Hsc expansion cocktail

Human MSCs were seeded and grown until 80% confluency in 6-well plates. HPSCs were exposed to ¹³⁷Cs γ-rays using a Gamma Cell-3000 irradiator (MDS Nordion International, Ontario, Canada) at a dose rate of 5 Gy/min. MSC medium was removed and non-irradiated or irradiated CD34+CD38− cells (1 × 10⁵ cells per well) were added to 2 mL of HSC culture medium containing HSC Expansion Cocktail (Miltenyi Biotech). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. The co-cultures were incubated for 3 and 6 days, at which HSCs were harvested and analyzed.

Human umbilical cord blood (UCB) was obtained from the umbilical vein immediately after delivery, with the informed consent of the mother; the protocol was approved by the Boramae Hospital Institutional Review Board (IRB) and the Korea Institute of Radiological & Medical Science IRB (IRB No. K-1501-002-022). Mononuclear cells (MNCs) were isolated from UCB using Ficoll-Hypaque (Sigma, St. Louis, MO, USA) gradient centrifugation. Next, cells were sorted from the MNCs using a magnetic cell-sorting MACS CD34+CD38− isolation kit (Miltenyi Biotech, Auburn, CA, USA) following the manufacturer’s protocol. CD34+CD38− cells were cultured with StemMACS HSC expansion media containing HSC Expansion Cocktail (Miltenyi Biotech). Umbilical cord blood-derived MSCs were purchased from the ATCC (Manassas, VA) and cultured with MSC growth medium MSCGM (Lonza, Walkersville, MD, USA).