Direct cd34 progenitor cell isolation kit

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).

Human CD34⁺ cells were isolated from the cord blood of 15 females after informed consent from the mother and IRB approval. Single-cell suspensions of 1 × 10⁵/0.3 mL of HUCBC were administered via the tail vein immediately after the termination of whole body heating (WBH). All protocols were approved by the Animal Ethics Committee of the Chi Mei Medical Center (Tainan, Taiwan) in accordance with the Guide for the Care and Use of Laboratory Animals of the National Science Council and the Guidelines of the Animal Welfare Act.
Human CD34⁺ cells were isolated from cord blood using a Direct CD34⁺ Progenitor Cell Isolation kit (Miltenyi Biotec, Bergisch Gladack, Germany) and CD34⁺ Multisort kit (Miltenyi Biotec) according to the manufacturer's protocol. In brief, human cord blood lymphocytes and monocytes were suspended in 300 μL of phosphate buffered saline (PBS) and 5 mM EDTA. These cells were labeled with a hapten-conjugated monoclonal antibody against CD34 (PharMingen, San Diego, CA), followed by an antihapten antibody coupled with microbeads, and were incubated with beads at ratios of 100 μL of beads per 10⁸ cells for 15 min at 4°C. FACS analysis using anti-CD34 antibodies (Phar Mingen) labeled with phycoerythrin (Becton Dickinson, Mountain View, CA) of MACS-sorted cells showed that 96 ± 3% of the selected cells were positive for CD34.