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Biology

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation

Published: May 20th, 2021

DOI:

10.3791/62499

1BPPS UMR-S 1255, FMTS, Université de Strasbourg, INSERM, EFS Grand Est

This protocol describes in detail all the steps involved in obtaining leukofilter-derived CD34+ hematopoietic progenitors and their in vitro differentiation and maturation into proplatelet-bearing megakaryocytes that are able to release platelets in the culture medium. This procedure is useful for in-depth analysis of cellular and molecular mechanisms controlling megakaryopoiesis.

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing platelets allows an in-depth study of the mechanisms underlying platelet biogenesis. Available culture protocols are mostly based on hematopoietic progenitors derived from bone marrow or cord blood raising a number of ethical, technical, and economic concerns. If there are already available protocols for obtaining CD34 cells from peripheral blood, this manuscript proposes a straightforward and optimized protocol for obtaining CD34+ cells from leukodepletion filters readily available in blood centers. These cells are isolated from leukodepletion filters used in the preparation of blood transfusion products, corresponding to eight blood donations. These filters are meant to be discarded. A detailed procedure to collect hematopoietic progenitors identified as CD34+ cells from these filters is described. The method to obtain mature megakaryocytes extending proplatelets while discussing their phenotypic evolution is also detailed. Finally, the protocol present a calibrated pipetting method, to efficiently release platelets that are morphologically and functionally similar to native ones. This protocol can serve as a basis for evaluating pharmacological compounds acting at various steps of the process to dissect the underlying mechanisms and approach the in vivo platelet yields.

Blood platelets come from specialized large polyploid cells, the megakaryocytes (MK), that originate from a constant and fine-tuned production process known as megakaryopoiesis (MKP). At the apex of this process are hematopoietic stem cells which, in contact with the bone marrow environment (cytokines, transcription factors, hematopoietic niche), will be able to proliferate and differentiate into hematopoietic progenitors (HP) able to commit toward the megakaryocytic pathway, giving rise to immature MKs1. Under the influence of various cytokines, and in particular thrombopoietin (TPO), which is the major cytokine of MKP; the MK will then underg....

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Control human samples were obtained from volonteer blood donors who gave written informed consent recruited by the blood transfusion center where the research was performed (Etablissement Français du Sang-Grand Est). All procedures were registered and approved by the French Ministry of Higher Education and Research and registered under the number AC_2015_2371.The donors gave their approval in the CODHECO number AC- 2008 - 562 consent form, in order for the samples to be used for research purposes. Human studies were.......

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Extraction and selection of CD34+ cells from LRFs
Here, the method, derived from Peytour et al.9, describes the extraction and selection of CD34+ cells from discarded LRFs available in blood banks after leukocyte removal. Following the backflush procedure, usually 1.03 x 109 ± 2.45 x 108 cells/LRF (Mean±SEM; n = 155) are recovered with a viability of 94.88 ± 0.10% (Figure 2A i.......

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This protocol describes a method for producing MK capable of emitting proplatelets from blood-derived HP and to release platelets from the culture medium. HP are obtained from LRF, a by-product of the blood banks, used to remove contaminating leukocytes from cellular blood products and avoid adverse reactions. Although this method is relatively simple, a few points deserve special attention.

Deposition of the cell suspension on the density gradient medium (step 1.3.1) has to be performed gentl.......

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This work has been supported by ANR (Agence National de la Recherche) Grant ANR- 17-CE14-0001-1.

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Name Company Catalog Number Comments
7-AAD Biolegend 558819
ACD EFS-Alsace NA
Anti-CD34-PE  Miltenyi biotec 130-081-002
Anti-CD34-PECy7 eBioscience 25-0349-42
Anti-CD41-Alexa Fluor 488 Biolegend 303724
Anti-CD42a-PE BD Bioscience 559919
Apyrase EFS-Alsace NA
BD Trucount Tubes BD Bioscience 340334
CD34 MicroBead Kit UltraPure, human  Miltenyi biotec 130-100-453
Centrifuge Heraeus Megafuge 1.OR Or equivalent material
Compteur ADAM  DiagitalBio NA Or equivalent material
Cryotubes Dutscher 55002 Or equivalent material
Dextran from leuconostoc spp  Sigma 31392-50g Or equivalent material
DMSO Hybri-max  Sigma D2650
EDTA 0.5 M  Gibco 15575-039
Eppendorf 1,5 mL  Dutscher 616201 Or equivalent material
Filtration unit Steriflip PVDF Merck Millipore Ltd SE1M179M6
Flow Cytometer BD Bioscience Fortessa
Human LDL Stemcell technologies #02698
ILOMEDINE 0,1 mg/1 mL Bayer MA038EX
Inserts Fenwal R4R1401 Or equivalent material
Laminar flow hood  Holten NA Archived product
LS Columms  Miltenyi Biotec 130-042-401 
Lymphoprep Stemcell 7861
Pen Strep Glutamine (100x) Gibco 10378-016
PBS (-) Life Technologies 14190-169  Or equivalent material
PGi2 Sigma P6188
Poches de transferts 600ml  Macopharma VSE4001XA
Pre-Separation Filters (30µm) Miltenyi Biotec 130-041-407
StemRegenin 1 (SR1) Stemcell technologies #72344
StemSpan Expansion Supplement (100x) Stemcell technologies #02696
StemSpan-SFEM  Stemcell technologies #09650
Stericup Durapore 0,22µm PVDF Merck Millipore Ltd SCGVU05RE
SVF Hyclone  Thermos scientific SH3007103
Syringues 30 mL  Terumo SS*30ESE1 Or equivalent material
Syringe filters Millex 0,22µM PVDF Merck Millipore Ltd SLGV033RB
TPO Stemcell technologies #02822
Tubes 50 mL Sarstedt 62.548.004 PP Or equivalent material
Tubes 15 mL  Sarstedt 62.554.001 PP Or equivalent material
Tubulures B Braun 4055137 Or equivalent material

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