Extended periods of neural induction and propagation of embryonic stem cell-derived neural progenitors with EGF and FGF2 enhances Lmx1a expression and neurogenic potential

Neural stem (NS) cells are multipotent cells defined by their capacity to proliferate and differentiate into all neuronal and glial phenotypes. NS cells can be obtained from specific regions of the adult brain, or generated from embryonic stem cells (ESCs). NS cells differentiate into neural progenitor (NP) cells and subsequently neural precursors, as transient steps towards terminal differentiation into specific mature neuronal or glial phenotypes. When cultured in EGF and FGF2, ESC-derived NS cells have been reported to be stable and multipotent. Conditions that enable differentiation of NS cells through the committed progenitor and precursor stages to specific neuronal subtypes have not been fully established. In this study we investigated, using Lmx1a reporter ESCs, whether the length of neural induction (NI) dictated the phenotypic potential of cultures of ESC-derived NS cells or NP cells. Following 4, 7 or 10 day periods of NI, ESCs in monolayer culture were harvested and cultured as neurospheres, prior to replating as monolayer cultures for several passages in EGF and FGF2. The NS/NP cultures were then directed towards mature neuronal fates over 16-17 days. 4 and 7-day NS cell cultures could not be differentiated towards dopaminergic, serotonergic or cholinergic fates as determined by the absence of tyrosine hydroxylase, 5-HT or choline acetyltransferase (ChAT) immunolabelling. In contrast NS/NP cultures derived after 10 days of NI were able to generate tyrosine hydroxylase and 5-HT positive neurons (24 ± 6 and 13 ± 1% of the βIII-tubulin positive population, respectively, n = 3). Our data suggest that extended periods of neural induction enhanced the potential of mouse ESC-derived NS/NP cells to generate specific subtypes of neurons. NS/NP cells derived after shorter periods of NI appeared to be lineage-restricted in relation to the neuronal subtypes observed after removal of EGF.     

Clonal analysis of adult human olfactory neurosphere forming cells

Olfactory neuroepithelium (ONe) is unique because it contains progenitor cells capable of mitotic division that replace damaged or lost neurons throughout life. We isolated populations of ONe progenitors from adult cadavers and patients undergoing nasal sinus surgery that were heterogeneous and consisted of neuronal and glial progenitors. Progenitor lines have been obtained from these cultures that continue to divide and form nestin positive neurospheres. In the present study, we used clonal and population analyses to probe the self-renewal and multipotency of the neurosphere forming cells (NSFCs). NSFCs plated at the single cell level produced additional neurospheres; dissociation of these spheres resulted in mitotically active cells that continued to divide and produce spheres as long as they were subcultured. The mitotic activity of clonal NSFCs was assessed using bromodeoxyuridine (BrdU) incorporation. Lineage restriction of the clonal cultures was determined using a variety of antibodies that were characteristic of different levels of neuronal commitment: β-tubulin isotype III, neural cell adhesion molecule (NCAM) and microtubule associated protein (MAP2), or glial restriction: astrocytes, glial fibrillary acidic protein (GFAP); and oligodendrocytes, galactocerebroside (GalC). Furthermore, nestin expression, a marker indicative of progenitor nature, decreased in defined medium compared to serum-containing medium. Therefore, adult human ONe-derived neural progenitors retain their capacity for self-renewal, can be clonally expanded, and offer multipotent lineage restriction. Therefore, they are a unique source of progenitors for future cell replacement strategies in the treatment of neurotrauma and neurodegenerative diseases.     

Clonal analysis of adult human olfactory neurosphere forming cells

Olfactory neuroepithelium (ONe) is unique because it contains progenitor cells capable of mitotic division that replace damaged or lost neurons throughout life. We isolated populations of ONe progenitors from adult cadavers and patients undergoing nasal sinus surgery that were heterogeneous and consisted of neuronal and glial progenitors. Progenitor lines have been obtained from these cultures that continue to divide and form nestin positive neurospheres. In the present study, we used clonal and population analyses to probe the self-renewal and multipotency of the neurosphere forming cells (NSFCs). NSFCs plated at the single cell level produced additional neurospheres; dissociation of these spheres resulted in mitotically active cells that continued to divide and produce spheres as long as they were subcultured. The mitotic activity of clonal NSFCs was assessed using bromodeoxyuridine (BrdU) incorporation. Lineage restriction of the clonal cultures was determined using a variety of antibodies that were characteristic of different levels of neuronal commitment: β-tubulin isotype III, neural cell adhesion molecule (NCAM) and microtubule associated protein (MAP2), or glial restriction: astrocytes, glial fibrillary acidic protein (GFAP); and oligodendrocytes, galactocerebroside (GalC). Furthermore, nestin expression, a marker indicative of progenitor nature, decreased in defined medium compared to serum-containing medium. Therefore, adult human ONe-derived neural progenitors retain their capacity for self-renewal, can be clonally expanded, and offer multipotent lineage restriction. Therefore, they are a unique source of progenitors for future cell replacement strategies in the treatment of neurotrauma and neurodegenerative diseases.     

Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1

Seladin-1 (SELective Alzheimer's Disease INdicator-1) is an anti-apoptotic gene, which is down-regulated in brain regions affected by Alzheimer's disease (AD). In addition, seladin-1 catalyzes the conversion of desmosterol into cholesterol. Disruption of cholesterol homeostasis in neurons may increase cell susceptibility to toxic agents. Because the hippocampus and the subventricular zone, which are affected in AD, are the unique regions containing stem cells with neurogenic potential in the adult brain, it might be hypothesized that this multipotent cell compartment is the predominant source of seladin-1 in normal brain. In the present study, we isolated and characterized human mesenchymal stem cells (hMSC) as a model of cells with the ability to differentiate into neurons. hMSC were then differentiated toward a neuronal phenotype (hMSC-n). These cells were thoroughly characterized and proved to be neurons, as assessed by molecular and electrophysiological evaluation. Seladin-1 expression was determined and found to be significantly reduced in hMSC-n compared to undifferentiated cells. Accordingly, the total content of cholesterol was decreased after differentiation. These original results demonstrate for the first time that seladin-1 is abundantly expressed by stem cells and appear to suggest that reduced expression in AD might be due to an altered pool of multipotent cells.     

人羊膜来源成体干细胞的多向分化潜能

干细胞治疗被认为是一种非常有潜力的治疗手段,其中成体干细胞由于不存在伦理问题,更为广大学者所青睐。本研究成功从人羊膜间质细胞中分离纯化出具有自我更新能力和多向分化潜能的成体干细胞。首先从羊膜间质细胞中通过极限稀释法进一步分离得到羊膜来源成体干细胞(Amnion-derived stemcells,ADSC),分析其形态、生长方式及主要的免疫表型,并在体外分别将其向脂肪、成骨、内皮、肝细胞及神经细胞诱导分化。结果发现,ADSC在适宜条件下能够向3个胚层的细胞分化,经连续传代30次,其形态及表型稳定,并仍保持多向分化潜能。证实了ADSC的干细胞特性,可能为细胞治疗及干细胞工程提供种子细胞的新来源。     

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.     

神经干细胞脑内移植后的存活、迁移和分化

从胚胎或成体大鼠脑组织、人胚脑组织均能分离到神经干细胞 ,将它们进行体外原代培养扩增或永生化后植入脑内 ,均能观察到其在脑内的迁移和分化现象。其分化能力主要取决于移植部位的脑内微环境 ,但这种影响作用是相对的。同时 ,体外培养环境如培养时间和细胞融合程度、维甲酸类诱导分化剂处理、NGF转导处理再移植或与嗜铬细胞 (分泌NGF)共移植等 ,也能决定神经干细胞脑内移植后向神经元方向分化的能力。神经干细胞移植为中枢神经系统功能重建和神经再生带来新的希望。     

Multipotent progenitor cells in the adult dentate gyrus

Neurogenesis persists in the adult dentate gyrus of rodents throughout the life of the organism. The factors regulating proliferation, survival, migration, and differentiation of neuronal progenitors are now being elucidated. Cells from the adult hippocampus can be propagated, cloned in vitro, and induced to differentiate into neurons and glial cells. Cells cultured from the adult rodent hippocampus can be genetically marked and transplanted back to the adult brain, where they survive and differentiate into mature neurons and glial cells. Although multipotent stem cells exist in the adult rodent dentate gyrus, their biological significance remains elusive. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 249–266, 1998.     

Transplantation of Stem/Progenitor Cells of Human Brain into Adult Rat Brain

We studied the development of stem/progenitor cells of the human brain transplanted in the adult rat brain after expansion in an in vitrotissue culture. It was preliminarily shown by the immunological methods that the stem cells grown in a medium with growth factors formed neurospheres, which were heterogenous and contained both stem and progenitor cells of the human brain. The cells were implanted in the hippocampus, striatum, or lateral ventricle of the rat brain as a suspension or aggregates (neurospheres) and their behavior and differentiation were studies within 10, 20, and 30 days using the morphological and immunochemical methods. The cultured cells of the human brain continued their development in the rat brain, migrated, and formed neurons and astrocytes. The white mater fibers, lateral ventricle wall, and perivascular spaces served as the main pathways of migration. The neuronal differentiation was shown by staining with antibodies to -tubulin III, neurofilaments-70, and calbindin. Some growing nerve cells had long processes with growth cones. At the same time, some transplanted cells retained the undifferentiated state within one month after the implantation, as shown by the vimentin expression.     

Growth factors regulate the survival and fate of cells derived from human neurospheres

Cells isolated from the embryonic, neonatal, and adult rodent central nervous system divide in response to epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2), while retaining the ability to differentiate into neurons and glia. These cultures can be grown in aggregates termed neurospheres, which contain a heterogeneous mix of both multipotent stem cells and more restricted progenitor populations. Neurospheres can also be generated from the embryonic human brain and in some cases have been expanded for extended periods of time in culture. However, the mechanisms controlling the number of neurons generated from human neurospheres are poorly understood. Here we show that maintaining cell-cell contact during the differentiation stage, in combination with growth factor administration, can increase the number of neurons generated under serum-free conditions from 8% to > 60%. Neurotrophic factors 3 and 4 (NT3, NT4) and platelet-derived growth factor (PDGF) were the most potent, and acted by increasing neuronal survival rather than inducing neuronal phenotype. Following differentiation, the neurons could survive dissociation and either replating or transplantation into the adult rat brain. This experimental system provides a practically limitless supply of enriched, non-genetically transformed neurons. These should be useful for both neuroactive drug screening in vitro and possibly cell therapy for neurodegenerative diseases.     

Subventricular zone astrocytes are neural stem cells in the adult mammalian brain.

Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. This germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. Here we show that SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, we show that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. We conclude that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain.     

Isolation and characterization of the CD133+ precursors from the ventricular zone of human fetal brain by magnetic affinity cell sorting

A fast and effective method to enrich large number of neural precursors from the ventricular zone of human fetus by magnetic affinity cell sorting (MACS) is reported. After incubation with phycoerythrin (PE)-conjugated anti-CD133 antibodies and anti-PE magnetic beads followed by one cycle of MACS, CD133(+) cells were harvested at 85% purity as confirmed by flow-cytometry and immunocytochemistry. In contrast to CD133(-) cells, these CD133(+) cells initiated primary and secondary neurospheres in culture, and the progeny of sorted cells could be differentiated into both neurons and glia, indicating that these highly enriched cells are capable of self-renewal and multi-lineage potential.     

Green fluorescent protein-labeled mapping of neural stem cells migrating towards damaged areas in the adult central nervous system

Neural stem cells, which are clonogenic cells with multilineage differentiation properties from regions of the fetal brain, cortex and hippocampus, are currently considered as powerful candidates for cell replacement therapy in neurodegenerative disorders, such as Parkinson's disease. A key issue is whether stem cells can survive, migrate and differentiate following transplantation into the adult CNS. Here, enhanced green fluorescent protein plasmid electroporation-transfected neural stem cells from the fetal cortex were grafted into the striatum of a rat model of Parkinson's disease. We found most of the grafted cells could survive in the adult parkinsonian rat brain and migrated towards damaged areas, while they moved randomly in the normal brain. Several grafted cells differentiated into neurons.     

Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.     

Effects of chitosan/collagen substrates on the behavior of rat neural stem cells

Spinal cord and brain injuries usually lead to cavity formation. The transplantation by combining stem cells and tissue engineering scaffolds has the potential to fill the cavities and replace the lost neural cells. Both chitosan and collagen have their unique characteristics. In this study, the effects of chitosan and collagen on the behavior of rat neural stem cells (at the neurosphere level) were tested in vitro in terms of cytotoxicity and supporting ability for stem cell survival, proliferation and differentiation. Under the serum-free condition, both chitosan membranes and collagen gels had low cytotoxicity to neurospheres. That is, cells migrated from neurospheres, and processes extended out from these neurospheres and the differentiated cells. Compared with the above two materials, chitosan-collagen membranes were more suitable for the co-culture with rat neural stem cells, because, except for low cytotoxicity and supporting ability for the cell survival, in this group, a large number of cells were observed to migrate out from neurospheres, and the differentiating percentage from neurospheres into neurons was significantly increased. Further modification of chitosan-collagen membranes may shed light on in vivo nerve regeneration by transplanting neural stem cells.     

Prospective isolation of late development multipotent precursors whose migration is promoted by EGFR

A simple procedure to isolate neural stem cells would greatly facilitate direct studies of their properties. Here, we exploited the increase in EGF receptor (EGFR) levels, that occurs in late development stem cells or in younger precursors upon exposure to FGF-2, to isolate cells expressing high levels of EGFR (EGFR(high)) from the developing and the adult brain. Independently of age and region of isolation, EGFR(high) cells were highly enriched in multipotent precursors and displayed similar antigenic characteristics, with the exception of GFAP and Lex/SSEA-1 that were mainly expressed in adult EGFR(high) cells. EGFR levels did not correlate with neurogenic potential, indicating that the increase in EGFR expression does not directly affect differentiation. Instead, in the brain, many EGFR(high) precursors showed tangential orientation and, whether isolated from the cortex or striatum, EGFR(high) precursors displayed characteristics of cells originating from the ventral GZ such as expression Dlx and Mash-1 and the ability to generate GABAergic neurons and oligodendrocytes. Moreover, migration of EGFR(high) cells on telencephalic slices required EGFR activity. Thus, the developmentally regulated increase in EGFR levels may affect tangential migration of multipotent precursors. In addition, it can be used as a marker to effectively isolate telencephalic multipotent precursors from embryonic and adult tissue.     

Neural stem/progenitor cells from the adult human spinal cord are multipotent and self-renewing and differentiate after transplantation

Neural stem/progenitor cell (NSPC) transplantation is a promising therapy for spinal cord injury (SCI). However, little is known about NSPC from the adult human spinal cord as a donor source. We demonstrate for the first time that multipotent and self-renewing NSPC can be cultured, passaged and transplanted from the adult human spinal cord of organ transplant donors. Adult human spinal cord NSPC require an adherent substrate for selection and expansion in EGF (epidermal growth factor) and FGF2 (fibroblast growth factor) enriched medium. NSPC as an adherent monolayer can be passaged for at least 9 months and form neurospheres when plated in suspension culture. In EGF/FGF2 culture, NSPC proliferate and primarily express nestin and Sox2, and low levels of markers for differentiating cells. Leukemia inhibitory factor (LIF) promotes NSPC proliferation and significantly enhances GFAP expression in hypoxia. In differentiating conditions in the presence of serum, these NSPC show multipotentiality, expressing markers of neurons, astrocytes, and oligodendrocytes. Dibutyryl cyclic AMP (dbcAMP) significantly enhances neuronal differentiation. We transplanted the multipotent NSPC into SCI rats and show that the xenografts survive, are post-mitotic, and retain the capacity to differentiate into neurons and glia.Together, these findings reveal that multipotent self-renewing NSPC cultured and passaged from adult human spinal cords of organ transplant donors, respond to exogenous factors that promote selective differentiation, and survive and differentiate after transplantation into the injured spinal cord.     

对比不同类型干细胞对于缺血性脑卒中治疗的研究进展

成人中枢神经系统存在着一定量的神经干细胞,其具有两大关键能力;自我更新和多向分化潜能。缺血性脑卒中是一种由于由脑血流的缺失或减少引起的脑动脉闭塞,进而导致脑组织梗死的脑血管疾病。虽然对于脑损伤的药物治疗已经取得了一定的成果,但目前以干细胞为基础的治疗方法仍成为了研究热点。无论是内源性神经干细胞还是外源性神经干细胞移植均可在脑损伤后向远端损伤区迁移并分化成新的神经细胞,从而在中枢神经系统疾病尤其是脑梗死后进行组织修复和功能恢复。因此在这篇综述中,我们主要探讨不同类型的干细胞对脑梗死介导的脑损伤的应用潜能,对比不同类型干细胞对缺血性脑卒中的治疗优缺点。     

Neural stem cells in the adult human brain

New neurons are continuously generated in certain regions of the adult brain. Studies in rodents have shown that new neurons are generated from self-renewing multipotent neural stem cells. Here we demonstrate that both the lateral ventricle wall and the hippocampus of the adult human brain harbor self-renewing cells capable of generating neurons, astrocytes, and oligodendrocytes in vitro, i.e., bona fide neural stem cells.