Human embryonic stem (hES) cells, due to their capacity of multipotency and self-renewal, may serve as a valuable experimental tool for human developmental biology and may provide an unlimited cell source for cell replacement therapy. The purpose of this study was to assess the developmental potential of hES cells to replace the selectively lost midbrain dopamine (DA) neurons in Parkinson's disease. Here, we report the development of an in vitro differentiation protocol to derive an enriched population of midbrain DA neurons from hES cells. Neural induction of hES cells co-cultured with stromal cells, followed by expansion of the resulting neural precursor cells, efficiently generated DA neurons with concomitant expression of transcriptional factors related to midbrain DA development, such as Pax2, En1 (Engrailed-1), Nurr1, and Lmx1b. Using our procedure, the majority of differentiated hES cells (> 95%) contained neuronal or neural precursor markers and a high percentage (> 40%) of TuJ1+ neurons was tyrosine hydroxylase (TH)+, while none of them expressed the undifferentiated ES cell marker, Oct 3/4. Furthermore, hES cell-derived DA neurons demonstrated functionality in vitro, releasing DA in response to KCl-induced depolarization and reuptake of DA. Finally, transplantation of hES-derived DA neurons into the striatum of hemi-parkinsonian rats failed to result in improvement of their behavioral deficits as determined by amphetamine-induced rotation and step-adjustment. Immunohistochemical analyses of grafted brains revealed that abundant hES-derived cells (human nuclei+ cells) survived in the grafts, but none of them were TH+. Therefore, unlike those from mouse ES cells, hES cellderived DA neurons either do not survive or their DA phenotype is unstable when grafted into rodent brains.
Summary Selective degeneration of midbrain dopaminergic (mDA) neurons is associated with Parkinson’s disease (PD), and thus an in-depth understanding of molecular pathways underlying mDA development will be crucial for optimal bioassays and cell replacement therapy for PD. In this study, we identified a novel Wnt1-Lmx1a autoregulatory loop during mDA differentiation of ES cells, and confirmed its in vivo presence during embryonic development. We found that the Wnt1-Lmx1a autoregulatory loop directly regulates Otx2 through the β-catenin complex and Nurr1 and Pitx3 through Lmx1a. We also found that Lmx1a and Lmx1b co-operatively regulate mDA differentiation with overlapping and cross-regulatory functions. Furthermore, co-activation of both Wnt1 and SHH pathways by exogenous expression of Lmx1a, Otx2 and FoxA2 synergistically enhanced the differentiation of ES cells to mDA neurons. Together with previous works, this study shows that two regulatory loops (Wnt1-Lmx1a and SHH-FoxA2) critically link extrinsic signals to cell-intrinsic factors and cooperatively regulate mDA neuron development.
Embryonic stem (ES) cells provide a potentially unlimited source of specialized cells for regenerative medicine. The ease of inducing stable genetic modifications in ES cells allows for in vitro manipulations to enhance differentiation into specific cell types and to optimize in vivo function of differentiated progeny in animal models of disease. We have generated mouse ES cells that constitutively express Bcl-XL, an antiapoptotic protein of Bcl-2 family. In vitro differentiation of Bcl-XL overexpressing ES (Bcl-ES) cells resulted in higher expression of genes related to midbrain dopamine (DA) neuron development and increased the number of ES-derived neurons expressing midbrain DA markers compared with differentiation of wild-type ES cells. Moreover, DA neurons derived from Bcl-ES cells were less susceptible to 1-methyl-4-phenylpyridium, a neurotoxin for DA neurons. On transplantation into parkinsonian rats, the Bcl-ES-derived DA neurons exhibited more extensive fiber outgrowth and led to a more pronounced reversal of behavioral symptoms than wild-type ES-derived DA neurons. These data suggest a role for Bcl-XL during in vitro midbrain DA neuron differentiation and provide an improved system for cell transplantation in a preclinical animal model of Parkinson's disease.
In this study, we have attempted to exploit the neurotrophic actions of astrocytes and Nurr1+Foxa2 functions in this cell type to improve the therapeutic outcomes of NPC transplantation using an animal model of PD. Our in vitro assays using a coculture system and conditioned media treatment revealed that astrocytes, especially those cultured from the ventral midbrain (VM), where HNF3β) is a potent cofactor that synergizes the Nurr1-mediated antiinflammatory roles in glia (16). Furthermore, forced coexpression of Nurr1 and Foxa2 (Nurr1+Foxa2) in glia promotes the synthesis and release of various neurotrophic factors, indicating that engineering of Nurr1 and Foxa2 in glia could become a powerful strategy for treating CNS disorders. Representative images of TH + DA neurons differentiated from VM-NPCs in the presence of Ctx-NPCs (control), Ctx-Ast, or VM-Ast. Scale bar: 100 μm. Insets, enlarged images of the boxed areas (original magnification, ×400). (C) DA neuronal yields at differentiation day 6 (D6). (D-H) Morphometric measurement of DA neuron maturity assessed by neurite outgrowth lengths (D), soma size (E), and by the Sholl test (F and G). In the Sholl analysis, the total number of neurite crossings was counted in each circle with the radius increasing in steps of 15 μm (F). The critical value (G) is the radius at which there was a maximum number of neurite crossings. *P < 0.05, significantly different from the control; # P < 0.05, significantly different from Ctx-Ast, 1-way ANOVA. n = 3-5 wells (C) and 100 (D and E) and 25-30 (F and G) TH + cells in each group.(H) Representative TH + neuronal morphologies reconstructed in 3D by Neurolucida. (I-L) Synaptic densities on TH + fibers. (I) Representative confocal images of synapsin + TH + fibers. Scale bar: 25 μm. Puncta positive for the synaptic vesicle-specific markers SV2 (J), synapsin (K), and bassoon (L) on TH + fibers were counted. *P < 0.05, significantly different from the control; # P < 0.05, significantly different from Ctx-Ast. n = 20 TH + fibers for each group. (M) Presynaptic DA release. n = 3 independent cultures. DA levels were measured in the media conditioned in the differentiated cultures for 24 hours (D13-D15) and in cultures evoked by KCl-mediated depolarization for 30 minutes. *P < 0.05; # P < 0.05, 1-way ANOVA. The Journal of Clinical Investigation R E S E A R C H A R T I C L E4 6 5 jci.org Volume 128 Number 1 January 2018 (Supplemental Figure 1B; supplemental material available online with this article; https://doi.org/10.1172/JCI93924DS1), immunocytochemical analyses revealed that major cell populations in the Ctx-and VM-astroglial cultures at day in vitro (DIV) 14 were positive for GFAP (85% and 82%), CD44 (79% and 76 %), GLT-1 (58% and 71 %), and GLAST protein (71% and 77%), respectively (Supplemental Figure 1A), except S100β (20% and 32%), a soluble protein associated with harmful reactivation of astrocytes (22). A few (0.24% ± 0.35% in Ctx-Ast and 0.43% ± 0.39% in VM-Ast, where Ast indicates astrocytes) and none of the cells in these c...
The steroid receptor-type transcription factor Nurr1 has a crucial role in the development of the mesencephalic dopamine (DA) neurons. Although ectopic expression of Nurr1 in cultured neural precursor cells is sufficient in establishing the DA phenotype, Nurr1-induced DA cells are morphologically and functionally immature, suggesting the necessity of additional factor(s) for full neuronal differentiation. In this study, we demonstrate that neurogenic basic helix-loop-helix (bHLH) factors Mash1, neurogenins (Ngns) and NeuroD play contrasting roles in Nurr1-induced DA neuronal differentiation. Mash1, but not Ngn2, spatially and temporally colocalized with aldehyde dehydrogenase 2 (AHD2), a specific midbrain DA neuronal progenitor marker, in the early embryonic ventral mesencephalon. Forced expression of Mash1 caused immature Nurr1-induced DA cells to differentiate into mature and functional DA neurons as judged by electrophysiological characteristics, release of DA, and expression of presynaptic DA neuronal markers. By contrast, atonal-related bHLHs, represented by Ngn1, Ngn2 and NeuroD, repressed Nurr1-induced expression of DA neuronal markers. Domain-swapping experiments with Mash1 and NeuroD indicated that the helix-loop-helix domain, responsible for mediating dimerization of bHLH transcription factors, imparts the distinct effect. Finally, transient co-transfection of the atonal-related bHLHs with Nurr1 resulted in an E-box-independent repression of Nurr1-induced transcriptional activation of a reporter containing Nurr1-binding element (NL3) as well as a reporter driven by the native tyrosine hydroxylase gene promoter. Taken together, these findings suggest that Mash1 contributes to the generation of DA neurons in cooperation with Nurr1 in the developing midbrain whereas atonal-related bHLH genes inhibit the process.
Loss-of-function mutations of DNAJC6, encoding HSP40 auxilin, have recently been identified in patients with early-onset Parkinson’s disease (PD). To study the roles of DNAJC6 in PD pathogenesis, we used human embryonic stem cells with CRISPR-Cas9–mediated gene editing. Here, we show that DNAJC6 mutations cause key PD pathologic features, i.e., midbrain-type dopamine (mDA) neuron degeneration, pathologic α-synuclein aggregation, increase of intrinsic neuronal firing frequency, and mitochondrial and lysosomal dysfunctions in human midbrain-like organoids (hMLOs). In addition, neurodevelopmental defects were also manifested in hMLOs carrying the mutations. Transcriptomic analyses followed by experimental validation revealed that defects in DNAJC6-mediated endocytosis impair the WNT-LMX1A signal during the mDA neuron development. Furthermore, reduced LMX1A expression during development caused the generation of vulnerable mDA neurons with the pathologic manifestations. These results suggest that the human model of DNAJC6-PD recapitulates disease phenotypes and reveals mechanisms underlying disease pathology, providing a platform for assessing therapeutic interventions.
Use of the physiological mechanisms promoting midbrain DA (mDA) neuron survival seems an appropriate option for developing treatments for Parkinson's disease (PD). mDA neurons are specifically marked by expression of the transcription factors Nurr1 and Foxa2. We show herein that Nurr1 and Foxa2 interact to protect mDA neurons against various toxic insults, but their expression is lost during aging and degenerative processes. In addition to their proposed cell-autonomous actions in mDA neurons, forced expression of these factors in neighboring glia synergistically protects degenerating mDA neurons in a paracrine mode. As a consequence of these bimodal actions, adeno-associated virus (AAV)-mediated gene delivery of Nurr1 and Foxa2 in a PD mouse model markedly protected mDA neurons and motor behaviors associated with nigrostriatal DA neurotransmission. The effects of the combined gene delivery were dramatic, highly reproducible, and sustained for at least 1 year, suggesting that expression of these factors is a promising approach in PD therapy.
Background: Mutation of PTEN-induced putative kinase 1 (PINK1) causes autosomal recessive early-onset Parkinson's disease (PD). Despite of its ubiquitous expression in brain, its roles in non-neuronal cells such as neural stem cells (NSCs) and astrocytes were poorly unknown.
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