ultrapure human epo protein  (R&D Systems)

Journal: Animal Cells and Systems

Article Title: Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

doi: 10.1080/19768354.2025.2492148

Figure Lengend Snippet: In vitro erythroid differentiation of canine PB MNCs with recombinant cEPO and recombinant hEPO. (a) Total cumulative cell numbers on differentiation days with cEPO at concentrations of 10 ng/mL ( n = 5), 20 ng/mL ( n = 5), 40 ng/mL ( n = 8), 80 ng/mL ( n = 5), and 160 ng/mL ( n = 2). *Indicates statistical significance with p ≤ 0.05. (b) Photographs of cell pellets on day 10 and day 14 of culture. (c) Total cumulative cell numbers with cEPO (40 and 80 ng/mL) compared to hEPO (40 and 80 ng/mL) ( n = 1). (d) Total cumulative cell numbers with cEPO (30, 40, and 50 ng/mL) compared to those with hEPO (30, 40, and 50 ng/mL) ( n = 1).

Article Snippet: The cultures were also supplemented with cytokines, including recombinant canine erythropoietin (cEPO) (10–160 ng/mL, R&D Systems, cat#3816-CE), recombinant human erythropoietin (hEPO) (40–80 ng/mL, Millipore, cat#329871), recombinant canine stem cell factor (cSCF) (100 ng/mL, R&D Systems, cat#2278-SC), and recombinant human interleukin-3 (hIL-3) (10 ng/mL, R&D Systems, cat#203-IL).

Techniques: In Vitro, Recombinant

Journal: Animal Cells and Systems

Article Title: Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

doi: 10.1080/19768354.2025.2492148

Figure Lengend Snippet: Characterization of cell types arising from in vitro erythroid differentiation of canine PB MNCs using cEPO at 40 ng/mL. (a) Representative Wright-Giemsa-stain images of cells during in vitro erythroid differentiation over 17 days. Orange arrowheads; Orthochromatic erythroblast, red arrowheads; Reticulocyte. (b) Identification of the major erythroid lineage cell types emerging from canine PB MNCs during in vitro differentiation, with a comparison to corresponding human cell types. (c) Quantification of cumulative erythroid lineage cell types during in vitro erythroid differentiation over 17 days. Erythroid cell types were quantified by counting over 200 cells per specific day of differentiation from Wright-Giemsa-stain images, and the stacked percentages of cell types were plotted. Scale bars: 10 μM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: The cultures were also supplemented with cytokines, including recombinant canine erythropoietin (cEPO) (10–160 ng/mL, R&D Systems, cat#3816-CE), recombinant human erythropoietin (hEPO) (40–80 ng/mL, Millipore, cat#329871), recombinant canine stem cell factor (cSCF) (100 ng/mL, R&D Systems, cat#2278-SC), and recombinant human interleukin-3 (hIL-3) (10 ng/mL, R&D Systems, cat#203-IL).

Techniques: In Vitro, Giemsa Stain, Comparison

Journal: Animal Cells and Systems

Article Title: Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

doi: 10.1080/19768354.2025.2492148

Figure Lengend Snippet: Quantification of cumulative erythroid lineage cell types during in vitro erythroid differentiation of canine PB MNCs with different concentrations of cEPO and hEPO. (a and b) Erythroid cell types on day 17 of the in vitro differentiation experiments presented in a were quantified by counting over 200 cells for each specific concentration of cEPO from Wright-Giemsa-stain images. Quantification of cumulative erythroid lineage cell types with 10, 20, 40, 80 and 160 ng/mL of cEPO (a), and the stacked percentages of cell types (b). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (c) Stacked percentages of erythroid cell types with cEPO (40 and 80 ng/mL) compared to those with hEPO (40 and 80 ng/mL). Erythroid cell types on day 21 of the in vitro differentiation experiments presented in (b) were quantified by counting over 100 cells for each specific concentration of cEPO and hEPO from Wright-Giemsa-stain images. RBC; red blood cells, Ortho-E; Ortho-erythroblasts, and Pro-/Baso-/Poly-E; pro-erythroblasts/baso-erythroblasts/poly-erythroblasts.

Article Snippet: The cultures were also supplemented with cytokines, including recombinant canine erythropoietin (cEPO) (10–160 ng/mL, R&D Systems, cat#3816-CE), recombinant human erythropoietin (hEPO) (40–80 ng/mL, Millipore, cat#329871), recombinant canine stem cell factor (cSCF) (100 ng/mL, R&D Systems, cat#2278-SC), and recombinant human interleukin-3 (hIL-3) (10 ng/mL, R&D Systems, cat#203-IL).

Techniques: In Vitro, Concentration Assay, Giemsa Stain

Journal: Animal Cells and Systems

Article Title: Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

doi: 10.1080/19768354.2025.2492148

Figure Lengend Snippet: Expression profiles of erythroid differentiation marker genes during in vitro erythroid differentiation of canine PB MNCs with cEPO (40 ng/mL) over 3 weeks. (a) mRNA expression profiles of canine erythroid markers: canine alpha hemoglobin stabilizing protein ( cAHSP ; n = 5), canine Krüppel-like factor 1 ( cKLF1 ; n = 8), canine erythropoietin receptor ( cEPOR ; n = 6), and canine transferrin receptor ( cTFRC ; n = 6). qRT-PCR was performed on total RNA extracted from specific days of differentiation, and the values (2^-ΔΔCt) were normalized to the expression levels at day 0. Blue lines represent the mean values, and the gray shades indicate the standard deviation (± SD). (b) A schematic representation of mRNA expression profiles of corresponding marker genes during human erythropoiesis. The mRNA expression data were obtained from previous studies (dos Santos et al. ; Marsee et al. ; Dzierzak and Philipsen ; Lee et al. ). It is noteworthy that hAHSP expression begins to increase in the baso-erythroblast stage, reaching its highest level in the reticulocyte. In contrast, hEPOR is expressed at the earliest stage of erythroid cell development, the proerythroblast, increases until the poly-erythroblast stage, but then starts to decrease and becomes undetectable in the reticulocyte. Although both hKLF1 and hTFRC start to be expressed in the baso-erythroblast stage, hKLF1 reaches its highest level in the ortho-erythroblast, while hTFRC peaks in the early reticulocyte stage.

Article Snippet: The cultures were also supplemented with cytokines, including recombinant canine erythropoietin (cEPO) (10–160 ng/mL, R&D Systems, cat#3816-CE), recombinant human erythropoietin (hEPO) (40–80 ng/mL, Millipore, cat#329871), recombinant canine stem cell factor (cSCF) (100 ng/mL, R&D Systems, cat#2278-SC), and recombinant human interleukin-3 (hIL-3) (10 ng/mL, R&D Systems, cat#203-IL).

Techniques: Expressing, Marker, In Vitro, Quantitative RT-PCR, Standard Deviation

Journal: Animal Cells and Systems

Article Title: Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

doi: 10.1080/19768354.2025.2492148

Figure Lengend Snippet: Functional analysis of day 17 cells from in vitro erythroid differentiation of canine PB MNCs with cEPO (40 ng/mL). (a) Western blot analysis of hemoglobin α (HBA) expression in differentiated cells from three different donors on day 21 of differentiation: lanes #1, #2, and #3. The lane labeled ‘RBC’ indicates peripheral blood RBCs collected from a reference dog. The arrow indicates the monomeric hemoglobin A (HBA), whereas the asterisk (*) denotes the HBA dimer. (b) Oxygen-binding capacity of the cells on day 18 of differentiation, demonstrating comparability to peripheral blood RBCs obtained from a different donor than the one used for in vitro differentiation.

Article Snippet: The cultures were also supplemented with cytokines, including recombinant canine erythropoietin (cEPO) (10–160 ng/mL, R&D Systems, cat#3816-CE), recombinant human erythropoietin (hEPO) (40–80 ng/mL, Millipore, cat#329871), recombinant canine stem cell factor (cSCF) (100 ng/mL, R&D Systems, cat#2278-SC), and recombinant human interleukin-3 (hIL-3) (10 ng/mL, R&D Systems, cat#203-IL).

Techniques: Functional Assay, In Vitro, Western Blot, Expressing, Labeling, Binding Assay

Journal: Animal Cells and Systems

Article Title: Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

doi: 10.1080/19768354.2025.2492148

Figure Lengend Snippet: Structural differences between human and canine EPO. (a) Amino acid sequence alignment of mature human EPO (hEPO, P01588) and canine EPO (cEPO, P33707). Multiple sequence alignment was performed using Clustal Omega (PDB id: clustalo-I20241209-014629-0930-11133740-p1 m). The dotted square regions indicate the predicted amphipathic α-helical structures. (b) Aligned 3D structures of hEPO (magenta) and cEPO (cyan) in the D3 and D4 amphipathic α-helical regions, visualized using ChimeraX. The amino acid sequences of D3 (residues 89–107) are as follows: hEPO: EPLQLHVDKAVSGLRSLTT; cEPO: ETPQLHVDKAVSSLRSLTS. The amino acid sequences of D4 (residues 131–152) are as follows: hEPO: RTITADTFRKLFRVYSNFLRGK; cEPO: RTFTVDTLCKLFRIYSNFLRGK. Residues in a significant difference in three-dimensional structure are highlighted with white circles in the enlarged images.

Article Snippet: The cultures were also supplemented with cytokines, including recombinant canine erythropoietin (cEPO) (10–160 ng/mL, R&D Systems, cat#3816-CE), recombinant human erythropoietin (hEPO) (40–80 ng/mL, Millipore, cat#329871), recombinant canine stem cell factor (cSCF) (100 ng/mL, R&D Systems, cat#2278-SC), and recombinant human interleukin-3 (hIL-3) (10 ng/mL, R&D Systems, cat#203-IL).

Techniques: Sequencing