Calcium and Neural Stem Cell Proliferation.

Intracellular calcium plays a pivotal role in central nervous system (CNS) development by regulating various processes such as cell proliferation, migration, differentiation, and maturation. However, understanding the involvement of calcium (Ca2+) in these processes during CNS development is challenging due to the dynamic nature of this cation and the evolving cell populations during development. While Ca2+ transient patterns have been observed in specific cell processes and molecules responsible for Ca2+ homeostasis have been identified in excitable and non-excitable cells, further research into Ca2+ dynamics and the underlying mechanisms in neural stem cells (NSCs) is required. This review focuses on molecules involved in Ca2+ entrance expressed in NSCs in vivo and in vitro, which are crucial for Ca2+ dynamics and signaling. It also discusses how these molecules might play a key role in balancing cell proliferation for self-renewal or promoting differentiation. These processes are finely regulated in a time-dependent manner throughout brain development, influenced by extrinsic and intrinsic factors that directly or indirectly modulate Ca2+ dynamics. Furthermore, this review addresses the potential implications of understanding Ca2+ dynamics in NSCs for treating neurological disorders. Despite significant progress in this field, unraveling the elements contributing to Ca2+ intracellular dynamics in cell proliferation remains a challenging puzzle that requires further investigation.

Obtaining Tissues of Human Amniotic Membrane and Identification of Pluripotent Markers.

The immunofluorescence technique has been used to identify pluripotent markers in the human amniotic epithelial cells (hAEC). hAEC belonging to human fetal membranes, specificamently to amnion layer, and are arising by epiblast, this sugest that the hAEC have characteristics of epiblast cells, in other words, characteristcs of pluripotent stem cells. Here we describe obtaining human amnion tissue and identifying pluripotent markers by immunofluorescence.

Pluripotent Stem Cells as a Model for Human Embryogenesis.

Pluripotent stem cells (PSCs; embryonic stem cells and induced pluripotent stem cells) can recapitulate critical aspects of the early stages of embryonic development; therefore, they became a powerful tool for the in vitro study of molecular mechanisms that underlie blastocyst formation, implantation, the spectrum of pluripotency and the beginning of gastrulation, among other processes. Traditionally, PSCs were studied in 2D cultures or monolayers, without considering the spatial organization of a developing embryo. However, recent research demonstrated that PSCs can form 3D structures that simulate the blastocyst and gastrula stages and other events, such as amniotic cavity formation or somitogenesis. This breakthrough provides an unparalleled opportunity to study human embryogenesis by examining the interactions, cytoarchitecture and spatial organization among multiple cell lineages, which have long remained a mystery due to the limitations of studying in utero human embryos. In this review, we will provide an overview of how experimental embryology currently utilizes models such as blastoids, gastruloids and other 3D aggregates derived from PSCs to advance our understanding of the intricate processes involved in human embryo development.

Increased Nuclear FOXP2 Is Related to Reduced Neural Stem Cell Number and Increased Neurogenesis in the Dorsal Telencephalon of Embryos of Diabetic Rats through Histamine H1 Receptors.

Diabetic rat embryos have increased cortical neurogenesis and neuron maturation, and their offspring presented altered neuron polarity, lamination, and diminished neuron excitability. The FOXP2 overexpression results in higher cortical neurogenesis by increasing the transition of radial glia to the intermediate progenitor. Similarly, histamine through H1-receptor activation increases cortical neuron differentiation. Indeed, blocking the H1-receptor by the systemic administration of chlorpheniramine to diabetic pregnant rats prevents increased neurogenesis. Here, we explore the relationship between the H1-receptor and FOXP2 on embryo neurogenesis from diabetic dams. Through qRT-PCR, Western blot, immunohistofluorescence, and flow cytometry, we showed an increased FOXP2 expression and nuclear localization, a reduced Nestin expression and -positive cells number, and a higher PKCα expression in the cortical neuroepithelium of fourteen-day-old embryos from diabetic rats. Interestingly, this scenario was prevented by the chlorpheniramine systemic administration to diabetic pregnant rats at embryo day twelve. These data, together with the bioinformatic analysis, suggest that higher H1-receptor activity in embryos under high glucose increases FOXP2 nuclear translocation, presumably through PKCα phosphorylation, impairing the transition of radial glia to intermediate progenitor and increasing neuron differentiation in embryos of diabetic rats.

The human amniotic epithelium confers a bias to differentiate toward the neuroectoderm lineage in human embryonic stem cells.

Human embryonic stem cells (hESCs) derive from the epiblast and have pluripotent potential. To maintain the conventional conditions of the pluripotent potential in an undifferentiated state, inactivated mouse embryonic fibroblast (iMEF) is used as a feeder layer. However, it has been suggested that hESC under this conventional condition (hESC-iMEF) is an artifact that does not correspond to the in vitro counterpart of the human epiblast. Our previous studies demonstrated the use of an alternative feeder layer of human amniotic epithelial cells (hAECs) to derive and maintain hESC. We wondered if the hESC-hAEC culture could represent a different pluripotent stage than that of naïve or primed conventional conditions, simulating the stage in which the amniotic epithelium derives from the epiblast during peri-implantation. Like the conventional primed hESC-iMEF, hESC-hAEC has the same levels of expression as the 'pluripotency core' and does not express markers of naïve pluripotency. However, it presents a downregulation of HOX genes and genes associated with the endoderm and mesoderm, and it exhibits an increase in the expression of ectoderm lineage genes, specifically in the anterior neuroectoderm. Transcriptome analysis showed in hESC-hAEC an upregulated signature of genes coding for transcription factors involved in neural induction and forebrain development, and the ability to differentiate into a neural lineage was superior in comparison with conventional hESC-iMEF. We propose that the interaction of hESC with hAEC confers hESC a biased potential that resembles the anteriorized epiblast, which is predisposed to form the neural ectoderm.

Prolactin from Pluripotency to Central Nervous System Development.

Prolactin (PRL) is a versatile hormone that exerts more than 300 functions in vertebrates, mainly associated with physiological effects in adult animals. Although the process that regulates early development is poorly understood, evidence suggests a role of PRL in the early embryonic development regarding pluripotency and nervous system development. Thus, PRL could be a crucial regulator in oocyte preimplantation and maturation as well as during diapause, a reversible state of blastocyst development arrest that shares metabolic, transcriptomic, and proteomic similarities with pluripotent stem cells in the naïve state. Thus, we analyzed the role of the hormone during those processes, which involve the regulation of its receptor and several signaling cascades (Jak/Mapk, Jak/Stat, and PI3k/Akt), resulting in either a plethora of physiological actions or their dysregulation, a factor in developmental disorders. Finally, we propose models to improve the knowledge on PRL function during early development.

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.

Differential localization of serotoninergic system elements in human amniotic epithelial cells†.

Serotonin or 5-hydroxytryptamine (5-HT) is a biogenic amine involved in regulating several functions, including development. However, its impact on human embryo development has been poorly studied. The present work investigated the expression and distribution of the main components of the serotoninergic system in human amniotic tissue and human amniotic epithelial cells (hAEC) in vitro, as an alternative model of early human embryo development. Amniotic membranes from full-term healthy pregnancies were used. Human amnion tissue or hAEC isolated from the amnion was processed for reverse transcription-polymerase chain reaction and immunofluorescence analyses of the main components of the serotoninergic system. We found the expression of tryptophan hydroxylase type 1 (TPH1), type 2 (TPH2), serotonin transporter (SERT), monoamine oxidase-A (MAOA), as well as HTR1D and HTR7 receptors at mRNA level in amnion tissue as well in hAEC. Interestingly, we found the presence of 5-HT in the nucleus of the cells in amnion tissue, whereas it was located in the cytoplasm of isolated hAEC. We detected TPH1, TPH2, and HTR1D receptor in both the nucleus and cytoplasm. SERT, MAOA, and HTR7 receptor were only observed in the cytoplasm. The results presented herein show, for the first time, the presence of the serotoninergic system in human amnion in vivo and in vitro.

Long-Term Functional and Cytoarchitectonic Effects of the Systemic Administration of the Histamine H1 Receptor Antagonist/Inverse Agonist Chlorpheniramine During Gestation in the Rat Offspring Primary Motor Cortex.

The transient histaminergic system is among the first neurotransmitter systems to appear during brain development in the rat mesencephalon/rhombencephalon. Histamine increases FOXP2-positive deep-layer neuron differentiation of cortical neural stem cells through H1 receptor activation in vitro. The in utero or systemic administration of chlorpheniramine (H1 receptor antagonist/inverse agonist) during deep-layer cortical neurogenesis decreases FOXP2 neurons in the developing cortex, and H1R- or histidine decarboxylase-knockout mice show impairment in learning and memory, wakefulness and nociception, functions modulated by the cerebral cortex. Due to the role of H1R in cortical neural stem cell neurogenesis, the purpose of this study was to evaluate the postnatal impact of the systemic administration of chlorpheniramine during deep-layer cortical neuron differentiation (E12-14) in the primary motor cortex (M1) of neonates (P0) and 21-day-old pups (P21). Chlorpheniramine or vehicle were systemically administered (5 mg/kg, i.p.) to pregnant Wistar rats at gestational days 12-14, and the expression and distribution of deep- (FOXP2 and TBR1) and superficial-layer (SATB2) neuronal cortical markers were analyzed in neonates from both groups. The qRT-PCR analysis revealed a reduction in the expression of Satb2 and FoxP2. However, Western blot and immunofluorescence showed increased protein levels in the chlorpheniramine-treated group. In P21 pups, the three markers showed impaired distribution and increased immunofluorescence in the experimental group. The Sholl analysis evidenced altered dendritic arborization of deep-layer neurons, with lower excitability in response to histamine, as evaluated by whole-cell patch-clamp recording, as well as diminished depolarization-evoked [3H]-glutamate release from striatal slices. Overall, these results suggest long-lasting effects of blocking H1Rs during early neurogenesis that may impact the pathways involved in voluntary motor activity and cognition.

Role of serotonin in vertebrate embryo development.

Since its discovery in 1937, serotonin (5-HT) has become one of the most studied biogenic amines due to its predominant role in regulating several physiological processes such as mood, sleep, and food intake. This amine and the main components of the serotoninergic system are in almost all cells of the body. The presence of 5-HT and the serotoninergic system has been observed in oocytes and in different embryo development stages of fish, amphibians, birds, and mammals. In several classes of vertebrates, the change in the concentration of 5-HT or the alteration of the serotoninergic system, interfere with early embryo development. These data suggest that 5-HT participates in embryo development of vertebrates.

Impaired Cortical Cytoarchitecture and Reduced Excitability of Deep-Layer Neurons in the Offspring of Diabetic Rats.

Maternal diabetes has been related to low verbal task scores, impaired fine and gross motor skills, and poor performance in graphic and visuospatial tasks during childhood. The primary motor cortex is important for controlling motor functions, and embryos exposed to high glucose show changes in cell proliferation, migration, and differentiation during corticogenesis. However, the existing studies do not discriminate between embryos with or without neural tube defects, making it difficult to conclude whether the reported changes are related to neural tube defects or other anomalies. Furthermore, postnatal effects on central nervous system cytoarchitecture and function have been scarcely addressed. Through molecular, biochemical, morphological, and electrophysiological approaches, we provide evidence of impaired primary motor cerebral cortex lamination and neuronal function in pups from diabetic rats, showing an altered distribution of SATB2, FOXP2, and TBR1, impaired cell migration and polarity, and decreased excitability of deep-layer cortical neurons, suggesting abnormalities in cortico-cortical and extra-cortical innervation. Furthermore, phase-plot analysis of action potentials suggests changes in the activity of potassium channels. These results indicate that high-glucose insult during development promotes complex changes in migration, neurogenesis, cell polarity establishment, and dendritic arborization, which in turn lead to reduced excitability of deep-layer cortical neurons.

Effects of progesterone on the cell number of gliomaspheres derived from human glioblastoma cell lines.

AIMS: The malignancy of the Glioblastomas (GBM), the most frequent and aggressive brain tumors, have been associated with the presence of glioma stem cells (GSCs) which can form gliomaspheres (GS) in vitro. Progesterone (P) increases the proliferation, migration, and invasion of GBM cell lines through the interaction with its intracellular receptor (PR). However, it is unknown if the PR is expressed and the possible effects of P in the formation/differentiation of GS. MAIN METHODS: GS were grown from U251 and U87 cell lines by selective culture with serum-free neural stem cell medium. GSCs were identified by the detection of Sox2, Ki67, Nestin, CD133, and CD15 by immunofluorescence. Additionally, the relative expression of PROM1, NES, SOX2, OLIG2, EZH2, BMI1 and PR genes was evaluated by RT-qPCR. The GS were treated with P, and the number of cells was quantified. By RT-PCR the ßIII-TUB and GFAP differentiation genes were evaluated. KEY FINDINGS: GS were maintained until passage four. The expression of all GSCs markers was significantly higher in GS as compared with the basal culture of U251 and U87 cells. We demonstrated for the first time that PR is expressed in GS and this expression was higher as compared with the U251 and U87 cells in basal conditions. Also, we observed that P increased the number of cells derived primary gliomaspheres (GS1) from the U251 line, as well as the expression of the neuronal differentiation marker ßIII-TUB. SIGNIFICANCE: These results suggest the participation of P in the growth of GSCs.

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane.

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

Central Histamine, the H3-Receptor and Obesity Therapy.

The brain histaminergic system plays a pivotal role in energy homeostasis, through H1- receptor activation, it increases the hypothalamic release of histamine that decreases food intake and reduces body weight. One way to increase the release of hypothalamic histamine is through the use of antagonist/inverse agonist for the H3-receptor. Histamine H3-receptors are auto-receptors and heteroreceptors located on the presynaptic membranes and cell soma of neurons, where they negatively regulate the synthesis and release of histamine and other neurotransmitters in the central nervous system. Although several compounds acting as H3-receptor antagonist/inverse agonists have been developed, conflicting results have been reported and only one has been tested as anti-obesity in humans. Animal studies revealed the opposite effect in food intake, energy expeditor, and body weight, depending on the drug, spice, and route of administration, among others. The present review will explore the state of art on the effects of H3-receptor ligands on appetite and body-weight, going through the following: a brief overview of the circuit involved in the control of food intake and energy homeostasis, the participation of the histaminergic system in food intake and body weight, and the H3-receptor as a potential therapeutic target for obesity.

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.

Establishment of human embryonic stem cell line Amicqui-2 using poor-quality embryos from Mexican population.

Although investigation with human embryonic stem cells (HESC) is not decreasing, the derivation of new lines has been diminished. The preeminence of only a few HESC lines in research is accompanied by lack of universal applicability of results as well as by genetic under-representation. We previously reported the derivation of one line with male karyotype from Mexican population. Here, we derived one HESC line (Amicqui-2) with female karyotype from poor-quality embryos. These line comply the pluripotent requirements (normal karyotype, detection of pluripotency-associated markers, mycoplasma test and teratoma formation) and could be a valuable model for studying diseases specific to under-represented population.

Pluripotency markers in tissue and cultivated cells in vitro of different regions of human amniotic epithelium.

Studies have described the presence of pluripotent markers in vivo and in vitro in human amnion. However, the amnion can be divided into reflected, placental and umbilical regions that are anatomically and functionally heterogeneous. Here, we evaluated the expression of pluripotency markers in tissue and cultivated cells in vitro of different regions of human amnion. To this end, we determined the presence of the core pluripotency factors OCT-4, NANOG and SOX-2 by immunofluorescence and RT-PCR and also performed transcriptome analysis of the different regions of amnion tissue. We identified the mRNA and protein of the pluripotency factors in the different regions of human amnion tissue. However, the OCT-4 and NANOG immunolocalization was cytoplasmic, whereas SOX-2 immunolocalization was nuclear regardless of the region analyzed. Moreover, we found three subpopulations of cells in the in vitro cultures of reflected and placental amnion: cells with immunostaining only in the nucleus, only in the cytoplasm, or in both compartments. Yet no statistically significant differences were found between the reflected and placental amnion. These results suggest a homogeneous distribution of the pluripotency transcription factors of the different regions of human amnion to isolate stem cells that can be used in regenerative medicine.

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.

Reference intervals for hemoglobin A1c (HbA1c) in healthy Mexican pregnant women: a cross-sectional study.

BACKGROUND: The reference intervals for hemoglobin A1c (HbA1c) in pregnant Mexican women without diabetes are not well defined. The study aims to determine the reference intervals for HbA1c at each trimester in healthy Mexican pregnant women. METHODS: This cross-sectional study included healthy Mexican pregnant women in trimester 1 (T1), 6-13.6 weeks of gestation (WG), trimester 2 (T2), 14-27 WG, and trimester 3 (T3), ≥27-36 WG, with a maternal age > 18 years, and pregestational body mass index (BMI) ranging between 18.5-24.9 kg/m2. Women with gestational diabetes mellitus, pregestational diabetes, anemia, a pregestational BMI < 18.5 or ≥ 25 kg/m2, and any hematologic, hepatic, immunological, renal, or cardiac disease were excluded. HbA1c was measured using high-performance liquid chromatography based on the National Glycohemoglobin Standardization Program-certified PDQ Primus guidelines. The HbA1c reference intervals were calculated in terms of the 2.5th to the 97.5th percentiles. RESULTS: We analyzed the HbA1c values of 725 women (T1 n = 84, T2 n = 448, and T3 n = 193). The characteristics of the participants were expressed as mean ± standard deviation and included: maternal age (28.2 ± 6.7 years), pregestational weight (54.8 ± 5.9 Kg), pregestational BMI (22.2 ± 1.7 Kg/m2), and glucose values using a 75 g-2 h oral glucose tolerance test; fasting 4.5 ± 0.3 mmol/L (81.5 ± 5.5 mg/dL), 1 h 6.4 ± 1.5 mmol/L (115.3 ± 26.6 mg/dL), and 2 h 5.7 ± 1.1 mmol/L (103.5 ± 19.6 mg/dL). Reference intervals for HbA1c, expressed as median and 2.5th to 97.5th percentile for each trimester were: T1: 5.1 (4.5-5.6%), T2: 5.0 (4.4-5.5%), and T3: 5.1 (4.5-5.6%). CONCLUSIONS: The reference range of HbA1C in healthy Mexican pregnant women during pregnancy was 4.4% to 5.6%. We suggest as upper limits of HbA1c value ≤5.6%, 5.5%, and 5.7% for T1, T2, and T3, respectively among Mexican pregnant women.

Secretion of heat shock -60, -70 kD protein, IL-1ß and TNFα levels in serum of a term normal pregnancy and patients with pre-eclampsia development.

The extracellular heat shock proteins (eHsp) family act as molecular chaperones regulating folding, transporting protein and are associated with immune modulation in different physiological and pathological processes. They have been localized in different gestational tissues and their concentration in amniotic fluid and serum has been determined. In the present study, we proposed to determine the concentration of eHsp-60, -70, IL-1ß and TNFα in the serum of pregnant patients with 34 weeks of gestation with and without clinical evidences of preeclampsia (PE). Our results indicate significant increase of these markers in patients with PE with respect to healthy pregnant patients without active labor. Finally, the concentration of eHsp-60 and -70 correlated positively with the hepatic dysfunction markers uric acid, lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT) glutamic pyruvic transaminase (GPT), and inflammatory IL-1ß and TNFα response. In conclusion, our results demonstrate a strong associated between Hsp and marker of hepatic dysfunction.