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Thrombopoietin-independent Megakaryocyte Differentiation of Hematopoietic Progenitor Cells from Patients with Myeloproliferative Neoplasms

来自骨髓增生性肿瘤患者的造血祖细胞的血小板生成素不依赖性巨核细胞分化

CT Chloe A. L. Thompson-Peach *
JF Johannes Foßelteder *
AR Andreas Reinisch
DT Daniel Thomas

 (*contributed equally to this work) 发布: 2023年01月20日第13卷第2期 DOI: 10.21769/BioProtoc.4592 浏览次数: 1047

评审: Salma MerchantAnonymous reviewer(s)

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Abstract

Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient–derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders.


Graphical abstract



Schematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation

Keywords: Hematopoietic stem and progenitor cells (造血干细胞和祖细胞) search Megakaryocyte differentiation (巨核细胞分化) search Thrombopoietin (血小板生成素) search TPO-independent (不依赖TPO) search Myeloproliferative neoplasm (骨髓增生性肿瘤) search Collagen (胶原蛋白) search Interleukin-6 (白细胞介素-6) search Interleukin-9 (白细胞介素-9) search

Background

Multipotent hematopoietic stem and progenitor cells (HSPCs) are not only responsible for life-long hematopoiesis but are also found to be the origin of many hematological malignancies (Bonnet and Dick, 1997; Jamieson et al., 2006; Woll et al., 2014; Reinisch et al., 2016). Myeloproliferative neoplasms (MPNs) are hematopoietic stem cell–derived diseases that are characterized by an aberrant proliferation of myeloid cells through constitutive activation of cytokine-signaling pathways (Tefferi and Pardanani, 2015; Spivak, 2017). In essential thrombocythemia (ET) and myelofibrosis (PMF), two common subtypes of MPNs, megakaryocyte lineage-biased differentiation and proliferation are hallmarks of disease pathogenesis (Vannucchi et al., 2013; Tefferi and Pardanani, 2019). Historically, thrombopoietin (TPO) was found to be a crucial growth factor in nearly all stages of megakaryocyte development with a profound effect on the survival, proliferation, and differentiation of committed HSPCs to the development of mature megakaryocytes (Debili et al., 1995; Choi et al., 1995; Broudy et al., 1995; Chen et al., 1995; Kojima et al., 1995). Thus, it is not surprising that the oncogenic drivers of MPNs were found to be activating mutations occurring in genes along the TPO signaling axis (MPL and JAK2), rendering cells TPO-independent and inducing increased megakaryopoiesis (Kralovics et al., 2005; Pikman et al., 2006; Woods et al., 2019). Additionally, mutations in the endoplasmic reticulum chaperone CALR, which has not been implicated in megakaryopoiesis to date, introduced structural changes, resulting in protein multimerization and binding to the TPO receptor (MPL). This binding results in an activation of downstream signaling in absence of TPO (Klampfl et al., 2013; Chachoua et al., 2016; Marty et al., 2016; Araki et al., 2016).


Despite recent advances in unveiling the oncogenic transformation of healthy HSPCs, detailed mechanisms of disease pathogenesis are still poorly understood. Current methods to investigate megakaryocyte-biased cellular transformation by MPN mutations either use TPO-dependent cell lines or costly HSPC differentiation assays that are not tailored to investigate TPO-independent cell growth (Lu et al., 2005; Araki et al., 2016; Elf et al., 2016; Pronier et al., 2018). Primary progenitor cells from patients with polycythemia vera, another MPN disease subtype, often form erythroid colonies in the absence of erythropoietin (Corre-Buscail et al., 2005), but assays to show TPO-independent growth or TPO-hypersensitive growth of ET- and PMF-derived cells are not well established. Current protocols for in vitro megakaryocyte differentiation focus on optimizing culture conditions to maximize the megakaryocyte output of healthy HSPCs, but do not consider MPN-related phenotypes. Here, we describe a reproducible and cost-effective step-by-step protocol for the isolation, culture, and TPO-independent megakaryocyte differentiation of primary MPN patient–derived CD34+ HSPCs to investigate disease-relevant characteristics. This approach uses fluorescence-activated cell sorting (FACS) to purify CD34+ HSPCs from the peripheral blood of MPN patients and an animal serum-free culture system for megakaryocyte differentiation in a clonogenic semi-solid media and collagen-based colony assay, as well as in liquid culture. This protocol facilitates future MPN research, providing an opportunity to test novel therapeutic interventions in a human pre-clinical setting (Tvorogov et al., 2022), but can be easily adapted to investigate other megakaryocyte-related diseases.

Materials and Reagents

  1. 50 mL conical tubes (Corning, FalconTM, catalog number: 352098)

  2. 1.8 mL cryogenic tubes (Thermo Fisher Scientific, NuncTM, catalog number: 368632)

  3. 5 mL round bottom tubes (Corning, FalconTM, catalog number: 352054)

  4. 5 mL round bottom tubes with cell strainer cap (Corning, FalconTM, catalog number: 352235)

  5. 1.5 mL screw cap micro tube (Sarstedt, catalog number: 72692005)

  6. 1.5 mL safe-lock tubes (Eppendorf, catalog number: 0030123328)

  7. Double chamber slides (Thermo Fisher Scientific, NuncTM, Lab-TekTM, catalog number: 177429)

  8. 100 mm culture dish (Corning, catalog number: 430591)

  9. 35 mm culture dish (Greiner Bio-One, catalog number: 627161)

  10. 48-well plate (tissue-culture treated) (Corning, CostarTM, catalog number: 3548)

  11. 3.5 mL transfer pipettes (Sarstedt, catalog number: 86.1171.001)

  12. Parafilm (Bemis, catalog number: PM996)

  13. Filter cards and spacers (Stemcell technologies, catalog number: 04911)

  14. Phosphate buffered saline (PBS) (Gibco, catalog number: 10010015)

  15. Lymphoprep (Stemcell technologies, catalog number: 07801)

  16. RPMI 1640 (Sigma-Aldrich, catalog number: R8758)

  17. IMDM (Pan-Biotech, catalog number: P04-20450)

  18. Fetal bovine serum (FBS) (Pan-Biotech, catalog number: P30-19475)

  19. Penicillin/streptomycin (Pan-Biotech, catalog number: P06-07100)

  20. Dimethyl sulfoxide (DMSO) (WAK-Chemie, catalog number: WAK-DMSO-10)

  21. DNase (Worthington, catalog number: LS002007)

  22. MegacultTM-C collagen and medium with lipids, without cytokines (Stemcell technologies, catalog number: 04974)

  23. Methanol (Merck, EMSURE®, catalog number: 106009)

  24. Acetone (Sigma-Aldrich, catalog number: 320110)

  25. Hydrochloric acid solution 6 M (HCl) (Merck, catalog number: 143007)

  26. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888)

  27. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A9434)

  28. Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: 237205)

  29. EDTA tetrasodium (Sigma-Aldrich, catalog number: 03699)

  30. TRIS (Roth, PUFFERAN®, catalog number: 5429.1)

  31. Tween 20 (Sigma-Aldrich, catalog number: P1379)

  32. Evans Blue (Sigma-Aldrich, catalog number: E2129)

  33. Serum-free expansion media II (SFEM II) (Stemcell technologies, catalog number: 09655) or StemPro 34 SFM + nutrient supplement (Thermo Fisher Scientific, catalog number: 10639011)

  34. L-Glutamine (200 mM) (Sigma-Aldrich, catalog number: 59202C)

  35. Recombinant human stem cell factor (SCF) (PeproTech, catalog number: 300-07

  36. Recombinant human thrombopoietin (TPO) (PeproTech, catalog number: 300-18)

  37. Recombinant human interleukin 3 (IL-3) (PeproTech, catalog number: 200-03)

  38. Recombinant human interleukin 6 (IL-6) (PeproTech, catalog number: 200-06)

  39. Recombinant human interleukin 9 (IL-9) (PeproTech, catalog number: 200-09)

  40. Lipids cholesterol rich (Sigma-Aldrich, catalog number: L4646)

  41. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906)

  42. EDTA (Thermo Fisher Scientific, catalog number: AM9260G, 0.5M solution)

  43. FcR blocking reagent human (Miltenyi, catalog number: 130-059-901)

  44. Mouse IgG1, κ isotype control (BioLegend, catalog number: 400101)

  45. Mouse anti-human CD41 (BioLegend, catalog number: 303702)

  46. ImmPRESS®-AP horse anti-mouse IgG polymer detection kit (Vector Laboratories, catalog number: MP-5402)

  47. Alkaline phosphatase (AP) substrate kit, Vector® Red (Vector Laboratories, catalog number: SK-5100)

  48. Mouse anti-human CD34 FITC (BD Biosciences, PharmingenTM, catalog number: 555821)

  49. Mouse anti-human CD45 V500-C (BD Biosciences, HorizonTM, catalog number: 655873)

  50. Mouse anti-human CD2 PE-Cy5 (BD Biosciences, PharmingenTM, catalog number: 555328)

  51. Mouse anti-human CD3 PE-Cy5 (BD Biosciences, PharmingenTM, catalog number: 555334)

  52. Mouse anti-human CD19 PE-Cy5 (BD Biosciences, PharmingenTM, catalog number: 555414)

  53. Mouse anti-human CD14 PerCP-Cy5.5 (BD Biosciences, PharmingenTM, catalog number: 562692)

  54. Mouse anti-human CD16 PE-Cy5 (BD Biosciences, PharmingenTM, catalog number: 555408)

  55. Mouse anti-human CD56 PE-Cy5 (BD Biosciences, PharmingenTM, catalog number: 555517)

  56. Mouse anti-human CD41 APC-Cy7 (BioLegend, catalog number: 303716)

  57. Mouse anti-human CD42b APC (BioLegend, catalog number: 303912)

  58. 7-Amino-Actinomycin D (7-AAD) (BD Biosciences, PharmingenTM, catalog number: 559925)

  59. Counting beads (Thermo Fisher Scientific, CountbrightTM, catalog number: C36950, LOT-specific concentration: 0.515 × 105 beads/50 µL)

  60. RBC lysis buffer (see Recipes)

  61. Freezing media (see Recipes)

  62. Thawing media (see Recipes)

  63. Staining buffer (SB) (see Recipes)

  64. Antibody cocktail (see Recipes)

  65. Megakaryocyte (Mk) colony media (see Recipes)

  66. Megakaryocyte differentiation (Mk-diff) media (see Recipes)

  67. Isotonic NaCl solution (0.15 M) (see Recipes)

  68. TRIS buffer (0.5 M, pH 7.6) (see Recipes)

  69. Wash buffer (see Recipes)

  70. Substrate buffer (see Recipes)

  71. Evans Blue solution (0.1%) (see Recipes)

  72. Staining tray (see Recipes)

Equipment

  1. Swing-bucket centrifuge (Eppendorf, model: 5810R)

  2. Tabletop centrifuge (Eppendorf, model: 5427R)

  3. Inverted light microscope (Zeiss, model: Primovert)

  4. Upright light microscope (Olympus, model: BX51)

  5. Fluorescence-activated cell sorter (BD Biosciences, model: Aria IIIu)

  6. Flow cytometer (Beckman Coulter, model: Cytoflex S or BD Biosciences FACS CantoII)

  7. Laminar flow workbench

  8. Fume hood

  9. Forceps

  10. Scalpel

Software

  1. FlowJo v10 (FlowJo LLC)

  2. Prism 9 (GraphPad)

Procedure

English
中文翻译

文章信息

版权信息

© 2023 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Thompson-Peach, C. A. L., Foßelteder, J., Reinisch, A. and Thomas, D. (2023). Thrombopoietin-independent Megakaryocyte Differentiation of Hematopoietic Progenitor Cells from Patients with Myeloproliferative Neoplasms. Bio-protocol 13(2): e4592. DOI: 10.21769/BioProtoc.4592.

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分类

干细胞 > 成体干细胞 > 造血干细胞

癌症生物学 > 癌症干细胞 > 细胞生物学试验

细胞生物学 > 细胞分离和培养 > 细胞分离

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