Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0107014 on July 12, 2007

Published online before print July 12, 2007
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(Journal of Leukocyte Biology. 2007;82:986-1002.)
© 2007 by Society for Leukocyte Biology

Differential gene expression in human hematopoietic stem cells specified toward erythroid, megakaryocytic, and granulocytic lineage

Xiao-Ling Liu*,1, Jin-Yun Yuan*,1, Jun-Wu Zhang*,2, Xin-Hua Zhang{dagger} and Rong-Xin Wang{dagger}

* National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; and
{dagger} The 303 Hospital, Nanning, China

2 Correspondence: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, and Peking Union Medical College, Beijing 100005, China. E-mail: junwu_zhang{at}pumc.edu.cn or junwu_zhang{at}hotmail.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To better understand the transcriptional program that accompanies orderly lineage-specific hematopoietic differentiation, we analyzed expression changes during the lineage-specific differentiation of human hematopoietic stem cells (HSC; CD34+/CD38–/CD33–); HSC and multipotent myeloid progenitors (MMP; CD34+/CD38–/CD33+) were isolated from the bone marrow of healthy individuals by MACS. CD34+ cells in semi-solid culture were stimulated with the cytokines erythropoietin, IL-6, and G-CSF to promote differentiation to committed erythroid, megakaryocytic, and granulocytic clones, respectively. Differential display RT-PCR analysis was performed to compare the mRNA transcripts in HSC, MMP, and the committed lineage-specific clones derived from these committed lineage-specific progenitors. Expressed sequence tags (n=256), which were differentially expressed, were identified. One hundred ninety-four were homologous to known genes, and some were associated with hematopoiesis. These known genes were classified as involved in transcription/translation, signal transduction, cell surface receptors/ligands, cell signaling, cell metabolism, cell cycle, cell apoptosis, and oncogenesis. We identified genes, which were up- or down-regulated specifically in the lineage-committed clones compared with HSC or/and MMP, suggesting that specific gene activation and repression might be necessary for specific lineage commitment and differentiation. Our data provide an extensive transcriptional profile of human hematopoiesis during in vitro, lineage-specific differentiation.

Key Words: differential display reverse transcription polymerase chain reaction (DDRT-PCR) • gene expression • hematopoietic differentiation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hematopoiesis involves the self-renewal of multipotential hematopoietic stem cells (HSC) and the commitment/differentiation of HSC to various lineages. It also refers to the development of the committed cells. Knowledge of the gene expression patterns during hematopoiesis is necessary for an understanding of the gene regulation in normal hematopoietic differentiation and would also provide insights into the consequences of gene expression alterations in hematopoietic diseases. The commitment and differentiation of HSC into various lineages involve a process governed by transcription factors, and hematopoietic growth factors are needed to sustain survival and proliferation [1 ]. It is generally believed that GATA-1, friend of GATA-1 (FOG 1), erythroid Krüppel-like factor (EKLF), and CREB-binding protein (CBP) are essential for early erythroid and megakaryocytic development [2 ]. Likewise, early neutrophil commitment depends on the transcription factors C/EBP-{alpha}, PU.1, retinoic acid receptor (RAR), core-binding factor (CBF), and c-Myb, and terminal neutrophil differentiation depends on C/EBP-{epsilon} and PU.1 [3 ]. Lineage-specific growth factors such as erythropoietin (EPO), G-CSF, GM-CSF, thrombopoietin, and IL-6 induce proliferation and differentiation into functionally active peripheral blood cells by activating multiple genes in an orchestrated way. Other growth factors such as stem cell factor (SCF), the ligand of the Flt3/Flt2 receptor tyrosine kinase, IL-1, and IL-3 support the growth and survival of primitive progenitor cells [4 5 6 7 ].

Currently, little is known about the complex molecular mechanisms underlying the commitment of human HSC and the differentiation of committed progenitors. As the phenotype of any given cell is ultimately the product of the genes, which it expresses or has expressed during the course of its development, describing the complete gene expression programs of self-renewing and differentiating cells is one approach that addresses how self-renewal and differentiation are regulated. However, most gene expression studies do not compare gene expression in HSC and multipotent myeloid progenitor cells (MMP) to the lineage-committed clones. We believe that it is important to perform and compare gene-expression profiling in these hematopoietic cell types to better understand the relationship between lineage commitment and gene expression as the determining features of hematopoietic differentiation. Therefore, we used three specific surface markers to isolate HSC (CD34+/CD38–/CD33–) and MMP (CD34+/CD38–/CD33+), respectively [8 9 10 ]. We also used a semi-solid culture, which supports the differentiation of normal human HSC from bone marrow to committed erythroid, megakaryocytic, or granulocytic progenitors. For the small number of cells, the differential display RT-PCR (DDRT-PCR) method was performed to analyze differential gene expression in HSC, MMP, clones derived from committed erythroid progenitors (EC), clones derived from committed megakaryocytic progenitors (MC), and clones derived from committed granulocytic progenitors (GC).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell isolation
Human adult bone marrow was obtained from 32 healthy volunteers after obtaining informed consent from the volunteers. We followed appropriate standards for human experimentation, and our experiment has been reviewed and approved by the Ethics Review Committee in the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (P.R.China). Mononuclear cell (MNC) fractions were isolated from the bone marrow by Percoll density gradient (d=1.077; Amersham Biotech, Germany). The MNCs from all participants were mixed and then divided into two portions for the next step. To obtain purified populations of HSC and MMP, CD34+ cells were enriched from MNCs through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Gladbach, Germany). CD34+ cells were subpopulated based on the coexpression of CD38 using negative immunomagnetic selection (anti-PE MicroBeads and PE-conjugated anti-CD38, Miltenyi Biotec). These CD34+/CD38– cells were fractioned further into CD33+ and CD33– subpopulations based on the coexpression of CD33 using positive and negative immunomagnetic selection (anti-PE MicroBeads and PE-conjugated anti-CD33, Miltenyi Biotec). Thus, we obtained HSC, which were CD34+/CD38–/CD33–, and MMP, which were CD34+/CD38–/CD33+ cells [8 9 10 ].

Semi-solid culture
For lineage-specific differentiation from HSC to committed erythropoietic, megakaryopoietic, and granulopoietic clones, we performed a semi-solid cell culture as described previously with some modifications [10 11 12 ]. In short, CD34+ cells from human adult bone marrow were plated at 105 cells/ml in six-well plates and cultured in IMDM supplemented with 30% FCS, 0.81% methylcellulose, 1% BSA, 100 µM 2-ME, 2 ng/ml recombinant human IL-3 (rhIL-3; Stem Cell Technologies, Vancouver, BC, Canada), 100 ng/ml rhSCF (Stem Cell Technologies), 60 mg/ml penicillin, and 100 mg/ml streptomycin. We obtained erythroid differentiation using 2 U/ml rhEPO (Eprex, Janssen-Cilag, High Wycombe, Buckinghamshire, UK) over 4 days. Megakaryocytic differentiation was obtained using 20 ng/ml IL-6 (Stem Cell Technologies) over 7 days, and granulocytic differentiation was obtained with 10 ng/ml G-CSF (Leucomax, Schering-Plough, Novartis, Kenilworth, NJ, USA) over 5 days. The lineage-specific cell clones from the corresponding, committed, lineage-specific progenitors were collected by a micropipette from cultures at the indicated times and stored at –80°C until purification of total RNA.

Flow cytometric analysis of the sorted cells and clones from the lineage-committed clones
Flow cytometric analysis was performed to ensure the purity of the isolated CD34+/CD38–/CD33– (HSC) and CD34+/CD38–/CD33+ (MMP) cells and EC, MC, and GC. Before sorting, the MNCs were labeled with FITC-conjugated mouse IgG and PE-conjugated mouse IgG as isotope controls. The enriched CD34+ cells were labeled with FITC-conjugated anti-CD34; CD38– cells and CD33+/CD33– cells were ready for flow cytometric analysis as a result of the PE-conjugated anti-CD38 and PE-conjugated anti-CD33 used for cell sorting. The collected erythroid clones were labeled with FITC-conjugated anti-CD71, the megakaryocytic clones with FITC-conjugated anti-CD41, and the granulocytic clones with FITC-conjugated anti-CD11b for flow cytometic analysis of cell surface markers specific for erythroid (CD71), megakaryocytic (CD41), and granulocytic (CD11b) cells, respectively.

RNA isolation and RT-PCR
Total RNA was extracted from the isolated CD34+/CD38–/CD33– and CD34+/CD38/-CD33+ cells and the collected clones using the RNeasy® micro kit (Qiagen, Hilden, Germany), according to the manufacturer's recommendations. To verify that chromosomal DNA was removed, amplifications were carried out with specific primers for the Bax promoter on mock-RT RNA samples, as described by Cavalloni et al. [13 ]. Then, first strand of cDNA was synthesized using SuperscriptTM III RT (Invitrogen, Carlsbad, CA, USA) and oligo d(T) primer (A2). The samples were incubated at 55°C for 60 min and then at 70°C for 10 min to inactivate the RT.

DDRT-PCR
DDRT-PCR was carried out as described previously with slight modifications [14 , 15 ]. A total of 15 random upstream primers (R1–R15; Table 1 ) was used in combination with a single downstream anchor primer (A2; Table 1 ) to amplify cDNAs representing a subset of mRNA. PCR reactions with {alpha}32P- labeled dCTP were carried out in a DNA Thermocycler Express (Perkin-Elmer Cetus, Norwalk, CT, USA) with the following parameters: one cycle at 94°C for 4 min, 40°C for 5 min, 72°C for 5 min; 35 cycles at 94°C for 1 min, 60°C for 2 min, 72°C for 1 min; and a final elongation step at 72°C for 5 min. Amplified cDNA fragments were separated on a 6% denaturing polyacrylamide/urea gel at 85W constant power. After electrophoresis, the gels were dried with fixation onto Whatman 3MM filter paper and exposed for 1–4 days to Kodak XAR-5 film with fluorescent ink orientation markers. All reactions were repeated twice with the same cDNA template. The relative mRNA expression was determined using the Cyclone Storage Phosphor System (Packard Instrument, Meriden, CT, USA) and normalized by GAPDH expression. The fragments of interest were excised from the gel, extracted, and reamplified for further analysis. The reamplified fragments were cloned into a TA-cloning vector [pMD 18-T; TaKaRa Biotechnology (Dalian) Co., P. R. China] and sequenced. The DNA sequences were compared with known sequences using the National Center for Biotechnology Information (NCBI) GenBank database.


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  		Table 1. Sequences of the Anchor (A2) and Random Primers (R1–R15) Used in This Study

 
Real time PCR
The mRNA expression level of target genes in isolated and cultured cells was quantified by real-time PCR analysis using the ABI PRISM® 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) and the SYBR® Premix Ex TaqTM kit [TaKaRa Biotechnology (Dalian) Co.]. The following PCR cycle parameters were used: 95°C for 10 s, 40 cycles at 95°C for 5 s, 60°C for 34 s. Each PCR reaction was performed in duplicate tubes with GAPDH as an internal control to standardize the amount of sample RNA. The quantification data were analyzed with the sequence detection system (SDS) software (Applied Biosystems). After PCR, baseline subtraction was performed using SDS software. The log-linear portion of the fluorescence-versus-cycle plot was extended to determine a fractional cycle number, at which threshold fluorescence was obtained [threshold cycle (CT)] for each analyzed cDNA and for GAPDH cDNA as the reference. This comparative CT method was used for quantification of the target cDNA relative to GAPDH cDNA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of HSC and MMP and differentiation of CD34+ cells toward specific lineages
MACS was used to isolate HSC and MMP using the appropriate markers. To check the purity of these two populations, they were examined by flow cytometry using FITC-conjugated anti-CD34 and PE-conjugated anti-CD38 and anti-CD33 (Fig. 1 ). The histograms show that before isolation, almost 1% of the MNCs were CD34+, 5.33% were CD38– (Fig. 1B) , 85.13% were CD33–, and 14.88% were CD33+ (Fig. 1C) . The purity of isolated CD34+/CD38– cells was 78.40% (Fig. 1D) , the purity of CD34+/CD33– was 85.52% (Fig. 1E) , and the purity of CD34+/CD33+ was 91.16% (Fig. 1F) . From these results, we concluded that the purity of isolated HSC (CD34+/CD38–/CD33–) and MMP (CD34+/CD38–/CD33+) was nearly 90%. Although the isolated CD34+/CD38–/CD33– and CD34+/CD38–/CD33+ cells were relatively pure, the two subsets were heterogeneous and included true HSC as well as myeloid progenitors and very primitive progenitors.


Figure 1
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  		Figure 1. Purification of hematopoietic cell populations from human bone marrow. The isolated HSC (CD34+/CD38–/CD33–) and MMP (CD34+/CD38–/CD33+) were examined by flow cytometry with FITC-conjugated anti-CD34 and PE-conjugated anti-CD38 and anti-CD33. Six thousand total events were analyzed for each sample. The percentage of surface marker-positive cells in the population is indicated.

 
CD34+ cells undergoing expansion and differentiation in a semi-solid culture system exhibit striking changes in cell shape, size, nuclear chromatin, and cytoplasmic volume. We found that erythroid clones were small and dense, and cells from the clones were large (Fig. 2A ). Megakaryocytic clones were large and dense, and cells from the clones were also large (Fig. 2B) . Finally, granulocytic clones were small and did not appear dense, and cells from the clones were also small (Fig. 2C) .


Figure 2
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  		Figure 2. Morphology of hematopoietic cell clones generated by in vitro differentiation.

 
Flow cytometric analysis was performed to monitor erythroid-specific cell surface marker expression using FITC-conjugated anti-CD71; megakaryocytic-specific cell surface marker expression was monitored using FITC-conjugated anti-CD41, and granulocytic-specific cell surface marker expression was monitored using FITC-conjugated anti-CD11b. The CD71 expression level of committed erythroid clones stimulated with EPO for 4 days in culture was greater than that without EPO (Fig. 3A ); ~59% cells expressed a high level of CD71. The expression level of CD41 of committed megakaryocytic clones stimulated with IL-6 for 7 days in culture was greater than that without IL-6; 60.8% of cells were CD41+ (Fig. 3B) . The expression level of CD11b of committed granulocytic clones stimulated with G-CSF for 5 days in culture was also much greater than that without G-CSF, and 97.8% cells were CD11b+ (Fig. 3C) . The expression level of lineage-specific markers was much higher, and almost 100% of cells were positive following longer stimulation (Fig. 3 , bottom panels). These results indicate that CD34+ cells can be stimulated specifically toward erythroid, megakaryocytic, and granulocytic differentiation in our semi-solid cultures.


Figure 3
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  		Figure 3. Fluorocytometric analysis of cell surface markers during lineage-specific differentiation. Erythroid differentiation was analyzed with FITC-conjugated anti-CD71, megakaryocytic differentiation with FITC-conjugated anti-CD41, and granulocytic differentiation with FITC-conjugated anti-CD11b. Cells were cultured for the indicated number of days in the absence (top row) and in the presence (middle and bottom rows) of lineage-specific, inducing cytokines: EPO for erythroid, IL-6 for megakaryocytic, and G-CSF for granulocytic differentiation. The histograms depict the fluorescence distribution (x-axis) and cell count (y-axis). Six thousand total events were analyzed for each sample. The percentage of surface marker-positive cells in the population is indicated.

 
Detection of differential gene expression among HSC, MMP, EC, MC, and GC using DDRT-PCR assay
DDRT-PCR was used to determine the changes in gene expression during the commitment and differentiation of HSC to MMP and then to erythroid, megakaryocytic, and granulocytic cells. Using a total of 15 different combinations of primers, we selected 300 bands, which were differentially expressed in the different populations. These bands were reamplified, cloned, sequenced, and analyzed using the NCBI BLAST computer program. We identified 256 expression sequence tags (ESTs) with differential expression. Figure 4 shows a part of one DDRT-PCR reaction, which contains several differential-expressed mRNA species (indicated with black arrows).


Figure 4
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  		Figure 4. Differential expression in HSC, MMP, and committed clones, as detected on a DDRT-PCR gel. Two parallel experiments were performed for each DDRT-PCR. Black arrows point out some bands showing differential expression.

 
Among the 256 ESTs with differential expression, 194 were homologous to known genes, some of which were associated with hematopoiesis. The relative expression of all the 256 ESTs in HSC, MMP, EC, MC, and GC with database matches is shown in Table 2 .


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  		Table 2. Differentially Expressed Genes in HSC, MMP, and the Committed Lineage-Specific Clones

 
Categorizing the differentially expressed genes in all five hematopoietic lineages, according to their biological or molecular functions, yielded 10 functional groups: genes associated with cell surface receptors/ligands; genes involved in cell signaling, signal transduction, transcription/translation, metabolism, cell cycle control, apoptosis, cancer, ribosomal genes, and unknown cDNA and genome sequences (Table 2) .

Characterization of mRNAs known to be lineage-specific
To confirm lineage-specific differentiation, we identified genes up-regulated markedly, which were already known to be associated with specific programs of hematopoietic differentiation. No expression of these genes in HSC (CD34+/CD38–/CD33–) and MMP (CD34+/CD38–/CD33+) was observed, but we identified specific, lineage-committed clones with increased expression. Figure 5 shows the expression of several specific genes for the erythroid cells (ANK1, GPA, EKLF1, KEL), megakaryocytic cells (GP1BA, LGALS, PECAM1, PF4), and granulocytic cells (FCGR, MIP2 {alpha}, S100A8). Some genes were not expressed in specific, lineage-committed clones but were expressed highly in HSC (HOXA9, ATF4) or MMP (CSF1R, ANXA1; Fig. 5 ).


Figure 5
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  		Figure 5. Confirmation of lineage-specific differentiation of CD34+ cells by DDRT-PCR. During lineage-specific differentiation of CD34+ cells in the semi-solid cultures, several well-known marker mRNAs for each hematopoietic lineage were expressed highly. The relative expression was determined using the Cyclone Storage Phosphor System and was normalized by GAPDH expression.

 
mRNAs specifically expressed in one or more lineages
HSC-specific mRNAs
Genes, which were specifically expressed in HSC, were associated mainly with self-renewal and the maintenance of multipotentiality. Examples include genes encoding general transcription factors such as TPT1, EBF2, STATA5B, CREBBP, ATF4, HOXA9, TES, SOX4, IRF2, PBX1, and FOXK2 and genes related to cell signal transduction such as MPI, VIPR2, MDR1, SPAG6, HPTA, NGFRAP1, WIBG, IMPACT, TNFAIP3, and GNB3 (Table 3 ).


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  		Table 3. The mRNAs That Are Specifically Expressed in One or Several Lineages

 
MMP-specific mRNAs
Genes, which were expressed specifically in MMP, included genes encoding many cell signal regulators such as IGFBP-4, CSF1R, MFAP4, CCL17, STK32B, TIGD1, MDCR1, RBBP-36, BIRC2, four transcription factors (RNPC2, BRD7, ZFY, FMNL3), and three apoptosis modulators [ANXA1, KPNA2, ubiquitin A-52 (UBA52); Table 3 ]. These genes may be functionally important for MMP commitment and maintenance of multipotentiality.

EC-specific mRNAs
The genes, which were specifically expressed in EC, contain some potentially novel and known, lineage-specific genes and markers of erythroid commitment and differentiation, as well as genes, which are involved in the signaling response to the stimulating cytokines. These include genes encoding cell signal regulators [CG25C, IL-15R{alpha}, ERMAP, SLAMF7, EPIM, CAPG, PDYN, FCN2, ARHGAP15, ANNEXIN1, ANK1, PLEKHM2, SFRP1, HAP1, CPLA2, CHP, follistatin (FST), and PLEKHH2], genes encoding transcription factors (TPTR, BPTF, CTNND1, HLF, and ZIMP10), and genes encoding other proteins (ENDOBREVIN, CFLAR, TGFBP, DNAJC8, SET, FALZ, and MLL5; Table 3 ).

MC-specific mRNAs
Some transcripts were uniquely detected only in MC. Examples include cell signal regulators such as ICAM2, 7TMR, PECAM1, LILRB1, IL8, FYB, SH3BP5, TRRAP, RIS1, TREM2, and TMF1; transcription factors such as SPCS1, TGFBR2, and MEF2C; and metabolic molecules such as TXNL1, PTGES3, SSR2, ADCY6, BAK1, and IG {kappa} (Table 3) . These genes may be critical for megakaryocytic commitment and differentiation.

GC-specific mRNAs
Genes, which were expressed specially in GC, included classic granulocytic-associated genes. They included many cell signal regulators such as GRIN2A, GPR86, PPARGC, FOLR3, CFH, PGR1, SDCBP2, IFIT5, MCP-3, PN-1, AXIN, SIRPB1, FLT3, TACSTD1, SPINK1, CALM1, NGAL, PIP15, and CDK5RAP2 and cancer regulators such as ß-TGL, EF-1-{alpha}, NPC2, and BMP6 (Table 3) . These genes may be involved in granulocytic commitment and differentiation.

mRNAs commonly expressed in all cell populations except HSC
Expression of a gene, which encodes F13A1 (coagulation factor XIII, A1 polypeptide), was commonly detected in all populations except HSC (Table 3) , indicating that this is a gene, which may be involved in the commitment of the MMP.

mRNAs expressed in HSC and MMP but not in the differentiated groups
The mRNAs for Myc-induced mitochondria protein (MIMITIN), CIITA, Wilm's tumor-related protein (QM), and ribosomal protein L26 (RPL26) were commonly detected in HSC and MMP but not in all the three specific, lineage-committed clones, suggesting that repression of these genes may be involved in commitment to the three lineages (Table 3) .

mRNAs commonly expressed in the three differentiated groups
The mRNAs for proteoglycan 1, secretory granule (PRG1), ankyrin repeat and SOCS box-containing 8 (ASB-8), prostaglandin E synthase 3 (PTGES3), destrin (actin depolymerizing factor; DSTN), NK-tumor recognition sequence (NKTR), and cylicin (basic protein of sperm head cytoskeleton 1; CYLC1) were identified in the three differentiated groups but not in HSC and MMP. This suggests that these genes may be involved in commitment and differentiation to all three lineages (Table 3) .

Validation of differential expression by real-time PCR
To validate the differential expression identified using DDRT-PCR, real-time PCR was performed for three selected mRNA, ZNF330, PLEKHH2, and ASB-8. Although slight discrepancies were observed between the two methods, DDRT-PCR and real-time PCR indicated that the mRNA of ZNF330 was expressed mainly in HSC, PLEKHH2 was expressed mainly in EC, and ASB-8 was expressed mainly in GC (Fig. 6 ). Pleckstrin homology (PH) domain is often present in many kinases, GTPases, and GTPase-activating proteins involved in interactions with GTP-binding proteins [16 ]. The mRNA of PLEKHH2 was expressed specially in EC, indicating that PH might play an important role in erythroid commitment and differentiation. The zinc finger protein 330 (ZNF330) contains nine cysteine-X-X-cysteine (GATA CXXC-type) motifs and is expressed in the nucleolus and the cytoplasm during interphase; it transiently associates with centromeres during mitosis [17 ]. HSC expresses a high level of ZNF330, which appears to be important for HSC proliferation. Down-regulation of ZNF330 might also be essential for the commitment of HSC to MMP and the lineage-specific progenitors. ASB-8 is a member of the SOCS family and possesses four ankyrin repeats at the N terminus and one SOCS box at the C terminus, which can interact with Elongin B and Elongin C [18 ]. The high expression of ASB-8 in GC suggests that ASB-8 might be involved in granulocytic commitment and differentiation.


Figure 6
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  		Figure 6. Real-time PCR was used to confirm the identification of ZNF330, PLEKHH2, and ASB-8 mRNA as differentially expressed mRNAs using DDRT-PCR. Both DDRT-PCR and real-time PCR found that ZNF330 was expressed mainly in HSC, PLEKHH2 in EC, and ASB-8 in GC. The real-time PCR experiments were performed in duplicate, and relative expression was normalized to GAPDH expression in each sample.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Various technical strategies, such as differential display, suppression PCR, oligo-based Affymetrix GeneChips, and micro-serial analysis of gene expression (micro-SAGE), have been used to characterize the genes associated with hematopoietic differentiation. Komor et al. [19 ] analyzed transcriptional profiling of human CD34+ cells recently during in vitro lineage-specific differentiation by microarray. Fujishima et al. [20 ] analyzed the transcriptional profile of human erythroid progenitors by micro-SAGE [20 ]. The DDRT-PCR method has also been applied previously to hematopoietic cells with some success. For instance, CD34+/CD38+ progenitor cells have been compared with CD34+/CD38– cells, resulting in the identification of a novel cDNA sequence specific to the CD34+/CD38– population [21 ]. Similarly, an analysis of gene expression in myelodysplastic patients compared with normal controls using the DDRT-PCR technique yielded a cDNA fragment, which is down-regulated in myelodyplastia [22 ].

However, a comprehensive transcription profile of human HSC differentiating into MMP and to the erythropoietic, megakaryopoietic, and granulopoietic lineages has not been performed. As CD34+ cells from human bone marrow include HSC and progenitors such as myeloid, erythroid, megakaryocytic, granulocytic, and lymphoid progenitors, we used three specific markers (CD34, CD38, and CD33) to acquire relatively homogeneous HSC and MMP populations. At the same time, we realized that committed, lineage-specific progenitors isolated from human bone marrow are also heterogeneous populations and that there are no specific markers to define lineage-specific progenitors at the early differentiation stage. To better understand transcriptional programs crucial to commitment and the differentiation of HSC to MMP and further to specific lineages, we adopted semi-solid culture systems combined with EPO, IL-6, and G-CSF to obtain erythroid, megakaryocytic, and granulocytic clones derived from each committed, lineage-specific progenitors.

The DDRT-PCR technique offers two major advantages for the study of hematopoietic cells: First, it permits analysis of small amounts of mRNA, thereby enabling the study of HSC and lineage-specific progenitors; second, it allows a comparative analysis of variations in subsets of the mRNA repertoire of HSC undergoing differentiation. As the qualities of purified HSC and MMP and the collected, committed lineage-specific clone cells were limited, and DDRT-PCR is more sensitive compared with cDNA microarray analysis, we chose the DDRT-PCR method to characterize and identify genes with differential expression. It was reported previously that mRNA RTs from one anchor primer (A2) represent 80% of the total cDNA obtained from all three anchor primers [15 ]; thus, we only used the anchor primer A2 to perform DDRT-PCR.

Gene expression in HSC, MMP, EC, MC, and GC was compared systematically in this study. Generally, mRNAs, which are involved in lineage commitment and differentiation, are highly expressed in specific committed lineages rather than in multipotential progenitors. Conversely, genes, which are involved in extensive self-renewal, are expressed only in multipotential progenitors rather than in committed lineages. Whereas lineage specificity has been viewed traditionally as strictly a positive event, the concomitant down-regulation of some genes in a multipotential progenitor upon activation of a program suggests that the manner in which multipotential progenitors choose a differentiation pathway is complex. For example, GATA-1 is expressed in early hematopoietic progenitors and is down-regulated specifically in myelomonocytic cells during lineage determination; its enforced expression could reprogram myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts [23 ]. Many genes were up-regulated specifically in EC, MC, or GC compared with HSC or/and MMP, and several genes, which were expressed highly in HSC or/and MMP, were down-regulated specifically in EC, MC, or GC in our study. This suggests that specific gene activation and specific gene repression are necessary for lineage-specific commitment and differentiation.

Proliferation and differentiation of HSC
A number of genes, which were reported previously to be associated with HSC self-renewal, were found to be expressed highly in HSC compared with the various committed, lineage-specific populations (Tables 2 and 3) . ATF4 is a transcription factor belonging to the CREB/ATF family, and its binding sites are present in a variety of cellular growth-regulating genes [24 ]. HOXA9 is preferentially expressed in subfractions, which are highly enriched for primitive, long-term, culture-initiating cells or myeloid progenitor cells [25 ]. It was reported that acute myeloid leukemia characterized by translocation t (8; 16)(p11; p13) and MYST3-CREBBP rearrangement also exhibited up-regulation of HOXA9 and HOXA10 [26 ]. Fujishima et al. [20 ] reported previously that HSC expresses high levels of forkhead box D3 by micro-SAGE. In this study, we found that the mRNA of another member of the forkhead box protein family, forkhead box K2 (FOXK2), was also enriched in HSC. FOXK2 binds to the purine-rich motifs of the HIV long-terminal repeat and to the similar purine-rich motif in the IL-2 promoters. FOXK2 is also involved in the regulation of viral and cellular promoter elements [27 ]. Immunofluorescence analysis shows that ZNF330 is present in the nucleolus and the cytoplasm during interphase and transiently associates with centromeres during mitosis [16 ]. In our study, high expression of many genes, including ATF4, HOXA9, CREBBP, FOXK2, and ZNF330, was detected in HSC. These genes were at low levels in MMP, EC, MC, and GC (Tables 2 and 3) , suggesting these genes might be important for HSC proliferation. Down-regulation of these genes may also be essential for the commitment of HSC to MMP and to the lineage-specific progenitors.

A c-Myc target mRNA, MIMITIN, was reported to be associated with cell proliferation and to be down-regulated in quiescent and differentiated cells [28 ]. MIMITIN mRNA was enriched in HSC and MMP, but almost no expression was found in EC, MC, or GC in our study (Table 2) . This suggests that this gene might be important for proliferation of HSC and MMP, and that repression of its expression might be important for MMP differentiation.

Myeloid commitment and differentiation
Several genes were expressed highly in MMP compared with HSC, suggesting the role of these genes in the commitment of HSC to MMP (Table 2) . UBA52 residue ribosomal protein fusion product 1 consists of ubiquitin at the N terminus and ribosomal protein L40 at the C terminus [29 ]. The ubiquitnation of CIITA by UBA52 enhances the association of CIITA with MHC class II transcription factors and the MHC class II promoter, resulting in an increase of transactivation function and in the expression of MHC class II mRNA [30 ]. The UBA52 and CIITA mRNAs were expressed at higher level in MMP compared with HSC in our study, which suggests that MHC class II plays an important role in MMP commitment and proliferation through the UBA52 pathway. Macrophage CSF1R mRNA was often found in monocytes/macrophages, seldom in erythroid and megakaryocytic lineage cells, and never in lymphocytes [31 ], which is consistent with our finding that M-CSF1R mRNA was enriched in MMP compared with in HSC. These data indicate the significance of M-CSF1R in MMP commitment and proliferation.

Erythroid commitment and differentiation
Many genes were expressed highly in EC (Table 3) , including some genes, which have been reported to be involved in erythtroid differentiation. EKLF, which has been shown extensively to play an important part in ß-globin gene regulation, also plays a role in erythtroid differentiation [32 ]. FST was reported to reduce activin A-mediated GATA2 and EPO-R expression to regulate erythroid differentiation [33 ]. Although the roles of the two PH domain-containing proteins (PLEKHM2 and PLEKHH2) in erythroid differentiation are not understood, they both contain an important PH domain. Another PH-containing protein, Rho GTPase-activating protein 15 (ArhGAP15) was expressed in K562 cells and was up-regulated during hemin-induced erythroid differentiation [34 ]. A recent experiment showed overexpression of ArhGAP15 coupled with accelerated erythroid differentiation and knock-down of ArhGAP15 delayed erythroid differentiation (Xiao-Fang Huo and J-W. Zhang, unpublished results). The mRNAs of EKLF, FST, PLEKHM2, PLEKHH2, and ArhGAP15 were found to be expressed specifically in EC in this study, suggesting the importance of these proteins in erythroid commitment and differentiation. Many members of the heat shock protein (HSP) family help cells respond to heat stimulation by regulating protein expression or degradation. Fujishima et al. [20 ] and Terskikh et al. [35 ] reported previously that EC expressed high levels of HSP86 and HSP90. The mRNA of HSP40 (DNAJC8) was also expressed specifically in EC, implying that it is important in regulating protein reconstruction or degradation during erythroid commitment and differentiation.

During HSC differentiation, the appearance and subsequent down-regulation of stem cell leukemia transcripts precede expression of GATA-2 and GATA-1 and are crucial to erythroid commitment and differentiation [36 ]. It is analogous that Septin 11 (SEPT11) mRNA decreased specifically in EC in this study, suggesting that repression of SEPT11 might be involved in specific erythroid commitment.

Megakaryocytic commitment and differentiation
Some genes were expressed specifically in MC, suggesting their role in megakaryocytic commitment and differentiation (Tables 2 and 3) . The signal sequence receptor (SSR) is a glycosylated endoplasmic reticulum (ER) membrane receptor associated with protein translocation across the ER membrane [37 ]. All ICAM proteins are type I transmembrane glycoproteins [38 ]. The mRNA of SSR2 and ICAM2 was expressed specifically in MC in our study (Table 3) , indicating that the regulation of glycosylation may be important to megakaryocytic commitment and differentiation. Shim et al. [39 ] reported previously that MC expressed high levels of coagulation factor XIII, A1 polypeptide (F13A1); lectin, galactoside-binding, soluble, 1 (GALECTIN1); and PF4 [39 ]. The mRNAs encoding these proteins were also up-regulated specifically in MC compared with HSC in our study, indicating that they might be involved in megakaryocytic commitment and differentiation (Table 2) .

Little is known about the down-regulated genes associated with megakaryocytic development in previous studies. We failed to identify mRNAs, which were undetectable only in MC in this study, but this does not rule out down-regulation of some genes, which play a part in megakaryocytic commitment and differentiation. The repressed or decreased expression of insulin-like growth factor-binding protein (IGFBP-4), hepatocyte growth factor (HPTA), tetratricopeptide repeat domain 3 (TTC3), CTG repeat (CTGR), ZNF281, COMM domain-containing 3 (COMMD3), lysozyme (LYZS), DiGeorge syndrome critical region (DGC2), ribosomal protein L31 (RPL31), and mitochondrial ribosomal protein L46 mRNAs was found in EC and MC, supporting the idea that erythroid and megakaryocytic precursors differentiate from one common progenitor. This further suggests their possible roles in commitment and differentiation of the common progenitor.

Granulocytic commitment and differentiation
Some mRNAs associated with granulocytic differentiation have been found to be GC-rich. One is the ASB-8 mRNA (Table 2) . The high expression of ASB-8 in GC was also confirmed by quantitative real-time RT-PCR (Fig. 5) . ASB-8 is a member of SOCS, possessing four ankyrin repeats in the N terminus and one SOCS box at the C terminus, which can interact with Elongin B and Elongin C [18 ]. SOCS3 is a key negative regulator of G-CSF signaling in myeloid cells and is of particular significance during G-CSF-driven emergency granulopoiesis [40 ]. ASB-2 mRNA is induced by all-trans RA in HL-60 cells and is regulated directly by RARK and RARE/RXRE elements in the ASB-2 promoter region [41 ]. The fact that ASB-2 expression in HL-60 cells limits cell proliferation and accelerates chromatin condensation strongly suggests that ASB-2 promotes commitment events at the early stages of RA-induced differentiation of leukemia cells [42 ]. A more recent study found that ASB-3 attenuates TNF-R2-mediated proteolysis [43 ]. The prominent expression of ASB-8 mRNA in GC in this study suggests that ASB-8 might be involved in granulocytic commitment and differentiation through negative regulation of G-CSF signaling. S100A8, also known as migration inhibitory factor-related protein-8 (MRP8), stimulates L-selectin shedding and activates membrane-activated complex 1/CD11b. It also induces neutrophil adhesion to fibrinogen in vitro and is involved in neutrophil migration to in vivo [44 ]. MRP8/14 heterodimers and MRP8/9 protein complexes are the exclusive, arachidonic acid-binding proteins in human neutrophils: They promote leukocyte–endothelial cell interactions and play an important role in leukocyte trafficking but do not affect neutrophil function [45 ]. In our study, the MRP8 (S100A8) mRNA level in GC was 16-fold higher than in HSC and the other cell populations, indicating that MRP8 may be important for granulocytic commitment and differentiation (Tables 2 and 3) .

Both HoxA9 and HoxA10 are highly expressed in human CD34+ cells, but are down-regulated during myeloid development. This down-regulation is important in granulocytic commitment and differentiation [46 ]. In this study, we observed suppressed expression of cell division cycle 14 homologue A (CDC14A) and Charcot-Leyden crystal protein (CLC) mRNAs in GC compared with MMC, EC, and GC (Table 2) , suggesting that the down-regulation of CDC14A and CLC is involved in granulocytic commitment and differentiation.

In conclusion, the data presented here describe changes in gene expression during the commitment and differentiation of HSC to MMP and further, to EC, MC, and GC specifically. These data suggest that gene activation and gene repression are necessary for specific lineage commitment and differentiation. These results contribute to understanding the complex mechanisms of hematopoietic commitment and differentiation.


   ACKNOWLEDGEMENTS
 
This work is supported by the Pre-Study Item of State Key Basic Research of China (2002CCA04300) and National Basic Research Program of China (No.2006CB504100).


   FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received January 5, 2007; revised June 15, 2007; accepted June 18, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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