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1.
Elife ; 92020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32011235

RESUMO

Generation of skeletal muscle cells with human pluripotent stem cells (hPSCs) opens new avenues for deciphering essential, but poorly understood aspects of transcriptional regulation in human myogenic specification. In this study, we characterized the transcriptional landscape of distinct human myogenic stages, including OCT4::EGFP+ pluripotent stem cells, MSGN1::EGFP+ presomite cells, PAX7::EGFP+ skeletal muscle progenitor cells, MYOG::EGFP+ myoblasts, and multinucleated myotubes. We defined signature gene expression profiles from each isolated cell population with unbiased clustering analysis, which provided unique insights into the transcriptional dynamics of human myogenesis from undifferentiated hPSCs to fully differentiated myotubes. Using a knock-out strategy, we identified TWIST1 as a critical factor in maintenance of human PAX7::EGFP+ putative skeletal muscle progenitor cells. Our data revealed a new role of TWIST1 in human skeletal muscle progenitors, and we have established a foundation to identify transcriptional regulations of human myogenic ontogeny (online database can be accessed in http://www.myogenesis.net/).

2.
Exp Neurol ; 323: 113086, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31639376

RESUMO

Muscular dystrophies are a group of genetic muscle disorders that cause progressive muscle weakness and degeneration. Within this group, Duchenne muscular dystrophy (DMD) is the most common and one of the most severe. DMD is an X chromosome linked disease that occurs to 1 in 3500 to 1 in 5000 boys. The cause of DMD is a mutation in the dystrophin gene, whose encoded protein provides both structural support and cell signaling capabilities. So far, there are very limited therapeutic options available and there is no cure for this disease. In this review, we discuss the existing cell therapy research, especially stem cell-based, which utilize myoblasts, satellite cells, bone marrow cells, mesoangioblasts and CD133+ cells. Finally, we focus on human pluripotent stem cells (hPSCs) which hold great potential in treating DMD. hPSCs can be used for autologous transplantation after being specified to a myogenic lineage. Over the last few years, there has been a rapid development of isolation, as well as differentiation, techniques in order to achieve effective transplantation results of myogenic cells specified from hPSCs. In this review, we summarize the current methods of hPSCs myogenic commitment/differentiation, and describe the current status of hPSC-derived myogenic cell transplantation.

3.
Stem Cells Transl Med ; 8(12): 1272-1285, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31631575

RESUMO

The ability to generate human-induced pluripotent stem cell (hiPSC)-derived neural cells displaying region-specific phenotypes is of particular interest for modeling central nervous system biology in vitro. We describe a unique method by which spinal cord hiPSC-derived astrocytes (hiPSC-A) are cultured with spinal cord hiPSC-derived motor neurons (hiPSC-MN) in a multielectrode array (MEA) system to record electrophysiological activity over time. We show that hiPSC-A enhance hiPSC-MN electrophysiological maturation in a time-dependent fashion. The sequence of plating, density, and age in which hiPSC-A are cocultured with MN, but not their respective hiPSC line origin, are factors that influence neuronal electrophysiology. When compared to coculture with mouse primary spinal cord astrocytes, we observe an earlier and more robust electrophysiological maturation in the fully human cultures, suggesting that the human origin is relevant to the recapitulation of astrocyte/motor neuron crosstalk. Finally, we test pharmacological compounds on our MEA platform and observe changes in electrophysiological activity, which confirm hiPSC-MN maturation. These findings are supported by immunocytochemistry and real-time PCR studies in parallel cultures demonstrating human astrocyte mediated changes in the structural maturation and protein expression profiles of the neurons. Interestingly, this relationship is reciprocal and coculture with neurons influences astrocyte maturation as well. Taken together, these data indicate that in a human in vitro spinal cord culture system, astrocytes support hiPSC-MN maturation in a time-dependent and species-specific manner and suggest a closer approximation of in vivo conditions. Stem Cells Translational Medicine 2019;8:1272&1285.

4.
Neuron ; 103(4): 627-641.e7, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31255487

RESUMO

Analysis of human pathology led Braak to postulate that α-synuclein (α-syn) pathology could spread from the gut to brain via the vagus nerve. Here, we test this postulate by assessing α-synucleinopathy in the brain in a novel gut-to-brain α-syn transmission mouse model, where pathological α-syn preformed fibrils were injected into the duodenal and pyloric muscularis layer. Spread of pathologic α-syn in brain, as assessed by phosphorylation of serine 129 of α-syn, was observed first in the dorsal motor nucleus, then in caudal portions of the hindbrain, including the locus coeruleus, and much later in basolateral amygdala, dorsal raphe nucleus, and the substantia nigra pars compacta. Moreover, loss of dopaminergic neurons and motor and non-motor symptoms were observed in a similar temporal manner. Truncal vagotomy and α-syn deficiency prevented the gut-to-brain spread of α-synucleinopathy and associated neurodegeneration and behavioral deficits. This study supports the Braak hypothesis in the etiology of idiopathic Parkinson's disease (PD).


Assuntos
Transporte Axonal , Transtornos Parkinsonianos/etiologia , Agregados Proteicos , Nervo Vago/metabolismo , alfa-Sinucleína/farmacocinética , Animais , Química Encefálica , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Duodeno/inervação , Duodeno/metabolismo , Humanos , Injeções Intramusculares , Corpos de Lewy/metabolismo , Aprendizagem em Labirinto , Transtornos da Memória/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Neurológicos , Músculo Liso/inervação , Músculo Liso/metabolismo , Comportamento de Nidação/fisiologia , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/prevenção & controle , Transtornos Parkinsonianos/psicologia , Fosforilação , Processamento de Proteína Pós-Traducional , Piloro/inervação , Piloro/metabolismo , Teste de Desempenho do Rota-Rod , Vagotomia , alfa-Sinucleína/administração & dosagem , alfa-Sinucleína/deficiência , alfa-Sinucleína/toxicidade
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.
Theriogenology ; 129: 70-76, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30825707

RESUMO

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-ß family and a physiological regulator. According to recent studies, GDF8 can be detected in follicular fluid and the uterus, suggesting that GDF8 may affect preimplantation embryonic development and act in a paracrine manner to improve the success of late-blastocyst implantation in vivo. We investigated the effect of GDF8 supplementation during in vitro culture (IVC) of porcine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) on cleavage, blastocyst formation rate, and total cell number and analysed gene transcription levels and cell linage specification in the resulting blastocysts. First, the concentration of GDF8 in porcine oviductal fluid was determined to be 139.8 pg/mL. Then, 0, 0.2, 2, or 20 ng/mL GDF8 was added to embryos throughout the entire IVC period. Our results showed that supplementation with GDF8 during porcine preimplantation embryo IVC enhanced blastocyst formation and total cell number and altered the transcriptional patterns of genes that regulate pluripotency and cavitation. Furthermore, using differential immunostaining, we demonstrated that supplementation with GDF8 enhanced the expression of the genuine inner cell mass (ICM) marker SOX2 and the ICM/trophectoderm ratio, improving IVF blastocyst quality. In conclusion, for the first time, we demonstrated the presence of the in vivo oviductal factor GDF8 in oviductal fluid. Furthermore, we found that GDF8 supplementation at 0.2 ng/mL increased the blastocyst total cell number and ICM/trophectoderm ratio by inducing the transcription of genes involved in developmental competence and the expression of genuine ICM marker SOX2 during porcine IVF embryo development in vitro.


Assuntos
Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacos , Miostatina/farmacologia , Suínos/embriologia , Animais , Biomarcadores/metabolismo , Linhagem da Célula , Técnicas de Cultura Embrionária/métodos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/veterinária , Suínos/metabolismo
7.
J Cell Physiol ; 2019 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-30779347

RESUMO

Canine mesenchymal stem cells (cMSCs) are gaining popularity in the veterinary field as a regenerative therapy. But, their limited culture lifespan makes it an obstacle for preclinical investigation and therapeutic use. In this study, primary canine adipose tissue-derived MSCs (PCAT-MSCs) were isolated from adipose tissue and were transfected with the SV40-T retrovirus resulting in a life-extended immortalized canine adipose tissue-derived MSCs (ICAT-MSCs). A comparison was made through the characterization of both PCAT-MSCs and ICAT-MSCs. Both showed a fibroblastic morphology; ICAT-MSCs showed a higher potential of colony formation compared with PCAT-MSCs and a reduced population doubling time; stem cell markers SOX2 and NANOG were expressed in both cell lines; karyotyping analysis showed no abnormalities in both PCAT-MSCs and ICAT-MSCs; both cell lines were CD90+ , CD44 + , and CD45 - ; both generated chondrogenic pellet; in osteogenic differentiation both showed upregulation of Osterix, a master transcriptome of osteogenesis, but in PCAT-MSCs, an upregulation of SOX2 was also observed. In conclusion, ICAT-MSCs showed similar characteristics with PCAT-MSCs, thus established as an easy to access platform for studies on better understanding about cMSCs nature.

8.
J Cell Mol Med ; 23(3): 2052-2063, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30609263

RESUMO

Prior to transplantation, preclinical study of safety and efficacy of neural progenitor cells (NPCs) is needed. Therefore, it is important to generate an efficient in vitro platform for neural cell differentiation in large animal models such as pigs. In this study, porcine-induced pluripotent stem cells (iPSCs) were seeded at high cell density to a neural induction medium containing the dual Sma- and Mad-related protein (SMAD) inhibitors, a TGF-ß inhibitor and BMP4 inhibitor. The dSMADi-derived NPCs showed NPC markers such as PLAG1, NESTIN and VIMENTIN and higher mRNA expression of Sox1 compared to the control. The mRNA expression of HOXB4 was found to significantly increase in the retinoic acid-treated group. NPCs propagated in vitro and generated neurospheres that are capable of further differentiation in neurons and glial cells. Gliobalstoma-cultured medium including injury-related cytokines treated porcine iPSC-NPCs survive well in vitro and showed more neuronal marker expression compared to standard control medium. Collectively, the present study developed an efficient method for production of neural commitment of porcine iPSCs into NPCs.

9.
BMC Vet Res ; 14(1): 331, 2018 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-30404643

RESUMO

BACKGROUND: The porcine brain is gyrencephalic with similar gray and white matter composition and size more comparable to the human rather than the rodent brain; however, there is lack of information about neural progenitor cells derived from this model. RESULTS: Here, we isolated GFAP-positive porcine neural stem cells (NSCs) from the brain explant of a transgenic piglet, with expression of CreERT2 under the control of the GFAP promoter (pGFAP-CreERT2). The isolated pGFAP-CreERT2 NSCs showed self-renewal and expression of representative NSC markers such as Nestin and Sox2. Pharmacological inhibition studies revealed that Notch1 signaling is necessary to maintain NSC identity, whereas serum treatment induced cell differentiation into reactive astrocytes and neurons. CONCLUSIONS: Collectively, these results indicate that GFAP promoter-driven porcine CreERT2 NSCs would be a useful tool to study neurogenesis of the porcine adult central nervous system and furthers our understanding of its potential clinical application in the future. ᅟ.


Assuntos
Proteína Glial Fibrilar Ácida/metabolismo , Células-Tronco Neurais/fisiologia , Suínos/anatomia & histologia , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Astrócitos/metabolismo , Diferenciação Celular , Suínos/genética
10.
Biomaterials ; 181: 347-359, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30098570

RESUMO

Peripheral nerve injuries often lead to incomplete recovery and contribute to significant disability to approximately 360,000 people in the USA each year. Stem cell therapy holds significant promise for peripheral nerve regeneration, but maintenance of stem cell viability and differentiation potential in vivo are still major obstacles for translation. Using a made-in-house 96-well vertical electrical stimulation (ES) platform, we investigated the effects of different stimulating pulse frequency, duration and field direction on human neural crest stem cell (NCSC) differentiation. We observed dendritic morphology with enhanced neuronal differentiation for NCSCs cultured on cathodes subject to 20 Hz, 100µs pulse at a potential gradient of 200 mV/mm. We further evaluated the effect of a novel cell-based therapy featuring optimized pulsatile ES of NCSCs for in vivo transplantation following peripheral nerve regeneration. 15 mm critical-sized sciatic nerve injuries were generated with subsequent surgical repair in sixty athymic nude rats. Injured animals were randomly assigned into five groups (N = 12 per group): blank control, ES, NCSC, NCSC + ES, and autologous nerve graft. The optimized ES was applied immediately after surgical repair for 1 h in ES and NCSC + ES groups. Recovery was assessed by behavioral (CatWalk gait analysis), wet muscle-mass, histomorphometric, and immunohistochemical analyses at either 6 or 12 weeks after surgery (N = 6 per group). Gastrocnemius muscle wet mass measurements in ES + NCSC group were comparable to autologous nerve transplantation and significantly higher than other groups (p < 0.05). Quantitative histomorphometric analysis and catwalk gait analysis showed similar improvements by ES on NCSCs (p < 0.05). A higher number of viable NCSCs was shown via immunochemical analysis, with higher Schwann cell (SC) differentiation in the NCSC + ES group compared to the NCSC group (p < 0.05). Overall, ES on NCSC transplantation significantly enhanced nerve regeneration after injury and repair, and was comparable to autograft treatment. Thus, ES can be a potent alternative to biochemical and physical cues for modulating stem cell survival and differentiation. This novel cell-based intervention presents an effective and safe approach for improved outcomes after peripheral nerve repair.


Assuntos
Regeneração Nervosa/fisiologia , Crista Neural/citologia , Traumatismos dos Nervos Periféricos/terapia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Estimulação Elétrica/métodos , Humanos , Imuno-Histoquímica , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Ratos , Transplante de Células-Tronco/métodos
11.
Theriogenology ; 113: 197-207, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29554602

RESUMO

Lysophosphatidic acid (LPA) is a phospholipid-derived signaling molecule with biological activities, such as stimulating cell proliferation, differentiation and migration. In the present study, we examined the effect of LPA on porcine oocytes during in vitro maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA) and in vitro fertilization (IVF). During IVM, the maturation medium was supplemented with various concentrations of LPA (0, 10, 30, and 60 µM). After 42 h of IVM, the 30 µM LPA-treated group showed a significant (P <0.05) increase in nuclear maturation and intracellular glutathione (GSH) levels compared with the other groups. The 30 µM LPA-treated group exhibited a significant decrease in intracellular reactive oxygen species (ROS) levels compared with the other groups. In PA, the 30 µM LPA-treated group had significantly higher cleavage (CL) and blastocyst (BL) rates compared with those of the other LPA-treated groups. In IVF, the 30 µM LPA-treated group had significantly higher CL and BL rates than the other LPA-treated groups. The expression of the developmental competence gene (proliferating cell nuclear antigen, PCNA) in the oocytes and cumulus cells of the individuals in the 30 µM LPA-treated group was significantly increased compared with the control group. In addition, the specific expression of urokinase Plasminogen Activator (uPA) and uPA Receptor (uPAR) in cumulus cells was significantly increased in the 30 µM LPA-treated group. The western blotting results revealed that LPA improves the activities of p38 mitogen-activated protein kinase (MAPK) and epidermal growth factor (EGF) by enhanced phosphorylation. In conclusion, treatment with 30 µM LPA during IVM promotes enhances the EGF-EGFR signaling pathway, resulting in cumulus cell expansion. And then, this treatment improves the developmental potential of PA and IVF porcine embryos by enhancing nuclear and cytoplasmic maturation and reducing ROS.


Assuntos
Células do Cúmulo/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Lisofosfolipídeos/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Suínos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Células do Cúmulo/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa , Partenogênese/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética
12.
Proc Natl Acad Sci U S A ; 115(4): 798-803, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29311330

RESUMO

Accumulating evidence suggests that α-synuclein (α-syn) occurs physiologically as a helically folded tetramer that resists aggregation. However, the mechanisms underlying the regulation of formation of α-syn tetramers are still mostly unknown. Cellular membrane lipids are thought to play an important role in the regulation of α-syn tetramer formation. Since glucocerebrosidase 1 (GBA1) deficiency contributes to the aggregation of α-syn and leads to changes in neuronal glycosphingolipids (GSLs) including gangliosides, we hypothesized that GBA1 deficiency may affect the formation of α-syn tetramers. Here, we show that accumulation of GSLs due to GBA1 deficiency decreases α-syn tetramers and related multimers and increases α-syn monomers in CRISPR-GBA1 knockout (KO) SH-SY5Y cells. Moreover, α-syn tetramers and related multimers are decreased in N370S GBA1 Parkinson's disease (PD) induced pluripotent stem cell (iPSC)-derived human dopaminergic (hDA) neurons and murine neurons carrying the heterozygous L444P GBA1 mutation. Treatment with miglustat to reduce GSL accumulation and overexpression of GBA1 to augment GBA1 activity reverse the destabilization of α-syn tetramers and protect against α-syn preformed fibril-induced toxicity in hDA neurons. Taken together, these studies provide mechanistic insights into how GBA1 regulates the transition from monomeric α-syn to α-syn tetramers and multimers and suggest unique therapeutic opportunities for PD and dementia with Lewy bodies.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Glucosilceramidase/deficiência , Glicoesfingolipídeos/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , 1-Desoxinojirimicina/análogos & derivados , Linhagem Celular Tumoral , Glucosilceramidase/genética , Humanos , Multimerização Proteica
13.
Nat Neurosci ; 20(9): 1209-1212, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28758997

RESUMO

Zika virus (ZIKV) infection is associated with neurological disorders of both the CNS and peripheral nervous systems (PNS), yet few studies have directly examined PNS infection. Here we show that intraperitoneally or intraventricularly injected ZIKV in the mouse can infect and impact peripheral neurons in vivo. Moreover, ZIKV productively infects stem-cell-derived human neural crest cells and peripheral neurons in vitro, leading to increased cell death, transcriptional dysregulation and cell-type-specific molecular pathology.


Assuntos
Morte Celular/fisiologia , Células-Tronco Neurais/patologia , Células-Tronco Neurais/virologia , Doenças do Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/virologia , Zika virus/fisiologia , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos ICR , Células Vero , Infecção por Zika virus/patologia
14.
J Virol ; 91(17)2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28637759

RESUMO

Mechanisms of neuronal infection by varicella-zoster virus (VZV) have been challenging to study due to the relatively strict human tropism of the virus and the paucity of tractable experimental models. Cellular mitogen-activated protein kinases (MAPKs) have been shown to play a role in VZV infection of nonneuronal cells, with distinct consequences for infectivity in different cell types. Here, we utilize several human neuronal culture systems to investigate the role of one such MAPK, the c-Jun N-terminal kinase (JNK), in VZV lytic infection and reactivation. We find that the JNK pathway is specifically activated following infection of human embryonic stem cell-derived neurons and that this activation of JNK is essential for efficient viral protein expression and replication. Inhibition of the JNK pathway blocked viral replication in a manner distinct from that of acyclovir, and an acyclovir-resistant VZV isolate was as sensitive to the effects of JNK inhibition as an acyclovir-sensitive VZV isolate in neurons. Moreover, in a microfluidic-based human neuronal model of viral latency and reactivation, we found that inhibition of the JNK pathway resulted in a marked reduction in reactivation of VZV. Finally, we utilized a novel technique to efficiently generate cells expressing markers of human sensory neurons from neural crest cells and established a critical role for the JNK pathway in infection of these cells. In summary, the JNK pathway plays an important role in lytic infection and reactivation of VZV in physiologically relevant cell types and may provide an alternative target for antiviral therapy.IMPORTANCE Varicella-zoster virus (VZV) has infected over 90% of people worldwide. While primary infection leads to the typically self-limiting condition of chickenpox, the virus can remain dormant in the nervous system and may reactivate later in life, leading to shingles or inflammatory diseases of the nervous system and eye with potentially severe consequences. Here, we take advantage of newer stem cell-based technologies to study the mechanisms by which VZV infects human neurons. We find that the c-Jun N-terminal kinase (JNK) pathway is activated by VZV infection and that blockade of this pathway limits lytic replication (as occurs during primary infection). In addition, JNK inhibition limits viral reactivation, exhibiting parallels with herpes simplex virus reactivation. The identification of the role of the JNK pathway in VZV infection of neurons reveals potential avenues for the development of alternate antiviral drugs.


Assuntos
Herpesvirus Humano 3/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Sistema de Sinalização das MAP Quinases , Ativação Viral , Latência Viral , Replicação Viral , Células Cultivadas , Varicela/virologia , Herpes Zoster/virologia , Células-Tronco Embrionárias Humanas/virologia , Humanos , Células-Tronco Neurais/virologia
15.
ALTEX ; 34(1): 75-94, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27463612

RESUMO

Migration of neural crest cells (NCCs) is one of the pivotal processes of human fetal development. Malformations arise if NCC migration and differentiation are impaired genetically or by toxicants. In the currently available test systems for migration inhibition of NCC (MINC), the manual generation of a cell-free space results in extreme operator dependencies, and limits throughput. Here a new test format was established. The assay avoids scratching by plating cells around a commercially available circular stopper. Removal of the stopper barrier after cell attachment initiates migration. This microwell-based circular migration zone NCC function assay (cMINC) was further optimized for toxicological testing of human pluripotent stem cell (hPSC)-derived NCCs. The challenge of obtaining data on viability and migration by automated image processing was addressed by developing a freeware. Data on cell proliferation were obtained by labelling replicating cells, and by careful assessment of cell viability for each experimental sample. The role of cell proliferation as an experimental confounder was tested experimentally by performing the cMINC in the presence of the proliferation-inhibiting drug cytosine arabinoside (AraC), and by a careful evaluation of mitotic events over time. Data from these studies led to an adaptation of the test protocol, so that toxicant exposure was limited to 24 h. Under these conditions, a prediction model was developed that allows classification of toxicants as either inactive, leading to unspecific cytotoxicity, or specifically inhibiting NC migration at non-cytotoxic concentrations.


Assuntos
Ensaios de Migração Celular/métodos , Movimento Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Crista Neural/efeitos dos fármacos , Proliferação de Células , Sobrevivência Celular , Humanos , Crista Neural/citologia , Células-Tronco , Teratogênios/toxicidade
16.
Nat Med ; 22(12): 1421-1427, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27841875

RESUMO

Familial dysautonomia (FD) is a debilitating disorder that affects derivatives of the neural crest (NC). For unknown reasons, people with FD show marked differences in disease severity despite carrying an identical, homozygous point mutation in IKBKAP, encoding IκB kinase complex-associated protein. Here we present disease-related phenotypes in human pluripotent stem cells (PSCs) that capture FD severity. Cells from individuals with severe but not mild disease show impaired specification of NC derivatives, including autonomic and sensory neurons. In contrast, cells from individuals with severe and mild FD show defects in peripheral neuron survival, indicating that neurodegeneration is the main culprit for cases of mild FD. Although genetic repair of the FD-associated mutation reversed early developmental NC defects, sensory neuron specification was not restored, indicating that other factors may contribute to disease severity. Whole-exome sequencing identified candidate modifier genes for individuals with severe FD. Our study demonstrates that PSC-based modeling is sensitive in recapitulating disease severity, which presents an important step toward personalized medicine.


Assuntos
Sistema Nervoso Autônomo/fisiopatologia , Disautonomia Familiar/fisiopatologia , Células-Tronco Pluripotentes Induzidas , Células Receptoras Sensoriais/citologia , Adolescente , Adulto , Sistema Nervoso Autônomo/citologia , Sistema Nervoso Autônomo/crescimento & desenvolvimento , Proteínas de Transporte/genética , Estudos de Casos e Controles , Sobrevivência Celular/genética , Criança , Disautonomia Familiar/genética , Feminino , Genótipo , Humanos , Masculino , Modelos Neurológicos , Mutação , Crista Neural/citologia , Neurônios/citologia , Fenótipo , Análise de Sequência de DNA , Índice de Gravidade de Doença , Fatores de Elongação da Transcrição , Adulto Jovem
17.
Proc Natl Acad Sci U S A ; 113(36): 10091-6, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27555588

RESUMO

Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/genética , Movimento Celular/genética , Fibroblastos/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Animais , Linhagem Celular , Polaridade Celular , Retroalimentação Fisiológica , Fibroblastos/citologia , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Células NIH 3T3 , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Optogenética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Polimerização , Ligação Proteica , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
18.
Cell Stem Cell ; 19(1): 95-106, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27320040

RESUMO

Neurons derived from human pluripotent stem cells (hPSCs) are powerful tools for studying human neural development and diseases. Robust functional coupling of hPSC-derived neurons with target tissues in vitro is essential for modeling intercellular physiology in a dish and to further translational studies, but it has proven difficult to achieve. Here, we derive sympathetic neurons from hPSCs and show that they can form physical and functional connections with cardiac muscle cells. Using multiple hPSC reporter lines, we recapitulated human autonomic neuron development in vitro and successfully isolated PHOX2B::eGFP+ neurons that exhibit sympathetic marker expression and electrophysiological properties and norepinephrine secretion. Upon pharmacologic and optogenetic manipulation, PHOX2B::eGFP+ neurons controlled beating rates of cardiomyocytes, and the physical interactions between these cells increased neuronal maturation. This study provides a foundation for human sympathetic neuron specification and for hPSC-based neuronal control of organs in a dish.


Assuntos
Diferenciação Celular , Miócitos Cardíacos/citologia , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Sistema Nervoso Simpático/citologia , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Ventrículos do Coração/citologia , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Neurônios/metabolismo , Optogenética , Fenótipo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/genética
19.
Cell Rep ; 15(10): 2301-2312, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27239027

RESUMO

Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our "chemical-compound-based" strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFß signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological "dual-SMAD" inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form "rescued" multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human "DMD-in-a-dish" model using hiPSC-based disease modeling.


Assuntos
Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Distrofia Muscular de Duchenne/patologia , Mioblastos/patologia , Animais , Linhagem Celular , Citometria de Fluxo , Humanos , Camundongos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Mioblastos/metabolismo , Fenótipo , Transdução de Sinais , Proteínas Smad/metabolismo
20.
Drug Discov Today ; 21(9): 1387-1398, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27109386

RESUMO

Engineered in vitro models using human cells, particularly patient-derived induced pluripotent stem cells (iPSCs), offer a potential solution to issues associated with the use of animals for studying disease pathology and drug efficacy. Given the prevalence of muscle diseases in human populations, an engineered tissue model of human skeletal muscle could provide a biologically accurate platform to study basic muscle physiology, disease progression, and drug efficacy and/or toxicity. Such platforms could be used as phenotypic drug screens to identify compounds capable of alleviating or reversing congenital myopathies, such as Duchene muscular dystrophy (DMD). Here, we review current skeletal muscle modeling technologies with a specific focus on efforts to generate biomimetic systems for investigating the pathophysiology of dystrophic muscle.


Assuntos
Músculo Esquelético/fisiologia , Distrofias Musculares/fisiopatologia , Engenharia Tecidual , Animais , Biomimética , Descoberta de Drogas , Humanos , Modelos Biológicos
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