In vitro and in vivo evidence for the long-term multilineage (myeloid, B, NK, and T) reconstitution capacity of ex vivo expanded human CD34(+) cord blood cells

Objective. The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic p rogenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity . Materials and Methods. CD34(+) cord blood (CB) cells were cultured in serum -free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 1 4 days, the primitive functions of expanded and nonexpanded cells were dete rmined in vitro using clonogenic cell (colony-forming cells, long-term cult ure initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis as says (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells al so was assessed by determining their telomere length and telomerase activit y. Results. Levels of expansion were up to 1,613-fold for total cells, 278-fol d for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 2 1-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they we re able to generate myeloid progenitors and lymphoid cells for 5 months. Th ese primitive progenitors engrafted the NOD-SCID bone marrow, which contain ed LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CD19(-) c ells sorted from the engrafted marrow were able to generate CD19(+) B-cells , CD56(+)CD3(-) NR. cells, and CD4(+)CD8(+)alpha beta TCR+ T-cells in speci fic cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 w eeks of culture. Conclusions. These experiments provide strong evidence that expanded CD34() CB cells retain their ability to support long-term hematopoiesis, as show n by their engraftment in the NOD-SCID model, and to undergo multilineage d ifferentiation along all myeloid and the B-, NK, and T-lymphoid pathways. T he expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical ap plications. (C) 2000 International Society for Experimental Hematology. Pub lished by Elsevier Science Inc.