Noninvasive Bioluminescent Imaging Demonstrates Long-Term Multilineage Engraftment of Ex Vivo-Expanded CD34-Selected Umbilical Cord Blood Cells

Noninvasive Bioluminescent Imaging Demonstrates Long-TermMultilineage Engraftment of Ex Vivo-Expanded CD34-SelectedUmbilical Cord Blood Cells

David Steinera, Juri Gelovanib, Barbara Savoldoc, Simon N. Robinsona, William K. Deckera,Nathalie Brouardd, Amer Najjarb, Dongxia Xinga, Hong Yanga, Sufang Lia, Frank Marinia,Patrick A. Zweidler-McKaya, Catherine M. Bollardc, Elizabeth J. Shpalla, Gianpietro Dottic,and Paul J. Simmonsd

aDepartment of Stem Cell Transplantation and Cellular Therapy, The University of Texas MDAnderson Cancer Center, Houston, Texas, USAbDepartment of Molecular Imaging, The University of Texas MD Anderson Cancer Center,Houston, Texas, USAcBaylor College of Medicine, Center for Cell and Gene Therapy, Houston, Texas, USAdThe University of Texas Health Science Center Brown Foundation Institute of MolecularMedicine, Houston, Texas, USA

AbstractThe use of umbilical cord blood (UCB) grafts for hematopoietic stem cell transplantation (HSCT)is a promising technique that permits a degree of human leukocyte antigen mismatch between thegraft and the host without the concomitant higher rate of graft-versus-host disease that would beobserved between an adult marrow graft and a mismatched host. A disadvantage to the use ofUCB for HSCT is that immune reconstitution may be significantly delayed because of the lowstem cell dose available in the graft. Ex vivo expansion of UCB CD34 cells would provide agreater number of stem cells; however, there are persistent concerns that ex vivo-expanded CD34cells may lose pluripotency and the ability to contribute meaningfully to long-term engraftment.To address this issue, we transduced CD34-selected UCB cells with a lentiviral constructexpressing luciferase, and determined homing and engraftment patterns in vivo by noninvasivebioluminescent imaging in sublethally irradiated NOD/SCID/IL-2Rγ−/− (NSG) mice. Graftcontribution to multilineage commitment was also confirmed by analysis of primary andsecondary transplants by flow cytometry and immunohistochemistry. Our results demonstrate that,

© AlphaMed Press

Correspondence: Elizabeth J. Shpall, M.D., Department of Stem Cell Transplantation and Cellular Therapy, The University of TexasMD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. Telephone: 713-745-2161; Fax:713-794-4902; [email protected].

Author contributions: D.S.: conception and design, collection and assembly of data, data analysis and interpretation, manuscriptwriting; J.G.: data analysis and interpretation, final approval of manuscript; B.S.: collection and assembly of data; S.R.: manuscriptwriting, provision of study materials; W.D.: provision of study materials, collection and assembly of data, data analysis andinterpretation, manuscript writing; N.B.: collection and assembly of data, data analysis and interpretation; A.N.: conception anddesign, provision of study materials; D.X.: collection and assembly of data; H.Y.: provision of study materials, data analysis andinterpretation; S.L.: collection and assembly of data; F.M.: provision of study materials, data analysis and interpretation, manuscriptwriting; P.Z.: conception and design, provision of study materials, data analysis and interpretation; C.B.: data analysis andinterpretation; E.S.: conception and design, financial support, provision of study materials, data analysis and interpretation, finalapproval of manuscript; G.D.: conception and design, collection and assembly of data, data analysis and interpretation; P.S.:conception and design, data analysis and interpretation, final approval of manuscript; D.S. and J.G. contributed equally to this work.

Disclosure of Potential Conflicts of InterestThe authors indicate no potential conflicts of interest.

NIH Public AccessAuthor ManuscriptStem Cells. Author manuscript; available in PMC 2012 May 09.

Published in final edited form as:Stem Cells. 2009 August ; 27(8): 1932–1940. doi:10.1002/stem.111.

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other than a mild delay at the onset of engraftment, there were no significant differences in lineagerepopulation or in long-term or secondary engraftment between culture-expanded and unexpandedUCB CD34-selected cells. The results suggest that multipotent stem cells can be expanded ex vivoand can contribute meaningfully to long-term hematopoietic engraftment.

KeywordsBioluminescent imaging; Umbilical cord blood; Hematopoiesis; Transplantation; Engraftment;Luciferase; Calvarium

IntroductionUmbilical cord blood (UCB) has become an important source of stem cells for use inhematopoietic stem cell (HSC) transplantation. The advantages of cord over mobilizedperipheral blood or bone marrow are numerous and include its ease of collection, storage,and distribution; its lower incidence and severity of post-transplant graft-versus-hostdisease; its tolerance of significant human leukocyte antigen mismatching between donorand recipient; and the ability of cord blood banks to obtain units that proportionatelyrepresent underserved minority populations. One downside, however, is the limited cell doseavailable for transplant in the UCB graft, especially for recipients >70 kg. This is asignificant issue and often results in delayed platelet and neutrophil engraftment and agreater risk for graft failure than with other sources of HSCs [1–11]. The hematopoieticprogenitor cell content of the UCB unit has been shown to be a reliable predictor of plateletand neutrophil engraftment [12]; hence, a number of strategies have been developed with thegoal of improving the dose of UCB hematopoietic progenitor cells in the graft. Thesestrategies have included the tandem transplantation of two different UCB units [13–15] aswell as the use of ex vivo expansion techniques, some of which have resulted in significantproliferation of pluripotent progenitors [16–25] and have also identified the importance ofinsulin-like and angiopoietin-like growth factors in the biology of expansion [26–29].

Many groups have attempted to identify ex vivo culture conditions that best preserve andexpand the “stem cell reserve” of a graft, thereby generating a product that might improveneutrophil and platelet engraftment for cord blood recipients; however, ex vivo cultureconditions drive hematopoietic differentiation and increase the numbers of maturehematopoietic progenitors at the expense of the most primitive, pluripotent hematopoieticprogenitors. The mature hematopoietic progenitors generated by an ex vivo expansionproduct may therefore allow for a rapid, transient short-term engraftment, but may lack thelong-term repopulating activity required for transfusion-independent hematopoiesis [16, 30–38]. And, although we have previously hypothesized that the culture conditions employedby our group do allow for the expansion of primitive, pluripotent progenitors [20], in vivoexperimental verification of this hypothesis is lacking.

Hematopoietic engraftment following transplantation is a poorly understood biologicalprocess. In an attempt to better understand the real-time dynamic nature of hematopoieticengraftment, we used a noninvasive bioluminescent-based imaging technique [39, 40] tofollow the in vivo engraftment kinetics of expanded and unexpanded UCB CD34+ cellsfollowing transplantation. Our results demonstrate long-term persistence of humanhematopoiesis in mice receiving both unexpanded and ex vivo-expanded UCB CD34+ cells.Further, we were also able to visualize human hematopoiesis in secondary recipients of bonemarrow from mice receiving ex vivo-expanded UCB CD34+ cells, demonstrating thepresence of primitive, pluripotent stem cells with long-term marrow repopulating activity inthe primary graft. The data confirm the hypothesis that our UCB CD34+ expansion protocol

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allows for the maintenance of primitive hematopoietic progenitors capable of long-termmultilineage engraftment.

Materials and MethodsMice

Six- to 12-week-old NOD.Cg-Prkdcscid IL2rgtmWjl/Sz (NSG) mice (Jackson Laboratory,Bar Harbor, ME, http://www.jax.org) were used for these studies. Unlike regular nonobesediabetic severe combined immunodeficient (NOD-SCID) mice, which possess some T andnatural killer (NK) cells, NSG mice are completely devoid of T-cell and NK-cell activity,allowing the robust detection of peripheral blood engraftment using as few as 102 totalprogenitor cells. Use of 105 progenitors can result in peripheral blood engraftment ≥70%[41–43].

Lentivirus ProductionThe plasmid pHIV-GFPFFLuc was constructed by insertion of the enhanced greenfluorescent protein (eGFP) firefly Luciferase (FFLuc) fusion cassette in place of the internalribosome entry site (IRES) enhanced yellow fluorescent protein (eYFP) sequence in theplasmid vector pHIV-IRES-eYFP (kindly provided by Dr. Richard Sutton, Baylor Collegeof Medicine, Houston, Texas). To produce the lentiviral supernatant, 293T cells werecotransfected with lentiviral vectors, pHIV-GFPFFLuc, and pDRF containing the sequencefor the RD114 envelope [44], using the Fugene6 transfection reagent (Roche, Indianapolis,IN, http://www.roche.-com) according to the manufacturer’s instructions. Lentiviralsupernatant was collected 48 and 72 hours later and immediately filtered and frozen.

Human UCB CD34 Selection and Lentiviral TransductionUCB units were obtained from the MD Anderson Cord Blood Bank as stipulated byinstitutional review board protocol LAB03-0796. Mononuclear cells (MNCs) were separatedfrom red cells using a Ficoll gradient. CD34+ cells were isolated from recovered UCBMNCs by magnetic selection (Miltenyi Biotec, Auburn, CA,http://www.miltenyibiotec.com). Percent purity was determined by flow cytometry. Fortransduction, 24-well nontissue culture plates precoated with retronectin (Takara Shuzo,Otsu, Japan, http://www.takara.co.jp) were incubated twice with 0.5 ml lentiviralsupernatant for 30 minutes. CD34-enriched cells were then plated in 0.5 ml completemedium—α-minimal essential medium (MEM) (Gibco-BRL, Gaithersburg, MD,http://www.gibcobrl.-com) supplemented with 10% fetal calf serum and 2 mM L-glutamine—at a concentration of 0.3–0.8 × 106 cells per well mixed with an additional 1.5 mllentiviral supernatant. After addition of the virus, the plates were centrifuged and incubatedat 37°C overnight. Subsequently, 50%–70% of the medium was removed and replaced withfresh HSC expansion medium. Transduction efficiency was determined 72 hours post-transduction by flow cytometric detection of CD34+GFP+ cells within the stem cell gate.Cells were then infused into mice or expanded further in culture.

UCB HSC ExpansionTransduced UCB cells were expanded for 10 days in HSC expansion medium consisting ofeither α-MEM (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) or SCGM CellGro(CellGenix, Antioch, IL, http://www.cellgenix.com) supplemented with 50 µg/mlgentamycin (Abbott Laboratories, Chicago, IL, http://www.abbott.com), 100 ng/ml stem cellfactor (CellGenix), 100 ng/ml Flt3-L (CellGenix), 100 ng/ml thrombopoietin (CellGenix),and 100 ng/ml G-CSF (Amgen, Thousand Oaks, CA, http://www.amgen.com).



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http://www.jax.org

http://www.roche.-com

http://www.miltenyibiotec.com

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http://www.gibcobrl.-com

http://www.invitrogen.com

http://www.cellgenix.com

http://www.abbott.com

http://www.amgen.com

Engraftment of Mice With CD34+ UCB CellsAdult NSG mice were irradiated with 270 cGy from a 137-Cs source. Mice were injectedi.v. through the tail vein with 0.8 × 105 to 3.5 × 105 transduced, unexpanded cells or theequivalent number (with regard to CD34+ content) of 10-day expanded cells.

Analysis of Engraftment by Flow CytometryMice were bled retroorbitally, and red cells were removed by treatment with PharmLyse(BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) MNCs were labeled withanti-mouse CD45 and anti-human CD45 monoclonal antibodies as well as a variety oflineage-specific antibodies including anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD19,anti-CD33, anti-CD34, and anti-CD56 (all from BD Biosciences). Labeled cells were fixedin 4% paraformaldehyde and analyzed on a FACScan flow cytometer using CellQuestsoftware (BD Biosciences).

Noninvasive Bioluminescent ImagingCultured, transduced, expanded or unexpanded human UCB cells were injected intoconditioned NSG mice, and human cell engraftment in the bone marrow of the recipientswas determined by bioluminescent imaging every other day during the first 2 weeks as wellas 3, 6, and 12 months post-transplant. For in vivo imaging of engrafted GFP-FFLuc-expressing cells, mice were injected i.p. with D-luciferin (150 mg/kg) and analyzed usingthe Xenogen-IVIS Imaging System (Caliper Life Sciences, Hopkinton, MA,http://www.caliperls.com). A constant region of interest was drawn and the intensity of thesignal was measured as total photons/second per cm2/sr.

ImmunohistochemistryMouse spines were dissected and fixed in 3% formaldehyde in phosphate-buffered saline(PBS) for 2 hours at 4°C, washed in PBS, and decalcified by immersion in a 10% solution ofEDTA changed daily for 10 days. Before embedding, spines were incubated in a Ca2+-containing solution for 1 hour. Spines were cut into two sections for embedding in OCT andin paraffin. For immunostaining, 8-µm cryosections were prepared on a Microm HM525cryostat (Microm, Walldorf, Germany, http://www.microm-online.com). Sections weredried overnight, rehydrated in PBS, washed in PBS-0.05% Tween-10 (PBS-T), andincubated in blocking buffer (5% bovine serum albumin, 5% skim milk powder, 0.05%Triton-X100, 2% normal donkey serum, and 2% rat anti-mouse CD16/32 in 4× SSC) for 30minutes. Sections were then incubated with mouse anti-human CD45 monoclonal antibodyand isotype control (BD Biosciences) overnight at 4°C in a humidified chamber, washed inPBS-T, and incubated with biotinylated donkey anti-mouse IgG (Jackson ImmunoResearchLaboratories, West Grove, PA, http://www.jacksonimmuno.com) for 1 hour at roomtemperature in a humidified chamber, and washed again in PBS-T. Endogenous peroxidaseactivity was blocked by incubation of the slides in 6% H2O2 in methanol for 30 minutes.Slides were washed in PBS-T, incubated for 1 hour at room temperature with streptavidin-biotinylated horseradish peroxidase complex, then washed in PBS. Peroxidase activity wasdetected by incubation with a solution of 0.25 mg/ml diaminobenzidine (Sigma-Aldrich, St.Louis, http://www.sigmaaldrich.com) in PBS supplemented with 3% H2O2 for 6 minutes.Slides were dehydrated in successive baths of ethanol and xylene and mounted in DPXmounting medium (Sigma-Aldrich). Images were visualized using a BX51 Olympusmicroscope and acquired by a DP71 Olympus camera (Olympus, Tokyo,http://www.olympus-global.com).



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http://www.caliperls.com

http://www.microm-online.com

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http://www.sigmaaldrich.com

http://www.olympus-global.com

ResultsCD34+ Human UCB Transduction

CD34+ UCB cells were isolated by magnetic selection to an average purity of 25%–50%;however, >95% of cells within the stem cell gate were CD34+. CD34-selected cells werethen transduced with lentivector expressing the gene marker eGFP or the fusion proteineGFPFFLuc. Transduction efficiency, as measured by flow cytometry, indicated apercentage of 10%–15% CD34+GFP+ cells on day 3 post-transduction (Fig. 1A). Thetransduced cells were divided into two equal fractions, and one fraction was injected directlyinto irradiated NSG mice while the other half was expanded ex vivo for an additional 7 days.The percentage of CD34+GFP+ cells was determined postexpansion (Fig. 1B), and expandedcells were injected into irradiated NSG mice. Typically, the number of expanded cellsinjected was three- to fourfold greater than the number of unexpanded cells, so that thenumber of CD34+ cells in the expanded cell fraction was equivalent to the number ofunexpanded CD34+ cells injected in any given experiment. We observed up to a tenfoldexpansion of CD34+ progenitors in some experiments; however, expansion data in aggregatewere quite similar to what we have reported previously [20].

Engraftment of Cultured Human UCB Cells in NSG MiceTo assess the engraftment capabilities of the transduced HSCs, we examined the mice bynoninvasive bioluminescent imaging. The first clear signals of engraftment were observedbetween days 7 and 9. Mice that received the expanded fraction exhibited engraftmentsignals 2–5 days later than those that received the unexpanded fraction; however, fewquantitative differences were discernable beyond day 20–25 (Fig. 2A, 2B). In contrast,peripheral blood engraftment of cord blood in the mice could not be detected by flowcytometry until day 21–28 at the earliest, >2 weeks after engraftment was apparent bybioluminescent imaging.

Expanded HSC EngraftmentThere was little variation between expanded and unexpanded UCB cells in terms of the sitesat which early engraftment was observed. All mice demonstrated early engraftment in thelong bones, sternum, spine, and pelvis with equal frequency; however, 45% of micereceiving unexpanded UCB cells exhibited an engraftment signal in the area of thecalvarium by day 14. Such a signal was never detected early among mice receivingexpanded UCB cells (representative mice shown in Fig. 2C, 2D; p < .001 by χ2). Calvarialengraftment signals were eventually seen at about post-transplant day 90 among mice whoreceived expanded UCB grafts. These differences were significant and observed exclusivelyin the calvarium. By harvesting tissues from mice and incubating them with D-luciferin andATP, we demonstrated that the source of the signal originated genuinely within thecalvarium (Fig. 3) and not within the brain (data not shown). This was the only temporal orspatial variation observed between expanded and unexpanded UCB grafts.

To determine lineage of engrafted cells, we analyzed the subset of human CD45+ cellspresent in the periphery of mice that received unexpanded or expanded UCB grafts. CD19was the dominant lineage observed within 3 months post-transplant. When peripheral humanCD45 was measured 3–6 months post-transplant, broad multilineage engraftment wasobserved, including human CD3, CD4, CD8, CD19, and CD14 (Fig. 4A). Over time, the T-cell subset gradually increased, so that by 10–12 months post-transplant the T-cellsubpopulation comprised the majority of peripheral human CD45+ cells in the engraftedmice (Fig. 4B). Thirteen mice analyzed for peripheral myeloid engraftment demonstrated anaverage of 4.6% ± 2.7% CD33+ cells (range, 0.4%–9.4%) (Fig. 4C). In some mice (but notall), low levels of CD56+ cells were observed; however, this population never comprised



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>2% of the total human cell population (data not shown). No significant differences inperipheral blood engraftment characteristics were observed between mice receivingexpanded and those receiving unexpanded cord blood cells.

Secondary TransplantationAt 1 year post-transplant, mice receiving both expanded and unexpanded grafts were stilldisplaying powerful engraftment signals (representative mouse shown in Fig. 2E). At thispoint, organs were harvested and the organ cell phenotype was determined by flowcytometry, and secondary mice were transplanted by reinfusion of 2–3 × 106 unmanipulatedchimeric bone marrow cells derived from expanded CD34 UCB primary transplants. Thephenotypic characteristics of a representative graft as determined by flow cytometry aredisplayed in Figure 5A. Myeloid engraftment characteristics of donors were typical of thegroup as a whole (data not shown). Engraftment signals in secondarily transplanted micewere detectable within 2–3 weeks post-transplant. Two months after secondary transplant,clear engraftment signals were detectable by imaging (Fig. 5B), but only low levels ofperipheral blood engraftment ([ltequ]5%) could be detected by flow cytometry.Nevertheless, we were able to verify the presence of CD45+ human cells in the marrowspaces of secondary transplants by immunohistochemistry (Fig. 6).

DiscussionHere, we have used in vivo bioluminescent imaging to compare the engraftment potential ofhuman cord blood CD34+ cells expanded in culture for 10 days with the equivalentunexpanded fraction. Our results validate the hypothesis that expanded UCB progenitors canproduce durable, multilineage engraftment, nearly comparable with that of unexpandedUCB progenitors.

A recent publication by Giassi et al. [45] demonstrated that cultured human UCB cells couldgenerate myeloid and erythroid lineages, but not lymphoid lineage cells in NSG mice unlessthe mice were pretreated with tumor necrosis factor α. In our model, the majority ofdetectable human cells were CD19+ following the onset of engraftment; however, as early as8 weeks post-transplant, we were able to detect CD3+ human cells as well. Furthermore, by10 months post-transplant, a majority of cells in the periphery were CD3+. Although theonly physical difference between our expansion protocol and that of Giassi et al. [45] is theuse of G-CSF, this cytokine is known to promote the enhancement of myeloid, notlymphoid, lineages. Given this, it is unlikely that this modification to our expansion protocolcontributed significantly to our differential results. More likely, Giassi et al. [45] did notreport results that extended beyond 8 weeks post-transplant. Because of the time that itmight take for T cells to mature in the thymus and for the thymus itself to become populatedwith de novo-generated human dendritic cells, it is unlikely that T cells would be observedprior to this time point. Indeed, we ourselves did not observe the development of T cellsuntil the later stages of engraftment. The shift from a predominantly CD19+ lymphoid subsetto a predominantly CD3+ subset approximately 10 months post-transplant was curious.Preliminarily, we have hypothesized that this shift may have been a result of physiologicevents related to aging and not necessarily a result of events that were hematopoietic innature. Histologic analysis of the long bones revealed an abnormal cellular distribution.Although we found human cells in the epiphysis, the diaphysis was calcified and completelya cellular, suggesting that the ability of older NSG mice to produce B cells could besignificantly impaired. However, we did not examine untransplanted controls, and wecannot rule out the hypothesis that the grafts themselves were the cause of the diaphysiscalcification.



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Our lentiviral transduction efficiency was consistent with that reported in a recentpublication by Liu et al. [46], which outlines the major parameters for efficient lentiviraltransduction and engraftment of human CD34+ UCB. Liu [46] demonstrated that optimaltransduction and engraftment efficiency is achieved by transducing unstimulated stem cellsfor 5 hours followed by 3 days of culture. We also observed the importance of incorporatinga short culture period subsequent to an overnight transduction. In order to allow the viralvector to survive an overnight incubation period, it would have been necessary to replace theRD114 viral envelope coat protein with vesicular stomatitis virus G (VSV-G). ThoughVSV-G is able to more efficiently transduce CD34+ stem cells, it is also much more toxic,and cell viability following a VSV-G transduction is unacceptable. Because RD114 is notable to form viable virus particles with any third-generation, self-inactivated lentiviralvectors, we did all the described studies with first-generation vectors. Such a protocol is finefor in vitro studies, but would be unacceptable in a clinical setting. If we are to use ourvectors clinically, we will likely have to adopt a transduction period similar to that of Liu[46], though they did not demonstrate the potential of their transduced cells to mediate long-term engraftment.

The sensitivity of luciferase-based bioluminescent imaging is much greater than that ofengraftment screening assays that detect the presence of human cells in peripheral blood.Although we were able to detect clear signals of engraftment in the marrow spaces no laterthan 10 days post-transplant and predict successful engraftment as early as day 6, we wereunable to detect human cells in the periphery by flow cytometry until post-transplant day 28at the earliest. Although bioluminescent imaging is not applicable clinically, we show, inprinciple, that engraftment failure can be detected very early. We determined thattransduction of as few as 3% of cells could still result in a detectable image (data notshown), and we speculate that the labeling of patient products prior to transplantation couldconceivably have predictive merit in a number of clinical protocols if used in conjunctionwith appropriate technologies that allow imaging of cells within human recipients.

In our imaging study, we were able to detect hematopoiesis in a defined area within thecalvarium. This area is not generally appreciated to be hematopoietically active; however, arecent report by Lo Celso et al [47]. demonstrates the importance of the calvarium as a focusof hematopoietic activity. By imaging, we demonstrated that UCB cells migrate to a specificcalvarial focus and expand significantly (per available volume niche). This area, which doesnot exist at the time of birth, develops during the postnatal period and may sustain uniquehematopoietic and niche migration characteristics. At 2 weeks post-transplant, there weresignificant differences in the calvarial engraftment signal between unexpanded andexpanded HSC grafts. With time, this difference diminished, and no significant differenceswere observed long term. The significance of this phenomenon is unclear and requiresfurther study. It is our hypothesis that stem cells lose the ability to migrate to the calvariumduring expansion, and that expanded grafts can only populate the calvarium after de novohematopoiesis occurs at other sites, that is, in the long bones or the spine. If this is indeedthe case, a better understanding of the mechanism of calvarial engraftment couldconceivably improve the rate of engraftment in expanded UCB clinical protocols.

In summary, with the exception of a small delay in early engraftment, human UCB cellsexpanded 10 days in culture could engraft and differentiate into multiple hematopoieticlineages in NSG mice in virtually the same manner as unexpanded human UCB cells. Theability to image transduced stem cells might allow the prediction of engraftment failure afew days after transplant, allowing the clinical decision-making process to be significantlyenhanced.



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AcknowledgmentsThis work was supported in part by NIH grant no. 5 R01 CA061508-13 (to E.J.S.).

References1. Barker JN, Davies SM, DeFor T, et al. Survival after transplantation of unrelated donor umbilical

cord blood is comparable to that of human leukocyte antigen-matched unrelated donor bonemarrow: Results of a matched-pair analysis. Blood. 2001; 97:2957–2961. [PubMed: 11342417]

2. Barker JN, Weisdorf DJ, DeFor TE, et al. Rapid and complete donor chimerism in adult recipientsof unrelated donor umbilical cord blood transplantation after reduced-intensity conditioning. Blood.2003; 102:1915–1919. [PubMed: 12738676]

3. Brunstein CG, Barker JN, Weisdorf DJ, et al. Umbilical cord blood transplantation afternonmyeloablative conditioning: Impact on transplantation outcomes in 110 adults with hematologicdisease. Blood. 2007; 110:3064–3070. [PubMed: 17569820]

4. Gluckman E, Rocha V, Boyer-Chammard A, et al. Outcome of cord-blood transplantation fromrelated and unrelated donors. Eurocord Transplant Group and the European Blood and MarrowTransplantation Group. N Engl J Med. 1997; 337:373–381. [PubMed: 9241126]

5. Gluckman E, Rocha V, Chevret S. Results of unrelated umbilical cord blood hematopoietic stemcell transplantation. Rev Clin Exp Hematol. 2001; 5:87–99. [PubMed: 11486656]

6. Gluckman E, Rocha V, Arcese W, et al. Factors associated with outcomes of unrelated cord bloodtransplant: Guidelines for donor choice. Exp Hematol. 2004; 32:397–407. [PubMed: 15050751]

7. Kurtzberg J, Laughlin M, Graham ML, et al. Placental blood as a source of hematopoietic stem cellsfor transplantation into unrelated recipients. N Engl J Med. 1996; 335:157–166. [PubMed:8657213]

8. Laughlin MJ, Barker J, Bambach B, et al. Hematopoietic engraftment and survival in adultrecipients of umbilical-cord blood from unrelated donors. N Engl J Med. 2001; 344:1815–1822.[PubMed: 11407342]

9. Laughlin MJ, Eapen M, Rubinstein P, et al. Outcomes after transplantation of cord blood or bonemarrow from unrelated donors in adults with leukemia. N Engl J Med. 2004; 351:2265–2275.[PubMed: 15564543]

10. Rubinstein P, Carrier C, Scaradavou A, et al. Outcomes among 562 recipients of placental-bloodtransplants from unrelated donors. N Engl J Med. 1998; 339:1565–1577. [PubMed: 9828244]

11. Wagner JE, Barker JN, DeFor TE, et al. Transplantation of unrelated donor umbilical cord blood in102 patients with malignant and nonmalignant diseases: Influence of CD34 cell dose and HLAdisparity on treatment-related mortality and survival. Blood. 2002; 100:1611–1618. [PubMed:12176879]

12. Migliaccio AR, Adamson JW, Stevens CE, et al. Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: Graft progenitor cell content is a better predictor thannucleated cell quantity. Blood. 2000; 96:2717–2722. [PubMed: 11023503]

13. Barker JN, Weisdorf DJ, Wagner JE. Creation of a double chimera after the transplantation ofumbilical-cord blood from two partially matched unrelated donors. N Engl J Med. 2001;344:1870–1871. [PubMed: 11407361]

14. Barker JN, Weisdorf DJ, DeFor TE, et al. Transplantation of 2 partially HLA-matched umbilicalcord blood units to enhance engraftment in adults with hematologic malignancy. Blood. 2005;105:1343–1347. [PubMed: 15466923]

15. De Lima M, St Johns LS, Wieder ED, et al. Double-chimaerism after transplantation of two humanleucocyte antigen mismatched, unrelated cord blood units. Br J Haematol. 2002; 119:773–776.[PubMed: 12437658]

16. McNiece IK, Almeida-Porada G, Shpall EJ, et al. Ex vivo expanded cord blood cells provide rapidengraftment in fetal sheep but lack long-term engrafting potential. Exp Hematol. 2002; 30:612–616. [PubMed: 12063029]

17. McNiece I, Harrington J, Turney J, et al. Ex vivo expansion of cord blood mononuclear cells onmesenchymal stem cells. Cytotherapy. 2004; 6:311–317. [PubMed: 16146883]



NIH


NIH


NIH


18. McNiece I, Kubegov D, Kerzic P, et al. Increased expansion and differentiation of cord bloodproducts using a two-step expansion culture. Exp Hematol. 2000; 28:1181–1186. [PubMed:11027837]

19. Shpall EJ, Quinones R, Giller R, et al. Transplantation of ex vivo expanded cord blood. Biol BloodMarrow Transplant. 2002; 8:368–376. [PubMed: 12171483]

20. Robinson SN, Ng J, Niu T, et al. Superior ex vivo cord blood expansion following co-culture withbone marrow-derived mesenchymal stem cells. Bone Marrow Transplant. 2006; 37:359–366.[PubMed: 16400333]

21. de Lima M, McMannis J, Gee A, et al. Transplantation of ex vivo expanded cord blood cells usingthe copper chelator tetraethylenepentamine: A phase I/II clinical trial. Bone Marrow Transplant.2008; 41:771–778. [PubMed: 18209724]

22. Xiao M, Broxmeyer HE, Horie M, et al. Extensive proliferative capacity of single isolated CD34human cord blood cells in suspension culture. Blood Cells. 1994; 20:455–466. discussion 466–467. [PubMed: 7538351]

23. Traycoff CM, Abboud MR, Laver J, et al. Ex vivo expansion of CD34+ cells from purified adulthuman bone marrow and umbilical cord blood hematopoietic progenitor cells. Prog Clin Biol Res.1994; 389:385–391. [PubMed: 7535446]

24. Moore MA, Hoskins I. Ex vivo expansion of cord blood-derived stem cells and progenitors. BloodCells. 1994; 20:468–479. discussion 479–481. [PubMed: 7538352]

25. Broxmeyer HE, Hangoc G, Cooper S, et al. Growth characteristics and expansion of humanumbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl AcadSci U S A. 1992; 89:4109–4113. [PubMed: 1373894]

26. Zhang CC, Kaba M, Iizuka S, et al. Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansionof human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation. Blood.2008; 111:3415–3423. [PubMed: 18202223]

27. Zhang CC, Kaba M, Ge G, et al. Angiopoietin-like proteins stimulate ex vivo expansion ofhematopoietic stem cells. Nat Med. 2006; 12:240–245. [PubMed: 16429146]

28. Zhang CC, Lodish HF. Murine hematopoietic stem cells change their surface phenotype during exvivo expansion. Blood. 2005; 105:4314–4320. [PubMed: 15701724]

29. Zhang CC, Lodish HF. Insulin-like growth factor 2 expressed in a novel fetal liver cell populationis a growth factor for hematopoietic stem cells. Blood. 2004; 103:2513–2521. [PubMed:14592820]

30. Abkowitz JL, Taboada MR, Sabo KM, et al. The ex vivo expansion of feline marrow cells leads toincreased numbers of BFU-E and CFU-GM but a loss of reconstituting ability. Stem Cells. 1998;16:288–293. [PubMed: 9708451]

31. Guenechea G, Segovia JC, Albella B, et al. Delayed engraftment of nonobese diabetic/severecombined immunodeficient mice transplanted with ex vivo-expanded human CD34(+) cord bloodcells. Blood. 1999; 93:1097–1105. [PubMed: 9920860]

32. Holyoake TL, Alcorn MJ, Richmond L, et al. CD34 positive PBPC expanded ex vivo may notprovide durable engraftment following myeloablative chemoradiotherapy regimens. Bone MarrowTransplant. 1997; 19:1095–1101. [PubMed: 9193752]

33. Peters SO, Kittler EL, Ramshaw HS, et al. Murine marrow cells expanded in culture with IL-3,IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts. Exp Hematol. 1995; 23:461–469. [PubMed: 7536685]

34. Peters SO, Kittler EL, Ramshaw HS, et al. Ex vivo expansion of murine marrow cells withinterleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiatedhosts. Blood. 1996; 87:30–37. [PubMed: 8547656]

35. Tisdale JF, Hanazono Y, Sellers SE, et al. Ex vivo expansion of genetically marked rhesusperipheral blood progenitor cells results in diminished long-term repopulating ability. Blood.1998; 92:1131–1141. [PubMed: 9694700]

36. Traycoff CM, Cornetta K, Yoder MC, et al. Ex vivo expansion of murine hematopoietic progenitorcells generates classes of expanded cells possessing different levels of bone marrow repopulatingpotential. Exp Hematol. 1996; 24:299–306. [PubMed: 8641356]



NIH


NIH


NIH


37. Von Drygalski A, Alespeiti G, Ren L, et al. Murine bone marrow cells cultured ex vivo in thepresence of multiple cytokine combinations lose radioprotective and long-term engraftmentpotential. Stem Cells Dev. 2004; 13:101–111. [PubMed: 15068698]

38. Williams DA. Ex vivo expansion of hematopoietic stem and progenitor cells–robbing Peter to payPaul? Blood. 1993; 81:3169–3172. [PubMed: 8507858]

39. De A, Lewis XZ, Gambhir SS. Noninvasive imaging of lentiviral-mediated reporter geneexpression in living mice. Mol Ther. 2003; 7(suppl 5):681–691. [PubMed: 12718911]

40. Lin Y, Molter J, Lee Z, et al. Bioluminescence imaging of hematopoietic stem cell repopulation inmurine models. Methods Mol Biol. 2008; 430:295–306. [PubMed: 18370307]

41. Ishikawa F, Yasukawa M, Lyons B, et al. Development of functional human blood and immunesystems in NOD/SCID/IL2 receptor γchain (null) mice. Blood. 2005; 106:1565–1573. [PubMed:15920010]

42. King M, Pearson T, Shultz LD, et al. A new Hu-PBL model for the study of human isletalloreactivity based on NOD-scid mice bearing a targeted mutation in the IL-2 receptor gammachain gene. Clin Immunol. 2008; 126:303–314. [PubMed: 18096436]

43. Shultz LD, Pearson T, King M, et al. Humanized NOD/LtSz-scid IL2 receptor common gammachain knockout mice in diabetes research. Ann N Y Acad Sci. 2007; 1103:77–89. [PubMed:17332083]

44. Kelly PF, Vandergriff J, Nathwani A, et al. Highly efficient gene transfer into cord blood nonobesediabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particlespseudo-typed with the feline endogenous retrovirus (RD114) envelope protein. Blood. 2000;96:1206–1214. [PubMed: 10942359]

45. Giassi LJ, Pearson T, Schultz LD, et al. Expanded CD34+ human umbilical cord blood cellsgenerate multiple lymphohematopoietic lineages in NOD-scid IL2rγ (null) mice. Exp Biol Med.2008; 233:997–1012.

46. Liu Y, Hangoc G, Campbell TB, et al. Identification of parameters required for efficient lentiviralvector transduction and engraftment of human cord blood CD34(+) NOD/SCID-repopulating cells.Exp Hematol. 2008; 36:947–956. [PubMed: 18640494]

47. Lo Celso C, Fleming HE, Wu JW, et al. Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature. 2009; 457:92–96. [PubMed: 19052546]



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Figure 1.Lentiviral transduction efficiency of CD34-selected UCB cells pre- and postexpansion.CD34-selected UCB cells were transduced with a lentiviral construct expressing both GFPand luciferase and were then cultured for 72 hours to allow reporter gene expression. (A):After 72 hours in culture, approximately 10%–15% of cells were GFP+ as determined byflow cytometry. All cells at this stage were typically still CD34+. (B): After 7–10 days ofexpansion in culture, approximately one third of CD34-selected UCB cells remained CD34+

and 3%–10% of the total cell population remained CD34+GFP+. x-axis, GFP content; y-axis, CD34 content. Abbreviations: GFP, green fluorescent protein; UCB, umbilical cordblood.



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Figure 2.Bioluminescent imaging demonstrates repopulation of hematopoietic niche compartmentsby transduced human CD34-selected umbilical cord blood (UCB) cells. By post-transplantday 20, differences in the kinetics of engraftment between unexpanded (A) and expanded(B) CD34-selected UCB cells are negligible in the long bones, spine, sternum, and pelvis.Calvarial engraftment (C), however, remained strikingly absent among recipients ofexpanded CD34-selected UCB cells (D) until 3–6 months post-transplant. At 1 year post-transplant (E), the signal intensity of repopulated niche areas remained very strong among



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mice transplanted with either expanded CD34 UCB cells (shown) or unexpanded CD34UCB cells. Dorsal, ventral, and lateral views are shown.



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Figure 3.Ex vivo bioluminescent imaging identifies a localized niche of calvarial hematopoiesis inthe mouse. As demonstrated by incubation of intact cranial bones in D-luciferin substrateand ATP, calvarial hematopoiesis in the mouse appears to be restricted to a specific nichelocated at the junction of the parietal and interparietal fissures. The calvarial hematopoieticniche is significant in that it is the only site at which expanded and unexpanded CD34-selected umbilical cord blood cells displayed differential engraftment kinetics.



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Figure 4.Multilineage engraftment of mice repopulated with expanded CD34-selected umbilical cordblood cells is dominated early by a CD19+ lymphoid subset and late by a CD3+ lymphoidsubset. (A): At 5 months post-transplant, >80% of human cells in the peripheral blood areCD19+. CD14+ and CD3+ (including both CD4+ and CD8+) subsets can also be identified.(B): By 1 year post-transplant, >90% of human cells in the peripheral blood are CD3+.Though the ratio of CD4+ to CD8+ human cells varied by recipient, both subsets wereadequately represented. (C): Typical myeloid engraftment characteristics are shown: 5.9%of total peripheral blood mononuclear cells are positive for human CD33. x-axis, human



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CD33-allophycocyanin (APC); y-axis, human CD45-peridinin-chlorophyll-protein complex(PerCP).



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Figure 5.Secondary transplantation. (A):. At 1 year post-transplant of expanded CD34-selectedumbilical cord blood cells, tissues were harvested from engrafted mice and characterized byflow cytometry (table shows composition of typical mouse). Total bone marrow washarvested and 2–3 × 106 marrow cells/mouse were used for secondary transplantation ofirradiated recipients. (B): Kinetics of secondary transplantation on days 14, 20, and 55.Dorsal and ventral views are shown.



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Figure 6.Demonstration of human CD45+ cells in the marrow spaces of secondary transplants byimmunohistochemistry. (A): Spines with intact marrow spaces were decalcified by EDTAimmersion, paraffin embedded, and sliced into 8-µm sections for analysis byimmunohistochemistry. Staining with an anti-human CD45 monoclonal antibodydemonstrated the engraftment of human cells in hematopoietic spaces. Black arrows point toexamples of positively stained cells. (B): Isotype (negative) control demonstrates theabsence of background and specificity of the anti-CD45 antibody.



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Noninvasive Bioluminescent Imaging Demonstrates Long-Term Multilineage Engraftment of Ex Vivo-Expanded CD34-Selected Umbilical Cord Blood Cells

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