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1.
Mol Psychiatry ; 26(7): 3558-3571, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-32839513

RESUMO

A homozygous mutation in the inositol monophosphatase 1 (IMPA1) gene was recently identified in nine individuals with severe intellectual disability (ID) and disruptive behavior. These individuals belong to the same family from Northeastern Brazil, which has 28 consanguineous marriages and 59 genotyped family members. IMPA1 is responsible for the generation of free inositol from de novo biosynthesis and recycling from inositol polyphosphates and participates in the phosphatidylinositol signaling pathway. To understand the role of IMPA1 deficiency in ID, we generated induced pluripotent stem cells (iPSCs) from patients and neurotypical controls and differentiated these into hippocampal dentate gyrus-like neurons and astrocytes. IMPA1-deficient neuronal progenitor cells (NPCs) revealed substantial deficits in proliferation and neurogenic potential. At low passage NPCs (P1 to P3), we observed cell cycle arrest, apoptosis, progressive change to a glial morphology and reduction in neuronal differentiation. These observations were validated by rescuing the phenotype with myo-inositol supplemented media during differentiation of patient-derived iPSCs into neurons and by the reduction of neurogenic potential in control NPCs-expressing shIMPA1. Transcriptome analysis showed that NPCs and neurons derived from ID patients have extensive deregulation of gene expression affecting pathways necessary for neurogenesis and upregulation of gliogenic genes. IMPA1 deficiency did not affect cell cycle progression or survival in iPSCs and glial progenitor cells or astrocyte differentiation. Therefore, this study shows that the IMPA1 mutation specifically affects NPC survival and neuronal differentiation.


Assuntos
Deficiência Intelectual , Neurogênese , Monoéster Fosfórico Hidrolases , Diferenciação Celular/genética , Humanos , Deficiência Intelectual/genética , Mutação , Neurogênese/genética , Monoéster Fosfórico Hidrolases/genética
2.
Hum Mol Genet ; 26(12): 2177-2191, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28334780

RESUMO

Biallelic loss-of-function mutations in the RNA-binding protein EIF4A3 cause Richieri-Costa-Pereira syndrome (RCPS), an autosomal recessive condition mainly characterized by craniofacial and limb malformations. However, the pathogenic cellular mechanisms responsible for this syndrome are entirely unknown. Here, we used two complementary approaches, patient-derived induced pluripotent stem cells (iPSCs) and conditional Eif4a3 mouse models, to demonstrate that defective neural crest cell (NCC) development explains RCPS craniofacial abnormalities. RCPS iNCCs have decreased migratory capacity, a distinct phenotype relative to other craniofacial disorders. Eif4a3 haploinsufficient embryos presented altered mandibular process fusion and micrognathia, thus recapitulating the most penetrant phenotypes of the syndrome. These defects were evident in either ubiquitous or NCC-specific Eif4a3 haploinsufficient animals, demonstrating an autonomous requirement of Eif4a3 in NCCs. Notably, RCPS NCC-derived mesenchymal stem-like cells (nMSCs) showed premature bone differentiation, a phenotype paralleled by premature clavicle ossification in Eif4a3 haploinsufficient embryos. Likewise, nMSCs presented compromised in vitro chondrogenesis, and Meckel's cartilage was underdeveloped in vivo. These findings indicate novel and essential requirements of EIF4A3 for NCC migration and osteochondrogenic differentiation during craniofacial development. Altogether, complementary use of iPSCs and mouse models pinpoint unique cellular mechanisms by which EIF4A3 mutation causes RCPS, and provide a paradigm to study craniofacial disorders.


Assuntos
Pé Torto Equinovaro/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Deformidades Congênitas da Mão/genética , Síndrome de Pierre Robin/genética , Animais , Osso e Ossos/metabolismo , Região Branquial/metabolismo , Diferenciação Celular/genética , Movimento Celular , Condrogênese/genética , Pé Torto Equinovaro/metabolismo , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Modelos Animais de Doenças , Deformidades Congênitas da Mão/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Crista Neural/crescimento & desenvolvimento , Crista Neural/metabolismo , Osteogênese/genética , Síndrome de Pierre Robin/metabolismo
3.
Am J Med Genet A ; 170A(2): 403-409, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26566760

RESUMO

Approximately a hundred patients with terminal 10q deletions have been described. They present with a wide range of clinical features always accompanied by delayed development, intellectual disability and craniofacial dysmorphisms. Here, we report a girl and a boy with craniosynostosis, developmental delay and other congenital anomalies. Karyotyping and molecular analysis including Multiplex Ligation dependent probe amplification (MLPA) and Array Comparative Genomic Hybridization (aCGH) were performed in both patients. We detected a 13.1 Mb pure deletion at 10q26.12-q26.3 in the girl and a 10.9 Mb pure deletion at 10q26.13-q26.3 in the boy, both encompassing about 100 genes. The clinical and molecular findings in these patients reinforce the importance of the DOCK1 smallest region of overlap I (SRO I), previously suggested to explain the clinical signs, and together with a review of the literature suggest a second 3.5 Mb region important for the phenotype (SRO II). Genotype-phenotype correlations and literature data suggest that the craniosynostosis is not directly related to dysregulated signaling in suture development, but may be secondary to alterations in brain development instead. Further, genes at 10q26 may be involved in the molecular crosstalk between brain and cranial vault.


Assuntos
Encéfalo/anormalidades , Deleção Cromossômica , Cromossomos Humanos Par 10/genética , Craniossinostoses/etiologia , Deficiências da Aprendizagem/etiologia , Suturas/efeitos adversos , Adulto , Encéfalo/patologia , Hibridização Genômica Comparativa , Craniossinostoses/patologia , Fácies , Feminino , Humanos , Recém-Nascido , Deficiências da Aprendizagem/patologia , Masculino , Prognóstico
4.
Stem Cell Res ; 71: 103181, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37595341

RESUMO

The DFNA58 locus contains a genomic duplication involving three protein-coding genes (CNRIP1, PLEK, and PPP3R1's exon 1) and other uncharacterized lncRNA genes (LOC101927723, LOC107985892 and LOC102724389). To clarify the role of these genes in hearing and precisely determine their role in hearing loss, four iPSC lines were generated from two carriers and two noncarriers of the duplication.


Assuntos
Perda Auditiva , Células-Tronco Pluripotentes Induzidas , Humanos , Leucócitos Mononucleares , Perda Auditiva/genética , Audição , Éxons
5.
Stem Cell Res Ther ; 10(1): 258, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31416480

RESUMO

BACKGROUND: Liver organoid technology holds great promises to be used in large-scale population-based drug screening and in future regenerative medicine strategies. Recently, some studies reported robust protocols for generating isogenic liver organoids using liver parenchymal and non-parenchymal cells derived from induced pluripotent stem cells (iPS) or using isogenic adult primary non-parenchymal cells. However, the use of whole iPS-derived cells could represent great challenges for a translational perspective. METHODS: Here, we evaluated the influence of isogenic versus heterogenic non-parenchymal cells, using iPS-derived or adult primary cell lines, in the liver organoid development. We tested four groups comprised of all different combinations of non-parenchymal cells for the liver functionality in vitro. Gene expression and protein secretion of important hepatic function markers were evaluated. Additionally, liver development-associated signaling pathways were tested. Finally, organoid label-free proteomic analysis and non-parenchymal cell secretome were performed in all groups at day 12. RESULTS: We show that liver organoids generated using primary mesenchymal stromal cells and iPS-derived endothelial cells expressed and produced significantly more albumin and showed increased expression of CYP1A1, CYP1A2, and TDO2 while presented reduced TGF-ß and Wnt signaling activity. Proteomics analysis revealed that major shifts in protein expression induced by this specific combination of non-parenchymal cells are related to integrin profile and TGF-ß/Wnt signaling activity. CONCLUSION: Aiming the translation of this technology bench-to-bedside, this work highlights the role of important developmental pathways that are modulated by non-parenchymal cells enhancing the liver organoid maturation.


Assuntos
Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Fígado/crescimento & desenvolvimento , Organoides/crescimento & desenvolvimento , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Adulto , Diferenciação Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Humanos , Fígado/metabolismo , Masculino , Organoides/metabolismo , Tecido Parenquimatoso/crescimento & desenvolvimento , Tecido Parenquimatoso/metabolismo , Proteoma/análise , Adulto Jovem
6.
Biofabrication ; 12(1): 015010, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31577996

RESUMO

The liver is responsible for many metabolic, endocrine and exocrine functions. Approximately 2 million deaths per year are associated with liver failure. Modern 3D bioprinting technologies allied with autologous induced pluripotent stem cells (iPS)-derived grafts could represent a relevant tissue engineering approach to treat end stage liver disease patients. However, protocols that accurately recapitulates liver's epithelial parenchyma through bioprinting are still underdeveloped. Here we evaluated the impacts of using single cell dispersion (i.e. obtained from conventional bidimensional differentiation) of iPS-derived parenchymal (i.e. hepatocyte-like cells) versus using iPS-derived hepatocyte-like cells spheroids (i.e. three-dimensional cell culture), both in combination with non-parenchymal cells (e.g. mesenchymal and endothelial cells), into final liver tissue functionality. Single cell constructs showed reduced cell survival and hepatic function and unbalanced protein/amino acid metabolism when compared to spheroid printed constructs after 18 days in culture. In addition, single cell printed constructs revealed epithelial-mesenchymal transition, resulting in rapid loss of hepatocyte phenotype. These results indicates the advantage of using spheroid-based bioprinting, contributing to improve current liver bioprinting technology towards future regenerative medicine applications and liver physiology and disease modeling.


Assuntos
Bioimpressão , Células-Tronco Pluripotentes Induzidas/citologia , Fígado/citologia , Esferoides Celulares/citologia , Bioimpressão/instrumentação , Bioimpressão/métodos , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fígado/metabolismo , Masculino , Impressão Tridimensional , Esferoides Celulares/metabolismo , Engenharia Tecidual
7.
Front Genet ; 9: 149, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29922329

RESUMO

Repeats in coding and non-coding regions have increasingly been associated with many human genetic disorders, such as Richieri-Costa-Pereira syndrome (RCPS). RCPS, mostly characterized by midline cleft mandible, Robin sequence and limb defects, is an autosomal-recessive acrofacial dysostosis mainly reported in Brazilian patients. This disorder is caused by decreased levels of EIF4A3, mostly due to an increased number of repeats at the EIF4A3 5'UTR. EIF4A3 5'UTR alleles are CG-rich and vary in size and organization of three types of motifs. An exclusive allelic pattern was identified among affected individuals, in which the CGCA-motif is the most prevalent, herein referred as "disease-associated CGCA-20nt motif." The origin of the pathogenic alleles containing the disease-associated motif, as well as the functional effects of the 5'UTR motifs on EIF4A3 expression, to date, are entirely unknown. Here, we characterized 43 different EIF4A3 5'UTR alleles in a cohort of 380 unaffected individuals. We identified eight heterozygous unaffected individuals harboring the disease-associated CGCA-20nt motif and our haplotype analyses indicate that there are more than one haplotype associated with RCPS. The combined analysis of number, motif organization and haplotypic diversity, as well as the observation of two apparently distinct haplotypes associated with the disease-associated CGCA-20nt motif, suggest that the RCPS alleles might have arisen from independent unequal crossing-over events between ancient alleles at least twice. Moreover, we have shown that the number and sequence of motifs in the 5'UTR region is associated with EIF4A3 repression, which is not mediated by CpG methylation. In conclusion, this study has shown that the large number of repeats in EIF4A3 does not represent a dynamic mutation and RCPS can arise in any population harboring alleles with the CGCA-20nt motif. We also provided further evidence that EIF4A3 5'UTR is a regulatory region and the size and sequence type of the repeats at 5'UTR may contribute to clinical variability in RCPS.

8.
PLoS Negl Trop Dis ; 4(11): e864, 2010 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-21072227

RESUMO

BACKGROUND: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature. METHODS: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied. FINDINGS AND CONCLUSIONS: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.


Assuntos
Doença de Chagas/parasitologia , Endotélio Vascular/parasitologia , Glicoproteínas/química , Glicoproteínas/metabolismo , Neuraminidase/química , Neuraminidase/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Tropismo , Trypanosoma cruzi/enzimologia , Motivos de Aminoácidos , Animais , Células Cultivadas , Doença de Chagas/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/parasitologia , Endotélio Vascular/metabolismo , Feminino , Glicoproteínas/genética , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neuraminidase/genética , Especificidade de Órgãos , Ligação Proteica , Proteínas de Protozoários/genética , Trypanosoma cruzi/química , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiologia
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