Anayansi Molina-Hernández

Histamine induces neural stem cell proliferation and neuronal differentiation by activation of distinct histamine receptors.

Histamine has neurotransmitter/neuromodulator functions in the adult brain, but its role during CNS development has been elusive. We studied histamine effects on proliferation, cell death and differentiation of neuroepithelial stem cells from rat cerebral cortex in vitro. RT‐PCR and Western blot experiments showed that proliferating and differentiated cells express histamine H1, H2 and H3 receptors. Treatments with histamine concentrations (100 nM–1 mM) caused significant increases in cell numbers without affecting Nestin expression. Cell proliferation was evaluated by BrdU incorporation; histamine caused a significant increase dependent on H2 receptor activation. Apoptotic cell death during proliferation was significantly decreased at all histamine concentrations, and cell death was promoted in a concentration‐dependent manner by histamine in differentiated cells. Immunocytochemistry studies showed that histamine increased 3‐fold the number of neurons after differentiation, mainly by activation of H1 receptor, and also significantly decreased the glial (astrocytic) cell proportion, when compared to control conditions. In summary, histamine increases cell number during proliferative conditions, and has a neuronal‐differentiating action on neural stem cells, suggesting that the elevated histamine concentration reported during development might play a role in cerebrocortical neurogenesis, by activation of H2 receptors to promote proliferation of neural precursors, and favoring neuronal fate by H1‐mediated stimulation.

Histamine in brain development.

J. Neurochem. (2012) 122, 872–882.

CHANGES IN THE CONTENT OF ESTROGEN α AND PROGESTERONE RECEPTORS DURING DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS TO DOPAMINE NEURONS

Embryonic stem cells (ESC) can differentiate to derivatives of the three embryonic germ layers. Dopamine neurons have been produced from mouse and human ESC. This in vitro induction mimics the developmental program followed by dopaminergic cells in vivo. Production of dopamine neurons might have clinical applications for Parkinsons disease, which has a higher incidence in men than in women, suggesting a protective role for sex hormones, particularly progesterone and estradiol. These hormones exert many of their effects through the interaction with their nuclear receptors. In this study, we used a described 5-stage protocol for dopamine neuron differentiation of ESC, allowing neuronal commitment as evidenced by specific markers and by behavioural recovery of hemiparkinsonian rats after grafting. We studied the expression of steroid hormone receptors by immunoblot during this procedure and found an increase in the content of both A and B isoforms of progesterone receptor (PR) and a decrease in estrogen receptor alpha (ER-alpha) when cells were at the neural/neuronal stages, when compared with the amount found in initial pluripotent conditions. We also found the same pattern of PR and ER-alpha expression by immunocytochemistry. Ninety-two percent of dopamine neurons expressed progesterone receptors and only 19% of these neurons co-expressed tyrosine hydroxylase and ER-alpha. These results show a differential expression pattern of ER-alpha and PR isoforms during neuronal differentiation of ESC.

Histamine is required during neural stem cell proliferation to increase neuron differentiation

Histamine in the adult central nervous system (CNS) acts as a neurotransmitter. This amine is one of the first neurotransmitters to appear during development reaching its maximum concentration simultaneously with neuron differentiation peak. This suggests that HA plays an important role in neurogenesis. We have previously shown that HA is able to increase neuronal differentiation of neural stem cells (NSCs) in vitro, by activating the histamine type 1 receptor. However the mechanism(s) by which HA has a neurogenic effect on NSCs has not been explored. Here we explore how HA is able to increase neuron phenotype. Cortex neuroepithelium progenitors were cultured and at passage two treatments with 100 μM HA were given during cell proliferation and differentiation or only during differentiation. Immunocytochemistry was performed on differentiated cultures to detect mature neurons. To explore the expression of certain important transcriptional factors involved on asymmetric cell division and commitment, RT-PCR and qRT-PCR were performed. Results indicate that HA is required during cell proliferation in order to increase neuron differentiation and suggest that this amine increases neuron commitment during the proliferative phase probably by rising prospero1 and neurogenin1 expression.

Activin A Promotes Neuronal Differentiation of Cerebrocortical Neural Progenitor Cells

Background 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. Methodology/Principal Findings 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. Conclusions Our results show an instructive neuronal effect of Activin A in cortical NPC in vitro, pointing out to a relevant role of this cytokine in the specification of NPC towards a neuronal phenotype.

Histamine up-regulates fibroblast growth factor receptor 1 and increases FOXP2 neurons in cultured neural precursors by histamine type 1 receptor activation: conceivable role of histamine in neurogenesis during cortical development in vivo

BackgroundDuring rat development, histamine (HA) is one of the first neuroactive molecules to appear in the brain, reaching its maximal value at embryonic day 14, a period when neurogenesis of deep layers is occurring in the cerebral cortex, suggesting a role of this amine in neuronal specification. We previously reported, using high-density cerebrocortical neural precursor cultures, that micromolar HA enhanced the effect of fibroblast growth factor (FGF)-2 on proliferation, and that HA increased neuronal differentiation, due to HA type 1 receptor (H1R) activation.ResultsClonal experiments performed here showed that HA decreased colony size and caused a significant increase in the percentage of clones containing mature neurons through H1R stimulation. In proliferating precursors, we studied whether HA activates G protein-coupled receptors linked to intracellular calcium increases. Neural cells presented an increase in cytoplasmic calcium even in the absence of extracellular calcium, a response mediated by H1R. Since FGF receptors (FGFRs) are known to be key players in cell proliferation and differentiation, we determined whether HA modifies the expression of FGFRs1-4 by using RT-PCR. An important transcriptional increase in FGFR1 was elicited after H1R activation. We also tested whether HA promotes differentiation specifically to neurons with molecular markers of different cortical layers by immunocytochemistry. HA caused significant increases in cells expressing the deep layer neuronal marker FOXP2; this induction of FOXP2-positive neurons elicited by HA was blocked by the H1R antagonist chlorpheniramine in vitro. Finally, we found a notable decrease in FOXP2+ cortical neurons in vivo, when chlorpheniramine was infused in the cerebral ventricles through intrauterine injection.ConclusionThese results show that HA, by activating H1R, has a neurogenic effect in clonal conditions and suggest that intracellular calcium elevation and transcriptional up-regulation of FGFR1 participate in HA-induced neuronal differentiation to FOXP2 cells in vitro; furthermore, H1R blockade in vivo resulted in decreased cortical FOXP2+ neurons.

Activated Notch1 is a stronger astrocytic stimulus than leukemia inhibitory factor for rat neural stem cells.

Neural stem cells (NSC) self-renew and generate specialized cell types. There are reports indicating that Notch and Leukemia Inhibitory Factor (LIF) signaling are involved in cell determination of NSC, either preventing differentiation or promoting astrocytic fate. In this work, we aimed to compare the astrocytogenic effect of activated Notch with that induced by LIF. To this end, rat cerebral cortex neural progenitors/NSC were transduced with retroviral vectors in order to express green fluorescent protein (GFP), or a fusion protein of GFP with the active Notch1 intracellular domain (NICD). In parallel, other cultures were treated with increasing concentrations of LIF. We confirmed, in proliferating NSC, that LIF activated intracellular effectors by measuring STAT3 phosphorylation and Socs3 transcription. In NICD-expressing cells, Hes5 mRNA was induced, an effect not present in GFP-transduced NSC. We quantified the proportion of cells expressing Nestin in the presence of Fibroblast Growth Factor-2 (FGF-2) with LIF or NICD treatments. LIF significantly increased the proportion of cells co-expresssing Nestin and Glial Fibrillary Acidic Protein (GFAP), an effect absent in cells with activated Notch. After FGF2 withdrawal to promote differentiation, Nestin was markedly down-regulated, and neuronal and glial markers appeared in control cultures. LIF treatment caused a significant increase in the proportion of GFAP-positive cells, but cells expressing NICD showed a significantly higher percentage of astrocytes than control and LIF-treated cultures. These experiments show that cells stimulated with NICD differentiate more readily to astrocytes than LIF-treated NSC.

MicroRNAs in central nervous system development.

Abstract During early and late embryo neurodevelopment, a large number of molecules work together in a spatial and temporal manner to ensure the adequate formation of an organism. Diverse signals participate in embryo patterning and organization synchronized by time and space. Among the molecules that are expressed in a temporal and spatial manner, and that are considered essential in several developmental processes, are the microRNAs (miRNAs). In this review, we highlight some important aspects of the biogenesis and function of miRNAs as well as their participation in ectoderm commitment and their role in central nervous system (CNS) development. Instead of giving an extensive list of miRNAs involved in these processes, we only mention those miRNAs that are the most studied during the development of the CNS as well as the most likely mRNA targets for each miRNA and its protein functions.

Markers of Pluripotency in Human Amniotic Epithelial Cells and Their Differentiation to Progenitor of Cortical Neurons

Human pluripotent stem cells (hPSC) have promise for regenerative medicine due to their auto-renovation and differentiation capacities. Nevertheless, there are several ethical and methodological issues about these cells that have not been resolved. Human amniotic epithelial cells (hAEC) have been proposed as source of pluripotent stem cells. Several groups have studied hAEC but have reported inconsistencies about their pluripotency properties. The aim of the present study was the in vitro characterization of hAEC collected from a Mexican population in order to identify transcription factors involved in the pluripotency circuitry and to determine their epigenetic state. Finally, we evaluated if these cells differentiate to cortical progenitors. We analyzed qualitatively and quantitatively the expression of the transcription factors of pluripotency (OCT4, SOX2, NANOG, KLF4 and REX1) by RT-PCR and RT-qPCR in hAEC. Also, we determined the presence of OCT4, SOX2, NANOG, SSEA3, SSEA4, TRA-1-60, E-cadherin, KLF4, TFE3 as well as the proliferation and epigenetic state by immunocytochemistry of the cells. Finally, hAEC were differentiated towards cortical progenitors using a protocol of two stages. Here we show that hAEC, obtained from a Mexican population and cultured in vitro (P0-P3), maintained the expression of several markers strongly involved in pluripotency maintenance (OCT4, SOX2, NANOG, TFE3, KLF4, SSEA3, SSEA4, TRA-1-60 and E-cadherin). Finally, when hAEC were treated with growth factors and small molecules, they expressed markers characteristic of cortical progenitors (TBR2, OTX2, NeuN and β-III-tubulin). Our results demonstrated that hAEC express naïve pluripotent markers (KLF4, REX1 and TFE3) as well as the cortical neuron phenotype after differentiation. This highlights the need for further investigation of hAEC as a possible source of hPSC.

Capturing the ephemeral human pluripotent state

During human development, pluripotency is present only in early stages of development. This ephemeral cell potential can be captured in vitro by obtaining pluripotent stem cells (PSC) with self‐renewal properties, the human embryonic stem cells (hESC). However, diverse studies suggest the existence of a plethora of human PSC (hPSC) that can be derived from both embryonic and somatic sources, depending on defined culture conditions, their spatial origin, and the genetic engineering used for reprogramming. This review will focus on hPSC, covering the conventional primed hESC, naïve‐like hPSC that resemble the ground‐state of development, region‐selective PSC, and human induced PSC (hiPSC). We will analyze differences and similarities in their differentiation potential as well as in the molecular circuitry of pluripotency. Finally, we describe the need for human feeder cells to derive and maintain hPSC, because they could emulate the interaction of in vivo pluripotent cells with extraembryonic structures that support development. Developmental Dynamics 245:762–773, 2016.