Summary:Delayed engraftment, graft failure, and adverse transplant-related events have been observed in unrelated umbilical cord blood (UCB) recipients, particularly in those receiving a low leukocyte cell dose and in CML patients. We report the outcomes of two older adult patients with high risk CML who received a low leukocyte cell dose of unmanipulated UCB cells supplemented with ex vivo expanded (AastromReplicell System) UCB cells. Each engrafted promptly and neither patient experienced GVHD or life-threatening infection. Both remain engrafted with cells exclusively of donor origin and are in cytogenetic remission at 19 and 8 months follow-up. Ex vivo expanded UCB cells appear to facilitate hematopoietic recovery and therefore may increase the number of CML patients eligible for unrelated UCB transplant. Bone Marrow Transplantation (2000) 25, 797-799. Keywords: chronic myelogenous leukemia; umbilical cord blood transplant; ex vivo expansion Transplantation of umbilical cord blood (UCB) from related and unrelated donors has been performed in an attempt to increase the number of potential allogeneic stem cell donors. 1,2 The limited availability of unrelated donor UCB units of adequate nucleated cell dose, however, restricts its application in older adult patients. Ex vivo expansion of UCB in small and clinical-scale regulated perfusion experiments (AastromReplicell System, Ann Arbor, MI, USA) has been proven capable of significantly increasing the number of total nucleated cells, CFU-GM, and longterm culture initiating cells. 3 Therefore, the effect of supplementing unrelated donor UCB with ex vivo expanded UCB cells from the same donor was evaluated in two older adult patients with high risk CML and no alternative donor.

Summary:Use of umbilical cord blood (CB) for stem cell transplantation has a number of advantages, but a major disadvantage is the relatively low cell number available. Ex vivo cell expansion has been proposed to overcome this limitation, and this study therefore evaluated the use of perfusion culture systems for CB cell expansion. CB was cryopreserved using standard methods and the thawed unpurified cells were used to initiate small-scale cultures supplemented with PIXY321, flt-3 ligand, and erythropoietin in serum-containing medium. Twelve days of culture resulted in the optimal output from most CB samples. Frequent medium exchange led to significant increases in cell (93%), CFU-GM (82%) and LTC-IC (350%) output as compared with unfed cultures. As the inoculum density was increased from 7.5 ؋ 10 4 per cm 2 to 6.0 ؋ 10 5 per cm 2 , the output of cells, CFU-GM, and LTC-IC increased. Cell and CFU-GM output reached a plateau at 6.0 ؋ 10 5 per cm 2 , whereas LTC-IC output continued to increase up to 1.2 ؋ 10 6 per cm 2 . Because the increase in culture output did not increase linearly with increasing inoculum density, expansion ratios were greatest at 1.5 ؋ 10 5 per cm 2 for cells (6.4-fold) and CFU-GM (192-fold). Despite the lack of adherent stroma, CB cultures expressed mRNA for many growth factors (G-CSF, GM-CSF, IL-1, IL-6, LIF, KL, FL, Tpo, TGF-␤, TNF-␣, and MIP-1␣) that were also found in bone marrow (BM) cultures, with the exception of IL-11 (found only in BM) and IL-3 (found in neither). Culture output was remarkably consistent from 10 CB samples (coefficient of variation 0.3) indicating that the procedure is robust and reproducible. Two commercial serum-free media were evaluated and found to support only approximately 25% of the culture output as compared with serum-containing medium. Implementation of optimal conditions in the clinical scale, automated cell production system (CPS) showed that the process scaled-up well, generating 1.7 ؋ 10 7 CFU-GM (298-fold expansion) from 1.2 ؋ 10 8 thawed viable nucleated CB cells (n = 3). The ability to generate Ͼ Ͼ Ͼ10 7 CFU-GM from Ͻ15 ml of CB within this closed, automated system without the need for extensive cell manipulations

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Summary:To reduce the number of apheresis procedures and maintain the usual rate of hematopoietic recovery in patients treated with high-dose chemotherapy, we studied the effect of adding a small volume of ex vivo expanded bone marrow to low doses of CD34 + blood stem cells. Thirty-four patients with breast cancer received G-CSF (10 g/kg/day) priming followed by a limited volume (50-100 ml) bone marrow aspiration and standard 10-liter aphereses. Marrow was expanded ex vivo using the AastromReplicell system and infused along with low doses of blood-derived CD34 + cells, collected in one apheresis. Thirty-one evaluable patients received a median CD34 + blood stem cell dose of 0.7 × 10 6 /kg (range, 0.2-2.5) and 4.7 × 10 7 nucleated cells/kg (range, 1.98-8.7) of ex vivo expanded marrow. All patients recovered with normal blood counts and engrafted 500 neutrophils/l and 20 000 platelets/l in a median of 10 and 13 days, respectively. Multivariate analysis revealed that, in addition to CD34 + lineage negative cell quantity, the quantity of stromal progenitors contained in the ex vivo expanded product correlated with engraftment outcome (r = 0.551, P = 0.004

Ex vivo culture of hematopoietic progenitor cells for autologous transplantation has generated world-wide interest, since it offers the prospect of using a limited cell number, and may allow more efficient gene transfer and passive elimination of contaminating tumor cells. In this study, we expanded bone marrow (BM) cells from 10 breast cancer patients to determine whether small BM aliquots can durably restore hematopoiesis, and whether thrombopoietin (TPO) improves hematopoietic reconstitution after myeloablative chemotherapy. We used the AastromReplicell System (ARS), performing a computer-controlled, stromal-based cell expansion process with frequent medium, cytokine and gas exchange. For the inoculation of 9 x 10(8) MNC, a median BM volume of 97.8 ml (range, 72.4-272) was harvested. We found a median 4.5-fold nucleated cell expansion, an 18-fold CFU-GM expansion, and 69% of input LTC-IC numbers. Nucleated and Lin-/CD34+ cells were infused with a median of 43.5 x 10(6)/kg (range, 34.1-71.7) and 2.8 x 10(5)/kg (range, 0.95-5.9), respectively. Despite tumor cell detection by immunocytochemical staining in 3/10 patients before expansion, tumor cells were not detectable in 9/10, and in one patient 1 log reduced post ARS culture. Following high-dose STAMP V chemotherapy, all patients received 12-day expanded BM cells. The median time to engraftment was 17 days (range, 11-20) for WBC >1000/microl, and 28 days (range, 21-55) for platelets >20,000/microl. A correlation between post-expansion Lin-/CD34+ cells and engraftment for ANC >500/microl, WBC >1000/microl and platelets >20,000/microl was observed. Hematopoiesis has been maintained for a median of 15 (range, 6-24) months. Our results demonstrate that transplantation of ex vivo expanded small BM aliquots allows hematopoietic reconstitution after myeloablative chemotherapy. Ex vivo generated ARS cells can reduce the risk of tumor cell reinoculation with autotransplants and may be valuable in settings in which only small stem cell doses are available, eg when using cord blood transplants or in non-mobilizing patients.