RESUMO
Human pluripotent stem cells (hPSCs) are a promising resource for the replacement of degenerated ventral midbrain dopaminergic (vmDA) neurons in Parkinson's disease. Despite recent advances in protocols for the in vitro generation of vmDA neurons, the asynchronous and heterogeneous nature of the differentiations results in transplants of surprisingly low vmDA neuron purity. As the field advances toward the clinic, it will be optimal, if not essential, to remove poorly specified and potentially proliferative cells from donor preparations to ensure safety and predictable efficacy. Here, we use two novel hPSC knock-in reporter lines expressing GFP under the LMX1A and PITX3 promoters, to selectively isolate vm progenitors and DA precursors, respectively. For each cell line, unsorted, GFP+, and GFP- cells were transplanted into male or female Parkinsonian rodents. Only rats receiving unsorted cells, LMX1A-eGFP+, or PITX3-eGFP- cell grafts showed improved motor function over 6 months. Postmortem analysis revealed small grafts from PITX3-eGFP+ cells, suggesting that these DA precursors were not compatible with cell survival and integration. In contrast, LMX1A-eGFP+ grafts were highly enriched for vmDA neurons, and importantly excluded expansive proliferative populations and serotonergic neurons. These LMX1A-eGFP+ progenitor grafts accelerated behavioral recovery and innervated developmentally appropriate forebrain targets, whereas LMX1A-eGFP- cell grafts failed to restore motor deficits, supported by increased fiber growth into nondopaminergic target nuclei. This is the first study to use an hPSC-derived reporter line to purify vm progenitors, resulting in improved safety, predictability of the graft composition, and enhanced motor function.SIGNIFICANCE STATEMENT Clinical trials have shown functional integration of transplanted fetal-derived dopamine progenitors in Parkinson's disease. Human pluripotent stem cell (hPSC)-derived midbrain progenitors are now being tested as an alternative cell source; however, despite current differentiation protocols generating >80% correctly specified cells for implantation, resultant grafts contain a small fraction of dopamine neurons. Cell-sorting approaches, to select for correctly patterned cells before implantation, are being explored yet have been suboptimal to date. This study provides the first evidence of using 2 hPSC reporter lines (LMX1A-GFP and PITX3-GFP) to isolate correctly specified cells for transplantation. We show LMX1A-GFP+, but not PITX3-GFP+, cell grafts are more predictable, with smaller grafts, enriched in dopamine neurons, showing appropriate integration and accelerated functional recovery in Parkinsonian rats.
Assuntos
Proteínas com Homeodomínio LIM/metabolismo , Mesencéfalo/metabolismo , Transtornos Parkinsonianos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/transplante , Transplante de Células-Tronco/métodos , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Feminino , Previsões , Humanos , Masculino , Mesencéfalo/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/terapia , Ratos , Ratos NusRESUMO
Glomerular injury and podocyte loss leads to secondary tubulointerstitial damage and the development of fibrosis. The possibility of genetically reprogramming adult cells, termed induced pluripotent stem cells (iPS), may pave the way for patient-specific stem-cell-based therapies. Here, we reprogrammed normal human mesangial cells to pluripotency by retroviral transduction using defined factors (OCT4, SOX2, KLF4 and c-Myc). The kidney iPS (kiPS) cells resembled human embryonic stem-cell-like colonies in morphology and gene expression: They were alkaline phosphatase-positive; expressed OCT3/4, TRA-1 to 60 and TRA-1 to 81 proteins; and showed downregulation of mesangial cell markers. Quantitative (qPCR) showed that kiPS cells expressed genes analogous to embryonic stem cells and exhibited silencing of the retroviral transgenes by the fourth passage of differentiation. Furthermore, kiPS cells formed embryoid bodies and expressed markers of all three germ layers. The injection of undifferentiated kiPS colonies into immunodeficient mice formed teratomas, thereby demonstrating pluripotency. These results suggest that reprogrammed kidney induced pluripotent stem cells may aid the study of genetic kidney diseases and lead to the development of novel therapies.
Assuntos
Células-Tronco Pluripotentes Induzidas , Rim/citologia , Células Mesangiais/fisiologia , Adolescente , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Transferência de Genes , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Masculino , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genéticaRESUMO
Despite heterogeneity across the six layers of the mammalian cortex, all excitatory neurons are generated from a single founder population of neuroepithelial stem cells. However, how these progenitors alter their layer competence over time remains unknown. Here, we used human embryonic stem cell-derived cortical progenitors to examine the role of fibroblast growth factor (FGF) and Notch signaling in influencing cell fate, assessing their impact on progenitor phenotype, cell-cycle kinetics, and layer specificity. Forced early cell-cycle exit, via Notch inhibition, caused rapid, near-exclusive generation of deep-layer VI neurons. In contrast, prolonged FGF2 promoted proliferation and maintained progenitor identity, delaying laminar progression via MAPK-dependent mechanisms. Inhibiting MAPK extended cell-cycle length and led to generation of layer-V CTIP2+ neurons by repressing alternative laminar fates. Taken together, FGF/MAPK regulates the proliferative/neurogenic balance in deep-layer corticogenesis and provides a resource for generating layer-specific neurons for studying development and disease.
Assuntos
Córtex Cerebral/embriologia , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Organogênese , Transdução de Sinais , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Organogênese/efeitos dos fármacos , Fator de Transcrição PAX6/metabolismo , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismoRESUMO
Dopaminergic neurons (DAns), generated from human pluripotent stem cells (hPSCs), are capable of functionally integrating following transplantation and have recently advanced to clinical trials for Parkinson's disease (PD). However, pre-clinical studies have highlighted the low proportion of DAns within hPSC-derived grafts and their inferior plasticity compared to fetal tissue. Here, we examined whether delivery of a developmentally critical protein, glial cell line-derived neurotrophic factor (GDNF), could improve graft outcomes. We tracked the response of DAns implanted into either a GDNF-rich environment or after a delay in exposure. Early GDNF promoted survival and plasticity of non-DAns, leading to enhanced motor recovery in PD rats. Delayed exposure to GDNF promoted functional recovery through increases in DAn specification, DAn plasticity, and DA metabolism. Transcriptional profiling revealed a role for mitogen-activated protein kinase (MAPK)-signaling downstream of GDNF. Collectively, these results demonstrate the potential of neurotrophic gene therapy strategies to improve hPSC graft outcomes.
Assuntos
Terapia Genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Doença de Parkinson , Transplante de Células-Tronco , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos , Humanos , Doença de Parkinson/terapia , Ratos , Ratos Sprague-DawleyRESUMO
Huntington disease (HD) is an incurable late-onset neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the HD gene (HTT). The major hallmark of disease pathology is neurodegeneration in the brain. Currently, there are no useful in-vitro human models of HD. Recently, two human embryonic stem cell (hESC) lines carrying partial (CAG(37)) and fully (CAG(51)) penetrant mutant alleles have been derived from affected IVF embryos identified following preimplantation genetic diagnosis (PGD). Fluorescence polymerase chain reaction (F-PCR) and Genescan analysis confirmed the original embryonic HD genotypes. Reverse transcription PCR (RT-PCR) analysis confirmed the expression of mutant transcripts and western blot analysis demonstrated expression of mutant huntingtin protein (HTT). After treatment with noggin, HD hESC formed neurospheres, which could be further differentiated into cells susceptible to neurodegeneration in HD, namely primary neurones and astrocytes. Small pool PCR analysis of neurosphere cells revealed instability of disease-length CAG repeats following differentiation. The presence of active HTT genes, neural differentiation capabilities and evidence of CAG repeat instability indicates these HD hESC lines may serve as valuable in-vitro human models of HD to better understand the mechanisms of neurodegeneration in patients, and for drug screening to identify new therapies for human clinical trials.
Assuntos
Células-Tronco Embrionárias/citologia , Doença de Huntington/patologia , Modelos Biológicos , Western Blotting , Diferenciação Celular , Linhagem Celular , Humanos , Proteína Huntingtina , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Repetições de TrinucleotídeosRESUMO
The capacity for induced pluripotent stem (iPS) cells to be differentiated into a wide range of neural cell types makes them an attractive donor source for autologous neural transplantation therapies aimed at brain repair. Translation to the in vivo setting has been difficult, however, with mixed results in a wide variety of preclinical models of brain injury and limited information on the basic in vivo properties of neural grafts generated from human iPS cells. Here we have generated a human iPS cell line constitutively expressing green fluorescent protein as a basis to identify and characterize grafts resulting from transplantation of neural progenitors into the adult rat brain. The results show that the grafts contain a mix of neural cell types, at various stages of differentiation, including neurons that establish extensive patterns of axonal growth and progressively develop functional properties over the course of 1 year after implantation. These findings form an important basis for the design and interpretation of preclinical studies using human stem cells for functional circuit re-construction in animal models of brain injury. Stem Cells Translational Medicine 2017;6:1547-1556.
Assuntos
Orientação de Axônios , Pedúnculo Cerebral/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Células-Tronco Neurais/citologia , Transplante de Células-Tronco/métodos , Animais , Células Cultivadas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Células-Tronco Neurais/transplante , RatosRESUMO
Development of safe and effective stem cell-based therapies for brain repair requires an in-depth understanding of the in vivo properties of neural grafts generated from human stem cells. Replacing dopamine neurons in Parkinson's disease remains one of the most anticipated applications. Here, we have used a human PITX3-EGFP embryonic stem cell line to characterize the connectivity of stem cell-derived midbrain dopamine neurons in the dopamine-depleted host brain with an unprecedented level of specificity. The results show that the major A9 and A10 subclasses of implanted dopamine neurons innervate multiple, developmentally appropriate host targets but also that the majority of graft-derived connectivity is non-dopaminergic. These findings highlight the promise of stem cell-based procedures for anatomically correct reconstruction of specific neuronal pathways but also emphasize the scope for further refinement in order to limit the inclusion of uncharacterized and potentially unwanted cell types.
Assuntos
Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Transplante de Células-Tronco , Fatores de Transcrição/metabolismo , Animais , Axônios/metabolismo , Diferenciação Celular , Linhagem Celular , Genes Reporter , Humanos , Masculino , Mesencéfalo/citologia , Atividade Motora , Rede Nervosa/metabolismo , Ratos NusRESUMO
Stem cell transplants offer significant hope for brain repair following ischemic damage. Pre-clinical work suggests that therapeutic mechanisms may be multi-faceted, incorporating bone-fide circuit reconstruction by transplanted neurons, but also protection/regeneration of host circuitry. Here, we engineered hydrogel scaffolds to form "bio-bridges" within the necrotic lesion cavity, providing physical and trophic support to transplanted human embryonic stem cell-derived cortical progenitors, as well as residual host neurons. Scaffolds were fabricated by the self-assembly of peptides for a laminin-derived epitope (IKVAV), thereby mimicking the brain's major extracellular protein. Following focal ischemia in rats, scaffold-supported cell transplants induced progressive motor improvements over 9 months, compared to cell- or scaffold-only implants. These grafts were larger, exhibited greater neuronal differentiation, and showed enhanced electrophysiological properties reflective of mature, integrated neurons. Varying graft timing post-injury enabled us to attribute repair to both neuroprotection and circuit replacement. These findings highlight strategies to improve the efficiency of stem cell grafts for brain repair.
Assuntos
Peptídeos/metabolismo , Transplante de Células-Tronco/métodos , Acidente Vascular Cerebral/genética , Animais , Atrofia , Diferenciação Celular , Humanos , Ratos , Acidente Vascular Cerebral/metabolismo , Alicerces TeciduaisRESUMO
Pluripotent stem cells (PSCs) are a valuable tool for interrogating development, disease modelling, drug discovery and transplantation. Despite the burgeoned capability to fate restrict human PSCs to specific neural lineages, comparative protocols for mouse PSCs have not similarly advanced. Mouse protocols fail to recapitulate neural development, consequently yielding highly heterogeneous populations, yet mouse PSCs remain a valuable scientific tool as differentiation is rapid, cost effective and an extensive repertoire of transgenic lines provides an invaluable resource for understanding biology. Here we developed protocols for neural fate restriction of mouse PSCs, using knowledge of embryonic development and recent progress with human equivalents. These methodologies rely upon naïve ground-state PSCs temporarily transitioning through LIF-responsive stage prior to neural induction and rapid exposure to regional morphogens. Neural subtypes generated included those of the dorsal forebrain, ventral forebrain, ventral midbrain and hindbrain. This rapid specification, without feeder layers or embryoid-body formation, resulted in high proportions of correctly specified progenitors and neurons with robust reproducibility. These generated neural progenitors/neurons will provide a valuable resource to further understand development, as well disorders affecting specific neuronal subpopulations.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular/fisiologia , Citometria de Fluxo , Imuno-Histoquímica , Mesencéfalo/citologia , Camundongos , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Fatores de Transcrição Otx/metabolismo , Fator de Transcrição PAX6/metabolismo , Células-Tronco Pluripotentes/metabolismo , Prosencéfalo/citologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Recent studies have shown evidence for the functional integration of human pluripotent stem cell (hPSC)-derived ventral midbrain dopamine (vmDA) neurons in animal models of Parkinson's disease. Although these cells present a sustainable alternative to fetal mesencephalic grafts, a number of hurdles require attention prior to clinical translation. These include the persistent use of xenogeneic reagents and challenges associated with scalability and storage of differentiated cells. In this study, we describe the first fully defined feeder- and xenogeneic-free protocol for the generation of vmDA neurons from hPSCs and utilize two novel reporter knock-in lines (LMX1A-eGFP and PITX3-eGFP) for in-depth in vitro and in vivo tracking. Across multiple embryonic and induced hPSC lines, this "next generation" protocol consistently increases both the yield and proportion of vmDA neural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. We identify the mechanism underlying these improvements and demonstrate clinical applicability with the first report of scalability and cryopreservation of bona fide vmDA progenitors at a time amenable to transplantation. Finally, transplantation of xeno-free vmDA progenitors from LMX1A- and PITX3-eGFP reporter lines into Parkinsonian rodents demonstrates improved engraftment outcomes and restoration of motor deficits. These findings provide important and necessary advancements for the translation of hPSC-derived neurons into the clinic. Stem Cells Translational Medicine 2017;6:937-948.
Assuntos
Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/transplante , Mesencéfalo/citologia , Atividade Motora , Doença de Parkinson/fisiopatologia , Doença de Parkinson/terapia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Células Cultivadas , Criopreservação , Dopamina/metabolismo , Células Alimentadoras/citologia , Fibroblastos/citologia , Humanos , Camundongos , Doença de Parkinson/patologia , Fenótipo , RatosRESUMO
The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that ß-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.
Assuntos
Expressão Gênica , Genes Reporter , Vetores Genéticos/genética , Células-Tronco Pluripotentes/metabolismo , Transgenes , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Células-Tronco Pluripotentes/citologia , Nucleases dos Efetores Semelhantes a Ativadores de TranscriçãoRESUMO
During development of the central nervous system, trophic, together with genetic, cues dictate the balance between cellular proliferation and differentiation. Subsequent to the birth of new neurons, additional intrinsic and extrinsic signals regulate the connectivity of these cells. While a number of regulators of ventral midbrain (VM) neurogenesis and dopaminergic (DA) axon guidance are known, we identify a number of novel roles for the secreted glycoprotein, Wnt7a, in this context. We demonstrate a temporal and spatial expression of Wnt7a in the VM, indicative of roles in neurogenesis, differentiation, and axonal growth and guidance. In primary VM cultures, and validated in Wnt7a-deficient mice, we show that the early expression within the VM is important for regulating VM progenitor proliferation, cell cycle progression, and cell survival, thereby dictating the number of midbrain Nurr1 precursors and DA neurons. During early development of the midbrain DA pathways, Wnt7a promotes axonal elongation and repels DA neurites out of the midbrain. Later, Wnt7a expression in the VM midline suggests a role in preventing axonal crossing while expression in regions flanking the medial forebrain bundle (thalamus and hypothalamus) ensured appropriate trajectory of DA axons en route to their forebrain targets. We show that the effects of Wnt7a in VM development are mediated, at least in part, by the ß-catenin/canonical pathways. Together, these findings identify Wnt7a as a new regulator of VM neurogenesis and DA axon growth and guidance.
Assuntos
Axônios/fisiologia , Mesencéfalo/embriologia , Neurogênese , Proteínas Wnt/fisiologia , Animais , Ciclo Celular , Forma Celular , Sobrevivência Celular , Células Cultivadas , Neurônios Dopaminérgicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos Knockout , Morfogênese , Fibras Nervosas/fisiologia , Células-Tronco Neurais/fisiologia , Neuritos/fisiologia , Especificidade de Órgãos , Ratos Sprague-DawleyRESUMO
Oligodendrocytes play a fundamental supportive role in the mammalian central nervous system (CNS) as the myelinating-glial cells. Disruption of fast axonal transport mechanisms can occur as a consequence of mature oligodendrocyte loss following spinal cord injury, stroke, or due to neuroinflammatory conditions, such as multiple sclerosis. As a result of the limited remyelination ability in the CNS after injury or disease, human embryonic stem cells (hESCs) may prove to be a promising option for the generation and replacement of mature oligodendrocytes. Moreover, hESC-derived oligodendrocytes may be experimentally utilized to unravel fundamental questions of oligodendrocyte development, along with their therapeutic potential through growth factor support of axons and neurons. However, an intensive characterization and examination of hESC-derived oligodendrocytes prior to preclinical or clinical trials is required to facilitate greater success in their integration following cellular replacement therapy (CRT). Currently, the protocols utilized to derive oligodendrocytes from hESCs consist of significant variations in culture style, time-length of differentiation, and the provision of growth factors in culture. Further, these differing protocols also report disparate patterns in the expression of oligodendroglial markers by these derived oligodendrocytes, throughout their differentiation in culture. We have comprehensively reviewed the published protocols describing the derivation of oligodendrocytes from hESCs and the studies that examine their efficacy to remyelinate, along with the fundamental issues of their safety as a viable CRT. Additionally, this review will highlight particular issues of concern and suggestions for troubleshooting to provide investigators critical information for the future improvement of establishing in vitro hESC-derived oligodendrocytes.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Doenças Desmielinizantes/terapia , Células-Tronco Embrionárias/metabolismo , Oligodendroglia/metabolismo , Transplante de Células-Tronco , Diferenciação Celular , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Doenças Desmielinizantes/metabolismo , Humanos , Fibras Nervosas Mielinizadas/metabolismo , Oligodendroglia/citologiaRESUMO
Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin (HTT) gene. Recently, induced pluripotent stem cell (iPSC) lines carrying atypical and aggressive (CAG60+) HD variants have been generated and exhibit disparate molecular pathologies. Here we investigate two human embryonic stem cell (hESC) lines carrying CAG37 and CAG51 typical late-onset repeat expansions in comparison to wildtype control lines during undifferentiated states and throughout forebrain neuronal differentiation. Pluripotent HD lines demonstrate growth, viability, pluripotent gene expression, mitochondrial activity and forebrain specification that is indistinguishable from control lines. Expression profiles of crucial genes known to be dysregulated in HD remain unperturbed in the presence of mutant protein and throughout differentiation; however, elevated glutamate-evoked responses were observed in HD CAG51 neurons. These findings suggest typical late-onset HD mutations do not alter pluripotent parameters or the capacity to generate forebrain neurons, but that such progeny may recapitulate hallmarks observed in established HD model systems. Such HD models will help further our understanding of the cascade of pathological events leading to disease onset and progression, while simultaneously facilitating the identification of candidate HD therapeutics.