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Journal ArticleDOI

Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease

TL;DR: A novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo is presented, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells.
Abstract: Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinson's disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinson's disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson's disease.

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Citations
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Journal ArticleDOI
26 Jan 2017-Nature
TL;DR: It is shown that activated microglia induce A1 astrocytes by secreting Il-1α, TNF and C1q, and that these cytokines together are necessary and sufficient to induce A2 astroCytes, which are abundant in various human neurodegenerative diseases.
Abstract: This work was supported by grants from the National Institutes of Health (R01 AG048814, B.A.B.; RO1 DA15043, B.A.B.; P50 NS38377, V.L.D. and T.M.D.) Christopher and Dana Reeve Foundation (B.A.B.), the Novartis Institute for Biomedical Research (B.A.B.), Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (B.A.B.), the JPB Foundation (B.A.B., T.M.D.), the Cure Alzheimer’s Fund (B.A.B.), the Glenn Foundation (B.A.B.), the Esther B O’Keeffe Charitable Foundation (B.A.B.), the Maryland Stem Cell Research Fund (2013-MSCRFII-0105-00, V.L.D.; 2012-MSCRFII-0268-00, T.M.D.; 2013-MSCRFII-0105-00, T.M.D.; 2014-MSCRFF-0665, M.K.). S.A.L. was supported by a postdoctoral fellowship from the Australian National Health and Medical Research Council (GNT1052961), and the Glenn Foundation Glenn Award. L.E.C. was funded by a Merck Research Laboratories postdoctoral fellowship (administered by the Life Science Research Foundation). W.-S.C. was supported by a career transition grant from NEI (K99EY024690). C.J.B. was supported by a postdoctoral fellowship from Damon Runyon Cancer Research Foundation (DRG-2125-12). L.S. was supported by a postdoctoral fellowship from the German Research Foundation (DFG, SCHI 1330/1-1).

4,326 citations

Journal ArticleDOI
03 Jul 2013-Nature
TL;DR: This is the first report demonstrating the generation of a functional human organ from pluripotent stem cells by transplantation of liver buds created in vitro (iPSC-LBs), and provides a promising new approach to study regenerative medicine.
Abstract: A critical shortage of donor organs for treating end-stage organ failure highlights the urgent need for generating organs from human induced pluripotent stem cells (iPSCs). Despite many reports describing functional cell differentiation, no studies have succeeded in generating a three-dimensional vascularized organ such as liver. Here we show the generation of vascularized and functional human liver from human iPSCs by transplantation of liver buds created in vitro (iPSC-LBs). Specified hepatic cells (immature endodermal cells destined to track the hepatic cell fate) self-organized into three-dimensional iPSC-LBs by recapitulating organogenetic interactions between endothelial and mesenchymal cells. Immunostaining and gene-expression analyses revealed a resemblance between in vitro grown iPSC-LBs and in vivo liver buds. Human vasculatures in iPSC-LB transplants became functional by connecting to the host vessels within 48 hours. The formation of functional vasculatures stimulated the maturation of iPSC-LBs into tissue resembling the adult liver. Highly metabolic iPSC-derived tissue performed liver-specific functions such as protein production and human-specific drug metabolism without recipient liver replacement. Furthermore, mesenteric transplantation of iPSC-LBs rescued the drug-induced lethal liver failure model. To our knowledge, this is the first report demonstrating the generation of a functional human organ from pluripotent stem cells. Although efforts must ensue to translate these techniques to treatments for patients, this proof-of-concept demonstration of organ-bud transplantation provides a promising new approach to study regenerative medicine.

1,653 citations

Journal ArticleDOI
09 Oct 2014-Cell
TL;DR: A scalable differentiation protocol is reported that can generate hundreds of millions of glucose-responsive β cells from hPSC in vitro that secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice.

1,596 citations


Cites background from "Dopamine neurons derived from human..."

  • ...Recent research has moved the stem cell field closer to that goal through development of strategies to generate cells that would otherwise be difficult to obtain, like neurons or cardiomyocytes (Kriks et al., 2011; Shiba et al., 2012; Son et al., 2011)....

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Journal ArticleDOI
19 May 2016-Cell
TL;DR: A miniaturized spinning bioreactor (SpinΩ) is developed to generate forebrain-specific organoids from human iPSCs that recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a distinct human-specific outer radial glia cell layer.

1,526 citations


Cites methods from "Dopamine neurons derived from human..."

  • ...Building upon a 2D differentiation protocol for generating midbrain dopaminergic (DA) neurons (Kriks et al., 2011), we applied Sonic hedgehog (SHH) agonists (recombinant SHH and Purmorphamine), FGF-8, SMAD inhibitors (SB431542 and LDN193189), and GSK3b inhibitor (CHIR99021) to induce floor-plate differentiation of human iPSCs, which were transferred to SpinU at day 14 (Figure 6A)....

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  • ...Building upon a 2D differentiation protocol for generating midbrain dopaminergic (DA) neurons (Kriks et al., 2011), we applied Sonic Hedgehog (SHH) agonists (recombinant SHH and Purmorphamine), FGF-8, SMAD inhibitors (SB431542 and LDN193189), and GSK3β inhibitor (CHIR99021) to induce floor plate…...

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Journal ArticleDOI
05 Jun 2013-Neuron
TL;DR: It is shown that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor.

1,111 citations


Cites background from "Dopamine neurons derived from human..."

  • ...…to examine specific subtypes of neurons, to generate human neurons for regenerative medicine, and to investigate changes in human neurons in neuropsychiatric disorders (e.g., see Cho et al., 2008; Fasano et al., 2010; Kriks et al., 2011; Shi et al., 2012; Chambers et al., 2012; Ma et al., 2012)....

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  • ...from Human Pluripotent Stem Cells...

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  • ...Kriks et al., 2011 22056989 NPCs 4 weeks ND ND 40–70...

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References
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Journal ArticleDOI
TL;DR: By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.
Abstract: DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The procedure first requires uploading a gene list containing any number of common gene identifiers followed by analysis using one or more text and pathway-mining tools such as gene functional classification, functional annotation chart or clustering and functional annotation table. By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.

31,015 citations

Journal ArticleDOI
TL;DR: Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.
Abstract: Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient to induce rapid and complete neural conversion of >80% of hES cells under adherent culture conditions. Temporal fate analysis reveals the appearance of a transient FGF5(+) epiblast-like stage followed by PAX6(+) neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.

3,152 citations

Journal ArticleDOI
04 Dec 2008-Nature
TL;DR: Modifications to xenotransplantation assays can dramatically increase the detectable frequency of tumorigenic cells, demonstrating that they are common in some human cancers.
Abstract: NOD/SCID interleukin-2 receptor gamma chain null (Il2rg 2/2 ) mice, can increase the detection of tumorigenic melanoma cells by several orders of magnitude. In limiting dilution assays, approximately 25% of unselected melanoma cells from 12 different patients, including cells from primary and metastatic melanomas obtained directly from patients, formed tumours under these more permissive conditions. In single-cell transplants, an average of 27% of unselected melanoma cells from fourdifferentpatientsformedtumours.Modificationstoxenotransplantationassayscanthereforedramaticallyincreasethe detectable frequency of tumorigenic cells, demonstrating that they are common in some human cancers.

1,666 citations

Journal ArticleDOI
06 Mar 2009-Cell
TL;DR: In this paper, the authors showed that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons using Cre-recombinase excisable viruses.

1,554 citations

Journal ArticleDOI
27 Oct 2000-Science
TL;DR: Data indicate that GDNF delivery using a lentiviral vector system can prevent nigrostriatal degeneration and induce regeneration in primate models of PD and might be a viable therapeutic strategy for PD patients.
Abstract: Lentiviral delivery of glial cell line-derived neurotrophic factor (lenti-GDNF) was tested for its trophic effects upon degenerating nigrostriatal neurons in nonhuman primate models of Parkinson's disease (PD). We injected lenti-GDNF into the striatum and substantia nigra of nonlesioned aged rhesus monkeys or young adult rhesus monkeys treated 1 week prior with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Extensive GDNF expression with anterograde and retrograde transport was seen in all animals. In aged monkeys, lenti-GDNF augmented dopaminergic function. In MPTP-treated monkeys, lenti-GDNF reversed functional deficits and completely prevented nigrostriatal degeneration. Additionally, lenti-GDNF injections to intact rhesus monkeys revealed long-term gene expression (8 months). In MPTP-treated monkeys, lenti-GDNF treatment reversed motor deficits in a hand-reach task. These data indicate that GDNF delivery using a lentiviral vector system can prevent nigrostriatal degeneration and induce regeneration in primate models of PD and might be a viable therapeutic strategy for PD patients.

1,254 citations


"Dopamine neurons derived from human..." refers methods in this paper

  • ...Primates: two adult (17–18 year old; 10–12 kg; female) rhesus monkeys were rendered hemiparkinsonian via carotid MPTP administration followed by weekly intravenous MPTP administration to create a bilateral parkinsonian syndrom...

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