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Induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (iMSCs) offer a promising alternative to primary mesenchymal stromal cells (MSCs) and their derivatives,particularly extracellular vesicles (EVs),for use in advanced therapy medicinal products. In this study we evaluated the immunomodulatory and regenerative potential of iMSCs as well as iMSC-EVs,alongside primary human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). Our findings demonstrate that iMSCs exhibit comparable abilities to hUCMSCs in regulating lymphocyte proliferation and inducing an anti-inflammatory phenotype in monocytes. We also observed decreased TNFα levels and increased IL-10 induction,indicating a potential mechanism for their immunomodulatory effects. Furthermore,iMSC-EVs also showed effective immunomodulation by inhibiting T cell proliferation and inducing macrophage polarization similar to their parental cells. Additionally,iMSC-EVs exhibited pro-regenerative potential akin to hUCMSC-EVs in in vitro scratch assays. Notably,priming iMSCs with pro-inflammatory cytokines significantly enhanced the immunomodulatory potential of iMSC-EVs. These results underscore the considerable promise of iMSCs and iMSCs-EVs as an alternate source for MSC-derived therapeutics,given their potent immunomodulatory and regenerative properties. The online version contains supplementary material available at 10.1038/s41598-024-75956-3. View Publication

Pericytes play a crucial role in controlling inflammation and vascular functions in the central nervous system,which are disrupted in Parkinson’s disease (PD). Still,there is a lack of studies on the impact of pericytes on neurodegenerative diseases,and their involvement in the pathology of PD is unclear. Our objective was to investigate the molecular and functional differences between healthy pericytes and pericytes with the LRRK2 G2019S mutation,which is one of the most common mutations associated with PD. Our study employed pericyte-like cells obtained from induced pluripotent stem cells produced from PD patients with the LRRK2 G2019S mutation as well as from healthy individuals. We examined the gene expression profiles of the cells and analyzed how the alterations reflect on their functionality. We have shown differences in the expression of genes related to inflammation and angiogenesis. Furthermore,we observe modified migration speed in PD pericyte-like cells as well as enhanced secretion of inflammatory mediators,such as soluble VCAM-1 and MCP-1,in these pericyte-like cells following exposure to proinflammatory stimuli. In summary,our findings support the notion that pericytes play a role in the inflammatory and vascular changes observed in PD. Further investigation of pericytes could provide valuable insight into understanding the pathogenesis of PD. The online version contains supplementary material available at 10.1186/s12987-024-00592-y. View Publication

Effective therapies for Alzheimer’s disease (AD) remain to be developed. APOE4 is the strongest genetic risk factor for late-onset AD. Pericyte degeneration and blood–brain barrier (BBB) disruption are thought to be early biomarkers of AD and contribute to cognitive decline in APOE4 carriers,representing potential therapeutic targets. Our previous studies have shown that pericyte transplantation is one of the most effective strategies for BBB restoration,exhibiting great therapeutic potential for APOE4-related BBB damage and AD phenotypes. Methods: APOE4/4 mice were treated with pericytes derived from APOE3/3 human induced pluripotent stem cells (hiPSCs). Behavioral tests,AD pathologies,and BBB integrity were assessed. Subsequently,temporal and spatial distribution of the transplanted pericytes was analyzed using tdTomato+ lentivirus labeling. Next,therapeutic effects of apoptotic vesicles (ApoVs) generated from APOE3/3 pericytes were evaluated in APOE4/4 pericytes in vitro. Additionally,transcriptomic and proteomic profiling were performed to identify key effector molecules in pericyte-derived ApoVs. Finally,the therapeutic effects of ApoVs derived from pericytes were evaluated in APOE4/4 mice. Results: Early,multiple transplantations of pericytes derived from APOE3/3 hiPSCs robustly rescued cognitive decline and AD pathologies,restored BBB integrity,and prevented in situ pericyte degeneration in aged APOE4/4 mice. Intriguingly,ApoVs released from the infused cells,rather than the transplanted pericytes,were predominantly distributed in the brain,which were ingested by in situ APOE4/4 pericytes and then promoted functional recovery. We further characterized insulin growth factor-2 (IGF-2) as a key factor in APOE3/3 pericyte-derived ApoVs. Infusion of the in vitro generated ApoVs from APOE3/3 pericytes demonstrated distinct therapeutic effects in APOE4/4 mice,which were reversed by IGF2 knockout. Conclusions: APOE3/3 pericytes or APOE3/3 pericyte-derived IGF2-rich ApoVs may offer promising therapeutic strategies for APOE4-associated AD. View Publication

Proteomics of dystrophic muscle samples is limited by the amount of protein that can be extracted from patient biopsies. Cells and tissues derived from patient-derived induced pluripotent stem cells (iPSCs) can be an expandable alternative source. We have patterned iPSCs from three Duchenne muscular dystrophy (DMD) patient lines into skeletal muscle cells using a two-dimensional as well as our three-dimensional organoid differentiation system. Probes with sufficient protein amounts could be extracted and prepared for mass spectrometry. In total,3007 proteins in 2D and 2709 proteins in 3D were detected in DMD patient probes. A total of 83 proteins in 2D and 338 proteins in 3D can be described as differentially expressed between DMD and control patient probes in a post hoc test. We have identified and we propose Myosin-9,Collagen 18A,Tropomyosin 1,BASP1,RUVBL1,and NCAM1 as proteins specifically altered in their expression in DMD for further investigation. Proteomics of skeletal muscle organoids resulted in greater consistency of results between cell lines in comparison to the two-dimensional myogenic differentiation protocol. View Publication

Chemotherapy-induced peripheral neuropathy (CIPN) affects up to two-thirds of cancer patients undergoing cytotoxic chemotherapy. Here,we used human iPSC-derived sensory neurons (iPSC-DSN) to model CIPN in vitro. Administration of various chemotherapeutic agents (i.e.,paclitaxel,vincristine,bortezomib and cisplatin) at clinically applicable concentrations resulted in reduced cell viability,axonal degeneration,electrophysiological dysfunction and increased levels of phosphorylated c-Jun in iPSC-DSN. Transcriptomic analyses revealed that the upregulation of c-Jun strongly correlated with the expression of genes of neuronal injury,apoptosis and inflammatory signatures. To test whether c-Jun plays a central role in the development of CIPN,we applied the small molecule inhibitor of the Jun N-terminal kinase,SP600125,to iPSC-DSN treated with neurotoxic chemotherapy. c-Jun inhibition prevented chemotherapy-induced neurotoxicity by preserving cell viability,axonal integrity and electrophysiological function of iPSC-DSN. These findings identify c-Jun as a key mediator of CIPN pathophysiology across multiple drug types and present preclinical evidence that c-Jun inhibition is an attractive therapeutic target to prevent CIPN. View Publication

Pancreatic β cells derived from human induced pluripotent stem cells (hiPSCs) represent a promising therapeutic avenue in regenerative medicine for diabetes treatment. However,current differentiation protocols lack the specificity and efficiency required to reliably produce fully functional β cells,limiting their clinical applicability. Epigenetic barriers,such as histone modifications,may hinder proper differentiation and the acquisition of essential maturation markers in these cells. Methods: hiPSCs were cultured under feeder-free conditions and subjected to lentiviral transduction with shRNA constructs to silence KDM4A. Differentiation into pancreatic β-like cells was performed using stepwise protocols,with or without doxycycline supplementation,to evaluate the effect of KDM4A suppression. Gene expression was quantified by RT-qPCR,protein expression was assessed by western blotting and immunofluorescence,and functional insulin release was determined by glucose-stimulated insulin secretion (GSIS) assays. Statistical analysis was conducted using unpaired two-tailed Student’s t-tests,with significance set at p < 0.05. Results: A reduction in pancreatic development proteins was observed in the different differentiation states evaluated,after blocking KDM4A expression. Knockdown of KDM4A significantly reduced the expression of pancreatic β-cell genes,such as PDX1,Nkx6.1,and Ins,by 50% compared to WT iPSCs differentiated under the same conditions. Similarly,glucose-stimulated insulin secretion was reduced by approximately 80% in KDM4A-deficient β-like cells. Conclusions: These results emphasize the critical role of histone demethylation in hiPSC differentiation toward β cells. Our findings identify KDM4A as a key epigenetic regulator,suggesting that its modulation could enhance the generation of functional β cells for regenerative medicine in diabetes. View Publication

Gastrointestinal (GI) dysfunction,characterized by severe diarrhea and dehydration,is a central contributor to morbidity and mortality in filovirus disease in patients,yet the role of the epithelium in this clinical outcome remains poorly defined. Here,we employ induced pluripotent stem cell (iPSC)-derived human intestinal (HIOs) and colonic organoids (HCOs) to model Ebola virus (EBOV) and Marburg virus (MARV) infection. These organoids are permissive to filovirus infection and support viral replication. Bulk RNA sequencing revealed distinct intestinal and colonic epithelial responses,including apical and junctional disruption and a delayed virus-specific induction of interferon-stimulated genes. Moreover,infection impaired adenylate cyclase signaling and CFTR-mediated ion transport,providing mechanistic insight into virus-induced secretory diarrhea. This platform recapitulates key features of human GI pathology in filoviral disease and serves as a powerful system to dissect host-pathogen interactions and identify therapeutic targets. Author summaryEbola virus (EBOV) and Marburg virus (MARV) are among the most lethal viruses known. Infection with these viruses leads to severe disease and death. One of their most harmful effects is damage to the gastrointestinal tract,causing intense diarrhea and life-threatening dehydration. Yet,how these viruses affect the gut remains poorly understood. In this study,we used human mini-guts—small,three-dimensional tissues grown from stem cells that mimic the human intestinal and colonic epithelium—to investigate how these viruses interact with gut epithelial cells. We found that both EBOV and MARV infect and replicate in these tissues,disrupt key barrier structures,and interfere with the cells’ ability to regulate fluid secretion. These effects mirror the severe symptoms seen in patients. Our study provides new insight into how EBOV and MARV damage the gut and identifies specific cellular pathways that may be targeted for treatment. This research not only improves our understanding of EBOV and MARV infections but also offers new infection platforms for testing therapies aimed at protecting the gastrointestinal system during filovirus outbreaks. View Publication

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease,characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene,resulting in the generation of progerin,a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin,and more importantly,lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs,progerin and its ageing-associated phenotypic consequences are restored. Specifically,directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally,our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs,also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing,our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing. View Publication

Demonstrates efficient reprogramming of iPS cells from CD34+ stem cells enriched from a small volume of peripheral blood. View Publication

Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of,for example,ion selectivity,gating mechanism,composition,or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs) and their somatic cell source,keratinocytes from plucked human hair. This comparison revealed that 26&x25; of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6&x25; were downregulated. Additionally,iPSCs express a much higher number of ion channels compared to keratinocytes. Further,to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny,namely,neurons and cardiomyocytes derived from iPS cells. To conclude,hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms. View Publication

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm,which is manifested by rosette formation,with consecutive differentiation into neural progenitors and early glial-like cells. In this study,we examined the involvement of early neural markers - OTX2,PAX6,Sox1,Nestin,NR2F1,NR2F2,and IRX2 - in the onset of rosette formation,during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation,which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation,when rosettes comprise no more than 3-5 cells,and that its expression precedes that of established markers of early neuronal differentiation. Importantly,the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly,we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro,and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice. ?? 2013 Elsevier B.V. View Publication

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the degeneration of motor neurons. Currently,there is no effective therapy for ALS. Stem cell transplantation is a potential therapeutic strategy for ALS,and the reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) represents a novel cell source. In this study,we isolated a specific neural stem cell (NSC) population from human iPSCs based on high aldehyde dehydrogenase activity,low side scatter and integrin VLA4 positivity. We assessed the therapeutic effects of these NSCs on the phenotype of ALS mice after intrathecal or intravenous injections. Transplanted NSCs migrated and engrafted into the central nervous system via both routes of injection. Compared with control ALS,treated ALS mice exhibited improved neuromuscular function and motor unit pathology and significantly increased life span,in particular with the systemic administration of NSCs (15%). These positive effects are linked to multiple mechanisms,including production of neurotrophic factors and reduction of micro- and macrogliosis. NSCs induced a decrease in astrocyte number through the activation of the vanilloid receptor TRPV1. We conclude that minimally invasive injections of iPSC-derived NSCs can exert a therapeutic effect in ALS. This study contributes to advancements in iPSC-mediated approaches for treating ALS and other neurodegenerative diseases. View Publication