Cord blood transplantation and stem cell regenerative potential

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1 Experimental Hematology 2011;39: Cord blood transplantation and stem cell regenerative potential Yanling Liao a, Mark B. Geyer a, Albert J. Yang a, and Mitchell S. Cairo a,b,c a Department of Pediatrics, New York-Presbyterian Morgan Stanley Children s Hospital, Columbia University, New York, NY., USA; b Department of Medicine, New York-Presbyterian Morgan Stanley Children s Hospital, Columbia University, New York, NY., USA; c Department of Pathology and Cell Biology, New York-Presbyterian Morgan Stanley Children s Hospital, Columbia University, New York, NY., USA (Received 28 September 2010; revised 6 January 2011; accepted 8 January 2011) The past 20 years of experience with umbilical cord blood transplantation have demonstrated that cord blood is effective in the treatment of a spectrum of diseases, including hematological malignancies, bone marrow failure, hemoglobinopathies, and inborn errors of metabolism. Cord blood can be obtained with ease and then safely cryopreserved for either public or private use without loss of viability. As compared to other unrelated donor cell sources, cord blood transplantation allows for greater human leukocyte antigen disparity without a corresponding increase in graft-vs.-host disease. Moreover, cord blood has a lower risk of transmitting infections by latent viruses and is less likely to carry somatic mutations than other adult cells. Recently, multiple populations of stem cells with primitive stem cell properties have been identified from cord blood. Meanwhile, there is an increasing interest in applying cord blood mononuclear cells or enriched stem cell populations to regenerative therapies. Accumulating evidence has suggested functional improvements after cord blood transplantation in various animal models for treatments of cardiac infarction, diabetes, neurological diseases, etc. In this review, we will summarize the most recent updates on clinical applications of cord blood transplantation and the promises and limitations of cell-based therapies for tissue repair and regeneration. Ó 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. Cord blood transplantation Allogeneic stem cell transplantation (AlloSCT) has been used for more than 40 years and offers the best potential for curing numerous malignant and nonmalignant diseases in children and adults. Although human leukocyte antigen (HLA) matched sibling bone marrow transplantation (BMT) was previously the predominant modality of transplantation, only some 25% of patients in need of AlloSCT have a fully matched sibling donor available. Related and unrelated umbilical cord blood (UCB) has emerged as an alternative source of hematopoietic stem cells (HSCs) to the majority of patients who are unable to identify a fully matched donor. The first umbilical cord blood transplantation (UCBT) was performed in 1988 in a 5-year-old boy with Fanconi anemia who underwent UCBT from his HLA-identical newborn sister [1]. Since that time, O10,000 total UCBTs have been performed with Offprint requests to: Mitchell S. Cairo, M.D., Division of Pediatric Blood and Marrow Transplantation, New York-Presbyterian Morgan Stanley Children s Hospital, Columbia University, 3959 Broadway, CHN 10-03, New York, NY 10032; mc1310@columbia.edu O300,000 UCB units (UCBU) available in O40 UCB banks [2]. The Center for International Blood and Marrow Transplant Research (CIBMTR) notes that an increasing percentage of CIBMTR-registered allogeneic transplants utilize UCB grafts. The number of CIBMTR-registered UCBTs has increased considerably since the 1990s, and in 2008, 898 UCBTs were facilitated by the National Marrow Donor Program [3,4]. In both pediatric and adult patients, the use of BM in unrelated donor transplantations has decreased. However, while some 40% of unrelated donor transplants in patients 20 years of age and younger now use UCB, use of UCB has increased more modestly in patients older than 20 years of age to only 7% of unrelated donor transplants [4]. Median search time for unrelated BM or peripheral blood stem cell (PBSC) donors through the National Marrow Donor Program is 51 days compared with!2 weeks for unrelated UCBCs [5]. Early experience with related and unrelated UCBT Early experiences with UCBT were reported by several groups in the mid-1990s. Wagner et al. reported early success in 44 children undergoing sibling UCBT for X/$ - see front matter. Copyright Ó 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: /j.exphem

2 394 Y. Liao et al./ Experimental Hematology 2011;39: leukemias, marrow failure syndromes, congenital immunodeficiencies, and inborn errors of metabolism, with 46% and 78% event-free survival (EFS) observed for those receiving 5/6 or 6/6 HLA-matched grafts, respectively [6]. Kurtzberg et al., as well as Wagner et al. and Cairo et al., subsequently reported on the use of unrelated UCBT in children and adolescents for malignant and nonmalignant diseases, noting delayed engraftment compared to BMT, low risk of severe graft-vs.-host disease (GVHD), delayed engraftment, and encouraging early survival [7 9]. In the late 1990s, the New York Blood Center (NYBC) and the Eurocord/European Blood and Marrow Transplant (EBMT) group reported more extensive experience with UCBT. The Eurocord/EBMT group, which analyzed outcomes after 143 related- and unrelated-donor UCBTs, demonstrated higher 1-year overall survival (OS) in patients with malignant and nonmalignant diseases undergoing related vs. unrelated donor UCBT (63 vs. 29%) [10]. A later review of 562 unrelated donor UCBTs revealed survival rates similar to those observed after unrelated donor BMT, with 1-year relapse risk of 30%, 18%, and 24% in children with acute myeloid leukemia (AML), chronic myeloid leukemia, and acute lymphoblastic leukemia (ALL), respectively. Day 100 transplant-related events (graft failure, retransplant, death) occurred in 46% [11]. UCBT in pediatric patients with malignant diseases Hematological malignancies are the most common indication for AlloSCT in children. Unrelated UCBT has been used successfully in the treatment of ALL, AML, chronic myeloid leukemia, myelodysplastic syndrome, neuroblastoma, Hodgkin lymphoma (HL), non-hodgkin lymphoma (NHL), and other malignant diseases. The Eurocord/EBMT group reported 2-year OS and leukemia-free survival (LFS) of 49% and 42%, respectively, in 95 children receiving unrelated UCBT for AML. Two-year LFS was significantly better among those transplanted in first (CR1) or second complete remission (CR2) (59% and 51%, respectively) than those in relapse (21%) [12]. However, Wall et al. reported 2-year LFS of only 28% among 32 infants and young children, with no significant differences in outcomes observed between those transplanted in CR1 or CR2 [13]. The Cord Blood Transplantation (COBLT) study, a prospective, multicenter study using public UCB banks, enrolled 191 pediatric patients with hematological malignancies, including 109 patients with ALL and 51 with AML. Cumulative incidence of relapse at 2 years was 19.9% and 2-year OS was 49.5%. Fully HLA-matched UCBT was associated with superior outcomes, although long-term survival was observed among some patients receiving only 2/6 or 3/6 matched UCBT [14]. Randomized studies of unrelated UCB vs. BM or PBSCs are lacking. However, several retrospective studies have suggested that outcomes of unrelated donor transplantation are similar, regardless of cell source (Table 1). Eapen et al. [15] reported outcomes in 267 patients diagnosed with ALL or AML before 18 months of age who subsequently underwent matched sibling marrow transplant (n 5 101), unrelated marrow transplant (n 5 85), or UCBT (n 5 81). Transplantrelated mortality (TRM) was significantly higher among UCBT recipients (31%) compared to related marrow (15%) or matched sibling marrow (6%) recipients, due in part to the higher risk of death from infections in the UCB group. However, no differences in OS were evident among these groups [15]. A larger retrospective study of 785 children ages 0 to 16 years with acute leukemia compared outcomes in 503 unrelated UCBT recipients to 282 unrelated marrow recipients. Five-year LFS was similar among patients receiving fully matched unrelated marrow transplant compared to those receiving one or two antigen-mismatched UCBT, and possibly higher in patients receiving fully matched UCBT (Fig. 1). However, two-antigen UCBT recipients had significantly higher risk of TRM when compared to recipients of fully matched BM [16]. These data support the efficacy of UCB grafts in the treatment of ALL and AML and suggest that mismatched UCB grafts lead to acceptable outcomes in patients with acute leukemia. The benefits derived from greater graft-vs.-leukemia effect in mismatched UCB transplants may balance the greater risks of TRM in mismatched UCBT recipients. UCBT in adults with malignant diseases Although early studies of UCBT focused predominantly on pediatric patients, UCBT has begun to establish itself as a feasible treatment strategy in adults with hematologic malignancies. Simultaneously published reports from the NYBC and the Eurocord/EBMT group described the feasibility of single UCBT in adults with leukemia or myelodysplastic syndrome (Table 1). Rocha et al. [17] compared 98 adults receiving UCBT to 568 adults receiving matched unrelated BMT. UCBT recipients were slower to engraft, had lower risk of grade II IV acute GVHD, and had similar risk of relapse to unrelated marrow recipients. While a trend toward superior 2-year OS (42% vs. 36%) and LFS (38% vs. 33%) for unrelated BM transplant vs. UCBT was observed in the univariate analysis, no difference was appreciated in the multivariate analysis [17]. Laughlin et al. [18] compared one- and two-antigen mismatched UCBT recipients (n 5 150) to those receiving matched (n 5 367) or one-antigen mismatched (n 5 83) unrelated BM. Patients receiving mismatched UCBT or unrelated BMT had slower engraftment and higher risk of TRM, treatment failure, and overall mortality compared to recipients of fully matched unrelated marrow; groups did not differ with respect to relapse [18]. However, median cell dose among UCBT recipients was only /kg in this report. The authors concluded that UCBT is an acceptable alternative to mismatched unrelated BMT in adults with hematological malignancies, with the advantage of faster availability of UCBUs. A very recent CIBMTR analysis of 1525 adults with acute leukemia also demonstrated

3 Table 1. Outcomes after unrelated umbilical cord blood transplantation vs. other stem cell sources in the treatment of hematologic malignancies First-named author, year Diseases Cell source and HLA match n Age range (y) (median) Median time to engraftment (d) Incidence of grade II IV agvhd (%) Incidence of TRM (%) Incidence of relapse (%) DFS (%) OS (%) Rocha, 2004 [17] * * * 2y: 2y: 2y: ALL, MUBMT (32) AML M/1 3MMUCBT (24.5) Laughlin, 2004 [18] * * * * *3 y: *3 y: ALL, MUBMT 367 All AML, 1MMUBMT CML, 1 2MMUCBT MDS Eapen, 2006 [15] * * * * *CR1/ADV 3 y CR1/ADV 3 y CR1/ADV ALL, MSDT 101!1.5, All 17 Unspecified 6 47/65 49/20 54/35 AML M/1MMUBMT 85!18 mos /45 54/30 62/33 M/1MMUCBT Eapen, 2007 [16] * * * * * *5 y: ALL, MUBMT 116 All!16 19 (all UBMT) Unspecified AML 1 2MMUBMT MUCBT (all UCBT) MMUCBT(h) MMUCBT(l) MMUCBT Eapen, 2010 [19] ALL, * * * * 2 y, in CR/not AML MUBMT 332 All O16 (33) /17 Unspecified 1MMUBMT /14 MUPBSCT 632 (39) /17 1MMUPBSCT /17 M/1 2MMUCBT 165 (28) /15 Y. Liao et al./ Experimental Hematology 2011;39: ADV 5 relapse or $CR2; agvhd 5 acute GVHD; CML 5 chronic myeloid leukemia; DFS 5 disease-free survival; (h) vs. (l) 5 cell dose O vs. # total nucleated cells/kg recipient body weight; MDS 5 myelodysplastic syndrome; M/MM matched/mismatched (MM preceded by number of HLA mismatches); MSDT 5 matched sibling donor stem cell transplant; OS 5 overall survival; TRM 5 transplant-related mortality; UBMT 5 unrelated bone marrow transplant; UPBSCT 5 unrelated peripheral blood stem cell transplant. *p! 0.05 between two or more compared groups. 395

4 396 Y. Liao et al./ Experimental Hematology 2011;39: Figure 1. Probability of leukemia-free survival after bone marrow and cord blood transplantation adjusted for disease status at transplantation. Reprinted from Eapen M, Rubinstein P, Zhang MJ, et al. Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: a comparison study. Lancet. 2007;369: [16], with permission. increased risk of TRM (37%) after matched or mismatched UCBT compared to fully matched unrelated BMT (22%) or PBSC transplant (24%), but similar to one antigenmismatched unrelated BMT (34%) and PBSC transplant (38%) (Fig. 2); no differences in risk of relapse or LFS were evident [19]. Another large study from Japan compared outcomes in 100 adult unrelated UCBT recipients (one- to four-antigen mismatched) to 71 matched or mismatched related marrow or PBSC transplantation recipients with hematologic malignancies after myeloablative conditioning (MAC). TRM, relapse, and disease-free survival did not differ with respect to graft source [20]. Recently, the Figure 2. Probabilities of transplant-related mortality by hematopoietic stem-cell source and donor recipient HLA matching in adults with acute leukemias. Reprinted from Eapen M, Rocha V, Sanz G, et al. Effect of graft source on unrelated donor haemopoietic stem-cell transplantation in adults with acute leukaemia: a retrospective analysis. Lancet Oncol. 2010;11: [19], with permission.

5 Y. Liao et al./ Experimental Hematology 2011;39: University of Minnesota has reported excellent 3-year OS (61%) among 19 adults treated with UCBT for ALL [21]. A report from the Japan Marrow Donor Program reported similar 2-year OS among 336 adults with ALL undergoing UCBT (52%) vs. matched related marrow (53%), further corroborating use of UCBT as a frontline alternative to unrelated marrow in these patients [22]. Ooi et al. noted 2-year and 5-year EFS of 73.5% and 62.8% in 77 adult UCBT recipients with AML [23]. Although the Japan Marrow Donor Program noted superior 2-year LFS in matched unrelated BMT recipients (54%) compared to UCBT recipients (36%) in 484 adults with AML, a significantly larger proportion of UCBT recipients had advanced leukemia [22]. Rodrigues et al. [24] recently reported 1-year and 2-year EFS of 40% and 36%, respectively, in 104 adult patients with HL, NHL, or chronic lymphocytic leukemia who underwent single or double UCBT. Superior 1-year progressionfree survival was observed in those with chemosensitive disease, those who received a higher total nucleated cell (TNC) dose per kilogram, and recipients of conditioning regimens containing low-dose total body irradiation (TBI) [24]. Earlier small studies of single-unit reduced-toxicity conditioning (RTC) UCBT in adults with advanced HL or NHL had demonstrated 1-year progression-free survival of 25% to 50% [25,26]. Although the place of UCB in selecting a graft source for adults with hematological malignancies is unknown, UCBT (up to two-antigen mismatched) appears to be acceptable when a fully matched unrelated adult donor is unavailable and, in some cases, might be considered a feasible alternative to unrelated BMT. UCBT in patients with nonmalignant diseases UCBT has been used successfully in the treatment of BM failure syndromes, hemoglobinopathies, and inborn errors of metabolism. Children with Fanconi anemia, Blackfan Diamond anemia, severe aplastic anemia, severe combined immunodeficiency, Wiskott-Aldrich syndrome, osteopetrosis, Hurler syndrome, adrenoleukodystrophy, and thalassemia were among the initial cohorts reported by the NYBC and Eurocord/EBMT groups [10,11]. In a retrospective review from Eurocord/EBMT of 93 children and adults with Fanconi anemia treated with UCBT, a high incidence of primary graft failure (40%) was observed, with 40% of patients alive at 3 years post-ucbt; younger age, higher cell dose, and fludarabine-based conditioning were associated with superior outcomes. The success of fludarabine in these patients may reflect the critical importance of adequate immunosuppression in patients heavily transfused before transplantation, such as those with Fanconi anemia [27]. A handful of reports have discussed UCBT from matched sibling donors in the treatment of sickle cell disease, after either MAC or RTC [10,28 31]. MAC followed by UCBT from a related donor appears to be associated with a low incidence of graft failure, high levels of donor chimerism, and excellent OS [29]. Unrelated UCBT in children with sickle cell disease remains a challenge due to nonengraftment and TRM [32]. Recently, investigators from Duke University Medical Center have reported their experience with UCBT in the treatment of inherited metabolic disorders, particularly the lysosomal and peroxisomal storage disorders [33,34]. In such patients, the goal of UCBT is replacement of the missing enzyme. One-year, 3-year, and 5-year survival probabilities of 79.0%, 62.7%, and 58.2%, respectively, were observed in a group of 159 pediatric patients with inherited metabolic disorders treated using UCBT. Among patients with diseases for which leukocyte or plasma enzyme level measurements exist, 97% achieved normal levels. Patients undergoing transplantation as newborns and those with less progressive juvenile forms of disease experienced superior functional outcomes to those transplanted with early infantile forms of disease and progressive symptoms. Specifically, among 45 children transplanted for severe Hurler syndrome, all experienced disease stabilization and most demonstrated cognitive improvement. The median age of patients at transplantation was 1.5 years, with 57% of patients younger than 2 years of age. Poorer survival outcomes were observed among those patients with worse baseline performance status and recipients of UCB grafts with fewer colony-forming units. Advantages and disadvantages (Table 2) Since the early days of UCBT, investigators have noted a higher risk of graft failure in UCBT recipients than in those receiving marrow or PBSCs. An early report from Wagner et al. noted an 18% risk of primary graft failure Table 2. Advantages and disadvantages of unrelated cord blood vs. unrelated adult donor stem cell transplantation Advantages of UCBT Decreased risk to donor Faster procurement Lower incidence of grade II IV acute GVHD Enhanced ability to cross donor-recipient HLA disparities Disadvantages of UCBT Difficult to achieve sufficient total nucleated cell dose in larger recipients using single cord blood unit Delayed engraftment and increased risk of graft failure Delayed T-cell immune reconstitution Increased risk of transplant-related mortality Increased costs of hospitalization No obvious cell source for post-transplant donor lymphocyte infusions

6 398 Y. Liao et al./ Experimental Hematology 2011;39: among 44 pediatric UCBT recipients [6]. The increased incidence of graft failure in UCBT recipients compared to marrow and PBSC recipients continued to be observed in larger studies, with the NYBC and EBMT groups reporting 18% to 19% of patients failed to engraft neutrophils [10,11]. Studies continue to note rates of neutrophil engraftment ranging from 60% to 87% after unrelated UCBT [12,14,16,19,24,27,33,35]. Eapen et al. noted that 85% of recipients of fully matched unrelated UCB engrafted neutrophils by 42 days post-transplantation, compared to 76% of recipients of two-antigen mismatched UCB [16]. Moreover, time to hematopoietic recovery is delayed after UCBT, with median times to neutrophil and platelet recovery of 25 and 59 days, respectively, compared to 19 and 27 days, respectively, after unrelated marrow transplantation [16]. Prasad et al. and Kurtzberg et al. reported neutrophil and platelet engraftment at a median of 27 and 174 days after UCBT in a large series of pediatric patients with hematological malignancies, and at 22 and 87 days, respectively, in a large series of pediatric recipients with nonmalignant diseases [14,33]. Delayed engraftment leaves the UCBT recipient in an extended state of profound immunocompromise, particularly after myeloablative conditioning. Indeed, Rocha et al. found that death from opportunistic infection and hemorrhage were more common after matched sibling UCBT than matched sibling BMT (33% and 21%, respectively; p ) [36]. Possible explanations for the delay in hematopoietic recovery after UCBT compared to BMT include decreased number of total [14] and CD34 þ cells in the graft [37], more immature CD34 þ progenitors (thus requiring more cell division for differentiation and engraftment), and lack of a subpopulation specializing in engraftment and homing effects [38]. A COBLT study led by Cairo et al. suggested that lower numbers of CD34 þ /CD41 þ cells in UCB compared to peripheral blood may be responsible for the delay in platelet recovery after UCBT [39]. Consistent with increased rates of graft failure and severe posttransplantation infection and prolonged inpatient stays, Majhail et al. further demonstrated that the cost of hospitalization after UCBT is greater than the cost after matched related BMT, with the median cost per day estimated to be $2082 vs. $1016, respectively [40]. In the COBLT study, TNC dose of O /kg was associated with significantly faster engraftment of neutrophils and platelets. TNC dose of! /kg was associated with significantly reduced OS as well [14]. Wagner et al. originally noted CD34 þ dose! /kg was associated with inferior outcomes in engraftment, TRM, and OS in 102 pediatric and adult UCBT recipients [41]. Stycynski et al. also reported greater CD34 þ dose was independently predictive of greater OS among 29 UCBT pediatric recipients (median CD34 þ dose, /kg) treated at Columbia University Medical Center [42]. As evidence suggests greater HLA disparity can be overcome, in part, by greater cell dose, different minimum cell doses have been proposed for differing levels of HLA disparity: 2.5 to /kg for fully matched UCBT, /kg for 5/6 HLA match, and /kg for 4/6 HLA match [43,44]. A recent review from the NYBC addressed the effects of cell dose and HLA disparity in 1061 recipients (mostly pediatric patients) of MAC, followed by single-unit UCBT [45]. TNC dose was associated with higher probability and rate of neutrophil and platelet engraftment in a dose-response manner. Recipients of fully matched UCBT had significantly greater neutrophil and platelet engraftment than mismatched UCBT recipients. However, two-antigen mismatched UCBT recipients showed similar rates of engraftment to oneantigen mismatched UCBT recipients [45]. Large studies have previously demonstrated a relationship between greater HLA disparity and higher TRM in patients undergoing UCBT [11]. In the NYBC study, TNC dose and HLA match were independently predictive of TRM and OS (Fig. 3). Increasing HLA disparity was associated with higher risk of severe (grade III IV) acute GVHD. There was no association between HLA disparity and relapse. The lowest risk of TRM was observed in patients receiving a fully matched UCBT, regardless of cell dose. Similar survival outcomes were observed among recipients of two-antigen mismatched UCBUs with TNC $ /kg and recipients of oneantigen mismatched UCBUs with TNC 2.5 to / kg [45]. Future studies will be needed to determine if prioritizing HLA match when selecting UCB grafts, while maximizing cell dose by transplanting two units, will allow for faster engraftment and preserve the benefits associated with lower HLA disparity. Unrelated marrow and PBSC transplantation is limited by a high risk of acute and chronic GVHD; this risk is further exacerbated with increasing HLA disparity [46]. UCBT allows for greater HLA disparity than other unrelated donor cell sources without a corresponding increase in GVHD. Eapen et al. noted a significantly reduced incidence of grade II to IV acute GVHD after matched unrelated UCBT (24%) compared to matched unrelated BMT (46%) or one- or two-antigen mismatched unrelated BMT (60%); the incidence of grade II to IV acute GVHD was 42% after oneantigen mismatched UCBT with high cell dose and 41% after two-antigen mismatched UCBT [16]. Similar risks were reported in other recent studies, with an overall incidence of grade II to IVacute GVHD of 25% to 45% in cohorts of patients with mainly one-antigen or two-antigen mismatched UCB grafts [12,14,17,18,24,33,34,42,47]. In utero trafficking of HLA molecules between mother and fetus may lead to tolerance to noninherited maternal HLA antigens; this has potential implications for mismatched UCBT when a donor s noninherited maternal antigen (NIMA) matches with the recipient s HLAdisparate antigen. van Rood et al. [48] reported on the impact of NIMA on transplantation outcomes in pediatric patients undergoing UCBT for hematological malignancies.

7 Y. Liao et al./ Experimental Hematology 2011;39: Figure 3. Cumulative incidence of 3-year TRM after UCBT. Data are shown by TNC dose (A), HLA-mismatch (B), TNC dose and HLA-mismatch combined (C), and the Kaplan-Meier probability of disease-free survival (D). Recipients of units with either 1 or 2 mismatches were analyzed by separate TNC-dose categories, whereas recipients of zero mismatched units and three mismatched units were not. Reprinted from Barker JN, Scaradavou A, Stevens CE. Combined effect of total nucleated cell dose and HLA match on transplantation outcome in 1061 cord blood recipients with hematologic malignancies. Blood. 2010;115: [45], with permission. Retrospectively, they identified 79 patients receiving mismatched unrelated UCBT that had a NIMA identical to the mismatched antigen of the recipient, and 980 recipients of no NIMA-matched UCBT. The probability of TRM was significantly lower in NIMA-matched UCBT recipients of all ages, although the difference was most striking in those patients 10 years of age and older, who also had a lower risk of overall mortality and treatment failure than no NIMA-matched UCBT recipients. A trend toward lower rates of relapse in patients with AML/chronic myeloid leukemia was appreciated in the NIMA-matched group as well. The authors speculate that upregulation of anti-nima immunity after re-exposure can reduce the risk of malignant relapse without increasing the risk of GVHD, specifically through fetal development of CD4 þ T cells exerting relapse-reducing effects, anti-minor histocompatibility antigen cytolytic T cells, and regulatory T-cells from the CD4 þ and CD8 þ compartments. Further studies may help to elucidate the role of NIMA matching in the selection of UCBUs [48]. Double UCBT Limited numbers of cells in single UCB grafts has limited the widespread use of UCBT in adult patients [4]. In a retrospective review of 102 pediatric and adult UCBT recipients, those receiving! CD34 þ cells/kg had significantly poorer survival outcomes [41]. Cotransplantation of two UCBUs (D-UCBT) from different donors increases the available cell dose and allows for UCBT in patients for whom no single UCBU would have sufficient cell dose. There is no universally accepted algorithm for unit selection in D-UCBT with respect to HLA matching. However, the published Minnesota algorithm recommends at least partial HLA matching between the transplanted units; HLA disparities between each unit and the recipient and between the two units need not be identical [49]. In practice, many studies have required units to be two or fewer locus HLA-mismatched with one another and with the patient [50 54]. Barker et al. reported 21 older adolescent and adult patients undergoing MAC and D-UCBT for hematological malignancies [50]. All patients

8 400 Y. Liao et al./ Experimental Hematology 2011;39: engrafted neutrophils; 65% developed grade II to IV acute GVHD [50]. Ballen et al. subsequently reported on 21 adult patients with predominantly malignant disease receiving reduced-intensity conditioning with fludarabine, melphalan, and anti-thymocyte globulin (ATG) followed by D-UCBT; O90% of patients engrafted neutrophils, 40% developed grade II to IV acute GVHD, TRM was 19% at 6 months, and 2-year DFS was 55% [51]. In a larger series of 110 adults with malignant and nonmalignant diseases receiving nonmyeloablative conditioning with cyclophosphamide, TBI, and ATG, significantly higher 3-year EFS was observed in D-UCBT recipients (39%) compared to single UCBT recipients (24%), with a lower incidence of relapse observed in D-UCBT recipients [52]. Additionally, among 177 adults and children receiving MAC followed by UCBT for acute leukemia, use of a double UCB graft compared to a single UCB graft was independently associated with significantly reduced incidence of relapse for patients in CR1 or CR2 [53]. Rodrigues et al. noted the use D-UCBT vs. single UCBT was the sole independent predictor of reduced risk of relapse (13% vs. 38%; p in univariate analysis, p in multivariate analysis) in 104 adults with HL, NHL, and chronic lymphocytic leukemia [24]. A higher incidence of grade II to IV acute GVHD has been noted in D-UCBT compared to single UCBT recipients (58% vs. 39%); however, no corresponding increase in TRM has been reported in those undergoing D-UCBT [55]. D-UCBT is a feasible strategy to extend the availability of UCBT to adolescents and adults. Moreover, larger ongoing studies may ultimately demonstrate significant long-term benefits in DFS and OS in patients receiving D-UCBT vs. single UCBT. After D-UCBT, a single unit generally ultimately serves as the source for hematopoiesis. Although initial engraftment of both units may be observed, engraftment usually skews progressively such that one unit predominates [50 52,55,56]. Despite the eventual predominance of a single UCBU, the nonengrafting unit may hasten and facilitate engraftment through as yet unknown immunologic mechanisms. The mechanism by which a single unit predominates is also incompletely understood. Multiple reports have noted that the first unit to be infused is more likely to dominate than the second; in addition, Haspel et al. noted that the predominant unit had significantly higher TNC and CD34 þ dose in a series of 38 adults undergoing reducedintensity D-UCBT [51,57]. Scaradavou et al. recently reported that among 44 D-UCBT recipients who engrafted a single unit, CD34 þ viability was significantly higher in the engrafting unit and that units with CD34 þ viability!75% were extremely unlikely to engraft [56]. One possible explanation for the predominance of the first unit is that this unit has the first opportunity to fill the HSC niche, reducing the niche space available for the second unit infused; increased cell dose may increase the probability of successful occupation of this niche space. This niche space can be thought of as the area of the marrow responsible for maintenance of HSCs; the osteoblast is the support cell of the niche [58 60]. Recently, Gutman et al. [61] described the development of CD8 þ T cells derived from the dominant UCBU specific for alloantigens present on the nonengrafting unit in 14 adult D-UCBT recipients. This population of effector cells produces interferon-g in response to the nondominant unit; no interferong secreting cells were identified when peripheral blood mononuclear cells (MNCs) were stimulated against cells derived from the engrafting unit; in patients with mixed chimerism, CD8 þ T cells tolerated cells from either unit without significant interferon-g production [61]. The exact antigenic specificity of this CD8 þ T-cell population and the role (if any) of CD4 þ T cells in graft-vs.-graft effects is unknown. Interactions between the two units infused may contribute to graft-vs.-leukemia effects by enhanced alloreactivity. Future studies are needed to understand the biology of D-UCBT to correlate with clinical observations of enhanced engraftment and reduced leukemic relapse in these patients. UCB expansion TNC and CD34 þ cell dose are strong predictors of clinical outcomes after UCBT as described previously. UCBUs contain approximately TNC on average and only 12% of the current public UCB inventory contains sufficient cell dosage for a 60-kg patient. The goal of UCB expansion is to increase the number of progenitor cells capable of rapid repopulation in vivo to improve the kinetics of hematopoietic recovery after UCBT. Currently, ex vivo expansion utilizes three methods: cytokinecontaining liquid expansion, coculture expansion with stromal cell hematopoietic microenvironment, and continuous perfusion in bioreactors rather than static cultures [62]. Ex vivo expansion of peripheral blood progenitor cells using growth factors in liquid culture has been shown to expedite neutrophil recovery after autologous stem cell transplantation [63]. However, most UCB ex vivo expansion studies to date have only shown that it is plausible, but have yet to yield a significant impact on myeloid engraftment rate [64 66]. This is thought to be secondary to cellular defects acquired during expansion, including cell-cycle abnormalities, homing defects, and induction of apoptosis [67]. However, Delaney and colleagues recently reported that transplantation of an unmanipulated UCBU along with Notch-mediated ex vivo expanded UCB progenitor cells resulted in faster neutrophil engraftment compared to a control group receiving unmanipulated D-UCBT (median 16 days vs. 26 days) [68]. Additional clinical trials are needed to confirm these encouraging results, and identification of molecular pathways that mediate cell homing and proliferation will need to be discovered to improve methods of ex vivo expansion.

9 Y. Liao et al./ Experimental Hematology 2011;39: Conditioning regimens Most studies of UCBT reported to date have employed MAC before transplantation. Successful engraftment has been observed with TBI containing and non TBI-containing regimens. The initial EBMT experience with UCBT used TBI/cyclophosphamide or busulfan/cyclophosphamide conditioning [10]. ATG and other anti T-cell antibodies have been incorporated into conditioning regimens for immunosuppression in unrelated UCBT recipients. While conditioning consisting of busulfan, melphalan, and ATG has been demonstrated to be well-tolerated among very young UCBT recipients, unusually delayed engraftment and a high incidence of graft failure were observed [13]. Day 100 TRM of 15% to 50% has been observed in MAC UCBT recipients, depending on the underlying disease and disease status before transplantation [7 9,11,12,14,29,36,42,47,69 72]. Significant causes of day-100 TRM include infection, organ failure, acute GVHD, and hepatic veno-occlusive disease. RTC and unrelated AlloSCT have been used successfully in the treatment of malignant and nonmalignant diseases in children and adults, with decreases in transplant-related morbidity and mortality observed. Our group previously reported preliminary results of RTC and UCBT in 21 children and adolescents with malignant and nonmalignant diseases [73]. We recently analyzed clinical outcomes among 88 patients undergoing MAC (n 5 49) or RTC (n 5 39) before UCBT at Columbia University Medical Center; all RTC regimens were fludarabine-based ( mg/m 2 ). Primary graft failure was observed in nine patients; MAC vs. RTC recipients did not differ with respect to graft failure or speed of engraftment. Day-100 TRM was only 2.6% in the RTC group, compared with 29.3% in the MAC group; in univariate and multivariate analyses, MAC vs. RTC was the only significant predictor of day-100 TRM and was a significant predictor of 1-year OS [73,74]. Other fludarabine-based reduced-intensity regimens, some of which incorporate low-dose TBI, have been successfully used in adult patients, with day-180 TRM!20% [25,26,51,52]. Immune reconstitution Immune reconstitution in pediatric AlloSCT recipients appears to take longer after MAC and UCBT than after matched sibling AlloSCT. Natural-killer and B-cell recovery is rapid and robust after UCBT, with a median of approximately 3 months and 6 months post-transplantation needed to achieve age-related normal levels, respectively. However, the median time needed to achieve normal cytolytic T-cell counts is approximately 8 to 9 months and for total and helper T-cell counts, approaches 12 months [75 78]. T-cell reconstitution after unrelated donor UCBT is further delayed compared to related donor UCBT. Cytolytic T-cell recovery after UCBT is markedly delayed compared to other stem cell sources, where CD8 þ T-cell recovery is often observed within 1 to 3 months post-transplantation [75,77]. T-cell recovery after AlloSCT derives from peripheral expansion of mature, post-thymic donor cells (the thymicindependent pathway) and prethymic cells that must undergo differentiation in the host (the thymic-dependent pathway) [79]. Given the low number of mature donor lymphocytes transferred in UCBT, less robust thymic-independent T-cell proliferation may be expected compared to related or unrelated BMT [75]. UCBT recipients are at particular risk of infection in the first 100 days post-transplantation. In a large comparison of patient outcomes after AlloSCT according to donor source, Laughlin et al. noted the proportion of infection-related outcomes within the first 100 days posttransplantation was significantly higher among UCBT recipients than among recipients of either matched or mismatched related BMT; proportions were similar among the groups after 100 days [18]. However, T-cell receptor diversity is ultimately greater at 2 years post-ucbt than after matched sibling donor BMT, as measured by T-cell receptor excision circles [80]. Parkman et al. [81] noted an association between successful immune reconstitution after UCBT in children and decreased risk of leukemic relapse and improved relapse-free survival. Specifically, robust antigen-specific T-cell proliferation in response to cytomegalovirus, herpes simplex virus, or varicella zoster virus was a strong predictor of relapse-free survival (Fig. 4). In addition, those without robust T-cell responses were more likely to die of infectious causes [81]. We recently analyzed immune reconstitution in 88 consecutive pediatric UCBT recipients at Columbia University Medical Center. At day þ100/180/365 posttransplantation, the percentages of patients who had achieved normal CD3, CD4, CD8, CD19, and CD56 counts according to age-specific reference ranges were 0/0/54%, 0/14%/71%, 3%/5%/58%, 53%/67%/92%, and 78%/71%/83%, respectively. Although most patients achieved normal naturalkiller cell counts by day 100 and normal B-cell counts by day 180, T-cell recovery was delayed. Lymphocyte subset counts and immunoglobulin levels did not differ significantly between patients receiving MAC vs. RTC before UCBT [82]. Strategies to enhance T-cell recovery in the early posttransplantation period in UCBT recipients warrant particular attention. Cord blood banking Discussion of specific UCB banking procedures is beyond the score of this review. A standard operating procedure of UCB collection, processing, and cryopreservation was developed by the COBLT study program (summarized in [83]). Matters of ongoing controversy include procedures for obtaining informed maternal consent, costto-benefit analysis of increasing the number of publically available UCBUs [84], the role of UCB directed donor (private) banking, cultural considerations regarding the placenta s role [85], and the use of preimplantation HLA typing [86].

10 402 Y. Liao et al./ Experimental Hematology 2011;39: Figure 4. Cumulative incidence of leukemic relapse by positive (solid line) vs. negative (dotted line) proliferative response status to the herpes viruses (cytomegalovirus, herpes simplex virus, varicella zoster virus) (p ). Those with antigen-specific proliferative responses to any of the three viruses at any time during the 3-year observation period were considered positive. Reprinted from Parkman R, Cohen G, Carter SL, et al. Successful immune reconstitution decreases leukemic relapse and improves survival in recipients of unrelated cord blood transplantation. Biol Blood Marrow Transplant. 2006;12: [81], with permission. Cord blood stem cell regenerative potential and properties Stem cell therapy has emerged as a novel and potential therapy for a variety of genetic, acquired, and degenerative diseases. Stem cells from various sources, embryonic, BM, UCB, and adult tissues, have been extensively studied preclinically in similar settings. Human embryonic stem (ES) cells, due to their indefinite capacity for self-renewal and pluripotency, hold great promise in regenerative medicine. However, in addition to ethical considerations, the therapeutic use of ES-derived cells is challenged with the high-risk of teratoma formation as a result of potential contamination with undifferentiated ES cells. Moreover, human ES cells can only be used as an allogeneic source [87]. The recent development of induced pluripotent stem (ips) cells has potentially circumvented the problems of ethical considerations and allogeneicity [88,89]. However, the standard production of ips cells involves viral transduction, which in itself creates the issue of insertional mutagenesis, although efforts are currently being made to excise the integrated viral constructs or to induce pluripotency by small molecules or proteins [90 93]. Meanwhile, the threat of teratoma formation remains. Adult tissue-specific stem cells are practical alternatives to ES cells for cell-based therapy. Compared to other adult stem cells, human UCB stem cells have unique properties; developmentally, they are only at 9 months of gestational age and have a longer telomere length, which correlates to their higher proliferative potential [94]. Importantly, UCB cells have not been exposed to immunological challenge and are less likely to carry somatic mutations than other adult cells. These unique properties have elicited special interest in using UCB for tissue regeneration and also as a starting cell type for the preparation of ips cells (Fig. 5). Indeed, the CD133 þ fraction of human UCB can be reprogrammed efficiently to ips cells with retroviral

11 Y. Liao et al./ Experimental Hematology 2011;39: Figure 5. Human cord blood as a stem cell source for cell-based regenerative therapies. Human cord blood is now routinely used as an autologous or allogeneic HSC source to treat both malignant and nonmalignant diseases. Cord blood mononuclear cells and enriched stem cell populations have both shown great potential in preclinical cell-based therapies for various degenerative diseases such as stroke and myocardial infarction. Cord blood contains multiple populations of tissue stem cells and progenitors, as well as primitive stem cells, which can further be differentiated to cells representative of all three embryonic germ layers. These primitive stem cells can therefore be a potential alternative to ES or ips cells for the derivation of somatic cells for tissue regeneration. transduction of only two factors, Oct4 and Sox2, as compared to four genes that are required in the case of reprogramming adult somatic cells [95]. ips cells can also be generated easily from CD34 þ CB stem cells through the addition of p53 inhibition under standard reprogramming conditions [96]. These data have demonstrated the plasticity and primitive nature of human UCB. UCB is a rich source of progenitors and stem cells (Fig. 5). In addition to HSCs, UCB also gives rise to another widely used stem cell population, mesenchymal stem cells (MSC). The therapeutic values of MSCs have been related to their ability to differentiate into cells mainly within mesoderm lineages, as well as their immunomodulatory function [97]. Moreover, MSCs exhibit low level of major histocompatibility complex I and are negative for major histocompatibility complex II antigens, indicating their immune evasion in the allogeneic setting [98]. These properties have elicited great interest in utilizing MSCs as an off-the-shelf product or as universal donors in cell-based therapy. However, the derivation efficiency of MSCs from UCB seemed to be lower than those from BM and adipose (30% vs. 100% of UCB units, respectively) [99]. This observation can be explained at least partially by a recent report that showed that MSCs were 10 times more frequent in the UCB of 24- to 28-week gestational age infants than 29- to 32-week gestational age UCB and even higher than 37- to 40-week gestational age UCB [100]. By comparison, endothelial colony-forming cells, identified based on the outgrowth of cobblestone-like adherent colonies between days 7 and 14 after plating of UCB MNCs on collagen-i coated plastics, are more enriched in full-term (37- to 40-week gestational age) UCB [100,101]. The endothelial colony-forming cells have shown the ability to form de novo blood vessels when seeded in either a collagen fibronectin or Matrigel matrix and implanted subcutaneously in immunocompromised mice, suggesting their potential therapeutic treatment of patients with impaired vascular function [102,103]. CB-derived primitive stem cells Besides these committed multipotent stem or progenitor cells, accumulating data have indicated the existence of multiple populations of more primitive stem cells in

12 404 Y. Liao et al./ Experimental Hematology 2011;39: UCB. These stem cells were identified using different methods. However, they carry similar capabilities to differentiate into cell types representative of all three germ layers, or express the embryonic stem cell markers, similar to human ES cells. Moreover, animal studies indicated that they do not have the risk to form teratomas, in contrast to human ES cells [94,104]. Therefore, these primitive stem cells could be made readily available from UCB and used as either an autologous source or HLA-matched allogeneic alternative to ES cells in tissue regeneration. The first identified UCB stem cell population with intrinsic pluripotent differentiation potential is named unrestricted somatic stem cells (USSCs) [104]. These CD45 neg / CD34 neg cells were isolated from UCB based on their outgrowth in the presence of dexamethasone. USSCs can be differentiated in vitro into bone; cartilage; adipocytes; hematopoietic cells; and neural cells, and in vivo into myocardial cells; Purkinje fibers; and hepatic cells [104]. After the identification of USSCs, extensive animal studies have shown these cells potential therapeutic applications in the promotion of bone healing, relief of neural injury, and improvement of recovery from myocardial infarction, although other investigations indicated a lack of functional efficacy after infusion of USSCs to an infarcted heart. These discrepancies might be due to the difference in timing and route of stem cell injection, as we will discuss later [ ]. USSCs have been considered an earlier cell type of MSCs and can be distinguished from MSCs by their broader differentiation capability, expression of DLK1 and a restricted adipogenic differentiation potential [111]. Recently, expression of a set of Hox genes, especially HOXA9, HOXB7, HOXC10, and HOXD8, has been proposed as candidate markers to discriminate MSCs from USSCs [112]. MSCs isolated from both BM and UCB are HOX-positive, whereas USSCs resemble H9 ES cells and are HOX-negative [112]. In addition, USSCs possess immunomodulatory effects, similar to MSCs [113,114]. They have also been shown to produce functionally significant amounts of hematopoiesis-supporting cytokines and are superior to the bone-marrow derived MSC in expansion of CD34- positive cells from UCB [115]. Moreover, cotransplantation of USSCs in nonobese diabetic/severe combined immunodeficient mice enhanced in vivo homing of both unselected and selectively amplified CD34-positive UCB cells, suggesting that USSCs could potentially be used clinically in UCB transplantation to facilitate homing and engraftment of donor cells [116]. As a step toward their future clinical application, USSCs can now be produced and expanded at a Good Manufacturing Practice grade [117]. In collaboration with BioE, Cairo et al. also characterized a population of multilineage progenitor cells (MLPC) from CB by nonparticular-based negative cell selection followed by plastic adherence [94]. MLPCs exhibit two distinct morphologies in cell surface phenotypes, a leukocyte-like morphology on freshly isolated cells (positive for CD45, CD34, CD133, CD13, CD29, CD44, CD73, CD90, CD105, CD9, and SSEA-3/4) and a fibroblastic morphology (CD45, CD34, CD133, CD13 þ, CD29 þ, CD44 þ, CD73 þ, CD90 þ, CD105 þ, CD9 þ ) upon continued culturing. MLPCs can be differentiated into cell types representative of all three germinal layers. Comparative microarray analyses indicated that MLPCs were also more quiescent and primitive, and less committed to lineage than MSCs. Moreover, standard in vitro culture in nondifferentiating media resulted in no instances of spontaneous differentiation and initial animal tests have not resulted in teratoma formation. Instead of deriving primitive stem cells by colony appearance from UCB MNCs as described here, other methods involve either positive or negative selection based on the expression of cell surface antigens. For example, embryonic-like stem cells can be isolated by immunomagnetic removal of CD45, CD33, CD7, and CD235apositive cells [ ]. The resultant lineage-negative cells, which represent 0.1% to 1% of total MNCs of each UCB, express embryonic stem cell markers Oct4, Sox2, and SSEA3/4, in addition to embryonic extracellular matrix components TRA-1-60 and TRA1-81. A similar stem cell fraction, isolated by depletion of CD34-positive cells (UCB-MNC CD34 ) was also characterized to express Oct4, Sox2, and Rex1 genes [121]. These cells further undergo spontaneous aggregation and differentiation toward neural lineage. From this point, neural stem cell lines could be derived by sequentially passaging only the floating cells from the epithelial growth factor stimulated culture [122]. Recently, a population of very small embryonic-like (VSEL) stem cells (CXCR4 þ lin CD45 ) has been identified from not only UCB, but also from human peripheral blood and BM by multiparameter fluorescence-activated cell sorting (FACS) analysis [ ]. This rare population is rich in the expression of CD34 and CD133 and represents about 0.02% of total MNCs. Single-cell characterization after FACS indicated that they express SSEA-4, Oct4, and Nanog. The promoter region of the Oct4 gene has consistently been shown to be hypomethylated and to adopt an open chromatin structure [125]. Moreover, it has been demonstrated that VSEL could be mobilized from BM into peripheral blood in patients after a stroke and acute myocardial infarction, implying their contribution to tissue regeneration [126,127]. However, VSELs purified from murine BM do not proliferate if cultured alone and can only be activated to form embryoid body-like structures and differentiate by coculturing with other cells, such as BM MNCs or myoblasts C2C12 [123]. This suggests that VSELs are a quiescent cell population, which could be further explained by its unique DNA methylation patterns at imprinted genes, similar to the epiblast-derived primordial germ cells [125]. In summary, several populations of primitive stem cells with multilineage differentiation capacity have been reported from UCB. This demonstrates the plasticity of UCB and underscores the value of UCB in cell-based