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Rationale: Brain pericytes can acquire multipotency to produce multi-lineage cells following injury. However,pericytes are a heterogenous population and it remains unknown whether there are different potencies from different subsets of pericytes in response to injury.Methods: We used an ischemic stroke model combined with pericyte lineage tracing animal models to investigate brain pericyte heterogeneity under both naïve and brain injury conditions via single-cell RNA-sequencing and immunohistochemistry analysis. In addition,we developed an NG2+ pericyte neural reprogramming culture model from both murine and humans to unveil the role of energy sensor,AMP-dependent kinase (AMPK),activity in modulating the reprogramming/differentiation process to convert pericytes to functional neurons by targeting a Ser 436 phosphorylation on CREB-binding protein (CBP),a histone acetyltransferase.Results: We showed that two distinct pericyte subpopulations,marked by NG2+ and Tbx18+,had different potency following brain injury. NG2+ pericytes expressed dominant neural reprogramming potential to produce newborn neurons,while Tbx18+ pericytes displayed dominant multipotency to produce endothelial cells,fibroblasts,and microglia following ischemic stroke. In addition,we discovered that AMPK modulators facilitated pericyte-to-neuron conversion by modulating Ser436 phosphorylation status of CBP,to coordinate an acetylation shift between Sox2 and histone H2B,and to regulate Sox2 nuclear-cytoplasmic trafficking during the reprogramming/differentiation process. Finally,we showed that sequential treatment of compound C (CpdC) and metformin,AMPK inhibitor and activator respectively,robustly facilitated the conversion of human pericytes into functional neurons.Conclusion: We revealed that two distinct subtypes of pericytes possess different reprogramming potencies in response to physical and ischemic injuries. We also developed a genomic integration-free methodology to reprogram human pericytes into functional neurons by targeting NG2+ pericytes. View Publication

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. View Publication

Peripheral nerve and large-scale muscle injuries result in significant disability,necessitating the development of biomaterials that can restore functional deficits by promoting tissue regrowth in an electroactive environment. Among these materials,graphene is favored for its high conductivity,but its low bioactivity requires enhancement through biomimetic components. In this study,we extrusion printed graphene-poly(lactide-co-glycolide) (graphene) lattice scaffolds,aiming to increase bioactivity by incorporating decellularized extracellular matrix (dECM) derived from mouse pup skeletal muscle. We first evaluated these scaffolds using human-induced pluripotent stem cell (hiPSC)-derived motor neurons co-cultured with supportive glia,observing significant improvements in axon outgrowth. Next,we tested the scaffolds with C2C12 mouse and human primary myoblasts,finding no significant differences in myotube formation between dECM-graphene and graphene scaffolds. Finally,using a more complex hiPSC-derived 3D motor neuron spheroid model co-cultured with human myoblasts,we demonstrated that dECM-graphene scaffolds significantly improved axonal expansion towards peripheral myoblasts and increased axonal network density compared to graphene-only scaffolds. Features of early neuromuscular junction formation were identified near neuromuscular interfaces in both scaffold types. These findings suggest that dECM-graphene scaffolds are promising candidates for enhancing neuromuscular regeneration,offering robust support for the growth and development of diverse neuromuscular tissues. View Publication

There has been a recent drive to replace in vivo studies with in vitro studies in the field of toxicity testing. Therefore,instead of conventional animal or planar cell culture models,there is an urgent need for in vitro systems whose conditions can be strictly controlled,including cell–cell interactions and sensitivity to low doses of chemicals. Neural organoids generated from human-induced pluripotent stem cells (iPSCs) are a promising in vitro platform for modeling human brain development. In this study,we developed a new tool based on various iPSCs to study and predict chemical-induced toxicity in humans. The model displayed several neurodevelopmental features and showed good reproducibility,comparable to that of previously published models. The results revealed that basic fibroblast growth factor plays a key role in the formation of the embryoid body,as well as complex neural networks and higher-order structures such as layered stacking. Using organoid models,pesticide toxicities were assessed. Cells treated with low concentrations of rotenone underwent apoptosis to a greater extent than those treated with high concentrations of rotenone. Morphological changes associated with the development of neural progenitor cells were observed after exposure to low doses of chlorpyrifos. These findings suggest that the neuronal organoids developed in this study mimic the developmental processes occurring in the brain and nerves and are a useful tool for evaluating drug efficacy,safety,and toxicity. View Publication

Huntington’s disease (HD) causes selective degeneration of striatal and cortical neurons,resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells,grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids,confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons,while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity,trajectories,synaptic density,and communication pathways upon cell-cell contact,while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells,emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment. Mosaic organoids where pathological and healthy cells are grown together,reveal the rescue of phenotypes in pathological cells due to communication with healthy cells without harming them,as demonstrated by single-cell RNA-sequencing data. View Publication

Purpose: CLN3 Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset,motor decline and premature death. Patient-derived CLN3 disease induced pluripotent stem cell-RPE cells show defective phagocytosis of photoreceptor outer segment (POS). Because modifier genes are implicated in CLN3 disease,our goal here was to investigate a direct link between CLN3 mutation and POS phagocytosis defect. Methods: Isogenic control and CLN3 mutant stem cell lines were generated by CRISPR-Cas9-mediated biallelic deletion of exons 7 and 8. A transgenic CLN3Δ7-8/Δ7-8 (CLN3) Yucatan miniswine was also used to study the impact of CLN3Δ7-8/Δ7-8 mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram,optical coherence tomography and histological analysis of CLN3Δ7-8/Δ7-8 and wild-type miniswine eyes were carried out at 6,36,or 48 months of age. Results: CLN3Δ7-8/Δ7-8 RPE (CLN3 RPE) displayed decreased POS binding and consequently decreased uptake of POS compared with isogenic control RPE cells. Furthermore,wild-type miniswine RPE cells phagocytosed CLN3Δ7-8/Δ7-8 POS less efficiently than wild-type POS. Consistent with decreased POS phagocytosis,lipofuscin/autofluorescence was decreased in CLN3 miniswine RPE at 36 months of age and was followed by almost complete loss of photoreceptors at 48 months of age. Conclusions: CLN3Δ7-8/Δ7-8 mutation (which affects ≤85% of patients) affects both RPE and POS and leads to photoreceptor cell loss in CLN3 disease. Furthermore,both primary RPE dysfunction and mutant POS independently contribute to impaired POS phagocytosis in CLN3 disease. View Publication

Human-induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) represent a promising and renewable cell source for therapeutic applications. A systematic evaluation of the immunological properties and engraftment potential of iMSCs generated from urine-derived iPSCs is lacking,which has impeded their broader application. In this study,we differentiated urine-derived iPSCs into iMSCs and assessed their fundamental MSC characteristics,immunogenicity,immunomodulatory capacity and in vivo engraftment. Compared to umbilical cord-derived MSCs (UCMSCs),iMSCs demonstrated an enhanced proliferative capacity,a higher level of regenerative gene expression,and lower immunogenicity,strengthening resistance to apoptosis induced by allogeneic peripheral blood mononuclear cells (PBMCs) and the NK-92 cell line. In addition,iMSCs exhibited a diminished ability to inhibit T cell proliferation and activation compared with UCMSCs. Transcriptomic analyses further revealed the decreased expression of immune regulatory factors in iMSCs. After transfusion into mouse models,iMSCs engrafted in the lungs,liver,and spleen and exhibited the ability to migrate to tumor tissues. Our results indicated that iMSCs generated from urine-derived iPSCs have a significant replicative capacity,low immunogenicity and unique immunomodulatory properties,and hence offer obvious advantages in immune privilege and allogenic therapeutic application prospects. View Publication

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study,we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg,HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV,thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection,we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients,we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions. View Publication

Angelman syndrome (AS) and Prader-Willi syndrome (PWS),two distinct neurodevelopmental disorders,result from loss of expression from imprinted genes in the chromosome 15q11-13 locus most commonly caused by a megabase-scale deletion on either the maternal or paternal allele,respectively. Each occurs at an approximate incidence of 1/15,000 to 1/30,000 live births and has a range of debilitating phenotypes. Patient-derived induced pluripotent stem cells (iPSCs) have been valuable tools to understand human-relevant gene regulation at this locus and have contributed to the development of therapeutic approaches for AS. Nonetheless,gaps remain in our understanding of how these deletions contribute to dysregulation and phenotypes of AS and PWS. Variability across cell lines due to donor differences,reprogramming methods,and genetic background make it challenging to fill these gaps in knowledge without substantially increasing the number of cell lines used in the analyses. Isogenic cell lines that differ only by the genetic mutation causing the disease can ease this burden without requiring such a large number of cell lines. Here,we describe the development of isogenic human embryonic stem cell (hESC) lines modeling the most common genetic subtypes of AS and PWS. These lines allow for a facile interrogation of allele-specific gene regulation at the chromosome 15q11-q13 locus. Additionally,these lines are an important resource to identify and test targeted therapeutic approaches for patients with AS and PWS. View Publication

BackgroundHuman pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show tremendous promise for cardiac regeneration following myocardial infarction (MI),but their transplantation gives rise to transient ventricular tachycardia (VT) in large-animal MI models,representing a major hurdle to translation. Our group previously reported that these arrhythmias arise from a focal mechanism whereby graft tissue functions as an ectopic pacemaker; therefore,we hypothesized that hPSC-CMs engineered with a dominant negative form of the pacemaker ion channel HCN4 (dnHCN4) would exhibit reduced automaticity and arrhythmogenic risk following transplantation.MethodsWe used CRISPR/Cas9-mediated gene-editing to create transgenic dnHCN4 hPSC-CMs,and their electrophysiological behavior was evaluated in vitro by patch-clamp recordings and optical mapping. Next,we transplanted WT and homozygous dnHCN4 hPSC-CMs in a pig MI model and compared post-transplantation outcomes including the incidence of spontaneous arrhythmias and graft structure by immunohistochemistry.ResultsIn vitro dnHCN4 hPSC-CMs exhibited significantly reduced automaticity and pacemaker funny current (If) density relative to wildtype (WT) cardiomyocytes. Following transplantation with either dnHCN4 or WT hPSC-CMs,all recipient hearts showed transmural infarct scar that was partially remuscularized by scattered islands of human myocardium. However,in contrast to our hypothesis,both dnHCN4 and WT hPSC-CM recipients exhibited frequent episodes of ventricular tachycardia (VT).ConclusionsWhile genetic silencing of the pacemaker ion channel HCN4 suppresses the automaticity of hPSC-CMs in vitro,this intervention is insufficient to reduce VT risk post-transplantation in the pig MI model,implying more complex mechanism(s) are operational in vivo. View Publication

BackgroundSpinal ventral root avulsion results in massive motoneuron degeneration with poor prognosis and high costs. In this study,we compared different isoforms of basic fibroblast growth factor 2 (FGF2),overexpressed in stably transfected Human embryonic stem cells (hESCs),following motor root avulsion and repair with a heterologous fibrin biopolymer (HFB).MethodsIn the present work,hESCs bioengineered to overexpress 18,23,and 31 kD isoforms of FGF2,were used in combination with reimplantation of the avulsed roots using HFB. Statistical analysis was conducted using GraphPad Prism software with one-way or two-way ANOVA,followed by Tukey’s or Dunnett’s multiple comparison tests. Significance was set at *p < 0.05,**p < 0.01,***p < 0.001,and ****p < 0.0001.ResultsFor the first set of experiments,rats underwent avulsion of the ventral roots with local administration of HFB and engraftment of hESCs expressing the above-mentioned FGF2 isoforms. Analysis of motoneuron survival,glial reaction,and synaptic coverage,two weeks after the lesion,indicated that therapy with hESCs overexpressing 31 kD FGF2 was the most effective. Consequently,the second set of experiments was performed with that isoform,so that ventral root avulsion was followed by direct spinal cord reimplantation. Motoneuron survival,glial reaction,synaptic coverage,and gene expression were analyzed 2 weeks post-lesion; while the functional recovery was evaluated by the walking track test and von Frey test for 12 weeks. We showed that engraftment of hESCs led to significant neuroprotection,coupled with immunomodulation,attenuation of astrogliosis,and preservation of inputs to the rescued motoneurons. Behaviorally,the 31 kD FGF2 - hESC therapy enhanced both motor and sensory recovery.ConclusionTransgenic hESCs were an effective delivery platform for neurotrophic factors,rescuing axotomized motoneurons and modulating glial response after proximal spinal cord root injury,while the 31 kD isoform of FGF2 showed superior regenerative properties over other isoforms in addition to the significant functional recovery.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03676-6. View Publication

Induced pluripotent stem cells (iPSCs) can be generated from various adult cells,genetically modified and differentiated into diverse cell populations. Type I interferons (IFN-Is) have multiple immunotherapeutic applications; however,their systemic administration can lead to severe adverse outcomes. One way of overcoming the limitation is to introduce cells able to enter the site of pathology and to produce IFN-Is locally. As a first step towards the generation of such cells,here,we aimed to generate human iPSCs overexpressing interferon-beta (IFNB,IFNB-iPSCs). IFNB-iPSCs were obtained by CRISPR/Cas9 editing of the previously generated iPSC line K7-4Lf. IFNB-iPSCs overexpressed IFNB RNA and produced a functionally active IFN-?. The cells displayed typical iPSC morphology and expressed pluripotency markers. Following spontaneous differentiation,IFNB-iPSCs formed embryoid bodies and upregulated endoderm,mesoderm,and some ectoderm markers. However,an upregulation of key neuroectoderm markers,PAX6 and LHX2,was compromised. A negative effect of IFN-? on iPSC neuroectoderm differentiation was confirmed in parental iPSCs differentiated in the presence of a recombinant IFN-?. The study describes new IFN-?-producing iPSC lines suitable for the generation of various types of IFN-?-producing cells for future experimental and clinical applications,and it unravels an inhibitory effect of IFN-? on stem cell neuroectoderm differentiation. View Publication