Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary,secondary, and tertiary multilineage repopulation in NOD/SCID mice. Nonobese diabetic/severe combined immunodeficient

The ability of advanced-generation lentiviral vectors to transfer the green fluorescent protein (GFP) gene into human hematopoietic stem cells (HSCs) was studied in culture conditions that allowed expansion of transplantable human HSCs. Following 96 hours' exposure to flt3/flk2 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), and interleukin-6 (IL-6) and overnight incubation with vector particles, cord blood (CB) CD34(+) cells were further cultured for up to 4 weeks. CD34(+) cell expansion was similar for both transduced and control cells. Transduction efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) was assessed by transplants into NOD/SCID mice. Mice that received transplants of transduced week 1 and week 4 expanded cells showed higher levels of human engraftment than mice receiving transplants of transduced nonexpanded cells (with transplants of 1 x 10(5) CD34(+) cells, the percentages of CD45(+) cells were 20.5 +/- 4.5 [week 1, expanded] and 27.2 +/- 8.2 [week 4, expanded] vs 11.7 +/- 2.5 [nonexpanded]; n = 5). The GFP(+)/CD45(+) cell fraction was similar in all cases (12.5% +/- 2.9% and 12.2% +/- 2.7% vs 12.7% +/- 2.1%). Engraftment was multilineage, with GFP(+)/lineage(+) cells. Clonality analysis performed on the bone marrow of mice receiving transduced and week 4 expanded cells suggested that more than one integrant likely contributed to the engraftment of GFP-expressing cells. Serial transplantations were performed with transduced week 4 expanded CB cells. Secondary engraftment levels were 10.7% +/- 4.3% (n = 12); 19.7% +/- 6.2% of human cells were GFP(+). In tertiary transplants the percentage of CD45(+) cells was lower (4.3% +/- 1.7%; n = 10); 14.8% +/- 5.9% of human cells were GFP(+), and human engraftment was multilineage. These results show that lentiviral vectors efficiently transduce HSCs, which can undergo expansion and maintain proliferation and self-renewal ability.     

Identification of parameters required for efficient lentiviral vector transduction and engraftment of human cord blood CD34(+) NOD/SCID-repopulating cells

OBJECTIVE: Human cord blood (CB) is a potential source of hematopoietic stem cells (HSC) for gene therapy to treat patients with hematopoietic disorders. However, limited numbers of CB CD34(+) cells, low transduction efficiency with lentiviral vectors (LVs), and low engraftment efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRC), a measure of HSC, are blocks to this procedure. To optimize culture and transduction conditions, we compared various lengths of time for prestimulation before transduction, transduction duration, and posttransduction cell culture. MATERIALS AND METHODS: We used a LV to transduce human CB CD34(+) cells followed by engraftment into NOD/SCID mice. We evaluated the effects of prestimulation and transduction time and optimized ex vivo cell culture duration before transplantation. RESULTS: We were able to achieve up to 40% transduction efficiency and up to 50% engraftment efficiency of SRC in CB CD34(+) cells when CB CD34(+) cells were either not prestimulated or prestimulated in 1% fetal bovine serum medium for 1 hour, followed by 5 hours transduction and 3 days culture in a cocktail of growth factors after transduction. No apparent functional changes of CB CD34(+) cells were noted under these conditions. CONCLUSION: This gene-transduction/cell-expansion protocol is the first systematic study to optimize prestimulation time, transduction time, and, very importantly, ex vivo culture time after transduction, and may be of use for LV gene transduction in a gene therapy setting.     

Lentiviral-mediated gene transfer into haematopoietic stem cells

OBJECTIVES: Lentiviral vectors can transduce nondividing cells. As most haematopoietic stem cells (HSCs) are nondividing in vivo, lentiviral vectors are promising viral vectors to transfer genes into HSCs. DESIGN AND SETTING: We have used HIV-1 based lentiviral vectors containing the green fluorescent protein (GFP) gene to transduce umbilical cord blood CD34+ and CD34+/CD38- cells prior to transplantation into NOD/SCID mice. RESULTS: High level engraftment of human cells was obtained and transgene expression was seen in both myeloid and lymphoid lineages. Bone marrow from the primary transplant recipients mice was transplanted into secondary recipients. GFP expression was seen in both lymphoid and myeloid cells in the secondary recipients 6 weeks posttransplantation. Human haematopoietic progenitor colonies were grown from both primary and secondary recipients. Over 50% of the haematopoietic colonies in these recipients were positive for the GFP transgene by PCR. Following inverse PCR, amplified fragments were sequenced and integration of the vector into human genomic DNA was demonstrated. Several vectors containing different internal promoters were tested in NOD/SCID mice that had been transplanted with transduced CD34+ and CD34+/CD38- cells. The elongation factor-1alpha (EF-1alpha) promoter gave the highest level of expression, both in the myeloid and lymphoid progeny of the engrafting cells. CONCLUSIONS: These data collectively indicate that candidate human HSCs can be efficiently transduced with lentiviral vectors and that the transgene is highly expressed in their progeny cells.     

Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis

Efficient vector transduction of hematopoietic stem cells is a requirement for successful gene therapy of hematologic disorders. We asked whether human umbilical cord blood CD34(+)CD38(lo) nonobese diabetic/severe combined immunodeficiency (NOD/SCID) repopulating cells (SRCs) could be efficiently transduced using lentiviral vectors, with a particular focus on the average number of vector copies integrating into these primitive progenitor cells. Mouse bone marrow was analyzed by fluorescence-activated cell-sorter scanner and by semiquantitative polymerase chain reaction (PCR) to determine the transduction efficiency into SRCs. Lentiviral vector transduction resulted in an average of 22% (range, 3%-90%) of the human cells expressing green fluorescent protein (GFP), however, multiple vector copies were present in human hematopoietic cells, with an average of 5.6 +/- 3.3 (n = 12) copies per transduced cell. To confirm the ability of lentiviral vectors to integrate multiple vector copies into SRCs, linear amplification mediated (LAM)-PCR was used to analyze the integration site profile of a selected mouse showing low-level engraftment and virtually all human cells expressing GFP. Individually picked granulocyte macrophage colony-forming unit colonies derived from the bone marrow of this mouse were analyzed and shown to have the same 5 vector integrants within each colony. Interestingly, one integration site of the 5 that were sequenced in this mouse was located in a known tumor-suppressor gene, BRCA1. Therefore, these findings demonstrate the ability of lentiviral vectors to transduce multiple copies into a subset of NOD/SCID repopulating cells. While this is efficient in terms of transduction and transgene expression, it may increase the risk of insertional mutagenesis.     

The modified International Prognostic Index can predict the outcome of localized primary intestinal lymphoma of both extranodal marginal zone B-cell and diffuse large B-cell histologies

This study aimed to assess the potential of human cord blood (CB) cells to engraft in the xenogenic non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model after in vitro expansion culture. We also studied the quality of human haemopoiesis arising from the transplantation of fresh or expanded cells in this model. Cord blood CD34(+) cells were cultured for 3, 7 or 10 d with stem cell factor, Flt3, thrombopoietin, interleukin 3 (IL-3), IL-6 and granulocyte colony-stimulating factor, all at 10 ng/ml in serum-replete conditions. Transplantation of mice with fresh CB containing 3 x 10(4) CD34(+) cells and 1-2 SCID repopulating cells (SRC) resulted in a median of 7.4% (0.4%-76.8%) human engraftment. When mice received the expanded product of 1-2 SRC, the ability to repopulate NOD/SCID mice was maintained even after 10 d of in vitro culture. Serial dilution of the expanded cells suggested that in vitro expansion had increased SRC numbers two- to fourfold. Expanded SRC produced long-term culture-initiating cells, clonogenic cells and CD34(+) cells in the same proportions as fresh cells after successful engraftment. Therefore, expanded SRC were able to differentiate in the same way as fresh SRC. There was a trend towards lower levels of engraftment when d 7 cultured cells were transplanted (median engraftment 0.8%, range 0.0-24.0%) compared with 1-2 fresh SRC. Our data suggest that this is owing to reduced proliferation of cultured cells in vivo. By utilizing limiting numbers of CB SRC, we confirmed that the engraftment potential of SRC in the NOD/SCID model was preserved after in vitro expansion. Furthermore, dilution experiments strongly suggest two- to fourfold expansion of SRC in vitro. These studies are relevant for developing clinical stem cell expansion strategies.     

Stem cell factor improves SCID-repopulating activity of human umbilical cord blood-derived hematopoietic stem/progenitor cells in xenotransplanted NOD/SCID mouse model

Poor in vivo homing capacity of hematopoietic stem/progenitor cells (HS/PCs) from umbilical cord blood (UCB) can be reversed by short-term ex vivo manipulation with recombinant human stem cell factor (rHuSCF). This study was designed to evaluate the effect of ex vivo manipulation of UCB-derived HS/PCs with rHuSCF on human cell engraftment rates in xenotransplanted NOD/SCID mouse model. The human cell engraftment rates in xenotransplanted primary and secondary NOD/SCID mice were characterized using four-color flow cytometric analysis and progenitor assay. Grafts of rHuSCF-treated UCB CD34(+) cells resulted in significantly higher levels of human cell engraftment than that of nontreated ones in both xenotransplanted primary and secondary NOD/SCID recipients. Fresh UCB CD34(+) cells did not express either of the matrix metalloproteinase (MMP) family members MMP-2 or MMP-9. rHuSCF-treated UCB CD34(+) cells expressed significant levels of MMP-2 and MMP-9. Pretreatment of UCB CD34(+) cells with the specific MMP inhibitor completely blocked human cell engraftment in xenotransplanted NOD/SCID recipients. Our results indicate that ex vivo manipulation of human HS/PCs with rHuSCF might provide an optimal approach to develop more effective stem cell-based therapies in situations where engraftment is delayed due to limiting HS/PCs number, for example, UCB transplantation.     

Concentrated RD114-pseudotyped MFGS-gp91phox vector achieves high levels of functional correction of the chronic granulomatous disease oxidase defect in NOD/SCID/beta -microglobulin-/- repopulating mobilized human peripheral blood CD34+ cells

In previous studies amphotropic MFGS-gp91phox (murine onco-retrovirus vector) was used in a clinical trial of X-linked chronic granulomatous disease (X-CGD) gene therapy to achieve transient correction of oxidase activity in 0.1% of neutrophils. We later showed that transduced CD34+ peripheral blood stem cells (CD34+ PBSCs) from this trial transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in correction of only 2.5% of human neutrophils. However, higher rates of transduction into stem cells are required. In the current study we demonstrate that the same vector (MFGS-gp91phox) pseudo-typed with RD114 envelope in a 4-day culture/transduction regimen results in a 7-fold increase in correction of NOD/SCID mouse repopulating X-CGD CD34+ PBSCs (14%-22% corrected human neutrophils; human cell engraftment 13%-67%). This increase may result from high expression of receptor for RD114 that we demonstrate on CD34+CD38- stem cells. Using RD114-MFGS encoding cyan fluorescent protein to allow similar studies of normal CD34+ PBSCs, we show that progressively higher levels of gene marking of human neutrophils (67%-77%) can be achieved by prolongation of culture/transduction to 6 days, but with lower rates of human cell engraftment. Our data demonstrate the highest reported level of functional correction of any inherited metabolic disorder in human cells in vivo with the NOD/SCID mouse system using onco-retrovirus vector.     

Behavior of CD34+ cells isolated from patients with polycythemia vera in NOD/SCID mice

OBJECTIVE: We investigated if polycythemia vera (PV) peripheral blood (PB) CD34+ cells contain cells capable of engrafting nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and if the JAK2V617F mutational burden of these cells alters their behavior in NOD/SCID mice. MATERIALS AND METHODS: CD34+ cells isolated from patients with PV, idiopathic myelofibrosis (IM), or granulocyte colony-stimulating factor-mobilized normal donors were transplanted into sublethally irradiated NOD/SCID mice. Cells engrafted into the NOD/SCID mice were analyzed flow cytometrically using lineage-specific antibodies. Genomic DNA was extracted from granulocytes, CD34+ cells, and sorted human CD45(+) cells purified from the bone marrow cells of these mice to examine their JAK2V617F mutational burdens. RESULTS: Multilineage human cell engraftment was observed in mice transplanted with CD34+ cells from mobilized normal volunteers, IM patients and PV patients with high JAK2V617F burden, but not in mice receiving grafts from PV patients with low JAK2V617F burden. The differentiation program of engrafting PV CD34+ cells with high JAK2V617F burden was remarkably different than that of IM CD34+ cells. The JAK2V617F allele frequency in the human CD45+ cells isolated from the mice receiving CD34+ cells was lower than that observed in the CD34+ cell grafts, indicating the persistence of a JAK2V617F negative compartment of stem cells. CONCLUSION: We conclude that PB CD34+ cells from PV patients with high JAK2V617F burden and patients with IM contain NOD/SCID repopulating cells, and that differentiation program of IM and PV CD34+ cells are dramatically different.     

Human CD34+ cell preparations contain over 100-fold greater NOD/SCID mouse engrafting capacity than do CD34- cell preparations.

OBJECTIVE: The CD34 cell surface marker is used widely for stem/progenitor cell isolation. Since several recent studies reported that CD34(-) cells also have in vivo engrafting capacity, we quantitatively compared the engraftment potential of CD34(+) vs CD34(-) cell preparations from normal human placental/umbilical cord blood (CB), bone marrow (BM), and mobilized peripheral blood (PBSC) specimens, using the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. METHODS: CD34(+) and CD34(-) cell preparations were purified by four different approaches in 14 individual experiments involving 293 transplanted NOD/SCID mice. In most experiments, CD34(+) cells were depleted twice (CD34(=)) in order to obtain efficient depletion of CD34(+) cells from the CD34(-) cell preparations. RESULTS: Dose-dependent levels of human hematopoietic cells were observed after transplantation of CD34(+) cell preparations. To rigorously assess the complementary CD34(-) cell preparations, cell doses 10- to 1000-fold higher than the minimum dose of the CD34(+) cell preparations necessary for engraftment were transplanted. Nevertheless, of 125 NOD/SCID mice transplanted with CD34(-) cell preparations purified from the same starting cells, only six mice had detectable human hematopoiesis, by flow cytometric or PCR assay. CONCLUSIONS: CD34(-) cells provide only a minor contribution to hematopoietic engraftment in this in vivo model system, as compared to CD34(+) cells from the same samples of noncultured human cells. Hematopoiesis derived from actual CD34(-) cells is difficult to distinguish from that due to CD34(+) cells potentially contaminating the preparations.     

In utero transplantation of human fetal haemopoietic cells in NOD/SCID mice

We have previously demonstrated that high levels of allogeneic, donor-derived mouse haemopoietic progenitor cells engraft following in utero transplantation in NOD/SCID mice. To evaluate whether the fetal NOD/SCID haemopoietic microenvironment supports the growth and development of human fetal haemopoietic progenitor cells, we injected fetal liver mononuclear cells (FL) or fetal bone marrow (FBM) derived CD34⁺ cells into NOD/SCID mice on day 13/14 of gestation. At 8 weeks of age 12% of FBM recipients and 10% of FL recipients were found to have been successfully engrafted with CD45⁺ human cells. CD45⁺ cells were present in the BM of all chimaeric animals; 5/6 recipients showed engraftment of the spleen, and 4/6 recipients had circulating human cells in the peripheral blood (PB). The highest levels of donor cells were found in the BM, with up to 15% of the nucleated cells expressing human specific antigens. Multilineage human haemopoietic engraftment, including B cells (CD19), myelomonocytic cells (CD13/33) and haemopoietic progenitor cells (CD34), was detected in the BM of chimaeric mice. In contrast, no human CD3⁺ cells were detected in any of the tissues evaluated. When the absolute number of engrafted human cells in the PB, BM and spleens of chimaeric mice was determined, a mean 16-fold expansion of human donor cells was observed. Although multilineage engraftment occurs in these fetal recipients, both the frequency and the levels of engraftment are lower than those previously reported when human cells are transplanted into adult NOD/SCID recipients.     

Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(+) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells.

Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34(+) cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34(+) and CD34(+)38(-) subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34(+) CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34(+) CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34(+) cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] +/- stem cell factor [SCF] +/- interleukin-6 [IL-6]). Mice that had been injected with >/=20,000 fresh or cryopreserved uncultured CD34(+) cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34(+) CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34(-) cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34(+) CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.     

Engraftment potential into NOD/SCID mice of CD34+ cells derived from human fetal liver as compared to fetal bone marrow

BACKGROUND AND OBJECTIVES: We hypothesized that qualitative or quantitative differences in hematopoietic stem cells from fetal liver (FL) and fetal bone marrow (FBM) may be the cause of their organ specificity. DESIGN AND METHODS: To analyze possible differences in vivo, we compared the engraftment potential of equal numbers of CD34+ cells isolated from human FL or FBM into immunodeficient NOD/SCID mice. RESULTS: Mice showing engraftment following transplantation of CD34+ cells from FL demonstrated 14% (range 2-76%) CD45+ cells of human origin in the bone marrow compared to significantly lower levels of engraftment (4%, range 2-20%, p < 0.04) of FBM CD34+ cells. Likewise, the percentage of CD34+ CD38- cells in FBM was 4 times lower than the percentage in FL (1.4+/-0.9% and 5.6+/-0.7%, respectively). Similar organ distribution of engrafted human cells was found. Subset analysis of human cells in bone marrow of engrafted mice revealed identical distribution of the lymphoid, myeloid and erythroid lineages after transplantation of CD34+ cells from FL or FBM. INTERPRETATION AND CONCLUSIONS: The FL CD34+ cells showed a four-fold higher content of the CD34+ CD38- subset coinciding with a four-fold higher engraftment of CD34+ cells into NOD/SCID mice. Since the organ distribution and differentiation potential of the cells engrafted were similar, we concluded that CD34+ hematopoietic cells derived from FL and FBM have quantitatively different, but qualitatively the same potential for engraftment into NOD/SCID mice.     

Human multidrug resistance-1 gene transfer to long-term repopulating human mobilized peripheral blood progenitor cells

Mobilized peripheral blood progenitor cells (PBPC) are an attractive target for the retrovirus-mediated transfer of cytostatic drug resistance genes. We analyzed NOD/SCID mouse repopulating CD34+ PBPC from cancer patients following retroviral Transwell transduction in various cytokine combinations with the FMEV-based (Friend-mink cell focus forming/murine embryonic stem cell virus) hybrid vector SF-MDR carrying the human multidrug resistance-1 (MDR1) gene. Five to 10 weeks following transplantation of 2.0 x 10(6) CD34+ PBPC into NOD/SCID mice we observed medium to high levels of human cell engraftment with up to 33%. The extent of vector-marked human cells was assessed by a quantitative real-time polymerase chain reaction (PCR). SF-MDR gene transfer into long-term in vivo repopulating human hematopoietic cells was optimal in the presence of either IL-3/IL-6/SCF/FL or FL/TPO/SCF resulting in three-fold (12.4% +/- 1.7%) or four-fold (16.5% +/- 6.8%) higher average proportions of gene-marked human cells in NOD/SCID mice as compared to IL-3 alone (P < 0.01). In conclusion, we could optimize the engraftment capacity and the retroviral gene transfer to CD34+ PBPC using cocktails of early acting cytokines in combination with the recombinant fibronectin fragment CH-296. Our data suggest that the NOD/SCID model provides a valid assay to estimate the gene transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation settings.     

Evaluation of different protocols for gene transfer into non-obese diabetes/severe combined immunodeficiency disease mouse repopulating cells

Purpose Although gene transfer with retroviral vectors has shown distinct clinical success in defined settings, efficient genetic manipulation of hematopoietic progenitor cells remains a challenge. To address this issue we have evaluated different transduction protocols and retroviral constructs in the non-obese diabetes (NOD)/severe combined immunodeficiency disease (SCID) xenograft model. Methods An extended transduction protocol requiring 144 h of in vitro manipulation was compared to a more conventional protocol requiring 96 h only. Result While pretransplantation analysis of cells transduced with a retroviral vector, expressing the enhanced green fluorescent protein (EGFP) marker gene, demonstrated significantly higher overall transduction rates for the extended protocol (33.6 ± 2.3 vs. 22.1 ± 3.8%), EGFP expression in CD34+ cells before transplantation (4.0 ± 0.9 vs. 11.6 ± 2.5%), engraftment of human cells in NOD/SCID bone marrow 4 weeks after transplantation (4.5 ± 1.7 vs. 36.5 ± 9.4%) and EGFP expression in these cells (0 ± 0 vs. 11.3 ± 2.8%) were significantly impaired. When the 96 h protocol was used in combination with the spleen focus forming virus (SFFV)/murine embryonic stem cell (MESV) hybrid vector SFβ11-EGFP, high transduction rates for CD45+ (41.0 ± 5.3%) and CD34+ (38.5 ± 3.7%) cells prior to transplantation, as well as efficient human cell engraftment in NOD/SCID mice 4 weeks after transplantation (32.4 ± 3.5%), was detected. Transgene expression was observed in B-lymphoid (15.9 ± 2.0%), myeloid (36.5 ± 3.5%) and CD34+ cells (10.1 ± 1.5%). Conclusion Our data show that CD34+ cells maintained in cytokines for multiple days may differentiate and loose their capacity to contribute to the haematological reconstitution of NOD/SCID mice. In addition, the SFFV/MESV hybrid vector SFβ11-EGFP allows efficient transduction of and gene expression in haematopoietic progenitor cells.     

Mesenchymal stem cells promote engraftment of human umbilical cord blood-derived CD34(+) cells in NOD/SCID mice

OBJECTIVE: Mesenchymal stem cells (MSC) have been implicated as playing an important role in hematopoietic stem cell engraftment. We identified and characterized a new population of MSC derived from human fetal lung. In cotransplantation experiments, we examined the homing of MSC as well as the effect on engraftment of human umbilical cord blood (UCB)-derived CD34(+) cells in NOD/SCID mice. MATERIALS AND METHODS: Culture-expanded fetal lung-derived CD34(+) cells were characterized by immune phenotyping and cultured under conditions promoting differentiation to osteoblasts or adipocytes. Irradiated (3.5 Gy) NOD/SCID mice (n = 51) were transplanted intravenously with 0.03 to 1.0 x 10(6) UCB CD34(+) cells in the presence or absence of 1 x 10(6) culture-expanded fetal lung-derived MSC, irradiated CD34(-) cells, B cells, or with cultured MSC only. RESULTS: Culture-expanded fetal lung CD34(+) cells were identified as MSC based on phenotype (CD105(+), SH3(+), SH4(+), CD160(+)) and their multilineage potential. Cotransplantation of low doses of UCB CD34(+) cells and MSC resulted in a three-fold to four-fold increase in bone marrow engraftment after 6 weeks, whereas no such effect was observed after cotransplantation of irradiated CD34(-) or B cells. Homing experiments indicated the presence of MSC in the lung, but not in the bone marrow, of NOD/SCID mice. CONCLUSIONS: We identified a population of MSC derived from human fetal lung. Upon cotransplantation, MSC, but not irradiated CD34(-) or B cells, promote engraftment of UCB CD34(+) cells in bone marrow, spleen, and blood by mechanisms that may not require homing of MSC to the bone marrow.     

Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice

In an attempt to develop efficient procedures of human hematopoietic gene therapy, retrovirally transduced CD34(+) cord blood cells were transplanted into NOD/SCID mice to evaluate the repopulating potential of transduced grafts. Samples were prestimulated on Retronectin-coated dishes and infected with gibbon ape leukemia virus (GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted recipients revealed a sustained engraftment of human hematopoietic cells expressing the EGFP transgene. On average, 33.6% of human CD45(+) cells expressed the transgene 90 to 120 days after transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of human CD45(+) cells expressing the EGFP transgene at this time. The transplantation of purified EGFP(+) cells increased the proportion of CD45(+) cells positive for EGFP expression to 57. 7% at 90 to 120 days after transplantation. At this time, 18.9% and 4.3% of NOD/SCID BM consisted of CD45(+)/EGFP(+) and CD34(+)/EGFP(+) cells, respectively. Interestingly, the transplantation of EGFP(-) cells purified at 24 hours after infection also generated a significant engraftment of CD45(+)/EGFP(+) and CD34(+)/EGFP(+) cells, suggesting that a number of transduced repopulating cells did not express the transgene at that time. Molecular analysis of NOD/SCID BM confirmed the high levels of engraftment of human transduced cells deduced from FACS analysis. Finally, the analysis of the provirus insertion sites by conventional Southern blotting indicated that the human hematopoiesis in the NOD/SCID BM was predominantly oligoclonal.     

Intra-bone marrow transplantation facilitates pauci-clonal human hematopoietic repopulation of NOD/SCID/beta2m(-/-) mice

OBJECTIVE: Intra-bone marrow transplantation (IBMT) has been shown to improve the limit of detection of primitive human SCID-repopulating cells (SRC) in NOD/SCID mice when compared to intravenous transplantation. We sought to further refine detection of SRC by comparing NOD/SCID mice to the more sensitive NOD/SCID/beta2m(-/-)strain as IBMT recipients of limiting numbers of purified primitive human hematopoietic cells. MATERIALS AND METHODS: Purified human Lin(-)CD34(+)CD38- cells at limiting doses were delivered by IBMT into NOD/SCID and NOD/SCID/beta2m(-/-) strains of recipient mice. Six weeks posttransplantation, injected and noninjected bones were analyzed separately for multilineage human hematopoietic chimerism. RESULTS: NOD/SCID/beta2m(-/-) mice are superior recipients for IBMT and show a trend toward increased levels of human hematopoietic engraftment. In addition, in contrast to NOD/SCID recipients, NOD/SCID/beta2m(-/-) mice were reconstituted with as few as five highly purified cells, indicative of pauci-clonal repopulation. Analysis of injected and noninjected bones demonstrated that engrafting cells were capable of in vivo migration and expansion. Although SRC hematopoietic reconstitution of NOD/SCID mice is commonly lymphoid-dominant, multilineage analysis of separate bone sites following IBMT of purified cells revealed that a subset of mice was repopulated with a myeloid-dominant graft in at least one bone site, revealing that SRC are developmentally heterogeneous among Lin(-)CD34(+)CD38- cells and capable of distinct differentiation potential. CONCLUSION: IBMT into NOD/SCID/beta2m(-/-) mice provides a highly sensitive experimental transplantation assay for the detection of human hematopoietic repopulating cells and demonstrates that Lin(-)CD34(+)CD38- cells are more highly enriched for human SRC than originally predicted.     

Human CD34+ hematopoietic cells transduced by retrovirus-mediated interferon alpha gene maintains regeneration capacity and engraftment in NOD/SCID mice.

To achieve long-term expression of human interferon alpha-5 (IFNalpha) gene in the bone marrow (BM) hematopoietic microenvironment, replication-deficient retroviral vector LSN-IFNalpha was used to deliver the IFNalpha gene into human BM CD34+ cells. After fibronectin-facilitated transduction, a fraction of CD34+ cells was plated in methylcellulose medium with or without G418 to assess transduction efficiency and the effect of IFNalpha gene transfer on colony formation. Colony-forming assay in the presence of G418 (400 microg/mL) revealed that 41% CFU-GM colonies are G418 resistant after infection with LSN-IFNalpha retrovirus. There was no significant difference in CFU-GM/BFU-E colony formation among IFNalpha gene-transduced CD34+ cells, control vector (LXSN) transduced-CD34+ cells and nontransduced CD34+ cells. Another portion of CD34+ cells was grown in liquid medium to measure IFNalpha production. RIA revealed that IFNalpha gene-transduced CD34+ cells produced 72.2 +/- 15.4 U/mL (10(6) cells/24 hours) of IFNalpha compared with 8.3 +/- 2.1 U/mL and 4.3 +/- 1.2 U/mL in LXSN-transduced or nontransduced CD34+ cells, respectively. The remaining portion of transduced CD34+ cells was transplanted into immunodeficient (NOD/SCID) mice to allow analysis of long-term expression of IFNalpha. Transplantation of 1x10(6) CD34+ cells into sublethally irradiated NOD/SCID mice showed that IFNalpha and neo(r) mRNA were detectable in engrafted mouse BM cells for up to 6 months. We conclude that continual local expression of IFNalpha in transduced CD34+ cells does not impair either CD34+ cell growth and differentiation or engraftment and long-term survival in NOD/SCID mice.     

Dynamic tracking of human hematopoietic stem cell engraftment using in vivo bioluminescence imaging

The standard approach to assess hematopoietic stem cell (HSC) engraftment in experimental bone marrow transplantation models relies on detection of donor hematopoietic cells in host bone marrow following death; this approach provides data from only a single time point after transplantation for each animal. In vivo bioluminescence imaging was therefore explored as a method to gain a dynamic, longitudinal profile of human HSC engraftment in a living xenogeneic model. Luciferase expression using a lentiviral vector allowed detection of distinctly different patterns of engraftment kinetics from human CD34+ and CD34+CD38- populations in the marrow NOD/SCID/beta 2mnull mice. Imaging showed an early peak (day 13) of engraftment from CD34+ cells followed by a rapid decline in signal. Engraftment from the more primitive CD34+CD38- population was relatively delayed but by day 36 increased to significantly higher levels than those from CD34+ cells (P <.05). Signal intensity from CD34+CD38-engrafted mice continued to increase during more than 100 days of analysis. Flow cytometry analysis of bone marrow from mice after death demonstrated that levels of 1% donor cell engraftment could be readily detected by bioluminescence imaging; higher engraftment levels corresponded to higher image signal intensity. In vivo bioluminescence imaging provides a novel method to track the dynamics of engraftment of human HSC and progenitors in vivo.