方法文章

Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells

DOI：

10.3791/64488

⸱

2023年5月19日

 ,  ,  ,  , 
1Hematopoietic Stem Cell Biology and Medical Innovation (HSCBMI), Department of Pediatrics, Kobe University Graduate School of Medicine, 2RIKEN Center for Biosystems Dynamics Research

本文内容

摘要

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We present a step-by-step protocol for the isolation of long-term hematopoietic stem cells (LT-HSCs) and short-term HSCs (ST-HSCs) using the Hoxb5 reporter system.

摘要

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Self-renewal capacity and multi-lineage differentiation potential are generally regarded as the defining characteristics of hematopoietic stem cells (HSCs). However, numerous studies have suggested that functional heterogeneity exists in the HSC compartment. Recent single-cell analyses have reported HSC clones with different cell fates within the HSC compartment, which are referred to as biased HSC clones. The mechanisms underlying heterogeneous or poorly reproducible results are little understood, especially regarding the length of self-renewal when purified HSC fractions are transplanted by conventional immunostaining. Therefore, establishing a reproducible isolation method for long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs), defined by the length of their self-renewal, is crucial for overcoming this issue. Using unbiased multi-step screening, we identified a transcription factor, Hoxb5, which may be an exclusive marker of LT-HSCs in the mouse hematopoietic system. Based on this finding, we established a Hoxb5 reporter mouse line and successfully isolated LT-HSCs and ST-HSCs. Here we describe a detailed protocol for the isolation of LT-HSCs and ST-HSCs using the Hoxb5 reporter system. This isolation method will help researchers better understand the mechanisms of self-renewal and the biological basis for such heterogeneity in the HSC compartment.

引言

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Hematopoietic stem cells (HSCs), which possess self-renewal capacity and multipotency, reside at the apex of the hematopoietic hierarchy1,2. In 1988, Weissman and colleagues demonstrated for the first time that the isolation of mouse HSCs could be achieved using flow cytometry3. Subsequently, a fraction defined by a combination of cell surface markers, Lineage−c-Kit+Sca-1+CD150+CD34−/loFlk2−, was reported to contain all HSCs in mice4,5....

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方案

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All the animal experiments described were approved by the RIKEN Center for Biosystems Dynamics Research.

1. Preconditioning of the recipient mice

  1. Prepare male C57BL/6 congenic mice aged 8-10 weeks old as recipient mice. The number of recipient mice depends on the experimental protocol. We typically prepare 10-20 mice for each condition.
    1. Feed the mice with sterilized water supplemented with enrofloxacin (170 mg/L). As irradiated recipient mice are highly susceptible to infection, keep the cages as clean as possible.
      NOTE: Supplementation with antibiotics starts 24 h prior to the irradiation an....

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结果

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Previously, self-renewal capacity has been measured using competitive transplantation assays, in which donor HSCs are thought to retain their self-renewal capacity only if multi-lineage donor cells in the recipient peripheral blood are observed17. In addition, several reports define LT-HSCs as cells that continue to produce peripheral blood cells several months after the second bone marrow transplantation10,18. Therefore, in order to compa.......

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讨论

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Traditionally, cell surface marker-defined HSCs have been prepared to study the functions of HSCs, such as self-renewal capacity and multi-potency19,20,21. However, the immunophenotypically defined (Lineage−c-Kit+Sca-1+CD150+CD34−/loFlk2−) HSC fraction contains two discrete HSC populations: LT-HSCs and ST-HSCs9<.......

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披露

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The authors declare no conflicts of interest associated with this study.

致谢

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We gratefully acknowledge Hiroshi Kiyonari for the animal care and for providing recipient mice at RIKEN BDR, as well as Hitomi Oga, Kayoko Nagasaka, and Masaki Miyahashi for laboratory management at Kobe University. The authors also greatly appreciate the ongoing support for this work. Masanori Miyanishi was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP17K07407 and JP20H03268, The Mochida Memorial Foundation for Medical and Pharmaceutical Research, The Life Science Foundation of Japan, The Takeda Science Foundation, The Astellas Foundation for Research on Metabolic Disorders, and AMED-PRIME, AMED under Grant Number JP18gm....

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材料

本文使用的材料清单
姓名公司目录编号评论
0.2 mL 8 联排管，圆顶盖SSIbio3230-00
0.5M EDTA，pH 8.0IinvtrogenAM9260G
100 &微量;m 细胞过滤器Falcon352360
30G 胰岛素注射器BD326668
40 µm 细胞过滤器Falcon352340
5 mL 圆底聚苯乙烯试管，带细胞过滤器卡口盖FALCON352235
7-AAD 活力染色溶液BioLegend420404
96 孔 U 型底FALCON351177
抗 APC-MicroBeads抗体 Milteny biotec130-090-855
带捕集阱的吸液器培养瓶BiosanFTA-1
B220-Alexa Fluor 700 （RA3-6B2）BioLegend103232
B220-生物素 （RA3-6B2）BioLegend103204
B220-BV786 （RA3-6B2）BD Biosciences563894
B6。CD45.1 同基因小鼠 Sankyo Labo 服务N/A
拜耳 10%拜耳341106546
BD FACS Aria II 特别订单系统 BD不适用
566349
CD11b-Alexa Fluor 700 （M1/70）BioLegend101222
CD11b-生物素 （M1/70）BioLegend101204
CD11b-BUV395 （M1/70）BD Biosciences 563553
CD11b-BV711 （M1/70）BD Biosciences563168
CD127-Alexa Fluor 700 （A7R34）Invitrogen56-1271-82
CD150-BV421 （TC15-12F12.2）BioLegend115943
CD16/CD32-Alexa Fluor 700 （93）Invitrogen56-0161-82
CD34-Alexa Fluor 647 （RAM34）BD Biosciences560230
CD34-FITC （RAM34）Invitrogen11034185
CD3-Alexa Fluor700 （17A2）BioLegend100216
CD3&ε; -生物素 （145-2C11）BioLegend100304
CD3&ε;-BV421 （145-2C11）BioLegend100341
CD45.1/CD45.2 同源小鼠N/AN/A在我们的实验室繁殖
CD45.1-FITC （A20）BD Biosciences553775
CD45.2-PE （104）BD Biosciences560695
CD4-Alexa Fluor 700 （GK1.5）BioLegend100430
CD4-生物素 （GK1.5）BioLegend100404
CD8a-Alexa Fluor 700 （53-6.7）BioLegend100730
CD8a-生物素 （53-6.7）BioLegend100704
离心管 15mlNICHIRYO00-ETS-CT-15
离心管 50mlNICHIRYO00-ETS-CT-50
c-Kit-APC-eFluor780 （2B8）Invitrogen47117182
D-PBS （-） 不含钙和镁，液体 Nacalai14249-24
牛血清Thermo Fisher10270106
Flk2-PerCP-eFluor710 （A2F10）eBioscience46135182
FlowJo版本10BD Biosciences 
Gmmacell 40 Exactor最佳电子N/A
Gr-1-Alexa Fluor 700 （RB6-8C5）BioLegend108422
Gr-1-生物素 （RB6-8C5）BioLegend108404
Hoxb5-tri-mCherry 小鼠（C57BL/6J 背景） N/AN/A在我们的实验室中饲养
的 IgG 来自大鼠血清，技术级，>=80% （SDS-PAGE），缓冲水溶液Sigma-AldrichI8015-100MG
异氟醚辉瑞4987-114-13340-3 
Kimwipes S200NIPPON PAPER CRECIA 6-6689-01
LS 色谱柱Milteny biotec130-042-401
裂解缓冲液 BD555899
MACS MultiStandMilteny biotec130-042-303
组织培养微孔板（用于粘附池） 6孔IWAKI3810-006
MidiMACS 分离器Milteny biotec130-042-302
小鼠派笼夏目制作所KN-331
多用途冷冻离心机TOMYEX-125
NARCOBIT-E （II）夏目制作所KN-1071-I
NK-1.1-PerCP-Cy5.5 （PK136）BioLegend108728
青霉素-链霉素混合溶液nacalai26253-84
瓷砂浆 φ120 mm，含 PestleAsone6-549-03
蛋白 LoBind 管 1.5 mL Eppendorf22431081
Sca-I-BUV395 （D7）BD Biosciences563990
不锈钢手术刀刀片FastGeneFG-B2010
链霉亲和素-BUV737BD Biosciences612775
SYTOX-redInvitrogenS34859
小鼠尾静脉抑制器标准BraintreeTV-150 STD
TCRb-BV421 （H57-597）BioLegend109230
Ter-119-Alexa Fluor 700 （TER-119）BioLegend116220
Ter-119-生物素 （TER-119）BioLegend116204
Terumo 5ml 同心鲁尔滑注射器TERUMOSS-05LZ
Terumo 皮下注射针 23G x 1TERUMONN-2325-R
胎https://www.flowjo.com/solutions/flowjo疗法

参考文献

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  1. Weissman, I. L., Shizuru, J. A. The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases. Blood. 112 (9), 3543-3553 (2008).
  2. Majeti, R., Park, C. Y., Weissman, I. L.

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