clinical application of hematopoietic progenitor cell expansion current

7/31/2019 Clinical Application of Hematopoietic Progenitor Cell Expansion Current

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Mini review

Clinical application of hematopoietic progenitor cell expansion: currentstatus and future prospects

SM Devine 1 , HM Lazarus 2 and SG Emerson 3

1 Section of Bone Marrow Transplantation and Leukemia, Division of Oncology, Department of Medicine, Siteman Cancer Center,Washington University School of Medicine, St Louis, MO, USA; 2 Department of Medicine, Comprehensive Cancer Center of CaseWestern Reserve University, Cleveland, OH, USA; and 3 Division of HematologyOncology, Departments of Medicine and Pediatrics,University of Pennsylvania School of Medicine, Philadelphia, PA, USA

Summary:

In the past decade, we have witnessed signicant advancesin ex vivo hematopoietic stem cell culture expansion,progressing to the point where clinical trials are beingdesigned and conducted. Preclinical milestone investiga-tions provided data to enable expansion of portions of hematopoietic grafts in a clinical setting, indicating safetyand feasibility of this approach. Data derived from currentclinical trials indicate successful reconstitution of hema-topoiesis after myeloablative chemoradiotherapy usinginfusion of ex vivo -expanded perfusion cultures. Futureavenues of exploration will focus upon rening preclinicaland clinical studies in which cocktails of availablecytokines, novel molecules and sophisticated expansionsystems will explore expansion of blood, marrow andumbilical cord blood cells.

Bone Marrow Transplantation (2003) 31, 241252.doi:10.1038/sj.bmt.1703813Keywords: bone marrow transplantation; stem cells

In the past decade, considerable progress has been made inour understanding of human hematopoietic stem cell(HSC) biology. 1,2 In parallel, advances in the supportivecare of patients undergoing autologous or allogeneic stemcell transplantion have signicantly reduced the mortalityrates associated with these procedures, resulting in anincrease in the proportion of potentially eligible patients. 3

Nevertheless, further progress has been hindered by theprofound and often prolonged pancytopenia associatedwith myelosuppressive conditioning, the toxicity of theconditioning regimens, graft-versus-host disease (GVHD),a myriad of opportunistic infections, and relapse of theoriginal disease. Unfortunately, patients with diseasesotherwise amenable to allogeneic HSC transplantationoften lack a suitably matched donor. Efforts to increasethe pool of available donors have led to clinical trialsevaluating alternative donor sources including volunteer

unrelated, mismatched related, and unrelated umbilicalcord blood (UCB). 410 While sometimes successful, theseprocedures are affected by an even higher risk of thetransplant-associated complications.

Recently, the dose of cells transplanted and patientoutcome have been shown to correlate positively. 4,619 Thisrelation holds for both the autologous and allogeneictransplant settings whether the variables analyzed includetotal nucleated cell dose, CD34+ cell dose, or hemato-poietic progenitor cell dose. As a logical extension of thesendings, it has been hypothesized that the capacity toexpand in vitro the quantity of hematopoietic stem andprogenitor cells available for transplantation would en-hance hematopoietic engraftment, reduce the risk of infection, and as a result increase the number of potentiallyavailable alternative donors. Moreover, the successful exvivo expansion of hematopoietic stem and progenitor cellscould be exploited for a variety of clinical applications,including the purging of contaminating tumor cells,increasing the number of cells available for geneticmodication, and the generation of large quantities of immunologically reactive cells (T cells, natural killer cells,and dendritic cells) for adoptive immunotherapeuticpurposes (Table 1). However, as many investigators beginto consider ex vivo HSC expansion for clinical applica-tions, a number of questions have emerged (Table 2).Successful clinical application of expanded hematopoiesiswill clearly require a greater understanding of humanstem cell biology, identication of the proper balanceand concentration of the available cytokine cocktails forex vivo culture, and hastening the slow pace of clinical

trials. In this review, we discuss the past, current, andfuture strategies of hematopoietic progenitor cell expan-sion, point out current pitfalls, and highlight potentialopportunities.

The path to the present

In the late 1980s several advances were made, whichsuggested that the ability of HSC to proliferate without lossof self-renewal might be harnessed ex vivo. Although themost concrete goal that surfaced was the controlledproduction of mature blood cells for transfusion therapy,

Correspondence: Dr SG Emerson, University of Pennsylvania School of Medicine, Maloney 510, 3600 Spruce Street, Philadelphia, PA 19010,USA

Bone Marrow Transplantation (2003) 31 , 241252& 2003 Nature Publishing Group All rights reserved 0268-3369/03$25.00

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the magnitude of cell production required was so large thatthe efciency requirements for a culture system that couldproduce red cells or platelets for clinical use immediatelywas seen as overly daunting. On the other hand, expansionof immature compartments, including hematopoietic stemand progenitor cells, sufcient for clinical application,seemed within reach; perhaps expansion to a degree as littleas 20100 fold would be useful.

Three related observations drove the eld forward.Elegant morphologic studies by Gail and Brian Naughtondemonstrated that restoration of a simile of the in vivo

three-dimensional bone marrow microenvironment withnylon mesh, allowing stromal cell tethering across openspace between attachments, encouraged the production of mature blood cells of all lineages, including red bloodcells. 20 Unfortunately, while this was qualitatively asignicant improvement over the rst generation of hematopoietic cell cultures performed on at dishes, theywere only effective in small culture systems. Multilayer,large-scale cultures built on such nylon meshes proved verydifcult to oxygenate and perfuse, such that very fewmature blood cells or progenitor cells could be produced.Also pursuing the notion of in vivo mimicry, Caldwell et al 21

and Schwartz et al 22,23 demonstrated that slow perfusionand local oxygenation of at hematopoietic cultures wouldallow stem cell survival with 1020-fold progenitor cellexpansion. The at architecture used by Schwartz et al could be manufactured to larger scales, so that sufcientnumbers of cells for clinical trials could be produced in oneor two closed culture vessels. 2123 Finally, Haylock et al showed that, if sufcient quantities of interleukin-1 b (IL-1),IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF),granulocytemacrophage-CSF (GM-CSF), and stem cellfactor (SCF) were added to cultures of CD34+-enrichedperipheral blood progenitor cells, progenitor cell expan-sions of 50-fold or more could be achieved. 24 Thesepreclinical data suggested that the incorporation of multi-ple stem cell active cytokines into systems that foster cell

survival and expansion potentially could be adapted forclinical use.

Three parallel areas of investigation followed on theheels of these tantalizing studies. First, literally hundreds of laboratories attempted to maximize the efciency of puried human progenitor cell cultures, by beginning withmore highly puried starting cell (CD34+ or CD34+lin-)populations, adding more or different cytokines or both.However, these studies were rarely accompanied by assaysof true stem cell repopulating cell numbers, largely becauseof the difculty of manipulating the human-to-NOD/SCID

or SCID/hu transplant assays quantitatively. Second,careful studies in murine stem cell transplants indicatedthat SCF, t-3 ligand, and thrombopoietin (TPO) likelywere the most important cytokines for promoting true stemcell expansion. 2532 Third, clinical trials began using ex vivoexpanded hematopoietic cells to augment or replacestandard autografts, and later allografts.

Clinical trials of ex vivo expansion

By and large, the published clinical trials have sought toexpand the number of short-term repopulating cells(STRC) and mature cells with the goal of abrogating orshortening the period of profound pancytopenia observedfollowing high-dose chemotherapy or chemoradiotherapy.Studies to date have not addressed whether true long-termrepopulating cells (LTRC) can be expanded and transfusedto shorten the period of pancytopenia and improve theoverall rate of engraftment. This distinction is critical sinceat present there are no convincing data to support thenotion that true HSC with LTRC potential can beexpanded substantially in culture. 2,33,34 The trials to datehave been designed to address two distinct but relatedquestions: (1) If a dose of cells sufcient to provide astandard autograft is cultured (expanded) ex vivo and usedas a sole source of hematopoietic support, will the patientsengraft? (2) Can neutrophil nadir, platelet nadir, or both be

Table 1 Potential applications and challenges for ex vivo hematopoietic expansion

Applications Challenges and current status

Accelerate hematologic recovery afterhematopoietic stem cell transplant

Supply sufcient numbers functional precursors andprogenitors (close at hand)

Reduction in the number of pluripotent stem cellsrequired for permanent reconstitution

Achieve sufcient multiplication (2050 ) of truepluripotent stem cells (not yet achieved)

Stem cells for tissue repair Understand how pluripotent stem cells commit to

tissue-specic lineages; direct this process in vitro(just beginning)

Hematopoietic stem cell gene therapy Force stem cell division, without loss of pluripotent stemcell repopulating ability, in vitro (some success withUCB, not yet for peripheral blood or bone marrow)

Table 2 Important questions regarding ex vivo hematopoietic stem cell expansion

1. Should investigators focus on expanding stem cells, progenitor cells, or both?2. Is there a need for the expanded graft to contain committed lymphoid cells?3. Will expansion of stem cells and committed progenitor cells affect homing to the bone marrow?4. Will the expansion of stem cells result in earlier senescence?5. Should investigators focus on the expansion of other types of stem cells to derive hematopoietic lineage stem cells,

for example, embryonic stem cells (ES), skeletal muscle stem cells, etc.

Clinical application of hematopoietic progenitor cell expansionSM Devineet al

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shortened signicantly by adding ex vivo cultured hemato-poietic cells to a standard graft? The studies have beenheterogeneous with respect to several variables likely toinuence outcome such as (1) the cell source chosen forhematopoietic rescue (bone marrow, mobilized peripheralblood (MPB), UCB) and (2) the conditions employed for invitro culture. The culture conditions have varied with

respect to the combination of cytokines employed, cytokineconcentrations, inclusion or exclusion of serum, duration of culture, initial cell density, the use of static vs dynamic (egperfusion) conditions, and the incorporation of accessory(eg stromal) cells. To critically analyze the results obtained,one must make note of each of these variables as they havea potentially profound inuence on the functional proper-ties of the cells emerging from these cultures (Table 3).

One of the earliest studies involving ex vivo-expandedcells was reported by Naparstek et al 35 in patients withhematologic malignancies receiving allogeneic bone mar-row transplants. Following conditioning, patients receivedtwo-thirds of the donor bone marrow unmanipulated onday 0 and one-third of the bone marrow following culturein GM-CSF and IL-3 for 4 days. No improvement inneutrophil recovery was observed and few data werereported as to the results of the ex vivo culture, such asfold expansion of colony-forming units granulocyte macrophage (CFU-GM) or total nucleated cells.

Later, Brugger et al 36 transfused 10 cancer patients withchemotherapy and G-CSF-mobilized autologous CD34-selected cells cultured in RPMI, 2% autologous plasma,SCF, interleukin-1 b, interleukin-3, interleukin-6, and ery-thropoietin (EPO) for 12 days. Six of the 10 patientsreceived expanded cells as the sole source of hematopoieticrescue. Although all patients had rapid and sustainedengraftment, recovery kinetics did not differ from historic

controls. Of some concern, no late follow-up was reportedand the publication was recently retracted. 37 Williams et al 38

expanded cells in PIXY321 (a GM-CSF/IL-3 fusionprotein) for 12 days and transfused these autologous cellsinto nine patients with metastatic breast cancer. Theexpanded cells were obtained following mobilization withchemotherapy plus G-CSF. The cells were subsequentlyCD34 selected with an immunomagnetic device andcultured in serum-free X-VIVO 10 medium (BioWhittaker,Walkersville, MD, USA) supplemented with 1% humanserum albumin and 100ng/ml PIXY321. An overall median26-fold cellular expansion was observed during the 12-dayculture and the median number of expanded cells infusedwas 44.6 106 /kg. No toxicity attributable to the expandedcells was observed, and clinical benet was seen. In another

trial reporting the use of cultured cells combined withunmanipulated PBPC for hematopoietic rescue, 10 patientswith nonmyeloid malignancies received expanded CD34+cells from cryopreserved PBPC. 39 Following thawing, cellswere CD34 selected using the Isolex 300I device (NexellInc., Irvine, CA, USA) and cultured for 8 days in mediumsupplemented with SCF, IL-3, IL-6, IL-1 b, and EPO. The

mean increase in total cell number (21-fold) and CFU-GM(139-fold) were substantial, there were no adverse eventsrelated to the infusion of expanded cells, and engraftmentwas comparable to historical controls receiving onlyunmanipulated grafts. The results of a second study bythe same group were sobering. Holyoake et al 40 infusedexpanded autologous CD34-selected cells as the sole sourceof hematopoietic rescue to four patients given a myeloa-blative conditioning regimen. Prolonged pancytopenia andfailure of engraftment was observed in all four patientsreceiving only the expanded cells. Each patient had to berescued with an autologous back-up stem cell collection.These results suggested that following a myeloablativeconditioning regimen, hematopoietic cells cultured underthese stroma-free conditions may be insufcient to supportthe maintenance of cells with both short- and long-termengraftment capacities.

Four recently published trials are more encouraging. 4144

McNiece et al 43 expanded CD34-selected G-CSF-mobilizedautologous peripheral blood hematopoietic cells in astroma-free culture, which included dened media (IMDM,1% human serum albumin, recombinant human transfer-rin, Amgen Inc., Thousand Oaks, CA, USA) supplementedwith SCF, megakaryocyte growth and development factor(MGDF), and G-CSF (all at 100 ng/ml; Amgen). The cellswere expanded in static culture for 10 days in Teon bags(American Fluoroseal, Gaithersburg, MD, USA) and

reinfused together with unexpanded CD34-selected cells.In a second cohort of 10 patients, only ex vivo-expandedcells were infused. Compared to historic controls, neutro-phil engraftment was hastened by 34 days, although therewas no effect on platelet recovery. Other recent studiessuggest that mobilized peripheral blood (MPB) cells placedin stroma-free expansion cultures, if provided in sufcientlyhigh numbers, may be useful for shortening neutropenicand thrombocytopenic nadirs when supplementing unma-nipulated peripheral (PB) grafts. Paquette et al 41 performedautografts in breast cancer patients with unmanipulated PBcells containing 5 106 CD34+ cells/kg supplemented withincreasing doses of unseparated PB cells cultured in G-CSF, SCF, and MGDF for 9 days. The study analyzed theimpact of varying the density of cells initially expanded.

Table 3 Variables affecting the results of hematopoietic expansion culture

Combination of cytokines used(SCF, Flt3 ligand, G-CSF, GM-CSF, EPO, IL-3, IL-3, IL-6, IL-11, PIXY321, MGDF, TPO, soluble IL6 receptor)

Concentrations of cytokinesInclusion/exclusion of serum-containing mediaInitial cell densityCD34 selection or unselected cells?Culture durationStatic vs dynamic (eg perfusion) systemsAccessory cells (eg stromal, mesenchymal stem cells) included?


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The fold expansion of CD34 cells following culture wasinversely proportional to the initial cell density. A dose-dependent shortening in both neutrophil and platelet nadirswas observed, with 29% of the patients having neutrophilnadirs o 3 days, and 38% experiencing no neutropenicfevers. This was signicantly improved in comparison to 78historical control patients. Of note, platelet engraftment

was improved in the recipients of expanded cells. However,it should also be noted that this is the only trial in whichcells were mobilized and collected following a combinationof G-CSF and SCF. The inuence of SCF on theengraftment function of the mobilized cells is unclear.Reiffers et al 42 similarly transplanted supplemented PBautografts in patients with multiple myeloma, but usingonly 2 106 unmanipulated CD34+ cells/kg and submit-ting the remainder of the leukapheresis to expansion cultureprior to infusion. As a result, many more progenitor cellsderived from the expanded cells were infused than from theunmanipulated PB collection. Neutrophil nadirs in thesepatients were extremely short, averaging only 2 days withneutrophils o 500/ ml. Validation of this preliminary reportwould represent a major step in the realization of nadirablation by expanded hematopoietic cell supplementation.

Current evidence supports the concept that localizationof hematopoiesis to marrow involves developmentallyregulated adhesive interactions between primitive lympho-hematopoietic cells and marrow stroma. In vitro and in vivostudies support the contention that stromal cells provide arich environment of signals (cytokines, extracellular matrixproteins, adhesion molecules) that control proliferation,survival, and differentiation of hematopoietic progenitorand stem cells. 2,45 Two groups of investigators began toincorporate the stromal components into the expansioncultures. 44,46 These two studies examined whether small

aliquots of steady-state bone marrow can be harvested,expanded ex vivo, and used as the sole source to supporthematopoietic recovery after high-dose chemotherapy.Both studies utilized a perfusion-based stromal cell-containing expansion device that employed frequentmedium, cytokine, and gas exchange. All patients receivedG-CSF following infusion of expanded cells. The publica-tion by Stiff et al 44 inoculated approximately 10% of astandard bone marrow mononuclear cell dose into perfu-sion culture vessels. When used as the sole source of hematopoietic support, this graft reconstituted neutrophilcount after a median 16 (range 1324) days and plateletcount at a median of 24 (range 1945) days. Engelhardtet al 46 found almost precisely the same results in a trial of similar design; neutrophil nadirs ranged from 9 to 18(median 16) days and platelet nadirs spanned 1649(median 23) days. These ndings are similar to unexpandedbone marrow, but slower than MPB. Therefore, theevidence thus far supports the ability of ex vivo-culturedbone marrow mononuclear cells, under dened environ-ments, to maintain its engraftment potential after a one-logexpansion. An added benet of such cultures is that manydifferent tumor types, including breast cancer and low-grade lymphoma, may die within these cultures, therebyproviding tumor cell purging as well. 47 On the other hand,there is as yet no convincing evidence that supplementationof an autograft with ex vivo-expanded hematopoietic cells,

at least under current culture conditions with the cytokinecombinations available, can completely eliminate hemato-poietic nadirs.

In all of these clinical trials, the relative contributions tohematopoietic recovery of expanded vs endogenous cellscannot be accurately assessed since autologous cells wereused and no gene marking was employed. The studies

reported by Paquette et al ,41

Reiffers et al ,42

and McNieceet al ,43 however, suggest that expanded mature or post-progenitor cells may make contributions to early hemato-poietic recovery. On the other hand, patients received largedoses of cells generated in culture, yet these subjects stillexperienced profound aplasia; such results are somewhatdisappointing and suggest that the culture conditionsmay affect the functional properties of at least a fractionof the cells infused. Earlier gene marking studies suggestedthat ex vivo culture may inhibit the capacity of in vitro-dened progenitor cells to contribute to early engraft-ment. 48,49 Recent studies in murine and nonhuman primatemodels strongly suggest the acquisition of functionaldefects acquired by progenitor cells following in vitroculture. 5055

Another complication pervading these early clinicalinvestigations was their use of suboptimal combinationsof stem cell-active cytokines in the hematopoietic cul-tures. 4044 Implementation of novel and potentially ad-vanced technology into expansion has been delayedbecause of many factors including FDA review, obtainingcorporate agreements, and other pragmatic but existinghurdles. Several laboratories have developed assays forlong-term culture-initiating cells (LTC-IC) or cobblestonearea forming cells (CAFC) performed in limiting dilution asa surrogate human stem cell assay. 27,29,30 Such assays detectcells capable of producing colony-forming cells (LTC-IC)

or tight clusters of blast cells (CAFC) after at least 5 weeksof stromal-based culture. These assays are useful tomonitor the expansion of primitive hematopoietic cells inculture. None of the clinical trials reported to date haveutilized combinations of the cytokines required for max-imal progenitor cell and LTC-IC expansion in vitro , norhave been combined with the stromal-based perfusionculture techniques that have shown the best preclinicalresults for LTC-IC survival and expansion. 56 Finally,except for the study by Holyoake et al 40 in which delayedengraftment was observed, none of the other trials usedconditioning regimens that would be considered trulymarrow-ablative. These aws weaken an assessment of the long-term engraftment capacity of the expanded cellstransplanted in these trials. The cumulative results of thepublished marrow and MPB ex vivo expansion trials arepresented in Table 4.

On a more pragmatic level, it remains to be seen whetherprogenitor cell expansion in support of autologoustransplantation is a useful clinical target. Is it worth theexpense and resource utilization to expand cells to achieve amarginal improvement in hematopoietic recovery, particu-larly when the risk of death during the early phase of pancytopenia after autografting is less than 1% in manycenters? Further, it is unclear whether a similar strategywould be benecial in the HLA-matched allogeneictransplant setting given that similar reductions in the


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duration of neutropenia have been observed simplyinfusing G-CSF-mobilized donor granulocytes. 57,58 Finally,the recent demonstration that reduced-intensity condition-ing in combination with donor MPB signicantly mayreduce the duration of pancytopenia after allogeneictransplantation suggests that a graft augmentation strategyin this setting may be unnecessary. 3,59,60 Notwithstanding,

several potentially useful clinical targets including UCBexpansion as well as expansion of hematopoietic cells forgenetic modication remain, and even modest improve-ments in these areas could be clinically meaningful.

Dening new clinical targets: UCB

UCB cells are an effective alternative donor source forpatients with hematological disorders requiring stemcell transplantation. 9,10,17 Overall survival in pediatricrecipients is comparable to that observed followingvolunteer unrelated marrow transplantation. 5,61 Both therates and kinetics of hematopoietic engraftment, however,are inferior to unrelated donor marrow and a signi-cant proportion of patients die because of opportunisticinfections during the period of prolonged neutro-penia. 5,10,12,17,61,62 Although the feasibility of UCBtransplantation has been demonstrated in adults, theperiods of pancytopenia are even longer and there is aclear relation between the dose of UCB cells transplantedand overall outcomes. 10 Given the fact that at least one logfewer of nucleated and CD34+ cells are infused comparedto conventional allografts as well as a much greater degreeof mismatching at HLA loci, it is remarkable thatengraftment occurs at all. Most heavily pretreated adultpatients simply do not tolerate such a prolonged period of neutropenia, and therefore studies designed to improve the

kinetics of hematopoietic engraftment are imperative. Oneapproach to increase the number of hematopoietic cellstransplanted involves combining multiple unrelated cordblood grafts. 9,63 It remains to be determined whether thistechnique will facilitate or hinder recovery, since immuneinteractions between infused units and the recipientconceivably could delay rather than augment donor cellengraftment. Alternatively, the in vitro expansion of all or aportion of a single UCB graft is being studied. Whether exvivo-cultured allogeneic cells can sufce for long-termreconstitution has not yet been dened.

UCB cells, which demonstrate the largest progenitor celland LTC-IC expansion potential in vitro , have only recentlybeen the subject of augmentation studies (Table 5). Threetrials evaluating the combination of unmanipulated cellswith ex vivo-expanded UCB grafts were reported inpreliminary fashion, but are difcult to interpret becauseof the study designs. 6466 In all three studies reported,expanded cells were always infused with unmanipulatedcells. Although one study suggested a potential hastening of platelet engraftment, 65 the others showed no benet. 64,66

One study involved the use of a static culture system indened media, while the other two used a perfusion cultureexpansion system. None of these systems have beendemonstrated to expand true LTRC and therefore the goalof these trials once again was to expand mature cells andSTRC with the hope of improving early engraftment. The T

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relative contributions of the unexpanded and expandedcells to hematopoietic recovery cannot be assessed in thesetrials. Pecora et al 67 published a report of two adult CMLpatients who experienced stable engraftment after receivingthe major portion (83 and 89%) of a UCB graft asunmanipulated cells, while the remaining cells were infusedafter 12 days in ex vivo culture. The relative contributions

of the expanded and unmanipulated cells to long-termrecovery were impossible to determine because most of theinfused CFU-GM were derived from expanded cells,whereas the majority of CD34+Lin-cells arose from theunexpanded portion of the graft. To date, there is noconvincing evidence that current approaches to ex vivoUCB expansion will lead to denable improvements inclinical outcomes.

Additional biologic advantages of UCB

Despite these caveats, the tremendous potential of UCBtransplantation for the correction of a variety of hemato-logical disorders warrants further studies of ex vivo UCBexpansion. Preclinical studies suggest signicant ontogeny-related differences in the in vivo repopulating potential of various human HSC sources. 31,6873 There appears to be acontinuum of engraftment capacity with greatest potentialresiding in fetal liver cells followed by UCB, adult marrow,and lastly MPB. 2,70,74,75 UCB possesses a greater capacityfor expansion of primitive progenitors in response tovarious early-acting cytokines in comparison with adultmarrow or MPB. 56,70,7480 The functional capacity of HSC,as assessed by the ability to engraft in various xenogenicmurine transplant models, is greater with UCB incomparison to marrow or MPB. 30,70,75,78 This nding mayrelate more to intrinsic differences in replicative history or

cell cycle status than to differences in homing capacity, asUCB may possess a limited capacity for self-renewaldivision in culture. 31,56,73,75,81,82 In competitive repopulationassays, UCB possesses superior engraftment capacitycompared to adult marrow. 78 Thus, preclinical datasuggests that UCB represents an optimum clinical targetfor ex vivo expansion strategies.

Current bottlenecks to clinical development

A signicant hindrance to efcient in vitro culture systemdevelopment has been the lack of reliable surrogatemarkers for HSC function. 33 Although surface phenotypehas been a useful surrogate means to dene a population of cells within adult marrow or MPB enriched for HSC, thereis an apparent dissociation between phenotype and func-tion after exposure to cytokines in vitro .79,83,84 Undernormal conditions, the CD34+, lineage negative(CD34+Lin-) or CD34+CD38 phenotype denes apopulation of human hematopoietic cells enriched forHSC. 1,2 Such markers, however, have been shown to be anunreliable surrogate for expansion of repopulating cellsafter culture. 33 Further complicating matters is the appar-ent disparity between surrogate in vitro assays of hemato-poietic progenitor and stem cell function and repopulatingpotential. Progenitor cell assays such as the CFU-GM havebeen useful to assess the quality of unexpanded grafts.

CFU-GM are distinct from the cells responsible formarrow repopulation, and therefore the prediction of repopulating potential based on ex vivo CFU-GM expan-sion will likely overestimate the functionality of thegraft. 33,48,85,86 Other surrogate assays of primitive humanhematopoietic progenitor cells such as the LTC-IC andCAFC may not accurately portray the expansion of an

LTRC, as demonstrated in the NOD/SCID mouse.85

Therefore, the demonstration of expansion of LTC-ICor CAFC after culture does not necessarily connoteexpansion of true hematopoietic repopulating cells, includ-ing HSC.

Many studies suggest that in vitro culture of any durationleads to the acquisition of an engraftment defect, related tochanges in the intrinsic program of the cells exposed tocytokines or to the development of a marrow homingdefect. Studies performed in rodent and nonhuman primatemodels provide clear evidence of an engraftment defectfollowing ex vivo expansion. 50,52,55,83,87 Recent investiga-tions suggest that this effect may be due, in part, to thedownregulation of various cellular adhesion molecules,beta integrins, or chemokine receptors crucial for homingof stem cells back into the bone marrow microenvironmentfollowing intravenous infusion. 32,55,8893 Moreover, it ispresently unclear whether the cytokine cocktails used incurrent expansion protocols promote expansion, mainte-nance, or potentially apoptosis of HSC during culture orshortly following in vivo transfer. If HSC are prone toreplicative senescence, it is possible that the culture will leadto the generation of exhausted progeny and diminishedfunctional capacity. 33,53 Whether in vitro -cultured cellsrequire continuous exposure to exogenous cytokines aftertransplantation is unclear, but data in baboons suggest thismaneuver will be required to optimize hematopoietic

recovery.94

The appropriate combination of exogenouscytokines for this purpose is unknown and clinical trials arehindered by a lack of reagents due, in part, to commercialconsiderations.

Recent data also suggest a relation between thereplicative history of an HSC and its engraftment capacity.All sources of HSC may possess some limited capacity forself-renewal. Both fetal liver and UCB appear capable of undergoing at least some self-renewal divisions whileretaining engraftment capacity in contrast to the morelimited repopulating capacity of adult marrow and MPBfollowing self-replication. 2,32,33,68,69,72,9597 There may alsobe ontogeny-associated differences in response to cytokinecombinations, and therefore the optimal combination forone source of HSC may not be optimal for a differentsource. 73 Further, the retention or reacquisition of cell cyclequiescence following culture may be required to retainrepopulating capacity; cells that have entered G 1 aftercytokine exposure may be depleted of the engraftmentcapacity. 93,98101 The phase of the cell cycle engaged at thetime of transplantation therefore may critically affecthoming and engraftment. Recent studies suggest that thiseffect may be reversible. 100 In summary, numerousquestions abound regarding the functional effects of invitro culture, which will require delineation in preclinicalsystems in order to ensure the likelihood of successclinically.


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Where do we go from here?

The engraftment of ex vivo-expanded HSCs requiresefcient homing, survival, in vivo expansion, and retentionof multilineage differentiative capacity. Expansion requiresconditions that induce self-renewal divisions and minimalapoptosis, differentiation, or loss of in vivo repopulating

capacity. To date, such an articial environment has notbeen achieved consistently, and therefore the identicationof signals and factors important for activating HSC self-renewal divisions without differentiation are crucial(Table 6). To improve the engraftment capacity of smallgrafts such as UCB, expansion of LTRC will likely berequired. 2 If successful, this would improve both early andlate engraftments as HSCs, when given in sufcientquantity, contribute to both short- and long-term engraft-ments in mice. 102104 This concept recently was proven inclinical trials involving high-speed sorting and transplanta-tion of cells dened by a primitive hematopoietic pheno-type. 105,106 Resources should be allocated towards thedevelopment of in vitro systems designed to expand LTRC.These culture conditions then could be studied rst inxenogeneic transplantation models and then optimally inlarge animal models before being taken for clinical trials.Sufcient evidence suggests the potential to improve cultureconditions and to enhance engraftment capacity of ex vivo-cultured cells. 107 For instance, Ueda et al 82 used acombination of early acting cytokines together with acomplex of interleukin-6 and soluble interleukin-6 receptorto upregulate CXCR4 expression and therefore improvecell homing ability. Improved capacity to engraft NOD/SCID mice was demonstrated under these culture condi-tions. Other strategies worthy of pursuit include targetingof antiapoptotic factors or the delta/jagged receptor

interaction.108110

Another interesting approach involvescopper chelation, as this element promotes hematopoieticdifferentiation in vitro .111 Clinical trials designed tocombine chelation in combination with ex vivo cytokineexposure are underway.

Additionally, Takatoku et al 121 noted retention of engraftment capacity if activating cytokines are combinedwith cell cycle inhibition in order to prevent dividing cellsfrom committing to lineage differentiation. Alternativestrategies include the isolation of novel cytokines with theability to amplify pluripotent stem cells, and developmentof soluble intracellular cell cycle activators or inhibitorsthat reversibly increase stem cell symmetric cycling andtherefore directly increase stem cell numbers. Candidatesmight include antisense oligonucleotide or RNA interfer-ence inhibition of p21 or p27, as well as soluble inhibitorsof transcription factors that suppress the transcription of HOX genes that stimulate stem cell cycling. 112,113 Of note,treatment with reversible modulators of stem cell cyclingraises concerns about inducing abnormally proliferativestem cells that could lead to the development of leukemia.Hence, such treatments must be studied in preclinicalanimal models before initiating clinical trials.

Efforts to recreate the in vivo hematopoietic microenvir-onment in culture systems have led to the generation of stromal cell lines from the aorto-gonado-mesonephrosregion, fetal liver, and bone marrow. 50,91,114 Postnatal

human marrow contains a second stem cell, the mesench-ymal stem cell (MSC), that gives rise to osteoblasts,chondrocytes, adipocytes, and myelosupportive stro-ma. 115,116 MSCs express adhesive ligands and solublefactors critical for hematopoiesis, and have been demon-strated to support hematopoiesis in vitro .115 Efforts toexpand UCB grafts in combination with expanded MSCare ongoing and preliminary data are encouraging. 117 Otherculture conditions designed to remove cells that mightinhibit the expansion of repopulating cells in a dynamicsystem are being pursued. 118 Finally, human embryonicstem cells can be induced to differentiate into hematopoie-tic cells 119,120 . If ethical and regulatory issues can beaddressed successfully, these cells may become a usefulsource of HSC with LTRC potential.

The past two decades have demonstrated that throughthe extraordinary efforts of cell and molecular biologists,bioengineers, and clinical investigators, at least some of theproliferative potential of HSCs have been harnessed forclinical therapy. Present evidence suggests that hemato-poietic augmentation strategies may able to signicantlyreduce postchemotherapy nadirs. Clinical trials based onthe most promising preclinical data should be pursued aslong as the target is appropriate and the study end pointsare well-dened. Each source of HSCs (BM, MPB, UCB)has its own, unique biologic properties in vitro as well asclinical efcacy in vivo, such that each tissue requires itsown thoughtful study. True clinically useful stem cellexpansion strategies that would allow the unlimited use of very small numbers of stem cells for full, rapid hemato-poietic reconstitution await further preclinical study.Future investigations hopefully will provide exponentialexpansion of stem cells for tissue repair in hematology andother disciplines.

References

1 Weissman IL. Translating stem and progenitor cell biology tothe clinic: barriers and opportunities. Science 2000; 287 :14421446.

Table 6 Future directions in ex vivo hematopoietic cell expansion

Identication of genes responsible for maintenance of stem cell phenotypeExpansion of long-term marrow repopulating cells

Targeting umbilical cord blood for expansion studiesImprove homing capacity of in vitro cultured cells

Upregulation of CXCR-4Maintenance of integrin expression

Targeting of antiapoptotic factors to preventsenescence in cultureCoordinate cell cycle activation with inhibition toprevent commitment

Novel cytokinesRNA interference (p21 or p27)

Continued efforts to recreate bone marrow microenvironmentMicrovascular endothelial cellsMesenchymal stem cellsBioengineered bone marrow organoids


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