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Jul 2019

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A Modified Semisolid Clonal Culture for Identification of B-1 and B-2 Progenitor Colony Forming Ability of Mouse Embryonic Hemogenic Endothelial Cells
改良半固态克隆培养鉴定小鼠胚胎造血内皮细胞B-1和B-2祖细胞集落形成能力   

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Abstract

The search for the origin of the first hematopoietic stem cells (HSCs) in the mouse embryo has been a hot topic in the field of developmental hematopoiesis. Detecting lymphoid potential is one of the supportive evidence to show the definitive hematopoietic activity of HSCs. However, the first B-lymphoid potential in the mouse embryos are reported to be biased to innate-like B-1 cell lineage that can develop from hemogenic endothelial cells (HECs) independently of HSCs. On the other hand, conventional adaptive immune B cells (B-2) cells are considered to be exclusively derived from HSCs. Therefore, segregating B-1 and B-2 progenitor potential is important to understand the developmental process of HSCs that are also produced from HECs through intermediate precursors referred to as pre-HSCs. Both HECs and pre-HSCs show endothelial surface phenotype and require stromal support to detect their hematopoietic activity. The method utilizing stromal cell culture followed by modified semisolid clonal culture enables us to detect the number of colony forming units for B-1/B-2 progenitors originally derived from HECs/pre-HSCs, which will reflect the potential of B-1 biased or multi-lineage repopulating HSCs.

Keywords: B-cell colony forming assay (B细胞集落形成试验), OP-9 (OP-9), B-1 and B-2 lymphoid progenitors (B-1和B-2淋巴样祖细胞), Mouse embryo (小鼠胚胎), Pre-HSC (造血干细胞前体), Hemogenic endothelial cell (造血内皮细胞), AGM region (AGM 区)

Background

Semisolid clonal culture (methylcellulose colony forming assay) is a traditional method to detect the number of hematopoietic progenitor cells. One colony is considered to be derived from a single progenitor cell (clonal origin). Added cytokines play important roles in the formation of yield colonies. For example, Epo enhances the colony forming unit of erythrocytes (CFU-E) or the burst forming unit of erythrocytes (BFU-E), while G-CSF/GM-CSF will enhance CFU-G (Granurocytes), M (Macrophages) or GM (Granulo-macrophages). SCF stimulate hematopoietic progenitor activity. The formation of B-lymphocyte progenitor colonies from mouse BM requires SCF, IL-7 and Flt3-ligand. Importantly, this assay can detect only the activity of CD45+ (or CD41+ in case of embryo-derived cells) hematopoietic progenitor cells, thus, cannot detect the hematopoietic activity of CD45-CD144+ hemogenic endothelial cells (HECs) that produce various hematopoietic cells in the mouse embryo. The detection of hematopoietic activity of HECs heavily depends on the stromal support or organ culture method. Therefore, it is challenging to determine the number of lymphoid progenitors produced from HECs. Recently, Montecino-Rodriguez et al. (2016) developed a modified semisolid clonal culture using S-17 stromal cells to detect B-1 and B-2 potential of hematopoietic progenitors in embryonic (E) day 10 yolk sac cells and fetal liver cells.

B-1 progenitor cells are mainly detected in the fetal liver and neonatal BM. B-1 cells belong to innate-like B-lymphocytes that produce natural IgM antibodies, while conventional adaptive immune B cells are referred to as B-2 cells. Montecino-Rodriguez et al., identified B-1 cell-specific progenitors in the mouse fetal liver and neonatal BM as lin-IgM-CD19+B220- (CD19 single positive) cells whereas B-2 specific progenitors (Pro-B cell) are lin-IgM-CD19-B220+ (B220 single positive) cells. These two populations quickly become CD19+B220+ double positive in vitro culture, thus, it is difficult to determine if B cells produced from embryonic tissues in the co-culture with stromal cells belong to B-1 or B-2 cells. This modified semisolid clonal culture enables us to detect B-1 and/or B-2 progenitor colonies derived from HECs/pre-HSC by utilizing stromal cells that support B lymphopoiesis (Kobayashi et al., 2019). We used OP9 stromal cells to support B-lymphopoiesis in the semisolid clonal culture, with which we have been successful to induce B lymphocytes from HECs (Yoshimoto et al., 2011; Kobayashi et al., 2019). Since B-2 progenitor colony forming ability seems to be one of the HSC activity, this method may be utilized to evaluate the HSC activity derived from HECs/pre-HSCs in the mouse embryo.

Materials and Reagents

  1. T-25 culture Flask (Corning, catalog number: 353109 )
  2. 6-well culture plates (Corning, Costar, catalog number: 3516 )
  3. 96-well Flat culture plates (Corning, Costar, catalog number: 3595 )
  4. Falcon Round bottom polypropylene14 ml tube (Corning, catalog number: 352059 )
  5. 1.5 ml Eppendorf tube
  6. 35 mm Petri dish (Falcon, catalog number: 351008 )
  7. 15 mm Petri dish
  8. 3 ml syringe (BD, catalog number: 309657 )
  9. 5 ml syringe
  10. 18 G needles (BD, catalog number: 305185 )
  11. 10 μl pipette tip
  12. Bottle top filter system (0.22 μm pore size)
  13. C57BL/6 male and females
  14. α-MEM (powder) (Gibco, catalog number: 11900-024 )
  15. Fetal Bovine Serum
    Note: We used FBS from Atlanta Biological for this publication. However, we always perform lot check from 5-10 test FBS samples to find the best FBS for OP9 maintenance culture. We usually purchase 20 bottles to maintain the same equality of experiments for 2-3 years.
  16. Fresh Milli-Q water
  17. PBS (Fisher Scientific, catalog number: BP665-1 )
  18. 2-Mercaptoethanol (Fisher Scientific, catalog number: O3446I-100 )
  19. 500 mM EDTA pH 8.0
  20. IMDM (Gibco, catalog number: 12440061 )
  21. Heparin (Sigma, catalog number: H3149-100KU )
  22. Endothelial mitogen 50 mg (Biomed Tech, catalog number: BT-203 )
  23. Penicillin-Streptomycin 10,000 U-10,000 µg/ml (Gibco, catalog number: 15140122 )
  24. X-VIVO 20 (Lonza, catalog number: 04-448Q )
  25. Methylcellulose-base medium for mouse pre-B lymphoid progenitor cells (Stemcell Technologies, MethocultTM, catalog number: M3630 )
  26. Recombinant murine IL-7 (Peprotech, catalog number: 217-17 )
  27. Recombinant murine Flt3-ligand (Peprotech, catalog number: 250-31L )
  28. 0.05% Trypsin/EDTA (Gibco, catalog number: 25300054 )
  29. 0.25% Collagenase Type I (Stemcell Technologies, catalog number: 0 7902 )
  30. DNase I Solution (1mg/ml) (Stemcell Technologies, catalog number: 0 7900 )
  31. Cell Dissociation Buffer (Hank’s balanced) (Gibco, catalog number: 13150-016 )
  32. Antibodies for sorting and detecting B cell colonies (Table 1)

    Table 1. Antibody list for flow cytometry


  33. OP9 stromal cells [obtained from Dr. Shin-Ichi Nishikawa (retired from Riken, Kobe, Japan), can be obtained from ATCC CRL-2749 (ideally < 30 passage)]
  34. Akt-expressing AGM-ECs (AGM-ECs): endothelial cells (ECs) obtained from aorta-gonod-mesonephros (AGM) region in the mouse embryo was established and induced Akt-overexpression by Dr. Brandon Hadland, Fred Hutchinson Cancer Center, Seattle, USA) (Hadland et al., 2015)
  35. Gelatin from porcine skin (Sigma, catalog number: G1890 )
  36. α-MEM (see Recipes)
  37. 2-mercaptoethanol (2-ME) stock (0.1 M) (see Recipes)
  38. OP9 maintenance medium (α-MEM + 20% FBS) (see Recipes)
  39. AGM-EC maintenance medium (see Recipes)
  40. Differentiation medium (α-MEM +10% FBS + 5 x 10-5 M 2ME) (see Recipes)
  41. Staining buffer (PBS + 5% FBS + 2 mM EDTA) (see Recipes)
  42. 0.1% Gelatin (see Recipes)

Equipment

  1. P1000 pipette
  2. Microcentrifuge
  3. FACS Sorter and analyzer with more than 6 fluorescent detectors are required. We used BD FACSAria for cell sorting, BD LSRII and BD LSR-Fortessa for analysis
  4. Standard cell culture CO2 incubator (CO2: 5%, O2: 20%)

Procedure

  1. Maintaining OP9 stromal cells and AGM-EC stromal cells

    Maintaining OP9 stromal cells
    1. Maintaining OP9 stromal cells in a good condition is an important key to obtain successful results. Make fresh OP9 maintenance medium from the powder every month. Test several lots of FBS for good growth of OP9 cells.
    2. Thaw OP9 in T25 Flask in freshly made OP9 medium. Split every 3 days, 1:3.
      Notes:
      1. OP9 cells are flat round or square shapes (Figure 1) if they become spindle shape, they are not good OP9 anymore because spindle shape cells tend to be transforming and losing B cell supporting ability.


        Figure 1. The morphology of OP9 cells (100x). Red arrows indicate good OP9 and blue arrows indicate bad OP9. Scale bar = 100 μm.

      2. Split one confluent OP9 in T25 flask into 3 flasks every 3 days. If they are not confluent in 3 days, the lot of FBS is not good for them.
      3. The density of OP9 needs to be appropriate (around 4 x 105/T25 flask at the starting point). When OP9 cells are confluent in T25 flask, the cell number is around 1.0 x 106-1.5 x 106.
      4. For splitting, aspirate medium, wash with PBS 2 times, and treat them with 0.05% Trypsin/EDTA for 3 min at 37 °C. OP9 cells will be peeled off from the flask and add medium and pipette to make a single cell suspension with 5 ml serological pipette (important). Make sure well pipetting and single cells, because leaving cell clumps may induce bad condition of OP9 cells at later passage. Spin 400 x g for 5 min, aspirate supernatant, loosen the pellets by tapping the bottom of the tube (important), add 1 ml OP9 maintenance medium and pipette very well with P1000 pipette to ensure single cell suspension. Add more OP9 medium and plate them into 3 x T25 flasks.
      5. OP9 must be maintained within 3 days (usually every 3 days). Even if they are not confluent in 3 days, they should be passed (1 to 1 or 1 to 2 to increase the cell density). If they are left confluent without passage, they can be used for co-culture, but they will not expand anymore.
      6. If OP9 cells are not in good condition, it is hard to obtain B cells from early stage of embryos (< E9.0-9.5).
      7. OP9 cells should not be maintained for more than 1 month.
      8. OP9 cells show good contact inhibition. If you need irradiation to stop the over-cell growth of OP9, they are already transformed and not good OP9 anymore.

    Maintaining AGM-EC stromal cells
    1. Coat 0.1% gelatin T25 Flask for more than 30 min.
    2. Aspirate gelatin before plating cells.
    3. Thaw AGM-EC on gelatin-coated T25 Flask in AGM-EC maintenance medium (near 5 x 105/flask). Pass every 3-4 days, 1:1-3 (Hadland et al., 2015).

  2. Harvest embryo tissues
    1. Set up timed mating of C57BL/6 male and females after 3:00 PM. The following early morning, check vaginal plugs of the female and separate plugged females into a different cage. Noon on this day is considered as embryonic (E) day 0.5.
    2. About 7 days before harvesting embryos, thaw OP9 and AGM-ECs in T25 flask and maintain.
    3. One day before harvesting embryos, prepare OP9 (1 x 104/well) and AGM-ECs (5 x 103/well) in 96-well plates respectively. In addition, keep maintaining OP9 in T25 Flask as needed for plating methylcellulose at 5-7 days later.
    4. Ten days later after timed mating (at E10.5), harvest embryos from pregnant mother (follow the procedure under the approved AICUC protocol at your institute) (Morgan et al., 2008).
    5. Under the stereomicroscope, open the uterus, separate yolk sac (YS) from embryos (Morgan et al., 2008). After removal of the YS, confirm the embryonic stage by counting somite pairs. Collect caudal half of embryos, containing AGM region into 14 ml tubes filled with 10 ml medium.
    6. Centrifuge the AGM tissues in the medium at 450 x g for 5 min and aspirate supernatant. Suspend the AGM tissues in 0.25% Collagenase Type I + DNase I (final 20 μg/ml) respectively [use 0.5-1 ml per embryo equivalent (e.e)].
    7. Incubate the tissue suspension at 37 °C. Pipette every 5 min and confirm dissociation under microscope. Once you see good single cells, stop the collagenase reaction by adding the same volume of Cell Dissociation Buffer. Usually this incubation will take 15-30 min depending on the age of embryos (the earlier embryo, the shorter incubation time).
    8. Add differentiation medium and filter them using 70 μm strainer, and centrifuge the cells at 450 x g for 5 min.
    9. Aspirate supernatant, suspend single cell pellets in 1 ml staining buffer, and count cell number. Normally around 5 x 105 cells /embryo will be harvested.

  3. Sorting HECs/pre-HSCs and co-culture
    When examining the B-lymphoid hematopoietic potential of HECs/pre-HSC, it is challenging to detect B-progenitor colony forming ability directly from these cells, since most of them are endothelial phenotype. In order to induce hematopoietic progenitors, sort HECs/pre-HSCs and plate them on OP9 (expand hematopoietic progenitors) or on AGM-ECs (let pre-HSCs mature into HSCs).

    Staining and cell sorting
    1. Suspend embryonic cells up to 1 x 107 cells in 100 μl staining buffer in 1.5 ml Eppendorf tube, take 5% volume first each for negative control and FMO staining.
    2. Add antibodies; anti-mouse CD45, CD144, c-kit, EPCR (CD201), and Ter119 at a ratio of 0.3 μl/106 cells and incubate them for 30 min on ice.
    3. Add 1 ml staining buffer for wash and centrifuge them at 800 x g for 2 min at microcentrifuge.
    4. Suspend the cells in staining buffer and sort Ter119-CD144+c-kit+EPCR+ cells on FACSAria (Figure 2).
    5. (optional) mix sorted Ter119-CD144+c-kit+EPCR+ cells with 1 x 105 OP9 cells in Methocult with IL-7 and Flt3 -ligand, and plate them onto a 35 mm Petri dish. However, direct plating of these cells will yield only a few B cell colonies.
    6. Plate 100-200 Ter119-CD144+c-kit+EPCR+ cells at one well of 96-well plate confluent with OP9 in differentiation medium or AGM-EC cells in X-vivo 20 with added IL-7 and Flt3-ligand (final concentration: 10 ng/ml for both).
    7. Five to seven days after co-culture, harvest all cells and stain them with anti-mouse CD45 and CD11b antibody.
    8. After staining, cells are washed with staining buffer and are suspended in staining buffer and sort CD11b-CD45+ cells from the co-cultured cells on FACSAria as shown in Figure 2B.


      Figure 2. Gating strategy for sorting pre-HSCs (A) and CD45+CD11b- cells after co-culture with AGM-ECs(B)

  4. Plating semisolid culture
    1. Thaw Methocult at room temperature or 4 °C over night.
    2. Always prepare N+1 samples for methylcellulose assays. Mix 200-600 CD45+ cells after co-culture with 1 x 105 OP9 cells/dish. For triplicates, mix 800-2,400 CD45+ cells and 4 x 105 OP9 cells in 14 ml round bottom tube and spin them down, aspirate the medium, and loosen the cell pellets by tapping the tube. Add 4 ml Methocult M3630 using 5 ml syringe with 18 G needle to the cell pellet (This way, you can plate 200-600 CD45+ cells with 1 x 105 OP9 cells per 35 mm dish.). In this scale, 20-80 colonies/dish will be expected.
    3. Add IL-7 (final 10 ng/ml) and Flit3-ligand (final 10 ng/ml), mix well by vortexing and leave it until all the bubbles are gone.
    4. Plate 1.1 ml of methylcellulose medium including cells into each 35 mm Petri dish x 3 dishes using 18 G needle with 3 ml syringe.
    5. Place 3 Petri dishes containing the Methocult and 1 Petri dish containing sterile H2O (preventing the dry out the medium) in a 15 mm Petri dish.
    6. Incubate them in a 5% CO2 incubator for 8-11 days and count colony numbers. The pictures in Figure 3 are representative B-cell colonies and their FACS analysis.


      Figure 3. B cell colonies and FACS analysis of picked-up colonies. A. B-1 progenitor (B-1-pro) colony. B. B-2 progenitor (B-2-pro) colony, confirmed by FACS analysis below. Scale bar = 200μm. C. FACS analysis of each picked up colony. Based on the gating of BM B-2 progenitors, same gating is applied to the analysis of each sample.

  5. Determine each colony as to B-1 or B-2 progenitors
    1. Prepare 10 μl pipette and 1 ml differentiation medium in sterile Eppendorf tubes. For picked up colonies, prepare 1.5 ml Eppendorf tubes containing 1 ml staining buffer. Prepare the same number of tubes as the colony number.
    2. Before picking up the colony, wet the 10 μl pipette tip with differentiation medium by pipetting several times to avoid the methylcellulose sticking to the inside of the pipette tips. Set 8 μl to pick up a colony. Under the microscope, pick up each colony using 10 μl pipette with a wet tip, and transfer each colony to the 1.5 ml tube containing staining buffer.
    3. Once all the colonies are picked up into each tube, centrifuge the tubes at 800 x g for 2 min in the microcentrifuge and aspirate the supernatant.
    4. Add 50 μl of staining buffer and vortex to make the pellet a single cell suspension.
    5. Stain them with the antibodies: anti-mouse AA4.1-APC, CD19-PE, B220-PEcy7, and Mac1-FITC at a ratio of 0.3 μl/106 cells for 20-30 min on ice.
    6. Wash with 1 ml staining buffer and centrifuge at 800 x g for 2 min.
    7. Aspirate the supernatant and suspend the cell pellet with staining buffer with PI or DAPI for dead cell exclusion and apply the samples to flow cytometry.
    8. Determine whether the colonies contain B-1 and/or B-2 progenitors (Figure 3) (Kobayashi et al., 2019). Prepare adult BM cells as a positive control for gating B-progenitors (Montecino-Rodriguez et al., 2006).

Data analysis

Data analysis and expected outcome
Based on the flow cytometric analysis, each dish should contain 1) only B-1 progenitors, 2) only B-2 progenitors, and 3) both B-1 and B-2 progenitors. Count number of colonies that contain B-1, B-2, or both B-1 and B-2 progenitors per plate and calculate mean and SD values for the results of three dishes.
  Usually, only B-2 colonies are expected from BM progenitors. B-1 progenitor colonies are dominant from embryonic tissues co-cultured with OP9, while B-1+B-2 progenitor colonies become dominant when these embryonic tissues are co-cultured with AGM-ECs or organ cultured (developing to HSCs) (Kobayashi et al., 2019).

Notes

  1. AGM-ECs can support the transition of pre-HSCs to HSCs, but they do not support B cell colony formation when they are plated together in Methylcellulose culture.
  2. The number of B cell colony forming cells varies depending on the age of the embryo and the condition of the OP9.
  3. Ter119-CD144+c-kit+EPCR+ cells are very rare, therefore, Ter119-CD144+c-kit+ should also be able to produce B cell colonies by support of OP9.
  4. Do not add SCF in the co-culture nor methylcellulose culture. Good OP9 support B-lymphopoiesis without SCF and SCF may enhance macrophage differentiation.

Recipes

Culture medium:

  1. α-MEM (with penicillin/streptomycin 50 U-50 μg/ml)
    1. Add powder α-MEM to 1,000 ml fresh Milli-Q water and stir until it is completely dissolved
    2. Add 2.2 g H2CO3 and stir until completely dissolved
    3. Add 5 ml Penicillin-Streptomycin
    4. Filter using 0.22 μm filtering system
  2. 2-mercaptoethanol (2-ME) stock (0.1 M)
    Mix 70 μl of 2-ME to 10 ml α-MEM and filter using 0.22 μm syringe filter
  3. OP9 maintenance medium (α-MEM + 20% FBS)
    Mix 400 ml α-MEM and 100 ml FBS and filter using 0.22 μm filtering system
  4. AGM-EC maintenance medium
    IMDM 400 ml
    FBS (Hyclone, heat inactivate) 100 ml
    Pen/Strep (100x) 5 ml
    Heparin 5 ml (10 mg/ml stock prepared fresh in IMDM)
    L-glutamine (100x) 5 ml
    Endothelial mitogen 50 mg
  5. Dissolve in 10 ml of above media mix
  6. Filter using 0.22 μm filtering system
  7. Differentiation medium (α-MEM + 10% FBS + 5 x 10-5 M 2-ME)
    450 ml α-MEM
    50 ml FBS
    250 μl of 0.1 M 2-ME
    Filter using 0.22 μm filtering system
  8. Staining buffer (PBS + 5% FBS + 2 mM EDTA)
    500 ml PBS 500 ml
    2.5 ml FBS + EDTA (final 2 mM)
    Filter using 0.22 μm filtering system
  9. 0.1% Gelatin
    0.5 g Gelatin
    Milli-Q water 500 ml
    Autoclave

Acknowledgments

This work is supported by NIAID R01AI121197. This protocol was derived from a recent report (Kobayashi et al., 2019).

Competing interests

There is no conflict of interest.

Ethics

The procedure of harvesting mouse embryos and other related animal works follows the Animal Welfare Committee (AWC) protocol approved by the Center for Laboratory Animal Medicine and Care (CLAMC) at UTHealth (AWC16-0124, 2016-2019).

References

  1. Hadland, B. K., Varnum-Finney, B., Poulos, M. G., Moon, R. T., Butler, J. M., Rafii, S. and Bernstein, I. D. (2015). Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derived hematopoietic stem cells. J Clin Invest 125(5): 2032-2045.
  2. Kobayashi, M., Tarnawsky, S. P., Wei, H., Mishra, A., Azevedo Portilho, N., Wenzel, P., Davis, B., Wu, J., Hadland, B. and Yoshimoto, M. (2019). Hemogenic Endothelial Cells Can Transition to Hematopoietic Stem Cells through a B-1 Lymphocyte-Biased State during Maturation in the Mouse Embryo. Stem Cell Reports 13(1): 21-30.
  3. Montecino-Rodriguez, E., Fice, M., Casero, D., Berent-Maoz, B., Barber, C. L. and Dorshkind, K. (2016). Distinct Genetic Networks Orchestrate the Emergence of Specific Waves of Fetal and Adult B-1 and B-2 Development. Immunity 45(3): 527-539.
  4. Montecino-Rodriguez, E., Leathers, H. and Dorshkind, K. (2006). Identification of a B-1 B cell-specified progenitor. Nat Immunol 7(3): 293-301.
  5. Morgan, K., Kharas, M., Dzierzak, E. and Gilliland, D. G. (2008). Isolation of early hematopoietic stem cells from murine yolk sac and AGM. J Vis Exp(16): pii: 789.
  6. Yoshimoto, M., Montecino-Rodriguez, E., Ferkowicz, M. J., Porayette, P., Shelley, W. C., Conway, S. J., Dorshkind, K. and Yoder, M. C. (2011). Embryonic day 9 yolk sac and intra-embryonic hemogenic endothelium independently generate a B-1 and marginal zone progenitor lacking B-2 potential. Proc Natl Acad Sci U S A 108(4): 1468-1473.

简介

[摘要 ] 在搜索起源的第一造血干细胞(HSCs)在小鼠胚胎一直是一个热门话题在该领域发展造血功能。检测淋巴潜力是一个支持性证据显示权威造血活动的造血干细胞然而,据报道,小鼠胚胎中的第一个B淋巴样电位偏向于先天性B-1细胞谱系,该谱系可以从造血内皮细胞(HEC)脱离HSC发育而来。 B细胞(B-2)细胞被认为仅来自HSC。因此,分离B-1和B-2祖细胞的潜力对于理解也由HEC通过中间前体(也称为HEC)产生的HSC的发育过程非常重要。 HECs和pre-HSCs均显示pre-HSCs显示内皮表面表型并需要基质支持以检测其造血活性。利用基质细胞培养后再进行改良的半固体克隆培养的方法使我们能够检测B-1的菌落形成单位数量/ B-2祖细胞最初源自HEC / HSC之前的细胞,将反映B-1偏倚或多谱系繁殖的HSC的潜力。

[背景 ] 半固体克隆培养(甲基纤维素集落形成测定法)是检测造血祖细胞数量的传统方法。一个集落被认为是来自单个祖细胞(克隆来源),添加的细胞因子在细胞的形成中起着重要作用。产量菌落,例如Epo增强了红细胞的菌落形成单位(CFU-E)或红细胞的爆发形成单位(BFU-E),而G-CSF / GM-CSF将增强CFU-G(粒细胞),M (巨噬细胞)或GM(粒巨噬细胞).SCF刺激造血祖细胞活性。从小鼠BM形成B淋巴细胞祖细胞集落需要SCF,IL-7和Flt3-配体。重要的是,该测定法只能检测CD45的活性+ (或CD41 + (如果是胚胎来源的细胞))造血祖细胞,因此无法检测CD45 - CD144 + 造血内皮细胞(HEC)的造血活性,而在小鼠胚胎中会产生各种造血细胞。离子造血活动的HEC很大程度上取决于基质支持或器官培养方法,因此,它是具有挑战性的挪威貂的数量生产从医院伦理委员会淋巴祖。近日,Montecino -罗德里格斯等人(2016)d Eveloped一种改进的半固体使用S-17基质细胞进行克隆培养,以检测胚胎(E)第10天卵黄囊细胞和胎儿肝细胞中造血祖细胞的B-1和B-2潜力。

B-1祖细胞主要在胎儿肝脏和新生儿BM中检测到。B-1细胞属于先天样B淋巴细胞,可产生天然IgM抗体,而传统的适应性免疫B细胞称为B-2细胞。 -Rodriguez 。等人,鉴定B-1细胞特异性祖细胞在小鼠胎肝和新生儿BM为lin - IgM抗体- CD19 Tasu B220 - (CD19单正)细胞,而B-2特异性祖细胞(原B细胞)是否lin - IgM - CD19 - B220 + (B220单阳性)细胞。这两个群体在体外培养中很快变成CD19 + B220 + 双阳性,因此,很难确定在与基质细胞属于B-1和B-2细胞。这个修改半流体克隆培养,使我们能够检测B-1和/或B-2祖殖民地来源于医院伦理委员会/预HSC利用基质细胞支持体B Lymphopoies 是(Kobayashi et al。,2019 )。我们使用OP9基质细胞来支持B淋巴管造血 半固态克隆文化,这是我们一直成功的诱导B淋巴细胞的HEC (吉等,2011;小林等人,2019 )。由于B-2祖集落形成能力似乎是其中的一个HSC活动,该方法可用于评估小鼠胚胎中源自HEC / pre-HSC的HSC活性。

关键字:B细胞集落形成试验, OP-9, B-1和B-2淋巴样祖细胞, 小鼠胚胎, 造血干细胞前体, 造血内皮细胞, AGM 区

材料和试剂


 


1. T-25培养瓶(Corning ,目录号:353109 )。      


2. 6 - 孔培养板(Corning公司,Costar公司,目录号:3516 )      


3. 96 - 孔平文化板(Corning ,Costar公司,目录号:3595)      


4. Falco n 圆底聚丙烯14 ml管(Corning ,目录号:352059)      


5. 1.5 mL Eppendorf管      


6. 35毫米的培养皿中(Falcon ,目录号:351008)      


7. 15毫米的培养皿      


8. 3毫升注射器(BD ,目录号:309657)      


9. 5毫升注射器      


10. 18 G针(BD ,目录号:305185)   


11. 10 Myueru 吸头   


12. 瓶口过滤系统(0.22 MYU 中号孔径)   


13. C57BL / 6男女   


14. Arufa -MEM(粉末)(g ^ IBCO,目录号:11900-024)   


15. 胎牛血清   


注意:我们使用亚特兰大生物公司的FBS作为出版物,但是我们总是从5-10个测试FBS样品中进行批次检查,以找到最适合OP9维护文化的FBS。我们通常购买20瓶以保持2次相同的实验-3年。


16. 新鲜的Milli-Q水   


17. PBS(Fisher Scientific,目录号:BP665-1)   


18. 2-巯基乙醇(Fisher Scientific ,目录号:O3446I-100)   


19.500 mM EDTA pH 8.0   


20. IMDM(ģ IBCO ,目录号:12440061)   


21. 肝素(Sigma ,目录号:H3149-100KU)                 


22. 内皮促分裂原50的Mg (生物医学科技,产品目录编号:BT-203)                                                           


23. 青霉素-链霉素10,000 U-10,000 µg / ml(G ibco,目录号:15140122)   


24. X-VIVO 20(Lonza ,目录号:04-448Q)   


25. 甲基纤维素的基础培养基小鼠前B淋巴样祖细胞(    Stemcell Technologies ,Methocult TM ,目录号:M3630 )。


26. 重组鼠IL-7(Peprotech ,目录号:217-17)   


27. 重组鼠Flt3- 配体(Peprotech ,目录号:250-31L)   


28. 0.05%胰蛋白酶/ EDTA(Gibco公司,目录编号:25300054)   


29. 0.25 Pasento I型胶原酶(干细胞技术,目录号:07902)   


30. DNase I溶液(1mg / ml)(Stemcell Technologies ,目录号:07900)   


31. 细胞解离缓冲液(汉克平衡)(Gibco ,目录号:13150-016)   


32. 用于分类和检测B细胞集落的抗体(表1)   


 


表1.流式细胞仪的抗体清单


名称


克隆


色泽


公司介绍


货号


CD144(VE-Cad)


11D4.1


亚历克斯面粉


蓝光


562242


CD201(EPCR)


eBio1560


聚乙烯


电子生物科学


12-2012-82


CD45


30-F11


FITC


电子生物科学


11-0451-82


Ter119


Ter119


PerCP-Cy5.5


生物传奇


116228


CD11b(Mac1)


M1 / 70


ACP-Cy7


生物传奇


101226


CD93(AA4.1)


AA4.1


装甲运兵车


生物传奇


136510


CD19


1D3


聚乙烯


生物传奇


152408


B220


RA3-6B2


PE-Cy7


生物传奇


103222


 


33. OP9 基质细胞(从Shin-Ichi Nishikawa博士(从日本神户市理研退休)获得,可以从ATCC CRL-2749获得(理想情况下<30代)]   


34. 表达Akt的AGM-EC(AGM-EC):建立了从小鼠胚胎的主动脉-中肾(AGM)区域获得的内皮细胞(EC),并由Fredon Hutchinson Cancer的Brandon Hadland博士诱导了Akt的过表达中心,美国西雅图)(Hadland 等,2015 )。   


35. 猪皮明胶(Sigma,目录号:G1890)   


36. Arufa -MEM (见配方)   


37. 2-巯基乙醇(2-ME)存量(0.1 M)(请参阅食谱)   


38. OP9维护介质(α-MEM+ 20%FBS)(请参阅食谱)   


39. AGM-EC维护介质(请参阅食谱)   


40. 分化培养基(α-MEM+ 10%FBS + 5 x 10 -5 M 2ME)(请参阅食谱)   


41. 染色缓冲液(PBS + 5个%FBS + 2mM EDTA)中(见配方)   


42.0.1 %明胶(请参阅食谱)   


 


配套设备


 


P1000移液器
微量离心机
FACS分选仪和分析仪 需要超过6个荧光探测器。 BD FACS Aria用于细胞分选,BD LSRII和BD LSR- Fortessa 用于分析
标准细胞培养CO 2 培养箱(CO 2 :5%,O 2 :20%)
 


程序


 


维持OP9基质细胞和AGM-EC基质细胞
 


维持OP9基质细胞


维持OP9基质细胞处于良好状态是获得成功结果的重要关键。每月从粉末中制成新鲜的OP9维持培养基。测试许多FBS以使OP9细胞良好生长。
用新鲜制得的OP9培养基将T25烧瓶中的OP9解冻,每3天,1:3分开。
注意事项:


细胞OP9扁圆形或方形的形状(图1)我˚F他们成为纺锤形,他们不擅长OP9了,因为梭形细胞往往是改造和丢失的B细胞保障能力。
 


D:\重新格式化\ 2020-3-2 \ 1902983--1369吉本桃子802297 \图jpg \图1.jpg


1.形态学图OP9细胞(100的X )。红色箭头表示良好OP9和蓝色箭头表示为OP9。量表乙氩= 100 MYU 中号。


 


每3天将它们在T25烧瓶中融合的OP9分成3个烧瓶,如果3天内没有融合,则大量的FBS对它们不利。
OP9的密度必须合适(起始点约为4 x 10 5 / T25烧瓶)。当OP9细胞在T25烧瓶中融合时,细胞数约为1.0 x 10 6 -1.5 x 10 6 。
为了分裂,使用吸气培养基,用PBS洗涤2次,然后在37℃下用0.05%胰蛋白酶/ EDTA处理3分钟。 °C。将OP9细胞从烧瓶中剥离,并加入培养基和移液器,用5 ml血清移液器(重要)制成单细胞悬液。确保移液和单细胞良好,因为留下细胞团可能会诱发OP9的不良状况细胞在后来的通道。旋400 XG 为5 敏,吸取上清液,松开颗粒通过点击底部的管(重要),加1 毫升OP9维持培养基吸取非常好,P 1000吸管确保单细胞悬液。加入更多OP9培养基,将其放入3 x T25烧瓶中。
OP9必须在3天之内(通常每3天)进行维护。即使它们在3天内未汇合,也应通过(1比1或1比2以增加细胞密度)。 ,它们可以用于共培养,但不会再扩展。
如果OP9细胞状态不佳,则很难从胚胎的早期阶段获得B细胞(
OP9细胞不应维持超过1个月。
OP9细胞显示出良好的接触抑制作用,如果需要照射以阻止OP9的过度细胞生长,它们已经被转化,不再是优质的OP9。
 


维持AGM-EC基质细胞


涂0.1%明胶T25烧瓶30分钟以上。
在铺板细胞之前吸出明胶。
在AGM-EC维持培养基(5 x 10 5 / 瓶)中,用明胶包被的T25烧瓶解冻AGM-EC 。每3-4天以1-3:1的速度通过(Hadland 等,2015 )。
 


收获胚胎组织
建立定时交配的C57BL / 从,女6直男3后:00 .. PM以下一些早期上午,检查阴道塞的女性和独立堵塞的女性成不同的笼子中午这一天被认为是胚胎(E)0.5天。
在收获胚胎前约7天,将OP9和AGM-ECs融化在T25烧瓶中并保持。
收获胚胎前一天,将准备OP9(1 X 10 4 /孔)和AGM-ECS(5 X 10 3 / 96孔)- 。孔板分别另外,,在T25保维护OP9烧瓶中的根据需要在镀敷甲基纤维素5-7天后。
定时交配十天后(在E10.5),从怀孕的母亲那里收获胚胎(按照您所在机构批准的AICUC协议中的步骤进行操作)(Morgan 等人,2008年)。
在立体显微镜下,打开子宫,从胚胎中分离卵黄囊(YS)(Morgan et al。,2008 )。去除YS后,通过计数so体对来确认胚胎阶段,收集包含AGM区的一半胚胎的尾部。 14 毫升试管充满10毫升培养基。
的AGM组织离心机于培养基450 XG 5分钟,吸去上清液。挂起的AGM组织在0.25 Pasento I型胶原酶Tasu DNA酶I(最终20 Myug / ml)的分别[ 使用0.5-1毫升,每胚当量(的Ee )] 。
将组织悬浮液在37 °C下孵育。每5分钟用移液管吸取并在显微镜下确认解离。一旦看到良好的单细胞,可通过添加相同体积的细胞解离缓冲液停止胶原酶反应。正常情况下,此孵育需要15-30分钟,具体取决于胚胎的年龄(胚胎越早,孵化时间越短)。
添加分化培养基并使用70μm滤网过滤,并以450 xg离心5分钟。 
吸出克隆,将单细胞沉淀物悬浮在1 ml染色缓冲液中,并计数细胞数量,每只胚胎大约收集5 x 10 5个细胞。
 


HEC / HSC之前的分类和共培养
在检查HECs / HSC前的B淋巴造血潜能时,直接从这些细胞中检测B祖细胞集落形成能力具有挑战性,因为它们大多数是内皮表型。为了诱导造血祖细胞,请对HECs / pre进行分类-HSC并将它们接种在OP9(扩增的造血祖细胞)或AGM-EC上(让HSC之前的成熟成熟为HSC)。


 


染色和细胞分选


在1.5 ml Eppendorf管中的100μl染色缓冲液中悬浮多达1 x 10 7个细胞,首先取5%的体积用于阴性对照和FMO染色。
抗体添加;抗-小鼠CD45,CD144,C-KIT,EPCR(CD201)和TER119在的比率0.3 MYU L / 10 6 细胞,并培育他们30分钟冰上。   
加入1 ml染色缓冲液进行洗涤,然后在微型离心机上以800 xg 的速度离心2分钟。
将细胞在挂起染色缓冲液和排序TER119 - CD144 Tasu 的c-kit Tasu EPCR Tasu 细胞在FACS 咏叹调(图茜2)。
(可选)将分选的Ter119 - CD144 + c-kit + EPCR + 细胞与1 x 10 5个OP9细胞在Methocult中与IL-7和Flt3-配体混合,然后将其铺在35 mm培养皿中,但是直接将其铺板细胞将仅产生少数B细胞集落。
100-200 TER119板- CD144 Tasu 的c-kit Tasu EPCR Tasu 细胞以一个井96 - 孔板汇合随着OP9在分化培养基中或AGM-EC细胞在X-20体内和增IL-7和FLT3-配体(最终浓度:两者均为10 ng / ml)。
共培养五至七天后,收获所有细胞并用抗小鼠CD45和CD11b抗体对其染色。
染色后,将细胞用染色缓冲液洗涤,并悬浮在染色缓冲液中,并从FACSAria 上的共培养细胞中分选出CD11b - CD45 + 细胞,如图2B 所示。
 


D:\重新格式化\ 2020-3-2 \ 1902983--1369吉本桃子802297 \图jpg \图2.jpg


2.门控STRA图Tegy用于分拣预造血干细胞(A)和CD45 Tasu CD11b的- 细胞船尾铒共培养AGM-EC的(B)


 


电镀半固态培养
在室温或4° C 过夜融化Methocu lt 。
始终准备N + 1个样品用于甲基纤维素测定。与1 x 10 5个OP9 细胞/ 培养皿共培养后混合200-600个CD45 + 细胞;一式三份,混合800-2个,400个CD45 + 细胞和4个10 5个OP9细胞将其倒入14 ml的圆底管中并旋转,吸出培养基,并通过敲打管使细胞沉淀松散。使用5 ml注射器和18 G针将4 ml Methocult M3630添加到细胞沉淀中(这样,您可以铺200 -35个培养皿中每600个CD45 + 细胞具有1 x 10 5个OP9细胞。)在这种规模下,预计每个培养皿将有20-80个菌落。
加入IL-7(最终10 ng / ml)和Flit3-配体(最终10 ng / ml),涡旋混合均匀,直至所有气泡消失。
使用18 ml针和3 ml注射器,将1.1 ml含细胞的甲基纤维素培养基(含细胞)倒入每个35 mm培养皿x 3个皿中。
3个将P ETRI菜含有METHOCULT和1个培养皿含有无菌ħ 2 O(防止该干燥出的介质)在一个15毫米的培养皿。
他们在一个孵育5 Pasento CO 2 培养箱中培养8-11天数和计数集落数。该照片在图茜3代表B细胞集落及其FACS分析。
 


D:\重新格式化\ 2020-3-2 \ 1902983--1369吉本桃子802297 \图jpg \图3.jpg


拾取保持的菌落。3. B细胞图菌落和FACS分析阿。B-1祖细胞(B-1-pro)的菌落。乙。B-2祖细胞(B-2-pro)的集落,在确认通过FACS分析下面。量表乙氩= 200 MYU M.C。 。FACS分析的每个捡起菌落。基于所述门控BM B-2祖细胞,同门控被施加到分析每个样品的。


 


确定每个B-1或B-2祖细胞的菌落
在无菌的Eppendorf管中准备10μl 移液管和1 ml分化培养基,对于收集的菌落,准备1.5 ml含1 ml染色缓冲液的Eppendorf管,准备与菌落数相同的试管数。
收集菌落前,用移液器吸几次以分化培养基浸湿10μl 移液器吸头,以避免甲基纤维素粘在移液器吸头内部。设置8μl 吸取菌落。在显微镜下,用吸管吸取每个菌落用湿尖端吸取10μl移液器,并将每个菌落转移至含有染色缓冲液的1.5 ml管中。
将所有菌落收集到每个试管中后,将试管在微型离心机中以800 xg离心2分钟,然后吸出上清液。
加入50μl染色缓冲液并涡旋以使沉淀物成为单细胞悬浮液。
它们与抗体染色:抗小鼠AA4.1-APC,CD19-PE,B220-PEcy7和Mac1的-FITC在的比率0.3 MYU L / 10 6 细胞20-30分钟冰上。
用1 ml染色缓冲液洗涤,并以800 xg离心2分钟。
吸出悬浮液,并用PI或DAPI染色缓冲液悬浮细胞沉淀以排除死细胞,然后将样品应用于流式细胞仪。
确定菌落中是否含有B-1和/或B-2祖细胞(图3 )(Kobayashi 等人,2019 )准备成年BM细胞作为门控B祖细胞的阳性对照(Montecino-Rodriguez 等人, 2006年)。
 


 


 


资料分析


 


数据分析和预期结果


根据流式细胞仪分析,每个培养皿应包含1)仅B-1祖细胞,2)仅B-2祖细胞和3)B-1和B-2祖细胞。每板B-2或B-1和B-2祖细胞,并计算三个培养皿的结果的平均值和SD值。


通常,BM祖细胞仅预期B-2菌落.B-1祖细胞菌落是与OP9共培养的胚胎组织的显性,而B-1 + B-2祖细胞菌落在这些胚胎组织的共培养中则占优势。带有AGM-EC或培养的器官(发展为HSC)(Kobayashi 等,2019 )。


 


注意事项


 


AGM-EC可以支持pre-HSC过渡到HSC,但是当它们在甲基纤维素培养物中一起铺板时,它们不支持B细胞集落形成。
B细胞集落形成细胞的数量取决于胚胎的年龄和OP9的状况。
Ter119 - CD144 + c-kit + EPCR + 细胞非常罕见,因此,Ter119 - CD144 + c-kit + 也应该能够在OP9的支持下产生B细胞集落。
请勿在共培养或甲基纤维素培养中添加SCF。良好的OP9支持不含SCF的B淋巴细胞生成,SCF可能会增强巨噬细胞分化。
 


菜谱


 


培养基:


阿尔法-MEM(具有青霉素/链霉菌CI Ñ50 ü - 50 亩克/ 毫升)
将粉末α -MEM 添加到1,000 ml 新鲜的Milli-Q水中,搅拌直至完全溶解
加入2.2 g H 2 CO 3 并搅拌直至完全溶解
加入5 毫升青霉素-链霉素
使用0.22过滤MYU 中号过滤系统
2-巯基乙醇(2-ME)库存(0.1 M)
70混合Myueru 2- ME至10ml Arufa -MEM和过滤使用0.22 MYU 中号针筒过滤器


OP9维护介质(α-MEM+ 20%FBS)
400毫升混合的α -MEM和100ml FBS和过滤用0.22 微米中号过滤系统


AGM-EC维护介质
IMDM 400 毫升                                                       


FBS(Hyclone,热灭活)100毫升             


钢笔/链球菌(100 x )5 毫升                                         


肝素5毫升(在IMDM中新鲜制备的10毫克/毫升储备液)


L-谷氨酰胺(100 x )5毫升                                         


内皮细胞有丝分裂原50毫克                           


D 溶解在10毫升上述培养基混合物中
˚F ILTER使用0.22 MYU 中号过滤系统
分化培养基(α -MEM + 10%FBS + 5 x 10 -5 M 2 - ME)
450毫升α -MEM


50毫升FBS


250 Myueru 0.1 M 2 - ME


˚F ILTER使用0.22 MYU 中号过滤系统


染色缓冲液(PBS + 5%FBS + 2 mM EDTA)
500毫升PBS 500 毫升


2.5 ml FBS + EDTA(最终2 mM )


˚F ILTER使用0.22 MYU 中号过滤系统


0.1%明胶
0.5 克明胶


Milli-Q水500 毫升


高压釜


 


致谢


 


这项工作得到了NIAID R01AI121197的支持。该协议源自最近的一份报告(Kobayashi 等,2019 )。


 


竞争利益


 


没有利益冲突。


 


道德规范


 


步骤收获小鼠胚胎和其他相关动物作品沿用了动物福利委员会(AWC)Protoc 011批准通过该中心对实验动物医学和护理(CLAMC)在UTHealth (AWC16-0124,2016-2019) 。


 


参考文献


 


1. Hadland,BK,Varnum-Finney,B.,Poulos,MG,Moon,RT,Butler,JM,Rafii,S.和Bernstein,ID(2015)。内皮细胞和NOTCH明确并扩增了源自主动脉-性腺-间苯二酚。 。造血干细胞临床研究杂志125(5):二零三二年至2045年。                                             


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引用:Kobayashi, M. and Yoshimoto, M. (2020). A Modified Semisolid Clonal Culture for Identification of B-1 and B-2 Progenitor Colony Forming Ability of Mouse Embryonic Hemogenic Endothelial Cells. Bio-protocol 10(9): e3601. DOI: 10.21769/BioProtoc.3601.
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