Best Practice & Research Clinical Haematology

Best Practice & Research Clinical Haematology 23 (2010) 245 257 Contents lists available at ScienceDirect Best Practice & Research Clinical Haematology journal homepage: www.elsevier.com/locate/beha 10 Ex vivo expansion of umbilical cord blood for transplantation S.S. Tung, S. Parmar, S.N. Robinson, M. De Lima, E.J. Shpall * Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 423, Houston, TX 77030, USA Keywords: umbilical cord blood transplant ex vivo expansion immune reconstitution Umbilical cord blood (UCB) has become an important source of hematopoietic stem cell transplant (HSCT) for hematologic malignancies in adults. Its ready availability, allowance of higher HLA disparity and lower incidence of graft-versus-host disease (GVHD) makes it a very attractive source especially for minority populations. The major limitation to a wider use of this source of HSCT is the relative low number of progenitor cells in the graft. For this reason, adult UCB transplants are usually associated with delayed engraftment and increased rates of infectious complications. CB ex vivo expansion holds the promise of delivering higher cell doses and improved outcomes. Here we discuss different methods of expansion, their shortcomings and future directions. Ó 2010 Elsevier Ltd. All rights reserved. Introduction Not surprisingly, the use of umbilical cord blood (UCB) as a hematopoietic stem cell (HSC) source has been increasing in recent years and has become an important source of HSC support following myeloablative and non-myeloablative therapies [1 6]. Unfortunately, it is not without restrictions. The major limiting factor to UCB application is the low cell dose available for transplantation. It is well documented that the total nucleated cell dose (TNC) transplanted per kilogram (kg) of body weight of the recipient correlates with outcomes. Patients with a total body weight of at least 45 kg who receive only a single unit of UCB have been shown to have a significant delay in time to neutrophil and platelet engraftment, as well as higher rates of engraftment failure [7 11]. For this reason, UCB transplantation remains significantly more successful in children [9,10,12]. However, even in small children who have * Corresponding author. E-mail address: eshpall@mdanderson.org (E.J. Shpall). 1521-6926/$ see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2010.06.004

246 S.S. Tung et al. / Best Practice & Research Clinical Haematology 23 (2010) 245 257 received adequate cell doses, a delay is evident in engraftment of all cell lines when compared to traditional stem cell sources [13 15] as well as some delay with immune reconstitution [16,17]. In an analysis of the effects of various demographic, graft related and treatment factors on clinical outcomes of 102 patients transplanted with a single UCB unit, the CD34 þ cell dose was significantly associated with rate of engraftment, transplant-related mortality (TRM), and survival [18]. Time to neutrophil engraftment was strongly associated with CD34 þ cell dose, and there was an inferior rate of engraftment and higher TRM in patients transplanted with less than 1.7 10 5 CD34 þ cells/kg. Based on these and other confirmatory data, guidelines advocating the selection of products with higher TNC and CD34 þ doses emerged. Recent recommendations are that a single unit should ideally contain a minimum TNC of 2.5 3 10 7 /kg for closely matched UCB units (5/6 or 6/6 matches, considering low-resolution HLA A and B matching and high-resolution matching at HLA-DRB1), with possibly greater TNC targets in the setting of a greater mismatch. Our improved understanding of optimal unit characteristics and the expansion of cord blood banks have improved clinical outcomes, especially for larger pediatric and adult recipients; however, more stringent selection criteria leave more potential recipients without an UCB unit of sufficient size and/or HLA matching. There are two general approaches aimed at overcoming the issue of low TNC associated with UCB transplantation. One approach would be infusion of more than one UCB unit in order to attain an elevated infusible cell number [19 23] and the second approach being ex vivo expansion of UCB. In a recent prospective randomized controlled trial, a double UCB transplant was compared to a transplant using one unmanipulated UCB unit combined with one unit that was expanded ex vivo (de Lima et al. Blood (ASH annual meeting abstracts) 2008 112: Abstract 154). In this trial, 71 patients with advanced hematologic malignancies were randomized receiving either a myeloablative preparative regimen (n ¼ 41) or a non-myeloablative regimen (n ¼ 30), depending on disease and clinical status. Thirty-four patients (48%) were alive at a median follow-up of 11.3 months (range, 2 49). Most of the patients on the expanded arm had some evidence of the expanded UCB chimerism post-transplant (7 82%), however by 14 months all patients had predominance of the unmanipulated cord. This suggests that expansion may affect the durability of engraftment by ex vivo-expanded cells. Ex vivo expansion is conducted on whole UCB units as well as selected portions; these expanded products can then be infused concurrently with an unmanipulated UCB, or sometime after infusion of the unmanipulated unit. Currently, clinical protocols aimed at proving the beneficial nature of this strategy are being conducted at the University of Texas MD Anderson Cancer Center as well as other clinical centers [24 27] (Table 1). Promising studies have been conducted examining the clinical impact of UCB progenitors expanded in liquid cultures [27,28] or on marrow-derived mesenchymal cells [29]. An updated version of the latter approach is now in clinical trials at MDAnderson Cancer Center and preliminary results demonstrate early neutrophil and platelet engraftment relative to historical norms [30]. Although promising to decrease early mortality related to prolonged neutropenia and/or thrombocytopenia, such approaches are not intended or expected to improve immune recovery [31]. Other groups are testing the clinical utility of ex vivo expansion on immobilized Notch-family ligands including Delta1 [32 34] and Delta4; these approaches may induce T lineage directed progenitor expansion that may enhance immune recovery. Ultimately, the goal of ex vivo expansion is the production of an optimal number of HSCs for graft transplantation as well as an appropriate number of specific progenitor cells for the purpose of rapid recovery from pancytopenia. A decrease in morbidity and mortality can be achieved if these goals can be met efficiently. Combination of expanded and unmanipulated UCB may prove to be the most efficient method for attaining these results. Expansion methods Liquid culture Cytokines UCB cells are cultured with combinations of cytokines, growth factors and other growth promoting compounds in liquid culture. Prior to expansion initiation, isolation of relatively primitive

Table 1 Summary of clinical trials evaluating ex vivo-expanded UCB. Expansion type Liquid Culture Stromal culture Investigator N ¼ (adults/ children) Days in culture TNC fold expansion CD34 þ fold expansion # of days to ANC >500 # of days to platelets >20,000 Cytokines Incidence of acute GVHD Shpall et al. [28] 37 10 56 4 28 106 SCF, TPO, G-CSF Grade II IV: 67% Grade III IV: 40% de Lima and Shpall 35 14 23 2.3 14 34 SCF, TPO, G-CSF Grade II IV: 43% (abstract, ASH 2008) Grade III IV: 7% de Lima and Shpall 10 21 219 6 30 48 SCF, FL, IL-6, TPO, Grade II: 44% G-CSF Grade III IV: 0% Delaney et al. 5 16 660 160 14 Notch ligand Delta1, (abstract ASH 2008) SCF, FL, IL-6, TPO, IL-3 de Lima and 6 14 12 12 14.5 30 SCF, TPO, G-CSF Grade II: 33% Shpall (abstract Grade III IV: 0% ASH 2009) 12 2.4 0.5 22 71 PIXY321, FL, EPO Grade II IV: 36% children) Grade III IV: 22% Bioreactor Jaroscak et al. [25] 27 (mostly Pecora et al. [26] 2 (both adults) 12 2.2 1.6 (No expansion in 2nd) Survival % (median survival) 32 (17 months) 48 (11 months) 30 (25 months) 83 (277 days) 83 (12 months) 39 (41 months) 28 56 PIXY321, FL, EPO None 100 (12 months) S.S. Tung et al. / Best Practice & Research Clinical Haematology 23 (2010) 245 257 247

248 S.S. Tung et al. / Best Practice & Research Clinical Haematology 23 (2010) 245 257 hematopoietic progenitor cells (primarily CD133 þ, or CD34 þ ) from UCB as well as bone marrow or mobilized peripheral blood progenitors has been required [35]. Miltenyi CliniMACS system and the Nexell Isolex device are techniques available to perform this isolation at a clinical grade. At this point, hematopoietic progenitor cells are incubated in a culture medium with growth factors including stem cell factor (SCF), interleukin (IL)-3, IL-6 and granulocyte colony-stimulating factor (G-CSF) [35]; SCF, thrombopoietin (TPO) and G-CSF [24,36]; and Flt-3 ligand (FL), SCF, IL-3, IL-6 and G-CSF [37,38]. McNiece et al. has developed a two-step, 14-day liquid suspension UCB expansion protocol for isolated CD34 þ UCB cells [39], which yields more effective ex vivo expansion (>400-fold increase in TNC and >20-fold increase in CD34 þ cells) [40] than does a single-step 10-day protocol [28]. Several modifications to this particular expansion technique includes: attempts to further optimize ex vivo culture conditions [41 47]; the development of serum-free culture systems [40,41,48]; the use of histone deacetylases, thought to promote HSC self-renewal [49]; the use of glycogen synthase kinase (GSK)-3 inhibitors reported to maintain pluripotency of stem cells [50] and the use of tetraethylenepentamine (TEPA), a copper-chelator thought to modulate the proliferation and differentiation of primitive hematopoietic progenitors [51 53]. Tetraethylenepentamine (TEPA) Tetraethylenepentamine has been shown to stimulate ex vivo expansion of hematopoietic progenitor cells by reducing their free copper content as well as by lowering their oxidative stress [54]. An investigation into the potential therapeutic efficacy of TEPA added in a 22-day liquid UCB expansion was conducted as a phase I/II trial by de Lima et al. [27]. In this study, ten heavily pre-treated patients were allocated UCB units that were frozen into two fractions: a smaller CD133 þ fraction and a larger CD133 fraction. Twenty-one days before transplantation the CD133 þ fraction was thawed and expanded using liquid culture technique in amem containing 10% FCS (Hyclone) and supplemented with SCF, FL, IL-6 and TPO and TEPA. Prior to transplant patients received myeloablative therapy, and on day 0 received an unmanipulated UCB fraction. Infusion of the expanded fraction followed on day 1. Nine of the ten engrafted at a median of 30 days (n ¼ 9; range, 16 46 days) with 100% donor chimerism despite the low TNC/kg infused in this study (mean ¼ 1.7 10 7 /kg). Platelet transfusion independence occurred at a median of 48 days (range, 35 105). Nine were alive at day 100; 3 died during the 180-day study period due to infectious complications. No grade III or IV graft-versus-host disease (GVHD) occurred. The average fold expansion of TNC in the expanded fraction was 219 with a CD34 þ cell mean increase of 6-fold over the CD34 þ cell content of the entire unit. It was impossible to determine a correlation between CFUs, CD34 þ cell dose or TNC count and engraftment due to a small sample size and heterogeneous make up of UCB units. Further studies are essential in order to determine the efficacy of TEPA in the expansion of UCB additional. There is a phase II multicenter trial with TEPA expanded UCB product and has enrolled upto 40 patients (personal communications Gamida Cell Ltd., Cell Therapy Technologies). Although liquid culture expansion is a promising technique, the optimal combination of cytokines and growth factors has yet to be defined. Liquid culture is furthermore limited by small volumes as well as the static nature of the culture. Notch ligand expansion Human Notch1 gene is detected in CD34 þ or CD34 þ lin human hematopoietic precursors and the retrovirus-mediated expression of a constitutively active form of Notch1 leads to enhanced selfrenewal of repopulating cells [55]. Furthermore, activation of endogenous Notch receptors by immobilized Notch ligand leads to profound effects on the growth and differentiation of mouse marrow progenitor cells with a multilog increase in the number of precursor cells with short-term lymphoid and myeloid repopulating ability [56]. Incubation of cord blood progenitors in the presence of immobilized ligand has been shown to generate an approximate 100-fold increase in the number of CD34 þ cells with enhanced repopulating ability in an immunodeficient mouse model [33]. In a variation of the liquid culture technique, Delaney et al. recently utilized an immobilized, engineered form of the Notch ligand Delta1 with recombinant cytokines (SCF, FL, IL-6, TPO and IL-3) to stimulate ex vivo UCB expansion [32,33]. CD34 þ CD38 precursors were cultured with different

S.S. Tung et al. / Best Practice & Research Clinical Haematology 23 (2010) 245 257 249 densities of immobilized Notch ligand, Delta1ext-IgG and relatively lower ligand densities of immobilized Delta1 promoted maximal generation of CD34 þ precursor cells, including those with NOD/SCID repopulating cell activity [33]. In contrast, whereas the generation of early lymphoid precursors was similar across all densities of ligand, higher densities were associated with decreased generation of myeloid cells and increased apoptosis of CD34 þ and repopulating cells. Moreover, further lymphoid maturation was seen as the density of ligand increased. These studies indicate an important role of ligand density in the differential promotion of cell-fate outcomes and also suggest a physiological role for the known in vivo variations of ligand density. Ten patients with high-risk acute leukemias in morphologic remission, with a median age of 27.5 years and median weight of 61.5 kg, received myeloablative preparative regimen followed by infusion of one unmanipulated and one ex vivo-expanded cord blood graft. All units were matched to the recipient at a minimum of 4/6 six loci and at least 3/6 matched to each other, with a minimum TNC dose in the unmanipulated graft of 2.5 10 7 TNCs/kg. There was a 164-fold average expansion of CD34 þ cells and 562-fold expansion of TNC. CD34 þ cell dose derived from the expanded UCB graft averaged 6 10 6 CD34 þ cells per kg (range 0.93 106 to 13 106) versus 0.24 10 6 CD34 þ cells per kg (range 0.06 10 6 to 0.54 10 6 )(P ¼ 0.0004) from the unmanipulated UCB graft. There was no significant difference, however, in the average number of TNCs per kilogram. The time to ANC 500 cells/ml was shortened significantly (P ¼ 0.002), with a median time of 16 days as opposed to a median time of 26 days (range 16 48; P ¼ 0.002) in a concurrent cohort of 20 patients undergoing double UCB transplantation with identical conditioning and post-transplant immunosuppressive regimen. Longer-term in vivo persistence of the expanded cell graft occurred in two subjects. In one subject, analysis at day 240 after transplant revealed that a portion (10 15%) of the donor CD14 þ, CD56 þ and CD19 þ cells were derived from the expanded graft but were no longer present by 1-year, at which point the donor engraftment was 100% from the unmanipulated cord blood graft. In the second subject, at day 180 after transplant, the contribution to engraftment from the expanded cell population in CD33 þ, CD14 þ, CD56 þ and CD19 þ cells ranged from 25% to 66% of total donor engraftment. Three of the four patients showed an IFN-g response by cells derived from the unmanipulated cord blood unit in response to cells derived from the cultured unit, and in these patients only cells from the unmanipulated unit survived long-term. In the fourth patient, failure to detect an IFN-g response was associated with persistence of the cultured as well as the unmanipulated unit. Notch-mediated expansion of UCB stem/progenitor cells results in a marked expansion of hematopoietic precursors capable of rapid multilineage in vivo NOD-SCID reconstitution, while possibly retaining longer term repopulating ability. Bioreactors Bioreactors are also being investigated for the ex vivo expansion of HSC as a continuous perfusion culture system, rather than the use of static culture (culture flasks or bags) [24 26,57 61]. These bioreactors are automated, continuous perfusion culture systems that have been designed to accommodate larger volumes as well as to improve gas exchange and nutrient delivery. The secreted products of mature granulocytes and macrophages are toxic to progenitors [62], and mature macrophages can directly damage cultured stroma and hematopoietic progenitors [63]. In order to eliminate these threats, a continuous perfusion of culture medium is provided that would remove these mature cells protecting the cultured cells from toxic byproducts. Although the hematopoietic reconstitution of UCB CD34 þ cells grown in static cultures were better than stirred cultures for cell expansion, the engraftment of stirred-culture HSCs was higher than static-culture HSCs. Stirred-culture HSCs had better multilineage reconstitution ability and colony-forming ability than static-culture HSCs. Static cultures thus favor the expansion of HSCs and stirred cultures are more effective in preserving functional HSCs. In one phase I trial [25], fractions of UCB were expanded ex vivo using Aastrom Replicell bioreactor technology and a growth factor cocktail (PIXY321, Flt-3 ligand) and erythropoietin (EPO). The expanded cells were administered 12 days after the transplant of unmanipulated fractions of UCB. No difference in the time to myeloid, erythroid or platelet engraftment was observed. In a second 2-patient study, ex vivo-expanded UCB cells (Aastrom Replicell bioreactor) generated to augment unmanipulated UCB

250 S.S. Tung et al. / Best Practice & Research Clinical Haematology 23 (2010) 245 257 appeared to facilitate hematopoietic recovery [24]. A newer bioreactor that uses serum-free medium, the Dideco Pluricell System, was used in recent preclinical and murine studies, where Astori et al. showed a MNC fold expansion of 230.4 91.5 and CD34 þ fold expansion of 21.0 11.9 at 12 days, as well as improved engraftment in NOD-SCID mouse model [64]. Other technologies such as rotating wall vessels which decrease sheer stress while maintaining consistent environment are being evaluated [65]. Stromal culture Another culture technique, stromal co-culture, attempts to produce a more natural hematopoietic microenvironment. When cells are expanded ex vivo, they lose the support and regulation provided by their natural hematopoietic microenvironment which is only partially replaced by specific cytokines and growth factors provided in the culture media. This microenvironment is composed of hematopoietic and non-hematopoietic (cellular and extra-cellular) components [66 68] that are partly responsible for regulation of differentiation and maturation of HSCs and also provide complex cues that direct hematopoiesis [69 85]. A loss of this regulation may have the undesired effect of differentiation at the expense of expansion or self-renewal. A possible addition to culture media is mesenchymal stem cells (MSC). In NOD-SCID mice, third party (neither donor nor recipient) allogeneic MSC have been shown to promote engraftment of UCB CD34 þ when co-administered [86,87] and also to possess immunomodulatory activity [88 95]. MSC can be isolated as plastic adherent cells from a variety of fetal and adult tissues [29,86,88,96 102] and are characterized by a spindle-shaped, plastic adherent morphology. Phenotypically MSC are characterized as HLA I (ABC), CD105, CD73, CD90 and CD166 positive and HLA-DR (II), CD80, CD31, CD34 and CD45 negative. Although UCB and MPB are poor sources of MSC [103], a recent study suggested that MSC from the Wharton s jelly of umbilical cords demonstrated surface receptors similar to those of other MSCs and may be able to support UCB expansion [104]. Partial restoration of the interaction between marrow stroma microenvironment and the HSC can be achieved with co-culture of UCB and MSC. Foci of hematopoiesis and cobblestone areas that were visible during co-culture [29] indicate the direct interaction between the HSC and MSC. For stromal co-culture, MNC are isolated by density separation and co-cultured with established MSC monolayers in medium containing FBS and a growth factor cocktail (e.g. SCF, TPO and G-CSF, as with liquid culture expansion) [29]. The non-adherent cells are removed from the co-culture after 7 days and subject to a secondary expansion on an additional MSC monolayer. The original adherent layer which is then composed of MSC and HSC is re-fed with fresh medium containing growth factors. Culture is then continued for an additional 7 days (total 14 days) (Fig. 1) [29]. A10 20-fold increase in total nucleated cells, 7 18-fold increase in committed progenitor cells (granulocyte-macrophage colony-forming cells, GM-CFC), 2 5-fold increase in primitive progenitor cells (high proliferative potential colony-forming cells, HPP-CFC) and a 16 37-fold increase in CD34 þ cells, has been reported using co-culture expansion [29]. Co-administration of third party MSC with the UCB-derived HSC may aid engraftment [86,87] and provide immunomodulatory benefits [89 94,105]; therefore, it may prove clinically beneficial to re-infuse both non-adherent and adherent cells from the expansion process. Promising data has also been presented which indicated that ex vivo co-culture of non-selected UCB cells with MSC will generate superior TNC and HPC expansion than would ex vivo liquid culture of CD133 þ selected UCB cells. Robinson et al. showed that this culture technique resulted in TNC numbers that were >10-fold higher (approximately 10 10 TNC, p ¼ 0.0005) than was noted with ex vivo liquid culture of CD133 þ cells. This data also showed that TNC and HPC appear to be superior when using UCB-MSC co-culture (without CD133 þ selection) as compared to ex vivo liquid culture of CD133 þ selected UCB cells. Robinson et al. also noted a benefit of this technique for patients >75 kg, reporting that MSC co-culture of UCB can yield a transplant dose sufficient enough for 10 8 TNC/kg (and 10 6 CD34 þ /kg) for a >75 kg patient [106]. At MD Anderson Cancer Center, a clinical trial is underway combining an unmanipulated UCB unit with an expanded UCB unit on a layer of related donor MSC member (minimum of 2/6 HLA match). Myeloablative therapy for this protocol is ATG plus fludarabine, melphalan and thiotepa, and nonmyeloablative therapy is ATG plus fludarabine, cyclophosphamide and 200 cgy TBI. On day 0, the unmanipulated UCB unit is infused, followed by the expanded UCB cells (from both the bags and the

Fig. 1. Ex vivo expansion of umbilical cord blood graft using mesenchymal stem cell (MSC) expansion technique. a: Third party derived bone marrow mononuclear cells are cultured in plastic flasks for 2 3 days to allow for 70% confluence. The non-adherent (hematopoietic cells) are removed and the MSCs are then passaged upto 12 times in 175 cm 2 flasks over a period of 7 10 days. b: MSCs show 70 80% confluence. c: The UCB to be expanded is then co-cultured in the separate flasks containing confluent MSCs for another 14 days for maximal expansion.

252 S.S. Tung et al. / Best Practice & Research Clinical Haematology 23 (2010) 245 257 co-culture flasks). A median 12-fold expansion was seen in both the TNC and the CD34 þ subsets. For the six recipients of myeloablative therapy, the median time to neutrophil engraftment has been 14.5 days (range 12 23) and platelet engraftment 30 days (range 25 51). Two of six patients developed Grade II acute GVHD which resolved with steroids. One patient died of pneumonia in remission at day 150. Five of the six patients are alive and in complete remission at a median follow up of one year with accrual continuing. (de Lima et al. Proc. from ASBMT 2009, in press) As with the liquid culture expansion, stromal co-culture lacks optimization of culture conditions such as growth factor cocktail utilized, the length of MSC and which hematopoietic cell to co-culture. The development of new, potentially more effective stromal cell lines to support HSC expansion may prove to be beneficial [107]. Challenges associated with cord blood expansion Differentiation of the progenitor cell Ex vivo expansion strategies are not unique to UCB, and could also be applied to bone marrow (BM) as well as mobilized peripheral blood (MPB)-derived HSC [35,36,39], as there is evidence of functional and phenotypic heterogeneity within the HSC population [108 112]. During expansion, a major concern is selective expansion of short-term (low quality) reconstituting HSC at the expense of long-term (higher quality) reconstituting HSC. This potential selectivity can lower long-term viability of the graft while initially demonstrating early hematopoietic recovery [113]. An inherent reduction in long-term hematopoietic reconstitution potential of ex vivo-expanded products may be evident under certain conditions [113 115]. However, the ability to manipulate UCB to produce a short-lived, albeit rapidly reconstituting, HSC profile can be clinically useful especially when it is combined with an unmanipulated UCB unit for transplantation. There is clinical data suggesting that ex vivo-expanded UCB tends to be the source of the rapid, initial hematopoietic reconstitution with the unmanipulated fraction being responsible for long-term hematopoietic sustainability [24]. On the other hand, there is also data suggesting the addition of the expanded fraction does not provide a beneficial long-term outcome. [24 26] The selective expansion of short-term reconstituting, lower quality HSC at the expense of longterm reconstituting, higher quality HSCs could potentially lead to earlier initial hematopoietic recovery with later graft failure, by diminishing the long-term reserve of the graft [113]. McNiece et al. report compromised long-term repopulating activity following ex vivo expansion in a fetal sheep model [114], Von Drygalski et al. report the loss of radioprotective and long-term engraftment potential with ex vivo expansion of murine bone marrow [116], and in a clinical study, Holyoake et al. report the absence of durable engraftment from ex vivo-expanded CD34 þ cells [115]. On the contrary, Piacibello et al. observe evidence of self-renewal and amplification of HSC during ex vivo expansion [117]; Lewis et al. report that UCB cells capable of engraftment in primary, secondary and tertiary xenogeneic recipients are preserved following ex vivo expansion [118]; and Guenechea et al. report a delay in engraftment in a mouse model, suggesting that potentially more primitive, less rapidly engrafting cells, are preserved during ex vivo expansion [119]. Homing of the cells after short-term ex vivo expansion does not seem to be impacted [120]; however, the overall durability of the grafts is an issue that will need to be clarified as more clinical expansion studies in humans progress. Ex vivo-expanded products may possess an inherent reduction in long-term hematopoietic reconstitution potential under certain conditions [113 115]. The potential skewing of the UCB product to a more rapidly reconstituting, but short-lived, HSC profile could potentially be exploited to provide a clinical advantage, especially when ex vivo-expanded and unmanipulated UCB fractions are combined for transplantation. Clinical data has suggested that UCB that has been subject to ex vivo expansion does provide more rapid initial hematopoietic reconstitution, while unmanipulated UCB is the source of the long-term, sustainable hematopoiesis [24]. There is other clinical data, however, that suggests that augmenting UCB with ex vivo-expanded cells may not provide any benefit in terms of outcomes [24 26]; a discrepancy further studies may elucidate. Graft contamination Manipulation of cell products carries an intrinsic risk for infectious contamination. Therefore, strict adherence to GMP protocols is necessary. Although, at MD Anderson Cancer Center, in a randomized

S.S. Tung et al. / Best Practice & Research Clinical Haematology 23 (2010) 245 257 253 setting, we compared expanded UCB units (n ¼ 44) to unmanipulated units (n ¼ 48) where liquid culture was utilized to expand the cord and no contamination was seen in either arm [121]. Similarly, no contamination was noted in another study of only expanded UCB, using Bioreactor (ViaCell; n ¼ 32) and MSCs (angioblasts technique; n ¼ 13) for UCB expansion. Timing of transplantation Another major challenge to ex vivo expansion is the unavoidable expansion phase which ultimately postpones transplantation by 2 3 weeks. Generally it takes 14 days for expansion, in addition to any required testing for the transplant itself. This delay may contribute to increased risk of disease progression or relapse. In high volume UCB transplant programs, timing of expansions can be an obstacle logistically. In order to overcome such challenges, the stem cell laboratory has to be adequately equipped with manpower and supporting technology. Summary Expansion of UCB progenitors ex vivo prior to infusion could potentially ameliorate inadequate hematopoietic recovery by generating higher numbers of the cells responsible for short-term reconstitution. Human UCB contains higher numbers of clonogenic progenitor cells [one to five per 1000 mononuclear cells (MNC)] than adult peripheral blood (1 5 per 20,000 MNC). CD34 þ UCB cells can generate several thousand more mature cells in culture without reducing the number of CD34 þ cells with which the cultures were inoculated, in contrast to cultures of adult bone marrow. UCB progenitor may therefore have an increased capacity to support self-renewal and might be a superior source of expanded stem and progenitor cells for clinical transplantation [1,4]. Even with the expansion, the UCB retains its power to engraft and primarily supports the patient during the initial post-transplant period. With advancement in the field of UCB transplantation, the aim is to utilize novel expansion methods to optimize the expansion within a minimal period of time leading to a maximal yield of progenitor cells. Conflict of interest statement No conflicts of interest to declare. References [1] Broxmeyer HE, Hangoc G, Cooper S, et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci U S A 1992;89:4109 13. [2] Kurtzberg J, Laughlin M, Graham ML, et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996;335:157 66. [3] Broxmeyer HE, Gluckman E, Auerbach A, et al. Human umbilical cord blood: a clinically useful source of transplantable hematopoietic stem/progenitor cells. Int J Cell Cloning 1990;8(Suppl. 1):76 89. discussion 89 91. [4] Broxmeyer HE, Douglas GW, Hangoc G, et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci U S A 1989;86:3828 32. [5] Stanevsky A, Goldstein G, Nagler A. Umbilical cord blood transplantation: pros, cons and beyond. Blood Rev 2009;23: 199 204. [6] Goldstein G, Toren A, Nagler A. Transplantation and other uses of human umbilical cord blood and stem cells. Curr Pharm Des 2007;13:1363 73. [7] Laughlin MJ, Barker J, Bambach B, et al. Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. N Engl J Med 2001;344:1815 22. [8] Migliaccio AR, Adamson JW, Stevens CE, et al. Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cell content is a better predictor than nucleated cell quantity. Blood 2000;96: 2717 22. [9] Gluckman E, Rocha V, Chevret S. Results of unrelated umbilical cord blood hematopoietic stem cell transplantation. Rev Clin Exp Hematol 2001;5:87 99. [10] Gluckman E, Rocha V, Arcese W, et al. Factors associated with outcomes of unrelated cord blood transplant: guidelines for donor choice. Exp Hematol 2004;32:397 407. [11] Rubinstein P, Carrier C, Scaradavou A, et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998;339:1565 77. [12] Gluckman E, Rocha V, Boyer-Chammard A, et al. Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. N Engl J Med 1997;337: 373 81.

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