Sustained proliferation, multi-lineage differentiation and maintenance of primitive human haemopoietic cells in NOD/SCID mice transplanted with human cord blood

Time course studies of sublethally irradiated non-obese mice with severe combined immunodeficiency (NOD/ SCID mice) transplanted intravenously with 10(7) human cord blood cells showed a rapid and parallel regeneration of human erythroid, granulopoietic, megakaryopoietic and B-lymphoid progenitors, as well as more primitive subpopulations of CD34+ cells (defined by their multi-lineage in vitro colony-forming ability, coexpression of Thy-1, or functional activity in long-term culture-initiating cell [LTC-IC] assays), in the marrow, spleen and blood. Maximum numbers of human cells were reached within 6 weeks and were then sustained for another 18-20 weeks. 3H-thymidine suicide studies showed all types of in vitro clonogenic human progenitors tested and the human LTC-IC to be proliferating in vitro throughout this period. A 2-week course of injections of human Steel factor, interleukin-3, granulocyte-macrophage colony-stimulating factor and erythropoietin given just prior to assessment of the mice had no effect on any of these human engraftment parameters. 4-6 weeks post-transplant, the marrow of primary NOD/SCID recipients contained human cells that were able to regenerate lymphopoiesis and/or myelopoiesis in secondary irradiated NOD/SCID mice. These findings establish a baseline for the kinetics of engraftment, multi-lineage differentiation and self-renewal of human cord blood stem cells in this xenogeneic transplant model and thus set the stage for future studies of their regulation in vivo.     

Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential

Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34(+) cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor, Flt3-ligand, and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells, colony-forming cells (CFC), or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period, and the cells infected under either condition regenerated similar numbers of human CD34(+) (myeloid) CFC and human CD19(+) (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However, the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant, 17% +/- 3% and 17% +/- 8%, G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected, the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% +/- 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM), or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17, respectively), whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a >/= 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols.     

Bone marrow repopulation by human marrow stem cells after long-term expansion culture on a porcine endothelial cell line

In vitro exposure of murine hematopoietic stem cells (HSCs) to cell cycle-inducing cytokines has been shown to result in a defect in the ability of these cells to engraft. We used a porcine microvascular endothelial cell (PMVEC) line in conjunction with exogenous interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) to expand human HSCs that express the CD34 and Thy-1 antigens but lack lineage-associated markers (CD34+Thy-1+Lin- cells). Ex vivo expansion of hematopoietic cells was evaluated in comparison to stromal cell-free, cytokine-supplemented cultures. Cells expressing the CD34+Thy-1+Lin- phenotype were detectable in both culture systems for up to 3 weeks. These cells were reisolated from the cultures and their ability to engraft human fetal bones implanted into SCID mice (SCID-hu bone) was tested. HSCs expanded in PMVEC coculture were consistently capable of competitive marrow repopulation with multilineage (CD19+ B lymphoid, CD33+ myeloid, and CD34+ cells) progeny present 8 weeks postengraftment. In contrast, grafts composed of cells expanded in stroma-free cultures did not lead to multilineage SCID-hu bone repopulation. Proliferation analysis revealed that by 1 week of culture more than 80% of the cells in the PMVEC cocultures expressing the primitive CD34+CD38- phenotype had undergone cell division. Fewer than 1% of the cells that proliferated in the absence of stromal cells remained CD34+CD38-. These data suggest that the proliferation of HSCs in the presence of IL-3, IL-6, GM-CSF, and SCF without stromal cell support may result in impairment of engraftment capacity, which may be overcome by coculture with PMVECs.     

Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays, knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38- cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38- cells and colony-forming cells, respectively, as well as a 2- to 4-fold increase in SRC after 4 d of culture. However, after 9 d of culture, all SRC were lost, despite further increases in total cells, CFC content, and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation, and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells.     

Circadian rhythm in the hypothermic response to serotonin1A receptor agonist 8-OH-DPAT in rats

The gene encoding the CD2 mouse cell surface antigen was retrovirally transduced into cord blood CD34+ cells. On infection by culture at the contact of retrovirus-packaging cells, the mCD2 marker was expressed by 30-40% CD34+ cells, that included the most primitive stem cell-enriched Thy-1+ and CD38- subsets. Accordingly, sorted cord blood CD34+Thy-1+ cells could be directly infected in the same conditions. mCD2- transgenic cord blood CD34+ cells were then used to reconstitute human fetal thymus implanted in SCID mice. Five to 8 weeks later, the mCD2 antigen was detected on approximately 10% of the human thymocytes repopulating the thymus grafts and the transgene genome was detected in graft cell DNA by Southern blot. These results demonstrate efficient gene transfer into primitive cord blood hematopoietic cells endowed with lymphoid potential and suggest gene therapy schemes in neonates suffering inherited or acquired-such as HIV infection-disorders of the T-cell lineage.     

Transplantation of human umbilical cord blood cells in macrophage-depleted SCID mice: evidence for accessory cell involvement in expansion of immature CD34+CD38- cells

In vivo expansion and multilineage outgrowth of human immature hematopoietic cell subsets from umbilical cord blood (UCB) were studied by transplantation into hereditary immunodeficient (SCID) mice. The mice were preconditioned with Cl2MDP-liposomes to deplete macrophages and 3.5 Gy total body irradiation (TBI). As measured by immunophenotyping, this procedure resulted in high levels of human CD45(+) cells in SCID mouse bone marrow (BM) 5 weeks after transplantation, similar to the levels of human cells observed in NOD/SCID mice preconditioned with TBI. Grafts containing approximately 10(7) unfractionated cells, approximately 10(5) purified CD34+ cells, or 5 x 10(3) purified CD34+CD38- cells yielded equivalent numbers of human CD45+ cells in the SCID mouse BM, which contained human CD34+ cells, monocytes, granulocytes, erythroid cells, and B lymphocytes at different stages of maturation. Low numbers of human GpA+ erythroid cells and CD41+ platelets were observed in the peripheral blood of engrafted mice. CD34+CD38+ cells (5 x 10(4)/mouse) failed to engraft, whereas CD34- cells (10(7)/mouse) displayed only low levels of chimerism, mainly due to mature T lymphocytes. Transplantation of graded numbers of UCB cells resulted in a proportional increase of the percentages of CD45+ and CD34+ cells produced in SCID mouse BM. In contrast, the number of immature, CD34+CD38- cells produced in vivo showed a second-order relation to CD34+ graft size, and mice engrafted with purified CD34+CD38- grafts produced 10-fold fewer CD34+ cells without detectable CD34+CD38- cells than mice transplanted with equivalent numbers of unfractionated or purified CD34+ cells. These results indicate that SCID repopulating CD34+CD38- cells require CD34+CD38+ accessory cell support for survival and expansion of immature cells, but not for production of mature multilineage progeny in SCID mouse BM. These accessory cells are present in the purified, nonrepopulating CD34+CD38+ subset as was directly proven by the ability of this fraction to restore the maintenance and expansion of immature CD34+CD38- cells in vivo when cotransplanted with purified CD34+CD38- grafts. The possibility to distinguish between maintenance and outgrowth of immature repopulating cells in SCID mice will facilitate further studies on the regulatory functions of accessory cells, growth factors, and other stimuli. Such information will be essential to design efficient stem cell expansion procedures for clinical use.     

High-level expression of a novel epitope of CD59 identifies a subset of CD34+ bone marrow cells highly enriched for pluripotent stem cells

To further define the hierarchy of human hematopoietic progenitor cells, we have attempted to identify antibodies to cell-surface molecules expressed on CD34+ progenitor cell subsets. Herein we describe the utility of a new monoclonal antibody, HCC-1, which binds to a novel epitope of CD59 differentially expressed among CD34+ progenitor cells. HCC-1 subdivides the adult marrow CD34+ population into HCC-1high and HCC-1low/- fractions of approximately equal size. Cobblestone area-forming cells (CAFC) in long-term bone marrow culture were enriched 10-30-fold in CD34+HCC-1high cells compared with CD34+HCC1-low/- cells and two-fold compared with CD34+ cells. When injected into fetal human bone fragments implanted in SCID mice, the CD34+HCC-1high population showed potent engrafting activity leading to the production of myeloid, lymphoid, and erythroid elements, as well as the retention of progenitor cell phenotype. These studies demonstrate that the CD34+HCC-1high population contains primitive pluripotent hematopoietic stem cells. No hematopoietic engrafting activity was detected in the CD34+HCC-1low/- population. Consistent with this finding, simultaneous five-color flow cytometric analysis revealed that HCC-1high cells include virtually all CD34+Thy-1+Lin- cells, a cell population previously characterized as highly enriched for primitive pluripotent hematopoietic stem cells. The ability of CD34+ cells divided into subsets by HCC-1 to produce T cells was assessed by transplantation of sorted cells into human fetal thymus implanted into SCID mice. A higher frequency of thymus-engrafting activity was observed in the CD34+HCC-1high than in the CD34+HCC-1low/- population. Consistent with the limited ability to engraft in the SCID-hu thymus model, the CD34+HCC-1low/- population was shown to contain a low frequency of CD34+CD10+ lymphoid progenitor cells. We conclude that the HCC-1 epitope is expressed at high levels on a subset of CD34+ cells that contain virtually all primitive pluripotent hematopoietic stem cells and that the population of CD59 molecules expressed on CD34+ cells is not homogeneous.     

Engraftment of ex vivo expanded and cycling human cord blood hematopoietic progenitor cells in SCID mice

The ability of human hematopoietic cells to engraft SCID mice provides a useful model in which to study the efficiency of retroviral gene transfer and expression in primitive stem cells. In this regard, it is necessary to determine whether SCID mice can be engrafted by cycling human hematopoietic progenitor cells. Human cord blood cells from 12 different donors were cultured in vitro for 6 days with interleukin-3 and stem cell factor. Phenotypic analysis indicated that hematopoietic cells were induced to cycle and the number of progenitors was expanded, thus making them targets for retroviral gene transfer. The cells were then transferred to SCID mice. Human hematopoietic progenitor cell engraftment was assessed up to 7 weeks later by growth of human progenitor cells in soft agar. After in vitro culture under conditions used for retroviral gene transfer, human cord blood hematopoietic cells engrafted the bone marrow and spleen of SCID mice. Interestingly, cultured cord blood cells engrafted after intraperitoneal but not after intravenous injection. Furthermore, engraftment of cord blood cells was observed in mice receiving no irradiation before transfer of the human cells, suggesting that competition for space in the marrow is not a limiting factor when these cells have been cultured. Administration of human cytokines after transfer of human cord blood cells to SCID mice was also not required for engraftment. Thus, engraftment of SCID mice with human hematopoietic cells cultured under conditions suitable for gene transfer may provide an in vivo assay for gene transfer to early human hematopoietic progenitor cells.     

Engraftment and retroviral marking of CD34+ and CD34+CD38- human hematopoietic progenitors assessed in immune-deficient mice

Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+ progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38- cells. Comparable extents of human engraftment and lineage development were obtained from 5 x 10(5) CD34+ cells and 2,000 CD34+CD38- cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38- cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38- cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.     

Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors

Efficient gene transfer into human hematopoietic stem cells (HSCs) is an important goal in the study of the hematopoietic system as well as for gene therapy of hematopoietic disorders. A lentiviral vector based on the human immunodeficiency virus (HIV) was able to transduce human CD34+ cells capable of stable, long-term reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-efficiency transduction occurred in the absence of cytokine stimulation and resulted in transgene expression in multiple lineages of human hematopoietic cells for up to 22 weeks after transplantation.     

Efficient gene transfer in primitive CD34+/CD38lo human bone marrow cells reselected after long-term exposure to GALV-pseudotyped retroviral vector

Successful retroviral gene transfer into human hematopoietic stem cells was demonstrated in preliminary clinical trials at low efficiency. We have shown previously that gene transfer into committed hematopoietic progenitor cells is more efficient using a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector instead of an amphotropic retroviral vector. Here, we have conducted a systematic study of human hematopoietic progenitor cells after extended transduction with a GALV-pseudotyped retroviral vector. CD34+/CD38lo Cells were transduced for 5 days and reselected according to phenotype after culture and analyzed for cell cycle status, long-term culture-initiating cell (LTC-IC) activity, and gene transfer. Reselection of rare, very primitive progenitor cells was successful. Equal to fresh CD34+/CD38lo cells, >90% of reselected CD34+/CD38lo cells were in G0/G1. CD34+/CD38lo reselection enriched for LTC-IC (10-fold), as compared to freshly isolated CD34+/CD38lo cells with excellent specificity (82.7% of total LTC-IC were recovered in the reselected CD34+/CD38lo population) and recovery (62% of initial LTC-IC number in CD34+/CD38lo cells were recovered in the reselected fraction after transduction). Gene transfer into primitive progenitor cells was efficient with 50.5% G418-resistant LTC-IC colonies and more than 40 copies of vector provirus detectable per 100 nuclei of CD34+/CD38lo cells. To our knowledge, this is the first systematic analysis of phenotype, function, and cell cycle demonstrating retroviral gene transfer into rare, very primitive human hematopoietic progenitor cells. The chosen strategy should be of considerable value for analyzing and improving gene therapy of the hematopoietic system.     

Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34(+) cord blood cells

The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo-expanded CB cells, purified CD34(+) cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo-expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34(+) cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo-expanded versus the fresh CB cells (CD45(+) repopulation in NOD/SCIDs BM: 3. 7% +/- 1.2% v 26.2% +/- 5.9%, respectively, at 20 days posttransplantation; P <.005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% +/- 1.7% of CD45(+) cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo-expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45(+) and CD45(+)/CD34(+) cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo-expanded samples were found. The analysis of the engrafted CD45(+) cells showed that both the fresh and the in vitro-incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.     

Importance of information following myocardial infarction: a study of the self-perceived information needs of patients and their spouse/partner compared with the perceptions of nursing staff

Time course studies revealed that the combination of Flt-3 ligand (FL), Steel factor (SF) and interleukin-3 (IL-3) did not elicit as large an amplification of the long-term culture-initiating cell (LTC-IC) population in serum-free cultures of CD34+ CD38- cord blood (CB) cells as was obtained in similar cultures of adult human CD34+ CD38- bone marrow (BM) cells (4- v 90-fold maximum increases), even though both total and colony-forming cell (CFC) numbers initially increased more rapidly in CB cultures. Multifactorial analysis of the short-term (10 d) effects of different cytokines identified FL and IL-6 in combination with the soluble IL-6 receptor (sIL-6R) as most important for expanding the CB LTC-IC population. In contrast, their counterparts in adult BM were most effectively stimulated by FL, SF and IL-3. For rapid generation of increased numbers of CFC, SF with either FL or IL-6/sIL-6R were found to be the most important contributors in cultures of CD34+ CD38- CB cells, whereas, in analogous BM cultures, IL-6/sIL-6R and TPO (in addition to FL, SF and IL-3) were required. These findings reinforce the principle of altered cytokine responsiveness as a hallmark of early haemopoietic cell differentiation and demonstrate how cytokine requirements may change during human ontogeny. Identification of conditions for optimizing the expansion of different subsets of primitive CB cells has additional important implications for clinical transplantation and gene transfer.     

Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse as a model system to study the engraftment and mobilization of human peripheral blood stem cells

Mobilized CD34(+) cells from human peripheral blood (PB) are increasingly used for hematopoietic stem-cell transplantation. However, the mechanisms involved in the mobilization of human hematopoietic stem and progenitor cells are largely unknown. To study the mobilization of human progenitor cells in an experimental animal model in response to different treatment regimens, we injected intravenously a total of 92 immunodeficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with various numbers of granulocyte colony-stimulating factor (G-CSF) -mobilized CD34(+) PB cells (ranging from 2 to 50 x 10(6) cells per animal). Engraftment of human cells was detectable for up to 6.5 months after transplantation and, depending on the number of cells injected, reached as high as 96% in the bone marrow (BM), displaying an organ-specific maturation pattern of T- and B-lymphoid and myeloid cells. Among the different mobilization regimens tested, human clonogenic cells could be mobilized from the BM into the PB (P = .019) with a high or low dose of human G-CSF, alone or in combination with human stem-cell factor (SCF), with an average increase of 4.6-fold over control. Therefore, xenotransplantation of human cells in NOD/SCID mice will provide a basis to further study the mechanisms of mobilization and the biology of the mobilized primitive human hematopoietic cell.     

The role of granulocyte colony-stimulating factor in mobilization and transplantation of peripheral blood progenitor and stem cells

The article provides a review of the role of granulocyte colony-stimulating factor (G-CSF) for mobilization and transplantation of peripheral blood progenitor and stem cells. Recombinant gene technology has permitted the production of highly purified material for therapeutic use in humans. Progenitor cells can be assessed using semisolid and liquid culture assays or direct immunofluorescence analysis of cells expressing CD34. This antigen is found on lineage-determined hematopoietic progenitor cells as well as on more primitive stem cells with extensive self-renewal capacity. Administration of G-CSF during steady-state hematopoiesis or following cytotoxic chemotherapy leads to an increase of hematopoietic progenitor cells in the peripheral blood. The level of circulating CD34+ cells post-chemotherapy is greater compared with G-CSF administration during steady state. On the other hand, CD34+ cells harvested post-chemotherapy contain a smaller proportion of more primitive progenitor cells (CD34+/HLA-DR- or CD34+/CD38-) compared with G-CSF treatment alone. Independent of the mobilization modality, the amount of previous cytotoxic chemo- and radiotherapy adversely affects the yield of hematopoietic progenitor cells. While continuous subcutaneous administration of G-CSF between 5 and 16 micrograms/kg bodyweight is preferred, additional dose-finding studies may be helpful to optimize current dose schedules. Adhesion molecules like L-selectin, VLA (very late antigen)-4 and LFA (leukocyte function antigen)-1 are likely to play a role in mobilization, since these antigens are expressed on CD34+ cells from bone marrow in different densities compared with blood-derived CD34+ cells collected following G-CSF-supported cytotoxic chemotherapy. It is also relevant for transplantation that during G-CSF-enhanced recovery post-chemotherapy, peripheral blood is enriched with a greater proportion of CD34+ cells expressing Thy-1 in comparison with CD34+ cells from bone marrow samples obtained on the same day or before the mobilization therapy was started. The early nature of the CD34+/Thy-1+ cells is very likely since this phenotype has been found on stem cells from human fetal liver and bone marrow and on cord blood cells. As a result, G-CSF-mobilized blood stem cells provide rapid and sustained engraftment following high-dose therapy, including myeloablative regimens. Positive selection of CD34+ cells as well as ex vivo expansion using different cytokines are currently being investigated for purging and improvement of short-term recovery post-transplantation. Future developments include the use of blood-derived hematopoietic stem cells for somatic gene therapy. The availability of growth factors has been an important prerequisite for the development of these new avenues for cell therapy.     

High efficiency gene transfer to human hematopoietic SCID-repopulating cells under serum-free conditions

Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34(+) cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45(+) cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions.     

Mature human hematopoietic cells in donor bone marrow complicate interpretation of stem/progenitor cell assays in xenogeneic hematopoietic chimeras

Xenogeneic hematopoietic chimeras have been used to assay the growth and differentiation of human stem/progenitor cells. The presence of human hematopoietic cells in immunodeficient mice transplanted with human marrow cells may be caused by proliferation and differentiation of early stem/progenitor cells and/or proliferation of mature cells. Unpurified human marrow mononuclear cells, T cell-depleted, or stem/progenitor cell-enriched (CD34+ or CD34+CD38-) populations were injected into sublethally irradiated NOD/LtSz scid/scid (NOD/SCID) mice. High levels of human cells were detected in mice (hu/mu chimeras) transplanted with each of the above human marrow populations. Large numbers of mature human T lymphocytes were found in marrow, spleens, and thymuses from hu/mu chimeras that had been transplanted with unpurified human mononuclear marrow cells. Human immunoglobulin was detected in sera from these chimeras, and some exhibited a clinical syndrome suggestive of graft-versus-host disease. In contrast, in hu/mu chimeras that had received T cell-depleted or stem/progenitor cell-enriched populations, multilineage hematopoiesis (myeloid, B lymphoid, and progenitor cells by immunophenotype) was detected but T lymphocytes and human immunoglobulin were not; in addition, no human cells were detected in the thymuses. Thus, injection of adult human marrow cells into immunodeficient mice can result in hematopoietic chimerism for at least 3 months after transplant. However, the types of cells present in hu/mu chimeras differ depending on the human cell population transplanted. This should be taken into account when hematopoietic chimeras are used to assess human stem/progenitor cell function.     

The potential role of FLT3 ligand in progenitor and primitive hematopoietic cell expansion

We have previously defined the experimental conditions for hematopoietic cell expansion. CD34+ human marrow cells were maintained in a serum-free, stroma-free liquid culture system, at a concentration of 10(3) cells/ml, for 10 days at 37 degrees C, in the presence of various cytokine combinations. The basic combination of early cytokines SCF (100 ng/ml), IL3 (5 ng/ml), IL6 (10 ng/ml), has a modest stimulating effect on all compartments: the number of total cells increased 56-fold and CD34+ cells 1-fold; CFU-GM, BFU-E and CFU-MK, increased 6-fold, 5-fold and 3-fold respectively. As far as CD34+ cells are concerned, the subpopulation CD34+/CD38- was only maintained. Interestingly, the addition of 100 ng/ml of Flt3 ligand (FL) significantly enhanced the amplification of total cells (276-fold), CFU-GM (54-fold) and BFU-E (15-fold). The number of CD34+ cells and the subpopulation CD34+/38- increased to 7-fold and 22-fold respectively. Moreover, long term culture-initiating cells (LTC-ICs) in limiting dilution assay (LDA) were found to increase 3-fold. Further addition of MGDF (10 ng/ml), G-CSF (10 ng/ml) and Epo (0.5 U/ml), in various combinations, acted synergically with the previous cytokine combination to support the formation of multiple types of hematopoietic colonies. As expected, the addition of MGDF increased the number of CFU-MK up to 5-fold expansion. Interestingly, MGDF addition was synergistic also for BFU-E and CFU-GM expansion. In the combination of SCF+ IL3+ IL6+ FL + MGDF, CFU-GM expanded to 73-fold and BFU-E to 17-fold. G-CSF in SCF + IL3 + IL6 + FL conditions stressed the expansion of the granulopoietic compartment doubling the number of CFU-GM and CD33+ cells, with no consequence on LTC-IC or BFU-E. Surprisingly, G-CSF induced the expansion of the megakaryocytic lineage up to 6-fold, in a similar way as MGDF. Epo in presence of SCF+ IL3+ IL6+/-FL dramatically increased total cell expansion (2300-2800-fold), mainly erythroblastic (70% glycoA) without exhaustion of all other compartments. The simultaneous use of these three cytokines (MGDF + G-CSF + Epo) in presence of four early cytokines (SCF + IL3 + IL6 + FL) clearly allows a significant expansion of all hematopoietic compartments, precursors, progenitors, and primitive stem cells. In conclusion, these data show the ability of a stroma-free, serum-free liquid system to expand all myeloid lineages, including CFU-MK and LTC-IC which are critical for clinical application of ex vivo expanded cells.     

CD34+, kit+, rhodamine123(low) phenotype identifies a marrow cell population highly enriched for human hematopoietic stem cells

We hypothesized that human hematopoietic cells displaying a CD34+, kit-, rhodamine123(low) phenotype would be highly enriched for cells with stem-like properties. To test this hypothesis, we employed fluorescence activated cell sorting (FACS) to isolate cells with this phenotype from normal light density marrow mononuclear cells (MNC). CD34+, kit+, rhodamine123(low) cells comprised from 0.05-0.01% of the total MNC population. They were small, had scant cytoplasm, and contained nuclei with dense, hyperchromatic chromatin and inconspicuous nucleoli. Additional immunophenotyping revealed that these cells were CD33-, CD38-, CD20-, and glycophorin A-. When plated in semisolid cultures containing optimal concentrations of IL-3, GM-CSF, KL, EPO, IL-6, and IL-1 these cells did not form colonies. However, when cultured over irradiated stromal cells, cobblestone areas were observed to form after 3 weeks, and harvested cells were able to initiate long-term cultures. To further demonstrate that these cells were indeed stem like, we also tested their ability to engraft and mature in immunocompromised (SCID) mice. Irradiated (400 cGy) SCID mice were transplanted with 2 x 10(3) candidate stem cells which were then injected with recombinant human growth factors every other day. Two months post-transplant the animals were sacrificed. PCR and FACS analysis of marrow and spleen cell samples revealed the presence of cells expressing human CD45 consistent with engraftment of human stem cells and the establishment of murine-human chimerism. Moreover, MNC isolated from transplanted mice formed unambiguously human BFU-E, CFU-GM and B cell colonies when stimulated with the appropriate growth factors. Accordingly, we have identified a relatively rapid and simple mechanism for isolating primitive human hematopoietic cells with stem cell-like properties. We anticipate that this strategy will be useful for experimental and therapeutic applications that require human stem cells in quantity.