Neurogenesis and cell death in olfactory epithelium

The olfactory epithelium (OE) of the mammal is uniquely suited as a model system for studying how neurogenesis and cell death interact to regulate neuron number during development and regeneration. To identify factors regulating neurogenesis and neuronal death in the OE, and to determine the mechanisms by which these factors act, investigators studied OE using two major experimental paradigms: tissue culture of OE; and ablation of the olfactory bulb or severing the olfactory nerve in adult animals, procedures that induce cell death and a subsequent surge of neurogenesis in the OE in vivo. These studies characterized the cellular stages in the olfactory receptor neuron (ORN) lineage, leading to the realization that at least three distinct stages of proliferating neuronal precursor cells are employed in generating ORNs. The identification of a number of factors that act to regulate proliferation and survival of ORNs and their precursors suggests that these multiple developmental stages may serve as control points at which cell number is regulated by extrinsic factors. In vivo surgical studies, which have shown that all cell types in the neuronal lineage of the OE undergo apoptotic cell death, support this idea. These studies, and the possible coregulation of neuronal birth and apoptosis in the OE, are discussed. © 1996 John Wiley & Sons, Inc.     

Regeneration of the amphibian intestinal epithelium under the control of stem cell niche

The epithelium of the mammalian digestive tract originates from stem cells and undergoes rapid cell-renewal throughout adulthood. It has been proposed that the microenvironment around the stem cells, called 'niche', plays an important role in epithelial cell-renewal through cell-cell and cell-extracellular matrix interactions. The amphibian intestine, which establishes epithelial cell-renewal during metamorphosis, serves as a unique and good model for studying molecular mechanisms of the stem cell niche. By using the organ culture of the Xenopus laevis intestine, we have previously shown that larval-to-adult epithelial remodeling can be organ-autonomously induced by thyroid hormone (TH) and needs interactions with the surrounding connective tissue. Thus, in this animal model, the functional analysis of TH response genes is useful for clarifying the epithelial-connective tissue interactions essential for intestinal remodeling at the molecular level. Recent progress in culture and transgenic technology now enables us to investigate functions of such TH response genes in the X. laevis intestine and sheds light on molecular aspects of the stem cell niche that are common to the mammalian intestine.     

Long noncoding RNA C21orf121/bone morphogenetic protein 2/microRNA-140-5p gene network promotes directed differentiation of stem cells from human exfoliated deciduous teeth to neuronal cells

Previous studies have revealed that long noncoding RNA (lncRNA) and microRNA play a crucial role in autism, which is a childhood neurodevelopmental disorder with complicated genetic origins. Hence, the study concerns whether lncRNA C21orf121/bone morphogenetic proteins 2 (BMP2)/miR-140-5p gene network affects directed differentiation of stem cells from human exfoliated deciduous teeth (SHED) to neuronal cells in rats with autism. Autism models were successfully established. The neuron cells that differentiated from SHED cell were identified. The expression of lncRNA C21orf121, miR-140-5p, BMP2, Nestin, βIII-tubulin, and microtubule-associated protein 2 (MAP2) and the expression of neuron-specific enolase (NSE) were examined. Besides, the gap junction (GJ) function of SHED, the intracellular free Ca ²⁺ concentration, and the social behavior and repetitive stereotyped movements of rats in autism were detected. The target relationship between lncRNA C21orf121 and miR-140-5p and that between miR-140-5p and BMP2 were also verified. Firstly, we successfully isolated SHED and identified the differentiated neurons of SHED. Besides, the expression of BMP2, MAP2, Nestin, βIII-tubulin, NSE positive rate, GJ function, and intracellular free Ca ²⁺ concentration were increased with the upregulation of C21orf121 and downregulation of miR-140-5p, and accumulated time of repetitive stereotyped movements decreased and the frequency of social behavior increased. The results indicate that lncRNA C21orf121 as a competing endogenous RNA competes with BMP2 binding to miR-140-5p, thereby promoting SHED to differentiate into neuronal cells via upregulating BMP2 expression.     

FGF2 suppresses neuronogenesis of a cell line derived from rat olfactory epithelium

Neurogenesis continues throughout adulthood in the mammalian olfactory epithelium (OE), and both neurons as well as nonneuronal cells are reconstituted following experimental injury. Underlying the capacity of the OE to replenish its mature elements is a population of progenitor basal cells. Although the precise lineage relationships among progenitor and mature cell types are incompletely understood, the population of globose basal cells (GBCs) contains immediate precursors to neurons as well as amplifying progenitors, and retroviral lineage analyses suggest that multipotential GBCs are activated following direct injury to the OE. To assess the controls on the process of epithelial regeneration, we have characterized a cell line derived from rat OE and studied the effects of serum and tissue extracts, fibroblast growth factor-2 (FGF2) and transforming growth factor-α (TGFα) on the cells. Using a panel of cell type-specific markers whose patterns of labeling in the OE are well defined, including recently developed markers for GBCs, we characterized the phenotype of the cell line under differing culture conditions. In complete medium, which contains serum and tissue extracts, the cell line displayed characteristics of GBCs that are prominent during regeneration. Serum and extract withdrawal induced the cells to differentiate into neurons. In contrast, FGF2 prevented neuronal differentiation and maintained a GBC phenotype. TGFα had a mitogenic or differentiative effect that was context dependent. Finally, we demonstrate here that FGF2 is contained in mature olfactory neurons and sustentacular cells in vivo, suggesting a physiologic role for this growth factor in OE cell regulation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 411–428, 1997     

Smad8 is expressed in the anterior necrotic zone: evidence for a role of bone morphogenetic proteins/SMAD signaling in the activation of a molecular cascade that culminates in cell death

Bone morphogenetic proteins (BMPs) play a crucial role in programmed cell death (PCD), a biological process required for the sculpturing of the embryonic limbs. However, it is unknown if BMP signaling directly promotes cell death, or if it induces a molecular cascade that culminates in cell death. Given that Smad8, which encodes one component of BMP signaling, is expressed during the regression of interdigital tissue and responds to BMPs, we presumed that it may be expressed in other cell death areas during chick limb development such as the anterior and posterior necrotic zones (ANZ and PNZ). The present study found that the Smad8 expression pattern in the anterior mesoderm of the hindlimb is very similar to that observed in limbs stained to detect cell death. Also, BMPs and retinoic acid, which act as apoptosis-promoting factors, induced expression of Smad8 before the onset of cell death, while sonic hedgehog protein, acting as a survival factor, inhibited Smad8 expression in the ANZ. However, although there was correlation between Smad8 expression patterns and PCD in the ANZ, phosphorylated forms of SMAD1/5/8 and TUNEL staining did not co-localize in dying cells. Interestingly, a short pulse of BMP was sufficient to trigger cell death. On the other hand, most dying cells were located in the avascular region, while many cells expressing Smad8 were located in the vascular region of the ANZ. These results suggest that BMPs mediated by SMAD signaling activate a molecular cascade that culminates in PCD.     

Upregulation of Flk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells （NSCs） constitute the cellular basis for embryonic brain development and neurogenesis. The process is regulated by NSC niche including neighbor cells such as vascular and glial cells. Since both vascular and glial cells secrete vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF）, we assessed the effect of VEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonic stem cells. VEGF alone did not have any significant effect. When bFGF was added, however, VEGF stimulated NSC proliferation in a dose-dependent manner, and this stimulation was inhibited by ZM323881, a VEGF receptor （Flk-1）- specific inhibitor. Interestingly, ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF, suggesting that VEGF autocrine plays a role in the proliferation of NSCs. The stimulatory effect of VEGF on NSC proliferation depends on bFGF, which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphorylation of ERK1/2. Collectively, this study may provide insight into the mechanisms by which microenvironmental niche signals regulate NSCs.     

Upregulation of FIk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells (NSCs) constitute the cellular basis for embryonic brain development and neurogenesis.The processis regulated by NSC niche including neighbor cells such as vascular and glial cells.Since both vascular and glial cellssecrete vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF),we assessed the effect ofVEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonicstem cells.VEGF alone did not have any significant effect.When bFGF was added,however,VEGF stimulated NSCproliferation in a dose-dependent manner,and this stimulation was inhibited by ZM323881,a VEGF receptor (Flk-1)-specific inhibitor.Interestingly,ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF,suggestingthat VEGF autocrine plays a role in the proliferation of NSCs.The stimulatory effect of VEGF on NSC proliferationdepends on bFGF,which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphoryla-tion of ERK1/2.Collectively,this study may provide insight into the mechanisms by which mieroenvironmental nichesignals regulate NSCs.     

A human endothelial cell feeder system that efficiently supports the undifferentiated growth of mouse embryonic stem cells

Feeder cells are commonly used to culture embryonic stem cells to maintain their undifferentiated and pluripotent status. Conventionally, mouse embryonic fibroblasts (MEFs), supplemented with leukemia inhibitory factor (LIF), are used as feeder cells to support the growth of mouse embryonic stem cells (mESCs) in culture. To prepare for fresh MEF feeder or for MEF-conditioned medium, sacrifice of mouse fetuses repeatedly is unavoidable in these tedious culture systems. Here we report the discovery of a human endothelial cell line (ECV-304 cell line) that efficiently supports growth of mESCs LIF-free conditions. mESCs that were successfully cultured for eight to 20 passages on ECV-304 feeders showed morphological characteristics similar to cells cultured in traditional feeder cell systems. These cells expressed the stem cell markers Oct3/4, Nanog, Sox2, and SSEA-1. Furthermore, cells cultured on the ECV-304 cell line were able to differentiate into three germ layers and were able to generate chimeric mice. Compared with traditional culture systems, there is no requirement for mouse fetuses and exogenous LIF does not need to be added to the culture system. As a stable cell line, the ECV-304 cell line efficiently replaces MEFs as an effective feeder system and allows the efficient expansion of mESCs.     

Chaperoning stem cells: a role for heat shock proteins in the modulation of stem cell self‐renewal and differentiation?

Self‐renewal and differentiation of stem cells are tightly regulated processes subject to intrinsic and extrinsic signals. Molecular chaperones and co‐chaperones, especially heat shock proteins (Hsp), are ubiquitous molecules involved in the modulation of protein conformational and complexation states. The function of Hsp, which are typically associated with stress response and tolerance, is well characterized in differentiated cells, while their role in stem cells remains unclear. It appears that embryonic stem cells exhibit increased stress tolerance and concomitant high levels of chaperone expression. This review critically evaluates stem cell research from a molecular chaperone perspective. Furthermore, we propose a model of chaperone‐modulated self‐renewal in mouse embryonic stem cells.     

Matrix regulators in neural stem cell functions

Background

Neural stem/progenitor cells (NSPCs) reside within a complex and dynamic extracellular microenvironment, or niche. This niche regulates fundamental aspects of their behavior during normal neural development and repair. Precise yet dynamic regulation of NSPC self-renewal, migration, and differentiation is critical and must persist over the life of an organism.

Scope of review

In this review, we summarize some of the major components of the NSPC niche and provide examples of how cues from the extracellular matrix regulate NSPC behaviors. We use proteoglycans to illustrate the many diverse roles of the niche in providing temporal and spatial regulation of cellular behavior.

Major conclusions

The NSPC niche is comprised of multiple components that include; soluble ligands, such as growth factors, morphogens, chemokines, and neurotransmitters, the extracellular matrix, and cellular components. As illustrated by proteoglycans, a major component of the extracellular matrix, the NSPC, niche provides temporal and spatial regulation of NSPC behaviors.

General significance

The factors that control NSPC behavior are vital to understand as we attempt to modulate normal neural development and repair. Furthermore, an improved understanding of how these factors regulate cell proliferation, migration, and differentiation, crucial for malignancy, may reveal novel anti-tumor strategies. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Regulation of neural stem cell by bone morphogenetic protein (BMP) signaling during brain development

Neurogenesis is the process in which neurons are generated from neural stem/progenitor cells (NSCs/NPCs). It involves the proliferation and neuronal fate specification/differentiation of NSCs, as well as migration, maturation and functional integration of the neuronal progeny into neuronal network. NSCs exhibit the two essential properties of stem cells: self-renewal and multipotency. Contrary to previous dogma that neurogenesis happens only during development, it is generally accepted now that neurogenesis can take place throughout life in mammalian brains. This raises a new therapeutic potential of applying stem cell therapy for stroke, neurodegenerative diseases and other diseases. However, the maintenance and differentiation of NSCs/NPCs are tightly controlled by the extremely intricate molecular networks. Uncovering the underlying mechanisms that drive the differentiation, migration and maturation of specific neuronal lineages for use in regenerative medicine is, therefore, crucial for the application of stem cell for clinical therapy as well as for providing insight into the mechanisms of human neurogenesis. Here, we focus on the role of bone morphogenetic protein (BMP) signaling in NSCs during mammalian brain development.     

Biphasic modulation of protein kinase C and enhanced cell toxicity by amyloid beta peptide and anoxia in neuronal cultures

A major feature of Alzheimer's disease is the deposition of the amyloid beta peptide (Abeta) in the brain by mechanisms which remain unclear. One hypothesis suggests that oxidative stress and Abeta aggregation are interrelated processes. Protein kinase C, a major neuronal regulatory protein is activated after oxidative stress and is also altered in the Alzheimer's disease brain. Therefore, we examined the effects of Abeta(1-40) peptide on the protein kinase C cascade and cell death in primary neuronal cultures following anoxic conditions. Treatment with Abeta(1-40) for 48 h caused a significant increase in the content and activity of Ca2+ dependent and Ca2+ independent protein kinase C isoforms. By 72 h various protein kinase C isoforms were down-regulated. Following 90 min anoxia and 6 h normoxia, a decrease in protein kinase C isoforms was noticed, independent of Abeta(1-40) treatment. A combination of Abeta(1-40) and 30-min anoxia enhanced cytotoxicity as noticed by a marked loss in the mitochondrial ability to convert 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and by enhanced 4',6-diamidino-2-phenylindole nuclear staining. Phosphorylation of two downstream protein kinase C substrates of apparent molecular mass 80 and 43 kDa, tentatively identified as the myristoyl alanine-rich C-kinase substrate (MARCKS), were gradually elevated up to 72 h upon incubation with Abeta(1-40). Anoxia followed by 30 min normoxia enhanced MARCKS phosphorylation in the membrane but not in the cytosolic fraction. In the presence of Abeta(1-40), phosphorylation of MARCKS was reduced. After 6 h normoxia, MARCKS phosphorylatability was diminished possibly because of protein kinase C down-regulation. The data suggest that a biphasic modulation of protein kinase C and MARCKS by Abeta(1-40) combined with anoxic stress may play a role in Alzheimer's disease pathology.     

IGF-1 and BMP-7 synergistically stimulate articular cartilage repairing in the rabbit knees by improving chondrogenic differentiation of bone-marrow mesenchymal stem cells

Knee injury is known as a frequently occurred damage related to sports, which may affect the function of cartilage. This study aims to explore whether Insulin-like growth factor 1 (IGF-1) and bone morphogenetic protein-7 (BMP-7)-modified bone-marrow mesenchymal stem cells (BMSCs) affect the repair of cartilage damage found in the knee. Primarily, BMSCs were treated with a series of pEGFP-C1, IGF-1, and BMP-7, followed by determination of IGF-1 and BMP-7 expression. A rabbit cartilage defect model was also established. Afterfward, cell morphology, viability, cartilage damage repair effect, and expression of collagen I and collagen II at the 6th and the 12th week were measured. BMSCs treated with pEGFP-C1/IGF-1, pEGFP-C1/BMP-7, and pEGFP-C1/BMP-7-IGF-1 exhibited elevated expression of BMP-7 and IGF-1. Besides, BMSCs in the P10 generation displayed decreased cell proliferation. Moreover, BMSCs treated with IGF-1, BMP-7, and IGF-1-BMP-7 showed reduced histological score and collagen I expression while elevated collagen II expression, as well as better repair effect, especially in those treated with IGF-1-BMP-7. Collectively, these results demonstrated a synergistic effect of IGF-1 and BMP-7 on the BMSC chondrogenic differentiation on the articular cartilage damage repair in the rabbit knees, highlighting its therapeutic potential for the treatment of articular cartilage damage.     

Embryonic stem cell differentiation: The role of extracellular factors

Embryonic stem (ES) cells have the capacity to self renew and to differentiate into cellular derivatives of the endodermal, ectodermal, and mesodermal lineages. Therefore, ES cells have been used to analyse the effects of exogenous factors on the developmental pattern during in vitro differentiation. By using an in vitro loss-of-function approach based on beta1 integrin-deficient ES cells, it was found that integrin-dependent mechanisms are involved in the regulation of Wnt-1 and BMP-4 expression. Antagonistic effects of the signalling molecules Wnt-1 and BMP-4, morphogens involved in early differentiation events, have been observed in vivo and in vitro: BMP-4 acts as a potent mesoderm inducer, whereas Wnt-1 plays a critical role in the determination of neuroectoderm. Here, we summarise data of ES cell-derived cardiac, myogenic, and neuronal differentiation of wild type and beta1 integrin-deficient ES cells. We present evidence that the interaction of cells with the extracellular matrix via integrins determines the expression of the signalling molecules BMP-4 and Wnt-1, resulting in the activation of the mesodermal and neuroectodermal lineage, respectively. The results support the idea that the influence of the extracellular 'niche' on the developmental fate of pluripotent stem cells is determined not only by soluble factors, but also by the extracellular matrix.