Successful function of autologous iPSC-derived dopamine neurons following transplantation in a non-human primate model of Parkinson's disease.

Autologous transplantation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons is a potential clinical approach for treatment of neurological disease. Preclinical demonstration of long-term efficacy, feasibility, and safety of iPSC-derived dopamine neurons in non-human primate models will be an important step in clinical development of cell therapy. Here, we analyzed cynomolgus monkey (CM) iPSC-derived midbrain dopamine neurons for up to 2 years following autologous transplantation in a Parkinson's disease (PD) model. In one animal, with the most successful protocol, we found that unilateral engraftment of CM-iPSCs could provide a gradual onset of functional motor improvement contralateral to the side of dopamine neuron transplantation, and increased motor activity, without a need for immunosuppression. Postmortem analyses demonstrated robust survival of midbrain-like dopaminergic neurons and extensive outgrowth into the transplanted putamen. Our proof of concept findings support further development of autologous iPSC-derived cell transplantation for treatment of PD.

Long-term health of dopaminergic neuron transplants in Parkinson's disease patients.

To determine the long-term health and function of transplanted dopamine neurons in Parkinson's disease (PD) patients, the expression of dopamine transporters (DATs) and mitochondrial morphology were examined in human fetal midbrain cellular transplants. DAT was robustly expressed in transplanted dopamine neuron terminals in the reinnervated host putamen and caudate for at least 14 years after transplantation. The transplanted dopamine neurons showed a healthy and nonatrophied morphology at all time points. Labeling of the mitochondrial outer membrane protein Tom20 and α-synuclein showed a typical cellular pathology in the patients' own substantia nigra, which was not observed in transplanted dopamine neurons. These results show that the vast majority of transplanted neurons remain healthy for the long term in PD patients, consistent with clinical findings that fetal dopamine neuron transplants maintain function for up to 15-18 years in patients. These findings are critically important for the rational development of stem-cell-based dopamine neuronal replacement therapies for PD.

Improved cell therapy protocols for Parkinson's disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons.

The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results, NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM(+) /CD29(low) DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future.

Characterization and criteria of embryonic stem and induced pluripotent stem cells for a dopamine replacement therapy.

Human pluripotent stem cells provide new choices for sources of A9-type dopaminergic (DA) neurons in clinical trials of neural transplantation for patients with Parkinson's disease (PD). For example, "self" and HLA-matched A9 DA neurons may improve the patient-to-patient variability observed in previous clinical trials using fetal DA neurons and obviate the need for long-term immunosuppression in the patient. Normal chromosomal structure and minimal somatic mutations in pluripotent stem cells are necessary criteria for assuring the safe and reproducible transplantation of differentiated DA neurons into patients with PD in clinical trials. However, with these new choices of cell source, the application of pluripotency assays as criteria to ensure pluripotent stem cell quality becomes less relevant. New more relevant standards of quality control, assurance, and function are required. We suggest that quality assurance measures for pluripotent stem cells need to focus upon readouts for authentic midbrain DA neurons, their integration and growth using in vivo assays, and their long-term functional stability.

Using stem cells and iPS cells to discover new treatments for Parkinson's disease.

Fetal cell transplantation can improve the symptoms of Parkinson's disease (PD) patients for more than a decade. In some patients, alpha-synuclein aggregates and Lewy bodies have been observed in the transplanted neurons without functional significance. Recently stem cells have emerged as an ethically acceptable source of cells for transplantation but, importantly, the type of stem cell matters. While the lineage restriction of adult neural stem cells limits their clinical applicability for patients with PD, human pluripotent stem cells provide an opportunity to replace specific types of degenerating neurons. Now, cellular reprogramming technology can provide patient-specific neurons for neural transplantation and problems with cell fate specification and safety are resolving. Induced pluripotent stem (iPS) cell-derived neurons are also a unique tool for interpreting the genetic basis for an individual's risk of developing PD into clinically meaningful information. For example, clinical trials for neuroprotective molecules need to be tested in presymptomatic individuals when the neurons can still be protected. Patient-specific neural cells can also be used to identify an individual's responsiveness to drugs and to understand the mechanisms of the disease. Along these avenues of investigation, stem cells are enabling research for new treatments in PD.

Oct4-induced reprogramming is required for adult brain neural stem cell differentiation into midbrain dopaminergic neurons.

Neural stem cells (NSCs) lose their competency to generate region-specific neuronal populations at an early stage during embryonic brain development. Here we investigated whether epigenetic modifications can reverse the regional restriction of mouse adult brain subventricular zone (SVZ) NSCs. Using a variety of chemicals that interfere with DNA methylation and histone acetylation, we showed that such epigenetic modifications increased neuronal differentiation but did not enable specific regional patterning, such as midbrain dopaminergic (DA) neuron generation. Only after Oct-4 overexpression did adult NSCs acquire a pluripotent state that allowed differentiation into midbrain DA neurons. DA neurons derived from Oct4-reprogrammed NSCs improved behavioural motor deficits in a rat model of Parkinson's disease (PD) upon intrastriatal transplantation. Here we report for the first time the successful differentiation of SVZ adult NSCs into functional region-specific midbrain DA neurons, by means of Oct-4 induced pluripotency.

Differentiated Parkinson patient-derived induced pluripotent stem cells grow in the adult rodent brain and reduce motor asymmetry in Parkinsonian rats.

Recent advances in deriving induced pluripotent stem (iPS) cells from patients offer new possibilities for biomedical research and clinical applications, as these cells could be used for autologous transplantation. We differentiated iPS cells from patients with Parkinson's disease (PD) into dopaminergic (DA) neurons and show that these DA neurons can be transplanted without signs of neurodegeneration into the adult rodent striatum. The PD patient iPS (PDiPS) cell-derived DA neurons survived at high numbers, showed arborization, and mediated functional effects in an animal model of PD as determined by reduction of amphetamine- and apomorphine-induced rotational asymmetry, but only a few DA neurons projected into the host striatum at 16 wk after transplantation. We next applied FACS for the neural cell adhesion molecule NCAM on differentiated PDiPS cells before transplantation, which resulted in surviving DA neurons with functional effects on amphetamine-induced rotational asymmetry in a 6-OHDA animal model of PD. Morphologically, we found that PDiPS cell-derived non-DA neurons send axons along white matter tracts into specific close and remote gray matter target areas in the adult brain. Such findings establish the transplantation of human PDiPS cell-derived neurons as a long-term in vivo method to analyze potential disease-related changes in a physiological context. Our data also demonstrate proof of principle of survival and functional effects of PDiPS cell-derived DA neurons in an animal model of PD and encourage further development of differentiation protocols to enhance growth and function of implanted PDiPS cell-derived DA neurons in regard to potential therapeutic applications.

Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid.

The cardinal motor symptoms of Parkinson's disease (PD) are caused by the vulnerability to dysfunction and degeneration of ventral midbrain (VM) dopaminergic (DA) neurons. A major limitation for experimental studies of current ES/iPS cell differentiation protocols is the lack of VM DA neurons with a stable phenotype as defined by an expression marker code of FOXA2/TH/ß-tubulin. Here we demonstrate a combination of three modifications that were required to produce VM DA neurons. Firstly, early and specific exposure to 10(-)(8)M (low dose) retinoic acid improved the regional identity of neural progenitor cells derived from human ES cells, PD or healthy subject-specific iPS cells. Secondly, a high activity form of human sonic hedgehog established a sizeable FOXA2(+) neural progenitor cell population in vitro. Thirdly, early exposure to FGF8a, rather than Fgf8b, and WNT1 was required for robust differentiation of the FOXA2(+) floor plate-like human neural progenitor cells into FOXA2(+) DA neurons. FOXA2(+) DA neurons were also generated when this protocol was adapted to feeder-free conditions. In summary, this new human ES and iPS cell differentiation protocol using FGF8a, WNT1, low dose retinoic acid and a high activity form of SHH can generate human VM DA neurons that are required for relevant new bioassays, drug discovery and cell based therapies for PD.

Klhl31 is associated with skeletal myogenesis and its expression is regulated by myogenic signals and Myf-5.

Klhl31 is an orthologue of Drosophila Kelch and belongs to a family of Kelch-like proteins in vertebrates. Members of this family contain multiple protein domains, including an amino-terminal broad complex/tram-track/bric-a-brac (BTB) or poxvirus and zinc finger (POZ) domain, carboxy-terminal Kelch repeats and a central linker region. We show that Klhl31 is highly expressed in the developing heart, the somite myotome and later in differentiated skeletal muscle and the myocardium. In developing somites expression of Klhl31 was initiated in the epaxial domain of the myotome, shortly after the skeletal muscle specific bHLH transcription factor, MyoD, was first expressed. Klhl31 remained expressed in skeletal muscle throughout embryonic and fetal development. Tissue ablations and rescue experiments that regulate myogenesis also govern expression of Klhl31 expression in somites. In particular, axial tissues, neural tube, floor plate and notochord, and surface ectoderm, provide combinatorial cues for myogenesis and the appropriate expression of Klhl31. We show that a combination of myogenic signals, Shh and either Wnt-1 or Wnt-6, are sufficient for Klhl31 expression in the dorsal somite. Furthermore, ectopic expression of Myf-5 led to expression of Klhl31 in the developing neural tube, indicating that Klhl31 is a novel and integral part of vertebrate myogenesis.

Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.

Expression of avian prickle genes during early development and organogenesis.

Chicken homologues of prickle-1 (pk-1) and prickle-2 (pk-2) were isolated to gain insight into the extent of planar cell polarity signaling during avian embryogenesis. Bioinformatics analyses demonstrated homology and showed that pk-1 and pk-2 exhibited conserved synteny with ADAMTS20 and ADAMTS9, GON-related zinc metalloproteases. Expression of pk-1 and pk-2 was established during embryogenesis and early organogenesis, using in situ hybridization and sections of chicken embryos. At early stages, pk-1 was expressed in Hensen's node, primitive streak, ventral neural tube, and foregut. In older embryos, pk-1 transcripts were detected in dorsolateral epithelial somites, dorsomedial lip of dermomyotomes, and differentiating myotomes. Furthermore, pk-1 expression was seen in lateral body folds, limb buds, and ventral metencephalon. pk-2 was expressed in Hensen's node and neural ectoderm at early stages. In older embryos, pk-2 expression was restricted to ventromedial epithelial somites, except in the most recently formed somite pair, and limb bud mesenchyme.

Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson's disease.

We report the first post-mortem analysis of two patients with Parkinson's disease who received fetal midbrain transplants as a cell suspension in the striatum, and in one case also in the substantia nigra. These patients had a favourable clinical evolution and positive 18F-fluorodopa PET scans and did not develop motor complications. The surviving transplanted dopamine neurons were positively identified with phenotypic markers of normal control human substantia nigra (n = 3), such as tyrosine hydroxylase, G-protein-coupled inward rectifying current potassium channel type 2 (Girk2) and calbindin. The grafts restored the cell type that provides specific dopaminergic innervation to the most affected striatal regions in the parkinsonian brain. Such transplants were able to densely reinnervate the host putamen with new dopamine fibres. The patients received only 6 months of standard immune suppression, yet by post-mortem analysis 3-4 years after surgery the transplants appeared only mildly immunogenic to the host brain, by analysis of microglial CD45 and CD68 markers. This study demonstrates that, using these methods, dopamine neuronal replacement cell therapy can be beneficial for patients with advanced disease, and that changing technical approaches could have a favourable impact on efficacy and adverse events following neural transplantation.

Context-dependent neuronal differentiation and germ layer induction of Smad4-/- and Cripto-/- embryonic stem cells.

Activation of transforming growth factor-beta (TGF-beta) receptors typically elicits mesodermal development, whereas inhibition of this pathway induces neural fates. In vitro differentiated mouse embryonic stem (ES) cells with deletion of the TGF-beta pathway-related factors Smad4 or Cripto exhibited increased numbers of neurons. Cripto-/- ES cells developed into neuroecto-/epidermal cell types, while Smad4-/- cells also displayed mesodermal differentiation. ES cell differentiation into catecholaminergic neurons showed that these ES cells retained their ability to develop into dopaminergic and serotonergic neurons with typical expression patterns of midbrain and hindbrain genes. In vivo, transplanted ES cells to the mouse striatum became small neuronal grafts, or large grafts with cell types from all germ layers independent of their ES cell genotype. This demonstrates that Smad4-/- and Cripto-/- ES cells favor a neural fate in vitro, but also express the mesodermal phenotype, implying that deletion of either Smad4 or Cripto is not sufficient to block nonneuronal tissue formation.