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.
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
2. Proplatelet Counting, Platelet Enumeration, and Platelet Activation
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 stained with anti-CD34-PE and anti-CD45 PerCP antibodies. The enriched CD34+ population is indicated in the figure (98.1%, right panel, upper right quadrant). (C) Phenotypic analysis of MK differentiated in vitro from CD34+ cells for 7 days or 11 days were stained with anti-CD41 and anti-CD42a antibodies. Mature MKs are positive for both CD41+ and CD42a+ (upper right quadrant).
Figure 2: MK staining, ploidy, and proplatelet formation in vitro. (A) Fluorescent images of day 11 MK stained with anti-CD41-PE and CD42b-APC antibodies. Nuclei were stained with DAPI. Yellow arrowhead indicates multi-nuclear MKs. Scale bar, 30 µm. (B) Fluorescent images of day 14 MK stained with anti-vWf (green), anti-CD62p (red), and anti-CD42b-APC (magenta) antibodies. Nuclei were stained with DAPI. Scale bar, 15 µm (C) Representative gating strategy showing the ploidy distribution of CD41+ events. The graph (lower panel) shows the observed distribution of ploidy classes (n = 4), error bars, SD. (D) The characteristic morphology of proplatelet-bearing MK is shown. The MK body is indicated by the arrow. The long cytoplasmic processes extending from the MK (proplatelets) are indicated by arrowheads. It may be unclear in some areas/fields of view whether one or two MKs are producing these proplatelet extensions. This should be counted as one proplatelet-bearing MK. Images taken with an inverted microscope, 10X objective. Scale Bar, 50 µm.
Figure 3: Abundance of platelets produced in vitro and platelet activation. (A) Human platelets from platelet-rich plasma were used to set the analytical gate using a log scale for forward and side scatter (left panel). The right panel shows cells from MK cultures. Platelets produced in vitro are observed in the analytical gate defined for human platelets. Cells and counting beads are indicated in the figure. Plt, platelet (B) Platelets produced in vitro were stained with anti-CD41 antibody. Human platelets from platelet-rich plasma were used to set the analytical gate using a log scale for forward and side scatter and to compare the profile within the CD41-FITC gate. C-FITC, FITC isotype control (C) Platelet activation following treatment by ADP to a final concentration of 20 µM. Human platelets from platelet-rich plasma were used as control (upper panels). Platelets produced in culture are shown in the lower panels. Binding of PAC1 antibody indicates platelet activation. C-FITC, FITC isotype control.
The authors have nothing to disclose.
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 |