Biomedical White Papers

Long-Term Engrafting Umbilical Cord Blood Cells Are Preserved after Ex Vivo Culture in Stroma-Free Culture

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• biomedical,
• term,
• culture,
• ex

Overview This article describes stroma-based and clinically applicable stroma-free cultures that maintain long-term engrafting umbilical cord blood (UCB) cells for at least 14 days ex vivo. UCB CD34+ cells were cultured in transwells above AFT024 feeders (AFT-NC) with Flt-3 ligand (FL), stem cell factor (SCF), interleukin (IL)-7, and/or thrombopoietin (TPO) or in stroma-free cultures with glycosaminoglycans (GAGs) and the same cytokines found in stromal supernatants (SF). Progeny were transplanted into NOD-SCID mice or preimmune fetal sheep. SCID repopulating cells (SRCs) with multilineage differentiation potential were maintained in AFT-NC culture with FL/SCF/IL- 7¨C or FL/TPO¨Ccontaining cultures for up to 28 days. Marrow from mice engrafted with high levels of uncultured or expanded cells induced multilineage human hematopoiesis in 50% of secondary but no tertiary recipients. Day-7 expanded cells engrafted primary, secondary, and tertiary fetal sheep recipients. Whereas day-14 expanded cells engrafted primary and to a lesser degree secondary fetal sheep, they failed to engraft tertiary recipients. Likewise, ¡Ý14- day SF cultures maintained SRCs that could be transferred to secondary NODSCID recipients. This is the first demonstration that ex vivo culture in AFT-NC and stroma-free cultures maintains long-term engrafting cells¡ªdefined by their capacity to engraft secondary or tertiary hosts¡ªand supports retroviral gene transfer into SRCs.

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