Midbrain dopaminergic (mDA) neuron development has been an intense area of research during recent years. This is due in part to a growing interest in regenerative medicine and the hope that treatment for diseases affecting mDA neurons, such as Parkinson's disease (PD), might be facilitated by a better understanding of how these neurons are specified, differentiated and maintained in vivo. This knowledge might help to instruct efforts to generate mDA neurons in vitro, which holds promise not only for cell replacement therapy, but also for disease modeling and drug discovery. In this Primer, we will focus on recent developments in understanding the molecular mechanisms that regulate the development of mDA neurons in vivo, and how they have been used to generate human mDA neurons in vitro from pluripotent stem cells or from somatic cells via direct reprogramming. Current challenges and future avenues in the development of a regenerative medicine for PD will be identified and discussed.

The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named “caudal neural progenitors” (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube. Stem Cells 2015;33:1759–1770

Myelination of the vertebrate nervous system has evolved to insulate axons and to promote rapid propagation of action potentials via saltatory conduction. During development of the CNS, committed oligodendrocyte progenitor cells (OPCs) arise from stereotyped germinal regions of the CNS, proliferate, and migrate throughout the CNS before differentiating into postmitotic premyelinating oligodendrocytes (Barres and Raff 1999;Richardson et al. 2000;Baumann and Pham-Dinh 2001). These cells subsequently either ensheath axons to form myelin membrane or undergo apoptosis. However, relatively little is known about the nature of the intracellular signals that control myelination or how these are regulated. Identifying such molecules and pathways has the potential to inform novel therapeutic approaches to promote remyelination.Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family of growth factors and signals through the tropomyosin-related kinase B (TrkB) receptor and the structurally unrelated p75 neurotrophin receptor (Huang and Reichardt 2001;Chao 2003). Recent studies have established that BDNF plays a key role in regulating CNS myelination, as both BDNF knockout and heterozygous Received May 17, 2012; revised manuscript received July 6, 2012; accepted July 9, 2012.Address correspondence and reprint requests to Dr Junhua Xiao, Centre for Neuroscience Research, The University of Melbourne, Victoria 3010, Australia. E-mail: xiaoj@unimelb.edu.auAbbreviations used: BDNF, brain-derived neurotrophic factor; BrdU, bromodeoxyuridine; CA, constitutively active; CNPase, 2¢,3¢-cyclic 11 nucleotide 3¢-phosphodiesterase; CREB, 2 cAMP-response element binding protein; DAPI, 4¢, 6¢-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; DN, dominant negative; DRG, dorsal root ganglion; Erk, extracellular signal-regulated kinase; GFAP, glial fibrillary acidic protein; JNK, c-Jun N-terminal kinase; MAG, myelin associated glycoprotein; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MEK, MAPK/Erk kinase; NGF, nerve growth factor; OPCs, oligodendrocyte progenitor cells; P, Postnatal; PDGFRa, platelet-derived growth factor receptor alpha; TrkB, tropomyosin-related kinase receptor B., , , We have previously shown that brain-derived neurotrophic factor (BDNF) promotes oligodendrocyte myelination. Here, we screened for the activation of candidate signaling pathways in in vitro myelination assays and found that extracellular signal-regulated kinase (Erk) signaling positively correlated with basal levels of oligodendrocyte myelination as well as BDNF-induced myelination in vitro. By selectively manipulating Erk1/2 activation in oligodendrocytes in vitro, we found that constitutive activation of Erk1/2 significantly increased myelination, mimicking the promyelinating effect of BDNF, and also caused myelination to occur earlier. Conversely, selective inhibition of Erk1/2 in oligodendrocytes significantly reduced the basal level of myelination and blocked the promyelinating effect of BDNF. Analysis of mye...

The great potential of induced pluripotent cells (iPS) cells is that it allows the possibility of deriving pluripotent stem cells from any human patient. Generation of patient-derived stem cells serves as a great source for developing cell replacement therapies and also for creating human cellular model systems of specific diseases or disorders. This is only of benefit if there are well-established differentiation assay systems to generate the cell types of interest. This chapter describes robust and well-characterized protocols for differentiating iPS cells to neural progenitors, neurons, glia and neural crest cells. These established assays can be applied to iPS cell lines derived from patients with neurodegenerative disorders to study cellular mechanisms associated with neurodegeneration as well as investigating the regenerative potential of patient derived stem cells.

The floor plate is one of the major organizers of the developing nervous system through its secretion of sonic hedgehog (Shh). Although the floor plate is located within the neural tube, the derivation of the floor plate during development is still debatable and some studies suggest that floor plate cells are specified by Shh in a temporarily restricted window different to neuroepithelial cells. Using human embryonic stem cells (hESC) as a model of neurogenesis, we sought to determine how floor plate cells may be temporarily specified by SHH signaling during human embryogenesis. We found that inhibition of both GSK3β and activin/nodal pathways in hESC induces a cellular state of SOX2+/PAX6− expression, we describe as “pre-neuroepithelial.” Exposure of SHH during this pre-neuroepithelial period causes the expression of GLI transcription factors to function as activators and consequently upregulate expression of the floor plate marker, FOXA2, while also supressing PAX6 expression to inhibit neuroepithelial fate. FOXA2+ cells were able to efficiently generate mesencephalic dopaminergic neurons, a floor plate derivative. Overall, this study demonstrates a highly efficient system for generating floor plate cells from hESC and, most importantly, reveals that specification of floor plate cells is temporally dependent, whereby it occurs prior to the onset of PAX6 expression, within a pre-neuroepithelial stage. Stem Cells2012;30:2400–2411

Human pluripotent stem cells have the capacity for directed differentiation into a wide variety of neuronal subtypes that may be useful for brain repair. While a substantial body of research has lead to a detailed understanding of the ability of neurons in fetal tissue grafts to structurally and functionally integrate after intra-cerebral transplantation, we are only just beginning to understand the in vivo properties of neurons derived from human pluripotent stem cells. Here we have utilized the human embryonic stem (ES) cell line Envy, which constitutively expresses green fluorescent protein (GFP), in order to study the in vivo properties of neurons derived from human ES cells. Rapid and efficient neural induction, followed by differentiation as neurospheres resulted in a GFP+ neural precursor population with traits of neuroepithelial and dorsal forebrain identity. Ten weeks after transplantation into neonatal rats, GFP+ fiber patterns revealed extensive axonal growth in the host brain, particularly along host white matter tracts, although innervation of adjacent nuclei was limited. The grafts were composed of a mix of neural cell types including differentiated neurons and glia, but also dividing neural progenitors and migrating neuroblasts, indicating an incomplete state of maturation at 10 weeks. This was reflected in patch-clamp recordings showing stereotypical properties appropriate for mature functional neurons, including the ability to generate action potentials, as well profiles consistent for more immature neurons. These findings illustrate the intrinsic capacity for neurons derived from human ES cells to integrate at a structural and functional level following transplantation.

Background & AimsHirschsprung disease (HSCR) is caused by failure of cells derived from the neural crest (NC) to colonize the distal bowel in early embryogenesis, resulting in absence of the enteric nervous system (ENS) and failure of intestinal transit postnatally. Treatment is by distal bowel resection, but neural cell replacement may be an alternative. We tested whether aneuronal (aganglionic) colon tissue from patients may be colonized by autologous ENS-derived cells.MethodsCells were obtained and cryopreserved from 31 HSCR patients from the proximal resection margin of colon, and ENS cells were isolated using flow cytometry for the NC marker p75 (nine patients). Aneuronal colon tissue was obtained from the distal resection margin (23 patients). ENS cells were assessed for NC markers immunohistologically and by quantitative reverse-transcription polymerase chain reaction, and mitosis was detected by ethynyl-2′-deoxyuridine labeling. The ability of human HSCR postnatal ENS-derived cells to colonize the embryonic intestine was demonstrated by organ coculture with avian embryo gut, and the ability of human postnatal HSCR aneuronal colon muscle to support ENS formation was tested by organ coculture with embryonic mouse ENS cells. Finally, the ability of HSCR patient ENS cells to colonize autologous aneuronal colon muscle tissue was assessed.ResultsENS-derived p75-sorted cells from patients expressed multiple NC progenitor and differentiation markers and proliferated in culture under conditions simulating Wnt signaling. In organ culture, patient ENS cells migrated appropriately in aneural quail embryo gut, and mouse embryo ENS cells rapidly spread, differentiated, and extended axons in patient aneuronal colon muscle tissue. Postnatal ENS cells derived from HSCR patients colonized autologous aneuronal colon tissue in cocultures, proliferating and differentiating as neurons and glia.ConclusionsNC-lineage cells can be obtained from HSCR patient colon and can form ENS-like structures in aneuronal colonic muscle from the same patient.

Generation of mesencephalic dopamine (mesDA) neurons from human embryonic stem cells (hESCs) requires several stages of signaling from various extrinsic and intrinsic factors. To date, most methods incorporate exogenous treatment of Sonic hedgehog (SHH) to derive mesDA neurons. However, we and others have shown that this approach is inefficient for generating FOXA2+ cells, the precursors of mesDA neurons. As mesDA neurons are derived from the ventral floor plate (FP) regions of the embryonic neural tube, we sought to develop a system to derive FP cells from hESC. We show that forced expression of the transcription factor GLI1 in hESC at the earliest stage of neural induction, resulted in their commitment to FP lineage. The GLI1+ cells coexpressed FP markers, FOXA2 and Corin, and displayed exocrine SHH activity by ventrally patterning the surrounding neural progenitors. This system results in 63% FOXA2+ cells at the neural progenitor stage of hESC differentiation. The GLI1-transduced cells were also able to differentiate to neurons expressing tyrosine hydroxylase. This study demonstrates that GLI1 is a determinant of FP specification in hESC and describes a highly robust and efficient in vitro model system that mimics the ventral neural tube organizer. Stem Cells 2010;28:1805–1815