-ordination with the Wnt/ß-catenin pathway during the regulation of several aspects of embryonic development, stem cell regulation and maintenance, as well as during the progression of several metabolic disorders.
Assuntos
Diferenciação Celular , Desenvolvimento Embrionário , Células-Tronco Embrionárias Humanas/metabolismo , Doenças Metabólicas/embriologia , PPAR delta/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Células-Tronco Embrionárias Humanas/patologia , Humanos , Doenças Metabólicas/patologia , Fator de Transcrição 4/metabolismoRESUMO
BACKGROUND: Coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Up to 20%-30% of patients hospitalized with COVID-19 have evidence of cardiac dysfunction. Xuebijing injection is a compound injection containing five traditional Chinese medicine ingredients, which can protect cells from SARS-CoV-2-induced cell death and improve cardiac function. However, the specific protective mechanism of Xuebijing injection on COVID-19-induced cardiac dysfunction remains unclear. METHODS: The therapeutic effect of Xuebijing injection on COVID-19 was validated by the TCM Anti COVID-19 (TCMATCOV) platform. RNA-sequencing (RNA-seq) data from GSE150392 was used to find differentially expressed genes (DEGs) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. Data from GSE151879 was used to verify the expression of Angiotensin I Converting Enzyme 2 (ACE2) and central hub genes in both human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) and adult human CMs with SARS-CoV-2 infection. RESULTS: A total of 97 proteins were identified as the therapeutic targets of Xuebijing injection for COVID-19. There were 22 DEGs in SARS-CoV-2 infected hiPSC-CMs overlapped with the 97 therapeutic targets, which might be the therapeutic targets of Xuebijing injection on COVID-19-induced cardiac dysfunction. Based on the bioinformatics analysis, 7 genes (CCL2, CXCL8, FOS, IFNB1, IL-1A, IL-1B, SERPINE1) were identified as central hub genes and enriched in pathways including cytokines, inflammation, cell senescence and oxidative stress. ACE2, the receptor of SARS-CoV-2, and the 7 central hub genes were differentially expressed in at least two kinds of SARS-CoV-2 infected CMs. Besides, FOS and quercetin exhibited the tightest binding by molecular docking analysis. CONCLUSION: Our study indicated the underlying protective effect of Xuebijing injection on COVID-19, especially on COVID19-induced cardiac dysfunction, which provided the theoretical basis for exploring the potential protective mechanism of Xuebijing injection on COVID19-induced cardiac dysfunction.
Assuntos
COVID-19/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , RNA-Seq , SARS-CoV-2/metabolismo , Linhagem Celular , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Células-Tronco Embrionárias Humanas/virologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/virologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/virologia , Tratamento Farmacológico da COVID-19RESUMO
Diabetes mellitus is a pandemic metabolic disorder that results from either the autoimmune destruction or the dysfunction of insulin-producing pancreatic beta cells. A promising cure is beta cell replacement through the transplantation of islets of Langerhans. However, donor shortage hinders the widespread implementation of this therapy. Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, represent an attractive alternative beta cell source for transplantation. Although major advances over the past two decades have led to the generation of stem cell-derived beta-like cells that share many features with genuine beta cells, producing fully mature beta cells remains challenging. Here, we review the current status of beta cell differentiation protocols and highlight specific challenges that are associated with producing mature beta cells. We address the challenges and opportunities that are offered by monogenic forms of diabetes. Finally, we discuss the remaining hurdles for clinical application of stem cell-derived beta cells and the status of ongoing clinical trials.
Assuntos
Diferenciação Celular , Diabetes Mellitus Tipo 1 , Células-Tronco Embrionárias Humanas , Células-Tronco Pluripotentes Induzidas , Células Secretoras de Insulina , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/terapia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Células-Tronco Embrionárias Humanas/transplante , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/transplante , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/transplanteRESUMO
Baicalin has been reported to protect mice against Salmonella typhimurium (S. typhimurium) infection, while its molecular mechanisms are unclear. In this study, multiplicity of infection (MOI) and observation time were measured. Cell viability and LDH levels were examined in RAW264.7 cells and H9 cells. RAW264.7 cells were stimulated with S typhimurium in the presence or absence of Baicalin, and the levels of pro-inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The changes in reactive oxygen species (ROS) production were determined by fluorescence microscopy and ELISA. The autophagy and TLR4/MAPK/NF-κB signalling pathway were examined by immunofluorescence microscopy, quantitative reverse transcription-polymerase chain reaction and Western blotting. The results indicated that MOI of 30 and duration of autophagy evident at 5 h were applicable to this study. Baicalin prevented death of macrophages, promoted bactericidal activity, decreased the levels of pro-inflammatory cytokines and ROS and reduced the changes of key biomarkers in autophagy and TLR4/MAPK/NF-κB signalling pathway infected by S typhimurium. TLR4-overexpressed cells, autophagy and TLR4/MAPK/NF-κB signalling pathway were activated by S typhimurium, which was suppressed by Baicalin. Our findings indicated that Baicalin exerts anti-inflammatory and cell-protective effects, and it mediates autophagy by down-regulating the activity of TLR4 infected by S typhimurium.
Assuntos
Anti-Inflamatórios/farmacologia , Autofagia/efeitos dos fármacos , Flavonoides/farmacologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Inflamação/prevenção & controle , Macrófagos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Infecções por Salmonella/prevenção & controle , Salmonella typhimurium/patogenicidade , Receptor 4 Toll-Like/metabolismo , Animais , Citocinas/metabolismo , Células-Tronco Embrionárias Humanas/enzimologia , Células-Tronco Embrionárias Humanas/microbiologia , Células-Tronco Embrionárias Humanas/patologia , Humanos , Inflamação/enzimologia , Inflamação/microbiologia , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Macrófagos/enzimologia , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , NF-kappa B/genética , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Infecções por Salmonella/enzimologia , Infecções por Salmonella/microbiologia , Infecções por Salmonella/patologia , Transdução de Sinais , Receptor 4 Toll-Like/genéticaRESUMO
Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene, where expansion size and somatic mosaicism correlates with disease severity and age of onset. While it is known that the mismatch repair protein MSH2 contributes to the unstable nature of the repeat, its role on other disease-related features, such as CpG methylation upstream of the repeat, is unknown. In this study, we investigated the effect of an MSH2 knock-down (MSH2KD) on both CTG repeat dynamics and CpG methylation pattern in human embryonic stem cells (hESC) carrying the DM1 mutation. Repeat size in MSH2 wild-type (MSH2WT) and MSH2KD DM1 hESC was determined by PacBio sequencing and CpG methylation by bisulfite massive parallel sequencing. We found stabilization of the CTG repeat concurrent with a gradual loss of methylation upstream of the repeat in MSH2KD cells, while the repeat continued to expand and upstream methylation remained unchanged in MSH2WT control lines. Repeat instability was re-established and biased towards expansions upon MSH2 transgenic re-expression in MSH2KD lines while upstream methylation was not consistently re-established. We hypothesize that the hypermethylation at the mutant DM1 locus is promoted by the MMR machinery and sustained by a constant DNA repair response, establishing a potential mechanistic link between CTG repeat instability and upstream CpG methylation. Our work represents a first step towards understanding how epigenetic alterations and repair pathways connect and contribute to the DM1 pathology.
Assuntos
Desmetilação , Instabilidade Genômica , Células-Tronco Embrionárias Humanas/patologia , Proteína 2 Homóloga a MutS/antagonistas & inibidores , Distrofia Miotônica/patologia , Miotonina Proteína Quinase/genética , Expansão das Repetições de Trinucleotídeos , Sistemas CRISPR-Cas , Metilação de DNA , Reparo do DNA , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Distrofia Miotônica/genéticaRESUMO
Tubulin α-1 A (TUBA1A) mutations cause a wide spectrum of brain abnormalities. Although many mutations have been identified and functionally verified, there are clearly many more, and the relationship between TUBA1A mutations and brain malformations remains unclear. The aim of this study was to identify a TUBA1A mutation in a fetus with severe brain abnormalities, verify it functionally, and determine the mechanism of the mutation-related pathogenesis. A de novo missense mutation of the TUBA1A gene, c.167C>G p.T56R/P.THR56Arg, was identified by exon sequencing. Computer simulations showed that the mutation results in a disruption of lateral interactions between the microtubules. Transfection of 293T cells with TUBA1A p.T56R showed that the mutated protein is only partially incorporated into the microtubule network, resulting in a decrease in the rate of microtubule re-integration in comparison with the wild-type protein. The mechanism of pathological changes induced by the mutant gene was determined by knockdown and overexpression. It was found that knockdown of TUBA1A reduced the generation of neural progenitor cells, while overexpression of wild-type or mutant TUBA1A promoted neurogenesis. Our identification and functional verification of the novel TUBA1A mutation extends the TUBA1A gene-phenotype database. Loss-of-function of TUBA1A was shown to play an important role in early neurogenesis of TUBA1A mutation-related brain malformations.
Assuntos
Encéfalo/anormalidades , Mutação com Perda de Função/genética , Neurogênese/genética , Tubulina (Proteína)/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Feminino , Feto/anormalidades , Células-Tronco Embrionárias Humanas/patologia , Humanos , Masculino , Microtúbulos/química , Modelos Moleculares , Neurônios/patologia , Polimerização , Tubulina (Proteína)/química , Sequenciamento do ExomaRESUMO
BACKGROUND AND AIMS: The COVID-19 pandemic has spread worldwide and poses a severe health risk. While most patients present mild symptoms, descending pneumonia can lead to severe respiratory insufficiency. Up to 50% of patients show gastrointestinal symptoms like diarrhea or nausea, intriguingly associating with prolonged symptoms and increased severity. Thus, models to understand and validate drug efficiency in the gut of COVID-19 patients are of urgent need. METHODS: Human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) have led, due to their complexity in mimicking human intestinal architecture, to an unprecedented number of successful disease models including gastrointestinal infections. Here, we employed PSC-HIOs to dissect SARS-CoV-2 pathogenesis and its inhibition by remdesivir, one of the leading drugs investigated for treatment of COVID-19. RESULTS: Immunostaining for viral entry receptor ACE2 and SARS-CoV-2 spike protein priming protease TMPRSS2 showed broad expression in the gastrointestinal tract with highest levels in the intestine, the latter faithfully recapitulated by PSC-HIOs. Organoids could be readily infected with SARS-CoV-2 followed by viral spread across entire PSC-HIOs, subsequently leading to organoid deterioration. However, SARS-CoV-2 spared goblet cells lacking ACE2 expression. Importantly, we challenged PSC-HIOs for drug testing capacity. Specifically, remdesivir effectively inhibited SARS-CoV-2 infection dose-dependently at low micromolar concentration and rescued PSC-HIO morphology. CONCLUSIONS: Thus, PSC-HIOs are a valuable tool to study SARS-CoV-2 infection and to identify and validate drugs especially with potential action in the gut.