Hematopoietic KG1a cells (derived from acute myeloid leukemia) and bone marrow stromal cell lines HS5 and HS27a were grown, propagated, and subjected to experiments between passages 8 and 24, as described previously [13 (link)]. The human MDS cell line SKM-1 cells were obtained from Prof. Xiao Li’s lab (Shanghai Jiaotong University Sixth People’s Hospital, Shanghai, China). Above cells were cultured in RPMI 1640 (Biological Industries, Kibutz Beit Haemek, Israel) with 10% fetal bovine serum (Biological Industries, Kibutz Beit Haemek, Israel). Primary CD34+ cord blood cells were derived from the cord blood (CB) of healthy babies from the People’s Hospital of Shaanxi Province. Blood was aseptically aspirated from placenta and umbilical CB veins immediately following normal obstetrical deliveries and placed into blood bags containing an anticoagulant (citrate, phosphate, dextrose, and adenine-1). Samples were processed within 8 h of collection. CD34+ cells were cultured in StemSpan™ SFEM II (StemCell Technologies, Canada) added with StemSpan™ Megakaryocyte Expansion Supplement (StemCell Technologies, Canada). Informed consent was obtained from all individual participants included in the study.
Stemspan megakaryocyte expansion supplement
Manufactured by STEMCELL
Sourced in Canada
StemSpan Megakaryocyte Expansion Supplement is a cell culture medium supplement designed to support the expansion and differentiation of megakaryocytes from hematopoietic stem and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Stemspan serum free expansion medium, by STEMCELL (2 mentions)
Stemdiff apel media, by STEMCELL (2 mentions)
Human collagen 4, by Advanced BioMatrix (2 mentions)
Matrigel, by BD (2 mentions)
Heparin, by Merck Group (2 mentions)
Stempro accutase, by Thermo Fisher Scientific (2 mentions)
Mtesr1, by STEMCELL (2 mentions)
Cd34 microbead kit, by Miltenyi Biotec (2 mentions)
Rpmi 1640, by Sartorius (1 mentions)
Stemspan sfem 2, by STEMCELL (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Direct cd34 progenitor cell isolation kit, by Miltenyi Biotec (1 mentions)
Cryostor cs10, by STEMCELL (1 mentions)
Dispase, by STEMCELL (1 mentions)
Stemspan, by STEMCELL (1 mentions)
Serum free expansion medium, by STEMCELL (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Relesr, by STEMCELL (1 mentions)
Stemspan acf, by STEMCELL (1 mentions)
9 protocols using stemspan megakaryocyte expansion supplement
1
Hematopoietic and Stromal Cell Cultures
2
Differentiation of hiPSCs into iHPCs and Megakaryocytes
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The hiPSCs, HA-iPSCs, and 2bF8-iPSCs were differentiated into hiHPCs, HA-iHPCs, and 2bF8-iHPCs using a STEMdiffTM hematopoietic kit (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions. Briefly, cells were dissociated to aggregates by 0.5 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA) and plated 40–80 aggregates/well on Matrigel-coated six-well plates in mTesR Plus. The cells were cultured in differentiation A medium on the second day, then the medium was half-refreshed. On day 3, the cells were cultured with differentiation B medium and then the medium was half-refreshed every other day, and cells were gradually suspended. The iHPCs were harvested after being cultured in differentiation B medium for 7 days.
The differentiated cells were sorted using the Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.
The sorted CD34 positive iHPCs were cultured in StemSpan™ Serum-Free Expansion Medium (STEMCELL Technologies, Vancouver, BC, Canada) contained with StemSpan™ Megakaryocyte Expansion Supplement (STEMCELL Technologies, Vancouver, BC, Canada) to differentiate into megakaryocyte on low-attachment surface plates (Corning).
The differentiated cells were sorted using the Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.
The sorted CD34 positive iHPCs were cultured in StemSpan™ Serum-Free Expansion Medium (STEMCELL Technologies, Vancouver, BC, Canada) contained with StemSpan™ Megakaryocyte Expansion Supplement (STEMCELL Technologies, Vancouver, BC, Canada) to differentiate into megakaryocyte on low-attachment surface plates (Corning).
3
Stem Cell Expansion and Differentiation
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STEMspan-ACF and STEMdiff-APEL Media, STEMspan Megakaryocyte Expansion Supplement, mTeSR1, Dispase, and CryoStor CS10 were purchased from STEMCELL Technologies. BMP4 was from HumanZyme. All other cytokines were obtained from PeproTech. Y-27632 was purchased from Stemgent. Human Collagen IV was from Advanced BioMatrix. Matrigel and antibodies for flow cytometry were obtained from BD Biosciences. I-BET 151(GSK1210151A) was purchased from ChemieTek. MMP-8 Inhibitor I (CAS 236403-25-1) was purchased from Millipore. StemPro Accutase was from Life Technologies. Heparin was purchased from Sigma.
4
Megakaryocytic Differentiation Assay
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HSPCs from MSC/CD34 + HSPC co-culture assay post treatment were harvested and seeded in StemSpan SFEM II medium containing StemSpan Megakaryocyte Expansion Supplement (Stem Cell Technologies, 02696) at a concentration of 1 × 104 cells/ml. Medium was changed every 7 days. After 21 days of incubation, megakaryocytic differentiation was assessed using flow cytometry.
5
Megakaryocyte Differentiation from CD34+ Cells
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Allcells Biotechnology (Shanghai, China) was the supplier of the Cord Blood-Stem/Progenitor (CD34+) cells that were used in this study. Serum-free expansion medium (StemCell Technologies, Canada) was used in the cultivation of CD34+ cells. To facilitate megakaryocyte differentiation, CD34+ cells were seeded at a density of 1×104 cells/mL in a volume of 1 mL each well in a 12-well plate. The medium used was SFEM II, and StemCell Technologies’ StemSpan Megakaryocyte Expansion Supplement was added.
Fresh megakaryocyte growth medium in the amount of 500 µL was added once every 2 days. Every 7 days, the cells were pelleted and then resuspended in fresh megakaryocyte growth media. At days 14 and 28, the cells were examined to determine the degree to which they expressed the megakaryocyte lineage markers CD41a and CD42b. Human megakaryoblastic leukaemia cell line MEG-01 was obtained through a request to the American Type Culture Collection. MEG-01 cells were cultivated at a temperature of 37°C in an atmosphere that was humidified and contained 5% carbon dioxide. The media the cells were grown in was RPMI 1640 medium (Thermo Fisher, USA), which was augmented with 10% fetal bovine serum (Biological Industries, Israel).
Fresh megakaryocyte growth medium in the amount of 500 µL was added once every 2 days. Every 7 days, the cells were pelleted and then resuspended in fresh megakaryocyte growth media. At days 14 and 28, the cells were examined to determine the degree to which they expressed the megakaryocyte lineage markers CD41a and CD42b. Human megakaryoblastic leukaemia cell line MEG-01 was obtained through a request to the American Type Culture Collection. MEG-01 cells were cultivated at a temperature of 37°C in an atmosphere that was humidified and contained 5% carbon dioxide. The media the cells were grown in was RPMI 1640 medium (Thermo Fisher, USA), which was augmented with 10% fetal bovine serum (Biological Industries, Israel).
6
Megakaryocyte Expansion Protocol
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STEMdiff APEL Media, STEMspan-ACF, STEMspan megakaryocyte expansion supplement, mTeSR1, and RelesR were procured from STEMCELL Technologies. All cytokines used were obtained from R&D systems. Y-27632 was purchased from Stemgent. Human Collagen IV was from Advanced BioMatrix. Matrigel and the antibodies for flow cytometry were obtained from BD Biosciences. StemPro Accutase was from Life Technologies. Heparin was purchased from Sigma.
7
Murine and Human Megakaryocyte Culture
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BM cells were isolated from mouse femurs and plated in DMEM containing Glutamax (Invitrogen) supplemented with 10% FBS (GE Hyclone), 50 units/mL of penicillin, and 50 μg/mL of streptomycin and indicated concentration of murine Thrombopoietin (TPO; BioLegend). Cells were incubated at 37°C with 5% CO2 for 5–6 days. Human MKs were cultured from CD34+ cells isolated from the BM of patients with multiple myeloma. Mononuclear cells were isolated using Ficoll-paque gradient centrifugation. CD34 cells were isolated using CD34 MicroBead kit (Miltenyi Biotech, RRID: AB_2848167) according to manufacturer's instructions. The isolated cells were plated in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 20% BIT9500 serum substitute and 1 × StemSpan Megakaryocyte Expansion Supplement (both from StemCell Technologies). The cultures were incubated at 37°C with 5% CO2 for 14 days. Cultured murine MKs were isolated using a BSA gradient as described previously (31 (link)).
8
Expansion of Megakaryocytes from MPN Patients
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CD34+ cells were sorted from MPN patient and HI cryopreserved bone marrow mononuclear cells (BMMCs) with MicroBeads (Miltenyi Biotec). Sorted cells (2 × 104) were plated in triplicate in serum-free media containing StemSpan Megakaryocyte Expansion Supplement (STEMCELL Technologies) for 10–12 days. Media were replaced every 5 days. Megakaryocyte cell surface markers CD41 (FITC-conjugated anti-CD41; X, X) and CD61 (PerCP-conjugated anti-CD61; X, X) were measured by flow cytometry. MEG-01 cells were treated with 10 ng/mL phorbol 12-myristate 13-acetate (PMA) to induce megakaryocyte differentiation (63 (link)).
9
Expansion and Differentiation of Human CD34+ Cells
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Human CD34 þ cells were isolated from the buffy coats of healthy adult human donors provided by the Geneva University Hospitals' blood bank using a CD34 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD34 þ cell quantification was evaluated using an anti-CD34 PE antibody (Miltenyi Biotec) followed by flow cytometry analysis. More than 95% of the cells were positive for CD34. CD34 þ cells were cultured for 7 days in StemSpan Serum-Free Expansion Medium (Stemcell Technologies, Vancouver, Canada) supplemented with 20 ng/mL human low-density lipoprotein (LDL, Stemcell Technologies), StemSpan Megakaryocyte Expansion Supplement (CC220, Stemcell Technologies), penicillin-streptomycin-glutamine (PSG, Gibco, Thermofisher, Waltham, Massachusetts, United States) and 1 μM StemRegenin 1 (SR1, Cellagen Technology, San Diego, California, United States), as described elsewhere. 15 Next, the cells were washed, seeded and cultured in the presence of 0.5 μg/mL thrombopoietin (TPO, Stemcell Technologies), LDL, PSG and SR1 for an additional 8 days (►Supplementary Fig. S1A, available in the online version).
All the experiments described below were repeated with different, independent batches of cells from different blood bank donors.
All the experiments described below were repeated with different, independent batches of cells from different blood bank donors.
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