Region-specific generation of functional neurons from naive embryonic stem cells in adult brain

Embryonic stem (ES) cells are multipotent progenitors with unlimited developmental potential, and in vitro differentiated ES cell-derived neuronal progenitors can develop into functional neurons when transplanted in the central nervous system. As the capacity of naive primary ES cells to integrate in the adult brain and the role of host neural tissue therein are yet largely unknown, we grafted low densities of undifferentiated mouse ES (mES) cells in adult mouse brain regions associated with neurodegenerative disorders; and we demonstrate that ES cell-derived neurons undergo gradual integration in recipient tissue and acquire morphological and electrophysiological properties indistinguishable from those of host neurons. Only some brain areas permitted survival of mES-derived neural progenitors and formed instructive environments for neuronal differentiation and functional integration of naive mES cells. Hence, region-specific presence of microenvironmental cues and their pivotal involvement in controlling ES cell integration in adult brain stress the importance of recipient tissue characteristics in formulating cell replacement strategies for neurodegenerative disorders.     

Developmental potential of defined neural progenitors derived from mouse embryonic stem cells

The developmental potential of a uniform population of neural progenitors was tested by implanting them into chick embryos. These cells were generated from retinoic acid-treated mouse embryonic stem (ES) cells, and were used to replace a segment of the neural tube. At the time of implantation, the progenitors expressed markers defining them as Pax6-positive radial glial (RG) cells, which have recently been shown to generate most pyramidal neurons in the developing cerebral cortex. Six days after implantation, the progenitors generated large numbers of neurons in the spinal cord, and differentiated into interneurons and motoneurons at appropriate locations. They also colonized the host dorsal root ganglia (DRG) and differentiated into neurons, but, unlike stem cell-derived motoneurons, they failed to elongate axons out of the DRG. In addition, they neither expressed the DRG marker Brn3a nor the Trk neurotrophin receptors. Control experiments with untreated ES cells indicated that when colonizing the DRG, these cells did elongate axons and expressed Brn3a, as well as Trk receptors. Our results thus indicate that ES cell-derived progenitors with RG characteristics generate neurons in the spinal cord and the DRG. They are able to respond appropriately to local cues in the spinal cord, but not in the DRG, indicating that they are restricted in their developmental potential.     

In vitro differentiation of transplantable neural precursors from human embryonic stem cells.

The remarkable developmental potential and replicative capacity of human embryonic stem (ES) cells promise an almost unlimited supply of specific cell types for transplantation therapies. Here we describe the in vitro differentiation, enrichment, and transplantation of neural precursor cells from human ES cells. Upon aggregation to embryoid bodies, differentiating ES cells formed large numbers of neural tube-like structures in the presence of fibroblast growth factor 2 (FGF-2). Neural precursors within these formations were isolated by selective enzymatic digestion and further purified on the basis of differential adhesion. Following withdrawal of FGF-2, they differentiated into neurons, astrocytes, and oligodendrocytes. After transplantation into the neonatal mouse brain, human ES cell-derived neural precursors were incorporated into a variety of brain regions, where they differentiated into both neurons and astrocytes. No teratoma formation was observed in the transplant recipients. These results depict human ES cells as a source of transplantable neural precursors for possible nervous system repair.     

猕猴神经干细胞的诱导分化、干性维持及同种脑内移植

为探索猕猴神经干细胞分化及特性维持,推进神经干细胞临床应用研究,该实验以绿色荧光蛋白(green fluorescence protein,GFP)为标记探讨猕猴胚胎干细胞向玫瑰花环(rosettes)结构神经干细胞的分化及其碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和表皮生长因子(epidermal growth factor,EGF)的扩增培养。结果表明:1)建立了稳定高效的猕猴神经干细胞分化体系,在该分化体系下,GFP标记猕猴胚胎干细胞在分化的第12天时,95%以上的细胞分化为神经干细胞;2)分化得到的Rosettes结构神经干细胞经bFGF/EGF扩增后,能够较好地维持其Rosettes结构;3)经bFGF/EGF扩增后的rosettes结构神经干细胞移植到猕猴脑内后能够较好的存活并向神经元分化,即bFGF/EGF扩增培养能较好地维持Rosettes结构的神经干细胞,且移植到猕猴脑内的该细胞亦能够较好地存活并向神经元分化,该结果为神经干细胞应用于临床提供了基础理论依据。     

小鼠胚胎干细胞移植入成体大鼠脑内的区域特异性存活与分化

全能区域非特异性的胚胎干细胞是研究成体不同脑区控制干细胞分化能力的十分有力的工具。胚胎干细胞源性神经前体细胞移植入成体脑后可分化为功能性神经元,但是未分化的胚胎干细胞在成体脑内各个部位的存活、生长与分化的潜能差异尚不清楚。本文旨在探讨成体脑组织对胚胎干细胞的影响及胚胎干细胞在成体脑内的一系列行为。将少量转绿色荧光蛋白未分化的小鼠胚胎干细胞移植入成体大鼠脑内不同部位,分别于移植5、14和28d后处死大鼠,进行形态学观察及免疫组化定性,以了解未分化的小鼠胚胎干细胞在大鼠脑内不同区域的存活、生长与分化。结果发现未分化的小鼠胚胎干细胞可逐步整合入受体组织并向nestin阳性神经前体细胞分化。移植细胞及其后裔在海马生长最为旺盛,而在隔区最差（P〈0．01）;移植细胞分化为神经干细胞的效率也是在海马最高,而在隔区最低（P〈0．01）。提示只有部分脑区适合胚胎干细胞及其后裔生存,并提供促进其分化的有益环境。因此,由于位置特异的微环境因子及环境因素的存在,宿主组织特性对决定中枢神经系统疾病的细胞替代疗法策略是相当重要的。     

Genetic selection of sox1GFP-expressing neural precursors removes residual tumorigenic pluripotent stem cells and attenuates tumor formation after transplantation

Because of their ability to proliferate and to differentiate into diverse cell types, embryonic stem (ES) cells are a potential source of cells for transplantation therapy of various diseases, including Parkinson's disease. A critical issue for this potential therapy is the elimination of undifferentiated cells that, even in low numbers, could result in teratoma formation in the host brain. We hypothesize that an efficient solution would consist of purifying the desired cell types, such as neural precursors, prior to transplantation. To test this hypothesis, we differentiated sox1-green fluorescent protein (GFP) knock-in ES cells in vitro, purified neural precursor cells by fluorescence-activated cell sorting (FACS), and characterized the purified cells in vitro as well as in vivo. Immunocytofluorescence and RT-PCR analyses showed that this genetic purification procedure efficiently removed undifferentiated pluripotent stem cells. Furthermore, when differentiated into mature neurons in vitro, the purified GFP+ cell population generated enriched neuronal populations, whereas the GFP- population generated much fewer neurons. When treated with dopaminergic inducing signals such as sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF8), FACS-purified neural precursor cells responded to these molecules and generated dopaminergic neurons as well as other neural subtypes. When transplanted, the GFP+ cell population generated well contained grafts containing dopaminergic neurons, whereas the GFP- population generated significantly larger grafts (about 20-fold) and frequent tumor-related deaths in the transplanted animals. Taken together, our results demonstrate that genetic purification of neural precursor cells using FACS isolation can effectively remove unwanted proliferating cell types and avoid tumor formation after transplantation.     

Neuronal differentiation of cryopreserved neural progenitor cells derived from mouse embryonic stem cells

Embryonic stem cells (ES cells) are developmentally pluripotent cells isolated from pre-implantation mammalian embryos. In cell culture ES cells can be easily differentiated to generate cultures of neural progenitors. We present a simple method for the cryopreservation of these ES-derived neural progenitors. Cryopreserved neural progenitor stocks can be thawed, expanded with FGF2, and differentiated into functional neurons. This method will facilitate studies using ES-derived neural progenitor cells as a cell culture model system for neural development and differentiation. It will also aid studies designed to test the ability of these progenitor cells to functionally engraft and repair damaged neural tissue.     

Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions

To model human neural-cell-fate specification and to provide cells for regenerative therapies, we have developed a method to generate human neural progenitors and neurons from human embryonic stem cells, which recapitulates human fetal brain development. Through the addition of a small molecule that activates canonical WNT signaling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. Ten days after initiation of differentiation, the progenitors could be transplanted to the adult rat striatum, where they formed neuron-rich and tumor-free grafts with maintained regional specification. Cells patterned toward a ventral midbrain (VM) identity generated a high proportion of authentic dopaminergic neurons after transplantation. The dopamine neurons showed morphology, projection pattern, and protein expression identical to that of human fetal VM cells grafted in parallel. VM-patterned but not forebrain-patterned neurons released dopamine and reversed motor deficits in an animal model of Parkinson's disease.     

ES cell-derived neuroepithelial cell cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells. Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).     

Neural differentiation of murine embryonic stem cells ES

Pluripotent murine embryonic stem (ES) cells can differentiate into all cell types both in vivo and in vitro. Based on their capability to proliferate and differentiate, these ES cells appear as a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the neural differentiation of the ES cells is a pre-requisite for selecting adequately the cells and conditions which will be able to correctly repair damaged brain and restore altered cognitive functions. Different methods allow obtaining neural cells from ES cells. Most of the techniques differentiate ES cells by treating embryoid bodies in order to keep an embryonic organization. More recent techniques, based on conditioned media, induce a direct differentiation of ES cells into neural cells, without going through the step of embryonic bodies. Beyond the fact that these techniques allow obtaining large numbers of neural precursors and more differentiated neural cells, these approaches also provide valuable information on the process of differentiation of ES cells into neural cells. Indeed, sequential studies of this process of differentiation have revealed that globally ES cells differentiating into neural cells in vitro recapitulate the molecular events governing the in vivo differentiation of neural cells. Altogether these data suggest that murine ES cells remain a highly valuable tool to obtain large amounts of precursor and differentiated neural cells as well as to get a better understanding of the mechanisms of neural differentiation, prior to a potential move towards the use of human ES cells in therapy.     

Embryonic stem cells and cell replacement therapies in the nervous system

Embryonic stem (ES) cells are pluripotential cells derived from the pre-implantation embryo. They can proliferate indefinitely in vitro while retaining pluripotency. ES cells can also be made to differentiate into a large variety of cell types in vitro. This has paved the way to research aimed at using ES-derived cells for cell replacement therapies. Hence, mouse ES cells can efficiently differentiate into neural precursors which can further generate functional neurons, astrocytes, and oligodendrocytes. Methods have also been developed to coax mouse ES-derived neural stem cells to differentiate into either dopaminergic neurons or motoneurons. Mouse ES-derived neural stem cells, or their fully differentiated progeny, have been shown to survive, integrate, and to some extent, function following transplantation within appropriate rodent host tissue. Research on human ES cells is still in its infancy. Considerable work has to be done: (1) to master growth and genetic manipulation of human ES cells; (2) to master their differentiation into specific cell types; and (3) to demonstrate that they can provide long term therapeutical benefits upon grafting into damaged tissues in humans. From the ethical point of view, the establishment of appropriate primate model will be an obligatory prerequisite to clinical trials based on ES cells derivatives grafting.     

Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration

The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.     

Differentiation of embryonic stem cells into retinal neurons

Mouse embryonic stem (ES) cells are continuous cell lines derived from the inner mass of blastocysts. Neural progenitors derived from these cells serve as an excellent model for controlled neural differentiation and as such have tremendous potential to understand and treat neurodegenerative diseases. Here, we demonstrate that ES cell-derived neural progenitors express regulatory factors needed for retinal differentiation and that in response to epigenetic cues a subset of them differentiate along photoreceptor lineage. During the differentiation, they activate photoreceptor regulatory genes, suggesting that ES cell-derived neural progenitors recruit mechanisms normally used for photoreceptor differentiation in vivo. These observations suggest that ES cells can serve as an excellent model for understanding mechanisms that regulate specification of retinal neurons and as an unlimited source of neural progenitors for treating degenerative diseases of the retina by cell replacement.     

Colloids as mobile substrates for the implantation and integration of differentiated neurons into the mammalian brain

Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons.     

一种有效区分移植细胞和宿主细胞脑损伤模型的建立

为了区分移植神经细胞和宿主细胞,便于将来在宿主体内对移植细胞进行在体的电生理记录以及其它方面的研究,通过机械损毁的方法,建立了一种特殊的脑损伤模型。结果发现,通过机械损毁的方法,在大鼠大脑皮层形成形态规则的损伤空洞,其模型稳定,重复性好;在空洞内进行干细胞移植,能够长时间存活,移植神经干细胞绝大部分细胞分化为神经元,只有少量细胞分化为胶质细胞,而且移植细胞与宿主细胞分界明显;对移植细胞进行单细胞电生理记录,记录到神经元放电信号。这些结果表明,通过机械损毁的方法,在大鼠大脑皮层成功建立了一个稳定、精确定位移植细胞与宿主细胞界限的脑损伤模型。     

Long-term survival of human neural stem cells in the ischemic rat brain upon transient immunosuppression

Understanding the physiology of human neural stem cells (hNSCs) in the context of cell therapy for neurodegenerative disorders is of paramount importance, yet large-scale studies are hampered by the slow-expansion rate of these cells. To overcome this issue, we previously established immortal, non-transformed, telencephalic-diencephalic hNSCs (IhNSCs) from the fetal brain. Here, we investigated the fate of these IhNSC's immediate progeny (i.e. neural progenitors; IhNSC-Ps) upon unilateral implantation into the corpus callosum or the hippocampal fissure of adult rat brain, 3 days after global ischemic injury. One month after grafting, approximately one fifth of the IhNSC-Ps had survived and migrated through the corpus callosum, into the cortex or throughout the dentate gyrus of the hippocampus. By the fourth month, they had reached the ipsilateral subventricular zone, CA1-3 hippocampal layers and the controlateral hemisphere. Notably, these results could be accomplished using transient immunosuppression, i.e administering cyclosporine for 15 days following the ischemic event. Furthermore, a concomitant reduction of reactive microglia (Iba1+ cells) and of glial, GFAP+ cells was also observed in the ipsilateral hemisphere as compared to the controlateral one. IhNSC-Ps were not tumorigenic and, upon in vivo engraftment, underwent differentiation into GFAP+ astrocytes, and β-tubulinIII+ or MAP2+ neurons, which displayed GABAergic and GLUTAmatergic markers. Electron microscopy analysis pointed to the formation of mature synaptic contacts between host and donor-derived neurons, showing the full maturation of the IhNSC-P-derived neurons and their likely functional integration into the host tissue. Thus, IhNSC-Ps possess long-term survival and engraftment capacity upon transplantation into the globally injured ischemic brain, into which they can integrate and mature into neurons, even under mild, transient immunosuppressive conditions. Most notably, transplanted IhNSC-P can significantly dampen the inflammatory response in the lesioned host brain. This work further supports hNSCs as a reliable and safe source of cells for transplantation therapy in neurodegenerative disorders.     

小鼠胚胎干细胞分化扩增期连续低密度传代对原始细胞中Oct-4阳性细胞比例及神经分化能力的影响

目的：探讨在分化扩增期采用连续低密度传代的方法是否能降低小鼠胚胎干细胞向神经细胞分化的前体细胞中Oct-4阳性细胞的比例,以及对神经分化能力的影响。方法：采用“五步法”将小鼠胚胎干细胞向神经元分化,进入扩增期后采用连续低密度传代的方法连续传10代。然后应用细胞免疫组化鉴定Oct-4阳性细胞、神经元与胶质细胞、流式细胞仪检测Oct-4阳性细胞比例、凋亡试剂盒测定细胞凋亡。结果：流式细胞仪检测出扩增期连续低密度传代得到的前体细胞中Oct-4阳性细胞的比例由16.17±4.8％降至4.33±1.63％,扩增期低密度传代细胞和正常高密度传代细胞的细胞凋亡率鉴定分别为15.16±3.64% 和11.88±2.63%,步骤5诱导分化后的细胞GFAP和Tuj-1免疫组化染色呈阴性。结论：低密度传代培养可以减少分化后Oct-4阳性细胞的比例,且该比例下降不是由Oct-4阳性细胞的凋亡引起的。同时可能是因为低密度传代培养和高密度相比引起了细胞的质变、或者改变了前体细胞向神经元分化的某种微环境,导致了前体细胞不能分化为神经细胞。提示高密度培养在前体细胞向神经元分化过程中具有重要作用,高密度和低密度培养的比较,提供了研究ES细胞向神经元分化机制的新平台和研究方向。     

N-cadherin is a useful marker for the progenitor of cardiomyocytes differentiated from mouse ES cells in serum-free condition

Cardiomyocytes are known to differentiate spontaneously from embryonic stem (ES) cells when they formed aggregates, so called "embryoid bodies", in the presence of serum. In this study, we explored the induction of cardiomyocytes from mouse ES cells in chemically defined serum-free medium by using a mesoderm-inducing factor, BMP4. Comparing the different inductive methods, we found a candidate cell surface marker, N-cadherin, for cardiomyocyte progenitors from ES cells. N-cadherin-positive cells highly expressed cardiogenic markers, Nkx2.5, Tbx5, and Isl1, and showed a high differentiation rate into cardiomyocyte lineage. These results indicate that N-cadherin can be a useful cell surface marker for the progenitors of cardiomyocyte differentiated from ES cells in the serum-free culture.