Accepted Manuscript

Title: Cord Blood Transplantation and Stem Cell Regenerative Potential Authors: Yanling Liao, Mark B. Geyer, Albert J. Yang, Mitchell S. Cairo PII: DOI: Reference: To appear in: S0301-472X(11)00009-9 10.1016/j.exphem.2011.01.002 EXPHEM 2716 Experimental Hematology

Received Date: 28 September 2010 Revised Date: 6 January 2011 Accepted Date: 8 January 2011

Please cite this article as: Liao Y, Geyer MB, Yang AJ, Cairo MS. Cord Blood Transplantation and Stem Cell Regenerative Potential, Experimental Hematology (2011), doi: 10.1016/j.exphem.2011.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cord Blood Transplantation and Stem Cell Regenerative Potential

Yanling Liao1, Mark B. Geyer1, Albert J. Yang1, and Mitchell S. Cairo1, 2, 3 Department of 1Pediatrics, 2Medicine and 3Pathology and Cell Biology, New YorkPresbyterian Morgan Stanley Childrens Hospital, Columbia University, New York, New York Correspondence and reprint requests: Mitchell S. Cairo, MD Chief, Division of Pediatric Blood and Marrow Transplantation Professor of Pediatrics, Medicine, Pathology and Cell Biology New York-Presbyterian Morgan Stanley Childrens Hospital Columbia University 3959 Broadway, CHN 10-03 New York, NY 10032 email: mc1310@columbia.edu Tel: 212-305-8316 Fax: 212-305-8428 Category: Stem Cell Transplantation Word count: 9,265

Abstract The past twenty years of experience with umbilical cord blood transplantation have demonstrated that cord blood is effective in the treatment 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 HLA disparity without a corresponding increase in graft-versus-host disease (GVHD). 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 following 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.

Keywords: Cord blood, regeneration, stem cells, transplantation, cord blood banking

Cord Blood Transplantation Allogeneic stem cell transplantation (AlloSCT) has been employed for more than 40 years and offers the best potential for curing numerous malignant and non-malignant diseases in children and adults. While 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 that are unable to identify a fully matched donor. The first umbilical cord blood transplant (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, more than 10,000 total UCBTs have been performed, with more than 300,000 UCB units (UCBU) available in more than 40 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 (NMDP)[3-4]. In both pediatric and adult patients, the use of bone marrow in unrelated donor transplants has decreased. However, while some 40% of unrelated donor transplants in patients 20 years of age now employ UCB, the use of UCB has increased more modestly in patients >20 years of age to only 7% of unrelated donor transplants [4]. Median search times for unrelated bone marrow or peripheral blood stem cell (PBSC) donors through the NMDP are 51 days, compared with less than two weeks for unrelated UCBCs [5].

Early Experience with Related and Unrelated UCBT Early experiences with UCBT were reported by several groups in the mid1990s. Wagner et al. reported early success in 44 children undergoing sibling UCBT for 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, as well as Wagner and Cairo, subsequently reported on the use of unrelated UCBT in children and adolescents for malignant and non-malignant diseases, noting delayed engraftment compared to bone marrow transplantation, low risk of severe graftversus-host disease (GVHD), delayed engraftment, and encouraging early survival [79]. In the late 1990s, the New York Blood Center (NYBC) and the Eurocord/European Blood and Marrow Transplant (EBMT) group reported more extensive experiences with UCBT. The Eurocord/EBMT group, which analyzed outcomes following 143 related- and unrelated-donor UCBTs, demonstrated higher 1year overall survival (OS) in patients with malignant and non-malignant 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 following unrelated donor BMT, with 1-year relapse risk of 30%, 18%, and 24% in children with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoblastic leukemia (ALL), respectively. Day 100 transplant-related events (graft failure, re-transplant, 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, CML, myelodysplastic syndrome, neuroblastoma, Hodgkin lymphoma (HL), nonHodgkin 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 or second complete remission (CR) (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, though long-term survival was observed among some patients receiving only 2/6 or 3/6-matched UCBT [14]. Randomized studies of unrelated UCB vs. bone marrow 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. reported outcomes in 267 patients diagnosed with ALL or AML before 18 months of age who subsequently underwent matched-sibling marrow transplant (n=101), unrelated marrow transplant (n=85), or UCBT (n=81). Transplant-related 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 between these groups [15]. A larger retrospective study of 785 children ages 0-16 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 (Figure 1). However, two-antigen UCBT recipients had significantly higher risk of TRM when compared to recipients of fully matched bone marrow [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-versus-leukemia effect in mismatched UCB transplant may balance the greater risks of TRM in mismatched UCBT recipients.

UCBT in Adults with Malignant Diseases While 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. 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 UBMT vs. UCBT was observed in the

univariate analysis, no difference was appreciated in the multivariate analysis [17]. Laughlin et al. compared one- and two-antigen mismatched UCBT recipients (n=150) to those receiving matched (n=367) or one-antigen mismatched (n=83) unrelated bone marrow. 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 2.2 x 107/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 increased risk of TRM (37%) following matched or mismatched UCBT compared to fully matched unrelated BMT (22%) or PBSCT (24%), but similar to one antigen-mismatched unrelated BMT (34%) and PBSCT (38%) (Figure 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 MAC. TRM, relapse, and disease-free survival (DFS) did not differ with respect to graft source [20]. Recently, the 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 (JMDP) reported similar 2-year OS among 336 adults with ALL undergoing UCBT (52%) vs. matched related marrow (53%), further corroborating the 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]. Though the JMDP 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. 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 1year progression free survival (PFS) was observed in those with chemosensitive disease, those who received a higher TNC dose/kg, and recipients of conditioning regimens containing low-dose TBI [24]. Small prior studies of single-unit RTC UCBT in adults with advanced HL or NHL had demonstrated 1-year PFS of 25-50% [25-26]. While 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 may be considered a feasible alternative to unrelated BMT.

UCBT in Patients with Non-Malignant Diseases UCBT has been used successfully in the treatment of bone marrow 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 transplant, 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, following either myeloablative conditioning (MAC) or reduced toxicity conditioning (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 non-engraftment 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 transplant was 1.5 years, with 57% of patients under 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 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 18-19% of patients failed to engraft neutrophils [10-11]. Studies continue to note rates of neutrophil engraftment ranging from 6087% following 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-transplant, compared to 76% of recipients of two-antigen mismatched UCB [16]. Moreover, time to hematopoietic recovery is delayed following UCBT, with median times to neutrophil and platelet recovery of 25 and 59 days, respectively, compared to 19 and 27 days, respectively, following unrelated marrow transplant [16]. Prasad et al. and Kurtzberg et al. reported neutrophil and platelet engraftment at a median of 27 and 174 days following 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 non-malignant diseases [14, 33]. Delayed engraftment leaves the UCBT recipient in an extended state of profound immunocompromise, particularly following myeloablative conditioning. Indeed, Rocha et al. found that death from opportunistic infection and hemorrhage were more common following

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matched sibling UCBT than matched sibling BMT, 33% and 21%, respectively (p=0.05)[36]. Possible explanations for the delay in hematopoietic recovery following UCBT compared to BMT include decreased number of total[14] and CD34+ cells in the graft[37], more immature CD34+ progenitors (thus requiring more cell division for differentiation and engraftment), and lack of a subpopulation specializing in engraftment and homing effects[38]. A COLBT study led by Cairo et al. suggested that lower numbers of CD34+/CD41+ cells in UCB compared to peripheral blood may be responsible for the delay in platelet recovery following UCBT[39]. Consistent with increased rates of graft failure and severe post-transplant infection and prolonged inpatient stays, Majhail et al. further demonstrated that the cost of hospitalization following UCBT is greater than the cost following matched related BMT, with the median cost per day estimated to be $2,082 vs. $1,016, respectively [40]. In the COBLT study, total nucleated cell (TNC) dose of greater than 5.1 x 107/kg was associated with significantly faster engraftment of neutrophils and platelets. TNC dose of less than 2.5 x 107/kg was associated with significantly reduced OS as well [14]. Wagner et al. originally noted CD34+ dose less than 1.7 x 105/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+ dose was independently predictive of greater OS among 29 UCBT pediatric recipients (median CD34+ dose 2.3 x 105/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: 2.5-3.0 x 107/kg for fully matched UCBT, 4.0 x 107/kg for 5/6 HLA match, and 5.0 x 107/kg for 4/6 HLA match [43-44].

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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 singleunit 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 one-antigen-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 (Figure 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 5.0 x 107/kg and recipients of oneantigen mismatched UCBUs with TNC 2.5 to 4.9 x 107/kg [45]. Future studies will be needed to determine whether prioritizing HLA match in selecting UCB grafts, while maximizing cell dose by transplanting two units, will allow for faster engraftment while preserving 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-IV acute GVHD following matched unrelated UCBT (24%)

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compared to matched unrelated BMT (46%) or one- or two-antigen mismatched unrelated BMT (60%); the incidence of grade II-IV acute GVHD was 42% following one-antigen mismatched UCBT with high cell dose and 41% following two-antigen mismatched UCBT [16]. Similar risks were reported in other recent studies, with an overall incidence of grade II-IV acute GVHD of 25-45% 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 non-inherited maternal HLA antigens; this has potential implications for mismatched UCBT when a donors noninherited maternal antigen (NIMA) matches with the recipients HLA-disparate antigen. Van Rood et al. reported on the impact of NIMA on transplant outcomes in pediatric patients undergoing UCBT for hematological malignancies. 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, though the difference was most striking in those patients 10 years of age, 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/CML was appreciated in the NIMA-matched group as well. The authors speculate that upregulation of anti-NIMA immunity following re-exposure may reduce the risk malignant relapse without increasing the risk of GVHD, specifically through fetal development of CD4+ T-cells exerting relapse-reducing effects, anti-minor histocompatibility antigen cytlolytic T-cells, and regulatory T-cells from the CD4+

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and CD8+ 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 less than 1.7 x 105 CD34+ cells/kg had significantly poorer survival outcomes [41]. Co-transplantation 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. All patients engrafted neutrophils; 65% developed grade II-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 ATG followed by D-UCBT; >90% of patients engrafted neutrophils, 40% developed grade II-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 non-malignant diseases receiving nonmyeloablative conditioning with cyclophosphamide, TBI, and ATG, significantly

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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=0.009 in univariate analysis, p=0.02 in multivariate analysis) in 104 adults with HL, NHL, and chronic lymphocytic leukemia [24]. A higher incidence of grade II-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 DUCBT vs. single UCBT. Following D-UCBT, a single unit generally ultimately serves as the source for hematopoiesis. Though initial engraftment of both units may be observed, engraftment usually skews progressively such that one unit predominates [50-52, 5556]. Despite the eventual predominance of a single UCBU, the non-engrafting unit may hasten and facilitate engraftment through 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+ dose in a series of 38

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adults undergoing reduced-intensity D-UCBT [51, 57]. Scaradavou et al. recently reported that among 44 D-UCBT recipients who engrafted a single unit, CD34+ viability was significantly higher in the engrafting unit and that units with CD34+ 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. described the development of CD8+ T-cells derived from the dominant UCBU specific for alloantigens present on the non-engrafting unit in 14 adult DUCBT recipients. This population of effector cells produces IFN- in response to the non-dominant unit; no IFN- 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+ T-cells tolerated cells from either unit without significant IFN- production [61]. The exact antigenic specificity of this CD8+ T-cell population and the role (if any) of CD4+ T-cells in graft-versus-graft effects is unknown. Interactions between the two units infused may contribute to graft-versus-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.

Umbilical Cord Blood Expansion TNC and CD34+ cell dose are strong predictors of clinical outcomes following UCBT

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as previously described. UCBUs contain approximately 1x109 TNC on average and only 12% of the current public UCB inventory contains sufficient cell dosage for a 60 kg patient. The goal of umbilical cord blood expansion is to increase the number of progenitor cells capable of rapid repopulation in vivo to improve the kinetics of hematopoietic recovery following UCBT. Currently, ex vivo expansion utilizes three methods: cytokine-containing liquid expansion, co-culture 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 following 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 DUCBT (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.

Conditioning Regimens Most studies of UCBT reported to date have employed MAC prior to transplant.

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Successful engraftment has been observed with total body irradiation (TBI)containing and non-TBI containing regimens. The initial EBMT experience with UCBT used TBI/cyclophosphamide or busulfan/cyclophosphamide conditioning [10]. Anti-thymocyte globulin (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 15-50% has been observed in MAC UCBT recipients, depending on the underlying disease and disease status prior to transplant [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 non-malignant 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 non-malignant diseases [73]. We recently analyzed clinical outcomes among 88 patients undergoing MAC (n=49) or RTC (n=39) prior to UCBT at Columbia University Medical Center; all RTC regimens were fludarabine-based (150-180 mg/m2). 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-

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based reduced intensity regimens, some of which incorporate low-dose TBI, have been successfully used in adult patients, with day 180 TRM less than 20% [25-26, 5152].

Immune Reconstitution Immune reconstitution in pediatric AlloSCT recipients appears to take longer following MAC and UCBT than following matched sibling AlloSCT. NK- and B-cell recovery is rapid and robust following UCBT, with a median of approximately 3 months and 6 months post-transplant needed to achieve age-related normal levels, respectively. However, the median time needed to achieve normal cytolytic T-cell counts is approximately 8-9 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+ T-cell recovery is often observed within 1-3 months post-transplant [75, 77]. T-cell recovery following AlloSCT derives from peripheral expansion of mature, post-thymic donor cells (the thymic-independent pathway) and pre-thymic 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-transplant. 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 post-transplant was significantly higher among UCBT recipients than among

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recipients of either matched or mismatched related BMT; proportions were similar among the groups after 100 days [18]. However, T-cell receptor (TCR) diversity is ultimately greater at 2 years post-UCBT than following matched sibling donor BMT, as measured by TCR excision circles [80]. Parkman et al. noted an association between successful immune reconstitution following 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 (Figure 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 +100/180/365 posttransplant, 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. While most patients achieved normal natural killer (NK) 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 prior to UCBT [82]. Strategies to enhance T-cell recovery in the early posttransplant 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

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was developed by the COBLT program (summarized in [83]). Matters of ongoing controversy include procedures for obtaining informed maternal consent, cost-benefit analysis of increasing the number of publically-available UCBUs [84], the role of UCB directed donor (private) banking, cultural considerations regarding the placentas role [85], and the use of pre-implantation HLA typing [86].

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, bone marrow, 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 cellbased therapy. Compared to other adult stem cells, human UCB stem cells have

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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 (Figure 5). Indeed, the CD133+ fraction of human UCB can be reprogrammed efficiently to iPS cells with retroviral 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+ 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 (Figure 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 MHC I and are negative for MHC II antigens, indicating their immune evasion in the allogeneic setting [98]. These properties have elicited great interest in utilizing MSCs as an offthe-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 bone marrow and adipose (30% verse 100% of UCB units, respectively) [99]. This observation can be explained at least partially by a recent report, which showed that MSCs were 10 times more frequent in the UCB of 24-28 wk gestational age infants

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than 29-32 wk gestational age UCB and even higher than 37-40 wk gestational age UCB [100]. By comparison, endothelial colony forming cells (ECFC), identified based on the outgrowth of cobblestone like adherent colonies between day 7 and 14 following plating of UCB MNCs on collagen I coated plastics, are more enriched in full term (37-40 wk gestational age) UCB [100-101]. The ECFCs 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 (Yoder MC, et.al. blood 2007, 109:1801-1809; Melero-Martin JM, etal. Blood 2007, 109:4761-4768).

CB-derived primitive stem cells Besides these committed multi-potent stem or progenitor cells, accumulating data have indicated the existence of multiple populations of more primitive stem cells in 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, 102]. Therefore, these primitive stem cells could be made readily available from UCB and used as either an autologous source or HLAmatched 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) [102]. These CD45neg/CD34neg cells were isolated from UCB based on their outgrowth in the

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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 [102]. Following 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 following infusion of USSCs to an infarcted heart. These discrepancies might be due to the difference in timing and route of stem cell injection, as will be discussed more below [103-108]. 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 [109]. 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 [110]. MSCs isolated from both bone marrow and UCB are HOX-positive, whereas USSCs resemble H9 ES cells and are HOX-negative [110]. In addition, USSCs possess immuno-modulatory effects, similar to MSCs [111112]. 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 [113]. Moreover, cotransplantation of USSCs in NOD/SCID 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 [114]. As a step further to their future clinical application, USSCs can now be produced and expanded at a GMP-grade [115].

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In collaboration with BioE, Cairo et al. also characterized a population of multi-lineage progenitor cells (MLPC) from CB by non-particular 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+, CD29+, CD44+, CD73+, CD90+, CD105+, CD9+) 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 above, 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 CD235a positive cells [116-118]. The resultant lineage negative cells, which represent 0.1-1% 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-MNCCD34-) was also characterized to express Oct4, Sox2 and Rex1 genes [119]. 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

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[120]. Recently, a population of very small embryonic-like (VSEL) stem cells (CXCR4+lin-CD45-) has been identified from not only UCB, but also from human peripheral blood and bone marrow by multiparameter FACS analysis [121-122]. 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 [123]. Moreover, it has been demonstrated that VSEL could be mobilized from bone marrow into peripheral blood in patients after a stroke and acute myocardial infarction, implying their contribution to tissue regeneration [124-125]. However, VSELs purified from murine bone marrow do not proliferate if cultured alone and can only be activated to form embroyoid body-like structures and differentiate by co-culturing with other cells such as bone marrow MNCs or myoblasts C2C12 [121]. 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 [123]. 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 therapies. However, it should be noted that these cells generally exist at low cell numbers and the efficiency of derivation may vary between different UCB samples. For instance, the generation efficiency of USSCs is about 35% from fresh UCB, and only one to eleven USSC colonies would be initiated in the cases of successful derivation, with a

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median frequency of four colonies per UCB [126]. The efficiency of isolating USSCs from cryopreserved UCB is even lower, and may be influenced by the procedures involved in cryopreservation (un-separated or volume- reduced) and after-thawing (Ficoll density gradient separation or ammonium chloride lysis) [126]. Therefore, the role of these rare stem cells in promoting tissue regeneration upon whole UCBT is yet to be determined. It can be speculated, however, that more significant therapeutic application of these primitive-potent stem cells is to be used as HLA typed universal donor cells alone or together with whole UCB in transplantation.

Regenerative application of UCB and UCB-derived stem cells As discussed earlier, UCB has now been routinely used to treat hematopoietic malignancies, marrow failure, metabolic diseases and immunodeficiency disorders. The plasticity of UCB and its readily accessibility have now encouraged its broader regenerative applications such as the treatment for spinal cord injury and chronic wounds [127-128]. Meanwhile, more clinical trials are in place to infuse UCB for the treatment of other non-hematopoietic diseases, such as epidermolysis bullosa (our center), and neonatal hypoxic ischemic encephalopathy (personal communications). Most of the clinical studies apply un-fractionated UCB with a primary goal to determine the safety and/or efficacy of UCB. The mechanism of action, the optimal therapeutic window and the best route of UCB infusion for each disease remain unknown and can only be inferred from animal studies. Recently, UCBT has been actively explored in animal models of cardiac infarction, diabetes and various neurological diseases including stroke, amyotrophic lateral sclerosis, spinal cord injury, Huntingtons disease, Parkinsons disease and

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hypoxic ischemic encephalopathy. In most of the studies, beneficial effects have been observed following UCBT, although inconsistent or in some cases conflicting results exist. Importantly, it has been clear that the route and timing of transplantation influence the mechanism of action for the transplanted cells and may explain the discrepant outcomes between different studies. Here we will discuss the multifaceted effects of UCB in the context of two most extensively studied experimental models, stroke and myocardial infarction.

Stroke UCBT in animal models for strokes was first reported in 2001. In that initial study, intravenous administration of UCB MNCs 24 hours after transient middle cerebral artery occlusion in a rat stroke model significantly improved neurological functioning [129]. Moreover, the UCB MNCs were able to migrate to the ischemic boundary of the injured hemisphere and some of these cells underwent neural differentiation as demonstrated by their immunoreactivity to neural markers such as GFAP (6%), MAP2 (2%) and NeuN (2%). However, even though surviving UCB cells were identified in the ischemic boundary, they only represented about 1% of the total MNCs administered. Since this initial report, more animal studies have been conducted to compare the effects of routes and timing of transplantation on graft survival and functional recovery. The route of cell delivery is an important factor to consider in determining the balance between the best therapeutic efficacy and minimal pain to the patients. Intravenous administration is noninvasive to patients and has resulted in functional improvement in various neurological diseases in both preclinical and clinical studies.

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However, it is also likely that cells could be trapped to other organs before entering the brain if delivered intravenously. Indeed, a non-quantitative comparative study indicated that more donor cells were found in the damaged striatum with intracerebral administration of neural precursors than with intracerebroventricular delivery and the intravenous injection yielded fewest donor cell engraftment [130-131]. Still, similar degrees of improvement were observed between the animals receiving UCB either intrastriatally or intravenously 24 hours after permanent middle cerebral artery occlusion [132]. Furthermore, no UCB cells were observed within striatum after either intravenous or intrastriatal delivery. Surprisingly, intravenous delivery appeared to be even more effective than striatal delivery in producing long-term functional benefits, as there were less cellular debris and infiltrate in the brains of the animals with intravenous injection. Therefore, according to this report, intravenous administration of CB has more advantages than intrastriatal delivery [132]. In addition to the route of injection, the timing of transplantation and stem cell dose also affect the efficacy of transplantation [133-134]. It has been demonstrated that stroke-induced chemokines, which attract stem cells to the site of injury, began to be up-regulated at about 24h post-stroke [135-136]. Therefore, intravenous injection of UCB immediately after stroke may not provide an optimal time window for the cells to migrate to the injured brain. However, the transplanted cells could still perform neuroprotective function by the releasing of trophic factors [137]. In support of these findings, it was later demonstrated that the timing of UCB intravenous delivery determines therapeutic efficacy and maximum improvements were observed with transplantation 48 hours post-stroke [138]. It appears that the transplanted cells at the acute phase play a neuroprotective role by relieving inflammation and/or

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promoting endogenous neurogenesis. In contrast, in a delayed stage with diminished inflammation, the cell engraftment may become important, in addition to the paracrine effect, in improving long-term neurological functions. Additionally, transplantation of bone marrow stromal cells even a month after a stroke has resulted in functional recovery [139]. Overall, multiple mechanisms have been postulated for functional recovery after transplantation of UCB. The first mechanism involves the release of trophic or neuroprotective factors, such as glial cell line-derived neurotrophic factor, nerve growth factor and brain-derived neurotrophic factor from UCB. It has been demonstrated that central nervous system entry of peripherally injected UCB cells was not required for neuroprotection in strokes, provided that neurotrophic factors released from UCB could enter the brain [137]. The elevated levels of trophic factors consequently promote neovascularization and/or induce sprouting of nerve fibers from the non-damaged hemisphere into the ischemic side of the brain [140-141]. The second mechanism is associated with the anti-inflammatory function of UCB. UCB transplantation has been shown to significantly decrease the number of CD45+/CD11b+ (microglia/macrophage) and CD45+/B220+ (B cell) cells in the brain that are usually at elevated levels after permanent middle cerebral artery occlusion [142]. A recent in vitro study further demonstrated that CD11b+- or CD19+-UCB cells decrease the survival of microglia [143]. Besides the anti-inflammatory effect, UCB has also been postulated to modulate stroke-induced peripheral immune responses, as measured by its ability to oppose stroke-induced spleen weight loss and alterations in spleen T-cell subset and proliferation [144]. The immuno-modulatory function of UCB was also implicated in an animal model of amyotrophic lateral sclerosis, where

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an increase in spleen mass was observed following UCBT [145]. The last potential mechanism relates to the ability of UCB to migrate, engraft and possibly differentiate at the sites of lesion. Similar migratory effects have also been observed with transplantation of neural precursor cells and in other animal models such as hypoxic ischemic encephalopathy, amyotrophic lateral sclerosis and Huntingtons disease [131, 146-149]. In addition to intravenous injection, intraperitoneal injection of UCB 24 hours after hypoxic infarction in a rat model of hypoxic ischemic encephalopathy also led to migration of the transplanted cells in the ischemic hemisphere [150]. The targeted migration is likely to be mediated by ischemia-induced chemokines, such as stromal-derived factor-1, monocytes chemoattractant protein-1 and macrophage inflammatory protein (MIP-alpha) [131, 146, 151-152]. However, as only a small percentage of the transplanted cells could reach the injury location and the number of cells undergoing neural differentiation was even lower, the mechanism by cell replacement may not play a major role in the observed functional improvement following UCBT. Moreover, graft survival does not guarantee neurological improvement, as suggested by a transplantation study with human neuroteratocarcinoma neurons. In that study, no functional improvement was observed in the experimental animals despite clear evidence of cell survival and neurite extension [153]. Therefore, functional integration of the transplanted cells into the resident tissue appears to be more important than mere graft survival in regenerating the damaged brain tissue.

Myocardial infarction (MI) Various cell sources, i.e., MSCs, HSCs, endothelial progenitor cells, skeletal

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myoblasts, cardiac stem cells, ES cells and unfractionated bone marrow MNCs have been explored in preclinical and clinical cell based therapy for MI [154]. Generally beneficial effects, such as angiogenesis, decrease of infarction size and improvement of left ventricular (LV) ejection fraction have been observed. However massive donor cell death has generally been observed. Accordingly, survival and further differentiation of the transplanted cells have been limited and the mechanisms of action have mainly been attributed to paracrine effects on injured myocardium. The use of UCB for the treatment of MI has also been actively investigated. In animal models of MI, direct injection of CD34+, CD133+ or MNCs from UCB into the necrotic myocardium or the border of the infarction following acute permanent coronary artery ligation, resulted in reduced infarction size and improved LV function [155-157]. Meanwhile, migration of the cells to the damaged heart has been observed following intravenous injection of UCB CD133+ cells or MNCs, 7 days and 1 day after MI, respectively [155, 158-159]. Moreover, enhanced angiogenesis and reduction in infarction size have also been achieved following intravenous injection [155, 158-159]. Later, an elegant study was conducted to compare the effects of the administration of UCB MNCs by intramyocardial, intracoronary or intravenous injections, as well as the dose, on the infarction size in rats with acute MI [160]. The authors concluded that all three administration methods resulted in significant reductions in infarct size without host immune suppression, as compared to Isolytetreated infracted hearts. Among these methods, intramyocardial injection of UCB most effectively reduced infarct size and required the least number of cells (4 x 106). In comparison, 16 x 106 cells are the optimal dose for intravenous injection. The authors also observed a plateau in infarction size reduction with each route of

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injection and suggested that use of extremely large number of cells is not necessary and in some cases may be detrimental. In the same study, intracoronary injection of 32 x 106 cells produced cardiac arrhythmias and sudden death in four of the experimental rats, most likely due to myocardial microinfarcts as a result of stem cell clumping and coronary arterial emboli. In addition to the CD34+ or CD133+ fraction and MNCs of UCB, UCB derived USSCs have also been applied to treat MI in animal models. In a chronic porcine model of MI, direct injection of 108 USSCs to the border of infarction 4 weeks post MI resulted in decrease in infarct size, reduction in end-diastolic volume and improvement in LV ejection fraction [106]. Meanwhile, the engrafted USSCs could be detected 4 weeks after transplantation. In contrast, in an acute porcine model of MI, myocardial injection of 1.3 x 107 USSCs led to a surprising, complete prevention of scar formation and dramatic functional improvements [161]. However, long-term survival of the transplanted cells and donor-based regeneration of myocardium were not observed. The authors concluded that differentiation, apoptosis and macrophage mobilization at the infarct site were not the mechanisms for the observed cardiac improvement. Instead, they suggested that the improvement was due to the paracrine effects on preserving/promoting the regeneration of recipient myocardium. Recently, encouraging results have also been reported on intramyocardial transplantation of USSCs into immunodeficient rats with acute MI [107]. In this study, transplantation of USSCs (105 or 106) dose-dependently preserved LV function. In addition, human specific cardiomyocytes, smooth muscle cells and endothelial cells have been detected in the ischemic rat myocardium, in a mechanism that is independent of cell fusion. This suggested vasculogenic and

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cardiomyogenic potential of USSCs and shed a light on their clinical application for MI. Despite all these promising results showing the efficacy of USSCs on MI, negative results have also been reported. In a similar porcine model of MI, Moelker and coworkers injected 108 USSCs into the infarct-related coronary artery 1 week post MI, mimicking the clinical scenario of an ongoing clinical trial of BMT [104]. MRI analysis failed to document any improvement in regional or global LV function. The transplanted cells survived only in the infarct border zone at 5 weeks and did not express any cardiomyocyte or endothelial markers. Indeed, intracoronary transplantation of USSCs resulted in more extensive infarcts, accompanied by increases in inflammatory cell infiltration and calcification in the infarct zone, compared to medium treated swine. The major difference between this negative study and previous positive studies is the route of administration. While intracoronary injection theoretically allows the transplanted cells to follow the blood flow to the perfused region, it also runs the risk of causing coronary obstruction. With a size of approximately 25m, which is about twice as big as freshly isolated MNCs from bone marrow or UCB, it is not surprising that USSCs cause the formation of microinfarctions. Similar observation has been reported with intracoronary injection of bone marrow derived MSCs [162]. To further explore the risk of USSCs in microembolization, Moelker and coworkers injected 50 x 106 USSCs in the artery of normal healthy myocardiums and observed similar extensive micro-infarction in the injected area [104]. Therefore, although it is likely safe to transplant MNCs via intracoronary injection using an optimal dose, transplantation of ex vivo expanded cells such as USSCs and MSCs needs to proceed with caution and administration by

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the route of intracoronary injection should be avoided. In a later study, the abilities of UCB-derived USSCs and MNCs to rescue MI were also evaluated in a rat model of MI by permanent coronary artery occlusion and by ischemia/reperfusion injury, following either intravenous or intramyocardial injection [108]. Except for a few changes in the expression of the extracellular matrix and cytokines in the infarct area, there was no sign of cardiac regeneration. Improvement in heart function or attenuation on LV dilatation was also not observed in this study.

Conclusions based on preclinical regenerative application of UCB While encouraging results have been obtained in many preclinical studies of UCB cell therapy, challenges remain and these challenges are not specific to UCB per se, but applicable to transplantation of other stem cell sources as well. Although the original idea of stem cell therapy is to replace the damaged cells and regenerate the affected organs, significant donor cell engraftment and functional integration of the survived donor cells have rarely been reported. While functional improvements have been observed in the absence of the evidence for donor cell engraftment, mainly in the studies with transplantation following acute insults, longterm survival of the transplanted cells and their functional integration to the recipient tissue will likely further enhance the beneficial effects of transplantation. Strategies to promote donor cell survival, such as preconditioning the cells with cytokines or genetically modifying the donor cells are now being actively explored [163]. It can also be expected that in a severe degenerative scenario, such as a cyst formation as a result of hypoxic ischemia, transplantation of stem cells supported by bioengineered matrix would help retain donor cells and enhance their reciprocal interactions with the

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recipient tissue, as has been suggested in an earlier study [164]. In addition, noninvasive measures for stem cell tracking via MRI, bioluminescence, or fluorescence resonance energy transfer (FRET), will also provide a quantitative evaluation on the survival and fate of the transplanted cells. Lastly, extensive animal studies have suggested that route of administration and timing of stem cell therapy affect the mechanism of action for the transplanted cells. Meanwhile, the dose and choice of the donor cells may also have immediate effects on the outcomes.

Summary UCB has now been routinely used as an alternative source of HSCs to bone marrow for the treatment of a variety of malignant and nonmalignant diseases, including hematologic malignancies (most prominently acute leukemias), marrow failure syndromes, hemoglobinopathies, and inherited lysosomal and peroxisomal storage diseases. Compared to UBMT, UCBT offers enhanced ability to cross donor-recipient HLA barriers without increased risk of GVHD, but is limited by increased risk of graft failure and life-threatening infection. Greater TNC dose and closer HLA match appear to be independently predictive of decreased TRM and enhanced OS. Strategies to expedite hematopoietic recovery and reduce TRM include D-UCBT and ex vivo expansion, which are under active clinical investigation and help extend UCBT to larger recipients; D-UCBT may additionally lower risks of malignant relapse. UCB stem cells offer many practical advantages such as relative ease of procurement, minimal risk to the donors and the ability to bank the screened and HLA-typed samples for public uses. Moreover, UCB also possesses a primitive ontogeny, has not

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been exposed to immunological challenges and is expected to carry minimal somatic mutations. Further, there has been accumulating evidence for the existence of very primitive stem cells that share ES cell properties yet do not pose the risk of teratoma formation upon transplantation. Therefore, these primitive UCB-derived stem cells could be a suitable alternative to ES cells for tissue regeneration.

Acknowledgements Supported in part by grants from the Pediatric Cancer Research Foundation, Dream.Discover.Cure., Marisa Fund, Sonia Scaramella Fund, Andrew J. Gargiso, Jr. Foundation, Paul Luisi Foundation, Brittany Barron Fund, and the Doris Duke Charitable Foundation.

Support and Financial Disclosure Declaration No financial interest/relationships with financial interest relating to the topic of this article have been declared.

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Table 1. Outcomes following unrelated umbilical cord blood transplantation vs. other stem cell sources in the treatment of hematologic malignancies
Median Time to Incidence of Incidence Incidence DFS OS Engraftment in Grade II-IV of TRM of Relapse Days aGVHD * * * 2 yr: 2 yr: 2 yr: Rocha et al., NEJM, ALL, AML MUBMT 584 15-59 (32) 19 39% 38% 23% 38% 42% 2004 (Ref. 17) M/1-3MMUCBT 98 15-55 (24.5) 26 26% 44% 23% 33% 36% * * * * *3yr: *3yr: Laughlin et al., NEJM, ALL, AML, MUBMT 367 All 16-60 18 48% 46% 23% 33% 35% 2004 (Ref. 18) CML, MDS 1MMUBMT 83 20 52% 65% 14% 19% 20% 1-2MMUCBT 150 27 41% 63% 17% 23% 26% * * * * *CR1 /ADV 3yr, CR1 /ADV 3yr, CR1 /ADV Eapen et al., JCO, 2006 ALL, AML MSDT 101 <1.5, All 17 Unspecified 6% 47% /65% 49% /20% 54% /35% (Ref. 15) M/1MMUBMT 85 <18 months 18 15% 24%/45% 54%/30% 62%/33% M/1MMUCBT 81 22 31% * * * * * *5yr: Eapen et al., Lancet, ALL, AML MUBMT 116 All <16 19 (all UBMT) 46% 21% 39% 38% Unspecified 2007 (Ref. 16) 1-2MMUBMT 166 60% 31% 31% 37% MUCBT 35 27 (all UCBT) 24% 6% 31% 60% 1MMUCBT(h) 201 42% 29% 29% 45% 1MMUCBT(l) 36% 43% 20% 36% 2MMUCBT 267 41% 46% 19% 33% * * * * 2 yr, in CR/not Eapen et al., Lancet ALL, AML MUBMT 332 All >16 (33) 19 39% 22% 34% 52%/17% Unspecified Oncology, 2010 (Ref. 1MMUBMT 140 46% 34% 30% 41%/14% 19) MUPBSCT 632 (39) 14 48% 24% 33% 50%/17% 1MMUPBSCT 256 52% 38% 30% 39%/17% M/1-2MMUCBT 165 (28) 24 30% 37% 26% 44%/15% *= p<0.05 between two or more compared groups, ALL= acute lymphoblastic leukemia, AML= acute myeloid leukemia, CML= chronic myeloid leukemia, MDS= myelodysplastic syndrome, CR1= first complete response, ADV= relapse or CR2, M/MM matched/mismatched (MM preceded by number of HLA mismatches), UBMT= unrelated bone marrow transplant, UCBT= umbilical cord blood transplant, MSDT= matched sibling donor stem cell transplant, UPBSCT= unrelated peripheral blood stem cell transplant, (h) vs. (l)= cell dose > vs. 3x107 total nucleated cells/kg recipient body weight, aGVHD= acute graft versus host disease, TRM= transplant-related mortality, DFS= disease free survival, OS= overall survival Report Diseases Cell Source and HLA Match N Age Range in Years (Median)

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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 donorrecipient 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

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Figure Legends Figure 1. Probability of leukemia-free survival after bone-marrow and cordblood transplantation adjusted for disease status at transplantation. Reprinted from The Lancet, Vol. 369, Eapen M, Rubinstein P, Zhang MJ, Stevens C, Kurtzberg J, Scaradavou A, et al. Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: a comparison study, pages 1947-54, Copyright (2007), with permission from Elsevier.[16]

Figure 2: Probabilities of transplant-related mortality by hematopoietic stem-cell source and donorrecipient HLA matching in adults with acute leukemias. Reprinted from Lancet Oncology, 11/7, Eapen M., Rocha V., Sanz G., Scaradavou A., Zhang M-J., Arcese W., Sirvent A., Champlin R.E., Chao N., Gee A.P., Isola L., Laughlin M.J., Marks D.I., Nabhan S., Ruggeri A., Soiffer R., Horowitz M.M., Gluckman E., Wagner J.E., Effect of graft source on unrelated donor haemopoietic stem-cell transplantation in adults with acute leukaemia: a retrospective analysis, 653660, Copyright (2010), with permission from Elsevier.

Figure 3. Cumulative incidence of 3-year TRM following 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 0-MM units and 3-MM units were not. Blood: journal of the American Society of Hematology by Barker JN, Scaradavou A,

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Stevens CE.. Copyright 2010 by AMERICAN SOCIETY OF HEMATOLOGY (ASH). Reproduced with permission of AMERICAN SOCIETY OF HEMATOLOGY (ASH) in the format Journal via Copyright Clearance Center.[45]

Figure 4. Cumulative incidence of leukemic relapse by positive (solid line) vs. negative (dotted line) proliferative response status to the herpes viruses (CMV, HSV, VZV), (p =0.003). Those with antigen-specific proliferative responses to any of the 3 viruses at any time during the 3-year observation period were considered positive. Reprinted from Biol Blood Marrow Transplant, Vol 12/9, Parkman R, Cohen G, Carter SL, Weinberg KI, Masinsin B, Guinan E et al. Successful immune reconstitution decreases leukemic relapse and improves survival in recipients of unrelated cord blood transplantation, pages 919-927, Copyright (2006), with permission from Elsevier.[81]

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 non-malignant 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.

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