Title: Hematopoietic stem cell expansion facilitates multilineage engraftment in a nonhuman primate cord blood transplantation model
Abstract: The use of umbilical cord blood for allogeneic transplantation has increased dramatically over the past years. However, the limited number of cells available in a single cord blood unit remains a serious obstacle. Here, we wished to establish a nonhuman primate cord blood transplantation model that would allow us to test various hematopoietic stem cell expansion and gene therapy strategies. We implemented HOXB4-mediated expansion based on our previous experience with HOXB4 in autologous cells. Cord blood units were divided into two equal parts; half of the cells were transduced with a yellow fluorescent protein control vector and cryopreserved, and half were transduced with a HOXB4GFP vector, expanded, and cryopreserved. Both fractions of cells were transplanted into Macaca nemestrina subjects. We found that neutrophil recovery occurred within 19 days in all animals, and both neutrophil and platelet recovery were substantially accelerated compared to human single unit cord blood transplants. In addition, HOXB4-transduced and expanded cells resulted in superior engraftment of all hematopoietic lineages in all animals over nonexpanded controls. In conclusion, we have successfully established a nonhuman primate cord blood transplantation model and demonstrated that HOXB4 stimulates expansion and engraftment of repopulating cells. The availability of such a model has significant implications for developing and testing strategies to improve clinical cord blood transplantation, as it will allow comparison of different stem cell expansion methodologies within a single animal. Furthermore, it can be used in long-term follow-up studies to determine how specific expansion techniques affect engraftment of various hematopoietic lineages. The use of umbilical cord blood for allogeneic transplantation has increased dramatically over the past years. However, the limited number of cells available in a single cord blood unit remains a serious obstacle. Here, we wished to establish a nonhuman primate cord blood transplantation model that would allow us to test various hematopoietic stem cell expansion and gene therapy strategies. We implemented HOXB4-mediated expansion based on our previous experience with HOXB4 in autologous cells. Cord blood units were divided into two equal parts; half of the cells were transduced with a yellow fluorescent protein control vector and cryopreserved, and half were transduced with a HOXB4GFP vector, expanded, and cryopreserved. Both fractions of cells were transplanted into Macaca nemestrina subjects. We found that neutrophil recovery occurred within 19 days in all animals, and both neutrophil and platelet recovery were substantially accelerated compared to human single unit cord blood transplants. In addition, HOXB4-transduced and expanded cells resulted in superior engraftment of all hematopoietic lineages in all animals over nonexpanded controls. In conclusion, we have successfully established a nonhuman primate cord blood transplantation model and demonstrated that HOXB4 stimulates expansion and engraftment of repopulating cells. The availability of such a model has significant implications for developing and testing strategies to improve clinical cord blood transplantation, as it will allow comparison of different stem cell expansion methodologies within a single animal. Furthermore, it can be used in long-term follow-up studies to determine how specific expansion techniques affect engraftment of various hematopoietic lineages. Umbilical cord blood transplantation provides a treatment option for patients suffering from a wide variety of hematologic and nonhematologic malignancies. Rapid accessibility and a reduced risk of graft-vs-host disease are distinct advantages in the choice of umbilical cord blood as a source of stem cells for transplantation. However, cell dose is a major issue, especially in adult patients and large pediatric patients, as total nucleated cell dose and CD34 cell dose are well documented to be predictors of cord blood transplantation success [1Gluckman E. Rocha V. Arcese W. et al.Factors associated with outcomes of unrelated cord blood transplant: guidelines for donor choice.Exp Hematol. 2004; 32: 397-407Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar]. Therefore, research has focused on overcoming the cell dose barrier, including transplantation of two cord blood units and ex vivo expansion of cells before transplantation, with the goal of generating clinically meaningful cell doses. The first strategy, double cord blood unit transplantation, is currently used by most centers. Although this technique has helped to overcome cell dose limitations, there continues to be delayed engraftment and immune reconstitution and the potential for increased complications from graft-vs-host disease. In addition, it is typical to see a single unit emerge as the dominant source of long-term hematopoiesis [2Ballen K.K. Spitzer T.R. Yeap B.Y. et al.Double unrelated reduced-intensity umbilical cord blood transplantation in adults.Biol Blood Marrow Transplant. 2007; 13: 82-89Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar]. Furthermore, the cost of cord blood for transplantation ranges between $25,000 and $45,000 per unit; thus, the expense involved in a double cord blood unit transplantation can be considerable. Therefore, even with double unit transplantations, there is the need to achieve faster engraftment and potentially better immune reconstitution to minimize infectious complications. For these reasons, many investigators have looked into novel stem cell expansion strategies. Unfortunately, expansion strategies that focus solely on the use of cytokines have not shown significant expansion of repopulating cells. In addition, these techniques are associated with an increased rate of differentiation, which leads to a loss of primitive cells. In short, these studies have not translated into improved engraftment in clinical trials [3Jaroscak J. Goltry K. Smith A. et al.Augmentation of umbilical cord blood (UCB) transplantation with ex vivo-expanded UCB cells: results of a phase 1 trial using the AastromReplicell System.Blood. 2003; 101: 5061-5067Crossref PubMed Scopus (278) Google Scholar, 4Pecora A.L. Stiff P. Jennis A. et al.Prompt and durable engraftment in two older adult patients with high risk chronic myelogenous leukemia (CML) using ex vivo expanded and unmanipulated unrelated umbilical cord blood.Bone Marrow Transplant. 2000; 25: 797-799Crossref PubMed Scopus (108) Google Scholar, 5Shpall E.J. Quinones R. Giller R. et al.Transplantation of ex vivo expanded cord blood.Biol Blood Marrow Transplant. 2002; 8: 368-376Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar]. Promising new leads to achieve stem cell expansion have emerged from the discovery of self-renewal genes, such as HOXB4 [6Sauvageau G. Thorsteinsdottir U. Eaves C.J. et al.Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo.Genes Dev. 1995; 9: 1753-1765Crossref PubMed Scopus (503) Google Scholar, 7Antonchuk J. Sauvageau G. Humphries R.K. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo.Cell. 2002; 109: 39-45Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, 8Krosl J. Austin P. Beslu N. Kroon E. Humphries R.K. Sauvageau G. In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein.Nat Med. 2003; 9: 1428-1432Crossref PubMed Scopus (252) Google Scholar]. We have recently exploited the large animal model to demonstrate a differential effect of HOXB4 overexpression on short- and long-term repopulating cells in vivo. Using a competitive repopulation assay in a large animal model (Macaca nemestrina), we found that HOXB4 overexpression resulted in superior engraftment over non-HOXB4 controls [9Zhang X.-B. Beard B.C. Beebe K. Storer B. Humphries R.K. Kiem H.-P. Differential effects of HOXB4 on nonhuman primate short- and long-term repopulating cells.PLoS Med. 2006 May; 3 (Epub 2006 May 2): e173Crossref PubMed Scopus (50) Google Scholar]. Interestingly, HOXB4 appears to have the most dramatic effect on short-term repopulating cells, resulting in 56-fold higher short-term engraftment when compared with control-transduced cells. This offers promise in the field of cord blood transplantation; HOXB4 expansion of a portion of a graft may promote short-term engraftment and provide hematopoietic rescue while awaiting engraftment of long-term repopulating cells. Furthermore, we have also demonstrated a differential effect of HOXB4 on cells from different species [10Zhang X.-B. Schwartz J.L. Humphries R.K. Kiem H.-P. Effects of HOXB4 overexpression on ex vivo expansion and immortalization of hematopoietic cells from different species.Stem Cells. 2007; 25: 2074-2081Crossref PubMed Scopus (26) Google Scholar]. In addition, we have recently shown that a combination of HOXB4 and the Notch ligand Delta-1 synergize to yield enhanced generation of cord blood nonobese diabetic/severe combined immune-deficient repopulating cells with higher levels of engraftment of human CD45+, CD34+, CD3+, CD20+, and CD41+ cells compared to either factor used alone [11Watts K.L. Delaney C. Humphries R.K. Bernstein I. Kiem H.-P. Combination of HOXB4 and delta-1 ligand improves expansion of cord blood cells.Blood. 2010; 116: 5859-5866Crossref PubMed Scopus (28) Google Scholar]. Therefore, by combining other factors with early-acting genes like HOXB4, it is possible to encourage differentiation along lineages that are often under-represented in populations of hematopoietic stem cells (HSCs) expanded with HOXB4 alone. Development of an efficient means for expanding stem cells has broad applications, not limited simply to umbilical cord blood stem cell transplantation. For example, for a large percentage of the population, especially minorities, availability of appropriate donors for allogeneic HSC transplantation is limited [12Johansen K.A. Schneider J.F. McCaffree M.A. Woods G.L. Efforts of the United States’ National Marrow Donor Program and Registry to improve utilization and representation of minority donors (Review).Transfusion Med. 2008; 18: 250-259Crossref PubMed Scopus (41) Google Scholar]; thus, alternate sources of HSCs are under investigation, such as umbilical cord blood. Gene therapy is another example of a field that would benefit from these techniques; investigators could increase the number of gene-modified cells in gene therapy protocols and boost cell numbers for transplantation after nonmyeloablative conditioning. However, one of the most significant obstacles facing researchers studying ex vivo expansion techniques is the lack of an appropriate model in which to study HSC biology and behavior. The availability of a large animal model would circumvent this limitation and allow the efficient evaluation of these strategies with long-term follow-up. Thus, in the current study, our goal was to establish a nonhuman primate cord blood transplantation model. Subsequently, we wished to use this model to determine if nonhuman primate cord blood cells could be expanded to numbers large enough to be of clinical significance and could engraft in a fully myeloablated nonhuman primate recipient. In order to test HOXB4-mediated expansion of cord blood cells in a clinically relevant setting, we developed a nonhuman primate competitive repopulation model (Fig. 1). Approximately 1 week before the due date, a cesarean section is performed and cord blood cells are collected from the infant. After processing, the cells are split into two fractions; half are transduced with a control gammaretroviral yellow fluorescent protein (YFP) vector during a 3-day transduction and then frozen. The remaining cells are transduced with a HOXB4GFP vector and expanded for an additional 6 days (for a total of 9 days of ex vivo culture), before being cryopreserved. The use of two different markers, green fluorescent protein (GFP) and YFP, allows for a competitive repopulation approach; cells from peripheral blood or bone marrow can easily be analyzed by flow cytometry for the presence of GFP+ and YFP+ cells. Both fractions of cells are maintained in liquid nitrogen for at least 6 months until the infant reaches an appropriate weight. On the day of transplantation, cells are pooled and intravenously infused into the myeloablated recipient. All pig-tailed macaques (Macaca nemestrina) were housed at the University of Washington National Primate Research Center under conditions approved by the American Association for Accreditation of Laboratory Animal Care. Experimental protocols were approved by the Institutional Animal Care and Use Committee. For 3 days before transplantation and continuing through day 56, animals received oral tacrolimus (FK-506) at a dose necessary to maintain serum trough levels between 10 and 15 ng/mL. During the 2 days before transplantation, animals were conditioned with fractionated, myeloablative total body irradiation of 1100 cGy [13Watts K.L. Beard B.C. Wood B.L. Kiem H.P. Myeloablative irradiation in non-human primates.J Med Primatol. 2009; 38: 425-432Crossref PubMed Scopus (3) Google Scholar] from a 6 MV x-ray beam of a single-source linear accelerator located at the Fred Hutchinson Cancer Research Center South Lake Union Facility. The animals underwent a training process to sit calmly in a specially modified cage. The cage provided clear access for the irradiation, while gently restricting excess movement by limiting space. The dose was administered at a rate of 7 cGy/min delivered as a midline tissue dose. The instrument was calibrated weekly to maintain accuracy. Beginning on the day of cell infusion, granulocyte colony-stimulating factor (G-CSF) was administered daily until the animals began to engraft, defined as absolute neutrophil count (ANC) >500/μL for 3 consecutive days. All animals received standard supportive care, including antibiotics, electrolytes, fluids, and transfusions. Daily complete blood counts were used to determine hematopoietic recovery. A total of three macaques were transplanted and followed for this study. Red cells from macaque umbilical cord blood were lysed in ammonium chloride red cell lysis buffer. Nucleated cells were incubated for 20 minutes with the 12.8 IgM anti-CD34 antibody, washed, and incubated for another 20 minutes with MACS IgM microbeads (Miltenyi Biotec, Auburn, CA, USA). CD34+ cells were enriched via magnetic column separation. Overall, samples ranged in purity from 80% to 99% CD34+ by flow cytometry. Transductions were carried out on fibronectin-coated, non−tissue culture-treated plates. Cells were prestimulated for 48 hours. After 48 hours, cells were exposed to virus-containing media for two 4-hour transductions (one exposure per day for 2 consecutive days). Cells were transduced at an multiplicity of infection of 0.3 (previously determined to cause minimal toxicity). The generation of Phoenix GALV-pseudotyped MSCV-HOXB4-ires-GFP and MSCV-ires-YFP viral vectors has been described previously [9Zhang X.-B. Beard B.C. Beebe K. Storer B. Humphries R.K. Kiem H.-P. Differential effects of HOXB4 on nonhuman primate short- and long-term repopulating cells.PLoS Med. 2006 May; 3 (Epub 2006 May 2): e173Crossref PubMed Scopus (50) Google Scholar, 14Antonchuk J. Sauvageau G. Humphries R.K. HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation.Exp Hematol. 2001; 29: 1125-1134Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 15Pineault N. Abramovich C. Ohta H. Humphries R.K. Differential and common leukemogenic potentials of multiple NUP98-Hox fusion proteins alone or with Meis1.Mol Cell Biol. 2004; 24: 1907-1917Crossref PubMed Scopus (79) Google Scholar]. Virus titers were assayed on HT1080 cells, and titers were obtained in the range of 1 × 105 to 2 × 105 IU/mL. Vector supernatant was filtered through a 0.45-μm filter and frozen at −80°C until used for transduction. Cord blood cells were cultured in Iscove’s modified Dulbecco’s medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The following growth factors were added at a concentration of 100 ng/mL: interleukin (IL)-3, IL-6, thrombopoietin, Flt3 ligand, stem cell factor, and G-CSF. Cultures were split as necessary to maintain cell concentrations in the range of 1 × 105 to 5 × 105 cells/mL. Flow cytometric data were collected using a Canto I (Becton Dickinson, San Jose, CA, USA) and analyzed using FlowJo software. At least 10,000 events were collected for each sample. Samples were analyzed for expression of the GFP marker (as an indicator of HOXB4 expression) and the YFP marker, as well as for CD3, CD4, CD8, CD13, CD14, CD20, and CD34. Nontransduced cells were used as a control for the gating of HOXB4GFP+ cells and YFP+ cells, and isotype control antibodies were used as a control for gating of positive populations among antibody-labeled cells. All antibodies were purchased from Becton Dickinson. Colony-forming unit assays were carried out in MethoCult H4230 methylcellulose media (Stem Cell Technologies, Vancouver, Canada) supplemented with 100 ng/mL erythropoietin, IL-3, IL-6, thrombopoietin, stem cell factor, G-CSF, and granulocyte-macrophage CSF. Plates were incubated at 37°C for 12 to 14 days; after this period of time, colonies of >50 cells were enumerated. At periodic intervals after transplantation in L09025, HOXB4GFP+ bone marrow cells were purified by fluorescence-activated cell sorting and analyzed by linear amplification−mediated polymerase chain reaction (PCR). The detailed protocol for linear amplification−mediated PCR has been reported previously [16Beard B.C. Keyser K.A. Trobridge G.D. et al.Unique integration profiles in a canine model of long-term repopulating cells transduced with gammaretrovirus, lentivirus, and foamy virus.Hum Gene Ther. 2007; 18: 423-434Crossref PubMed Scopus (69) Google Scholar]. In brief, PCR products were cloned and sequenced. The sequences with legitimate linker and long terminal repeat (LTR) were subjected to BLAST-like alignment tool (BLAT) analysis using the University of California, Santa Cruz Genome Browser Web site. The rhesus genome from the January 2006 assembly was used for analysis. For analysis of retroviral integration sites in T09214, we used an improved analytical technique involving nebulization-mediated (NM) PCR. Briefly, 300 ng to 3 μg DNA were nebulized with pressurized nitrogen for 60 seconds. Fragmented DNA was isolated and polished, and modified linkers were ligated following standard procedures (454/Roche-GS 20 DNA Library Preparation Kit, Branford, CT, USA). To amplify the vector-genome junction, double-stranded DNA was amplified in sequential, nested exponential PCR. Successful integration sites amplified by NM-PCR were gel purified to isolate DNA fragments ∼800 through ∼1500 bp in length. Gel purified samples were shipped to the University of Illinois at Urbana-Champaign, quality control checked, sequenced on the 454/Roche Titanium system, and FASTA format sequence reads were deposited on a secure server for downstream processing. For NM-PCR−based amplified vector LTR-chromosome junctions, DNA sequences were processed as described previously [16Beard B.C. Keyser K.A. Trobridge G.D. et al.Unique integration profiles in a canine model of long-term repopulating cells transduced with gammaretrovirus, lentivirus, and foamy virus.Hum Gene Ther. 2007; 18: 423-434Crossref PubMed Scopus (69) Google Scholar, 17Beard B.C. Dickerson D. Beebe K. et al.Comparison of HIV-derived lentiviral and MLV-based gammaretroviral vector integration sites in primate repopulating cells.Mol Ther. 2007; 15: 1356-1365Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 18Trobridge G.D. Miller D.G. Jacobs M.A. et al.Foamy virus vector integration sites in normal human cells.PNAS. 2006; 103: 1498-1503Crossref PubMed Scopus (206) Google Scholar]. The LTR proximal genomic sequences were aligned to the rhesus genome using a stand-alone version of BLAT [19Kent W.J. BLAT—the BLAST-like alignment tool.Genome Res. 2002; 12: 656-664Crossref PubMed Scopus (6117) Google Scholar] that generated a basic local alignment search tool alignment score. Rhesus genome alignments were converted to the human genome position and PERL programs were used to compare localized integration sites to various chromosomal features by using tables available from the University of California at Santa Cruz database as described previously [20Karolchik D. Baertsch R. Diekhans M. et al.The UCSC Genome Browser Database.Nucleic Acids Res. 2003; 31: 51-54Crossref PubMed Scopus (1200) Google Scholar]. We first wished to establish a cord blood harvest procedure. During the course of our study, we implemented several improvements to our cord blood collection procedure, including heparin coating of the catheters used to collect blood, use of heparinized saline to flush the vein, massage of the placenta to collect additional cells, as well as other techniques that have been shown to aid in maximizing cell collection during clinical cord blood harvest [21Bornstein R. Flores A.I. Montalban M.A. del Rey M.J. de la S.J. Gilsanz F. A modified cord blood collection method achieves sufficient cell levels for transplantation in most adult patients.Stem Cells. 2005; 23: 324-334Crossref PubMed Scopus (55) Google Scholar]. By utilizing these practices, we were able to increase cell numbers collected by nearly threefold (Fig. 2); in addition, successful transduction and expansion of these cells allowed engraftment in our nonhuman primate recipients. Next, we wished to determine whether M nemestrina cord blood cells could be efficiently transduced and expanded. As shown in Table 1, transduction efficiencies ranged from 35% to 46% for the YFP control arm, and from 42% to 46% for the HOXB4GFP arm. During the 3-day transduction period, YFP cells expanded between 3- and 19-fold. HOXB4GFP cells were expanded for an additional 6 days after transduction, for a total of 9 days in culture. These cells expanded between 78- and 204-fold. Transplantation doses ranged from 1.0 × 105 to 7.8 × 105 YFP+CD34+ cells per kilogram for the control arm, and 2.6 × 106 to 3.4 × 106 HOXB4GFP+CD34+ cells per kilogram for the expanded arm. (The difference in YFP+ and GFP+ cell doses is due to the fact that the GFP+ cells were expanded for 6 additional days, thus accounting for an increase in overall cell numbers; however, it is important to reiterate that both experimental arms (GFP+ and YFP+) consisted of identical cell numbers prior to expansion, and thus the HOXB4GFP+ dose consisted of the progeny of an equivalent number of HSCs as the YFP+ dose. Prefreeze and post-thaw colony forming unit plating showed that there was no significant loss of repopulating cell viability during cryopreservation (data not shown). These data demonstrate that cord blood cells from macaques can be transduced and expanded to clinically relevant doses.Table 1Pre-transplantation and post-transplantation data from L09025, K09175, and T09214∗This table provides a summary of relevant information, including age and weight at time of transplantation, transduction efficiencies, fold expansion of YFP and GFP experimental arms, transplantation dose, time to neutrophil recovery, and post-transplantation survival.L09025K09175T09214Age (mos)678Weight (kg)1.21.01.3SexMaleFemaleFemaleTransduction efficiency (%)46 (YFP)35 (YFP)4 (YFP)46 (GFP)45 (GFP)4 (GFP)Fold-expansion YFP arm (over 3 days)31319Fold-expansion GFP arm (over 9 days)78155204Transplantation dose1.05 YFP+CD34+/kg1.85 YFP+CD34+/kg7.85 YFP+CD34+/kg2.66 GFP+CD34+/kg3.36 GFP+CD34+/kg3.46 GFP+CD34+/kgTime to ANC >500/μL (days)19710Survival post-transplantation (days)8245270+OutcomeTTP-like syndromeViral pneumoniaAliveTTP = thrombotic thrombocytopenic purpura.∗ This table provides a summary of relevant information, including age and weight at time of transplantation, transduction efficiencies, fold expansion of YFP and GFP experimental arms, transplantation dose, time to neutrophil recovery, and post-transplantation survival. Open table in a new tab TTP = thrombotic thrombocytopenic purpura. After confirming that we were able to successfully expand cord blood cells ex vivo, we wished to study the hematopoietic recovery kinetics of macaques transplanted with genetically modified cord blood cells. All animals attained neutrophil recovery within the first 3 weeks after transplantation. An ANC ≥500 cells per μL was reached by L09025 at day 19, by K09175 at day 7, and by T09214 at day 10. G-CSF was discontinued at day 23 for L09025, day 18 for K09175, and day 15 for T09214. ANC levels remained >500 cells per μL for the remainder of the study for each animal, although occasional drops in neutrophil counts necessitated an additional dose of G-CSF to boost neutrophil production. The effects of these additional doses can be seen in Figure 3 (top panel) as points when the ANC rises. All animals demonstrated a decline in platelet counts after total body irradiation, and remained thrombocytopenic for at least 2 to 3 weeks after transplantation. However, Figure 3 (lower panel) shows that T09214 and L09025 began to resume normal thrombopoiesis by the end of the first month post-transplantation. K09175 continued to experience thrombocytopenia, and required frequent transfusions during the course of her time in the study. Overall, these data demonstrate that we are able to achieve recovery of neutrophils and platelets in a timely manner after myeloablative conditioning. The inclusion of the GFP or YFP reporter gene in our retroviral vectors allowed us to track the contribution of gene-marked cells in the animals. All animals demonstrated a similar pattern in engraftment of gene-modified granulocytes. This trend is illustrated in Figure 4. In each case, gene marking in the HOXB4GFP arm climbed rapidly during the first 2 weeks, reached a peak around 2 to 3 weeks post-transplantation, and then began to decline until eventually stabilizing after approximately the first month. HOXB4GFP marking levels in L09025 and K09175 stabilized around 10%; in T09214, HOXB4GFP marking levels stabilized around 20%. Gene marking in the YFP control arm was detectable in all three animals, but at low levels. In both L09025 and T09214, only about 0.5% to 1.5% of granulocytes were YFP+ by flow cytometric analysis. In K09175, this level was significantly higher and remained relatively consistent at about 5%. Thus, we were able to show improved engraftment kinetics and durability of HOXB4-transduced and expanded cells compared to control-transduced cells. To determine the percentages of, and gene marking within, various subsets contributing to hematopoietic recovery, we performed subset stain assays. Full, comprehensive subset stains were performed on L09025 (approximately 3 months post-transplantation) and T09214 (1, 3, 6, and 9 months post-transplantation). Peripheral blood cells were stained for CD3, CD13, CD14, and CD20, and analyzed by flow cytometry to determine the contributions of different subsets to overall hematopoiesis (Fig. 5) and the contributions of GFP+/YFP+ cells to different subsets (Fig. 6). One month after transplantation, T09214 had lower percentages of CD3, CD13, and CD20 cells compared to a nontransplanted control animal. However, by 3 and 9 months post-transplantation, T09214 had regained normal hematopoiesis (Fig. 5), thus showing that HOXB4-mediated expansion did not lead to any long-term skewing of hematopoiesis.Figure 6Percent of GFP+ and YFP+ cells among hematopoietic lineages at various time points post-transplantation. Over time, the percentage of GFP+ cells is dropping among all lineages, showing that HOXB4GFP+ cells are conferred with a short-term, but not long-term, repopulating advantage. At each time point, GFP+ cells outnumber YFP+ cells in all four lineages; this effect is most dramatic among myeloid populations. FACS = fluorescence-activated cell sorting.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The percentage of gene marking within individual subsets is represented in Figure 6. Here, we show that, at each time point, the percentage of GFP+ cells is higher than the percentage of YFP+ cells within each subset. However, this effect is much more exaggerated among the myeloid lineages (a 10−30 percentage point difference) than among the lymphoid lineages (typically a 1−3 percentage point difference). Thus, we show that the HOXB4-transduced and expanded cells are more prevalent than control-transduced cells among multiple lineages, with the most pronounced effect on myeloid populations. Retroviral integration site analysis was performed on L09025 at day 82 post-transplantation using a linear amplification−mediated PCR-based approach. We used this technique to analyze three separate populations of cells (GFP-sorted cells from the bone marrow, lymphocyte-like cells from the peripheral blood, and granulocyte-like cells from the peripheral blood). The presence of multiple bands on the acrylamide gel shown in Figure 7A indicates a polyclonal population. Furthermore, the table in Figure 7B lists several of the most frequently identified integration sites, and it is noted that several of these show up among more than one of the three populations studied, thus indicating that true hematopoietic repopulating cells have been transduced and expanded. When analyzing retroviral integration sites in T09214, we modified our approach and opted to use an improved technique involving NM-PC