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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Discussion

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.......

Materials

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