Wnt信号在胚胎干细胞自我更新和分化中的作用

胚胎干细胞分离自胚泡内细胞团,具有无限自我更新和多向分化潜能,有很大的医学应用前景。Wnt家族是一类分泌型的细胞信号传导蛋白,可以通过复杂的信号传递通路调控胚胎的早期发育,对细胞的分化、增殖及生长具有重要的调节作用。该文就Wnt信号通路调节胚胎干细胞的自我更新和分化作一综述。     

Genome-wide gene amplification during differentiation of neural progenitor cells in vitro

DNA sequence amplification is a phenomenon that occurs predictably at defined stages during normal development in some organisms. Developmental gene amplification was first described in amphibians during gametogenesis and has not yet been described in humans. To date gene amplification in humans is a hallmark of many tumors. We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells. Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells. We identified several amplified genes in neural progenitor cells that are known to be amplified in malignant tumors. There is also a striking overlap of amplified chromosomal regions between differentiating neural progenitor cells and malignant tumor cells derived from astrocytes. Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.     

Spatiotemporal expression of connexin 39 and -43 during myoblast differentiation in cultured cells and in the mouse embryo

Connexin39 (Cx39) and connexin43 (Cx43) are known to be expressed during development of skeletal muscles. Here we have compared the expression pattern of both connexins during differentiation of established C(2)C(12) mouse myoblasts and in the mouse embryo. Cx43 is highly abundant in undifferentiated myoblasts, but no Cx39 protein was detected in these cells. Upon differentiation into myotubes, Cx39 expression increased. The consecutive expression of these connexins was also observed in the mouse embryo. Cx39 and Cx43 were found in different plaques in accordance with the notion that Cx43 is exclusively expressed in myoblasts and Cx39 in myotubes. Thus, differentiating C(2)C(12) cells in culture can serve to study the involvement of gap junctions in myogenesis, since expression of corresponding Cx39 and Cx43 proteins appears to be very similar as in the mouse embryo.     

Involvement of gangliosides in proliferation of immortalized neural progenitor cells

The CNS consists of neuronal and glial cells generated from common neural progenitor cells during development. Cellular events for neural progenitor cells, such as proliferation and differentiation, are regulated by multiple intrinsic and extrinsic cell signals. Although much is known on the importance of the proteinous factors in regulating the fate of neural progenitor cells, the involvement of other molecules such as gangliosides, sialic acid-containing glycosphingolipids, remains to be clarified. To elucidate the biological functions of gangliosides in neural progenitor cells, we transfected an immortalized neural progenitor cell line, C17.2, which does not express GD3 ganglioside, with a fusion protein of GD3-synthase (ST-II) and enhanced green fluorescent protein (ST-II-EGFP). Analysis of the ST-II transfectants revealed the ectopic expression of b- and c-series gangliosides. In the ST-II transfectants, proliferation induced by epidermal growth factor (EGF) was severely retarded. EGF-induced proliferation of C17.2 cells was dependent on the Ras-mitogen-activated protein kinase (Ras-MAPK) pathway, and the EGF-induced activation of this pathway was significantly repressed in the transfectants. Thus, ST-II overexpression retarded proliferation of C17.2 cells via repression of the Ras-MAPK pathway. The result supports the concept that gangliosides may play an important role in regulating the proliferation of neural progenitor cells.     

Erythropoietin and the effect of oxygen during proliferation and differentiation of human neural progenitor cells

Background  

Hypoxia plays a critical role in various cellular mechanisms, including proliferation and differentiation of neural stem and progenitor cells. In the present study, we explored the impact of lowered oxygen on the differentiation potential of human neural progenitor cells, and the role of erythropoietin in the differentiation process.     

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.     

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.     

Cdk2 is critical for proliferation and self-renewal of neural progenitor cells in the adult subventricular zone

We investigated the function of cyclin-dependent kinase 2 (Cdk2) in neural progenitor cells during postnatal development. Chondroitin sulfate proteoglycan (NG2)–expressing progenitor cells of the subventricular zone (SVZ) show no significant difference in density and proliferation between Cdk2^−/− and wild-type mice at perinatal ages and are reduced only in adult Cdk2^−/− mice. Adult Cdk2^−/− SVZ cells in culture display decreased self-renewal capacity and enhanced differentiation. Compensatory mechanisms in perinatal Cdk2^−/− SVZ cells, which persist until postnatal day 15, involve increased Cdk4 expression that results in retinoblastoma protein inactivation. A subsequent decline in Cdk4 activity to wild-type levels in postnatal day 28 Cdk2^−/− cells coincides with lower NG2⁺ proliferation and self-renewal capacity similar to adult levels. Cdk4 silencing in perinatal Cdk2^−/− SVZ cells abolishes Cdk4 up-regulation and reduces cell proliferation and self- renewal to adult levels. Conversely, Cdk4 overexpression in adult SVZ cells restores proliferative capacity to wild-type levels. Thus, although Cdk2 is functionally redundant in perinatal SVZ, it is important for adult progenitor cell proliferation and self-renewal through age-dependent regulation of Cdk4.     

Involvement of de novo synthesized palmitate and mitochondrial EGFR in EGF induced mitochondrial fusion of cancer cells

Increased expressions of fatty acid synthase (FASN) and epidermal growth factor receptor (EGFR) are common in cancer cells. De novo synthesis of palmitate by FASN is critical for the survival of cancer cells via mechanisms independent of its role as an energy substrate. Besides the plasma membrane and the nucleus, EGFR can also localize at the mitochondria; however, signals that can activate mitochondrial EGFR (mtEGFR) and the functions of mtEGFR of cancer cells remain unknown. The present study characterizes mtEGFR in the mitochondria of cancer cells (prostate and breast) and reveals that mtEGFR can promote mitochondrial fusion through increasing the protein levels of fusion proteins PHB2 and OPA1. Activation of plasma membranous EGFR (pmEGFR) stimulates the de novo synthesis of palmitate through activation of FASN and ATP-citrate lyase (ACLy). In vitro kinase assay with isolated mitochondria shows that palmitate can activate mtEGFR. Inhibition of FASN blocks the mtEGFR phosphorylation and palmitoylation induced by EGF. Mutational studies show that the cysteine 797 is important for mtEGFR activation and palmitoylation. Inhibition of FASN can block EGF induced mitochondrial fusion and increased the sensitivity of prostate cancer cells to EGFR tyrosine kinase inhibitor. In conclusion, these results suggest that mtEGFR can be activated by pmEGFR through de novo synthesized palmitate to promote mitochondrial fusion and survival of cancer cells. This mechanism may serve as a novel target to improve EGFR-based cancer therapy.     

CREB signalling in neural stem/progenitor cells: recent developments and the implications for brain tumour biology

This paper discusses the evidence for the role of CREB in neural stem/progenitor cell (NSPC) function and oncogenesis and how these functions may be important for the development and growth of brain tumours. The cyclic-AMP response element binding (CREB) protein has many roles in neurons, ranging from neuronal survival to higher order brain functions such as memory and drug addiction behaviours. Recent studies have revealed that CREB also has a role in NSPC survival, differentiation and proliferation. Recent work has shown that over-expression of CREB in transgenic animals can impart oncogenic properties on cells in various tissues and that aberrant CREB expression is associated with tumours in patients. It is the central position of CREB, downstream of key developmental and growth signalling pathways, which give CREB the ability to influence a spectrum of cell activities, such as cell survival, growth and differentiation in both normal and cancer cells.     

Akhirin regulates the proliferation and differentiation of neural stem cells/progenitor cells at neurogenic niches in mouse brain

Specialized microenvironment, or neurogenic niche, in embryonic and postnatal mouse brain plays critical roles during neurogenesis throughout adulthood. The subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus in the mouse brain are two major neurogenic niches where neurogenesis is directed by numerous regulatory factors. Now, we report Akhirin (AKH), a stem cell maintenance factor in mouse spinal cord, plays a pivotal regulatory role in the SVZ and in the DG. AKH showed specific distribution during development in embryonic and postnatal neurogenic niches. Loss of AKH led to abnormal development of the ventricular zone and the DG along with reduction of cellular proliferation in both regions. In AKH knockout mice (AKH^−/−), quiescent neural stem cells (NSCs) increased, while proliferative NSCs or neural progenitor cells decreased at both neurogenic niches. In vitro NSC culture assay showed increased number of neurospheres and reduced neurogenesis in AKH^−/−. These results indicate that AKH, at the neurogenic niche, exerts dynamic regulatory role on NSC self-renewal, proliferation and differentiation during SVZ and hippocampal neurogenesis.     

N-cadherin-based adherens junction regulates the maintenance,proliferation, and differentiation of neural progenitor cells during development

This review addresses our current understanding of the regulatory mechanism by which N-cadherin, a classical cadherin, affects neural progenitor cells (NPCs) during development. N-cadherin is responsible for the integrity of adherens junctions (AJs), which develop in the sub-apical region of NPCs in the neural tube and brain cortex. The apical domain, which contains the sub-apical region, is involved in the switching from symmetric proliferative division to asymmetric neurogenic division of NPCs. In addition, N-cadherin-based AJ is deeply involved in the apico-basal polarity of NPCs and the regulation of Wnt-β-catenin, hedgehog (Hh), and Notch signaling. In this review, we discuss the roles of N-cadherin in the maintenance, proliferation, and differentiation of NPCs through components of AJ, β-catenin and αE-catenin.     

GDNF augments survival and differentiation of TH-positive neurons in neural progenitor cells

GDNF plays an important role in the survival and differentiation of primary dopaminergic neurons, but it requires multiple factors for its entire range of activities. This study investigated the effects of GDNF and its cofactors on the development of bFGF-responsive neural progenitor cells (NPCs), mesencephalic and cortical progenitor cells (MP and CP). Various factors were found to have significant inductive effects on the survival and maintenance of these cells in late developmental stages. MP had greater potential than CP to differentiate into dopaminergic neurons. Treatment of NPCs with GDNF and its cofactors enhanced MAP-2 and TH expression, particularly the latter. These findings suggest that NPCs, particularly MP, could develop into more specific neurons if the appropriate factors were applied during the final cell fate specification. They might thus become beneficial sources of donor cells in the treatment of neurological disorders.     

DISP3 promotes proliferation and delays differentiation of neural progenitor cells

DISP3 (PTCHD2), a sterol-sensing domain-containing protein, is highly expressed in neural tissue but its role in neural differentiation is unknown. In the present study we used a multipotent cerebellar progenitor cell line, C17.2, to investigate the impact of DISP3 on the proliferation and differentiation of neural precursors. We found that ectopically expressed DISP3 promotes cell proliferation and alters expression of genes that are involved in tumorigenesis. Finally, the differentiation profile of DISP3-expressing cells was altered, as evidenced by delayed expression of neural specific markers and a reduced capacity to undergo neural differentiation.     

ADAM23 promotes neuronal differentiation of human neural progenitor cells

Background

ADAM23 is widely expressed in the embryonic central nervous system and plays an important role in tissue formation.

Results

In this study, we showed that ADAM23 contributes to cell survival and is involved in neuronal differentiation during the differentiation of human neural progenitor cells (hNPCs). Upregulation of ADAM23 in hNPCs was found to increase the number of neurons and the length of neurite, while its downregulation decreases them and triggers cell apoptosis. RNA microarray analysis revealed mechanistic insights into genes and pathways that may become involved in multiple cellular processes upon up- or downregulation of ADAM23.

Conclusions

Our results suggest that ADAM23 regulates neuronal differentiation by triggering specific signaling pathways during hNPC differentiation.