Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Summary

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34⁺ cells. A method for CD34⁺ cell isolation and megakaryocyte differentiation is described here.

Abstract

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34⁺ cells under suitable conditions. The protocol detailed here describes isolation of CD34⁺ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Introduction

Isolation of adequate numbers of primary human megakaryocytes (MK) for regular laboratory use is not feasible due to their low frequency in the bone marrow, where they account for ~0.01% of nucleated cells¹. A convenient alternative is the ex vivo expansion and differentiation of hematopoietic stem and progenitor cells in the presence of specific growth factors. A number of cytokines including stem cell factor (SCF; c-kit ligand) and interleukin (IL)-3 and IL-11 have been employed in culture systems to produce MKs. Thrombopoietin (TPO) is the most effective growth and differentiation factor for megakaryocytic cultures and is effective alone or with other cytokines, such as SCF and IL-3². TPO can act on stem cell populations to result in both the proliferation and maturation of MKs².

MK produce platelets from cytoplasmic protrusions called proplatelets and, in vivo, approximately 1 x 10¹¹ platelets are formed daily to sustain platelet counts of 150 - 400 x 10⁹/L. Platelet production in vitro is up to 1000-fold lower than the in vivo estimates³, and this has given rise to numerous culture conditions using CD34⁺ hematopoietic progenitor cells to improve MK and platelet production in vitro. The initial source of CD34⁺ cells used for MK differentiation was human peripheral blood⁴. Other cell sources include bone marrow^5,6, embryonic stem cells/induced pluripotent stem cells (ESC/iPSC)⁷, and umbilical cord blood (UCB)^8,9,10. Human bone marrow CD34^{+ 11} and mouse lineage negative bone marrow cells⁵ produce MK and platelets in vitro; nevertheless, the lack of availability of human bone marrow limits its use as a source of CD34⁺ cells. In contrast, ESC and iPSC represent an unlimited source of cells for in vitro platelet production. Platelet production from these cells requires feeder cells such as murine OP9 cells and longer culture periods. Platelets derived in feeder-free conditions appear to be less functional¹². iPSC-derived platelets are likely to be of use in clinical settings since they can be expanded to a large scale. This process requires lentiviral-mediated transduction of transcription factors and long-term cell culture¹³.

UCB is an accessible source of CD34⁺ cells that can be readily used in research settings. TPO alone can promote differentiation of cord blood-derived CD34⁺ cells and this gives rise to highly pure, mature MKs without the need for serum supplementation or co-culture with feeder cells. Other cytokines such as SCF may decrease differentiation from UCB CD34⁺ cells, while Flt-3 ligand and IL-11 promote the production of immature megakaryocytes¹⁴. This protocol describes the production of highly pure MK cultures from cord blood CD34⁺ cells in serum-free conditions.

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Protocol

This protocol was approved by the South Eastern Sydney Human Research Ethics Committee and ratified by the University of New South Wales' Human Research Ethics Committee. Umbilical cord blood obtained from healthy donors was provided by the Sydney Cord Blood Bank (Sydney, NSW, Australia). Volumes of approximately 100 mL were used for this procedure.

NOTE: Work in a Class II biosafety cabinet using aseptic technique. Decontaminate the exterior of the cord blood bag with 70% ethanol. Use sterile instruments (scissors, tweezers) for this procedure.

1. Cord Blood Cell Preparation and Isolation of CD34⁺ Cells

1. Prepare sterile separation buffer (SB) with phosphate buffered saline (PBS), at pH 7.2, and containing 0.5% bovine serum albumin and 2 mM EDTA.
2. Dispense 10 mL of SB into a 50 mL conical tube (one tube per 10 mL of blood is required). Using a G18 blunt needle mounted on a 10 mL syringe, draw 10 mL of blood from the bag and dispense into the 50 mL tubes containing 10 mL of SB.
3. Add 15 mL of lymphocyte separation media (see Table of Materials) to the bottom of the tube containing the diluted blood, creating two layers.
   NOTE: Dispense media slowly at the bottom of the tube to avoid mixing the different layers.
4. Centrifuge tubes at 1,200 × g for 30 min at room temperature (RT) with no break and no acceleration.
5. Transfer the layer near the center of the tube containing mononuclear cells (approximately 5 - 10 mL from each tube) into a new 50 mL tube using a Pasteur pipette. Add SB to each tube to a total volume of 50 mL.
6. Centrifuge at 400 × g for 10 min at RT. Discard the supernatant.
7. Resuspend cell pellets carefully with a Pasteur pipette in 5 - 10 mL of SB. Combine the suspended cells and bring volume to 50 mL with SB.
8. Count cells using Trypan blue staining and a hemocytometer or automated cell counter (see Table of Materials). To determine the percentage of CD34⁺ cells in the sample, resuspend 2.5 × 10⁵ cells in 100 µL of SB and stain by adding 10 µL of anti-CD34-PE antibody for 15 - 30 min at 4 °C. Analyze by flow cytometry (Figure 1A).
9. Centrifuge tubes at 400 × g for 10 min at 4 °C. Discard supernatant.
   NOTE: If not required immediately, mononuclear cells can be frozen at this stage.
10. Resuspend cells in 300 µL of SB per 10⁸ cells. Add 100 µL of FcR human IgG (blocking reagent, see Table of Materials) and 100 µL of CD34 magnetic beads per 10⁸ cells. Mix gently and incubate at 4 °C for 30 - 40 min.
    NOTE: A single cell suspension is required (if necessary, pass the cells though a 30 µm filter before adding reagents). Use the reagent volumes described here for 10⁸ cells or less. For more than 10⁸ cells, scale up the reagents (SB, blocking solution, and CD34 magnetic beads) accordingly.
11. Prepare the LS separation column during the incubation period by placing the LS column in a magnetic holder and washing with 3 mL of SB. Discard the effluent.
    NOTE: LS separation columns can be loaded with up to 2 × 10⁸ total cells. Smaller and larger columns are available.
12. Add 5 - 10 mL of SB to the cell mixture and centrifuge at 400 × g for 10 min. Discard the supernatant.
13. Resuspend cells in 1.5 mL of SB and load the cell suspension (1.5 mL) onto the LS column. Collect the flow through containing the unlabeled cells in a 15 mL collection tube (negative fraction #1). Let the liquid drain and wash the column with 1.5 mL of SB.
14. Load the collected flow through (3 mL) back into the column and collect the flow through in the same 15 mL collection tube (negative fraction #1). Wash the LS column 3 times with 3 mL of SB.
    NOTE: If required, cells in the negative fraction (12 mL) can be stained with anti-CD34 antibody to ascertain capture of CD34⁺ cells by the LS column.
15. Remove column from the magnetic separator and flush cells slowly with a syringe plunger into a new 15 mL tube with 2 mL of SB.
16. Place column back on magnetic separator and load the 2 mL of cells back into the column. Collect the flow through and wash with 2 mL SB. This is the negative fraction #2 (4 mL).
17. Remove LS column from magnetic separator and add 2 mL of SB. Flush cells steadily and firmly with a syringe plunger to collect the CD34⁺ cell fraction. Count the cells using trypan blue staining and a hemocytometer or automated cell counter.
18. Centrifuge tube with the positive fraction at 400 × g for 15 min at 4 °C. Discard the supernatant. Resuspend cells in serum-free media for CD34⁺ cells (SFM, see Table of Materials).
    NOTE: If not required immediately, cells can be frozen in liquid nitrogen at this stage.

2. Purity Check of Isolated CD34⁺ Cells

1. Stain 2 x 10⁴ cells from the positive fraction with 10 µL anti-CD34-PE antibody and 20 µL anti-CD45-PerCP antibody for 15 min at 4 °C (use a separate tube for isotype controls) (Figure 1B).
2. Add 1 mL of SB to wash. Centrifuge at 400 × g for 10 min. Resuspend pellet in 200 µL of SB.
3. Perform flow cytometric analysis and gate the live cell population to exclude debris (Figure 1B). Set the gate for PE and PerCP positive populations using the PE and PerCP isotype controls (Figure 1B) and determine the percentage of CD34⁺/CD45⁺ cells.

3. Megakaryocyte Differentiation

1. Seed 5 × 10⁵ cells/mL CD34⁺ cells in 2 mL of SFM supplemented with 50 ng/mL recombinant human thrombopoietin (rhTPO) per well in a 12-well plate. Incubate cells at 37 °C, 5% CO₂ in a humidified atmosphere. If cells are confluent before they are required for analysis, harvest the cells and split into multiple wells with fresh media and rhTPO.
   NOTE: Two or three wells should be prepared specifically to monitor differentiation at different time points (e.g., days 7, 9, and 10).
2. Harvest cells from the wells set aside to monitor differentiation without disturbing the cells in the other wells.
3. Stain cells with 20 µL anti-GPIIb/CD41-FITC antibody and 10 µL anti-GPIX/CD42a-Alexa Fluor 647 antibody in a final volume of 100 µL. Set up a control tube using the respective isotype control antibodies. Incubate for 15 - 30 min at 4 °C.
4. Add 1 mL of SB to wash. Centrifuge at 400 × g for 10 min. Discard the supernatant and resuspend the pellet in 200 - 300 µL of SB. Analyze by flow cytometry.
5. For each fluorophore, analyze the isotype controls to set the gating for FITC and Alexa Flour 647 positive populations. Analyze the stained cell samples to determine the percent of CD41⁺/CD42a⁺ double positive cells, which represent mature MK (Figure 1C).
6. For microscopic visualization of cell surface markers stain cells as described in 3.3.
   1. Add 1 mL of SB to wash. Centrifuge at 400 × g for 10 min. Resuspend the pellet in 100 µL of SB and spin onto a glass slide at 1,000 x g for 5 min. Fix cells on the slide by dipping in methanol for 30 s. Air dry, add 20 µL of mounting media containing DAPI (see Table of Materials), cover with a coverslip, and visualize using a fluorescent microscope (Figure 2A).
7. For visualization of intracellular antigens, resuspend cells in PBS and fix with paraformaldehyde (1% final concentration) for 15 min at room temperature. To permeabilize the cells, add triton-X 100 (0.1%) and incubate for 15 min. Wash cells with 2 mL PBS/0.1% triton-X 100. Resuspend in 100 µL of PBS/0.1% triton-X 100, add anti vWf and anti CD62p antibodies (1:200 dilution) and incubate for 30 min at room temperature.
   1. Wash with 2 mL PBS/0.1% triton-X 100 and centrifuge at 400 × g for 10 min, resuspend in 100 µL of the same buffer, and add anti-mouse IgG-Alexa 594 and anti-rabbit IgG-Alexa 488 (1:100). Incubate for 30 min at room temperature, wash with 2 mL PBS/0.1% triton-X 100, resuspend in 100 µL of the same buffer, and add 20 µL of anti CD42b-APC. Then spin the cells onto glass slides as described in step 3.6.1 and prepare samples for microscopic visualization as described in step 3.6.1.
8. For ploidy determination, harvest cells at days 12 or 13 of differentiation. Add 1 mL of SB to wash. Centrifuge at 400 × g for 10 min and stain with 20 µL anti-GPIIb/CD41-FITC antibody in a final volume of 100 µL. Incubate at 4 °C for 30 min.
   1. Wash once with 1 mL of SB and resuspend pellet in 300 µL of hypotonic citrate buffer (1.25 mM sodium citrate, 2.5 mM sodium chloride, 3.5 mM dextrose) containing 20 µg/ml propidium iodide and 0.05% Triton-X 100. Incubate for 15 min at 4 °C protected from light.
   2. Add RNase to a final concentration of 20 µg/mL and incubate for 30 min at 4 °C protected from light. Determine the intensity of propidium iodide by flow cytometry by collecting 30,000 to 50,000 events of the CD41-FITC⁺ population (Figure 2C).

4. Proplatelet Counting, Platelet Enumeration, and Platelet Activation

1. Harvest cells (from step 3.1) at days 8 or 9 of differentiation and seed at 1 × 10⁴ cells/well in 48-well plates in 200 µL of fresh SFM supplemented with 50 ng/mL rhTPO. Culture for 5 days at 37 °C, 5% CO_2.
   NOTE: For quantitation purposes, seed wells in triplicate. This low density is required for visualization and counting of proplatelet-bearing MK. Proplatelets usually start appearing after 2 days of culture. The peak is between days 4 and 5.
2. Count the number of proplatelet-bearing MK in the whole well on an inverted light microscope using 10X or 20X objectives.
   NOTE: A heated (37 °C) microscope stage is preferable, since keeping the cells at room temperature for extended periods causes shrinkage of the proplatelet extensions. Proplatelets are observed as long extensions from the MK body. Each MK may have several proplatelet protrusions. As proplatelets develop, the body of the MK decreases in size.
3. Harvest cells and centrifuge at 400 x g for 10 min at room temperature. Stain cells with 20 µL anti-human CD41-FITC antibody, as described in steps 3.3 and 3.4. Calculate the percentage of proplatelet-bearing MK (pbMK): pbMK (%) = [(Proplatelet-bearing MKs/ well) / (Total CD41⁺ cells/ well)] x 100
4. To count platelets released into the culture medium, gently mix the cells with a Pasteur pipette and collect 100 µL at days 14 or 15 of culture.
   1. Stain with 20 µL anti-human CD41-FITC antibody for 20 - 30 min at 4 °C. Set up a control tube using the respective isotype control antibody.
   2. Add 150 µL of SB and 50 µL of counting beads.
   3. For flow cytometric analysis, set the FSC and SSC to log scale. Use normal human blood platelets from platelet-rich plasma stained with CD41-FITC as described in section 4.4.1 to set the gating for platelets (Figure 3A).
   4. Analyze the stained platelets with counting beads by flow cytometry. Collect 1,000 events of counting beads using the FSC versus SSC scatter plot (Figure 3A). Calculate the number of platelets based on CD41-FITC positive events (Figure 3B) using the formula:
      Platelets per µL=[(number of CD41-FITC positive events)/1,000 beads)] x [(number of beads in 50 µL)/sample volume)]
5. To analyze platelet activation, gently mix the cells with a Pasteur pipette and collect 100 µL at days 14 or 15 of culture. Add 1 mL of Tyrode's buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl_2, 0.2 mM Na₂HPO₄, 12 mM NaHCO₃, 5.5 mM D-glucose, pH 6.5) and centrifuge at 200 x g for 5 min to pellet cells.
   1. Collect the supernatant and centrifuge at 800 x g for 10 min to pellet platelet-sized particles.
   2. Discard supernatant and resuspend in 100 µL of Tyrode's buffer. Add 20 µL of PAC1-FITC antibody and adenosine diphosphate (ADP) to a final concentration of 20 µM. Incubate at room temperature for 20 min. Analyze by flow cytometry to determine the percentage of FITC positive events. Use fresh human platelets treated in the same manner as a positive control (Figure 3C).

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Results

This protocol allows the preparation of highly pure MK cultures from cord blood-derived CD34⁺ cells. The percentage of CD34⁺ cells in cord blood is approximately 1.3%¹⁵ (Figure 1A) and the total number of mononuclear cells (step 1.8) ranges from 90 - 300 x 10⁶ per UCB unit. The purity of CD34+/CD45+ cells after isolation ranges from 90 to 99% (Figure 1B). MK (defined a...

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Discussion

The protocol described here is suitable for consistent production of MK and platelets in culture from umbilical cord blood. These cells can be used to study various processes such as the effect of drugs or biological activities on MK proliferation, differentiation, proplatelet formation, and platelet production.

A variety of culture media and cytokine combinations have been presented in the literature. Addition of cytokines such as stem cell factor, Flt-3 ligand, IL-3, and IL-6 supports CD34

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Materials

Name	Company	Catalog Number	Comments
Cell Culture Reagents
Recombinant Human TPO	Miltenyi Biotec	130-094-013
StemSpan SFEM II	Stem Cell Technologies	9605	Serum-free media for CD34+ cells
Name	Company	Catalog Number	Comments
CD34 Isolation Reagents
CD34 MicroBead kit ultrapure	Miltenyi Biotec	130-100-453	This kit includes the FcR human IgG blocking reagent and CD34 microbeads. These beads contain the anti-CD34 antibody clone QBEND/10. Use a different anti-CD34 clone for purity check (e.g. clone 8G12).
Lymphoprep	Alere Technologies	1114545	Lymphocyte separation media (density 1.077 g/mL)
Sterile separation buffer (SB)	Miltenyi Biotec	130-091-221	This buffer contains phosphate buffered saline (PBS), pH 7.2 containing 0.5% bovine serum albumin and 2 mM EDTA. It can be prepared using sterile, cell culture grade components. De-gas before use because air bubbles can block the column.
Name	Company	Catalog Number	Comments
Flow Cytometry and Cell Staining Reagents
PE Mouse anti-Human CD34	BD Biosciences	340669	Clone 8G12. This can be used for CD34 purity check. Final antibody concentration 1:10 dilution.
PerCP mouse anti-human CD45	BD Biosciences	347464	1:10 dilution
PerCP isotype control	BD Biosciences	349044	1:10 dilution
FITC Mouse anti-Human CD41a	BD Biosciences	340929	Final antibody concentration 1:5 dilution.
APC Mouse anti-Human CD42b	BD Biosciences	551061	This antibody can also be used to detect mature MK (the percentage of positive cells in usually lower than with anti CD42a). Final antibody concentration 1:10 dilution.
Alexa Fluor 647 Mouse anti-Human CD42a	AbD Serotec	MCA1227A647T	Currently distributed by Bio-Rad. Final antibody concentration 1:10 dilution.
Alexa Fluor 647 Mouse Negative Control	AbD Serotec	MCA928A647	Currently distributed by Bio-Rad. Isotype control antibody
Anti von Willebrand factor rabbit polyclonal	Abcam	AB6994	1:200 dilution
V450 mouse anti-humna CD41a	BD Biosciences	58425	1: 20 dilution
V450 isotype control	BD Biosciences	580373	1:20 dilution
PAC1-FITC antibody	BD Biosciences	340507	1:10 dilution
Anti CD62p mouse monoclonal	Abcam	AB6632	1:200 dilution
Alexa Fluor 488 goat anti rabbit IgG	Invitrogen	A11008	1:100 dilution
Alexa Fluor 594 goat anti mouse IgG	Invitrogen	A11020	1:100 dilution
Ig Isotype Control cocktail-C	BD Biosciences	558659	Isotype control antibody
Propidium iodide	Sigma Aldrich	P4864
CountBright Absolute Counting Beads	Molecular Probes, Invitrogen	C36950	Counting beads
Name	Company	Catalog Number	Comments
Materials
LS columns	Miltenyi Biotec	130-042-401	Smaller and larger columns are also commercially available
MidiMACS Separator magnet	Miltenyi Biotec	130-042-302
MACS MultiStand	Miltenyi Biotec	130-042-303
Falcon 5mL round bottom polypropylene FACS tubes, with Snap Cap, Sterile	In Vitro technologies	352063
Glass slides	Menzel-Glaser	J3800AMNZ
Mounting media with DAPI	Vector Laboratories	H-1200	Antifade mounting medium with DAPI
Name	Company	Catalog Number	Comments
Equipment
Inverted microscope	Leica	DMIRB inverted microscope
Fluorescent microscope	Zeiss	Vert.A1
Cell analyser	BD Biosciences	FACS Canto II
Cytospin centrifuge	ThermoScientific	Cytospin 4
Name	Company	Catalog Number	Comments
Software
Cell analyser software	BD Biosciences	FACS Diva Software
Single cell analysis software	Tree Star	FlowJo
Fluorescent microscope software	Zeiss	Zen 2 blue edition