RESUMO
Idiopathic cholangiopathies are diseases that affect cholangiocytes, and they have unknown etiologies. Currently, orthotopic liver transplantation is the only treatment available for end-stage liver disease. Limited access to the bile duct makes it difficult to model cholangiocyte diseases. In this study, by mimicking the embryonic development of cholangiocytes and using a robust, feeder- and serum-free protocol, we first demonstrate the generation of unique functional 3D organoids consisting of small and large cholangiocytes derived from human pluripotent stem cells (PSCs), as opposed to traditional 2D culture systems. At day 28 of differentiation, the human PSC-derived cholangiocytes expressed markers of mature cholangiocytes, such as CK7, CK19, and cystic fibrosis transmembrane conductance regulator (CFTR). Compared with the 2D culture system-generated cholangiocytes, the 3D cholangiocyte organoids (COs) showed higher expression of the region-specific markers of intrahepatic cholangiocytes YAP1 and JAG1 and extrahepatic cholangiocytes AQP1 and MUC1. Furthermore, the COs had small-large tube-like structures and functional assays revealed that they exhibited characteristics of mature cholangiocytes, such as multidrug resistance protein 1 transporter function and CFTR channel activity. In addition to the extracellular matrix supports, the epidermal growth factor receptor (EGFR)-mediated signaling regulation might be involved in this cholangiocyte maturation and differentiation. These results indicated the successful generation of intrahepatic and extrahepatic cholangiocytes by using our 3D organoid protocol. The results highlight the advantages of our 3D culture system over the 2D culture system in promoting the functional differentiation and maturation of cholangiocytes. In summary, in advance of the previous works, our study provides a possible concept of small-large cholangiocyte transdifferentiation of human PSCs under cost-effective 3D culture conditions. The study findings have implications for the development of effective cell-based therapy using COs for patients with cholangiopathies.
RESUMO
BACKGROUND: Hirschsprung disease (HSCR) is a complex genetic disorder characterized by the lack of ganglion cells in the intestines. A current study showed that the NRG1 rare variant frequency in Indonesian patients with HSCR is only 0.9%. Here, we investigated the impact of NRG1 expressions and methylation patterns on the pathogenesis of HSCR. METHODS: This cross-sectional study determined NRG1 type I (HRGα, HRGß1, HRGß2, HRGß3, HRGγ, and NDF43 isoforms), type II and type III expressions in both ganglionic and aganglionic colons of 20 patients with HSCR and 10 control colons by real-time polymerase chain reaction (qPCR). For methylation studies, we treated the extracted gDNA from 16 HSCR patients' and 17 control colons with sodium bisulfate and analyzed the methylation pattern of NRG1 exon 1 with methylation-specific PCR. The samples were collected and analyzed at our institution from December 2018 to December 2020. RESULTS: NRG1 types I, II and III expressions were upregulated (17.2-, 3.2-, and 7.2-fold, respectively) in the ganglionic colons compared with control colons (type I: 13.32 ± 1.65 vs. 17.42 ± 1.51, p < 0.01; type II: 13.73 ± 2.02 vs. 16.29 ± 2.19, p < 0.01; type III: 13.47 ± 3.01 vs. 16.32 ± 2.58, p = 0.03; respectively); while only type I (7.7-fold) and HRGß1/HRGß2 (3.3-fold) isoforms were significantly upregulated in the aganglionic colons compared to the controls (type I: 14.47 ± 1.66 vs. 17.42 ± 1.51, p < 0.01; HRGß1/HRGß2: 13.62 ± 3.42 vs 14.75 ± 1.26, p = 0.01). Moreover, the frequency of partially methylated NRG1 was higher in the ganglionic (81%) and aganglionic (75%) colons than in the controls (59%). CONCLUSIONS: Our study provides further insights into the aberrant NRG1 expression in the colons of patients with HSCR, both ganglionic and aganglionic bowel, which might contribute to the development of HSCR, particularly in Indonesia. Furthermore, these aberrant NRG1 expressions might be associated with its methylation pattern.
Assuntos
Doença de Hirschsprung , Estudos Transversais , Doença de Hirschsprung/genética , Doença de Hirschsprung/metabolismo , Humanos , Metilação , Neuregulina-1/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Background: The semaphorin 3D (SEMA3D) gene has been implicated in the pathogenesis of Hirschsprung disease (HSCR), a complex genetic disorder characterized by the loss of ganglion cells in varying lengths of gastrointestinal tract. We wished to investigate the role of SEMA3D variants, both rare and common variants, as well as its mRNA expression in Indonesian HSCR patients. Methods: Sanger sequencing was performed in 54 HSCR patients to find a pathogenic variant in SEMA3D. Next, we determined SEMA3D expression in 18 HSCR patients and 13 anorectal malformation colons as controls by quantitative real-time polymerase chain reaction (qPCR). Results: No rare variant was found in the SEMA3D gene, except one common variant in exon 17, p.Lys701Gln (rs7800072). The risk allele (C) frequency at rs7800072 among HSCR patients (23%) was similar to those reported for the 1,000 Genomes (27%) and ExAC (28%) East Asian ancestry controls (p = 0.49 and 0.41, respectively). A significant difference in SEMA3D expression was observed between groups (p = 0.04). Furthermore, qPCR revealed that SEMA3D expression was strongly up-regulated (5.5-fold) in the ganglionic colon of HSCR patients compared to control colon (ΔCT 10.8 ± 2.1 vs. 13.3 ± 3.9; p = 0.025). Conclusions: We report the first study of aberrant SEMA3D expressions in HSCR patients and suggest further understanding into the contribution of aberrant SEMA3D expression in the development of HSCR. In addition, this study is the first comprehensive analysis of SEMA3D variants in the Asian ancestry.
RESUMO
BACKGROUND: MicroRNAs (miRNAs) have been associated with the Hirschsprung disease (HSCR) pathogenesis, however, the findings are still inconclusive. We aimed to investigate the effect of miRNA-206 and its targets, fibronectin 1 (FN1), serum deprivation response (SDPR), and paired box 3 (PAX3) expressions on multifactorial HSCR in Indonesia, a genetically distinct group within Asia. METHODS: We determined the miRNA-206, FN1, SDPR and PAX3 expressions in both the ganglionic and aganglionic colon of HSCR patients and control colon by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Twenty-one sporadic HSCR patients and thirteen controls were ascertained in this study. The miRNA-206 expression was up-regulated (2-fold) in the ganglionic colon and down-regulated (0.5-fold) in the aganglionic colon compared to the control group (ΔCT 12.4 ± 3.0 vs. 14.1 ± 3.9 vs. 13.1 ± 2.7), but these differences did not reach significant levels (p = 0.48 and p = 0.46, respectively). Interestingly, the FN1 expression was significantly increased in both the ganglionic (38-fold) and aganglionic colon (18-fold) groups compared to the control group ΔCT 5.7 ± 3.0 vs. 6.8 ± 2.3 vs. 11.0 ± 5.0; p = 0.001 and p = 0.038, respectively). Furthermore, the expressions of SDPR were similar in the ganglionic, aganglionic and control colon groups (ΔCT 2.4 ± 0.6 vs. 2.2 ± 0.4 vs. 2.1 ± 0.6; p = 0.16 and p = 0.39, respectively), while no change was observed in the PAX3 expression between the ganglionic, aganglionic, and control colon groups (ΔCT 3.8 ± 0.8 vs. 4.1 ± 0.8 vs. 3.7 ± 1.1; p = 0.83 and p = 0.44, respectively). CONCLUSION: Our study is the first report of aberrant FN1 expressions in the colon of patients with HSCR and supplies further insights into the contribution of aberrant FN1 expression in the HSCR pathogenesis.
Assuntos
Fibronectinas/genética , Doença de Hirschsprung/genética , MicroRNAs/genética , Adolescente , Adulto , Proteínas de Transporte/genética , Criança , Pré-Escolar , Colo/metabolismo , Feminino , Humanos , Indonésia , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Fator de Transcrição PAX3/genética , Proteínas de Ligação a Fosfato , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
BACKGROUND: Adenocarcinoma derived from umbilicus is very rare. Most adenocarcinomas in umbilicus are secondary events. Carcinoma derived from sweat glands is sporadic, highly radioresistant and has a clinical appearance that is difficult to predict. CASE PRESENTATION: A 37-year-old woman presented with recurrent umbilicus adenocarcinoma after a history of umbilicus tumor surgery 14 months earlier and Capecitabine chemotherapy six times. Malignant cells were found in Fine Needle Aspiration Biopsy (FNAB) examination. A colonoscopy examination found pathological colitis without any colonic mass. The patient underwent wide excision and reconstruction surgery using a composite attachment visceral mesh with a size of 30 × 30 cm. Histopathologic examination of the surgery diagnosed adenocarcinoma of the sudoriferous gland with adjacent tissue free of tumor cells. Six months post operation, Positron Emission Tomography (PET) scan was performed and found neither residue nor recurrence. CONCLUSIONS: Wide excision and reconstruction surgery for recurrent sweat gland umbilical adenocarcinoma followed by chemotherapy can be an alternative to prevent recurrences.
Assuntos
Adenocarcinoma/cirurgia , Neoplasias das Glândulas Sudoríparas/cirurgia , Umbigo/patologia , Adulto , Antimetabólitos Antineoplásicos/administração & dosagem , Biópsia por Agulha Fina , Capecitabina/administração & dosagem , Feminino , Humanos , Recidiva Local de Neoplasia/patologia , Procedimentos de Cirurgia Plástica/métodos , RiscoRESUMO
BACKGROUND: Hirschsprung disease (HSCR) is a heterogeneous genetic disorder characterized by absence of ganglion cells along the intestines resulting in functional bowel obstruction. Mutations in neuregulin 1 (NRG1) gene have been implicated in some cases of intestinal aganglionosis. This study aims to investigate the contribution of the NRG1 gene to HSCR development in an Indonesian population. METHODS: We analyzed the entire coding region of the NRG1 gene in 54 histopathologically diagnosed HSCR patients. RESULTS: All patients were sporadic non-syndromic HSCR with 53/54 (98%) short-segment and 1/54 (2%) long-segment patients. NRG1 gene analysis identified one rare variant, c.397G > C (p.V133 L), and three common variants, rs7834206, rs3735774, and rs75155858. The p.V133 L variant was predicted to reside within a region of high mammalian conservation, overlapping with the promoter and enhancer histone marks of relevant tissues such as digestive and smooth muscle tissues and potentially altering the AP-4_2, BDP1_disc3, Egr-1_known1, Egr-1_known4, HEN1_2 transcription factor binding motifs. This p.V133 L variant was absent in 92 non-HSCR controls. Furthermore, the rs7834206 polymorphism was associated with HSCR by case-control analysis (p = 0.037). CONCLUSIONS: This study is the first report of a NRG1 rare variant associated with HSCR patients of South-East Asian ancestry and provides further insights into the contribution of NRG1 in the molecular genetic pathogenesis of HSCR.
Assuntos
Predisposição Genética para Doença , Variação Genética , Doença de Hirschsprung/genética , Neuregulina-1/genética , Adulto , Povo Asiático/genética , Estudos de Casos e Controles , Feminino , Humanos , Indonésia , Masculino , Reação em Cadeia da Polimerase , Ligação Proteica , Análise de Sequência de DNA , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Some Hirschsprung's disease (HSCR) patients showed persistent bowel symptoms following an appropriately performed pull-through procedure. The mechanism is presumed to be down-regulated small-conductance calcium-activated potassium channel 3 (SK3) expression in the HSCR ganglionic intestines. We aimed to investigate the SK3 expression's impact in HSCR patients after a properly performed pull-through surgery in an Indonesian population, a genetically distinct group within Asia. METHODS: We assessed SK3 gene expression in both the ganglionic and aganglionic colon of HSCR patients and controls colon by quantitative real-time polymerase chain reaction (RT-PCR). RESULTS: We ascertained fourteen sporadic HSCR patients and six anorectal malformation patients as controls. Quantitative RT-PCR showed that the SK3 expression was significantly lower (23-fold) in the ganglionic colon group compared to the control group (9.9 ± 4.6 vs. 5.4 ± 3.4; p = 0.044). The expression of SK3 in the aganglionic colon group was also significantly lower (43-fold) compared to the control group (10.8 ± 4.4 vs. 5.4 ± 3.4; p = 0.015). CONCLUSION: Our study shows that the down-regulated SK3 expression in ganglionic intestines might contribute to the persistent bowel symptoms following a properly performed pull-through surgery in Indonesian HSCR patients. Furthermore, this study is the first report of SK3 expression in a sample population of Asian ancestry.