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Stem Cells, Vol. 16, No. 5, 334-342, September 1998
© 1998 AlphaMed Press

Insulin-Like Growth Factor-1 (IGF-1) has a Costimulatory Effect on Proliferation of Committed Progenitors Derived from Human Umbilical Cord CD34+ Cells

S. Frostada, R. Bjerknesb, J.F. Abrahamsenc, J. Olweusd, Ø. Bruserude

a Division for Endocrinology and
e Division for Haematology, Medical Department B;
b Pediatric Department;
c The Gade Institute, Department of Pathology, University of Bergen, Haukeland Hospital, Norway;
d Becton Dickinson Immunocytometry Systems, San Jose, California, USA

Key Words. Umbilical cord hematopoietic stem cells • Insulin-like growth factor-1 (IGF-1) • Granulocyte colony stimulating factor

Dr. Stein Frostad, Division for Endocrinology, Medical Department B, Haukeland University Hospital, N-5021 Bergen, Norway.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of insulin-like growth factor-1 (IGF-1) on highly enriched human umbilical cord CD34+ cells were investigated in vitro. CD34+ cells were cultured in serum-free medium containing stem cell factor (SCF), GM-CSF, and interleukin-3 (IL-3). Culture of CD34+ cells for one week in the presence of these cytokines resulted in a dose-dependent increase in total cell number. Addition of G-CSF together with SCF+IL-3+GM-CSF increased the proliferation of myelopoietic cells as determined by the number of cells expressing the myelomonocytic marker CD64 and the granulocytic marker CD15 without significantly altering the number of CD34+ cells in the cultures. In the presence of G-CSF, IGF-1 induced a dose-dependent increase in the total cell number and a moderate but significant increase in the percentages of CD15+, CD64+ cells with sustained CD34+ cell proliferation. We conclude that IGF-1 can enhance the in vitro proliferation of committed progenitor cells derived from umbilical cord CD34+ cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Allogeneic stem cell transplantation is used in the treatment of several hematologic malignancies. Because HLA-matched family donors are available only for a minority of these patients, HLA-matched unrelated adults are increasingly used as stem cell donors [1, 2]. It is usually required that unrelated donors and the recipients should be matched for the HLA-A, -B, and -DRB1 loci, because even minor mismatches on these loci are associated with an increased risk of serious graft-versus host disease (GVHD) [1, 2]. However, during the last years, umbilical cord blood has become an alternative source of transplantable hematopoietic stem cells, and at least for children, the risk of serious GVDH seems to be acceptable even when umbilical cord stem cells are transplanted against HLA/B/DR mismatches [3, 4]. The use of umbilical cord stem cells may therefore make allotransplantation available for more patients.

The clinical use of umbilical cord stem cells is associated with two major problems: A) the number of available stem cells from each donor is limited and may not be sufficient for adult recipients [5], and B) due to late hematopoietic reconstitution, these patients have an increased risk of bleeding and infections [3, 4, 6, 7]. The time until reconstitution correlates with the number of transplanted cells, and cytokine-dependent ex vivo expansion of noncommitted umbilical cord stem cells may therefore be used to make sufficient stem cells available for adult recipients [4, 5]. An alternative approach is to culture a fraction of the umbilical cord stem cells in vitro to achieve cellular expansion and differentiation [8, 9]. Transplantation of such ex vivo differentiated cells together with native umbilical cord stem cells may then result in an early hematopoietic reconstitution caused by the ex vivo differentiated cells and a later permanent reconstitution caused by the native noncommitted cells [8].

A number of different cytokine combinations have been tested for the ability to expand progenitor cells. A cytokine that has not been extensively studied in this regard is insulin-like growth factor (IGF-1). This cytokine has complex regulatory functions on adult human hematopoietic progenitor cells and can stimulate the proliferation of both myeloid and erythroid progenitors [10, 11]. IGF-1 is released in the bone marrow by stromal cells; it can also be released by distant organs into serum/extracellular fluid and thereby function as a systemic regulatory mechanism of hematopoiesis [12].

Because IGF-1 has effects on adult bone marrow progenitor cells [10, 11], we have investigated whether IGF-1 can be used for ex vivo expansion of committed hematopoietic progenitors derived from umbilical cord progenitor cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Preparation
Umbilical cord blood was collected from normal full-term deliveries. Immediately after dissection of the umbilical cord, 40 ml venous blood was harvested into a syringe containing 4 ml 0.11 M acid citrate dextrose solution (Baxter; Thetford, UK). The blood was then diluted 10-fold in a solution containing 0.9% NH4Cl, 0.1% KHCO3 and 0.0037% tetrasodium EDTA in double distilled water (pH 7.2 at 20°C) to lyse erythrocytes. Cells were centrifuged five min later, and washed twice in phospate buffered saline (PBS) with 2% inactivated fetal calf serum (FCS) (Hyclone Sterile Systems; Logan, UT).

CD34+ cells were purified from the nucleated umbilical cord cells by an immunomagnetic isolation technique as described previously [13]. Briefly, leukocytes were labeled with murine CD34-specific antibodies. The cells were then incubated for 15 min at 4°C before incubation with a secondary anti-murine IgG antibody bound to magnetic beads (Minimacs; Miltenyi Biotech; Auburn, CA). CD34+ cells were thereafter separated on a magnetic column. The percentage of CD34+ cells was estimated by flow cytometric analysis (see below).

Suspension Cultures
Culture medium was Iscove's modified Dulbecco's medium supplemented with 100 U/ml of penicillin and 100 mg/ml of streptomycin (all from BioWhittaker; Walkersville, MD), 200 mg/L human iron saturated holo-transferrin (ICN Biomedicals; Costa Mesa, CA), 5 x 10–5 M 2-mercaptoethanol, 2% w/w bovine serum albumin, and 0.05 mg/ml low-density lipoprotein (all from Sigma; St. Louis, MO).

Recombinant human IGF-1 was purchased from Pepro Tech Inc (Rocky Hill, NJ). The other recombinant human cytokines were used at the following concentrations: GM-CSF 10 ng/ml, (Sandoz; Basel, Switzerland), stem cell factor (SCF) 40 ng/ml, interleukin 3 (IL-3) 10 ng/ml (both from Pepro Tech) and G-CSF 10 ng/ml (Roche; Basel, Switzerland).

The purified CD34+ cells were cultured in quadruplicates at a concentration of 5,000 cells/ml in 24-well tissue culture plates (Costar; Cambridge, MA) with each well containing 1 ml medium. Cultures were incubated for seven days in 5% CO2 in humidified air at 37°C. The cell number was then determined for each well separately using a Coulter Counter (Coulter Electronics; Luton, UK) before staining for flow cytometry. In selected series, the Coulter Counter assessment of total cell number was validated by light microscopy in a Bürker chamber.

Colony Assay
Enriched CD34+ umbilical cord stem cells were cultured for seven days as described above, and the cells were then washed and the concentrations adjusted before cells were dissolved in methylcellulose-containing medium (StemCell Technology; Vancouver, Canada). The cells were seeded at a concentration of 2-6 x 103 cells/well (0.5 ml medium/well) in 24-well tissue culture plates (Costar). Cultures were prepared in triplicates and colonies counted using a cell concentration resulting in 100-200 colonies/well. Colony assays were performed in two different media: one containing conditioned medium as the source of colony-stimulating factor (Methocult H4433, StemCell Technology), and another medium containing recombinant human growth factors (Methocult GF H4434 containing SCF, IL-3, GM-CSF, and erythropoietin). Both media support the growth of colony-forming unit erythroid (CFU-E), colony forming unit-granulocyte-macrophage (CFU-GM), CFU-mixed, and BFU-E. Colonies were scored by light microscopy after 10 and 15 days and the number of colonies were expressed as colonies per 1,000 seeded cells. CFU-E were assayed after 10 days of culture, and after 15 days, the numbers of total erythroid, CFU-GM, and CFU-mix were estimated.

Flow Cytometry

Monoclonal Antibodies (mAbs)   All antibodies were conjugated either to fluorescein-isothiocyanate (FITC) or phycoerythrin (PE). The mAbs used in the study were specific for CD3 (Leu-4), CD19 (Leu-12) (both from Dako; Glostrup, Denmark), CD15 (DU-HL60-3, Sigma), CD64 (M22, Fc{gamma}-receptor I, Medarex Inc.; Annandale, NJ), CD14 (Leu-M3), CD34 (HPCA-2), CD45 (anti-HLe-1), and CD71 (L01.1, transferrin receptor) (all from Becton Dickinson Immunocytometry Systems; San Jose, CA). Isotype control mAbs included anti-keyhole limpet hemocyanin (mouse IgG1 and IgG2a, Becton Dickinson Immunocytometry Systems) and mouse IgM (Sigma).

Cell Staining   Directly fluorochrome-conjugated monoclonal antibodies were added simultaneously at concentrations giving optimal fluorescent staining and minimal cell aggregation. All incubation steps were performed at 4°C for 25 min. Cells were then washed with PBS with 10% inactivated FCS. Immunostained cells were either kept on ice and analysed immediately by flow cytometric analysis or fixed in 0.5% paraformaldehyde and analyzed within 24 h.

Flow Cytometric Analysis   Umbilical cord stem cells were stained simultaneously with an FITC-conjugated CD45-specific antibody and a PE-conjugated CD34-specific antibody. Enumeration of CD 34+ cells was performed as described by Sutherland et al. [14]. Briefly, the incidence of CD34+ cells was determined by "back gating" for CD45 expression and side scatter, and true CD34+ events were characterized by low-density CD45 expression and low side scatter on a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems) ( Fig. 1).



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  		Figure 1. Estimation of CD34+ cells in enriched umbilical cord stem cells by two-color immunofluorescence flow cytometry analysis. Cells from one donor were labeled with PE-conjugated anti-CD34 and FITC-conjugated anti-CD45 monoclonal antibodies. Positive cells were determined by reference to nonspecific staining by isotype control antibodies. The figure shows CD34 PE versus CD45 FITC fluorescence of purified umbilical cord stem cells. These results demonstrate that 97% of the enriched umbilical cord stem cells were CD34+/CD45+ progenitor cells.

 
After in vitro culture, cells were harvested, the total cell number in each well was determined, and cells from parallel wells were pooled and stained for flow cytometric analysis. Cells expressing CD64 were regarded as immature myelomonocytic cells [15], while CD15+ cells were regarded as mature granulocytes or monocytes. Cells were also stained with anti-CD71 antibodies to document erythroid differentiation. In all experiments, a distinct cell population of CD71high/CD64– cells could be demonstrated ( Fig. 2), and in accordance with previous studies performed on fetal bone marrow cells, these CD71high/CD64– cells were regarded as erythropoietic progenitors [15, 16]. Stained cells were analyzed on a Coulter Epics XL flow cytometer (Coulter Electronics).



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  		Figure 2. Analysis of CD64+ and CD71+ cells among in vitro cultured umbilical cord CD34+ cells. Cells were labeled with PE-conjugated anti-CD64 and FITC-conjugated anti-CD71. The CD71high/64– populations were regarded as erythroid, while the 64+ cells were regarded as myelomonocytic. For each sample, 5,000 events were acquired. Positive cells were determined by reference to nonspecific staining by isotypic control. A) Control culture cells stimulated with SCF+IL-3+GM-CSF. B) Addition of IGF-1 200 ng/ml caused an increase in total cell number, but the percentages of CD71high/64– and CD64+ cells were not altered. C) In cultures stimulated with SCF+IL-3+GM-CSF the addition of G-CSF 10 ng/ml induced a significant increase in total cell number and in number and percentage of CD64+ cells. D) When IGF-1 200 ng/ml were added to the stimulation with SCF+IL-3+GM-CSF+G-CSF, an additional increase in total cell number and percentages of CD64+ was detected, but the percentage of CD71high/64– cells was not altered.

 

Viability Analysis   To allow for discrimination of dead cells, samples were incubated with propidium iodide ([PI], Immunotech; Marseille, France) at a final concentration of 2 mg/106 cells. For each sample 10,000 events were acquired.

Morphological Examination
The morphology of in vitro cultured cells was examined by light microscopy of May-Grünwald Giemsa stained cytospin preparations. Cells were classified according to generally accepted criteria as undifferentiated blast cells, erythroid precursors, or cells with cytoplasmic granulation consistent with neutrophil differentiation. The neutrophils were subclassified as either immature (promyelocytes/myelocytes/metamyelocytes) or mature forms (band forms and polymorphonuclear cells).

After harvesting of the nonadherent cells, the number of adherent cells per well was determined by light microscopy of May-Grünwald Giemsa-stained wells.

Analysis of G-CSF Production
G-CSF concentrations were determined in supernatants using ELISA assay (Quantikine ELISA kits; R&D Systems Europe; Abingdon, UK). All assays were performed strictly according to the manufacturer's instructions. Briefly, standard samples were prepared in culture medium and standard curves then determined using the mean of duplicate determinations. The minimal detectable G-CSF concentration was 7.2 pg/ml.

Presentation of the Data
Number of cells after culture was expressed as fold expansions of the seeded 5,000 cells per well (number of cells after culture relative to the number of seeded cells). The data were analyzed using a two-tailed Student's t-test for paired samples. p-values were corrected for the number of comparisons and differences were regarded as statistically significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of Purified CD34+ Cells
Proliferation of CD34+ umbilical cord stem cells in suspension cultures was investigated for seven individuals. Immunomagnetic purification of CD34+ umbilical cord blood stem cells resulted in a highly enriched population as determined by flow cytometric analysis (n = 7; mean number of CD34+ cells 95%, variation 89%-99%). The results from one typical experiment are presented in Figure 1. Less than 5% of enriched CD34+ cells expressed the differentiation markers CD3, CD14, CD15, CD19, CD64, and CD71. The fraction of dead cells as determined by PI uptake was less than 5% when tested after CD34+ cell enrichment.

Effects of IGF-1 on Cytokine-Dependent Proliferation of Umbilical Cord Stem Cells
CD34+ umbilical cord cells did not proliferate when cultured in medium alone or in medium only containing IGF-1. In contrast, when exogenous SCF 40 ng/ml + IL-3 10 ng/ml + GM-CSF 10 ng/ml were added to the cultures, the number of cells increased significantly during seven days of in vitro culture. The average cell proliferation corresponded to 15.9-fold expansions of the seeded 5,000 cells/well (n = 7, range 4.2- to 43.2-fold expansion; p = 0.023, see also Fig. 3). The cell viability after seven days of culture in the presence of these exogenous cytokines corresponded to 86%-90% as evaluated by PI uptake (data not shown). In all the following experiments, umbilical cord CD34+ cells were cultured in the presence of SCF+IL-3+GM-CSF, and this is referred to as cytokine-dependent proliferation.



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  		Figure 3. Effect of IGF-1 with or without G-CSF 10 ng/ml on the proliferation of committed progenitors from umbilical cord stem cells. The total cell number, numbers of CD71high/64– cells, CD15+ cells, CD34+, and CD64+ cells after seven days of culture with SCF+IL-3+GM-CSF are expressed as a percentage of total cell number in the control. In the absence of G-CSF, the controls were cultured with SCF+IL-3+GM-CSF, and the corresponding control cultures contained SCF+IL-3+GM-CSF+G-CSF, respectively. The figure presents data of four separate experiments (four separate umbilical cords stem cell donors), and the results are presented as the means + standard deviation.

 
Cytokine-dependent stem cell proliferation was compared for cultures with and without IGF-1 in seven experiments. Addition of exogenous IGF-1 (2, 20, 200 ng/ml) caused a dose-dependent enhancement of cytokine-dependent umbilical cord stem cell proliferation in all experiments. The results for four of these experiments are included in Figure 3. For IGF-1 200 ng/ml, the mean increase in overall cell number corresponded to 31.7-fold expansions (variation range 15.1- to 59.5-fold expansions; Student's t-test; p = 0.02 when compared with cultures without IGF-1), and the proliferation could not be further increased by adding IGF-1 600 ng/ml (data not shown). In the following experiments we only investigated IGF-1 2, 20, and 200 ng/ml.

A high percentage (>86%) of viable cells was detected for all these cultures (data not shown).

Effects of IGF-1 + G-CSF on Umbilical Cord Stem Cell Proliferation
G-CSF could not be detected (<7.2 pg/ml) in culture supernatants when umbilical cord CD34+ cells were cultured in the presence of SCF+IL-3+GM-CSF, and addition of IGF-1 did not induce detectable G-CSF levels (data not shown). The presence of G-CSF 10 ng/ml significantly increased cytokine-dependent progenitor cell proliferation, and the mean cell number after seven days of culture corresponded to 22.9-fold expansions of the seeded 5,000 cells per well (n = 6, range 10.1- to 50.3-fold expansion, Student's t-test; p = 0.0001). The effect of IGF-1 (2, 20, and 200 ng/ml) on total cell proliferation was investigated for umbilical cord stem cells cultured in the presence of G-CSF+SCF+IL-3+GM-CSF for seven days. Umbilical cord cells derived from six donors were studied. IGF-1 had a dose-dependent stimulatory effect on cytokine-dependent proliferation also in the presence of exogenous G-CSF, and IGF-1 increased the number of cells for all the donors. The results from four of these experiments are also included in Figure 3. For IGF-1 200 ng/ml, the mean increase in overall cell number corresponded to 50.1-fold expansions (range 35.7- to 80.3-fold expansions; Student's t-test; p = 0.007 when compared with corresponding cultures without IGF-1).

In Vitro Effects of IGF-1 on Differentiated Umbilical Cord Stem Cells
The percentages of cells expressing differentiation markers were determined after seven days of cytokine (SCF+IL-3+GM-CSF)-dependent proliferation of umbilical CD34+ cells, and results were compared for cultures with and without IGF-1. The percentages of different cell subsets among cultured umbilical cord CD34+ cells are presented in Table 1, and the cell numbers of different subsets are shown as percentage of controls in Figure 3. No major difference in the percentages of CD15+, CD64+, or CD71high/CD64– cells was detected when comparing cultures with and without IGF-1, although in the presence of IGF-1 200 ng/ml, a small but significant increase in the percentage of cells expressing the marker CD15 was observed (n = 4, Student's t-test; p = 0.007). In the absence of G-CSF, neither the number nor the percentages of CD64+ cells were significantly altered by IGF-1 2-200 ng/ml ( Fig. 3).


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  		Table 1. The effects of IGF-1 on differentiated (CD15+, CD64+, CD71high/CD64–) hematopoietic cells derived from cultured umbilical cord CD34+ cells
 
CD34+ cells could still be detected after seven days of cytokine-dependent culture (16%-37%). Both for cultures with and without IGF-1, less than 5% of the cells expressed the monocyte marker CD14, and the lymphocyte markers CD3 and CD19 were detected on less than 2% of the cultured cells (data not shown).

The majority of cells (>70%), still had a morphological appearance consistent with immature hematopoietic cells even when examined after seven days of cytokine-dependent proliferation. Cells with a morphological appearance consistent with erythroid differentiation or mature granulocytes constituted less than 5% of the cells, and morphological signs of megakaryocyte differentiation could not be detected. No difference was detected between cultures with and without IGF-1. Light microscopy of culture wells demonstrated fewer than 100 adherent cells per well, both for cultures with and without IGF-1 (data not shown).

Differentiation During Cytokine-Dependent Growth of Umbilical Cord Stem Cells, Effects of IGF-1+G-CSF
When umbilical cord stem cells were cultured in vitro for seven days in the presence of G-CSF 10 ng/ml and SCF+IL-3+GM-CSF, the number of CD15+ cells ( Fig. 3, n = 4, Student's t-test; p = 0.02) and CD64+ cells ( Fig. 3, n = 4, p = 0.001) were significantly enhanced, but number and percentage of erythropoietic cells (CD71high/64– cells) were unaltered. Addition of IGF-1 (2, 20, 200 ng/ml) in conjunction with G-CSF caused a further dose-dependent and statistically significant increase in the number of CD64+ and CD15+ cells compared with cultures containing only G-CSF although the increase in CD64 expression was small ( Fig. 3, Table 1). CD34+ cells could still be detected after in vitro culture with G-CSF alone and G-CSF+IGF-1 (range 30%-64%).

An increased percentage showing morphological signs of neutrophil differentiation was detected in the presence of G-CSF, but mature neutrophil forms and erythroid cells still constituted less than 5% of the cells. Morphological signs of megakaryocyte differentiation could not be observed in any cultures. Fewer than 100 adherent cells were counted per well in the presence of G-CSF. Addition of IGF-1 together with G-CSF did not cause any alteration in number of adherent cells.

Colony-Forming Cells in Expanded Umbilical Cord Stem Cells
Enriched CD34+ cells derived from three donors were expanded for seven days before colony formation was assayed. The cells were expanded in IL3+SCF+GM-CSF alone or with these three cytokines and additional IGF-1 (20, 200, and 600 ng/ml) or IGF-1+G-CSF 10 ng/ml. Addition of IGF-1 did not alter the number of CFU-E compared with corresponding control cultures (data not shown).

The results for total cell number and total number of colonies, BFU-E, CFU-GM, and CFU-Mix for one of the donors are presented in Table 2. Either unaltered or decreased frequencies of the different colony-forming cells (BFU-E, CFU-GM-CFU-mix) were observed in the presence of IGF-1. Thus, the major determinant for the increased cell numbers in the presence of IGF-1 and/or G-CSF is expansion of lineage-committed cells with a decreasing frequency of colony-forming progenitors. However, despite this preferential expansion of differentiated cells, a high number of colony-forming cells was maintained during culture. This is illustrated by the unaltered total number of colony-forming cells in the presence of IGF-1/G-CSF. Similar results were observed for all three cell donors and for both colony-forming assays.


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  		Table 2. Colony-forming cells after cytokine-dependent expansion of CD34+ umbilical cord stem cells; studies of IGF-1 effects
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have demonstrated that IGF-1 can enhance in vitro growth of human hematopoietic progenitor cells derived from normal bone marrow [10]. Although IGF-1 receptor expression on umbilical cord stem cells has not been demonstrated, our present study demonstrates that IGF-1 has an enhancing effect also on the in vitro proliferation of hematopoietic progenitor cells derived from umbilical cord CD34+ cells. This enhancing effect could be demonstrated in the presence of various combinations of exogenous cytokines. Although both in vitro characterization and clinical experience from allogeneic stem cell transplantation indicate that functional differences exist between umbilical cord and normal bone marrow CD34+ cells [17], the effects of IGF-1 thus seem to be similar for these two stem cell populations. In our present experiments the combination of IGF-1 and G-CSF induced the highest increase both in the total cell number and in the number of differentiated myelopoietic cells.

We investigated IGF-1 effects on cytokine dependent progenitor cell proliferation using a modified version of the well-characterized in vitro models developed by Olweus et al. [15, 16, 18]. In all the experiments, the umbilical cord CD34+ cells were cultured in a defined serum-free medium supplemented with SCF+IL-3+GM-CSF [18], and our present results can therefore be compared with previous studies of stem cells derived from other tissues.

In most of our experiments we investigated IGF-1 at the concentrations 2, 20, and 200 ng/ml. These concentrations were chosen because: A) increasing IGF-1 up to 600 ng/ml did not further increase total cell proliferation, and B) taking into account the serum-free conditions, the concentrations correspond to the variation in normal serum levels of free IGF-1 and with 200 ng/ml exceeding the upper limit of the normal serum levels (see detailed discussion below). In all cultures, a distinct population of CD71high/64– and CD71high/15– cells was easily detectable. Culture progeny of bone marrow progenitors with the corresponding phenotype has been demonstrated to consist of erythroid progenitors [15, 16]. Previous studies have demonstrated that IGF-1 stimulates proliferation of erythroid progenitors derived from bone marrow [11], and the present results demonstrate that IGF-1 has a similar effect on erythroid CD71high/CD64– progenitors derived from umbilical cord stem cells.

Signs of differentiation with expression of both myeloid and erythroid markers were detected after cytokine-dependent culture of the umbilical cord cells. However, erythroid or mature neutrophil forms could not be recognized by morphological criteria. Thus, only limited signs of differentiation could be induced in our in vitro model, and this was similar both for cultures with and without IGF-1.

IGF-1 did not induce proliferation of progenitors expressing monocyte (CD14) or lymphocyte (CD3, CD19) markers. However, our results do not exclude an IGF-1 effect on the proliferation of lymphoid progenitors, because such IGF-1 effect may depend on the presence of other cytokines with the capability to induce the early events of differentiation towards lymphocytes [19, 20].

The normal serum IGF-1 levels increase from 20 to 200 ng/ml in infants to 200-1,000 ng/ml in the last part of puberty, and the serum levels gradually decline thereafter [21]. Serum levels of IGF-1 in allogenic stem cell transplant recipients do not differ from those of normal individuals [22, 23]. The level of free IGF-1 corresponds to 1%-2% of the total serum level [24]. Although the effects of IGF-1 described in the present study were detected at concentrations corresponding to normal serum levels (2-200 ng/ml), they were observed under serum-free conditions, and similar serum levels of free IGF-1 would be expected to occur only in adolescents and young adults.

The rate of engraftment correlates with the number of transplanted umbilical cord stem cells per kilogram body weight of the recipient [4]. One major problem in the clinical use of cord cells for allotransplantation in adults is therefore late engraftment due to the limited number of available cells. The majority of patients transplanted with umbilical cord cells have therefore been children, although successful engraftment has also been reported in adults [4, 25]. After seven days of in vitro culture, we achieved up to 59.5-fold expansions of the seeded umbilical cord stem cells. By using different cytokine combinations and two weeks of in vitro culture, up to 200-fold expansions of human cord-blood derived progenitors can be achieved [5, 26]. However, taking into account the high viability of our cells after culture, the expansion achieved in our system should be sufficient to allow transplantation in a majority of adult patients when used in combination with an optimal method for stem cell cryopreservation [27]. The advantage of our in vitro system is that this is a well-characterized serum-free model where stem cells can be expanded in a well-characterized microenvironment [15, 16].

Due to the late engraftment after allogeneic umbilical cord stem cell transplantation, prophylactic platelet transfusions have to be given for a prolonged period to reduce the risk of serious bleedings [4]. However, prophylactic therapy to reduce the risk of complicating infections during leucopenia is less effective. It has therefore been suggested that a part of the umbilical cord stem cell graft should be cultured ex vivo prior to transplantation to increase the number of committed progenitors which would become responsible for an earlier hematopoietic reconstitution [9]. These committed progenitors should then be transplanted together with native umbilical cord stem cells which would be responsible for the long-term engraftment [9]. Our results indicate that IGF-1 in combination with other cytokines can be used for expansion of both erythroid and myeloid progenitors derived from umbilical cord stem cells.

Long-term hematopoietic reconstitution after cord stem cell transplantation would be expected to depend on the presence of noncommitted stem cells in the allograft. After seven days of culture, a majority of umbilical cord cells expressed lineage-specific membrane molecules, but a CD34+ population could still be detected ( Fig. 3). These results indicate that a population of CD34+ stem cells is conserved even after in vitro expansion. This is also supported by the observation that a high number (although a decreased frequency/percentage) of colony-forming cells were detected in the expanded cell population. However, further studies are clearly required to characterize the noncommitted umbilical cord stem cells which remain after in vitro culture with IGF-1. These studies should probably include investigation of different subsets of CD34+ cells as well as the ability of expanded cells to reconstitute hematopoiesis in nude mice [28, 29].

We conclude that IGF-1 stimulates cytokine-dependent proliferation of lineage-committed umbilical cord progenitor cells. These results indicate a potential role for IGF-1 in ex vivo manipulation of umbilical cord progenitors prior to allotransplantation.


   Acknowledgments
 
The study was supported by the Norwegian Cancer Society.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication August 18, 1998.



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