1、544AFlow Cytometry Reagents流式在细胞治疗上的应用细胞治疗概述1第一章 流式细胞仪在干细胞治疗的应用1第一节 造血干细胞表型鉴定中的应用1第二节 外周血干细胞移植中的应用2第三节 造血干细胞保存中的应用2第四节 间充质干细胞表型鉴定中的应用3第二章 流式细胞仪在肿瘤免疫细胞治疗的应用7第一节 细胞因子介导的杀伤细胞（cytokine induced killer，CIK）疗法8第二节 树突状细胞（DC）免疫治疗9第三节 树突状细胞联合细胞因子诱导的杀伤细胞免疫治疗 (DC- CIK)10第 四 节 T 细 胞 疗 法 10第五节 T 细胞受体疗法（TCR）12第六节

2、CAR-T 治疗13第三章 流式细胞仪对细胞治疗的疗效评价的应用17第四章 流式细胞仪分选在细胞治疗的应用18目录1流式在细胞治疗上的应用细胞治疗概述细胞治疗是近几年兴起的疾病治疗新技术，是指利用某些具有特定功能的细胞的特性，采用生物工程方法获取和 / 或通过体外扩增、特殊培养等处理后，使这些细胞具有增强免疫、杀死病原体和肿瘤细胞、促进组织器官再生和机体康复等治疗功效，从而达到治疗疾病的目的。细胞治疗以其良好的疗效，副作用小，更个体化、个性化等独特的优势，为一些难治性疾病的治疗，提供了一种选择，有时甚至是最后的选择。在当前和今后很长的历史阶段，细胞治疗都将在临床治疗中担当重要的角色，二十一世纪

3、将是细胞治疗发挥重要作用的时代。细胞治疗分为两大类：干细胞治疗；免疫细胞治疗。第一章 流式细胞仪在干细胞治疗的应用干细胞技术，又称为再生医疗技术，是指通过对于干细胞进行分离、体外培养、定向诱导、甚至基因修饰等过程，在体外繁育出全新的、正常的甚至更年轻的细胞、组织或器官，并最终通过细胞组织或器官的移植实现对临床疾病的治疗。目前临床应用的主要是成体干细胞，这类细胞包括造血干细胞、间充质干细胞、神经干细胞。第一节 造血干细胞表型鉴定中的应用随着近年来造血干细胞移植病例的日益增多，造血干细胞的流式定量越来越成为一个突出的话题。如何准确地定量干细胞数量，什么是有效的质量控制成了各大实验室的话题。1995

4、 年， 血 液 病 治 疗 及 移 植 国 际 联 合 会 (the International Society of Hematotherapy and Graft Engineering，ISHAGE) 成立了干细胞绝对计数 (Enumeration) 小组，致力于寻求一种简单、快速、灵敏的流式细胞仪检测方法去定量外周血中的造血干细胞数量。这一方法对于临床实验室中不同的流式细胞仪都有效，而且在不同的移植中心具有可比性。这一方案即在 96 年问世的 ISHAGE 方案，至今为止，其他各种方案改进均以此方案为基础。干细胞移植对于白血病等多种疾病的治疗具有重要意义，而准确的干细胞绝对计数对于干细

5、胞移植的成败又十分重要。流式细胞仪结合荧光单抗计数为干细胞绝对计数提供了一种快速、定量、重复性好的方法。文章：1. Single Platform Flow Cytometric Absolute CD341 Cell Counts Based on the ISHAGE GuidelinesMichael Keeney,el Cytometry (Communications in Clinical Cytometry) 34:6170 (1998)在 EPICS XL 上使用使用 Stem-Kit 试剂盒运用 ISHAGE 法定量检测造血干细胞2第二节 外周血干细胞移植中的应用正常情况下外

6、周血干细胞含量很少，需要进行动员处理方可有效增加外周血干细胞含量，以供移植时采集。准确计数 CD34+ 细胞的数量，可判定骨髓动员方案是否成功，何时开始采集及何时终止采集 PBSC。第三节 造血干细胞保存中的应用造血干细胞保存是造血干细胞治疗技术的重要组成部分，现已广泛用于临床。脐血干细胞来源广泛，采集方便，作为造血干细胞容易获得，但需冻存保存，保存条件要求高，规模要求大，需相当投资规模的脐血库来进行保存。文章：脐血造血干细胞不同温度长期冻存效果的实验研究（许利民，王劲等 . 中国现代医学杂志2007 年 第 24 期）3第四节 间充质干细胞表型鉴定中的应用间充质干细胞是一种来源于中胚层具有自

7、我复制更新和多向分化的多能干细胞，具有独特的免疫表型和免疫调节能力。因此，间充质干细胞被广泛应用于干细胞移植、组织工程和器官移植免疫治疗等方面并且作为组织工程良好的种子细胞被运用于一系列基础和临床研究中。目前比较通用的是国际细胞治疗协会（ISCT）2006 年推荐的一套定义 MSC 的最低标准。但对于不同来源的 MSC 的特异性鉴别，尚无任何方法和结论。不同实验室分别尝试应用各种不同的表面标记来定义 MSC。流式细胞术以其高灵敏、高速度、多参数测量、获取形态学信息等方面的优势在血液学、微生物学、分子生物学等领域中也得到广泛的应用。应用这一技术，不仅可以用于原代细胞或较低代次细胞的表型分析，还可

8、以应用到细胞传代后的表型稳定性研究中。4文章：1. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statementM Dominici，el Cytotherapy (2006) Vol. 8, No. 4, 315 317国际细胞治疗协会提出了一个鉴定 MSC 的最低标准：1、在正常培养条件下，MSC 必须贴壁生长；2、必须表达 CD105, CD73, CD90，同时不表达 CD45，

9、CD34 ，CD14 或 CD11b，CD79 或 CD19， HLA -DR 表面分子；3、在体外可以向成骨细胞，脂肪细胞，软骨细胞分化。2. Human Mesenchymal Stem Cells: From Immunophenotyping by Flow Cytometry to Clinical ApplicationsArthur A. Nery,el Cytometry Part A 83A: 48-61, 2013总结分析不同来源的间充质干细胞的免疫表型。Table 1. (Continued)CELLTYPESHMSCHMSCHMSC 1PLACENTAHMSC 1PLA

10、CENTAHASCHASCHASCSYNOVIALTISSUEUMBILICALCORDUMBILICALCORDDENTALPULPCD19XCD27XCD28XCD31XXXXXCD33XCD34XXXXXXCD36XCD38CD45XXXXXXCD50XCD71CD102XCD105XCD106XCD117XCD133XXXCD144XCD243XHLA-DRXXXFLK-1XMOPC-21, 2735XFLA-1XReference2026272829Expression patterns were compiled from works cited in the

11、 table.hMSC: bone marrow human mesenchymal stem cells; hASC: human adipose mesenchymal stem cells.REVIEW ARTICLECytometry Part A ? 83A: 48?61, 201351Table 1. Immunophenotypic characterization of MSCs of different originsCELLTYPESHMSCHMSCHMSC 1PLACENTAHMSC 1PLACENTAHASCHASCHASCSYNOVIALTISSUEUMBILICAL

12、CORDUMBILICALCORDDENTALPULPPositive antigensCD9XXXXXCD10XXCD13XXCD18XCD29XXXXXXXXCD34XXCD38XCD44XXXXXXCD49 a,b,c,e,fXXXCD51XCD54XXXXCD55XCD56XCD58XCD61XCD63XCD71XXXXCD73XXXXXCD90XXXXXXXXXCD97XCD98XCD99XCD104XCD105XXXXCD106XXCD146XXCD155XCD166XXXXXCD276XCD304XCD324XLNGFRXHLA-IXHLA-DRXHLA-ABCXSTRO-1XB

13、MPRIAXIntegrin a11XIntegrin b5XIntegrin b7XIntegrin b8XABCG2XSurvivinXBcl-2XReference2026272829Negative antigensCD3XCD14XXXXCD16XREVIEW ARTICLE50hMSCs: From FCM Immunophenotyping to Clinical Studiespreparation beg the question of whether the resulting cellsare sufficiently similar to allo

14、w for a direct comparison ofreported biologic properties and experimental outcomes,especially in the context of cell therapy. This question ofcell equivalence is, in part, because of the lack ofuniversally accepted criteria to define MSC. Most im-portantly, the inability to compare and contrast stud

15、iesfrom different groups is likely to hinder progress in thefield.To address this problem, the Mesenchymal and TissueStem Cell Committee of the ISCT proposes a set ofstandards to define human MSC for both laboratory-basedscientific investigations and for pre-clinical studies. Theseidentifying criter

16、ia should not be confused with releasespecifications for clinical studies, as the current proposal isintended solely as identifying criteria for research pur-poses. The aim of this position statement is to provide thescientific community with a standard set of criteria, basedon the best currently av

17、ailable data, to define the identityof MSC, recognizing that future research will probablymandate a revision of the criteria as new data emerge.We propose three criteria to define MSC:?/adherence to plastic?/specific surface antigen (Ag) expression?/multipotent differentiation potential (Table 1).Fi

18、rst, MSC must be plastic-adherent when maintained instandard culture conditions using tissue culture flasks.Second, /95% of the MSC population must expressCD105, CD73 and CD90, as measured by flow cytometry.Additionally, these cells must lack expression (5/2%positive) of CD45, CD34, CD14 or CD11b, C

19、D79a orCD19 and HLA class II. Third, the cells must be able todifferentiate to osteoblasts, adipocytes and chondroblastsunder standard in vitro differentiating conditions.Plastic adherence is a well-described property of MSC,and even unique subsets of MSC that have been describedmaintain this proper

20、ty 4,5. While MSC may be main-tained, and possibly expanded, without adherence 6,these protocols typically require very specific cultureconditions, and these cells, if maintained under morestandard conditions, would be expected to demonstrateadherence if the cells are to be considered a population o

21、fMSC.Surface Ag expression, which allows for a rapididentification of a cell population, has been used exten-sively in immunology and hematology. To identify MSC,we propose that cells should express CD105 (known asendoglin and originally recognized by the MAb SH2),CD73 (known as ecto 5? nucleotidase

22、 and originallyrecognized by the MAb SH3 and SH4) and CD90 (alsoknown as Thy-1). Novel surface markers that may beidentified in the future could lead to modifications of thesecriteria. To assure that studies of heterogeneous popula-tions of MSC are not confounded by other cells, werecommend that lac

23、k of expression of hematopoietic Ag beused as additional criteria for MSC as they are not knownto express these Ag. For this purpose, we recommend thata panel of Ag be used to exclude the cells most likely to befound in MSC cultures. CD45 is a pan-leukocyte marker;CD34 marks primitive hematopoietic

24、progenitors andendothelial cells; CD14 and CD11b are prominentlyexpressed on monocytes and macrophages, the most likelyhematopoietic cells to be found in an MSC culture;CD79a and CD19 are markers of B cells that may alsoadhere to MSC in culture and remain vital throughstromal interactions; and HLA-D

25、R molecules are notexpressed on MSC unless stimulated, e.g. by IFN-g.Only one of the two macrophage and B-cell markersneeds to be tested. Each group of investigators shouldselect the marker(s) that is (are) most reliable in theirlaboratory.Finally, the biologic property that most uniquelyidentifies

26、MSC is their capacity for trilineage mesenchy-mal differentiation. Thus, cells must be shown to differ-entiate to osteoblasts, adipocytes and chondroblasts usingstandard in vitro tissue culture-differentiating conditions.Differentiation to osteoblasts can be demonstrated bystaining with Alizarin Red

27、 or von Kossa staining. Adipo-cyte differentiation is most readily demonstrated bystaining with Oil Red O. Chondroblast differentiation isdemonstrated by staining with Alcian blue or immunohis-Table 1. Summary of criteria to identify MSC1 Adherence to plastic in standard culture conditions2 Phenotyp

28、ePositive (/95%/)Negative (5/2%/)CD105CD45CD73CD34CD90CD14 or CD11bCD79a or CD19HLA-DR3 In vitro differentiation: osteoblasts, adipocytes, chondroblasts(demonstrated by staining of in vitro cell culture)53. Life-Sparing Efect of Human Cord Blood-Mesenchymal Stem Cells in Experimental Acute Kidney In

29、juryMARINA MORIGI,el STEM CELLS 2010;28:513522从脐带血中提取间充质干细胞用于急性肾损伤治疗，在 FC500 上检测人脐带血来源间充质干细胞（CB-MSCs）的表型。表 达 CD105，CD44，CD90，CD73，HLA class I，CD146，SMA，SSEA4； 弱 表 达 CD56，desmin，CK3/12, CK19 和 BB9；不表达 SSEA4，CD56，HLA class II，NGFR，CD34，NG2。Figure 1.Characterization ofhuman (h) cord blood-mesen-chymal

30、stem cells (CB-MSCs).(A):RepresentativeimageofhCB-MSCs with typical spindle-shapedmorphology.Originalmagnification, ?10. (B): Osteo-genicdifferentiationofhCB-MSCs is evidenced by AlizarinRed staining with area of miner-alization. Original magnification,?20. (C): Proteoglycans stainedby Alcian Blue r

31、eveals chondro-genicdifferentiation.Originalmagnification, ?20. (D): FlowcytometriccharacterizationofhCB-MSCs. Representative dotplots showing the expression ofCD105,CD44,CD90,CD73,HLA class I, CD146, aSMA ofhCB-MSCs at passage 6. The re-spective isotype control is shownasaredline.Abbreviations:HLA,

32、 hyuman leukocyte antigen;aSMA, a-smooth muscle actin;SSEA, stage-specific embryonicantigen.Table 1. Immunophenotype of hCB-MSCsAntigensPercentage of fluorescent cellsCD10555.3 6 37.3CD4462.8 6 35.1CD9090.4 6 6.9CD7398.8 6 1.9SSEA43.9 6 4.1HLA cl I96.6 6 2.9CD14635 6 18.1a SMA53.5 6 19.6CD562.1 6 1.

33、4DESMIN0.4 6 0.3CK3/121 6 0.7CK192.6 6 1.7BB90.2 6 0.1Data are expressed as mean 6 SD.Abbreviation:hCB-MSCs,humancordblood-mesenchymalstemcells.516Cord Blood-Mesenchymal Stem Cells in AKI ModelThe expression of cell-surface antigens was evaluated byflow cytometry on all hCB-MSC cultures. As shown in

34、 Figure1D(representativeanalysis)andTable1,hCB-MSCsexpressed CD105, CD44, CD90, CD73, SSEA4, and HLAclass I. hCB-MSCs were weakly positive for CD56, desmin,CK3/12, CK19, and BB9, and negative for HLA class II,NGFR, CXCR4, and VE-Cadherin. These cells also expressedCD146 and alpha SMA (but not CD34,

35、CD45, and NG2),which altogether characterize a multipotent adult stem cellpopulation consistent with a perivascular/pericyte-like pheno-type (Fig. 1D; Table 1) 44. The colocalization of embryonicmarker SSEA4 with CD146 was also observed by immunoflu-orescence (Supporting Information Fig. 1).The mole

36、cular profile of hCB-MSCs by reverse transcrip-tase-polymerase chain reaction (RT-PCR) analysis showed thatthe cells expressed some genes including RUNX1 and OCT4mRNA, markers that characterize the undifferentiated stem cellstate (Supporting Information Fig. 2). hCB-MSCs did notexpress PPAR-2, VE-Ca

37、dherin, ABCG2, REX1, FGF4, hTERT,SOX2, PAX7, and renal genes such as PAX2 and GATA3.Therapeutic Effect of hCB-MSCs in Cisplatin-Micewith AKIThe renoprotective potential of hCB-MSCs was investigatedin immunodeficient mice with cisplatin-induced AKI. Previousexperiments 13 indicated that subcutaneous

38、injection of cis-platin (12.7 mg/kg) in NOD-SCID mice caused renal functionimpairment, as indicated by increased levels of BUN, whichpeaked at 4 days and stabilized to high values until animaldeath within 57 days. Here, we tested the effect of i.v. injec-tion of hCB-MSCs or saline in NOD-SCID mice,

39、1 day aftercisplatin treatment, when tubular ultrastructural changes werealready present 13. Renal function and histology were stud-ied at 4 days, the time at which all animals with AKI werestill alive. As shown in Figure 2A, hCB-MSC infusion mark-edly improved renal function, as reflected by the si

40、gnificant (p .01) reduction of BUN levels at 3 and 4 days in respect tomice given saline. Renal histology of mice with cisplatin-induced AKI on day 4 showed focal and severe tubular celldegenerative changes with necrosis and luminal casts (Fig.2B; Table 2). In kidneys of cisplatin-mice injected with

41、 hCB-MSCs, renal tubular damage was markedly attenuated asreflected by the significant reduction of numbers of necrotictubules and casts (Fig. 2B; Table 2). Electron microscopyanalysis of renal tissue of cisplatin-treated mice sacrificed at 4days confirmed the presence of necrotic epithelial cellswi

42、th detachment from the underlying basement membrane,and showed peritubular capillary changes consisting of endo-thelialcellswelling,cytoplasmicvacuolisation,nucleardegeneration, and detachment, occasionally associated withFigure 1.Characterization ofhuman (h) cord blood-mesen-chymal stem cells (CB-M

43、SCs).(A):RepresentativeimageofhCB-MSCs with typical spindle-shapedmorphology.Originalmagnification, ?10. (B): Osteo-genicdifferentiationofhCB-MSCs is evidenced by AlizarinRed staining with area of miner-alization. Original magnification,?20. (C): Proteoglycans stainedby Alcian Blue reveals chondro-g

44、enicdifferentiation.Originalmagnification, ?20. (D): FlowcytometriccharacterizationofhCB-MSCs. Representative dotplots showing the expression ofCD105,CD44,CD90,CD73,HLA class I, CD146, aSMA ofhCB-MSCs at passage 6. The re-spective isotype control is shownasaredline.Abbreviations:HLA, hyuman leukocyt

45、e antigen;aSMA, a-smooth muscle actin;SSEA, stage-specific embryonicantigen.Table 1. Immunophenotype of hCB-MSCsAntigensPercentage of fluorescent cellsCD10555.3 6 37.3CD4462.8 6 35.1CD9090.4 6 6.9CD7398.8 6 1.9SSEA43.9 6 4.1HLA cl I96.6 6 2.9CD14635 6 18.1a SMA53.5 6 19.6CD562.1 6 1.4DESMIN0.4 6 0.3

46、CK3/121 6 0.7CK192.6 6 1.7BB90.2 6 0.1Data are expressed as mean 6 SD.Abbreviation:hCB-MSCs,humancordblood-mesenchymalstemcells.64. Adipose TissueDerived Human Mesenchymal Stem Cells Mediated Prodrug Cancer Gene TherapyLucia Kucerova，el Cancer Res 2007; 67: (13). July 1, 2007使用 EPICS ALTRA flow cyto

47、meter (Beckman Coulter) 分析改造前后脂肪来源的 MSC 表面标记 CD29，CD44，CD90，CD105 的表达情况。通过对 MSC 进行基因改造后用于个体化的肿瘤靶向治疗。Figure 2. Retrovirus transduction doesnot affect cell surface marker expressionand differentiation properties and directedmigration toward colon carcinoma cellsHT-29 in transgene-expressing CD-AT-MSC

48、derived from cultured AT-MSC.A, AT-MSC and CD-AT-MSC were labeledwith fluorophore-conjugated antibodiesand analyzed by flow cytometry (redhistograms). Mouse isotype antibodiesIgG1 and IgG2a served as respectivecontrols (white histograms). Both cell typeswere positive for CD29, CD44, CD90,and CD105.

49、B, AT-MSC and CD-AT-MSCwere cultured in medium supplementedwith components for adipogenicdifferentiation for 28 d. Cells werestained with Oil Red-O. Red-stainedoil droplets visible in AT-MSC andCD-AT-MSC were indicative of adipogenicdifferentiation. Both cell types were foundto be capable of adipoge

50、nic differentiation.Magnification, ?80. C, AT-MSC andCD-AT-MSC were cultured in mediumsupplemented with components forosteogenic differentiation. Cells werestained with Alizarin red S 28 d later.Red-stained calcium deposits weredetected in differentiated cultured AT-MSCand CD-AT-MSC. Both transgene-