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1、Current status and prospect of therapeutic clone studyIslet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimenA.M. James Shapiro, et al.A.M. James Shapiro, et al. University of Alberta Hospital, Edmonton, CanadaNew Engl J Med 2000，34
2、3(4):230-238核心内容如下：核心内容如下：核心内容如下：核心内容如下：n n胰岛分离纯化后即刻进行移植胰岛分离纯化后即刻进行移植胰岛分离纯化后即刻进行移植胰岛分离纯化后即刻进行移植n n移植足够数量的胰岛组织（两名或以移植足够数量的胰岛组织（两名或以移植足够数量的胰岛组织（两名或以移植足够数量的胰岛组织（两名或以上供体）上供体）上供体）上供体）n n抗免疫排斥反应采用非类固醇激素的抗免疫排斥反应采用非类固醇激素的抗免疫排斥反应采用非类固醇激素的抗免疫排斥反应采用非类固醇激素的药物和单克隆抗体药物和单克隆抗体药物和单克隆抗体药物和单克隆抗体胰岛移植示意图Edmonton方案的最新进展l
3、国际多中心临床试验结果显示：一年以上不依赖胰岛素治疗者达64%三年以上不依赖胰岛素治疗者达53%血清中能检测到C肽者达72%l值得一提的是，有四家开始参与该研究的中心未能获得任何一例的成功l胰岛移植是治愈糖尿病的潜在希望ADA annual Meeting, 2004, Orlando胰岛移植面临的两大挑战l胰岛组织来源：远远满足不了需要 l免疫排斥反应：移植失败的重要原因 Definition of Stem CellsSelf-renewalMulti-lineage differentiation CorneaPancreasNerveLiverSkinClassification of
4、 Stem CellslDifferentiation capacitynTotipotent stem cellsnPluripotent (multipotent) stem cellsnOligopotent (unipotent) stem cellslSourcenEmbryonic stem (ES) cellsnSomatic (adult) stem cellsWhat is ES cells?Primitive (undifferentiated) cells from the embryo that have the potential to become a wide v
5、ariety of specialized cell types.ES cellsPluripotent stem cellsIn vitro fertilizationTotipotent cellsBlastocystInner cell mass(ICM)-NIHs definition胚胎干细胞的研究历程胚胎干细胞的研究历程1981年，Martin从小鼠囊胚中分离出胚胎干细胞并在体外培养1995年，Thomson从恒河猴囊胚中分离并建立了第一个灵长类动物的胚胎干细胞系Martin GR. PNAS, 1981, 78 (12):7634-7638Thomson JA, et al. P
6、NAS, 1995, 92 (17):7844-7848n1999年，杂志将干细胞研究评为世界十大科学成就之首, 列在了人类基因组计划之前里程碑里程碑n1998年，美国学者Thomson和Gearhart分别从体外受精形成的囊胚中及从5-9周龄流产胎儿的性腺脊和肠系膜中建立了人胚胎干细胞系Thomson和Gearhart采用不同的途径成功培养干细胞的过程 Thomson JA, et al. Science, 1998, 282 (5391):1145-1147Shamblott MJ, et al. PNAS, 1998, 95 (23):13726-13731Gearhart JD. Sc
7、ience, 1998, 282 (5391):1061-1062MilestonenIn 2004, Scientists from Korea and U.S.A reported the derivation of a pluripotent embryonic stem cell line (SCNT-hES-1) from a cloned human blastocystHwang WS, et al. Science 2004, 303(5664):1669-1674 人胚胎干细胞的来源人胚胎干细胞的来源Gearhart取自终止妊娠的胎儿早期性器官组织Thomson取自体外受精胚
8、胎囊胚期的内细胞团Moon通过体细胞核转移技术获得Thomson JA, et al. Science, 1998, 282 (5391):1145-1147Shamblott MJ, et al. PNAS, 1998, 95 (23):13726-13731Hwang WS, et al. Science 2004, 303(5664):1669-1674GearhartThomsonMoon人胚胎干细胞人胚胎干细胞(hES)的特点的特点形态：圆形或椭圆，体积较小，核质比较大，体外抑制分化培养时成鸟巢状，集落生长并紧密堆积，细胞界限不明显Thomson JA, et al. Science
9、, 1998, 282 (5391):1145-1147Hwang WS, et al. Science 2004, 303(5664):1669-1674彭红梅等. 北京大学学报（医学版）北京大学学报（医学版） 2004, 36(6):605-608n n 增殖速度：1824h分裂增殖一次uhES体外培养要解决的关键问题是维持细胞的分裂增殖而抑制其分化uhES必须在含有白血病抑制因子（LIF）的培养基及成纤维细胞饲养层（freeder cells）的条件下才能保持增殖而不分化n具有分化成外、中、内三个胚层的潜能n能形成嵌合体动物，从而成为联系细胞和个体之间的桥梁Thomson JA, et
10、al. Science, 1998, 282 (5391):1145-1147Hwang WS, et al. Science 2004, 303(5664):1669-1674彭红梅等. 北京大学学报（医学版）北京大学学报（医学版） 2004, 36(6):605-608人人胚胚胎胎干干细细胞胞的的鉴鉴定定l人胚胎干细胞具有正常稳定的二倍体核型和带型l胚胎干细胞具有较高的端粒酶活性及碱性磷酸酶的表达n人胚胎干细胞系端粒酶活性都很高，说明其可在体外未分化状态下进行长期培养Thomson JA, et al. Science, 1998, 282 (5391):1145-1147Hwang WS
11、, et al. Science 2004, 303(5664):1669-1674lhES细胞有特异性表面抗原的表达细胞有特异性表面抗原的表达鼠鼠和和人人胚胚胎胎干干细细胞胞表表达达的的表表面面抗抗原原有有种种属属差差异异。Gearhart等等认认为为SSEA-1SSEA-1阳阳阳阳性性性性可可能能是是源源于于原原始始生殖细胞的多能干细胞分化的标志生殖细胞的多能干细胞分化的标志Hwang WS, et al. Science 2004, 303(5664):1669-1674Thomson JA, et al. Science, 1998, 282 (5391):1145-1147Shamb
12、lott MJ, et al. PNAS, 1998, 95 (23):13726-13731Thomson JA, et al. PNAS, 1995, 92 (17):7844-7848l具有转录因子Oct-4的表达n小鼠Oct-4只限定在多潜能细胞中表达。人和小鼠的胚胎干细胞都表达Oct-4，当胚胎干细胞分化时，其表达水平大大降低nOct-4可能是哺乳动物不同发育阶段多潜能细胞所特有的少数特异性调控分子之一Thomson JA, et al. Science, 1998, 282 (5391):1145-1147Hwang WS, et al. Science 2004, 303(566
13、4):1669-1674彭红梅等. 北京大学学报（医学版）北京大学学报（医学版） 2004, 36(6):605-608l l人胚胎干细胞具分化的多潜能性人胚胎干细胞具分化的多潜能性hES注射小鼠皮下混混合合组组织织瘤瘤EctodermMesodermEndodermNerveSkinAdrenalBloodHeartMuscle& BonePancreasLiverBoth in vivo and in vitro彭红梅等. 北京大学学报（医学版）北京大学学报（医学版） 2004, 36(4):431-434碱性磷酸酶染色SSEA-1染色裸鼠皮下接种腺样体结构神经组织结构软骨组织结构彭红梅等
14、. 北京大学学报（医学版）北京大学学报（医学版） 2004, 36(6):605-608拟胚体（EB）培养分子标志的表达1周2周3周4周hES 1周 2周 3周 4周 MEFLanesOct-4NestinNSEGFAPSNAPNFHPDX-1InsulinGlucagonAFPGlobulinGAPDHMEF = mouse embryonic fibroblastsNSE = neuron specific enolaseGFAP= glial fibrillary acidic proteinSNAP= synaptosome-associated proteinNFH = neurof
15、ilament heavy chainFromEmbryonic Stem Cells to Insulin-Secreting Cells胚胎干细胞分化为胰岛胚胎干细胞分化为胰岛 细胞的路径细胞的路径ES cellsEctodermFrom mouse ES cells to insulin-secreting pancreatic islet cluster (Five-stage protocol)Lumelsky N, et al. Science 2001, 292(5520):1389-1394Stage 1Expansion of ES cellsStage 2Generatio
16、n of EBsStage 3Selection of nestincellsStage 4Expansion of pancreatic endocrine progenitor cells Stage 5Induction ofdifferentiation and morphogenesisof insulin-secreting islet cluster 2-3 days4 days6-7 days6 days6 daysLumelsky N, et al. Science 2001, 292(5520):1389-1394Lumelsky N, et al. Science 200
17、1, 292(5520):1389-1394主要结论主要结论 l从小鼠ES细胞诱导分化产生可表达胰岛素和其它胰腺内分泌激素的细胞l该细胞可自聚集形成类似正常胰岛的三维结构l葡萄糖可促发这些细胞群释放胰岛素，其机制类似体内的刺激作用l移植到糖尿病小鼠体内时，这些胰岛素产生细胞出现快速的血管化，并保持胰岛样结构，但没有明显的降血糖作用Lumelsky N, et al. Science 2001, 292(5520):1389-1394From human ES cells to insulin-secreting cellsSchuldiner M, et al. PNAS 2000, 97(2
18、1):11307-11312Assady S, et al. Diabetes 2001, 50(8):1691-1697Segev H, et al. Stem Cells 2004, 22(3):265-2745 day10 dayFurtherinduction?Assady S, et al. Diabetes 2001, 50(8):1691-1697InsulinGKGlut-2Glut-1Oct-4Ngn-3PDX-1b-actinDifferentiation (days)071522281030uhESEBdhESNHF NCAssady S, et al. Diabetes
19、 2001, 50(8):1691-1697NHF = normal human fibroblastsDifferentiation of Human Embryonic Stem Cells into Insulin-Producing ClustersHANNA SEGEV, BETTINA FISHMAN, ANNA ZISKIND, MARGARITA SHULMAN, JOSEPH ITSKOVITZ-ELDORSTEM CELLS 2004;22(3):265-274 www.StemCInsulinstainingTUNEL+nucleiDAPI stainfor nuclei
20、Merge(A-C)InsulinstainingFITC-InsulinuptakeInsulin staining of ES cell progeny from insulin uptakeRajagopal J, et al. Science 2003, 299(5605):363Insulin/C-peptide/DNAInsulin /Caspase-3Insulin/DNATUNELInsulin/DNAInsulin/FITC-Ins/DNAArtifactual insulin release from differentiated embryonic stem cellsH
21、ansson M, et al. Diabetes 2004, 53(10):2603-2609Hansson M, et al. Diabetes 2004, 53(10):2603-2609Key Messages from these two StudieslES cells differentiated in media without exogenous insulin did not stain for insulin, and differentiated ES cells subsequently cultured in insulin-deficient media lost
22、 insulin staininglAlthough differentiated cells contained immunoreactive insulin, they did not contain C-peptidelVariable insulin release from these cells upon glucose challenge could be found, but C-peptide release was not detectedlMany of the insulin-immunoreactive cells were undergoing apoptosis
23、or necrosislThese cells took up fluorescein isothiocyanate (FITC)-labeled insulin from the culture mediumSuggesting that containing insulin in these cells is not as a result of biosynthesis but from the uptake of exogenous insulinRajagopal J, et al. Science 2003, 299(5605):363Hansson M, et al. Diabe
24、tes 2004, 53(10):2603-2609ConclusionInsulin detecting alone can overestimate genuine b cell differentiation when exogenous insulin is present. Therefore, several methods should be combined for reliable analysis of insulin expression includingl lC-peptide staining and C-peptide staining and releasing
25、releasingl lElectron microscopyElectron microscopyl lNorthern blotNorthern blotl lIn situIn situ hybridization hybridizationl lMetabolic labelingMetabolic labelingl lDemonstration Demonstration of of bi-bi-phasic insulin secretionphasic insulin secretionl lTransplantation Transplantation assays assa
26、ys for for b b cell cell function function that that demonstrate demonstrate rescue rescue of of the the diabetic diabetic phenotype phenotype for more than a monthfor more than a monthRajagopal J, et al. Science 2003, 299(5605):363Hansson M, et al. Diabetes 2004, 53(10):2603-2609What is therapeutic
27、 cloning？Therapeutic cloningPatientCell biopsyEnucleated oocyteand fusionNucleus transferReprogramming Development into blastocystsES cells establishedIn vitro differentiationAutologous tissue graftProspectTo normalize all steps and relevant techniques for establishing hES cell lines from cloned blastocystTo optimize the culture condition for inducing ES cells to differentiate into genuine islet b cells To evaluate the long-term bio-safety of transplantation of insulin-secreting structure derived from ES cellsThank you
