Successful Expansion of Umbilical Cord Blood Cells as a Source of Hematopoietic Progenitors for Adult Transplantation.

The low yield of progenitor CD34⁺ cells recovered from umbilical cord blood (UCB) limits the utility of this source for transplantation in adults. This limitation has triggered investigations into how ex vivo expansion of hematopoietic stem cells (HSC) could be achieved to allow for transplantation in larger recipients. In the present study, the incubation of MNC and not selected CD34⁺cells in the presence of SCF 25ng/ml+MGDF 10ng/ml+FLt-3 25ng/ml+IL-6 20ng/ml, and 10% human serum in stroma-free liquid culture generated long-term expansion of transplantable UCB HSC. In vitro, HSC expansion from 13 UCB lasted >7 months giving 39, 1.3x10⁴, and 4.7 x10⁹ fold increase in total cell count after 14, 70 and 217 d of expansion as compared to d0 (10⁵/ml). Similarly, CD34⁺ and CD34⁺/CD38⁻ cell populations increased reaching 229 and 2.2x10⁵ and 91 and 2.2x10⁴ fold after 14 and 70d of expansion. Examination of cell morphology and analysis by flow cytometry showed the presence of primitive and mature cells belonging to all hematopoietic cell lineages. Similarly, multilineage colonies with recloning capacity were generated in culture. Erythroid, myeloid and mixed colonies increased by 116 and 1.8x10⁴ fold and megakaryocytic colonies by 8 and 527 fold after 14 and 70d. Expanded cells were karyotypically normal and lacked the most common chromosome translocations seen in AML and CML t (15,17) and t (9, 22). HSC expanded for 6 and 13 weeks and cropreserved for 6–11 months were able to re-expand in liquid culture and generate colonies capable of recloning and multi-lineage differentiation. We estimated the frequency of SCID repopulating cells (SRC) in UCB samples expanded for 2 and 12 w using 1000,500,250 and 125 unselected CD34⁺ cells injected intravenously into sublethally irradiated NOD/SCID mice. Mice were sacrificed 20 weeks after transplantation. Human cell engraftment measured as CD45⁺ (HuCD45⁺) was detected in all mice (x3mice/dilution) (0.1–9.8%). In addition, HuCD45⁺ cells with multilineage phenotype were present, (CD19 (lymphoid), CD33 (myeloid), CD71and Glycophorin-A (erythroid) as well as CD34⁺/CD38⁻ cells). SRC increased by 90 fold after 2 weeks of expansion and by 187 fold after 12 weeks compared to unexpanded CD34⁺cells. Additional proof of human cell engraftment was documented using semisolid culture (MethoCult^TM GF H4434 Stem Cell Technologies). Human myeloid and erythroid colonies were generated from all dilutions, and counts ranged between 63–271/500,000 MNC.

Initial studies to test the relative magnitude of UCB HSC expansion from 24-well plates to culture bags (OptiCyte ^TM, Baxter) using one UCB, the total cell count increased by (6.3 and 20 (bags) Vs 2.3 and 3.3 fold (wells) after 7 and 14d) and CD34⁺ subpopulations including CD34⁺/CD38⁻. Based on these ongoing results, a phase II clinical trial using ex vivo expanded UCB for 14d in a setting of sub ablative Conditioning is planned.