研究生課程移植

1、小鼠造血干細(xì)胞移植技術(shù)/ 2013-11-7內(nèi)容小鼠造血干細(xì)胞表型常用流式分析方法和功能實(shí)驗(yàn)常用移植方法小鼠造血干細(xì)胞表型Immunophenotype of HSC/HPCsCFU-S (Till & McCulloch),證實(shí)造血干細(xì)胞存在Thy1.1lo Lin-Sca-1+(Weissman)Lin-Sca-1+c-kit+ , LKS+ (Suda)SP cells (Gol)LKS+CD34-(Nakauchi)Side population(Ho33342 blue/redlow)LKS+CD34-Flt3-(Jacobsen)LKS+CD150+CD244-CD48-(

2、Slam family) (Morrison)LKS+CD150+CD244-CD48-CD229- (Morrison)LKS+CD34-CD150+CD48- or LKS+CD34-Flt3-Current201320052001199619921988Lineage： CD3，CD4，CD8 B220Ter119 Mac-1 Gr-1 (IL7R)1961Hematopoietic HierarchyWang, L. D. and Wagers, A. J. 2011, Nat Rev Mol Cell BiolJörgen Adolfsson,. 2005, CellLKS

3、+LKS-E. Passegue,. 2005, JEMLT-HSCCD34-Flk2-LKS+ CD150+CD48-LKS+ST-HSCCD34+Flk2-LKS+MPPCD34+Flk2+LKS+GMPCD34+CD16/32hiLKS-CMPCD34+CD16/32loLKS-MEPCD34-CD16/32-LKS-CLPLin- IL-7R+Sca-1loc-KitloLT-HSCMPPCD34-Flk2-STCD34FSCSca-1CD150+CD48-MPPLTMEPCD150CD34LTCD34-150+CD34Linc-KitCD16/32CD150CD48Flk2CLPCM

4、PGMPLTLKS+LKS-Side population cellsAllison Mayle,. 2005, CytometryHoechst 33342細(xì)胞周期 (Cell cycle)和細(xì)胞增殖(Proliferation)細(xì)胞凋亡 (Apoptosis)活性氧簇 (ROS, reactive oxygen species)磷酸化 (Phosphorylation)分選 (Sorting)常用流式分析方法和功能實(shí)驗(yàn)細(xì)胞周期DNA dyes：PI, Hoechst (Ho), DAPI, 7-AADKi67 and DNA dyeRNA dye and DNA dyePyronin Y

5、(PY) and Ho 33342 G1S/G2/MG1S/G2/M G0Ho 33342G0DAPIPI細(xì)胞固定; 活細(xì)胞(Verapamil)細(xì)胞固定細(xì)胞固定，破膜Ki67Pyronin Y細(xì)胞增殖CFSE (CFDA-SE)BrdU (or EdU) and DNA dyeSIn vitro: culture 1-2 hIn vivo (HSC):Short-term: i.p. - 12-16 h Long-term: drink water- 7 days常用于示蹤細(xì)胞凋亡Annexin V and DNA dyeTUNEL and DNA dye流式檢測細(xì)胞熒光計(jì)數(shù)Annexin

6、V碎片晚期活細(xì)胞早期Richard XuFeng,. 2009, PNAS7-AAD胞內(nèi)ROS分析ROS：氧離子，過氧化氫等DCF-DA (Invitrogen)：FITC通道MitoTracker (Invitrogen)線粒體HSCHPC磷酸化分析Kenneth D. Gibbs,. 2011, Blood分選 (Population level)Colony forming unit(集落形成)LTC-IC(Long-Term Culture-Initiating Cell) (長期培養(yǎng)-啟動細(xì)胞)CAFC (cobblestone area forming cell )(鵝卵石樣區(qū)域形

7、成細(xì)胞)Transplant(移植) Homing (歸巢)PB analysis every 4wMicroarray / RNA-seq 等qPCRGene Function功能分析)(Over-expression (cDNA)or Knock-down (shRNA)分選 (Single cell level)14 DaysSingle-cell colony1. Cytospin2. Flow analysis(Mac-1, Gr-1, Ter119, CD41)Transplant96x96Single cell qPCRSymmetrical/asymmetrical divis

8、ion對稱/不對稱Ryo Yamamoto,. 2013, Cell常用移植方法骨髓重建移植和連續(xù)移植BM transplantation (BMT) and Serial BMT (sBMT)競爭性骨髓移植和連續(xù)移植Competitive BM transplantation(cBMT) coupledwith serial transfer單細(xì)胞移植Single cell transplantation極限稀釋移植Limiting-dilution transplantation其他移植Hui Cheng,. 2013, Methods Mol BiolCongenic strain of

9、 miceB6-Ly5.1B6.SJL-PtprcaPepcb/BoyJB6-Ly5.2 C57BL/6J, B6CD45.2CD45.1221F1CD45.1/21/221CD45.1CD45.2DonorLy5.1Ly5.1CompetitorF1Ly5.2RecipientLy5.2Ly5.2Define HSCs in transplant assayLT-HSC ：1st transplantation > 6 month 2nd transplantation abilityMulti-lineageST-HSC1st transplantation < 3 month

10、or 1st transplantation > 3 month, but not multi-lineageNo 2nd transplantation abilityHPC1st transplantation < 1 month No multi-lineageRyo Yamamoto,. 2013, Cell骨髓重建移植和連續(xù)移植第一次移植：反應(yīng)HSC重建能力連續(xù)移植：反應(yīng)HSC自我更新能力移植細(xì)胞數(shù)量(全骨髓細(xì)胞)：1、half a femur 2、1x106-1x107移植次數(shù)： 3移植體積：300 -500LM檢測：每隔4周檢測外周血CD45.1比例，及T,B,M的重

11、建率。外周血20-50L。PBS+2mM EDTA。16w 或 24w，測骨髓各系及干細(xì)胞比例， 移植。SS+MSS:stem cellM: mature cellsCD45.1+ cellsEngraftment in BM (%)CD45.1CD45.1BMCD45.2BMBMWTKO10050I° II° III°競爭性骨髓移植和連續(xù)移植供體細(xì)胞：全骨髓細(xì)胞或HSC 競爭細(xì)胞：全骨髓細(xì)胞(LKS+ or LT-HSC)CD45.1+ Lin- Sca-1+ c-kit+ CD34-CD45.1+ Lin- Sca-1+ c-kit+ CD34- CD150+

12、 CD48-Method 1:移植次數(shù)： 2Method 2:PBCD45.2B220Mac-1CD3Miyamoto K,. 2007, Cell Stem CellCD45.1CD45.1BTMPBB220CD3Mac-1CD45.2CD45.2CD45.2CD45.2Irene M. Min,. 2008, Cell Stem CellCD45.1SSCCD45.1SSCCD45.1SSCCD45.1RUs (Repopulating Units): indicate the amount of populating activity (functionalquality of HSCs

13、), measure the functional potential of the unknown source of HSCs against a set knownnumberof HSCs, providequalitative orat bestsemiquantitative information about the HSCs withinagivenpopulationitcannotdistinguishbetweenthenumberof HSCsproduced per HSC)定性，非定量ortheirquality(progeny1x 105 WT BM cells

14、= 1 RUDonor percentageCompetitor cell numberx=Donor RUsCompetitor percentage1x 105CD45.1 cell RUs 33.6 65.1x5 =2.58 RUsCD45.25x105 BM cellsCD45.1單細(xì)胞移植CD45.2Modified from Ryo Yamamoto,. 2013, CellHideo Ema,. 2005, Development CellCD45.1PrimaryPositive : one lineage reconstitution > 0.1SecondaryYoh

15、ei Morita,. 2010, JEM極限稀釋移植目的：計(jì)算HSC數(shù)量（定量）> 8 mice per cell dose (每個(gè)劑量大于8只小鼠)> 3 cell doses (大于3個(gè)劑量)Differentdoses of cells9.5GyLy5.1Analysis(16 weeks)Ly5.22x105 BM cellsLy5.2細(xì)胞(Source)：1、Whole BM cells: 8 x 103 - 1 x 106 + 2 x 105 competitors (BM cells)2、Purified cells (e.g. LKS+ cells): ? (Tr

16、y) + 2 x 105 competitors (BM cells)Positive: All lineages (T,B,M) reconstitution > 0.1全部positive沒有意義。所選的劑量中，必須要有negative組。所選劑量最好是部分positive，部分negative。（negative 頻率要有>37%，也要有< 37%） KOPoisson statistics (泊松分布): 當(dāng)率為37%時(shí)，每個(gè)孔或者小鼠分配到一個(gè)干細(xì)胞。因此，此時(shí)的細(xì)胞劑量(dose)中含有1個(gè)干細(xì)胞(CRU)。CRUs (competitive repopulati

17、ng units):number of stem cells or quantity of HSCsCRU計(jì)算：L-Calc (StemCell Technologies Inc.)其他移植GFP normalize to 1If GFP+=20%-50%BMTSortingcBMT+ competitorJianwei Wang,. 2012, CellHSC NicheDavid Scadden Sean Morrison Linheng Li Toshio Suda Paul FrenetteHSC Self-renewal vs. DifferentiationIrving Weissman Hiromitsu