Umbilical cord blood cells capable of engrafting in primary secondary, andtertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system

This report describes stroma-based and stroma-free cultures that maintain l ongterm engrafting hematopoietic cells for at least 14 days ex vivo. Umbili cal cord blood (UCB) CD34(+) cells were cultured in transwells above AFT024 feeders with fetal-liver-tyrosine-kinase (FL) + stem cell factor (SCF) + i nterleukin 7 (IL-7), or FL + thrombopoietin (Tpo), CD34(+) progeny were tra nsplanted into nonobese diabetic-severe combined immunodeficiency (NOD-SCID ) mice or preimmune fetal sheep. SCID repopulating cells (SRC) with multili neage differentiation potential were maintained in FL-SCF-IL-7 or FL-Tpo co ntaining cultures for up to 28 days. Marrow from mice highly engrafted with uncultured or expanded cells induced multilineage human hematopoiesis in 5 0% of secondary but not tertiary recipients. Day 7 expanded cells engrafted primary, secondary, and tertiary fetal sheep. Day 14 expanded cells, altho ugh engrafting primary and to a lesser degree secondary fetal sheep, failed to engraft tertiary recipients. SRC that can be transferred to secondary r ecipients were maintained for at least 14 days in medium containing glycosa minoglycans and cytokines found in stromal supernatants, This is the first demonstration that ex vivo culture in stroma-noncontact and stroma-free cul tures maintains "long-term" engrafting cells, defined by their capacity to engraft secondary or tertiary hosts.(Blood.2001; 97:3441-3449) (C) 2001 by The American Society of Hematology.