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...

Induced pluripotent stem cells (iPS cells) are a promising source for a cell-based therapy to treat Parkinson's disease (PD), in which midbrain dopaminergic neurons progressively degenerate. However, long-term analysis of human iPS cell-derived dopaminergic neurons in primate PD models has never been performed to our knowledge. Here we show that human iPS cell-derived dopaminergic progenitor cells survived and functioned as midbrain dopaminergic neurons in a primate model of PD (Macaca fascicularis) treated with the neurotoxin MPTP. Score-based and video-recording analyses revealed an increase in spontaneous movement of the monkeys after transplantation. Histological studies showed that the mature dopaminergic neurons extended dense neurites into the host striatum; this effect was consistent regardless of whether the cells were derived from patients with PD or from healthy individuals. Cells sorted by the floor plate marker CORIN did not form any tumours in the brains for at least two years. Finally, magnetic resonance imaging and positron emission tomography were used to monitor the survival, expansion and function of the grafted cells as well as the immune response in the host brain. Thus, this preclinical study using a primate model indicates that human iPS cell-derived dopaminergic progenitors are clinically applicable for the treatment of patients with PD.

SummaryHuman induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (DA) neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN+ cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN+ cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN+ cells in a NURR1+ cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application.

SummaryConsiderable progress has been made in generating fully functional and transplantable dopamine neurons from human embryonic stem cells (hESCs). Before these cells can be used for cell replacement therapy in Parkinson’s disease (PD), it is important to verify their functional properties and efficacy in animal models. Here we provide a comprehensive preclinical assessment of hESC-derived midbrain dopamine neurons in a rat model of PD. We show long-term survival and functionality using clinically relevant MRI and PET imaging techniques and demonstrate efficacy in restoration of motor function with a potency comparable to that seen with human fetal dopamine neurons. Furthermore, we show that hESC-derived dopamine neurons can project sufficiently long distances for use in humans, fully regenerate midbrain-to-forebrain projections, and innervate correct target structures. This provides strong preclinical support for clinical translation of hESC-derived dopamine neurons using approaches similar to those established with fetal cells for the treatment of Parkinson’s disease.