Stem Cells http://www.stemcellsportal.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online December 22, 2005
Stem Cells Vol. 24 No. 4 April 2006, pp. 1087 -1094
doi:10.1634/stemcells.2005-0396; www.StemCells.com
© 2006 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0396v1
24/4/1087    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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wagers, A. J.
Right arrow Articles by Weissman, I. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wagers, A. J.
Right arrow Articles by Weissman, I. L.

TISSUE-SPECIFIC STEM CELLS

Differential Expression of {alpha}2 Integrin Separates Long-Term and Short-Term Reconstituting Lin–/loThy1.1loc-kit+ Sca-1+ Hematopoietic Stem Cells

Amy J. Wagersa, Irving L. Weissmana,b

a Departments of Pathology and
b Developmental Biology, Stanford University School of Medicine, Stanford, California, USA

Key Words. Cell surface markers • Integrins • Hematopoietic stem cell • Hematopoietic stem cell transplantation

Correspondence: Amy J. Wagers, Ph.D., Section on Developmental and Stem Cell Biology, Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215, USA. Telephone: 617-732-2590; Fax: 617-732-2593; e-mail: amy.wagers{at}joslin.harvard.edu

Received August 16, 2005; accepted for publication December 14, 2005.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
 References
 
Self-renewing, multipotent hematopoietic stem cells are highly enriched within the Lin– Thy1.1loc-kit+ Sca-1+ subset of mouse bone marrow. However, heterogeneous expression within this population of certain cell surface markers raises the possibility that it may be further fractionated phenotypically and perhaps functionally. We previously identified {alpha}2-integrin (CD49b) as a surface marker with heterogeneous expression on Lin– /loThy1.1loc-kit+ Sca-1+ stem cells. To determine whether differences in {alpha}2 expression were indicative of differences in stem cell function, we purified {alpha}2– and {alpha}2hi stem cells by fluorescence-activated cell sorting and analyzed their function in long- and short-term hematopoietic reconstitution assays. Both {alpha}2– and {alpha}2hi cells could give rise to mature lymphoid and myeloid cells after transplantation into lethally irradiated congenic recipients. However, {alpha}2hi cells supported hematopoiesis for only a short time (<4 weeks), whereas {alpha}2– cells reproducibly yielded robust, long-term (>20 weeks) reconstitution, suggesting that {alpha}2– cells represent a more primitive population than do {alpha}2hi cells. Consistent with this idea, {alpha}2– Lin– /loThy1.1loc-kit+ Sca-1+ cells exhibited an approximately sixfold decreased frequency of spleen colony-forming units (day 12) versus {alpha}2hi cells. Furthermore, bone marrow cells isolated from animals transplanted >20 weeks previously with 20 {alpha}2– Lin– /loThy1.1loc-kit+ Sca-1+ cells included both {alpha}2– and {alpha}2hi stem cells of donor origin, indicating that {alpha}2hi cells are likely lineal descendents of {alpha}2– cells. Interestingly, {alpha}2 integrin expression is significantly reduced on lineage-restricted oligopotent progenitors in the marrow, suggesting that high level expression of {alpha}2 selectively marks a subset of primitive hematopoietic cells which retains multilineage reconstitution potential but exhibits reduced self-renewal capacity.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
 References
 
Life-long production of mature hematopoietic cells depends critically on the continuing function of hematopoietic stem cells (HSCs). These HSCs reside primarily in the bone marrow (BM) of adult mammals and generate all of the lineages of mature blood cells through a process involving the sequential restriction of lineage potential [1]. Successful hematopoietic cell transplantation, therefore, relies critically on the function of these cells to durably repopulate the ablated hematolymphoid system of transplant recipients, and characterization of cell surface markers that identify primitive HSCs is of interest to facilitate their purification, further understand their biological properties, and improve strategies for their clinical application. Earlier studies have phenotypically and functionally defined both long-term (LT) and short-term (ST) reconstituting HSCs and also multipotent progenitor (MPP) cells [2–4]. Although each of these subsets of primitive BM cells retains the capacity for multilineage hematopoietic differentiation, forming each and every type of adult mature blood cell, they differ dramatically in their self-renewal potential. In mice, LT HSCs self-renew for at least the lifetime of the animal, ST HSCs self-renew for approximately 4–8 weeks, and MPPs show little or no self-renewal potential [2, 4]. Phenotypically, LT and ST HSCs are contained within the Lin– /loThy1.1loc-kit+ Sca-1+ Flk-2– subset of murine BM; LT HSCs are Lin–, whereas ST HSCs are Linlo (Mac-1lo). The more differentiated MPPs are contained within the Lin– /lo Thy1.1loc-kit+ Sca-1+ Flk-2+ subset of BM [2, 4].

In an effort to identify adhesion molecules involved in HSC migration, we previously examined the expression of integrin receptors by HSCs [5]. Integrins are a large family of cell surface–expressed heterodimeric adhesion molecules, consisting of noncovalently associated {alpha}- and ß-subunits [6]. Normal mouse BM HSCs express high levels of many ß1 integrins, including {alpha}4ß1, {alpha}5ß1, and {alpha}6ß1, and low levels of {alpha}2ß1 [5]. In normal BM, {alpha}2 integrin is heterogeneously expressed by highly enriched Lin– /loThy1.1loSca-1+c-kit+ mouse HSCs (~85%–95% of HSCs express {alpha}2) [5]; however, expression of {alpha}2 integrin is downregulated on populations of HSCs that exhibit decreased engraftment potentials, including HSCs from mobilized, aged, and recently transplanted (4 weeks after transplant) animals [5]. To evaluate the potential role of {alpha}2-integrin in HSC function, we compared the engraftment kinetics of {alpha}2hi and {alpha}2– HSCs. Transplantation of either {alpha}2hi or {alpha}2– HSCs into lethally irradiated congenic recipients supported multilineage (T, B, and myeloid) donor-derived hematopoiesis, but surprisingly, while animals receiving {alpha}2– HSCs maintained high levels of donor-derived hematopoiesis, granulocyte production from {alpha}2hi HSCs was not detected past 6 weeks after transplant, indicating that these cells provide ST, but not LT, hematopoietic function. Interestingly, day-12 spleen colony-forming unit (CFU-S12) activity was higher in {alpha}2hi than in {alpha}2– subsets of HSCs. Together, these data suggest that in unperturbed animals, upregulation of expression of {alpha}2-integrin accompanies a loss of self-renewal potential and marks the maturation of mouse BM Lin– /loThy1.1loSca-1+ c-kit+ HSCs.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Summary
 Disclosures
 References
 
Mice and Antibodies
C57BL/Ka and C57BL/Ka-Thy1.1 mouse strains were bred and maintained at Stanford University’s Research Animal Facility. Enhanced green fluorescent protein (GFP) transgenic mice were generated as described [7] and were back-crossed to C57BL/Ka-Thy1.1 for at least nine generations. Mice used in these studies were generally 6–12 weeks old, unless otherwise indicated. The monoclonal antibodies (mAb) used in these studies included 19XE5 (anti-Thy1.1, phycoerythrin [PE] or fluorescein isothiocyanate [FITC] conjugate), 2B8 (anti-c-kit, allophycocyanin [APC] conjugate), E13–161.7 (anti-Sca-1, Ly6A/E, Texas Red [TR] conjugate), and Hm{alpha}2 (anti–{alpha}2-integrin, PE conjugate; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen). The cocktail of lineage marker antibodies included KT31.1 (anti-CD3), GK1.5 (anti-CD4), 53–7.3 (anti-CD5), 53–6.7 (anti-CD8), Ter119 (anti-erythrocyte specific antigen), 6B2 (anti-B220), 8C5 (anti-Gr-1), and M1/70 (anti-Mac-1). In addition, the following mAbs were used for evaluation of multilineage engraftment in the peripheral blood: A20.1 (anti-Ly5.2, TR conjugate), AL1-4A2 (anti-Ly5.1, FITC conjugate; BD Pharmingen), M1/70 and 8C5 (PE conjugates), 6B2 (TRICOLOR conjugate; BD Pharmingen), and KT31.1, GK1.5, and 53–6.7 (APC conjugates). Unless otherwise indicated, mAbs were produced and purified in the I.L.W. laboratory.

Flow Cytometry
To visualize HSCs, nucleated cells were isolated from whole BM (WBM), spleen, or blood after ammonium chloride–mediated lysis of RBCs. Cells were stained first with purified lineage cocktail (described above) followed by PE- or TRICOLOR-conjugated goat anti-rat immunoglobulin G antibody (CALTAG Laboratories, Burlingame, CA, http://www.caltag.com), FITC-or PE-conjugated 19XE5, TR-conjugated E13–161.7, APC-conjugated 2B8, and PE-conjugated anti–{alpha}2-integrin mAb. TRICOLOR-conjugated secondary antibodies were used in all instances in which PE-conjugated anti–{alpha}2-mAb was also used or when GFP transgenic animals were used as HSC donors. Fluorescence-activated cell sorting (FACS) data were collected on a modified FACSVantage cytometer (Becton, Dickinson and Company, Mountain View, CA, http://www.bd.com) maintained at the Stanford University Shared FACS Facility, and data were analyzed using FlowJo software (Tree Star, Inc., San Carlos, CA, http://www.treestar.com). Data are presented as either histograms or contour plots of fluorescence intensity.

HSC and BM Transplantation
For transplantation of sorted HSCs, the indicated number of Ly5.1+ HSCs, together with 3 x 105 WBM cells from congenic Ly5.2+ C57BL/Ka-Thy1.1 animals, were transplanted into lethally irradiated (950 rad, 3 hours prior to transplantation) Ly5.2+ C57BL/Ka-Thy1.1 recipients via retro-orbital injection. Transplanted animals were maintained on acidified water containing 106 U/l polymixin B sulfate and 1.1 g/l neomycin sulfate. Multilineage engraftment was monitored by flow cytometric analysis of samples of peripheral blood, collected via tail vein, and stained for lineage markers and Ly5, as indicated above. A subset of animals was sacrificed at 2 weeks or 6 months after transplant, and peripheral blood, spleen and/or BM cells were isolated and stained for lineage and congenic markers or for HSC markers and integrins, as indicated above.

Spleen CFU Assays
CFU-S12 assays were performed as described previously [8]. One hundred to 200 {alpha}2– or {alpha}2hi HSCs were injected intravenously into lethally irradiated recipients. Twelve days after transplant, mice were sacrificed and spleens were harvested and fixed in Tellyesniczky’s solution. Spleen colonies were counted and normalized for input cell number.

Statistics
Data were analyzed for statistical significance using the Student’s t test (Microsoft Excel; Microsoft Corporation, Seattle, WA, http://www.microsoft.com). Differences were considered significant at p < .05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Summary
 Disclosures
 References
 
{alpha}2–, but Not {alpha}2hi, KTLS HSCs Provide Robust, LT, Multilineage Hematopoiesis
In previous studies [5], we described the heterogeneous expression by Lin– Thy1.1loSca-1+ c-kit+ (KTLS) HSCs of {alpha}2 integrin, one subunit of the integrin cell adhesion protein, very late antigen-2 (VLA-2) [9]. Whereas the majority (85–95%) of BM Lin– /loThy1.1loSca-1+ c-kit+ HSCs express low levels of {alpha}2, a minor subset of HSCs do not express detectable surface levels of this protein (Fig. 1Go). To determine whether differences in expression of {alpha}2 integrin had any influence on HSC engraftment activity, we separated {alpha}2– from {alpha}2hi HSCs by FACS and assayed their function after transplantation into lethally irradiated congenic recipient animals. For these competitive repopulation experiments, {alpha}2– Lin– /loThy1.1loSca-1+ c-kit+ HSCs (representing ~5%–15% of total KTLS HSCs) and {alpha}2hi Lin– /lo Thy1.1loSca-1+ c-kit+ HSCs (representing ~50% of total Lin– /loThy1.1loSca-1+ c-kit+ HSCs) were sorted from the BM of C57BL6/Ka-Thy1.1/Ly5.1 donors and transferred at 20 HSCs per animal to C57BL6/Ka-Thy1.1/Ly5.2 recipients, together with syngeneic (Ly5.2+) competitor WBM cells. Peripheral blood hematopoietic chimerism of the recipient animals was assessed at multiple time points from 4 weeks to more than 20 weeks after transplant. Transplanted {alpha}2– Lin– /loThy1.1loSca-1+ c-kit+ HSCs showed significantly more robust LT engraftment capacity as compared with {alpha}2hi HSCs, and only {alpha}2– HSCs gave sustained production of granulocytes (Fig. 2Go). In all animals receiving 20 {alpha}2– HSCs, donor-derived B cells, T cells, and granulocytes were easily detected at 4–6 weeks after transplant, and their frequency increased with time; in contrast, animals receiving 20 {alpha}2hi HSCs showed only low-level hematopoietic chimerism, predominantly of the lymphoid lineages, which was detectable at early time points in only a fraction (3 of 5) of transplanted animals and declined with time (Fig. 2Go).


Figure 1
View larger version (36K):
[in this window]
[in a new window]
  		Figure 1. Sorting parameters for {alpha}2hi and {alpha}2– BM HSCs. (A): Lin– /loThy1.1loSca-1+ c-kit+ HSCs were identified in undepleted WBM by flow cytometry. (B): c-kit+Thy1.1+ Lin– /loSca-1+ HSCs were identified in WBM depleted of mature hematopoietic cells by negative magnetic selection for lineage markers (lineage-depleted BM). Representative FACS plots show sequential gating from left to right. The frequency of {alpha}2– HSCs among the total HSC population varied from 5%–15% among independent experiments (n = 5). The {alpha}2hi population was gated as the top approximately 50% of the total HSC population. Abbreviations: BM, bone marrow; FACS, fluorescence-activated cell sorting; HSC, hematopoietic stem cell; WBM, whole bone marrow.

 

Figure 2
View larger version (20K):
[in this window]
[in a new window]
  		Figure 2. Kinetics of engraftment of lymphoid and myeloid lineages by {alpha}2– or {alpha}2hi HSCs. Lethally irradiated adult Ly5.2+ recipients (five per group) were transplanted with 20 {alpha}2– (circles) or 20 {alpha}2hi (squares) Ly5.1+ HSCs, together with 2 x 105 Ly5.2+ WBM cells. (A): At the indicated time points, from 5–22 weeks, recipient mice were bled via the tail vein and the percentage of Ly5.1+ (donor-derived) cells among granulocytes (Mac-1+ Gr-1+) in the peripheral blood was determined. (B): At the same time points, the percentage of Ly5.1+ (donor-derived) cells among B cells (B220+) in the peripheral blood was determined. (C): Likewise, the percentage of Ly5.1+ (donor-derived) cells among T cells (CD3/4/8+) in the peripheral blood was determined. Each line represents an individual mouse. Results are representative of three independent experiments. Abbreviations: HSC, hematopoietic stem cell; WBM, whole bone marrow.

 
{alpha}2hi Lin– /loThy1.1loSca-1+ c-kit+ HSCs Are ST Engrafting Cells
Although low-level B- and T-cell chimerism was detectable in a subset (3 of 5) of animals transplanted with {alpha}2hi HSCs, donor-derived granulocytes were rarely detected in these animals, analyzed 4 weeks or more after transplant. This finding might indicate that the differentiation potential of {alpha}2hi HSCs is skewed toward lymphoid development and that these cells have lost granulocytic, and thus multilineage, differentiation capacity. Alternatively, these data could indicate that {alpha}2 integrin expression enriches for a less primitive subset of Lin– /loThy1.1loSca-1+ c-kit+ HSCs, with reduced self-renewal potential, that provides only ST, multilineage hematopoietic engraftment. Because granulocytes are short-lived cells (average half-life of 1–2 days [10]), they may be undetectable at 4 weeks after transplant in recipients of {alpha}2hi HSCs if ST engrafting {alpha}2hi HSCs have exhausted their self-renewal potential by this time and therefore are no longer able to sustain daily replacement of mature peripheral cells. In contrast, lymphoid cells are relatively long-lived, and so, lymphoid chimerism could be maintained in such mice from cells generated earlier post-transplant. To distinguish between these possibilities, lymphoid and myeloid engraftment from {alpha}2– or {alpha}2hi HSCs was analyzed in recipient mice at 2 weeks after transplant. Because overall levels of hematopoietic regeneration are quite low at this time, transplanted animals were sacrificed and their total blood volume was collected for analysis by transcardiac perfusion. Hemato-poietic chimerism was also assessed in the BM and spleen of recipient mice. Donor-derived granulocytes were easily detectable in the blood, BM, and spleens of animals transplanted with {alpha} 2hi or {alpha}2– Lin– /loThy1.1loSca-1+ c-kit+ HSCs (Fig. 3A, 3CGo). Low-level chimerism of B cells was also evident in both {alpha}2– HSC– and {alpha}2hi HSC–transplanted animals (Fig. 3BGo). These data demonstrate that {alpha}2hi HSCs rapidly generate both lymphoid and myeloid lineage cells after transplantation into lethally irradiated recipients. The inability of {alpha}2hi HSCs to provide sustained contribution to granulocyte production likely indicates that this population is enriched for ST reconstituting HSCs with limited self-renewal potential.


Figure 3
View larger version (29K):
[in this window]
[in a new window]
  		Figure 3. {alpha}2hi HSCs provide short-term production of granulocytes. The presence of donor-derived (Ly5.1+) B and myeloid cells in the peripheral blood was determined 14 days after transplant of Ly5.2+ recipients with 20 {alpha}2hi, 30 {alpha}2hi, or 30 {alpha}2– Ly5.1+ HSCs, together with 2 x 105 Ly5.2+ WBM cells. (A): Results are shown as dot plots of relative fluorescence intensity for Ly5.1 (donor, shown on the x-axis) versus Ly5.2 (host, shown on the y-axis) granulocytes (Mac-1+ Gr-1+) for two individual recipients in a representative experiment. (B): Results are shown as dot plots of relative fluorescence intensity for Ly5.1 (donor, shown on the x-axis) versus Ly5.2 (host, shown on the y-axis) B cells (B220+) for two individual recipients in a representative experiment. Untransplanted control Ly5.1+ and Ly5.2+ animals are also shown for comparison. (C): The percentage of gated Ly5.1+ (donor-derived) cells is indicated on each plot, and data on myeloid chimerism are summarized as the average percentage of donor-derived Mac-1+ Gr-1+ granulocytes (± SD) in the blood, BM, or spleen at 14 days after transplant for all animals in these experiments (n = 6). Background staining of negative control animals was less than 0.1% in all cases. Abbreviations: BM, bone marrow; HSC, hematopoietic stem cell; WBM, whole bone marrow.

 
{alpha}2– Lin– /loThy1.1loSca-1+ c-kit+ HSCs Regenerate {alpha}2hi Lin– /loThy1.1loSca-1+ c-kit+ HSCs After Transplantation
To investigate the lineage relationship between {alpha}2hi and {alpha}2– HSCs, we analyzed the integrin expression profile of donor-derived Lin– /loThy1.1loSca-1+ c-kit+ HSCs in the BM of recipient animals transplanted with {alpha}2– HSCs approximately 6 months previously. Donor-derived Lin– Thy1.1loSca-1+ c-kit+ HSCs exhibited a normal profile of {alpha}2-integrin expression, with approximately 90% of cells expressing {alpha}2-integrin (Fig. 4Go). It should be noted that this expression pattern differs from that observed immediately after transplantation (4 weeks after transplant), when Lin– /loThy1.1loSca-1+ c-kit+ HSCs exhibit a substantial reduction in the fraction of {alpha}2-expressing cells [5], suggesting that, as has been noted for HSC proliferative status [11], a significant amount of time is required to restore ho-meostasis in the HSC compartment after transplant. {alpha}4, {alpha}5, {alpha}6, and ß1 integrins were also expressed by Lin– /loThy1.1loSca-1+ c-kit+ HSCs at normal levels at 6 months after transplant (as compared with age-matched, untransplanted animals). Notably, both in untransplanted animals and in animals transplanted 6 months previously, Lin– /loThy1.1loSca-1+ c-kit+ HSCs express uniformly high levels of {alpha}4, {alpha}5, {alpha}6, and ß1 integrins, indicating that differences in the expression levels of these other integrins are unlikely to account for the observed differences in the engraftment functions of {alpha}2– and {alpha}2hi HSCs. These transplant data indicate that LT reconstituting {alpha}2– HSCs give rise to ST reconstituting {alpha}2hi HSCs after transplant and suggest that {alpha}2– cells represent a less mature subset of HSCs. Consistent with this hypothesis, {alpha}2hi Lin– /loThy1.1loSca-1+ c-kit+ HSCs exhibit an approximately sixfold increased frequency of CFU-S12 (Fig. 5Go). Spleen colony-forming activity is largely regarded as a measure of hematopoietic stem/progenitor cell activity, how-ever, among very primitive BM populations, CFU-S12 activity correlates inversely with LT reconstituting activity [12]. Thus, taken together, these data strongly suggest that {alpha}2hi Lin– /loThy1.1loSca-1+ c-kit+ cells lie downstream of {alpha}2– HSCs in hematopoietic ontogeny and that among Lin– /loThy1.1loSca-1+ c-kit+ BM HSCs, upregulation of {alpha}2 integrin expression may mark a transition from LT to ST reconstituting potential. Interestingly, {alpha}2 integrin is expressed at substantially reduced levels (only 2%–25% of progenitor cells are {alpha}2+) on committed oligolineage progenitor cells, identified by previously defined cell surface markers [13, 14] (Fig. 6Go), suggesting that {alpha}2 integrin may specifically enrich for a subset of Lin– /lo BM cells that retain multilineage differentiation potential but have substantially reduced self-renewal potential.


Figure 4
View larger version (24K):
[in this window]
[in a new window]
  		Figure 4. Transplanted {alpha}2– HSCs give rise to {alpha}2hi HSCs in vivo. BM was harvested from mice transplanted 6 months previously with 20 {alpha}2– Ly5.1+ HSCs. Integrin expression by Ly5.1+ Lin– /loThy1.1loSca-1+ c-kit+ HSCs was determined by FACS analysis. Daughter HSCs derived from {alpha}2– HSCs (bottom row) express levels of {alpha}2, {alpha}4, {alpha}5, {alpha}6, and ß1 integrins similar to wild-type, untransplanted HSCs (middle row). Data are shown as histograms of fluorescence intensity for isotype control (red) or anti-integrin (blue) monoclonal antibody (n = 5). Abbreviations: BM, bone marrow; FACS, fluorescence-activated cell sorting; HSC, hemato-poietic stem cell; WBM, whole bone marrow.

 

Figure 5
View larger version (28K):
[in this window]
[in a new window]
  		Figure 5. {alpha}2hi HSCs are enriched for CFU-S12 activity as compared with {alpha}2– HSCs. One hundred to 200 HSCs (either {alpha}2– or {alpha}2hi) were transplanted into lethally irradiated adult recipients (2–10 mice per group). Twelve days after transplants, recipient spleens were harvested and fixed in Tellyesniczky’s solution. Macroscopic spleen colonies were counted and normalized for input cell number. Data are plotted as the mean number of colonies per input cell (± SD) for {alpha}2– or {alpha}2hi HSCs. Data combined from two independent experiments. Abbreviations: CFU-S12, day-12 spleen colony-forming unit; HSC, hematopoietic stem cell.

 

Figure 6
View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. Oligolineage progenitor cells downregulate {alpha}2 integrin expression. Lineage-depleted BM was stained and analyzed by FACS for {alpha}2 integrin on (1) HSCs (Lin– /loThy1.1loSca-1+ c-kit+), (2) CMPs (Lin– /loc-kit+Sca-1– CD34+ Fc-{gamma}-Rlo), GMPs (Lin– /loc-kit+Sca-1–CD34+ Fc-{gamma}-Rhi), and MEPs (Lin– /loc-kit+Sca-1–CD34– Fc-{gamma}-Rlo), or (3) CLPs (c-kitloThy1.1–Lin– Sca-1loIL7R-{alpha}+). Data are shown as histograms of fluorescence intensity with anti-{alpha}2 staining shown in red and staining with isotype control monoclonal antibody shown in blue. Approximately 90% of HSCs, 25% of CMPs, and 2% of GMPs, MEPs, and CLPs were {alpha}2 integrin+. Representative of two independent experiments. Abbreviations: CLP, common lymphocyte progenitor; CMP, common myeloid progenitor; GMP, granulocyte/macrophage progenitor; HSC, hematopoietic stem cell; MEP, megakaryocyte/erythrocyte progenitor.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Summary
 Disclosures
 References
 
HSCs are rare, clonogenic, multipotent cells that maintain both extensive self-renewal capacity and complete multilineage differentiation potential. HSCs are the functional units in BM transplantation and allow rescue from otherwise lethal irradiation of transplant recipients. Thus, a careful and deterministic approach to the purification of HSCs is essential to a complete understanding of their biological properties and improved application of these cells in clinical transplantation. Many methods to enrich for HSCs from unfractionated BM have been described [2–4, 15–18] and have allowed a refined understanding of the heterogeneity of the "HSC" pool, which is now known to be comprised of a lineage of multipotent cells with LT reconstituting, ST reconstituting, and MPP properties [2, 4].

Here, we describe the heterogeneous expression of the cell surface–expressed protein {alpha}2 integrin by highly enriched mouse HSCs and further demonstrate that this antigen provides an effective means of separating enriched populations of LT or ST reconstituting cells from among the Lin– /loThy1.1loSca-1+ c-kit+ HSC population. When paired with ß1 integrin, {alpha}2 forms an adhesion receptor (VLA-2) that binds with high affinity to the extracellular matrix components collagen and laminin. Among mature hematopoietic cells, {alpha}2ß1 is expressed by activated T lymphocytes, megakaryocytes, and platelets and plays a nonredundant role the in vitro adhesion of platelets to soluble collagen [19, 20]. {alpha}2ß1 is also expressed by fibroblasts and epithelial cells and appears to promote increased complexity of branching morphogenesis of breast epithelial tissue [20].

Among highly enriched Lin– /loThy1.1loSca-1+c-kit+ mouse HSCs, {alpha}2-integrin is expressed by approximately 85%–95% of cells. In response to pharmacological mobilization of HSCs, which induces increased migration of HSCs from the BM into the blood [21, 22], {alpha}2-integrin expression by HSCs is substantially decreased, both by HSCs that remain in the BM and by HSCs that traffic to the blood and spleen [5]. This finding initially led us to hypothesize that downregulation of {alpha} 2-integrin may be involved in HSC egress from BM and that {alpha}2ß1 may be an important homing receptor for transplanted HSCs. Surprisingly, however, the functional analyses described here indicate that, contrary to expectations, expression of {alpha}2 integrin divides HSCs into enriched populations of {alpha}2hi ST and {alpha} 2– LT reconstituting subsets. Sorted {alpha}2hi HSCs exhibit decreased LT engraftment potential, but increased CFU-S12 activity, suggesting that they are less primitive as compared with {alpha}2– HSCs, which maintain robust multilineage LT engraftment and reduced CFU-S12 activity. The temporally restricted production of mature granulocytes from transplanted {alpha}2hi HSCs further supports their limited self-renewal capacity. Although at present we cannot completely exclude the possibility that some LT engrafting cells may be present at low levels in the {alpha}2hi subset of HSCs, it is clear from these data that {alpha}2hi cells are substantially enriched for rapid reconstituting ST HSCs, whereas {alpha}2– HSCs are highly enriched for LT reconstituting cells.

The anti-{alpha}2 antibody used in this study, Hm{alpha}2, has been reported to block the function of {alpha}2ß1 for collagen binding and for co-stimulation of mature T lymphocytes [23] and to block the invasive migration of mouse mammary carcinoma cells [24]. Thus, it is possible that the observed differences in LT hema-topoietic engraftment capacity of {alpha}2– and {alpha}2hi Lin– /lo Thy1.1loSca-1+c-kit+ HSCs result from functional blockade of {alpha}2 integrin activity on {alpha}2hi HSCs. However, using a ST in vivo homing assay, we previously demonstrated [5] that Hm{alpha}2 does not inhibit the initial homing of intravenously infused HSCs to the BM, measured 3 hours after transplant. In addition, Hm{alpha}2 does not block ST hematopoietic engraftment, as demonstrated by the observation that {alpha}2hi Lin– /loThy1.1loSca-1+c-kit+ HSCs, with bound anti-{alpha}2 mAb, contribute to both lymphoid and myeloid lineages at 2 weeks after transplant (Fig. 3Go). Thus, differences in the hematopoietic reconstituting capacity of {alpha}2– and {alpha}2hi Lin– /loThy1.1loSca-1+c-kit+ HSCs likely relate to intrinsic properties of these populations rather than to a direct effect of antibody binding by one of these HSC subsets.

Interestingly, among Lin– BM cells, highly enriched for hematopoietic stem and progenitor cells, {alpha}2-integrin appears to be a relatively specific marker of multilineage repopulating cells with limited self-renewal activity. {alpha}2– LT HSCs give rise to {alpha}2hi ST HSCs after transplantation; however, expression of {alpha}2 integrin is subsequently lost or substantially reduced (to ~2%–25% of cells) on more committed oligolineage progenitor cells, which lack self-renewal and multilineage reconstitution potential, downstream of HSCs. This pattern of expression differs from that described for other markers (including Flk-2 and Mac-1) that are also differentially expressed by LT versus ST reconstituting HSCs [2, 4]. Cell surface expression of both Flk-2 and Mac-1, though likewise lacking on the most primitive LT HSCs, is maintained on some or all oligolineage-committed progenitor cells after differentiation of Flk2+ MPP or Mac-1+ ST HSCs [13, 25]).

Whether changes in {alpha}2-integrin expression lead directly to functional differences in {alpha}2hi versus {alpha}2– HSCs cannot be established from these studies. Although genetic ablation of {alpha}2 integrin does cause specific deficiencies in platelet function in vitro and mast cell-mediated inflammatory responses in vivo, {alpha}2 is dispensable for platelet production and normal hemostasis, and other hematopoietic lineages in these mice appear to be grossly unaffected [20, 26]. These data suggest that {alpha}2 deficiency does not result in obvious defects in HSC function; however, direct analysis of {alpha}2 integrin–/– HSC and hematopoietic progenitor cell frequency and function in transplantation assays has not been reported. In other systems, ligand binding by {alpha}2ß1 receptors can induce activation of matrix metalloproteinases [27–29], promote cell migration and growth factor–dependent cell cycle progression [30, 31], or restore differentiation potential [32]. To determine whether {alpha}2 functions similarly to promote motility, proliferation, or differentiation of {alpha}2hi ST HSCs will require further analysis of these processes in {alpha}2-deficient HSCs; however, it is clear from our previous studies that blockade of {alpha}2-integrin adhesive function does not significantly impede the initial homing of HSCs to the BM, analyzed 3 hours after transplant [5], or ST hematopoietic engraftment by {alpha}2hi Lin– /loThy1.1loSca-1+ c-kit+ HSCs (Fig. 3Go). Nonetheless, it remains possible that interactions of {alpha}2ß1 integrin expressed by ST HSCs with extracellular matrix components of the BM environment may direct their localization to particular microenvironments, which may in turn determine their fate.


   SUMMARY
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
Disclosures
 References
 
We show here that among Lin– /loThy1.1loSca-1+ c-kit+ BM cells, the only subset of BM containing cells capable of LT, multilineage hematopoietic reconstitution [33], expression of the cell surface adhesion receptor {alpha}2-integrin effectively distinguishes populations enriched for LT reconstituting ({alpha}2–) or ST reconstituting ({alpha}2hi) cells. {alpha}2– Lin– /loThy1.1loSca-1+ c-kit+ HSCs give rise to {alpha}2hi Lin– /loThy1.1loSca-1+c-kit+ BM cells after transplantation; however, {alpha}2 expression is not maintained at high levels as ST HSCs lose self-renewal potential and differentiate into oligolineage progenitor cells. Thus, although the precise role, if any, of {alpha}2-integrin in HSC function remains to be defined, expression of this receptor represents a useful phenotypic marker for separating highly enriched populations of LT and ST HSCs in normal BM, which should facilitate studies aimed at defining molecular determinants of stem cell self-renewal.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
 References
 
We thank L. Jerabek for excellent laboratory management, S. Smith for antibody preparation, and L. Hidalgo, J. Dollaga, and D. Escoto for animal care. This work was supported in part by NIH (grant 2 RO1 HL58770, and grant CA86065 to I.L.W.), and American Cancer Society (grant PF-00-017-01-LBC), and the Frederick Frank/Lehman Brothers, Inc. Irvington Institute Fellowship to A.J.W.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
References
 
I.L.W. has a financial interest in or has served as an officer or member of the Board of Amgen, Cellerant, and Stem Cells, Inc.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
 References
 

  1. Wagers AJ, Christensen JL, Weissman IL. Cell fate determination from stem cells. Gene Ther 2002;9:606–612.[CrossRef][Medline]

  2. Morrison SJ, Wandycz AM, Hemmati HD et al. Identification of a lineage of multipotent hematopoietic progenitors. Development 1997; 124:1929–1939.[Abstract]

  3. Wiesmann A, Phillips RL, Mojica M et al. Expression of CD27 on murine hematopoietic stem and progenitor cells. Immunity 2000;12:193–199.[CrossRef][Medline]

  4. Christensen JL, Weissman IL. Flk-2 is a marker in hematopoietic stem cell differentiation: A simple method to isolate long-term stem cells. Proc Natl Acad Sci U S A 2001;98:14541–14546.[Abstract/Free Full Text]

  5. Wagers AJ, Allsopp RC, Weissman IL. Changes in integrin expression are associated with altered homing properties of Lin(–/lo)Thy1.1(lo)Sca-1(+)c-kit(+) hematopoietic stem cells following mobilization by cyclophosphamide/granulocyte colony-stimulating factor. Exp Hematol 2002;30:176–185.[CrossRef][Medline]

  6. Hynes RO. Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11–25.[CrossRef][Medline]

  7. Wright DE, Cheshier SH, Wagers AJ et al. Cyclophosphamide/granulocyte colony-stimulating factor causes selective mobilization of bone marrow hematopoietic stem cells into the blood after M phase of the cell cycle. Blood 2001;97:2278–2285.[Abstract/Free Full Text]

  8. Morrison SJ, Wandycz AM, Akashi K et al. The aging of hematopoietic stem cells. Nat Med 1996;2:1011–1016.[CrossRef][Medline]

  9. Santoro SA, Zutter MM. The alpha 2 beta 1 integrin: A collagen receptor on platelets and other cells. Thromb Haemost 1995;74:813–821.[Medline]

  10. Lagasse E, Weissman IL. bcl-2 inhibits apoptosis of neutrophils but not their engulfment by macrophages. J Exp Med 1994;179:1047–1052.[Abstract/Free Full Text]

  11. Allsopp RC, Cheshier SH, Weissman IL. Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells. J Exp Med 2001;17:15.

  12. Na Nakorn T, Traver D, Weissman IL et al. Myeloerythroid-restricted progenitors are sufficient to confer radioprotection and provide the majority of day 8 CFU-S. J Clin Invest 2002;109:1579–1585.[CrossRef][Medline]

  13. Akashi K, Traver D, Miyamoto T et al. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 2000;404:193–197.[CrossRef][Medline]

  14. Kondo M, Weissman IL, Akashi K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 1997;91:661–672.[CrossRef][Medline]

  15. Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1994;1:661–673.[CrossRef][Medline]

  16. Spangrude GJ, Johnson GR. Resting and activated subsets of mouse multipotent hematopoietic stem cells. Proc Natl Acad Sci U S A 1990; 87:7433–7437.[Abstract/Free Full Text]

  17. Jones RJ, Wagner JE, Celano P et al. Separation of pluripotent haematopoietic stem cells from spleen colony- forming cells. Nature 1990;347: 188–189.[CrossRef][Medline]

  18. 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]

  19. Holtkotter O, Nieswandt B, Smyth N et al. Integrin alpha 2-deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen. J Biol Chem 2002;277:10789–10794.[Abstract/Free Full Text]

  20. Chen J, Diacovo TG, Grenache DG et al. The alpha(2) integrin subunit-deficient mouse: A multifaceted phenotype including defects of branching morphogenesis and hemostasis. Am J Pathol 2002;161:337–344.[Abstract/Free Full Text]

  21. Neben S, Marcus K, Mauch P. Mobilization of hematopoietic stem and progenitor cell subpopulations from the marrow to the blood of mice following cyclophosphamide and/or granulocyte colony-stimulating factor. Blood 1993;81:1960–1967.[Abstract/Free Full Text]

  22. Morrison SJ, Wright DE, Weissman IL. Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate prior to mobilization. Proc Natl Acad Sci U S A 1997;94:1908–1913.[Abstract/Free Full Text]

  23. Miyake S, Sakurai T, Okumura K et al. Identification of collagen and laminin receptor integrins on murine T lymphocytes. Eur J Immunol 1994;24:2000–2005.[Medline]

  24. Lochter A, Navre M, Werb Z et al. alpha1 and alpha2 integrins mediate invasive activity of mouse mammary carcinoma cells through regulation of stromelysin-1 expression. Mol Biol Cell 1999;10:271–282.[Abstract/Free Full Text]

  25. Karsunky H, Merad M, Cozzio A et al. Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo. J Exp Med 2003;198:305–313.[Abstract/Free Full Text]

  26. Edelson BT, Li Z, Pappan LK et al. Mast cell-mediated inflammatory responses require the alpha 2 beta 1 integrin. Blood 2004;103:2214–2220.[Abstract/Free Full Text]

  27. Langholz O, Rockel D, Mauch C et al. Collagen and collagenase gene expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins. J Cell Biol 1995;131(6 Pt 2):1903–1915.[Abstract/Free Full Text]

  28. Ravanti L, Heino J, Lopez-Otin C et al. Induction of collagenase-3 (MMP-13) expression in human skin fibroblasts by three-dimensional collagen is mediated by p38 mitogen-activated protein kinase. J Biol Chem 1999;274:2446–2455.[Abstract/Free Full Text]

  29. Zigrino P, Drescher C, Mauch C. Collagen-induced proMMP-2 activation by MT1-MMP in human dermal fibroblasts and the possible role of alpha2beta1 integrins. Eur J Cell Biol 2001;80:68–77.[CrossRef][Medline]

  30. Klekotka PA, Santoro SA, Wang H et al. Specific residues within the alpha 2 integrin subunit cytoplasmic domain regulate migration and cell cycle progression via distinct MAPK pathways. J Biol Chem 2001;276: 32353–32361.[Abstract/Free Full Text]

  31. Klekotka PA, Santoro SA, Ho A et al. Mammary epithelial cell-cycle progression via the alpha(2)beta(1) integrin: Unique and synergistic roles of the alpha(2) cytoplasmic domain. Am J Pathol 2001;159: 983–992.[Abstract/Free Full Text]

  32. Zutter MM, Santoro SA, Staatz WD et al. Re-expression of the alpha 2 beta 1 integrin abrogates the malignant phenotype of breast carcinoma cells. Proc Natl Acad Sci U S A 1995;92:7411–7415.[Abstract/Free Full Text]

  33. Uchida N, Weissman IL. Searching for hematopoietic stem cells: Evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. J Exp Med 1992;175:175–184.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
G. E. Desanti, A. Cumano, and R. Golub
Identification of CD4int progenitors in mouse fetal spleen, a source of resident lymphoid cells
J. Leukoc. Biol., May 1, 2008; 83(5): 1145 - 1154.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
A. Terunuma, V. Kapoor, C. Yee, W. G. Telford, M. C. Udey, and J. C. Vogel
Stem Cell Activity of Human Side Population and {alpha}6 Integrin-Bright Keratinocytes Defined by a Quantitative In Vivo Assay
Stem Cells, March 1, 2007; 25(3): 664 - 669.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
J. B. Gilner, W. G. Walton, K. Gush, and S. L. Kirby
Antibodies to Stem Cell Marker Antigens Reduce Engraftment of Hematopoietic Stem Cells
Stem Cells, February 1, 2007; 25(2): 279 - 288.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
T. Umemoto, M. Yamato, Y. Shiratsuchi, M. Terasawa, J. Yang, K. Nishida, Y. Kobayashi, and T. Okano
Expression of Integrin beta3 Is Correlated to the Properties of Quiescent Hemopoietic Stem Cells Possessing the Side Population Phenotype
J. Immunol., December 1, 2006; 177(11): 7733 - 7739.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
P. E. Hall, J. D. Lathia, N. G. A. Miller, M. A. Caldwell, and C. Ffrench-Constant
Integrins Are Markers of Human Neural Stem Cells
Stem Cells, September 1, 2006; 24(9): 2078 - 2084.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0396v1
24/4/1087    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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wagers, A. J.
Right arrow Articles by Weissman, I. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wagers, A. J.
Right arrow Articles by Weissman, I. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS
http://www.stemcellsportal.com/