大鼠脑神经干细胞系(RNSC-FMU 1)的建立和鉴定

采用无血清培养液分离和培养新生SD 大鼠脑的神经干细胞,以机械分散的方法传代,成功地建立了大鼠脑神经干细胞系(RSNC-FMU 1)。该细胞系可在体外长期传代,至今已在体外连续生长超过21个月(>100代),保持了神经干细胞的特性和正常的核型,经诱导可分化成为神经元、星形胶质细胞和少突胶质细胞,具有较旺盛的自新能力,倍增时间约为20h,并可冷冻保存,裸鼠体内移植证实其不具有成瘤性。该细胞系为神经干细胞研究提供了一个良好的工具。     

Cellular Environment Directs Differentiation of Human Umbilical Cord Blood�CDerived Neural Stem Cells In Vitro

Cord blood–derived neural stem cells (NSCs) are proposed as an alternative cell source to repair brain damage upon transplantation. However, there is a lack of data showing how these cells are driven to generate desired phenotypes by recipient nervous tissue. Previous research indicates that local environment provides signals driving the fate of stem cells. To investigate the impact of these local cues interaction, the authors used a model of cord blood–derived NSCs co-cultured with different rat brain–specific primary cultures, creating the neural-like microenvironment conditions in vitro. Neuronal and astro-, oligo-, and microglia cell cultures were obtained by the previously described methods. The CMFDA-labeled neural stem cells originated from, non-transformed human umbilical cord blood cell line (HUCB-NSCs) established in a laboratory. The authors show that the close vicinity of astrocytes and oligodendrocytes promotes neuronal differentiation of HUCB-NSCs, whereas postmitotic neurons induce oligodendrogliogenesis of these cells. In turn, microglia or endothelial cells do not favor any phenotypes of their neural commitment. Studies have confirmed that HUCB-NSCs can read cues from the neurogenic microenvironment, attaining features of neurons, astrocytes, or oligodendrocytes. The specific responses of neurally committed cord blood–derived cells, reported in this work, are very much similar to those described previously for NSCs derived from other “more typical” sources. This further proves their genuine neural nature. Apart from having a better insight into the neurogenesis in the adult brain, these findings might be important when predicting cord blood cell derivative behavior after their transplantation for neurological disorders.     

Fate of amnion-derived stem cells transplanted to the fetal rat brain: migration, survival and differentiation

We have recently characterized a stem cell population isolated from the rodent amniotic membrane termed amnion-derived stem cells (ADSCs). In vitro ADSCs differentiate into cell types representing all three embryonic layers, including neural cells. In this study we evaluated the neuroectodermal potential of ADSCs in vivo after in utero transplantation into the developing rat brain. A clonal line of green fluorescent protein-expressing ADSCs were infused into the telencephalic ventricles of the developing embryonic day 15.5 rat brain. At E17.5 donor cells existed primarily as spheres in the ventricles with subsets fused to the ventricular walls, suggesting a mode of entry into the brain parenchyma. By E21.5 green fluorescent protein (GFP) ADSCs migrated to a number of brain regions. Examination at postnatal time points revealed that donor ADSCs expressed vimentin and nestin. Subsets of transplanted ADSCs attained neuronal morphologies, although there was no immunohistochemical evidence of neural or glial differentiation. Some donor cells migrated around blood vessels and differentiated into putative endothelial cells. Donor ADSCs transplanted in utero were present in recipients into adulthood with no evidence of immunological rejection or tumour formation. Long-term survival may suggest utility in the treatment of disorders where differentiation to a neural cell type is not required for clinical benefit.     

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。     

Adult vascular smooth muscle cells in culture express neural stem cell markers typical of resident multipotent vascular stem cells

Differentiation of resident multipotent vascular stem cells (MVSCs) or de-differentiation of vascular smooth muscle cells (vSMCs) might be responsible for the SMC phenotype that plays a major role in vascular diseases such as arteriosclerosis and restenosis. We examined vSMCs from three different species (rat, murine and bovine) to establish whether they exhibit neural stem cell characteristics typical of MVSCs. We determined their SMC differentiation, neural stem cell marker expression and multipotency following induction in vitro by using immunocytochemistry, confocal microscopy, fluorescence-activated cell sorting analysis and quantitative real-time polymerase chain reaction. MVSCs isolated from rat aortic explants, enzymatically dispersed rat SMCs and rat bone-marrow-derived mesenchymal stem cells served as controls. Murine carotid artery lysates and primary rat aortic vSMCs were both myosin-heavy-chain-positive but weakly expressed the neural crest stem cell marker, Sox10. Each vSMC line examined expressed SMC differentiation markers (smooth muscle α–actin, myosin heavy chain and calponin), neural crest stem cell markers (Sox10⁺, Sox17⁺) and a glia marker (S100β⁺). Serum deprivation significantly increased calponin and myosin heavy chain expression and decreased stem cell marker expression, when compared with serum-rich conditions. vSMCs did not differentiate to adipocytes or osteoblasts following adipogenic or osteogenic inductive stimulation, respectively, or respond to transforming growth factor-β1 or Notch following γ-secretase inhibition. Thus, vascular SMCs in culture express neural stem cell markers typical of MVSCs, concomitant with SMC differentiation markers, but do not retain their multipotency. The ultimate origin of these cells might have important implications for their use in investigations of vascular proliferative disease in vitro.     

Adherent self-renewable human embryonic stem cell-derived neural stem cell line: functional engraftment in experimental stroke model

Background

Human embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.

Methods/Principal Findings

We isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.

Conclusions/Significance

The SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research.     

Isolation and characterization of a neural progenitor cell line from tilapia brain

Astroglial cell lines have many applications for advancing neural developmental and functional studies. However, few astroglial cell lines have been reported from fish. In this study, we report the characterization of the immortal cell line TB2 isolated from adult tilapia brain tissue. The cell line was established at 25 degrees C in L15 medium supplemented with 15% fetal bovine serum. Most of the cells displayed a fibrous morphology and were immunoreactive for A2B5 antigen, glial fibrillary acidic protein (GFAP), keratin, vimentin, and the gap junction protein connexin 43 (Cx43). They weakly expressed glutamine synthetase (GS), S100 protein, and the neural stem cell markers Sox2 and brain lipid binding protein (BLBP). In contrast to astroglia in vivo, most TB2 cells also expressed galactocerebroside (GalC), substance P (SP), and tyrosine hydroxylase (TH). By immunoblot and RT-PCR, the cells also expressed myelin basic protein (MBP), proteolipid protein (PLP), and Cx35. On a poly-L-lysine-coated substrate in vitro, TB2 cells showed increases in neuronal dopamine decarboxylase (DDC) and microtubule-associated protein 2 (MAP2), indicating that they can initiate differentiation into neurons. Taken together, the results suggest that TB2 cells are astroglial progenitor cells (neural stem cells) and may develop into oligodendrocytes and neurons in a suitable environment. The present study advances our knowledge of fish astroglia. However, the factors that affect neural development in fish remain unknown, as do the characteristics of the intermediate differentiation stages between stem cells and mature nerve cells. The TB2 cell line will promote these investigations.     

Isolation and characterization of a neural progenitor cell line from tilapia brain.

Astroglial cell lines have many applications for advancing neural developmental and functional studies. However, few astroglial cell lines have been reported from fish. In this study, we report the characterization of the immortal cell line TB2 isolated from adult tilapia brain tissue. The cell line was established at 25 degrees C in L15 medium supplemented with 15% fetal bovine serum. Most of the cells displayed a fibrous morphology and were immunoreactive for A2B5 antigen, glial fibrillary acidic protein (GFAP), keratin, vimentin, and the gap junction protein connexin 43 (Cx43). They weakly expressed glutamine synthetase (GS), S100 protein, and the neural stem cell markers Sox2 and brain lipid binding protein (BLBP). In contrast to astroglia in vivo, most TB2 cells also expressed galactocerebroside (GalC), substance P (SP), and tyrosine hydroxylase (TH). By immunoblot and RT-PCR, the cells also expressed myelin basic protein (MBP), proteolipid protein (PLP), and Cx35. On a poly-L-lysine-coated substrate in vitro, TB2 cells showed increases in neuronal dopamine decarboxylase (DDC) and microtubule-associated protein 2 (MAP2), indicating that they can initiate differentiation into neurons. Taken together, the results suggest that TB2 cells are astroglial progenitor cells (neural stem cells) and may develop into oligodendrocytes and neurons in a suitable environment. The present study advances our knowledge of fish astroglia. However, the factors that affect neural development in fish remain unknown, as do the characteristics of the intermediate differentiation stages between stem cells and mature nerve cells. The TB2 cell line will promote these investigations.