The breakthrough of reprogramming human somatic cells was achieved in 2006 by the work of Yamanaka and Takahashi. From this point, fibroblasts are the most commonly used primary somatic cell type for the generation of induced pluripotent stem cells (iPSCs). Various characteristics of fibroblasts supported their utilization for the groundbreaking experiments of iPSC generation. One major advantage is the high availability of fibroblasts which can be easily isolated from skin biopsies. Furthermore, their cultivation, propagation, and cryoconservation properties are uncomplicated with respect to nutritional requirements and viability in culture. However, the required skin biopsy remains an invasive approach, representing a major drawback for using fibroblasts as the starting material. More and more studies appeared over the last years, describing the reprogramming of other human somatic cell types. Cells isolated from blood samples or urine, as well as more unexpected cell types, like pancreatic islet beta cells, synovial cells, or mesenchymal stromal cells from wisdom teeth, show promising characteristics for a reprogramming strategy. Here, we want to highlight the advantages of keratinocytes from human plucked hair as a widely usable, noninvasive harvesting method for primary material in comparison with other commonly used cell types.

Despite decades of research on amyotrophic lateral sclerosis (ALS), there is only one approved drug, which minimally extends patient survival. Here, we investigated pathophysiological mechanisms underlying ALS using motor neurons (MNs) differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying mutations in FUS or SOD1. Patient-derived MNs were less active and excitable compared to healthy controls, due to reduced Na 1 /K 1 ratios in both ALS groups accompanied by elevated potassium channel (FUS) and attenuated sodium channel expression levels (FUS, SOD1). ALS iPSC-derived MNs showed elevated endoplasmic reticulum stress (ER) levels and increased caspase activation. Treatment with the FDA approved drug 4-Aminopyridine (4AP) restored ion-channel imbalances, increased neuronal activity levels and decreased ER stress and caspase activation. This study provides novel pathophysiological data, including a mechanistic explanation for the observed hypoexcitability in patient-derived MNs and a new therapeutic strategy to provide neuroprotection in MNs affected by ALS. STEM CELLS 2016;34:1563-1575 SIGNIFICANCE STATEMENTOur primary objective was to characterize the neurophysiology of iPSC-based models of amyotrophic lateral sclerosis (ALS) and, based on these results, to prevent neurodegeneration using targeted pharmacological intervention. We observed that mutant FUS and SOD1 iPSC-derived motor neurons displayed hypoexcitability, which recently has been shown to be a promising target to increase resilience against ALS-associated neurodegeneration. We identified the molecular mechanisms causing this phenotype, which enabled us to define a new therapeutic strategy using the FDA-approved drug 4-Aminopyridine. 4-Aminopyridine is a potassium channel blocker that raised motor neuronal activity and decreased endoplasmic reticulum stress and caspase activation. Our results provide novel pathophysiological data and an innovative treatment concept for symptomatic and neuroprotective therapy in ALS that stands in contrast to current strategies.

Evidence is mounting that the novel corona virus SARS-CoV2 inflicts neurological symptoms in a subgroup of COVID-19 patients. While plenty of theories on the route of neuroinvasion have been proposed, little histological evidence has been presented supporting any of these hypotheses. Therefore, we carried out immunostainings for ACE2 and TMPRSS2, two proteinases crucial for the entry of SARS-CoV2 into host cells, in the human enteric nervous system (ENS), as well as in the choroid plexus of the lateral ventricles. Both of these sites are important, yet often neglected entry gates to the nervous system. We found that ACE2 and TMPRSS2 are expressed by enteric neurons and glial cells of the small and large intestine, as well as choroid plexus epithelial cells, indicating that these cells meet the molecular requirements for viral entry. Together, our results are fundamental histological evidence substantiating current theories of neuroinvasion by SARS-CoV2.

Mutations within the FUS gene (Fused in Sarcoma) are known to cause Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease affecting upper and lower motoneurons. The FUS gene codes for a multifunctional RNA/DNA-binding protein that is primarily localized in the nucleus and is involved in cellular processes such as splicing, translation, mRNA transport and DNA damage response. In this study, we analyzed pathophysiological alterations associated with ALS related FUS mutations (mFUS) in human induced pluripotent stem cells (hiPSCs) and hiPSC derived motoneurons. To that end, we compared cells carrying a mild or severe mFUS in physiological- and/or stress conditions as well as after induced DNA damage. Following hyperosmolar stress or irradiation, mFUS hiPS cells recruited significantly more cytoplasmatic FUS into stress granules accompanied by impaired DNA-damage repair. In motoneurons wild-type FUS was localized in the nucleus but also deposited as small punctae within neurites. In motoneurons expressing mFUS the protein was additionally detected in the cytoplasm and a significantly increased number of large, densely packed FUS positive stress granules were seen along neurites. The amount of FUS mislocalization correlated positively with both the onset of the human disease (the earlier the onset the higher the FUS mislocalization) and the maturation status of the motoneurons. Moreover, even in non-stressed post-mitotic mFUS motoneurons clear signs of DNA-damage could be detected. In summary, we found that the susceptibility to cell stress was higher in mFUS hiPSCs and hiPSC derived motoneurons than in controls and the degree of FUS mislocalization correlated well with the clinical severity of the underlying ALS related mFUS. The accumulation of DNA damage and the cellular response to DNA damage stressors was more pronounced in post-mitotic mFUS motoneurons than in dividing hiPSCs suggesting that mFUS motoneurons accumulate foci of DNA damage, which in turn might be directly linked to neurodegeneration.

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