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

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Abstract
This report describes stroma-based and stroma-free cultures that maintain long-term engrafting hematopoietic cells for at least 14 days ex vivo. Umbilical cord blood (UCB) CD34+ cells were cultured in transwells above AFT024 feeders with fetal-liver-tyrosine-kinase (FL) + stem cell factor (SCF) + interleukin 7 (IL-7), or FL + thrombopoietin (Tpo). CD34+ progeny were transplanted into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice or preimmune fetal sheep. SCID repopulating cells (SRC) with multilineage differentiation potential were maintained in FL-SCF-IL-7 or FL-Tpo containing cultures for up to 28 days. Marrow from mice highly engrafted with uncultured or expanded cells induced multilineage human hematopoiesis in 50% of secondary but not tertiary recipients. Day 7 expanded cells engrafted primary, secondary, and tertiary fetal sheep. Day 14 expanded cells, although engrafting primary and to a lesser degree secondary fetal sheep, failed to engraft tertiary recipients. SRC that can be transferred to secondary recipients were maintained for at least 14 days in medium containing glycosaminoglycans and cytokines found in stromal supernatants. This is the first demonstration that ex vivo culture in stroma-noncontact and stroma-free cultures maintains “long-term” engrafting cells, defined by their capacity to engraft secondary or tertiary hosts.

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