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

Published: July 31, 2023

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 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% CO…

Representative 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 determine the percentage of the CD34+ cells in the sample (1.3%, right panel). (B) After separation, the positive fraction was staine…

Açıklamalar

The authors have nothing to disclose.

Materials

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

Etiketler

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Bu Makaleden Alıntı Yapın
An In Vitro Method for the Differentiation of Megakaryocytes and Platelet Formation. J. Vis. Exp. (Pending Publication), e21532, doi: (2023).

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