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1.
Diabetologia ; 2024 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-39080045

RESUMEN

AIMS/HYPOTHESIS: Homozygous mutations in RFX6 lead to neonatal diabetes accompanied by a hypoplastic pancreas, whereas heterozygous mutations cause MODY. Recent studies have also shown RFX6 variants to be linked with type 2 diabetes. Despite RFX6's known function in islet development, its specific role in diabetes pathogenesis remains unclear. Here, we aimed to understand the mechanisms underlying the impairment of pancreatic islet development and subsequent hypoplasia due to loss-of-function mutations in RFX6. METHODS: We examined regulatory factor X6 (RFX6) expression during human embryonic stem cell (hESC) differentiation into pancreatic islets and re-analysed a single-cell RNA-seq dataset to identify RFX6-specific cell populations during islet development. Furthermore, induced pluripotent stem cell (iPSC) lines lacking RFX6 were generated using CRISPR/Cas9. Various approaches were then employed to explore the consequences of RFX6 loss across different developmental stages. Subsequently, we evaluated transcriptional changes resulting from RFX6 loss through RNA-seq of pancreatic progenitors (PPs) and endocrine progenitors (EPs). RESULTS: RFX6 expression was detected in PDX1+ cells in the hESC-derived posterior foregut (PF). However, in the PPs, RFX6 did not co-localise with pancreatic and duodenal homeobox 1 (PDX1) or NK homeobox 1 (NKX6.1) but instead co-localised with neurogenin 3, NK2 homeobox 2 and islet hormones in the EPs and islets. Single-cell analysis revealed high RFX6 expression levels in endocrine clusters across various hESC-derived pancreatic differentiation stages. Upon differentiating iPSCs lacking RFX6 into pancreatic islets, a significant decrease in PDX1 expression at the PF stage was observed, although this did not affect PPs co-expressing PDX1 and NKX6.1. RNA-seq analysis showed the downregulation of essential genes involved in pancreatic endocrine differentiation, insulin secretion and ion transport due to RFX6 deficiency. Furthermore, RFX6 deficiency resulted in the formation of smaller islet organoids due to increased cellular apoptosis, linked to reduced catalase expression, implying a protective role for RFX6. Overexpression of RFX6 reversed defective phenotypes in RFX6-knockout PPs, EPs and islets. CONCLUSIONS/INTERPRETATION: These findings suggest that pancreatic hypoplasia and reduced islet cell formation associated with RFX6 mutations are not due to alterations in PDX1+/NKX6.1+ PPs but instead result from cellular apoptosis and downregulation of pancreatic endocrine genes. DATA AVAILABILITY: RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: https://doi.org/10.5281/zenodo.10656891 ).

2.
Cell Mol Life Sci ; 80(6): 176, 2023 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-37270452

RESUMEN

Recent studies reported that pancreatic ß-cells are heterogeneous in terms of their transcriptional profiles and their abilities for insulin secretion. Sub-populations of pancreatic ß-cells have been identified based on the functionality and expression of specific surface markers. Under diabetes condition, ß-cell identity is altered leading to different ß-cell sub-populations. Furthermore, cell-cell contact between ß-cells and other endocrine cells within the islet play an important role in regulating insulin secretion. This highlights the significance of generating a cell product derived from stem cells containing ß-cells along with other major islet cells for treating patients with diabetes, instead of transplanting a purified population of ß-cells. Another key question is how close in terms of heterogeneity are the islet cells derived from stem cells? In this review, we summarize the heterogeneity in islet cells of the adult pancreas and those generated from stem cells. In addition, we highlight the significance of this heterogeneity in health and disease conditions and how this can be used to design a stem cell-derived product for diabetes cell therapy.


Asunto(s)
Diabetes Mellitus , Células Secretoras de Insulina , Islotes Pancreáticos , Humanos , Adulto , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Células Secretoras de Insulina/metabolismo , Diabetes Mellitus/metabolismo , Células Madre
3.
Semin Cancer Biol ; 87: 1-16, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36354097

RESUMEN

The interplay between microRNAs (miRNAs) and pluripotency transcription factors (TFs) orchestrates the acquisition of cancer stem cell (CSC) features during the course of malignant transformation, rendering them essential cancer cell dependencies and therapeutic vulnerabilities. In this review, we discuss emerging themes in tumor heterogeneity, including the clonal evolution and the CSC models and their implications in resistance to cancer therapies, and then provide thorough coverage on the roles played by key TFs in maintaining normal and malignant stem cell pluripotency and plasticity. In addition, we discuss the reciprocal interactions between miRNAs and MYC, OCT4, NANOG, SOX2, and KLF4 pluripotency TFs and their contributions to tumorigenesis. We provide our view on the potential to interfere with key miRNA-TF networks through the use of RNA-based therapeutics as single agents or in combination with other therapeutic strategies, to abrogate the CSC state and render tumor cells more responsive to standard and targeted therapies.


Asunto(s)
MicroARNs , Neoplasias , Humanos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , MicroARNs/genética , Neoplasias/genética , Neoplasias/patología , Células Madre Neoplásicas/patología , Factores de Transcripción/genética
4.
Cell Commun Signal ; 21(1): 229, 2023 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-37670346

RESUMEN

BACKGROUND: Our recent studies have demonstrated the crucial involvement of FOXA2 in the development of human pancreas. Reduction of FOXA2 expression during the differentiation of induced pluripotent stem cells (iPSCs) into pancreatic islets has been found to reduce α-and ß-cell masses. However, the extent to which such changes are linked to alterations in the expression profile of long non-coding RNAs (lncRNAs) remains unraveled. METHODS: Here, we employed our recently established FOXA2-deficient iPSCs (FOXA2-/- iPSCs) to investigate changes in lncRNA profiles and their correlation with dysregulated mRNAs during the pancreatic progenitor (PP) and pancreatic islet stages. Furthermore, we constructed co-expression networks linking significantly downregulated lncRNAs with differentially expressed pancreatic mRNAs. RESULTS: Our results showed that 442 lncRNAs were downregulated, and 114 lncRNAs were upregulated in PPs lacking FOXA2 compared to controls. Similarly, 177 lncRNAs were downregulated, and 59 lncRNAs were upregulated in islet cells lacking FOXA2 compared to controls. At both stages, we observed a strong correlation between lncRNAs and several crucial pancreatic genes and TFs during pancreatic differentiation. Correlation analysis revealed 12 DE-lncRNAs that strongly correlated with key downregulated pancreatic genes in both PPs and islet cell stages. Selected DE-lncRNAs were validated using RT-qPCR. CONCLUSIONS: Our data indicate that the observed defects in pancreatic islet development due to the FOXA2 loss is associated with significant alterations in the expression profile of lncRNAs. Therefore, our findings provide novel insights into the role of lncRNA and mRNA networks in regulating pancreatic islet development, which warrants further investigations. Video Abstract.


Asunto(s)
Células Madre Pluripotentes Inducidas , Células Secretoras de Insulina , ARN Largo no Codificante , Humanos , Páncreas , Diferenciación Celular , ARN Mensajero , Factor Nuclear 3-beta del Hepatocito
5.
Int J Mol Sci ; 24(9)2023 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-37175824

RESUMEN

Dementia is a progressive and debilitating neurological disease that affects millions of people worldwide. Identifying the minimally invasive biomarkers associated with dementia that could provide insights into the disease pathogenesis, improve early diagnosis, and facilitate the development of effective treatments is pressing. Proteomic studies have emerged as a promising approach for identifying the protein biomarkers associated with dementia. This pilot study aimed to investigate the plasma proteome profile and identify a panel of various protein biomarkers for dementia. We used a high-throughput proximity extension immunoassay to quantify 1090 proteins in 122 participants (22 with dementia, 64 with mild cognitive impairment (MCI), and 36 controls with normal cognitive function). Limma-based differential expression analysis reported the dysregulation of 61 proteins in the plasma of those with dementia compared with controls, and machine learning algorithms identified 17 stable diagnostic biomarkers that differentiated individuals with AUC = 0.98 ± 0.02. There was also the dysregulation of 153 plasma proteins in individuals with dementia compared with those with MCI, and machine learning algorithms identified 8 biomarkers that classified dementia from MCI with an AUC of 0.87 ± 0.07. Moreover, multiple proteins selected in both diagnostic panels such as NEFL, IL17D, WNT9A, and PGF were negatively correlated with cognitive performance, with a correlation coefficient (r2) ≤ -0.47. Gene Ontology (GO) and pathway analysis of dementia-associated proteins implicated immune response, vascular injury, and extracellular matrix organization pathways in dementia pathogenesis. In conclusion, the combination of high-throughput proteomics and machine learning enabled us to identify a blood-based protein signature capable of potentially differentiating dementia from MCI and cognitively normal controls. Further research is required to validate these biomarkers and investigate the potential underlying mechanisms for the development of dementia.


Asunto(s)
Enfermedad de Alzheimer , Disfunción Cognitiva , Humanos , Proteómica , Proyectos Piloto , Biomarcadores
6.
Cell Mol Life Sci ; 78(6): 2459-2483, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33242105

RESUMEN

Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycemia as a result of progressive loss of pancreatic ß cells, which could lead to several debilitating complications. Different paths, triggered by several genetic and environmental factors, lead to the loss of pancreatic ß cells and/or function. Understanding these many paths to ß cell damage or dysfunction could help in identifying therapeutic approaches specific for each path. Most of our knowledge about diabetes pathophysiology has been obtained from studies on animal models, which do not fully recapitulate human diabetes phenotypes. Currently, human pluripotent stem cell (hPSC) technology is a powerful tool for generating in vitro human models, which could provide key information about the disease pathogenesis and provide cells for personalized therapies. The recent progress in generating functional hPSC-derived ß cells in combination with the rapid development in genomic and genome-editing technologies offer multiple options to understand the cellular and molecular mechanisms underlying the development of different types of diabetes. Recently, several in vitro hPSC-based strategies have been used for studying monogenic and polygenic forms of diabetes. This review summarizes the current knowledge about different hPSC-based diabetes models and how these models improved our current understanding of the pathophysiology of distinct forms of diabetes. Also, it highlights the progress in generating functional ß cells in vitro, and discusses the current challenges and future perspectives related to the use of the in vitro hPSC-based strategies.


Asunto(s)
Diabetes Mellitus/patología , Diferenciación Celular , Reprogramación Celular , Diabetes Mellitus/metabolismo , Edición Génica , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Modelos Biológicos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Medicina de Precisión , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
7.
Int J Mol Sci ; 23(22)2022 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-36430529

RESUMEN

Cofactor flavin adenine dinucleotide (FAD), a compound with flavin moiety and a derivative of riboflavin (vitamin B2), is shown to bind to Sox9 (a key transcription factor in early pancreatic development) and, subsequently, induce a large increase in markers of pancreatic development, including Ngn3 and PTF1a. Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, also binds to Sox9 and results in a similar increase in pancreatic development markers. Sox9 is known to be specifically important for pancreatic progenitors. Previously, there was no known link between FAD, PLP, or other co-factors and Sox9 for function. Thus, our findings show the mechanism by which FAD and PLP interact with Sox9 and result in the altered expression of pancreatic progenitor transcription factors involved in the pancreas development.


Asunto(s)
Flavina-Adenina Dinucleótido , Páncreas , Flavina-Adenina Dinucleótido/metabolismo , Páncreas/metabolismo , Hormonas Pancreáticas/metabolismo , Riboflavina/metabolismo , Fosfato de Piridoxal/metabolismo , Fosfatos/metabolismo , Vitaminas/metabolismo
8.
Int J Mol Sci ; 23(6)2022 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-35328807

RESUMEN

Ischemic strokes are associated with significant morbidity and mortality, but currently there are no reliable prognostic or diagnostic blood biomarkers. MicroRNAs (miRNAs) regulate various molecular pathways and may be used as biomarkers. Using RNA-Seq, we conducted comprehensive circulating miRNA profiling in patients with ischemic stroke compared with healthy controls. Samples were collected within 24 h of clinical diagnosis. Stringent analysis criteria of discovery (46 cases and 95 controls) and validation (47 cases and 96 controls) cohorts led to the identification of 10 differentially regulated miRNAs, including 5 novel miRNAs, with potential diagnostic significance. Hsa-miR-451a was the most significantly upregulated miRNA (FC; 4.8, FDR; 3.78 × 10-85), while downregulated miRNAs included hsa-miR-574-5p and hsa-miR-142-3p, among others. Importantly, we computed a multivariate classifier based on the identified miRNA panel to differentiate between ischemic stroke patients and healthy controls, which showed remarkably high sensitivity (0.94) and specificity (0.99). The area under the ROC curve was 0.97 and it is superior to other current available biomarkers. Moreover, in samples collected one month following stroke, we found sustained upregulation of hsa-miR-451a and downregulation of another 5 miRNAs. Lastly, we report 3 miRNAs that were significantly associated with poor clinical outcomes of stroke, as defined by the modified Rankin scores. The clinical translation of the identified miRNA panel may be explored further.


Asunto(s)
MicroARN Circulante , Accidente Cerebrovascular Isquémico , MicroARNs , Accidente Cerebrovascular , Biomarcadores , MicroARN Circulante/genética , Perfilación de la Expresión Génica , Humanos , Accidente Cerebrovascular Isquémico/diagnóstico , Accidente Cerebrovascular Isquémico/genética , MicroARNs/genética , Curva ROC , Accidente Cerebrovascular/genética
9.
Int J Mol Sci ; 24(1)2022 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-36613546

RESUMEN

Transient ischemic attack (TIA) refers to a momentary neurologic deficit caused by focal cerebral, spinal or retinal ischemic insult. TIA is associated with a high risk of impending acute ischemic stroke (AIS), a neurologic dysfunction characterized by focal cerebral, spinal or retinal infarction. Understanding the differences in molecular pathways in AIS and TIA has merit for deciphering the underlying cause for neuronal deficits with long-term effects and high risks of morbidity and mortality. In this study, we performed comprehensive investigations into the circulating microRNA (miRNA) profiles of AIS (n = 191) and TIA (n = 61) patients. We performed RNA-Seq on serum samples collected within 24 hrs of clinical diagnosis and randomly divided the study populations into discovery and validation cohorts. We identified a panel of 11 differentially regulated miRNAs at FDR < 0.05. Hsa-miR-548c-5p, -20a-5p, -18a-5p, -484, -652-3p, -486-3p, -24-3p, -181a-5p and -222-3p were upregulated, while hsa-miR-500a-3p and -206 were downregulated in AIS patients compared to TIA patients. We also probed the previously validated gene targets of our identified miRNA panel to highlight the molecular pathways affected in AIS. Moreover, we developed a multivariate classifier with potential utilization as a discriminative biomarker for AIS and TIA patients. The underlying molecular pathways in AIS compared to TIA may be explored further in functional studies for therapeutic targeting in clinical translation.


Asunto(s)
MicroARN Circulante , Ataque Isquémico Transitorio , Accidente Cerebrovascular Isquémico , MicroARNs , Accidente Cerebrovascular , Humanos , Biomarcadores , MicroARN Circulante/genética , Ataque Isquémico Transitorio/genética , Accidente Cerebrovascular Isquémico/genética , MicroARNs/metabolismo , Accidente Cerebrovascular/terapia
10.
Diabetes Metab Res Rev ; 37(5): e3400, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-32857429

RESUMEN

AIM: Beta cell replacement strategies are a promising alternative for diabetes treatment. Human pluripotent stem cells (hPSCs) serve as a scalable source for producing insulin-secreting cells for transplantation therapy. We recently generated novel hPSC-derived pancreatic progenitors, expressing high levels of the transcription factor NKX6.1, in the absence of PDX1 (PDX1- /NKX6.1+ ). Herein, our aim was to characterize this novel population and assess its ability to differentiate into insulin-secreting beta cells in vitro. MATERIALS AND METHODS: Three different hPSC lines were differentiated into PDX1- /NKX6.1+ progenitors, which were further differentiated into insulin-secreting cells using two different protocols. The progenitors and beta cells were extensively characterized. Transcriptome analysis was performed at different stages and compared with the profiles of various pancreatic counterparts. RESULTS: PDX1- /NKX6.1+ progenitors expressed high levels of nestin, a key marker of pancreatic islet-derived progenitors, in the absence of E-cadherin, similar to pancreatic mesenchymal stem cells. At progenitor stage, comparison of the two populations showed downregulation of pancreatic epithelial genes and upregulation of neuronal development genes in PDX1- /NKX6.1+ cells in comparison to the PDX1+ /NKX6.1+ cells. Interestingly, on further differentiation, PDX1- /NKX6.1+ cells generated mono-hormonal insulin+ cells and activated pancreatic key genes, such as PDX1. The transcriptome profile of PDX1- /NKX6.1+ -derived beta (3D-beta) was closely similar to those of human pancreatic islets and purified hPSC-derived beta cells. Also, the 3D-beta cells secreted C-peptide in response to increased glucose concentrations indicating their functionality. CONCLUSION: These findings provide a novel source of insulin-secreting cells that can be used for beta cell therapy for diabetes.


Asunto(s)
Células Secretoras de Insulina , Células Madre Pluripotentes , Péptido C , Diabetes Mellitus , Proteínas de Homeodominio/genética , Humanos , Transactivadores/genética
11.
J Transl Med ; 16(1): 276, 2018 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-30305089

RESUMEN

New technologies and therapies designed to facilitate development of personalized treatments are rapidly emerging in the field of biomedicine. Strikingly, the goal of personalized medicine refined the concept of therapy by developing cell-based therapies, the so-called "living drugs". Breakthrough advancements were achieved in this regard in the fields of gene therapy, cell therapy, tissue-engineered products and advanced therapeutic techniques. The Advanced Therapies in Healthcare symposium, organized by the Clinical Research Center Department of Sidra Medicine, in Doha, Qatar (October 2017), brought together world-renowned experts from the fields of oncology, hematology, immunology, inflammation, autoimmune disorders, and stem cells to offer a comprehensive picture of the status of worldwide advanced therapies in both pre-clinical and clinical development, providing insights to the research phase, clinical data and regulatory aspects of these therapies. Highlights of the meeting are provided in this meeting report.


Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Medicina de Precisión , Terapia Genética , Humanos , Inmunoterapia , Terapia Molecular Dirigida , Neoplasias/inmunología , Neoplasias/terapia , Qatar
12.
Diabetes Metab Res Rev ; 34(7): e3026, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29774648

RESUMEN

The aim of this study was to conduct a systematic review and meta-analysis and determine the prevalence of diabetic nephropathy (DN) among Arab patients with T1D. A systematic literature search was conducted using 4 different literature databases (PubMed, ScienceDirect, Web of Science, and Embase) to capture all relevant data about Arab patients with T1D that had DN. Meta-analysis and systematic review were performed using the random effect model, and the heterogeneity of the studies was assessed using the Q-test, I2, and Tau-squared statistics. Publication bias was assessed using the funnel-plot test. Our search strategy captured 372 studies in only 10 out of the 22 Arab countries in a period of 48 years (1969-2017); of which, 41 met our inclusion criteria for full article analysis, of those, 15 were eligible for meta-analysis. We estimated the prevalence of DN among Arab people with T1D to be 18.2% (95% confidence interval 13.1%-24.8%). In conclusion, DN prevalence is underexplored among Arab patients with T1D and represents a significant risk for the well-being of Arab patients with T1D. Therefore, there is an urgent need for comprehensive epidemiological studies for DN among Arab patients with T1D.


Asunto(s)
Árabes/estadística & datos numéricos , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/epidemiología , Nefropatías Diabéticas/epidemiología , Mundo Árabe , Humanos , Medio Oriente/epidemiología , Prevalencia
13.
Stem Cell Reports ; 2024 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-39059374

RESUMEN

Advancements in in vitro human embryo research prompt a reconsideration of the 14-day rule, highlighting the integration of global religious perspectives, particularly Islam. Through analyzing classical Muslim scholars' perspectives and modern interdisciplinary Islamic bioethical discussions, we advocate extending the 14-day limit to at least 40 days, with specified conditions.

14.
Stem Cell Rev Rep ; 2024 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-38916841

RESUMEN

The pathogenesis of diabetes involves complex changes in the expression profiles of mRNA and non-coding RNAs within pancreatic islet cells. Recent progress in induced pluripotent stem cell (iPSC) technology have allowed the modeling of diabetes-associated genes. Our recent study using FOXA2-deficient human iPSC models has highlighted an essential role for FOXA2 in the development of human pancreas. Here, we aimed to provide further insights on the role of microRNAs (miRNAs) by studying the miRNA-mRNA regulatory networks in iPSC-derived islets lacking the FOXA2 gene. Consistent with our previous findings, the absence of FOXA2 significantly downregulated the expression of islet hormones, INS, and GCG, alongside other key developmental genes in pancreatic islets. Concordantly, RNA-Seq analysis showed significant downregulation of genes related to pancreatic development and upregulation of genes associated with nervous system development and lipid metabolic pathways. Furthermore, the absence of FOXA2 in iPSC-derived pancreatic islets resulted in significant alterations in miRNA expression, with 61 miRNAs upregulated and 99 downregulated. The upregulated miRNAs targeted crucial genes involved in diabetes and pancreatic islet cell development. In contrary, the absence of FOXA2 in islets showed a network of downregulated miRNAs targeting genes related to nervous system development and lipid metabolism. These findings highlight the impact of FOXA2 absence on pancreatic islet development and suggesting intricate miRNA-mRNA regulatory networks affecting pancreatic islet cell development.

15.
Theriogenology ; 224: 107-118, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38761667

RESUMEN

Mesenchymal stromal cells (MSCs) have regenerative and immunomodulatory potential and may be used to treat injured tissues. Pregnancy has been associated with increased MSCs in the peripheral circulation in multiple species, but to date, there are no reports on this matter in horses. This study aimed to evaluate the effect of pregnancy on isolation efficiency and proliferation capacity of equine MSCs derived from the peripheral blood (PB) of mares. Venous blood samples were collected at the 11th month of gestation and 1 month after delivery from clinically healthy Arabian mares that presented normal pregnancies. Blood samples were processed for in vitro cellular culture and hormonal and metabolic profiles. MSCs were isolated and characterized by trilineage differentiation potential, immunophenotyping, analyzed by gene sequencing and proliferation assays. The isolation of peripheral blood mononuclear cells (PBMCs) of pregnant mares were associated with higher isolation efficiency and proliferative capacity of MSCs derived from peripheral blood (PB-MSCs) recovered pre-partum than those isolated post-partum. Although fetal gender, parity, 5α-reduced pregnanes, insulin, and cortisol were shown to affect cellular proliferation, individual factors and the small population studied must be considered. This study suggests that PB-MSCs from pregnant mares could be a valuable alternative source of MSCs for therapeutic purposes.


Asunto(s)
Proliferación Celular , Células Madre Mesenquimatosas , Animales , Femenino , Caballos , Embarazo , Células Madre Mesenquimatosas/fisiología , Células Madre Mesenquimatosas/citología , Preñez , Leucocitos Mononucleares/fisiología , Diferenciación Celular , Células Cultivadas
16.
Stem Cell Rev Rep ; 19(4): 942-952, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36707464

RESUMEN

The multipotent pancreatic progenitor cells (MPCs) co-expressing the transcription factors, PDX1 and NKX6.1, are the source of functional pancreatic ß-cells. The aim of this study was to examine the effect of p53 inhibition in MPCs on the generation of PDX1+/NKX6.1+ MPCs and pancreatic ß-cell generation. Human embryonic stem cells (hESCs) were differentiated into MPCs and ß-cells. hESC-MPCs (stage 4) were treated with different concentrations of p53 inhibitors, and their effect was evaluated using different approaches. NKX6.1 was overexpressed during MPCs specification. Inhibition of p53 using pifithrin-µ (PFT-µ) at the MPC stage resulted in a significant increase in the number of PDX1+/NKX6.1+ cells and a reduction in the number of CHGA+/NKX6.1- cells. Further differentiation of MPCs treated with PFT-µ into pancreatic ß-cells showed that PFT-µ treatment did not significantly change the number of C-Peptide+ cells; however, the number of C-PEP+ cells co-expressing glucagon (polyhormonal) was significantly reduced in the PFT-µ treated cells. Interestingly, overexpression of NKX6.1 in hESC-MPCs enhanced the expression of key MPC genes and dramatically suppressed p53 expression. Our findings demonstrated that the p53 inhibition during stage 4 of differentiation enhanced MPC generation, prevented premature endocrine induction and favored the differentiation into monohormonal ß-cells. These findings suggest that adding a p53 inhibitor to the differentiation media can significantly enhance the generation of monohormonal ß-cells.


Asunto(s)
Células Madre Pluripotentes , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Diferenciación Celular/genética
17.
Stem Cell Rev Rep ; 19(4): 1082-1097, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36749553

RESUMEN

Recently, we reported that forkhead box A2 (FOXA2) is required for the development of human pancreatic α- and ß-cells. However, whether miRNAs play a role in regulating pancreatic genes during pancreatic development in the absence of FOXA2 expression is largely unknown. Here, we aimed to capture the dysregulated miRNAs and to identify their pancreatic-specific gene targets in pancreatic progenitors (PPs) derived from wild-type induced pluripotent stem cells (WT-iPSCs) and from iPSCs lacking FOXA2 (FOXA2-/-iPSCs). To identify differentially expressed miRNAs (DEmiRs), and genes (DEGs), two different FOXA2-/-iPSC lines were differentiated into PPs. FOXA2-/- PPs showed a significant reduction in the expression of the main PP transcription factors (TFs) in comparison to WT-PPs. RNA sequencing analysis demonstrated significant reduction in the mRNA expression of genes involved in the development and function of exocrine and endocrine pancreas. Furthermore, miRNA profiling identified 107 downregulated and 111 upregulated DEmiRs in FOXA2-/- PPs compared to WT-PPs. Target prediction analysis between DEmiRs and DEGs identified 92 upregulated miRNAs, predicted to target 1498 downregulated genes in FOXA2-/- PPs. Several important pancreatic TFs essential for pancreatic development were targeted by multiple DEmiRs. Selected DEmiRs and DEGs were further validated using RT-qPCR. Our findings revealed that FOXA2 expression is crucial for pancreatic development through regulating the expression of pancreatic endocrine and exocrine genes targeted by a set of miRNAs at the pancreatic progenitor stage. These data provide novel insights of the effect of FOXA2 deficiency on miRNA-mRNA regulatory networks controlling pancreatic development and differentiation.


Asunto(s)
Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 3-beta del Hepatocito , Células Madre Pluripotentes Inducidas , Islotes Pancreáticos , MicroARNs , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/fisiología , MicroARNs/genética , Humanos , Islotes Pancreáticos/citología , Islotes Pancreáticos/crecimiento & desarrollo , Islotes Pancreáticos/metabolismo , Diferenciación Celular/genética , Línea Celular
18.
Cells ; 12(23)2023 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-38067110

RESUMEN

Targeting tumour metabolism through glucose transporters is an attractive approach. However, the role these transporters play through interaction with other signalling proteins is not yet defined. The glucose transporter SLC2A3 (GLUT3) is a member of the solute carrier transporter proteins. GLUT3 has a high affinity for D-glucose and regulates glucose uptake in the neurons, as well as other tissues. Herein, we show that GLUT3 is involved in the uptake of arsenite, and its level is regulated by peroxiredoxin 1 (PRDX1). In the absence of PRDX1, GLUT3 mRNA and protein expression levels are low, but they are increased upon arsenite treatment, correlating with an increased uptake of glucose. The downregulation of GLUT3 by siRNA or deletion of the gene by CRISPR cas-9 confers resistance to arsenite. Additionally, the overexpression of GLUT3 sensitises the cells to arsenite. We further show that GLUT3 interacts with PRDX1, and it forms nuclear foci, which are redistributed upon arsenite exposure, as revealed by immunofluorescence analysis. We propose that GLUT3 plays a role in mediating the uptake of arsenite into cells, and its homeostatic and redox states are tightly regulated by PRDX1. As such, GLUT3 and PRDX1 are likely to be novel targets for arsenite-based cancer therapy.


Asunto(s)
Arsenitos , Transportador de Glucosa de Tipo 3 , Arsenitos/toxicidad , Glucosa/metabolismo , Transportador de Glucosa de Tipo 3/genética , Transportador de Glucosa de Tipo 3/metabolismo , Linfocitos Nulos/efectos de los fármacos , Linfocitos Nulos/metabolismo , Peroxirredoxinas/metabolismo , Humanos , Células HEK293
19.
Stem Cells Transl Med ; 11(7): 704-714, 2022 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-35640144

RESUMEN

Although genome profiling provides important genetic and phenotypic details for applying precision medicine to diabetes, it is imperative to integrate in vitro human cell models, accurately recapitulating the genetic alterations associated with diabetes. The absence of the appropriate preclinical human models and the unavailability of genetically relevant cells substantially limit the progress in developing personalized treatment for diabetes. Human pluripotent stem cells (hPSCs) provide a scalable source for generating diabetes-relevant cells carrying the genetic signatures of the patients. Remarkably, allogenic hPSC-derived pancreatic progenitors and ß cells are being used in clinical trials with promising preliminary results. Autologous hiPSC therapy options exist for those with monogenic and type 2 diabetes; however, encapsulation or immunosuppression must be accompanied with in the case of type 1 diabetes. Furthermore, genome-wide association studies-identified candidate variants can be introduced in hPSCs for deciphering the associated molecular defects. The hPSC-based disease models serve as excellent resources for drug development facilitating personalized treatment. Indeed, hPSC-based diabetes models have successfully provided valuable knowledge by modeling different types of diabetes, which are discussed in this review. Herein, we also evaluate their strengths and shortcomings in dissecting the underlying pathogenic molecular mechanisms and discuss strategies for improving hPSC-based disease modeling investigations.


Asunto(s)
Diabetes Mellitus Tipo 2 , Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Diferenciación Celular , Diabetes Mellitus Tipo 2/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Células Madre Pluripotentes/metabolismo , Medicina de Precisión
20.
Methods Mol Biol ; 2454: 351-363, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-33190184

RESUMEN

Diabetes is a complex metabolic disorder, with no available treatment. Islet transplantation is currently practiced beta cell replacement therapy option, however, with major limitations. Human pluripotent stem cells (hPSCs) can be used as a scalable source for generation of insulin-secreting cells as hPSCs have high proliferative capacity and can differentiate into any tissue type. In vitro stepwise protocols have been designed for differentiating hPSCs into pancreatic lineages that finally give rise to beta cells; however, these hPSC-derived beta cells are dissimilar to adult human beta cells in key aspects of gene expression and functionality. Alternatively, pancreatic progenitors, when transplanted in the body, have been shown to mature into functional insulin-secreting beta cells, capable of reversing hyperglycemia. These pancreatic progenitors require the co-expression of PDX1, a transcription factor (TF) regulating pancreatic development, and NKX6.1, another TF key for beta cell maturation and function, to produce glucose-responsive beta cells. Given the crucial role played by NKX6.1, we optimized an in vitro differentiation protocol to enhance the generation of pancreatic progenitors co-expressing PDX1 and NKX6.1 by modulating cell density, matrix availability, and cellular dissociation.


Asunto(s)
Proteínas de Homeodominio , Células Secretoras de Insulina , Células Madre Pluripotentes , Transactivadores , Diferenciación Celular/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Páncreas , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
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