Expansion and homing of umbilical cord blood hematopoietic stem and progenitor cells for clinical transplantation.

Biol Blood Marrow Transplant xxx (2015) 1e12

1 2 3 4 5 journal homepage: www.bbmt.org 6 7 8 9 10 11 12 13 1, 2 14 , Kevin Kwee Hong Seah 3, Zhiyong Poon 4, Alice Man Sze Cheung 5, Xiubo Fan 6, Q 12 Sudipto Bari 15 1 Shin-Yeu Ong , Shang Li 5, Liang Piu Koh 7, William Ying Khee Hwang 1, 5, 8, * 16 1 17 Department of Hematology, Singapore General Hospital, Singapore 2 Department of Pharmacy, National University of Singapore, Singapore 18 3 Sydney Medical School, The University of Sydney, Australia 19 4 BioSystems and Micromechanics, Singapore-MIT Alliance for Research and Technology, Singapore 20 5 Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore 6 21 Department of Clinical Research, Singapore General Hospital, Singapore 7 Department of Hematology-Oncology, National University Cancer Institute, Singapore 22 8 23 Q 1 Singapore Cord Blood Bank, Singapore 24 25 a b s t r a c t Article history: 26 Received 19 November 2014 The successful expansion of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB) 27 Accepted 22 December 2014 for transplantation could revolutionize clinical practice by improving transplantation-related outcomes and 28 making available UCB units that have suboptimal cell doses for transplantation. New cytokine combinations 29 Key Words: appear able to promote HSPC growth with minimal differentiation into mature precursors and new agents, 30 Ex vivo expansion such as insulin-like growth factorebinding protein 2, are being used in clinical trials. Molecules that simulate 31 Hematopoietic stem and the HSPC niche, such as Notch ligand, have also shown promise. Further improvements have been made with Q2 progenitor cells 32 the use of mesenchymal stromal cells, which have made possible UCB expansion without a potentially Hematopoietic stem cell 33 deleterious prior CD34/CD133 cell selection step. Chemical molecules, such as copper chelators, nicotinamide, transplantation 34 and aryl hydrocarbon antagonists, have shown excellent outcomes in clinical studies. The use of bioreactors Umbilical cord blood 35 could further add to HSPC studies in future. Drugs that could improve HSPC homing also appear to have Clinical trials potential in improving engraftment times in UCB transplantation. Technologies to expand HSPC from UCB and 36 Engraftment to enhance the homing of these cells appear to have attained the goal of accelerating hematopoietic recovery. 37 Further discoveries and clinical studies are likely to make the goal of true HSPC expansion a reality for many 38 applications in future. 39 Ó 2015 American Society for Blood and Marrow Transplantation. 40 41 42 43 44 45 annually, and this number is expected to plateau in the next INTRODUCTION 46 few decades. Compared with use of bone marrow (BM) or Since the first umbilical cord blood transplant (UCBT) was 47 mobilized peripheral blood stem cells (PBSC), the use of UCB performed in 1988 to successfully treat a patient with Fan48 for transplantation has several advantages, including greater coni’s anemia [1], over 2 decades of clinical and preclinical 49 ease of finding a donor and prompt availability (with over work have been established umbilical cord blood (UCB) as a 50 700,000 units stored worldwide) [3,4]. More importantly, Food and Drug Administrationeapproved source of he51 the greater tolerance across human leukocyte antigen (HLA) matopoietic stem and progenitor cells (HSPCs) [2]. To date, 52 barriers and lower incidence of graft-versus-host-disease over 30,000 UCBT have been performed worldwide to treat 53 (GVHD) [5] makes UCB an ideal alternative for a significant various blood diseases and genetic disorders. Among all 54 number of HSCT patients (approximately 40% of Caucasians hematopoietic stem cell transplantations (HSCT) performed 55 and up to 55% to 80% of non-Caucasians) who do not have worldwide, approximately 10% of patients undergo UCBT 56 access to a HLA-matched donor [6,7]. Furthermore, in several 57 meta-analyses, UCBT has also been shown to lead to equivFinancial disclosure: See Acknowledgments on page 10. 58 alent outcomes to fully matched BM transplantations in both * Correspondence and reprint requests: William Ying Khee Hwang, FRCP, 59 adult and pediatric patients, thus serving as an effective FAMS, MMed, MRCP, MBBS, The Academia, Level 3, 20 College Road, 60 donor source for allogeneic HSCT for patients without a Singapore 169856. 61 matched sibling donor [8,9]. E-mail address: [email protected] (W.Y. Khee Hwang). 62 63

Biology of Blood and Marrow Transplantation

Expansion and Homing of Umbilical Cord Blood Hematopoietic Stem and Progenitor Cells for Clinical Transplantation

http://dx.doi.org/10.1016/j.bbmt.2014.12.022 1083-8791/Ó 2015 American Society for Blood and Marrow Transplantation.

REV 5.2.0 DTD YBBMT53732_proof 9 January 2015 2:19 pm ce LS

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S. Bari et al. / Biol Blood Marrow Transplant xxx (2015) 1e12

Table 1 Clinical Strategies to Enhance Expansion and Homing of UCB Grafts 1. Ex vivo expansion Hematopoietic cytokines in liquid culture SCF, TPO, and Flt-3L is the most potent cocktail for HSPC maintenance, whereas cytokines such as IL-3, IL-6, IL-11, and G-CSF rapidly generate differentiated cells. Growth factors secreted by differentiated cells deter self-renewal capacity of HSPC. Novel growth factors include IGFBP-2, Angptl proteins, and pleiotrophin. MSC cocultures recreate the microenvironment of the BM Allogeneic BM-derived MSC eliminate the need to perform stem cell selection and control vital functions of HSPC through direct cell-to-cell contact and proteomic secretion. Chemical molecules and proteins that regulate signaling pathways of HSPC Molecules, such as tetraethylenepentamine TEPA (copper chelator), NAM (sirtuin 1 inhibitor), SR1 (aryl hydrocarbon receptor antagonist), and immobilized delta-1 (Notch ligand). Bioreactors and devices that allows continuous perfusion culture Large-scale expansion that maintains an optimal concentration of the potent cytokines while minimizing inhibitory factors. 2. Homing Inhibition of CD26/Dipeptidylpeptidase IV (DPP-4) Interfere in the chemotaxis of CD34þ human hematopoietic cells through DPP-4 enzymatic cleavage in SDF-1. Complement fragment 3a (C3a) priming Anaphylatoxin C3a receptor, which binds to C3a (modulates SDF-1 dependent chemotaxis of CD34þ cells) to allow influx of calcium into the CD34þ cells. Dimethyl-prostaglandin E2 Regulates Wnt pathway via degradation of b-catenin and increases expression of CXCR4. Fucosylation UCB CD34þ cells exhibit poor homing due to a defect in binding BM endothelial selectin as its ligand possess inadequate alpha13 fucose required to form glycan determinants, such as sialyl Lewis x (sLeX). 3. Other clinical strategies Choosing UCB unit that has higher TNC dosage and the optimal HLA match. High TNC is the most critical factor in mediating a successful UCBT; however, it lowers chances of finding such units for patients who belong to racial minorities. Improving methods of UCB collection along with minimizing postthaw cell loss Placental perfusion increases collected cells but requires specialized technical expertise to perform. Increasing the number of infused TNC using either dUCB units or a single UCB unit along with haploidentical CD34þ cells. Dual UCB units or coinfusion of haploidentical CD34þ cells enhances UCB engraftment but requires complex HLA matching with potentially increased incidence of GVHD. Improved homing by targeted transplantation of UCB graft via intra-bone or intraosseous infusion Compared with i.v. infusion, intraosseous infusion is an invasive procedure that causes site morbidity. Coinfusion with accessory cells, such as allogeneic BM-MSC Enhances homing and engraftment of the transplanted HSPC by creating a suitable microenvironment and alleviating symptoms of GVHD. Infusion of mature immune cells derived from part or whole UCB unit Overcomes the current limitation of DLI in UCBT, which could treat post-transplantation complications, such as relapse, infections, and GVHD.

TEPA indicates tetraethylenepentamine; DLI, donor lymphocyte infusion.

UCBT has a characteristically slower rate of hematopoietic recovery, relative to transplants of BM or PBSC, as a consequence of a lower absolute HSPC content [4]. Median time to neutrophil recovery is typically more than 25 days for

unmanipulated UCB grafts versus a median of less than 20 and 24 days, respectively, for PBSC or BM grafts [10,11]. The profound delay in hematopoietic reconstitution increases risk of opportunistic microbial and viral infection in the pancytopenic recipients, thus contributing to the high transplantation-related mortality of >30% after UCBT [12]. However, the mortality risks appear to be lower with a higher infused cell dose for transplantation [13]. Current strategies for improving the clinical outcome of UCBT for adult patients, as outlined in Table 1, focus on increasing the effective cell dose of the graft in an effort to enhance hematopoietic cell engraftment. Although the simultaneous administration of 2 unmanipulated UCB grafts has demonstrated some possible improvement over single UCBT, the rate of hematopoietic recovery still remains suboptimal, with the reported median time to neutrophil and platelet repopulation ranging widely between 12 and 32 and 41 and 105 days, respectively [14]. In view of this, approaches for ex vivo expansion of a single UCB unit to achieve cell numbers for a successful adult transplantation have been widely sought after. In this review, we present emerging data from preclinical and clinical studies of UCB expansion with recently developed agents (including cytokine combinations, proteins, and chemicals) in combination with novel strategies, such as the use of mesenchymal stromal cell (MSC) coculture and bioreactors. Additionally, methods to enhance homing of UCB to facilitate hematopoietic recovery, as well as the crucial cellular pathways controlling HSPC survival and proliferation, will also be discussed. Although some preclinical data will be reviewed, including animal models using severe combined immunodeficiency (SCID) mice, this paper will focus mainly on strategies that have been, or are close to being, brought to clinical trials.

RECENT STRATEGIES TO EXPAND UCB GRAFTS Cytokines In the past 2 decades, various cytokines have been identified to play an important role in regulating HSPC survival, proliferation, and differentiation [15,16]. Although numerous studies have attempted to use these factors for HSPC expansion in vitro, the optimal combination and concentration are yet to be established, possibly because of the limited desired effects of these cytokines alone on the target population. Today, HSPC expansion cytokine cocktails vary among the various preclinical and clinical protocols but they generally include 3 key factors: stem cell factor (SCF), thrombopoietin (TPO), and Flt-3 ligand (Flt-3L) [17,18]. This cytokine combination has been shown to maintain hematopoietic cell viability [19] and telomere length coupled with elevated telomerase activity [20], as well as to regulate expression of adhesion molecules and enhancers of stem cell expansion [21,22]. The use of other cytokines, such as erythropoietin, granulocyte colonyestimulating factor (GCSF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 11 (IL-11), macrophage colonyestimulating factor, and plateletderived growth factor, is more variable and with a tendency to promote cell differentiation [17,18,23]. For example, IL-3 supports rapid total cell expansion in vitro but the expanded cells possess reduced in vivo hematopoietic regenerative potential [24]. An important expansion study by Levac et al. showed that when the serum-free media were supplemented with the potent cocktail of SCF, TPO, and Flt3L, withdrawal of several differentiation-promoting cytokines, such as IL-3, IL-6, and G-CSF from the expansion


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Table 2 Brief Description of Protocols that Enhanced Expansion of UCB Grafts in Completed Clinical Trials Approach/System

Cell Selection of Manipulated Unit

Growth Factors

Infusion of Unmanipulated Cells

Details of UCB Unit Manipulation

Ref.

Cytokine

CD34 selected

G-CSF, SCF, and MGDF

Larger fraction of the same UCB unit

[26]

Continuous perfusion culture

Not performed

FBS, glutamine, HS, hydrocortisone, PIXY321, EPO, Flt-3L

Copper chelation

CD133 selected

FCS, TPO, IL-6, Flt-3L, SCF, and TEPA

Notch mediated

CD34 selected

MSC coculture

Not performed

Fibronectin, IL-3, IL-6, TPO, Flt-3L, and SCF SCF, Flt-3L, TPO, and G-CSF

Nicotinamide

CD133 selected

UCB unit frozen in 2 fractions, 1 of which was thawed, selected, and expanded for 10 d and infused into patients followed by the unmanipulated fraction. The majority of the UCB unit was transplanted into the patients, while remaining cells were expanded for 12 d in the AastromReplicell system and infused. The smaller fraction was thawed, selected, and expanded for 21 d and infused into the patients 24 h after infusion of the unmanipulated fraction. The CD34þ cells from the smaller unit were cultured for 16 d and transplanted. Smaller UCB unit cocultured with MSC feeder layer for 7 d, after which the nonadherent cells were transferred to culture bags while the flasks were replenished with growth media. On day 14, all the nonadherent cells in the bags and flasks were pooled, washed, and transplanted. The CD133þ cells from the smaller UCB unit were cultured for 19 to 23 d while the CD133 fraction was cryopreserved. Patients consecutively received the expanded fraction and rethawed T cell replete.

Separate UCB unit with higher TNC, which was transplanted before the expanded cells

FBS, Flt-3L, SCF, TPO, IL-6, and NAM

[27]

[28]

[29] [30]

[31]

MGDF indicates megakaryocyte growth and development factor; HS, horse serum; EPO, erythropoietin.

culture had a negligible negative impact on the number of SCID mouse repopulating cells (SRCs) [25]. A clinical trial involving expanded UCB (Table 2), conducted at the MD Anderson Cancer Center, made use of CD34þ selected cells from a fraction of the graft for expansion with a combination of SCF, G-CSF, and TPO over a 10-day period [26]. UCB units with quite low cell doses were used and the protocol led to a median 4- and 56-fold increase in the CD34þ and total nucleated cell (TNC) numbers, respectively (Table 4). The study enrolled 37 patients suffering from hematological malignancies and reported median time to neutrophil and platelet recovery to be 28 and 106 days, respectively (Table 5). Approximately 63% of patients exhibited extensive symptoms of chronic GVHD and only 35% patients survived beyond 30 months after transplantation. Recent studies have reported a range of growth factors that are capable of inducing HSPCs expansion in UCB. Examples include insulin-like growth factorebinding protein-2 (IGFBP-2) [35], angiopoietin-like (Angptl) proteins [36], and pleiotrophin [37]. The addition of IGFBP-2 and Angplt5 to UCB expansion cultures containing SCF, TPO, and fibroblast growth factor-1 have been demonstrated to significantly increase the number of SRC by 20 fold [35]. Thorough experimentation demonstrated that IGFBP-2 can synergize

with neurotrophin-3 to further enhance HSPCs expansion [38], whereas Angptl-like proteins preserved stem cells during expansion by binding to the immune-inhibitory receptor human leukocyte immunoglobulin-like receptor B2 [39,40]. Conversely, Ventura et al. reported that combination of IGFBP-2 and Angptl-5 impaired short-term lymphoid reconstitution [41]. Pleiotrophin had a milder expansion effect on human LinCD34þCD38 cells, which was likely mediated via the phosphoinositide 3-kinase and Notch signaling pathways [37]. In an ongoing clinical trial in Singapore, nonenriched UCB units are expanded in an MSC coculture system, which is supplemented with IGFBP-2, SCF, TPO, and Flt-3L for 11 days [42] before infusion into myeloablated patients in combination with an unmanipulated unit. Preliminary data (unpublished) suggest that the expanded unit is likely to contribute to the long-term hematopoiesis (>6 months after transplantation) of the recipients. The complete pilot trial results are expected to be reported by mid-2015. There is also recent demonstration that rapidly differentiating cell populations in the expansion culture secrete cytokines and chemokines, such as tumor necrosis factor-a, macrophage inflammatory protein-1a [43], monocyte chemotactic protein-1 [44], CCL2, CCL3, CCL4, and CXCL10

Table 3 Brief Description of Protocols that Enhanced Homing of UCB Grafts in Completed Clinical Trials Homing Enhancement Strategy

Infusion of Separate Unmanipulated UCB Unit

Details of UCB Unit Manipulation and Infusion into Patient

Ref.

CD26/DPP-4 inhibition

No

[32]

C3a priming

Yes

PGE2 exposure

Yes

Conventional single unit UCBT where the patients were conditioned with the DPP-4 inhibitor Sitagliptin at every 24 h starting at days -1 to þ2 of the transplantation. A UCB unit was primed with C3a for 15 min at room temperature and infused into the patients without any further washing within 30 min of infusing the unmanipulated unit. The larger of the 2 units was incubated with PGE2 for 120 min at 37 C, which was washed and infused in the patients followed by the smaller unmanipulated unit.

DPP-4 indicates dipeptidylpeptidase IV; C3a, complement fragment 3a.


[33] [34]

322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386


Nicotinamide

MAC indicates myeloablative conditioning; TBI, total body irradiation; Melph, melphalan; Bu, busulfan; Cy, cyclophosphamide; Ara-C, cytarabine; ATG, antithymocyte globulin; UMn, unmanipulated UCB unit or fraction; Ex, expanded UCB unit or fraction; Fluda, fludarabine; Thio, thiotepa; MTX, methotrexate; MMF, mycophenolate mofetil.

[31] UMn: 7.0 (3.0-48.0) Ex: 350 (90.0-1830.0) 72.0 (16-186) 4/6 Tacrolimus þ MMF

486 (171-463)

4/6 MSC coculture

MAC: Melph, Thio, Fluda, ATG MAC: TBI, Fluda, Cy

Tacrolimus þ MMF

12.2 (1.0-29.8)

30.1 (0-37.8)

UMn: 2.6 (1.9-4.3) Ex: 2.5 (1.7-3.8)

[30]

[29]

UMn: 24 (6-54) Ex: 603 (92-1330) UMnþEx: 181 (9.0-988.0) 91.5 (41-471) *Mean 562 (146-1496) 4/6 Notch mediated

Continuous perfusion Copper chelation

Tacrolimus þ MTX

e

161 (2-620)

2.26 (.67-19.18)

UMn: 3.3 (1.5-6.3) Ex: 4.6 (.6-9.1) UMnþEx: 8.34 (1.66-19.19)

[28]

[27]

UMn: 7.8 (2.0-159.9) Ex: 1.0 (1.0-16.6) UMnþEx: 15.0 (5.0-463.0) 3/6 (HLA-DRB1 prioritized) 4/6 Prednisolone þ Cyclosporine

2.4 (1.0-8.5)

.5 (.09-2.45)

UMn: 2.05 (1.1-5.5) Ex: 2.05 (.06-10.19) UMnþEx: 1.8 (1.12-5.63)

[26] UMnþEx: 10.4 (.97-311.0) UMnþEx: .99 (.28-8.50) 4/6 Prednisolone þ Cyclosporine

MAC: TBI, Melph, Bu, Cy, Ara-C, ATG MAC: TBI, Cy, Bu, Melph, ATG MAC: Melph, Fluda, Bu, Thio, ATG MAC: TBI, Cy, Fluda Cytokine

56 (1.03-278)

4.0 (.1-20.0)

CD34 Dosage (cells/kg, 104) TNC Dosage (cells/kg, 107) CD34 Expansion (Median, Fold) TNC Expansion (Median, Fold) Minimum HLA Matching (Loci) GVHD Prophylaxis Conditioning Regimen Study

Table 4 Clinical Transplantation Details of the Expanded UCB Grafts

387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451

Ref.

Q 13

4

[45], which induce a detrimental effect on the in vitro HSPCs expansion. It is thought that the inhibitory factors either negatively regulate HSPCs survival and proliferation or they alter the function of the stimulatory cytokines. To address this issue, Zandstra et al. developed a computer-monitored fed-batch culture system, which targets to maintain the concentrations of cytokines within an optimal range via systematic growth media dilution [46]. This method led to an 80-fold increase of CD34þ cells in a 12-day expansion culture with enhanced SRC. Current data suggest that the mere addition of cytokines may not suffice in achieving the optimal HSPC growth factor milieu; rather, specific cytokine types and their concentrations may need to be tweaked routinely, depending on culture duration and cell population composition, to achieve the desired ex vivo expansion effect [47]. Molecular Simulation of HSPC Niche Over the years, widespread effort in understanding HSPC regulation has resulted in the identification of a myriad of potential molecular targets that await further validation for clinical expansion. Indeed, a number of these have demonstrated promising effects on UCB cells in the laboratory. The addition of soluble forms of sonic hedgehog protein to cytokine-supplemented cultures of human C34þCD38 CB cells has been shown to increase both primitive repopulating cells as well as myeloid colony-forming progenitors [48]. Interestingly, this expansion appears to be dependent on bone morphogenetic protein 4, implicating the bone morphogenetic protein pathway as a potential target of intervention. Another signaling target of interest is the Wnt pathway. An early study by Murdoch et al. showed that although in vitro exposure of human UCB HSPCs to Wnt-5A did not affect cell proliferation and differentiation, infusion of Wnt-5Aecontaining media into mice engrafted with human repopulating cells gave rise to enhanced multilineage hematopoietic reconstitution [49]. The molecular mechanisms for Wnt-5A’s effect on HSPCs are not known and need to be further investigated. Recent studies involving UCB progenitors have shown that overexpression of Wnt5a, coupled with inhibition of glycogen synthase kinase-3b, enhanced in vivo engraftment but failed to increase cell numbers in expansion cultures. In addition, seminal genetic studies also showed that overexpression of HOXB4 in human UCB increased the frequency of both long-term cultureinitiating cells as well as SRC [50]. More importantly, a similar advantage on human hematopoietic repopulation was also noted in the nonhuman primate model subsequently [51]. Encouraged by these results, researcher’s have attempted to enhance cellular HOXB4 activity via direct delivery of HOXB4 homeoproteins and demonstrated similar efficacy in expanding UCB cells [52]. It is yet to be determined whether HOXB4’s potency in human UCB expansion can be translated into clinical benefits. Notch-mediated expansion In-depth studies involving various types of stem cells have shown that the 5 Notch ligands are essential for determining self-renewal, survival, and differentiation [53]. Initial studies involving human HSPCs demonstrated the presence of Notch1 gene in the CD34þ cells [54]. Retroviral overexpression of Notch1 enhanced the self-renewal capacity of the repopulating cells [55]. Early studies suggested Jagged-1 to be a novel growth factor for human HSPCs [56]. Although soluble or cell-bound Notch ligand produced


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Table 5 Outcome of Completed Clinical Trials Involving Manipulated UCB Grafts Days to Platelet > 20,000

Survival

GVHD

Engrafting Unit in Majority of Patients

Ref.

28 (15-49)

106 (38-345)

OS at 17 mo, 32%

NA

[26]

1

22 (13-40)

71 (39-139)

EFS at 41 mo, 39% OS at 100 d, 65%

aGVHD at day > 28, 66.7% cGVHD at day > 100, 74% aGVHD grade 0-I, 63.6% aGVHD grade II-IV, 36.3%

NA

[27]

10

1

30 (16-46)

48 (35-105)

OS at 100 d, 90%

NA

[28]

10

2

16 (7-34)

Not reported

OS at 354 d, 70%

Unmanipulated

[29]

31

2

15 (9-42)

42 (15-62)

OS at 1 yr, 32%

Unmanipulated

[30]

11

2

13 (7-26)

33 (26-49)

Manipulated

[31]

24

1

21 (13-50)

77 (48-220)

NA

[32]

29

2

7 (6-26)

Unmanipulated

[33]

12

2

Avg. CI of Plt > 20,000 at day 60, 86.5% 43 (26-60)

OS at 1 yr, 82% PFS at 1 yr, 73% EFS at 1 yr, 3% OS at 1 yr, 59% NRM CI at 1 yr, 10% PFS at 1 yr, 52%

aGVHD grade II, 44.4% cGVHD at day > 100, 50% aGVHD grade II, 85.7% aGVHD grade III, 14.2% CI of aGVHD grade II to IV, 42% CI of aGVHD grade III or IV, 13% cGVHD, 13% aGVHD grade II, 50% cGVHD, 20% aGVHD grade II, 5.9% cGVHD at day > 100, 9.1% Not reported

aGVHD grade I, 25% aGVHD grade II, 16.7% cGVHD, 8.3%

Manipulated

[34]

Study

Subjects

No. of UCB Units Infused

Cytokine expansion Continuous perfusion expansion Copper chelation expansion Notch-mediated expansion MSC coculture expansion

37

1

27

Nicotinamide expansion CD26/DPP-4 inhibition C3a priming

PGE2 exposure

Days to ANC > 500

17.5 (14-31)

PFS at 1 yr, 61.7% OS at 1 yr, 75%

ANC indicates absolute neutrophil count; aGVHD, acute GVHD; cGVHD, chronic GVHD; NA, not applicable; EFS, event-free survival; CI, cumulative incidence; Plt, platelet; NRM, nonrelapse mortality.

minimal HSPCs expansion, engineered and immobilized Delta-1, along with fibronectin fragments, produced a 100fold expansion of CD34þCD38 progenitors when supplemented with a cytokine cocktail of IL-3, Il-6, TPO, FLT-3L, and SCF [29]. Unlike others, this expansion protocol limited myeloid differentiation and increased the absolute number of lymphoid precursors. HSPCs expanded in low concentrations of Notch1 retained the ability to repopulate the NOD/ SCID mice, whereas high concentration promoted apoptosis. In the phase I clinical trial (Table 2), the 16-day expansion of the T celledepleted unit produced 164- and 562-fold mean expansion of CD34þ and TNC, respectively. The manipulated units with a median CD34þ and TNC dosage of 6.0 106 and 4.6 107 cells/kg, respectively, were infused in to the 10 eligible, myeloablated patients along with an unmanipulated unit (Table 4). This transplantation procedure proved to be safe, with no reports of engraftment failures or increased incidence of GVHD. The median time to neutrophil engraftment was 16 days, which was significantly lower than the control of 26 days (Table 5). Analysis of hematopoietic reconstitution in the long-term showed that only one half of the patients engrafted with the manipulated unit. This raises concerns that the expansion protocol was either depleting the HSPCs or was merely assisting in rapid engraftment of the unexpanded unit [29]. Interim results from an ongoing multicenter trial involving 23 patients receiving an unmanipulated graft along with a partially HLA-matched Notchexpanded UCB showed median neutrophil and platelet engraftment of 13 and 38 days, respectively, with overall survival (OS) of 62% at 4 years [57]. The current data suggest that neutrophil recovery is dependent on the CD34þ cell dosage and could significantly reduce 100-day nonrelapse mortality. In another ongoing single center trial, the Notchexpanded UCB is administered as a non-HLA matched, offthe-shelf cells to boost hematopoietic recovery in the setting of a conventional single or dual UCBT [58]. Provisional data involving 15 patients have shown the median time for neutrophil and platelet recovery of 19 and 35 days,

respectively, with no transplantation-related mortality or grade III and IV acute GVHD [58]. Further investigation of the myeloablated patients in these 2 ongoing trials has shown significantly reduced risk of bacteremia in the first 100 days, likely due to the Notch-expanded graft homing into the sites of mucosal injury induced by the preparative total body irradiation [59].

Mesenchymal Stromal Cell Coculture In BM, HSPCs reside in a complex stem cell niche that provides pivotal cues for its self-renewal, apoptosis, proliferation, and differentiation into mature blood cells [60]. There are at least 3 different types of marrow niches (endosteal, reticular, and vascular) that impart differential biological and physiological effects on HSPCs [61]. Within these HSPC niches, MSC play important hematopoiesis supporting roles and have thus been extensively used in ex vivo coculture system to recapitulate the natural marrow environment [62]. MSC controls vital functions of HSPCs through direct cell-to-cell contact [63,64], secretion or suppression of endogenous cytokines and the formation of an extracellular matrix network [65]. Among the various factors secreted by MSC, the chemokine CXCL12 (also known as SDF-1) is a prominent regulator of hematopoiesis and HSPCs homing [66]. Elevated levels of SDF-1 in MSC coculture system have resulted in down-regulation of a range of cytokines and chemokines that promote HSPC differentiation and deter self-regeneration [67]. The use of a feeder layer coculture system using MSCs could eliminate the need to perform a prior enrichment of CD34þ or CD133þ cells to achieve significant HSPC expansion [68]. The major advantages of this are reduced loss of HSPCs during the selection process and avoidance of the potential negative effect of positive cell surface marker selection on HSPC activities. MSCs can be further enhanced to express surface markers, such as STRO-1 [30] and angiopoietin-like 5 [69], or genetically manipulated to express


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prohematopoiesis proteins, like hTERT, which have also been shown to possess enhanced HSPCs supporting ability [70,71]. In a recently completed dual UCBT (dUCBT) trial (Table 2), investigators from the MD Anderson Cancer Center performed ex vivo expansion of the smaller UCB unit on a MSC coculture system in the presence of SCF, TPO, FLT-3L, and G-CSF and transplanted it with a separate nonmanipulated unit into 31 myeloablated patients suffering from varying hematologic malignancies [30]. The MSCs were either obtained from a related donor via BM aspiration or an “off-the-shelf” source. The expansion protocol resulted in 12-, 31-, and 17.5-fold increases in the number of TNC, CD34þ, and colony-forming cells, respectively (Table 4). The combined transplantation of the expanded and the unexpanded unit resulted in an increased dosage of TNC, CD34þ cells, and megakaryocyte progenitors compared with historical unmanipulated dUCBT, resulting in the median times to neutrophil and platelet engraftment of 15 and 42 days, respectively (Table 5). This study demonstrated that, compared with recipients of conventional dUCBT, a higher proportion of patients achieved cumulative incidence of neutrophil and platelet engraftment by days 26 and 30, respectively. However, prolonged follow-up (>6 months) of the patients showed that only the unmanipulated UCBs contributed to the long-term donor-derived hematopoiesis, which is in line with several of the xenotransplantation studies performed in mice. The cumulative incidence of acute GVHD (grade III or IV) was observed in 13% of the patients, whereas 45% demonstrated chronic GVHD [30].

Chemical Molecules Copper chelator Studies have shown that the cellular content of copper could be vital in regulating the proliferation and differentiation of UCB HSPCs [72]. The lack of copper arrested the differentiation and maturation of hematopoietic cells, with negligible effect on the development of progenitor cells [73]. Peled et al. performed ex vivo expansion of CD34þCD38 UCB cells in the presence of a known copper chelator, tetraethylenepentamine, along with TPO, FLT-3L, IL-6, and SCF, and saw an 89-fold increase of CD34þ cells that possessed an enhanced SRC [74,75]. The protocol was implemented in a phase I/II clinical trial by investigators in the MD Anderson Cancer Center [28]. Unlike other reported trials, this study involved only 1 UCB unit that was cryopreserved in 2 fractions (Table 2). Before infusion, CD133þ cells from the smaller fraction were expanded in culture for 21 days in the presence of tetraethylenepentamine and resulted in average increases in TNC and CD34þ cells of 219-fold and 6-fold, respectively. Myeloablated patients suffering from hematological malignancies received the nonexpanded fraction folQ 5 lowed by the expanded fraction (StemEx) of the same UCB at a relatively low combined median TNC of 1.8 107 cells/kg (Table 4). Nine of 10 patients achieved UCB engraftment, with median times to neutrophil and platelet recovery of 30 and 48 days, respectively (Table 5). No significant increase in the rates of acute GVHD (grade III or above) and nonrelapse mortality were noted. This study established that transplantation of a single expanded unit was able to attain engraftment with recovery times comparable to that of traditional single unmanipulated UCBT [28]. Interim reports from an ongoing multicenter phase II trial have shown that in 101 patients who underwent transplantation with the StemEx graft, times to neutrophil and platelet recovery were 21

and 54 days, respectively, which are significantly faster than after conventional dUCBT [76]. Nicotinamide Recent studies have implicated nicotinamide (NAM) as a novel agent to expand CD34/CD133-selected UCB cells in vitro [77]. NAM is a form of vitamin B3 that acts as a potent inhibitor for both NADþ-dependent enzymes and the sirtuin family of histone/protein deacetylase [77]. It has been shown to delay differentiation of the CD34þ cells by inhibiting the SIRT1 deacetylase. Further investigation revealed that CD34þ cells cultured in presence of NAM along with TPO, IL-6, FLT3L, and SCF possessed an enhanced ability to home and repopulate NOD/SCID mice and resulted in 9-fold and 7.6fold increases in short-term repopulating cells compared with fresh CD34þ cells and those harvested from NAMnegative control cultures, respectively [77]. These preclinical data laid the foundation for a small-scale phase I clinical trial involving 11 patients that was carried out by investigators in Duke University Medical Center [31]. In the clinical expansion protocol (Table 2), CD133þ cells from a single unit of UCB were expanded ex vivo in the presence of NAM and subsequently coinfused together with the remaining previously frozen thawed noncultured T cell fraction from the same UCB unit, along with a second unmanipulated UCB unit. The expansion resulted in 486-fold and 72-fold increases of TNC and CD34þ cells, respectively, resulting in a median transplanted dose of 3.5 106 CD34þ cells/kg (Table 4). The median times to neutrophil and platelet engraftment were 13 and 33 days, respectively, which were significantly shorter than the historical controls (Table 5). Only 2 patients developed chronic GVHD and 5 patients showed acute grade II GVHD, with 1-year OS and progression-free survival (PFS) rates of 82% and 73%, respectively, at a median follow-up of 21 months. More importantly, the expanded UCB unit in this study (NiCord) Q 6 was reported to contribute to both short-term (6 months) hematopoiesis, which is not commonly observed in other similar trial designs [31]. Such promising data have now prompted investigators to initiate a multicenter trial where patients are designated to receive only the NAM-expanded UCB without the infusion of the second unmanipulated unit. Aryl hydrocarbon receptor antagonist In a recent high throughput screen of an extensive array of small molecules, Boitano et al. identified an aryl hydrocarbon receptor antagonist, StemRegenin 1 (SR1), as a potent agent for in vitro HSPCs expansion [78]. In contrast to many of the studied agents, low concentrations of SR1 were found to induce proliferation only in presence of the cytokine cocktail (TPO, SCF, FLT-3L, and IL-6), whereas a high concentration of it exhibited an antiproliferative response. In vitro culture of CD34þ cells from UCB in the presence of SR1 resulted in 669-fold and 17,100-fold expansions in short (3 week) and long-term (5 week) cultures, respectively. When the UCB CD34þ cells were expanded for 3 weeks in SR1 and cytokine-supplemented cultures, the result was a more than a 10-fold increase in long-term SRC compared with noncultured or cytokine-expanded controls [78]. Although this study established that aryl hydrocarbon receptor is implicated in regulating HSPCs activities, the exact mechanism of action for SR1 is yet to be identified. Currently, SR1-expanded UCB (HSC835) is being evaluated in a clinical trial conducted and sponsored by Novartis


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International AG (Switzerland) [79,80]. In this trial, each patient receives 2 UCB units: (1) a larger unmanipulated unit, and (2) a smaller unit that had its CD34þ cells expanded in culture with SR1 and infused along with the noncultured, frozen-thawed CD34 fraction. The SR1-supplemented cultures resulted in a median of 328-fold expansion of CD34þ cells [80]. The median time for neutrophil recovery in all 18 patients was 14.5 days. When HSC835 dominated, neutrophil recovery occurred at a median of 10.5 days compared with 23 days in patients with unmanipulated graft dominance [80]. The fastest median time for neutrophil recovery of 7 days was noted in patients with dual myeloid chimerism. Based on these data, investigators have started to administer HSC835 as a stand-alone expanded unit, with the first 2 patients showing neutrophil recovery at days 8 and 12 [80]. In the future, the trial will evaluate the engraftment potential of HSC835, which possesses a very high dose of CD34þ cells in nonmyeloablated patients. Bioreactor Systems Several of the technologies for UCB expansion reviewed above have been developed as static, small-scale systems for laboratory and early clinical studies. To enable expanded UCB-based therapies for routine clinical use, the scaling of these culture systems in an affordable and good manufacturing practiceerelevant manner, while maintaining its therapeutic potency, is an important technical hurdle that must be overcome. To achieve this, bioreactors systems that allow integration of UCB expansion technologies are currently under development. During culture expansion, UCB cells are typically suspended in a stirred tank or within hollow fibers while being supplemented with the necessary cytokines that are automatically regulated in the presence of stromal cells or biomaterials [81,82]. A significant advantage of bioreactors compared with static cultures is their ability to support growth of high cell densities, due to improved nutrient/waste exchange rates, while regulating global levels of signaling and growth factors in the culture media. An example of this is the application of controlled media dilution during UCB cell culture in a bioreactor [46]. As discussed above, this approach provided the means for stabilizing global concentrations of cell-secreted factors such that a constant ratio of inhibitory to stimulatory factors was achieved, in contrast to the periodic fluctuations of factor concentrations when cells are cultured using a standard media change protocol. After 12 days in culture, limiting dilution transplantation analyses showed 7.6-fold versus a 3.6-fold increase in the number of long-term SRC relative to fresh cells, for the fed-batch versus complete media change control, respectively [46]. Such media control strategies in bioreactor systems are amenable for large-scale cell expansion towards clinical use and can also be combined with other expansion protocols. The addition of a newly identified pyrimidoindole derivative, UM171, which is an agonist of HSPCs self-renewal to this bioreactor system, further enhanced in vitro and in vivo expansion of the UCB CD34þ cells [83]. To date, only 1 clinical study at Duke University Medical Center evaluated the use of a bioreactor, known as the AastromReplicell System, to expand a small fraction of an UCB unit (Table 2) [27]. The majority of the unmanipulated cells from the same unit was infused into the 27 patients suffering from hematological malignancies or inherited disorders on day 0 with a median cell dose of 2.05 107 cells/kg. The expansion cultures were supplemented with bovine and

7

horse serum, erythropoietin, FLT-3L, and PIXY321, and the media was continuously perfused for 12 days into the bioreactor; resulting in 2.4-, 82-, and .5-fold expansions of TNC, granulocyte-macrophage colony-forming units, and Q 8 CD34þ cells, respectively (Table 4). Although the investigators established the safety and feasibility of the expanded cells that had median transplantation dosage of 2.05 107 cells/kg, the data did not suggest an improvement for the rate of hematopoietic recovery. The median times to neutrophil and platelet engraftment were 22 and 94 days, respectively, with about 36% of the evaluable patients developing moderate to severe GVHD (Table 5) [27]. In this study, the total absolute number of CD34þ cells was reduced after the expansion compared with the unmanipulated graft, which could possibly explain the suboptimal clinical outcome. RECENT CLINICAL STRATEGIES TO IMPROVE HOMING OF UCB GRAFTS A successful UCBT, as determined by the speed of neutrophil recovery, has been shown to be correlated with the TNC, CD34, and granulocyte-macrophage colony-forming unit dosage. However, defective homing of the transplanted UCB cells also hinders hematopoietic reconstitution and remains to be solved. Homing of HSPCs after conventional intravenous infusion involves the successful extravasation and lodging at specialized niches within the BM to allow for subsequent cell repopulation to occur. In the past, various strategies to improve homing efficiency of HSPCs have been explored, including intra-bone (i.b.) infusion of UCB grafts [84,85] and coinfusion of UCB cells with other accessory cells, such as MSC [86]. A phase I/II study carried out by San Martino Hospital, Italy involved 32 myeloablated patients who received washed UCB grafts via the i.b. route with median time to neutrophil and platelet recovery of 23 and 36 days, respectively [85]. A subsequent study involving positron emission topography of 21 i.b. UCBT recipients showed that platelet recovery could be predicted by the increased FDG uptake at the injection sites [87]. In- Q 9 vestigators from University of Minnesota carried out a dual UCBT in 10 myeloablated patients, where 1 unit was infused via the intravenous route (i.v.) and other was introduced via i.b. injections [88]. Five patients engrafted with i.v. unit, whereas 4 patients engrafted with the i.b. unit, with median times to neutrophil and platelet engraftment of 21 and 69 days, respectively [88]. A recently completed trial in Japan reported neutrophil recovery at a median of 17 days when unwashed UCB grafts were injected via the i.b. route into 10 patients receiving reduced-intensity conditioning [89]. A retrospective analysis of single unit i.b. versus dual unit i.v. UCBT have shown the medial time to neutrophil recovery to be 23 and 28 days, respectively. Multivariate analysis at 4.7 months after transplantation showed that i.b. UCBT was associated with lower relapse and GVHD incidence and improved disease-free survival [90]. These approaches have only led to moderate improvement in HSPC homing, leaving room for further development. This section will highlight the recent developments to enhance HSPC homing to the BM as an alternative means to increase the overall hematopoietic repopulation of transplanted UCB cells. CD26/Dipeptidylpeptidase IV The cell surface peptidase CD26/dipeptidylpeptidase IV (DPP-4) has been shown to interfere in the chemotaxis of CD34þ human hematopoietic cells through DPP-4 enzymatic


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cleavage in the penultimate proline or alanine unit of SDF-1, thus incapacitating its chemotactic activity [91,92]. Truncated SDF-1 is unable to activate C-X-C chemokine receptor type 4 (CXCR4) and also antagonizes signals from full-length SDF-1 [91]. Murine studies demonstrated that DPP-4 inhibition during G-CSF mobilization resulted in fewer number of progenitors mobilized into the peripheral blood compared with controls [93]. Additionally, the inhibition or deletion of DPP-4 enhanced engraftment of systemically delivered human CD34þ UCB cells in immunodeficient mice [94]. These data generated interest in the use of DPP-4 inhibitors to precondition UCB cells or transplant recipients before cell infusion. A recently completed pilot trial evaluated the safety, efficacy, and feasibility of orally administering a Food and Drug Administrationeapproved DPP-4 inhibitor, sitagliptin, to enhance the engraftment of a single-unit UCBT in adults suffering from hematological malignancies (Table 3) [32]. All 24 patients received a myeloablative conditioning regimen with sitagliptin administered from day -1 to day þ2 of transplantation. The infused UCB grafts were red cell depleted and had a median cell dose of 3.6 107 cells/kg. The median time to neutrophil engraftment was 21 days, which was comparable to historical single UCBT (Table 5). A positive effect of the sitagliptin treatment to donor UCB cell engraftment was suggested by the significant correlation between the engraftment of the transplanted cells and the area under the DPP-4 activity-time curve. The event-free survival and 1-year OS of the red celledepleted recipients were 53% and 59%, respectively (Table 5) [32]. This study demonstrated the modest benefits of CD26 inhibition and further work is required to optimize the sitagliptin dosing to achieve further improvements in UCB transplantation outcomes. Complement Fragment 3a Priming Human CD34þ cells express the complement anaphylatoxin C3a receptor (C3aR), which binds to C3a to allow influx of calcium into the cells [95]. Although C3a does not directly affect the survival and proliferation of HSPCs, it modulates SDF-1edependent chemotaxis of CD34þ cells [96]. G-CSF treatment of C3aR and C3 knockout mice showed significantly increased mobilization of progenitors in the peripheral blood [97]. Similarly, wild-type mice treated with C3aR antagonist also showed enhanced mobilization [98]. Additional experiments showed that C3a priming of the CD34þ cells allowed greater incorporation of CXCR4 receptor into the lipid rafts, which resulted in improved HSPC homing and repopulation after transplantation [99,100]. These studies demonstrated that C3a fragments modulate the responsiveness of CXCR4þ HSPCs to the SDF-1 gradient, which is a key mechanism for HSPC homing into the BM. A pilot trial included 29 patients with hematological malignancies who underwent nonmyeloablative conditioning and dUCBT with the smaller UCB unit being primed with commercially available human C3a complement before infusion (Table 3) [33]. The median TNC doses of the primed and unmanipulated unit were 1.5 107 and 2.5 107 cells/ kg, respectively, resulting in the median time to neutrophil engraftment of 7 days, which was faster than the matched transplantation center historical control of 12 days. Two thirds of the study patients engrafted with the C3a-primed unit and had a disease PFS of 52% (Table 5). The cumulative incidence of nonrelapse mortality at 1 year was 10% [33]. Although the data suggested comparable outcomes with

historical controls, a large cohort study is required to establish the benefit of C3a priming of UCBT. Dimethyl-prostaglandin E2 A screening study of biologically active compounds in zebra fish embryos showed that chemicals that enhance prostaglandin E2 (PGE2) synthesis increased HSPC numbers, whereas those that block its synthesis have an opposite effect [101]. Additional mechanistic studies showed that the Wnt pathway in HSPCs is regulated by PGE2 via degradation of beta-catenin [102]. Both human and murine HSPCs express PGE2 receptors and short-term exposure of these cells to PGE2 increases the cell viability, chemotaxis towards SDF1, and proliferative capacity as determined by in vitro functional assays [103]. PGE2 also increased expression of CXCR4 and survivin, which are involved in homing and survival of HSPCs, respectively [103]. PGE2-treated cells performed better in competitive transplantation studies and maintained serial transplantation capacity. Preclinical studies involving UCB showed that PGE2 treatment increased the number of both in vitro colony forming units and in vivo SRCs [104]. However, recent findings implicated PGE2 treatment with a transient increase in HSPCs homing and repopulation, as indicated by serial xenotransplantation experiments [105]. In a recently completed phase 1 trial, 21 patients suffering from hematological disorders underwent dUCBT after reduced-intensity conditioning [34]. One of the UCB units was incubated with PGE2 for at least 1 hour and washed before infusion into patients who were divided into 2 cohorts (Table 3). In the first cohort, the smaller UCB unit was exposed to PGE2 and resulted in median neutrophil engraftment at day 24, with only 2 of the 9 patients showing long-term hematopoiesis from the manipulated unit. For the second cohort, the larger UCB unit was treated with PGE2 that had a median cell dosage of 1.8 107 cells/kg but was comparable to the unmanipulated smaller unit. The median time to neutrophil engraftment was 17.5 days, which was at least 3.5 days earlier than the defined control (Table 5). Median time for platelet engraftment was 43 days and the manipulated unit dominated in 9 of the 11 evaluable patients of cohort 2. Grade I and II acute GVHD was observed in 5 patients, whereas only 1 patient had chronic GVHD. The 1and 2-year PFS rates were 61.7% and 31.3%, respectively, and the 2-year OS rate was 38.9% (Table 5) [34]. Currently a phase II randomized trial is underway with plans of initiating new phase I studies, which would involve single-unit UCBT after PGE2 exposure. Fucosylation Extravasation of transplanted hematopoietic cells to the BM is a multi-step process involving interaction with a number of cell surface molecules on endothelial cells including the P- and E-selectins [106]. Binding to selectins requires the formation of cell surface glycan determinants, such as sialyl Lewis x (sLeX) via glycoprotein fucosylation [106]. It was shown that selectin ligands on CD34þ UCB cells display significantly lower levels of alpha 1-3 fucosylation compared with CD34þ cells from BM or peripheral blood, leading to reduced binding to the endothelial selectins [106,107]. This fucosylation deficiency has been hypothesized to contribute to a decreased efficiency of UCB cells to home to the BM. Indeed, comparative studies that involved isolating and transplanting either fucosylated or nonfucosylated UCB CD34þ cells into NSG mice demonstrated that only the fucosylated fraction resulted in detectable


Q 10

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Table 6 Ongoing Clinical Trials Involving Manipulated UCB Grafts Responsible Organization

ClinicalTrials.gov Identifier

Gamida Cell Ltd.

NCT01590628

Phase

Brief Outline of Ongoing Study

Primary Outcome

10

I/II

(1) Safety, tolerability. and infusional toxicity of NiCord cells and (2) CI of donor-derived neutrophil engraftment

NCT01816230

10

I/II

Cotransplantation of an unmanipulated UCB graft along with a NAM-expanded UCB unit (NiCord) for sickle cell disease patients A single unit of NAM-expanded UCB, NiCord, will be transplanted

NCT01690520 (Multicenter)

160

II

dUCBT with or without Notch-mediated ex vivo UCB expansion

NCT00498316 (Multicenter)

125

I

NCT01471067

50

I

University of Pennsylvania

NCT00891592

36

I

Novartis Pharmaceuticals

NCT01474681

27

I/II

Singapore General Hospital

NCT01624701

10

I/II

dUCBT where 1 UCB graft is expanded in presence of a MSC feeder layer compared to control where patients are receiving both unmanipulated grafts dUCBT where the larger UCB unit is infused without any manipulation while the smaller graft will undergo ex vivo fucosylation before infusion. Part of an unmanipulated UCB graft will be infused while rest of the cells will be ex vivo expanded to obtain CD3/CD28 costimulated T cells, which will be coinfused dUCBT where the smaller unit is ex vivo expanded in presence of the small molecule, SR1, while the larger unit is infused without any manipulation Mesenchymal coculture expanded UCB graft without prior CD34/CD133 selection will be transplanted along with an unmanipulated UCB unit

Fred Hutchinson Cancer Research Center MD Anderson Cancer Center

Estimated Enrollment

CI of patient engrafted with NiCord-derived neutrophil at day 42 after transplantation Time to engraftment of ANC 500

Engraftment and time to engraftment

(1) Mean time to engraftment and (2) No. of patients with engraftment by day 42 Grade IV GVHD within 90 d of transplantation as a measure of dose-limiting toxicity Safety and tolerability of HSC835

Safety, infusional toxicities, and potential immunological competition

Information obtained from ClinicalTrials.gov as December 21, 2014. All trials are for patients with hematological malignancies unless mentioned otherwise.

hematopoietic cell engraftment [108]. In addition, xenotransplantation of UCB CD34þ cells that endogenously express sLeX demonstrated a significantly higher rate and magnitude of multi-lineage engraftment compared with sLeX-deficient CD34 cells [108]. To overcome this inherent fucosylation deficiency, several research groups have attempted to expose UCB CD34þ cells to recombinant human fucosyltransferase VI (FTVI) or FTVII and its substrate GDPfucose to increase the fucosylation level [109,110]. In comparison, FTVII proved to be better in fucosylating not only CD34þ cells, but also T and B cells, which could favorably modulate both GVHD and graft-versus-tumor effects [110]. Preclinical studies have shown that exogenously fucosylated UCB CD34þ cells contributed to both significantly earlier BM homing and multilineage hematopoietic reconstitution of sublethally irradiated NSG mice. In an ongoing dUCBT clinical trial in MD Anderson Cancer Center, an unmanipulated UCB unit with the higher cell dosage is being infused into the patients followed by the smaller unit that has undergone fucosylation using FTVI and GDP-fucose for 30 minutes [111]. The fucosylation level of the CD34þ cells in the manipulated unit increased from a median of 33% to 99%. To date, 10 patients have undergone this transplantation regimen after a nonmyeloablative conditioning. The interim median times to neutrophil and platelet recovery are reported to be 14 and 33 days, respectively. Among the evaluable patients, 4 had engraftment from the fucosylated smaller unit, whereas 2 patients engrafted with the larger unmanipulated unit. Initial observations showed minimal infusion related toxicity and 2 patients developed grade II acute GVHD [111]. Currently, the study is recruiting patients and the outcome will be reported in the near future.

FUTURE OF EXPANDED UCB IN HSCT Unmanipulated UCB have already been established as the preferred choice of HSPCs for transplantation into pediatric patients. New developments in UCB expansion have translated into significant clinical benefits over the last decades and are believed to continue to improve in the years to come for adult transplantation (Table 6). The proliferation of true hematopoietic stem cells without differentiation is considered to be the gold standard for achieving an ultimate ex vivo expansion. However, currently available techniques are likely expanding more progenitors rather than true stem cells as, in most dUCBT studies, long-term hematopoiesis is imparted by the unmanipulated unit. Nonetheless, expansion and infusion of high-dose hematopoietic progenitors is also an important achievement as it reduces the period of pancytopenia in the UCBT patients; hence, decreasing the chances of infection-related morbidity and mortality. Also, recent developments have shown that it is feasible and cost effective to obtain clinically relevant numbers of differentiated immune cells, such as natural killer [112], regulatory, and cytotoxic T cells from UCB [113], which could overcome the current lack of donor lymphocyte infusion in UCBT. In conclusion, the true expansion of hematopoietic stem cells can be achieved by addressing some of the burgeoning questions, which include improved UCB acquisition and selection based on better predictive markers, identification of various specific cell subsets for applications in different clinical situations, implementation of an optimal in vitro expansion condition, as well as the subsequent development of a cost-effective clinical-scale expansion protocol. Further development with regards to identifying and regulating pathways involved in maintaining the viability and symmetric stem cell division would further improve HSPCs


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expansion. With our increasing understanding of HSPC biology from the emerging genetic, cellular, and clinical studies (Table 6), there is no doubt that even better cell expansion strategies will be devised in near future.

Q 11

ACKNOWLEDGMENTS This work was supported by Singapore General Hospital Research Grant 2014 (SRG/C3/04/2014) which is under the National Medical Research Council Center Grant, Singapore. Laboratory space was provided by Duke-NUS Graduate Medical School, Singapore and Singapore General Hospital, Singapore. Financial disclosure: ---. Conflict of interest statement: There are no conflicts of interest to report. Authorship statement: S.B. reviewed the literature, wrote, and revised the manuscript, and constructed the tables; K.K.H.S. reviewed the literature and wrote the first draft; Z.P. and A.M.S.C. reviewed literature, wrote relevant sections, and performed major revisions; X.B.F. reviewed the literature and wrote relevant section; S.Y.O., S.L., and L.P.K. revised the manuscript; W.Y.K.H. conceived the idea, reviewed literature, and wrote and revised the manuscript and the tables. Z.P. and A.M.S.C. contributed equally to this work. REFERENCES 1. Gluckman E, Devergié A, Bourdeau-Esperou H, et al. Transplantation of umbilical cord blood in Fanconi’s anemia. Nouv Rev Fr Hematol. 1990;32:423-425. 2. Voelker R. FDA grants approval for first cord blood product. JAMA. 2011;306:2442. 3. Norkin M, Lazarus HM, Wingard JR. Umbilical cord blood graft enhancement strategies: has the time come to move these into the clinic? Bone Marrow Transplant. 2013;48:884-889. 4. Ballen KK, Gluckman E, Broxmeyer HE. Umbilical cord blood transplantation: the first 25 years and beyond. Blood. 2013;122:491-498. 5. Rocha V, Wagner JE, Sobocinski KA, et al. Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. N Engl J Med. 2000;342:1846-1854. 6. Cunha R, Loiseau P, Ruggeri A, et al. Impact of HLA mismatch direction on outcomes after umbilical cord blood transplantation for hematological malignant disorders: a retrospective Eurocord-EBMT analysis. Bone Marrow Transplant. 2014;49:24-29. 7. Barker JN, Byam CE, Kernan NA, et al. Availability of cord blood extends allogeneic hematopoietic stem cell transplant access to racial and ethnic minorities. Biol Blood Marrow Transplant. 2010;16: 1541-1548. 8. Hwang WYK, Samuel M, Tan D, et al. A meta-analysis of unrelated donor umbilical cord blood transplantation versus unrelated donor bone marrow transplantation in adult and pediatric patients. Biol Blood Marrow Transplant. 2007;13:444-453. 9. Hwang WYK, Ong SY. Allogeneic haematopoietic stem cell transplantation without a matched sibling donor: current options and future potential. Ann Acad Med Singapore. 2009;38:340-346. 10. Rocha V, Labopin M, Sanz G, et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med. 2004;351:2276-2285. 11. Laughlin MJ, Eapen M, Rubinstein P, et al. Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med. 2004;351:2265-2275. 12. Oran B, Shpall E. Umbilical cord blood transplantation: a maturing technology. Hematology Am Soc Hematol Educ Program. 2012;2012: 215-222. 13. Michel G, Rocha V, Chevret S, et al. Unrelated cord blood transplantation for childhood acute myeloid leukemia: a Eurocord Group analysis. Blood. 2003;102:4290-4297. 14. Sideri A, Neokleous N, Brunet De La Grange P, et al. An overview of the progress on double umbilical cord blood transplantation. Haematologica. 2011;96:1213-1220. 15. Zhang CC, Lodish HF. Cytokines regulating hematopoietic stem cell function. Curr Opin Hematol. 2008;15:307-311. 16. Chou S, Chu P, Hwang W, Lodish H. Expansion of human cord blood hematopoietic stem cells for transplantation. Cell Stem Cell. 2010;7: 427-428.

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Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells from Umbilical Cord Blood.

Umbilical cord blood progenitor cells for clinical transplantation.

progenitor cells.

Expansion of CD133(+) Umbilical Cord Blood Derived Hematopoietic Stem Cells on a Biocompatible Microwells.

progenitor cells in three-dimensional Nanoscaffold coated with Fibronectin.

Non-hematopoietic stem cells in umbilical cord blood.

progenitor cells in vitro.

Pharmacological preconditioning for short-term ex vivo expansion of human umbilical cord blood hematopoietic stem cells by filgrastim.

Derivation, expansion and characterization of clinical grade mesenchymal stem cells from umbilical cord matrix using cord blood serum.

Progenitor Cells.

Umbilical cord blood for transplantation.

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

progenitor cells with different cytokine combinations.

Human umbilical cord blood-derived stromal cells: A new source of stromal cells in hematopoietic stem cell transplantation.

Umbilical cord and placental blood hematopoietic stem cells: collection, cryopreservation, and storage.

Advances in umbilical cord blood stem cell expansion and clinical translation.

CD34(+) stem cells from umbilical cord blood.

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood.

Expanded umbilical cord blood T cells used as donor lymphocyte infusions after umbilical cord blood transplantation.

Umbilical cord blood transplantation and unmanipulated haploidentical hematopoietic SCT for pediatric hematologic malignances.

Umbilical Cord Blood Platelet Lysate as Serum Substitute in Expansion of Human Mesenchymal Stem Cells.

Apoptosis, DAP-Kinase1 Expression and the Influences of Cytokine Milieu and Mesenchymal Stromal Cells on Ex Vivo Expansion of Umbilical Cord Blood-Derived Hematopoietic Stem Cells.

Infusion of allogeneic umbilical cord blood hematopoietic stem cells in patients with chemotherapy-related myelosuppression.

Endothelium-targeted human Delta-like 1 enhances the regeneration and homing of human cord blood stem and progenitor cells.