Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease

Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinson's disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinson's disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson's disease.     

Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells, whereas mural cells are believed to derive from mesoderm, neural crest or epicardial cells and migrate to form the vessel wall. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation, whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system.     

Human embryonic stem cell lines derived from single blastomeres

The derivation of human embryonic stem (hES) cells currently requires the destruction of ex utero embryos. A previous study in mice indicates that it might be possible to generate embryonic stem (ES) cells using a single-cell biopsy similar to that used in preimplantation genetic diagnosis (PGD), which does not interfere with the embryo's developmental potential. By growing the single blastomere overnight, the resulting cells could be used for both genetic testing and stem cell derivation without affecting the clinical outcome of the procedure. Here we report a series of ten separate experiments demonstrating that hES cells can be derived from single blastomeres. In this proof-of-principle study, multiple biopsies were taken from each embryo using micromanipulation techniques and none of the biopsied embryos were allowed to develop in culture. Nineteen ES-cell-like outgrowths and two stable hES cell lines were obtained. The latter hES cell lines maintained undifferentiated proliferation for more than eight months, and showed normal karyotype and expression of markers of pluripotency, including Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, nanog and alkaline phosphatase. These cells retained the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. The ability to create new stem cell lines and therapies without destroying embryos would address the ethical concerns of many, and allow the generation of matched tissue for children and siblings born from transferred PGD embryos.     

Derivation of embryonic germ cells and male gametes from embryonic stem cells

Egg and sperm cells (gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac. Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo. Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a property characteristic of the germ lineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis.     

小鼠胚胎干细胞对病毒性心肌炎的影响

目的使用小鼠验证这样一个假设:外界病毒浸入诱发心肌炎时,机体的干细胞将进入心脏提高心肌的抗病毒能力。方法雄性BALB/c小鼠分为三组:小鼠胚胎干细胞对照组(ES),心肌炎病毒组(EM CV)及EM-CV加ES治疗组。通过尾静脉注射,令小鼠立即感染病毒。小鼠死亡率,炎性细胞浸润及心肌坏死等为观察指征。干细胞的游走及分化等通过免疫荧光法来验证。结果给予干细胞后的小鼠的存活率明显高于生理盐水对照组,炎性细胞侵润及心肌坏死亦明显低于生理盐水对照组。免疫荧光法表明,干细胞进入心肌并分化成新的心肌细胞。结论干细胞能明显提高心肌炎小鼠的存活率,减少心肌组织的坏死。同时,亦证明当心脏遭受病毒的侵入后,干细胞通过某种机理修复或再生心肌细胞,从而提高组织的抗病毒能力。     

An intrinsic mechanism of corticogenesis from embryonic stem cells

The cerebral cortex develops through the coordinated generation of dozens of neuronal subtypes, but the mechanisms involved remain unclear. Here we show that mouse embryonic stem cells, cultured without any morphogen but in the presence of a sonic hedgehog inhibitor, recapitulate in vitro the major milestones of cortical development, leading to the sequential generation of a diverse repertoire of neurons that display most salient features of genuine cortical pyramidal neurons. When grafted into the cerebral cortex, these neurons develop patterns of axonal projections corresponding to a wide range of cortical layers, but also to highly specific cortical areas, in particular visual and limbic areas, thereby demonstrating that the identity of a cortical area can be specified without any influence from the brain. The discovery of intrinsic corticogenesis sheds new light on the mechanisms of neuronal specification, and opens new avenues for the modelling and treatment of brain diseases.     

Derivation of haploid embryonic stem cells from mouse embryos

Most animals are diploid, but haploid-only and male-haploid (such as honeybee and ant) species have been described. The diploid genomes of complex organisms limit genetic approaches in biomedical model species such as mice. To overcome this problem, experimental induction of haploidy has been used in fish. Haploid development in zebrafish has been applied for genetic screening. Recently, haploid pluripotent cell lines from medaka fish (Oryzias latipes) have also been established. In contrast, haploidy seems less compatible with development in mammals. Although haploid cells have been observed in egg cylinder stage parthenogenetic mouse embryos, most cells in surviving embryos become diploid. Here we describe haploid mouse embryonic stem cells and show their application in forward genetic screening.     

Pluripotency of mesenchymal stem cells derived from adult marrow

We report here that cells co-purifying with mesenchymal stem cells--termed here multipotent adult progenitor cells or MAPCs--differentiate, at the single cell level, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. When injected into an early blastocyst, single MAPCs contribute to most, if not all, somatic cell types. On transplantation into a non-irradiated host, MAPCs engraft and differentiate to the haematopoietic lineage, in addition to the epithelium of liver, lung and gut. Engraftment in the haematopoietic system as well as the gastrointestinal tract is increased when MAPCs are transplanted in a minimally irradiated host. As MAPCs proliferate extensively without obvious senescence or loss of differentiation potential, they may be an ideal cell source for therapy of inherited or degenerative diseases.     

Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer （SCNT）, holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells （hESCs） have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmis- sion, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi- or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer- ESC （ntESC） lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results in- dicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.     

'Rejuvenation' protects neurons in mouse models of Parkinson's disease

Why dopamine-containing neurons of the brain's substantia nigra pars compacta die in Parkinson's disease has been an enduring mystery. Our studies suggest that the unusual reliance of these neurons on L-type Ca(v)1.3 Ca2+ channels to drive their maintained, rhythmic pacemaking renders them vulnerable to stressors thought to contribute to disease progression. The reliance on these channels increases with age, as juvenile dopamine-containing neurons in the substantia nigra pars compacta use pacemaking mechanisms common to neurons not affected in Parkinson's disease. These mechanisms remain latent in adulthood, and blocking Ca(v)1.3 Ca2+ channels in adult neurons induces a reversion to the juvenile form of pacemaking. Such blocking ('rejuvenation') protects these neurons in both in vitro and in vivo models of Parkinson's disease, pointing to a new strategy that could slow or stop the progression of the disease.     

Induction of embryonic stem cells to hematopoietic cells in vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright's staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34+ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34-CD38+. Wright's staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

Properties and applications of embryonic stem cells

Mouse embryonic stem (ES) cells are pluripotent cells derived from the early embryo and can be propagated stably in undifferentiated state in vitro. They retain the ability to differentiate into all cell types found in the embryonic and adult body in vivo, and can be induced to differentiate into many cell types under appropriate culture conditions in vitro. Using these properties, people have set up various differentiated systems of many cell types and tissues in vitro. Through analysis of these systems, one can identify novel bioactive factors and reveal mechanisms of cell differentiation and organogenesis. ES cell-derived differentiated cells can also be applied to cell transplantation therapy. In addition, we summarized the features and potential applications of human ES cells.     

Acidic pH transiently prevents the silencing of self-renewal and dampens microRNA function in embryonic stem cells

Enhanced glycolysis is a distinct feature associated with numerous stem cells and cancer cells.However,little is known about its regulatory roles in gene expres...     

A Drosophila model of Parkinson's disease

Parkinson's disease is a common neurodegenerative syndrome characterized by loss of dopaminergic neurons in the substantia nigra, formation of filamentous intraneuronal inclusions (Lewy bodies) and an extrapyramidal movement disorder. Mutations in the alpha-synuclein gene are linked to familial Parkinson's disease and alpha-synuclein accumulates in Lewy bodies and Lewy neurites. Here we express normal and mutant forms of alpha-synuclein in Drosophila and produce adult-onset loss of dopaminergic neurons, filamentous intraneuronal inclusions containing alpha-synuclein and locomotor dysfunction. Our Drosophila model thus recapitulates the essential features of the human disorder, and makes possible a powerful genetic approach to Parkinson's disease.     

哺乳动物胚胎干细胞的特性及利用

哺乳动物胚胎干细胞（ES细胞）是由动物早期胚胎发育的内细胞团（ICM）或原始生殖细胞（PGC）分离得到的。人们利用ES细胞所具有的全能性、体外分化以及稳定的遗传性能等特点，展示了ES细胞在建立哺乳动物的早期胚胎体外分化模型、转基因动物模型、器官和组织的修复和移植治疗、克隆动物的生产、发育生物学的研究等方面广阔的应用前景。但是，由于哺乳动物错综复杂的基因调控和环境因素的影响，对于胚胎干细胞的研究还存在诸多问题，还需作更深入细致的研究。     

Maintenance of human embryonic stem cells on gelatin

Matrigel is routinely used as a coating material in the feeder-free culture system of human embryonic stem cells （hESCs）. However, matrigel is costive and inconvenient to use. In this study, the possibility of using gelatin as an alternative coating material was investigated. The results showed that, after trypsinization, hESCs were maintained undifferentiated on gelatin. These hESCs expressed pluripotent markers, formed teratoma and maintained a normal karyotype. As measured at passage 10, the hESCs expressed a high level of Oct4 on both gelatin and Matrigeh hESCs growing on gelatin formed AP-positive colonies in similar size and number to those growing on Matrigel （P〉 0.05）. Moreover, hESCs growing on gelatin contained a comparable percentage of SSEA-4-positive cells to those growing on Matrigel （95.1% vs.94.3%, P〉 0.05）. H-1 hESCs were maintained undifferentiated on gelatin for 20 passages and remained the stable normal karyotype. This gelatin-based culture protocol may allow us to propagate hESCs in large scale, with less cost.     

Differentiation of embryonic stem cells transfected by ibeB gene

We have previously identified an E. coli determinant, ibeB gene locus contributing to invasion of human brain microvascular endothelial cells. In the present study, we established embryonic stem (ES) cell lines overexpressing IbeB and found that exogenic ibeB gene could start-up expression of a neural stem cell specific marker, nestin, and give rise to polar changes. In analysis of IbeB location, it was found that GFP-IbeB fusion protein targeted at the ES cell nucleus. These data suggests that ibeB gene may play an important role in the regulation of nestin expression.     

用高代数小鼠胚胎干细胞制作嵌合体小鼠

为了检验胚胎干的全能性,通过用1日龄卵巢移植后,经体外受精得到的囊胚的饲养建立了小鼠的ES系,并在较高的代数下(第41代)制出了5只毛色嵌合体小鼠,但是没有得到性腺嵌合的小鼠。研究结果证实,高代数的ES细胞仍然具有构建嵌合体的能力。