Journal: American journal of hematology

Article Title: Erythropoietic properties of human induced pluripotent stem cells-derived red blood cells in immunodeficient mice.

doi: 10.1002/ajh.26410

Figure Lengend Snippet: FIGURE 1 Generation of human induced pluripotent stem cells (iPSC)-derived erythroblasts and iPSC-red blood cells (RBCs). Human iPSC clones were generated by introduction of OCT4, SOX2, KLF4, MYC, and BCL-XL genes into primary erythroblasts from health donors. (A) Human iPSC clones were photographed during culture in E8 medium (20X). (B) Human iPSC were collected after treatment with accutase, and subjected to flow cytometry analyses with anti-human TRA-1-60, SSEA4, and Nanog antibodies, or isotype. (C) Alternatively, Human iPSC were seeded and differentiated into hematopoietic progenitor cells (HPC) with STEMdiff Hematopoietic kit. After 12-day culture, the cells in suspension were harvested and subjected to flow cytometry analysis with anti-human CD45 and CD34 antibodies. The resulting dot plot is presented. (D) Human iPSC-derived HPC were continuously differentiated into erythroblasts with StemSpan erythroid expansion kit for 14 days. The numbers of human iPSC-derived HPC and erythroblasts were counted, and the cell number fold increase was calculated based on seeded iPSC number. (E) Human iPSC-RBCs were harvested after 8-day maturation culture in the presence of human serum (Serum), human platelet lysate (hPL), or human plasma albumin (Albumin). The cells were subjected to flow cytometry analyses with anti-human CD34, CD49d, CD71, CD233, and CD235a antibodies. Normal human RBCs and iPSC-derived erythroblasts before maturation served as control cells. Flow cytometry histograms are presented. (F) iPSC-RBCs were also stained with the DRAQ5 dye. Normal human RBCs served as DRAQ5 cells. (G) The cell number fold-increase of iPSC- RBCs after 8-day maturation in the presence of human serum, platelet lysate or albumin was calculated based on initial iPSC number. Data were from three independent experiments

Article Snippet: iPSC were profiled with phycoerythrin-conjugated (PE) anti-human SSEA4 (Millipore Corporation), TRA-1-60 and Nanog antibodies (1:100; BD Pharmingen) on a FACSCalibur Flow Cytometer (BD). iPSC-derived cells were analyzed with: fluorescein isothiocyanateconjugated (FITC) anti-human CD235a (1:100; Caprico Biotechnologies); PE anti-human CD45 (1:100; BioLegend) and CD233 antibodies (1:100; Miltenyi Biotech); allophycocyanin-conjugated (APC) anti-human CD34, CD49d (Biolegend) and CD71 antibodies (1:100; Caprico Biotechnologies); and DNA-binding dye DRAQ5 (1:500; BioLegend).

Techniques: Derivative Assay, Clone Assay, Generated, Flow Cytometry, Suspension, Clinical Proteomics, Control, Staining

Journal: American journal of hematology

Article Title: Erythropoietic properties of human induced pluripotent stem cells-derived red blood cells in immunodeficient mice.

doi: 10.1002/ajh.26410

Figure Lengend Snippet: FIGURE 3 Human induced pluripotent stem cells-red blood cells (iPSC-RBCs) survival in clodronate liposomes (CL)- and cobra venom factor (CVF)-treated NOD scid gamma (NSG) mice. (A) Human iPSC-RBCs were transfused into NSG mice treated with the combination of CL and CVF before and after transfusion. Murine peripheral blood was collected after transfusion at different time points for flow cytometry analyses with anti-human CD235a antibody. Gated CD235a+ cells represent transfused human iPSC-RBCs. (B) The percentages of circulating human iPSC- RBCs in murine peripheral blood at selected time points were calculated from three independent experiments. (C) Murine peripheral blood cells were further stained with dye DRAQ5. Nucleated human cells were gated on DRAQ5+ population, and enucleated human iPSC-RBCs were DRAQ5. Flow cytometry dot plots at different time points were representative from three independent experiments. (D) Percentage of circulating enucleated human iPSC-RBCs at different time points after transfusion was calculated accordingly. (E) Alternatively, murine peripheral blood samples on day 24 after transfusion were also stained with anti-human CD235a, CD49d, CD71, CD233 antibodies, or isotype control. Flow cytometry histograms were represented from one of three independent experiments

Article Snippet: iPSC were profiled with phycoerythrin-conjugated (PE) anti-human SSEA4 (Millipore Corporation), TRA-1-60 and Nanog antibodies (1:100; BD Pharmingen) on a FACSCalibur Flow Cytometer (BD). iPSC-derived cells were analyzed with: fluorescein isothiocyanateconjugated (FITC) anti-human CD235a (1:100; Caprico Biotechnologies); PE anti-human CD45 (1:100; BioLegend) and CD233 antibodies (1:100; Miltenyi Biotech); allophycocyanin-conjugated (APC) anti-human CD34, CD49d (Biolegend) and CD71 antibodies (1:100; Caprico Biotechnologies); and DNA-binding dye DRAQ5 (1:500; BioLegend).

Techniques: Liposomes, Combined Bisulfite Restriction Analysis Assay, Flow Cytometry, Staining, Control