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.

Recent reports demonstrate that somatic mouse cells can be directly converted to other mature cell types by using combined expression of defined factors. Here we show that the same strategy can be applied to human embryonic and postnatal fibroblasts. By overexpression of the transcription factors Ascl1, Brn2, and Myt1l, human fibroblasts were efficiently converted to functional neurons. We also demonstrate that the converted neurons can be directed toward distinct functional neurotransmitter phenotypes when the appropriate transcriptional cues are provided together with the three conversion factors. By combining expression of the three conversion factors with expression of two genes involved in dopamine neuron generation, Lmx1a and FoxA2, we could direct the phenotype of the converted cells toward dopaminergic neurons. Such subtypespecific induced neurons derived from human somatic cells could be valuable for disease modeling and cell replacement therapy. C ellular reprogramming, the process by which somatic cells can be converted into induced pluripotent stem (iPS) cells and subsequently differentiated to mature cells, including specific types of neurons, has opened up new possibilities for disease modeling and cellular repair (1-5). Recently, it was shown that somatic cells can also be directly converted to other mature cell types by expression of a specific combinations of genes (6-9). Expression of Ascl1, Brn2, and Myt1l efficiently converted mouse embryonic fibroblasts (MEFs) and postnatal fibroblasts into functional neurons (induced neurons, or iN cells) (10). Cells generated via direct conversion do not pass through a pluripotent state, are probably not tumorigenic, and may serve as an interesting alternative to iPS cells for generating patient-and/or disease-specific neurons.Here, we show the direct conversion of human fibroblasts into functional neurons using the same combination of neural conversion factors used for iN conversion of mouse fibroblasts (10). We also demonstrate that the expression of additional transcription factors leads to the generation of cells with properties of dopaminergic neurons, which is the cell type lost in Parkinson's disease. Our findings provide proof of principle that specific subtypes of iN cells can be produced from human somatic cells by transcription factor-mediated fate instruction combined with the three neural conversion factors. ResultsTo investigate whether direct conversion into neurons from human somatic cells is possible, we established fibroblast cultures from human embryos aged 5.5-7 wk postconception (for details see Table S1). The head, the dorsal part of the embryo containing the spinal cord, and all red organs were removed, and the remaining tissue was dissociated and plated under standard fibroblast conditions (Fig. 1A). After one passage followed by a freeze-thaw cycle, the fibroblast identity and the absence of the neural crest marker SOX10 in the resulting cell lines were confirmed (Fig. 1B, Figs. S1 and S2, and Tables S2 and S3). The cells were then...

Embryonic stem cells (ESCs IntroductionEmbryonic stem cells (ESCs) represent a potentially unlimited source of cells for applications in regenerative medicine. One promising strategy is the derivation of midbrain dopaminergic (DA) neurons for cell replacement therapy in Parkinson disease (PD). Several protocols have been developed to obtain ESC-derived midbrain DA neurons in vitro, though current technologies are far from perfect and cell preparations often contain contaminating non-DA cell populations and potentially tumorigenic cell types (1, 2). Furthermore, despite more than 40 years of research on developing DA cell replacement paradigms, many fundamental questions critical for effective clinical translation have remained unanswered, such as the stage of DA neuron development most appropriate for transplantation; whether other non-dopamine cell types within the graft, such as glia, are necessary for in vivo DA neuron survival and function; and how closely in vitro-generated cells match the properties of authentic midbrain DA neurons generated in vivo. The use of ESC technology has the potential to address all these questions and presents a virtually unlimited source of fully defined DA neurons.One strategy to obtain highly enriched populations of midbrain DA neurons is the purification of ESC-derived progeny prior to transplantation through the use of fluorescence-activated cell

Human pluripotent stem cells possess the potential to generate cells for regenerative therapies in patients with neurodegenerative diseases, and constitute an excellent cell source for studying human neural development and disease modeling. Protocols for neural differentiation of human pluripotent stem cells have undergone significant progress during recent years, allowing for rapid and synchronized neural conversion. Differentiation procedures can further be combined with accurate and efficient positional patterning to yield regionalized neural progenitors and subtype-specific neurons corresponding to different parts of the developing human brain. Here, we present a step-by-step protocol for neuralization and regionalization of human pluripotent cells for transplantation studies or in vitro analysis.

In this study, we have used a microRNA-regulated lentiviral reporter system to visualize and segregate differentiating neuronal cells in pluripotent cultures. Efficient suppression of transgene expression, specifically in undifferentiated pluripotent cells, was achieved by using a lentiviral vector expressing a fluorescent reporter gene regulated by microRNA-292. Using this strategy, it was possible to track progeny from murine ES, human ES cells, and induced pluripotent stem cells as they differentiated toward the neural lineage. In addition, this strategy was successfully used to FACS purify neuronal progenitors for molecular analysis and transplantation. FACS enrichment reduced tumor formation and increased survival of ES cell-derived neuronal progenitors after transplantation. The properties and versatility of the microRNA-regulated vectors allows broad use of these vectors in stem cell applications.ES | induced pluripotent stem cells | stem cells | transplantation