Stem Cells 2003;21:236-244
www.StemCells.com
© 2003 AlphaMed Press
Interleukin-3 Promotes Proliferation and Differentiation of Human Hematopoietic Stem Cells but Reduces Their Repopulation Potential in NOD/SCID Mice
Andreas Nitschea,b,
Ilse Junghahnc,
Stefanie Thulkea,
Jutta Aumannc,
Aleksandar Radoni
a,
Iduna Fichtnerc,
Wolfgang Siegerta
a Medizinische Klinik II, Charité-Campus Charité Mitte, Humboldt Universität zu Berlin, Germany;
b Robert Koch-Institute, Berlin, Germany;
c Department of Experimental Pharmacology, Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany
Key Words. NOD/SCID mice • Hematopoietic • Stem cell • Engraftment • Interleukin 3 • Immunochecmistry
Correspondence:
Wolfgang Siegert, M.D., Medizinische Klinik II, Charite-Campus Charite Mitte, Schumannstr. 20/21, 10117 Berlin, Germany. Telephone: 49-30-450-513144 or 49-30-450-513002 (Secretariat); Fax: 49-30-450-513952; e-mail: wolfgang.siegert{at}charite.de
 |
ABSTRACT
|
|---|
In the present study we explored systematically the influence of human interleukin-3 (IL-3) on the cord blood (CB) cell-derived production of human hematopoietic cells in the bone marrow, blood, and spleen of chimeric nonobese/severe combined immunodeficient mice ((NOD/SCID) mice. CB mononuclear cells and MACS-enriched CB CD34+ cells were injected into irradiated NOD/SCID mice. The mice were additionally transplanted with a stably transfected rat fibroblast cell line expressing the human IL-3 gene (Rat-IL-3) constitutively, or with the nontransfected rat fibroblast cell line as a control (Rat-1). Rat-IL-3 mice displayed a higher engraftment of human hematopoietic cells in bone marrow, spleen, and peripheral blood compared with mice with Rat-1 cotransplantation. When we transplanted their total bone marrow cell population into secondary mice, surprisingly, mice transplanted with bone marrow cells from Rat-1 mice displayed a higher proportion of human hematopoietic cells compared with Rat-IL-3 mice. As expected, bone marrow cultures (BMCs) from Rat-IL-3 mice contained a higher proportion of human cells than Rat-1 bone marrow cells. However, when BMCs were passaged to new flasks, we observed a higher proportion of human cells in BMCs from Rat-1 mice compared with BMCs from Rat-IL-3 mice. IL-3 promotes the proliferation and differentiation of hematopoietic stem cells in chimeric bone marrow. In addition, IL-3 may play a role in the depletion of hematopoietic stem cells in chimeric bone marrow. In the absence of IL-3, the hematopoietic stem cells may remain in a quiescent state and proliferation can be induced by stimuli, including secondary transplantation or cell passage.
|
INTRODUCTION
|
|---|
The characterization of the pluripotent hematopoietic stem cell is of significant interest to hematological investigators [1]. However, the detailed investigation and characterization of these cells have been hampered by a lack of accessibility to candidate cells. Routinely used methods are cell culture based and therefore provide only limited information about stem cell characteristics in vivo [2]. Therefore, during the past years, several immunodeficient mouse models have been established [3, 4]. They provide an environment that more closely resembles the human bone marrow than any other model system known to date [5].
Various immunodeficient mouse strains and transplantation conditions have been investigated so far [6]. Finally, the nonobese/severe combined immunodeficient (NOD/ SCID) mouse has been proven to provide a test system that allows high engraftment rates of human hematopoietic stem cells by maintaining a reasonable lifespan of the mice [7]. The transplantation of hematopoietic stem cells from sources including bone marrow, peripheral blood, and cord blood resulted in detectable engraftment rates in bone marrow, spleen, and the peripheral blood of transplanted mice [8, 9].
To optimize engraftment rates, supplementation with human cytokines seems to be beneficial [10, 11]. While several hematopoietic active cytokines are cross-reactive between mice and humans and can provide human hematopoietic cells with growth factors, interleukin-3 (IL-3), formerly known as multi-colony-stimulating factor, is highly species specific [12]. The support of early human hematopoiesis in the chimeric bone marrow with IL-3 is probably crucial for human cell engraftment. Moreover, it may have an impact on multilineage hematopoiesis, promoting the development of certain cell types and inhibiting the development of others [13].
Results of recent studies on the influence of IL-3 are controversial. While it is widely accepted that IL-3 supports the expansion of CD34+ cells and leads to high expansion rates of post-progenitor and mature cells in vitro, as well as a reduced formation of B-lymphoid cells [14, 15], it is still discussed whether the repopulating potential of IL-3-treated CD34+ cells is reduced [16, 17]. All studies to date have been based on a stem cell manipulation using human IL-3 in vitro prior to transplantation [18].
Therefore, we examined the influence of human IL-3 quasi in vivo in the NOD/SCID mouse on human hematopoiesis after primary and secondary transplantations of nonfractionated cord blood mononuclear cells (CB-MNCs) and purified CD34+ cells. In contrast to cell culture studies, our approach allows a more reliable evaluation of IL-3 effects on hematopoietic activity in the complex stromal cell environment.
|
MATERIALS AND METHODS
|
|---|
CB Cells
Human umbilical CB was obtained from the umbilical vein immediately after vaginal delivery in uncomplicated full-term pregnancies. CB-MNCs were prepared from heparinized CB from a single parturient by Ficoll-Hypaque density centrifugation according to standard procedures.
CD34+ cells were isolated from the CB-MNC fraction using the MACS magnetobead separation system (Miltenyi Biotec; Bergisch-Gladbach, Germany; http://www.miltenyibiotec.com). The procedure was carried out according to the instructions of the manufacturer. After two successive fractionation steps, selected CD34+ cells had a purity of 90%-95% as determined by fluorescence-activated cell sorting (FACS) analysis using a CD34-specific monoclonal antibody, anti-HPCA-2 (Pharmingen; San Diego, California; http://www.bdbiosciences.com/pharmingen.
Mice
Breeding pairs of NOD/LtSz-SCID/SCID mice (originally obtained from Dr. Leonard Schultz, Jackson Laboratories; Bar Harbor, Maine) were expanded and maintained under pathogen-free conditions in the animal facility of the Max Delbrück Center of Molecular Medicine (Berlin, Germany). They were fed with a sterilized standard diet purchased from Sniff (Soest, Germany) and acidified drinking water ad libitum. Mice were irradiated sublethally at 6-8 weeks of age with a dose of 160 cGy of a 137Cs-gamma source and transplanted with human cells within 3-5 hours thereafter. For transplantation, 0.3-ml samples of 1 x 107 CB-MNCs or 1 x 105 purified CD34+ cells were injected into the lateral tail vein. For certain experiments, mice were cotransplanted with a stably transfected rat fibroblast cell line (Rat-IL-3) expressing the human IL-3 gene as described previously [19]. Briefly, transfected cells were propagated in Dulbecco’s-modified Eagle’s medium (GIBCO BRL; Paisley, United Kingdom; http://www.invitrogen.com) with 10% fetal calf serum. Trypsinized cells were washed, resuspended in ice-cold culture medium without additives, and mixed with matrigel (Basement Membrane Matrix 40234; Becton Dickinson; Bedford, Massachusetts; http://www.bd.com) as described. Five million cells were injected subcutaneously into the lateral abdomen, producing 3-5 ng/ml human IL-3 detectable in mouse peripheral blood.
For secondary transplantations, mice belonging to one group were sacrificed 6 weeks after primary transplantation; the bone was pooled and analyzed for chimerism by flow cytometry, and subsequently, aliquots of 107 total (chimeric) cells were injected into secondary mice. These were analyzed as described above.
Cell Preparation, Staining, and FACS Analysis
Mice were killed by cervical dislocation 6-9 weeks after transplantation. Their blood and bone marrow cells were collected for analysis. Single-cell suspensions were prepared, cell counts were performed, and viability was determined by trypan-blue exclusion. Following blocking of Fc receptors by pretreatment of the cells with human serum and an antimouse IgG receptor antibody (clone 2.4G2; Pharmingen), cell staining was carried out as described. Human- or mouse-specific monoclonal antibodies conjugated with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) were used in combinations to identify cells of human and murine origin: mouse antihuman CD45; antihuman HLA class-I; antihuman IL-3 receptor (Pharmingen); anti-CD2, -CD3, -CD4, -CD10, -CD13, -CD14, -CD15, -CD19, -CD33, -CD61; anti-HLA-DR (Immunotech; Marseille, France; http://www.immunotech.cz); anti-CD34 and -CD38 (Becton Dickinson); and rat-antimouse-CD45 (Pharmingen). In each experiment, cells from a NOD/SCID mouse not transplanted with human cells were stained with the same antibodies as a control. Background fluorescence was assessed by including isotype-matched antibodies conjugated with FITC or PE. Cell analysis was performed with FACSCalibur system (Becton Dickinson) using CellQuest software. Each measurement contained 10,000 events. Dead cells were excluded by outgating of propidium iodide-stained cells. FACS analysis was performed on freshly prepared cell suspensions of bone marrow and spleen or on peripheral blood. As a control for most experiments, immunocytochemistry was also performed on these tissues.
Bone Marrow Cultures (BMCs) and Immunohistochemistry
Bone marrow cells were prepared from mice 6-9 weeks after transplantation and from nontransplanted control animals. Cells were seeded at 1-2 x 106/ml into slide flasks (NUNC; Wiesbaden, Germany; http://www.nuncbrand.com) using previously described culture conditions [20]. The cultured cells were examined by light microscopy for monolayer formation and morphology. The cultures were terminated 2-3 weeks after seeding by removal of the culture medium and staining as previously described. Slides were screened for the presence of human cells by light microscopy. In representative fields, 400 individual cells were counted to determine the percentage of positive cells. Selected slides, especially areas with high densities of positive-staining cells, were also documented by microphotography. BMCs were analyzed by immunocytochemistry only.
Statistical Analysis
Statistical significance was tested with the Mann-Whitney test.
|
RESULTS
|
|---|
Transplantation of Nonfractionated CB-MNCs
Between 6-9 weeks after transplantation of 10 x 106 CB-MNCs into irradiated NOD/SCID mice, several different human cell types could be detected in the bone marrow by FACS analysis and immunocytochemistry. HLA-I was used as a marker for human cells, with CD45 as a marker to prove hematopoietic origin. The spectrum of detectable human cell types included CD34+ cells as putative stem cells; lymphocyte progenitors; more mature lymphocytes (CD2+, CD4+, CD10+, CD19+, or CD38+, respectively); CD33+ myeloid progenitor cells; CD13+, CD14+, or CD15+ myelomonocytic cells; granulocytes, and CD61+ megakaryocytic cells. Moreover, the human IL-3 receptor molecule could be detected, while CD3+ T cells were found only rarely. Thus, multilineage hematopoiesis could be observed in the bone marrow, spleen, and peripheral blood of all transplanted mice. The proportion of human cells in spleen and peripheral blood was generally lower than in bone marrow.
To examine the influence of human IL-3 on CB cell-derived multilineage hematopoiesis, we injected CB-MNCs into mice that had been either cotransplanted with rat fibroblast cells that were stably transfected with the human IL-3 gene (Rat-IL-3), or with the nontransfected rat fibroblast cells as a negative control (Rat-1). In contrast to the injection of human IL-3 solutions into the mice, the cotransplantation of Rat-IL-3 cell guarantees a lifelong, constitutive expression of IL-3 in the mice.
Several independent experiments showed that the presence of IL-3 resulted in significantly higher proportions of various human hematopoietic cell types in the bone marrow (Fig. 1
), spleen, and blood (data not shown). The increasing proportion of human cells could be observed for all of the cell types that were analyzed. Figure 1 shows a selection of detectable cell markers, including the pan-hematopoietic marker, CD45, as well as the lymphoid markers, myeloid markers, and the putative stem cell marker, CD34.
View larger version (20K):
[in this window]
[in a new window]
|
Figure 1. Influence of human IL-3 on the CB-MNC-derived engraftment of human cells in NOD/SCID mice. Individual portions of various human hematopoietic cell types in the chimeric bone marrow are shown for mice that were either cotransplanted with Rat-1 (open squares) or Rat-IL-3 (black squares). Portions of human cells were analyzed by FACS. Significant differences are indicated by asterisks (p < 0.05).
|
|
To find out whether the presence of IL-3 causes changes in the composition of human hematopoietic cells produced in the chimeric bone marrow, the proportions of particular cell types were related to the total number of human HLA-I+ cells. As summarized in Table 1, the proportion of the identified human cell types was not significantly changed under the influence of human IL-3. For lymphocytic CD2+ and CD19+ cells, a nonsignificant reduction could be observed in the presence of human IL-3. However, because not all human HLA-I+ were also CD45+, we assume that this difference can be explained by the development of nonhematopoietic cells in the mouse bone marrow.
Transplantation of Purified CB-Derived CD34+ Cells
The injection of 1 x 105 CD34+ cells, enriched by MACS to a purity of >90%, also led to clearly detectable amounts of human cells in chimeric bone marrow (Fig. 2), blood, and spleen (data not shown). The amount of human cells in chimeric bone marrow derived from purified CD34+ cells was comparable to the transplantation of nonfractionated CB-MNCs and was also consistently higher when IL-3-producing Rat-IL-3 cells were cotransplanted (Fig. 2). Although the IL-3-derived increase of CD2+ and CD34+ cells was clearly visible, it did not reach statistical significance (Fig. 2), while the production of all remaining cell types was significantly higher under the influence of IL-3 compared with controls without IL-3.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 2. Influence of human IL-3 on the CB CD34 cell-derived engraftment of human cells in NOD/SCID mice. Individual portions of various human hematopoietic cell types in the chimeric bone marrow are shown for mice that were either cotransplanted with Rat-1 (open squares) or Rat-IL-3 (black squares). Portions of human cells were analyzed by FACS. Significant differences are indicated by asterisks (p < 0.05).
|
|
Again, in analogy to the transplantation of nonfractionated CB-MNCs, following CD34 cell transplantation, the increased production of human cells was not restricted to individual cell types. As shown in Table 1
, no significant change in the development of individual cell types could be observed when Rat-IL-3 cells were cotransplanted. Though not significant, we found a slight increase of CD33 myeloid progenitor cells in these mice (Table 1). In contrast to the transplantation of CB-MNCs that resulted in a clearly detectable human hematopoiesis in every experiment, the transplantation of CD34+ cells required the presence of Rat-IL-3 cells to lead to human cell engraftment reliably. The importance of Rat-IL-3 cotransplantation seemed to increase with the purity of the injected CD34+ cell population (data not shown). Figure 3 shows representative results for Rat-IL-3 chimeric bone marrow cells (Fig. 3A) and a spleen cryosection (Fig. 3B) stained with an antibody specific for human HLA-I.
View larger version (90K):
[in this window]
[in a new window]
|
Figure 3. Photomicrograph of chimeric bone marrow cells from a single cell suspension (A) and a spleen cryosection (B) stained with a human specific HLA-I antibody. Human cells appear in red. The high proportion of human cells in chimeric bone marrow and the growth of human cells in islets in the spleen are clearly visible.
|
|
BMCs
In order to characterize the proliferative potential of chimeric bone marrow cells following CD34 cell transplantation, we performed BMCs of each mouse routinely. As expected, BMCs grown from the bone marrow of Rat-1 cotransplanted mice (Rat-1-BMC), which contained fewer human cells in the bone marrow, resulted in a reduced number of human cells compared with BMCs that were grown from bone marrow of Rat-IL-3 cotransplanted mice (Rat-IL-3-BMC), which contained a higher number of human cells. Although clearly detectable, the number of human cells in Rat-IL-3-BMC was only significantly increased in the HLA-I cell fraction because of the high variation. Figure 4A summarizes the results for human HLA-I, CD45+, and CD34+ cells as determined by immunocytochemistry.
View larger version (17K):
[in this window]
[in a new window]
|
Figure 4. Bone marrow cultures from mice cotransplanted with Rat-1 (open squares) or Rat-IL-3 cells (closed squares). The mean portion of human cells is shown before cell passage (4A) and after cell passage (4B) as determined by immunocytochemistry. Results are from five independent experiments including 11 mice. Significant differences are indicated by asterisks (p < 0.05).
|
|
Interestingly, when exactly the same BMCs were passaged from one flask to a second, even without a change in culture conditions, we obtained completely opposite results. After passage of Rat-IL-3-BMC cells we found a reduced proportion of human cells in the resulting BMCs, indicating a loss of proliferative potential of these cells. However, when Rat-1-BMCs were transferred to a new flask, the proportion of human cells in the second flask increased to a level where it significantly exceeded the proportion of human cells in the original Rat-1-BMC (Fig. 4B).
Secondary Transplantation of Chimeric Bone Marrow
Finally, we tried to confirm the results obtained in BMC experiments in vivo using NOD/ SCID mice. As described above, mice were transplanted with purified CB CD34+ cells and then cotransplanted with Rat-1 or Rat-IL-3 cells, respectively. Six weeks after transplantation, two mice were sacrificed; their bone marrow was pooled, an aliquot was kept for chimerism analysis, and a total of 1 x 107 cells were transplanted into one new mouse. All secondary transplanted mice were cotransplanted with Rat-IL-3 cells.
Consistent with the results shown previously, cotransplantation of Rat-IL-3 cells resulted in an elevated production of human cells in the bone marrow of primary transplanted mice compared with Rat-1 mice, but not in the amount of total cell numbers in the mice bone marrow. Interestingly, when the same number of chimeric bone marrow cells was injected into secondary mice, we observed higher engraftment rates in mice that received Rat-1 bone marrow cells, compared with mice that received Rat-IL-3 bone marrow cells. Figure 5 shows one representative out of four independently performed experiments. As shown, the secondary transplantation of Rat-IL-3 bone marrow cells resulted in a hardly detectable engraftment in the secondary mouse, while the Rat-1 bone marrow secondary transplanted mouse still displayed engraftment rates up to 40%. This is remarkable because the CD34 cells that had been used for the primary transplantation in Rat-1 and Rat-IL-3 mice, respectively, were taken from the same CB source and had been treated identically until injected in primary mice.
View larger version (20K):
[in this window]
[in a new window]
|
Figure 5. Secondary transplantation of Rat-1 and Rat-IL-3 chimeric bone marrow cells. The portion of human hematopoietic cells in the chimeric bone marrow is shown for primary mice that were either cotransplanted with Rat-1 (light gray filled bars) or Rat-IL-3 (black filled bars). Results from secondary transplanted mice are shown as dark gray and white filled bars, respectively. This figure summarizes the results from two primary and two secondary transplanted mice. It is one representative example out of four independently performed experiments.
|
|
|
DISCUSSION
|
|---|
The present study was designed to examine the influence of human IL-3 in an in vivo environment that resembles the bone marrow of patients undergoing hematopoietic stem cell transplantation. Since recent reports were restricted to an in vitro manipulation of cultured stem cell candidates prior to the examination of their repopulating potential in immunodeficient mice [16, 18, 21], we performed the treatment of candidate stem cells already in vivo. To achieve this, CB-MNCs or CB CD34+ cells were injected into NOD/SCID mice that received either a human IL-3 expressing rat cell line or the nontransfected cell line as a negative control. It has been shown previously that due to the continuous constitutive expression of human IL-3, this approach guarantees a more consistent impact on hematopoiesis compared with repetitive IL-3 injections, even when levels of human IL-3 may be supraphysiological [1922].
In the first series of experiments, we showed a significantly improved engraftment rate for human cells in NOD/SCID mice under the influence of human IL-3. This effect was seen after transplantation of nonfractionated CB-MNCs as well as after transplantation of purified CB CD34+ cells. As with the transplantation of CB-MNCs, the injection of even a 100-fold reduced dose of CD34+ cells resulted in distinctly pronounced multilineage hematopoiesis consisting of mainly B-lymphoid cells, myeloid cells, and putative stem cells, indicating the presence of a pluripotent hematopoietic stem cell in the transplanted cell fraction.
In all experiments, the proportion of human cells in the spleen and the blood mirrored the content and the composition of human cells in the bone marrow, with the lowest amounts found in the peripheral blood.
Routinely performed immunocytochemistry on BMCs consequently confirmed the results we obtained from direct FACS analysis of the bone marrow. Interestingly, the increased production of human cells in mouse bone marrow was observed for all cell types examined, indicating an IL-3-mediated effect either on all cells or on a cell that is still oligopotent enough to form all the types of cells we were able to detect. To date, several attempts have been made to find cell culture conditions that allow the expansion of bone marrow repopulating stem cells up to therapeutically useful amounts [23–26]. Most of these studies agree with the finding that IL-3 supports the proliferation of nucleated cells in vitro. With our in vivo test system, we were able to confirm these findings; IL-3 supported the total production of all human cell types analyzed in primary transplanted mice. However, when looked at in context of the human HLA-I+ cells that were formed in the mice, a nonsignificant decrease of CD19+ B-lymphoid cells was found following the transplantation of CB-MNCs and was even less pronounced when CD34+ cells were used for transplantation. Again, this is in agreement with in vitro studies that show that IL-3 exerts an inhibitory effect on the early developmental stages of B lymphopoiesis in mice and humans [14, 15, 27]. In one study, a bifunctional influence of IL-3 has been shown in the mouse system. On the one hand, IL-3 stimulated the proliferative activity of B-cell progenitors; on the other hand, the differentiation into the B-cell lineage was inhibited by the same cytokine. In addition, several other studies pointed to an inhibition of T lymphopoiesis. Unfortunately, due to low T-lymphopoietic activity, we were not able to verify these in vitro data in our in vivo model.
An even more contradictory discussion describes the influence of IL-3 on the repopulating potential of hematopoietic stem cells. While some studies show that the repopulating potential of in vitro expanded cells is maintained in the presence of IL-3 and decreases with the ex vivo expansion period [2], others report a complete abrogation of the repopulating potential by IL-3, independent of the treatment duration [17]. The results we obtained with our in vivo model indicate that IL-3 at least reduces the repopulating capability of hematopoietic stem cells. All experiments showed that the repopulating activity of chimeric bone marrow cells was reduced in secondary transplanted mice when the cells were exposed to IL-3 in primary mice. These modified repopulation and proliferation characteristics must have been generated in the primary mice as a consequence of IL-3 supplementation. Depending on the engraftment rates in primary mice, this inhibited human hematopoiesis was more or less pronounced. However, this effect is not necessarily directly caused by IL-3. It can also be the consequence of a still to be characterized IL-3-mediated effect on the hematopoietic environment in the mouse bone marrow. The portion of CD34+ cells in the bone marrow of Rat-IL-3 mice was slightly increased compared with Rat-1 mice, resulting in higher numbers of CD34+ putative stem cells injected into secondary transplanted mice. However, their repopulating activity was reduced. Since the pluripotent, bone marrow repopulating stem cell is supposed to be among the CD34 cell fraction [28, 29], it is still not completely characterized and will certainly only represent a subpopulation. In this context, it will be demanding to discover which subpopulations of CD34+ cells are elevated in Rat-1 mice and in which state of the cell cycle these cells exist.
Because various cell types have been affected by IL-3, we suggest either a general effect of IL-3 on several hematopoietic cells or, and this has been postulated previously, an inhibitory effect of IL-3 on the more primitive hematopoietic progenitor cells. Finally, the secondary transplantation experiments show that the proliferative and probably the repopulation potential of hematopoietic stem cells is at least inhibited by IL-3 in vivo.
Interestingly, similar results could be observed with BMCs from primary transplanted mice. In this setting, the passage of cultured cells into a new flask acted as a trigger of hematopoietic proliferation. Cells that have experienced IL-3 in the bone marrow of the primary mice resulted in higher proportions of human cells before cell passage, but lower proportions of human cells after cell passage. With bone marrow cells from Rat-1 mice, we obtained the opposite results. Although showing lower proportions in the cell culture before passage, the proportion of human cells increased clearly after transferring the cells to a new flask. These results suggest that although in the absence of IL-3, the proliferation of human cells is reduced, and their potential to proliferate and differentiate is still maintained and can be exhausted by an external stimulus such as cell passaging.
|
CONCLUSION
|
|---|
Our results confirm that IL-3 promotes the proliferative potential of CB-derived hematopoietic stem cells even in vivo, but reduces their repopulating potential as demonstrated in secondary transplanted NOD/SCID mice. Further characterization of the hematopoietic stem cell will be necessary to elucidate the remaining questions regarding the mechanisms of IL-3 action in human hematopoiesis.
|
ACKNOWLEDGMENT
|
|---|
We gratefully acknowledge the expert technical assistance of Delia Barz, Monika Becker, and Margit Lemm, as well as Ian M. Mackay, Brisbane, Australia, for critical reading of the manuscript. The Rat-IL-3 and Rat-1 cell lines were established and kindly provided by Ursula Just (GSF; Munich, Germany). This work was supported by a grant from the Deutsche Krebshilfe (10-1362-Si I).
|
REFERENCES
|
|---|
- Fliedner TM. The role of blood stem cells in hematopoietic cell renewal. stem cells 1998;16 (suppl 1):13–29.
- Mobest D, Goan SR, Junghahn I et al. Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells. stem cells 1999;17:152–161.[Abstract/Free Full Text]
- Vormoor J, Lapidot T, Pflumio F et al. Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice. Blood 1994;83:2489–2497.[Abstract/Free Full Text]
- Vormoor J, Lapidot T, Pflumio F et al. High-level multilineage engraftment of human cord blood cells in SCID mice. J Hematother 1993;2:215–216.[Medline]
- Vormoor J, Lapidot T, Pflumio F et al. SCID mice as an in vivo model of human cord blood hematopoiesis. Blood Cells 1994;20:316–320.[Medline]
- Greiner DL, Hesselton RA, Shultz LD. SCID mouse models of human stem cell engraftment. stem cells 1998;16:166–177.[Abstract/Free Full Text]
- Hogan CJ, Shpall EJ, McNulty O et al. Engraftment and development of human CD34(+)-enriched cells from umbilical cord blood in NOD/LtSz-scid/scid mice. Blood 1997;90:85–96.[Abstract/Free Full Text]
- Cashman J, Bockhold K, Hogge DE et al. Sustained proliferation, multi-lineage differentiation and maintenance of primitive human haemopoietic cells in NOD/SCID mice transplanted with human cord blood. Br J Haematol 1997;98:1026–1036.[CrossRef][Medline]
- Cashman JD, Lapidot T, Wang JC et al. Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice. Blood 1997;89:4307–4316.[Abstract/Free Full Text]
- Bonnet D, Bhatia M, Wang JC et al. Cytokine treatment or accessory cells are required to initiate engraftment of purified primitive human hematopoietic cells transplanted at limiting doses into NOD/SCID mice. Bone Marrow Transplant 1999;23:203–209.[CrossRef][Medline]
- Bock TA, Orlic D, Dunbar CE et al. Improved engraftment of human hematopoietic cells in severe combined immunodeficient (SCID) mice carrying human cytokine transgenes. J Exp Med 1995;182:2037–2043.[Abstract/Free Full Text]
- Burger H, van Leen RW, Dorssers LC et al. Species specificity of human interleukin-3 demonstrated by cloning and expression of the homologous rhesus monkey (Macaca mulatta) gene. Blood 1990;76:2229–2234.[Abstract/Free Full Text]
- Liang D, Shih LY, Kuo MC et al. The synergistic effect of thrombopoietin in erythropoiesis and myelopoiesis from human bone marrow cells. Acta Haematol 2000;102:135–139.[CrossRef][Medline]
- Matsunaga T, Hirayama F, Yonemura Y et al. Negative regulation by interleukin-3 (IL-3) of mouse early B-cell progenitors and stem cells in culture: transduction of the negative signals by betac and betaIL-3 proteins of IL-3 receptor and absence of negative regulation by granulocyte-macrophage colony-stimulating factor. Blood 1998;92:901–907.[Abstract/Free Full Text]
- Miyamoto K, Tsuji K, Maekawa T et al. Inhibitory effect of interleukin 3 on early development of human B-lymphopoiesis. Br J Haematol 2001;114:690–697.[CrossRef][Medline]
- Guenechea G, Segovia JC, Albella B et al. Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34(+) cord blood cells. Blood 1999;93:1097–1105.[Abstract/Free Full Text]
- Yonemura Y, Ku H, Hirayama F et al. Interleukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem cells. Proc Natl Acad Sci USA 1996;93:4040–4044.[Abstract/Free Full Text]
- Rossmanith T, Schroder B, Bug G et al. Interleukin 3 improves the ex vivo expansion of primitive human cord blood progenitor cells and maintains the engraftment potential of scid repopulating cells. stem cells 2001;19:313–320.[Abstract/Free Full Text]
- Goan SR, Schwarz K, von Harsdorf S et al. Fibroblasts retrovirally transfected with the human IL-3 gene initiate and sustain multilineage human hematopoiesis in SCID mice: comparison of CD34-enriched vs CD34-enriched and in vitro expanded grafts. Bone Marrow Transplant 1996;18:513–519.[Medline]
- Goan SR, Junghahn I, Wissler M et al. Donor stromal cells from human blood engraft in NOD/SCID mice. Blood 2000;96:3971–3978.[Abstract/Free Full Text]
- Novelli EM, Cheng L, Yang Y et al. Ex vivo culture of cord blood CD34+ cells expands progenitor cell numbers, preserves engraftment capacity in nonobese diabetic/severe combined immunodeficient mice, and enhances retroviral transduction efficiency. Hum Gene Ther 1999;10:2927–2940.[CrossRef][Medline]
- Dao MA, Pepper KA, Nolta JA. Long-term cytokine production from engineered primary human stromal cells influences human hematopoiesis in an in vivo xenograft model. stem cells 1997;15:443–454.[Abstract/Free Full Text]
- Ueda T, Tsuji K, Yoshino H et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. J Clin Invest 2000;105:1013–1021.[Medline]
- Fibbe WE, Noort WA, Schipper F et al. Ex vivo expansion and engraftment potential of cord blood-derived CD34+ cells in NOD/SCID mice. Ann NY Acad Sci 2001;938:9–17.[Medline]
- Douay L. Experimental culture conditions are critical for ex vivo expansion of hematopoietic cells. J Hematother Stem Cell Res 2001;10:341–346.[CrossRef][Medline]
- Nakahata T. Ex vivo expansion of human hematopoietic stem cells. Int J Hematol 2001;73:6–13.[Medline]
- Ball TC, Hirayama F, Ogawa M. Modulation of early B lymphopoiesis by interleukin-3. Exp Hematol 1996;24:1225–1231.[Medline]
- Silvestri F, Banavali S, Baccarani M et al. The CD34 hemopoietic progenitor cell associated antigen: biology and clinical applications. Haematologica 1992;77:265–273.[Medline]
- Srour EF, Brandt JE, Briddell RA et al. Human CD34+ HLA-DR- bone marrow cells contain progenitor cells capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis. Blood Cells 1991;17:287–295.[Medline]
Received on August 27, 2002;
accepted for publication on December 2, 2002.
Top
Abstract
Introduction
