An Advocacy for the Use of 3D Stem Cell Culture Systems for the Development of Regenerative Medicine: An Emphasis on Photoreceptor Generation

The availability of stem cells is of great promise to study early developmental stages and to generate adequate cells for cell transfer therapies. Although many researchers using stem cells were successful in dissecting intrinsic and extrinsic mechanisms and in generating specific cell phenotypes, few of the stem cells or the differentiated cells show the capacity to repair a tissue. Advances in cell and stem cell cultivation during the last years made tremendous progress in the generation of bona fide differentiated cells able to integrate into a tissue after transplantation, opening new perspectives for developmental biology studies and for regenerative medicine. In this review, we focus on the main works attempting to create in vitro conditions mimicking the natural environment of CNS structures such as the neural tube and its development in different brain region areas including the optic cup. The use of protocols growing cells in 3D organoids is a key strategy to produce cells resembling endogenous ones. An emphasis on the generation of retina tissue and photoreceptor cells is provided to highlight the promising developments in this field. Other examples are presented and discussed, such as the formation of cortical tissue, the epithelial gut or the kidney organoids. The generation of differentiated tissues and well-defined cell phenotypes from embryonic stem (ES) cells or induced pluripotent cells (iPSCs) opens several new strategies in the field of biology and regenerative medicine. A 3D organ/tissue development in vitro derived from human cells brings a unique tool to study human cell biology and pathophysiology of an organ or a specific cell population. The perspective of tissue repair is discussed as well as the necessity of cell banking to accelerate the progress of this promising field.

Pattern of Wnt ligand expression during chick eye development

The dorsoventral axis of the eye is determined prior to optic cup invagination. A variety of signaling pathways have been implicated in the maintenance of the optic dorsoventral axis, including, but not limited to, bone morphogenetic protein 4, Sonic Hedgehog and retinoic acid. Here, we investigated the possible contribution of Wnt ligands to the establishment or maintenance of the optic axis by analyzing their expression pattern during early chick optic development. We performed in situ hybridization of Wnt-1, Wnt-3a, Wnt-4, and Wnt-5a during the optic vesicle, early optic cup and established optic cup stages and focused our analysis on the optic region. Our data showed that Wnt-5a, but none of the others, is expressed in the dorsal region of the eye starting from the Hamburger and Hamilton stage 14 (HH14). These results are supported by cryosections of the labeled optic region, which further reveal that Wnt-5a is expressed only in the dorsal retinal pigmented epithelium. Thus, we propose that Wnt-5a is a marker for dorsal retinal pigmented epithelium in chick embryos from HH14 to HH19.

La prise d’acide folique en période périconceptionnelle : une étude sur la concordance aux directives cliniques canadiennes et sur l’impact sur la prévalence des malformations congénitales au Québec

La prise d’un supplément d’acide folique en période préconceptionnelle réduit le risque d’une anomalie du tube neural (ATN), une malformation du système nerveux. Dans le but d’en réduire la prévalence, la Société des Obstétriciens et Gynécologues du Canada a émis de nouvelles directives cliniques en 2007 qui tenaient compte de différents facteurs de risque pour les ATN et pour qui la dose recommandée variait selon le profil de risque de la femme, allant de 0,4 à 5,0 mg d’acide folique. Jusqu’à présent, peu de données sont disponibles sur les effets de la prise d’une haute dose d’acide folique. Les objectifs de cette étude étaient: 1) d’évaluer la concordance entre la supplémentation en acide folique chez les femmes enceintes et les nouvelles recommandations canadiennes; 2) d’identifier les déterminants d’une utilisation concordante et 3) d’évaluer si la prise de hautes doses d’acide folique en période périconceptionnelle réduisait le risque de malformations congénitales autre que les ATN. Pour répondre à ces objectifs, une étude transversale et une étude écologique ont été effectuées. La première incluait 361 femmes enceintes recrutées aux cliniques d’obstétriques du CHU Sainte-Justine et la deuxième utilisait le Registre Québécois des Grossesses, issu du jumelage de trois banques de données administratives au Québec (RAMQ, Med-Écho et ISQ), où 152 392 couples mère-enfant ont été identifiés. Seul 27% des femmes enceintes ayant participé à l’étude transversale avaient une supplémentation en acide folique, avec ou sans ordonnance, concordante aux lignes directrices canadiennes. La concordance variait selon leur profil de facteurs de risque pour les ATN. Notre étude écologique montre que la prévalence annuelle de l’utilisation de haute dose d’acide folique (avec ordonnance) en période périconceptionnelle a augmenté de 0,17% à 0,80% (p < 0,0001) entre 1998 et 2008 et que la prévalence des malformations congénitales majeures a augmenté de 15% au cours de la même période (3,35% à 3,87%, p<0,0001). Les résultats de nos deux études montrent que l’acide folique n’est pas largement utilisé par les femmes en âge de procréer et ce, peu importe la dose. De nouvelles campagnes de santé publique devront être mises sur pied, afin d’inciter les femmes à consommer de l’acide folique avant et pendant leur grossesse. Également, la prise de haute dose d’acide folique ne semble pas avoir diminué le risque de malformations congénitales, à l’échelle populationnelle.

Preliminary population study of Methylenetetrahydrofolate Reductase (MTHFR) C677T polymorphism determination in a pilot group of students from the University of Rosario

 Introduction: the 5, 10-methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme in folate metabolism; their polymorphisms have been associated with heart disease risk increase, obstetric problems, neural tube defects in fetuses and cancer susceptibility. This gene has a single nucleotide polymorphism, a C-T change at nucleotide 677, which affects significantly its enzymatic activity. Objective: because of the biological importance of this enzyme and the Colombian population genetic heterogeneity characteristic, a study was performed to determine allele and genotype frequencies of MTHFR C677T polymorphism in healthy individuals, taking into account that in Colombia there are only studies that have involved case-control methodology. Methods: we analyzed this polymorphism trough the amplification of the DNA of a 206 students sample population. Additionally, Colombian overall frequencies were calculated, using data from healthy controls reported in other studies. Results: a Hardy-Weinberg disequilibri m was found in the sample tested. For the Colombian data, we found that the global population was in equilibrium. Conclusion: T allele population frequency seems to be under positive selection pressure, which is reflected in the population allele increase, despite its deleterious effect. A Spanish study reported similar results and identified folic acid supplementation on expectant mothers as a probably cause of this change. 

Búsqueda de selección en el polimorfismo 677C>T (c.665C>T) del gen de la metilentetrahidrofolato reductasa (MTHFR) en una población Colombiana

Se realizó un estudio genético – poblacional en dos grupos etarios de población colombiana con la finalidad de evaluar las diferencias genéticas relacionadas con el polimorfismo MTHFR 677CT en busca de eventos genéticos que soporten la persistencia de este polimorfismo en la especie humana debido que este ha sido asociado con múltiples enfermedades. De esta manera se genotipificaron los individuos, se analizaron los genotipos, frecuencias alélicas y se realizaron diferentes pruebas genéticas-poblacionales. Contrario a lo observado en poblaciones Colombianas revisadas se identificó la ausencia del Equilibrio Hardy-Weinberg en el grupo de los niños y estructuras poblacionales entre los adultos lo que sugiere diferentes historias demográficas y culturales entre estos dos grupos poblacionales al tiempo, lo que soporta la hipótesis de un evento de selección sobre el polimorfismo en nuestra población. De igual manera nuestros datos fueron analizados junto con estudios previos a nivel nacional y mundial lo cual sustenta que el posible evento selectivo es debido a que el aporte de ácido fólico se ha incrementado durante las últimas dos décadas como consecuencia de las campañas de fortificación de las harinas y suplementación a las embarazadas con ácido fólico, por lo tanto aquí se propone un modelo de selección que se ajusta a los datos encontrados en este trabajo se establece una relación entre los patrones nutricionales de la especie humana a través de la historia que explica las diferencias en frecuencias de este polimorfismo a nivel espacial y temporal.  

Expression and regulation of Nkd-1, an intracellular component of Wnt signalling pathway in the chick embryo

The Wnt family of secreted signalling molecules control a wide range of developmental processes in all metazoans. The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement. Recent work has identified the protein Naked Cuticle to act as an intracellular switch to promote the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked Cuticle-1 (cNkd-1) and show that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly, we show that the expression of cNkd-1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

Amphioxus molecular biology: insights into vertebrate evolution and developmental mechanisms

The cephalochordate amphioxus is the best available proxy for the last common invertebrate ancestor of the vertebrates. During the last decade, the developmental genetics of amphioxus have been extensively examined for insights into the evolutionary origin and early evolution of the vertebrates. Comparisons between expression domains of homologous genes in amphioxus and vertebrates have strengthened proposed homologies between specific body parts. Molecular genetic studies have also highlighted parallels in the developmental mechanisms of amphioxus and vertebrates. In both groups, a similar nested pattern of Hox gene expression is involved in rostrocaudal patterning of the neural tube, and homologous genes also appear to be involved in dorsoventral neural patterning. Studies of amphioxus molecular biology have also hinted that the protochordate ancestor of the vertebrates included cell populations that modified their developmental genetic pathways during early vertebrate evolution to yield definitive neural crest and neurogenic placodes. We also discuss how the application of expressed sequence tag and gene-mapping approaches to amphioxus have combined with developmental studies to advance our understanding of chordate genome evolution. We conclude by considering the potential offered by the sequencing of the amphioxus genome, which was completed in late 2004.

Pax gene expression in the developing central nervous system of Ciona intestinalis

We compare the expression patterns in Ciona intestinalis of three members of the Pax gene family, CiPax3/7, CiPax6 and Cipax2/5/8. All three genes are expressed in restricted patterns in the developing central nervous system. At the tailbud stage, CiPax3/7 is present in three patches in the brain and along the posterior neural tube, CiPax6 throughout the anterior brain and along the posterior neural tube and CiPax2/5/8 in a restricted region of the posterior brain. Double in situ hybridisations were used to identify areas of overlap between the expression of different genes. This showed that CiPax3/7 overlaps with the boundaries of CiPax6 expression in the anterior brain, and with CiPax2/5/8 in the posterior brain. The overlap between CiPax3/7 and CiPax2/5/8 is unlike that described in the ascidian Halocynthia rorezti. (C) 2003 Elsevier B.V. All rights reserved.

Rho Signaling Pathway and Apical Constriction in the Early Lens Placode

Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase activity. Here we investigate apical constriction during chick lens placode invagination. Inhibition of actin polymerization and myosin II activity by cytochalasin D or blebbistatin prevents lens invagination. To further verify if lens placode invaginate through apical constriction, we analyzed the role of Rho-ROCK pathway. Rho GTPases expression at the apical portion of the lens placode occurs with the same dynamics as that of the cytoskeleton. Overexpression of the pan-Rho inhibitor C3 exotoxin abolished invagination and had a strong effect on apical myosin II enrichment and a mild effect on apical actin localization. In contrast, pharmacological inhibition of ROCK activity interfered significantly with apical enrichment of both actin and myosin. These results suggest that apical constriction in lens invagination involves ROCK but apical concentration of actin and myosin are regulated through different pathways upstream of ROCK. genesis 49: 368-379, 2011. (C) 2011 Wiley-Liss, Inc.

The possible role of selenium status in adverse pregnancy outcomes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Ultrasonographic monitoring of early pregnancy development in Murrah buffalo heifers (Bubalus bubalis)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural analysis of the embryonic development in Brycon cephalus (Gunther, 1869)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sonic hedgehog (SHH) mutation in patients within the spectrum of holoprosencephaly

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Os histricomorfos sul-americanos: uma análise comparativa do desenvolvimento embriológico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Oral clefts and vitamin supplementation

Objectives: the evidence linking low levels of folic acid and orofacial clefting (OFC) is presently equivocal, There is stronger evidence for the role of folic acid supplementation in protection against the occurrence and recurrence of neural tube defects. The present investigation tested the hypotheses that cleft lip, cleft palate, or both are inversely associated with maternal intake of dietary and supplemental vitamins during the periconceptional period and first 4 months of pregnancy in a Brazilian population.Design: A population-based, case-control study of cleft lip with or without cleft palate (CL(P)) and isolated cleft palate (CP) in a Brazilian population. in structured interviews, case histories were taken from the mothers of a consecutive sample of 450 infants born with nonsyndromic OFC,Results: Mothers who had children with CL(P) were less likely to have been supplemented during the periconceptional period. The statistical significance of the difference in prevalence of the use of supplements between mothers of patients and of controls was greater for the CL(P) group: p < .05 for CP and p < .001 for CL(P). Multivariate analysis confirmed this finding of a protective effect for both types of orofacial cleft,Conclusions: the use of vitamin supplements in the first 4 months of pregnancy was suggestive of a protective effect against the occurrence of CP and CL(P) in this population, the significance of an association between multivitamin supplementation and OFC and the possible role of gene/environment interaction are discussed.