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First published online June 13, 2005
Stem Cells Vol. 23 No. 8 September 2005, pp. 1170 -1179
doi:10.1634/stemcells.2004-0322; www.StemCells.com
© 2005 AlphaMed Press

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Growth Hormone–Induced Stimulation of Multilineage Human Hematopoiesis

Mary B. Hanleya, Laura A. Napolitanoa,b, Joseph M. McCunea,b,c

a Gladstone Institute of Virology and Immunology, San Francisco, California, USA;
b Department of Medicine, University of California, San Francisco, California, USA;
c Department of Microbiology and Immunobiology, University of California, San Francisco, California, USA

Key Words. Hematopoiesis • Growth factor • Hematopoietic progenitor • In vitro • Marrow stromal cells • Bone marrow cells

Correspondence: Joseph M. McCune, M.D., Ph.D., Gladstone Institute of Virology and Immunology, 1650 Owens Street, San Francisco, California 94158-2261, USA. Telephone: 415-734-5060; Fax: 415-826-8449; e-mail: mmccune{at}gladstone.ucsf.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Growth hormone (GH) has been shown to have significant positive effects on hemato-lymphopoiesis in rodent models and, more recently, to increase thymic mass and circulating naïve CD4+ T cells in humans infected with the human immunodeficiency virus, type 1. To determine whether the latter effects on human T lymphopoiesis might be due, at least in part, to effects on the bone marrow (BM), we examined the specific effects of GH and its proximal mediator, insulin-like growth factor I (IGF-I), on human multilineage hematopoiesis in fetal BM (FBM). Using in vitro analysis, we found that GH and IGF-I each stimulated the expansion of primitive multilineage CD34+CD38– hematopoietic progenitor cells and increased yields of several hematopoietic subpopulations, including CD34+CD38+CD10+ lymphoid progenitor cells. Additionally, GH and IGF-I had direct effects on FBM stromal elements, inducing the expansion of myeloid-like CD45+CD14+ FBM stromal cells and enhancing production of the hematopoietic cytokine interleukin-3 by fibroblast-like CD45–CD10+ FBM stromal cells. Surface expression of GH and type-I IGF receptors correlated with the observed biologic responses to these hormones. Whereas GH enhanced the proliferation of FBM progenitors and stroma, IGF-I exerted a predominantly antiapoptotic effect. Finally, both GH and IGF-I stimulated the generation of hematopoietic colony forming cells. These findings identify specific targets of GH and IGF-I within human FBM, and demonstrate direct and indirect effects that may contribute to GH-mediated enhancement of human hemato-lymphopoiesis.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Growth hormone (GH) and its proximal mediator, insulin-like growth factor I (IGF-I), have been shown to play an important role in T lymphopoiesis [1]. Administration of GH or IGF-I reverses thymic involution and enhances T lymphopoiesis in older rodents [2–4] and accelerates immune reconstitution in immunodeficient animals [2, 4–7]. Recently, we extended these observations to humans, demonstrating that GH treatment is associated with increases in thymic mass and circulating naïve CD4+ T cells in adults infected with human immunodeficiency virus, type 1 (HIV-1) [8]. These data suggest that de novo T-cell production may be inducible in immunodeficient humans.

T lymphopoiesis is dependent upon the migration of prethymic bone marrow (BM) progenitor cells to the thymus [9], and age-related declines in T lymphopoiesis have been attributed to both decreased hematopoietic capacity of BM progenitors and involution of the thymus (reviewed in Miller [10] and Globerson [11]). Thus, GH may enhance human T lymphopoiesis by acting on cellular targets in the BM and/or in the thymus. BM appears to be an important target of GH action, as demonstrated by rodent studies showing a significant effect of GH on multilineage hematopoiesis. In studies of mice and aging rats, administration of GH and IGF-I facilitates early stages of T-cell development by increasing the number of multilineage BM progenitors [12, 13] and by enhancing the migration and engraftment of progenitor cells into the thymus [2, 4, 14]. Similarly, in vitro analyses showed that GH stimulates erythroid [15, 16] and myeloid [17] colony formation from murine and human BM progenitors. These effects appear to be mediated, at least in part, by IGF-I [16–18].

In the current study, we sought to more specifically identify cellular targets of GH and IGF-I action within the human fetal BM (FBM). We hypothesized that GH may stimulate hematopoiesis either through direct effects upon multilineage or lineage-restricted hematopoietic progenitor cells or, indirectly, by inducing FBM stromal cells to produce cytokines that facilitate the survival or maturation of FBM progenitors. We found that GH and IGF-I have direct effects on the proliferation and survival of both multilineage and lineage-committed progenitor cells, and that these hormones also enhance cytokine production by FBM stroma. These findings further delineate the effects of GH and IGF-I within human BM and provide insight into mechanisms that may contribute to GH– and IGF-I–mediated enhancement of hemato-lymphopoiesis.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
FBM Progenitor Cells
Human fetal bones (20–24 weeks’ gestation) were acquired in RPMI media (Mediatech, Inc., Herndon, VA, http://www.cell-gro.com/shop/customer/home.php) from Advanced Biosciences Resources (Alameda, CA). The mononuclear cell layer was isolated by centrifugation on a ficoll (Histopaque-1077) (Sigma Chemical Co., St. Louis, http://www.sigmaaldrich.com) gradient and resuspended in Iscove’s Modified Dulbecco’s Medium (IMDM; Mediatech) containing 2% bovine serum albumin (BSA; Sigma Chemical Co.), 10 µg/ml transferrin (Gibco, Carlsbad, CA, http://www.invitrogen.com), 50 U/ml penicillin (Gibco), 50 µg/ml streptomycin (Gibco), and 2 mM L-glutamine (Gibco) (BT medium). FBM mononuclear cells were seeded in six-well plates (BD Falcon, San Jose, CA, http://www.bdbiosciences.com) at 1–1.5 x 106 cells per ml in 2 ml of BT medium. Recombinant human GH (Research Diagnostics, Inc., Flanders, NJ, http://www.researchd.com, or Serono, Rockland, MA, http://www.serono.com) or recombinant human IGF-I (R&D Systems, Minneapolis, http://www.rndsystems.com) was added at concentrations ranging from 0–250 ng/ml. In concordance with previous in vitro studies [16, 19], the optimal dosage to generate consistent effects in our experiments was found to be 100 ng/ml, and this is the dosage that is usually reported in the text. For cultures maintained longer than 4 days, 1 ml of supplemental medium was added on day 4. In experiments using enriched CD34+ cells, positive selection of CD34+ cells from freshly isolated FBM mononuclear cells was performed by indirect isolation using the MiniMACS system (Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com) according to manufacturer’s instructions. Upon reanalysis post isolation, the CD34 purity was always greater than 90%.

FBM Stromal Cells
Mononuclear cells from FBM were plated in IMDM with 15% fetal bovine serum (FBS; Gemini Bio-Products, Woodland, CA, http://www.gembio.com), 15% horse serum (Gibco), 1 µM 2-mercaptoethanol (Sigma Chemical Co.), 1 µM hydrocortisone (Sigma Chemical Co.), 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine. Subconfluent layers of primary stromal cells were split by trypsinization and plated in DMEM (Mediatech) containing 10% FBS at a density of 1–3 x 105 cells per well in a six-well plate. FBM stromal cells were allowed to adhere overnight prior to addition of GH or IGF-I. For analysis of GH– or IGF-I–treated stroma, cells were trypsinized and washed in phosphate-buffered saline (PBS) prior to phenotypic analysis.

Phenotypic Analysis of FBM Cells
Four-color phenotypic analysis was performed on fresh lyisolated FBM cells, using fluorochrome-conjugated monoclonal antibodies (MAbs) antiCD19 and antiCD14 (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com); antiCD38 and antiCD34 (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen); antiCD10 (Caltag, Burlingame, CA, http://www.caltag.com), and antitype I IGF receptor (IGFR) (R&D Systems). Fluoresceinated mouse IgG1 isotypic controls (BD Biosciences) were used to set gates. Cells were first preincubated for 10 minutes on ice with 1 mg/ml human gamma globulin (Gemini Bio-Products) to block nonspecific Fc receptor binding. After incubation with antibodies for 30 minutes at 4°C in the dark, the cells were washed in PBS containing 2% FBS (PBS-FBS), resuspended, and fixed in 1% paraformaldehyde (Sigma Chemical Co.). A minimum of 10,000 events was collected for analysis. Samples were analyzed within 48 hours by flow cytometry (FACSCalibur; BD Biosciences). For phenotypic analysis of stromal cells, cells were first trypsinized using 0.05% trypsin with 0.53 mM EDTA (Gibco) and then washed with PBS-FBS prior to antibody incubation. In addition to the monoclonal antibodies listed above, antiCD45 (BD Pharmingen) was also included.

GH Receptor Staining
A MAb specific for GH receptor (GHR) (MAb-263; Research Diagnostics, Inc.) was biotinylated using the Mini-Biotin-XX protein labeling kit (Molecular Probes, Eugene, OR, http://probes.invitrogen.com). Cells were incubated with the biotinylated GHR MAb or a biotinylated IgG isotype (DakoCytomation, Carpinteria, CA, http://www.dakocytomation.us), each at 10 µg/ml, for 30 minutes at 4°C. After washing in PBS-FBS, a directly conjugated secondary reagent (either fluorescein isothiocyanate [FITC]– or allophycocyanin-conjugated streptavidin [BD Biosciences]) was added at 1:50 along with directly conjugated MAbs to other phenotypic lineage markers (see above). A minimum of 50,000 events was collected for analysis.

Proliferation Measurement
5-Bromo-2'-deoxyuridine (BrdU; Roche Applied Science, Indianapolis, http://www.roche-applied-science.com) was added (final concentration 100 µM) 18–24 hours prior to harvest of FBM or FBM stromal cultures. Cells were first stained for phenotypic surface markers, as described above, and then fixed and permeabilized in a solution containing 0.1% Tween-20 (USB, Cleveland, http://www.usbweb.com/index.asp?flash=Y) and 1% paraformaldehyde, for 1 hour at room temperature (RT) in the dark. After washing with PBS-FBS, antiBrdU FITC containing DNase (BD Biosciences) was added for 30 minutes at RT in the dark. As a negative control, cells were incubated without BrdU. A minimum of 10,000 events was collected for analysis.

Apoptosis Measurement
Apoptotic cells were visualized using annexin V-green fluorescent protein (GFP) (kindly provided by Joel Ernst, New York University). Cell suspensions were incubated first in PBS with 2% BSA and 1.5 mM CaCl2 (Sigma Chemical Co.) with fluoresceinated phenotypic MAbs, as described above, and then with annexin V-GFP (0.38 µg/ml final concentration) for 10 minutes. Cells were washed twice, resuspended, and fixed in 1% paraformaldehyde prior to acquisition on the BD FACSCalibur. A minimum of 10,000 events was collected for analysis.

IGF-I Enzyme-Linked Immunosorbent Assay
Culture supernatants were assayed using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) to quantitate levels of IGF-I.

Intracellular Cytokine Detection
Trypsinized stromal cells were resuspended in media containing 10% FBS and incubated with 10 µg/ml brefeldin A (Sigma Chemical Co.) for 4–5 hours at 37°C. After washing with cold PBS, cells were labeled for phenotypic markers using fluoresceinated MAbs (as described above), and then resuspended in 2% paraformaldehyde for 5 minutes at 37°C. After washing with PBS-BSA, cells were incubated in BD FACS Lysing solution followed by BD FACS Permeabilizing solution. Intracellular cytokines were visualized using phycoerythrin-conjugated antibodies against interleukin (IL)-6 (BD Biosciences) and IL-3 (BD Pharmingen), or with biotinylated antibodies against IL-7 and stem cell factor (SCF) (both from R&D Systems). After washing, biotinylated antibodies were followed by incubation with FITC-conjugated streptavidin. After 30 minutes at RT, cells were again washed and resuspended in 1% paraformaldehyde for acquisition and analysis. A minimum of 50,000 events was collected for analysis.

Colony-Forming Units-Culture Assay (CFU-C)
Hematopoietic colony-forming cells were enumerated in semi-solid methylcellulose cultures. FBM cells post culture were added to methylcellulose medium (Methocult; Stem Cell Technologies, Vancouver, British Columbia, Canada, http://www.stemcell.com/default.asp) supplemented with 2 U/ml erythropoietin, 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF), 100 ng/ml SCF, 10 ng/ml IL-3, and 10 ng/ml IL-6 (all from R&D Systems). After 14 days at 37°C, triplicate plates were scored for colony-forming units-granulocyte, macrophage (CFU-GM), burst-forming units-erythroid (BFU-E), and colony-forming units-granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM), using standard criteria for their detection with an inverted microscope (Nikon, Tokyo, http://www.nikon.com) [20].

Data Analysis
Flow cytometric analysis was performed using FlowJo (Tree Star, Inc., San Carlos, CA, http://www.treestar.com). Statistical analysis was performed using StatView 5.0 (SAS Institute, Inc., Cary, NC, http://www.sas.com). p values were determined using the Mann-Whitney U-test (in cases in which data were pooled from different experiments) or the unpaired Student’s t-test (in cases in which samples from individual experiments were compared).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH Stimulates Proliferation of Primitive Multilineage Hematopoietic Progenitor Cells
To identify potential GH targets within the FBM, the expression of cell-surface GHR was assessed using a biotinylated antiGHR antibody on freshly acquired FBM mononuclear cells. Various subpopulations of multilineage and lineage-restricted hematopoietic progenitor cells were discriminated on the basis of their phenotypic markers, as described in Figure 1Go. The frequency of each of these subpopulations in the FBM was determined for six donors (Table 1Go). As shown in Figure 2AGo (left panel), GHR was expressed on a sizeable fraction of total FBM mononuclear cells (mean 19.4%, range 15%–28.8% in seven independent experiments). Using antibodies against the lineage markers shown in Figure 1Go, we found that these progenitor subpopulations expressed varying levels of GHR (Fig. 2AGo). Incubation of total FBM mononuclear cells or CD34+ cells with GH for at least 4 days resulted in a higher yield of total cells relative to untreated controls (p < .05) (Fig. 2BGo, left panel). Amongst the subpopulations included within FBM, statistically significant increases were observed in the primitive CD34+CD38– multilineage progenitors as well as the CD34+CD38+CD10+ lymphoid progenitors (Fig. 2BGo). There was also a strong positive correlation between the fraction of cells expressing GHR within a given subpopulation and the subsequent recovery of that subpopulation after the addition of GH (Fig. 2CGo).



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  		Figure 1. CD34+ progenitor cell subpopulations in FBM. (A): Flow diagram representing multilineage human hematopoiesis. Cell surface markers designate those used to identify the different progenitor cell populations [22, 34, 42]. (B): Flow cytometry plots show gating strategy used for phenotypic data collection. The first panel represents gating for CD34/CD38 populations, after gating on mononuclear cells gated by forward and side scatter (not shown). The large dashed box shows the total CD34+ gate. This CD34+ population was subdivided using CD38 (small boxes, labeled 34+38– and 34+38+). The CD34+CD38+ population was first subdivided with CD10 (next panel) into CD34+CD38+CD10+ and CD34+CD38+CD10– subpopulations. The remaining panels show gating used to discriminate CD34+CD19+, CD34+CD14+, and BrdU+ cell subpopulations, respectively, after first gating on mononuclear cells (not shown). Abbreviations: BFU-E, burst-forming unit-erythroid; CFU-GEMM, colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte; CFU-GM, colony-forming unit-granulocyte-monocyte; DC, dendritic cell; FBM, fetal bone marrow; NK, natural killer.

 

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  		Table 1. Frequency of FBM progenitor subpopulations
 


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  		Figure 2. Effects of GH on FBM progenitor cells. (A): Expression of GHR (shaded histograms) relative to isotype control staining (open histograms) after first gating on a mononuclear cell gate. These results are representative of seven independent experiments carried out on FBM progenitor cells from seven different donors. The numbers above the bars represent the percentage of GHR+ cells, that is, the percentage of cells in given subpopulation with levels of GHR staining above those found with the isotype control. (B): Effect of GH on the yield of cells within different FBM subpopulations as a mean percentage of untreated control cultures (unfilled bars). An increase in the absolute number of many subpopulations is seen after the addition of GH (100–250 ng/ml for 4–8 days) (filled bars) to either total FBM mononuclear cells (BM) or purified CD34+ cells (*p < .05). No difference was noted between using 100 ng/ml of GH versus 250 ng/ml; these two concentrations have accordingly been grouped for the analyses shown here. These results are pooled from seven independent experiments carried out on FBM cells from seven different donors. (C): Relationship between the percentage of GHR+ cells in different hematopoietic subpopulations (as shown in A) and the mean cell yield (as shown in B) after incubation of CD34+ cells with GH (r2 = 0.767, p < .0001). (D): BrdU incorporation of CD34+38– cells after stimulation of total FBM or CD34+ cells with GH (100–250 ng/ml for 4–7 days) (filled bars) compared with untreated controls (unfilled bars). Data are pooled from six independent experiments using six different donors and are represented as a percentage of control. The increase is significant after stimulation of CD34+ cells (*p < .05). Abbreviations: BM, bone marrow; FBM, fetal bone marrow; GH, growth hormone; GHR, growth hormone receptor.

 
GH-associated increases in the total number of hematopoietic progenitor cells could be due to enhanced proliferation or decreased levels of apoptosis. To discriminate between these possibilities, total FBM mononuclear cells or CD34+ cells were treated with GH and analyzed by flow cytometry to detect proliferation (BrdU incorporation) and apoptosis (annexin-V binding). Levels of proliferation were significantly increased in the CD34+CD38– subpopulation after GH stimulation of CD34+ cells (Fig. 2DGo), whereas none of the FBM subpopulations showed significant changes in apoptosis (data not shown).

To confirm that GH stimulation resulted in an increase in functionally competent human hematopoietic progenitor cells, total FBM mononuclear cells were cultured for 6 days in the presence or absence of GH and then plated into methylcellulose cultures. As shown in Table 2Go, GH treatment resulted in a statistically significant (p < .05) increase in the number of CFU-GM and CFU-GEMM, but no change in the number of BFU-E.


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  		Table 2. GH and IGF-I effects on CFU-Cs
 
GH Stimulates Proliferation of FBM Stromal Cells
The above data indicate that GH may interact directly with and induce the proliferation of primitive multilineage human hematopoietic progenitor cells as well as of lineage-restricted progenitor cells. Given the high expression levels of GHR on CD34+CD14+ myeloid progenitor cells, some of which might mature into FBM stromal macrophages [21, 22], we hypothesized that GH might also indirectly regulate hematopoiesis through the FBM stroma. As a first test of this hypothesis, expression levels of GHR were assessed on recently plated (<four passages) FBM stromal cells. Analyses by our lab (data not shown) and others [23] have found that the majority of BM stromal cells are positive for intracellular GHR. To identify those cells that might be most responsive to GH, we sought to examine surface expression of GHR in the hematopoietic (CD45+) and nonhematopoietic (CD45–, which includes fibroblasts, adipocytes, and epithelial cells) compartments of the FBM stroma. In cultures from nine different donors, a variably sized (mean 3.4%, range 0.91%–11.5%) subpopulation of cells expressing surface GHR (Fig. 3AGo, left panel) was observed. Many of these GHR+ cells were also positive for the hematopoietic lineage marker, CD45 (Fig. 3AGo, left panel). IGFR (Fig. 3BGo, middle panel) and CD14 (macrophages) [24] (Fig. 3BGo, right panel) were highly expressed on the surface of these GHR+CD45+ stromal cells. Additionally, some GHR+ cells were found to be mature stromal cells (CD10+CD45–; n = 4, mean 21.2%, range 9%–39%, data not shown) that were fibroblast-like in appearance.



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  		Figure 3. Effects of GH on FBM stromal cells. (A): Representative surface staining of GHR on FBM stromal cells (n = 9, a mean 3.4% of total, range 0.91%–11.5%, were positive for GHR) (second panel). Gates were set using the isotype control (left panel). Many of the GHR+ cells were also CD45+ (n = 9, mean 65.3%, range 25%–97%). GHR+CD45+ FBM stromal cells are IGFR+ (n = 3, range 76%–99%) (solid line) whereas GHR+CD45– FBM stromal cells are IGFR low or negative (dashed line) (third panel). CD45+GHR+ FBM stromal cells are also CD14+ (n = 3, range 68%–93%) (solid line, right panel). (B): Incubation of FBM stromal cells with GH (100 ng/ml) results in an increase in the total number of total FBM stromal cells, stromal cells that are GHR+IGFR+CD45+, and stromal cells that are CD14+IGFR+CD45+. These panels represent pooled data from four separate experiments. Cell yields in GH-treated cultures are expressed as a mean percentage of untreated control cultures. (C): BrdU incorporation into the FBM stromal cell populations after 1 day in culture with GH addition (100 ng/ml). *p < .05 in (B, C). Abbreviations: FBM, fetal bone marrow; GH, growth hormone; GHR, growth hormone receptor; IGFR, antitype I insulin-like growth factor receptor.

 
Incubation of FBM stromal cultures with exogenous GH for 1 day resulted in an increased yield of stromal cells relative to untreated control cultures (p < .05) (Fig. 3BGo). Additionally, there was an increase of GHR+IGFR+CD45+ and CD14+IGFR+CD45+ stromal cells compared with untreated controls (Fig. 3BGo). Labeling studies with BrdU or annexin-V showed that these increases in cell yield were associated with increased levels of proliferation (p < .05) (Fig. 3CGo) without changes in apoptosis (data not shown). Thus, GH appears to induce the proliferation of myeloid-like FBM stromal cells.

IGF-I Effects on Human Multilineage Hematopoiesis
GH effects are often mediated through IGF-I [16, 25]. Indeed, in three FBM stromal cultures treated with exogenous GH (100 ng/ml), a significant increase (p < .05) in IGF-I production was observed relative to untreated controls (999 pg/ml versus 1,421 pg/ml, respectively; data not shown). These findings suggest that the effects of GH might be partly attributable to IGF-I.

To determine potential cellular targets of IGF-I, IGFR expression was assessed on FBM mononuclear and stromal cells. Surface expression of IGFR was detected on CD45+CD14+ myeloid stromal cells (see above, Fig. 3AGo) and on a large fraction of FBM progenitor cells (Fig. 4AGo). Additionally, mature CD10+CD45– fibroblast-like stromal cells expressed a low level of IGFR (data not shown).



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  		Figure 4. Effects of IGF-I on FBM progenitor cells. (A): Expression of IGFR (shaded histograms) on FBM mononuclear subpopulations relative to isotype control staining (open histograms). These results are representative of three independent experiments carried out on FBM cells from three different donors. The numbers above the bars represent the percentage of IGFR+ cells, that is, the percentage of cells in given subpopulation with levels of IGFR staining above those found with the isotype control. (B): Effect of IGF-I on the yield of cells within different FBM mononuclear subpopulations as a percentage of the mean of the untreated control cultures (unfilled bars). An increase in the absolute number of many subpopulations is seen after the addition of IGF-I (100 ng/ml) (filled bars) to either total FBM mononuclear cells or CD34+ cells. These results represent pooled data from eight independent experiments carried out on FBM progenitor cells from eight different donors. (C): Effect of IGF-I on the percentage of apoptotic (annexin-V staining) cells within FBM mononuclear subpopulations as a percentage of the mean of the untreated control cultures (unfilled bars). These results represent pooled data from eight independent experiments carried out on FBM progenitor cells from eight different donors. In (B, C), *p < .05. Abbreviations: FBM, fetal bone marrow; IGF-I, insulin-like growth factor I; IGFR, antitype I insulin-like growth factor receptor.

 
To determine whether these cells were functionally responsive to IGF-I, total FBM mononuclear cells or purified CD34+ mononuclear cells were incubated with IGF-I. Treatment with IGF-I for 4–8 days resulted in an increase in the yield of FBM CD34+ progenitor cells relative to untreated control cultures (p < .05) (Fig. 4BGo, second panel from left). As in the case of GH stimulation, this increase in cell number was reflected by an increased yield of multiple hematopoietic subpopulations, including primitive, multilineage CD34+CD38– progenitor cells (Fig. 4BGo, remaining panels). In contradistinction to the effects of GH, the increased cell numbers observed after IGF-I treatment appeared to be primarily due to increased cell survival, with substantially lower levels of apoptosis (annexin-V staining) observed in multiple subpopulations after 4–8 days of culture (Fig. 4CGo). No consistent changes in the proliferation of these subpopulations (as assessed by BrdU incorporation) were observed (data not shown). IGF-I treatment of FBM stroma resulted in an increase in proliferation (as assessed by BrdU incorporation) of CD45+CD14+GHR+IGFR+ stromal cells (data not shown). This cell subpopulation also revealed an IGF-I–associated decrease in apoptosis (as assessed by annexin-V staining, data not shown).

In contrast to the effects of GH, treatment of FBM cells with IGF-I resulted not only in an increase in CFU-GM and CFU-GEMM but also in an increase in BFU-E (p < .005 in each case) (Table 2Go).

GH and IGF-I Induce Cytokine Secretion by FBM Stromal Cells
We hypothesized that GH and IGF-I might facilitate multilineage hematopoiesis by inducing the secretion of key hematopoietic cytokines from FBM stromal cells. To investigate this possibility, FBM stroma cultures were plated in replicate, stimulated with exogenous GH or IGF-I for 1 day, treated with brefeldin A to block secretion of induced cytokines, and then assessed by flow cytometry for the presence of intracellular IL-3. Relative to untreated cultures (Fig. 5Go, upper panels), IL-3 production was consistently stimulated by IGF-I (Fig. 5Go, middle panels) and, to a more variable and lesser degree, by GH (Fig. 5Go, lower panels). When lineage markers were included in the analysis, most IL-3–producing cells were found to be nonhematopoietic (CD45–CD10+) mature fibroblast-like FBM stromal cells (data not shown). Although increases in IL-3 production were observed in four of five stromal cultures incubated with GH (average = 2.2-fold) and in five of five cultures incubated with IGF-I (average = 5.9-fold), these changes were variable, even within a given experiment (and as evidenced in the replicates of Fig. 5Go). In addition to IL-3, we observed sporadic increases in the expression of other hematopoietic cytokines (e.g., IL-6, IL-7, and SCF; data not shown). In aggregate, these results suggest that GH may act, directly or indirectly, to induce both IGF-I and IL-3 from FBM stroma, but that the stromal cell(s) making these secondary mediators are rare.



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  		Figure 5. Induction of IL-3 production by GH and IGF-I. FBM stromal cells were incubated in triplicate with medium alone (upper panels), 100 ng/ml IGF-I (middle panels), or 100 ng/ml GH (lower panels) for 24 hours, and then assessed by flow cytometry for the presence of intracellular IL-3. Triplicate results from a single donor are displayed. This experiment is representative of five independent experiments carried out with cells from five different donors. Abbreviations: FBM, fetal bone marrow; GH, growth hormone; IGF-I, insulin-like growth factor I; IL, interleukin.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have examined the specific effects of GH and IGF-I on multilineage and lineage-restricted progenitor cells in human FBM. We demonstrate that multiple cell subpopulations therein express receptors for GH and for IGF-I, and are responsive to stimulation with these hormones in vitro. Notably, the most responsive subpopulations included primitive multilineage CD34+CD38– hematopoietic progenitor cells and progenitor cells to the lymphoid (CD34+CD38+CD10+) lineage. In the case of GH stimulation, these populations responded with increased levels of proliferation. In the case of IGF-I, lower levels of apoptosis were observed. We have additionally shown that GH and IGF-I significantly increase the number of human FBM colony forming units in methylcellulose culture. This finding suggests that GH and IGF-I directly increase the production of lineage-committed human FBM progenitors. More specifically, our results imply that the proliferative and antiapoptotic effects of GH and IGF-I lead to the expansion of multilineage human FBM progenitors, and that this population then gives rise to increased numbers of lineage- committed progenitor cells. Last, we have established that FBM stromal cells may also serve as targets for GH and IGF-I, as demonstrated by the direct effects of these hormones on the expansion of myeloid-like (CD45+CD14+) stromal cells and on the enhanced secretion of the hematopoietic cytokine IL-3 by fibroblast-like (CD45–CD10+) stroma. By exerting such effects on FBM stroma, GH and IGF-I may stimulate human hemato-lymphoid development in an indirect manner.

These results confirm previous in vitro studies [13–15] demonstrating the ability of GH and IGF-I to enhance human hematopoiesis and extend these findings to provide a detailed look at the specific effects of GH and IGF-I on cellular components within human FBM. Our results document a clear effect of GH and IGF-I on the expansion of the primitive multipotential CD34+CD38– progenitor population. Because this subpopulation has been shown to contain the majority of BM hematopoietic stem cell activity [26–28], these results indicate that GH is able to function as a hematopoietic cytokine. Whereas the role of IGF-I as a hematopoietic survival factor has been previously studied in hematopoietic cell lines [29, 30], our finding of a potent antiapoptotic effect on an early subset of primary human FBM progenitor cells provides direct evidence of IGF-I effects on human hematopoiesis.

Although it is not possible to directly extend our in vitro results to in vivo processes, the finding that GH and IGF-I significantly expand primitive multipotential and lineage-committed FBM progenitors is consistent with in vivo findings from several rodent models. For instance, Tian and colleagues [31] found that administration of GH to mice after syngeneic BM transplantation (BMT) was associated with significant increases in hematopoietic progenitor cells and in improved post-transplant recovery of leukocytes, platelets, and erythrocytes. Similarly, our finding that GH and IGF-I enhance the survival and proliferation of CD34+CD38+CD10+ lymphoid-committed progenitor cells is consistent with rodent studies demonstrating that GH and IGF-I can accelerate T-cell recovery after BMT [7] and can also reverse age-associated changes in hematopoiesis and thymic involution in rodents [4, 11, 19, 32, 33]. The CD34+CD38+CD10+ population was shown by Galy et al. to have T-lineage potential in adult, as well as fetal, BM [34]. We hypothesize that these lymphoid progenitors, responsive to stimulation with GH and IGF-I in vitro, are likely to be inclusive of cells that migrate into and give rise to T cell–restricted intrathymic progenitors. To the extent that these cells are stimulated to divide by GH or IGF-I, it is to be expected that thymopoiesis will be augmented. Because CD34+CD14+ myeloid cells may be able to populate the functional human thymus, the stimulation of this cell subpopulation by GH and by IGF-I may serve to enhance thymopoiesis as well. We speculate that our in vitro findings may correspond to in vivo mechanisms that mediate enhanced human T lymphopoiesis.

Also in conjunction with previous studies [35, 36], our results indicate that GH and IGF-I have stimulatory effects on the B-cell compartment, primarily through an increase in CD34+CD19+ preB cells in vitro (Figs. 2BGo and 5BGo). IGF-I has been shown to activate B-cell differentiation, though not to directly stimulate B-cell proliferation [35]. Additionally, GH has also been implicated in B-cell stimulation and migration (reviewed in [36]). The data here confirm the stimulatory effects previously seen in B-cell progenitors in vivo and suggest that the in vivo increases induced by IGF-I may be due to an increase in cell survival (Fig. 4CGo).

A stromal network of supporting cells, including fibroblasts and macrophages, sustains hematopoiesis in the BM. Upon direct analysis of such stroma, receptors for GH and for IGF-I were found to be most highly expressed on CD45+CD14+ macrophages; in parallel, the yield of CD45+CD14+ macrophages was increased in GH-treated FBM stromal cultures. Due to the high level of GHR on the surface of CD34+CD14+ progenitor cells and to the subsequent increase seen in this population after GH addition (Fig. 2BGo), it is possible that GH stimulates the CD34+CD14+ progenitor cells to differentiate into stromal macrophages [21, 22].

GH addition to FBM stromal cultures increased IGF-I production in the supernatant (as detected by ELISA), suggesting that the stromal effects of GH might be mediated through IGF-I. However, it is notable that the levels of IGF-I induced by GH (average = 1,421 pg/ml) were lower than those required to attain a response in the hematopoietic populations studied (100 ng/ml). Possibly, the low levels of IGF-I in the stromal cell supernatant reflect rapid uptake of IGF-I by cells bearing IGFR. Alternatively, the requirement for higher (100 ng) effective concentrations of IGF-I may be attributable to the presence of IGF-I binding proteins within the culture medium (see below). Nevertheless, we cannot conclusively state that GH effects on human BM are mediated through IGF-I.

Despite the similarities between our in vitro findings with in vivo reports, it is important to discuss several potential limitations of our in vitro analyses. For instance, the majority of the experiments reported here were performed with fetal tissues, and extrapolation of results to the adult case is made with uncertainty. We have, however, performed similar experiments using adult BM (using three different donors) with findings similar to those seen in FBM, suggesting that GH-responsive counterparts also exist in adult BM (data not shown). Further experimentation is required to confirm these preliminary data. Consideration should also be given to the fact that our FBM cultures contained serum to sustain the survival and function of stromal cells. Because IGF-I, GH binding proteins, and IGF-I binding proteins can be present in media containing 10% serum at concentrations sufficient to interfere with assays of GH or IGF-I function [37–39], the presence of these factors may obscure the effects of exogenously added GH or IGF-I. Thus, even though we were able to detect effects of GH and IGF-I on FBM stromal cell expansion and IL-3 production, the presence of serum may have contributed to the variable results that we obtained when examining the production of other stromal cytokines and IGF-I. Another consideration of our in vitro design relates to the concentrations of GH and IGF-I used for these studies. Although the IGF-I concentrations used in the described experiments were within physiologic range, the concentrations of GH required to induce measurable effects in vitro were above physiologic concentrations [40, 41]. These GH concentrations, however, are in concordance with previous in vitro studies [16, 19]. It is possible that the higher GH concentrations might be attributed to suboptimal culture conditions for certain accessory cell types, or might be necessary to overcome the presence of GH-binding proteins within serum. Finally, and in contrast to previous results [16], GH treatment did not result in an increased yield of BFU-E in our study. This disparity may be explained by different experimental conditions. For instance, Merchav et al. [17] added GH during the cell plating in the colony assay, whereas in the studies described here, cells were first treated in liquid culture and plated after 4 days as a method to confirm our phenotypic progenitor analysis post liquid culture.

In summary, GH appears to have multiple direct and indirect effects on the human FBM compartment, all of them facilitating the proliferation and maturation of multilineage progenitor cells. These effects are consistent with the possibility that GH may serve as an adjunct in immune reconstitution of certain immunodeficient hosts and may also facilitate multilineage reconstitution in individuals with hematopoietic deficiencies.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study was supported by National Institutes of Health (NIH) grants R37 AI40312 and R01 AI43864 (to J.M.M.) and K08 AI01597 (to L.A.N). J.M.M. is an Elizabeth Glaser Pediatric AIDS Foundation Scientist and a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research and of the NIH Director’s Pioneer Award.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received on November 19, 2004; accepted for publication on April 11, 2005.




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