RESUMO
The central nervous system is particularly susceptible to DNA repair deficiency, which renders a variety of neurodevelopmental and neurodegenerative disorders in humans. It is generally believed that DNA damage occurs upon repetitive replication and oxidative stress in highly proliferating neuroprogenitor cells (NPs), or due to high rates of metabolism and active neuronal activity in terminally differentiated neurons. DNA double-stranded breaks (DSBs) and single-stranded breaks (SSBs) constitute the most prevalent forms of DNA damage, which can result in neuronal apoptosis if unrepaired. Despite these notions, there are still gaps in our knowledge regarding the mechanism and specificity of DNA damage and repair in the neural development and the homeostasis of neural tissues. Recent studies have identified recurrent DSBs within neuronal long genes in NPs and 'programmed' SSBs in neuronal activity genes. However, the physiological function of these DNA breakages in the nervous system has not been so far explored. In this review, we summarise the recent advances in the field of DNA damage and DNA repair in neural development and neuropathies.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Neurônios/metabolismo , Dano ao DNA , DNA/metabolismoRESUMO
BACKGROUND: The etiology of Parkinson's disease (PD) is only partially understood despite the fact that environmental causes, risk factors, and specific gene mutations are contributors to the disease. Biallelic mutations in the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) gene involved in mitochondrial homeostasis, vesicle trafficking, and autophagy are sufficient to cause PD. OBJECTIVES: We sought to evaluate the difference between controls' and PINK1 patients' derived neurons in their transition from neuroepithelial stem cells to neurons, allowing us to identify potential pathways to target with repurposed compounds. METHODS: Using two-dimensional and three-dimensional models of patients' derived neurons we recapitulated PD-related phenotypes. We introduced the usage of midbrain organoids for testing compounds. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), we corrected the point mutations of three patients' derived cells. We evaluated the effect of the selected compound in a mouse model. RESULTS: PD patient-derived cells presented differences in their energetic profile, imbalanced proliferation, apoptosis, mitophagy, and a reduced differentiation efficiency to tyrosine hydroxylase positive (TH+) neurons compared to controls' cells. Correction of a patient's point mutation ameliorated the metabolic properties and neuronal firing rates as well as reversing the differentiation phenotype, and reducing the increased astrocytic levels. Treatment with 2-hydroxypropyl-ß-cyclodextrin increased the autophagy and mitophagy capacity of neurons concomitant with an improved dopaminergic differentiation of patient-specific neurons in midbrain organoids and ameliorated neurotoxicity in a mouse model. CONCLUSION: We show that treatment with a repurposed compound is sufficient for restoring the impaired dopaminergic differentiation of PD patient-derived cells. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Doença de Parkinson , 2-Hidroxipropil-beta-Ciclodextrina/metabolismo , Animais , Encéfalo/metabolismo , Neurônios Dopaminérgicos/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Organoides/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , FenótipoRESUMO
Increasing evidence suggests that neurodevelopmental alterations might contribute to increase the susceptibility to develop neurodegenerative diseases. We investigate the occurrence of developmental abnormalities in dopaminergic neurons in a model of Parkinson's disease (PD). We monitor the differentiation of human patient-specific neuroepithelial stem cells (NESCs) into dopaminergic neurons. Using high-throughput image analyses and single-cell RNA sequencing, we observe that the PD-associated LRRK2-G2019S mutation alters the initial phase of neuronal differentiation by accelerating cell-cycle exit with a concomitant increase in cell death. We identify the NESC-specific core regulatory circuit and a molecular mechanism underlying the observed phenotypes. The expression of NR2F1, a key transcription factor involved in neurogenesis, decreases in LRRK2-G2019S NESCs, neurons, and midbrain organoids compared to controls. We also observe accelerated dopaminergic differentiation in vivo in NR2F1-deficient mouse embryos. This suggests a pathogenic mechanism involving the LRRK2-G2019S mutation, where the dynamics of dopaminergic differentiation are modified via NR2F1.
Assuntos
Encéfalo/enzimologia , Fator I de Transcrição COUP/metabolismo , Neurônios Dopaminérgicos/enzimologia , Células-Tronco Pluripotentes Induzidas/enzimologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Células-Tronco Neurais/enzimologia , Neurogênese , Doença de Parkinson/enzimologia , Animais , Encéfalo/patologia , Fator I de Transcrição COUP/genética , Ciclo Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Neurônios Dopaminérgicos/patologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Mutação , Células-Tronco Neurais/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fenótipo , RNA-Seq , Transdução de Sinais , Análise de Célula Única , Fatores de TempoRESUMO
MCPH1 is a causal gene for the neurodevelopmental disorder, human primary microcephaly (MCPH1, OMIM251200). Most pathogenic mutations are located in the N-terminal region of the gene, which encodes a BRCT domain, suggesting an important function of this domain in brain size determination. To investigate the specific function of the N-terminal BRCT domain in vivo, we generated a mouse model lacking the N'-BRCT domain of MCPH1 (referred as Mcph1-ΔBR1). These mutant mice are viable, but exhibit reduced brain size, with a thinner cortex due to a reduction of neuroprogenitor populations and premature neurogenic differentiation. Mcph1-ΔBR1 mice (both male and female) are infertile; however, almost all female mutants develop ovary tumours. Mcph1-ΔBR1 MEF cells exhibit a defect in DNA damage response and DNA repair, and show the premature chromosome condensation (PCC) phenotype, a hallmark of MCPH1 patient cells and also Mcph1 knockout cells. In comparison with Mcph1 complete knockout mice, Mcph1-ΔBR1 mice faithfully reproduce all phenotypes, indicating an essential role of the N-terminal BRCT domain for the physiological function of MCPH1 in the control of brain size and gonad development as well as in multiple cellular processes.
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Encéfalo/fisiologia , Proteínas de Ciclo Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Fertilidade/fisiologia , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Feminino , Masculino , Camundongos , Domínios ProteicosRESUMO
MRE11, RAD50, and NBS1 form the MRN complex in response to DNA damage to activate ATM, a gene responsible for Ataxia-Telangiectasia (A-T). Loss of any components of the MRN complex compromises cell life. Mutations in MRE11, RAD50, and NBS1 cause human genomic instability syndromes Ataxia-Telangiectasia-like disorder (A-TLD), NBS-like disorder (NBSLD), and Nijmegen Breakage Syndrome (NBS), respectively. Among other pathologies, neuronal deficits, including microcephaly, intellectual disabilities, and progressive cerebellar degeneration, are common in these disorders. Nbs1 deletion in neural stem cells of mouse models resulted in cerebellar atrophy and ataxia, mimicking the A-T syndrome suggesting an etiological function of MRN-mediated DDR in neuronal homeostasis and neuropathology. Here we show that deletion of Nbs1 or Mre11 specifically in Purkinje neurons of mouse models (Nbs1-PCΔ and Mre11-PCΔ, respectively) is compatible with cerebellar development. Deleting Nbs1 in Purkinje cells disrupts the cellular localization pattern of MRE11 or RAD50 without inducing apparent DNA damage, albeit impaired DNA damage response (judged by 53BP1 focus formation) to ionizing radiation (IR). However, neither survival nor morphology of Purkinje cells and thus locomotor capabilities is affected by Nbs1 deletion under physiological conditions. Similarly, deletion of Mre11 in Purkinje cells does not affect the numbers or morphology of Purkinje cells and causes no accumulation of DNA damage. Mre11-deleted Purkinje cells have regular intrinsic neuronal activity. Taken together, these data indicate that the MRN complex is not essential for the survival and functionality of postmitotic neurons such as Purkinje cells. Thus, cerebellar deficits in MRN defect-related disorders and mouse models are unlikely to be a direct consequence of loss of these factors compromising DDR in postmitotic neurons such as Purkinje cells.
RESUMO
Parkinson's disease (PD) is a multifactorial disorder with complex etiology. The most prevalent PD associated mutation, LRRK2-G2019S is linked to familial and sporadic cases. Based on the multitude of genetic predispositions in PD and the incomplete penetrance of LRRK2-G2019S, we hypothesize that modifiers in the patients' genetic background act as susceptibility factors for developing PD. To assess LRRK2-G2019S modifiers, we used human induced pluripotent stem cell-derived neuroepithelial stem cells (NESCs). Isogenic controls distinguish between LRRK2-G2019S dependent and independent cellular phenotypes. LRRK2-G2019S patient and healthy mutagenized lines showed altered NESC self-renewal and viability, as well as impaired serine metabolism. In patient cells, phenotypes were only partly LRRK2-G2019S dependent, suggesting a significant contribution of the genetic background. In this context we identified the gene serine racemase (SRR) as a novel patient-specific, developmental, genetic modifier contributing to the aberrant phenotypes. Its enzymatic product, d-serine, rescued altered cellular phenotypes. Susceptibility factors in the genetic background, such as SRR, could be new targets for early PD diagnosis and treatment.
Assuntos
Autorrenovação Celular/genética , Doença de Parkinson/genética , Racemases e Epimerases/genética , Serina/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Sobrevivência Celular/genética , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Células-Tronco Neurais , Doença de Parkinson/metabolismo , FenótipoRESUMO
Class IIa histone deacetylases (HDACs) are a subfamily of HDACs with important functions in development and adult tissue homeostasis. As opposed to other HDACs, they lack catalytic function and bind transcription factors to recruit transcriptional co-regulators, mostly co-repressors such as nuclear receptor co-repressor (NCoR)/silencing mediator of retinoid and thyroid hormone receptor (SMRT). Class IIa HDACs enhance mouse somatic cell reprogramming to induced pluripotent stem cells (iPSCs) by repressing the function of the pro-mesenchymal transcription factor myocyte enhancer factor 2 (MEF2), which is upregulated during this process. Here, we describe, using HDAC4 and 7 as examples, that class IIa HDACs exhibit nuclear-cytoplasmic trafficking in reprogramming, being mostly cytoplasmic in donor fibroblasts and intermediate cells but translocating to the nucleus in iPSCs. Importantly, over-expressing a mutant form of HDAC4 or 7 that becomes trapped in the nucleus enhances the early phase of reprogramming but is deleterious afterwards. The latter effect is mediated through binding to the exogenous reprogramming factors at pluripotency loci, and the subsequent recruitment of NCoR/SMRT co-repressors. Thus, our findings uncover a context-dependent function of class IIa HDACs in reprogramming and further reinforce the idea that recruitment of co-repressors by the exogenous factors is a major obstacle for reactivating the pluripotency network in this process.
RESUMO
The development of new and easy-to-use nucleases, such as CRISPR/Cas9, made tools for gene editing widely accessible to the scientific community. Cas9-based gene editing protocols are robust for creating knock-out models, but the generation of single nucleotide transitions or transversions remains challenging. This is mainly due to the low frequency of homology directed repair, which leads to the screening of a high number of clones to identify positive events. Moreover, lack of simultaneous biallelic modifications, frequently results in second-allele indels. For example, while one allele might undergo homology directed repair, the second can undergo non-homologous end joining repair. Here we present a step-wise protocol for biallelic gene editing. It uses two donors carrying a combination of fluorescent reporters alongside homology arms directed to the same genomic region for biallelic targeting. These homology arms carry the desired composite of modifications to be introduced (homozygous or heterozygous changes). Plus, the backbone of the plasmid carries a third fluorescent reporter for negative selection (to discard random integration events). Fluorescent selection of non-random biallelic targeted clones can be performed by microscopy guided picking or cell sorting (FACS). The positive selection module (PSM), carrying the fluorescence reporter and an antibiotic resistance, is flanked by inverted terminal repeats (ITR) that are recognized by transposase. Upon purification of the clones correctly modified, transfection of the excision-only transposase allows the removal of the PSM resulting in the integration of only the desired modifications.
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Modeling Parkinson's disease (PD) using advanced experimental in vitro models is a powerful tool to study disease mechanisms and to elucidate unexplored aspects of this neurodegenerative disorder. Here, we demonstrate that three-dimensional (3D) differentiation of expandable midbrain floor plate neural progenitor cells (mfNPCs) leads to organoids that resemble key features of the human midbrain. These organoids are composed of midbrain dopaminergic neurons (mDANs), which produce and secrete dopamine. Midbrain-specific organoids derived from PD patients carrying the LRRK2-G2019S mutation recapitulate disease-relevant phenotypes. Automated high-content image analysis shows a decrease in the number and complexity of mDANs in LRRK2-G2019S compared to control organoids. The floor plate marker FOXA2, required for mDAN generation, increases in PD patient-derived midbrain organoids, suggesting a neurodevelopmental defect in mDANs expressing LRRK2-G2019S. Thus, we provide a robust method to reproducibly generate 3D human midbrain organoids containing mDANs to investigate PD-relevant patho-mechanisms.
RESUMO
Emerging evidence suggests that Parkinson's disease (PD), besides being an age-associated disorder, might also have a neurodevelopment component. Disruption of mitochondrial homeostasis has been highlighted as a crucial cofactor in its etiology. Here, we show that PD patient-specific human neuroepithelial stem cells (NESCs), carrying the LRRK2-G2019S mutation, recapitulate key mitochondrial defects previously described only in differentiated dopaminergic neurons. By combining high-content imaging approaches, 3D image analysis, and functional mitochondrial readouts we show that LRRK2-G2019S mutation causes aberrations in mitochondrial morphology and functionality compared with isogenic controls. LRRK2-G2019S NESCs display an increased number of mitochondria compared with isogenic control lines. However, these mitochondria are more fragmented and exhibit decreased membrane potential. Functional alterations in LRRK2-G2019S cultures are also accompanied by a reduced mitophagic clearance via lysosomes. These findings support the hypothesis that preceding mitochondrial developmental defects contribute to the manifestation of the PD pathology later in life.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mitocôndrias/genética , Mutação , Células-Tronco Neurais/metabolismo , Doença de Parkinson/genética , Idoso de 80 Anos ou mais , Diferenciação Celular/genética , Neurônios Dopaminérgicos/metabolismo , Feminino , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologiaRESUMO
Parkinson's disease (PD)-specific neurons, grown in standard 2D cultures, typically only display weak endophenotypes. The cultivation of PD patient-specific neurons, derived from induced pluripotent stem cells carrying the LRRK2-G2019S mutation, is optimized in 3D microfluidics. The automated image analysis algorithms are implemented to enable pharmacophenomics in disease-relevant conditions. In contrast to 2D cultures, this 3D approach reveals robust endophenotypes. High-content imaging data show decreased dopaminergic differentiation and branching complexity, altered mitochondrial morphology, and increased cell death in LRRK2-G2019S neurons compared to isogenic lines without using stressor agents. Treatment with the LRRK2 inhibitor 2 (Inh2) rescues LRRK2-G2019S-dependent dopaminergic phenotypes. Strikingly, a holistic analysis of all studied features shows that the genetic background of the PD patients, and not the LRRK2-G2019S mutation, constitutes the strongest contribution to the phenotypes. These data support the use of advanced in vitro models for future patient stratification and personalized drug development.
RESUMO
Genome editing and human induced pluripotent stem cells hold great promise for the development of isogenic disease models and the correction of disease-associated mutations for isogenic tissue therapy. CRISPR-Cas9 has emerged as a versatile and simple tool for engineering human cells for such purposes. However, the current protocols to derive genome-edited lines require the screening of a great number of clones to obtain one free of random integration or on-locus non-homologous end joining (NHEJ)-containing alleles. Here, we describe an efficient method to derive biallelic genome-edited populations by the use of fluorescent markers. We call this technique FACS-assisted CRISPR-Cas9 editing (FACE). FACE allows the derivation of correctly edited polyclones carrying a positive selection fluorescent module and the exclusion of non-edited, random integrations and on-target allele NHEJ-containing cells. We derived a set of isogenic lines containing Parkinson's-disease-associated mutations in α-synuclein and present their comparative phenotypes.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Parkinson/genética , alfa-Sinucleína/genética , Alelos , Células Cultivadas , Reparo do DNA por Junção de Extremidades/genética , Citometria de Fluxo/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologiaRESUMO
The p.G2019S mutation of the leucine-rich repeat kinase 2 (LRRK2) has been identified as the most prevalent genetic cause of familial and sporadic Parkinson's disease (PD). The Cre-LoxP recombination system has been used to correct the LRRK2-G2019S mutation in patient derived human induced pluripotent stem cells (hiPSCs) in order to generate isogenic controls. However, the remaining LoxP site can influence gene expression. In this study, we report the generation of a footprint-free LRRK2-G2019S isogenic hiPS cell line edited with the CRISPR/Cas9 and piggyBac technologies. We observed that the percentage of Tyrosine Hydroxylase (TH) positive neurons with a total neurite length of >2000µm was significantly reduced in LRRK2-G2019S dopaminergic (DA) neurons. The average branch number in LRRK2-G2019S DA neurons was also decreased. In addition, we have shown that in vitro TH positive neurons with a total neurite length of >2000µm were positive for Serine 129 phosphorylated (S129P) alpha-Synuclein (αS) and we hypothesize that S129P-αS plays a role in the maintenance or formation of long neurites. In summary, our footprint-free LRRK2-G2019S isogenic cell lines allow standardized, genetic background independent, in vitro PD modeling and provide new insights into the role of LRRK2-G2019S and S129P-αS in the pathogenesis of PD.
Assuntos
Sistemas CRISPR-Cas/genética , Neurônios Dopaminérgicos/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação/genética , alfa-Sinucleína/metabolismo , Idoso de 80 Anos ou mais , Diferenciação Celular , Linhagem Celular , Elementos de DNA Transponíveis/genética , Feminino , Vetores Genéticos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pessoa de Meia-Idade , Neuritos/metabolismo , Fenótipo , Plasmídeos/metabolismo , Transfecção , Transposases/metabolismoRESUMO
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic (DAergic) neurons in the substantia nigra and the gradual depletion of dopamine (DA). Current treatments replenish the DA deficit and improve symptoms but induce dyskinesias over time, and neuroprotective therapies are nonexistent. Here we report that Nuclear receptor-related 1 (Nurr1):Retinoid X receptor α (RXRα) activation has a double therapeutic potential for PD, offering both neuroprotective and symptomatic improvement. We designed BRF110, a unique in vivo active Nurr1:RXRα-selective lead molecule, which prevents DAergic neuron demise and striatal DAergic denervation in vivo against PD-causing toxins in a Nurr1-dependent manner. BRF110 also protects against PD-related genetic mutations in patient induced pluripotent stem cell (iPSC)-derived DAergic neurons and a genetic mouse PD model. Remarkably, besides neuroprotection, BRF110 up-regulates tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), and GTP cyclohydrolase I (GCH1) transcription; increases striatal DA in vivo; and has symptomatic efficacy in two postneurodegeneration PD models, without inducing dyskinesias on chronic daily treatment. The combined neuroprotective and symptomatic effects of BRF110 identify Nurr1:RXRα activation as a potential monotherapeutic approach for PD.
Assuntos
Antiparkinsonianos/farmacologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Doença de Parkinson/tratamento farmacológico , Receptor X Retinoide alfa/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Antiparkinsonianos/química , Antiparkinsonianos/farmacocinética , Encéfalo/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Dopamina/genética , Estabilidade de Medicamentos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Terapia de Alvo Molecular , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/fisiologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/agonistas , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Multimerização Proteica , Ratos , Receptor X Retinoide alfa/agonistas , Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/genéticaRESUMO
Class IIa histone deacetylases (HDACs) and myocyte enhancer factor 2 (MEF2) proteins compose a signaling module that orchestrates lineage specification during embryogenesis. We show here that this module also regulates the generation of mouse induced pluripotent stem cells by defined transcription factors. Class IIa HDACs and MEF2 proteins rise steadily during fibroblast reprogramming to induced pluripotent stem cells. MEF2 proteins tend to block the process by inducing the expression of Tgfß cytokines, which impairs the necessary phase of mesenchymal-to-epithelial transition (MET). Conversely, class IIa HDACs endeavor to suppress the activity of MEF2 proteins, thus enhancing the MET and colony formation efficiency. Our work highlights an unexpected role for a developmental axis in somatic cell reprogramming and provides new insight into how the MET is regulated in this context.
Assuntos
Desdiferenciação Celular/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Histona Desacetilases/metabolismo , Fatores de Regulação Miogênica/metabolismo , Animais , Desdiferenciação Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células HEK293 , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Humanos , Camundongos , Fatores de Regulação Miogênica/genética , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genéticaRESUMO
Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. At the transcriptional level, Sox2/Oct4 suppress the EMT mediator Snail, c-Myc downregulates TGF-beta1 and TGF-beta receptor 2, and Klf4 induces epithelial genes including E-cadherin. Blocking MET impairs the reprogramming of fibroblasts whereas preventing EMT in epithelial cells cultured with serum can produce iPSCs without Klf4 and c-Myc. Our work not only establishes MET as a key cellular mechanism toward induced pluripotency, but also demonstrates iPSC generation as a cooperative process between the defined factors and the extracellular milieu. PAPERCLIP: