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
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
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
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
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
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
Postoperative cognitive dysfunction(POCD)is a common surgical complication.Diabetes mellitus(DM)increases risk of developing POCD after surgery.DM patients with POCD seriously threaten the quality of patients’life,ho...Postoperative cognitive dysfunction(POCD)is a common surgical complication.Diabetes mellitus(DM)increases risk of developing POCD after surgery.DM patients with POCD seriously threaten the quality of patients’life,however,the intrinsic mechanism is unclear,and the effective treatment is deficiency.Previous studies have demonstrated neuronal loss and reduced neurogenesis in the hippocampus in mouse models of POCD.In this study,we constructed a mouse model of DM by intraperitoneal injection of streptozotocin,and then induced postoperative cognitive dysfunction by transient bilateral common carotid artery occlusion.We found that mouse models of DM-POCD exhibited the most serious cognitive impairment,as well as the most hippocampal neural stem cells(H-NSCs)loss and neurogenesis decline.Subsequently,we hypothesized that small extracellular vesicles secreted by induced pluripotent stem cell-derived mesenchymal stem cells(iMSC-sEVs)might promote neurogenesis and restore cognitive function in patients with DM-POCD.iMSC-sEVs were administered via the tail vein beginning on day 2 after surgery,and then once every 3 days for 1 month thereafter.Our results showed that iMSC-sEVs treatment significantly recovered compromised proliferation and neuronal-differentiation capacity in H-NSCs,and reversed cognitive impairment in mouse models of DM-POCD.Furthermore,miRNA sequencing and qPCR showed miR-21-5p and miR-486-5p were the highest expression in iMSC-sEVs.We found iMSC-sEVs mainly transferred miR-21-5p and miR-486-5p to promote H-NSCs proliferation and neurogenesis.As miR-21-5p was demonstrated to directly targete Epha4 and CDKN2C,while miR-486-5p can inhibit FoxO1 in NSCs.We then demonstrated iMSC-sEVs can transfer miR-21-5p and miR-486-5p to inhibit EphA4,CDKN2C,and FoxO1 expression in H-NSCs.Collectively,these results indicate significant H-NSC loss and neurogenesis reduction lead to DM-POCD,the application of iMSC-sEVs may represent a novel cell-free therapeutic tool for diabetic patients with postoperative cognitive dysfunction.展开更多
Brain diseases affect 1 in 6 people worldwide.These diseases range from acute neurological conditions such as stroke to chronic neurodegenerative disorders such as Alzheimer’s disease.Recent advancements in tissue-en...Brain diseases affect 1 in 6 people worldwide.These diseases range from acute neurological conditions such as stroke to chronic neurodegenerative disorders such as Alzheimer’s disease.Recent advancements in tissue-engineered brain disease models have overcome many of the different shortcomings associated with the various animal models,tissue culture models,and epidemiologic patient data that are commonly used to study brain disease.One innovative method by which to model human neurological disease is via the directed differentiation of human pluripotent stem cells(hPSCs)to neural lineages including neurons,astrocytes,and oligodendrocytes.Three-dimensional models such as brain organoids have also been derived from hPSCs,offering more physiological relevance due to their incorporation of various cell types.As such,brain organoids can better model the pathophysiology of neural diseases observed in patients.In this review,we will emphasize recent developments in hPSC-based tissue culture models of neurological disorders and how they are being used to create neural disease models.展开更多
BACKGROUND Current evidence shows that human induced pluripotent stem cells(hiPSCs)can effectively differentiate into keratinocytes(KCs),but its effect on skin burn healing has not been reported.AIM To observe the eff...BACKGROUND Current evidence shows that human induced pluripotent stem cells(hiPSCs)can effectively differentiate into keratinocytes(KCs),but its effect on skin burn healing has not been reported.AIM To observe the effects of hiPSCs-derived KCs transplantation on skin burn healing in mice and to preliminarily reveal the underlying mechanisms.METHODS An analysis of differentially expressed genes in burn wounds based on GEO datasets GSE140926,and GSE27186 was established.A differentiation medium containing retinoic acid and bone morphogenetic protein 4 was applied to induce hiPSCs to differentiate into KCs.The expression of KCs marker proteins was detected using immunofluorescence staining.A model of a C57BL/6 mouse with deep cutaneous second-degree burn was created,and then phosphate buffered saline(PBS),hiPSCs-KCs,or hiPSCs-KCs with knockdown of COL7A1 were injected around the wound surface.The wound healing,re-epithelialization,engraftment of hiPSCs-KCs into wounds,proinflammatory factor level,and the NF-κB pathway proteins were assessed by hematoxylin-eosin staining,carboxifluorescein diacetate succinimidyl ester(CFSE)fluorescence staining,enzyme linked immunosorbent assay,and Western blotting on days 3,7,and 14 after the injection,respectively.Moreover,the effects of COL7A1 knockdown on the proliferation and migration of hiPSCs-KCs were confirmed by immunohistochemistry,EdU,Transwell,and damage repair assays.RESULTS HiPSCs-KCs could express the hallmark proteins of KCs.COL7A1 was down-regulated in burn wound tissues and highly expressed in hiPSCs-KCs.Transplantation of hiPSCs-KCs into mice with burn wounds resulted in a significant decrease in wound area,an increase in wound re-epithelialization,a decrease in proinflammatory factors content,and an inhibition of NF-κB pathway activation compared to the PBS group.The in vitro assay showed that COL7A1 knockdown could rescue the inhibition of hiPSCs-KCs proliferation and migration,providing further evidence that COL7A1 speeds up burn wound healing by limiting cell proliferation and migration.CONCLUSION In deep,second-degree burn wounds,COL7A1 can promote KC proliferation and migration while also suppressing the inflammatory response.展开更多
Cardiomyopathy is a pathological condition characterized by cardiac pump failure due to myocardial dysfunction and the major cause of advanced heart failure requiring heart transplantation.Although optimized medical t...Cardiomyopathy is a pathological condition characterized by cardiac pump failure due to myocardial dysfunction and the major cause of advanced heart failure requiring heart transplantation.Although optimized medical therapies have been developed for heart failure during the last few decades,some patients with cardiomyopathy exhibit advanced heart failure and are refractory to medical therapies.Desmosome,which is a dynamic cell-to-cell junctional component,maintains the structural integrity of heart tissues.Genetic mutations in desmo-somal genes cause arrhythmogenic cardiomyopathy(AC),a rare inheritable disease,and predispose patients to sudden cardiac death and heart failure.Recent advances in sequencing technologies have elucidated the genetic basis of cardiomyopathies and revealed that desmosome-related cardiomyopathy is concealed in broad cardiomyopathies.Among desmosomal genes,mutations in PKP2(which encodes PKP2)are most frequently identified in patients with AC.PKP2 deficiency causes various pathological cardiac phenotypes.Human cardiomyocytes differentiated from patient-derived induced pluripotent stem cells(iPSCs)in combination with genome editing,which allows the precise arrangement of the targeted genome,are powerful experimental tools for studying disease.This review summarizes the current issues associated with practical medicine for advanced heart failure and the recent advances in disease modeling using iPSC-derived cardiomyocytes targeting desmosome-related cardiomyopathy caused by PKP2 deficiency.展开更多
BACKGROUND Induced pluripotent stem cells(iPSCs)show great ability to differentiate into any tissue,making them attractive candidates for pathophysiological investigations.The rise of organ-on-a-chip technology in the...BACKGROUND Induced pluripotent stem cells(iPSCs)show great ability to differentiate into any tissue,making them attractive candidates for pathophysiological investigations.The rise of organ-on-a-chip technology in the past century has introduced a novel way to make in vitro cell cultures that more closely resemble their in vivo environments,both structural and functionally.The literature still lacks consensus on the best conditions to mimic the blood-brain barrier(BBB)for drug screening and other personalized therapies.The development of models based on BBB-on-achip using iPSCs is promising and is a potential alternative to the use of animals in research.AIM To analyze the literature for BBB models on-a-chip involving iPSCs,describe the microdevices,the BBB in vitro construction,and applications.METHODS We searched for original articles indexed in PubMed and Scopus that used iPSCs to mimic the BBB and its microenvironment in microfluidic devices.Thirty articles were identified,wherein only 14 articles were finally selected according to the inclusion and exclusion criteria.Data compiled from the selected articles were organized into four topics:(1)Microfluidic devices design and fabrication;(2)characteristics of the iPSCs used in the BBB model and their differentiation conditions;(3)BBB-on-a-chip reconstruction process;and(4)applications of BBB microfluidic three-dimensional models using iPSCs.RESULTS This study showed that BBB models with iPSCs in microdevices are quite novel in scientific research.Important technological advances in this area regarding the use of commercial BBB-on-a-chip were identified in the most recent articles by different research groups.Conventional polydimethylsiloxane was the most used material to fabricate in-house chips(57%),whereas few studies(14.3%)adopted polymethylmethacrylate.Half the models were constructed using a porous membrane made of diverse materials to separate the channels.iPSC sources were divergent among the studies,but the main line used was IMR90-C4 from human fetal lung fibroblast(41.2%).The cells were differentiated through diverse and complex processes either to endothelial or neural cells,wherein only one study promoted differentiation inside the chip.The construction process of the BBB-on-a-chip involved previous coating mostly with fibronectin/collagen Ⅳ(39.3%),followed by cell seeding in single cultures(36%)or co-cultures(64%)under controlled conditions,aimed at developing an in vitro BBB that mimics the human BBB for future applications.CONCLUSION This review evidenced technological advances in the construction of BBB models using iPSCs.Nonetheless,a definitive BBB-on-a-chip has not yet been achieved,hindering the applicability of the models.展开更多
Extracellular vesicles(EVs)are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools.Accordingly,in various biological contexts,EVs are re...Extracellular vesicles(EVs)are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools.Accordingly,in various biological contexts,EVs are reported to engage in immune modulation,senescence,and cell proliferation and differentiation.Therefore,EVs could be key elements for potential off-the-shelf cell-free therapy.Little has been studied regarding EVs derived from human pluripotent stem cells(hPSC-EVs),even though hPSCs offer good opportunities for induction of tissue regeneration and unlimited proliferative ability.In this review article,we provide an overview of studies using hPSC-EVs,focusing on identifying the conditions in which the cells are cultivated for the isolation of EVs,how they are characterized,and applications already demonstrated.The topics reported in this article highlight the incipient status of the studies in the field and the significance of hPSC-EVs’prospective applications as PSC-derived cell-free therapy products.展开更多
A century has passed since the Nobel Prize winning discovery of insulin,which still remains the mainstay treatment for type 1 diabetes mellitus(T1DM)to this day.True to the words of its discoverer Sir Frederick Banti...A century has passed since the Nobel Prize winning discovery of insulin,which still remains the mainstay treatment for type 1 diabetes mellitus(T1DM)to this day.True to the words of its discoverer Sir Frederick Banting,“insulin is not a cure for diabetes,it is a treatment”,millions of people with T1DM are dependent on daily insulin medications for life.Clinical donor islet transplantation has proven that T1DM is curable,however due to profound shortages of donor islets,it is not a mainstream treatment option for T1DM.Human pluripotent stem cell derived insulin-secreting cells,pervasively known as stem cell-derivedβcells(SC-βcells),are a promising alternative source and have the potential to become a T1DM treatment through cell replacement therapy.Here we briefly review how isletβcells develop and mature in vivo and several types of reported SC-βcells produced using different ex vivo protocols in the last decade.Although some markers of maturation were expressed and glucose stimulated insulin secretion was shown,the SC-βcells have not been directly compared to their in vivo counterparts,generally have limited glucose response,and are not yet fully matured.Due to the presence of extra-pancreatic insulin-expressing cells,and ethical and technological issues,further clarification of the true nature of these SC-βcells is required.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
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
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,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 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 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 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.
基金supported by the National Natural Science Foundation of China,No.82101463(to GWH)Natural Science Foundation of Jiangxi Provincial Science and Technology Department,No.20202BAB216013(to HLL)+1 种基金Jiangxi Provincial Health Commission General Science and Technology Project,No.202130370(to HLL)The Second Affiliated Hospital of Nanchang University’s Youth Innovation Team of Science and Technology Program,No.2019YNQN12009(to HLL)。
文摘Postoperative cognitive dysfunction(POCD)is a common surgical complication.Diabetes mellitus(DM)increases risk of developing POCD after surgery.DM patients with POCD seriously threaten the quality of patients’life,however,the intrinsic mechanism is unclear,and the effective treatment is deficiency.Previous studies have demonstrated neuronal loss and reduced neurogenesis in the hippocampus in mouse models of POCD.In this study,we constructed a mouse model of DM by intraperitoneal injection of streptozotocin,and then induced postoperative cognitive dysfunction by transient bilateral common carotid artery occlusion.We found that mouse models of DM-POCD exhibited the most serious cognitive impairment,as well as the most hippocampal neural stem cells(H-NSCs)loss and neurogenesis decline.Subsequently,we hypothesized that small extracellular vesicles secreted by induced pluripotent stem cell-derived mesenchymal stem cells(iMSC-sEVs)might promote neurogenesis and restore cognitive function in patients with DM-POCD.iMSC-sEVs were administered via the tail vein beginning on day 2 after surgery,and then once every 3 days for 1 month thereafter.Our results showed that iMSC-sEVs treatment significantly recovered compromised proliferation and neuronal-differentiation capacity in H-NSCs,and reversed cognitive impairment in mouse models of DM-POCD.Furthermore,miRNA sequencing and qPCR showed miR-21-5p and miR-486-5p were the highest expression in iMSC-sEVs.We found iMSC-sEVs mainly transferred miR-21-5p and miR-486-5p to promote H-NSCs proliferation and neurogenesis.As miR-21-5p was demonstrated to directly targete Epha4 and CDKN2C,while miR-486-5p can inhibit FoxO1 in NSCs.We then demonstrated iMSC-sEVs can transfer miR-21-5p and miR-486-5p to inhibit EphA4,CDKN2C,and FoxO1 expression in H-NSCs.Collectively,these results indicate significant H-NSC loss and neurogenesis reduction lead to DM-POCD,the application of iMSC-sEVs may represent a novel cell-free therapeutic tool for diabetic patients with postoperative cognitive dysfunction.
基金CTSA Award from the National Center for Advancing Translational Sciences to the Vanderbilt Institute for Clinical and Translational Research,No.UL1 TR002243A Pilot and Feasibility Award from the NIDDK to the Vanderbilt Diabetes Research and Training Center,No.DK020593a VA MERIT Award,No.BX004845.
文摘Brain diseases affect 1 in 6 people worldwide.These diseases range from acute neurological conditions such as stroke to chronic neurodegenerative disorders such as Alzheimer’s disease.Recent advancements in tissue-engineered brain disease models have overcome many of the different shortcomings associated with the various animal models,tissue culture models,and epidemiologic patient data that are commonly used to study brain disease.One innovative method by which to model human neurological disease is via the directed differentiation of human pluripotent stem cells(hPSCs)to neural lineages including neurons,astrocytes,and oligodendrocytes.Three-dimensional models such as brain organoids have also been derived from hPSCs,offering more physiological relevance due to their incorporation of various cell types.As such,brain organoids can better model the pathophysiology of neural diseases observed in patients.In this review,we will emphasize recent developments in hPSC-based tissue culture models of neurological disorders and how they are being used to create neural disease models.
基金Supported by the Hospital Research Fund,No.SDFEYBS1805,No.SDFEYGJ2013 and No.XKTJ-HRC20210015Suzhou Science and Technology Development Project,No.SYS2020105,No.SKJY2021078 and No.2022SS43+2 种基金the Special Project of“Technological Innovation”Project of CNNC Medical Industry Co.Ltd,No.ZHYLZD2021002CNNC Elite Talent Program2022 State Key Laboratory of Radiological Medicine and Radiation Protection jointly built by Province and Ministry,No.GZK1202244.
文摘BACKGROUND Current evidence shows that human induced pluripotent stem cells(hiPSCs)can effectively differentiate into keratinocytes(KCs),but its effect on skin burn healing has not been reported.AIM To observe the effects of hiPSCs-derived KCs transplantation on skin burn healing in mice and to preliminarily reveal the underlying mechanisms.METHODS An analysis of differentially expressed genes in burn wounds based on GEO datasets GSE140926,and GSE27186 was established.A differentiation medium containing retinoic acid and bone morphogenetic protein 4 was applied to induce hiPSCs to differentiate into KCs.The expression of KCs marker proteins was detected using immunofluorescence staining.A model of a C57BL/6 mouse with deep cutaneous second-degree burn was created,and then phosphate buffered saline(PBS),hiPSCs-KCs,or hiPSCs-KCs with knockdown of COL7A1 were injected around the wound surface.The wound healing,re-epithelialization,engraftment of hiPSCs-KCs into wounds,proinflammatory factor level,and the NF-κB pathway proteins were assessed by hematoxylin-eosin staining,carboxifluorescein diacetate succinimidyl ester(CFSE)fluorescence staining,enzyme linked immunosorbent assay,and Western blotting on days 3,7,and 14 after the injection,respectively.Moreover,the effects of COL7A1 knockdown on the proliferation and migration of hiPSCs-KCs were confirmed by immunohistochemistry,EdU,Transwell,and damage repair assays.RESULTS HiPSCs-KCs could express the hallmark proteins of KCs.COL7A1 was down-regulated in burn wound tissues and highly expressed in hiPSCs-KCs.Transplantation of hiPSCs-KCs into mice with burn wounds resulted in a significant decrease in wound area,an increase in wound re-epithelialization,a decrease in proinflammatory factors content,and an inhibition of NF-κB pathway activation compared to the PBS group.The in vitro assay showed that COL7A1 knockdown could rescue the inhibition of hiPSCs-KCs proliferation and migration,providing further evidence that COL7A1 speeds up burn wound healing by limiting cell proliferation and migration.CONCLUSION In deep,second-degree burn wounds,COL7A1 can promote KC proliferation and migration while also suppressing the inflammatory response.
基金Supported by JSPS KAKENHI,No.20K21602,No.21H02915,and No.22K19526the Japan Agency for Medical Research and Development,No.21bm0804008h0005+2 种基金the Cell Science Research Foundationthe Grant for Basic Research of the Japanese Circulation Society(2018)SENSHIN Medical Research Foundation.
文摘Cardiomyopathy is a pathological condition characterized by cardiac pump failure due to myocardial dysfunction and the major cause of advanced heart failure requiring heart transplantation.Although optimized medical therapies have been developed for heart failure during the last few decades,some patients with cardiomyopathy exhibit advanced heart failure and are refractory to medical therapies.Desmosome,which is a dynamic cell-to-cell junctional component,maintains the structural integrity of heart tissues.Genetic mutations in desmo-somal genes cause arrhythmogenic cardiomyopathy(AC),a rare inheritable disease,and predispose patients to sudden cardiac death and heart failure.Recent advances in sequencing technologies have elucidated the genetic basis of cardiomyopathies and revealed that desmosome-related cardiomyopathy is concealed in broad cardiomyopathies.Among desmosomal genes,mutations in PKP2(which encodes PKP2)are most frequently identified in patients with AC.PKP2 deficiency causes various pathological cardiac phenotypes.Human cardiomyocytes differentiated from patient-derived induced pluripotent stem cells(iPSCs)in combination with genome editing,which allows the precise arrangement of the targeted genome,are powerful experimental tools for studying disease.This review summarizes the current issues associated with practical medicine for advanced heart failure and the recent advances in disease modeling using iPSC-derived cardiomyocytes targeting desmosome-related cardiomyopathy caused by PKP2 deficiency.
基金CNPq,Nos.308901/2020-7 and 400856/2016-6FAPESP,Nos.2019/21070-3,2017/17868-4,and 2016/21470-3+1 种基金SisNANO 2.0/MCTIC,No.442539/2019-3the National Institute of Science and Technology Complex Fluids,INCT-FCx.
文摘BACKGROUND Induced pluripotent stem cells(iPSCs)show great ability to differentiate into any tissue,making them attractive candidates for pathophysiological investigations.The rise of organ-on-a-chip technology in the past century has introduced a novel way to make in vitro cell cultures that more closely resemble their in vivo environments,both structural and functionally.The literature still lacks consensus on the best conditions to mimic the blood-brain barrier(BBB)for drug screening and other personalized therapies.The development of models based on BBB-on-achip using iPSCs is promising and is a potential alternative to the use of animals in research.AIM To analyze the literature for BBB models on-a-chip involving iPSCs,describe the microdevices,the BBB in vitro construction,and applications.METHODS We searched for original articles indexed in PubMed and Scopus that used iPSCs to mimic the BBB and its microenvironment in microfluidic devices.Thirty articles were identified,wherein only 14 articles were finally selected according to the inclusion and exclusion criteria.Data compiled from the selected articles were organized into four topics:(1)Microfluidic devices design and fabrication;(2)characteristics of the iPSCs used in the BBB model and their differentiation conditions;(3)BBB-on-a-chip reconstruction process;and(4)applications of BBB microfluidic three-dimensional models using iPSCs.RESULTS This study showed that BBB models with iPSCs in microdevices are quite novel in scientific research.Important technological advances in this area regarding the use of commercial BBB-on-a-chip were identified in the most recent articles by different research groups.Conventional polydimethylsiloxane was the most used material to fabricate in-house chips(57%),whereas few studies(14.3%)adopted polymethylmethacrylate.Half the models were constructed using a porous membrane made of diverse materials to separate the channels.iPSC sources were divergent among the studies,but the main line used was IMR90-C4 from human fetal lung fibroblast(41.2%).The cells were differentiated through diverse and complex processes either to endothelial or neural cells,wherein only one study promoted differentiation inside the chip.The construction process of the BBB-on-a-chip involved previous coating mostly with fibronectin/collagen Ⅳ(39.3%),followed by cell seeding in single cultures(36%)or co-cultures(64%)under controlled conditions,aimed at developing an in vitro BBB that mimics the human BBB for future applications.CONCLUSION This review evidenced technological advances in the construction of BBB models using iPSCs.Nonetheless,a definitive BBB-on-a-chip has not yet been achieved,hindering the applicability of the models.
文摘Extracellular vesicles(EVs)are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools.Accordingly,in various biological contexts,EVs are reported to engage in immune modulation,senescence,and cell proliferation and differentiation.Therefore,EVs could be key elements for potential off-the-shelf cell-free therapy.Little has been studied regarding EVs derived from human pluripotent stem cells(hPSC-EVs),even though hPSCs offer good opportunities for induction of tissue regeneration and unlimited proliferative ability.In this review article,we provide an overview of studies using hPSC-EVs,focusing on identifying the conditions in which the cells are cultivated for the isolation of EVs,how they are characterized,and applications already demonstrated.The topics reported in this article highlight the incipient status of the studies in the field and the significance of hPSC-EVs’prospective applications as PSC-derived cell-free therapy products.
基金Supported by the Juvenile Diabetes Research Foundation,No.4-2006-1025Diabetes Australia Research TrustTelethon Perth Children’s Hospital Research Fund(TPCHRF)grant to Jiang FX.
文摘A century has passed since the Nobel Prize winning discovery of insulin,which still remains the mainstay treatment for type 1 diabetes mellitus(T1DM)to this day.True to the words of its discoverer Sir Frederick Banting,“insulin is not a cure for diabetes,it is a treatment”,millions of people with T1DM are dependent on daily insulin medications for life.Clinical donor islet transplantation has proven that T1DM is curable,however due to profound shortages of donor islets,it is not a mainstream treatment option for T1DM.Human pluripotent stem cell derived insulin-secreting cells,pervasively known as stem cell-derivedβcells(SC-βcells),are a promising alternative source and have the potential to become a T1DM treatment through cell replacement therapy.Here we briefly review how isletβcells develop and mature in vivo and several types of reported SC-βcells produced using different ex vivo protocols in the last decade.Although some markers of maturation were expressed and glucose stimulated insulin secretion was shown,the SC-βcells have not been directly compared to their in vivo counterparts,generally have limited glucose response,and are not yet fully matured.Due to the presence of extra-pancreatic insulin-expressing cells,and ethical and technological issues,further clarification of the true nature of these SC-βcells is required.
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.
