Search

Nanowire arrays used as cell culture substrates build a potent tool for advanced biological applications such as cargo delivery and biosensing. The unique topography of nanowire arrays, however, renders them a challenging growth environment for cells and explains why only basic cell lines have been employed in existing studies. Here, we present the culturing of human induced pluripotent stem cell-derived neural progenitor cells on rectangularly arranged nanowire arrays: In detail, we mapped the impact on proliferation, viability, and topography-induced membrane deformation across a multitude of array pitches (1, 3, 5, 10 μm) and nanowire lengths (1.5, 3, 5 μm). Against the intuitive expectation, a reduced proliferation was found on the arrays with the smallest array pitch of 1 μm and long NWs. Typically, cells settle in a fakir-like state on such densely-spaced nanowires and thus experience no substantial stress caused by nanowires indenting the cell membrane. However, imaging of F-actin showed a distinct reorganization of the cytoskeleton along the nanowire tips in the case of small array pitches interfering with regular proliferation. For larger pitches, the cell numbers depend on the NW lengths but proliferation generally continued although heavy deformations of the cell membrane were observed caused by the encapsulation of the nanowires. Moreover, we noticed a strong interaction of the nanowires with the nucleus in terms of squeezing and indenting. Remarkably, the cell viability is maintained at about 85% despite the massive deformation of the cells. Considering the enormous potential of human induced stem cells to study neurodegenerative diseases and the high cellular viability combined with a strong interaction with nanowire arrays, we believe that our results pave the way to apply nanowire arrays to human stem cells for future applications in stem cell research and regenerative medicine.

High-throughput phenotypic screening enables the identification of new therapeutic targets even when the molecular mechanism underlying the disease is unknown. In the case of neurodegenerative disease, there is a dire need to identify new targets that can ameliorate, halt, or reverse degeneration. Stem cell-based disease models are particularly powerful tools for phenotypic screening because they use the same cell type affected in patients. Here, we describe the expansion of mouse stem cells and human induced pluripotent stem cells as well as the differentiation of these cells into neural lineages that, when exposed to neuroinflammatory stress, can be used for compound screening followed by hit identification, validation, and target deconvolution.

Summary Aberrant hedgehog (Hh) signaling contributes to the pathogenesis of multiple cancers. Available inhibitors target Smoothened (Smo), which can acquire mutations causing drug resistance. Thus, compounds that inhibit Hh signaling downstream of Smo are urgently needed. We identified dynarrestin, a novel inhibitor of cytoplasmic dyneins 1 and 2. Dynarrestin acts reversibly to inhibit cytoplasmic dynein 1-dependent microtubule binding and motility in vitro without affecting ATP hydrolysis. It rapidly and reversibly inhibits endosome movement in living cells and perturbs mitosis by inducing spindle misorientation and pseudoprometaphase delay. Dynarrestin reversibly inhibits cytoplasmic dynein 2-dependent intraflagellar transport (IFT) of the cargo IFT88 and flux of Smo within cilia without interfering with ciliogenesis and suppresses Hh-dependent proliferation of neuronal precursors and tumor cells. As such, dynarrestin is a valuable tool for probing cytoplasmic dynein-dependent cellular processes and a promising compound for medicinal chemistry programs aimed at development of anti-cancer drugs.

SummaryThe LRRK2 mutation G2019S is the most common genetic cause of Parkinson’s disease (PD). To better understand the link between mutant LRRK2 and PD pathology, we derived induced pluripotent stem cells from PD patients harboring LRRK2 G2019S and then specifically corrected the mutant LRRK2 allele. We demonstrate that gene correction resulted in phenotypic rescue in differentiated neurons and uncovered expression changes associated with LRRK2 G2019S. We found that LRRK2 G2019S induced dysregulation of CPNE8, MAP7, UHRF2, ANXA1, and CADPS2. Knockdown experiments demonstrated that four of these genes contribute to dopaminergic neurodegeneration. LRRK2 G2019S induced increased extracellular-signal-regulated kinase 1/2 (ERK) phosphorylation. Transcriptional dysregulation of CADPS2, CPNE8, and UHRF2 was dependent on ERK activity. We show that multiple PD-associated phenotypes were ameliorated by inhibition of ERK. Therefore, our results provide mechanistic insight into the pathogenesis induced by mutant LRRK2 and pointers for the development of potential new therapeutics.

Rapid and efficient protocols to generate oligodendrocytes (OL) from human induced pluripotent stem cells (iPSC) are currently lacking, but may be a key technology to understand the biology of myelin diseases and to develop treatments for such disorders. Here, we demonstrate that the induction of three transcription factors (SOX10, OLIG2, NKX6.2) in iPSC-derived neural progenitor cells is sufficient to rapidly generate O4+ OL with an efficiency of up to 70% in 28 d and a global gene-expression profile comparable to primary human OL. We further demonstrate that iPSC-derived OL disperse and myelinate the CNS of Mbpshi/shiRag-/- mice during development and after demyelination, are suitable for in vitro myelination assays, disease modeling, and screening of pharmacological compounds potentially promoting oligodendroglial differentiation. Thus, the strategy presented here to generate OL from iPSC may facilitate the studying of human myelin diseases and the development of high-throughput screening platforms for drug discovery.

SummaryStem cells, through their ability to both self-renew and differentiate, can produce a virtually limitless supply of specialized cells that behave comparably to primary cells. We took advantage of this property to develop an assay for small-molecule-based neuroprotection using stem-cell-derived motor neurons and astrocytes, together with activated microglia as a stress paradigm. Here, we report on the discovery of hit compounds from a screen of more than 10,000 small molecules. These compounds act through diverse pathways, including the inhibition of nitric oxide production by microglia, activation of the Nrf2 pathway in microglia and astrocytes, and direct protection of neurons from nitric-oxide-induced degeneration. We confirm the activity of these compounds using human neurons. Because microglial cells are activated in many neurological disorders, our hit compounds could be ideal starting points for the development of new drugs to treat various neurodegenerative and neurological diseases.