For secondary co-culture, floating cells from day-10 iPSC-OP9 co-culture were harvested and 2 × 105 cells were seeded onto fresh OP9 in differentiating medium in the presence of 50 ng/ml SCF, 20 ng/ml TPO, 20 ng/ml IL-3, 20 ng/ml IL-6, and 0.5 U/ml erythropoietin (R&D Systems). Floating cells were harvested after 3 days and analyzed by Wright-Giemsa staining. For myeloid expansion, day-10 floating cells were recultured on fresh OP9 monolayer in differentiating medium with 100 ng/ml GM-CSF. Flow cytometry was performed after 3 days. For erythroid differentiation, day-10 floating cells were seeded onto fresh OP9 in differentiating medium with 50 ng/ml SCF, 20 ng/ml TPO, and 0.5 U/ml erythropoietin for 7 days and analyzed by flow cytometry. For megakaryocyte differentiation, day-10 floating CD34+CD45+ cells were seeded onto ultralow attachment plates (Corning) in differentiating medium containing 50 ng/ml SCF, 20 ng/ml TPO, 20 ng/ml IL-3, 20 ng/ml IL-6, and 20 ng/ml IL-11.
Erythropoietin
Manufactured by R&D Systems
Sourced in United States
Erythropoietin is a glycoprotein hormone produced by the kidneys that stimulates the production of red blood cells in the bone marrow. It plays a crucial role in the regulation of erythropoiesis, the process of red blood cell formation.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 6 (il 6), by R&D Systems (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (2 mentions)
C reactive protein (crp), by R&D Systems (2 mentions)
Adiponectin, by R&D Systems (2 mentions)
Insulin, by Mercodia (2 mentions)
Ferritin, by Abcam (2 mentions)
Gdf 15, by R&D Systems (2 mentions)
Interleukin 3 (il 3), by Corning (1 mentions)
Ultra low attachment plate, by Corning (1 mentions)
Interleukin 3 (il 3), by R&D Systems (1 mentions)
Stem cell factor (scf), by Miltenyi Biotec (1 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (1 mentions)
Insulin like growth factor 1, by R&D Systems (1 mentions)
Interleukin 6 il 6, by R&D Systems (1 mentions)
Il 11, by R&D Systems (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Stempro 34, by Thermo Fisher Scientific (1 mentions)
12 protocols using erythropoietin
1
Directed Differentiation of iPSCs into Hematopoietic Lineages
2
Embryoid Body Generation from hESCs
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To generate EBs, we treated hESCs with collagenase B (1 mg/mL; Roche) for 20 min. Cells were gently scraped with a cell scraper to form small aggregates (10–20 cells). Aggregates were resuspended in StemPro-34 (Invitrogen), supplemented with L-glutamine (2 mM), ascorbic acid (1 mM), monothioglycerol (4 × 10−4 M; Sigma-Aldrich), and transferrin (150 mg/mL; Roche). BMP4 (10 ng/mL; R&D), basic fibroblast growth factor (5 ng/mL; Peprotech), SB431542 (6 μM; Tocris), vascular endothelial growth factor (15 ng/mL; R&D), interleukin-6 (IL-6) (10 ng/mL; R&D), insulin-like growth factor 1 (25 ng/mL; R&D), IL-11 (5 ng/mL; R&D), stem cell factor (SCF) (50 ng/mL; Miltenyi), erythropoietin (2 U/mL), thrombopoietin (30 ng/mL; R&D), IL-3 (30 ng/mL; R&D), and FMS-like tyrosine kinase 3 ligand (FLT3L) (10 ng/mL; Miltenyi) were added as indicated (see Figure 3 A). Cultures were maintained in a 5% CO2/5% O2/90% N2 environment. On the day of assay, EBs were harvested and dissociated to single cells by a 40-min treatment with 0.2% collagenase IV. Afterward, 1 mL of medium with serum was added and the EBs were dissociated to single cells by passaging six times through a 20-gauge needle.
3
Isolation of Peripheral Blood Mononuclear Cells
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PBMNCs were isolated by Ficoll-Paque Premium (GE Healthcare, Little Chalfont, UK)
according to the method published by Bin-Kuan Chou et al.28 (link) In brief, mononuclear cells (MNCs) were isolated by loading 20 mL diluted PB onto a
layer of 15ml of Filcoll-Paque Premium, then the layer of MNCs was harvested, washed, and
resuspended in mononuclear cell (MNC) medium, which consisted of 50% Iscove's Modified
Dulbecco's Medium (Gibco, Grand Isle, NY, USA), 50% Ham's F12 (Gibco),
Insulin-Transferrin-Selenium-Ethanolamine (Gibco), lipid concentrate (Gibco), 5 mg/mL
bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA), 50 µg/mL of L-ascorbic acid
(Sigma), 2 mM Glutamax (Gibco), 200 µM 1-thioglycerol (Sigma), supplemented with 10 ng/mL
interleukin-3 (PeproTech, Rocky Hill, NJ, USA), 100 ng/mL human stem cell factor
(PeproTech), 40 ng/mL insulin-like growth factor 1 (PeproTech), 2 U/mL erythropoietin
(R&D Systems, Minneapolis, USA), 100 mg/mL human holo-transferrin (R&D Systems),
and 20 mM dexamethasone (Sigma). Cells were seeded at low-attachment six-well plates and
maintained in MNC medium for 8–12 days before reprograming, and culture medium was changed
every other day.
according to the method published by Bin-Kuan Chou et al.28 (link) In brief, mononuclear cells (MNCs) were isolated by loading 20 mL diluted PB onto a
layer of 15ml of Filcoll-Paque Premium, then the layer of MNCs was harvested, washed, and
resuspended in mononuclear cell (MNC) medium, which consisted of 50% Iscove's Modified
Dulbecco's Medium (Gibco, Grand Isle, NY, USA), 50% Ham's F12 (Gibco),
Insulin-Transferrin-Selenium-Ethanolamine (Gibco), lipid concentrate (Gibco), 5 mg/mL
bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA), 50 µg/mL of L-ascorbic acid
(Sigma), 2 mM Glutamax (Gibco), 200 µM 1-thioglycerol (Sigma), supplemented with 10 ng/mL
interleukin-3 (PeproTech, Rocky Hill, NJ, USA), 100 ng/mL human stem cell factor
(PeproTech), 40 ng/mL insulin-like growth factor 1 (PeproTech), 2 U/mL erythropoietin
(R&D Systems, Minneapolis, USA), 100 mg/mL human holo-transferrin (R&D Systems),
and 20 mM dexamethasone (Sigma). Cells were seeded at low-attachment six-well plates and
maintained in MNC medium for 8–12 days before reprograming, and culture medium was changed
every other day.
4
Erythropoietin-Induced CFU-E Formation
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Single cell suspensions were prepared from CBA/Pk, Char10C and C57BL/6J spleen and bone marrow in IMDM (Life Technologies, ON, Canada) supplemented with 10% FBS and 100U/mL penicillin/streptomycin (Thermo Scientific, UT, USA). Spleen or bone marrow were harvested from 3 mice per group and pooled. Cells were plated into 1% methylcellulose medium supplemented with 15% FBS, 1% BSA, 10 μg/mL insulin and 200 μg/mL transferrin (M3234, Stem Cell Technologies, BC, Canada). Various concentrations (0–200 mU/mL) of erythropoietin (R&D Systems, Minneapolis, MN, USA) as well as 2 mM of L-glutamine (Life Technologies, ON, Canada) were added to the medium. Cells were plated in 35 mm dishes (Sarstedt, Montreal, Canada) at a density of 4x104 cells/mL for spleen and 2x104 cells/mL for bone marrow. CFU-E were scored after 2.5 days of culture in a 5% CO2 humidified incubator kept at 37°C.
5
In Vitro Hematopoietic Differentiation
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The sorted mouse bone marrow cells were cultured in Iscove's Modified Dulbecco's Medium supplemented with 20% heat-inactivated fetal calf serum, 2 mM L-glutamine, 10 mM HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 μg/mL gentamicin, 1 × MEM nonessential amino acids, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate. The medium was supplemented with a myelo-erythroid cytokine cocktail consisting of 20 ng/mL each of recombinant mouse IL-3, IL-6, IL-7, IL-9, IL-11, thrombopoietin, stem cell factor, granulocyte-macrophage colony-stimulating factor purchased from Peprotech (Rocky Hill, NJ), IL-5 and erythropoietin from R&D Systems (Minneapolis, MN). The medium and the supplements were from Sigma-Aldrich (St. Louis, MO). The cells were cultured at 37°C with 5% CO2.
6
Erythropoietin Improves TSHI Outcomes
7
Quantification of Serum Biomarkers
8
Generation of iPSC Lines from PBMCs
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The iPSC line iPSC-UkB-Ctrl-XX was generated from PBMCs via expansion to a cellular intermediate of EPCs, using Stem Span SFEM II (Stem Cell Technologies) and addition of Erythropoietin (R&D Systems), Stem Cell Factor (PeproTech), Interleukin-3 (PeproTech), Insulin Growth Factor-1 (PeproTech), and dexamethasone (Sigma-Aldrich). Following expansion, EPCs were electroporated with Epi5 Reprogramming (Thermo Fisher Scientific) Lonza 4D-Nucleofector X-Unit (Lonza). Cells were plated on Corning ESC-grade Matrigel (Corning) in ReproTeSR (Stem Cell Technologies) before changing to mTeSR 1 (Stem Cell Technologies) when colonies first appeared. Following iPSC clones were picked and expanded before characterization. iPSC-UkB-Ctrl-XX line is cultured on Corning ESC-grade Matrigel and in mTeSR 1. Cells are passaged when colonies are too large using 0.5 mM EDTA in PBS and cultured in a humidified incubator at 37°C and 5% CO2.
9
Canine Bone Marrow Cell Proliferation Assay
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Canine bone marrow cells were isolated under sterile conditions from the humerus of beagle dogs and placed in warmed DMEM medium (Gibco Life Technologies). The sample was filtered through a stainless steel wire mesh (Sigma-Aldrich) and gently ground with a glass pestle. The suspension was then passed through a 100-μm cell strainer and washed three times in warmed Alsever's solution (Sigma-Aldrich). The final pellet was resuspended in 20 mL of DMEM complete medium. Cell suspensions were plated in 96-well plates (Costar Corning) at a density of 2 × 105 cells per well in DMEM complete medium containing oclacitinib (0.001–10 μm ) or vehicle control, and Erythropoietin (0.2 U per well Erythropoietin; R&D Systems). Plates were incubated at 37 °C for 48 h. Tritiated thymidine, 0.4 μCi per well (Perkin Elmer), was added for 20 additional hours. Plates were frozen and then thawed, washed, and filtered using a Brandel MLR-96 cell harvester and prewet filter mats (Perkin Elmer). Filters were dried at 60 °C for 1 h and placed into filter sample bags (Perkin Elmer) with 10 mL of scintillant (Perkin Elmer). Sealed filters were counted on an LKB Wallac 1205 Betaplate liquid scintillation counter. Data were expressed as percent control, and dose–response data were then analyzed using a 4-parameter logistic equation.
10
Quantification of Serum Biomarkers
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Serum samples were used for estimation of the following analytes, using commercially available ELISA kits: insulin (#10–1247-01, Mercodia, Sweden), hepcidin (#S-1465.0001, Peninsula Laboratories, USA), ferritin (#Ab157713, Abcam, UK), adiponectin (#MRP300, R&D Systems, USA), erythropoietin (#MEP00B, R&D Systems, USA), C-reactive protein (#MCRP00, R&D Systems, USA), GDF-15 (#DT6385, R&D Systems, USA) and interleukin-6 (#DY406–05, R&D Systems, USA). All assays were carried out as per manufacturers' instructions. Serum levels of iron were estimated, using a colorimetric assay based on bathophenanthrolene dye-binding as described earlier [41 (link)]. Serum triglyceride was estimated using a commercially available kit (#10010303, Cayman Chemicals, USA).
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