Stem Cells
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online December 13, 2007
Stem Cells Vol. 26 No. 3 March 2008, pp. 646 -655
doi:10.1634/stemcells.2007-0623; www.StemCells.com
© 2008 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
2007-0623v1
26/3/646    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Noda, S.
Right arrow Articles by Miyoshi, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Noda, S.
Right arrow Articles by Miyoshi, H.

TISSUE-SPECIFIC STEM CELLS

Repopulating Activity of Ex Vivo-Expanded Murine Hematopoietic Stem Cells Resides in the CD48–c-Kit+Sca-1+Lineage Marker– Cell Population

Shinichi Nodaa,b, Kana Horiguchia, Hitoshi Ichikawac, Hiroyuki Miyoshia

aSubteam for Manipulation of Cell Fate, RIKEN BioResource Center, Tsukuba, Japan;
bGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan;
cCancer Transcriptome Project, National Cancer Center Research Institute, Tokyo, Japan

Key Words. Hematopoietic stem cells • Mouse • Ex vivo expansion • Cell surface markers • Long-term repopulation • Microarray

Correspondence: Correspondence: Hiroyuki Miyoshi, Ph.D., Subteam for Manipulation of Cell Fate, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. Telephone: 81-29-836-9056; Fax: 81-29-836-9014; e-mail: miyoshi{at}brc.riken.jp

Received on August 6, 2007; accepted for publication on December 4, 2007.

First published online in STEM CELLS EXPRESS  December 13, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
A better understanding of the biology of cultured hematopoietic stem cells (HSCs) is required to achieve ex vivo expansion of HSCs. In this study, clonal analysis of the surface phenotype and repopulating activity of ex vivo-expanded murine HSCs was performed. After 7 days of culture with stem cell factor, thrombopoietin, fibroblast growth factor-1, and insulin-like growth factor-2, single CD34–/lowc-Kit+Sca-1+lineage marker– (CD34–KSL) cells gave rise to various numbers of cells. The proportion of KSL cells decreased with increasing number of expanded cells. Transplantation studies revealed that the progeny containing a higher percentage of KSL cells tended to have enhanced repopulating potential. We also found that CD48 was heterogeneously expressed in the KSL cell population after culture. Repopulating activity resided only in the CD48–KSL cell population, which had a relatively long intermitotic interval. Microarray analysis showed surprisingly few differences in gene expression between cultured CD48–KSL cells (cycling HSCs) and CD48+KSL cells (cycling non-HSCs) compared with freshly isolated CD34–KSL cells (quiescent HSCs), suggesting that the maintenance of stem cell activity is controlled by a relatively small number of genes. These findings should lead to a better understanding of ex vivo-expanded HSCs.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Hematopoietic stem cells (HSCs) are defined as self-renewing cells with the capacity to differentiate into all blood lineages. HSC transplantation is used to treat a number of blood disorders, including leukemia, aplastic anemia, and severe combined immunodeficiency [1, 2]. In this context, ex vivo expansion of HSCs would have widespread clinical applications, but the amplification of HSCs without loss of stem cell activity has been difficult to achieve. Therefore, clinical trials to date have sought to expand the number of progenitors and mature cells to improve hematopoietic recovery following high-dose chemotherapy or myeloablative chemoradiotherapy [3].

A large number of attempts at ex vivo expansion of HSCs with cytokines or stromal cells have resulted in limited success, with a severalfold increase in HSC numbers under the optimal culture conditions [4, 5]. Recently, some progress has been made with the use of signaling molecules or transcription factors that can promote the proliferation and survival of HSCs, such as bone morphogenetic protein 4, Wnt3A, fibroblast growth factor-1 (FGF-1), and HoxB4 [6–9]. However, a major problem is that ex vivo culture conditions established thus far have induced HSC proliferation coupled with differentiation, leading to the massive production and accumulation of differentiated cells [10–15]. Consequently, stem cell activity is lost rapidly during ex vivo culture. Numerous factors are secreted by differentiated cells [16], and some have been shown to act as negative regulators that limit HSC expansion [17]. Therefore, a crucial challenge is to identify extrinsic regulators that facilitate only symmetrical self-renewal and survival of HSCs. Overcoming this challenge will demand a better understanding of the biology of ex vivo-expanded HSCs.

Prospective isolation of HSCs has been achieved using a combination of cell surface markers and flow cytometry, and HSC activity is evaluated by in vivo transplantation assays. Substantial progress has been made in the isolation of murine HSCs compared with human HSCs. For example, single-cell transplantation assays identified 20%–40% of CD34–/lowc-Kit+Sca-1+lineage marker– (CD34–KSL) cells in adult mouse bone marrow as long-term repopulating cells [15, 18]. The direct effect of various cytokines on cell division and the asymmetry of initial cell divisions have been demonstrated at the single-cell level [15, 19]. However, little is known about the physical characteristics of ex vivo-expanded HSCs. Several studies have suggested that the relationship between cell surface phenotype and stem cell function is not maintained after in vitro culture [20, 21]. Therefore, characterization of cell surface markers that identify ex vivo-expanded HSCs is required for their purification and for developing culture conditions that allow their amplification.

In the present study, the expression of cell surface markers after culture of single CD34–KSL cells was analyzed. Simultaneously, the clonal progeny were subjected to a competitive repopulation assay. The progeny containing a higher percentage of KSL cells showed a higher level of engraftment. We also found that CD48 was heterogeneously expressed in the KSL cell population after culture, and only the CD48–KSL fraction of expanded cells could engraft in vivo. These findings will facilitate the selective isolation of stem cells from a large number of expanded cells and the elucidation of their biological properties. Furthermore, microarray analysis was performed on CD48–KSL and CD48+KSL cells, as well as on freshly isolated CD34–KSL cells. The gene expression profiling data facilitate the exploration of factors and pathways that might enhance ex vivo expansion of HSCs.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mice
C57BL/6 (B6-Ly5.2) mice were purchased from Charles River Laboratories Japan (Yokohama, Japan, http://www.criver.com). C57BL/6 mice congenic for the Ly5 locus (B6-Ly5.1) were obtained from RIKEN BioResource Center (BRC) (Tsukuba, Japan, http://www.brc.riken.jp). B6-Ly5.1/Ly5.2 F1 mice were obtained from mating pairs of B6-Ly5.1 and B6-Ly5.2 mice. All animal experiments were approved by the Animal Experiment Committee at the RIKEN Tsukuba Institute.

Purification and Culture of CD34–KSL Cells
CD34–KSL cells were purified as described, with minor modifications [18]. Briefly, bone marrow cells isolated from 10–16-week-old B6-Ly5.2 mice were stained with a lineage marker antibody cocktail consisting of biotinylated anti-Gr-1, anti-Mac-1, anti-B220, anti-IgM, anti-CD4, anti-CD8, and anti-Ter119 antibodies (eBioscience Inc., San Diego, http://www.ebioscience.com). Lineage marker+ cells were depleted using streptavidin-coupled Dynabeads M-280 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The remaining cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD34, phycoerythrin (PE)-conjugated anti-Sca-1, and allophycocyanin (APC)-conjugated anti-c-Kit antibodies (all from BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). The biotinylated antibodies were developed with streptavidin-APC-Cy7 (BD Biosciences). Fluorescence-activated cell sorting (FACS) was performed with a FACSVantage SE (BD Biosciences). CD34–KSL cells were sorted into individual wells of a 96-well plate containing 200 µl of StemSpan Serum-Free Expansion Medium (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) supplemented with 10 ng/ml mouse stem cell factor (SCF) (Peprotech, Rocky Hill, NJ, http://www.peprotech.com), 100 ng/ml human thrombopoietin (TPO) (Peprotech), 10 ng/ml mouse FGF-1 (Invitrogen), and 20 ng/ml mouse insulin-like growth factor-2 (IGF-2) (R&D Systems Inc., Minneapolis, http://www.rndsystems.com).

Analysis of Ex Vivo-Expanded Cells
Total cell numbers of ex vivo-expanded cells were counted using a microscope equipped with a charge-coupled device camera (≤1,000) or a FACSCalibur (BD Biosciences) (>1,000). The surface phenotypes of cells after culture were analyzed by staining with a biotinylated lineage antibody cocktail: PE-conjugated anti-Sca-1, APC-conjugated anti-c-Kit, FITC-conjugated anti-CD34, FITC-conjugated anti-CD48, FITC-conjugated anti-CD150, biotinylated anti-Flt3 (CD135), and biotinylated anti-CD244 antibodies (all from eBioscience). The biotinylated antibodies were developed with streptavidin-peridinin chlorophyll-a protein (PerCP) (BD Biosciences) or streptavidin-APC-Cy7. The cells were analyzed with a FACSCalibur flow cytometer and sorted on a FACSVantage SE cell sorter. For cell cycle analysis, prior to antibody staining, the cells were incubated with 20 µg/ml Hoechst 33342 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 60 minutes at 37°C.

Competitive Repopulation Assay
The competitive repopulation assay was performed by using the congenic Ly5 mouse system as described previously [15]. The indicated numbers of freshly isolated CD34–KSL cells from B6-Ly5.2 mice or CD34–KSL progeny after culture were mixed with 2 x 105 total bone marrow competitor cells from B6-Ly5.1/5.2 F1 mice and transplanted into lethally (9.5 Gy) irradiated B6-Ly5.1 mice. Twelve weeks after transplantation, peripheral blood cells of the recipient mice were collected by retro-orbital bleeding. After lysis of red blood cells with ammonium chloride buffer, the remaining nucleated cells were stained with FITC-conjugated anti-Ly5.2, PE-conjugated anti-Ly5.1, biotinylated anti-Mac1, and biotinylated anti-Gr1 antibodies, followed by addition of streptavidin-PerCP. The cells were stained simultaneously with APC-conjugated anti-B220 antibody or a mixture of APC-conjugated anti-CD4 and anti-CD8 antibodies. FACS analysis was performed with a FACSCalibur flow cytometer. Donor chimerism was determined as the percentage of Ly5.2+ cells.

Microarray Analysis
Total RNA was isolated from 4,000–5,000 FACS-sorted cells using an ISOGEN reagent (Nippon Gene, Tokyo, http://www.nippongene.com). Biotin-labeled cRNA was prepared from the total RNA equivalent to 3,000 cells with a two-cycle cDNA synthesis kit and 3'-amplification reagents for in vitro transcription labeling (Affymetrix, Santa Clara, CA, http://www.affymetrix.com) and was hybridized to an Affymetrix GeneChip Mouse Genome 430 2.0 array (Affymetrix), which contained approximately 45,000 probe sets for analyzing the expression levels of more than 34,000 mouse genes. After washing and staining with the antibody amplification procedure, the microarrays were scanned with an Affymetrix GeneChip Scanner 3000 7G. All these procedures were carried out according to the manufacturer's instructions. The expression value (signal) and detection call (present, absent, or marginal) for each probe set were calculated using GeneChip Operating Software version 1.4 (Affymetrix). The signal values were normalized so that their mean in each experiment was 100 to adjust for minor differences between the experiments. The change value (signal log ratio) and change call (increase, marginal increase, no change, marginal decrease, or decrease) for each probe set were calculated by comparison analysis of the software. All experiments were performed in duplicate using two FACS-sorted cell samples that were independently prepared from separate experiments. To identify differentially expressed genes, we selected probe sets that showed a change call of increase and a signal log ratio value of ≥1 (more than twofold upregulation) or a change call of decrease and a signal log ratio value of ≤–1 (more than twofold downregulation) in each of the two independent experiments. Gene Ontology analysis was performed using FatiGO web-based software (http://fatigo.bioinfo.cipf.es) [22].


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Effects of Cytokines on Ex Vivo Expansion of CD34–KSL Cells
To verify the effects of cytokines on ex vivo expansion of HSCs, 100 CD34–KSL cells were cultured in serum-free medium with the following combinations of cytokines: SCF/TPO, SCF/TPO/FGF-1, SCF/TPO/IGF-2, or SCF/TPO/FGF-1/IGF-2. After 7 days of culture, the progeny were analyzed for the expression of c-Kit, Sca-1, and lineage markers. Although the total numbers of expanded cells did not differ significantly, the proportion of KSL cells (the primitive hematopoietic cell population) increased most when CD34–KSL cells were cultured with SCF/TPO/FGF-1/IGF-2 (Fig. 1A). An in vivo competitive repopulation assay was performed to compare cultured and freshly isolated CD34–KSL cells. The results showed increased repopulating ability resulting from culture with SCF/TPO/FGF-1/IGF-2 (Fig. 1B). In contrast, low levels of engraftment were observed from culture with SCF/TPO. In agreement with the results reported by Zhang et al. [23], we observed a modest expansion of HSCs with a combination of SCF, TPO, FGF-1, and IGF-2. Thus, this cytokine combination was used for subsequent analyses.


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		Figure 1. Effects of cytokines on ex vivo expansion and repopulating activity of CD34–KSL cells. (A): One hundred freshly isolated CD34–KSL cells were cultured for 7 days in serum-free medium with the indicated combination of cytokines. The total number of expanded cells was counted, and the fold increase was calculated. Simultaneously, the expression of c-Kit, Sca-1, and lineage markers was analyzed by flow cytometry. The data shown represent the mean ± SD (n = 9–10). p values were calculated by Student's t test. p = .0001, SCF/TPO versus SCF/TPO/FGF-1/IGF-2. (B): One hundred freshly isolated CD34–KSL cells from B6-Ly5.2 mice, or the progeny of CD34–KSL cells after 7 days of culture with the indicated combination of cytokines, were transplanted together with 2 x 105 total bone marrow competitor cells from B6-Ly5.1/5.2 F1 mice into lethally irradiated B6-Ly5.1 mice (n = 6 or 9). Twelve weeks after transplantation, peripheral blood cells of the recipient mice were analyzed by flow cytometry, and donor chimerism was determined as the percentage of Ly5.2+ cells. Horizontal lines indicate the mean values. p = .0021, fresh versus SCF/TPO/FGF-1/IGF-2. p = .0001, SCF/TPO versus SCF/TPO/FGF-1/IGF-2. Abbreviations: FGF, fibroblast growth factor; IGF, insulin-like growth factor; KSL, c-Kit+Sca-1+lineage marker–; SCF, stem cell factor; TPO, thrombopoietin.

 
Clonal Analysis of the Surface Phenotype and Repopulating Activity of Ex Vivo-Expanded Cells Derived from Single CD34–KSL Cells
For clonal analysis, single CD34–KSL cells were individually plated and cultured for 7 days in serum-free medium with SCF, TPO, FGF-1, and IGF-2, and a total of 184 individual progeny were analyzed for the expression of primitive cell surface markers. Single CD34–KSL cells gave rise to various numbers (4–3,846) of cells. The vast majority of expanded cells remained c-Kit+ and lineage marker–, but Sca-1 was expressed heterogeneously after culture. The proportion of KSL cells appeared to decrease as the number of expanded cells increased (Fig. 2A). We divided the progeny into four groups on the basis of the percentage of KSL cells (0%–25%, 26%–50%, 51%–75%, and 76%–100%) (Fig. 2B). Approximately half of the progeny analyzed (98 of 184) contained less than 25% KSL cells. In this group, the number of expanded cells varied (6–3,846), and large cells with megakaryocytic morphology were frequently seen. For progeny containing a higher percentage (≥51%) of KSL cells, the number of expanded cells was consistently small (≤516) (Fig. 2A).


Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		Figure 2. Clonal analysis of the KSL phenotype of ex vivo-expanded cells derived from single CD34–KSL cells. Single CD34–KSL cells were individually plated and cultured for 7 days in serum-free medium with stem cell factor, thrombopoietin, fibroblast growth factor-1, and insulin-like growth factor-2. The total number of progeny was counted, and the expression of c-Kit, Sca-1, and lineage markers was analyzed by flow cytometry. (A): The proportion of KSL cells tended to decrease as the number of progeny increased. Representative morphologies and fluorescence-activated cell sorting profiles are shown. (B): Analyzed individual progeny (n = 184) were divided into four groups on the basis of the percentage of KSL cells (0%–25%, 26%–50%, 51%–75%, and 76%–100%). Black columns represent the KSL phenotype. Abbreviation: KSL, c-Kit+Sca-1+lineage marker–.

 
To assess the long-term repopulating activity of ex vivo-expanded cells derived from single CD34–KSL cells, the cell surface phenotype was analyzed in half of the progeny, and a competitive repopulation assay was performed on the other half. As shown in Table 1, the progeny containing a higher percentage of KSL cells appeared to have greater repopulating potential. Single-cell transplantation assays identified only ~30% of freshly isolated CD34–KSL cells as long-term repopulating cells (data not shown), which is consistent with previous reports [15, 18, 19]. These results suggest that the proportion of KSL cells in ex vivo-expanded cells reflects the functional heterogeneity of CD34–KSL cells and correlates with the in vivo repopulating potential.


View this table:
[in this window]
[in a new window]

  		Table 1. Repopulating activity of ex vivo-expanded cells derived from single CD34–KSL cells

 
Repopulating Activity of Ex Vivo-Expanded Cells Resides in the CD48–KSL Cell Population
We next analyzed the expression of CD34, Flt3 (CD135), CD150, CD48, and CD244 on ex vivo-expanded cells because these markers have been shown to be useful for purification of noncultured HSCs. After 7 days of culture of CD34–KSL cells, almost all expanded cells remained Flt3– and CD150+ but became CD34+ and CD244+ (Fig. 3A). On the other hand, CD48 was expressed heterogeneously in the KSL cell population. To test whether CD48 expression status can be used to enrich ex vivo-expanded HSCs, CD48–KSL, CD48+KSL, and Sca-1– cells purified from ex vivo-expanded cells were subjected to a competitive repopulation assay. As shown in Figure 3B, repopulating activity was found to reside only in the CD48–KSL cell population.


Figure 3
View larger version (26K):
[in this window]
[in a new window]

  		Figure 3. Expression of cell surface markers and repopulating activity of ex vivo-expanded cells derived from CD34–KSL cells. (A): The expression of CD34, Flt3, CD150, CD48, and CD244 on freshly isolated CD34–KSL cells and their progeny after 7 days of culture with stem cell factor (SCF), thrombopoietin (TPO), fibroblast growth factor-1 (FGF-1), and insulin-like growth factor-2 (IGF-2) was analyzed by flow cytometry. (B): One thousand CD34–KSL cells (100 cells per well) were cultured with SCF, TPO, FGF-1, and IGF-2. After 7 days of culture, CD48–KSL, CD48+KSL, or Sca-1– cells were purified from ex vivo-expanded cells, and the indicated number of cells from each subpopulation was subjected to a competitive repopulation assay. Twelve weeks after transplantation, peripheral blood cells of the recipient mice were analyzed by flow cytometry, and donor chimerism was determined. The data shown were obtained from three independent experiments. Horizontal lines indicate the mean values. Abbreviation: KSL, c-Kit+Sca-1+lineage marker–.

 
Growth Kinetics and Cell Cycle Status of the CD48–KSL Cell Population in Ex Vivo-Expanded Cells
To further characterize the CD48–KSL cell population in ex vivo-expanded cells, growth kinetics and cell cycle status were analyzed. As shown in Figure 4A, the majority of expanded cells remained CD48– and Sca-1+ after 2 days of culture, at which time ~90% of cultured CD34–KSL cells had divided at least once, as detected by microscopy at the single cell level. After more than 4 days of culture, the proportion of CD48–KSL cells decreased with time in culture, whereas the number of CD48–KSL cells increased dramatically, along with the other populations. Cell cycle analysis showed a decreased proportion of CD48–KSL cells in the S/G2/M phases compared with CD48+KSL cells or Sca-1– cells (Fig. 4B). After 7 days of culture, CD48–KSL and CD48+KSL cells were purified and cultured independently, and then growth kinetics and cell surface phenotype were analyzed. The results showed that the total numbers of expanded cells after 2 days of culture were significantly lower in CD48–KSL cells compared with CD48+KSL cells, whereas a large increase in total cell numbers was detected after 7 days of culture, ~2.8 times greater than total cell numbers in CD48+KSL cells (supplemental online Fig. 1A). In addition, the progeny derived from CD48–KSL cells contained a significantly higher percentage of KSL cells after 4 and 7 days of culture (supplemental online Fig. 1B). Taken together, these results indicate that CD48–KSL cells have a relatively long intermitotic interval and a high proliferative potential.


Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		Figure 4. Growth kinetics and cell cycle status of the CD48–KSL population in ex vivo-expanded cells. (A): CD34–KSL cells were cultured with stem cell factor, thrombopoietin, fibroblast growth factor-1, and insulin-like growth factor-2. The expression of CD48, c-Kit, Sca-1, and lineage markers was analyzed by flow cytometry, and the total number of CD48–KSL cells was counted at the indicated time points. Fold increase in the number of CD48–KSL cells was calculated relative to the input number of CD34–KSL cells at day 0. Data represent the mean ± SD (n = 3–10). (B): After 7 days of culture, cell cycle analysis of the indicated cell populations was performed by Hoechst staining. Representative fluorescence-activated cell sorting histograms showing the DNA content and the percentage of cells in the S/G2/M phases are shown. Data represent the mean ± SD (n = 3). p values were calculated by Student's t test. p = .0004, CD48–KSL versus CD48+KSL. p = .0005, CD48–KSL versus Sca-1–. Abbreviation: KSL, c-Kit+Sca-1+lineage marker–.

 
Gene Expression Profiling of Ex Vivo-Expanded HSCs
To identify genes specifically expressed in the ex vivo-expanded CD48–KSL cell population, microarray analysis was performed on CD48–KSL cells, CD48+KSL cells, and freshly isolated CD34–KSL cells by using an Affymetrix GeneChip array containing approximately 45,000 probe sets representing more than 34,000 mouse genes. Two independently prepared samples were examined in each cell population. The signal values and detection calls for this analysis are provided in the supplemental online data. A total of 17,788 probe sets were scored as "present" in the CD34–KSL population, of which 90.6% and 93.3% were also scored as present in the CD48–KSL and CD48+KSL populations, respectively, suggesting substantial transcriptional homogeneity of these cell populations. Unsupervised hierarchical clustering analysis of the six samples showed that two samples of each cell population exhibited nearly same gene expression and that the difference between the CD48–KSL and CD48+KSL populations was relatively small compared with the CD34–KSL population (supplemental online Fig. 2). Genes that were differentially expressed by more than twofold between the two cell populations were selected and classified according to function by using simplified ontologies.

The comparison of CD48–KSL and CD34–KSL populations identified 756 differentially expressed genes (353 upregulated and 403 downregulated genes in the CD48–KSL population) (Table 2). In contrast, only 198 genes were differentially expressed between the CD48–KSL and CD48+KSL populations. This might reflect greater homogeneity in expression of genes associated with proliferation than those associated with maintenance of in vivo repopulating activity. Of particular interest in this regard are the expression profiles of cell cycle regulatory genes (Table 3). Among cyclins and cyclin-related genes, cyclin D1, cyclin D3, and p57/Cdkn1c were upregulated in the CD34–KSL population (quiescent HSCs). In contrast, cyclin A2, cyclin B1, cyclin B2, cyclin E2, Cdk4, Cdc2a, Cdc20, and p21/Cdkn1a were upregulated in both the CD48–KSL (cycling HSC) and CD48+KSL (cycling non-HSC) populations. Intriguingly, cyclin D3 and p57 were upregulated in the CD48–KSL population compared with the CD48+KSL population. These differences may be responsible for the prolonged cell cycle of cycling HSCs.


View this table:
[in this window]
[in a new window]

  		Table 2. Summary of the numbers of differentially expressed genes

 


View this table:
[in this window]
[in a new window]

  		Table 3. Genes differentially expressed in CD48–KSL cells compared with CD48+KSL or CD34–KSL cells

 
Other differentially expressed genes between the CD48–KSL and CD48+KSL populations might be involved in maintaining stem cell activity or suppressing differentiation. Gfi1 (growth factor-independent 1), a zinc-finger transcriptional repressor, was highly and preferentially expressed in the CD48–KSL population. Gfi1 is implicated in self-renewal of HSCs by restraining their proliferation [24, 25]. Genes downregulated during culture but still highly expressed in the CD48–KSL population compared with the CD48+KSL population include Klf4 (Krüppel-like factor 4), Ndn (necdin), Runx1t1, NPDC-1 (neural proliferation, differentiation, and control 1), and Neo1 (neogenin). KLF4 functions as a tumor suppressor, and ectopic expression of KLF4 suppresses cell proliferation through p21-dependent cell cycle arrest [26]. Necdin and NPDC-1 are potent mitotic suppressors expressed predominantly in the nervous system [27–29].

Membrane and extracellular space proteins that were differentially expressed in the CD48–KSL and CD48+KSL populations, such as CD28, CD83, CD140b/Pdgfrb (platelet-derived growth factor receptor β), CD144 (VE-cadherin)/Cdh5, CD201/Procr (endothelial protein C receptor), and CD244, are potential markers for further purification of cycling HSCs. Although CD144 is thought to be an endothelial marker, the expression of CD144 has been demonstrated recently in fetal liver HSCs but not in adult bone marrow HSCs [30]. It has also been shown that CD201 is specifically expressed at high levels in bone marrow HSCs [31].


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
In this study, we describe a link between cell surface phenotype and stem cell function after ex vivo expansion of murine HSCs from CD34–KSL cells. Although CD34–KSL cells are highly enriched for HSCs, single-cell transplantation studies by others revealed that only 20%–40% of CD34–KSL cells were able to reconstitute, indicating that CD34–KSL cells are still not a pure population of HSCs [15, 18, 19]. Our clonal analysis also showed functional heterogeneity of CD34–KSL cells: progeny of single CD34–KSL cells that contained a higher percentage of KSL cells were able to attain a higher level of engraftment. Matsuzaki et al. reported that CD34–KSL cells with the Hoechst dye-effluxing side population (SP) phenotype represent an almost homogeneous HSC population [32]. Although inconsistent findings have also been reported [33, 34], it would be interesting to characterize the surface phenotype of ex vivo-expanded cells derived from single SP CD34–KSL cells to examine the homogeneity. Our preliminary clonal analysis data of SP CD34–KSL cells showed an increased proportion of progeny containing a higher percentage of KSL cells.

Our results confirm the report by Zhang et al. that a combination of SCF, TPO, FGF-1, and IGF-2 led to a modest expansion of HSCs (approximately eightfold expansion after 10-day culture of SP cells) [23]. In contrast, culture of CD34–KSL cells with SCF and TPO resulted in no expansion of repopulating HSCs and decreased HSC activity, despite induction of cell proliferation at the same level as SCF, TPO, FGF-1, and IGF-2 (Fig. 1). TPO has been shown to act directly on HSCs and induce cell proliferation in combination with SCF, but this combination leads to asymmetric division, resulting in a substantial decrease in HSC number [15, 19, 35, 36]. It has been demonstrated that IGF-2 binds to fetal liver and adult bone marrow HSCs and stimulates their ex vivo expansion, in combination with other cytokines [37]. On the other hand, FGF-1 is able to preserve HSC activity of unfractionated bone marrow cells but not of purified HSCs, although receptors for FGF-1 are present on primitive hematopoietic cell subsets [8, 38]. These findings suggest that IGF-2 acts synergistically with SCF and TPO to stimulate the growth and maintenance of HSCs, whereas FGF-1 maintains HSCs through activating differentiated cells.

Recently, the use of SLAM family markers CD150 and CD48 has been shown to improve the purity of noncultured murine HSCs, which can be highly enriched in the CD150+CD48– cell population [39–41]. In contrast, Venezia et al. reported that the expression of CD48 was detected on quiescent HSCs and increased in cycling HSCs after 5-fluorouracil treatment, although these authors did not show a direct correlation between CD48 expression and cycling HSC activity [42]. In the present study, we demonstrated that the HSC activity of ex vivo-expanded cells was contained in the CD48–KSL cell population, whereas the vast majority of expanded cells remained CD150+. The number of CD48–KSL cells increased ~20-fold after 7 days of culture (Fig. 4A), whereas the repopulating activity of 400 CD48–KSL cells (Fig. 3B) is comparable to that of 100 freshly isolated CD34–KSL cells (Fig. 1B). Therefore, the number of cells with in vivo repopulating potential after culture increased only approximately fivefold, indicating that CD48–KSL cells are still not a pure population of HSCs. Zhang et al. reported that the long-term repopulating activity of total bone marrow cells after 10 days of culture was found in the Sca-1+, c-Kit+, prion protein PrP–, CD62L–, IGF2-hFc+, CD31+, Tie-2–, and prominin-1– population [34]. It would be interesting to test whether these markers, together with our potential markers identified by microarray analysis, can subdivide the CD48–KSL cell population to enhance ex vivo-expanded HSC purity. Currently, precise evaluation of HSC function is assessed by in vivo long-term repopulation assays, but in vivo assays are cumbersome and time-consuming. Therefore, once the relationship between cell surface phenotype and repopulating activity of ex vivo-expanded HSCs is defined, flow cytometry would provide a surrogate in vitro assay for the rapid evaluation of HSC function.

Clonal analysis of ex vivo-expanded cells showed that the progeny containing low numbers of expanded cells with a higher percentage of KSL cells have relatively high HSC activity. We also found a prolonged cell cycle for the CD48–KSL cell population compared with the other ex vivo-expanded cell populations. This observation is in agreement with the study by Nygren et al., in which CD34–KSL cells were cultured for 6 days with SCF, Flt3 ligand, TPO, and interleukin-3, and expanded cells were sorted into G1 and S/G2/M fractions and evaluated for in vivo repopulating activity [43]. The results showed that HSC activity resides predominantly in the G1 population [43]. Furthermore, time-lapse video monitoring of single HSC cultures coupled with an in vivo functional assay revealed that HSC activity is associated with smaller clone sizes and longer cell cycle times [44]. Similar findings were reported by Srour et al. using highly purified human HSC cells placed in single-cell cultures and tracked for cell division and maintenance of primitive hematopoietic function [45]. In addition, numerous studies have indicated that HSCs have a limited self-renewal capacity, and HSC function is gradually lost with each cell division. Thus, a relatively low cycling activity may be required for the maintenance of HSC function in vitro.

Microarray analysis of ex vivo-expanded HSCs has not been widely performed. Unexpectedly, our gene expression profile analysis revealed small differences between CD48–KSL cells (cycling HSCs) and CD48+KSL cells (cycling non-HSCs) compared with freshly isolated CD34–KSL cells (quiescent HSCs). Although this result may be due in part to the heterogeneity of the CD48–KSL population as cycling HSCs, it suggests that the maintenance of stem cell activity is controlled by a relatively small number of genes. On the other hand, HSC quiescence may be in a more tightly regulated state than HSC proliferation. As expected, significant changes in the expression of cell cycle regulatory genes were observed upon proliferation induced by the combination of cytokines. In terms of cell cycle control of the G1 phase, cyclin D3 and p57/Cdkn1c were downregulated during culture but still highly expressed in cycling HSCs compared with cycling non-HSCs. Of particular interest is the significantly higher level of p21/Cdkn1a expression in cycling HSCs compared with quiescent HSCs. p21 and p57, members of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, may inhibit a broad range of cyclins and CDK complexes, resulting in slowly cycling HSCs. p21 is implicated in sustaining HSC quiescence. In p21-deficient mice, the proliferation of HSCs is dramatically increased, leading to exhaustion of the HSC pool [46, 47]. Our microarray analysis identified several genes, such as Gfi1, Klf4, Ndn, and NPDC-1, that may play roles in regulating the cell cycle of ex vivo-expanded HSC.

Molecular mechanisms that regulate proliferation and preservation of the functional integrity of ex vivo-expanded HSCs are largely unknown. Our findings should facilitate a better understanding of ex vivo-expanded HSCs at the molecular level and the exploration of pathways that promote HSC survival and proliferation.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Drs. J. Hayashi and Y. Obata for encouragement and discussion and RIKEN BRC Experimental Animal Division for animal care. S.N. is a Junior Research Associate at RIKEN.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. Kondo M, Wagers AJ, Manz MG et al. Biology of hematopoietic stem cells and progenitors: Implications for clinical application. Annu Rev Immunol 2003;21:759–806.[CrossRef][Medline]

  2. Shizuru JA, Negrin RS, Weissman IL. Hematopoietic stem and progenitor cells: Clinical and preclinical regeneration of the hematolymphoid system. Annu Rev Med 2005;56:509–538.[CrossRef][Medline]

  3. Devine SM, Lazarus HM, Emerson SG. Clinical application of hematopoietic progenitor cell expansion: Current status and future prospects. Bone Marrow Transplant 2003;31:241–252.[CrossRef][Medline]

  4. Sauvageau G, Iscove NN, Humphries RK. In vitro and in vivo expansion of hematopoietic stem cells. Oncogene 2004;23:7223–7232.[CrossRef][Medline]

  5. Sorrentino BP. Clinical strategies for expansion of haematopoietic stem cells. Nat Rev Immunol 2004;4:878–888.[CrossRef][Medline]

  6. Sadlon TJ, Lewis ID, D'Andrea RJ. BMP4: Its role in development of the hematopoietic system and potential as a hematopoietic growth factor. STEM CELLS 2004;22:457–474.[Abstract/Free Full Text]

  7. Willert K, Brown JD, Danenberg E et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 2003;423:448–452.[CrossRef][Medline]

  8. de Haan G, Weersing E, Dontje B et al. In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1. Dev Cell 2003;4:241–251.[CrossRef][Medline]

  9. Krosl J, Austin P, Beslu N et al. In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein. Nat Med 2003;9:1428–1432.[CrossRef][Medline]

  10. Knobel KM, McNally MA, Berson AE et al. Long-term reconstitution of mice after ex vivo expansion of bone marrow cells: Differential activity of cultured bone marrow and enriched stem cell populations. Exp Hematol 1994;22:1227–1235.[Medline]

  11. Peters SO, Kittler EL, Ramshaw HS et al. Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts. Exp Hematol 1995;23:461–469.[Medline]

  12. Traycoff CM, Cornetta K, Yoder MC et al. Ex vivo expansion of murine hematopoietic progenitor cells generates classes of expanded cells possessing different levels of bone marrow repopulating potential. Exp Hematol 1996;24:299–306.[Medline]

  13. Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J Exp Med 1997;186:619–624.[Abstract/Free Full Text]

  14. Glimm H, Oh IH, Eaves CJ. Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G(2)/M transit and do not reenter G(0). Blood 2000;96:4185–4193.[Abstract/Free Full Text]

  15. Ema H, Takano H, Sudo K et al. In vitro self-renewal division of hematopoietic stem cells. J Exp Med 2000;192:1281–1288.[Abstract/Free Full Text]

  16. Majka M, Janowska-Wieczorek A, Ratajczak J et al. Numerous growth factors, cytokines, and chemokines are secreted by human CD34(+) cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an autocrine/paracrine manner. Blood 2001;97:3075–3085.[Abstract/Free Full Text]

  17. Madlambayan GJ, Rogers I, Kirouac DC et al. Dynamic changes in cellular and microenvironmental composition can be controlled to elicit in vitro human hematopoietic stem cell expansion. Exp Hematol 2005;33:1229–1239.[CrossRef][Medline]

  18. Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996;273:242–245.[Abstract]

  19. Takano H, Ema H, Sudo K et al. Asymmetric division and lineage commitment at the level of hematopoietic stem cells: Inference from differentiation in daughter cell and granddaughter cell pairs. J Exp Med 2004;199:295–302.[Abstract/Free Full Text]

  20. Bunting KD, Galipeau J, Topham D et al. Effects of retroviral-mediated MDR1 expression on hematopoietic stem cell self-renewal and differentiation in culture. Ann N Y Acad Sci 1999;872:125–140 discussion 140–141.[CrossRef][Medline]

  21. Dorrell C, Gan OI, Pereira DS et al. Expansion of human cord blood CD34(+)CD38(–) cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: Dissociation of SRC phenotype and function. Blood 2000;95:102–110.[Abstract/Free Full Text]

  22. Al-Shahrour F, Diaz-Uriarte R, Dopazo J. FatiGO: A web tool for finding significant associations of Gene Ontology terms with groups of genes. Bioinformatics 2004;20:578–580.[Abstract/Free Full Text]

  23. Zhang CC, Lodish HF. Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood 2005;105:4314–4320.[Abstract/Free Full Text]

  24. Zeng H, Yucel R, Kosan C et al. Transcription factor Gfi1 regulates self-renewal and engraftment of hematopoietic stem cells. EMBO J 2004;23:4116–4125.[CrossRef][Medline]

  25. Hock H, Hamblen MJ, Rooke HM et al. Gfi-1 restricts proliferation and preserves functional integrity of haematopoietic stem cells. Nature 2004;431:1002–1007.[CrossRef][Medline]

  26. Rowland BD, Peeper DS. KLF4, p21 and context-dependent opposing forces in cancer. Nat Rev Cancer 2006;6:11–23.[CrossRef][Medline]

  27. Yoshikawa K. Cell cycle regulators in neural stem cells and postmitotic neurons. Neurosci Res 2000;37:1–14.[CrossRef][Medline]

  28. Galiana E, Vernier P, Dupont E et al. Identification of a neural-specific cDNA, NPDC-1, able to down-regulate cell proliferation and to suppress transformation. Proc Natl Acad Sci U S A 1995;92:1560–1564.[Abstract/Free Full Text]

  29. Sansal I, Dupont E, Toru D et al. NPDC-1, a regulator of neural cell proliferation and differentiation, interacts with E2F-1, reduces its binding to DNA and modulates its transcriptional activity. Oncogene 2000;19:5000–5009.[CrossRef][Medline]

  30. Kim I, Yilmaz OH, Morrison SJ. CD144 (VE-cadherin) is transiently expressed by fetal liver hematopoietic stem cells. Blood 2005;106:903–905.[Abstract/Free Full Text]

  31. Balazs AB, Fabian AJ, Esmon CT et al. Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow. Blood 2006;107:2317–2321.[Abstract/Free Full Text]

  32. Matsuzaki Y, Kinjo K, Mulligan RC et al. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Immunity 2004;20:87–93.[CrossRef][Medline]

  33. Camargo FD, Chambers SM, Drew E et al. Hematopoietic stem cells do not engraft with absolute efficiencies. Blood 2006;107:501–507.[Abstract/Free Full Text]

  34. Morita Y, Ema H, Yamazaki S et al. Non-side-population hematopoietic stem cells in mouse bone marrow. Blood 2006;108:2850–2856.[Abstract/Free Full Text]

  35. Sitnicka E, Lin N, Priestley GV et al. The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells. Blood 1996;87:4998–5005.[Abstract/Free Full Text]

  36. Ku H, Yonemura Y, Kaushansky K et al. Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice. Blood 1996;87:4544–4551.[Abstract/Free Full Text]

  37. Zhang CC, Lodish HF. Insulin-like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells. Blood 2004;103:2513–2521.[Abstract/Free Full Text]

  38. Yeoh JS, van Os R, Weersing E et al. Fibroblast growth factor-1 and -2 preserve long-term repopulating ability of hematopoietic stem cells in serum-free cultures. STEM CELLS 2006;24:1564–1572.[Abstract/Free Full Text]

  39. Kiel MJ, Yilmaz OH, Iwashita T et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005;121:1109–1121.[CrossRef][Medline]

  40. Yilmaz OH, Kiel MJ, Morrison SJ. SLAM family markers are conserved among hematopoietic stem cells from old and reconstituted mice and markedly increase their purity. Blood 2006;107:924–930.[Abstract/Free Full Text]

  41. Kim I, He S, Yilmaz OH et al. Enhanced purification of fetal liver hematopoietic stem cells using SLAM family receptors. Blood 2006;108:737–744.[Abstract/Free Full Text]

  42. Venezia TA, Merchant AA, Ramos CA et al. Molecular signatures of proliferation and quiescence in hematopoietic stem cells. PLoS Biol 2004;2:e301.[CrossRef][Medline]

  43. Nygren JM, Bryder D, Jacobsen SE. Prolonged cell cycle transit is a defining and developmentally conserved hemopoietic stem cell property. J Immunol 2006;177:201–208.[Abstract/Free Full Text]

  44. Dykstra B, Ramunas J, Kent D et al. High-resolution video monitoring of hematopoietic stem cells cultured in single-cell arrays identifies new features of self-renewal. Proc Natl Acad Sci U S A 2006;103:8185–8190.[Abstract/Free Full Text]

  45. Srour EF, Tong X, Sung KW et al. Modulation of in vitro proliferation kinetics and primitive hematopoietic potential of individual human CD34+CD38-/lo cells in G0. Blood 2005;105:3109–3116.[Abstract/Free Full Text]

  46. Cheng T, Rodrigues N, Shen H et al. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 2000;287:1804–1808.[Abstract/Free Full Text]

  47. Yu H, Yuan Y, Shen H et al. Hematopoietic stem cell exhaustion impacted by p18 INK4C and p21 Cip1/Waf1 in opposite manners. Blood 2006;107:1200–1206.[Abstract/Free Full Text]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
2007-0623v1
26/3/646    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Noda, S.
Right arrow Articles by Miyoshi, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Noda, S.
Right arrow Articles by Miyoshi, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS