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Stem Cells 2005;23:335-346 www.StemCells.com
© 2005 AlphaMed Press

Rac2-Deficient Hematopoietic Stem Cells Show Defective Interaction with the Hematopoietic Microenvironment and Long-Term Engraftment Failure

Michael Jansena, Feng-Chun Yangb, José A. Cancelasa,c, Jeff R. Baileya, David A. Williamsb

a Division of Experimental Hematology, Cincinnati Children’s Research Foundation, Cincinnati, OH, USA;
b Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA;
c Hoxworth Blood Center, University of Cincinnati Medical Center, Cincinnati, OH, USA

Key Words. Stem Cells • Rho GTPase • RAC • Engraftment • Homing

Correspondence: David A. Williams, M.D., Division of Experimental Hematology, Cincinnati Children’s Hospital Research Foundation, 3333 Burnet Avenue, ML 7013, Cincinnati, OH 45215. Telephone: 513-636-0364; Fax: 513-636-3768; e-mail: David.Williams{at}cchmc.org


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The hematopoietic-specific Rho GTPase, Rac2, regulates a variety of cellular functions including cell shape changes, motility, integrin-dependent adhesion, and apoptosis. In the study reported here, we demonstrate that wild-type (WT) hematopoietic stem cells/progenitors (HSC/P) preferentially engraft in nonablated Rac2–/– bone marrow. In addition, primitive Rac2–/– HSC/P transplanted into lethally irradiated WT recipients showed a significant competitive defect compared with WT cells. These defects appeared to be related to HSC/P-intrinsic defective microenvironment interactions, since Rac2–/– cells showed less adhesion to the femur bone marrow density 1 (FBMD-1) stromal cell line, a lower frequency of cobblestone area–forming cells, and lower performance in long-term marrow cultures in vitro when compared with WT cells. In contrast, primitive Rac2–/– hematopoietic cells exhibited normal progenitor colony formation in semisolid medium in vitro and normal proliferation in the steady state in vivo when compared with WT cells. Taken together, these data suggest that Rac2–/– stem/progenitor cells exhibit abnormal interaction with the hematopoietic microenvironment, which leads to defective long-term engraftment.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hematopoietic stem cells (HSC) are comprised of a pluripotent cell population residing near the endosteum of the bone medullary cavity of adult mammals [1, 2]. These cells are responsible for maintaining multilineage hematopoiesis by generating committed progenitors that ultimately differentiate into mature elements of the blood. HSC are also capable of self-renewal divisions that maintain the stem cell compartment throughout the lifetime of the individual [3]. Interaction with a variety of cells, extracellular matrix components, and growth factors, making up the hematopoietic microenvironment, appears to be crucial for maintenance of the stem cell compartment [4, 5]. These interactions include adhesion to the mesenchymal cells, possibly osteoblasts [6] of the bone marrow stroma that display membrane-associated growth factors [6–14] such as stem cell factor (SCF) and produce extracellular matrix proteins, including collagen, laminins, and fibronectin. In addition, matrix proteins also provide a reservoir of secreted growth factors such as interleukin-3 (IL-3) [15, 16] and chemoattractants, such as stromal cell–derived factor-1 alpha (SDF-1{alpha}), which appears to have a pivotal role in stem cell migration [17, 18].

Adhesion receptors expressed on hematopoietic cells mediate direct contact with mesenchymal cells and matrix proteins, and are therefore, highly relevant to localization of these cells in the hematopoietic microenvironment. These same receptors may also initiate signaling pathways following adhesion [19]. The heterodimeric beta-1 integrins, particularly the {alpha}4ß1 (VLA-4) and {alpha}5ß1 (VLA-5) integrins, and the adhesion molecule CD44 appear to be of critical importance for homing and localization of HSC to the hematopoietic microenvironment [20–27] mediating binding to fibronectin, as well as the vascular cell adhesion molecule (VCAM-1) [28, 29] and proteoglycans. The localization of HSC in the hematopoietic microenvironment is of particular importance in transplantation biology and cellular therapy since after infusion or mobilization of HSC into the bloodstream these cells must translocate from the peripheral blood into the bone marrowspace. The migration of HSC into the appropriate location in the medullary cavity is thought to be a nonrandom process and is termed "homing."

Analogous to the well-described process involved in egress of leukocytes from the bloodstream, homing of HSC presumably requires a complex series of interactions with the vascular endothelium and bone marrow extracellular matrix. Leukocyte egress from the blood is initiated by capture and rolling of cells on the endothelium, then mediated via interaction of selectins on the hematopoietic cells to proteoglycans expressed on endothelial cells. This is followed by firm adhesion of these cells to the blood vessel wall via activated integrins and cytokine/chemokine/chemoattractant-induced transendothelial transmigration [30–32]. For HSC this process is thought to be directed in part by SDF-1{alpha}, which binds to the cellular receptor CXCR4 expressed on hematopoietic cells [33].

The migration of HSC in and out of the hematopoietic microenvironment likely requires significant changes in cell shape, adhesion or de-adhesion to different ligands, and activation of directed cellular migration [34]. In addition, it has been hypothesized that open "space" in the hematopoietic microenvironment, so-called stem cell niches, favor successful engraftment of transplanted cells [35–37]. Appropriate localization of HSC in specific niches of the hematopoietic microenvironment likely requires orchestrated binding to the extracellular matrix proteins and interactions with a variety of soluble and membrane-bound growth factors [38–41].

The process of migration is regulated in many cell types by the small Rho GTPase family [42–45], and downstream effectors (reviewed by Symons [46]). In addition, in some cells, both integrins and growth factor receptors have been shown to transduce signals that converge on Rho GTPases, leading to cross-talk between these surface receptors [19, 47–49]. Such converging signals lead to migration of mast cells on fibronectin after stimulation by stem cell factor (SCF) via activation of Vav, a guanine exchange factor (GEF) for Rac, and subsequent Rac activation [19]. Rho GTPases act as molecular switches by cycling between an active GTP-bound form and an inactive GDP-bound state. Upon activation via specific but multiple different receptors, GTP-bound Rho GTPases have been implicated in a variety of cellular responses that ultimately regulate significant cell shape changes. Actin polymerization leading to cytoskeleton remodeling, integrin clustering, and integrin-mediated adhesion and motility are regulated via Rho GTPases [42, 43, 50–56].

We previously demonstrated that the hematopoietic-specific Rac2 protein, a member of the Rac subfamily of Rho-GTPases [57–60] has unique roles in blood cell development and function, including regulation of migration, phagocytosis, degranulation, and adhesion of a variety of differentiated myeloid cells [52, 61, 62]. More recently, Rac2 has also been implicated in adhesion, migration, and cell shape changes of primitive HSC [51, 63]. Furthermore, the highly homologous Rho GTPase Rac1 has been shown to regulate both overlapping and unique functions in blood cells and HSC. The cellular changes associated with Rac deficiency lead to increased in vivo trafficking of HSC, as measured by elevated numbers of these cells in the peripheral blood of Rac2–/– mice and more dramatically in Rac1–/–; Rac2–/– cells. To date, the effect of Rac2 deficiency on homing and long-term engraftment of HSC has not been examined.

Because adhesion is thought to play a critical part in the localization of HSC in the hematopoietic microenvironment and is defective in Rac2-deficient HSC, we hypothesized that the increased circulating HSC seen in Rac2–/– mice was associated with increased availability of stem cell niches in the medullary hematopoietic microenvironment of these mice. The experiments described here were designed to address this hypothesis by determining if wild-type (WT) HSC preferentially engraft in nonablated Rac2–/– bone marrow. Here we report that HSC from WT mice, defined both phenotypically and by secondary transplantation, display significantly higher multiline age repopulation in nonablated Rac2–/–recipients than in WT recipients. We also observe long-term defects of Rac2–/– HSC in engraftment studies. These data suggest that Rac2 has a significant physiologic role in the localization of HSC to the hematopoietic microenvironment and provides quantitative evidence of the central role of Rac GTPases in the long-term engraftment process.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Mouse strains C57BL/6J (WT), B6.SJL-Ptprca-Pep3b/BoyJ (BoyJ) (both Jackson Laboratories Bar Harbor, ME, http://www.jax.org) and 129/BL6 Rac2–/– (B6.129Rac2<tm1mddw) [52] backcrossed at least 12 generations in C57BL/6 (N12) were kept in specific pathogen-free conditions. Male and female 6- to 12-week-old mice were used as either bone marrow donors or transplant recipients. All animal procedures used approved protocols in accordance with the regulations of the Laboratory Animal Research Committee, Indiana University School of Medicine, and the Animal Use Committee, Cincinnati Children’s Hospital Medical Center.

Cell Cultures
The murine stroma cell line femur bone marrow density 1 (FBMD-1) [64], (a gift of Dr. J. Cancelas-Perez), was used in adhesion assays and cobblestone area–forming cell (CAFC) assays. Cells were maintained in Myelo Cult M5300 (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) with 10 µM L-hydrocortisone (Sigma Chemical Corp., St. Louis, MO, http://www.sigma-aldrich.com) and 100 IU/ml penicillin, 0.1 mg/ml streptomycin (P/S) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) on tissue culture–treated flasks (Corning, Acton, MA, http://www.corning.com) at 33°C, 10% CO2.

Homing Studies
Low-density mononuclear bone marrow (LDBM) cells were prepared from whole bone marrow of WT C57BL/6J and Rac2–/– mice. Bone marrow was harvested by flushing femorae, tibiae, and iliac crests with Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen), 10% fetal calf serum (FCS), 1% P/S followed by isolation on a density gradient (Histopaque 1083; Sigma) by standard methods. The resulting LDBM cells were stained with 5 µM 5- (and 6-)-carboxyfluorescein succinimidyl ester (CFSE) mixed isomers (Molecular Probes, Eugene, OR, http://www.probes.com) for 10 minutes according to the manufacturer’s recommendations. Stained cells were resuspended in IMDM, and 200 µl cell suspension containing 2–4x 106 cells were infused by intravenous tail vein injection into 6- to 10-week-old female WT recipient mice that were lethally irradiated (11 Gy, split dose with 3 hours minimum between doses) using a 137Cs (Cesium) irradiator (JL Shepherd & Associates, San Fernando, CA, www.jlshepherd.com) 24 hours prior to cell infusion. Mice were sacrificed 24 hours after injection. Bone marrow was harvested, stained with anti-Sca-1 phycoerythrin (PE) and anti-c-Kit antigen-presenting cells (APCs) monoclonal antibodies (all antibodies from BD Bioscience, Franklin Lakes, NJ, http://www.bdbioscience.com) and analyzed by flow cytometry using a FACS-calibur (BD Bioscience) for the presence of CFSE+ cells.

Engraftment Studies
Engraftment experiments in nonablated recipients were carried out as described by Quesenberry et al [65]. In brief, LDBM cells were prepared from hind limbs of B6. SJL-Ptprca-Pep3b/BoyJ (Boy/J) mice, as described above, and 2.5 to 2.7 x 107 were transplanted into either Rac2–/– or WT recipients. Peripheral blood (PB) from recipient animals was collected monthly (for 4 months) by tail vein bleeding. Blood counts were determined using the F-820 instrument (Sysmex, Long Grove, IL, www.sysmex.com). Leukocytes were stained, after red blood cell lysis, with anti-mCD45.1 fluorescein isothiocyanate (FITC) and either anti-mCD11b/Gr-1 PE, anti-mCD45R/B220, or anti-mCD3{varepsilon} (all antibodies by Pharmingen, San Diego, CA, www.bdbiosciences.com/pharmingen) for 30 minutes at 4°C for flow cytometric analysis. Animals were sacrificed after 4 months; bone marrow (BM) from hind limbs and cells from spleen (S) and lymph nodes (LN) were harvested, and single-cell suspensions were generated, then analyzed by flow cytometry, as described above.

In one experiment splenocytes were also labeled with anti-mCD21 FITC, anti-mCD23 PE, and anti-mCD45.1 biotin/streptavidin APCs to analyze donor-derived marginal zone B lymphocytes, as described by Croker et al [43].

Alternatively, WT or Rac2–/– LDBM cells were also transplanted into nonablated Boy/J recipient animals. Analysis of PB and organs was carried out in identical fashion as above, except that anti-mCD45.2 FITC mAb was used to determine donor cell chimerism.

Secondary Transplantation
Primary recipient animals from one experiment were sacrificed 4 months after transplantation, and 2 x 106 whole BM cells from five animals with the highest donor cell chimerism from both experimental and control mice were transplanted into lethally irradiated secondary female WT recipients. Animals were bled according to the schedule described in the engraftment studies above to determine blood counts and donor cell chimerism of granulocytes, B cells, and T cells by flow cytometry. As described above, donor cell presence was also determined for BM, S, and LN after the final blood sampling.

Competitive Repopulation
Competitive repopulation experiments using congenic mice differing only at the Ly5 leukocyte surface antigen were performed similar to the procedure previously described by Harrison et al [67]. Lethally irradiated 8- to 10-week-old female WT recipient mice were transplanted with 1 x106 WT or Rac2–/– LDBM, or mixtures of both at different ratios (3:1, 1:1, 1:3). Beginning 4 weeks after transplantation, animals were bled monthly for 4 months to determine blood counts and donor cell chimerism, as described above.

As controls, mice were irradiated but not transplanted. Bone marrow from these irradiated nontransplanted animals was plated in methylcellulose (Metho Cult 3434; Stem Cell Technologies) to confirm the absence of surviving progenitor cells.

Limiting Dilution CAFC Assay
The frequency of cobblestone area–forming cells (CAFCs) was determined, as previously described [68]. The murine bone marrow stroma–derived cell line FBMD-1 [64] wasused to generate a confluent stromal layer in 96-well flat-bottom plates. Cells from logarithmic phase cultures were plated at 1 x 103 cells per well in 100-µl FBMD-1 medium (see Cell Cultures) into individual wells and grown to confluency. Twelve dilutions of LDBM cells or purified populations derived from LDBM cells were plated into 15 wells for each concentration on confluent FBMD-1. Each well was examined weekly (for 5 weeks) for the presence or absence of cobblestones. The number of CAFCs per 1 x 105 plated cells was calculated from the number of cobblestone-negative wells for each dilution using Poisson statistics, as described [68].

In Vitro BrdU Labeling of Bone Marrow Cultures
FBMD-1 cells were grown to confluency in 24-well flat-bottom plates in MyeloCult 5300. Fluorescence-activated cell sorted (FACS) lineage–, c-Kit+ (LK) cells were plated at a concentration of 1 x 105 cells per well on top of the confluent FBMD-1 cell layer. Cells were cocultured for 20 or 44 hours. After this incubation period, BrdU was added to each well to a final concentration of 20 µM. Cells were cultured for another 4 hours in the presence of BrdU. The plates were then agitated and rinsed twice with phosphate-buffered solution (PBS) to remove nonadherent LK cells from the wells. The remaining cells were trypsinized and washed with culture medium to inactivate trypsin. The samples were then stained with an APC-conjugated monoclonal {alpha}–mCD45 antibody (Pharmingen) for 30 minutes on ice. Following permeabilization with the Cyto Fix/Perm Kit (Pharmingen), samples were split in halves and stained with either an FITC-conjugated monoclonal {alpha}–BrdU antibody or a matching isotype control. Prior to flow cytometric analysis, cells were stained with 7-amino-actinomycin (7-ADD; Molecular Probes, Invitrogen, Eugene, OR, http://www.probes.com) at 2 µg/ml for a minimum of 10 minutes. The percentages of LK cells (CD45+) in the different stages of the cell cycle were determined using flow cytometry (FACS calibur; BD Biosciences).

HPP-CFC Assay
High proliferative potential colony-forming cell (HPP-CFC) assay was performed using lineage–, Sca-1+, c-Kit+ (LSK) cells in double-layer agar cultures, as described previously [69]. Recombinant hematopoietic growth factors murine granulocyte/macrophage-colony-stimulating factor (mGM-CSF) (20 ng/mL), IL-3 (10 ng/mL), IL-1{alpha} (4,000 U/mL) (all Pepro Tech, Rocky Hill, NJ, http://www.peprotech.com), and recombinant rat SCF (100 ng/mL) (Amgen, Thousand Oaks, CA, http://www.amgen.com) were added to 300 cells in 10 x 35 mm gridded tissue culture dishes. Triplicate cultures of fractionated cells were plated with the multiple growth factor combination and incubated in a 5% O2, 10% CO2, and 85% N2 humidified environment. On day 14 of culture, colonies were scored for HPP-CFCs, defined as colonies larger than 5 mm in diameter.

Adhesion Assay
In a 24-well plate, FMBD-1 cells were grown to confluency in MyeloCult M5300. After 10 minutes of CFSE staining (5 µM) at 37°C, 1 x 105 LK cells from WT or Rac2–/– bone marrow were added to FBMD-1–containing wells in 500 µ1 IMDM, 10% FCS, 1% P/S with 100 ng/ml SCF. Cells were incubated at 37°C in 5% CO2. After 1 hour the supernatant and nonadherent cells were removed, and adherent cells were washed once with PBS. The wash solution was added to the supernatant cells. Adherent cell fractions were then harvested using 0.25% trypsin (Invitrogen) solution. Trypsin was inactivated, and cells were washed once in PBS + 5% FCS. Adherent and suspension cell fractions were analyzed by flow cytometry, and total cell number per sample was determined by aspirating the complete sample volume (~300 µ1) and recording the number of events counted for live cells (according to forward versus side scatter plots).

Statistical Analysis
For statistical comparison of results from different groups, we used the Student’s t-test unless otherwise stated.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
WT HSC Preferentially Engraft in Nonablated Rac2–/– Mice
We previously demonstrated an increase in the circulation of hematopoietic stem cells/progenitors HSC/P (day 12 colony-forming unit, spleen) in Rac2–/– mice at baseline and after stimulation with granulocyte colony-stimulating factor (G-CSF) [51]. We hypothesized that increased circulating HSC/P would be associated with increased frequency of empty stem cell niches in the medullary cavity and that defective adhesion would be associated with a lower capacity of Rac2–/– hematopoietic stem cells (HSC) to provide long-term engraftment. To test this hypothesis, WT (CD45.1+) low-density bone marrow (LDBM) cells were transplanted into nonablated WT or Rac2–/– (both CD45.2+) recipients, and engraftment was followed in the peripheral blood and hematopoietic organs over 4 months. In each of three independent experiments, the number of peripheral blood cells derived from WT donor HSC/P was significantly higher in nonablated Rac2–/– recipient mice (KO), than in WT recipient mice (Fig. 1AGo). These differences occurred despite similar total bone marrow cellularity of naive WT and Rac2–/– mice (data not shown). WT donor engraftment in Rac2–/– recipients remained significantly higher at all time points analyzed, reaching a maximum level of 8- to 10-fold higher in Rac2–/– recipients than in WT recipients at 4 months. These data suggest a more pronounced defect in the primitive stem cell compartment of these mice compared with the progenitor compartment, which gives rise to blood cells early and transiently after engraftment. WT donor cells contributed to multilineage reconstitution in the blood of recipient mice (Fig. 1CGo), suggesting a defect in multipotential HSC/P in Rac2–/– mice. Engraftment of WT cells was also significantly higher in all other hematopoietic organs examined at 4 months in Rac2–/– mice compared with WT mice, including bone marrow, spleen, and lymph nodes (Fig. 1BGo).



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  		Figure 1. Engraftment and lineage contribution of donor-derived LDBM cells in nonablated recipient mice. In Figure 1A to 1C, data for transplantation of WT (CD45.1+) donor cells into nonablated WT or Rac2–/– (KO) recipient mice are shown, while Figure 1D and 1E represent data from transplantation of WT or Rac2–/– cells (both CD45.2+) into nonablated WT (CD45.1+) recipient animals. (A): The data show percentage of WT donor-derived (CD45.1+) cells in the peripheral blood in the first 4 months post-transplant for a representative experiment (n = 8–10 animals per group). Lineage analysis includes granulocytes (Gr-1+; white bars), B-lymphocytes (CD45R/B220+; black bars), and T cells (CD3+;striped bars). Cells not characterized by the antibodies used are indicated as stippled bars (*p < .05). Parts A, B, D, and E of this figure share the legend for cell type labeling. (B): Combined data from three independent experiments for Rac2–/– (KO) (n = 30) and WT (n = 24) recipients, showing peripheral blood, bone marrow, spleen, and lymph nodes at 4 months post-transplant (*p < .001). (C): Peripheral blood flow cytometry analysis of a highly engrafted Rac2–/– animal (engraftment IV, animal no. 20) transplanted with WT (CD45.1+) donor cells at 4 months post-transplant. Demonstrated is the contribution of donor-derived WT cells to different lineages in the peripheral blood. The numbers in the dot blot quadrants correspond to the percentage of this population within the population of viable peripheral blood leukocytes. (D): Flow cytometric analysis of the percentage of WT or Rac2–/– (KO) donor-derived (CD45.2+) cells in the peripheral blood of WT recipients (n = 10 animals per group) in the first 4 months post-transplant (*p < .05). (E): Flow cytometry analysis of donor-derived chimerism in bone marrow, spleen, and lymph nodes of the recipient animals in (D) at 4 months post-transplant (*p < .05). Abbreviations: KO, Knockout; LDBM, Low-density mononuclear bone marrow; WT, wild-type.

 
While the contribution of WT donor cells was multilineage in nonablated Rac2–/– recipients, WT chimerism was highest in the spleen and in lymph nodes. These data are consistent with the previously described defect in recirculating B-cell populations in Rac2–/– mice, which includes a pronounced deficiency of splenic marginal zone B cells [43]. We further analyzed the presence of WT donor B cells in the spleen of reconstituted, nonablated recipient mice. A significantly higher percentage of WT donor-derived marginal zone B cells (CD21high, CD23low) were present in the spleens of Rac2–/– recipients in comparison with WT recipients (24.6% ± 6.5% vs. 16.7% ± 7.1%, p < .05). Thus, these data demonstrate both a defect in the primitive stem/progenitor cell compartment of Rac2–/– mice and a defect in specific B-cell populations in lymphoid tissues.

The evidence of a defect in Rac2–/– mice at the level of the stem cell is further supported by experiments that study engraftment of Rac2–/– hematopoietic cells (CD45.2+) in nonablated congenic WT-recipient mice. In these experiments, Rac2–/– cells show significantly lower engraftment, than WT cells, as measured by contribution of donor-derived cells to the peripheral blood (Fig. 1DGo) over a 4-month period. This defect was not the result of a difference of HSC/P content in the transplant, as lineage–, c-Kit+ (LK) and lineage–, Sca-1+, c-Kit+ (LSK) frequencies in the harvested bone marrow of untreated WT and Rac2–/– animals do not show statistically significant differences (data not shown). The ~two-fold reduction in engraftment is observed at both early and late time points post-transplant. The donor cell contribution from both genotypes is multilineage, providing evidence that the engraftment is the result of donor-derived HSC. Furthermore, the lower engraftment capability of Rac2–/– HSC is not only reflected in the peripheral blood but is also seen in all other hematopoietic tissues examined (Fig. 1EGo).

As demonstrated above, donor-derived WT cells engrafted in nonablated Rac2–/– recipients are capable of producing multilineage mature progeny over a period of 4 months after transplantation. To confirm a defect in the stem cell compartment in these mice, we transplanted whole bone marrow cells from nonablated primary WT and Rac2–/– recipients into lethally irradiated WT secondary recipient animals. As seen in Figure 2AGo, the donor-derived WT cells that preferentially engrafted in Rac2–/– (KO) mice reconstituted multilineage hematopoiesis (consisting of granulocytes, B cells, and T lymphocytes) for up to 4 months in the secondary recipients. In addition, donor-derived WT cells were present in the bone marrow, lymph nodes, and spleens of these secondary recipients when examined at 4 months after transplant (Fig. 2BGo). As expected, due to the low level of donor-derived engraftment in the BM of nonablated primary WT recipients, the mice transplanted with these bone marrow cells showed an overall low level of donor-derived WT cells in all hematopoietic tissues tested, never exceeding 0.5%. In contrast, WT donor-derived cells from primary Rac2–/– recipients averaged ~3% in the peripheral blood, equaling an approximately sixfold higher engraftment. The level of WT donor-derived cells from primary Rac2–/– recipients in different tissues of secondary transplanted mice 4 months after transplant resembled the distribution in primary animals, with the highest chimerism seen in the spleen followed by lymph nodes, peripheral blood, and bone marrow.



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  		Figure 2. Secondary transplantation of WT or Rac2–/– (KO); nonablated primary recipients engrafted with WT LDBM cells. (A): The data show the percentage of WT cells derived from the primary recipients (CD45.1+) in the peripheral blood of secondary recipients over a period of 4 months postsecondary transplantation. Given are the mean overall percentage (height of the bar) of WT donor cells and the relative percentage of lineage marker positive cells (shaded areas). Lineage analysis includes granulocytes (Gr-1+; white), B-lymphocytes (CD45R/B220+; black), and T cells (CD3+; striped). Cells not characterized by the antibodies used are indicated as stippled bars (*p < .05). (B): Data from bone marrow, lymph nodes, and spleen of the same experiment (n = 15 animals per group) (*p < .05). For a legend of the different cell types represented, see part A of this figure. Abbreviations: KO, Knockout; LDBM, low-density mononuclear bone marrow; WT, wild-type.

 
Defective Long-Term Reconstitution in Competitive Repopulation Assays Despite Normal Homing
These data demonstrate that Rac2–/– stem cells have an engraftment defect compared with WT stem cells. To measure this defect precisely, we performed competitive repopulation assays. Mixtures of congenic WT (CD45.1+) and Rac2–/– (CD45.2+) low-density bone marrow cells were infused at ratios of 3:1, 1:1, and 1:3 into lethally irradiated WT recipient animals. As shown in Figure 3AGo, at all ratios of infused cells, WT stem cells had a significant repopulation advantage over Rac2–/– stem cells, demonstrated by an increased ratio of WT:Rac2–/– cells in the peripheral blood over 4 months after transplant. At 4 months after transplant, the contribution of WT cells was increased ~threefold (range: 2.58 to 3.06) over Rac2–/– cells for each mixture of starting cells (to 9.17:1, 2.8:1, and 0.85:1 from 3:1, 1:1, and 1:3, respectively, p < .05).



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  		Figure 3. Competitive repopulation capacity of WT and Rac2–/– KO low-density LDBM cells. (A): Time course of chimerism in the peripheral blood after transplantation of WT (CD45.1+) and Rac2–/– (CD45.2+) cells. Data are plotted in comparison with pretransplant mixtures of each genotype at different ratios. Shown are the mean and standard deviation error bars. The asterisk indicates statistically significant differences of pre-transplant ratios versus the ratios at 4 months post-transplant with p < .05. The initial contribution of WT versus Rac2–/– cells to the pretransplant mixture includes 75% WT /25% Rac2–/– (3:1; squares), 50% WT plus 50% Rac2–/– (1:1; circles), and 25% WT plus 75% Rac2–/– (1:3; triangles). Also shown are the contribution and lineage composition of donor WT and Rac2–/– cells to the (B) peripheral blood, (C) bone marrow, and (D) spleen at 4 months post-transplant. The stacked bars show the mean values of lineage contribution for each donor cell type (n = 8 for each group). Lineage analysis includes granulocytes (Gr-1+; white), B lymphocytes (CD45R/B220+; black), and T cells (CD3+; striped). Cells not characterized by the antibodies used are indicated as stippled bars. The categories on the X-axis indicate the initial portion (percentage) of WT and Rac2–/– cells (see A) in the pre-transplant mixture. The figure shows data from one of two independent experiments with similar results. Abbreviations: KO, Knockout; LDBM, Low-density mononuclear bone marrow; WT, wild-type.

 
The increased ratio of WT: Rac2–/– cells was seen in all lineages, consistent with the preferential engraftment of pluripotent WT cells (Fig. 3BGo). The number of WT cells was also higher than Rac2–/– (KO) cells in the bone marrow of the recipient mice at 4 months post-transplantation (Fig. 3CGo) where the ratio of WT vs. Rac2–/– cells in the three groups increased to 6.18:1, 2:1, and 0.4:1, representing an average engraftment advantage of WT cells of 1.8 (range: 1.33–2.06). At 4 months post-transplantation, WT cells represented up to 65% of bone marrow cells in mice infused with a 1:1 ratio of WT: Rac2–/– cells (n = 5, p < .05). Again the highest engraftment of WT cells was in the spleen, where WT cells demonstrated a 4.92- to 6.08-fold (p < .01) increase in repopulation in comparison with Rac2-deficient cells (Fig. 3DGo). These data demonstrate that Rac2–/– stem cells have a modest, but significant and highly reproducible, defect in long-term reconstitution compared with WT cells.

Engraftment of HSC in vivo is a multistep process that depends initially on appropriate homing of infused cells to the bone marrow medullary cavity and adhesion in the hematopoietic microenvironment, specifically in the stem cell niche. Since Rac2–/– cells showed defective adhesion to fibronectin (FN) in vitro, [51, 63] and Rac2–/– neutrophils demonstrate defects in endothelial capture and rolling [52], we compared the homing capacity of WT and Rac2–/– cells in vivo. Lethally irradiated recipients were transplanted with WT or Rac2–/– LDBM cells stained with the carboxyfluorescein succinimidyl ester (CFSE) dye. Surprisingly, as seen in Figure 4Go, there was no significant difference in the percentage of labeled WT versus Rac2–/– (KO) donor cells in the bone marrow 24 hours post-transplantation. In addition, no differences were detected specifically in the homing of the more primitive Sca-1+ (Fig. 4Go) or c-Kit+ (data not shown) cells to the bone marrow. These data suggest that Rac2-deficient cells home normally to the bone marrow cavity compared with WT cells, as measured by the currently used "homing" assay.



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  		Figure 4. Homing of WT and Rac2–/– (KO) low-density LDBM cells to the bone marrow cavity. Recovery of diacetate CFSE+ (light dotted bars) and CFSE+, Sca-1+ (dark dotted bars) cells from the hind limbs of lethally irradiated recipients 24 hours after infusion of 4 x 106 CFSE-labeled WT or Rac2–/–LDBM cells. The data show the means ± SD of one representative experiment (n = 8 per group) out of four independent experiments with similar results. Abbreviations: CFSE, carboxyfluorescein succinmidyl ester; KO, Knockout; LDBM, Low-density mononuclear bone marrow; WT, wild-type.

 
Differential Growth Abnormalities in Stromal-Independent Cultures Associated with Defective Adhesion to the Hematopoietic Microenvironment
Since Rac2–/– cells appeared to home normally to the medullary cavity of the bone marrow, we next determined if increased engraftment of WT cells in nonablated Rac2–/– mice and defects in competitive engraftment of Rac2–/– bone marrow cells was due to reduced intrinsic proliferation of primitive Rac2–/– hematopoietic cells. The growth of high proliferative potential colony-forming cells (HPP-CFCs), a measure of the number and proliferation of primitive hematopoietic stem and progenitor cells, from purified LSK bone marrow cells of Rac2–/– and WT mice was examined in response to SCF, GM-CSF, IL-1{alpha}, and IL-3 in double-layer soft agar assays. As seen in Figure 5AGo, the growth of HPP-CFCs was equivalent for Rac2–/– (KO) and WT bone marrow cells.



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  		Figure 5. Growth of primitive WT and Rac2–/– (KO) hematopoietic cells in vitro. (A): Growth of HPP (white bars) and LPP (black bars) CFCs from WT and Rac2–/– lineage–, Sca-1+, c-Kit+ (LSK) cells, in response to SCF, GM-CSF, IL-3, and IL-1{alpha}, is shown. The bars represent the frequency of CFC/300 plated in a double-layer agar assay. Data represent mean ± SD (n = 3). (B) and (C): Frequency of CAFCs in lineage–, c-Kit+ (LK) and LDBM cells from WT and Rac2–/– mice. Figure 5C shares the same y-axis label as Figure 5B. The frequency of CAFCs per 1 x 105 LK cells of WT (black bars) and Rac2–/– (white bars) from FACS-purified (B) LK cells or (C) LDBM was measured in a limiting dilution assay. Cells were plated on confluent monolayers of the FBMD-1 cell line in 96-well cluster plates. Displayed is the mean frequency (± standard error of the mean) of CAFCs at culture days 28, 35, and 42, calculated using Poisson statistics. Data are shown on a logarithmic scale. The figure shows data from one of two independent experiments with similar results (*p < .01 for paired t-test). Abbreviations: CAFCs, cobblestone area-forming cells; CFCs, colony-forming cells; HPP, high proliferative potential; IL-3, interleukin-3; KO, knockout; LDBM, low-density mononuclear bone marrow; LPP, low proliferative potential; SCF, stem cell factor; WT, wild-type.

 
In contrast, the frequency of HSC/P measured in the stromal-dependent, limiting dilution CAFC assays, which requires coordinated adhesion and migration of HSC/P under a layer of supporting stromal cells, was consistently abnormal in Rac2–/– mice. As seen in Figure 5BGo, Rac2–/– LK cells (white bars) showed a 2.7- to 3.1-fold decrease in the frequency of d28, d35, and d42 CAFCs compared with WT cells (black bars). A similar reduction in the frequency of CAFCs was present in Rac2–/– LDBM cells (Fig. 5CGo). This defect is intrinsic to the Rac2–/– HSC as WT stromal cells do not express Rac2, as determined by reverse transcription polymerase chain reaction (data not shown).

However, when analyzing cell cycle status of LK cells that successfully adhered to FBMD-1 cells after 24 and 48 hours of coculture, there was no significant difference between WT and Rac2–/– cells in the percentage of cells in different stages of the cell cycle (Table 1Go). These data, together with the growth of cells in HPP-CFC assays, suggests that there is no defect in the intrinsic proliferative capacity in Rac2–/– HSC/P but implicate defective stromal interactions of Rac2–/– HSC/P. Furthermore, the ability of Rac2–/– bone marrow to establish robust long-term marrow (Dexter) cultures was significantly reduced, even in the presence of WT stromal cells. In contrast, stromal cells from Rac2–/– animals were capable of supporting long-term marrow cultures initiated with WT hematopoietic cells (data not shown).


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  		Table 1. Cell cycle analysis of bone marrow cells (percentage) cultured on confluent femur bone marrow density 1 (FBMD-1)
 
Taken together, the normal growth of Rac2–/– HPP-CFCs compared with WT bone marrow but reduced growth of immature hematopoietic cells in CAFC assays and long-term cultures suggests defective interactions with the hematopoietic microenvironment. To directly measure this interaction, we next determined the adhesion of Rac2–/–hematopoietic cells to bone marrow–derived stromal cells in vitro. Rac2–/– LDBM cells and more purified LK cells showed 2.1- and 8.7-fold reduction (LDBM: Rac2–/– 9.3% ± 2.5% vs. WT 19.7% ± 5.6%; LK: Rac2–/– 7.9% ± 1.8% vs. WT 68.9% ± 5.7%) in adhesion to stromal cells, respectively, compared with WT cells (p < .005).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The deficiency of Rac2 in hematopoietic cells has been demonstrated to result in lower adhesion to the extracellular matrix protein fibronectin, greater nondirected motility of HSC, and greater stem cell trafficking of HSC/P into the peripheral blood [51]. The data presented here strongly suggest that these changes in Rac2-deficient hematopoietic cells lead to an increase in empty stem cell niches in the bone marrow cavity that can be occupied in nonablated Rac2–/– mice by transplanted WT cells. The ability of engrafted WT cells from primary Rac2–/– recipients to produce multilineage progeny in secondary recipients strongly suggests that this empty niche can be occupied by primitive stem cell populations.

While reduced adhesion related to defective ß1 integrin function is likely the primary cause of this abnormality in Rac2–/– HSC/P, another possible mechanism might be deregulated transcriptional activation of matrix metalloproteases (MMPs) in Rac2-deficient cells, possibly bone marrow neutrophils, as described in bone marrow–derived mast cells [70]. Recent data suggest cleavage of cell surface proteins—including VCAM-1, an {alpha}4ß1 receptor ligand—by proteases has an important role in stem cell mobilization [71]. Thus, altered neutrophil function could contribute to enhanced trafficking of HSC in Rac2–/– mice, even though {alpha} 4 ß 1expression, as measured by mean fluorescence intensity, is equivalent on the surface of Rac2–/– LSK cells compared with WT cells (data not shown).

Data presented here indicate that HSC/P from Rac2–/–micealso display defective interactions with the hematopoietic microenvironment, including reduced adhesion of primitive hematopoietic cells to stromal cells and reduced frequency of CAFCs, which depend on the adhesive and migration behavior of HSC in collaboration with stromal cells, as well as reduced growth in long-term (Dexter) marrow cultures, aphysiological relevant primary culture system. Reduced signaling of adhesion receptors via Rac GTPases may lead to defective assembly of focal adhesion complexes in HSC, as has been described for Bac1 macrophages injected with dominant negative Rac1 or Cdc42 [72], or diminished integrin clustering at the site of contact of the HSC to the hematopoietic microenvironment [73]. Both of these effects could explain the observed defects in CAFC formation and elevated engraftment of WT HSC in nonablated Rac2–/– mice.

However, once established in the appropriate location, Rac2–/– HSC are capable of functioning similar to WT HSC with respect to proliferation and self-renewal, as demonstrated by the data from BrdU labeling of FBMD-1 and LK cell coculture (Table 1Go) and the long-term contribution to hematopoiesis by those Rac2–/– cells that did engraft in competitive repopulation experiments. Previous studies have supported the view that inhibition of {alpha}4ß1 is related to the migration of immature hematopoietic cells out of the bone marrow into the periphery [74–76]. These observations are consistent with the published data on increased mobilization of Rac2–/– progenitors in response to G-CSF, stressing the presence of perturbed inside-out signaling in Rac2–/–HSC.

Homing of transplanted Rac2–/– bone marrow cells to the medullary cavity was similar to that of WT cells. Homing requires the rolling of the hematopoietic cells along the endothelial wall, followed by tethering and firm adhesion of the bone marrow cells to the endothelial cells. The lack of differences in homing of Rac2–/– cells, which have previously been shown to be defective in some of these processes in vitro, suggests that the assay performed in irradiated mice may not measure these functions in a sensitive manner. Other possible explanations for this observation might also be compensation for Rac2-deficiency by Rac1 in vivo in combination with compensatory overexpression of Rac1 and Cdc42, which has been demonstrated in Rac2–/– bone marrow–derived cultured mast cells [51]. Furthermore, the reduced signaling through {alpha}4ß1 during homing may not be sufficient to abolish the homing function of the hematopoietic cells, as other pathways involving selectins, ß2-integrins, and LFA-1 contribute to HSC localization to the marrow. Combined inhibition of several adhesion receptors that lead to synergistic reduction in homing has previously been observed [29, 77]. In either case, we hypothesize that once in the medullary space, migration of HSC to the endosteum, as well as interaction with matrix proteins and mesenchymal cells in this location, is defective in Rac2–/– cells. This hypothesis is supported by lower CAFC frequencies and lower performance in long-term marrow cultures.

The presence of a defect in Rac2–/– HSC interaction with the hematopoietic microenvironment is also supported by competitive repopulation experiments. Rac2–/– bone marrow cells transplanted into lethally irradiated WT recipients were capable of multilineage reconstitution, but they demonstrated a significant competitive disadvantage over WT HSC. Rac2–/– HSC showed less contribution of mature progeny in the bone marrow, peripheral blood, spleen, and lymph nodes after reconstitution. Since in vitro proliferation in semisolid medium of immature HPP-CFCs and the more committed LPP-CFC population was unchanged in Rac2–/–hematopoietic cells compared with WT cells, these data also suggest a crucial defect in Rac2–/– HSC interaction with the hematopoietic microenvironment in vivo, which affects a long-term reconstituting cell, probably involving the function of {alpha}4ß1 and {alpha}5ß1 in HSC localization to the endosteum. The occurrence of open niches is clearly not restricted to the bone marrow. As has been shown by Croker et al. [43], Rac2–/– animals exhibit fewer spleen marginal zone B cells, bone marrow recirculating B cells, and B1a peritoneal B cells. In the studies reported here, transplanted WT stem cells that successfully engrafted in the bone marrow of nonablated Rac2–/– mice and differentiated into lymphocytes were able to successfully reconstitute the splenic marginal zone.

Thus, we have demonstrated that the hematopoietic-specific Rho GTPase, Rac2, mediates interactions of HSC to the hematopoietic microenvironment in vitro and that Rac2 deficiency is associated with preferential engraftment of WT HSC into nonablated Rac2–/– mice in vivo. These data, together with previous observations showing increased mobilization of HSC in Rac2–/– and Rac1–/–; Rac2–/– mice [51, 70], suggest that Rac proteins are key regulators of the engraftment and mobilization function of HSC.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Jamie Siefring, Aparna Jasti, and Victoria Summey-Harner for technical support. We are also grateful to the members of the laboratory for helpful discussion and to Keisha Steward and Eva Meunier for administrative assistance. This work was supported by National Institutes of Health grant


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received August 31, 2004; accepted for publication November 30, 2004.



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