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:

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.

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.

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.Parkinson's disease | target validation | neuroprotection