Rhepo

Freshly sorted cells were plated at 200 cells/ml in 1% methylcellulose culture medium supplemented with 30% FBS (Fisher Scientific), 2 mM L-Glutamine (Lonza), 0.1 mM hemin (Sigma-Aldrich), 50 ng/ml rmSCF (R&D Systems), 1 U/ml recombinant human erythropoietin (rhEPO, Amgen), and 5% vol/vol pokeweed mitogen mouse spleen cell conditioned medium. Cells were cultured at 5% CO₂, 5% O₂ in a humidified incubator, and colonies were scored on day 6 based on morphologies of CFU-GM, BFU-E, and CFU-GEMM progenitors. For high specific activity tritiated thymidine kill assay to determine % CFU in S-phase of the cell cycle, cells were incubated in IMDM media containing either control diluent or 50 mCi high specific activity tritiated thymidine for 30 minutes in the same incubator for cell culture aforementioned before washing and plating. For hCB CFU assay, cells were plated in 1% methylcellulose culture medium supplemented with 30% FBS, 2 mM L-Glutamine, human recombinant EPO (1U/ml), SCF (50 ng/ml), GM-CSF (10 ng/ml) and IL-3 (10 ng/ml) and cultured under the same conditions as for mouse cells. Human CFU-GM and CFU-GEMM colonies were scored on day 14 after culture, as this combination of growth factors does not pick up BFU-E.

The dual MK and E colony assay was performed as previously described (Sanada et al., 2016 (link)). Liquid culture of sorted MEPs was performed in StemSpan Serum Free Expansion Medium (Stem Cell Technologies) with rhEPO (3.0 U/mL), rhIL-3 (10 ng/mL), rhIL-6 (10 ng/mL), and rhSCF (25 ng/mL) with/without rhTPO (50 ng/mL) for 48h. All cytokines purchased from ConnStem (Cheshire, CT) except rhEPO (Amgen).

Male mice were injected intraperitoneally (i.p.) with 5000 U/kg rhEPO (Amgen, Thousand Oaks, CA), the most common dose utilized in murine models [1 (link), 9 (link)]. Mice received a total of 6 of rhEPO, with the first dose of rhEPO administrated 8 h before hypoxemia. Daily doses were given for the next 2 consecutive days, followed by weekly rhEPO injections up to 1 month. Injured animals were randomized to receive rhEPO or saline vehicle administration using computer-generated number randomization.

For HPC colony assays, BM cells flushed from femurs of indicated mice were plated at 5 × 10⁴ cells/mL in 1% methylcellulose culture medium with 0.1 mM hemin (Sigma-Aldrich; St. Louis, MO, USA), 30% FBS, 1 U/mL recombinant human erythropoietin (rhEPO; Amgen; Thousand Oaks, CA, USA), 50 ng/mL recombinant mouse stem cell factor (rmSCF; R&D Systems; cat. # 455-MC), and 5% vol/vol pokeweed mitogen mouse spleen cell conditioned medium. Colonies were scored after 6 days of incubation at 5% CO₂ and lowered 5% O₂ in a humidified chamber, and granulocyte-macrophage colony-forming units (CFU-GM), erythrocyte burst-forming units (BFU-E), and granulocyte, erythrocyte, macrophage, megakaryocyte colony-forming units (CFU-GEMM) were distinguished by morphology of colonies. Total numbers of colonies per femur were calculated. For high specific activity tritiated thymidine kill assays, BM cells were treated with 50 μCi of high specific activity [3H]Tdr (20 Ci/mmol; DuPont NEN) at room temperature for 40 minutes then washed twice prior to plating in HPC colony assays. These assays were performed exactly as reported [14 (link)–16 ].