Differential Expression of 2 Integrin Separates Long-Term and Short-Term Reconst - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:07 |只看该作者 |倒序浏览 |打印

作者：Amy J. Wagersa, Irving L. Weissmana,b 6 O' S. _: H/ V/ d+ Z1 F


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      【摘要】
   Self-renewing, multipotent hematopoietic stem cells are highly enriched within the Lin¨C 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 2-integrin (CD49b) as a surface marker with heterogeneous expression on Lin¨C /loThy1.1loc-kit  Sca-1  stem cells. To determine whether differences in 2 expression were indicative of differences in stem cell function, we purified 2¨C and 2hi stem cells by fluorescence-activated cell sorting and analyzed their function in long- and short-term hematopoietic reconstitution assays. Both 2¨C and 2hi cells could give rise to mature lymphoid and myeloid cells after transplantation into lethally irradiated congenic recipients. However, 2hi cells supported hematopoiesis for only a short time (<4 weeks), whereas 2¨C cells reproducibly yielded robust, long-term (>20 weeks) reconstitution, suggesting that 2¨C cells represent a more primitive population than do 2hi cells. Consistent with this idea, 2¨C Lin¨C /loThy1.1loc-kit  Sca-1  cells exhibited an approximately sixfold decreased frequency of spleen colony-forming units (day 12) versus 2hi cells. Furthermore, bone marrow cells isolated from animals transplanted >20 weeks previously with 20 2¨C Lin¨C /loThy1.1loc-kit  Sca-1  cells included both 2¨C and 2hi stem cells of donor origin, indicating that 2hi cells are likely lineal descendents of 2¨C cells. Interestingly, 2 integrin expression is significantly reduced on lineage-restricted oligopotent progenitors in the marrow, suggesting that high level expression of 2 selectively marks a subset of primitive hematopoietic cells which retains multilineage reconstitution potential but exhibits reduced self-renewal capacity.
      【关键词】 Cell surface markers Integrins Hematopoietic stem cell Hematopoietic stem cell transplantation
   INTRODUCTION9 a# {( V" {/ W
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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 .! F2 F( F5 F& t" y3 N" ~) h
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In an effort to identify adhesion molecules involved in HSC migration, we previously examined the expression of integrin receptors by HSCs . To evaluate the potential role of 2-integrin in HSC function, we compared the engraftment kinetics of 2hi and 2¨C HSCs. Transplantation of either 2hi or 2¨C HSCs into lethally irradiated congenic recipients supported multilineage (T, B, and myeloid) donor-derived hematopoiesis, but surprisingly, while animals receiving 2¨C HSCs maintained high levels of donor-derived hematopoiesis, granulocyte production from 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 2hi than in 2¨C subsets of HSCs. Together, these data suggest that in unperturbed animals, upregulation of expression of 2-integrin accompanies a loss of self-renewal potential and marks the maturation of mouse BM Lin¨C /loThy1.1loSca-1  c-kit  HSCs.
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MATERIALS AND METHODS. v" i% b/ g% L2 M4 o: A
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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  conjugate), and Hm2 (anti¨C2-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¨C7.3 (anti-CD5), 53¨C6.7 (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¨C6.7 (APC conjugates). Unless otherwise indicated, mAbs were produced and purified in the I.L.W. laboratory.0 Z* J5 K; E- t' B& x
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Flow Cytometry
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To visualize HSCs, nucleated cells were isolated from whole BM (WBM), spleen, or blood after ammonium chloride¨Cmediated 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¨C161.7, APC-conjugated 2B8, and PE-conjugated anti¨C2-integrin mAb. TRICOLOR-conjugated secondary antibodies were used in all instances in which PE-conjugated anti¨C2-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.  o( I  n  ?* u1 Z  i

HSC and BM Transplantation0 d6 d) C) h6 K8 c9 d5 q
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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.
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Spleen CFU Assays

CFU-S12 assays were performed as described previously . One hundred to 200 2¨C or 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.. \  L0 P3 y& X  s
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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

RESULTS+ R8 @+ E! T9 z4 w
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2¨C, but Not 2hi, KTLS HSCs Provide Robust, LT, Multilineage Hematopoiesis/ }2 j- }. t6 `4 P% I
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In previous studies . Whereas the majority (85¨C95%) of BM Lin¨C /loThy1.1loSca-1  c-kit  HSCs express low levels of 2, a minor subset of HSCs do not express detectable surface levels of this protein (Fig. 1). To determine whether differences in expression of 2 integrin had any influence on HSC engraftment activity, we separated 2¨C from 2hi HSCs by FACS and assayed their function after transplantation into lethally irradiated congenic recipient animals. For these competitive repopulation experiments, 2¨C Lin¨C /loThy1.1loSca-1  c-kit  HSCs (representing ~5%¨C15% of total KTLS HSCs) and 2hi Lin¨C /lo Thy1.1loSca-1  c-kit  HSCs (representing ~50% of total Lin¨C /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 2¨C Lin¨C /loThy1.1loSca-1  c-kit  HSCs showed significantly more robust LT engraftment capacity as compared with 2hi HSCs, and only 2¨C HSCs gave sustained production of granulocytes (Fig. 2). In all animals receiving 20 2¨C HSCs, donor-derived B cells, T cells, and granulocytes were easily detected at 4¨C6 weeks after transplant, and their frequency increased with time; in contrast, animals receiving 20 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. 2).
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Figure 1. Sorting parameters for 2hi and 2¨C BM HSCs. (A): Lin¨C /loThy1.1loSca-1  c-kit  HSCs were identified in undepleted WBM by flow cytometry. (B): c-kit Thy1.1  Lin¨C /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 2¨C HSCs among the total HSC population varied from 5%¨C15% among independent experiments (n = 5). The 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.
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Figure 2. Kinetics of engraftment of lymphoid and myeloid lineages by 2¨C or 2hi HSCs. Lethally irradiated adult Ly5.2  recipients (five per group) were transplanted with 20 2¨C (circles) or 20 2hi (squares) Ly5.1  HSCs, together with 2 x 105 Ly5.2  WBM cells. (A): At the indicated time points, from 5¨C22 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.3 ~" s; n: ~/ ]4 M+ F/ ]3 l( |
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2hi Lin¨C /loThy1.1loSca-1  c-kit  HSCs Are ST Engrafting Cells& T  i( X% {, z* L+ a2 \9 H
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Although low-level B- and T-cell chimerism was detectable in a subset (3 of 5) of animals transplanted with 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 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 2 integrin expression enriches for a less primitive subset of Lin¨C /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¨C2 days ), they may be undetectable at 4 weeks after transplant in recipients of 2hi HSCs if ST engrafting 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 2¨C or 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  2hi or 2¨C Lin¨C /loThy1.1loSca-1  c-kit  HSCs (Fig. 3A, 3C). Low-level chimerism of B cells was also evident in both 2¨C HSC¨C and 2hi HSC¨Ctransplanted animals (Fig. 3B). These data demonstrate that 2hi HSCs rapidly generate both lymphoid and myeloid lineage cells after transplantation into lethally irradiated recipients. The inability of 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. 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 2hi, 30 2hi, or 30 2¨C 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 (¡À 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.7 V3 T& ]2 V' J) s

2¨C Lin¨C /loThy1.1loSca-1  c-kit  HSCs Regenerate 2hi Lin¨C /loThy1.1loSca-1  c-kit  HSCs After Transplantation5 ?# }  h: ]# p% K) P# M

To investigate the lineage relationship between 2hi and 2¨C HSCs, we analyzed the integrin expression profile of donor-derived Lin¨C /loThy1.1loSca-1  c-kit  HSCs in the BM of recipient animals transplanted with 2¨C HSCs approximately 6 months previously. Donor-derived Lin¨C Thy1.1loSca-1  c-kit  HSCs exhibited a normal profile of 2-integrin expression, with approximately 90% of cells expressing 2-integrin (Fig. 4). It should be noted that this expression pattern differs from that observed immediately after transplantation (4 weeks after transplant), when Lin¨C /loThy1.1loSca-1  c-kit  HSCs exhibit a substantial reduction in the fraction of 2-expressing cells  (Fig. 6), suggesting that 2 integrin may specifically enrich for a subset of Lin¨C /lo BM cells that retain multilineage differentiation potential but have substantially reduced self-renewal potential.
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Figure 4. Transplanted 2¨C HSCs give rise to 2hi HSCs in vivo. BM was harvested from mice transplanted 6 months previously with 20 2¨C Ly5.1  HSCs. Integrin expression by Ly5.1  Lin¨C /loThy1.1loSca-1  c-kit  HSCs was determined by FACS analysis. Daughter HSCs derived from 2¨C HSCs (bottom row) express levels of 2, 4, 5, 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.  M6 T# _3 w5 w5 N5 P

Figure 5. 2hi HSCs are enriched for CFU-S12 activity as compared with 2¨C HSCs. One hundred to 200 HSCs (either 2¨C or 2hi) were transplanted into lethally irradiated adult recipients (2¨C10 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 (¡À SD) for 2¨C or 2hi HSCs. Data combined from two independent experiments. Abbreviations: CFU-S12, day-12 spleen colony-forming unit; HSC, hematopoietic stem cell.

Figure 6. Oligolineage progenitor cells downregulate 2 integrin expression. Lineage-depleted BM was stained and analyzed by FACS for 2 integrin on (1) HSCs (Lin¨C /loThy1.1loSca-1  c-kit ), (2) CMPs (Lin¨C /loc-kit Sca-1¨C CD34  Fc--Rlo), GMPs (Lin¨C /loc-kit Sca-1¨CCD34  Fc--Rhi), and MEPs (Lin¨C /loc-kit Sca-1¨CCD34¨C Fc--Rlo), or (3) CLPs (c-kitloThy1.1¨CLin¨C Sca-1loIL7R- ). Data are shown as histograms of fluorescence intensity with anti-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 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.2 L  w" ]1 }$ v7 D+ X+ U6 a8 A% }

DISCUSSION
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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 .
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Here, we describe the heterogeneous expression of the cell surface¨Cexpressed protein 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¨C /loThy1.1loSca-1  c-kit  HSC population. When paired with ß1 integrin, 2 forms an adhesion receptor (VLA-2) that binds with high affinity to the extracellular matrix components collagen and laminin. Among mature hematopoietic cells, 2ß1 is expressed by activated T lymphocytes, megakaryocytes, and platelets and plays a nonredundant role the in vitro adhesion of platelets to soluble collagen .
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Among highly enriched Lin¨C /loThy1.1loSca-1 c-kit  mouse HSCs, 2-integrin is expressed by approximately 85%¨C95% of cells. In response to pharmacological mobilization of HSCs, which induces increased migration of HSCs from the BM into the blood . This finding initially led us to hypothesize that downregulation of  2-integrin may be involved in HSC egress from BM and that 2ß1 may be an important homing receptor for transplanted HSCs. Surprisingly, however, the functional analyses described here indicate that, contrary to expectations, expression of 2 integrin divides HSCs into enriched populations of 2hi ST and  2¨C LT reconstituting subsets. Sorted 2hi HSCs exhibit decreased LT engraftment potential, but increased CFU-S12 activity, suggesting that they are less primitive as compared with 2¨C HSCs, which maintain robust multilineage LT engraftment and reduced CFU-S12 activity. The temporally restricted production of mature granulocytes from transplanted 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 2hi subset of HSCs, it is clear from these data that 2hi cells are substantially enriched for rapid reconstituting ST HSCs, whereas 2¨C HSCs are highly enriched for LT reconstituting cells.3 z% a5 a' ^* L! A/ X8 Z5 n7 d
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The anti-2 antibody used in this study, Hm2, has been reported to block the function of 2ß1 for collagen binding and for co-stimulation of mature T lymphocytes  that Hm2 does not inhibit the initial homing of intravenously infused HSCs to the BM, measured 3 hours after transplant. In addition, Hm2 does not block ST hematopoietic engraftment, as demonstrated by the observation that 2hi Lin¨C /loThy1.1loSca-1 c-kit  HSCs, with bound anti-2 mAb, contribute to both lymphoid and myeloid lineages at 2 weeks after transplant (Fig. 3). Thus, differences in the hematopoietic reconstituting capacity of 2¨C and 2hi Lin¨C /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.) @, A' n, _' @: \$ q6 r

Interestingly, among Lin¨C BM cells, highly enriched for hematopoietic stem and progenitor cells, 2-integrin appears to be a relatively specific marker of multilineage repopulating cells with limited self-renewal activity. 2¨C LT HSCs give rise to 2hi ST HSCs after transplantation; however, expression of 2 integrin is subsequently lost or substantially reduced (to ~2%¨C25% 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 ).* O! }8 x/ V! l4 Q. p9 _
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Whether changes in 2-integrin expression lead directly to functional differences in 2hi versus 2¨C HSCs cannot be established from these studies. Although genetic ablation of 2 integrin does cause specific deficiencies in platelet function in vitro and mast cell-mediated inflammatory responses in vivo, 2 is dispensable for platelet production and normal hemostasis, and other hematopoietic lineages in these mice appear to be grossly unaffected , or ST hematopoietic engraftment by 2hi Lin¨C /loThy1.1loSca-1  c-kit  HSCs (Fig. 3). Nonetheless, it remains possible that interactions of 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.9 w/ q, }: u% l, ]6 @; V

SUMMARY
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We show here that among Lin¨C /loThy1.1loSca-1  c-kit  BM cells, the only subset of BM containing cells capable of LT, multilineage hematopoietic reconstitution , expression of the cell surface adhesion receptor 2-integrin effectively distinguishes populations enriched for LT reconstituting (2¨C) or ST reconstituting (2hi) cells. 2¨C Lin¨C /loThy1.1loSca-1  c-kit  HSCs give rise to 2hi Lin¨C /loThy1.1loSca-1 c-kit  BM cells after transplantation; however, 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 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.7 W) u+ O1 X6 k( k+ W1 m

ACKNOWLEDGMENTS9 C5 o" r6 m  ~' }
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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

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.
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