Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Oct 3.
Published in final edited form as: Platelets. 2020 Sep 16;32(7):928–935. doi: 10.1080/09537104.2020.1817359

In vitro megakaryocyte culture from human bone marrow aspirates as a research and diagnostic tool

Kirill R Butov 1,3,*, Elena Y Osipova 1, Nikita B Mikhalkin 3, Natalia M Trubina 1, Mikhail A Panteleev 3,4,5, Kellie R Machlus 2
PMCID: PMC9295913  NIHMSID: NIHMS1625380  PMID: 32936668

Abstract

Megakaryocytes (MKs) are relatively rare in bone marrow, comprising <0.05% of the nucleated cells, which makes direct isolation from human bone marrow impractical. As such, in vitro expansion of primary MKs from patient samples offers exciting fundamental and clinical opportunities. As most of the developed ex vivo methods require a substantial volume of biomaterial, they are not widely performed on young patients. Here we propose a simple, robust, and adapted method of primary human MK culture from 1 mL of bone marrow aspirate. Our technique uses a small volume of bone marrow per culture, uses straightforward isolation methods, and generates approximately 6x105 mature MKs per culture. The relative high cell purity and yield achieved by this technique, combined with efficient use of low volumes of bone marrow, make this approach suitable for diagnostic and basic research of human megakaryopoiesis.

Method

Materials:

  1. Recombinant human thrombopoietin (TPO) – Gibco Catalog # PHC9514

  2. Fetal Bovine Serum (FBS) (Performance Plus) – Gibco Catalog #16000–044

  3. Penicillin-Streptomycin 10,000 U/mL 100 ml sterile-filtered – Gibco Catalog # 15140122

  4. Bovine Serum Albumin (BSA) 7.5% – Sigma Catalog # A8412

  5. Lympholyte-H – Cedarline Catalog # CL5015

  6. Corning® 0.2 µm syringe filters PES membrane – Corning Catalog # CLS431229

  7. Trypan Blue 0.4% solution – Gibco Catalog # 15250061

  8. 60 mm petri dish non-treated, sterile – Eppendorf Catalog #0030701011

  9. Cell imaging dish 35 mm, glass-bottom, sterile – Eppendorf Catalog # 0030740009

  10. Sterile 15-ml conical-bottom centrifuge tubes – Corning Catalog # 430052

  11. Sterile 50 ml conical-bottom centrifuge tubes – Corning Catalog # 352098

  12. PBS without Ca and Mg, sterile – Gibco Catalog # 10010015

  13. MEM Vitamin Solution (100X) – Gibco Catalog # 11120052

  14. DPBS without Ca and Mg, sterile – Gibco Catalog #14190144

  15. Human recombinant SCF (Stem cell factor) – Stemcell Catalog # 78062.1

Optional:

  1. DRAQ5™ – Biolegend Catalog # 424101

  2. Helix NP™ Blue – Biolegend Catalog # 425305

  3. Anti-CD41a antibody (HIP8 clone) conjugated with FITC – Invitrogen Catalog #11–0419-42

  4. Anti-CD42b antibody (HIP1 clone) conjugated with PE-Cy7 – Invitrogen Catalog #25–0429-42

  5. FxCycle™ PI/RNAse staining solution – Invitrogen Catalog # F10797

  6. Hoescht 33342 10 mg/ml solution – Invitrogen Catalog # H3570

  7. MethoCult™ H4100 – Stemcell Catalog # 04100

  8. FluoroBrite DMEM – Gibco Catalog # A1896701

  9. Poly-L-Lysine solution – Sigma-Aldrich Catalog # P8920–100ML

  10. Wright-Giemsa Stain, Modified – Sigma-Aldrich Catalog # WG32–1L

Herein, we describe a protocol that is optimized for differentiation and maturation of human megakaryocytes (MKs) from 1 mL of human bone marrow. In this protocol, we used bone marrow cultures expanded from routine bone marrow biopsies that were obtained from pediatric patients with confirmed ALL remission as a part of their diagnostic work-up. Of note, we chose only patients with no history of platelet disorders who had platelet counts within the normal range. As such, we consider these results to be representative of healthy ‘control’ bone marrow.

To adapt our method for larger or smaller volumes, the steps described below can be followed with Lympholyte-H and DPBS volumes adjusted as recommended in Figure 1A. Bone marrow samples should be drawn into K3EDTA or K4EDTA tubes. Do not overfill the tube, as it promotes clotting; tubes with clots are not suitable for further processing by this method. During culture, we recommend using flow cytometry to assess megakaryocyte differentiation and maturation on Days 0, 7, and 14 using the method described below. We do not recommend performing a regular DNA content check, as large amounts of cells are needed to prepare the sample for flow cytometry.

Figure 1. Simplified method flow chart and volume conversion table.

Figure 1.

Open in a new tab

Schematic image of the method described. (A) Step 1 – Layer diluted bone marrow on the Lympholyte-H. Step 2 – Centrifugation to isolate mononuclear cells. Step 3 – Seeding cells from the Lympholyte-H layer. Step 4 – Layering cells after 14-day culture on one-step BSA gradient. Step 5 – Isolation procedure. Step 6 – Seeding isolated MK for further analysis and observing proplatelet formation. (B) Volume conversion table for different starting volumes of bone marrow samples

Isolating cells from the bone marrow (Day 0):

  1. Place DPBS (supplemented with 2% FBS), IMDM (with 10% FBS, 1x GlutaMAX, 100 U/mL Penicillin-Streptomycin and 1x MEM vitamins, which will hereafter be referred to as ‘fully supplemented’) in 37°C water bath. Let Lympholyte-H equilibrate to room temperature.

  2. Visually inspect the bone marrow for any clots present. Gently swirl the tube with the bone marrow 2–3 times. Bone marrow samples may be kept in a vertical position for up to 24 hours without any significant changes in cell culture viability.

  3. Transfer heated DPBS, IMDM, Lympholyte-H, and the bone marrow tube to a sterile biosafety cabinet.

  4. Gently resuspend bone marrow with a serological pipette and transfer 1 mL of bone marrow to a 5 mL tube.

  5. Prepare a 5- or 15-mL tube with 1.5 mL of Lympholyte-H. Using a 5 mL tube is preferable for this volume, but a 15 mL may be used if no appropriate centrifuge rotors are available for 5 mL tubes.

  6. Dilute bone marrow sample in a 1:1 ratio with DPBS (supplemented with 2% FBS) and resuspend gently 2–3 times with serological pipette and carefully layer on the Lympholyte-H. (Figure 1B, step 1)

  7. Centrifuge the 5 mL tubes at room temperature for 30 min at 800 x g with deceleration set to 0. (Acceleration may be set as high as 2–4). (Figure 1B, step 2)

  8. Carefully transfer centrifuged tubes to a sterile biosafety cabinet, holding them vertically. Clear layer separation should be evident with erythrocytes at the bottom, followed by a Lympholyte-H layer and plasma. Between the plasma and Lympholyte-H layers, a small band of cells should be visible. (Figure 1B, step 3)

  9. Using a sterile 100–1000 µL tip, carefully transfer cells to a new 5 mL tube trying not to disturb the plasma layer, as it contains many platelets that promote cell aggregation in culture.

  10. Add 2–3 mL of DPBS to aspirated cells and centrifuge at 300 x g for 5 minutes (SL40R ThermoFisher Scientific, Waltham, MA)

  11. Aspirate the supernatant, resuspend cell pellet in 2–3 mL of DPBS, and centrifuge at 300 x g for 5 minutes.

  12. Aspirate the supernatant and rigorously resuspend the pellet in 1 mL of fully supplemented IMDM.

  13. Count cells manually or using an automatic cell counter.

  14. Prepare 5 mL of fully supplemented IMDM, adding TPO to the final concentration of 50 ng/mL and SCF to a final concentration of 20 ng/mL

  15. Seed cells with an approximate density of 0.5 x 106 in 5 mL of fully supplemented IMDM with cytokines into appropriate number of 60 mm cell culture dishes and place culture dishes in 37°C 5% CO2 incubator.

B). Changing media (Day 3)

  1. Place IMDM (supplemented with 10% FBS, 100 U/mL Penicillin-Streptomycin, 1x GlutaMAX and 1x MEM vitamins) in 37°C water bath.

  2. Transfer the cell culture dishes into a sterile biosafety cabinet and gently swirl media to mix.

  3. Resuspend cells 2–3 times and transfer to a 15 mL conical tube.

  4. Centrifuge the 15 mL tube at 250 x g for 5 min.

  5. Resuspend the pellet with 5 mL of IMDM

  6. Re-seed cells back into a 60 mm cell culture dish and dilute to approximate density of 1 x 106 in 5 mL of fully supplemented IMDM and add TPO to a final concentration of 50 ng/mL and SCF to a final concentration of 20 ng/mL. Place dishes in 37°C, 5% CO2 incubator for 4 days.

C). Changing media (Days 7 and 10)

  1. Place IMDM (supplemented with 10% FBS, 100 U/mL Penicillin-Streptomycin, 1x GlutaMAX and 1x MEM vitamins) in 37°C water bath.

  2. Transfer cell culture dish to a sterile biosafety cabinet and gently swirl the dish to mix.

  3. Carefully aspirate 1–2 mL of cell suspension and transfer it to a 5 mL tube. You can use these cells for Wright-Giemsa, immunofluorescent microscopy or flow cytometry analysis.

  4. Add 2–3 mL of fresh media supplemented with TPO and SCF to the cell culture dish and place it in 37°C, 5% CO2 incubator. We do not perform any correction of cell density on these days to preserve cells as little cell proliferation is typically observed.

  5. Repeat the procedure on Day 10.

D). Isolating megakaryocytes (Day 14)

  1. Prepare 3% and 1.5% BSA (w/v in DPBS) solutions by diluting 7.5% BSA stock solution and place with fully supplemented IMDM in 37°C water bath.

  2. Sterile-filter BSA solutions into the new tubes through 0.2 um filter in a sterile biosafety cabinet.

  3. Pipette 1 mL of 3% BSA into a new 15 mL conical tube.

  4. Tilt the tube, and slowly layer 1.5% BSA solution on top, pressing the tip against the wall near the tube neck.

  5. Slowly re-orient tubes vertically and place aside. Retrieve culture dishes from the incubator.

  6. Gently resuspend cells with a 5 mL serological pipette and transfer to a 15 mL conical tube

  7. Centrifuge the 15 mL tube at 200 × g for 5 min.

  8. Decant the supernatant and resuspend the pellet in 1 mL of IMDM.

  9. Layer cell suspension as described in step 4 (Figure 1B, step 4).

  10. Place the 15 mL tube vertically into the incubator for 30 minutes (Figure 1B, step 5).

  11. Gently retrieve the tube. A loose, white MK pellet should be observed.

  12. Aspirate top two layers by drawing 1 mL sequentially. Aspirate only 500 µL of the last layer (Figure 1B, step 6).

  13. Add 3 mL of fully supplemented IMDM and seed in tissue-culture treated culture vessels. We recommend seeding cells into 24-well plates with approximate density of 0.05 x 106 cells per well.

  14. Mature MKs will produce proplatelets over the next 24–48 hours. These cells can be used for further analysis as described in examples below

Flow cytometry

For flow cytometry analysis, we recommend including anti-CD41 as well as anti-CD42b antibodies. The former is an early marker and the latter a late marker of MK development. We recommend using any suitable Live/Dead exclusion dyes to determine cell culture viability. In our protocol Helix NP™ Blue (Sytox™ Blue) is used. Performing flow cytometry on Days 0, 7, and 14 allows for adequate monitoring of the culture to detect any defects in megakaryopoiesis. For determining the percentage of other cell types such as leukocytes and some types of hematopoietic stem cells, CD45 and CD34 staining may be used as shown in Figure 2E, F. Plotting CD45 against SSC allows for gating of granulocytes, monocytes, and lymphocytes based on their granularity and level of CD45 expression. Blasts are defined as cells in intermediate stages of differentiation. This gating strategy has been described in greater detail in previous publications (1,2).

Figure 2. Flow cytometry analysis of MK cultures obtained from patients in an acute lymphoid leukemia (ALL) remission.

Figure 2.

Open in a new tab

(A) 5x105 cells were stained with FITC-anti-CD41, PE-Cy7-anti-CD42b and Helix NP Blue and analyzed on a FACS Canto II (BD, San Diego, CA, USA). Viable cells were gated as Helix NP Blue negative and representative plots are shown. 85 000 events were collected for the analysis for all the samples (B) Megakaryocytes (CD41+CD42b+ events) comprise 1.2% ± 0.5% events on Day 0, 10.8% ± 2.1% on Day 7 and 68% ± 18% after isolation on Day 14. N=8 (C) Cells were fixed in ethanol and stained with FxCycle PI/RNAse staining solution. Representative DNA content on Day 0 and Day 7 is shown. (D) Graph shows >4n events comprising 1.1% ± 0.5% on Day 0, 11.25% ± 2.1%. N=8. (E) A representative example of CD45/SSC gating strategy on viable cells for enumerating main cell types after Lympholyte-H isolation. PMN – Granulocytes, Mono – Monocytes, Lymphocytes, Blasts – other cell types. The gating strategy briefly explained in the Flow Cytometry section of the paper. (F) A flow cytometry plot of CD34 staining of viable Helix NP Blue negative cells on Day 0 (G) After Lympholyte-H isolation granulocyte represent 28.7% ± 7.8% cells, 9.51% ± 2.6% monocytes, 30.75% ± 4.2% lymphocytes, 28.3% ± 11.3% blasts and 9% ±1.55% CD34+ cells of all viable Helix NP Blue negative cells. N=3. The data are the means ± SEM from duplicates of experiments. *p<0.05, **p<0.01, ***p<0.001, or non-significant (ns) by Mann-Whitney test

A). Cell Surface Marker Profiling

  1. Prepare flow cytometry buffer containing 1x PBS / 2% FBS/BSA / 0.1% Sodium azide/ 2 mM EDTA.

  2. Count live cells manually with a hemocytometer or with an automatic cell counter using trypan blue exclusion dye.

  3. Centrifuge 1–3 x 105 cells at 320g x 5 min.

  4. Decant the supernatant and rigorously resuspend cells in 100 µL ice-cold flow cytometry buffer.

  5. Add an optimized amount of anti-CD41 and anti-CD42b antibodies. We use 0.125 µg/test of anti-CD41 FITC and 0.25 µg/test of anti-CD42b PE-Cy7. However, we recommend performing antibody titration for freshly obtained antibodies to achieve optimal resolution.

  6. Put the tubes on ice or at +4°C for 30 minutes in the dark.

  7. After incubation, add 500 µL of flow cytometry buffer and centrifuge at 320g x 5 min.

  8. Decant the supernatant and rigorously resuspend cells in 500 µL of PBS

  9. Dilute stock solution of Helix NP Blue (Sytox™ Blue) in a 1:4 ratio.

  10. Add 1 µL of Helix NP Blue and incubate for 5 minutes at room temperature.

  11. Run flow cytometry. Figure 2A shows examples of flow cytometry plots of CD41+/CD42b+ events gated on viable, Helix NP Blue negative events. Figure 2B shows the average percentage of megakaryocytes at different days of culture.

B). DNA content

  1. Prepare a 70% ethanol solution and cool it to −20°C.

  2. Centrifuge 7–10 x 105 cells at 320g x 5 min.

  3. Decant the supernatant.

  4. Slowly, by drops, add 200 µL of the 70% ethanol solution, constantly vortexing the tube.

  5. Resuspend vigorously.

  6. Incubate for no less than 30 minutes at −20°C. Cells are stable for 24 hours.

  7. For the flow cytometry analysis, centrifuge the cell suspension at 600g x 5 min

  8. Aspirate the supernatant and resuspend the pellet in 250 µL of FxCycle PI/RNAse staining solution.

  9. Run flow cytometry. Propidium iodide signal should be analyzed using the PE or PE-Cy7 channel. Figure 2D shows a significant increase in high ploidy cells on Day 7.

Microscopy

Performing microscopy is optional to confirm the morphology of generated MKs. Therefore, we briefly describe these methods and possible example uses for these techniques in different conditions. Of note, we strongly recommend assessing proplatelet forming ability by live cell microscopy, as this is the main physiological processes of mature MKs.

A). Immunofluorescence microscopy

  1. Immerse coverslips into 70% ethanol solution for 15 minutes and let them air dry.

  2. Coat coverslips with 0.1% poly-L-lysine solution for 5 minutes and let them dry at room temperature. Coated coverslips may be kept for up to one year if protected from dust.

  3. Cytospin 5–10 x 104 cells onto poly-L-lysine coated coverslips at 200g 5 min.

  4. Fix cells in 4% formaldehyde solution for 15 minutes at room temperature.

  5. Carefully wash cells with PBS/HBSS 3 times for 2 minutes each.

  6. Block and permeabilize the specimen in a buffer containing 1x PBS / 1% BSA / 0.1% Sodium azide/ 0.1% Saponin for 1 hour.

  7. Wash cells with PBS/HBSS 3 times for 2 minutes.

  8. Follow the general antibody staining procedure.

  9. Image cells using epifluorescent or confocal microscopy techniques. Figure 3A shows an example of an epifluorescent picture of a large CD41 positive MK with a multilobular nucleus, surrounded by several CD14+ granulocytes. Figure 3C shows an example confocal picture obtained revealing the details of the tubulin cytoskeleton, a multilobular nucleus, and CD41 expression in an MK.

Figure 3. Microscopy analysis of MK cultures obtained from patients in an acute lymphoid leukemia (ALL) remission.

Figure 3.

Open in a new tab

(A-B) 6x104 cells from a Day 14 culture were cytospun onto coverslips before isolation and stained as described with FITC-antiCD41, Alexa647-antiCD14 or Alexa647-antiCD62p, and DAPI. Images were taken on a Nikon Ni-U (Melville, NY, USA) or Zeiss Axio Observer Z1 microscope (Carl Zeiss, Jena, Germany) equipped with a Yokogawa spinning disc confocal device (CSU-X1; Yokogawa Corporation of America, Sugar Land, TX) and a 1.3 numerical aperture ×100 objective. Scale bar – 10 µm (A) Epifluorescent microscopy reveals a large CD41+/CD14− MK with a multilobular nucleus, surrounded by several CD14 granulocytes. (B) Confocal microscopy shows the tubulin cytoskeleton, multilobular nucleus, and CD41 expression in a MK. (D) Brighfield illumination of Wright-Giemsa stained slides show megakaryocytes at different stages of maturation. Staging as described by Levine, et al (3).

B). Wright-Giemsa Staining

  1. Coat slides with 0.1% poly-L-lysine solution for 5 minutes and let them dry at room temperature. Coated slides may be kept for up to one year if protected from dust.

  2. Cytospin 7–10 x 104 cells onto poly-L-lysine coated slide at 200g x 5 minutes

  3. Dry the slide for 1 hour at room temperature.

  4. Immerse the slide in Wright-Giemsa stain for 1 minute.

  5. Wash slide in deionized water for 3 minutes. Do not agitate.

  6. Rinse in fresh deionized water and air dry for 20 minutes.

  7. Image cells by brightfield microscopy. Four types of megakaryocyte morphologies can be identified representing different maturation stages, as seen in Figure 3C. Staging can be performed using criteria developed by Levine, et al. (3).

C). Live cell imaging

For live cell imaging we used 35 mm 170 µm sterile glass bottom imaging dishes coated with BSA. We recommend using freshly coated dishes as uncoated glass may promote megakaryocyte spreading. Proplatelets are characterized as large, nonadherent, pseudopodia-like structures floating in the media which may elongate, thin, and branch with a uniform diameter of 2–4 µm (4).

  1. Place FluoroBrite™ DMEM (Supplemented with 10% FBS and 1x GlutaMAX) in a 37°C water bath. We recommend using FluoroBrite™ DMEM if further live staining is required. Otherwise, it can be substituted with any other no-HEPES, no-phenol red media.

  2. Working in a sterile biosafety cabinet, coat glass bottom imaging dish with 3% BSA solution for 30 minutes.

  3. Centrifuge 1–3 x 105 isolated Day 14 cells at 250g x 5 minutes.

  4. Decant the supernatant and gently resuspend the pellet in 1 mL of FluoroBrite™ DMEM.

  5. Add 600 uL of the cell suspension into the image area of the dish and fill margins of the imaging dish with 1–1.5 mL of FluoroBrite DMEM.

  6. MethoCult™ H4100 can be added as a medium thickener to reduce cellular movement.

  7. Image the cells using a microscope with a heated stage at 37°C and humidified CO2 incubator set at 5% CO2. Figure 4 shows examples live MKs, with and without proplatelets, at different magnifications.

Figure 4. Live cell imaging analysis of isolated MK.

Figure 4.

Open in a new tab

(A-C) 1x105 cells isolated on Day 14 were prepared as described and imaged on a Zeiss Axio Observer Z1 microscope (Carl Zeiss, Jena, Germany) equipped with a 1.3 numerical aperture ×100 objective or a 0.83 numerical aperture ×20 objective. Scale bar – (A,C) 20 µm, and (B) 10 µm. (A) Nuclei were stained with DRAQ5 for 10 minutes without washing. Low magnification (x20) epifluorescent microscopy allows simultaneously visualize cell morphology in DIC with clear DRAQ5+ nuclei identification (B) Using high magnification (x100) DIC microscopy to visualize proplatelet dynamics (Supplementary Video 1) (C) Low magnification (x20) DIC microscopy of a megakaryocyte making proplatelets (D) Spontaneous proplatelet formation was observed in 8.6% ± 2.3 % (N=3) of megakaryocytes when imaged in a 35 mm tissue culture-treated plastic dish 24 hours after BSA gradient isolation. Cells were imaged and counted using low magnification (x20) live DIC microscopy and proplatelet bearing MK were counted as stated earlier.

Discussion

Herein, we describe a method to culture MKs from 1 mL of human bone marrow aspirate and then characterize their surface marker expression, DNA content, and morphology. This method promotes MK development using a combination of TPO and SCF; SCF increases viability of CD34+ cells during culture and TPO promotes efficient MK development (5). As defects in megakaryopoiesis and platelet production are typical hallmarks of inherited thrombocytopenias, the ability to directly investigate MKs expanded from patient bone marrow is of great interest. Although the direct effects of known genetic defects on megakaryopoiesis are currently undergoing extensive research, these studies are predominantly done in murine models (6–8). While murine models are the most versatile and accessible system, they sometimes do not express the same phenotype as in humans, leading to results that are not directly translatable to human disease (9). Likewise, several known mutations that have been assessed in humans revealed differences in proplatelet formation, intracellular signalling, or megakaryocyte development, revealing a more complex more phenotype than was observed in mouse models (10–12). Therefore, studies using human MKs are necessary. Human MKs are commonly differentiated from CD34+ progenitors isolated from peripheral blood. Although a convenient and ethical technique, it requires a relatively large volume of blood, not only from the patient but also from healthy donors to perform a control culture. Because many congenital thrombocytopenias are diagnosed at a very young age, the large volumes of biomaterial needed for peripheral blood progenitor collection cannot be obtained from these children (13,14). Therefore, this protocol provides a practical and ethical method to directly study human MK development in the context of thrombocytopenic children.

There are several other commonly-used techniques of generating primary human MKs that use CD34+ cells derived from cord or peripheral blood and adult bone marrow. However, bone marrow from children is one of the most inefficient sources of MKs (15). Most commonly for adult patients, CD34+ cells from peripheral blood are cultured with a combination of TPO and other cytokines to differentiate these progenitor cells into MKs. To estimate the efficiency of MK production, MK yield per CD34+ cell input is calculated and MKs are further assessed by their DNA content analysis (16). Our technique produced ~12 MK per input CD34+ cell, with 11.25% ± 2.1% of >4n cells on Day 7 of culture, which is consistent with published data on the CD34+ cells derived from peripheral blood, bone marrow, and some cord blood culture protocols (17–21). Moreover, in our preliminary experiments we observed approximately 8% (Figure 4D) of MKs producing proplatelet-like protrusions, which is also consistent with previously published data on human MKs (22). Therefore, this technique provides comparable efficiency in MK expansion and maturation, despite the very low sample volume.

Although a relatively high yield of mature MKs can be obtained using this isolation procedure, the purity may not be good enough for very sensitive applications. Moreover, MKs isolated by the one-step BSA gradient are not synchronized (23), but using non-treated (low-attachment) surfaces promotes relative synchronization as was previously shown (24,25). In addition, another drawback of the current method is the lack of ‘control’ samples. Bone marrow biopsies are performed on thrombocytopenic children as a part of their diagnostic workup, providing the opportunity to use a small amount of the sample for research. As it is not ethically and clinically justified to obtain a BM sample from a healthy pediatric patient, we suggest that small volumes of BM can be obtained from other groups of patients which require BM biopsy to establish their diagnosis or prognosis, as was done in this manuscript with ALL remission patients. We propose that patient samples may be considered representative if 1) no significant platelet count change is observed before the biopsy procedure and 2) the disease for which the biopsy is performed has no known direct connection with platelet function and/or count/or the disease is in confirmed remission. Using these samples as a control group may help to establish our method as a clinical test for platelet and MK disorders in the future. Therefore, we suggest that our method can be widely used for basic and clinical research on the generated MKs.

Conclusions

Our system provides a robust technique to culture primary MKs from patient bone marrow samples. The proposed method is devoid of time-intensive techniques of immunomagnetic isolation, flow cytometry sorting, and using expensive and complex cytokine cocktails and media combinations. It is adapted for use with a small amount of biomaterial with a relatively high level of MK expansion efficiency, which is a significant benefit in a pediatric cohort.

Supplementary Material

Figure 6
Download video file (49.9MB, avi)

Sources of Funding

The work was supported by the grant from the endowment fund Doctors, Innovations, Science for Children to MAP. KRM is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (K01DK111515) at the National Institutes of Health and is an American Society of Hematology Scholar.

Footnotes

Ethics Comittee

The research was approved by the Center for Theoretical Problems of Physicochemical Pharmacology Ethics Committee. Prior to all collections, written informed consent was obtained from the parents.

Declaration of Interest

None

References

  • 1.Nicholson JKA, Hubbard M, Jones BM. Use of CD45 fluorescence and side-scatter characteristics for gating lymphocytes when using the whole blood lysis procedure and flow cytometry. Commun Clin Cytom 1996;26(1):16–21. [DOI] [PubMed] [Google Scholar]
  • 2.Lacombe F, Durrieu F, Briais A, Dumain P, Belloc F, Bascans E, et al. Flow cytometry CD45 gating for immunophenotyping of acute myeloid leukemia. Leukemia 1997;11(11):1878–86. [DOI] [PubMed] [Google Scholar]
  • 3.Williams N, Levine RF. Annotation: The Origin, Development and Regulation of Megakaryocytes. Br J Haematol [Internet]. 1982. Oct;52(2):173–80. Available from: 10.1111/j.1365-2141.1982.tb03878.x [DOI] [PubMed] [Google Scholar]
  • 4.Italiano JEJ, Patel-Hett S, Hartwig JH. Mechanics of proplatelet elaboration. J Thromb Haemost 2007. Jul;5 Suppl 1:18–23. [DOI] [PubMed] [Google Scholar]
  • 5.Machlus KR, Italiano JE. The incredible journey: From megakaryocyte development to platelet formation. J Cell Biol 2013;201(6):785–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Pleines I, Dütting S, Cherpokova D, Eckly A, Meyer I, Morowski M, et al. Defective tubulin organization and proplatelet formation in murine megakaryocytes lacking Rac1 and Cdc42. Blood. 2013;122(18):3178–87. [DOI] [PubMed] [Google Scholar]
  • 7.Bender M, Stritt S, Nurden P, Van Eeuwijk JMM, Zieger B, Kentouche K, et al. Megakaryocyte-specific Profilin1-deficiency alters microtubule stability and causes a Wiskott-Aldrich syndrome-like platelet defect. Nat Commun 2014;5. [DOI] [PubMed] [Google Scholar]
  • 8.Marconi C, DI Buduo CA, LeVine K, Barozzi S, Faleschini M, Bozzi V, et al. Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia. Blood. 2019;133(12):1346–57. [DOI] [PubMed] [Google Scholar]
  • 9.Sabri S, Foudi A, Boukour S, Franc B, Charrier S, Jandrot-Perrus M, et al. Deficiency in the Wiskott-Aldrich protein induces premature proplatelet formation and platelet production in the bone marrow compartment. Blood. 2006;108(1):134–40. [DOI] [PubMed] [Google Scholar]
  • 10.Bury L, Malara A, Momi S, Petito E, Balduini A, Gresele P. Mechanisms of thrombocytopenia in platelet-type von Willebrand disease. Haematologica 2019;104(7):1473–81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Pecci A, Cannobio I, Balduini A, Stefanini L, Cisterna B, Marseglia C, et al. Pathogenetic mechanisms of hematological abnormalities of patients with MYH9 mutations. Hum Mol Genet 2005;14(21):3169–78. [DOI] [PubMed] [Google Scholar]
  • 12.Bluteau D, Balduini A, Balayn N, Currao M, Nurden P, Deswarte C, et al. Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation. J Clin Invest 2014;124(2):580–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Noris P, Pecci A. Hereditary thrombocytopenias: A growing list of disorders. Hematology. 2017;2017(1):385–99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Drachman JG. Review in translational hematology Inherited thrombocytopenia : when a low platelet count does not mean ITP. Blood [Internet]. 2004;103(2):390–8. Available from: http://www.ncbi.nlm.nih.gov/pubmed/14504084 [DOI] [PubMed] [Google Scholar]
  • 15.van den Oudenrijn S, von dem Borne AEG., de Haas M. Differences in megakaryocyte expansion potential between CD34+ stem cells derived from cord blood, peripheral blood, and bone marrow from adults and children. Exp Hematol [Internet]. 2000. Sep;28(9):1054–61. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0301472X00005178 [DOI] [PubMed] [Google Scholar]
  • 16.Reems JA, Pineault N, Sun S. In Vitro Megakaryocyte Production and Platelet Biogenesis: State of the Art. Transfus Med Rev [Internet]. 2010;24(1):33–43. Available from: 10.1016/j.tmrv.2009.09.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Guerriero R, Testa U, Gabbianelli M, Mattia G, Montesoro E, Macioce G, et al. Unilineage megakaryocytic proliferation and differentiation of purified hematopoietic progenitors in serum-free liquid culture. Blood. 1995. Nov;86(10):3725–36. [PubMed] [Google Scholar]
  • 18.Norol F, Vitrat N, Cramer E, Guichard J, Burstein SA, Vainchenker W, et al. Effects of cytokines on platelet production from blood and marrow CD34+ cells. Blood. 1998. Feb;91(3):830–43. [PubMed] [Google Scholar]
  • 19.Mazur EM, Basilico D, Newton JL, Cohen JL, Charland C, Sohl PA, et al. Isolation of large numbers of enriched human megakaryocytes from liquid cultures of normal peripheral blood progenitor cells. Blood [Internet]. 1990;76(9):1771–82. Available from: http://www.ncbi.nlm.nih.gov/pubmed/2224127 [PubMed] [Google Scholar]
  • 20.Tao H, Gaudry L, Rice A, Chong B. Cord blood is better than bone marrow for generating megakaryocytic progenitor cells. Exp Hematol 1999;27(2):293–301. [DOI] [PubMed] [Google Scholar]
  • 21.Shim MH, Hoover A, Blake N, Drachman JG, Reems JA. Gene expression profile of primary human CD34+CD38locells differentiating along the megakaryocyte lineage. Exp Hematol 2004;32(7):638–48. [DOI] [PubMed] [Google Scholar]
  • 22.Takahashi R, Sekine N, Nakatake T. Influence of monoclonal antiplatelet glycoprotein antibodies on in vitro human megakaryocyte colony formation and proplatelet formation. Blood. 1999;93(6):1951–8. [PubMed] [Google Scholar]
  • 23.Vijey P, Posorske B, Machlus KR. In vitro culture of murine megakaryocytes from fetal liver-derived hematopoietic stem cells. Platelets [Internet]. 2018;29(6):583–8. Available from: https://www.tandfonline.com/doi/full/10.1080/09537104.2018.1492107 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Shivdasani R a, Schulze H. Culture, expansion, and differentiation of murine megakaryocytes. Curr Protoc Immunol [Internet]. 2005;Chapter 22:Unit 22F.6. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18432953 [DOI] [PubMed]
  • 25.Schlinker AC, Duncan MT, DeLuca TA, Whitehead DC, Miller WM. Megakaryocyte polyploidization and proplatelet formation in low-attachment conditions. Biochem Eng J [Internet]. 2016;111:24–33. Available from: 10.1016/j.bej.2016.03.001 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure 6
Download video file (49.9MB, avi)

RESOURCES