Developmental and Functional Nature of Human iPSC Derived Motoneurons

Stockmann, Marianne; Linta, Leonhard; Föhr, Karl J.; Boeckers, Anja; Ludolph, Albert C.; Kuh, Georges Ful; Udvardi, Patrick T; Proepper, Christian; Storch, Alexander; Kleger, Alexander; Liebau, Stefan; Boeckers, Tobias M.

doi:10.1007/s12015-011-9329-4

Cited by 36 publications

(48 citation statements)

References 204 publications

(336 reference statements)

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“…Human keratinocytes were reprogrammed as previously described (Stockmann et al, 2013) using a lentiviral polycystronic STEMCCA cassette encoding octamer-binding transcription factor 4 ( OCT4 ), sex determining region Y-box 2 ( SOX2 ), Kruppel-like factor 4 ( KLF4 ), and c-MYC (Somers et al, 2012). Briefly, keratinocytes (75% confluence) were treated with 5 × 10 5 proviral genome copies in EpiLife medium (Invitrogen, Carlsbad, CA, USA) supplemented with 8 μg/ml polybrene (Sigma-Aldrich, St. Louis, MO, USA) on two subsequent days.…”

Section: Methodsmentioning

confidence: 99%

“…Post mortem tissues are also difficult to obtain and mouse models have been useful but they do not reflect the complete pathology seen in humans. In this study, we took advantage of the generation of human induced pluripotent stem cells (hiPSCs) by reprogramming somatic cells (Aasen et al, 2008; Patel and Yang, 2010) and derived into various lineages, including spinal MNs (Stockmann et al, 2013). More importantly, patient-derived iPSCs lines expressing endogenous mFUS were analyzed.…”

Section: Introductionmentioning

confidence: 99%

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FUS Mislocalization and Vulnerability to DNA Damage in ALS Patients Derived hiPSCs and Aging Motoneurons

Higelin

Demestre

Putz

et al. 2016

Front. Cell. Neurosci.

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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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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

FUS Mislocalization and Vulnerability to DNA Damage in ALS Patients Derived hiPSCs and Aging Motoneurons

Higelin

Demestre

Putz

et al. 2016

Front. Cell. Neurosci.

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“…HEK293T and HeLa cells were acquired from ATCC. Control MEFs (C57BL/6) and PKD2 S707A/S711A -MEFs were generated according to standard protocols (37). Homozygous PKD2 S707A/S711A mice (35,36) were kindly provided by D. Cantrell, Dundee, UK.…”

Section: Methodsmentioning

confidence: 99%

Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion

Eiseler

Wille

Koehler

et al. 2016

Journal of Biological Chemistry

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Vesicle formation and fission are tightly regulated at the trans-Golgi network (TGN) during constitutive secretion. Two major protein families regulate these processes: members of the adenosyl-ribosylation factor family of small G-proteins (ARFs) and the protein kinase D (PKD) family of serine/threonine kinases. The functional relationship between these two key regulators of protein transport from the TGN so far is elusive. We here demonstrate the assembly of a novel functional protein complex at the TGN and its key members: cytosolic PKD2 binds ARF-like GTPase (ARL1) and shuttles ARL1 to the TGN. ARL1, in turn, localizes Arfaptin2 to the TGN. At the TGN, where PKD2 interacts with active ARF1, PKD2, and ARL1 are required for the assembly of a complex comprising of ARF1 and Arfaptin2 leading to secretion of matrix metalloproteinase-2 and -7. In conclusion, our data indicate that PKD2 is a core factor in the formation of this multiprotein complex at the TGN that controls constitutive secretion of matrix metalloproteinase cargo.

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“…Keratinocytes from plucked human (hiPSC1: female 40 years old, hiPSC2: male 35 years old) hair were generated as described previously Stockmann et al, 2011;Illing et al, 2013). Keratinocytes were grown on 20 μg/mL collagen IV-coated dishes and cultured in EpiLife medium supplemented with HKGS (both Life technologies, USA).…”

Section: Generation Of Hipscsmentioning

confidence: 99%

Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes

Demestre

Orth

Föhr³

et al. 2015

Stem Cell Research

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Striated skeletal muscle cells from humans represent a valuable source for in vitro studies of the motoric system as well as for pathophysiological investigations in the clinical settings. Myoblasts can readily be grown from human muscle tissue. However, if muscle tissue is unavailable, myogenic cells can be generated from human induced pluripotent stem cells (hiPSCs) preferably without genetic engineering. Our study aimed to optimize the generation of hiPSCs derived myogenic cells by employing selection of CD34 positive cells and followed by distinct, stepwise culture conditions. Following the expansion of CD34 positive single cells under myogenic cell culture conditions, serum deprived myoblast-like cells finally fused and formed multinucleated striated myotubes that expressed a set of key markers for muscle differentiation. In addition, these myotubes contracted upon electrical stimulation, responded to acetylcholine (Ach) and were able to generate action potentials. Finally, we co-cultured motoneurons and myotubes generated from identical hiPSCs cell lines. We could observe the early aggregation of acetylcholine receptors in muscle cells of immature co-cultures. At later stages, we identified and characterised mature neuromuscular junctions (NMJs). In summary, we describe here the successful generation of an iPS cell derived functional cellular system consisting of two distinct communicating cells types. This in vitro co-culture system could therefore contribute to research on diseases in which the motoneurons and the NMJ are predominantly affected, such as in amyotrophic lateral sclerosis or spinal muscular atrophy.

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Developmental and Functional Nature of Human iPSC Derived Motoneurons

Cited by 36 publications

References 204 publications

FUS Mislocalization and Vulnerability to DNA Damage in ALS Patients Derived hiPSCs and Aging Motoneurons

FUS Mislocalization and Vulnerability to DNA Damage in ALS Patients Derived hiPSCs and Aging Motoneurons

Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion

Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes

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