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Differential Kinetics of Primitive Hematopoietic Cells Assayed In Vitro and In Vivo During Serum-Free Suspension Culture of CD34⁺ Blood Progenitor Cells

^a Experimental Hematology Group, Department of Hematology/Oncology, University Medical Center, Freiburg, Germany;
^b Department of Experimental Pharmacology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany;

Key Words. Hematopoietic progenitor cells • Serum-free medium • Transplantation • Ex vivo expansion

Dr. Reinhard Henschler, Department of Hematology/Oncology, University Medical Center, Hugstetter Strasse 55, 79106 Freiburg, Germany.

	Abstract

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So far, blood progenitor cells (BPC) expanded ex vivo in the absence of stromal cells have not been demonstrated to reconstitute hematopoiesis in myeloablated patients. To characterize the fate of early hematopoietic progenitor cells during ex vivo expansion in suspension culture, human CD34⁺-enriched BPC were cultured in serum-free medium in the presence of FLT3 ligand (FL), stem cell factor (SCF) and interleukin 3 (IL-3). Both CD34 surface expression levels and the percentage of CD34⁺ cells were continuously downregulated during the culture period. We observed an expansion of colony-forming units granulocyte-macrophage (CFU-GM) and BFU-E beginning on day 3 of culture, reaching an approximate 2-log increase by days 5 to 7. Limiting dilution analysis of primitive in vitro clonogenic progenitors was performed through a week 6 cobblestone-area-forming cell (CAFC) assay, which has previously been shown to detect long-term bone marrow culture-initiating cells (LTC-IC). A maintenance or a slight (threefold) increase of week 6 CAFC/LTC-IC was found after one week of culture. To analyze the presence of BPC mediating in vivo engraftment, expanded CD34⁺ cells were transplanted into preirradiated NOD/SCID mice at various time points. Only CD34⁺ cells cultured for up to four days successfully engrafted murine bone marrow with human cells expressing myeloid or lymphoid progenitor phenotypes. In contrast, five- and seven-day expanded human BPC did not detectably engraft NOD/SCID mice. When FL, SCF and IL-3-supplemented cultures were performed for seven days on fibronectin-coated plastic, or when IL-3 was replaced by thrombopoietin, colony forming cells and LTC-IC reached levels similar to those of control cultures, yet no human cell engraftment was recorded in the mice. Also, culture in U-bottom microplates resulting in locally increased CD34⁺ cell density had no positive effect on engraftment. These results indicate that during ex vivo expansion of human CD34⁺ cells, CFC and LTC-IC numbers do not correlate with the potential to repopulate NOD/SCID mice. Our results suggest that ex vivo expanded BPC should be cultured for limited time periods only, in order to preserve bone-marrow-repopulating hematopoietic stem cells.

	Introduction

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Peripheral blood progenitor cells (BPC) have been used for both autologous, and more recently, allogeneic hematopoietic stem cell transplantation [1, 2]. Hematopoietic cytokines have been used to expand hematopoietic cells in ex vivo culture [3, 4]. The main rationales for ex vivo expansion have been: A) to generate amplified numbers of progenitor cell populations such as colony-forming cells of the granulocyte-macrophage or megakaryocytic lineages or their progeny, in an attempt to shorten neutrophil or platelet regeneration during the recovery phase from bone marrow aplasia [3-5]; B) to provide a prolonged time period for retroviral infection in the course of developing improved protocols for gene transfer into hematopoietic progenitors [6-8], or C) to purge contaminating tumor cells from autografts [5, 9, 10].

Clinical protocols have previously shown that bone marrow transplantation with cultured hematopoietic cells is principally feasible; Barnett et al. [11] and Chang et al. [12] transplanted bone marrow cultured under Dexter-type conditions in patients with acute or chronic leukemia. Purging of leukemic cells was observed, and numbers of Philadelphia-chromosome positive colony forming cells (CFC) decreased in patients with chronic myelogenous leukemia; however, hematopoietic regeneration was generally delayed compared with transplantation using noncultured cells. Later, cytokine-supported stroma-free suspension culture protocols were developed [3, 13-15]. The efficiency of the progenitor expansion as well as the maintenance of the transplantation potential have been assessed using semisolid clonogenic assays, numbers of CD34⁺ cells, or the incidence of cells capable of initiating long-term bone marrow cultures [3, 13-15]. In mice, the ability of ex vivo-expanded hematopoietic progenitor cells to repopulate the bone marrow has been studied in congenic recipients [16-18]. More recently, immunodeficient mice have been used as a model for the bone marrow repopulation by human ex vivo expanded hematopoietic cells [19-21].

For the clinical use of cultured progenitor cells in hematopoietic transplantation, serum-free culture medium has been proposed to avoid some disadvantages of serum, such as the risk of infection by viruses, variation between individual batches, and regulatory problems [15, 17, 22]. Data comparing the incidence of primitive cells assayed in vitro and their value to predict for the presence of cells capable of repopulating the bone marrow in vivo are scarce but are of primary importance when developing clinical protocols. We used transplantation into NOD/SCID mice in addition to CFC and long-term culture-initiating cells (LTC-IC) assays to determine the levels of progenitor cells during a serum-free expansion protocol. We used stem cell factor (SCF), FLT3-ligand (FL), and interleukin 3 (IL-3) as cytokines, since they have been shown to be efficient mediators of primitive cell maintenance and expansion in studies employing in vitro progenitor cell assays [15, 23]. Our data indicate that hematopoietic bone-marrow-repopulating cells follow different kinetics in cytokine-supported suspension culture as CFC and LTC-IC.

	Materials and Methods

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Patients and Cells
Patients with a diagnosis of a solid tumor or a lymphoid neoplasia were included after informed consent according to the declaration of Helsinki and underwent BPC mobilization by administration of combination chemotherapy and daily s.c. injection of G-CSF. Patients had received previous chemotherapy consisting of between two and eight cycles of doxorubicin, cyclophosphamide, vincristine, bleomycin, etoposide, prednisolone (VACOP-B); cyclophosphamide, adriamycin, vincristine, prednisolone (CHOP) in lymphoma; high-dose dexamethasone in multiple myeloma; cyclophosphamide, methotrexate, 5-fluorouracil (CMF); or cyclophosphamide, epirubicin, cis-platinum (PEC) in mammary carcinoma; etoposide, ifosfamide, cis-platinum, epirubicin (VIP) in sarcoma; cis-platinum, etoposide, ifosfamide (PEI); or cis-platinum, etoposide, bleomycin (PEB) in carcinoma testis ( Table 1). Mobilization was achieved by once daily s.c. application of 300 or 480 µg G-CSF on days 1-12 and a one-day course of etoposide, ifosfamide, cis-platinum with or without eipirubicin on day 0. BPC were separated from whole blood after anticoagulation with acid citrate dextrose by leukapheresis in a CS-3000 cell separator (Baxter; Munich, Germany). Aliquots taken from the leukapheresis harvests were separated on Ficoll gradients (density 1.077 g/ml, Biochrom KG; Berlin, Germany). Anti-CD34-antibody (CellPro; Bothell, WA) was added to the cell suspensions and CD34⁺ separation was subsequently performed on Ceprate^TM LC columns (CellPro) according to the manufacturer's instructions. In some cases, CD34⁺ cells were aliquots from clinical scale CD34⁺ cell separations (Ceprate^TM SC columns). CD34⁺ cell purity was above 70% in all cases. Viability of CD34⁺ BPC preparations was >95% as determined by flow cytometric analysis of propidium iodide (Sigma; Deisenhofen, Germany) stained cells using a FACScan^® flow cytometer (Becton Dickinson; Heidelberg, Germany). CD34⁺-enriched BPC were suspended at a concentration of 2 to 6 x 10⁶/ml in the CD34⁺ column elution buffer containing phosphate-buffered saline. Ex vivo expansion was started immediately after cell isolation.

Methylcellulose Clonogenic Assays and Long-Term Bone Marrow Cultures
Nucleated cell counts were obtained using a hemocytometer and trypan blue dead cell exclusion. Colony-forming cells were assayed in 0.9% methylcellulose (WAK Chemie; Bad Homburg, Germany) in Iscove's modified Dulbecco's medium (IMDM), 20% fetal calf serum (FCS), 10% bovine serum albumin in the presence of 100 ng/ml rh-IL-3, rh-GM-CSF (both from Genzyme) and 2 U/ml erythropoietin (Janssen-Cilag; Neuss, Germany). Numbers of inoculated cells per ml were 2,000 to 5,000 (for CD34⁺) or 2,000 to 10,000 (for expanded cells). Cultures were incubated at 37°C, 5 % CO₂ in a humidified atmosphere for 14 days in 35-mm petri dishes (Nunc; Wiesbaden, Germany). Colonies >50 cells were counted under an inverted microscope and classified according to the presence of GM cells and red hemoglobinized BFU-E. For generation of preformed stroma, LTBMC were established as described, using bone marrow aspirations from healthy donors [24, 26]. Briefly, red cells were depleted from bone marrow by density gradient sedimentation at 1g in 0.1% methylcellulose/IMDM for 1 h, and the remaining cells were seeded at 2 x 10⁶/ml into 96-well plates (Becton Dickinson) in IMDM, 10% preselected horse serum (Sigma), 10% FCS (GIBCO; Paisley, UK), and 5 x 10^–7 M hydrocortisone. Cultures were incubated at 33°C in an atmosphere of 5% CO₂ in air. When a confluent stroma layer had formed after three to four weeks, cultures were irradiated using a ¹³⁷Cs-gamma source with 15 Gy at a dose rate of 3-4 Gy/min. After removal of most of the culture medium, irradiated cultures were reseeded with CD34⁺ or expanded cells as starting cell populations in fresh medium at limiting dilution using 10-20 replicates per dilution step. Dilutions were chosen at 1,000, 300, 100, 30, and 10 cells per well for CD34⁺ fractions, and at 30,000, 10,000, 3,000, 1,000, 300, and 100 cells for expanded cells. After six weeks' incubation at 33°C with weekly half medium exchanges, cultures were evaluated under a light microscope by scoring for the presence or absence of at least one cobblestone area consisting of more than 15 closely attached, homogeneously formed bright cells. Previous experiments have demonstrated that under these culture conditions, the LTC-IC/week 6 cobblestone area forming cell (CAFC) frequencies measured are equivalent to the incidences obtained by replating microcultures into a methylcellulose progenitor assay at week 6 [14, 27] and have therefore been termed "LTC-IC" here. The incidence of LTC-IC was calculated by plotting the percentage of positive wells against the values of cell numbers, fitting a linear regression through the data points and choosing the 37% intercept, according to Poisson statistics.

Mice
A NOD/LtSz-scid/scid mouse colony (originally obtained from Dr. Leonard Schultz, Jackson Laboratories; Bar Harbor, ME) was expanded and maintained under pathogen-free conditions in the animal facility of the Max Delbrück Center of Molecular Medicine (Berlin, Germany). They were fed a standard diet purchased from Sniff (Soest, Germany) and acidified drinking water ad libitum. Mice were irradiated at six to eight weeks of age with a dose of 250 to 300 cGy of gamma irradiation and transplanted with human cells within 3 to 5 h. For transplantation, 0.2-ml samples of BPC at graft doses of 3-5 x 10⁵ cells or the expanded progeny thereof were injected into the lateral tail vein. Mice with BPC grafts were additionally transplanted with a stably transfected rat fibroblast cell line (Rat-hIL-3) expressing the human IL-3 gene [28]. Briefly, transfected cells were propagated in Dulbecco's modified Eagle's medium (GIBCO) with 10% FCS. Trypsinized cells were washed, resuspended in cold medium without additives, and mixed with Matrigel (Basement Membrane Matrix 40234; Becton Dickinson; Bedford, MA) as previously described [29]. Five million cells were injected s.c. into the lateral abdomen.

At five to eight weeks post-transplantation, mice were killed by cervical dislocation, and blood as well as 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 anti-mouse IgG receptor antibody (2.4G2, Pharmingen; San Diego, CA), cell staining was carried out as described earlier [29]. Human- or mouse-specific monoclonal antibodies conjugated with either FITC or PE were used in combinations to identify cells of human and murine origin: mouse anti-human CD45 (clone HI30) and anti-human HLA class I (clone G46-2.6, Pharmingen), anti-CD10 (clone ALB1), anti-CD13 (clone SJ1D1), and anti-CD33 (clone D3HL60.251, Immunotech), anti-CD14 (clone MP9, Becton Dickinson; Heidelberg, Germany); rat anti-mouse CD45 (LCA; Pharmingen). As a control, in each experiment, cells from a NOD/SCID mouse not transplanted with human cells were used and stained with the same antibodies. Background fluorescence was assessed by including isotype-matched antibodies conjugated with FITC or PE. Cell analysis was performed with a FACSCalibur system (Becton Dickinson; Heidelberg) using CellQuest software. Each measurement contained 10,000 events. Dead cells were excluded by outgating of propidium-iodide-stained cells.

DNA Analysis
The presence of human-specific DNA within the bone marrow of transplanted mice was confirmed by polymerase chain reaction (PCR) using primers complementary to sequences of an 850-bp DNA fragment of the -satellite DNA of the human chromosome 17 [30]. Genomic DNA was extracted through a QIAmp tissue kit (Qiagen; Hilden, Germany). Briefly, the DNA of lysed cells was adsorbed to a silica matrix, washed and eluted with QIAAmp elution buffer by centrifugation. Amplification of the centromere-specific human fragments of chromosome 17 was performed using primers corresponding to the primer pair 17a1/17a2 as described by Warburton et al. [31]. The primers were elongated to 25 nucleotides each for use at a higher annealing temperature. The 5' primer (5' GGG ATA ATT TCA GCT GAC TAA ACA G 3') covers the positions 15 to 39, and the 3'primer (5' TTC CGT TTA GTT AGG TGC AGT TAT C 3') covers the positions 867 to 891 of the sequence HSSATA17 (Gene Bank #M13882). For PCR, the AmpliTaq-Gold polymerase and related reagents from Perkin Elmer (Applied Biosystems GmbH; Weiterstadt, Germany) were used. The PCR reaction contained 200 µM each of the respective nucleotides, 250 nM of each primer, 2 mM MgCl2, and 250 ng of genomic DNA were reacted. Following an initial DNA denaturation and Taq activation at 94°C for 10 min, 35 1-min cycles of denaturation at 94°C and annealing/extension at 60°C were performed followed by a final elongation step at 72°C for 10 min. Amplified DNA fragments were electrophoresed through 1.75% agarose gels and subsequently visualized through ultraviolet light after staining with ethidium bromide. Genomic DNA samples from both a human breast carcinoma line (MaCa 3366) as a positive control and NOD/SCID mouse liver tissue as a negative control were processed in parallel.

	Results

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Analysis of Cellular Phenotypes During Ex Vivo Expansion
We have analyzed the potential of ex vivo expanded peripheral blood CD34⁺ progenitors to initiate clonogenic semisolid assays, LTBMC, and human hematopoiesis in xenografted NOD/SCID mice. The CD34⁺ cell expansion cultures resulted in variable increases in total cell numbers over the culture period of one week ( Table 1). The proportion of CD34-expressing cells decreased during this culture period ( Fig. 1). The majority of the CD34⁺ cells also initially expressed CD38 and HLA-DR antigens. During the seven-day expansion period, numbers of CD34⁺ CD38⁺ cells fell to almost zero levels, whereas a CD38^– population still expressing CD34 remained ( Fig. 1). HLA-DR expression decreased from highly positive to lower levels ( Fig. 1). Flow cytometric determination of expanded cells also revealed development of CD14⁺ and CD15⁺ monocyte/macrophage and granulocytic cells (approximately 10%) during ex vivo expansion; the development of myeloid cells was confirmed by light microscopical analysis of May-Grünwald-Giemsa stained samples (not shown). Megakaryocytic and erythrocytic cells, as determined by morphological analysis of May-Grünwald-Giemsa stained samples, were present at very low levels. In contrast, T and B lymphocytes, as assessed by expression of CD3 or CD19, respectively, did not develop. Therefore, CD34⁺-enriched blood progenitor cells from cancer patients downregulated expression of the CD34 antigen, and predominantly developed along a myeloid differentiation pathway in serum-free medium in the presence of SCF, FL, and IL-3 over a seven-day culture period.

View larger version (46K): [in this window] [in a new window]   Figure 1. Flow cytometric analysis of CD34+ BPC during ex vivo expansion in serum-free medium. CD34+ BPC were cultured in the presence of SCF, FL, and IL-3 (100, 300, and 100 ng/ml, respectively) for the indicated time periods. Aliquots were analyzed after staining with FITC- or PE-conjugated antibodies, shown on the x-axis and y-axis, respectively, for the indicated cell surface markers, or IgG control antibodies (isotype). The gate of the analyzed cells is framed in the left panels. The analysis of a representative experiment is shown. (SSC = side scatter; FSC = forward scatter).

View larger version (19K): [in this window] [in a new window]   Figure 2. Analysis of total cell numbers, CFU-GM, BFU-E colonies, and LTC-IC (assessed through week 6 CAFC) during CD34+ cells in suspension culture. CD34+ BPC were cultured in the presence of SCF, FL, and IL-3 (100, 300, and 100 ng/ml, respectively) for the indicated time periods, and aliquots were analyzed in the indicated assay systems. Different symbols indicate analysis of cells from different donors. Duplicate symbols for total cells, CFU-GM, and BFU-E values represent duplicate determinations. LTC-IC incidences were determined by limiting dilution analysis. LTC-IC/ml were calculated from the total cell number and the LTC-IC incidence values. Numbers attached to symbols of LTC-IC-incidences are r2 values of the regression analysis. Each symbol represents an individual donor. Symbols represent the donors as shown in Table 1.

View larger version (51K): [in this window] [in a new window]   Figure 3. Flow cytometric analysis of human hematopoietic cells in the bone marrow of NOD/SCID mice. A total of 3 to 5 x 105 CD34+ BPC were cultured in the presence of SCF, FL, and IL-3 (100, 300, and 100 ng/ml, respectively) for the indicated time periods and injected i.v. into each animal. Engraftment was analyzed five to eight weeks after transplantation. Bone marrow cells from the mice were stained with the indicated FITC- or PE-conjugated antibodies, shown on the x-axis and y-axis, respectively, and analyzed by flow cytometry.

View larger version (15K): [in this window] [in a new window]   Figure 4. Engraftment of human cells in the bone marrow or blood of NOD/SCID mice after culture in serum-free medium and SCF, FL, and IL-3 (100, 300, and 100 ng/ml, respectively). Three to 5 x 105 CD34+ cells per mouse were precultured for the indicated time periods, and engraftment was analyzed five to eight weeks after transplantation by staining with human-specific antibodies for HLA-I (bone marrow cells) or CD45 (peripheral blood cells) and subsequent flow cytometric analysis. Each symbol represents an individual donor. Duplicate or triplicate use of a symbol represents analysis in different mice. The different symbols represent the donors as shown in Table 1.

View larger version (19K): [in this window] [in a new window]   Figure 5. Development of CFC, LTC-IC, and hematopoietic repopulation potential in NOD/SCID mice after seven-day ex vivo culture of CD34+ BPC using variations in culture conditions. FS3, control conditions as in experiments shown in Figures 2 to 4 using FL, SCF, and IL-3 (300, 100, and 100 ng/ml, respectively) in flat-bottom flasks; FS3/Fn, conditions as in FS3 but with fibronectin (10 µg/cm2) precoated culture surface; FST, conditions as in FS3, except that IL-3 was substituted by TPO (100 ng/ml); FS3-U, conditions as in FS3 except that culture was performed in U-bottomed 96-well plates. Positive control mice transplanted with noncultured CD34+ BPC (3 to 5 x 105 per mouse) were the same as shown in Figure 4 (day 0) and showed efficient human engraftment. Results represent values from individual cultures from different donors. Duplicate symbols for CFU-GM and BFU-E values represent duplicate determinations; LTC-IC incidences were determined by limiting dilution analysis. LTC-IC/ml were calculated from the total cell number and the LTC-IC incidence values. Numbers attached to symbols of LTC-IC incidences are r2 values of the regression analysis. Engraftment in NOD/SCID mice was analyzed five to seven weeks after transplantation by staining of bone marrow cells with human-specific antibodies for HLA-I; values are means from two to three mice per experiment. *) values obtained from all experiments (n = 2-3) range from 0 to 0.1%. Each symbol represents an individual donor. Symbols represent the donors as shown in Table 1.

	Discussion

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Ex vivo expansion of hematopoietic progenitors has been investigated in a number of previous studies, mostly using CD34-enriched cell populations [3-5, 13-15, 22, 35]. However, these studies have been restricted to in vitro endpoints (CFC, LTC-IC) to assess the transplantation potential of cultured cells, and systematic analyses of in vitro and in vivo repopulation potential over several time points have not been performed. In our serum-free expansion protocol, as well as in a study by Zandstra et al. [35], SCF and FL were present at relatively high concentrations (100 to 300 ng/ml). The resulting CFC amplification was in a range similar to that obtained by Petzer et al. [15], who also used these cytokines at high concentrations and obtained optimal LTC-IC expansion from bone marrow CD34⁺ CD38^– cells. The relatively lower degree of LTC-IC expansion in our cultures compared with the study by Petzer et al. [15] coincides with the fact that we have not added a further enrichment procedure for primitive progenitors, such as the additional CD38^– separation, which may preselect for a more primitive subpopulation of LTC-IC with higher expansion potential.

Gan et al. [19] have described a loss in the ability of human hematopoietic cells cultured on bone marrow stromal feeders to repopulate irradiated NOD/SCID mice. Our results after expansion of CD34⁺ BPC in the absence of stroma show that after a similar period of seven days in stroma-free suspension culture, the ability of CD34⁺ BPC to repopulate the bone marrow is also abrogated. Similar results were obtained by Bhatia et al. [36], who cultured human enriched CD34⁺CD38^– cord blood cells in the presence of cytokines without stroma support. These authors noted that after a four-day suspension culture period, the repopulation potential of expanded cells in NOD/SCID mice was preserved or slightly augmented; however, after eight days it was generally lost. Interestingly, FL was also present in these cultures at high levels. Dao et al., in their gene transduction work, noted that the presence of FL was required to maintain the repopulation potential of human CD34⁺ cells for a 72-h period (assessed in humanized bnx mice) [8] . Larochelle et al. found loss of human hematopoietic repopulating potential of CD34⁺CD38^–-enriched cord blood cells within 48 h after cytokine incubation; in that study, all experiments except one were conducted in the absence of FL [7].

Murine progenitor cells have also been transplanted into lethally irradiated recipient mice after cytokine-supported stroma-free expansion cultures. These studies used either enriched or nonenriched cells, various cell densities, and different cytokine combinations. In some studies, maintenance of the repopulation potential was achieved [16, 37], but other authors showed that early loss of stem cells occurred during the expansion period [38]. Whereas the former researchers all used serum-supplemented media, Rebel and coworkers used serum-free expansion of murine highly enriched progenitors and showed that, although the numbers of cells with a primitive (lineage-negative wheat germ agglutinin-positive) phenotype expanded, the engraftment potential of the cultured product was not amplified but could at best be maintained [17]. Similar results were also reported by Brown et al. [39] after expansion of murine bone marrow cells in the presence of SCF and GM-CSF. Recently, however, an increase in murine reconstituting cells could be demonstrated in a culture system starting from Sca-1⁺ lin^– bone marrow cells [40].

Our studies did not reveal a major advantage of using U-bottom vessels or fibronectin coating for the maintenance of transplantation potential. The substitution of IL-3 by TPO did not lead to major alterations in CFC or LTC-IC numbers within the expansion cultures on day 7, showing that substitution of IL-3 by TPO in SCF and FL-containing cultures is not sufficient to preserve NOD/SCID repopulating stem cells. Taken together, we have demonstrated that progenitor cells with the ability to engraft in vivo may be maintained ex vivo for a time period of three to four days with cytokines and serum-free suspension culture. The short-term culture protocol presented here should be of benefit for the clinical introduction of gene marking and tumor cell purging protocols, which both require maintenance of repopulating stem cells during in vitro culture. Other work has shown that for effective tumor cell purging with recombinant immunotoxins, short time periods may be sufficient [10]. Also, incubation of CD34⁺ Thy⁺ lin^– progenitors in SCF, FL, and TPO has been shown to result in cell division in very primitive populations capable of repopulating immunodeficient mice during the first two to four days of culture [41], which leads to the expectation that retroviral gene transfer into primitive progenitors may be achieved under such conditions. Further studies will have to compare the repopulation in NOD/SCID mice by expanded BPC with human hematopoietic regeneration.

	Acknowledgments

 
We gratefully acknowledge the technical help of J. Aumann, D. Wider, E. Samek, B. Gross, and F. Luquiaud. We also thank Dr. M. Engelhardt for critical reading of the manuscript.

This work has been financially supported by the "Deutsche Forschungsgemeinschaft", through SFB 364, Project A1, and grant Fi-527/1; the "Bundesministerium für Bildung und Forschung" through grant A 4.2F (Verbund Klinische Pharmakologie Berlin/Brandenburg), and the EU Strukturfonds.

	Footnotes

 
^c Present address: Medical Center III; Friedrich-Alexander-University, Erlangen, Germany

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. To LB, Haylock DN, Simmons PJ et al. The biology and clinical uses of blood stem cells. Blood 1997;89:2233-2258.[Free Full Text]

2. Berenson RJ, Andrews RG, Bensinger WI et al. Antigen CD34⁺ marrow cells engraft lethally irradiated baboons. J Clin Invest 1988;81:951-955.

3. Haylock DN, To LB, Dowse TL et al. Ex vivo expansion and maturation of peripheral blood CD34⁺ cells and unseparated peripheral blood progenitor cells. Blood 1992;80:1405-1412.[Abstract/Free Full Text]

4. Brugger W, Heimfeld S, Berenson R et al. Ex vivo expanded peripheral blood CD34⁺ cells mediate hematopoietic recovery after high dose chemotherapy. N Engl J Med 1995;333:283-287.[Abstract/Free Full Text]

5. Purdy MH, Hogan CJ, Hami L. Large volume ex vivo expansion of CD34-positive hematopoietic progenitor cells for transplantation. J Hematother 1995;4:515-525.[Medline]

6. Hanenberg H, Xiao XL, Dilloo D et al. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nat Med 1996;2:876-882.[Medline]

7. Larochelle A, Vormoor J, Hanenberg H et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med 1996;2:1329-1337.[Medline]

8. Dao MA, Hannum CH, Kohn DB et al. FLT3 ligand preserves the ability of human CD34⁺ progenitors to sustain long-term hematopoiesis in immunodeficient mice after retroviral-mediated transduction. Blood 1997;89:446-456.[Abstract/Free Full Text]

9. Vogel W, Behringer D, Scheding S et al. Ex vivo expansion of CD34⁺ peripheral blood progenitor cells: implications for the expansion of contaminating epithelial tumor cells. Blood 1996;88:2707-2713.[Abstract/Free Full Text]

10. Spyridonidis A, Schmidt M, Bernhardt W et al. Purging of mammary carcinoma cells during ex vivo expansion of CD34⁺ hematopoietic progenitor cells with recombinant immunotoxins. Blood 1998;91:1820-1827.[Abstract/Free Full Text]

11. Barnett M, Eaves CJ, Phillips GL et al. Successful autografting in chronic myeloid leukemia after maintenance of marrow in culture. Bone Marrow Transplant 1989;4:345-351.[Medline]

12. Chang J, Morgenstern GR, Coutinho LH et al. The use of bone marrow cells grown in long-term culture for autologous bone marrow transplantation in acute myeloid leukemia: an update. Bone Marrow Transplant 1989;4:5-9.[Medline]

13. Brugger W, Moecklin W, Heimfeld S et al. Ex vivo expansion of peripheral blood CD34⁺ progenitor cells by stem cell factor, interleukin-1beta (IL-1beta), IL-6, IL-3, interferon-gamma, and erythropoietin. Blood 1993;81:2579-2584.[Abstract/Free Full Text]

14. Henschler R, Brugger W, Luft T et al. Maintenance of transplantation potential in ex vivo expanded CD34⁺ peripheral blood progenitor cells. Blood 1994;84:2898-2903.[Abstract/Free Full Text]

15. Petzer AL, Zandstra PW, Piret JM et al. Differential cytokine effects on primitive (CD34⁺CD38^–) human hematopoietic cells: novel responses to Flt3-ligand and thrombopoietin. J Exp Med 1996;183:2551-2558.[Abstract/Free Full Text]

16. Muench MO, Firpo MT, Moore MAS. Bone marrow transplantation with interleukin-1 plus kit-ligand ex vivo expanded bone marrow accelerates hematopoietic reconstitution in mice without loss of stem cell lineage and proliferative potential. Blood 1993;81:3463-3473.[Abstract/Free Full Text]

17. Rebel VI, Dragowska W, Eaves CJ et al. Amplification of Sca-1⁺ Lin^– WGA⁺ cells in serum-free cultures containing steel factor, interleukin-6, and erythropoietin with maintenance of cells with long-term in vivo reconstituting potential. Blood 1994;83:128-136.[Abstract/Free Full Text]

18. Rebel VI, Lansdorp PM. Culture of purified stem cells from fetal liver results in loss of in vivo repopulating potential. J Hematother 1996;5:25-37.[Medline]

19. Gan OI, Larochelle A, Dick JE. Differential maintenance of primitive human SCID-repopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells. Blood 1997;90:641-650.[Abstract/Free Full Text]

20. Dao MA, Hannum CH, Kohn DB et al. FLT3 ligand preserves the ability of human CD34⁺ progenitors to sustain long-term hematopoiesis in immune-deficient mice after ex vivo retroviral-mediated transduction. Blood 1997;89:446-456.

21. Conneally E, Eaves CJ, Humphries RK. Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential. Blood 1998;91:3487-3493.[Abstract/Free Full Text]

22. Möbest D, Mertelsmann R, Henschler R. Ex vivo expansion of CD34⁺ blood progenitor cells in serum-free medium. Biotechnol Bioeng 1998;60:341-347.[Medline]

23. Petzer AL, Hogge DE, Lansdorp PM et al. Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium. Proc Natl Acad Sci USA1996;93:1470-1474.[Abstract/Free Full Text]

24. Coutinho LH, Gilleece MH, DeWynter EA et al. Clonal and long term cultures using human bone marrow. In: Testa NG, Molineux G, eds. Hemopoiesis, A Practical Approach. Oxford, New York, Tokyo: IRL Press, 1993:75-106.

25. Strobel ES, Möbest D, von Kleist S et al. Adhesion and migration are differentially regulated in hematopoietic progenitor cells by cytokines and extracellular matrix. Blood 1997;90:3524-3532.[Abstract/Free Full Text]

26. Gartner S, Kaplan HS. Long-term culture of human bone marrow cells. Proc Natl Acad Sci USA 1980;77:4756-4759.[Abstract/Free Full Text]

27. Pettengell R, Luft T, Henschler R et al. Direct comparison by limiting dilution analysis of long-term bone marrow culture-initiating cells in human bone marrow, umbilical cord blood, and blood stem cells. Blood 1994;84:3653-3659.[Abstract/Free Full Text]

28. 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]

29. Goan SR, Fichtner I, Just U et al. The severe combined immunodeficient-human peripheral blood stem cell (SCID-huPBSC) mouse: a xenotransplant model for hu-PBSC-initiated hematopoiesis. Blood 1995;86:89-100.[Abstract/Free Full Text]

30. Waye JS, Willard HF. Structure, organization and sequence of alpha-satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X-chromosome. Mol Cell Biol 1986;6:3156-3165.[Abstract/Free Full Text]

31. Warburton PE, Greig GM, Haaf T et al. PCR amplification of chromosome-specific alpha-satellite DNA: definition of centromeric STS markers and polymorphic analysis. Genomics 1991;11:324-333.[Medline]

32. Testa NG, Dexter TM. The biology of long-term bone marrow cultures and its application to bone marrow transplantation. Curr Opin Oncol 1991;3:272-278.[Medline]

33. Eaves CJ, Sutherland HJ, Udomsakdi C. The human hematopoietic stem cell in vitro and in vivo. Blood cells 1992;18:301-307.[Medline]

34. Kaushansky K. Thrombopoietin and the hematopoietic stem cell. Blood 1998;92:1-3.[Free Full Text]

35. Zandstra PW, Conneally E, Petzer AL et al. Cytokine manipulation of primitive human hematopoietic cell self-renewal. Proc Natl Acad Sci USA 1997;94:4698-4703.[Abstract/Free Full Text]

36. Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J Exp Med 1997;186:619-624.[Abstract/Free Full Text]

37. Holyoake TL, Freshney MG, McNair L et al. Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow. Blood 1996;87:4589-4595.[Abstract/Free Full Text]

38. Peters SO, Kittler ELW, Ramshaw HS et al. Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts. Blood 1996;87:30-37.[Abstract/Free Full Text]

39. Brown RL, Sheng F, Dusing SK et al. Serum-free culture conditions for cells capable of producing long-term survival in lethally irradiated mice. STEM CELLS 1997;15:237-245.[Abstract/Free Full Text]

40. Miller CL, Eaves CJ. Expansion in vitro of adult murine hematopoietic stem cells with transplantable lymphomyeloid reconstituting ability. Proc Natl Acad Sci USA 1997;94:13648-13653.[Abstract/Free Full Text]

41. Luens KM, Travis MA, Chen BP et al. Thrombopoietin, kit ligand, and flk2/flt3 ligand together induce increased numbers of primitive hematopoietic progenitors from human CD34⁺Thy-1⁺Lin^– cells with preserved ability to engraft SCID-hu mice. Blood 1998;91:1206-1215.[Abstract/Free Full Text]

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