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 undergo two major stages of maturation: endomitosis and the development of demarcation membranes (DMS). This fully mature MK then appears close to a sinusoid vessel in which it can emit cytoplasmic extensions, the proplatelets, which will be released under the blood flow and subsequently remodeled into functional platelets². The cloning of TPO in 1994³ provided a boost in the study of MKP by accelerating the development of in vitro culture techniques allowing HP differentiation and MK maturation.

There are many pathologies affecting blood platelets, both in terms of platelet number (increase or decrease) and function^4,5. Being able to recapitulate MKP in vitro from human HP could improve understanding of the molecular and cellular mechanisms underlying this process and ultimately the therapeutic management of patients.

Various sources of human HP are suitable: cord blood, bone marrow, and peripheral blood^6,7,8. Harvesting HP from peripheral blood raises less logistical and ethical problems than their recovery from cord blood or the bone marrow. HP can be recovered from leukapheresis or buffy coat, but these sources are expensive and not always available in blood centers. Other protocols, less expensive and easier to perform, allow direct recovery of human peripheral blood mononuclear cells (PBMCs) without the need for prior CD34 driven isolation^4,8. However, the purity of megakaryocytes is not satisfactory with this method and a selection of CD34+ cells from PBMC is recommended for optimal differentiation into MK. This led us to implement a HP purification from leukoreduction filters (LRF), routinely used in blood banks to remove white blood cells and thus avoid adverse immunological reactions⁹. Indeed, since 1998, platelet concentrates have been automatically leukodepleted in France. At the end of this process, LRF are discarded and all the cells retained in the LRF are destroyed. Cells in LRFs are, therefore, readily available at no additional cost. LRFs have a cellular content close to that obtained by leukapheresis or in buffy coats, notably in their composition of CD34+ HP making them a remarkably attractive source¹⁰. LRF as a human HP source has already been demonstrated to provide cells with intact functional capacities¹¹. This source has the advantage of being abundant and affordable for laboratory research. In this context, this article describes successively: i) the extraction and selection of CD34⁺ HP from LRFs; ii) a two-phase optimized culture, which recapitulates the commitment of HP into the megakaryocytic pathway and the maturation of MK capable of emitting proplatelets; iii) a method for efficiently releasing platelets from these MK; and iv) a procedure for phenotyping MK and cultured platelets.

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 performed according to Helsinki declaration.

1. Extraction and selection of CD34+ cells (HP) from LRF

1. Reagent's preparation (for one LRF)
   1. Prepare 25 mL of filtered elution buffer: 21.25 mL of Phosphate Buffered Saline (PBS), 2.5 mL of Acid-Citrate-Dextrose (ACD) and 1.25 mL of decomplemented Fetal Bovine Serum (FBS). Filter on 0.22 µm and place at 37 °C.
   2. Prepare 500 mL of PBS with 2 mM of ethylenediaminetetraacetic-acid (EDTA).
   3. Dispose 25 mL of density gradient medium (DGM) (1.077 g/mL) in two 50 mL tubes.
2. LRF back-flushing modalities (Figure 1)
   NOTE: This step requires a sterile tube welding machine, allowing sterile connection of thermoplastic tubing.
   1. First, connect the LRF to an empty 600 mL transfer bag and the LRF to a tubing set (Figure 1A). Under the biosafety cabinet, inject the total volume of filtered and prepared elution buffer corresponding to the number of LRFs handled (xLRF x 25 mL) into the empty bag. Then, using a 30 mL syringe to gently aspirate the entire contents of the bag through the LRF, backflush and transfer the cells into a new 50 mL tube (Figure 1B).
   2. To the red blood cells sedimentation, dilute the cell suspension by half with Dextran 2% and mix well to aggregate red blood cells. Wait for 30 min at room temperature (RT).

Figure 1: LRF back flushing modalities. (A) Representative scheme of (i) the sterile connection of the transfer bag to the LRF and (ii) the tubing set to the LRF. (B) Representative scheme of the connection of the syringes for cell collection. Please click here to view a larger version of this figure.

3. PBMC collection
   1. Following the red blood cells sedimentation, remove and transfer the supernatant into a 50 mL tube and fill it with PBS-EDTA 2 mM. Gently overlay the supernatant onto the above-prepared DGM. Let the supernatant flow gently without breaking the surface plane of the density gradient. Centrifuge at 400 x g at RT for 30 min in brake off mode.
   2. Collect the PBMC layer with a disposable transfer pipette. Transfer the cells from each DGM tube into a new sterile 50 mL tube. Fill each tube with PBS-EDTA 2 mM and wash twice in 50 mL of PBS-EDTA 2 mM at 200 x g for 10 min at RT in brake on mode.
   3. Pipette off and pool the cell pellet with 50 mL PBS-EDTA 2 mM.
      NOTE: There is a possibility to stop the procedure by maintaining the collected cells under agitation at 4 °C during the night. Then, filter the suspension with a 40 µm cell strainer to remove the aggregates formed.
4. CD34+ cells selection
   1. Determine the cell number and centrifuge at 400 x g at room temperature for 10 min with the break on.
   2. Aspirate the supernatant completely and resuspend in the appropriate volume of PBS-EDTA 2 mM detailed by the manufacturer of the CD34 selection kit (300 µL of PBS-EDTA for 10⁸ cells). Add the FcR blocking Reagent and the CD34 Microbeads in the appropriate concentration (50 µL for 10⁸ cells).
   3. After 30 min at 4 °C, wash the cell suspension and resuspend in the appropriate volume of PBS-EDTA 2 mM detailed by the manufacturer of the CD34 selection kit (500 µL per 10⁸ cells).
      ​NOTE: The selection is made on sorting columns for the passage of maximum 2 x 10⁹ cells per column.
   4. Pass the sample over the wet column of the magnet. Wash twice with 3 mL of PBS-EDTA 2 mM and elute the cells with 5 mL of PBS-EDTA 2 mM. A second run on a new column following the same procedure is necessary to improve the purity of the sample.
      NOTE: An expected number of 6.1 x 10⁵ cells/LRF (Figure 2A). For higher LRF numbers, scale up reagents and methods accordingly.
5. Evaluation of CD34+ cell purity
   1. Add to an aliquot of 100 µL of suspension obtained after the CD34⁺ selection, 2 µL of human CD34-PE antibody or 2 µL of IgG - PE (control). Mix well and incubate for 15 min at 4 °C.
   2. Wash cells by adding 2 mL of PBS and centrifuge at 400 x g for 5 min. Aspirate supernatant completely and resuspend in 200 µL of PBS.
   3. Analyze the purity by flow cytometry as shown in Figure 2A and in the Discussion.
      ​NOTE: A purity of CD34+ cells above 90% is expected (Figure 2Bii).
   4. Use the CD34+ cells directly or freeze for further use.
6. CD34+ cells freezing
   NOTE: CD34+ cells freezing is done at a density of 10⁶ cells per mL.
   1. Following the CD34+ cell number determination, prepare the following cryopreservation media: (1) 60% Stemspan + 40% FBS, (2) 40% Stemspan + 40% FBS + 20% Dimethyl Sulfoxide (DMSO) and allow cooling at 4 °C.
   2. Centrifuge the CD34+ cells at 400 x g at room temperature for 5 min and resuspend the pellet in cold solution 1 and then immediately add to the cold solution 2 (v/v).
   3. Place the cryotubes immediately into a -80 °C freezer for 24 h and then transfer cryotubes into the liquid nitrogen tank.

2. Culture and differentiation of CD34+ cells to produce mature proplatelet-bearing megakaryocytes

NOTE: Cell culture protocol (Figure 3A), representative scheme of the cell culture procedure are detailed in this section.

1. CD34+ cells thawing (if required)
   1. Prepare the thawing solution: 13 mL of PBS-20% FBS and place at 37 °C for 15 min. Quickly transfer the cryotubes to a 37 °C water bath until only one small ice crystal. Under the biological culture cabinet, pipette the whole content, and slowly transfer in 13 mL of prewarmed thawing solution.
   2. Determine the cell number and cell viability.
2. Culture protocol, step 1: from day 0 to day 7
   NOTE: Usually cultures are made in 24-well plates with 1 mL of medium per well at a density of 40,000 viable cells/mL, corresponding to 20,000 viable cells/cm². It is crucial to respect this density if any scale up is planned.
   1. Growth medium preparation : In serum-free hematopoietic cell expansion media (previously heated to 37 °C) add Penicillin-Streptomycin-Glutamine (PSG) 1x, human Low-Density Lipoprotein (hLDL) at 20 µg/mL, cytokine cocktail of megakaryocyte expansion 1x and Stemregenin 1 (SR1) at 1 µM.
   2. Cell seeding : Centrifuge the thawed CD34+ cells at 400 x g at room temperature for 5 min. Thoroughly remove the supernatant and resuspend the cell pellet in 1 mL of culture media and performa cell numeration and viability to determine the appropriate volume to seed the cells.
   3. Centrifuge the cells 400 x g at room temperature for 5 min and resuspend the pellet in the appropriate volume of the warm growth medium. Incubate the cells at 37 °C with 5% CO₂ for 7 days.
3. Culture protocol, step 2: from day 7 to day 13 (Figure 3B, representative images at day 13)
   NOTE: Usually cultures are made in 24-well plates with 1 mL of medium per well at a density of 50,000 viable cells/mL, corresponding to 25,000 viable cells/cm². It is crucial to respect this density if any scale up is planned.
   1. Maturation medium preparation: In serum-free hematopoietic cell expansion media (previously heated to 37 °C) add PSG 1x, hLDL at 20 µg/mL, TPO at 50 ng/mL, and SR1 at 1 µM.
   2. Examine the cells under the microscope. At day 7, cells display a round and homogeneous appearance by filling the wells or the flasks without being too confluent.
   3. Under a biosafety cabinet, gently transfer the cells in a 15 mL tube. Wash wells with PBS. Then, determine the number of cells and their viability to calculate the appropriate volume to seed the cells.
   4. Centrifuge the cells at 400 x g at room temperature for 5 min. Remove the supernatant and resuspend the cells in the appropriate volume of warm media calculated in the previous step. Incubate the cells at 37 °C, 5% CO₂ for 6 days.
4. Cultured platelet release at day 13
   1. Add 0.5 µM of prostaglandine I₂ (PGI₂₎ and 0.02 U/mL of apyrase to the culture and perform successive pipetting five times with a 1 mL pipette.
      ​NOTE: Platelets are now released into the medium.

3. Flow cytometry analysis (MK phenotyping and cultured platelets count)

NOTE: This protocol can be applied to the phenotyping of the cells on the selected culture days. It also allows the determination of the number of the cultured platelet release (Figure 4A,B).

1. Preparation for MK analysis
   1. Label four sets of microcentrifuge tubes as follows: Unlabeled cells as control, cells + 5 µL CD41 - Alexa Fluor 488, Cells + 5 µL CD34 - PECy7, Cells + 5 µL CD41 - Alexa Fluor 488 + 5 µL CD34 - PECy7. Use a minimum of 1.10⁵ cells per tube, do not exceed 1.10⁶ cells per tube. Add to 100 µL of cell suspension per cytometry tube and the different antibodies. Incubate in the dark for 30 min at 4 °C.
   2. Then, add 2 mL of PBS-EDTA 2 mM per tube and centrifuge at 400 x g for 5 min at room temperature. During centrifugation, prepare a solution of PBS-EDTA 2 mM + 7-Aminoactinomycin-D (7AAD) (1/100), allow for 300 µL solution per tube.
   3. Remove the supernatant and take up the pellet in 300 µL of PBS-EDTA 2 mM with 7AAD. Run samples through the flow cytometer within 30 min.
      NOTE: Analysis strategy for flow cytometry is shown in the Figure 3C and in the Discussion.
2. Tube preparation for cultured platelet analysis
   1. Label four sets of microcentrifuge tubes as follows: Unlabeled cells as control, Cells + 5 µL CD41 - Alexa Fluor 488, Cells + 20 µL CD42a - PE, Cells + 5 µL CD41 - Alexa Fluor 488 + 20 µL CD42a - PE. Following five successive pipetting in culture well, transfer 300 µL of the suspension in tube for cytometry containing a calibrated number of fluorescent beads.
   2. Add antibodies and incubate in the dark at RT for 30 min.
   3. Run the samples through the flow cytometer within 30 min and set the acquisition for the passage of 5,000 beads.
      NOTE: Analysis strategy for flow cytometry is shown in the Figure 4B and in the Discussion.

Results

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...

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