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 ....

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⁺ C....

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 as CD4.......

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

Materials

List of materials used in this article

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