European Journal of Haematology 83 (183–190)
ORIGINAL ARTICLE
Differential gene expression in umbilical cord blood and maternal peripheral blood Michaela Merkerova1, Alzbeta Vasikova1, Hana Bruchova1, Helena Libalova2, Jan Topinka2, Ivan Balascak3, Radim J. Sram2, Radim Brdicka1 1 3
Institute of Hematology and Blood Transfusion, Prague 2, Czech Republic; 2Institute of Experimental Medicine, Prague 4, Czech Republic; Faculty Hospital Motol, Prague 5, Czech Republic
Abstract Objectives: Umbilical cord blood (UCB) has become a useful alternative source of hematopoietic stem cells for clinical and research applications. UCB represents neonatal blood and differs from adult blood in many aspects, displaying different cell composition and various features of cellular immaturity. To understand molecular basis of phenotypic differences between neonatal and adult blood, we studied variations in transcriptome of UCB and maternal peripheral blood (PB). Methods: Using Illumina microarrays, we determined gene expression profiles of UCB and PB samples obtained from 30 mothers giving birth to living baby. Results: Out of 20 589 tested genes, 424 genes were down-regulated and 417 genes were up-regulated in UCB compared with PB. Reduced expression of many immunity-related pathways (e.g. TLR pathway, Jak-STAT pathway, cytokine)cytokine receptor interaction) in neonatal blood cells may contribute to the poor response to antigens, increasing susceptibility to infections at the time of disappearance of protective maternal antibodies. On the other hand, overexpression of erythropoiesis-related genes (glycophorins, fetal hemoglobins, enzymes catalysing heme synthesis and erythrocyte differentiation) in UCB probably enforces red cell production in newborns. Conclusions: Our study demonstrates that neonatal and maternal bloods show specific gene expression profiles, likely reflecting differences in phenotypes of immunologically immature and fully evolved hematopoietic cells. Key words gene expression; umbilical cord blood; peripheral blood; hematopoiesis; immune system Correspondence Radim Brdicka, Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20 Prague 2, Czech Republic. Tel: +420221977219; Fax: +420221977371; e-mail:
[email protected] Accepted for publication 17 May 2009
Umbilical cord blood (UCB) is the blood of newborn that is found in umbilical cord and returns to the neonatal circulation if the cord is not prematurely clamped. Previously considered a biological waste product, UCB has recently emerged as an important alternative source of hematopoietic stem cells (HSCs) employed especially for allogenic transplantations. The use of UCB grafts as stem cell source provides both benefits and disadvantages for transplant recipients (compared with transplantation of bone marrow or G-CSF-mobilized peripheral blood stem cells), such as permission of higher degree of HLA disparity, reduction in the incidence and severity of graft-vs.-host disease (GvHD) (1–4), delayed engraftment (5) or more frequent and severe post-transplantation infections (6, 7). These variations result from unique characteristics of
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doi:10.1111/j.1600-0609.2009.01281.x
UCB, i.e. different cell composition (e.g. reduced lymphocyte population, predominance of neutrophils, higher erythrocyte count) or immunological naivety of the cells (e.g. B- ⁄ T-lymphocytes) caused by altered signal transduction mechanisms (8–10). While different biological properties of UCB are well known, their molecular basis is still poorly understood. Ng et al. (11) described variations in gene expression profiles of CD34+ HSCs from various sources including UCB, reflecting functional differences in their activity. However, expression profiles of another UCB cells and their peripheral blood counterparts have not been unravelled yet. In this study, we analyzed gene expression profiles of UCB and maternal peripheral blood (PB) using Illumina microarrays to indentify differences in their transcriptome
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Gene expression in UCB and maternal PB
and assess signaling pathways that may regulate development of hematopoietic cells. Materials and methods Cell samples
Samples of UCB and maternal PB were obtained from 30 mothers giving birth to living baby at the Faculty Hospital Motol in Prague (Czech Republic). All blood samples were acquired with donor’s written informed consent in accordance with the Declaration of Helsinki. The study was approved by the Institutional Review Board of the Faculty Hospital Motol. For immediate stabilization of expression profiles, blood samples were drawn into PAXgene Blood RNA Tubes (PreAnalytiX, Hombrechtikon, Switzerland) and stored at )80C until RNA isolation.
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(undetected genes) in more than 50% of arrays were filtered out. Limma package was used for identification of differentially expressed genes. Linear model was fitted for each gene given a series of arrays using lmFit function. The empirical Bayes method was applied to rank differential expression of genes using eBayes function. Multiple testing correction was performed using Benjamini & Hochberg method. DAVID database (http://david.abcc.ncifcrf.gov) was used for the functional annotation of differentially expressed genes. Quantitative real-time reverse transcriptase-polymerase chain reaction
Total RNA from the whole blood samples was isolated by PAXgene Blood RNA Kit (PreAnalytiX). RNA integrity was verified using Agilent 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA). The RNA quality was comparable among all samples; only the samples with RNA integrity number (RIN) > 7.5 were used for gene expression profiling.
Validation of microarray data was performed using qRTPCR with TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). Following genes were quantified: ADA (adenosine deaminase), FECH (ferrochelatase), E2F2 (E2F transcription factor 2), CSF3R (colony stimulating factor 3 receptor), IFNAR1 (interferon alpha receptor 1), TLR4 (toll-like receptor 4). All measurements were performed in duplicates according to the manufacturer’s instruction on RotorGene 3000 apparatus (Corbett Research, Sydney, Australia). The data were normalized to an endogenous control B2M (beta-2-microglobulin) and relative gene expression levels were calculated by the DDCT method (12).
Gene expression profiling
Results
Total RNA extraction
HumanRef-8 v2 Expression BeadChips (Illumina, San Diego, CA, USA) were used for determination of gene expression profiles (20 589 transcripts, complete gene annotation file at http://www.switchtoi.com/annotation files.ilmn). Total RNA (200 ng) was reverse transcribed into cDNA, then amplified and biotinylated by in vitro transcription (Illumina TotalPrep RNA Amplification Kit, Ambion, Foster City, CA, USA). Labelled cRNA (750 ng) was hybridized onto microchips and labelled with Cy-3-streptavidin conjugate. All procedures were performed according to manufacturer protocols. Microarrays were scanned on BeadStation 500 (Illumina) and raw data were extracted using BeadStudio Software (Illumina). Microarray data analysis
Quality control and normalization of the data were performed in R statistical environment (http://www.r-project.org) using Lumi package, which is part of the Bioconductor project (http://www.bioconductor.org). Bead-summary data from BeadStudio were log-transformed and processed using quantile normalization. To reduce false positives, genes with detection P > 0.01
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Hierarchical cluster analysis of UCB and PB samples
Gene expression profiles were determined in whole blood samples of UCB and PB obtained from mothers at the time of delivery. For the analysis, 27 UCB and 29 PB samples were selected based on RNA integrity (RIN > 7.5). Out of 20 589 genes on the Illumina chips, 12 240 were undetected (detection P > 0.01 in more than 50% of arrays) and excluded from further data analyses. An unsupervised sample hierarchical clustering of the ‘present’ genes determined two clusters according to the tissue origin (UCB cluster and PB cluster, Fig. 1). Table 1 enumerates 20 genes with the highest transcript levels in both UCB and PB samples; out of these, mostly genes coding for hemoglobins (HBB, HBA2, HBG1, HBG2) or housekeeping genes (H3F3A, ACTB, UBB, RPS29, B2M) were present. Differentially expressed genes between UCB and PB
To identify statistically significant differences in gene expression between UCB and PB, a series of statistical
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Gene expression in UCB and maternal PB
Figure 1 Hierarchical clustering of UCB and PB samples according to the expression of 8 349 detected genes. UCB, umbilical cord blood; PB, peripheral blood.
Table 1 Genes with the highest transcript levels in all samples
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1
RefSeq_ID
Symbol
Definition
AVG signal1
NM_0005184 NM_0041522 NM_0005173 NM_0001463 NM_0211092 NM_0029643 NM_0014025 NM_0021073 NM_0011012 NM_0005592 NM_0001842 NM_0011754 NM_0064352 NM_0189552 NM_0010323 NM_0160161 NM_0040482 NM_0032952 NM_0054372 NM_0027272
HBB OAZ1 HBA2 FTL TMSB4X S100A8 EEF1A1 H3F3A ACTB HBG1 HBG2 ARHGDIB IFITM2 UBB RPS29 SLC25A39 B2M TPT1 NCOA4 SRGN
Hemoglobin beta Ornithine decarboxylase antizyme 1 Hemoglobin, alpha 2 Ferritin, light polypeptide Thymosin, beta 4, X-linked S100 calcium binding protein A8 Eukaryotic translation elongation factor 1 alpha 1 H3 histone, family 3A Actin beta Hemoglobin gamma A Hemoglobin gamma G Rho GDP dissociation inhibitor (GDI) beta Interferon- induced transmembrane protein 2 (1-8D) Ubiquitin B Ribosomal protein S29 (RPS29) Solute carrier family 25, member 39 Beta-2-microglobulin Tumor protein, translationally-controlled 1 Nuclear receptor coactivator 4 Serglycin
1.96E+04 1.93E+04 1.91E+04 1.82E+04 1.78E+04 1.71E+04 1.64E+04 1.58E+04 1.52E+04 1.45E+04 1.44E+04 1.41E+04 1.38E+04 1.37E+04 1.28E+04 1.23E+04 1.22E+04 1.19E+04 1.17E+04 1.16E+04
Average signal intensity detected for the given gene measured by Illumina microarrays
tests were applied to the microarray data and 841 genes were found (> 2-fold, adjusted P < 0.001). Out of these, 424 genes were down-regulated and 417 genes were up-regulated in UCB compared with PB (heat map in Fig. 2). In Table 2, the list of 10 genes with the highest increase and reduction of expression level is shown. The list of 10 most down-regulated genes in UCB included genes with functions in immune system (IFIT1, IFIT2,
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IFIT3, IFI44, IL18R1) or coagulation factor 5 (factor V Leiden, F5). On the other hand, the most up-regulated genes in UCB were, e.g. fetal hemoglobin zeta (HBZ) and three members of glycophorins (GYPA, GYPB, GYPE), major sialoproteins of erythrocyte membranes. The DAVID annotation analysis revealed the most represented gene ontology (GO) biological process categories in all differentially expressed genes (Table 3).
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Figure 2 Hierarchical clustering of differentially expressed genes in umbilical cord blood and peripheral blood samples. Heat map shows 841 genes with significant differences in expression level (> 2-fold, adjusted P < 0.001). Each row represents a single gene and each column a separate blood sample. UCB, umbilical cord blood; PB, peripheral blood.
Table 2 Genes with the highest decrease (A) and increase (B) of expression level in UCB compared with PB RefSeq_ID (A). Down-regulated in UCB 1 NM_001011666.1 2 NM_001548.3 3 NM_001031683.1 4 NM_000902.3 5 NM_004833.1 6 NM_003855.2 7 NM_006417.3 8 NM_016323.2 9 NM_001547.4 10 NM_000130.4 (B). Up-regulated in UCB 1 NM_005332.2 2 NM_002100.3 3 NM_002102.3 4 NM_001010987.1 5 NM_003126.2 6 NM_199186.1 7 NM_002099.3 8 NM_001972.2 9 NM_181738.1 10 NM_175842.1
AVG ratio1 (UCB ⁄ PB)
Symbol
Gene
CREB5 IFIT1 IFIT3 MME AIM2 IL18R1 IFI44 HERC5 IFIT2 F5
cAMP responsive element binding protein 5 Interferon-induced protein with tetratricopeptide repeats 1 Interferon-induced protein with tetratricopeptide repeats 3 Membrane metallo-endopeptidase Absent in melanoma 2 Interleukin 18 receptor 1 Interferon-induced protein 44 Hect domain and RLD 5 Interferon-induced protein with tetratricopeptide repeats 2 Coagulation factor V (proaccelerin, labile factor)
HBZ GYPB GYPE IFIT1L SPTA1 BPGM GYPA ELA2 PRDX2 SMOX
Hemoglobin, zeta Glycophorin B (MNS blood group) Glycophorin E Interferon-induced protein with tetratricopeptide repeats 1-like Spectrin, alpha, erythrocytic 1 (elliptocytosis 2) 2,3-Bisphosphoglycerate mutase Glycophorin A (MNS blood group) Elastase 2, neutrophil Peroxiredoxin 2, nuclear gene encoding mitochondrial protein Spermine oxidase
Adjusted P-value2
0.1127 0.1574 0.1683 0.1724 0.1750 0.1771 0.2304 0.2320 0.2348 0.2363
5.39E-36 3.15E-18 4.80E-22 2.20E-25 2.97E-29 1.45E-13 1.23E-21 6.59E-18 3.35E-20 6.08E-21
20.5024 18.5898 15.8308 15.7173 14.8663 13.2785 12.8027 12.0073 11.4185 9.0278
8.36E-17 4.36E-30 5.48E-30 8.77E-31 3.68E-30 1.03E-30 1.92E-34 1.19E-20 1.72E-30 7.23E-23
1
Average ratio of signal intensities of the gene between UCB and PB. 2P-value indicating significance of differential expression adjusted using Benjamini & Hochberg method (for elimination of false positive calls). UCB, umbilical cord blood; PB, peripheral blood.
Majority of the genes with reduced expression in UCB was assigned to the categories associated with immunity (categories with the lowest P-values) ) immune system process (72 genes, P = 1.8E-17), response to wounding
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(43 genes, 2.0E-17), inflammatory response (36 genes, 4.9E-17), response to external stimulus (51 genes, 1.8E16), etc. Annotation of the up-regulated genes in UCB resulted in the detection of other GO biological process
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Gene expression in UCB and maternal PB
Table 3 Gene Ontology biological processes deregulated in UCB (compared with PB) assessed by DAVID annotation tool. The genes are listed according to P-values (significance of gene-enrichment within the annotation category) and 20 of the most significant processes are shown Gene Ontology ID (A). Down-regulated in UCB GO:0002376 GO:0009611 GO:0006954 GO:0009605 ⁄ GO:0051707 ⁄ GO:0009615 GO:0006950 GO:0007243 GO:0007242 GO:0001816 GO:0006935 GO:0007249 (B). Up-regulated in UCB GO:0006783 ⁄ GO:0006779 ⁄ GO:0046148 GO:0006512 GO:0051186 GO:0015669 GO:0019748 GO:0044249 GO:0030218 GO:0046483 GO:0006916 GO:0006979
Biological process
No. of genes
P-value
Immune system process Response to wounding Inflammatory response Response to external stimulus ⁄ other organism ⁄ virus
72 43 36 51
1.8E-17 2.0E-17 4.9E-17 1.8E-16
Response to stress Protein kinase cascade Intracellular signaling cascade Cytokine production Chemotaxis I-kappaB kinase ⁄ NF-kappaB cascade
59 33 66 15 16 15
7.6E-12 2.5E-11 2.7E-9 1.7E-7 3.9E-7 7.9E-7
Heme ⁄ porphyrin ⁄ pigment biosynthetic process
8
3.0E-9
Ubiquitin cycle Cofactor metabolic process Gas transport Secondary metabolic process Cellular biosynthetic process Erythrocyte differentiation Heterocycle metabolic process Anti-apoptosis Response to oxidative stress
27 17 6 8 43 7 9 12 9
9.4E-6 1.6E-5 3.3E-5 4.2E-5 6.3E-5 8.5E-5 2.3E-4 4.8E-4 5.7E-4
UCB, umbilical cord blood; PB, peripheral blood.
categories ) e.g. heme ⁄ porphyrin metabolic process (8 genes, 2.5E-8), ubiquitin cycle (27 genes, 9.4E-6), cofactor metabolic process (17 genes, 1.5E-5), gas transport (6 genes, 3.2E-5), and erythrocytic differentiation (7 genes, 8.1E-5). In the gas transport deregulated genes, five genes coding for hemoglobins expressed mainly in fetus (HBD, HBG2, HBE1, HBQ1, HBZ) were present. Further, the DAVID database detected 13 significantly different pathways in UCB (Table 4). The down-regulated genes were linked to seven cellular pathways toll-like receptor (TLR) signaling pathway, cytokine)cytokine receptor interaction, hematopoietic cell lineage, natural killer cell-mediated cytotoxicity, Jak-STAT signaling pathway, adipocytokine signaling pathway, pathogenic Escherichia coli infection). The up-regulated genes were categorized into six pathways (porphyrin metabolism, ubiquitin-mediated proteolysis, cell cycle, renal cell carcinoma, purine metabolism, amyotrophic lateral sclerosis). Validation of microarray data using qRT-PCR
For validation of microarray data, we have focused on differentially expressed genes that had been described in hematopoietic tissues and ⁄ or have function of particular
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interest: ADA (adenosine deaminase), FECH (ferrochelatase), E2F2 (E2F transcription factor 2), CSF3R (colony stimulating factor 3 receptor), IFNAR1 (interferon alpha receptor 1), TLR4 (toll-like receptor 4). We re-evaluated array data using qRT-PCR and high correlation between both the methods was observed for all tested genes (r = 0.92, P < 0.0001) (Fig. 3). Discussion
Molecular basis of hematopoiesis has been systematically studied in adult blood cells. Nowadays, an extensive research focuses on neonatal blood, represented particularly by UCB, which becomes a significant source of stem cells. It is known that neonatal hematopoietic system differs in many quantitative and qualitative aspects and its immaturity influences almost all cellular components of the blood (e.g. ineffective immune system, distinct composition of hemoglobin, hyporeactive platelets). In this study, we investigated molecular bases of UCB features at the gene expression level. The comparative analysis of expression profiles of UCB and PB cells determined high proportion of differentially expressed genes between neonatal and adult blood. Out of the 20 589 tested genes, 841 genes (4%)
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Table 4 Signaling pathways deregulated in UCB (compared to PB) assessed by DAVID annotation tool (KEGG pathways) Signaling pathways (A). Down-regulated in umbilical cord blood 1 Toll-like receptor signaling pathway
No. of genes
P-value
Genes
14
2.0E-5
2
Cytokine)cytokine receptor interaction
22
6.1E-5
3 4
Hematopoietic cell lineage Natural killer cell mediated cytotoxicity
9 10
5.9E-3 2.4E-2
5
Jak-STAT signaling pathway
10
5.8E-2
6 5
8.7E-2 9.2E-2
CD14, FOS, IFNAR1, IFNAR2, IL1B, LY96, MAPK14, MYD88, STAT1, TLR2, TLR4, TLR5, TLR6, TLR8 CCR1, CSF3R, CXCL16, IFNAR1, IFNAR2, IFNGR1, IL1B, IL1R2, IL4R, IL8RA, IL8RB, IL10RB, IL17RA, IL18R1, IL18RAP, LTBR, OSM, PLEKHQ1, TNFRSF1A, TNFSF10, TNFSF13B, TNFSF14 ANPEP, CD14, CD55, CSF3R, IL1B, IL1R2, IL4R, ITGAM, MME FCER1G, FCGR3B, HLA-C, HLA-E, IFNAR1, IFNAR2, IFNGR1, TNFSF10, TYROBP, VAV1 CSF3R, IFNAR1, IFNAR2, IFNGR1, IL4R, IL10RB, OSM, SOCS3, STAT1, STAT3 ACSL1, ACSL4, IRS2, SOCS3, TNFRSF1A, STAT3 CD14, HCLS1, LY96, TLR4, TLR5
9 9 8 6 8 3
5.1E-6 1.5E-2 2.2E-2 2.4E-2 6.6E-2 7.9E-2
ALAD, ALAS2, BLVRB, CPOX, FECH, PPOX, UROD, UROS CDC16, CDC20, CDC34, CUL4A, DDB1, RBX1, TCEB2, UBE2H, UBE2O CCNB2, CDC16, CDC20, E2F2, MCM7, PTTG1, RBX1, TFDP1 ETS1, FLCN, PIK3R2, RBX1, SLC2A1, TCEB2 ADA, AK1, DCK, GMPR, GUK1, NME4, NP, POLR1D BCL2L1, CAT, SOD1
6 Adipocytokine signaling pathway 7 Pathogenic Escherichia coli infection (B). Up-regulated in umbilical cord blood 1 Porphyrin metabolism 2 Ubiquitin mediated proteolysis 3 Cell cycle 4 Renal cell carcinoma 5 Purine metabolism 6 Amyotrophic lateral sclerosis
The pathways are listed according to P-values (significance of gene-enrichment in the pathway). The gene names are available at http:// www.ncbi.nlm.nih.gov/. UCB, umbilical cord blood; PB, peripheral blood.
Figure 3 Validation of microarray data. The array data of selected genes (ADA, FECH, E2F2, TLR4, IFNAR1, and CSF3R) were validated by qRT-PCR. The bar graphs represent the comparison of fold change differences detected by both methods. The data are presented as the mean plus standard error. UCB, umbilical cord blood; PB, peripheral blood.
were differentially expressed between UCB and PB, and the distribution of down-regulated and up-regulated genes was almost equal (424 down-regulated genes vs.
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417 up-regulated genes in UCB). The hierarchical clustering analysis clearly grouped the samples into two clusters according to their origin (UCB cluster and PB cluster), demonstrating that neonatal and adult blood display specific expression patterns. Annotation of the genes revealed several biological processes and signaling pathways differentially regulated between neonatal and adult blood. The substantial part of the down-regulated genes in UCB was involved in the regulation of immune system. We found the reduced transcription of genes involved in defense response to various stimuli such as stress, wounding, external stimuli (e.g. viruses), or inflammation. Moreover, we detected the down-regulation of pathways related to the immune system, i.e. TLR pathway, cytokine-cytokine receptor interaction, hematopoietic cell lineage, natural killer cell mediated cytotoxicity, Jak-STAT signaling pathway, adipocytokine signaling pathway and pathogenic Escherichia coli infection. The TLR signaling pathway was the most significantly down-regulated pathway in UCB; the decrease in transcript levels was observed particularly for five genes coding for TLRs (TLR2 ⁄ 4 ⁄ 5 ⁄ 6 ⁄ 8). TLRs recognize conserved motifs predominantly found in microorganisms, stimulating in phagocytes immediate defensive response against pathogens (i.e. innate immunity) (13). Moreover, CD-antigens specific for phagocytes (i.e. neutrophils, macrophages, and dendritic cells) were down-regulated in UCB (CD11b ⁄ 13 ⁄ 14 ⁄ 114 ⁄ 121 ⁄ 124), suggesting reduced population of these cells that may
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limit possibility of newborns’ blood to phagocyte various pathogens or impaired cells. Another down-regulated immunity-related pathway in UCB was Jak-STAT pathway. This pathway plays a central role in cell fate decision, regulating the processes of cell proliferation, differentiation and apoptosis. In hematopoiesis, Jak-STAT pathway is particularly important for production of blood cells (14–16). Proper signaling by Jak-STAT is also crucial in immunity system development and response, and congenital mutations in the pathway genes are known to be associated with immunodeficiency disorders (severe combined immunodeficiency or reduced response to viral infection) (16–18). Jak-STAT pathway is the principal signaling mechanism of a wide array of cytokines and growth factors (16). In UCB, we found the down-regulation of many genes coding for cytokines and their receptors e.g. interferon receptors (IFNAR1, IFNAR2, IFNGR1), interleukins and their receptors (IL1B, IL1, R2, IL4R, IL8RA, IL8RB, IL10RB, IL17RA, IL18R1, IL18RAP) and tumor necrosis factors and their receptors (TNFRSF1A, TNFSF10, TNFSF13B, TNFSF14), suggesting undeveloped activation mechanisms of immune system. Downregulation of several chemokins and their receptors (e.g. CCR1, CKLF, CXCL16, FPR1, FPRL1) implies that chemotaxis, which is primarily relevant to circulating phagocytic cells (14), is reduced in neonatal blood, supporting evidences of insufficiency of phagocytes. Taken together, we found overall down-regulation of genes related to the immune system in UCB compared to adult PB. Particularly, innate immunity system seems not to be fully evolved. Immaturity of neonatal phagocytes was previously demonstrated and impaired response of neonatal monocytes and macrophages to multiple TLR ligands was shown (19–21). In addition to large number of immunity-related genes, we also detected down-regulation of many genes involved in intracellular signaling cascade (66 genes) or protein kinase cascade (33 genes) in UCB. Multitude of different signal transduction pathways within the cells is required for general coordination of cell behavior; therefore, the decrease in signal transduction may be associated with various biological processes. In context of this complexity, the down-regulation of signaling cascades in UCB demonstrates different regulation of biological processes in neonatal hematopoiesis. Nevertheless, limited signal transduction may partially reflect the insufficiency of immune system in newborns, supporting thus the large down-regulation of immunity-related genes. Set of the up-regulated genes in UCB comprised of another functional categories. Large group of the genes plays key roles in erythropoiesis; we found overexpression of genes coding for glycophorins, fetal hemoglobins
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Gene expression in UCB and maternal PB
or enzymes catalysing heme synthesis and erythrocyte differentiation. These findings may reflect physiological erythrocytosis and macrocytosis observed at birth. Similarly, it is known that fetal hemoglobin F (a2c2) has still high level in newborns (22). Large up-regulation of cell cycle, anti-apoptosis, ubiquitin-mediated proteolysis and various biosynthetic processes (purines, cofactors, etc.) in UCB compared with adult PB may be connected with the increased production of blood cells with a shortened life span in newborns (23). The up-regulation of biosynthesis probably reflects changes in functional properties of neonatal blood cells with respect to requirements of higher activity of cellular metabolism during development of early hematopoiesis. To sum up, neonatal and adult bloods widely differ in gene expression profiles. Based on the expression data, the blood samples can be clustered into the groups according to their origin (PB vs. UCB samples). Reduced expression of many immunity-related genes in neonatal blood cells likely represents molecular mechanisms of the poor response to antigens, increasing susceptibility to infections at the time of disappearance of protective maternal antibodies. On the other hand, overexpression of erythrocyte-related genes may be associated with enforced red cell production in newborns. Our results provide insights into regulatory network of neonatal hematopoiesis that displays differential regulation of particular pathways. However, further determination of gene expression profiles in separated UCB cell lineages will be needed to completely understand the molecular basis of neonatal blood cell phenotypes. Acknowledgements
The authors wish to thank Mgr. Viktor Stranecky (Institute of Inherited Metabolic Disorders, 1st Faculty of Medicine of Charles University, Prague) for the array data analyses. The study was supported by the Czech Ministry of Education Youth and Sports (Grant No. 2B06088). References 1. Rocha V, Wagner JE Jr, Sobocinski KA, Klein JP, Zhang MJ, Horowitz MM, Gluckman E. Graft-versushost disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. N Engl J Med 2000;342:1846–54. 2. Rocha V, Labopin M, Sanz G, et al. Transplants of umbilical-cord blood or bone marrow from unrelated
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