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.     

Long-term molecular and cellular stability of human neural stem cell lines

Human Neural Stem Cells (hNSCs) are excellent candidates for in vitro and in vivo molecular, cellular, and developmental research, and also for ex-vivo gene transfer and cell therapy in the nervous system. However, hNSCs are mortal somatic cells, and thus invariably enter an irreversible growth arrest after a finite number of cell divisions in culture. It has been proposed that this is due to telomere shortening. Here, we show that long-term cultured (up to 4 years) v-myc perpetuated hNSC lines do preserve short but stable and homogeneous telomeres (TRF and Q-FISH determinations). hNSC lines (but not strains) express high levels of telomerase activity, which is activated by v-myc, as demonstrated here. Telomerase activity is not constitutive, becoming non-detectable after differentiation (in parallel to v-myc down-regulation). hNSC lines also maintain a stable cell cycle length, mitotic potential, differentiation and neuron generation capacity, and do not express senescence-associated beta-galactosidase over years, as studied here. These data, collectively, help to explain the immortal nature of v-myc-perpetuated hNSC lines, and to establish them as excellent research tools for basic and applied neurobiological and translational studies.     

Bcl-XL modulates the differentiation of immortalized human neural stem cells

Understanding basic processes of human neural stem cell (hNSC) biology and differentiation is crucial for the development of cell replacement therapies. Bcl-X(L) has been reported to enhance dopaminergic neuron generation from hNSCs and mouse embryonic stem cells. In this work, we wanted to study, at the cellular level, the effects that Bcl-X(L) may exert on cell death during differentiation of hNSCs, and also on cell fate decisions and differentiation. To this end, we have used both v-myc immortalized (hNS1 cell line) and non-immortalized neurosphere cultures of hNSCs. In culture, using different experimental settings, we have consistently found that Bcl-X(L) enhances neuron generation while precluding glia generation. These effects do not arise from a glia-to-neuron shift (changes in fate decisions taken by precursors) or by only cell death counteraction, but, rather, data point to Bcl-X(L) increasing proliferation of neuronal progenitors, and inhibiting the differentiation of glial precursors. In vivo, after transplantation into the aged rat striatum, Bcl-X(L) overexpressing hNS1 cells generated more neurons and less glia than the control ones, confirming the results obtained in vitro. These results indicate an action of Bcl-X(L) modulating hNSCs differentiation, and may be thus important for the future development of cell therapy strategies for the diseased mammalian brain.     

Physical passaging of embryoid bodies generated from human pluripotent stem cells

Spherical three-dimensional cell aggregates called embryoid bodies (EBs), have been widely used in in vitro differentiation protocols for human pluripotent stem cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Recent studies highlight the new devices and techniques for hEB formation and expansion, but are not involved in the passaging or subculture process. Here, we provide evidence that a simple periodic passaging markedly improved hEB culture condition and thus allowed the size-controlled, mass production of human embryoid bodies (hEBs) derived from both hESCs and hiPSCs. hEBs maintained in prolonged suspension culture without passaging (>2 weeks) showed a progressive decrease in the cell growth and proliferation and increase in the apoptosis compared to 7-day-old hEBs. However, when serially passaged in suspension, hEB cell populations were significantly increased in number while maintaining the normal rates of cell proliferation and apoptosis and the differentiation potential. Uniform-sized hEBs produced by manual passaging using a 1∶4 split ratio have been successfully maintained for over 20 continuous passages. The passaging culture method of hEBs, which is simple, readily expandable, and reproducible, could be a powerful tool for improving a robust and scalable in vitro differentiation system of human pluripotent stem cells.     

Pluripotent stem cells: Maintenance of genetic and epigenetic stability and prospects of cell technologies

Permanent lines of pluripotent stem cells can be obtained from humans and monkeys using different techniques and from different sources—inner cell mass of the blastocyst, primary germ cells, parthenogenetic oocytes, and mature spermatogonia—as well as by transgenic modification of various adult somatic cells. Despite different origin, all pluripotent lines demonstrate considerable similarity of the major biological properties: active self-renewal and differentiation into various somatic and germ cells in vitro and in vivo, similar gene expression profiles, and similar cell cycle structure. Ten years of intense studies on the stability of different human and monkey embryonic stem cells demonstrated that, irrespective of their origin, long-term in vitro cultures lead to the accumulation of chromosomal and gene mutations as well as epigenetic changes that can cause oncogenic transformation of cells. This review summarizes the research data on the genetic and epigenetic stability of different lines of pluripotent stem cells after long-term in vitro culture. These data were used to analyze possible factors of the genome and epigenome instability in pluripotent lines. The prospects of using pluripotent stem cells of different origin in cell therapy and pharmacological studies were considered.     

Gene marking of human neural stem/precursor cells using green fluorescent proteins

BACKGROUND: Ex vivo gene therapy and cell replacement in the nervous system may provide therapeutic opportunities for neurodegenerative disorders. The development of optimal gene marking procedures for human neural stem cells (hNSCs) is crucial for the success of these strategies, in order to provide a correct understanding of the biology of transplanted cells. METHODS: hNSCs were modified to express various members of the green fluorescent protein family of proteins. Both DNA and retroviral expression vectors were used. Cells were analyzed for transgene expression under transient and stable expression schemes, and in the presence or absence of drug selection, by fluorescence microscopy, histochemistry, immunocytochemistry, immunoblotting, RT-PCR and flow cytometry. Genetically marked cells were analyzed in vivo after intrastriatal transplantation in neonatal rats. RESULTS: Using the same experimental procedures, we have compared Aequorea victoria enhanced green fluorescent protein (Av-eGFP) and Renilla raniformis GFP (Rh-GFP, h- from humanized) for the purpose of gene marking of hNSCs. Our findings revealed practical problems for the derivation of stable Av-eGFP-expressing hNSCs, whereas Rh-GFP could be well expressed. In a second phase of the study, stable Rh-GFP-expressing clonal hNSCs were derived. Rh-GFP did not interfere with the differentiation potential of the cells, and expression levels were identical between division and differentiation conditions. Thirdly, in vivo, we have confirmed the usefulness of Rh-GFP for the study of the transplant performance of hNSCs, and demonstrated that Rh-GFP does not interfere with multipotency and differentiation. CONCLUSIONS: Searching for suitable and useful reporter genes, we have found that Rh-GFP works efficiently for the purpose of stable gene marking of hNSCs, and is highly useful in vivo. The nature, properties, and possible side effects of marker genes are discussed, since these are important parameters to consider in gene marking studies involving hNSCs.     

人胚与鼠胚神经干细胞体外培养的差异

为比较人胚与鼠胚神经干细胞体外培养的差异。实验采用具有丝裂原作用的细胞生长因子。结合无血清细胞培养技术从人胚和鼠胚皮层分离神经干细胞。在连续传代过程中观察其体外培养特性,免疫荧光染色检测Nestin抗原和分化后特异性成熟神经细胞抗原的表达,并用流式细胞仪检测神经干细胞分化情况。结果表明：（1）使用单一生长因子即可从鼠胚皮层分离神经干细胞,但在人胚却需同时使用多种生长因子,协同使用bFGF,EGF和LIF是人胚神经干细胞体外培养的较佳条件;（2）鼠胚皮层神经干细胞在连续传代过程中增殖速度快于人胚,其Nestin阳性率和BrdU标记的阳性率亦高于人胚,表明其增殖能力明显高于人胚,（3）人胚神经干细胞较鼠胚更易分化为神经元。     

Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells

In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs) we isolated human fetal liver stromal cells (hFLSCs) from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days). Basic fibroblast growth factor (bFGF) is known to play an important role in promoting self-renewal of human embryonic stem (hES) cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2), and transforming growth factor β (TGF-β), thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.     

Early Cell Fate Decisions of Human Embryonic Stem Cells and Mouse Epiblast Stem Cells Are Controlled by the Same Signalling Pathways

Human embryonic stem cells have unique value for regenerative medicine, as they are capable of differentiating into a broad variety of cell types. Therefore, defining the signalling pathways that control early cell fate decisions of pluripotent stem cells represents a major task. Moreover, modelling the early steps of embryonic development in vitro may provide the best approach to produce cell types with native properties. Here, we analysed the function of key developmental growth factors such as Activin, FGF and BMP in the control of early cell fate decisions of human pluripotent stem cells. This analysis resulted in the development and validation of chemically defined culture conditions for achieving specification of human embryonic stem cells into neuroectoderm, mesendoderm and into extra-embryonic tissues. Importantly, these defined culture conditions are devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Importantly, the growth factor roles defined using these culture conditions similarly drove differentiation of mouse epiblast stem cells derived from post implantation embryos, thereby reinforcing the hypothesis that epiblast stem cells share a common embryonic identity with human pluripotent stem cells. Therefore the defined growth factor conditions described here represent an essential step toward the production of mature cell types from pluripotent stem cells in conditions fully compatible with clinical use ant also provide a general approach for modelling the early steps of mammalian embryonic development.     

Comparative study of mouse and human feeder cells for human embryonic stem cells

Various types of feeder cells have been adopted for the culture of human embryonic stem cells (hESCs) to improve their attachment and provide them with stemness-supporting factors. However, feeder cells differ in their capacity to support the growth of undifferentiated hESCs. Here, we compared the expression and secretion of four well-established regulators of hESC pluripotency and/or differentiation among five lines of human foreskin fibroblasts and primary mouse embryonic fibroblasts throughout a standard hESC culture procedure. We found that human and mouse feeder cells secreted comparable levels of TGF beta 1. However, mouse feeder cells secreted larger quantities of activin A than human feeder cells. Conversely, FGF-2, which was produced by human feeder cells, could not be detected in culture media from mouse feeder cells. The quantity of BMP-4 was at about the level of detectability in media from all feeder cell types, although BMP-4 dimers were present in all feeder cells. Production of TGF beta 1, activin A, and FGF-2 varied considerably among the human-derived feeder cell lines. Low- and high-producing human feeder cells as well as mouse feeder cells were evaluated for their ability to support the undifferentiated growth of hESCs. We found that a significantly lower proportion of hESCs maintained on human feeder cell types expressed SSEA3, an undifferentiated cell marker. Moreover, SSEA3 expression and thus the pluripotent hESC compartment could be partially rescued by addition of activin A. Cumulatively, these results suggest that the ability of a feeder layer to promote the undifferentiated growth of hESCs is attributable to its characteristic growth factor production.     

Establishment and maintenance of three Chinese human embryonic stem cell lines

To establish a potential resource for cell therapy and a developmental model for human diseases, we had isolated three Chinese human embryonic stem cell lines from the inner cell mass of human blastocysts in 2002. All the three cell lines were grown on mouse embryonic fibroblasts as feeder cells; one of these cell lines, chHES-3, has maintained its normal karyotype even after being cultured in vitro for more than 100 passages, after the standardization of mouse feeder preparation. Each hES cell line has been completely characterized. All the three cell lines expressed hES-specific markers and pluripotency-related genes. These cells maintained their normal karyotype during long-term culture and displayed a high telomerase activity. When differentiated in vivo and in vitro, the derivatives representing the three germ layers could be observed. Human leukocyte antigen, ABO blood type, and DNA fingerprinting were also performed to provide a unique identity to each cell line. By establishing these hES cell lines, we provide an appropriate in vitro model to study human development and regeneration. All the three cell lines can be obtained for research purposes by placing a request at our website at www.hescbank.cn.     

Human Neural Stem Cells Genetically Modified to Overexpress Akt1 Provide Neuroprotection and Functional Improvement in Mouse Stroke Model

In a previous study, we have shown that human neural stem cells (hNSCs) transplanted in brain of mouse intracerebral hemorrhage (ICH) stroke model selectively migrate to the ICH lesion and induce behavioral recovery. However, low survival rate of grafted hNSCs in the brain precludes long-term therapeutic effect. We hypothesized that hNSCs overexpressing Akt1 transplanted into the lesion site could provide long-term improved survival of hNSCs, and behavioral recovery in mouse ICH model. F3 hNSC was genetically modified with a mouse Akt1 gene using a retroviral vector. F3 hNSCs expressing Akt1 were found to be highly resistant to H₂O₂-induced cytotoxicity in vitro. Following transplantation in ICH mouse brain, F3.Akt1 hNSCs induced behavioral improvement and significantly increased cell survival (50–100% increase) at 2 and 8 weeks post-transplantation as compared to parental F3 hNSCs. Brain transplantation of hNSCs overexpressing Akt1 in ICH animals provided functional recovery, and survival and differentiation of grafted hNSCs. These results indicate that the F3.Akt1 human NSCs should be a great value as a cellular source for the cellular therapy in animal models of human neurological disorders including ICH.     

Dedifferentiated adult articular chondrocytes: a population of human multipotent primitive cells

OBJECTIVE: To test the hypothesis that dedifferentiated adult human cartilage chondrocytes (HAC) are a true multipotent primitive population. METHODS: Studies to characterize dedifferentiated HAC included cell cycle and quiescence analysis, cell fusion, flow-FISH telomere length assays, and ABC transporter analysis. Dedifferentiated HAC were characterized by flow cytometry, in parallel with bone marrow mesenchymal stem cells (MSC) and processed lipoaspirate (PLA) cells. The in vitro differentiation potential of dedifferentiated HAC was studied by cell culture under several inducing conditions, in multiclonal and clonal cell populations. RESULTS: Long-term HAC cultures were chromosomically stable and maintained cell cycle dynamics while showing telomere shortening. The phenotype of dedifferentiated HAC was quite similar to that of human bone marrow MSC. In addition, this population expressed human embryonic stem cell markers. Multiclonal populations of dedifferentiated HAC differentiated to chondrogenic, osteogenic, adipogenic, myogenic, and neurogenic lineages. Following VEGF induction, dedifferentiated HAC expressed characteristics of endothelial cells, including AcLDL uptake. A total of 53 clonal populations of dedifferentiated HAC were efficiently expanded; 17 were able to differentiate to chondrogenic, osteogenic, and adipogenic lineages. No correlation was observed between telomere length or quiescent population and differentiation potential in the clones assayed. CONCLUSION: Dedifferentiated HAC should be considered a human multipotent primitive population.     

Effects of DMEM and RPMI 1640 on the biological behavior of dog periosteum-derived cells

Periosteum-derived cells (PDCs) are being extensively studied as potential tissue engineering seed cells and have demonstrated tremendous promise to date. There is convincing evidence that culture medium could modulate the biological behavior of cultured cells. In this study, we investigate the effects of DMEM (low glucose) and RPMI 1640 on cell growth and cell differentiation of PDCs in vitro. PDCs isolated from Beagle dogs were maintained in DMEM and RPMI 1640, respectively. Then, the cell migration rate of periosteum tissues was analyzed. PDCs of the third passage were harvested for the study of proliferation and osteogenic activity. Proliferation was detected by MTT assay. Alkaline phosphatase activity and mineralized nodules were measured to investigate osteogenic differentiation. Our data demonstrated that DMEM induced alkaline phosphatase activity and strongly stimulated matrix mineralization in cell culture, while similar cell migration rates and proliferation behaviors were observed in the two culture conditions. Interestingly, the osteogenic differentiation of PDCs could be enhanced in DMEM compared with that in RPMI 1640. Thus, it can be ascertained that DMEM may serve as a suitable culture condition allowing osteogenic differentiation of dog PDCs.     

长期培养小鼠胚胎干细胞拟胚体(EB)的观察

胚胎干细胞在体外培养条件下能够维持自我更新,并具有向多种细胞类型分化的能力,因此被广泛用于研究细胞分化的分子机理以及药物筛选.形成拟胚体(Embryoid body,EB)是胚胎干细胞分化常用的技术手段.为了便于今后利用EB做进一步的药物筛选及分化研究,严格规范了形成EB的条件,得到了分化状态均一性很高的EB.利用这一条件,观察到在分化条件下长期培养(长达60 d)的EB中仍有表达各项多能性指标的细胞集落.有关这一现象的进一步分析工作正在进行中.     

Establishment of a microcarrier culture system with serial sub-cultivation for functionally active human endothelial cells

A microcarrier culture system was established for a large-scale production of functional human endothelial cells. It has been difficult to cultivate human endothelial cells in large quantities for the reasons that specific growth factor and extracellular matrix are required for the survival and proliferation of the cells and the life span of the primary cells are limited. A lot of studies have reported that the shear stress gives significant influences on the structure, growth rate and biological functions of endothelial cells. We aimed to develop a convenient microcarrier culture system for human endothelial cells which can reproduce the flow effects experienced in vivo or in vitro. In 200 mL volume culture, human umbilical vein endothelial cells (HUVEC) could be serially sub-cultivated by optimizing the culture conditions such as shear strength, growth factor, beads and seeding cell concentration, serum concentration, and passage timing. The growth rate was enhanced depending on the shear strength and the life span of the cells was elongated until over 43PDL which is much longer than those of monolayer cultures. The cells maintained the diploidy of over 80% without obvious abnormal changes in the chromosomes. The serially sub-cultured microcarrier cells maintained various endothelial cell functions such as the syntheses of von Willebrand factor (vWf), prostacyclin and other biological substances, the expression of CD31, and the VEGF(165) dependent growth characteristic. The synthesis of biological products was affected by shear strength. In the case of prostacyclin, a different synthesis response was observed between steady flow and transiently reduced shear strength. The synthesis of endothelin-1 (ET-1) was down-regulated by increase of shear strength different from those of other products. The culture system was scaled up until 2 L volume under the optimum DO control. The cells synthesized IL-6 in response to shear strength. These results indicate that the established microcarrier system might be able to contribute to the supply of functional human endothelial cells for various medical applications such as the reconstruction of injured blood vessels caused by atherosclerosis or restenosis of coronary arteries after angioplasty, and the construction of an anti-coagulable artificial blood vessel or an artificial skin with good transplant-ability.