Milodistim(Amgen, Inc.)

Allogeneic stem cell transplantation with umbilical cord blood (UCB) cells is limited by the cell dose a single unit provides recipients. Ex vivo expansion is one strategy to increase the number of cells available for transplantation. Aastrom Biosciences developed an automated continuous perfusion culture device for expansion of hematopoietic stem cells (HSCs). Cells are expanded in media supplemented with fetal bovine serum, horse serum, PIXY321, flt-3 ligand, and erythropoietin. We performed a phase 1 trial augmenting conventional UCB transplants with ex vivo–expanded cells. The 28 patients were enrolled on the trial between October 8, 1997 and September 30, 1998. UCB cells were expanded in the device, then administered as a boost to the conventional graft on posttransplantation day 12. While expansion of total cells and colony-forming units (CFUs) occurred in all cases, the magnitude of expansion varied considerably. The median fold increase was 2.4 (range, 1.0-8.5) in nucleated cells, 82 (range, 4.6-266.4) in CFU granulocyte-macrophages, and 0.5 (range, 0.09-2.45) in CD34+ lineage negative (lin–) cells. CD3+ cells did not expand under these conditions. Clinical-scale ex vivo expansion of UCB is feasible, and the administration of ex vivo–expanded cells is well tolerated. Augmentation of UCB transplants with ex vivo–expanded cells did not alter the time to myeloid, erythroid, or platelet engraftment in 21 evaluable patients. Recipients of ex vivo–expanded cells continue to have durable engraftment with a median follow-up of 47 months (range, 41-51 months). A randomized phase 2 study will determine whether augmenting UCB transplants with ex vivo–expanded UCB cells is beneficial.

Prolific cultures of human bone marrow mononuclear cells (BM MNCs) were recently developed that include a full spectrum of hematopoietic and accessory cells, with the presence of autofluorescent cells indicating adequate cell expansion. However, phenotypic and functional clonogenic characterizations of the autofluorescent cells and the various other subpopulations present in these cultures have not been carried out.

Cells from a continuously perfused bioreactor inoculated with BM MNCs and cultured for 12 days in serum‐containing medium with PIXY321, erythropoietin, and with or without FLT3‐L were evaluated by using flow cytometry.

Two antibodies, CD71 and CD13, allowed the separation of the autofluorescent cells into two distinct populations. The CD71+CD13++ autofluorescent population contained the colony‐forming unit (CFU) fibroblast, and the CD71++CD13++ autofluorescent population contained macrophage/dendritic like cells. The CFU‐granulocyte/macrophage (CFU‐GM) could not be thoroughly evaluated with CD71 and CD13. However, the number of CD13+/++Lin− cells correlated with the number of CFU‐GM (r = 0.83), with approximately 1 CFU‐GM for every 30 CD13+/++Lin− cells.

The data showed that CD71 and CD13 antibodies separate the autofluorescent cells into two populations but do not separate hematopoietic cells into specific phenotypic populations. The data also showed that the number of CD13+/++Lin− cells correlated with the number of CFU‐GM. These data present the initial step toward detailed phenotypic analysis of ex vivo expanded human BM MNC cultures. Cytometry Part A 53A:22–27, 2003. © 2003 Wiley‐Liss, Inc.