Clinimacs cell selection device

All UCB units were thawed using standard methods (Rubinstein et al., (1995) (link)). On day −15, the lower dosed unit was CD34 enriched using the CliniMACS Cell Selection Device (Miltenyi Biotec) following manufacturer’s instructions and placed in expansion media and the CD34 negative fraction was cryopreserved. The expansion culture media consisted of SCF, FLT-3L, TPO and IL-6 (all at 50 ng/mL) and SR-1 without the addition of antibiotics. Prior to lot release, the product was required to have a cell viability ≥70%, negative gram stain, endotoxin <5EU/kg, and negative in-process aerobic and anaerobic bacterial/fungal cultures (Table S1). On day 0, the unmanipulated unit was infused followed 4 hours later by the infusion of HSC835. On day +1, the cryopreserved CD34 depleted cells were thawed and infused.

Leukopaks containing G-CSF mobilized peripheral blood CD34⁺ HSPCs were purchased (Apheresis Care Group, Inc. San Francisco, CA) and CD34⁺ HSPCs purified by magnetic bead selection using a CliniMACS cell selection device (Miltenyi Biotec, Auburn, CA). The enriched CD34⁺ HSPCs were resuspended in mobilized blood CD34 maintenance media: X-Vivo 10 (Lonza, Basel, Switzerland) supplemented with 2 mM L-glutamine, 1% penicillin/streptomycin/amphotericin B (PSA) (Sigma Aldrich, St Louis, MO), and 100 ng/mL each of stem cell factor (SCF), fms-like tyrosine kinase 3 (flt-3) ligand and thrombopoietin (TPO) (PeproTech, Rocky Hill, NJ,).
Fetal liver samples were obtained from Advanced Bioscience Resources (Alameda, CA) or Novogenix Laboratories (Los Angeles, CA), as anonymous waste samples, with approval of the University of Southern California’s Institutional Review Board. Human CD34⁺ HSPCs were isolated from the tissues following physical disruption and incubation in collagenase to give single cell suspensions, followed by magnetic-activated cell sorting (MACS) (Miltenyi Biotec), as previously described⁴⁷ . Fetal liver–derived HSPCs were cultured in fetal liver CD34 maintenance media consisting of X-Vivo 15 (Lonza) supplemented with 50ng/ml each of SCF, Flt3 ligand and TPO (R&D Systems, Minneapolis, MN), plus 1% PSA.