Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD, and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs, using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected, cell lines harboring the PSEN2 N141I mutation displayed an increase in the Aβ42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 N141I lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit, restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased Aβ42/40 was also normalized following CRISPR/Cas-mediated correction of the PSEN2 N141I mutation. The genome editing data confirms the robust consistency of mutation-related changes in Aβ42/40 ratio while also showing a PSEN2-mutation-related alteration in electrophysiology.Electronic supplementary materialThe online version of this article doi: (10.1186/s40478-017-0475-z) contains supplementary material, which is available to authorized users.

DYT6 dystonia is caused by mutations in THAP1 [Thanatos-associated (THAP) domain-containing apoptosis-associated protein] and is autosomal dominant and partially penetrant. Like other genetic primary dystonias, DYT6 patients have no characteristic neuropathology, and mechanisms by which mutations in THAP1 cause dystonia are unknown. Thap1 is a zinc-finger transcription factor, and most pathogenic THAP1 mutations are missense and are located in the DNA-binding domain. There are also nonsense mutations, which act as the equivalent of a null allele because they result in the generation of small mRNA species that are likely rapidly degraded via nonsense-mediated decay. The function of Thap1 in neurons is unknown, but there is a unique, neuronal 50-kDa Thap1 species, and Thap1 levels are auto-regulated on the mRNA level. Herein, we present the first characterization of two mouse models of DYT6, including a pathogenic knockin mutation, C54Y and a null mutation. Alterations in motor behaviors, transcription and brain structure are demonstrated. The projection neurons of the deep cerebellar nuclei are especially altered. Abnormalities vary according to genotype, sex, age and/or brain region, but importantly, overlap with those of other dystonia mouse models. These data highlight the similarities and differences in age- and cell-specific effects of a Thap1 mutation, indicating that the pathophysiology of THAP1 mutations should be assayed at multiple ages and neuronal types and support the notion of final common pathways in the pathophysiology of dystonia arising from disparate mutations.

Adipose mesenchymal stem cells (ASC) are considered minimally immunogenic. This is due to the low expression of human leukocyte antigens I (HLA-I), lack of HLA-II expression and low expression of co-stimulatory molecules such as CD40 and CD80. The low rate of observed immunological rejection as well as the immunomodulatory qualities, position ASC as a promising cell-based therapy for the treatment of a variety of inflammatory indications. Yet, few studies have addressed relevant aspects of immunogenicity such as ASC donor-to-patient HLA histocompatibility or assessment of immune response triggered by ASC administration, particularly in the cases of presensitization. The present study aims to assess allo-immune responses in a cohort of Crohn's disease patients administered with allogeneic ASC (darvadstrocel formerly Cx601) for the treatment of complex perianal fistulas. We identified donor-specific antibodies (DSA) generation in a proportion of patients and observed that patients showing preexisting immunity were prone to generating DSA after allogeneic therapy. Noteworthy, naïve patients generating DSA at week 12 (W12) showed a significant reduction in DSA titer at week 52 (W52), whereas DSA titer was reduced in pre-sensitized patients only with no specificities against the donor administered. Remarkably, we did not observe any correlation of DSA generation with ASC therapeutic efficacy. In vitro complement-dependent cytotoxicity (CDC) studies have revealed limited cytotoxic levels based upon HLA-I expression and binding capacity even in pro-inflammatory conditions. We sought to identify CDC coping mechanisms contributing to the limited cytotoxic killing observed in ASC in vitro . We found that ASC express membrane-bound complement regulatory proteins (mCRPs) CD55, CD46, and CD59 at basal levels, with CD46 more actively expressed in pro-inflammatory conditions. We demonstrated that CD46 is a main driver of CDC signaling; its depletion significantly enhances sensitivity of ASC to CDC. In summary, despite relatively high clearance, DSA generation may represent a major challenge for allogeneic cell therapy management. Sensitization may be a significant concern when evaluating re-treatment or multi-donor trials. It is still unknown whether DSA generation could potentially be the consequence of donor-to-patient interaction and, therefore, subsequently link to efficacy or biological activity. Lastly, we propose that CDC modulators such as CD46 could be used to ultimately link CDC specificity with allogeneic cell therapy efficacy.

BackgroundType 2 diabetes (T2D) is a recognized risk factor for the development of cognitive impairment (CI) and/or dementia, although the exact nature of the molecular pathology of T2D-associated CI remains obscure. One link between T2D and CI might involve decreased insulin signaling in brain and/or neurons in either animal or postmortem human brains as has been reported as a feature of Alzheimer’s disease (AD). Here we asked if neuronal insulin resistance is a cell autonomous phenomenon in a familial form of AD.MethodsWe have applied a newly developed protocol for deriving human basal forebrain cholinergic neurons (BFCN) from skin fibroblasts via induced pluripotent stem cell (iPSC) technology. We generated wildtype and familial AD mutant PSEN2N141I (presenilin 2) BFCNs and assessed if insulin signaling, insulin regulation of the major AD proteins Aβ and/or tau, and/or calcium fluxes is altered by the PSEN2N141I mutation.ResultsWe report herein that wildtype, PSEN2N141I and CRISPR/Cas9-corrected iPSC-derived BFCNs (and their precursors) show indistinguishable insulin signaling profiles as determined by the phosphorylation of canonical insulin signaling pathway molecules. Chronic insulin treatment of BFCNs of all genotypes led to a reduction in the Aβ42/40 ratio. Unexpectedly, we found a CRISPR/Cas9-correctable effect of PSEN2N141I on calcium flux, which could be prevented by chronic exposure of BFCNs to insulin.ConclusionsOur studies indicate that the familial AD mutation PSEN2N141I does not induce neuronal insulin resistance in a cell autonomous fashion. The ability of insulin to correct calcium fluxes and to lower Aβ42/40 ratio suggests that insulin acts to oppose an AD-pathophysiology. Hence, our results are consistent with a potential physiological role for insulin as a mediator of resilience by counteracting specific metabolic and molecular features of AD.Electronic supplementary materialThe online version of this article (10.1186/s13024-018-0265-5) contains supplementary material, which is available to authorized users.

Background Mesenchymal stem cells (MSCs) activate the endogenous immune regulatory system, inducing a therapeutic effect in recipients. MSCs have demonstrated the ability to modulate the differentiation of myeloid cells toward a phagocytic and anti-inflammatory profile. Allogeneic, adipose-derived MSCs (ASCs) have been investigated for the management of complex perianal fistula, with darvadstrocel being the first ASC therapy approved in Europe in March 2018. Additionally, ASCs are being explored as a potential treatment in other indications. Yet, despite these clinical advances, their mechanism of action is only partially understood. Methods Freshly isolated human monocytes from the peripheral blood were differentiated in vitro toward M0 non-polarized macrophages (Mphs), M1 pro-inflammatory Mphs, M2 anti-inflammatory Mphs, or mature dendritic cells (mDCs) in the presence or absence of ASCs, in non-contact conditions. The phenotype and function of the differentiated myeloid populations were determined by flow cytometry, and their secretome was analyzed by OLINK technology. We also investigated the capacity of ASCs to modulate the phenotype and function of terminally differentiated M1 Mphs. The role of soluble factors interleukin (IL)-6 and prostaglandin E2 (PGE2) on the ability of ASCs to modulate myeloid cells was assessed using neutralization assays, CRISPR/Cas9 knock-down of cyclooxygenase 2 (COX-2), and ASC-conditioned medium assays using pro-inflammatory stimulus. Results Co-culture of monocytes in the presence of ASCs resulted in the polarization of Mphs and mDCs toward an anti-inflammatory and phagocytic phenotype. This was characterized by an increase in phagocytic receptors on the cell surface of Mphs (M0, M1, and M2) and mDCs, as well as modulation of chemokine receptors and reduced expression of pro-inflammatory, co-stimulatory molecules. ASCs also modulated the secretome of Mphs and mDCs, demonstrated by reduced expression of pro-inflammatory factors and increased expression of anti-inflammatory and reparative factors. Chemical inhibition of PGE2 with indomethacin abolished this modulatory effect, whereas treatment with a neutralizing anti-IL-6 antibody resulted in a partial abolishment. The knock-down of COX-2 in ASCs and the use of IL-1β-activated ASC-conditioned media confirmed the key role of PGE2 in ASC-mediated myeloid modulation. In our in vitro experimental settings, ASCs failed to modulate the phenotype and function of terminally polarized M1 Mphs. Conclusions The results demonstrate that ASCs are able to modulate the in vitro differentiation of myeloid cells toward an anti-inflammatory and reparative profile. This modulatory effect was mediated mainly by PGE2 and, to a lesser extent, IL-6.