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 subtype-specific induced neurons derived from human somatic cells could be valuable for disease modeling and cell replacement therapy.

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.

Human 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.

Considerable 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.

Cellular reprogramming is a new and rapidly emerging field in which somatic cells can be turned into pluripotent stem cells or other somatic cell types simply by the expression of specific combinations of genes. By viral expression of neural fate determinants, it is possible to directly reprogram mouse and human fibroblasts into functional neurons, also known as induced neurons. The resulting cells are nonproliferating and present an alternative to induced pluripotent stem cells for obtaining patient- and disease-specific neurons to be used for disease modeling and for development of cell therapy. In addition, because the cells do not pass a stem cell intermediate, direct neural conversion has the potential to be performed in vivo. In this study, we show that transplanted human fibroblasts and human astrocytes, which are engineered to express inducible forms of neural reprogramming genes, convert into neurons when reprogramming genes are activated after transplantation. Using a transgenic mouse model to specifically direct expression of reprogramming genes to parenchymal astrocytes residing in the striatum, we also show that endogenous mouse astrocytes can be directly converted into neural nuclei (NeuN)-expressing neurons in situ. Taken together, our data provide proof of principle that direct neural conversion can take place in the adult rodent brain when using transplanted human cells or endogenous mouse cells as a starting cell for neural conversion.