An In Vitro Method for the Differentiation of Megakaryocytes and Platelet Formation

Abstract

Source: Perdomo, J., et al. Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells. J. Vis. Exp. (2017)

This video demonstrates a technique for the differentiation of megakaryocytes and the formation of platelets. Hematopoietic stem progenitor cells — isolated from human umbilical cord blood — differentiate into mature megakaryocytes in the presence of thrombopoietin. The mature megakaryocytes produce cytoplasmic extensions termed proplatelets, which give rise to individual platelets.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. 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 them into multiple wells....

Results

Figure 1: Flow cytometry plots of CD34+ cell isolation and MK differentiation in culture. (A) Mononuclear cells (gated as shown in the left panel) purified from human cord blood were stained with anti-CD34-PE antibody to determin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
<strong>Cell Culture Reagents</strong>
Recombinant Human TPO	Miltenyi Biotec	130-094-013
StemSpan SFEM II	Stem Cell Technologies	9605	Serum-free media for CD34+ cells
<strong>Flow Cytometry and Cell Staining Reagents</strong>
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
<strong>Materials</strong>
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
<strong>Equipment</strong>
Inverted microscope	Leica	DMIRB inverted microscope
Fluorescent microscope	Zeiss	Vert.A1
Cell analyser	BD Biosciences	FACS Canto II
Cytospin centrifuge	ThermoScientific	Cytospin 4
<strong>Software</strong>
Cell analyser software	BD Biosciences	FACS Diva Software
Single cell analysis software	Tree Star	FlowJo
Fluorescent microscope software	Zeiss	Zen 2 blue edition