85-23, revised 1996), and all University requirements. Human cell purification Umbilical Cord Blood (UCB) that failed to meet the selleck chemicals MG132 minimal total nucleated cell count was obtained from the cord blood banking facility at Cardinal Glennon Children’s Hospital, St Louis, MO, and used in accordance with the ethical guidelines at Washington University School of Medicine and the principles outlined in the Declaration of Helsinki. Mononuclear cells (MNCs) were isolated from UCB by Hypaque-Ficoll centrifugation (Pharmacia Biotech, Uppsala, Sweden). MNCs from different cord blood samples were pooled (24 cords were used in total) and lineage depleted or enriched for CD34+ cells as previously described[8].

Briefly, UCB MNCs were incubated with a human-specific lineage depletion antibody cocktail or anti human CD34 antibody followed by magnetic bead labeling before negative or positive selection, respectively, on an immunomagnetic separation column, according to the manufacturer’s directions (Stem Cell Technologies, Vancouver, BC, Canada). FACS sorting of aldehyde dehydrogenase high and low expressing cells Cells to be sorted were cultured overnight in X-Vivo 15 media (Lonza Group, Basel, Switzerland) on RetroNectin coated plates (25 ��g/cm2; Takara Bio INC., Otsu, Japan) in the presence of recombinant human SCF, Flt3-L and TPO (all 10 ng/ml, R&D Systems, Minneapolis, MN) and nano-particles in selected experiments as indicated below. Total cells were detached on the following day by gentle washing with Cell Dissociation Buffer (CDB, Invitrogen, Carlsbad, CA) and purified according to their levels of ALDH activity by staining with the Aldefluor reagent (Aldagen, Durham, NC), according to the manufacturer’s specifications.

Briefly, Aldefluor substrate (0.625 ��g/mL) was added to 1 to 5 �� 106 Lin- cells/mL suspended in Aldefluor assay buffer and incubated for 20 to 30 minutes at 37��C. Cells were then FACS sorted on a MoFlo (BD, San Jose, CA) according to high and low Aldefluor signal as described [8]. Whole organ fluorescent imaging 655 nm fluorescent emitting nano-particle labeling Human UCB Lin- or CD34+ cells were incubated with 655 nm fluorescent Quantum Dot nano crystals (QD655, Invitrogen) in cell media (X-Vivo with recombinant human SCF, Flt3-L and TPO (all 10 ng/ml)) in the presence of 0.

1 nM protamine sulphate for 15 min followed by overnight incubation in cell media at 106 cells/well on Retronectin coated non-tissue culture treated 24 well plates at 37��C and 5% CO2. The following day the GSK-3 Lin- cells were then detached by gentle washing with CDB and resuspended in PBS and sorted according to high or low expression of ALDH as described above. The cells were then subjected to a second round of labeling overnight as described. CD34+ sorted cells were labeled in parallel but without sorting for ALDH activity.