Sarcoglycans of the zebrafish: orthology and localization to the sarcolemma and myosepta of muscle

The zebrafish is an established model of vertebrate development and is also receiving increasing attention in terms of human disease modelling. In order to provide experimental support to realize this modelling potential, we report here the identification of apparent orthologues of many critical members of the dystrophin-associated glycoprotein complex (DGC) that have been implicated in a diverse range of neuromuscular disorders. In addition, immunohistochemical studies show the localization of the DGC to the sarcolemma of adult zebrafish muscle and in particular the myosepta. Together, these data suggest that the DGC in adult zebrafish may play a highly conserved functional role in muscle architecture that, when disrupted, could offer insight into human neuromuscular disease processes.     

The IGF-I splice variant MGF increases progenitor cells in ALS, dystrophic, and normal muscle

The effects of muscle splice variants of insulin-like growth factor I (IGF-I) on proliferation and differentiation were studied in human primary muscle cell cultures from healthy subjects as well as from muscular dystrophy and ALS patients. Although the initial numbers of mononucleated progenitor cells expressing desmin were lower in diseased muscle, the E domain peptide of IGF-IEc (MGF) significantly increased the numbers of progenitor cells in healthy and diseased muscle. IGF-I significantly enhances myogenic differentiation whereas MGF E peptide blocks this pathway, resulting in an increased progenitor (stem) cell pool and thus potentially facilitating repair and maintenance of this postmitotic tissue.     

Mutated fukutin-related protein (FKRP) localises as wild type in differentiated muscle cells

The mechanism of disease in forms of congenital and limb girdle muscular dystrophy linked to mutations in the gene encoding for Fukutin-related protein (FKRP) has previously been associated with the mis-localisation of FKRP from the Golgi apparatus. In the present report, we have transfected V5-tagged Fukutin-related protein expression constructs into differentiated C2C12 myotubes and the tibialis anterior of normal mice. The transfection of either wild type (WT) or several mutant constructs (P448L, C318Y, L276I) into myotubes consistently showed clear co-localisation with GM130, a Golgi marker. In contrast, whilst WT and the L276I localised to the Golgi of Cos-7 cells, the P448L and C318Y was mis-localised in the majority of these undifferentiated cells. The injection of the same constructs into the tibialis anterior of mice resulted in similar localisation of both the WT and all the mutants. Immunolabelling of FKRP in the muscle of MDC1C and LGMD2I patients was found to be indistinguishable from normal controls. Overall, these data suggest that retention in the endoplasmic reticulum of FKRP is not the main mechanism of disease but that this may instead relate to a disruption of the functional activity of this putative enzyme with its substrate(s) in the Golgi.     

High expression of the gamma5 isoform of G protein in neuroepithelial cells and its replacement of the gamma2 isoform during neuronal differentiation in the rat brain

High concentrations of G proteins, which include multiple isoforms of each subunit, alpha, beta, and gamma, are expressed in the adult brain. In this study, we concentrated attention on changes of these isoforms during embryonic development in the rat brain. Concentrations of gamma2 as well as GoAalpha, GoBalpha, and beta2 were low in early embryogenesis and then increased, whereas expression of gamma5, in contrast, was initially high followed by a drop, with only very low levels observed throughout postnatal development. Among the other isoforms, Gi1alpha, G(s)alpha-short, G12alpha, G13alpha, beta4, gamma3, gamma7, and gamma12 were present in the embryonic brain at low levels, but their levels markedly increased after birth. In contrast, the levels of Gi2alpha, G(s)alpha-long, Gq/11alpha, and beta1 were essentially constant throughout. Immunohistochemical staining of the brain vesicles in the embryos showed gamma5 to be specifically expressed in the proliferative region of the ventricular zone, whereas gamma2 was mainly present in differentiated neuronal cells of the marginal zone. Furthermore, differentiation of P19 mouse embryonal carcinoma cells to neuronal cells with retinoic acid induced the expression of gamma2 and a decrease of gamma5, the major isoform in the undifferentiated state. These results suggest that neuronal differentiation is responsible for the on/off switch of the expression of gamma2 and gamma5 subunits.     

Glycine regulates proliferation and differentiation of salivary-gland-derived progenitor cells

Amino acids have various physiological activities that influence processes such as intestinal regeneration, EGF secretion, protein synthesis, and cell growth. Salivary glands are exposed to nutrients that influence their proliferation and regeneration. Glycine is included in saliva in large quantities and reportedly has important roles in antibacterial activities and the inhibition of tumor growth and as a precursor of nucleotide synthesis in cell proliferation. We have investigated the effects of glycine on the proliferation and differentiation of salivary glands by using mouse salivary-gland-derived progenitor (mSGP) cells. In cultures of mSGP cells, cell proliferation is suppressed in the presence of glycine, whereas it is promoted by its removal. Glycine promotes three-dimensional formations of mSGP cells, which are negative for immature markers and positive for differentiation markers. In cell-cycle analysis, cell-cycle progression is delayed at the S-phase by glycine supplementation. Glycine also suppresses the phosphorylation of p42/p44MAPK. These results suggest that glycine suppresses the proliferation and promotes the differentiation of mSGP cells, and that it has inhibitory effects on growth factor signaling and cell-cycle progression. Glycine might therefore be a physiological activator that regulates the proliferation and differentiation of salivary glands. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported in part by the Advanced Education Program for Integrated Clinical, Basic and Social Medicine, Graduate School of Medical Sciences, Kumamoto University (Support Program for Improving Graduate School Education, MEXT,　Japan).     

猕猴神经干细胞的诱导分化、干性维持及同种脑内移植

为探索猕猴神经干细胞分化及特性维持,推进神经干细胞临床应用研究,该实验以绿色荧光蛋白(green fluorescence protein,GFP)为标记探讨猕猴胚胎干细胞向玫瑰花环(rosettes)结构神经干细胞的分化及其碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和表皮生长因子(epidermal growth factor,EGF)的扩增培养。结果表明:1)建立了稳定高效的猕猴神经干细胞分化体系,在该分化体系下,GFP标记猕猴胚胎干细胞在分化的第12天时,95%以上的细胞分化为神经干细胞;2)分化得到的Rosettes结构神经干细胞经bFGF/EGF扩增后,能够较好地维持其Rosettes结构;3)经bFGF/EGF扩增后的rosettes结构神经干细胞移植到猕猴脑内后能够较好的存活并向神经元分化,即bFGF/EGF扩增培养能较好地维持Rosettes结构的神经干细胞,且移植到猕猴脑内的该细胞亦能够较好地存活并向神经元分化,该结果为神经干细胞应用于临床提供了基础理论依据。     

DCC is specifically required for the survival of retinal ganglion and displaced amacrine cells in the developing mouse retina

Netrin-1 and DCC are well known for their roles in neurite growth, axonal guidance, and neuronal migration. Recently, a number of studies showed that DCC is involved in the induction of apoptosis, and this proapoptotic activity can be blocked in the presence of Netrin-1. However, here, we found that DCC is required for the survival of two types of neurons selectively in the developing mouse retina where DCC is abundantly expressed. Our results showed that the DCC^−/− retina displayed a reduced ganglion cell layer with relatively normal neuroblastic layer. Immunostaining assays revealed that in DCC^−/− mice, initial neurogenesis within retina was unchanged while the numbers of differentiated retinal ganglion cells and displaced amacrine cells in ganglion cell layer were greatly reduced due to increased apoptosis. By contrast, other neuronal types including horizontal cells, bipolar cells, amacrine cells, photoreceptors, and Müller cells appeared normal in DCC mutant retinas. Moreover, DCC^kanga mice that lack the intracellular P3 domain of DCC receptor displayed the same defects as DCC^−/− mice. Thus, our findings suggest that DCC is a key regulator for the survival of specific types of neurons during retinal development and that DCC-P3 domain is essential for this developing event.     

Wnt/BMP signal integration regulates the balance between proliferation and differentiation of neuroepithelial cells in the dorsal spinal cord

Multiple signaling pathways regulate proliferation and differentiation of neural progenitor cells during early development of the central nervous system (CNS). In the spinal cord, dorsal signaling by bone morphogenic protein (BMP) acts primarily as a patterning signal, while canonical Wnt signaling promotes cell cycle progression in stem and progenitor cells. However, overexpression of Wnt factors or, as shown here, stabilization of the Wnt signaling component beta-catenin has a more prominent effect in the ventral than in the dorsal spinal cord, revealing local differences in signal interpretation. Intriguingly, Wnt signaling is associated with BMP signal activation in the dorsal spinal cord. This points to a spatially restricted interaction between these pathways. Indeed, BMP counteracts proliferation promoted by Wnt in spinal cord neuroepithelial cells. Conversely, Wnt antagonizes BMP-dependent neuronal differentiation. Thus, a mutually inhibitory crosstalk between Wnt and BMP signaling controls the balance between proliferation and differentiation. A model emerges in which dorsal Wnt/BMP signal integration links growth and patterning, thereby maintaining undifferentiated and slow-cycling neural progenitors that form the dorsal confines of the developing spinal cord.     

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.     

In vitro proliferation and differentiation potential of bone marrow-derived mesenchymal stem cells from ovariectomized rats

Bone marrow-derived mesenchymal stem cells (BMMSCs) from the patients suffering from age-related osteoporosis were found to have numerous degeneration, such as decreased growth rate, impaired capacity of differentiating into local tissue, and repressed telomerase activity. However, it is not clear whether post-menopausal osteoporotic bone is either subject to such decline in cellular function. In the present study, bone marrow cells were harvested from ovariectomized (OVX) and Sham rats and cultured in vitro at 3 months post-surgery. MTT assay indicated that the proliferation potential of ^OVXBMMSCs was always higher than that of ^ShamBMMSCs, no matter cultured in basic, osteoblastic or adipogenic medium. Alkaline phosphatase activity assay, Alizarin red S staining, Oil red O staining and real-time RT-PCR analysis further demonstrated that bilateral ovariectomization positively influenced the osteoblastic and adipogenic differentiation potential of BMMSCs, this action may be partly mediated through up-regulation of osteoblastic special markers core binding factor a1, collagen type I and low-density lipoprotein receptor-related protein 5, as well as adipogenic special markers peroxisome proliferators activated receptor gamma, CCAAT/enhancer binding protein alpha and adipocyte lipid-binding protein 2. These results may hold great promise for using post-menopausal osteoporotic bone as an attractive autologous marrow source for tissue engineering and cell-based therapies.     

c-kit marks late retinal progenitor cells and regulates their differentiation in developing mouse retina

Retinal progenitor cells are believed to display altered proliferation and differentiation during retinal development, suggesting that retinal progenitor cell populations are not homogeneous. However, the composition of progenitor cell populations is not known, due in part to the lack of known surface markers identifying distinct stages of retinal progenitor cells. We found a dramatic change in the expression profile of the cell surface antigens c-kit and stage-specific embryonic antigen-1 (SSEA-1) in retinal progenitor cells during development. While SSEA-1 was expressed early in development, c-kit expression peaked in late stage progenitor cells. The identification of these developmental markers enabled us to characterize distinct sub-populations of retinal progenitor cells. Progenitor cell subpopulations expressing either SSEA-1, c-kit, or both showed different proliferation and differentiation abilities. Although SSEA-1-positive cells were augmented by beta-catenin signaling, c-kit-positive cells were positively regulated by Notch signaling. Taken together, our data suggest that c-kit and SSEA-1 can be used to spatiotemporally differentiate retinal progenitor populations that have intrinsically distinct characteristics. Prolonged expression of c-kit by a retrovirus resulted in the promotion of proliferation and the appearance of nestin-positive cells in the presence of the c-kit ligand, stem cell factor (SCF). This suggests a role for c-kit, Notch, and the beta-catenin signaling network in retinal development.     

The CNTF/LIF signaling pathway regulates developmental programmed cell death and differentiation of rod precursor cells in the mouse retina in vivo

Natural cell death is critical for normal development of the nervous system, but the extracellular regulators of developmental cell death remain poorly characterized. Here, we studied the role of the CNTF/LIF signaling pathway during mouse retinal development in vivo. We show that exposure to CNTF during neonatal retinal development in vivo retards rhodopsin expression and results in an important and specific deficit in photoreceptor cells. Detailed analysis revealed that exposure to CNTF during retinal development causes a sharp increase in cell death of postmitotic rod precursor cells. Importantly, we show that blocking the CNTF/LIF signaling pathway during mouse retinal development in vivo results in a significant reduction of naturally occurring cell death. Using retroviral lineage analysis, we demonstrate that exposure to CNTF causes a specific reduction of clones containing only rods without affecting other clone types, whereas blocking the CNTF/LIF receptor complex causes a specific increase of clones containing only rods. In addition, we show that stimulation of the CNTF/LIF pathway positively regulates the expression of the neuronal and endothelial nitric oxide synthase (NOS) genes, and blocking nitric oxide production by pre-treatment with a NOS inhibitor abolishes CNTF-induced cell death. Taken together, these results indicate that the CNTF/LIF signaling pathway acts via regulation of nitric oxide production to modulate developmental programmed cell death of postmitotic rod precursor cells.     

On the formation of germ cells: The good,the bad and the ugly

Mammalian germ cells are powerful cells, the only ones that transmit information to the next generation ensuring the continuation of the species. But “with great power, comes great responsibility”, meaning that germ cells are only a few steps away from turning carcinogenic. Despite recent advances little is known about germ cell formation in mammals, predominantly because of the inaccessibility of these cells. Moreover, it is difficult to pin down what in essence is characteristic of a germ cell, as germ cells keep changing place, morphology, expression markers and epigenetic identity. Formation of (primordial) germ cells in primate ES cell cultures would therefore be helpful to identify molecular signalling pathways associated with germ cell differentiation and to study epigenetic changes in germ cells. In addition, the in vitro derivation of functional germ cells from ES cells could be used in combination with therapeutic cloning to generate patient-specific ES cell lines, and can have applications in animal breeding. In this review we present the state-of-the-art on how mouse and human germ cells are formed in vivo (the good), we discuss the link between germ cells, pluripotency and germ cell tumours (the bad) and show that despite continuous progress in trying to differentiate germ cells in vitro (the ugly) the generation of functional germ cells is still a real challenge.     

Insulin-like growth factor 2 regulates the proliferation and differentiation of rat adipose-derived stromal cells via IGF-1R and IR

BackgroundInsulin-like growth factor 2 (IGF2), an essential component of the stem cell niche, has been reported to modulate the proliferation and differentiation of stem cells. Previously, a continuous expression of IGF2 in tissues was reported to maintain the self-renewal ability of several types of stem cells. Therefore, in this study, we investigated the expression of IGF2 in adipose tissues and explored the effects of IGF2 on adipose-derived stromal cells (ADSCs) in vitro.MethodsThe expression pattern of IGF2 in rat adipose tissues was determined by gene expression and protein analyses. The effect of IGF2 on proliferation, stemness-related marker expression and adipogenic and osteogenic differentiation was systematically investigated. Furthermore, antagonists of IGF2-specific receptors—namely, BMS-754807 and picropodophyllin—were added to explore the underlying signal transduction mechanisms.ResultsIGF2 levels displayed a tendency to decrease with age in rat adipose tissues. After the addition of IGF2, isolated ADSCs displayed higher proliferation and expression of the stemness-related markers NANOG, OCT4 and SOX2 and greater differentiation potential to adipocytes and osteoblasts. Additionally, both type 1 insulin-like growth factor receptor (IGF-1R) and insulin receptor (IR) participated in the IGF2-mediated promotion of stemness in ADSCs.ConclusionsOur findings indicate that IGF2 could enhance the stemness of rat ADSCs via IGF-1R and IR and may highlight an effective method for the expansion of ADSCs for clinical application.     

Role of a new member of IGFBP superfamily, IGFBP-rP10, in proliferation and differentiation of osteoblastic cells

Bone regeneration is critically regulated by various molecules. To identify the new genes involved in bone regeneration, we performed microarray-based gene expression analysis using a mouse bone regeneration model. We identified a new member of the IGFBP superfamily, designated IGFBP-rP10, whose expression is up-regulated at the early phase of bone regeneration. IGFBP-rP10 consists of an IGFBP homologous domain followed by a Kazal-type protein inhibitor domain and an immunoglobulin G-like domain. A real-time-based RT-PCR analysis demonstrated that various tissues including bone expressed IGFBP-rP10 mRNA in various degrees, and confirmed an up-regulation at the early phase of bone regeneration. In situ hybridization revealed that osteoblastic cells expressed IGFPB-rP10 mRNA during bone regeneration. Bone morphogenetic protein-2 increased the expression level of IGFBP-rP10 mRNA in various cells including C3H10T1/2, MC3T3-E1, C2C12, and primary murine osteoblastic cells. The addition of recombinant mouse IGFBP-rP10 promoted the proliferation of these cells but failed to stimulate alkaline phosphatase activity. These results suggest that IGFBP-rP10 is involved in the proliferation of osteoblasts during bone formation and bone regeneration.     

Akhirin regulates the proliferation and differentiation of neural stem cells in intact and injured mouse spinal cord

Although the central nervous system is considered a comparatively static tissue with limited cell turnover, cells with stem cell properties have been isolated from most neural tissues. The spinal cord ependymal cells show neural stem cell potential in vitro and in vivo in injured spinal cord. However, very little is known regarding the ependymal niche in the mouse spinal cord. We previously reported that a secreted factor, chick Akhirin, is expressed in the ciliary marginal zone of the eye, where it works as a heterophilic cell‐adhesion molecule. Here, we describe a new crucial function for mouse Akhirin (M‐AKH) in regulating the proliferation and differentiation of progenitors in the mouse spinal cord. During embryonic spinal cord development, M‐AKH is transiently expressed in the central canal ependymal cells, which possess latent neural stem cell properties. Targeted inactivation of the AKH gene in mice causes a reduction in the size of the spinal cord and decreases BrdU incorporation in the spinal cord. Remarkably, the expression patterns of ependymal niche molecules in AKH knockout (AKH?/?) mice are different from those of AKH+/+, both in vitro and in vivo. Furthermore, we provide evidence that AKH expression in the central canal is rapidly upregulated in the injured spinal cord. Taken together, these results indicate that M‐AKH plays a crucial role in mouse spinal cord formation by regulating the ependymal niche in the central canal. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 494–504, 2015     

Effect of lithium chloride on cell proliferation and osteogenic differentiation in stem cells from human exfoliated deciduous teeth

Lithium Chloride (LiCl) has been used as a canonical Wnt pathway activator due to its ability to inhibit a glycogen synthase kinase-3. The aim of the present study was to investigate the effect of LiCl on cell proliferation and osteogenic differentiation in stem cells isolated from human exfoliated deciduous teeth (SHEDs). SHEDs were isolated and cultured in media supplemented with LiCl at 5, 10, or 20 mM. The results demonstrated that LiCl significantly decreased SHEDs colony forming unit ability in a dose dependent manner. LiCl significantly enhanced the percentage of cells in the sub G0 phase, accompanied by a reduction of the percentage of cells in the G1 phase at day 3 and 7 after treatment. Further, LiCl markedly decreased OSX and DMP1 mRNA expression after treating SHEDs in an osteogenic induction medium for 7 days. In addition, no significant difference in alkaline phosphatase enzymatic activity or mineral deposition was found. Together, these results imply that LiCl influences SHEDs behavior.