Effects of growth hormone therapeutic supplementation on hematopoietic stem/progenitor cells in children with growth hormone deficiency: focus on proliferation and differentiation capabilities

Abstract

We investigated the direct effects of growth hormone (GH) replacement therapy (GH-RT) on hematopoiesis in children with GH deficiency (GHD) with the special emphasis on proliferation and cell cycle regulation. Peripheral blood (PB) was collected from sixty control individuals and forty GHD children before GH-RT and in 3rd and 6th month of GH-RT to measure hematological parameters and isolate CD34(+)-enriched hematopoietic progenitor cells (HPCs). Selected parameters of PB were analyzed by hematological analyzer. Moreover, collected HPCs were used to analyze GH receptor (GHR) and IGF1 expression, clonogenicity, and cell cycle activity. Finally, global gene expression profile of collected HPCs was analyzed using genome-wide RNA microarrays. GHD resulted in a decrease in several hematological parameters related to RBCs and significantly diminished clonogenicity of erythroid progenies. In contrast, GH-RT stimulated increases in clonogenic growth of erythroid lineage and RBC counts as well as significant up-regulation of cell cycle-propagating genes, including MAP2K1, cyclins D1/E1, PCNA, and IGF1. Likewise, GH-RT significantly modified GHR expression in isolated HPCs and augmented systemic IGF1 levels. Global gene expression analysis revealed significantly higher expression of genes associated with cell cycle, proliferation, and differentiation in HPCs from GH-treated subjects. (i) GH-RT significantly augments cell cycle progression in HPCs and increases clonogenicity of erythroid progenitors; (ii) GHR expression in HPCs is modulated by GH status; (iii) molecular mechanisms by which GH influences hematopoiesis might provide a basis for designing therapeutic interventions for hematological complications related to GHD.

Fig. 1

Expression of GHR mRNA in CD34⁺ HPCs of controls and GHD patients at different time points. Results are expressed as the mean value ± SD, *P < 0.05; ***P < 0.001 vs. GHD patients before therapy

Fig. 2

The percentage of GHR⁺ HPCs (a) and GHR⁺ lymphocytes (b) of controls and GHD patients at different time points. The results are expressed as the mean value ± SD. *P < 0.05; **P < 0.01 vs. GHD patients before therapy

Fig. 3

Comparative analysis of BFU-E (a), CFU-GM (b), and CFU-B lymph (c) clonogenicity of CD34⁺ HPCs from controls and GHD patients at different time points. The results are expressed as the percentage of the control value, which was set at 100 %. The results are presented as the mean value ± SD. *P < 0.05 vs. GHD patients before therapy; ^# P < 0.05 vs. control group

Fig. 4

Analysis of the cell cycle phases (G1, S, and G2+M) in CD34⁺ HPCs of controls and GHD patients at different time points. The results are expressed as the mean value ± SD. *P < 0.05; **P < 0.01 vs. GHD patients before therapy

Fig. 5

Expression of cell cycle-regulating genes in PB-derived CD34⁺ HPCs of controls and GHD patients at different time points. mRNA expression of the MAP2K1 (a), PCNA (b), CCND1 (c), and CCNE1 (d) genes was determined. mRNA levels are expressed in arbitrary units as the mean value ± SD. **P < 0.01; ***P < 0.001 vs. GHD patients before therapy

Fig. 6

The levels of IGF1 in peripheral blood (a) and the expression of IGF1 mRNA in CD34⁺ HPCs (b) of controls and GHD patients at different time points. The results are expressed as the mean value ± SD. **P < 0.01; ***P < 0.001 vs. GHD patients before therapy; ^# P < 0.05 vs. control group

Fig. 7

Global gene expression changes in CD34⁺ HPCs of controls and GHD patients at different time points (before GH-RT and in the 3rd and 6th months of GH-RT). The heatmap represents the expression levels of highly over-expressed genes (fold change >2). Individual genes are designated according to the GO classification of specific biological processes listed on the left side of the graph. Each column comprises a set of horizontal lines, each representing a single gene. The levels of gene expression are indicated on a color scale, with yellow corresponding to the highest level of expression and blue corresponding to the lowest level. The expression range of the analyzed genes is shown below the graph. The upper diagram (a) depicts large-scale alterations in gene expression between HPCs collected in the 3rd and 6th months of GH-RT compared to those collected before GH-RT. The lower diagram (b) presents large-scale alterations in gene expression between HPCs collected during the course of GH-RT compared to those collected before GH-RT as well as HPCs obtained from controls