Previous research has demonstrated the feasibility of repairing nerve defects through acellular allogeneic nerve grafting with bone marrow mesenchymal stem cells.However,adult tissue–derived mesenchymal stem cells en...Previous research has demonstrated the feasibility of repairing nerve defects through acellular allogeneic nerve grafting with bone marrow mesenchymal stem cells.However,adult tissue–derived mesenchymal stem cells encounter various obstacles,including limited tissue sources,invasive acquisition methods,cellular heterogeneity,purification challenges,cellular senescence,and diminished pluripotency and proliferation over successive passages.In this study,we used induced pluripotent stem cell-derived mesenchymal stem cells,known for their self-renewal capacity,multilineage differentiation potential,and immunomodulatory characteristics.We used induced pluripotent stem cell-derived mesenchymal stem cells in conjunction with acellular nerve allografts to address a 10 mm-long defect in a rat model of sciatic nerve injury.Our findings reveal that induced pluripotent stem cell-derived mesenchymal stem cells exhibit survival for up to 17 days in a rat model of peripheral nerve injury with acellular nerve allograft transplantation.Furthermore,the combination of acellular nerve allograft and induced pluripotent stem cell-derived mesenchymal stem cells significantly accelerates the regeneration of injured axons and improves behavioral function recovery in rats.Additionally,our in vivo and in vitro experiments indicate that induced pluripotent stem cell-derived mesenchymal stem cells play a pivotal role in promoting neovascularization.Collectively,our results suggest the potential of acellular nerve allografts with induced pluripotent stem cell-derived mesenchymal stem cells to augment nerve regeneration in rats,offering promising therapeutic strategies for clinical translation.展开更多
Cerebral edema caused by blood-brain barrier injury after intracerebral hemorrhage is an important factor leading to poor prognosis.Human-induced pluripotent stem cell-derived neural stem cell exosomes(hiPSC-NSC-Exos)...Cerebral edema caused by blood-brain barrier injury after intracerebral hemorrhage is an important factor leading to poor prognosis.Human-induced pluripotent stem cell-derived neural stem cell exosomes(hiPSC-NSC-Exos)have shown potential for brain injury repair in central nervous system diseases.In this study,we explored the impact of hiPSC-NSC-Exos on blood-brain barrier preservation and the underlying mechanism.Our results indicated that intranasal delivery of hiPSC-NSC-Exos mitigated neurological deficits,enhanced blood-brain barrier integrity,and reduced leukocyte infiltration in a mouse model of intracerebral hemorrhage.Additionally,hiPSC-NSC-Exos decreased immune cell infiltration,activated astrocytes,and decreased the secretion of inflammatory cytokines like monocyte chemoattractant protein-1,macrophage inflammatory protein-1α,and tumor necrosis factor-αpost-intracerebral hemorrhage,thereby improving the inflammatory microenvironment.RNA sequencing indicated that hiPSC-NSC-Exo activated the PI3K/AKT signaling pathway in astrocytes and decreased monocyte chemoattractant protein-1 secretion,thereby improving blood-brain barrier integrity.Treatment with the PI3K/AKT inhibitor LY294002 or the monocyte chemoattractant protein-1 neutralizing agent C1142 abolished these effects.In summary,our findings suggest that hiPSC-NSC-Exos maintains blood-brain barrier integrity,in part by downregulating monocyte chemoattractant protein-1 secretion through activation of the PI3K/AKT signaling pathway in astrocytes.展开更多
Neuronal cell death and the loss of connectivity are two of the primary pathological mechanisms underlying Alzheimer's disease.The accumulation of amyloid-βpeptides,a key hallmark of Alzheimer's disease,is be...Neuronal cell death and the loss of connectivity are two of the primary pathological mechanisms underlying Alzheimer's disease.The accumulation of amyloid-βpeptides,a key hallmark of Alzheimer's disease,is believed to induce neuritic abnormalities,including reduced growth,extension,and abnormal growth cone morphology,all of which contribute to decreased connectivity.However,the precise cellular and molecular mechanisms governing this response remain unknown.In this study,we used an innovative approach to demonstrate the effect of amyloid-βon neurite dynamics in both two-dimensional and three-dimensional cultu re systems,in order to provide more physiologically relevant culture geometry.We utilized various methodologies,including the addition of exogenous amyloid-βpeptides to the culture medium,growth substrate coating,and the utilization of human-induced pluripotent stem cell technology,to investigate the effect of endogenous amyloid-βsecretion on neurite outgrowth,thus paving the way for potential future applications in personalized medicine.Additionally,we also explore the involvement of the Nogo signaling cascade in amyloid-β-induced neurite inhibition.We demonstrate that inhibition of downstream ROCK and RhoA components of the Nogo signaling pathway,achieved through modulation with Y-27632(a ROCK inhibitor)and Ibuprofen(a Rho A inhibitor),respectively,can restore and even enhance neuronal connectivity in the presence of amyloid-β.In summary,this study not only presents a novel culture approach that offers insights into the biological process of neurite growth and inhibition,but also proposes a specific mechanism for reduced neural connectivity in the presence of amyloid-βpeptides,along with potential intervention points to restore neurite growth.Thereby,we aim to establish a culture system that has the potential to serve as an assay for measuring preclinical,predictive outcomes of drugs and their ability to promote neurite outgrowth,both generally and in a patient-specific manner.展开更多
In recent years,the progression of stem cell therapies has shown great promise in advancing the nascent field of regenerative medicine.Considering the non-regenerative nature of the mature central nervous system,the c...In recent years,the progression of stem cell therapies has shown great promise in advancing the nascent field of regenerative medicine.Considering the non-regenerative nature of the mature central nervous system,the concept that“blank”cells could be reprogrammed and functionally integrated into host neural networks remained intriguing.Previous work has also demonstrated the ability of such cells to stimulate intrinsic growth programs in post-mitotic cells,such as neurons.While embryonic stem cells demonstrated great potential in treating central nervous system pathologies,ethical and technical concerns remained.These barriers,along with the clear necessity for this type of treatment,ultimately prompted the advent of induced pluripotent stem cells.The advantage of pluripotent cells in central nervous system regeneration is multifaceted,permitting differentiation into neural stem cells,neural progenitor cells,glia,and various neuronal subpopulations.The precise spatiotemporal application of extrinsic growth factors in vitro,in addition to microenvironmental signaling in vivo,influences the efficiency of this directed differentiation.While the pluri-or multipotency of these cells is appealing,it also poses the risk of unregulated differentiation and teratoma formation.Cells of the neuroectodermal lineage,such as neuronal subpopulations and glia,have been explored with varying degrees of success.Although the risk of cancer or teratoma formation is greatly reduced,each subpopulation varies in effectiveness and is influenced by a myriad of factors,such as the timing of the transplant,pathology type,and the ratio of accompanying progenitor cells.Furthermore,successful transplantation requires innovative approaches to develop delivery vectors that can mitigate cell death and support integration.Lastly,host immune responses to allogeneic grafts must be thoroughly characterized and further developed to reduce the need for immunosuppression.Translation to a clinical setting will involve careful consideration when assessing both physiologic and functional outcomes.This review will highlight both successes and challenges faced when using human induced pluripotent stem cell-derived cell transplantation therapies to promote endogenous regeneration.展开更多
The progressive loss of dopaminergic neurons in affected patient brains is one of the pathological features of Parkinson's disease,the second most common human neurodegenerative disease.Although the detailed patho...The progressive loss of dopaminergic neurons in affected patient brains is one of the pathological features of Parkinson's disease,the second most common human neurodegenerative disease.Although the detailed pathogenesis accounting for dopaminergic neuron degeneration in Parkinson's disease is still unclear,the advancement of stem cell approaches has shown promise for Parkinson's disease research and therapy.The induced pluripotent stem cells have been commonly used to generate dopaminergic neurons,which has provided valuable insights to improve our understanding of Parkinson's disease pathogenesis and contributed to anti-Parkinson's disease therapies.The current review discusses the practical approaches and potential applications of induced pluripotent stem cell techniques for generating and differentiating dopaminergic neurons from induced pluripotent stem cells.The benefits of induced pluripotent stem cell-based research are highlighted.Various dopaminergic neuron differentiation protocols from induced pluripotent stem cells are compared.The emerging three-dimension-based brain organoid models compared with conventional two-dimensional cell culture are evaluated.Finally,limitations,challenges,and future directions of induced pluripotent stem cell–based approaches are analyzed and proposed,which will be significant to the future application of induced pluripotent stem cell-related techniques for Parkinson's disease.展开更多
BACKGROUND Type 1 diabetes(T1D)results from the autoimmune-mediated loss of pancreatic β-cells.Current insulin therapies offer symptomatic relief but fall short of providing a definitive cure.Islet cell transplantati...BACKGROUND Type 1 diabetes(T1D)results from the autoimmune-mediated loss of pancreatic β-cells.Current insulin therapies offer symptomatic relief but fall short of providing a definitive cure.Islet cell transplantation,while promising,faces limitations related to donor scarcity,procedural complexities,and the necessity for long-term immunosuppression.Consequently,there is an urgent need for innovative strategies aimed at β-cell regeneration.Patient-derived induced pluripotent stem cells(iPSCs),obtained from peripheral blood mononuclear cells(PBMCs)of T1D patients,hold great potential as a source of cells for therapeutic purposes.Therefore,the differentiation of T1D-iPSCs into functional pancreatic β-cells is a critical step toward effective β-cell replacement therapy.AIM To assess the potential of patient-derived T1D-β-like cells(differentiated from T1D-iPSCs reprogrammed from T1D-PBMCs)for restoring β-cell function in T1D.METHODS T1D-iPSCs were reprogrammed from T1D-PBMCs using an episomal vectorbased approach.Pluripotency was confirmed by flow cytometry(FCM),quantitative real-time polymerase chain reaction,genomic stability analysis,and teratoma formation assays.Differentiation into pancreatic β-cells was optimized using triiodothyronine(T3),vitamin C(Vc),and an adenovirus(M3C)encoding pancreatic duodenal homeobox-1,neurogenin 3(Ngn3),and MAF bZIP transcription factor A(MafA).Following characterization of β-cell features by immunofluorescence,quantitative real-time polymerase chain reaction,and flow cytometry,therapeutic efficacy was assessed through blood glucose monitoring after transplantation under the renal capsule of streptozotocin-induced diabetic mice.RESULTS T1D-iPSCs were successfully generated from T1D-PBMCs.These cells exhibited the hallmark characteristics of pluripotent stem cells,including appropriate morphology,differentiation potential,genomic integrity,and expression of pluripotency-associated genes.Differentiation yielded insulin-positive(insulin+)pancreatic β-like cells that,at the mRNA level,expressed key β-cell markers such as pancreatic duodenal homeobox-1,Ngn3,MafA,NeuroD,glucagon-like peptide-1 receptor,Nkx6.1,glucose transporter 2,and Kir6.2.Notably,the T3+Vc group displayed the lowest Ngn3 expression(1.31±0.38 vs 1.96±0.25 vs 2.51±0.24,P<0.01),while the M3C+T3+Vc group exhibited the highest MafA expression(0.49±0.11 vs 0.32±0.06 vs 0.29±0.08,P<0.05).Both in vitro and in vivo assessments confirmed the insulin secretion ability of the generated β-like cells;however,they did not demonstrate appropriate modulation of insulin release in response to variations in extracellular glucose concentrations.CONCLUSION T1D-iPSCs derived from T1D-PBMCs can be differentiated into insulin+β-like cells,albeit with functional immaturity.These cells represent a potential source of seed cells for β-cell replacement therapy in T1D.展开更多
BACKGROUND The therapeutic potential of induced pluripotent stem cells(iPSCs)for Parkinson’s disease(PD)has been demonstrated.Exercise can also modulate metabolism to improve motor dysfunction in PD patients.AIM To i...BACKGROUND The therapeutic potential of induced pluripotent stem cells(iPSCs)for Parkinson’s disease(PD)has been demonstrated.Exercise can also modulate metabolism to improve motor dysfunction in PD patients.AIM To investigate the therapeutic effect of exercise combined with iPSCs in a PD mouse model and explore the underlying mechanisms.METHODS In this study,we included 10 normal mice and 40 PD model mice,which were divided into five groups:The control group(n=10),the sedentary PD group(St group,n=10),the exercise PD group(E group,n=10),the iPSC-treated PD group(T group,n=10),and the combined exercise and iPSC-treated PD group(ET group,n=10).The T and ET groups received cell injection therapy,while the E and ET groups underwent an 8-week exercise intervention.After the intervention,behavioral tests were performed on mice from all groups.Serum levels of epinephrine(EPI)and nerve growth factor were measured,and the expression of Wnt1,Lmx1a,and other factors related to the Wnt signaling pathway in the midbrain of mice were assessed.RESULTS The motor ability of the T group was higher than that of the St group,but the difference was not significant.However,the protein and gene expression levels of Wnt1,Lmx1a,Neurog2,and TH in the T group were significantly higher than those in the St group(P<0.01).Compared with the T group,the motor ability of the E group was significantly enhanced(P<0.01),and the gene expression level of Wnt1 in the midbrain of the E group was significantly higher than that of the T group(P<0.05).The levels of EPI and nerve growth factor were increased in both the E and ET groups.Exercise can improve motor dysfunction in PD,increase EPI levels,and elevate Wnt1 levels.However,western blot results revealed no significant change in the TH level of the E group,which may be because exercise does not cause a noticeable change in the number of neurons.Compared with the St group,both the E and ET groups showed improved motor function(P<0.01).The results showed that compared with the St group,the protein and gene expression levels of Wnt1,Lmx1a,and Neurog2 were significantly increased in the E,T,and ET groups(P<0.05).Compared with the T and E groups,the protein and gene expression levels of Wnt1,Lmx1a,and Neurog2 were significantly increased in the ET group(P<0.05).CONCLUSION Exercise increases EPI levels,activates the Wnt signaling pathway throughβ2 receptors,enhances the Wnt1-Lmx1a regulatory loop,and promotes the differentiation of iPSCs into dopaminergic neurons,thereby increasing the number of neurons.展开更多
This article focused on the recent contribution by Jiang et al,who demonstrated that voluntary exercise can significantly potentiate the effects of induced pluripotent stem cell transplantation in a Parkinson’s disea...This article focused on the recent contribution by Jiang et al,who demonstrated that voluntary exercise can significantly potentiate the effects of induced pluripotent stem cell transplantation in a Parkinson’s disease(PD)model through activation of the Wnt1-Lmx1a signaling cascade.Jiang et al’s findings highlight the role of exercise as a molecular modulator of neurogenesis and support the development of integrated strategies combining physical activity,stem cell transplantation,and biomaterials to improve outcomes in PD.We highlight exercise as a molecular modulator that fosters a neurogenic milieu,recommend examining additional developmental signals(sonic hedgehog,fibroblast growth factor 8,bone morphogenetic protein),and suggest biomaterial-based strategies to support graft survival and integration.We also stress the need to optimize exercise regimens in relation to transplantation,framing these insights within a translational strategy for advancing regenerative therapies in PD.展开更多
BACKGROUND The discovery of induced pluripotent stem cells revolutionized regenerative medicine,providing a source for generating induced pluripotent stem cell-derived mesenchymal stem cells(iMSCs).AIM To evaluate and...BACKGROUND The discovery of induced pluripotent stem cells revolutionized regenerative medicine,providing a source for generating induced pluripotent stem cell-derived mesenchymal stem cells(iMSCs).AIM To evaluate and compare five iMSC differentiation protocols,assessing their efficiency,phenotypic characteristics,and functional properties relative to primary mesenchymal stem cells(MSCs).METHODS Five iMSC differentiation protocols were assessed:SB431542-based differentiation(iMSC1,iMSC3),an iMatrix-free method(iMSC2),growth factor supplementation(iMSC4),and embryoid body formation with retinoic acid(EB-iMSC).iMSC identity was confirmed according to the International Society for Cell&Gene Therapy 2006 criteria,requiring expression of surface markers(CD105,CD73,CD90)and absence of pluripotency markers.Functional assays were conducted to evaluate differentiation potential(osteogenic and adipogenic),proliferation,mitochondrial function,reactive oxygen species,senescence,and migration.RESULTS All iMSC types expressed MSC markers and lacked pluripotency markers.EBiMSC and iMSC2 showed enhanced osteogenesis(runt-related transcription factor 2;P≤0.01 and P≤0.0001,respectively),while adipogenic potential was reduced in iMSC2(Adipsin;P≤0.01)and EB-iMSC(Adipsin and peroxisome proliferatoractivated receptor gamma;P≤0.0001 and P≤0.01,respectively).Proliferation was comparable or superior to bone marrow MSCs,except in iMSC1,with iMSC4 showing the highest rate(MTT assay;P values ranged from 0.01 to 0.001).Despite reduced mitochondrial health in iMSC3 and iMSC4(P≤0.001),reactive oxygen species levels were lower in all iMSCs(P values ranged from 0.001 to 0.0001),and senescence was significantly reduced in all iMSCs with the exception of iMSC1(P values ranged from 0.01 to 0.0001).Migration was most reduced in iMSC4(P≤0.001 at 24 hours and P≤0.0001 at 48 hours).CONCLUSION While all protocols generated functional iMSCs,variations in differentiation,proliferation,and function emphasize the impact of protocol selection.These findings contribute to optimizing iMSC generation for research and clinical applications.展开更多
Pluripotent stem cells(PSCs)are useful for developmental and translational research because they have the potential to differentiate into all cell types of an adult individual.Pigs are one of the most important domest...Pluripotent stem cells(PSCs)are useful for developmental and translational research because they have the potential to differentiate into all cell types of an adult individual.Pigs are one of the most important domestic ungulates,commonly used for food and as bioreactors.Generating stable pluripotent porcine PSC lines remains challenging.So far,the pluripotency gene network of porcine PSCs is poorly understood.Here we found that TBX3-derived induced pluripotent stem cells(iPSCs)closely resemble porcine 4-cell embryos with the capacity of totipotent-like stem cells(TLSCs).Interestingly,our data suggest that TBX3 facilitates the activation of H3K4me3 methyltransferase,specifically MLL1.Subsequent investigations revealed that the porcine 4-cell specific gene,MCL1,is a key downstream effector of the TBX3-MLL1 axis.Together,our study of the TBX3 regulatory network is helpful in the understanding of the totipotency characteristics of pigs.展开更多
BACKGROUNDMesenchymal stem cell(MSC)extracellular vesicles,particularly exosomes(Exos),are gaining recognition aspromising therapeutic tools for cancer due to their capacity to modulate tumor cell biology.Induced plur...BACKGROUNDMesenchymal stem cell(MSC)extracellular vesicles,particularly exosomes(Exos),are gaining recognition aspromising therapeutic tools for cancer due to their capacity to modulate tumor cell biology.Induced pluripotentstem cell-derived MSCs(iMSCs)revealed therapeutic characteristics compared with conventional MSCs due totheir proliferative capacity and enhanced differentiation potential.AIMTo study the impact of Exos derived from iMSCs(iMSC-Exos)and bone marrow MSCs(BMSC-Exos)on PANC1and MDA-MB-231 cancer cells.METHODSThe iMSCs and BMSCs were characterized based on the International Society for Cellular Therapy(2006)criteria byverifying the expression of MSC-specific markers and their differentiation potential.Exos were isolated from 48-hour conditioned media using sequential ultracentrifugation and characterized based on size,morphology,andexpression of surface markers including CD9,CD81,and CD63.PANC1 and MDA-MB-231 cells were treated withthe isolated Exos,and their effects on cell proliferation,apoptosis,senescence,and invasion were assessed.RESULTSIn PANC1 cells iMSC-Exos sustained antiproliferative activity for 48 hours(35%reduction,P<0.01)while BMSCExoshad a transient effect.In MDA-MB-231 cells,both Exos lowered proliferation significantly after 48 hours(~28%and~22%reduction,P<0.05).Notably,these antiproliferative effects were not associated with apoptosis,but an increase in senescence-like tumor cells was identified as the primary response with iMSC-Exos inducingapproximately 2.3-fold higher number of senescence-associatedβ-galactosidase-positive cells compared withBMSC-Exos across both cancer cell lines.Tumor cell invasion was markedly inhibited in PANC1 and MDA-MB-231cells in response to iMSC-Exos(~60%and~45%reduction,respectively,P<0.001),and only in PANC1 cells inresponse to BMSC-Exos.CONCLUSIONiMSC-Exos effectively inhibited tumor proliferation and invasion via a senescence-like mechanism.These resultsindicated that iMSC-Exos could serve as a cell-free cancer therapy and merit further animal model evaluation.展开更多
The potential of induced pluripotent stem cells(iPSCs)for modeling and treating metabolic associated fatty liver disease(MAFLD)and metabolic associated steatohepatitis(MASH)is emerging.MAFLD is a growing global health...The potential of induced pluripotent stem cells(iPSCs)for modeling and treating metabolic associated fatty liver disease(MAFLD)and metabolic associated steatohepatitis(MASH)is emerging.MAFLD is a growing global health concern,currently with limited treatment options.While primary mesenchymal stem cells hold promise,iPSCs offer a versatile alternative due to their ability to differentiate into various cell types,including iPSC-derived mesenchymal stem cells.However,challenges remain,including optimizing differentiation protocols,ensuring cell safety,and addressing potential tumorigenicity risks.In addition,iPSCs offer the possibility to generate complex cellular models,including three-dimensional organoid models,which are closer representations of the human disease than animal models.Those models would also be valuable for drug discovery and personalized medicine approaches.Overall,iPSCs and their derivatives offer new perspectives for advancing MAFLD/MASH research and developing novel therapeutic strategies.Further research is needed to overcome current limitations and translate this potential into effective clinical applications.展开更多
Mesenchymal stem cells(MSCs)have received significant attention in recent years due to their large potential for cell therapy.Indeed,they secrete a wide variety of immunomodulatory factors of interest for the treatmen...Mesenchymal stem cells(MSCs)have received significant attention in recent years due to their large potential for cell therapy.Indeed,they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases.MSCs can be extracted from multiple tissues of the human body.However,several factors may restrict their use for clinical applications:the requirement of invasive procedures for their isolation,their limited numbers,and their heterogeneity according to the tissue of origin or donor.In addition,MSCs often present early signs of replicative senescence limiting their expansion in vitro,and their therapeutic capacity in vivo.Due to the clinical potential of MSCs,a considerable number of methods to differentiate induced pluripotent stem cells(iPSCs)into MSCs have emerged.iPSCs represent a new reliable,unlimited source to generate MSCs(MSCs derived from iPSC,iMSCs)from homogeneous and well-characterized cell lines,which would relieve many of the above mentioned technical and biological limitations.Additionally,the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells.In this review,we analyze the main current protocols used to differentiate human iPSCs into MSCs,which we classify into five different categories:MSC Switch,Embryoid Body Formation,Specific Differentiation,Pathway Inhibitor,and Platelet Lysate.We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization.Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added.The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.展开更多
Mesenchymal stem cells(MSCs)have the potential for use in cell-based regenerative therapies.Currently,hundreds of clinical trials are using MSCs for the treatment of various diseases.However,MSCs are low in number in ...Mesenchymal stem cells(MSCs)have the potential for use in cell-based regenerative therapies.Currently,hundreds of clinical trials are using MSCs for the treatment of various diseases.However,MSCs are low in number in adult tissues;they show heterogeneity depending upon the cell source and exhibit limited proliferative potential and early senescence in in vitro cultures.These factors negatively impact the regenerative potential of MSCs and therefore restrict their use for clinical applications.As a result,novel methods to generate induced MSCs(iMSCs)from induced pluripotent stem cells have been explored.The development and optimization of protocols for generation of iMSCs from induced pluripotent stem cells is necessary to evaluate their regenerative potential in vivo and in vitro.In addition,it is important to compare iMSCs with primary MSCs(isolated from adult tissues)in terms of their safety and efficacy.Careful investigation of the properties of iMSCs in vitro and their long term behavior in animals is important for their translation from bench to bedside.展开更多
Non-human primates play a key role in the preclinical validation of pluripotent stem cell-based cell replacement therapies.Pluripotent stem cells used as advanced therapy medical products boost the possibility to rege...Non-human primates play a key role in the preclinical validation of pluripotent stem cell-based cell replacement therapies.Pluripotent stem cells used as advanced therapy medical products boost the possibility to regenerate tissues and organs affected by degenerative diseases.Therefore,the methods to derive human induced pluripotent stem cell and embryonic stem cell lines following clinical standards have quickly developed in the last 15 years.For the preclinical validation of cell replacement therapies in non-human primates,it is necessary to generate non-human primate pluripotent stem cell with a homologous quality to their human counterparts.However,pluripotent stem cell technologies have developed at a slower pace in non-human primates in comparison with human cell systems.In recent years,however,relevant progress has also been made with non-human primate pluripotent stem cells.This review provides a systematic overview of the progress and remaining challenges for the generation of non-human primate induced pluripotent stem cells/embryonic stem cells for the preclinical testing and validation of cell replacement therapies.We focus on the critical domains of(1)reprogramming and embryonic stem cell line derivation,(2)cell line maintenance and characterization and,(3)application of non-human primate pluripotent stem cells in the context of selected preclinical studies to treat cardiovascular and neurodegenerative disorders performed in non-human primates.展开更多
Efforts have been made to establish various human pluripotent stem cell lines.However,such methods have not yet been duplicated in non-human primate cells.Here,we introduce a multiplexed single-cell sequencing techniq...Efforts have been made to establish various human pluripotent stem cell lines.However,such methods have not yet been duplicated in non-human primate cells.Here,we introduce a multiplexed single-cell sequencing technique to profile the molecular features of monkey pluripotent stem cells in published culture conditions.The results demonstrate suboptimized maintenance of pluripotency and show that the selected signaling pathways for resetting human stem cells can also be interpreted for establishing monkey cell lines.Overall,this work legitimates the translation of novel human cell line culture conditions to monkey cells and provides guidance for exploring chemical cocktails for monkey stem cell line derivation.展开更多
Insulin resistance(IR)is associated with several metabolic disorders,including type 2 diabetes(T2D).The development of IR in insulin target tissues involves genetic and acquired factors.Persons at genetic risk for T2D...Insulin resistance(IR)is associated with several metabolic disorders,including type 2 diabetes(T2D).The development of IR in insulin target tissues involves genetic and acquired factors.Persons at genetic risk for T2D tend to develop IR several years before glucose intolerance.Several rodent models for both IR and T2D are being used to study the disease pathogenesis;however,these models cannot recapitulate all the aspects of this complex disorder as seen in each individual.Human pluripotent stem cells(hPSCs)can overcome the hurdles faced with the classical mouse models for studying IR.Human induced pluripotent stem cells(hiPSCs)can be generated from the somatic cells of the patients without the need to destroy a human embryo.Therefore,patient-specific hiPSCs can generate cells genetically identical to IR individuals,which can help in distinguishing between genetic and acquired defects in insulin sensitivity.Combining the technologies of genome editing and hiPSCs may provide important information about the genetic factors underlying the development of different forms of IR.Further studies are required to fill the gaps in understanding the pathogenesis of IR and diabetes.In this review,we summarize the factors involved in the development of IR in the insulin-target tissues leading to diabetes.Also,we highlight the use of hPSCs to understand the mechanisms underlying the development of IR.展开更多
Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by ...Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.展开更多
Neurodegenerative diseases,including Parkinson＇s disease,Alzheimer＇s disease and Amyotrophic Lateral Sclerosis,are characterized by idiopathic neuron loss in different regions of the central nervous system,which con...Neurodegenerative diseases,including Parkinson＇s disease,Alzheimer＇s disease and Amyotrophic Lateral Sclerosis,are characterized by idiopathic neuron loss in different regions of the central nervous system,which contributes to the relevant dysfunctions in the patients.The application of cell replacement therapy using human embryonic stem（hES） cells,though having attracted much attention,has been hampered by the intrinsic ethical problems.It has been demonstrated that adult somatic cells can be reprogrammed into the embryonic state,called induced pluripotent stem（iPS） cells.It is soon realized that iPS cells may be an alternative source for cell replacement therapy,because it raises no ethical problems and using patient-specific iPS cells for autologous transplantation will not lead to immunological rejection.What＇s more,certain types of neurons derived from patient-specific iPS cells may display disease-relevant phenotypes.Thus,patientspecific iPS cells can provide a unique opportunity to directly investigate the pathological properties of relevant neural cells in individual patient,and to study the vulnerability of neural cells to pathogenic factors in vitro,which may help reveal the pathogenesis of many neurodegenerative diseases.In this review,the recent development in cellular treatment of neurodegenerative diseases using iPS cells was summarized,and the potential value of iPS cells in the modeling of neurodegenerative disease was discussed.展开更多
Human induced pluripotent stem （iPS） cells are similar to embryonic stem （ES） cells, and can proliferate intensively and differentiate into a variety of cell types. However, the hepatic differentiation of human iP...Human induced pluripotent stem （iPS） cells are similar to embryonic stem （ES） cells, and can proliferate intensively and differentiate into a variety of cell types. However, the hepatic differentiation of human iPS cells has not yet been reported. In this report, human iPS cells were induced to differentiate into hepatic cells by a stepwise protocol. The expression of liver cell markers and liver-related functions of the human iPS cell-derived cells were monitored and compared with that of differentiated human ES cells and primary human hepatocytes. Approximately 60% of the differentiated human iPS cells at day 7 expressed hepatic markers alpha fetoprotein and Alb. The differentiated cells at day 21 exhibited liver cell functions including albumin Asecretion, glycogen synthesis, urea production and inducible cytochrome P450 activity. The expression of hepatic markers and fiver-related functions of the iPS cellderived hepatic ceils were comparable to that of the human ES cell-derived hepatic cells. These results show that human iPS cells, which are similar to human ES cells, can be efficiently induced to differentiate into hepatocyte-like cells.展开更多
基金supported by the National Natural Science Foundation of China,No.32171356(to YW)Self-Support Research Projects of Shihezi University,No.ZZZC2021105(to WJ)+1 种基金Capital Medical University Natural Science Cultivation Fund,No.PYZ23044(to FQM)Beijing Municipal Natural Science Foundation,No.7244410(to JHD)。
文摘Previous research has demonstrated the feasibility of repairing nerve defects through acellular allogeneic nerve grafting with bone marrow mesenchymal stem cells.However,adult tissue–derived mesenchymal stem cells encounter various obstacles,including limited tissue sources,invasive acquisition methods,cellular heterogeneity,purification challenges,cellular senescence,and diminished pluripotency and proliferation over successive passages.In this study,we used induced pluripotent stem cell-derived mesenchymal stem cells,known for their self-renewal capacity,multilineage differentiation potential,and immunomodulatory characteristics.We used induced pluripotent stem cell-derived mesenchymal stem cells in conjunction with acellular nerve allografts to address a 10 mm-long defect in a rat model of sciatic nerve injury.Our findings reveal that induced pluripotent stem cell-derived mesenchymal stem cells exhibit survival for up to 17 days in a rat model of peripheral nerve injury with acellular nerve allograft transplantation.Furthermore,the combination of acellular nerve allograft and induced pluripotent stem cell-derived mesenchymal stem cells significantly accelerates the regeneration of injured axons and improves behavioral function recovery in rats.Additionally,our in vivo and in vitro experiments indicate that induced pluripotent stem cell-derived mesenchymal stem cells play a pivotal role in promoting neovascularization.Collectively,our results suggest the potential of acellular nerve allografts with induced pluripotent stem cell-derived mesenchymal stem cells to augment nerve regeneration in rats,offering promising therapeutic strategies for clinical translation.
基金supported by the National Natural Science Foundation of China,No.8227050826(to PL)Tianjin Science and Technology Bureau Foundation,No.20201194(to PL)Tianjin Graduate Research and Innovation Project,No.2022BKY174(to CW).
文摘Cerebral edema caused by blood-brain barrier injury after intracerebral hemorrhage is an important factor leading to poor prognosis.Human-induced pluripotent stem cell-derived neural stem cell exosomes(hiPSC-NSC-Exos)have shown potential for brain injury repair in central nervous system diseases.In this study,we explored the impact of hiPSC-NSC-Exos on blood-brain barrier preservation and the underlying mechanism.Our results indicated that intranasal delivery of hiPSC-NSC-Exos mitigated neurological deficits,enhanced blood-brain barrier integrity,and reduced leukocyte infiltration in a mouse model of intracerebral hemorrhage.Additionally,hiPSC-NSC-Exos decreased immune cell infiltration,activated astrocytes,and decreased the secretion of inflammatory cytokines like monocyte chemoattractant protein-1,macrophage inflammatory protein-1α,and tumor necrosis factor-αpost-intracerebral hemorrhage,thereby improving the inflammatory microenvironment.RNA sequencing indicated that hiPSC-NSC-Exo activated the PI3K/AKT signaling pathway in astrocytes and decreased monocyte chemoattractant protein-1 secretion,thereby improving blood-brain barrier integrity.Treatment with the PI3K/AKT inhibitor LY294002 or the monocyte chemoattractant protein-1 neutralizing agent C1142 abolished these effects.In summary,our findings suggest that hiPSC-NSC-Exos maintains blood-brain barrier integrity,in part by downregulating monocyte chemoattractant protein-1 secretion through activation of the PI3K/AKT signaling pathway in astrocytes.
基金supported by a BBSRC CASE training studentship,No.BB/K011413/1(to KG)。
文摘Neuronal cell death and the loss of connectivity are two of the primary pathological mechanisms underlying Alzheimer's disease.The accumulation of amyloid-βpeptides,a key hallmark of Alzheimer's disease,is believed to induce neuritic abnormalities,including reduced growth,extension,and abnormal growth cone morphology,all of which contribute to decreased connectivity.However,the precise cellular and molecular mechanisms governing this response remain unknown.In this study,we used an innovative approach to demonstrate the effect of amyloid-βon neurite dynamics in both two-dimensional and three-dimensional cultu re systems,in order to provide more physiologically relevant culture geometry.We utilized various methodologies,including the addition of exogenous amyloid-βpeptides to the culture medium,growth substrate coating,and the utilization of human-induced pluripotent stem cell technology,to investigate the effect of endogenous amyloid-βsecretion on neurite outgrowth,thus paving the way for potential future applications in personalized medicine.Additionally,we also explore the involvement of the Nogo signaling cascade in amyloid-β-induced neurite inhibition.We demonstrate that inhibition of downstream ROCK and RhoA components of the Nogo signaling pathway,achieved through modulation with Y-27632(a ROCK inhibitor)and Ibuprofen(a Rho A inhibitor),respectively,can restore and even enhance neuronal connectivity in the presence of amyloid-β.In summary,this study not only presents a novel culture approach that offers insights into the biological process of neurite growth and inhibition,but also proposes a specific mechanism for reduced neural connectivity in the presence of amyloid-βpeptides,along with potential intervention points to restore neurite growth.Thereby,we aim to establish a culture system that has the potential to serve as an assay for measuring preclinical,predictive outcomes of drugs and their ability to promote neurite outgrowth,both generally and in a patient-specific manner.
基金supported by Ohio State Start Up FundNational Institutes of Health(NIH)+12 种基金Department of Defense(DoD)Wings for Life Spinal Cord Research Foundation,Wings for Life Spinal Cord Research Foundation(Austria)California Institute of Regenerative Medicine(CIRM)International Spinal Research Trust(United Kingdom)Stanford University Bio-X Program Interdisciplinary Initiatives Seed Grant IIP-7Dennis Chan FoundationKlein Family FundLucile Packard Foundation for Children's HealthStanford Institute for Neuro-Innovation and Translational Neurosciences(SINTN)Saunders Family Neuroscience FundJames Doty Neurosurgery FundHearst Neuroscience FundEileen Bond Research Fund(to GP)。
文摘In recent years,the progression of stem cell therapies has shown great promise in advancing the nascent field of regenerative medicine.Considering the non-regenerative nature of the mature central nervous system,the concept that“blank”cells could be reprogrammed and functionally integrated into host neural networks remained intriguing.Previous work has also demonstrated the ability of such cells to stimulate intrinsic growth programs in post-mitotic cells,such as neurons.While embryonic stem cells demonstrated great potential in treating central nervous system pathologies,ethical and technical concerns remained.These barriers,along with the clear necessity for this type of treatment,ultimately prompted the advent of induced pluripotent stem cells.The advantage of pluripotent cells in central nervous system regeneration is multifaceted,permitting differentiation into neural stem cells,neural progenitor cells,glia,and various neuronal subpopulations.The precise spatiotemporal application of extrinsic growth factors in vitro,in addition to microenvironmental signaling in vivo,influences the efficiency of this directed differentiation.While the pluri-or multipotency of these cells is appealing,it also poses the risk of unregulated differentiation and teratoma formation.Cells of the neuroectodermal lineage,such as neuronal subpopulations and glia,have been explored with varying degrees of success.Although the risk of cancer or teratoma formation is greatly reduced,each subpopulation varies in effectiveness and is influenced by a myriad of factors,such as the timing of the transplant,pathology type,and the ratio of accompanying progenitor cells.Furthermore,successful transplantation requires innovative approaches to develop delivery vectors that can mitigate cell death and support integration.Lastly,host immune responses to allogeneic grafts must be thoroughly characterized and further developed to reduce the need for immunosuppression.Translation to a clinical setting will involve careful consideration when assessing both physiologic and functional outcomes.This review will highlight both successes and challenges faced when using human induced pluripotent stem cell-derived cell transplantation therapies to promote endogenous regeneration.
基金supported by Singapore National Medical Research Council(NMRC)grants,including CS-IRG,HLCA2022(to ZDZ),STaR,OF LCG 000207(to EKT)a Clinical Translational Research Programme in Parkinson's DiseaseDuke-Duke-NUS collaboration pilot grant(to ZDZ)。
文摘The progressive loss of dopaminergic neurons in affected patient brains is one of the pathological features of Parkinson's disease,the second most common human neurodegenerative disease.Although the detailed pathogenesis accounting for dopaminergic neuron degeneration in Parkinson's disease is still unclear,the advancement of stem cell approaches has shown promise for Parkinson's disease research and therapy.The induced pluripotent stem cells have been commonly used to generate dopaminergic neurons,which has provided valuable insights to improve our understanding of Parkinson's disease pathogenesis and contributed to anti-Parkinson's disease therapies.The current review discusses the practical approaches and potential applications of induced pluripotent stem cell techniques for generating and differentiating dopaminergic neurons from induced pluripotent stem cells.The benefits of induced pluripotent stem cell-based research are highlighted.Various dopaminergic neuron differentiation protocols from induced pluripotent stem cells are compared.The emerging three-dimension-based brain organoid models compared with conventional two-dimensional cell culture are evaluated.Finally,limitations,challenges,and future directions of induced pluripotent stem cell–based approaches are analyzed and proposed,which will be significant to the future application of induced pluripotent stem cell-related techniques for Parkinson's disease.
基金Supported by the Nonprofit Research Institutes Foundation of Fujian Province,China,No.2020R1011003 and No.2022R1012001the Talents Training Project for the Key Young Scholars of Fujian Provincial Health Commission,China,No.2021GGA056.
文摘BACKGROUND Type 1 diabetes(T1D)results from the autoimmune-mediated loss of pancreatic β-cells.Current insulin therapies offer symptomatic relief but fall short of providing a definitive cure.Islet cell transplantation,while promising,faces limitations related to donor scarcity,procedural complexities,and the necessity for long-term immunosuppression.Consequently,there is an urgent need for innovative strategies aimed at β-cell regeneration.Patient-derived induced pluripotent stem cells(iPSCs),obtained from peripheral blood mononuclear cells(PBMCs)of T1D patients,hold great potential as a source of cells for therapeutic purposes.Therefore,the differentiation of T1D-iPSCs into functional pancreatic β-cells is a critical step toward effective β-cell replacement therapy.AIM To assess the potential of patient-derived T1D-β-like cells(differentiated from T1D-iPSCs reprogrammed from T1D-PBMCs)for restoring β-cell function in T1D.METHODS T1D-iPSCs were reprogrammed from T1D-PBMCs using an episomal vectorbased approach.Pluripotency was confirmed by flow cytometry(FCM),quantitative real-time polymerase chain reaction,genomic stability analysis,and teratoma formation assays.Differentiation into pancreatic β-cells was optimized using triiodothyronine(T3),vitamin C(Vc),and an adenovirus(M3C)encoding pancreatic duodenal homeobox-1,neurogenin 3(Ngn3),and MAF bZIP transcription factor A(MafA).Following characterization of β-cell features by immunofluorescence,quantitative real-time polymerase chain reaction,and flow cytometry,therapeutic efficacy was assessed through blood glucose monitoring after transplantation under the renal capsule of streptozotocin-induced diabetic mice.RESULTS T1D-iPSCs were successfully generated from T1D-PBMCs.These cells exhibited the hallmark characteristics of pluripotent stem cells,including appropriate morphology,differentiation potential,genomic integrity,and expression of pluripotency-associated genes.Differentiation yielded insulin-positive(insulin+)pancreatic β-like cells that,at the mRNA level,expressed key β-cell markers such as pancreatic duodenal homeobox-1,Ngn3,MafA,NeuroD,glucagon-like peptide-1 receptor,Nkx6.1,glucose transporter 2,and Kir6.2.Notably,the T3+Vc group displayed the lowest Ngn3 expression(1.31±0.38 vs 1.96±0.25 vs 2.51±0.24,P<0.01),while the M3C+T3+Vc group exhibited the highest MafA expression(0.49±0.11 vs 0.32±0.06 vs 0.29±0.08,P<0.05).Both in vitro and in vivo assessments confirmed the insulin secretion ability of the generated β-like cells;however,they did not demonstrate appropriate modulation of insulin release in response to variations in extracellular glucose concentrations.CONCLUSION T1D-iPSCs derived from T1D-PBMCs can be differentiated into insulin+β-like cells,albeit with functional immaturity.These cells represent a potential source of seed cells for β-cell replacement therapy in T1D.
基金Supported by the Central Government Guides Local Science and Technology Development Funds,No.YDZX2022091General Program of Natural Sciences of Qilu Medical University,No.X24ZKQN03.
文摘BACKGROUND The therapeutic potential of induced pluripotent stem cells(iPSCs)for Parkinson’s disease(PD)has been demonstrated.Exercise can also modulate metabolism to improve motor dysfunction in PD patients.AIM To investigate the therapeutic effect of exercise combined with iPSCs in a PD mouse model and explore the underlying mechanisms.METHODS In this study,we included 10 normal mice and 40 PD model mice,which were divided into five groups:The control group(n=10),the sedentary PD group(St group,n=10),the exercise PD group(E group,n=10),the iPSC-treated PD group(T group,n=10),and the combined exercise and iPSC-treated PD group(ET group,n=10).The T and ET groups received cell injection therapy,while the E and ET groups underwent an 8-week exercise intervention.After the intervention,behavioral tests were performed on mice from all groups.Serum levels of epinephrine(EPI)and nerve growth factor were measured,and the expression of Wnt1,Lmx1a,and other factors related to the Wnt signaling pathway in the midbrain of mice were assessed.RESULTS The motor ability of the T group was higher than that of the St group,but the difference was not significant.However,the protein and gene expression levels of Wnt1,Lmx1a,Neurog2,and TH in the T group were significantly higher than those in the St group(P<0.01).Compared with the T group,the motor ability of the E group was significantly enhanced(P<0.01),and the gene expression level of Wnt1 in the midbrain of the E group was significantly higher than that of the T group(P<0.05).The levels of EPI and nerve growth factor were increased in both the E and ET groups.Exercise can improve motor dysfunction in PD,increase EPI levels,and elevate Wnt1 levels.However,western blot results revealed no significant change in the TH level of the E group,which may be because exercise does not cause a noticeable change in the number of neurons.Compared with the St group,both the E and ET groups showed improved motor function(P<0.01).The results showed that compared with the St group,the protein and gene expression levels of Wnt1,Lmx1a,and Neurog2 were significantly increased in the E,T,and ET groups(P<0.05).Compared with the T and E groups,the protein and gene expression levels of Wnt1,Lmx1a,and Neurog2 were significantly increased in the ET group(P<0.05).CONCLUSION Exercise increases EPI levels,activates the Wnt signaling pathway throughβ2 receptors,enhances the Wnt1-Lmx1a regulatory loop,and promotes the differentiation of iPSCs into dopaminergic neurons,thereby increasing the number of neurons.
文摘This article focused on the recent contribution by Jiang et al,who demonstrated that voluntary exercise can significantly potentiate the effects of induced pluripotent stem cell transplantation in a Parkinson’s disease(PD)model through activation of the Wnt1-Lmx1a signaling cascade.Jiang et al’s findings highlight the role of exercise as a molecular modulator of neurogenesis and support the development of integrated strategies combining physical activity,stem cell transplantation,and biomaterials to improve outcomes in PD.We highlight exercise as a molecular modulator that fosters a neurogenic milieu,recommend examining additional developmental signals(sonic hedgehog,fibroblast growth factor 8,bone morphogenetic protein),and suggest biomaterial-based strategies to support graft survival and integration.We also stress the need to optimize exercise regimens in relation to transplantation,framing these insights within a translational strategy for advancing regenerative therapies in PD.
文摘BACKGROUND The discovery of induced pluripotent stem cells revolutionized regenerative medicine,providing a source for generating induced pluripotent stem cell-derived mesenchymal stem cells(iMSCs).AIM To evaluate and compare five iMSC differentiation protocols,assessing their efficiency,phenotypic characteristics,and functional properties relative to primary mesenchymal stem cells(MSCs).METHODS Five iMSC differentiation protocols were assessed:SB431542-based differentiation(iMSC1,iMSC3),an iMatrix-free method(iMSC2),growth factor supplementation(iMSC4),and embryoid body formation with retinoic acid(EB-iMSC).iMSC identity was confirmed according to the International Society for Cell&Gene Therapy 2006 criteria,requiring expression of surface markers(CD105,CD73,CD90)and absence of pluripotency markers.Functional assays were conducted to evaluate differentiation potential(osteogenic and adipogenic),proliferation,mitochondrial function,reactive oxygen species,senescence,and migration.RESULTS All iMSC types expressed MSC markers and lacked pluripotency markers.EBiMSC and iMSC2 showed enhanced osteogenesis(runt-related transcription factor 2;P≤0.01 and P≤0.0001,respectively),while adipogenic potential was reduced in iMSC2(Adipsin;P≤0.01)and EB-iMSC(Adipsin and peroxisome proliferatoractivated receptor gamma;P≤0.0001 and P≤0.01,respectively).Proliferation was comparable or superior to bone marrow MSCs,except in iMSC1,with iMSC4 showing the highest rate(MTT assay;P values ranged from 0.01 to 0.001).Despite reduced mitochondrial health in iMSC3 and iMSC4(P≤0.001),reactive oxygen species levels were lower in all iMSCs(P values ranged from 0.001 to 0.0001),and senescence was significantly reduced in all iMSCs with the exception of iMSC1(P values ranged from 0.01 to 0.0001).Migration was most reduced in iMSC4(P≤0.001 at 24 hours and P≤0.0001 at 48 hours).CONCLUSION While all protocols generated functional iMSCs,variations in differentiation,proliferation,and function emphasize the impact of protocol selection.These findings contribute to optimizing iMSC generation for research and clinical applications.
基金funded by the National Basic Research Program of China(2022YFD1302200,2023YFF1000904,2023ZD0404303 and 2021YFD1200301)the National Natural Science Foundation of China(32072806,32372970 and 32002246)+2 种基金the Program of Shaanxi Province Science and Technology Innovation Team,China(2019TD-036)the Key Technologies Demonstration of Animal Husbandry in Shaanxi Province,China(20221086 and 20230978)the Inner Mongolia Autonomous Region Open Competition Project,China(2022JBGS0025).
文摘Pluripotent stem cells(PSCs)are useful for developmental and translational research because they have the potential to differentiate into all cell types of an adult individual.Pigs are one of the most important domestic ungulates,commonly used for food and as bioreactors.Generating stable pluripotent porcine PSC lines remains challenging.So far,the pluripotency gene network of porcine PSCs is poorly understood.Here we found that TBX3-derived induced pluripotent stem cells(iPSCs)closely resemble porcine 4-cell embryos with the capacity of totipotent-like stem cells(TLSCs).Interestingly,our data suggest that TBX3 facilitates the activation of H3K4me3 methyltransferase,specifically MLL1.Subsequent investigations revealed that the porcine 4-cell specific gene,MCL1,is a key downstream effector of the TBX3-MLL1 axis.Together,our study of the TBX3 regulatory network is helpful in the understanding of the totipotency characteristics of pigs.
文摘BACKGROUNDMesenchymal stem cell(MSC)extracellular vesicles,particularly exosomes(Exos),are gaining recognition aspromising therapeutic tools for cancer due to their capacity to modulate tumor cell biology.Induced pluripotentstem cell-derived MSCs(iMSCs)revealed therapeutic characteristics compared with conventional MSCs due totheir proliferative capacity and enhanced differentiation potential.AIMTo study the impact of Exos derived from iMSCs(iMSC-Exos)and bone marrow MSCs(BMSC-Exos)on PANC1and MDA-MB-231 cancer cells.METHODSThe iMSCs and BMSCs were characterized based on the International Society for Cellular Therapy(2006)criteria byverifying the expression of MSC-specific markers and their differentiation potential.Exos were isolated from 48-hour conditioned media using sequential ultracentrifugation and characterized based on size,morphology,andexpression of surface markers including CD9,CD81,and CD63.PANC1 and MDA-MB-231 cells were treated withthe isolated Exos,and their effects on cell proliferation,apoptosis,senescence,and invasion were assessed.RESULTSIn PANC1 cells iMSC-Exos sustained antiproliferative activity for 48 hours(35%reduction,P<0.01)while BMSCExoshad a transient effect.In MDA-MB-231 cells,both Exos lowered proliferation significantly after 48 hours(~28%and~22%reduction,P<0.05).Notably,these antiproliferative effects were not associated with apoptosis,but an increase in senescence-like tumor cells was identified as the primary response with iMSC-Exos inducingapproximately 2.3-fold higher number of senescence-associatedβ-galactosidase-positive cells compared withBMSC-Exos across both cancer cell lines.Tumor cell invasion was markedly inhibited in PANC1 and MDA-MB-231cells in response to iMSC-Exos(~60%and~45%reduction,respectively,P<0.001),and only in PANC1 cells inresponse to BMSC-Exos.CONCLUSIONiMSC-Exos effectively inhibited tumor proliferation and invasion via a senescence-like mechanism.These resultsindicated that iMSC-Exos could serve as a cell-free cancer therapy and merit further animal model evaluation.
基金American Heart Association Award,No.24IVPHA1288417and FCT Fellowships,No.2022.13253.BDANA.
文摘The potential of induced pluripotent stem cells(iPSCs)for modeling and treating metabolic associated fatty liver disease(MAFLD)and metabolic associated steatohepatitis(MASH)is emerging.MAFLD is a growing global health concern,currently with limited treatment options.While primary mesenchymal stem cells hold promise,iPSCs offer a versatile alternative due to their ability to differentiate into various cell types,including iPSC-derived mesenchymal stem cells.However,challenges remain,including optimizing differentiation protocols,ensuring cell safety,and addressing potential tumorigenicity risks.In addition,iPSCs offer the possibility to generate complex cellular models,including three-dimensional organoid models,which are closer representations of the human disease than animal models.Those models would also be valuable for drug discovery and personalized medicine approaches.Overall,iPSCs and their derivatives offer new perspectives for advancing MAFLD/MASH research and developing novel therapeutic strategies.Further research is needed to overcome current limitations and translate this potential into effective clinical applications.
文摘Mesenchymal stem cells(MSCs)have received significant attention in recent years due to their large potential for cell therapy.Indeed,they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases.MSCs can be extracted from multiple tissues of the human body.However,several factors may restrict their use for clinical applications:the requirement of invasive procedures for their isolation,their limited numbers,and their heterogeneity according to the tissue of origin or donor.In addition,MSCs often present early signs of replicative senescence limiting their expansion in vitro,and their therapeutic capacity in vivo.Due to the clinical potential of MSCs,a considerable number of methods to differentiate induced pluripotent stem cells(iPSCs)into MSCs have emerged.iPSCs represent a new reliable,unlimited source to generate MSCs(MSCs derived from iPSC,iMSCs)from homogeneous and well-characterized cell lines,which would relieve many of the above mentioned technical and biological limitations.Additionally,the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells.In this review,we analyze the main current protocols used to differentiate human iPSCs into MSCs,which we classify into five different categories:MSC Switch,Embryoid Body Formation,Specific Differentiation,Pathway Inhibitor,and Platelet Lysate.We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization.Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added.The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.
文摘Mesenchymal stem cells(MSCs)have the potential for use in cell-based regenerative therapies.Currently,hundreds of clinical trials are using MSCs for the treatment of various diseases.However,MSCs are low in number in adult tissues;they show heterogeneity depending upon the cell source and exhibit limited proliferative potential and early senescence in in vitro cultures.These factors negatively impact the regenerative potential of MSCs and therefore restrict their use for clinical applications.As a result,novel methods to generate induced MSCs(iMSCs)from induced pluripotent stem cells have been explored.The development and optimization of protocols for generation of iMSCs from induced pluripotent stem cells is necessary to evaluate their regenerative potential in vivo and in vitro.In addition,it is important to compare iMSCs with primary MSCs(isolated from adult tissues)in terms of their safety and efficacy.Careful investigation of the properties of iMSCs in vitro and their long term behavior in animals is important for their translation from bench to bedside.
基金supported by the German Centre for Cardiovascular Research(DZHK)the German Primate Center-Leibniz Institute for Primate Research,which is financed by the Bundesrepublik Deutschland and the Bundesländer(Federal states)(Grant number 81Z0300201 to RB).
文摘Non-human primates play a key role in the preclinical validation of pluripotent stem cell-based cell replacement therapies.Pluripotent stem cells used as advanced therapy medical products boost the possibility to regenerate tissues and organs affected by degenerative diseases.Therefore,the methods to derive human induced pluripotent stem cell and embryonic stem cell lines following clinical standards have quickly developed in the last 15 years.For the preclinical validation of cell replacement therapies in non-human primates,it is necessary to generate non-human primate pluripotent stem cell with a homologous quality to their human counterparts.However,pluripotent stem cell technologies have developed at a slower pace in non-human primates in comparison with human cell systems.In recent years,however,relevant progress has also been made with non-human primate pluripotent stem cells.This review provides a systematic overview of the progress and remaining challenges for the generation of non-human primate induced pluripotent stem cells/embryonic stem cells for the preclinical testing and validation of cell replacement therapies.We focus on the critical domains of(1)reprogramming and embryonic stem cell line derivation,(2)cell line maintenance and characterization and,(3)application of non-human primate pluripotent stem cells in the context of selected preclinical studies to treat cardiovascular and neurodegenerative disorders performed in non-human primates.
基金supported by the National Key R&D Program of China(Nos.2021YFA0805700 and 2021YFA1102000)the National Natural Science Foundation of China(No.U2102204)the Natural Science Foundation of Yunnan Province,China(Nos.202001BC070001 and 202102AA100053)。
文摘Efforts have been made to establish various human pluripotent stem cell lines.However,such methods have not yet been duplicated in non-human primate cells.Here,we introduce a multiplexed single-cell sequencing technique to profile the molecular features of monkey pluripotent stem cells in published culture conditions.The results demonstrate suboptimized maintenance of pluripotency and show that the selected signaling pathways for resetting human stem cells can also be interpreted for establishing monkey cell lines.Overall,this work legitimates the translation of novel human cell line culture conditions to monkey cells and provides guidance for exploring chemical cocktails for monkey stem cell line derivation.
基金the Qatar National Research Fund,No.NPRP10-1221-160041.
文摘Insulin resistance(IR)is associated with several metabolic disorders,including type 2 diabetes(T2D).The development of IR in insulin target tissues involves genetic and acquired factors.Persons at genetic risk for T2D tend to develop IR several years before glucose intolerance.Several rodent models for both IR and T2D are being used to study the disease pathogenesis;however,these models cannot recapitulate all the aspects of this complex disorder as seen in each individual.Human pluripotent stem cells(hPSCs)can overcome the hurdles faced with the classical mouse models for studying IR.Human induced pluripotent stem cells(hiPSCs)can be generated from the somatic cells of the patients without the need to destroy a human embryo.Therefore,patient-specific hiPSCs can generate cells genetically identical to IR individuals,which can help in distinguishing between genetic and acquired defects in insulin sensitivity.Combining the technologies of genome editing and hiPSCs may provide important information about the genetic factors underlying the development of different forms of IR.Further studies are required to fill the gaps in understanding the pathogenesis of IR and diabetes.In this review,we summarize the factors involved in the development of IR in the insulin-target tissues leading to diabetes.Also,we highlight the use of hPSCs to understand the mechanisms underlying the development of IR.
基金Supported by the National Science and Technology Major Project(2011ZX09102-010-04)
文摘Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.
基金supported by grants of the Key Project of Shanghai Science and Technology Committee(No. 08411951100,10ZR1425800)National Major Special Project of Science and Technology from the Ministry of Science and Technology,China (No. 2008ZX09312)
文摘Neurodegenerative diseases,including Parkinson＇s disease,Alzheimer＇s disease and Amyotrophic Lateral Sclerosis,are characterized by idiopathic neuron loss in different regions of the central nervous system,which contributes to the relevant dysfunctions in the patients.The application of cell replacement therapy using human embryonic stem（hES） cells,though having attracted much attention,has been hampered by the intrinsic ethical problems.It has been demonstrated that adult somatic cells can be reprogrammed into the embryonic state,called induced pluripotent stem（iPS） cells.It is soon realized that iPS cells may be an alternative source for cell replacement therapy,because it raises no ethical problems and using patient-specific iPS cells for autologous transplantation will not lead to immunological rejection.What＇s more,certain types of neurons derived from patient-specific iPS cells may display disease-relevant phenotypes.Thus,patientspecific iPS cells can provide a unique opportunity to directly investigate the pathological properties of relevant neural cells in individual patient,and to study the vulnerability of neural cells to pathogenic factors in vitro,which may help reveal the pathogenesis of many neurodegenerative diseases.In this review,the recent development in cellular treatment of neurodegenerative diseases using iPS cells was summarized,and the potential value of iPS cells in the modeling of neurodegenerative disease was discussed.
基金We thank Dr Zicai Liang and Huang Huang （Institute of Molecular Medicine, Peking University） for their kind help with BioTek Multi-Detection Microplate Reader and Yizhe Zhang for technical support on real-time PCR. We also thank Chengyan Wang, Pengbo Zhang, Pingping Hou, Haisong Liu, Chun Liu and other colleagues in our laboratory for technical assistance and advice in carrying out these experiments. This study was supported by a Bill ＆ Melinda Gates Foundation Grant （37871）, a Ministry of Education grant （705001）, the National Basic Research Program of China （973 program, 2009CB522502, 2009CB941200 and 2007CB947901）, National Natural Science Foundation of China for Creative Research Groups （30421004）, the Chinese Science and Technology Key Project （2008zx10002-014, 2008zx10002- 011 and 2009ZX 10004-403） and a 111 Project to Deng H.
文摘Human induced pluripotent stem （iPS） cells are similar to embryonic stem （ES） cells, and can proliferate intensively and differentiate into a variety of cell types. However, the hepatic differentiation of human iPS cells has not yet been reported. In this report, human iPS cells were induced to differentiate into hepatic cells by a stepwise protocol. The expression of liver cell markers and liver-related functions of the human iPS cell-derived cells were monitored and compared with that of differentiated human ES cells and primary human hepatocytes. Approximately 60% of the differentiated human iPS cells at day 7 expressed hepatic markers alpha fetoprotein and Alb. The differentiated cells at day 21 exhibited liver cell functions including albumin Asecretion, glycogen synthesis, urea production and inducible cytochrome P450 activity. The expression of hepatic markers and fiver-related functions of the iPS cellderived hepatic ceils were comparable to that of the human ES cell-derived hepatic cells. These results show that human iPS cells, which are similar to human ES cells, can be efficiently induced to differentiate into hepatocyte-like cells.
