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
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
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
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
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
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
Human pluripotent stem cell(hPSC)-derived kidney organoids share similarities with the fetal kidney.However,the current hPSC-derived kidney organoids have some limitations,including the inability to perform nephrogene...Human pluripotent stem cell(hPSC)-derived kidney organoids share similarities with the fetal kidney.However,the current hPSC-derived kidney organoids have some limitations,including the inability to perform nephrogenesis and lack of a corticomedullary definition,uniform vascular system,and coordinated exit path-way for urinary filtrate.Therefore,further studies are required to produce hPSC-derived kidney organoids that accurately mimic human kidneys to facilitate research on kidney development,regeneration,disease modeling,and drug screening.In this review,we discussed recent advances in the generation of hPSC-derived kidney organoids,how these organoids contribute to the understanding of human kidney development and research in disease modeling.Additionally,the limitations,future research focus,and applications of hPSC-derived kidney organoids were highlighted.展开更多
Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vi...Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development.Emerging evidence and impressive advances in human induced pluripotent stem cells,with tuned neural induction and differentiation protocols,makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible.Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol,we obtained multiple subtypes of neurons,including 20%tyrosine hydroxylase-positive dopaminergic neurons.To obtain more dopaminergic neurons,we next added sonic hedgehog(SHH)and fibroblast growth factor 8(FGF8)on day 8 of induction.This increased the proportion of dopaminergic neurons,up to 75%tyrosine hydroxylase-positive neurons,with 15%tyrosine hydroxylase and forkhead box protein A2(FOXA2)co-expressing neurons.We further optimized the induction protocol by applying the small molecule inhibitor,CHIR99021(CHIR).This helped facilitate the generation of midbrain dopaminergic neurons,and we obtained 31-74%midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining.Thus,we have established three induction protocols for dopaminergic neurons.Based on tyrosine hydroxylase and FOXA2 immunostaining analysis,the CHIR,SHH,and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons,which could be an ideal resource for tackling midbrain-related diseases.展开更多
Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal mus...Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal muscular atrophy-like clinical phenotype.The aims of this study were to determine the mechanism of the severe phenotype caused by the MORC2 p.S87L mutation and to explore potential treatment strategies.Epithelial cells were isolated from urine samples from a spinal muscular atrophy(SMA)-like patient[MORC2 p.S87L),a CMT2Z patient[MORC2 p.Q400R),and a healthy control and induced to generate pluripotent stem cells,which were then differentiated into motor neuron precursor cells.Next-generation RNA sequencing followed by KEGG pathway enrichment analysis revealed that differentially expressed genes involved in the PI3K/Akt and MAP K/ERK signaling pathways were enriched in the p.S87L SMA-like patient group and were significantly downregulated in induced pluripotent stem cells.Reduced proliferation was observed in the induced pluripotent stem cells and motor neuron precursor cells derived from the p.S87L SMA-like patient group compared with the CMT2Z patient group and the healthy control.G0/G1 phase cell cycle arrest was observed in induced pluripotent stem cells derived from the p.S87L SMA-like patient.MORC2 p.S87Lspecific antisense oligonucleotides(p.S87L-ASO-targeting)showed significant efficacy in improving cell prolife ration and activating the PI3K/Akt and MAP K/ERK pathways in induced pluripotent stem cells.Howeve r,p.S87L-ASO-ta rgeting did not rescue prolife ration of motor neuron precursor cells.These findings suggest that downregulation of the PI3K/Akt and MAP K/ERK signaling pathways leading to reduced cell proliferation and G0/G1 phase cell cycle arrest in induced pluripotent stem cells might be the underlying mechanism of the severe p.S87L SMA-like phenotype.p.S87L-ASO-targeting treatment can alleviate disordered cell proliferation in the early stage of pluripotent stem cell induction.展开更多
BACKGROUND Human induced pluripotent stem cell(hiPSC)technology is a valuable tool for generating patient-specific stem cells,facilitating disease modeling,and invest-igating disease mechanisms.However,iPSCs carrying ...BACKGROUND Human induced pluripotent stem cell(hiPSC)technology is a valuable tool for generating patient-specific stem cells,facilitating disease modeling,and invest-igating disease mechanisms.However,iPSCs carrying specific mutations may limit their clinical applications due to certain inherent characteristics.AIM To investigate the impact of MERTK mutations on hiPSCs and determine whether hiPSC-derived extracellular vesicles(EVs)influence anomalous cell junction and differentiation potential.METHODS We employed a non-integrating reprogramming technique to generate peripheral blood-derived hiPSCs with and hiPSCs without a MERTK mutation.Chromo-somal karyotype analysis,flow cytometry,and immunofluorescent staining were utilized for hiPSC identification.Transcriptomics and proteomics were employed to elucidate the expression patterns associated with cell junction abnormalities and cellular differentiation potential.Additionally,EVs were isolated from the supernatant,and their RNA and protein cargos were examined to investigate the involvement of hiPSC-derived EVs in stem cell junction and differentiation.RESULTS The generated hiPSCs,both with and without a MERTK mutation,exhibited normal karyotype and expressed pluripotency markers;however,hiPSCs with a MERTK mutation demonstrated anomalous adhesion capability and differentiation potential,as confirmed by transcriptomic and proteomic profiling.Furthermore,hiPSC-derived EVs were involved in various biological processes,including cell junction and differentiation.CONCLUSION HiPSCs with a MERTK mutation displayed altered junction characteristics and aberrant differentiation potential.Furthermore,hiPSC-derived EVs played a regulatory role in various biological processes,including cell junction and differentiation.展开更多
Human pluripotent stem cell(hPSC)models provide unprecedented opportunities to study human neurological disorders by recapitulating human-specific disease mechanisms.In particular,hPSC-based human–animal brain chimer...Human pluripotent stem cell(hPSC)models provide unprecedented opportunities to study human neurological disorders by recapitulating human-specific disease mechanisms.In particular,hPSC-based human–animal brain chimeras enable the study of human cell pathophysiology in vivo.In chimeric brains,human neural and immune cells can maintain human-specific features,undergo maturation,and functionally integrate into host brains,allowing scientists to study how human cells impact neural circuits and animal behaviors.The emerging human–animal brain chimeras hold promise for modeling human brain cells and their interactions in health and disease,elucidating the disease mechanism from molecular and cellular to circuit and behavioral levels,and testing the efficacy of cell therapy interventions.Here,we discuss recent advances in the generation and applications of using human–animal chimeric brain models for the study of neurological disorders,including disease modeling and cell therapy.展开更多
Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood ve...Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood vessels are related to many disorders like stroke,myocardial infarction,aneurysm,and diabetes,which are important causes of death worldwide.Translational research for new appro-aches to disease modeling and effective treatment is needed due to the huge socio-economic burden on healthcare systems.Although mice or rats have been widely used,applying data from animal studies to human-specific vascular physiology and pathology is difficult.The rise of induced pluripotent stem cells(iPSCs)provides a reliable in vitro resource for disease modeling,regenerative medicine,and drug discovery because they carry all human genetic information and have the ability to directionally differentiate into any type of human cells.This review summarizes the latest progress from the establishment of iPSCs,the strategies for differentiating iPSCs into vascular cells,and the in vivo trans-plantation of these vascular derivatives.It also introduces the application of these technologies in disease modeling,drug screening,and regenerative medicine.Additionally,the application of high-tech tools,such as omics analysis and high-throughput sequencing,in this field is reviewed.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
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 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 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.
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.
基金the National Natural Science Foundation of China,No.82360148Guizhou Science&Technology Department,No.QKHPTRC2018-5636-2 and No.QKHPTRC2020-2201.
文摘Human pluripotent stem cell(hPSC)-derived kidney organoids share similarities with the fetal kidney.However,the current hPSC-derived kidney organoids have some limitations,including the inability to perform nephrogenesis and lack of a corticomedullary definition,uniform vascular system,and coordinated exit path-way for urinary filtrate.Therefore,further studies are required to produce hPSC-derived kidney organoids that accurately mimic human kidneys to facilitate research on kidney development,regeneration,disease modeling,and drug screening.In this review,we discussed recent advances in the generation of hPSC-derived kidney organoids,how these organoids contribute to the understanding of human kidney development and research in disease modeling.Additionally,the limitations,future research focus,and applications of hPSC-derived kidney organoids were highlighted.
基金supported by the National Natural Science Foundation of China,No.81771222(to LS)Guangzhou Key Research Program on Brain Science,Nos.202007030011,202206060001(to LS)the Program of Introducing Talents of Discipline to Universities of China,No.B14036(to KFS)。
文摘Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development.Emerging evidence and impressive advances in human induced pluripotent stem cells,with tuned neural induction and differentiation protocols,makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible.Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol,we obtained multiple subtypes of neurons,including 20%tyrosine hydroxylase-positive dopaminergic neurons.To obtain more dopaminergic neurons,we next added sonic hedgehog(SHH)and fibroblast growth factor 8(FGF8)on day 8 of induction.This increased the proportion of dopaminergic neurons,up to 75%tyrosine hydroxylase-positive neurons,with 15%tyrosine hydroxylase and forkhead box protein A2(FOXA2)co-expressing neurons.We further optimized the induction protocol by applying the small molecule inhibitor,CHIR99021(CHIR).This helped facilitate the generation of midbrain dopaminergic neurons,and we obtained 31-74%midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining.Thus,we have established three induction protocols for dopaminergic neurons.Based on tyrosine hydroxylase and FOXA2 immunostaining analysis,the CHIR,SHH,and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons,which could be an ideal resource for tackling midbrain-related diseases.
基金supported by the National Natural Science Foundation of China,Nos.82171172(to RZ)and 81771366(to RZ)Fundamental Research Funds for the Central Universities of Central South University,Nos.2021zzts1095(to SZ)and 2022zzts0832(to HY)。
文摘Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal muscular atrophy-like clinical phenotype.The aims of this study were to determine the mechanism of the severe phenotype caused by the MORC2 p.S87L mutation and to explore potential treatment strategies.Epithelial cells were isolated from urine samples from a spinal muscular atrophy(SMA)-like patient[MORC2 p.S87L),a CMT2Z patient[MORC2 p.Q400R),and a healthy control and induced to generate pluripotent stem cells,which were then differentiated into motor neuron precursor cells.Next-generation RNA sequencing followed by KEGG pathway enrichment analysis revealed that differentially expressed genes involved in the PI3K/Akt and MAP K/ERK signaling pathways were enriched in the p.S87L SMA-like patient group and were significantly downregulated in induced pluripotent stem cells.Reduced proliferation was observed in the induced pluripotent stem cells and motor neuron precursor cells derived from the p.S87L SMA-like patient group compared with the CMT2Z patient group and the healthy control.G0/G1 phase cell cycle arrest was observed in induced pluripotent stem cells derived from the p.S87L SMA-like patient.MORC2 p.S87Lspecific antisense oligonucleotides(p.S87L-ASO-targeting)showed significant efficacy in improving cell prolife ration and activating the PI3K/Akt and MAP K/ERK pathways in induced pluripotent stem cells.Howeve r,p.S87L-ASO-ta rgeting did not rescue prolife ration of motor neuron precursor cells.These findings suggest that downregulation of the PI3K/Akt and MAP K/ERK signaling pathways leading to reduced cell proliferation and G0/G1 phase cell cycle arrest in induced pluripotent stem cells might be the underlying mechanism of the severe p.S87L SMA-like phenotype.p.S87L-ASO-targeting treatment can alleviate disordered cell proliferation in the early stage of pluripotent stem cell induction.
基金Supported by the National Natural Science Foundation of China,No.82125007Beijing Natural Science Foundation,No.Z200014.
文摘BACKGROUND Human induced pluripotent stem cell(hiPSC)technology is a valuable tool for generating patient-specific stem cells,facilitating disease modeling,and invest-igating disease mechanisms.However,iPSCs carrying specific mutations may limit their clinical applications due to certain inherent characteristics.AIM To investigate the impact of MERTK mutations on hiPSCs and determine whether hiPSC-derived extracellular vesicles(EVs)influence anomalous cell junction and differentiation potential.METHODS We employed a non-integrating reprogramming technique to generate peripheral blood-derived hiPSCs with and hiPSCs without a MERTK mutation.Chromo-somal karyotype analysis,flow cytometry,and immunofluorescent staining were utilized for hiPSC identification.Transcriptomics and proteomics were employed to elucidate the expression patterns associated with cell junction abnormalities and cellular differentiation potential.Additionally,EVs were isolated from the supernatant,and their RNA and protein cargos were examined to investigate the involvement of hiPSC-derived EVs in stem cell junction and differentiation.RESULTS The generated hiPSCs,both with and without a MERTK mutation,exhibited normal karyotype and expressed pluripotency markers;however,hiPSCs with a MERTK mutation demonstrated anomalous adhesion capability and differentiation potential,as confirmed by transcriptomic and proteomic profiling.Furthermore,hiPSC-derived EVs were involved in various biological processes,including cell junction and differentiation.CONCLUSION HiPSCs with a MERTK mutation displayed altered junction characteristics and aberrant differentiation potential.Furthermore,hiPSC-derived EVs played a regulatory role in various biological processes,including cell junction and differentiation.
文摘Human pluripotent stem cell(hPSC)models provide unprecedented opportunities to study human neurological disorders by recapitulating human-specific disease mechanisms.In particular,hPSC-based human–animal brain chimeras enable the study of human cell pathophysiology in vivo.In chimeric brains,human neural and immune cells can maintain human-specific features,undergo maturation,and functionally integrate into host brains,allowing scientists to study how human cells impact neural circuits and animal behaviors.The emerging human–animal brain chimeras hold promise for modeling human brain cells and their interactions in health and disease,elucidating the disease mechanism from molecular and cellular to circuit and behavioral levels,and testing the efficacy of cell therapy interventions.Here,we discuss recent advances in the generation and applications of using human–animal chimeric brain models for the study of neurological disorders,including disease modeling and cell therapy.
文摘Blood vessels constitute a closed pipe system distributed throughout the body,transporting blood from the heart to other organs and delivering metabolic waste products back to the lungs and kidneys.Changes in blood vessels are related to many disorders like stroke,myocardial infarction,aneurysm,and diabetes,which are important causes of death worldwide.Translational research for new appro-aches to disease modeling and effective treatment is needed due to the huge socio-economic burden on healthcare systems.Although mice or rats have been widely used,applying data from animal studies to human-specific vascular physiology and pathology is difficult.The rise of induced pluripotent stem cells(iPSCs)provides a reliable in vitro resource for disease modeling,regenerative medicine,and drug discovery because they carry all human genetic information and have the ability to directionally differentiate into any type of human cells.This review summarizes the latest progress from the establishment of iPSCs,the strategies for differentiating iPSCs into vascular cells,and the in vivo trans-plantation of these vascular derivatives.It also introduces the application of these technologies in disease modeling,drug screening,and regenerative medicine.Additionally,the application of high-tech tools,such as omics analysis and high-throughput sequencing,in this field is reviewed.
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.
