CD133-Positive Hematopoietic Stem Cell "Stemness" Genes Contain Many Genes Mutated or Abnormally Expressed in Leukemia
http://www.100md.com
《干细胞学杂志》
a Department of Pediatric Hemato-Oncology, Safra Children’s Hospital and
b Department of Obstetrics and Gynecology, The Sheba Medical Center, Tel-Hashomer, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel;
c Department of Molecular Cell Biology and
d Department of Complex Systems, Weizmann Institute of Science, Rehovot, Israel;
e Department of Obstetrics and Gynecology, Rambam Medical Center, Bruce Rappaport Institute of Technology, Technion-Israel Institute of Technology, Haifa, Israel
Key Words. Hematopoietic stem cells ? Stemness ? Peripheral blood ? Cord blood ? Gene expression
Correspondence: Amos Toren, M.D., Ph.D., Department of Pediatric Hematology-Oncology, Sheba Medical Center, Tel-Hashomer, Israel 52621. Telephone: 972-3-5303037; Fax: 972-3-5303031; e-mail: amost@post.tau.ac.il
ABSTRACT
A unique characteristic of stem cells is their ability for self-renewal and multipotential differentiation, the mechanism of which is poorly understood. Hematopoietic stem cells (HSCs) are currently used in clinical stem cell transplantation. In addition, they hold great promise for future regenerative medicine, tissue repair, and gene therapy. Deciphering the mechanisms regulating proliferation and differentiation may improve our ability for rational usage of HSCs for these purposes.
Umbilical cord blood (CB) and mobilized peripheral blood (PB) are relatively new sources of HSCs that have been increasingly used in clinical transplantations. These two sources show considerable differences in their proliferative capacity, engraftment kinetics, and differentiation potential . Differences in these biological properties may stem from the expression of a different set of genes in these two groups of HSCs. Despite these differences, these stem cells, similar to stem cells derived from other tissues, share many biological properties. This may result from "stemness" gene expression profile characteristic of stem cells.
Recent studies using different DNA microarray technologies (oligonucleotide-based or cDNA arrays) for the analysis of gene expression profiles of HSCs were performed mainly in mice using antibodies reactive to CD34 with a combination of other markers to enrich for stem cells. Some studies focused on the differences between primitive HSCs, the more differentiated progenitors, and the mature cells. Other studies concentrated on the differences and similarities in gene expression between stem cells from different tissues (hematopoietic, neuronal, embryonic, etc.) .
The CD34 antigen was usually served for the isolation of HSCs both for the purpose of stem cell transplantation and for laboratory studies. However, recent studies show that the CD34– fraction also has a repopulating capacity and includes cells that are precursors of CD34+ cells . The CD133 antigen (also known as AC133 or prominin-1) was found to be coexpressed with CD34 but also found in CD34–CD38–Lin– precursors . A small, rare population within the CD34–Lin– cells that expresses CD133 has a high progenitor activity and was capable of giving rise to CD34+ cells , and coexpression of CD133+ and CD34+ led to a higher clonogenic capacity compared with the CD133–CD34+ cells . Another recent study pointed out that among the CD34+/CD38– cells, the fraction of slow dividing cells that is associated with primitive function and self-renewal expresses high levels of the CD133 gene in contrast with the fast dividing cells. CD133 may have a central role in the asymmetric division that is believed to characterize true stemness . Altogether, these studies suggest that CD133+ may provide a more appropriate marker to enrich stem cells and therefore was used as an isolation target in our study.
The aim of this study was to identify specific genes that are upregulated in progenitors and HSCs originating from CB and mobilized PB and may represent the stemness genes in HSCs. Because the CD133+ cell fraction used in this study is far from being a pure HSC fraction, the term stemness is used in this work merely for simplicity, and many of the stemness genes defined in the study are in fact stem/progenitor or progenitor rather than stem cell–proper genes. In addition, we looked for genes differentially expressed in HSCs from either PB or CB.
MATERIALS AND METHODS
Affymetrix Hu133A oligonucleotide arrays covering 22,215 PSs were used to determine the gene expression profile of CD133+ HSCs derived from CB and PB. The CD133+ cells were highly enriched (85% purity). CD133-expressing cells represent the stem cell–enriched population, and the CD133-negative cells represent the differentiated cells at various stages. Our analysis focused on expressed genes that are enriched in CB and PB CD133+ cells compared with CD133– cells from both sources as well as in either CB or PB alone.
An unsupervised hierarchical clustering of 14,025 valid PSs out of the total 22,215 PSs present on the microarray (Fig. 1A) showed a clear distinction between the CD133-expressing and -nonexpressing cells, each type clustered together on different sides of the tree. The comparison between the CD133+ samples and CD133– samples identified a list of 584 differentially expressed PSs, of which 297 were upregulated and 287 were downregulated (Fig. 1C, supplemental online Table 2). CB CD133+ cells were compared with CB CD133– cells, and 1,689 PSs were identified as differentially expressed by at least twofold. Of these PSs, 789 (672 genes) were upregulated and 900 (708 genes) were downregulated (Fig. 1D, supplemental online Table 2). PB CD133+ cells were compared with PB CD133– cells. A total of 1,336 PSs differentiate between the two populations by at least twofold; 690 (596 genes) PSs were upregulated and 646 (495 genes) were downregulated (Fig. 1D, supplemental online Table 2).
Figure 1. Comparative gene expression profiles of cell populations originating from human CB and PB. Red indicates high relative expression; green, low expression. Each column represents a sample and each row a gene. Two samples originating from PB-enriched CD133+ cells, two samples from CB-enriched CD133+ cells, two samples from PB CD133– fraction, and two samples from CB CD133– fraction are shown. (A): The matrix of the unsupervised hierarchal clustering of 14,025 valid PSs out of the total 22,215. A clear distinction was found between the CD133+ and the CD133–cells. (B): Clustering demonstrates 1,053 PSs upregulated in CD133+ cells from PB or CB compared with the CD133– cells and 1,092 PSs downregulated in CD133+ cells from PB or CB. (C): Clustering of 297 PSs upregulated in CD133+ cells from both PB and CB compared with CD133– cells and 287 probe sets downregulated in CD133+ cells from PB and CB. (D): Upregulated PSs and genes (italics and underlined) in CD133+ cells. The 1,053 PSs upregulated in HSCs were combined from 789 (672 genes) in CB and 690 (596 genes) in PB minus the common 426 PSs (367 genes) as calculated by the average fold change of the samples of CB or PB compared with the differentiated cells. Out of the 426 PSs, 297 (244 genes) showed a fold change above 2 for each of the samples. The numbers in the crescents represent the source-specific (PB or CB) upregulated PSs (genes). Abbreviations: CB, cord blood; HSC, hematopoietic stem cell; PB, peripheral blood; PS, probe set.
The intersection between the upregulated PSs in CD133+ CB cells and CD133+ PB cells creates a group of 426 PSs (367 genes) calculated by the average fold change of the samples of CB or PB compared with the differentiated cells. Out of these, 297 PSs (244 genes) showed a fold change above two for each compared with the differentiated cells and were the subject of further analysis.
Of these genes, a list of 132 that were upregulated by at least five are presented in Table 1. The genes common to Ivanova et al. , Ramalho-Santos et al. , and Georgantas et al. are marked in specific lanes.
Table 1. Genes upregulated by more than fivefold in CD133+ CB and PB cells compared with CD133– cells
A total of 363 PSs (304 genes) are upregulated in CB only, and 264 PSs (218 genes) are upregulated in PB only (Fig. 1D, supplemental online Tables 4B, 4C).
To further validate the hybridization results, QRT-PCR analysis was performed on eight genes selected from the 244 stemness genes. A good correlation between the two methods was observed, as shown in Figure 2 and supplemental online Table 6.
Figure 2. Verification of gene expression results using quantitative real-time polymerase chain reaction (QRT-PCR). The upregulated genes (FZD6, HOXA9, FLT3, MLLT3, TIE1, HLF, SPINK2, and MEIS1) in the CD133+ versus CD133– cells in cord blood (CB) and peripheral blood (PB) according to the results obtained by the array analysis were compared with QRT-PCR results. Each column represents the expectation value of the natural logarithm of the ratio of CD133+ signal to the CD133– signal. The signal values as well as the logarithm of the fold change and its SD are represented in supplemental online Table 6.
We compared this list of genes with those provided by Ivanova et al. and Ramalho-Santos et al. , who performed a similar analysis mainly in mice, and found 65 and 33 common genes, respectively. A comparison with the list of genes provided by Georgantas et al. that studied human HSCs yielded 24 common stemness genes (Fig. 3, Tables 2 and 3, supplemental online Table 3).
Figure 3. Venn diagram showing similarities between HSC populations isolated by (A) our study from human CB and PB, (B) Ivanova et al. from murine BM and fetal liver, (C) Ramalho-Santos et al. from murine BM, and (D) Georgantas et al. from human CB and PB. A total of 1,905 genes were found to be upregulated in at least one of the stem cell populations by at least twofold (active genes), determining HSC-related genes from Ivanova et al. . A total of 922 genes from the HSC-enriched genes that were described by Ramalho-Santos et al. were found to have human orthologs. A total of 109 upregulated probe sets (99 genes) in human CB and PB HSCs were described by Georgantas et al. . A total of 244 genes were determined to be HSC-related genes from the CD133+ versus CD133– comparison (stemness) done in our study. Sixty-five genes are common to our stemness group and to the 1,905 genes originating from the date of Ivanova et al. , 33 genes are common to our stemness group and to the 922 HSC-enriched genes from Ramalho-Santos et al. data, and 24 genes are common to the common HSC genes described by Georgantas et al. . Abbreviations: BM, bone marrow; CB, cord blood; HSC, hematopoietic stem cell; PB, peripheral blood.
DISCUSSION
A.T., B.B., J.J.-H., and T.F. contributed equally to this study. We would like to thank the Kahn Family Foundation for supporting this research. This research was partially supported by the Israel Science Foundation.
REFERENCES
Gluckman E, Rocha V, Chastang C. Peripheral stem cells in bone marrow transplantation. Cord blood stem cell transplantation. Baillieres Best Pract Res Clin Haematol 1999;12:279–292.
Ivanova NB, Dimos JT, Schaniel C et al. A stem cell molecular signature. Science 2002;298:601–604.
Ramalho-Santos M, Yoon S, Matsuzaki Y et al. "Stemness": transcriptional profiling of embryonic and adult stem cells. Science 2002;298:597–600.
Terskikh AV, Miyamoto T, Chang C et al. Gene expression analysis of purified hematopoietic stem cells and committed progenitors. Blood 2003;102:94–101.
Steidl U, Kronenwett R, Rohr UP et al. Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow-derived and circulating human CD34+ hematopoietic stem cells. Blood 2002;99:2037–2044.
Graf L, Heimfeld S, Torok-Storb B. Comparison of gene expression in CD34+ cells from bone marrow and G-CSF-mobilized peripheral blood by high-density oligonucleotide array analysis. Biol Blood Marrow Transplant 2001;7:486–494.
Ng YY, van Kessel B, Lokhorst HM et al. Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity. J Leukoc Biol 2004;75:314–323.
Shojaei F, Gallacher L, Bhatia M. Differential gene expression of human stem progenitor cells derived from early stages of in utero human hematopoiesis. Blood 2004;103:2530–2540.
Georgantas RW, Tanadve V, Malehorn M et al. Microarray and serial analysis of gene expression analyses identify known and novel transcripts over-expressed in hematopoietic stem cells. Cancer Res 2004;64:4434–4441.
Bhatia M, Bonnet D, Murdoch B et al. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med 1998;4:1038–1045.
Gallacher L, Murdoch B, Wu DM et al. Isolation and characterization of human CD34– Lin– and CD34+ Lin– hematopoietic stem cells using cell surface markers AC133 and CD7. Blood 2000;95:2813–2820.
Bhatia M. AC133 expression in human stem cells. Leukemia 2001;15:1685–1688.
Gordon PR, Leimig T, Babarin-Dorner A et al. Large-scale isolation of CD133+ progenitor cells from G-CSF mobilized peripheral blood stem cells. Bone Marrow Transplant 2003;31:17–22.
Lang P, Bader P, Schumm M et al. Transplantation of a combination of CD133+ and CD34+ selected progenitor cells from alternative donors. Br J Haematol 2004;124:72–79.
Wagner W, Ansorge A, Wirkner U et al. Molecular evidence for stem cell function of the slow-dividing fraction among human hematopoietic progenitor cells by genome-wide analysis. Blood 2004;104:675–686.
Dennis G Jr, Sherman BT, Hosack DA et al. DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 2003;4:P3.
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b Department of Obstetrics and Gynecology, The Sheba Medical Center, Tel-Hashomer, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel;
c Department of Molecular Cell Biology and
d Department of Complex Systems, Weizmann Institute of Science, Rehovot, Israel;
e Department of Obstetrics and Gynecology, Rambam Medical Center, Bruce Rappaport Institute of Technology, Technion-Israel Institute of Technology, Haifa, Israel
Key Words. Hematopoietic stem cells ? Stemness ? Peripheral blood ? Cord blood ? Gene expression
Correspondence: Amos Toren, M.D., Ph.D., Department of Pediatric Hematology-Oncology, Sheba Medical Center, Tel-Hashomer, Israel 52621. Telephone: 972-3-5303037; Fax: 972-3-5303031; e-mail: amost@post.tau.ac.il
ABSTRACT
A unique characteristic of stem cells is their ability for self-renewal and multipotential differentiation, the mechanism of which is poorly understood. Hematopoietic stem cells (HSCs) are currently used in clinical stem cell transplantation. In addition, they hold great promise for future regenerative medicine, tissue repair, and gene therapy. Deciphering the mechanisms regulating proliferation and differentiation may improve our ability for rational usage of HSCs for these purposes.
Umbilical cord blood (CB) and mobilized peripheral blood (PB) are relatively new sources of HSCs that have been increasingly used in clinical transplantations. These two sources show considerable differences in their proliferative capacity, engraftment kinetics, and differentiation potential . Differences in these biological properties may stem from the expression of a different set of genes in these two groups of HSCs. Despite these differences, these stem cells, similar to stem cells derived from other tissues, share many biological properties. This may result from "stemness" gene expression profile characteristic of stem cells.
Recent studies using different DNA microarray technologies (oligonucleotide-based or cDNA arrays) for the analysis of gene expression profiles of HSCs were performed mainly in mice using antibodies reactive to CD34 with a combination of other markers to enrich for stem cells. Some studies focused on the differences between primitive HSCs, the more differentiated progenitors, and the mature cells. Other studies concentrated on the differences and similarities in gene expression between stem cells from different tissues (hematopoietic, neuronal, embryonic, etc.) .
The CD34 antigen was usually served for the isolation of HSCs both for the purpose of stem cell transplantation and for laboratory studies. However, recent studies show that the CD34– fraction also has a repopulating capacity and includes cells that are precursors of CD34+ cells . The CD133 antigen (also known as AC133 or prominin-1) was found to be coexpressed with CD34 but also found in CD34–CD38–Lin– precursors . A small, rare population within the CD34–Lin– cells that expresses CD133 has a high progenitor activity and was capable of giving rise to CD34+ cells , and coexpression of CD133+ and CD34+ led to a higher clonogenic capacity compared with the CD133–CD34+ cells . Another recent study pointed out that among the CD34+/CD38– cells, the fraction of slow dividing cells that is associated with primitive function and self-renewal expresses high levels of the CD133 gene in contrast with the fast dividing cells. CD133 may have a central role in the asymmetric division that is believed to characterize true stemness . Altogether, these studies suggest that CD133+ may provide a more appropriate marker to enrich stem cells and therefore was used as an isolation target in our study.
The aim of this study was to identify specific genes that are upregulated in progenitors and HSCs originating from CB and mobilized PB and may represent the stemness genes in HSCs. Because the CD133+ cell fraction used in this study is far from being a pure HSC fraction, the term stemness is used in this work merely for simplicity, and many of the stemness genes defined in the study are in fact stem/progenitor or progenitor rather than stem cell–proper genes. In addition, we looked for genes differentially expressed in HSCs from either PB or CB.
MATERIALS AND METHODS
Affymetrix Hu133A oligonucleotide arrays covering 22,215 PSs were used to determine the gene expression profile of CD133+ HSCs derived from CB and PB. The CD133+ cells were highly enriched (85% purity). CD133-expressing cells represent the stem cell–enriched population, and the CD133-negative cells represent the differentiated cells at various stages. Our analysis focused on expressed genes that are enriched in CB and PB CD133+ cells compared with CD133– cells from both sources as well as in either CB or PB alone.
An unsupervised hierarchical clustering of 14,025 valid PSs out of the total 22,215 PSs present on the microarray (Fig. 1A) showed a clear distinction between the CD133-expressing and -nonexpressing cells, each type clustered together on different sides of the tree. The comparison between the CD133+ samples and CD133– samples identified a list of 584 differentially expressed PSs, of which 297 were upregulated and 287 were downregulated (Fig. 1C, supplemental online Table 2). CB CD133+ cells were compared with CB CD133– cells, and 1,689 PSs were identified as differentially expressed by at least twofold. Of these PSs, 789 (672 genes) were upregulated and 900 (708 genes) were downregulated (Fig. 1D, supplemental online Table 2). PB CD133+ cells were compared with PB CD133– cells. A total of 1,336 PSs differentiate between the two populations by at least twofold; 690 (596 genes) PSs were upregulated and 646 (495 genes) were downregulated (Fig. 1D, supplemental online Table 2).
Figure 1. Comparative gene expression profiles of cell populations originating from human CB and PB. Red indicates high relative expression; green, low expression. Each column represents a sample and each row a gene. Two samples originating from PB-enriched CD133+ cells, two samples from CB-enriched CD133+ cells, two samples from PB CD133– fraction, and two samples from CB CD133– fraction are shown. (A): The matrix of the unsupervised hierarchal clustering of 14,025 valid PSs out of the total 22,215. A clear distinction was found between the CD133+ and the CD133–cells. (B): Clustering demonstrates 1,053 PSs upregulated in CD133+ cells from PB or CB compared with the CD133– cells and 1,092 PSs downregulated in CD133+ cells from PB or CB. (C): Clustering of 297 PSs upregulated in CD133+ cells from both PB and CB compared with CD133– cells and 287 probe sets downregulated in CD133+ cells from PB and CB. (D): Upregulated PSs and genes (italics and underlined) in CD133+ cells. The 1,053 PSs upregulated in HSCs were combined from 789 (672 genes) in CB and 690 (596 genes) in PB minus the common 426 PSs (367 genes) as calculated by the average fold change of the samples of CB or PB compared with the differentiated cells. Out of the 426 PSs, 297 (244 genes) showed a fold change above 2 for each of the samples. The numbers in the crescents represent the source-specific (PB or CB) upregulated PSs (genes). Abbreviations: CB, cord blood; HSC, hematopoietic stem cell; PB, peripheral blood; PS, probe set.
The intersection between the upregulated PSs in CD133+ CB cells and CD133+ PB cells creates a group of 426 PSs (367 genes) calculated by the average fold change of the samples of CB or PB compared with the differentiated cells. Out of these, 297 PSs (244 genes) showed a fold change above two for each compared with the differentiated cells and were the subject of further analysis.
Of these genes, a list of 132 that were upregulated by at least five are presented in Table 1. The genes common to Ivanova et al. , Ramalho-Santos et al. , and Georgantas et al. are marked in specific lanes.
Table 1. Genes upregulated by more than fivefold in CD133+ CB and PB cells compared with CD133– cells
A total of 363 PSs (304 genes) are upregulated in CB only, and 264 PSs (218 genes) are upregulated in PB only (Fig. 1D, supplemental online Tables 4B, 4C).
To further validate the hybridization results, QRT-PCR analysis was performed on eight genes selected from the 244 stemness genes. A good correlation between the two methods was observed, as shown in Figure 2 and supplemental online Table 6.
Figure 2. Verification of gene expression results using quantitative real-time polymerase chain reaction (QRT-PCR). The upregulated genes (FZD6, HOXA9, FLT3, MLLT3, TIE1, HLF, SPINK2, and MEIS1) in the CD133+ versus CD133– cells in cord blood (CB) and peripheral blood (PB) according to the results obtained by the array analysis were compared with QRT-PCR results. Each column represents the expectation value of the natural logarithm of the ratio of CD133+ signal to the CD133– signal. The signal values as well as the logarithm of the fold change and its SD are represented in supplemental online Table 6.
We compared this list of genes with those provided by Ivanova et al. and Ramalho-Santos et al. , who performed a similar analysis mainly in mice, and found 65 and 33 common genes, respectively. A comparison with the list of genes provided by Georgantas et al. that studied human HSCs yielded 24 common stemness genes (Fig. 3, Tables 2 and 3, supplemental online Table 3).
Figure 3. Venn diagram showing similarities between HSC populations isolated by (A) our study from human CB and PB, (B) Ivanova et al. from murine BM and fetal liver, (C) Ramalho-Santos et al. from murine BM, and (D) Georgantas et al. from human CB and PB. A total of 1,905 genes were found to be upregulated in at least one of the stem cell populations by at least twofold (active genes), determining HSC-related genes from Ivanova et al. . A total of 922 genes from the HSC-enriched genes that were described by Ramalho-Santos et al. were found to have human orthologs. A total of 109 upregulated probe sets (99 genes) in human CB and PB HSCs were described by Georgantas et al. . A total of 244 genes were determined to be HSC-related genes from the CD133+ versus CD133– comparison (stemness) done in our study. Sixty-five genes are common to our stemness group and to the 1,905 genes originating from the date of Ivanova et al. , 33 genes are common to our stemness group and to the 922 HSC-enriched genes from Ramalho-Santos et al. data, and 24 genes are common to the common HSC genes described by Georgantas et al. . Abbreviations: BM, bone marrow; CB, cord blood; HSC, hematopoietic stem cell; PB, peripheral blood.
DISCUSSION
A.T., B.B., J.J.-H., and T.F. contributed equally to this study. We would like to thank the Kahn Family Foundation for supporting this research. This research was partially supported by the Israel Science Foundation.
REFERENCES
Gluckman E, Rocha V, Chastang C. Peripheral stem cells in bone marrow transplantation. Cord blood stem cell transplantation. Baillieres Best Pract Res Clin Haematol 1999;12:279–292.
Ivanova NB, Dimos JT, Schaniel C et al. A stem cell molecular signature. Science 2002;298:601–604.
Ramalho-Santos M, Yoon S, Matsuzaki Y et al. "Stemness": transcriptional profiling of embryonic and adult stem cells. Science 2002;298:597–600.
Terskikh AV, Miyamoto T, Chang C et al. Gene expression analysis of purified hematopoietic stem cells and committed progenitors. Blood 2003;102:94–101.
Steidl U, Kronenwett R, Rohr UP et al. Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow-derived and circulating human CD34+ hematopoietic stem cells. Blood 2002;99:2037–2044.
Graf L, Heimfeld S, Torok-Storb B. Comparison of gene expression in CD34+ cells from bone marrow and G-CSF-mobilized peripheral blood by high-density oligonucleotide array analysis. Biol Blood Marrow Transplant 2001;7:486–494.
Ng YY, van Kessel B, Lokhorst HM et al. Gene-expression profiling of CD34+ cells from various hematopoietic stem-cell sources reveals functional differences in stem-cell activity. J Leukoc Biol 2004;75:314–323.
Shojaei F, Gallacher L, Bhatia M. Differential gene expression of human stem progenitor cells derived from early stages of in utero human hematopoiesis. Blood 2004;103:2530–2540.
Georgantas RW, Tanadve V, Malehorn M et al. Microarray and serial analysis of gene expression analyses identify known and novel transcripts over-expressed in hematopoietic stem cells. Cancer Res 2004;64:4434–4441.
Bhatia M, Bonnet D, Murdoch B et al. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med 1998;4:1038–1045.
Gallacher L, Murdoch B, Wu DM et al. Isolation and characterization of human CD34– Lin– and CD34+ Lin– hematopoietic stem cells using cell surface markers AC133 and CD7. Blood 2000;95:2813–2820.
Bhatia M. AC133 expression in human stem cells. Leukemia 2001;15:1685–1688.
Gordon PR, Leimig T, Babarin-Dorner A et al. Large-scale isolation of CD133+ progenitor cells from G-CSF mobilized peripheral blood stem cells. Bone Marrow Transplant 2003;31:17–22.
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