Unraveling the Spatiotemporal Human Pluripotency in Embryonic Development

There have been significant advances in understanding human embryogenesis using human pluripotent stem cells (hPSCs) in conventional monolayer and 3D self-organized cultures. Thus, in vitro models have contributed to elucidate the molecular mechanisms for specification and differentiation during development. However, the molecular and functional spectrum of human pluripotency (i.e., intermediate states, pluripotency subtypes and regionalization) is still not fully understood. This review describes the mechanisms that establish and maintain pluripotency in human embryos and their differences with mouse embryos. Further, it describes a new pluripotent state representing a transition between naïve and primed pluripotency. This review also presents the data that divide pluripotency into substates expressing epiblast regionalization and amnion specification as well as primordial germ cells in primates. Finally, this work analyzes the amnion’s relevance as an “signaling center” for regionalization before the onset of gastrulation

Maternal Diabetes and Fetal Programming Toward Neurological Diseases: Beyond Neural Tube Defects

The purpose of this review was to search for experimental or clinical evidence on the effect of hyperglycemia in fetal programming to neurological diseases, excluding evident neural tube defects. The lack of timely diagnosis and the inadequate control of diabetes during pregnancy have been related with postnatal obesity, low intellectual and verbal coefficients, language and motor deficits, attention deficit with hyperactivity, problems in psychosocial development, and an increased predisposition to autism and schizophrenia. It has been proposed that several childhood or adulthood diseases have their origin during fetal development through a phenomenon called fetal programming. However, not all the relationships between the outcomes mentioned above and diabetes during gestation are clear, well-studied, or have been related to fetal programming. To understand this relationship, it is imperative to understand how developmental processes take place in health, in order to understand how the functional cytoarchitecture of the central nervous system takes place; to identify changes prompted by hyperglycemia, and to correlate them with the above postnatal impaired functions. Although changes in the establishment of patterns during central nervous system fetal development are related to a wide variety of neurological pathologies, the mechanism by which several maternal conditions promote fetal alterations that contribute to impaired neural development with postnatal consequences are not clear. Animal models have been extremely useful in studying the effect of maternal pathologies on embryo and fetal development, since obtaining central nervous system tissue in humans with normal appearance during fetal development is an important limitation. This review explores the state of the art on this topic, to help establish the way forward in the study of fetal programming under hyperglycemia and its impact on neurological and psychiatric disorders

The Systemic Administration of the Histamine H1 Receptor Antagonist/Inverse Agonist Chlorpheniramine to Pregnant Rats Impairs the Development of Nigro-Striatal Dopaminergic Neurons

The dopaminergic and histaminergic systems are the first to appear during the development of the nervous system. Through the activation of H1 receptors (H1Rs), histamine increases neurogenesis of the cortical deep layers, while reducing the dopaminergic phenotype (cells immunoreactive to tyrosine hydroxylase, TH+) in embryo ventral mesencephalon. Although the function of histamine in neuronal differentiation has been studied, the role of H1Rs in neurogenesis has not been addressed. For this purpose, the H1R antagonist/inverse agonist chlorpheniramine was systemically administered (5 mg/kg, i.p.) to pregnant Wistar rats (gestational days 12–14, E12–14), and control and experimental embryos (E14 and E16) and pups (21-day-old) were evaluated for changes in nigro-striatal development. Western blot and immunohistochemistry determinations showed a significant increase in the dopaminergic markers’ TH and PITX3 in embryos from chlorpheniramine-treated rats at E16. Unexpectedly, 21-day-old pups from the chlorpheniramine-treated group, showed a significant reduction in TH immunoreactivity in the substantia nigra pars compacta and dorsal striatum. Furthermore, striatal dopamine content, evoked [3H]-dopamine release and methamphetamine-stimulated motor activity were significantly lower compared to the control group. These results indicate that H1R blockade at E14–E16 favors the differentiation of dopaminergic neurons, but hampers their migration, leading to a decrease in dopaminergic innervation of the striatum in post-natal life

Cerebrocortical neural progenitor cells are responsive to addition of Activin A.

<p>Passage 2 neural progenitor cells (NPC) were obtained from E14 rat embryos and kept in proliferation with FGF2. <b>A:</b> RNA was isolated from these cultures to perform RT-PCR to detect Activin and TGF-β type I and type II receptors. <b>B:</b> Cells were stimulated with 3 ng/ml Activin A at the indicated times. Protein from each sample was obtained and immunoprecipitated with anti-Smad 2/3 antibodies, followed by immunoblot detection of phosphorylated (p)Smad 2, pSmad 3 and total Smad 2/3. The molecular weight (Mol. Wt.) markers are indicated on the right side. <b>C:</b> Immunocytochemistry for Smad 2/3 in cells either untreated or treated during 30 minutes with Activin A. <b>D:</b> Quantification of nuclear translocation of Smad 2/3 from 10 random fields. The values are represented as mean ±S.D. expressed as the percent of total cells (detected by Hoechst staining in blue) with predominant nuclear staining for Smad 2/3 (green label). **<i>p<0</i>.01 versus untreated condition. Scale bar = 50 µm.</p

Activin A increases neuronal differentiation in clonal NPC cultures.

<p>Cells were grown at low density and the areas with isolated cells were identified with circles with the aid of a marking objective. Cultures were maintained with FGF2 during 6 days and differentiated by 8 additional days. Cells received continuous treatment with Activin A and were immunocytochemically stained for β–III Tubulin and GFAP. <b>A:</b> In controls, a few differentiated neurons were found, whereas Activin A (<b>B</b>) increased the number of β–III Tubulin⁺ cells. <b>A’</b> and <b>B’</b> are magnifications of control and Activin-treated cells, respectively. <b>C:</b> Quantification of the percentage of neuronal (β-III Tubulin-positive) or astrocytic (GFAP-positive) cells relative to total cell number in each clone. Results are mean ±S.D. *<i>p<0</i>.05 versus control condition. Scale bar = 100 µm.</p

Activin A Promotes Neuronal Differentiation of Cerebrocortical Neural Progenitor Cells

<div><h3>Background</h3><p>Activin A is a protein that participates principally in reproductive functions. In the adult brain, Activin is neuroprotective, but its role in brain development is still elusive.</p> <h3>Methodology/Principal Findings</h3><p>We studied if Activin A influences proliferation, differentiation or survival in rat cerebrocortical neural progenitor cells (NPC). After stimulation of NPC with Activin A, phosphorylation and nuclear translocation of Smad 2/3 were induced. In proliferating NPC, Activin produced a significant decrease in cell area and also a discrete increase in the number of neurons in the presence of the mitogen Fibroblast Growth Factor 2. The percentages of cells incorporating BrdU, or positive for the undifferentiated NPC markers Nestin and Sox2, were unchanged after incubation with Activin. In differentiating conditions, continuous treatment with Activin A significantly increased the number of neurons without affecting astroglial differentiation or causing apoptotic death. In cells cultured by extended periods, Activin treatment produced further increases in the proportion of neurons, excluding premature cell cycle exit. In clonal assays, Activin significantly increased neuronal numbers per colony, supporting an instructive role. Activin-induced neurogenesis was dependent on activation of its receptors, since incubation with the type I receptor inhibitor SB431542 or the ligand-trap Follistatin prevented neuronal differentiation. Interestingly, SB431542 or Follistatin by themselves abolished neurogenesis and increased astrogliogenesis, to a similar extent to that induced by Bone Morphogenetic Protein (BMP)4. Co-incubation of these Activin inhibitors with the BMP antagonist Dorsomorphin restored neuronal and astrocytic differentiation to control levels.</p> <h3>Conclusions</h3><p>Our results show an instructive neuronal effect of Activin A in cortical NPC <em>in vitro,</em> pointing out to a relevant role of this cytokine in the specification of NPC towards a neuronal phenotype.</p> </div

Blockade of Alk receptor activation abolishes neuronal differentiation due to Activin A treatment.

<p>Cells were cultured 4 days with FGF2 and 6 days without FGF2. Neuronal and astrocytic differentiation was analyzed by immunocytochemistry and quantitative RT-PCR. Addition of growth factors and inhibitors was made every other day. SB431542 (Alk4, Alk5 and Alk7 inhibitor, 5 µM), Follistatin (Activin A ligand-trap, 20 ng/ml) or Dorsomorphin (BMP antagonists, 5 µM) were added to NPC cultures 1 hour previous to Activin A. <b>A:</b> Representative micrographs showing the antagonistic effect of SB431542 or Follistatin on neuronal differentiation caused by Activin A; either of these compounds had the same antagonistic effects in the absence of Activin A, and also increased astrocyte differentiation. Dorsomorphin by itself did not change neuronal/glial proportions relative to controls. Co-treatment with Dorsomorphin and SB431542, however, prevented SB-induced astrocyte differentiation. <b>B:</b> Quantification of the total number of β-III Tubulin-positive or GFAP-positive cells in experiments performed by duplicate from 3 independent cultures. In addition to antagonizing the neurogenic effects of Activin A, SB431542 and Follistatin by themselves, decreased the number of neurons and also increased astroglial differentiation. When cells received co-treatment with both inhibitors (Dorsomorphin and SB431542) the proportion of neurons was similar to untreated cells, while the number of astrocytes was not as high as that obtained with Activin A and SB431542. <b>C:</b> Real time RT-PCR from cells treated with Activin A plus inhibitors. The represented values were normalized by GAPDH expression level and represented relative to the control condition, set to 1. Co-treatment with Activin A and SB431542 or Activin A plus Follistatin decreased to the half mRNA expression for neuronal markers, while increased GFAP expression. Combined treatment with Dorsomorphin plus SB431542 have neuronal and astrocytic transcript indistinguishable from control values. Results are means ±S.D. The Student-Newman-Keuls was used as a post-hoc test after one-way ANOVA. *<i>p<0</i>.05, **<i>p<0</i>.01 and ***<i>p<0</i>.001 versus control condition. ^#<i>p<0</i>.05, ^{# #}<i>p<0</i>.01, ^###<i>p<0</i>.001 versus the indicated conditions. Scale bar = 50 µm.</p