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Cardiac disease modeling using induced pluripotent stem cell-derived human cardiomyocytes

World Journal of Stem Cells 2015年第2期7卷 329-342页

作者： Patrizia Dell'Era Patrizia Benzoni Elisabetta Crescini Matteo Valle Er Xia Antonella Consiglio Maurizio Memo fibroblast reprogramming unit Department of Molecular and Translational MedicineUniversity of Brescia Department of Molecular and Translational Medicine University of Brescia

Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alterationhas been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes(CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells(i PSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since i PSC preserve the entire patients' genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledg

关键词： Cardiomyopathies Cardiac arrhythmias Induced pluripotent stem cells Human cardiomyocytes

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Small molecules facilitate single factor-mediated sweat gland cell reprogramming

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Military Medical Research 2022年第6期9卷 655-667页

作者： Shuai-Fei Ji Lai-Xian Zhou Zhi-Feng Sun Jiang-Bing Xiang Shao-Yuan Cui Yan Li Hua-Ting Chen Yi-Qiong Liu Huan-Huan Gao Xiao-Bing Fu Xiao-Yan Sun Research Center for Tissue Repair and Regeneration Affiliated To Medical Innovation Research Department and 4th Medical Center PLA General Hospital and PLA Medical CollegePLAKey Laboratory of Tissue Repairand Regenerative Medicine and BeijingKey Research Laboratoryof Skin InjuryRepair and Regeneration28 FuXing RoadBeijing 100853China Research unit of Trauma Care Tissue Repair and RegenerationChinese Academy of Medical Sciences2019RU051Beijing 100048China Departmentof Respiratory the Second Medical CenterChinese PLA General HospitalBeijing 100036China Bioengineering College of Chongqing University Chongqing 400044China Department of Nephrology the First Medical CenterChinese PLA General HospitalState Key Laboratory of Kidney DiseasesBeijing 100048China

Background: Large skin defects severely disrupt the overall skin structure and can irreversibly damage sweat glands(SGs), thus impairing the skin’s physiological function. This study aims to develop a stepwise reprogramming strategy to convert fibroblasts into SG lineages, which may provide a promising method to obtain desirable cell types for the functional repair and regeneration of damaged ***: The expression of the SG markers cytokeratin 5(CK5), cytokeratin 10(CK10), cytokeratin 18(CK18), carcinoembryonic antigen(CEA), aquaporin 5(AQP5) and α-smooth muscle actin(α-SMA) was assessed with quantitative PCR(qPCR), immunofluorescence and flow cytometry. Calcium activity analysis was conducted to test the function of induced SG-like cells(iSGCs). Mouse xenograft models were also used to evaluate the in vivo regeneration of ***/c nude mice were randomly divided into normal group, SGM treatment group and iSGC transplantation *** analyses and starch-iodine sweat tests were used to confirm the in vivo regeneration of ***: Ectodermal dysplasia antigen(EDA) overexpression drove human dermal fibroblast(HDF) conversion into i SGCs in SG culture medium(SGM). qPCR indicated significantly increased mRNA levels of the SG markers CK5, CK18and CEA in iSGCs, and flow cytometry data demonstrated(4.18±0.04)% of iSGCs were CK5 positive and(4.36±0.25)%of iSGCs were CK18 positive. The addition of chemical cocktails greatly accelerated the SG fate program. qPCR results revealed significantly increased mRNA expression of CK5, CK18 and CEA in iSGCs, as well as activation of the duct marker CK10 and luminal functional marker AQP5. Flow cytometry indicated, after the treatment of chemical cocktails,(23.05±2.49)% of iSGCs expressed CK5^(+) and(55.79±3.18)% of iSGCs expressed CK18^(+), respectively. Calcium activity analysis indicated that the reactivity of iSGCs to acetylcholine was close to that of primary SG cells [(60.79±7.71)% vs.(70.59±0.34)%

关键词： Direct reprogramming Human dermal fibroblasts Sweat gland Regeneration

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Gene targeting and Calcium handling efficiencies in mouse embryonic stem cell lines

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World Journal of Stem Cells 2010年第6期2卷 127-140页

作者： Solomon Mamo Julianna Kobolak Istvan Borbíró Tamás Bíró Istvan Bock Andras Dinnyes Genetic reprogramming Group Agricultural Biotechnology CenterSzent-Gyorgyi Albert ut.4H-2100 GdllHungary Solomon MamoLyons Research FarmUniversity College DublinNewcastleCo DublinIreland Department of Physiology Research Center for Molecular MedicineMedical and Health Science CenterUniversity of DebrecenNagyerdei krt.98H-4032 DebrecenHungary Abiol Ltd. Nagyerdei krt.98H-4032 DebrecenHungary BioTalentum Ltd. Aulich l.26GdllHungary Andras DinnyesMolecular Animal Biotechnology LaboratorySzent Istvan UniversityPáter K.u.1H-2103 GdllHungary

AIM:To compare gene targeting efficiencies,expression profiles,and Ca2+ handling potentials in two widely used mouse embryonic stem cell ***:The two widely used mouse embryonic stem cell lines,R1 and HM-1,were cultured and maintained on Mitomycin C treated mouse embryonic fibroblast feeder cell layers,following standard culture *** were incubated with primary and secondary antibodies before fluorescence activated cell sorting analysis to compare known pluripotency ***,cells were harvested by trypsinization and transfected with a kinase-inactive murine Tyk2 targeting construct,following the BioRad and Amaxa transfection ***,the cells were cultured and neomycin-resistant cells were picked after 13 d of *** clones were screened twice by polymerase chain reaction(PCR) and finally confirmed by Southern blot analysis before *** gene expression profiles of more than 20 400 probes were also compared and significantly regulated genes were conf irmed by real time PCR *** handling potentials of these cell lines were also compared using various ***:We found signif icant differences in transfection eff iciencies of the two cell lines(91% ± 6.1% vs 75% ± 4.2%,P = 0.01).Differences in the targeting efficiencies were also significant whether the Amaxa or BioRad platforms were used for *** did not observe signif icant differences in the levels of many known pluripotency ***,our genomewide expression analysis using more than 20 400 spotted cDNA arrays identified 55 differentially regulated transcripts(P < 0.05) implicated in various important biological processes,including binding molecular functions(particularly Ca2+ binding roles).Subsequently,we measured Ca2+ signals in these cell lines in response to various calcium agonists,both in high and low Ca2+ solutions,and found signif icant differences(P < 0.05) in the regulation of Ca2+ homeostasis betwee

关键词： Embryonic stem cells Microarray Calcium Agonists Transfection Cell culture Pluripotency Gene targeting

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