CBF
  1. Ex vivo expansion of umbilical cord blood hemopoietic stem and progenitor cells. McNiece I, Gluckman E, Wagner JE, Verfaillie CM. Exp Hematol. 2004;32:409-413.

In a series of three short editorials the authors review the current status of ex-vivo expansion of umbilical cord blood stem and progenitor cells. McNiece points out that clinical trials have demonstrated that ex-vivo-expanded cells can be cultured and safely infused, but no significant clinical benefit has been demonstrated to date. A number of challenges remain regarding ex-vivo expansion of cord blood (CB) cells: (1) The CB products in the majority of CB banks are frozen as single products so that the infusion of the expanded cells occurs 10 to 14 days after the unmanipulated product, thus minimizing the potential to demonstrate a significant effect on engraftment, (2) CD34 selection is required for optimal ex-vivo expansion, (3) clinical grade growth factors are required by regulatory agencies, and (4) regulation by the FDA will increase the complexity and cost of undertaking ex-vivo expansion trials. No reports have demonstrated the feasibility of expanding human stem cells (HSCs) and, in contrast, the clinical ex-vivo expansion studies performed to date most likely result in decreased levels of HSCs. However, since neutrophils, platelets and the cells involved in immune recovery may arise from distinct cells, the optimal graft may require a mixture of neutrophil progenitors, megakaryocyte progenitors and T cell precursors.

Gluckman points out that several options are under discussion to improve the speed of engraftment of cord blood transplants: (1) ex-vivo expansion with cytokine cocktails, (2) multi-cord transplants or intra-bone infusion of CD34 cells, and (3) addition of mesenchymal cells which may facilitate cell proliferation and engraftment both in vitro and in vivo and decrease the risk of GVHD. They state that there are very few studies ongoing and that there is a clear need for prospective clinical trials.

Wagner and Verfaillie point out that it is unknown which cell should be expanded (i.e., stem cell, primitive progenitor, or more committed progenitor) but, in any case, it would seem optimal that stem cells not be lost at the expense of generating progenitors incapable of self-renewal. They insist that it is critical that the manufacture of the expansion culture media be performed in a GMP cell therapy facility staffed with technologists experienced in clinical cell therapy. They further emphasize that it has been difficult to determine how expansion culture might be tested clinically, since studies have placed only a proportion of the UCB graft into expansion culture, and the lack of a genetic marker prevents any ability to track the expanded population. To solve this problem the authors suggest transplanting two partially HLA-matched UCB units as a model for testing the safety and potential efficacy of expansion culture.

  1. Pre-clinical development of cord blood-derived progenitor cell graft expanded ex vivo with cytokines and the polyamine copper chelator tetraethylenepentamine. Peled T, Mandel J, Goudsmid RN, et al. Cytotherapy 2004;6:344-355. Abstract

The authors indicate that they have previously demonstrated that the copper chelator tetraethylenepentamine (TEPA) enables preferential expansion of early hematopoietic progenitor cells (CD34+ CD38-, CD34+CD38-Lin-) in human umbilical cord blood-derived CD34+ cell cultures. In the present study they established a clinically applicable protocol for large-scale ex vivo expansion of cord blood-derived progenitors. CD133+ cells purified from cord bloods were cultured for 3 weeks in a clinical-grade closed culture bag system, using the chelator-based technology in combination with early-acting cytokines (SCF, thrombopoietin, IL-6 and FLT-3 ligand). The median output value of CD34+ cells increased 89-fold, CD34+CD38- cells increased by 30-fold, and CFUc increased by 172-fold over the input value. Transplantation into sublethally irradiated NOD/SCID mice indicated that the engraftment potential was significantly superior to that of unexpanded cells. The chelator-based ex-vivo expansion technology is currently being tested in a phase 1 clinical trial in patients undergoing cord blood transplantation for hematological malignancies.

  1. Improved haematopoietic recovery following transplantation with ex vivo-expanded mobilized blood cells. Prince HM, Simmons PJ, Whitty G, Wall DP, Barber L, Toner GC, Seymour JF, Richardson G, Mrongovius R, Haylock DN. Br J Haematol. 2004;126:536-45. Abstract

The authors demonstrated that CD34+ cells expanded ex vivo with the cytokine combination of G-CSF, SCF and PEG-rHuMGDFDF resulted in a 33-fold and 2.8 fold expansion of total cells and CD34+ cells, respectively. They tested the efficacy of the ex-vivo-expanded cells in three cohorts of three patients with breast cancer who received three cycles of repetitive high dose chemotherapy supported by either unmanipulated and/or ex-vivo-expanded cells. Efficacy was assessed by an internal comparison of each patient's consecutive high dose chemotherapy cycles, and to 106 historical controls using unmanipulated cells.

Twenty-one cycles were supported by ex-vivo-expanded cells and six by unmanipulated cells alone. Infusions of ex-vivo-expanded cells resulted in fewer days with an absolute neutrophil count (ANC) <0.1 x 10(9)/l (median 2 vs. 4 d, P = 0.002) and 3 d faster ANC recovery to >0.1 x 10(9)/l (median 5 vs. 8 d, P = 0.0002). This resulted in a major reduction in the incidence of febrile neutropenia compared with unmanipulated cycles (0% vs. 83%; P = 0.008) and in 66% of historical unmanipulated cycles (P = 0.01) and a marked reduction in hospital re-admission. There were also fewer platelet transfusions required (43% vs. 100%; P = 0.009). The investigators considered that the most important effect was the markedly reduced depth of thrombocytopenia following high dose chemotherapy, which resulted in 43% of the cycles not requiring any platelet transfusions.

The authors pointed out that utilization of "internal" controls, with six patients having one of their three high dose chemotherapy cycles supported by unmanipulated cells with the remaining two cycles supported by ex-vivo-expanded cells, provides compelling evidence that the ex-vivo-expanded cells enhanced neutrophil recovery. This finding was further supported by data provided by the historical control group.

The investigators concluded that ex-vivo-expanded cells enhance both neutrophil and platelet recovery and reduce febrile neutropenia, platelet transfusion and hospital re-admission.

  1. Elevated telomerase activity and minimal telomere loss in cord blood long-term cultures with extensive stem cell replication. Gammaitoni L, Weisel KC, Gunetti M, Wu KD, Bruno S, Pinelli S, Bonati A, Aglietta M, Moore MA, Piacibello W. Blood. 2004;103:4440-8. Abstract

The authors indicate that there is clearly a clinical value for a system that provides extensive ex-vivo stem cell expansion without concomitant telomere erosion. They evaluated telomerase activity, telomere length, stem/progenitor cell production, and function of CD34(+) cells from cord blood (CB), bone marrow, and mobilized peripheral blood in long-term cultures. CB cells were cultured either on OP-9 stromal cells transduced with an adenovector expressing thrombopoietin (TPO) or stimulated by a cytokine cocktail in the absence of stroma, with, in one method, CD34(+) cells re-isolated at monthly intervals for passage. Despite extensive proliferation, telomere length initially increased and only at late stages of culture was evidence of telomere shortening noted. This telomere stabilization correlated with maintenance of high levels of telomerase activity in the CD34(+) cell population for prolonged periods of culture.

  1. Clinical application of hematopoietic progenitor cell expansion: current status and future prospects. Devine SM, Lazarus HM, Emerson SG. Bone Marrow Transplant 2003; 31:241-252.Abstract

In this review of ex vivo expansion of hematopoietic progenitor cells, the authors point out that in the past decade, there have been significant advances in ex vivo hematopoietic stem cell culture expansion, progressing to the point where clinical trials are being designed and conducted. Reported clinical trials are reviewed in depth; other topics reviewed include, "the path to the present", "defining new clinical targets – umbilical cord blood", "additional biologic advantages of cord blood", "current bottlenecks to clinical development", and "where do we go from here?" They point out that successful clinical application of expanded hematopoiesis will clearly require a greater understanding of human stem cell biology, identification of the proper balance and concentration of the available cytokine cocktails for ex vivo culture, and hastening the slow pace of clinical trials.

  1. Ex vivo expansion of hematopoietic progenitor cells and mature cells.  McNiece I, Briddell R.  Exp Hematol. 2001;29:3-11. Abstract 

The authors of this review state that many investigators have explored methods to culture hematopoietic cells in vitro to increase the numbers of these cells. Studies attempting to expand hematopoietic stem cells, progenitor cells, and mature cells in vitro have become possible over the past decade due to the availability of recombinant growth factors and cell selection technologies. However, the authors state that, as of the date of this publication, no studies have demonstrated convincing data on the expansion of true stem cells. A number of clinical studies have been performed using a variety of culture conditions and there are evolving data that suggest that there are real clinical benefits associated with the use of the expanded cells. The authors suggest that the next decade should determine what culture conditions and what cell populations are needed for a range of clinical applications.

  1. Ex-vivo expansion of cord blood mononuclear cells on mesenchymal stem cells. McNiece I, Harrington J, Turney J, Kellner J, Sphall EJ. Cytotherapy 2004;6:311-317. Abstract

The authors point out that the current clinical conditions for ex vivo expansion of cord blood (CB) cells require selection of the CD34+ subset or culture in perfusion systems, as unfractionated or even mononuclear cells (MNC) do not expand well in static culture. CD34+ cell selection of thawed CB units often results in low CD34+ cell recoveries (median recovery of 50%) and suboptimal purities, resulting in lower expansion overall. A number of studies have demonstrated the supportive role of stromal cells for hematopoietic stem cells and progenitor cells, and clinical studies have evaluated the potential of mesenchymal stem cells (MSCs) to facilitate engraftment and possibly decrease the incidence of GVHD. Accordingly, the authors evaluated the potential of MSCs to support ex vivo expansion of unselected CB products. Their results indicated that ex vivo expansion of CB MNC on MSC resulted in 10- to 20-fold expansion of total nucleated cells, seven- to 18-fold expansion of committed progenitor cells, two- to five-fold expansion of primitive progenitor cells and 16- to 37- fold expansion of CD34+ cells. The authors' current focus is to initiate clinical trials to evaluate the in vivo potential of CB cells expanded with these conditions.

  1. Augmentation of umbilical cord blood (UCB) transplantation with ex vivo-expanded UCB cells: results of a phase 1 trial using the AastromReplicell System.  Jaroscak J, Goltry K, Smith A, Waters-Pick B, Martin PL, Driscoll TA, Howrey R, Chao N, Douville J, Burhop S, Fu P, Kurtzberg J.  Blood. 2003;101:5061-5067. Full Text

The authors performed a phase 1 trial augmenting conventional UCB transplants with ex vivo-expanded cells. Twenty-eight patients were enrolled on the trial between October 8, 1997 and September 30,1998. UCB cells were expanded, 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. Augmentation of UCB transplants with ex vivo-expanded cells did not alter the time to myeloid, erythroid, or platelet engraftment in 21 evaluable patients.

  1. Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system.  Lewis ID, Almeida-Porada G, Du J, Lemischka IR, Moore KA, Zanjani ED, Verfaillie CM.  Blood. 2001;97:3441-3449. Full Text

The authors describe the ability of the murine fetal liver cell line, AFT024, to maintain/expand human umbilical cord blood CD34+ repopulating cells assayed in the NOD-SCID mouse and the fetal sheep model. They found that culture of cord blood CD34+ cells for up to 28 days in an AFT 024 noncontact system supplemented with a combination of early acting cytokines, including SCF, FL, Tpo, and IL-7, maintains repopulating cells in NOD/SCID as well as fetal sheep. They concluded that their data indicate that ex-vivo expanded cells continue to contain “long-term” repopulating cells because they can be serially passaged to secondary NOD-SCID mice and secondary and tertiary fetal sheep.

  1. Ex vivo expansion of megakaryocyte precursors from umbilical cord blood CD34 cells in a closed liquid culture system.  Shaw PH, Gilligan D, Wang XM, Thall PF, Corey SJ.  Biol Blood Marrow Transplant. 2003 Mar;9(3):151-6. Abstract

The authors propose to hasten platelet engraftment by expanding the number of megakaryocyte (MK) precursors (CD34/CD41 cells) through cytokine stimulation within a closed, pre-clinical liquid culture system. Thirteen UCB samples from full-term births were Ficoll-separated and frozen for subsequent use. On thawing, the mononuclear cell population was positively selected for CD34+ expression. The cells were cultured in gas-permeable Teflon-coated bags in serum-free medium containing the following cytokines: recombinant human interleukin-3, recombinant human Flt3 ligand, recombinant human stem cell factor, and recombinant human thrombopoietin. MK lineage cell expansion was assessed using mononuclear cell count and flow cytometry (CD34/41, CD41, CD34/61, and CD61 expression) on days 7, 11, and 14. Optimal expansion of CD34/41 and CD41 cells was observed at day 11, with a median 6-fold and 33-fold increase in the starting cell doses, respectively. CD34/61 and CD61 cell expansion at day 11 was 7-fold and 14-fold, respectively. The authors concluded that MK precursors can be successfully expanded from CD34+ UCB cells in a closed liquid culture system. However, they stated that their ex vivo expansion technique needs to be further optimized before it can be used in a pilot UCB transplantation trial.

  1. Transplantation of ex vivo expanded cord blood.  Shpall EJ, Quinones R, Giller R, Zeng C, Baron AE, Jones RB, Bearman SI, Nieto Y, Freed B, Madinger N, Hogan CJ, Slat-Vasquez V, Russell P, Blunk B, Schissel D, Hild E, Malcolm J, Ward W, McNiece IK. Biol Blood Marrow Transplant. 2002;8:368-376.Abstract

The authors 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 this feasibility study, 37 patients (25 adults, 12 children) with hematologic malignancies (n = 34) or breast cancer (n = 3) received high-dose therapy followed by unrelated allogeneic CB transplantation. A fraction of each patient's CB allograft was CD34-selected and cultured ex vivo for 10 days prior to transplantation in defined media with stem cell factor, granulocyte colony-stimulating factor, and megakaryocyte growth and differentiation factor. The remainder of the CB graft was infused without further manipulation. Patients received a median of 0.99 x 10(7) total nucleated cells (expanded plus unexpanded) per kilogram. The median time to engraftment of neutrophils was 28 days (range, 15-49 days) and of platelets was 106 days (range, 38-345 days). All evaluable patients who were followed for 28 days or longer achieved engraftment of neutrophils. Grade III/IV acute GVHD was documented in 40% and extensive chronic GVHD in 63% of patients. At a median follow-up of 30 months, 13 (35%) of 37 of patients survived. The authors concluded that their study demonstrates that the CD34 selection and ex vivo expansion of CB prior to transplantation of CB is feasible. Additional accrual will be required to assess the clinical efficacy of expanded CB progenitors.

  1. Human stem-progenitor cells from neonatal cord blood have greater hematopoietic expansion capacity than those from mobilized adult blood.   Tanavde VM, Malehorn MT, Lumkul R, Gao Z, Wingard J, Garrett ES, Civin CI.  Exp Hematol. 2002;30:816-823. Abstract

The authors compared the hematopoietic capacity of CD34+ cell preparations from neonatal cord blood (CB) vs adult mobilized peripheral blood (PBSC) before and after ex vivo culture. CD34+ cell preparations purified from CB or PBSC were cultured in serum-free medium containing FKT: FLT-3 ligand (FL), KIT ligand (KL), and thrombopoietin (TPO). After 1-4 weeks ex vivo culture, CB CD34+ cell preparations had greatly increased numbers of total cells, CD34+ cells, and colony-forming cells (CFC). In contrast, ex vivo-cultured PBSC CD34+ cell preparations generated far less in vitro assessed hematopoietic capacity. Nonobese diabetic severe combined immunodeficient mouse (NOD/SCID) engrafting potential (SEP) was maintained in ex vivo-cultured CB CD34+ cell preparations, whereas it was lost in ex vivo-cultured PBSC. CB CD34+ cells continued to proliferate throughout 3 weeks ex vivo, whereas after 1 week, no additional cell divisions were detected in PBSC CD34+ cells. After 3 weeks in culture, the average CB CD34+ cell had divided more than 5 times, as compared to only 2 times for the average PBSC CD34+ cell. The authors concluded that CB CD34+ cell preparations generated massively increased in vitro assessed hematopoietic capacity and maintained SEP during 1- to 4-week ex vivo cultures. In contrast, ex vivo-cultured PBSC CD34+ cell preparations generated far less in vitro assessed hematopoietic capacity and decreased SEP.

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