Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Garone, Maria Giovanna; Turris, Valeria de; Soloperto, Alessandro; Brighi, Carlo; Santis, Riccardo De; Pagani, Francesca; Angelantonio, Silvia Di; Rosa, Alessandro

doi:10.3791/59321

Cited by 22 publications

(28 citation statements)

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“…The experiment was performed in differentiating spinal MNs-expressing high levels of nHOTAIRM1 (Fig. S6A) -derived from human iPSCs through a fast method 48,49 . To assess whether RAP worked properly, it was applied in parallel to U1 snRNA (Fig.…”

Section: Identification Of Nhotairm1 Protein Interactorsmentioning

confidence: 99%

HOTAIRM1 regulates neuronal differentiation by modulating NEUROGENIN 2 and the downstream neurogenic cascade

et al. 2020

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Neuronal differentiation is a timely and spatially regulated process, relying on precisely orchestrated gene expression control. The sequential activation/repression of genes driving cell fate specification is achieved by complex regulatory networks, where transcription factors and noncoding RNAs work in a coordinated manner. Herein, we identify the long noncoding RNA HOTAIRM1 (HOXA Transcript Antisense RNA, Myeloid-Specific 1) as a new player in neuronal differentiation. We demonstrate that the neuronal-enriched HOTAIRM1 isoform epigenetically controls the expression of the proneural transcription factor NEUROGENIN 2 that is key to neuronal fate commitment and critical for brain development. We also show that HOTAIRM1 activity impacts on NEUROGENIN 2 downstream regulatory cascade, thus contributing to the achievement of proper neuronal differentiation timing. Finally, we identify the RNA-binding proteins HNRNPK and FUS as regulators of HOTAIRM1 biogenesis and metabolism. Our findings uncover a new regulatory layer underlying NEUROGENIN 2 transitory expression in neuronal differentiation and reveal a previously unidentified function for the neuronal-induced long noncoding RNA HOTAIRM1.

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Section: Identification Of Nhotairm1 Protein Interactorsmentioning

confidence: 99%

HOTAIRM1 regulates neuronal differentiation by modulating NEUROGENIN 2 and the downstream neurogenic cascade

et al. 2020

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“…It is used in non-viral vectors for transgenesis 17 , 18 , gene therapy 7 , 19 , insertional mutagenesis 20 , and genetic screens 21 – 23 . It has also found application in novel therapeutic strategies including CAR T-cell engineering 24 – 26 , CRISPR/Cas-mediated gene therapy 27 – 29 , and human induced pluripotent stem cells (iPSC) engineering 30 – 32 . Useful variants have been developed through random mutation including a hyperactive pB transposase called hyPBase 33 as well as one that can excise pB but cannot integrate it 34 .…”

Section: Introductionmentioning

confidence: 99%

Structural basis of seamless excision and specific targeting by piggyBac transposase

et al. 2020

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The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates specifically at TTAA tetranucleotides. We present cryo-EM structures of piggyBac transpososomes: a synaptic complex with hairpin DNA intermediates and a strand transfer complex capturing the integration step. The results show that the excised TTAA hairpin intermediate and the TTAA target adopt essentially identical conformations, providing a mechanistic link connecting the two unique properties of piggyBac. The transposase forms an asymmetric dimer in which the two central domains synapse the ends while two C-terminal domains form a separate dimer that contacts only one transposon end. In the strand transfer structure, target DNA is severely bent and the TTAA target is unpaired. In-cell data suggest that asymmetry promotes synaptic complex formation, and modifying ends with additional transposase binding sites stimulates activity.

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“…In order to gain insights into HuD regulation in ALS, we took advantage of spinal MNs derived from isogenic pairs of FUS WT and P525L hiPSC lines (hereafter FUS WT and FUS P525L ) by inducible expression of a “programming module” consisting of the transcription factors Ngn2, Isl1 and Lhx3 (NIL) [31, 32]. The P525L mutation, localized in the PY-NLS domain (Supplementary Figure S2A), causes severe mislocalization of the FUS protein in the cytoplasm and is often associated to juvenile ALS [2].…”

Section: Resultsmentioning

confidence: 99%

“…The spinal MN differentiation protocol is detailed in [31, 32]. Briefly, epB-NIL-containing cells were differentiated upon induction with 1 μg/ml doxycycline (Thermo Fisher Scientific) in DMEM/F12 (Sigma-Aldrich), supplemented with 1X Glutamax (Thermo Fisher Scientific), 1X NEAA (Thermo Fisher Scientific) and 0.5X Penicillin/Streptomycin (Sigma-Aldrich) for 2 days and Neurobasal/B27 medium (Neurobasal Medium, Thermo Fisher Scientific; supplemented with 1X B27, Thermo Fisher Scientific; 1X Glutamax, Thermo Fisher Scientific; 1X NEAA, Thermo Fisher Scientific; and 0.5X Penicillin/Streptomycin, Sigma Aldrich), containing 5 μM DAPT and 4 μM SU5402 (both from Sigma-Aldrich) for additional 3 days.…”

Section: Methodsmentioning

confidence: 99%

ALS-FUS mutation affects the activities of HuD/ELAVL4 and FMRP leading to axon phenotypes in motoneurons

Garone

Birsa

Rosito

et al. 2020

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Mutations in RNA-binding proteins (RBPs) have been genetically associated with the motoneuron disease Amyotrophic Lateral Sclerosis (ALS). Using both human induced Pluripotent Stem Cells and mouse models, we found that FUS-ALS causative mutations have a profound impact on a network of RBPs, including two relevant factors with important roles in neuronal RNA metabolism: HuD and FMRP. Mechanistically, cytoplasmic localization of mutant FUS leads to upregulation of HuD levels through competition with FMRP for HuD 3’UTR binding. In turn, increased HuD levels overly stabilize the transcript levels of its targets, NRN1 and GAP43. As a consequence, mutant FUS motoneurons show altered axon branching and growth upon injury. Abnormal axon branching and regrowth in FUS mutant motoneurons could be rescued by dampening NRN1 levels. Since similar phenotypes have been previously described in SOD1 and TDP-43 mutant models, aberrant axonal growth and branching might represent broad early events in the pathogenesis of ALS.

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Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Cited by 22 publications

References 0 publications

HOTAIRM1 regulates neuronal differentiation by modulating NEUROGENIN 2 and the downstream neurogenic cascade

HOTAIRM1 regulates neuronal differentiation by modulating NEUROGENIN 2 and the downstream neurogenic cascade

Structural basis of seamless excision and specific targeting by piggyBac transposase

ALS-FUS mutation affects the activities of HuD/ELAVL4 and FMRP leading to axon phenotypes in motoneurons

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