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AbstractBackgroundParkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, which promotes a sustained inflammatory environment in the central nervous system. Regulatory T cells (Tregs) play an important role in the control of inflammation and might play a neuroprotective role. Indeed, a decrease in Treg number and function has been reported in PD. In this context, pramipexole, a dopaminergic receptor agonist used to treat PD symptoms, has been shown to increase peripheral levels of Treg cells and improve their suppressive function. The aim of this work was to determine the effect of pramipexole on immunoregulatory Treg cells and its possible neuroprotective effect on human dopaminergic neurons differentiated from human embryonic stem cells.MethodsTreg cells were sorted from white blood cells of healthy human donors. Assays were performed with CD3/CD28?activated and non?activated Treg cells treated with pramipexole at concentrations of 2 or 200 ng/mL. These regulatory cells were co?cultured with in vitro?differentiated human dopaminergic neurons in a cytotoxicity assay with 6?hydroxydopamine (6?OHDA). The role of interleukin?10 (IL?10) was investigated by co?culturing activated IL?10?producing Treg cells with neurons. To further investigate the effect of treatment on Tregs, gene expression in pramipexole?treated, CD3/CD28?activated Treg cells was determined by Fluidigm analysis.ResultsPramipexole?treated CD3/CD28?activated Treg cells showed significant protective effects on dopaminergic neurons when challenged with 6?OHDA. Pramipexole?treated activated Treg cells showed neuroprotective capacity through mechanisms involving IL?10 release and the activation of genes associated with regulation and neuroprotection.ConclusionAnti?CD3/CD28?activated Treg cells protect dopaminergic neurons against 6?OHDA?induced damage. In addition, activated, IL?10?producing, pramipexole?treated Tregs also induced a neuroprotective effect, and the supernatants of these co?cultures promoted axonal growth. Pramipexole?treated, activated Tregs altered their gene expression in a concentration?dependent manner, and enhanced TGF??related dopamine receptor regulation and immune?related pathways. These findings open new perspectives for the development of immunomodulatory therapies for the treatment of PD. Pramipexole?treated, activated regulatory T cells protect dopaminergic neurons against 6 OHDA damage and promote primary neurite length. This could be due to the production of the regulatory cytokine IL?10 and an increased expression of genes related to regulation and neuroprotection.

BackgroundStem-cell-derived therapy is a promising option for tissue regeneration. Human iPSC-derived progenitors of smooth muscle cells (pSMCs) exhibit limited proliferation and differentiation, which minimizes the risk of tumor formation while restoring smooth muscle cells (SMCs). Up to 29% of women suffer from recurrence of vaginal prolapse after prolapse surgery. Therefore, there is a need for therapies that can restore vaginal function. SMCs contribute to vaginal tone and contractility. We sought to examine whether human pSMCs can restore vaginal function in a rat model.MethodsFemale immunocompromised RNU rats were divided into 5 groups: intact controls (n?=?12), VSHAM (surgery?+?saline injection, n?=?35), and three cell-injection groups (surgery?+?cell injection using pSMCs from three patients, n?=?14/cell line). The surgery to induce vaginal injury was analogous to prolapse surgery. Menopause was induced by surgical ovariectomy. The vagina, urethra, bladder were harvested 10 weeks after surgery (5 weeks after cell injection). Organ bath myography was performed to evaluate the contractile function of the vagina, and smooth muscle thickness was examined by tissue immunohistochemistry. Collagen I, collagen III, and elastin mRNA and protein expressions in tissues were assessed.ResultsVaginal smooth muscle contractions induced by carbachol and KCl in the cell-injection groups were significantly greater than those in the VSHAM group. Collagen I protein expression in the vagina of the cell-injections groups was significantly higher than in the VSHAM group. Vaginal elastin protein expression was similar between the cell-injection and VSHAM groups. In the urethra, gene expression levels of collagen I, III, and elastin were all significantly greater in the cell-injection groups than in the VSHAM group. Collagen I, III, and elastin protein expression of the urethra did not show a consistent trend between cell-injection groups and the VSHAM group.ConclusionsHuman iPSC-derived pSMCs transplantation appears to be associated with improved contractile function of the surgically injured vagina in a rat model. This is accompanied by changes in extracellular protein expression the vagina and urethra. These observations support further efforts in the translation of pSMCs into a treatment for regenerating the surgically injured vagina in women who suffer recurrent prolapse after surgery.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03900-3.

The immune-modulatory effects of mesenchymal stromal cells (MSCs) are widely used to treat inflammatory disorders, with indoleamine 2,4-dioxygenase-1 (IDO-1) playing a pivotal role in suppressing stimulated T-cell proliferation. Taking that three-dimensional (3D) cultures enhance MSCs’ anti-inflammatory properties compared with two-dimensional (2D) cultures, the differentially expressed miRNAs were examined. Thus, we identified hsa-miR-4662a-5p (miR-4662a) as a key inducer of IDO-1 via its suppression of bridging integrator-1 (BIN-1), a negative regulator of the IDO-1 gene. The IDO-1-inducing potential of miR-4662a was conserved across primary MSCs from various donors and sources but exhibited variability. Notably, iPSC-derived MSCs (iMSCs) demonstrated superior IDO-1 induction and immune-modulatory efficacy compared with their donor-matched primary MSCs. Accordingly, iMSCs expressing miR-4662a (4662a/iMSC) exhibited stronger suppressive effects on T-cell proliferation and more potent suppressive effects on graft-versus-host disease (GVHD), improving survival rates and reducing tissue damage in the liver and gut. Our results point to the therapeutic potential of standardized, off-the-shelf 4662a/iMSC as a robust immune-modulating cell therapy for GVHD.

New approach methodologies (NAMs) for predicting embryotoxicity and developmental toxicity are urgently needed for generating human relevant data, while reducing turnover time and costs, and alleviating ethical concerns related to the use of animal models. We have previously developed the PluriLum assay, a NKX2.5-reporter gene 3D model using human-induced pluripotent stem cells (hiPSCs) that are genetically modified to enable the assessment of adverse effects of chemicals on the early-stage embryo. Aiming at improving the predictive value of the PluriLum assay for future screening purposes, we sought to introduce standardization steps to the protocol, improving the overall robustness of the PluriLum assay, as well as a shortening of the assay protocol. First, we showed that the initial size of embryoid bodies (EBs) is crucial for a proper differentiation into cardiomyocytes and overall reproducibility of the assay. When the starting diameter of the EBs exceeds 500 µm, robust differentiation can be anticipated. In terms of reproducibility, exposure to the fungicide epoxiconazole at smaller initial diameters resulted in a larger variation of the derived data, compared to more reliable concentration–response curves obtained using spheroids with larger initial diameters. We further investigated the ideal length of the differentiation protocol, resulting in a shortening of the PluriLum assay by 24 h to 7 days. Following exposure to the teratogens all-trans and 13-cis retinoic acid, both cardiomyocyte contraction and measurement of NKX2.5-derived luminescence were recorded with a similar or increased sensitivity after 6 days of differentiation when compared to the original 7 days. Finally, we have introduced an efficient step for enzymatic dissociation of the EBs at assay termination. This allows for an even splitting of the individual EBs and testing of additional endpoints other than the NKX2.5-luciferase reporter, which was demonstrated in this work by the simultaneous assessment of ATP levels. In conclusion, we have introduced standardizations and streamlined the PluriLum assay protocol to improve its suitability as a NAM for screening of a large number of chemicals for developmental toxicity testing.