Neural precursors derived from embryonic stem cells, but not those from fetal ventral mesencephalon, maintain the potential to differentiate into dopaminergic neurons after expansion in vitro

Neural precursors (NPs) derived from ventral mesencephalon (VM) normally generate dopaminergic (DA) neurons in vivo but lose their potential to differentiate into DA neurons during mitogenic expansion in vitro, hampering their efficient use as a transplantable and experimental cell source. Because embryonic stem (ES) cell-derived NPs (ES NP) do not go through the same maturation process during in vitro expansion, we hypothesized that expanded ES NPs may maintain their potential to differentiate into DA neurons. To address this, we expanded NPs derived from mouse embryonic day-12.5 (E12.5) VM or ES cells and compared their developmental properties. Interestingly, expanded ES NPs fully sustain their ability to differentiate to the neuronal as well as to the DA fate. In sharp contrast, VM NPs almost completely lost their ability to become neurons and tyrosine hydroxylase-positive (TH(+)) neurons after expansion. Expanded ES NP-derived TH(+) neurons coexpressed additional DA markers such as dopa decarboxylase and DAT (dopamine transporter). Furthermore, they also expressed other midbrain DA markers, including Nurr1 and Pitx3, and released significant amounts of DA. We also found that these ES NPs can be cryopreserved without losing their proliferative and developmental potential. Finally, we tested the in vivo characteristics of the expanded NPs derived from J1 ES cells with low passage number. When transplanted into the mouse striatum, the expanded NPs as well as control NPs efficiently generated DA neurons expressing mature DA markers, with approximately 10% tumor formation in both cases. We conclude that ES NPs maintain their developmental potential during in vitro expansion, whereas mouse E12.5 VM NPs do not.     

Building Brains: Neural Chimeras in the Study of Nervous System Development and Repair

The ability to isolate multipotential neuroepithelial precursor cells from the mammalian nervous system provides exciting perspectives for the in vitro analysis of early nervous system development and the generation of donor cells for neural repair. New models are needed to study the properties of these cells in vivo. Neural chimeras have revealed a remarkable degree of plasticity in the developmental potential of neuroepithelial precursor cells. Following transplantation into the cerebral ventricle of embryonic hosts, precursors derived from various brain regions and developmental stages participate in host brain development and undergo region-specific differentiation into neurons and glia. These findings indicate that in the developing nervous system, migration and differentiation of neural precursors cells are regulated to a large extent by extrinsic signals. Neural chimeras composed of genetically modified cells will permit the study of the molecular mechanisms underlying these guidance cues, which may eventually be exploited for cell replacement strategies in the adult brain. A key problem in neural transplantation is the availability of suitable donor tissue. Neural chimeras composed of embryonic stem (ES) cell-derived neurons and glia depict ES cells as a versatile and virtually unlimited donor source for neural repair. Generation of interspecies neural chimeras composed of human and rodent cells facilitates the translation of these advances into clinical strategies for human nervous system repair.     

Comparison of neuron-like cells derived from bone marrow stem cells to those differentiated from adult brain neural stem cells

Bone marrow-derived stem/progenitor cells have been shown by independent investigators to give rise to neural-like cells (neurons and glia) both in vitro and in vivo. The objective of the present study was to determine whether nestin-enriched cells derived from bone marrow can differentiate into cells with the same morphological and functional characteristics as neurons derived from adult brain neurogenic zones. Cell culture methods were used for generation of adult bone marrow and brain stem/progenitor cells and for studying their differentiation into neural-like cells. The proportion of cells expressing neuronal markers was greater in cultures derived from adult hippocampal neural stem cells than in the bone marrow-derived cells, but the electrophysiological and functional characteristics of the cells were similar. Action potentials with electrical characteristics corresponding to those exhibited by adult neural stem cell-derived neurons were recorded from approximately 2.5% of patched neuron-like cells differentiated from bone marrow cells. The active uptake of tritium-labeled neurotransmitters gamma-aminobutyric acid ([(3)H]GABA) and dopamine ([(3)H]DA) was measured in both sets of cultures. [(3)H]GABA uptake, but not [(3)H]DA, was significantly increased in differentiated neurons in both neural stem cell cultures and bone marrow-derived cultures. [(3)H]GABA uptake was greater in differentiated neurons derived from brain neural stem cells. In summary, both the nestin-expressing bone marrow and the adult brain neural stem/progenitors developed into cells with morphological, immunocytochemical, and functional characteristics of neurons. Even though a smaller proportion of neuron-like cells was generated from bone marrow-derived progenitors than from brain-derived neural stem cells, these cells may be useful in the cellular therapy of neurodegenerative diseases and traumatic brain and spinal cord injury.     

Expression of angiogenic and neurotrophic factors in the progenitor cell niche of adult monkey subventricular zone

The subventricular zone along the anterior horn (SVZa) of the cerebral lateral ventricle of adult mammals contains multipotent progenitor cells, which supposedly exist in an angiogenic niche. Numerous signals are known to modulate the precursor cell proliferation, migration or differentiation, in rodent models. In contrast, the data on signals regulating the primate SVZa precursors in vivo are scarce. We analyzed the expression at protein level of a panel of angiogenic and/or neurotrophic factors and their receptors in SVZa of adult macaque monkeys, under normal condition or after transient global ischemia which enhances endogenous progenitor cell proliferation. We found that fms-like tyrosine kinase 1 (Flt1), a receptor for vascular endothelial cell growth factor, was expressed by over 30% of the proliferating progenitors, and the number of Flt1-positive precursors was significantly increased by the ischemic insult. Smaller fractions of mitotic progenitors were positive for the neurotrophin receptor tropomyosin-related kinase (Trk) B or the hematopoietic receptor Kit, while immature neurons expressed Flt1 and the neurotrophin receptor TrkA. Further, SVZa astroglia, ependymal cells and blood vessels were positive for distinctive sets of ligands/receptors, which we characterized. The presented data provide a molecular phenotypic analysis of cell types comprising adult monkey SVZa, and suggest that a complex network of angiogenic/neurotrophic signals operating in an autocrine or paracrine manner may regulate SVZa neurogenesis in the adult primate brain.     

Axonal growth regulation of fetal and embryonic stem cell-derived dopaminergic neurons by Netrin-1 and Slits

The physical restoration of dopamine circuits damaged or lost in Parkinson disease by implanting embryonic stem (ES)-derived cells may become a treatment. It is critical to understand responses of ES-derived dopamine (DA) neurons to guidance signals that determine axonal path and targeting. Using a collagen gel culture system, we examined effects of secreted molecules Netrin-1 and Slits on neurite outgrowth of fetal DA neurons and murine ES-differentiated DA neurons. We have previously shown that fetal DA neurons express DCC and Robo1/2 receptors and that Netrin-1 and Slit2 function as an attractant and a repellent for DA neurite outgrowth. In the present study, we observe that both Slit1 and Slit3 repel and inhibit neurite growth of fetal DA neurons. Here, we also demonstrate that ES-differentiated neurons including DA neurons express the Netrin receptor DCC and Slit receptor Robo proteins. In the gel culture system of ES cells, Netrin-1 promoted neurite outgrowth mediated by DCC receptor, and Slit1 and Slit3 were inhibitory for neurite outgrowth through Robo receptors. Slit2 appeared to exert inhibitory as well as repulsive effects in the coculture assay. However, unlike fetal DA neurites, no directed neurite outgrowth was observed in the cocultures of ES-derived DA neurons with Netrin-1-, Slit1-, and Slit3-producing cells. The findings suggest that ES-derived DA neurons generated by current protocols can respond to guidance cues in vitro in a similar manner to fetal cells but also exhibit distinct responses. This may result from developmental differences generated by present in vitro methods of cell patterning or conditioning during ES cell differentiation.     

Chemically defined sequential culture media for TH+ cell derivation from human embryonic stem cells

During the past few years several differentiation protocols to derive midbrain dopamine (DA) neurons from human embryonic stem (hES) cells have been developed, but the production of sufficient amounts of the 'right' therapeutic DA cells has not yet been accomplished. The aim of this study was to efficiently generate tyrosine hydroxylase (TH)-positive cells in vitro from our hES cells using a chemically defined culture system. At the end of differentiation, the vast majority of cells (>90%) were positive for both TH and beta-tubulin isotype III (TuJ1). Other markers of dopaminergic cells, like dopamine transporter (DAT) and Nurr1 were also detected by immunofluorescence or RT-PCR. The functions of these cells were confirmed by measurements of DA release in vitro and by transplantation of derived cells into Parkinson's disease (PD) rats in vivo. We found these cells were able to release DA when depolarized by high K(+). Moreover, 4 weeks after transplantation, the hES-derived cells could survive and reduce the apomorphine-induced rotation behaviour of the rats. In conclusion, the experimental system presented here provided a reliable protocol to produce a large number of hES-derived TH(+) cells which may be used in cell therapy for PD in future.     

Proteomic signatures and aberrations of mouse embryonic stem cells containing a single human chromosome 21 in neuronal differentiation: an in vitro model of Down syndrome

Neurodegeneration in fetal development of Down syndrome (DS) patients is proposed to result in apparent neuropathological abnormalities and to contribute to the phenotypic characteristics of mental retardation and premature development of Alzheimer disease. In order to identify the aberrant and specific genes involved in the early differentiation of DS neurons, we have utilized an in vitro neuronal differentiation system of mouse ES cells containing a single human chromosome 21 (TT2F/hChr21) with TT2F parental ES cells as a control. The paired protein extracts from TT2F and TT2F/hChr21 cells at several stages of neuronal differentiation were subjected to two-dimensional polyacrylamide gel electrophoresis protein separation followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to identify the proteins differentially expressed between TT2F and TT2F/hChr21 cells. We provide here a novel set of specific gene products altered in early differentiating DS neuronal cells, which differs from that identified in adult or fetal brain with DS. The aberrant protein expression in early differentiating neurons, due to the hChr21 gene dosage effects or chromosomal imbalance, may affect neuronal outgrowth, proliferation and differentiation, producing developmental abnormalities in neural patterning, which eventually leads to formation of a suboptimal functioning neuronal network in DS.     

Derivation and cloning of a novel rhesus embryonic stem cell line stably expressing tau-green fluorescent protein

Embryonic stem cells (ESC) have the ability of indefinite self-renewal and multilineage differentiation, and they carry great potential in cell-based therapies. The rhesus macaque is the most relevant preclinical model for assessing the benefit, safety, and efficacy of ESC-based transplantations in the treatment of neurodegenerative diseases. In the case of neural cell grafting, tracing both the neurons and their axonal projections in vivo is essential for studying the integration of the grafted cells in the host brain. Tau-Green fluorescent protein (tau-GFP) is a powerful viable lineage tracer, allowing visualization of cell bodies, dendrites, and axons in exquisite detail. Here, we report the first rhesus monkey ESC line that ubiquitously and stably expresses tau-GFP. First, we derived a new line of rhesus monkey ESC (LYON-ES1) that show marker expression and cell cycle characteristics typical of primate ESCs. LYON-ES1 cells are pluripotent, giving rise to derivatives of the three germ layers in vitro and in vivo through teratoma formation. They retain all their undifferentiated characteristics and a normal karyotype after prolonged culture. Using lentiviral infection, we then generated a monkey ESC line stably expressing tau-GFP that retains all the characteristics of the parental wild-type line and is clonogenic. We show that neural precursors derived from the tau-GFP ESC line are multipotent and that their fate can be precisely mapped in vivo after grafting in the adult rat brain. Disclosure of potential conflicts of interest is found at the end of this article.     

Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson's disease

For the safe clinical application of embryonic stem cells (ESCs) for neurological diseases, it is critical to evaluate the tumorigenicity and function of human ESC (hESC)-derived neural cells in primates. We have herein, for the first time, compared the growth and function of hESC-derived cells with different stages of neural differentiation implanted in the brains of primate models of Parkinson's disease. We herein show that residual undifferentiated cells expressing ESC markers present in the cell preparation can induce tumor formation in the monkey brain. In contrast, a cell preparation matured by 42-day culture with brain-derived neurotrophic factor/glial cell line-derived neurotrophic factor (BDNF/GDNF) treatment did not form tumors and survived as primarily dopaminergic (DA) neurons. In addition, the monkeys with such grafts showed behavioral improvement for at least 12 months. These results support the idea that hESCs, if appropriately matured, can serve as a source for DA neurons without forming any tumors in a primate brain.     

Midbrain dopaminergic development in vivo and in vitro from embryonic stem cells

The midbrain dopaminergic (mDA) neurons play a key role in the function of a variety of brain systems, including motor control and reward pathways. This has led to much interest in these neurons as targets for intervention in human disorders such as Parkinson's disease and schizophrenia. A major area of interest is to direct embryonic stem (ES) cells to differentiate into mDA neurons in vitro, which can then be used for cell therapy or drug screening. At present, our understanding of mDA development in vivo is limited. However, recent studies have identified a number of regulatory factors that influence the development of mDA neurons in vivo. Such studies will not only increase our understanding of mDA development in vivo, they may also promote new paradigms for regulating mDA production from ES cells in vitro. Here we review the current knowledge on mDA development in vivo and mDA differentiation.     

Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson's disease: effect of in vitro differentiation on graft survival and teratoma formation

Human embryonic stem cells (hESCs) have been proposed as a source of dopamine (DA) neurons for transplantation in Parkinson's disease (PD). We have investigated the effect of in vitro predifferentiation on in vivo survival and differentiation of hESCs implanted into the 6-OHDA (6-hydroxydopamine)-lesion rat model of PD. The hESCs were cocultured with PA6 cells for 16, 20, or 23 days, leading to the in vitro differentiation into DA neurons. Grafted hESC-derived cells survived well and expressed neuronal markers. However, very few exhibited a DA neuron phenotype. Reversal of lesion-induced motor deficits was not observed. Rats grafted with hESCs predifferentiated in vitro for 16 days developed severe teratomas, whereas most rats grafted with hESCs predifferentiated for 20 and 23 days remained healthy until the end of the experiment. This indicates that prolonged in vitro differentiation of hESCs is essential for preventing formation of teratomas.     

Iris as a recipient tissue for pigment cells: Organized in vivo differentiation of melanocytes and pigmented epithelium derived from embryonic stem cells in vitro

Regenerative transplantation of embryonic stem (ES) cell-derived melanocytes into adult tissues, especially skin that includes hair follicles or the hair follicle itself, generally not possible, whereas that of ES cell-derived pigmented epithelium was reported previously. We investigated the in vivo differentiation of these two pigment cell types derived from ES cells after their transfer into the iris. Melanocytes derived from ES cells efficiently integrated into the iris and expanded to fill the stromal layer of the iris, like those prepared from neonatal skin. Transplanted pigmented epithelium from either ES cells or the neonatal eye was also found to be integrated into the iris. Both types of these regenerated pigment cells showed the correct morphology. Regenerated pigment epithelium expressed its functional marker. Functional blocking of signals required for melanocyte development abolished the differentiation of transplanted melanocytes. These results indicate successful in vivo regenerative transfer of pigment cells induced from ES cells in vitro. Developmental Dynamics 237:2394-2404, 2008. (c) 2008 Wiley-Liss, Inc.     

Subventricular zone cells remain stable in vitro after brain injury

Subventricular zone (SVZ) cells emigrate toward brain injury but relatively few survive. Thus, if they are to be used for repair, ex vivo expansion and autologous transplantation of SVZ cells may be necessary. Since it is unclear how brain injury alters SVZ cell culture, we studied neurosphere formation, differentiation, and migration, after cortical lesions. The number of neurosphere forming cells from lesioned mice was comparable to controls. Also, the proportion of astrocytes and neurons generated in vitro remained unchanged after cortical lesions. Cell emigration from neurospheres was characterized by increased cell-cell contact after injury in adults and neonates. However, neither molecules implicated in SVZ migration nor the extent of migration changed after injury. Thus, neurospheres can be successfully cultured after extensive brain damage, and they are remarkably stable in vitro, suggesting suitability for ex vivo expansion and autologous transplantation.     

Development of human brain neural/progenitor cells after transplantation into the brain of adult rats

The purpose of the present study was the investigation of human neural stem/progenitor cells (SPC) cultured in vitro, with special reference to their capacity for grafting, migration and differentiation after transplantation into adult rat brain. SPC were isolated from the brain of 9-week-old human embryos and were cultured in a selective medium for 3 weeks. For transplantation, cell suspension or whole neurospheres were used; they were studied 4 weeks following the transplantation in hippocampus, striatum and lateral ventricle of adult rat brain. For the analysis of transplanted SPC, various histological and immunohistochemical staining methods were applied (bisbenzidine, BrdU, antibodies against human nuclei, vimentin, beta-tubulin, neurofilaments, GFAP), that allowed an independent evaluation of their state and differentiation. Transplanted human brain SPC were shown to survive well for one month in all the areas of adult rat brain without immunosuppression. Cells from suspension transplants actively migrated and differentiated into neurons and glial cells. Meanwhile, cell migration from the transplanted whole neurospheres was limited or absent due to the formation of glial barrier.     

Persistent dopamine functions of neurons derived from embryonic stem cells in a rodent model of Parkinson disease

The derivation of dopamine neurons is one of the best examples of the clinical potential of embryonic stem (ES) cells, but the long-term function of the grafted neurons has not been established. Here, we show that, after transplantation into an animal model, neurons derived from mouse ES cells survived for over 32 weeks, maintained midbrain markers, and had sustained behavioral effects. Microdialysis in grafted animals showed that dopamine (DA) release was induced by depolarization and pharmacological stimulants. Positron emission tomography measured the expression of presynaptic dopamine transporters in the graft and also showed that the number of postsynaptic DA D(2) receptors was normalized in the host striatum. These data suggest that ES cell-derived neurons show DA release and reuptake and stimulate appropriate postsynaptic responses for long periods after implantation. This work supports continued interest in ES cells as a source of functional DA neurons.     

小鼠胚胎干细胞植入大鼠脑内分化的研究

观察小鼠胚胎干细胞植入大鼠隔区和海马内之后的分化状况。以 SD大鼠为宿主 ,将胚胎干细胞移植入宿主隔区和海马内 ,移植后在 l、2、3、4和 8周取脑 ,冰冻切片 ,进行 Nissl染色和 M6、NSE、GFAP免疫组织化学反应。胚胎干细胞移植入大鼠隔区和海马之后 ,从第 2周开始表达 M6、GFAP、NSE等抗原 ,持续至第 4周 ,主要位于移植区内 ,较少迁移。小鼠胚胎干细胞移植入大鼠隔区和海马内之后 ,分化为神经元和神经胶质细胞     

Fluorescence-activated cell sorting-based purification of embryonic stem cell-derived neural precursors averts tumor formation after transplantation

The differentiation of dopaminergic (DA) neurons from mouse embryonic stem cells (ESCs) can be efficiently induced, making these neurons a potential source for transplantation as a treatment for Parkinson's disease, a condition characterized by the gradual loss of midbrain DA neurons. One of the major persistent obstacles to the successful implementation of therapeutic ESC transplantation is the propensity of ESC-derived grafts to form tumors in vivo. To address this problem, we used fluorescence-activated cell sorting to purify mouse ESC-derived neural precursors expressing the neural precursor marker Sox1. ESC-derived, Sox1+ cells began to express neuronal cell markers and differentiated into DA neurons upon transplantation into mouse brains but did not generate tumors in this site. In contrast, Sox1- cells that expressed ESC markers frequently formed tumors in vivo. These results indicate that Sox1-based cell sorting of neural precursors prevents graft-derived tumor formation after transplantation, providing a promising strategy for cell transplantation therapy of neurodegenerative disorders.