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Serial Transfer of Human Hematopoietic and Hepatic Stem/progenitor Cells
人造血干细胞和肝干细胞/祖细胞的串行转移   

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Abstract

A range of assays have been developed to determine the stemness or stem cell activity of human stem cells. The key assays of stem cells are functional: they must show self-renewal and the ability to generate the appropriate tissue. The best assays available to study this property in putative human stem cells involve xeno-transplantation into immune-deficient mice. Demonstration of both long-term (2-3 months) multi-lineage reconstitution of human blood or liver in a murine host and the ability of the putative stem cells to mediate reconstitution of a secondary host upon re-isolation from the primary mouse are generally accepted as the gold standard for demonstrating the presence of human hematopoietic and hepatic stem cells. Here, we describe a method of reconstituting NOD-scid IL-2Rγ-/-(NSG) mice with CD34+ stem cells from human fetal liver and repurification of CD34+ cells for serial transplantation.

Keywords: Hematopoietic progenitor(Hematopoietic祖), Hepatic progenitor(肝祖), Immune system(免疫系统), Human Hepatocyte(人肝细胞), Chimera mouse(嵌合体小鼠)

Materials and Reagents

  1. NOD.Cg-Prkdcscid //2rgtm1Wjl/SzJ (The Jackson Laboratory, stock number: 00 5557 )
  2. CD34+ fetal liver cells
  3. StemSpanTM SFEM (STEMCELL Technologies, catalog number: 0 9650 )
  4. ACK Lysing Buffer (Life Technologies, catalog number: A1049201 )
  5. Liver perfusion medium (Life Technologies, catalog number: 17701-038 )
  6. Liver digestion medium (Life Technologies, catalog number: 17703-034 )
  7. RoboSepTM Buffer (STEMCELL Technologies, catalog number: 20104 )
  8. Trypan blue solution (Sigma-Aldrich, catalog number: T8154 )
  9. EasySepTM Human Cord Blood CD34 Positive Selection Kit (STEMCELL Technologies, catalog number: 18096 )
  10. DMEM

Equipment

  1. Biosafety cabinet
  2. Petri dishes (100-mm2)
  3. 137 Cs gamma irradiator
  4. Insulin syringe (29 G 1 cc) (BD Biosciences, catalog number: 320310 )
  5. Syringe (5 ml) (BD, catalog number: 309646 )
  6. Heating pad or warming lamp
  7. Butterfly needle (BD, catalog number: 368659 )
  8. Cell strainer (100 μm) (BD Biosciences, catalog number: 352360 )
  9. Falcon tube(15 ml)

Procedure

  1. Engraftment of primary recipient mouse
    1. CD34+ fetal liver cells are purified based on the protocol “Isolation of CD34+ Cells from Human Fetal Liver and Cord Blood” (Chen and Chen, 2013)
    2. Monitor breeder pairs for the birth of new litters.
      Note: Engraftment procedures should be performed on newborn pups 24 to 48 h post-natal.
    3. Prepare CD34+ fetal liver cells suspend in StemSpan at 2.5 x 105 cells/50 μl/pup.
      Note: Freshly prepared or previously frozen preparations may be used.
    4. Place 24- to 48-h post-natal pups from a single litter into a 100 mm2 petri dish along with a small amount of bedding material from the breeder cage.
    5. The petri dish is put into a 137 Cs gamma irradiator. Irradiate pups with 1 Gy whole body irradiation.
    6. The petri dish is then brought back into biosafety cabinet. A second sterile petri dish is prepared with cotton nestlet from parent cage.
    7. One pup is taken at a time from the irradiated dish and held firmly, yet with great care, by thumb and index finger of one hand. Tilt the pup back so that abdomen is exposed. The liver will be visible on the right flank of the pup (Figure 1). Disinfect area with alcohol pad.


      Figure 1. Liver: site of injection

    8. The other hand holds a 29 G 1 cc insulin syringe loaded with 50 μl of fetal liver cells.
    9. The needle (perpendicular to body) will be inserted straight in, with bevel facing upwards, approximately 3 mm into the pup.
    10. The cells are then carefully and slowly injected into the pup's liver.
    11. Once injected, the needle is removed and gentle pressure is applied to the area. The injected pup is then placed in the second petri-dish.
    12. Steps A7-11 are repeated until all the pups have been injected.
    13. The pups are carefully placed back into their parents’ cage and covered with the cotton nestlet so they will smell familiar to parents.
    14. The pups will be monitor everyday for seven days. Any pups exbihiting severe weight loss, dehyration, dyspnea should be euthanized immediately. From experience, the pups are mostly unaffected by the injection.

  2. Cell repurification and reconstitution of secondary recipient mouse
    1. 8 to 10 weeks later, the primary mice are used for repurification of human hematopoietic stem cells and hepatic progenitor cells respectively.

      For repurification of hematopoietic stem cells from femurs
    2. Femurs are harvested and Remove as much muscle as possible around the femur bone.
    3. Attach a 27 G needle to a 5 ml syringe filled with PBS.
    4. Place the needle into the bone marrow (red middle of the bone), and flush out cells onto a 100 μm cell strainer in a 5 cm petri dish.
    5. Mesh cells and transfer the pass-through to a 15 ml Falcon tube.
    6. Pellet cells at 400 x g for 5 min.
    7.  Lyze red blood cells with ACK lysis buffer.
    8. Re-purify CD34+ cells by magnetic selection with EasySepTM Human Cord Blood CD34 Positive Selection Kit.

      For repurification of hepatic progenitor cells from livers
    9. Cannulate portal vein (Figure 2) with a 27 G buffer fly needle.
    10. Make incision in inferior vena cava (Figure 2).


      Figure 2. Portal vein and inferior vena cava

    11. Mouse livers were first perfused with pre-warmed liver perfusion medium at 0.7 ml/min for 10 min, then with pre-warmed liver digestion medium for 10 min.
    12. Carefully transfer the digested liver to a petri dish and dis-associate liver cells into single cell suspensions with curved forceps.
    13. The cell suspensions were washed with ice-cold DMEM at 50 x g for 5 min.
    14. Assay cell viability by Trypan Blue dye.
    15. CD34+ cells were re-purified from the cell suspensions with EasySepTM Human Cord Blood CD34 Positive Selection Kit.

  3. Reconstitution of secondary recipients
    1. CD34+ cells of desired numbers were injected into sublethally irradiated newborn NSG pups.
    2. After 8 to 10 weeks, samples e.g. blood and livers are harvested for analysis.

Acknowledgments

This protocol was developed and adapted from the previous publication Chen et al (2013a) and (2013b).

References

  1. Chen, Q., Khoury, M., Limmon, G., Choolani, M., Chan, J. K. and Chen, J. (2013a). Human fetal hepatic progenitor cells are distinct from, but closely related to, hematopoietic stem/progenitor cells. Stem Cells 31(6): 1160-1169.
  2. Chen, Q. and Chen, J. (2013b). Isolation of CD34+ cells from human fetal lLiver and cord blood. Bio-protocol 3(23): e991.

简介

已经开发了一系列测定来确定人类干细胞的干细胞或干细胞活性。 干细胞的关键测定是功能性的:它们必须显示自我更新和产生适当组织的能力。 用于在推定的人类干细胞中研究该特性的最佳测定涉及到免疫缺陷小鼠的异种移植。 在鼠宿主中长期(2-3个月)多谱系重建人血液或肝脏以及当从初级小鼠再分离时推定干细胞介导次级宿主重建的能力一般 被接受为证明人造血干细胞和肝干细胞存在的金标准。 在这里,我们描述了用来自人胎儿肝脏的CD34 +干细胞重建NOD-scid IL-2Rγ(NSG)小鼠的方法和CD34 + 细胞进行连续移植。

关键字:Hematopoietic祖, 肝祖, 免疫系统, 人肝细胞, 嵌合体小鼠

材料和试剂

  1. NOD.Cg- Prkdc scid // 2rg tm1Wjl /SzJ 实验室,库存号:005557)
  2. CD34 + 胎儿肝细胞
  3. StemSpan TM SFEM(STEMCELL Technologies,目录号:09650)
  4. ACK裂解缓冲液(Life Technologies,目录号:A1049201)
  5. 肝灌注培养基(Life Technologies,目录号:17701-038)
  6. 肝消化培养基(Life Technologies,目录号:17703-034)
  7. RoboSep TM Buffer(STEMCELL Technologies,目录号:20104)
  8. 台盼蓝溶液(Sigma-Aldrich,目录号:T8154)
  9. EasySep TM 人脐血CD34阳性选择试剂盒(STEMCELL Technologies,目录号:18096)
  10. DMEM

设备

  1. 生物安全柜
  2. 培养皿(100-mm 2
  3. 137 Cs伽玛辐射器
  4. 胰岛素注射器(29G 1cc)(BD Biosciences,目录号:320310)
  5. 注射器(5ml)(BD,目录号:309646)
  6. 加热垫或加温灯
  7. 蝴蝶针(BD,目录号:368659)
  8. 细胞过滤器(100μm)(BD Biosciences,目录号:352360)
  9. Falcon管(15ml)

程序

  1. 原始受体小鼠的移植
    1. CD34 + 胎儿肝细胞是基于方案"隔离CD34 + 来自人胎儿肝和细胞的细胞 血"(陈和陈,2013)
    2. 监控繁殖者对为新的胎儿的诞生。
      注意:应在产后24至48小时对新生小狗进行移植手术。
    3. 制备CD34 +胎儿肝细胞以2.5×10 5个细胞/50μl/只悬浮于StemSpan中。
      注意:可以使用新鲜制备的或以前冷冻的制剂。
    4. 将来自单个窝的24-至48小时出生后幼崽与来自育种笼的少量垫料一起放入100mm 2培养皿中。
    5. 将培养皿放入137 Csγ辐照器中。 用1 Gy全身照射辐照幼仔。
    6. 然后将培养皿放回生物安全柜。 第二个无菌培养皿用来自母体笼的棉花巢制备
    7. 每次从被照射的皿中取出一只小狗,并用非常小心,用一只手的拇指和食指牢牢地握住。 倾斜小狗回到腹部暴露。 肝脏将在小狗的右侧可见(图1)。 用酒精垫消毒区域。


      图1.肝脏:注射部位

    8. 另一只手拿着装有50μl胎肝细胞的29G 1cc胰岛素注射器
    9. 针头(垂直于身体)将直接插入,斜面朝上,大约3毫米进入小狗。
    10. 然后将细胞小心地并缓慢地注射到小狗的肝脏中
    11. 一旦注射,针被移除并且对该区域施加缓和的压力。 然后将注射的小狗放入第二个培养皿中。
    12. 重复步骤A7-11,直到所有幼犬被注射
    13. 幼仔被仔细地放回他们父母的笼子里,用棉花毯子覆盖,所以他们会闻到父母的熟悉。
    14. 幼崽将每天监测七天。 任何严重减肥,痉挛,呼吸困难的幼崽应立即安乐死。 根据经验,幼仔几乎不受注射的影响。

  2. 细胞再纯化和二次受体小鼠的重建
    1. 8至10周后,将原代小鼠分别用于人造血干细胞和肝祖细胞的再纯化。

      用于补充来自股骨的造血干细胞。
    2. 收集股骨,并在股骨周围尽可能多地去除肌肉
    3. 将27G针头连接到装有PBS的5ml注射器中
    4. 将针放入骨髓(骨的红色中部),并冲洗出细胞到5厘米培养皿中的100μm细胞过滤器。
    5. 网孔细胞,并将通过转移到15毫升Falcon管
    6. 将细胞以400×g离心5分钟。
    7.  用ACK裂解缓冲液冲洗红细胞。
    8. 使用EasySep TM 人脐带血CD34阳性选择试剂盒通过磁选择重新纯化CD34 + 细胞。

      用于从肝脏再补充肝脏祖细胞
    9. 用27 G缓冲针针刺入门静脉(图2)
    10. 在下腔静脉切开(图2)。


      图2.门静脉和下腔静脉

    11. 首先用预热的肝灌注培养基以0.7ml/min灌注小鼠肝脏10分钟,然后用预热的肝脏消化培养基灌注10分钟。
    12. 小心地将消化的肝脏转移到培养皿中,并将肝细胞分离成具有弯曲镊子的单细胞悬浮液。
    13. 将细胞悬浮液用冰冷的DMEM以50×g洗涤5分钟。
    14. 通过台盼蓝染料测定细胞活力。
    15. 用EasySep TM人脐带血CD34阳性选择试剂盒从细胞悬浮液中重新纯化CD34 +细胞。

  3. 二级受助人的重组
    1. 将所需数目的CD34 +细胞注射到亚致死辐射的新生儿NSG幼仔中。
    2. 8至10周后,收集样品例如血液和肝脏用于分析

致谢

该协议是从以前的出版物Chen等人(2013a)和(2013b)开发和改编的。

参考文献

  1. Chen,Q.,Khoury,M.,Limmon,G.,Choolani,M.,Chan,J.K.and Chen,J.(2013a)。 人胎儿肝祖细胞与造血干/祖细胞不同,但密切相关。/a> 干细胞 31(6):1160-1169。
  2. Chen,Q.和Chen,J。(2013b)。 从人胎儿肝脏和脐带血中分离CD34 + 细胞 。 生物协议 3(23):e991。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, Q. and Chen, J. (2013). Serial Transfer of Human Hematopoietic and Hepatic Stem/progenitor Cells. Bio-protocol 3(23): e992. DOI: 10.21769/BioProtoc.992.
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