Unmanipulated Cord Blood Research Articles

Transplantation of Cord Blood Expanded Ex Vivo with Copper Chelator.

Cord blood (CB) is used to restore hematopoiesis in transplant patients lacking marrow donors. CB is associated with higher rates of delayed/failed engraftment. Peled et al developed an expansion technology using the copper chelator tetraethylenepentamine (TEPA), which enhanced the expansion of primitive CB populations when combined with early acting cytokines. A phase I clinical trial employing this technology was initiated. 10 patients with high-risk, heavily pre-treated hematologic malignancies (AML-2, ALL-5, HD-2, and NHL-1) have been enrolled with CB units that were cryopreserved in 2 fractions [20:80% (n=2), 40:60% (n=5) or 50:50% (n=3)]. 21days prior to infusion, AC133+ cells were isolated from the smaller (if unequal) or 50% CB fractions using the CliniMACS device and cultured for 21 days in media containing 10% FBS and SCF, FLT-3, IL6, TPO plus the copper chelator TEPA (Gamida). Patients then received myeloablative therapy with ATG and either fludara and busulfan (AML), or fludara, melphalan, thiotepa (ALL, HD, NHL) with infusion of the unmanipulated CB fraction on day 0, and the expanded fraction on day +1. GVHD prophylaxis was methotrex 5 mg/m2 days 2, 4,7, and tacrolimus for 6 months. The median age was 21 (range 7–51) and weight 69 (range 31–156) kg. The CB units were matched at 4/6 (n=8) or 5/6 (n=2) HLA antigens. The pre-thaw total nucleated cell (TNC) dose of the CB units was a median of 2.5x107/kg with post-thaw TNC of 2.4 x107/kg. Following AC133-selection, the manipulated CB fractions were a median of 73 (range 38–95)% AC133+ with a median of 0.650 (range 0.16–2.7) x106 TNCs, which were placed in culture. After 21 days of culture the expanded fraction had 69 (range 2–1638) x106 TNCs representing a 207 (range 2–616) fold TNC expansion. Patients received a total (expanded plus unmanipulated) median of 1.8 (range 1.1–6.1) x107 TNC/kg and 1.6x105 (range 0.4–49.9) CD34+ cells/kg. Two patients have CB cultures in progress. Of the 8 patients transplanted, 1 had autologous recovery with relapse of AML on day 30 and death. Of the remaining patients, 7 were evaluable for neutrophil engraftment and 4 of them for platelet engraftment (2 too early for platelet evaluation and 1 early death). The median time to engraftment was 27 days for neutrophils (range 16–46) and 48 days for platelets (range 27–96). Preliminary analysis suggest a correlation between a shorter time to neutrophil engraftment and total TNC/kg infused (p=0.02), and a trend for CD34+ cells/kg infused (p=0.09). Three patients have developed grade ≤2 acute skin GVHD and one had chronic extensive GVHD of the skin and GI tract; all resolved with steroids. One patient (without GVHD) died of a systemic viral infection on day 56, despite adequate neutrophil recovery (not platelets). All of the remaining patients are all alive and free of malignancy at a median follow-up of 4 (range 1–16) months.Conclusion: There was no toxicity associated with infusion of the TEPA-expanded CB cells. Additional data is necessary to determine the efficacy of this approach. Future directions include the expansion of the entire CB unit and removal of methotrex from the GVHD regimen to improve time to neutrophil engraftment, as well as comprehensive assessment of immune reconstitution.

Transplantation of ex vivo expanded cord blood

Umbilical cord blood (CB) from unrelated donors is increasingly used to restore hematopoiesis after myeloablative or more recently, nonmyeloablative therapy. CB transplants are associated with higher rates of delayed and failed engraftment than are bone marrow transplants, particularly for adult patients. At the University of Colorado, we studied the ex vivo expansion of CB in an attempt to improve time to engraftment and reduce the graft failure rate in the recipients. In the study, 43 patients with hematologic malignancies (n = 40) or breast cancer (n = 3) received high-dose therapy followed by unrelated allogeneic CB transplantation [1Shpall E.J. Quinones R. Giller R. et al.Transplantation of ex vivo expanded cord blood.Biol Blood Marrow Transplant. 2002; 8: 368-376Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar]. A fraction of each patient’s CB allograft was CD34-selected and cultured ex vivo for 10 or 14 days prior to transplantation in defined media with stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), and megakaryocyte growth and differentiation factor (MGDF). The remainder of the CB graft was infused without further manipulation. Two sequential cohorts of patients were accrued to the study. The first cohort had 40% and the second cohort had 60% of their CB graft expanded. This study demonstrated that the CD34 selection and ex vivo expansion of CB prior to transplantation is feasible. The engraftment failure rate was relatively low, and the time to neutrophil in large adults was in the range reported for pediatric recipients of much larger CB cell doses. In the successor cord expansion trial being conducted at the M.D. Anderson Cancer Center, several changes in the approach have been made. Patients with hematologic malignancies are now being randomized to receive either two unmanipulated CB units or one unmanipulated unit and one unit from which all the cells are expanded ex vivo. For the patients randomized to expansion, on day −14 one of their cord units is AC133-selected using the CliniMACS device. The AC133-negative fraction containing the T cells is frozen and the AC133+ fraction is cultured ex vivo for 14 days in media containing SCF, G-CSF and thrombopoietin (TPO). Following administration of the preparative regimen, on Day 0, the second unmanipulated CB unit is infused, followed by the thawed AC133-negative fraction and finally, the fraction which has been in culture for two weeks. This trial was recently initiated and accrual is in progress. Another expansion trial technology involves the use of a copper chelating agent, which has been shown by Peled et al to enhance the expansion of a more primitive CD34+ CB population when combined with early acting growth factors. A clinical trial employing this technology is in progress at M.D. Anderson. Patients with hematologic malignancies undergoing CB transplantation, with a unit that is cryopreserved in two fractions are eligible for the study. Twenty one days prior to infusion, the AC133+ cells are isolated from the smaller cord fraction using the CliniMACS device and cultured for three weeks in media containing interleukin-6 TPO, FLT-3, SCF, and the copper chelator tetraethylenepentamine (TEPA). The patients then receive high-dose therapy with the infusion of the larger, unmanipulated CB fraction on day 0, and infusion of the smaller expanded fraction of day +1. Thus far eight patients have been enrolled on the study and the results will be forthcoming. Future directions include the use of mesenchymal stem cells as a supportive stroma for the expansion of CB mononuclear cell progenitors, developed by McNiece et al. This approach would obviate the need for CD34 or AC133-selection, as those procedures are associated with substantial losses of CD34+ CB cells

Quantitative analysis demonstrates expansion of SCID-repopulating cells and increased engraftment capacity in human cord blood following ex vivo culture with human brain endothelial cells.

Initial clinical trials examining the transplantation of ex vivo expanded cord blood (CB) cells have failed to demonstrate an impact on hematopoietic recovery compared with historical unmanipulated CB controls. In this study, we tested whether coculture with primary human brain endothelial cells (HUBECs) could increase the engraftment capacity and repopulating cell frequency within CB CD34+ cells. Quantitative analysis demonstrated that HUBEC coculture for 7 days supported a 19-fold greater number of CD34+ cells and 3.4-fold and 2.6-fold greater severe combined immunodeficient (SCID)-repopulating cell (SRC) frequencies than fresh CB CD34+ cells and liquid suspension-cultured cells. Mice transplanted with day-14 HUBEC-cultured cells showed 4.2-fold higher levels of human engraftment than mice transplanted with day-7 HUBEC-cultured cells, indicating that SRC enrichment continued to occur through day 14. Noncontact HUBEC cultures also maintained SRCs at levels comparable with contact HUBEC cultures, demonstrating that HUBEC-secreted soluble factors critically supported SRC self-renewal. Seeding efficiency studies demonstrated that HUBEC-cultured CB CD34+ cells engrafted nonobese diabetic/SCID marrow at significantly higher levels than either fresh CB CD34+ cells or liquid suspension-cultured CD34+ cells. These studies indicate that the application of HUBEC coculture or HUBEC-conditioned media can potentially improve upon current strategies for the clinical expansion of CB stem cells.

Ex Vivo Expansion of Bone Marrow and Cord Blood Cells to Produce Stem and Progenitor Cells for Hematopoietic Reconstitution

Ex vivo expansion of a small volume of bone marrow offers an alternate approach to cellular support for repair of a hematopoietic system damaged by radiation exposure. Unpurified bone marrow cells cultured using frequent exchange of medium (perfusion) results in (1) the growth of an adherent layer that provides a supportive microenvironment for hematopoiesis and (2) the growth of hematopoietic stem and progenitor cells. Similar increases in stem and progenitor cells were also observed in cord blood cultures using frequent medium exchange methods. An automated clinical scale system, the AastromReplicell Cell Production System, has been developed to implement the biology of this ex vivo expansion process. Clinical use of ex vivo expanded bone marrow alone or in augmenting a low dose of mobilized peripheral blood stem cells has successfully reconstituted hematopoiesis. Ex vivo expanded cord blood cells combined with unmanipulated cord blood cells have also shown significant benefits in overall survival and engraftment in pediatric and adult patients, respectively. These results suggest that ex vivo expansion of hematopoietic cells provides a mechanism for generating cells capable of hematopoietic reconstitution.

Transplantation of adult and pediatric cancer patients with ex vivo expanded cord blood

Cord blood (CB) cells are being used increasingly as a source of allogeneic hematopoietic support, but have been associated with delays in engraftment. In this study we evaluated ex vivo expansion of CB from unrelated donors in 29 patients (21 adult, 8 children) with AML (n = 5), ALL (n = 8), CML (n = 3), CLL (n = 3), NHL (n = 5), HD (n= 2), and breast ca (n = 3). The patients received high-dose melphalan and ATG plus TBI (n = 17) or busulfan (n = 4), or TBI-cytoxan-ARA-C (n = 8 children). Strata A patients (n = 22) had CB frozen in one fraction (fx). On day 0 to transplant, the CB was thawed and 60% of the CB was infused without manipulation. The CD34+ cells in the remaining 40% of the CB were isolated and expanded in teflon bags in 800 ml of media containing 100 ng/ml each of rhSCF, rhG-CSF and rhMGDF. After 10 days in culture the expanded product was harvested, washed and reinfused. Strata B patients (n = 7), whose CB was frozen in two fx, had the 40% fx thawed on day −10 and cultured for 10 days as described above. Both the expanded and the unmanipulated CB were infused on day 0. The cultures generated a median 89 (range 42-270) fold expansion of cells and 4 (range 2-13) fold expansion of CD34+ cells. To date, all 19 of 20 (95%) patients, evaluable for engraftment, achieved recovery of neutrophils (ANC ≥500/mm3) in a median of 26 (range 15–45) days. Fourteen of 15 (93%) patients engrafted platelets (≥20,000/mm3, unsupported) in a median of 58 (range 27–380) days. All patients tested at day +60 (n = 11) have been 98–100% donor. Eight of 13 patients (62%) experienced acute and 7/10 (70%) extensive chronic GVHD. Preliminary analysis suggests that the neutrophil engraftment failure rate of 5% in our patients may be lower than the 15–60% failure rate reported for recipients of unexpanded CB transplants. The median time to neutrophil and platelet engraftment rates in adult patients appears to be comparable to that in smaller pediatric patients despite significantly lower CB cell doses.