研究生课程移植

1、小鼠造血干细胞移植技术/ 2013-11-7内容小鼠造血干细胞表型常用流式分析方法和功能实验常用移植方法小鼠造血干细胞表型Immunophenotype of HSC/HPCsCFU-S (Till & McCulloch),证实造血干细胞存在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细胞周期 (Cell cycle)和细胞增殖(Proliferation)细胞凋亡 (Apoptosis)活性氧簇 (ROS, reactive oxygen species)磷酸化 (Phosphorylation)分选 (Sorting)常用流式分析方法和功能实验细胞周期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细胞固定; 活细胞(Verapamil)细胞固定细胞固定，破膜Ki67Pyronin Y细胞增殖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常用于示踪细胞凋亡Annexin V and DNA dyeTUNEL and DNA dye流式检测细胞荧光计数Annexin

6、V碎片晚期活细胞早期Richard XuFeng,. 2009, PNAS7-AAD胞内ROS分析ROS：氧离子，过氧化氢等DCF-DA (Invitrogen)：FITC通道MitoTracker (Invitrogen)线粒体HSCHPC磷酸化分析Kenneth D. Gibbs,. 2011, Blood分选 (Population level)Colony forming unit(集落形成)LTC-IC(Long-Term Culture-Initiating Cell) (长期培养-启动细胞)CAFC (cobblestone area forming cell )(鹅卵石样区域形

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

8、ion对称/不对称Ryo Yamamoto,. 2013, Cell常用移植方法骨髓重建移植和连续移植BM transplantation (BMT) and Serial BMT (sBMT)竞争性骨髓移植和连续移植Competitive BM transplantation(cBMT) coupledwith serial transfer单细胞移植Single cell transplantation极限稀释移植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骨髓重建移植和连续移植第一次移植：反应HSC重建能力连续移植：反应HSC自我更新能力移植细胞数量(全骨髓细胞)：1、half a femur 2、1x106-1x107移植次数： 3移植体积：300 -500LM检测：每隔4周检测外周血CD45.1比例，及T,B,M的重

11、建率。外周血20-50L。PBS+2mM EDTA。16w 或 24w，测骨髓各系及干细胞比例， 移植。SS+MSS:stem cellM: mature cellsCD45.1+ cellsEngraftment in BM (%)CD45.1CD45.1BMCD45.2BMBMWTKO10050I° II° III°竞争性骨髓移植和连续移植供体细胞：全骨髓细胞或HSC 竞争细胞：全骨髓细胞(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:移植次数： 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单细胞移植CD45.2Modified from Ryo Yamamoto,. 2013, CellHideo Ema,. 2005, Development CellCD45.1PrimaryPositive : one lineage reconstitution > 0.1SecondaryYoh

15、ei Morita,. 2010, JEM极限稀释移植目的：计算HSC数量（定量）> 8 mice per cell dose (每个剂量大于8只小鼠)> 3 cell doses (大于3个剂量)Differentdoses of cells9.5GyLy5.1Analysis(16 weeks)Ly5.22x105 BM cellsLy5.2细胞(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没有意义。所选的剂量中，必须要有negative组。所选剂量最好是部分positive，部分negative。（negative 频率要有>37%，也要有< 37%） KOPoisson statistics (泊松分布): 当率为37%时，每个孔或者小鼠分配到一个干细胞。因此，此时的细胞剂量(dose)中含有1个干细胞(CRU)。CRUs (competitive repopulati

17、ng units):number of stem cells or quantity of HSCsCRU计算：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