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Roles of cell fusion, hybridization and polyploid cell formation in cancer metastasis

World Journal of clinical Oncology 2020年第3期11卷 121-135页

作者： Ivan Shabo Joar Svanvik Annelie Lindström Tanguy Lechertier Sara Trabulo James Hulit Tim Sparey John Pawelek Endocrine and Sarcoma Surgery Unit Department of Molecular Medicine and SurgeryKarolinska InstituteStockholm SE 17177Sweden Patient Area of Breast Cancer Sarcoma and Endocrine TumoursTheme CancerKarolinska University HospitalStockholm SE 17176Sweden The Transplant Institute Sahlgrenska University HospitalGothenburg SE 41345Sweden Division of Surgery Department of Biomedical and Clinical SciencesFaculty of Medicine and Health SciencesLinköping UniversityLinköping SE 58183Sweden Division of Cell Biology Department of Biomedical and Clinical SciencesFaculty of Medicine and Health SciencesLinköping UniversityLinköping SE 58185Sweden Novintum Bioscience Ltd London Bioscience Innovation CentreLondon NW10NHUnited Kingdom Department of Dermatology and the Yale Cancer Center Yale University School of MedicineNew HavenCT 06520United States

Cell-cell fusion is a normal biological process playing essential roles in organ formation and tissue differentiation,repair and *** cell fusion somatic cells undergo rapid nuclear reprogramming and epigenetic modifications to form hybrid cells with new genetic and phenotypic properties at a rate exceeding that achievable by random *** that stimulate cell fusion are inflammation and *** of cancer cells with non-neoplastic cells facilitates several malignancy-related cell phenotypes,e.g.,reprogramming of somatic cell into induced pluripotent stem cells and epithelial to mesenchymal *** is now considerable in vitro,in vivo and clinical evidence that fusion of cancer cells with motile leucocytes such as macrophages plays a major role in cancer *** the many changes in cancer cells after hybridizing with leucocytes,it is notable that hybrids acquire resistance to chemo-and radiation *** phenomenon that has been largely overlooked yet plays a role in these processes is *** of the mechanism of polyploid cell formation,it happens in response to genotoxic stresses and enhances a cancer cell’s ability to *** we summarize the recent progress in research of cell fusion and with a focus on an important role for polyploid cells in cancer *** addition,we discuss the clinical evidence and the importance of cell fusion and polyploidization in solid tumors.

关键词： Cell fusion Hybrid formation Polyploidization Macrophage Cancer progression Oncologic treatment resistance

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Effects of Angelica Polysaccharide on telomere length in mice with benzene-induced aplastic anemia

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TMR Modern Herbal Medicine 2020年第1期3卷 6-12页

作者： Runjie Sun Xing Cui Qingsong Wang Yanyu Zhang Xiaoqi Sun Institute of Traditional Chinese Medicine Shandong University of Traditional Chinese MedicineJinanChina Department of Hematology Affiliated Hospital of Shandong University of Traditional Chinese MedicineJinanChina First clinical College of clinical Shandong University of Traditional Chinese MedicineJinanChina

Objective:To investigate the changes in telomere length and the level of burst-forming units-erythrocyte(BFU-Es)and colony-forming unit-erythrocytes(CFU-Es)in mice with benzene-induced aplastic anemia(AA),and follow-up the therapeutic effects of Angelica Polysaccharide(AP).Methods:Male BALB/c mice(n=120)were randomly divided into three groups(1,2,3):normal control(n=24),AA control(n=48),and treated AA(n=48),*** in Group 2 received benzene inhalation for 2.5 months and 1 ml distilled water *** day for 2 weeks after the establishment of AA *** procedure was applied to the mice in Group 3 and AP was given for 2 weeks after the establishment of AA ***-time polymerase chain reaction was used to survey relative telomere length measurement of the bone marrow cells.A BFU-Es and CFU-Es survey was done to follow-up the therapeutic effects of ***:Compared with normal control,significant reductions of RBC,WBC,and platelet counts were found in peripheral blood of AA *** treatment with AP,counts of BFU-Es and CFU-Es were restored up to 66.8%and 77.25%,respectively,and length of telomere was restored up to 76.34%,of the normal *** telomere length in treated AA group was higher than the control AA ***:The AP can protect the telomere length and differentiation of hemopoietic stem/progenitor cells,accelerate the recovery of BFU-Es and CFU-Es of AA mice,and then improve the bone marrow failure.

关键词： Aplastic anemia Telomere Bone marrow failure Angelica polysaccharide

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Investigation of bone reconstruction using an attenuated immunogenicity xenogenic composite scaffold fabricated by 3D printing

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Bio-Design and Manufacturing 2020年第4期3卷 396-409页

作者： Qiongxi Pan Chenyuan Gao Yingying Wang Yili Wang Cong Mao Quan Wang Sophia N.Economidou Dennis Douroumis Feng Wen Lay Poh Tan Huaqiong Li School of Biomedical Engineering School of Ophthalmology and Optometry and Eye HospitalWenzhou Medical UniversityWenzhou 325035Zhejiang ProvincePeople’s Republic of China Engineering Research Center of clinical Functional Materials and Diagnosis and Treatment Devices of Zhejiang Province Wenzhou InstituteUniversity of Chinese Academy of SciencesWenzhou 325011Zhejiang ProvincePeople’s Republic of China Key Laboratory of Orthopedics of Zhejiang Province Department of OrthopedicsThe Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical UniversityWenzhou 325027Zhejiang ProvincePeople’s Republic of China Medway School of Pharmacy University of KentMedway CampusCentral AvenueChatham MaritimeChathamKent ME44TBUK CIPER Centre for Innovation and Process Engineering ResearchKent ME44TBUK School of Chemical and Biomedical Engineering Nanyang Technological UniversitySingapore 637459Singapore School of Materials Science and Engineering Nanyang Technological UniversitySingapore 639798Singapore

Bone is known to have a natural function to heal ***,if the bone damage is beyond a critical degree,intervention such as bone grafting may be *** this work,the fabrication of a novel bone scaffold composed of natural bone components and polycaprolactone(PCL)using 3D printing is put forward.α1,3-galactosyltransferase deficient pigs were used as the donor source of a *** porcine bone(DCB)with attenuated immunogenicity was used as the natural component of the scaffold with the aim to promote bone *** 3D printed DCB-PCL scaffolds combined essential advantages such as uniformity of the interconnected macropores and high porosity and enhanced compressive *** biological properties of the DCB-PCL scaffolds were evaluated by studying cell adhesion,viability,alkaline phosphatase activity and osteogenic gene expression of human bone marrow-derived mesenchymal stem *** in vitro results demonstrated that the DCB-PCL scaffolds exhibit an enhanced performance in promoting bone differentiation,which is correlated to the DCB ***,critical-sized cranial rat defects were used to assess the effect of DCB-PCL scaffolds on bone regeneration in *** results confirm that in comparison with PCL scaffolds,the DCB-PCL scaffolds can significantly improve new bone formation in cranial ***,the proposed 3D printed DCB-PCL scaffolds emerge as a promising regeneration alternative in the clinical treatment of large bone defects.

关键词： Polycaprolactone 3D printing Decellularized porcine bone Cranial bone regeneration Attenuated immunogenicity

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The deubiquitylase UCHL3 maintains cancer stem-like properties by stabilizing the aryl hydrocarbon receptor

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Signal Transduction and Targeted Therapy 2020年第1期5卷 1609-1622页

作者： Lianlian Ouyang Bin Yan Yating Liu Chao Mao Min Wang Na Liu Zuli Wang Shouping Liu Ying Shi Ling Chen Xiang Wang Yan Cheng Ya Cao Desheng Xiao Lingqiang Zhang Shuang Liu Yongguang Tao Department of Oncology Institute of Medical SciencesNational Clinical Research Center for Geriatric DisordersXiangya HospitalCentral South University410008 ChangshaHunanChina Key Laboratory of Carcinogenesis and Cancer Invasion Ministry of EducationDepartment of PathologyXiangya HospitalCentral South University410078 ChangshaHunanChina Key Laboratory of Carcinogenesis of the Ministry of Health Cancer Research InstituteSchool of Basic MedicineCentral South University410078 ChangshaHunanChina Department of Thoracic Surgery Hunan Key Laboratory of Tumor Models and Individualized MedicineSecond Xiangya HospitalCentral South UniversityChangshaChina Xiangya School of Pharmaceutical Sciences Central South University410008 ChangshaChina Department of Pathology Xiangya HospitalCentral South University410008 ChangshaHunanChina State Key Laboratory of Proteomics National Center for Protein Sciences(Beijing)Beijing Institute of Lifeomics100850 BeijingChina

Cancer stem cells(CSCs)exhibit highly aggressive and metastatic features and resistance to chemotherapy and *** hydrocarbon receptor(AhR)expression varies among non-small cell lung cancers(NSCLCs),and the mechanisms that support abnormal AhR expression in CSCs remain ***,we identified ubiquitin carboxyl terminal hydrolase L3(UCHL3),a DUB enzyme in the UCH protease family,as a bona fide deubiquitylase of the AhR in ***3 was shown to interact with,deubiquitylate,and stabilize AhR in a manner dependent on its deubiquitylation ***,we showed that UCHL3 promotes the stem-like characteristics and potent tumorigenic capacity of NSCLC ***3 increased AhR stability and the binding of AhR to the promoter regions of the“stemness”genes ATP-binding cassette subfamily G member 2(ABCG2),KLF4,and *** of UCHL3 markedly downregulated the“stemness”genes ABCG2,KLF4,and c-Myc,leading to the loss of selfrenewal and tumorigenesis in ***,the UCHL3 inhibitor TCID induced AhR degradation and exhibited significantly attenuated efficacy in NSCLC cells with stem cell-like ***,UCHL3 was shown to indicate poor prognosis in patients with lung *** general,our results reveal that the UCHL3 deubiquitylase is pivotal for AhR protein stability and a potential target for NSCLC-targeted therapy.

关键词： lung markedly chemotherapy

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Deciphering transcriptome alterations in bone marrow hematopoiesis at single-cell resolution in immune thrombocytopenia

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Signal Transduction and Targeted Therapy 2022年第11期7卷 4190-4207页

作者： Yan Liu Xinyi Zuo Peng Chen Xiang Hu Zi Sheng Anli Liu Qiang Liu Shaoqiu Leng Xiaoyu Zhang Xin Li Limei Wang Qi Feng Chaoyang Li Ming Hou Chong Chu Shihui Ma Shuwen Wang Jun Peng Department of Hematology Qilu HospitalCheeloo College of MedicineShandong UniversityJinan250012China State Key Laboratory of Experimental Hematology National Clinical Research Center for Blood DiseasesHaihe Laboratory of Cell EcosystemInstitute of Hematology&Blood Diseases HospitalChinese Academy of Medical Sciences&Peking Union Medical CollegeTianjin300020China Department of Hematology the Affiliated Hospital of Qingdao UniversityQingdao266000China Advanced Medical Research Institute Shandong UniversityJinan250012China Shangdong Key Laboratory of Immunochematology Qilu HospitalCheeloo College of MedicineShandong UniversityJinan250012China Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Chinese Ministry of HealthQilu HospitalCheeloo College of MedicineShandong UniversityJinan250012China Department of Biomedical Informatics Harvard Medical SchoolBoston02115MAUSA

Immune thrombocytopenia(ITP)is an autoimmune disorder,in which megakaryocyte dysfunction caused by an autoimmune reaction can lead to thrombocytopenia,although the underlying mechanisms remain ***,we performed single-cell transcriptome profiling of bone marrow CD34+hematopoietic stem and progenitor cells(HSPCs)to determine defects in megakaryopoiesis in *** expression,cell-cell interactions,and transcriptional regulatory networks varied in HSPCs of ITP,particularly in immune cell *** expressed gene(DEG)analysis indicated that there was an impaired megakaryopoiesis of *** cytometry confirmed that the number of CD9+and HES1+cells from Lin−CD34+CD45RA−HSPCs decreased in *** culture assays demonstrated that CD9+Lin−CD34+CD45RA−HSPCs tended to differentiate into megakaryocytes;however,this tendency was not observed in ITP patients and more erythrocytes were *** percentage of megakaryocytes differentiated from CD9+Lin−CD34+CD45RA−HSPCs was 3-fold higher than that of the CD9−counterparts from healthy controls(HCs),whereas,in ITP patients,the percentage decreased to only 1/4th of that in the HCs and was comparable to that from the CD9−***,when co-cultured with pre-B cells from ITP patients,the differentiation of CD9+Lin−CD34+CD45RA−HSPCs toward the megakaryopoietic lineage was *** analysis revealed that megakaryocytic progenitors(MkP)can be divided into seven subclusters with different gene expression patterns and *** ITP-associated DEGs were MkP subtype-specific,with most DEGs concentrated in the subcluster possessing dual functions of immunomodulation and platelet *** study comprehensively dissects defective hematopoiesis and provides novel insights regarding the pathogenesis of ITP.

关键词： impaired alterations progenitor

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Surgical and nutritional interventions for endometrial receptivity:A case report and review of literature

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World Journal of clinical Cases 2022年第33期10卷 12295-12304页

作者： Dinorah Hernández-Melchor Cecilia Palafox-Gómez Ivan Madrazo Ginna Ortiz America Padilla-Viveros Esther López-Bayghen Science Technology and Society ProgramCentro de Investigación y de Estudios Avanzados del Instituto Politécnico NacionalMéxico City 07360CDMXMéxico clinical Research Instituto Regenera SCMéxico City 05320CDMXMéxico Departamento de Toxicología Centro de Investigación y de Estudios Avanzados del Instituto Politécnico NacionalMéxico City 07360CDMXMéxico Investigación Clínica Instituto de Infertilidad y Genética México SCINGENESMéxico City 05320CDMXMéxico Departamento de Toxicología Centro de Investigación y de Estudios Avanzados del Instituto Politécnico NacionalMexico City 07360CDMXMéxico

BACKGROUND Polycystic ovary syndrome(PCOS)is an endocrine disease that combines metabolic,reproductive,and psychological *** disorders and impaired endometrial receptivity in PCOS can cause *** resistance(IR)is a pathological state of inadequate response to insulin that affects reproduction in PCOS,as damage caused by IR at the endometrial level becomes an obstacle for embryo *** IR resulted in spontaneous pregnancies in PCOS patients,indicating that metabolic corrections improve endometrial *** stem-cell treatment has also corrected endometrial quality and lead to pregnancies in patients with Asherman’s *** propose a combination of nutritional intervention with the surgical placement of stem cells to improve endometrial quality to achieve pregnancy in a PCOS patient undergoing in vitro fertilization(IVF)*** SUMMARY After two failed IVF cycles,a metabolic intervention,consisting of a ketogenic diet with daily consumption of 50 g of carbohydrates(CH),was indicated until *** Syndrome was assessed using the Harmonizing Definition(3 of 5 pathologies:Central obesity,hypertension,hyperglycemia,hypertriglyceridemia,and dyslipidemia),and the Homeostatic Model Assessment of IR(HOMA-IR)was used to measure the level of *** IR improved,endometrial quality ***,two day 5-thawed embryos(euploid,donated oocyte–partner's sperm)failed to implant,suggesting endometrial quality improvement was ***,transmyometrial implantation of mesenchymal stem cells from the stromal vascular fraction of adipose tissue was performed to enrich the endometrial stem cell *** endometrial mean thickness for embryo transfer(6.9 mm)was achieved three months after stem cell treatment and continuous dietary control of *** euploid-day 5-thawed embryos(donated oocyte–partner's sperm)were transferred,and embryo implantation was confirmed on day 14 byβ-hCG s

关键词： Polycystic ovarian syndrome Insulin resistance Nutritional intervention Endometrial quality stem cell treatment Case report

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Double‑Layer Nanofibrous Sponge Tube via Electrospun Fiber and Yarn for Promoting Urethral Regeneration

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Advanced Fiber Materials 2023年第2期5卷 662-680页

作者： Kaile Zhang Muhammad Aqeel Bhutto Liyang Wang Kai Wang Jie Liu Wenyao Li Wenguo Cui Qiang Fu The Department of Urology Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghai Jiao Tong UniversityShanghai 200233People’s Republic of China Institute of Biotechnology and Genetic Engineering University of SindhJamshoro 76080Pakistan School of Materials Science and Engineering Shanghai University of Engineering ScienceShanghai 201620People’s Republic of China clinical Research Center Shanghai Chest HospitalShanghai Jiao Tong UniversityShanghai 200233People’s Republic of China Regenerative Medicine Research Center The First People’s Hospital of Yunnan ProvinceThe Affiliated Hospital of Kunming University of Science and TechnologyKunming 650000People’s Republic of China Shanghai Institute of Traumatology and Orthopaedics Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint DiseasesRuijin HospitalShanghai Jiao Tong University School of MedicineShanghai 200025People’s Republic of China

Insufficient bionic performance is a structural obstacle and makes urethral repair *** overcome this challenge,we mimicked the urethral matrix and applied two electrospinning techniques to build a double-layer sponge tube of nanofib-ers and ***,silk fibroin(SF)and vitamin B5(VitB5)could be introduced to increase the elasticity of the outer layer and reduce the hydrophobicity to further improve mesenchymal cell *** experiments validated the bionic structure,biocompatibility,and exosome delivery capacity in this *** achieved scarless urethral repair by delivering the bioactive growth factors from adipose-derived stem cell exosomes by physical *** regeneration of the urethra can be accomplished with continuous epithelium in ***,bioinformatics studies revealed that the expression of cell proliferation and fibrotic genes(e.g.,Wnt7a,cfa-miR-574)was responsible for the bio-logical regeneration of the adipose-derived stem cells exosomes(ADSC-exos)by delivering poly l-lactide-co-caprolactone/SF/VitB5 bilayer sponge(PSVBS)via reduced fibrosis gene expression,as well as improved epithelial formation and blood vessel ***,the PSVBS design appeared to be an instructive approach for urethral and other tubular organ regeneration.

关键词： Electrospinning Nanofiber Nanoyarn Urethral stricture Urethral reconstruction

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T cell-depleting nanoparticles ameliorate bone loss by reducing activated T cells and regulating the Treg/Th17 balance

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Bioactive Materials 2021年第10期6卷 3150-3163页

作者： Xiaoshan Yang Fuxing Zhou Pingyun Yuan Geng Dou Xuemei Liu Siying Liu Xiangdong Wang Ronghua Jin Yan Dong Jun Zhou Yajie Lv Zhihong Deng Shiyu Liu Xin Chen Ying Han Yan Jin State Key Laboratory of Military Stomatology&National clinical Research Center for Oral Diseases&Shaanxi International Joint Research Center for Oral Diseases Center for Tissue EngineeringSchool of StomatologyFourth Military Medical UniversityXi’anShaanxi710032China Xijing Hospital of Digestive Diseases&State Key Laboratory of Cancer Biology Fourth Military Medical UniversityXi’anShaanxi710032China Department of Gynecology and Obstetrics Xijing HospitalFourth Military Medical UniversityXi’anShaanxi710032China School of Chemical Engineering and Technology Shaanxi Key Laboratory of Energy Chemical Process IntensificationInstitute of Polymer Science in Chemical EngineeringXi’an Jiao Tong UniversityXi’anShaanxi710049China Department of Dermatology Tangdu HospitalFourth Military Medical UniversityXi’anShaanxi710038China

Estrogen deficiency is one of the most frequent causes of osteoporosis in postmenopausal *** chronic inflammatory conditions caused by estrogen deficiency,activated T cells contribute to elevated levels of proinflammatory cytokines,impaired osteogenic differentiation capabilities of bone marrow mesenchymal stem cells(BMMSCs),and disturbed regulatory T cell(Treg)/Th17 cell ***,therapeutic strategies that re-establish immune homeostasis in this disorder have not been well ***,we produced T cell-depleting nanoparticles(TDNs)that ameliorated the osteopenia phenotype and rescued the osteogenic deficiency of BMMSCs in ovariectomized(OVX)*** consist of monocyte chemotactic protein-1(MCP-1)-encapsulated mesoporous silica nanoparticles as the core and Fas-ligand(FasL)as the *** showed that the delicate design of the TDNs enables rapid release of MCP-1 to recruit activated T cells and then induces their apoptosis through the conjugated FasL both in vitro and in *** signals recognized by macrophages help skew the Treg/Th17 cell balance and create an immune tolerant state,further attenuating the osteogenic deficiency of BMMSCs and the osteopenia ***,we found that the therapeutic effects of TDNs were partially mediated by apoptotic T cell-derived extracellular vesicles(ApoEVs),which promoted macrophage transformation towards the M2 *** findings demonstrate that TDNs may represent a promising strategy for treating osteoporosis and other immune disorders.

关键词： Nanoparticles Activated T cell Immune tolerance Apoptotic extracellular vesicles Osteoporosis

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Characterization of mesenchymal stem cells in human fetal bone marrow by single-cell transcriptomic and functional analysis

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Signal Transduction and Targeted Therapy 2023年第4期8卷 2022-2034页

作者： Ping Zhang Ji Dong Xiaoying Fan Jun Yong Ming Yang Yunsong Liu Xiao Zhang Longwei Lv Lu Wen Jie Qiao Fuchou Tang Yongsheng Zhou Department of Prosthodontics Peking University School and Hospital of Stomatology100081 BeijingChina National Center for Stomatology&National clinical Research Center for Oral Diseases&National Engineering Reasearch Center of Oral Biomaterials and Digital Medical Devices&Beijing Key Laboratory of Digital Stomatology&National Health Commission Key Laboratory of Digital Technology of Stomatology 100081 BeijingChina Guangzhou Laboratory 510005 GuangzhouChina Biomedical Pioneering Innovation Center and Center for Reproductive Medicine Ministry of Education Key Laboratory of Cell Proliferation and Differentiation100871 BeijingChina Beijing Advanced Innovation Center for Genomics School of Life SciencesDepartment of Obstetrics and GynecologyThird HospitalPeking University100871 BeijingChina Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology 100191 BeijingChina Peking-Tsinghua Center for Life Sciences Peking University100871 BeijingChina

Bone marrow mesenchymal stromal/stem cells (MSCs) are a heterogeneous population that can self-renew and generate stroma,cartilage, fat, and bone. Although a significant progress has been made toward recognizing about the phenotypic characteristics ofMSCs, the true identity and properties of MSCs in bone marrow remain unclear. Here, we report the expression landscape of humanfetal BM nucleated cells (BMNCs) based on the single-cell transcriptomic analysis. Unexpectedly, while the common cell surfacemarkers such as CD146, CD271, and PDGFRa used for isolating MSCs were not detected, LIFR+PDGFRB+ were identified to bespecific markers of MSCs as the early progenitors. In vivo transplantation demonstrated that LIFR+PDGFRB+CD45-CD31-CD235a-MSCs could form bone tissues and reconstitute the hematopoietic microenvironment (HME) effectively in vivo. Interestingly, wealso identified a subpopulation of bone unipotent progenitor expressing TM4SF1+CD44+CD73+CD45-CD31-CD235a-, which hadosteogenic potentials, but could not reconstitute HME. MSCs expressed a set of different transcription factors at the different stagesof human fetal bone marrow, indicating that the stemness properties of MSCs might change during development. Moreover,transcriptional characteristics of cultured MSCs were significantly changed compared with freshly isolated primary MSCs. Ourcellular profiling provides a general landscape of heterogeneity, development, hierarchy, microenvironment of the human fetal BMderivedstem cells at single-cell resolution.

关键词： CD146 fetal CD45

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Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method

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World Journal of Gastroenterology 2007年第11期13卷 1672-1679页

作者： Han-Tso Lin Chung-Lan Kao Kun-Hsiung Lee Yuh-Lih Chang Shih-Hwa Chiou Fu-Ting Tsai Tung-Hu Tsai Dey-Chyi Sheu Larry LT Ho Hung-Hai Ku Department of Medical Research and Education Taipei Veterans General Hospital Department of Physical Medicine and Rehabilitation Taipei Veterans General Hospital and National Yang-Ming University Animal Technology Institute Taiwan Chunan Miaoli Department of Pharmacy Taipei Veterans General Hospital and National Yang-Ming University Department of Medical Research and Education Taipei Veterans General Hospital and Institute of Clinical Medicine National Yang-Ming University Department of Medical Research and Education Taipei Veterans General Hospital and Department of Bioengineering Tatung University Institute of Traditional Medicine National Yang-Ming University and Department of Education and Research Taipei City Hospital Department of Bioengineering Tatung University Division of Endocrinology and Metabolism Department of Internal Medicine Taipei Veterans General Hospital Taipei Institute of Anatomy and Cell Biology National Yang-Ming University and Department of Education and Research Taipei City Hospital

AIM： To enhance the differentiation of insulin producing cell （IPC） ability from embryonic stem （ES） cells in vitro. METHODS： Four-day embryoid body （EB）-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of NucleofectorTM electroporator （Amaxa biosystems, Germany） in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectincoated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before *** differentiation status of these cells was monitored by semi-quantitative PCR （SQ-PCR） detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixll, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry （IHC）, and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease （SCID） pretreated with streptozotocin （STZ） were used to eliminate plasma glucose restoration after pax4^＋ ES implantation. RESULTS： A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixll, pdxl, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin

关键词： Diabetes mellitus Nucleofection pax4 Embryonic stem cells Insulin producing cells

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