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1.
Cell Stem Cell ; 25(4): 514-530.e8, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31543366

RESUMO

Cellular senescence is a mechanism used by mitotic cells to prevent uncontrolled cell division. As senescent cells persist in tissues, they cause local inflammation and are harmful to surrounding cells, contributing to aging. Generally, neurodegenerative diseases, such as Parkinson's, are disorders of aging. The contribution of cellular senescence to neurodegeneration is still unclear. SATB1 is a DNA binding protein associated with Parkinson's disease. We report that SATB1 prevents cellular senescence in post-mitotic dopaminergic neurons. Loss of SATB1 causes activation of a cellular senescence transcriptional program in dopamine neurons both in human stem cell-derived dopaminergic neurons and in mice. We observed phenotypes that are central to cellular senescence in SATB1 knockout dopamine neurons in vitro and in vivo. Moreover, we found that SATB1 directly represses expression of the pro-senescence factor p21 in dopaminergic neurons. Our data implicate senescence of dopamine neurons as a contributing factor in the pathology of Parkinson's disease.

3.
Cell Stem Cell ; 25(1): 120-136.e10, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31155483

RESUMO

Current challenges in capturing naive human pluripotent stem cells (hPSCs) suggest that the factors regulating human naive versus primed pluripotency remain incompletely defined. Here we demonstrate that the widely used Essential 8 minimal medium (E8) captures hPSCs at a naive-to-primed intermediate state of pluripotency expressing several naive-like developmental, bioenergetic, and epigenomic features despite providing primed-state-sustaining growth factor conditions. Transcriptionally, E8 hPSCs are marked by activated lipid biosynthesis and suppressed MAPK/TGF-ß gene expression, resulting in endogenous ERK inhibition. These features are dependent on lipid-free culture conditions and are lost upon lipid exposure, whereas short-term pharmacological ERK inhibition restores naive-to-primed intermediate traits even in the presence of lipids. Finally, we identify de novo lipogenesis as a common transcriptional signature of E8 hPSCs and the pre-implantation human epiblast in vivo. These findings implicate exogenous lipid availability in regulating human pluripotency and define E8 hPSCs as a stable, naive-to-primed intermediate (NPI) pluripotent state.

4.
Nat Biotechnol ; 37(4): 436-444, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936566

RESUMO

Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.


Assuntos
Proteínas Hedgehog/metabolismo , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Animais , Biotecnologia , Padronização Corporal , Diferenciação Celular , Colesterol/metabolismo , Humanos , Camundongos , Modelos Neurológicos , Células-Tronco Neurais/metabolismo , Neurogênese , Organoides/citologia , Células-Tronco Pluripotentes/metabolismo , Prosencéfalo/citologia , Transdução de Sinais
5.
Nat Biomed Eng ; 3(7): 571-582, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30962586

RESUMO

Patient-specific human-induced pluripotent stem cells (hiPSCs) hold great promise for the modelling of genetic disorders. However, these cells display wide intra- and interindividual variations in gene expression, which makes distinguishing true-positive and false-positive phenotypes challenging. Data from hiPSC phenotypes and human embryonic stem cells (hESCs) harbouring the same disease mutation are also lacking. Here, we report a comparison of the molecular, cellular and functional characteristics of three congruent patient-specific cell types-hiPSCs, hESCs and direct-lineage-converted cells-derived from currently available differentiation and direct-reprogramming technologies for use in the modelling of Charcot-Marie-Tooth 1A, a human genetic Schwann-cell disorder featuring a 1.4 Mb chromosomal duplication. We find that the chemokines C-X-C motif ligand chemokine-1 (CXCL1) and macrophage chemoattractant protein-1 (MCP1) are commonly upregulated in all three congruent models and in clinical patient samples. The development of congruent models of a single genetic disease using somatic cells from a common patient will facilitate the search for convergent phenotypes.

6.
Nat Protoc ; 14(4): 1261-1279, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30911172

RESUMO

The enteric nervous system (ENS) represents a vast network of neuronal and glial cell types that develops entirely from migratory neural crest (NC) progenitor cells. Considerable improvements in the understanding of the molecular mechanisms underlying NC induction and regional specification have recently led to the development of a robust method to re-create the process in vitro using human pluripotent stem cells (hPSCs). Directing the fate of hPSCs toward the enteric NC (ENC) results in an accessible and scalable in vitro model of ENS development. The application of hPSC-derived enteric neural lineages provides a powerful platform for ENS-related disease modeling and drug discovery. Here we present a detailed protocol for the induction of a regionally specific NC intermediate that occurs over the course of a 15-d interval and is an effective source for the in vitro derivation of functional enteric neurons (ENs) from hPSCs. Additionally, we introduce a new and improved protocol that we have developed to optimize the protocol for future applications in regenerative medicine, in which components of undefined activity have been replaced with fully defined culture conditions. This protocol provides access to a broad range of human ENS lineages within a 30-d period.


Assuntos
Técnicas de Cultura de Células , Sistema Nervoso Entérico/citologia , Intestino Delgado/citologia , Crista Neural/citologia , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula/fisiologia , Sistema Nervoso Entérico/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Intestino Delgado/metabolismo , Crista Neural/metabolismo , Neurônios/metabolismo , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Células-Tronco Pluripotentes/metabolismo , Medicina Regenerativa/métodos
7.
Nat Biotechnol ; 37(3): 267-275, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30804533

RESUMO

The mechanistic basis of gliogenesis, which occurs late in human development, is poorly understood. Here we identify nuclear factor IA (NFIA) as a molecular switch inducing human glial competency. Transient expression of NFIA is sufficient to trigger glial competency of human pluripotent stem cell-derived neural stem cells within 5 days and to convert these cells into astrocytes in the presence of glial-promoting factors, as compared to 3-6 months using current protocols. NFIA-induced astrocytes promote synaptogenesis, exhibit neuroprotective properties, display calcium transients in response to appropriate stimuli and engraft in the adult mouse brain. Differentiation involves rapid but reversible chromatin remodeling, glial fibrillary acidic protein (GFAP) promoter demethylation and a striking lengthening of the G1 cell cycle phase. Genetic or pharmacological manipulation of G1 length partially mimics NFIA function. We used the approach to generate astrocytes with region-specific or reactive features. Our study defines key mechanisms of the gliogenic switch and enables the rapid production of human astrocytes for disease modeling and regenerative medicine.


Assuntos
Diferenciação Celular/genética , Fatores de Transcrição NFI/genética , Neurogênese/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Astrócitos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Fatores de Transcrição NFI/metabolismo , Células-Tronco Neurais/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/metabolismo , Regiões Promotoras Genéticas
8.
Nat Commun ; 9(1): 4815, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30446643

RESUMO

Common disorders, including diabetes and Parkinson's disease, are caused by a combination of environmental factors and genetic susceptibility. However, defining the mechanisms underlying gene-environment interactions has been challenging due to the lack of a suitable experimental platform. Using pancreatic ß-like cells derived from human pluripotent stem cells (hPSCs), we discovered that a commonly used pesticide, propargite, induces pancreatic ß-cell death, a pathological hallmark of diabetes. Screening a panel of diverse hPSC-derived cell types we extended this observation to a similar susceptibility in midbrain dopamine neurons, a cell type affected in Parkinson's disease. We assessed gene-environment interactions using isogenic hPSC lines for genetic variants associated with diabetes and Parkinson's disease. We found GSTT1-/- pancreatic ß-like cells and dopamine neurons were both hypersensitive to propargite-induced cell death. Our study identifies an environmental chemical that contributes to human ß-cell and dopamine neuron loss and validates a novel hPSC-based platform for determining gene-environment interactions.

9.
Nat Commun ; 9(1): 4345, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30341316

RESUMO

Environmental and genetic risk factors contribute to Parkinson's Disease (PD) pathogenesis and the associated midbrain dopamine (mDA) neuron loss. Here, we identify early PD pathogenic events by developing methodology that utilizes recent innovations in human pluripotent stem cells (hPSC) and chemical sensors of HSP90-incorporating chaperome networks. We show that events triggered by PD-related genetic or toxic stimuli alter the neuronal proteome, thereby altering the stress-specific chaperome networks, which produce changes detected by chemical sensors. Through this method we identify STAT3 and NF-κB signaling activation as examples of genetic stress, and phospho-tyrosine hydroxylase (TH) activation as an example of toxic stress-induced pathways in PD neurons. Importantly, pharmacological inhibition of the stress chaperome network reversed abnormal phospho-STAT3 signaling and phospho-TH-related dopamine levels and rescued PD neuron viability. The use of chemical sensors of chaperome networks on hPSC-derived lineages may present a general strategy to identify molecular events associated with neurodegenerative diseases.

10.
Genes Dev ; 32(17-18): 1161-1174, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30115631

RESUMO

Alternative splicing (AS) plays important roles in embryonic stem cell (ESC) differentiation. In this study, we first identified transcripts that display specific AS patterns in pluripotent human ESCs (hESCs) relative to differentiated cells. One of these encodes T-cell factor 3 (TCF3), a transcription factor that plays important roles in ESC differentiation. AS creates two TCF3 isoforms, E12 and E47, and we identified two related splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNPs) H1 and F (hnRNP H/F), that regulate TCF3 splicing. We found that hnRNP H/F levels are high in hESCs, leading to high E12 expression, but decrease during differentiation, switching splicing to produce elevated E47 levels. Importantly, hnRNP H/F knockdown not only recapitulated the switch in TCF3 AS but also destabilized hESC colonies and induced differentiation. Providing an explanation for this, we show that expression of known TCF3 target E-cadherin, critical for maintaining ESC pluripotency, is repressed by E47 but not by E12.


Assuntos
Processamento Alternativo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Caderinas/metabolismo , Células-Tronco Embrionárias/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Antígenos CD , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caderinas/genética , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/citologia , Éxons , Regulação da Expressão Gênica , Humanos , Precursores de RNA/química , RNA Mensageiro/química , Sequências Reguladoras de Ácido Ribonucleico
11.
Dis Model Mech ; 11(9)2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-30061297

RESUMO

Transgenic animals are invaluable for modeling cancer genomics, but often require complex crosses of multiple germline alleles to obtain the desired combinations. Zebrafish models have advantages in that transgenes can be rapidly tested by mosaic expression, but typically lack spatial and temporal control of tumor onset, which limits their utility for the study of tumor progression and metastasis. To overcome these limitations, we have developed a method referred to as Transgene Electroporation in Adult Zebrafish (TEAZ). TEAZ can deliver DNA constructs with promoter elements of interest to drive fluorophores, oncogenes or CRISPR-Cas9-based mutagenic cassettes in specific cell types. Using TEAZ, we created a highly aggressive melanoma model via Cas9-mediated inactivation of Rb1 in the context of BRAFV600E in spatially constrained melanocytes. Unlike prior models that take ∼4 months to develop, we found that TEAZ leads to tumor onset in ∼7 weeks, and these tumors develop in fully immunocompetent animals. As the resulting tumors initiated at highly defined locations, we could track their progression via fluorescence, and documented deep invasion into tissues and metastatic deposits. TEAZ can be deployed to other tissues and cell types, such as the heart, with the use of suitable transgenic promoters. The versatility of TEAZ makes it widely accessible for rapid modeling of somatic gene alterations and cancer progression at a scale not achievable in other in vivo systems.

12.
Proc Natl Acad Sci U S A ; 115(37): E8775-E8782, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30154162

RESUMO

Herpes simplex virus type 1 (HSV-1) encephalitis (HSE) is the most common sporadic viral encephalitis in Western countries. Some HSE children carry inborn errors of the Toll-like receptor 3 (TLR3)-dependent IFN-α/ß- and -λ-inducing pathway. Induced pluripotent stem cell (iPSC)-derived cortical neurons with TLR3 pathway mutations are highly susceptible to HSV-1, due to impairment of cell-intrinsic TLR3-IFN immunity. In contrast, the contribution of cell-intrinsic immunity of human trigeminal ganglion (TG) neurons remains unclear. Here, we describe efficient in vitro derivation and purification of TG neurons from human iPSCs via a cranial placode intermediate. The resulting TG neurons are of sensory identity and exhibit robust responses to heat (capsaicin), cold (icilin), and inflammatory pain (ATP). Unlike control cortical neurons, both control and TLR3-deficient TG neurons were highly susceptible to HSV-1. However, pretreatment of control TG neurons with poly(I:C) induced the cells into an anti-HSV-1 state. Moreover, both control and TLR3-deficient TG neurons developed resistance to HSV-1 following pretreatment with IFN-ß but not IFN-λ. These data indicate that TG neurons are vulnerable to HSV-1 because they require preemptive stimulation of the TLR3 or IFN-α/ß receptors to induce antiviral immunity, whereas cortical neurons possess a TLR3-dependent constitutive resistance that is sufficient to block incoming HSV-1 in the absence of prior antiviral signals. The lack of constitutive resistance in TG neurons in vitro is consistent with their exploitation as a latent virus reservoir in vivo. Our results incriminate deficiencies in the constitutive TLR3-dependent response of cortical neurons in the pathogenesis of HSE.


Assuntos
Imunidade/imunologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Receptor 3 Toll-Like/metabolismo , Antivirais/farmacologia , Diferenciação Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Criança , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/fisiologia , Humanos , Imunidade/genética , Células-Tronco Pluripotentes Induzidas/citologia , Interferon beta/farmacologia , Mutação , Neurônios/efeitos dos fármacos , Neurônios/virologia , Poli I-C/farmacologia , Receptor 3 Toll-Like/genética , Gânglio Trigeminal/citologia
13.
Nat Mater ; 17(7): 633-641, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29784997

RESUMO

Classic embryological studies have successfully applied genetics and cell biology principles to understand embryonic development. However, it remains unresolved how mechanics, as an integral driver of development, is involved in controlling tissue-scale cell fate patterning. Here we report a micropatterned human pluripotent stem (hPS)-cell-based neuroectoderm developmental model, in which pre-patterned geometrical confinement induces emergent patterning of neuroepithelial and neural plate border cells, mimicking neuroectoderm regionalization during early neurulation in vivo. In this hPS-cell-based neuroectoderm patterning model, two tissue-scale morphogenetic signals-cell shape and cytoskeletal contractile force-instruct neuroepithelial/neural plate border patterning via BMP-SMAD signalling. We further show that ectopic mechanical activation and exogenous BMP signalling modulation are sufficient to perturb neuroepithelial/neural plate border patterning. This study provides a useful microengineered, hPS-cell-based model with which to understand the biomechanical principles that guide neuroectoderm patterning and hence to study neural development and disease.

14.
Cell ; 172(5): 952-965.e18, 2018 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-29474921

RESUMO

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.

15.
Proc Natl Acad Sci U S A ; 114(51): E10972-E10980, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29196523

RESUMO

Progeroid syndromes are rare genetic disorders that phenotypically resemble natural aging. Different causal mutations have been identified, but no molecular alterations have been identified that are in common to these diseases. DNA replication timing (RT) is a robust cell type-specific epigenetic feature highly conserved in the same cell types from different individuals but altered in disease. Here, we characterized DNA RT program alterations in Hutchinson-Gilford progeria syndrome (HGPS) and Rothmund-Thomson syndrome (RTS) patients compared with natural aging and cellular senescence. Our results identified a progeroid-specific RT signature that is common to cells from three HGPS and three RTS patients and distinguishes them from healthy individuals across a wide range of ages. Among the RT abnormalities, we identified the tumor protein p63 gene (TP63) as a gene marker for progeroid syndromes. By using the redifferentiation of four patient-derived induced pluripotent stem cells as a model for the onset of progeroid syndromes, we tracked the progression of RT abnormalities during development, revealing altered RT of the TP63 gene as an early event in disease progression of both HGPS and RTS. Moreover, the RT abnormalities in progeroid patients were associated with altered isoform expression of TP63 Our findings demonstrate the value of RT studies to identify biomarkers not detected by other methods, reveal abnormal TP63 RT as an early event in progeroid disease progression, and suggest TP63 gene regulation as a potential therapeutic target.


Assuntos
Período de Replicação do DNA , Progéria/genética , Idoso de 80 Anos ou mais , Biomarcadores , Criança , Fibroblastos/metabolismo , Expressão Gênica , Genômica/métodos , Humanos , Recém-Nascido , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Progéria/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética
16.
Cell Stem Cell ; 21(5): 569-573, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29100010

RESUMO

Stem cell-based therapies for Parkinson's disease are moving into a new and exciting era, with several groups pursuing clinical trials with pluripotent stem cell (PSC)-derived dopamine neurons. As many groups have ongoing or completed GMP-level cell manufacturing, we highlight key clinical translation considerations from our recent fourth GForce-PD meeting.


Assuntos
Ensaios Clínicos como Assunto , Neurônios Dopaminérgicos/transplante , Doença de Parkinson/terapia , Transplante de Células-Tronco , Adulto , Idoso , Humanos , Pessoa de Meia-Idade
17.
Cell Stem Cell ; 21(3): 399-410.e7, 2017 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-28886367

RESUMO

Directing the fate of human pluripotent stem cells (hPSCs) into different lineages requires variable starting conditions and components with undefined activities, introducing inconsistencies that confound reproducibility and assessment of specific perturbations. Here we introduce a simple, modular protocol for deriving the four main ectodermal lineages from hPSCs. By precisely varying FGF, BMP, WNT, and TGFß pathway activity in a minimal, chemically defined medium, we show parallel, robust, and reproducible derivation of neuroectoderm, neural crest (NC), cranial placode (CP), and non-neural ectoderm in multiple hPSC lines, on different substrates independently of cell density. We highlight the utility of this system by interrogating the role of TFAP2 transcription factors in ectodermal differentiation, revealing the importance of TFAP2A in NC and CP specification, and performing a small-molecule screen that identified compounds that further enhance CP differentiation. This platform provides a simple stage for systematic derivation of the entire range of ectodermal cell types.


Assuntos
Diferenciação Celular , Linhagem da Célula , Ectoderma/citologia , Células-Tronco Pluripotentes/citologia , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Crista Neural/citologia , Placa Neural/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Fenantrolinas/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Transcrição AP-2/metabolismo
18.
Cell Stem Cell ; 21(2): 274-283.e5, 2017 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-28736217

RESUMO

Zika virus (ZIKV) infects fetal and adult human brain and is associated with serious neurological complications. To date, no therapeutic treatment is available to treat ZIKV-infected patients. We performed a high-content chemical screen using human pluripotent stem cell-derived cortical neural progenitor cells (hNPCs) and found that hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ) can inhibit ZIKV infection in hNPCs. Further validation showed that HH also rescues ZIKV-induced growth and differentiation defects in hNPCs and human fetal-like forebrain organoids. Finally, HH and AQ inhibit ZIKV infection in adult mouse brain in vivo. Strikingly, HH suppresses viral propagation when administered to adult mice with active ZIKV infection, highlighting its therapeutic potential. Our approach highlights the power of stem cell-based screens and validation in human forebrain organoids and mouse models in identifying drug candidates for treating ZIKV infection and related neurological complications in fetal and adult patients.


Assuntos
Antivirais/uso terapêutico , Encéfalo/virologia , Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Organoides/virologia , Infecção por Zika virus/tratamento farmacológico , Zika virus/fisiologia , Adolescente , Alcaloides de Amaryllidaceae/farmacologia , Amodiaquina/farmacologia , Animais , Antivirais/farmacologia , Linhagem Celular , Criança , Feminino , Feto/efeitos dos fármacos , Feto/virologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos SCID , Células-Tronco Neurais/efeitos dos fármacos , Organoides/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Infecção por Zika virus/patologia
19.
Prog Brain Res ; 230: 191-212, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28552229

RESUMO

Over the last 10 years, there has been significant progress in defining culture conditions to derive bona fide human midbrain dopamine (mDA) neurons from human embryonic stem cells or from human-induced pluripotent stem cells, two cell sources referred to as human pluripotent stem cells (hPSCs). Those developments have made it possible to manufacture mDA neurons with at sufficient scale and precision to contemplate their use in cell replacement therapy for the treatment of Parkinson's disease. Our group is one of the several teams that are in the process of initiating the first human clinical trials based on the use of mDA neurons derived from hPSCs. With support from the NY state stem cell program (NYSTEM), we have implemented protocols for deriving mDA neurons under current good manufacturing practice-compliant conditions for regulatory approval of the cell-based product for human applications. We have been able to demonstrate that in vitro-derived mDA neurons can be generated under defined conditions and at large scale; that they can be cryopreserved prior to transplantation; and that the cryopreserved product is capable of reversing PD symptoms in rodent models of PD. We have further demonstrated the ability of the cells to engraft in the brain of PD monkeys and defined factors such as the shelf-life of the cells prior to and the viability of the cells after thawing and observed a lack of tumorigenic cells in the preparation. The final steps prior to initiating human trails include extensive safety studies using the fully qualified and cryopreserved mDA neuron products to get data from the exactly same batch of cells to be used for early-stage human studies. Our work sets the stage for developing an off-the-shelf cell therapy for Parkinson's disease that may develop into a valid therapeutic option for PD patients in the future.


Assuntos
Neurônios Dopaminérgicos/citologia , Doença de Parkinson/terapia , Células-Tronco Pluripotentes/citologia , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mesencéfalo/citologia
20.
Stem Cell Reports ; 8(2): 191-193, 2017 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-28199825

RESUMO

As stem cell products are increasingly entering early stage clinical trials, we are learning from experience about how cell products may be best assessed for safety and efficacy. In two papers published in this issue of Stem Cell Reports, a human neural stem cell product, HuCNS-SC, failed to demonstrate efficacy in central nervous system repair in two different animal models (Anderson et al., 2017; Marsh et al., 2017), although closely related research-grade cell products showed evidence of efficacy. This indicates the need for increased cell characterization to determine comparability of lots proposed for pre-clinical and clinical use. Without such improvements, pre-clinical data supporting a clinical study might not adequately reflect the performance of subsequent batches of cells intended for use in patients.

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