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Abstract We present Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a scalable platform producing autologous organotypic iPS cell-derived induced skin composite (iSC) grafts for definitive treatment. Clinical-grade manufacturing integrates CRISPR-mediated genetic correction with reprogramming into one step, accelerating derivation of COL7A1-edited iPS cells from patients. Differentiation into epidermal, dermal and melanocyte progenitors is followed by CD49f-enrichment, minimizing maturation heterogeneity. Mouse xenografting of iSCs from four patients with different mutations demonstrates disease modifying activity at 1 month. Next-generation sequencing, biodistribution and tumorigenicity assays establish a favorable safety profile at 1-9 months. Single cell transcriptomics reveals that iSCs are composed of the major skin cell lineages and include prominent holoclone stem cell-like signatures of keratinocytes, and the recently described Gibbin-dependent signature of fibroblasts. The latter correlates with enhanced graftability of iSCs. In conclusion, DEBCT overcomes manufacturing and safety roadblocks and establishes a reproducible, safe, and cGMP-compatible therapeutic approach to heal lesions of DEB patients.

Apical neural progenitors (aNPs) drive neurogenesis by means of a program consisting of self-proliferative and neurogenic divisions. The balance between these two manners of division sustains the pool of apical progenitors into late neurogenesis, thereby ensuring their availability to populate the brain with terminal cell types. Using knockout and in utero electroporation mouse models, we report a key role for the microtubule-associated protein 600 (p600) in the regulation of spindle orientation in aNPs, a cellular event that has been associated with cell fate and neurogenesis. We find that p600 interacts directly with the neurogenic protein Ndel1 and that aNPs knockout for p600, depleted of p600 by shRNA or expressing a Ndel1-binding p600 fragment all display randomized spindle orientation. Depletion of p600 by shRNA or expression of the Ndel1-binding p600 fragment also results in a decreased number of Pax6-positive aNPs and an increased number of Tbr2-positive basal progenitors destined to become neurons. These Pax6-positive aNPs display a tilted mitotic spindle. In mice wherein p600 is ablated in progenitors, the production of neurons is significantly impaired and this defect is associated with microcephaly. We propose a working model in which p600 controls spindle orientation in aNPs and discuss its implication for neurogenesis.

During interphase, the spindle assembly factor TPX2 is compartmentalized in the nucleus where its roles remain largely uncharacterized. Recently, we found that TPX2 regulates the levels of serine 139-phosphoryated H2AX (γ-H2AX) at chromosomal breaks induced by ionizing radiation. Here, we report that TPX2 readily associates with the chromatin in the absence of ionizing radiation. Overexpression of TPX2 alters the DAPI staining pattern of interphase cells and depletion of TPX2 constitutively decreases the levels of histone H4 acetylated at lysine16 (H4K16ac) during G1-phase. Upon ionizing irradiation, this constitutive TPX2 depletion-dependent decrease in H4K16ac levels correlates with increased levels of γ-H2AX. The inversely correlated levels of H4K16ac and γ-H2AX can also be modified by altering the levels of SIRT1, herein identified as a novel protein complex partner of TPX2. Furthermore, we find that TPX2 depletion also interferes with formation of 53BP1 ionizing radiation-induced foci, known to depend on γ-H2AX and the acetylation status of H4K16. In brief, our study is the first indication of a constitutive control of TPX2 on H4K16ac levels, with potential implications for DNA damage response.

Cytoskeleton dynamics, membranes trafficking and positioning are essential for the proper functioning of any mammalian cell. The identification of the molecules and mechanisms that allow these cellular processes to interface is vital for understanding cell behaviors. Ndel1, the mammalian homolog of the Aspergillus nidulans NudE, organizes the cytoskeleton and regulates molecular motors, thereby impacting on the positioning of membranes. Hypothetically, Ndel1 can act in concert with enzymes controlling membrane trafficking (vesicle-mediated transport) per se, but this idea has never been investigated. We now report that a pool of Ndel1 associates directly with Dynamin 2 (Dyn2), a large cytosolic GTPase involved in the trafficking of the AMPA receptor subunit GluR1. In vitro, Ndel1 enhances Dyn2 GTPase activity in its unassembled and assembled forms, without promoting oligomerization of the enzyme. In cells, gain and loss of function of Ndel1 recapitulate the effects of overexpression of Dyn2 and Dyn2 dominant negative with reduced GTPase activity on the intracellular localization of GluR1, respectively, without affecting the stability of microtubules. Together, these results indicate that Ndel1 regulates Dyn2 GTPase activity and impacts GluR1-containing membranes distribution in a manner reminiscent of Dyn2.

Failure of axons to regenerate following acute or chronic neuronal injury is attributed to both the inhibitory glial environment and deficient intrinsic ability to re-grow. However, the underlying mechanisms of the latter remain unclear. In this study, we have investigated the role of the mammalian homologue of aspergillus nidulans NudE, Ndel1, emergently viewed as an integrator of the cytoskeleton, in axon regeneration. Ndel1 was synthesized de novo and upregulated in crushed and transected sciatic nerve axons, and, upon injury, was strongly associated with neuronal form of the intermediate filament (IF) Vimentin while dissociating from the mature neuronal IF (Neurofilament) light chain NF-L. Consistent with a role for Ndel1 in the conditioning lesion-induced neurite outgrowth of Dorsal Root Ganglion (DRG) neurons, the long lasting in vivo formation of the neuronal Ndel1/Vimentin complex was associated with robust axon regeneration. Furthermore, local silencing of Ndel1 in transected axons by siRNA severely reduced the extent of regeneration in vivo. Thus, Ndel1 promotes axonal regeneration; activating this endogenous repair mechanism may enhance neuroregeneration during acute and chronic axonal degeneration.