The isolation or in vitro derivation of many human cell types remains challenging and inefficient. Direct conversion of human pluripotent stem cells (hPSCs) by forced expression of transcription factors provides a potential alternative. However, deficient inducible gene expression in hPSCs has compromised efficiencies of forward programming approaches. We have systematically optimized inducible gene expression in hPSCs using a dual genomic safe harbor gene-targeting strategy. This approach provides a powerful platform for the generation of human cell types by forward programming. We report robust and deterministic reprogramming of hPSCs into neurons and functional skeletal myocytes. Finally, we present a forward programming strategy for rapid and highly efficient generation of human oligodendrocytes.

Background: Parkinson's disease (PD) is a common neurodegenerative disorder. The contribution of the immune system to its pathogenesis remains incompletely understood. Methods: In this study, we performed comprehensive immune cell profiling in the cerebrospinal fluid (CSF) and peripheral blood (PB) of PD patients. Ten PD patients were diagnosed according to brain bank criteria and underwent detailed clinical examination, magnetic resonance imaging, PB and CSF immune cell profiling by multiparameter flow cytometry, and cytokine and chemokine measurements by bead-based arrays. Thirteen healthy elderly volunteers served as control population. Results: The proportions of activated T-lymphocytes and non-classical monocytes in the CSF were increased in patients with PD compared to the control group. In accordance, we found increased levels of the pro-inflammatory cytokines IL-2, IL-6 and TNFα and of the monocyte chemoattractant protein 1 (MCP-1) in the CSF of the included PD patients. Conclusions: Our data provide novel evidence for a response of the innate and adaptive immune system in the central nervous system of patients with PD.

Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development.

Epileptic seizures are well known sequelae of patients with stroke but only little is known about the different risk factors and about the influence of the different types of stroke including sinus thrombosis and bleedings on developing such seizures. Further, the association of post-stroke seizures and conventional vascular risk factors has not been evaluated to date. We performed a cohort study on a sample of 593 consecutive patients with different types of cerebrovascular events. In 421 patients, sufficient data were obtained in a personal interview over a mean observation period of 30 months. Data regarding the clinical history were recorded from the patients' charts. The total prevalence of epileptic seizures was 11.6%, the total annual risk was 4.6%. We detected the following significant risk factors: younger age at stroke; higher NIH stroke scale score; any coagulopathy. TIA was found significantly less frequent as a cause of seizures as compared to infarction, bleeding, and sinus thrombosis. Patients with bleeding (14.3%) and with sinus thrombosis (16.3%) were significantly more frequent in the seizure group than in the non-seizure group (6.7% and 1.6%, respectively). The location of stroke, including cortical versus subcortical, did not influence the risk of seizures. The majority of patients developed secondary generalized seizures (57.1%). In adjusted analyses, the two major risk factors for post-stroke epilepsy were a higher NIH stroke scale and a sinus thrombosis as the initial cerebrovascular event. Common lifestyle, vascular, and metabolic risk factors of stroke and for dementia were not associated with the development of seizures. In conclusion, our data show that epileptic seizures occur in particular after major strokes and in sinus thrombosis. Interestingly, conventional vascular risk factors were not associated with the occurrence of post-stroke seizures. Considering the risk for seizures after certain types of cerebrovascular events might help to early identify patients for anticonvulsive treatment. In the future, it should be investigated whether these patients might benefit from pre-emptive anticonvulsant treatment.