c-Kit-Mediated Functional Positioning of Stem Cells to Their Niches Is Essential for Maintenance and Regeneration of Adult Hematopoiesis

The mechanism by which hematopoietic stem and progenitor cells (HSPCs) through interaction with their niches maintain and reconstitute adult hematopoietic cells is unknown. To functionally and genetically track localization of HSPCs with their niches, we employed novel mutant loxPs, lox66 and lox71 and Cre-recombinase technology to conditionally delete c-Kit in adult mice, while simultaneously enabling GFP expression in the c-Kit-deficient cells. Conditional deletion of c-Kit resulted in hematopoietic failure and splenic atrophy both at steady state and after marrow ablation leading to the demise of the treated adult mice. Within the marrow, the c-Kit-expressing GFP+ cells were positioned to Kit ligand (KL)-expressing niche cells. This c-Kit-mediated cellular adhesion was essential for long-term maintenance and expansion of HSPCs. These results lay the foundation for delivering KL within specific niches to maintain and restore hematopoiesis.


Introduction
The molecular pathways involved in HSPC-niche cell interactions that maintain hematopoiesis during adulthood and promotes reconstitution after bone marrow (BM) suppression are not fully defined. Certain stem cell active factors are important for embryonic hematopoiesis. However, conditional deletions of these factors in adult mice do not manifest any major impairment in the HSPC maintenance, although they may serve essential functions for hematopoietic recovery after marrow suppression [1,2,3].
KL is expressed by BM niches raising the possibility that c-Kit/ KL signaling might play a critical role in the modulation of steady state hematopoiesis [4]. In this regard, naturally-occurring W/c-Kit mutant mice have served as a great model for study of hematopoietic cell development [5]. Indeed, the encoding protein, c-Kit receptor tyrosine kinase (RTK) is employed in combination with other cell surface molecules, such as lineage 2 (Lin 2 ) and Sca-1 + as Lin 2 Sca1 + c-Kit + (LSK) cells, to identify enriched-population of hematopoietic stem cells [6,7]. Nonetheless, due to the late embryonic and early neonatal lethality by the germline c-Kit deficiency [5], it remains to be determined whether c-Kit is essential for the maintenance and function of HSPCs in adult hematopoiesis. To address this question by genetic approach, we aimed to generate c-Kit conditional knock out (KO) mice to interrogate the role of c-Kit in hematopoietic function in adult mice. Moreover, to unravel the mechanism by which c-Kit regulates interaction of HSPCs with BM microenvironments, we develop a strategy to locate the position of the c-Kit-expressing HSPCs within the BM after conditional c-Kit deletion. To achieve these two goals, we employed mutant loxPs, lox66 and lox71 [8,9] to create a c-Kit conditional KO GFP-reporter mouse, in which deletion of c-Kit in cells results in the expression of GFP driven by the c-Kit native promoter. This approach allowed us to track the location of the c-Kit-deficient HSPCs within various BM niches and assess their fate at steady state conditions and during hematopoietic recovery from BM suppression.
Here, we demonstrate that c-Kit is required for hematopoiesis in the BM and spleen and the in vivo maintenance and regeneration of HSPCs in adult mice. Moreover, we identify that c-Kit is essential for mediating the interactions of HSPCs with niche cells in vitro and functional positioning of the regenerating HSPCs with KL + VEGFR3 + marrow sinusoidal endothelial cells (SECs) in vivo.

Generation of c-Kit conditional KO GFP-reporter mice
We generated a targeting cassette containing genes, a splice acceptor (SA) site followed by IRES-GFP-polyadenylations that were flanked by lox66 and lox71 in head-to-head orientation (Fig. 1A). The cassette was inserted in anti-sense orientation into the intron 8 of the c-Kit up-stream from the exon 10, which encodes the transmembrane domain of c-Kit RTK. The targeted c-Kit lox66-71 allele will transcribe a full-length c-Kit transcript, since the inserted genes remain silenced due to the anti-sense orientation relative to the c-Kit gene. Upon activation of Cre recombinase, the inserted genes will be flipped, but not excised, by recombination of lox66 and lox71 [8,9,10]. Therefore, activation of the c-Kit promoter leads to a splicing event with the inserted SA initiating the IRES-dependent expression of GFP, while the polyadenylation signals will silence the downstream genes, which encode the transmembrane and kinase domains of the c-Kit RTK resulting in the loss of c-Kit cell surface expression.
To generate c-Kit conditional KO GFP-reporter mice, the mutant W allele, which codes for a defective molecule lacking the transmembrane domain of c-Kit thereby resulting in no cell surface expression of the molecule [11], was introduced by breeding W heterozygotes (c-Kit +/W mice) to the germlinetransmitted c-Kit +/lox66-71 mice ( Fig. 1B-C). The introduction of Green dots in the Sca-1 and c-Kit profiles gated on Lin 2 cells represent Lin 2 Sca-1 + c-Kit::GFP + cells. Right panels show that after tamoxifen injections GFP expression is initiated in heterozygous c-Kit +/DGFP and c-Kit-deficient c-Kit W/DGFP mice. Data are representative of more than three experiments with three mice per genotype. doi:10.1371/journal.pone.0026918.g001 the mutant allele for gene targeting in the system of mutant loxPs is required for circumvention of mosaic alleles which could be produced from Cre-mediated recombinations of the mutant loxPs between the different alleles. The W heterozygotes hemizygously carried the ROSA-CreERT2 transgene (c-Kit +/W ; ROSA-CreERT2 +/Tg mice), allowing recombination of lox66 and lox71 sites upon tamoxifen treatment [12]. To account for any possible off-targeted phenotypes and Cre toxicity, all mice used for the following studies hemizygously carried the ROSA-CreERT2 transgene in control c-Kit +/+ , heterozygous c-Kit +/DGFP , heterozygous c-Kit +/W , and c-Kit-deficient c-Kit W/DGFP mice (Fig. 1C, Fig.  S1A). All of the control CreERT2 c-Kit +/+ , CreERT2 c-Kit +/ lox66-71 , CreERT2 c-Kit +/W , and CreERT2 c-Kit W/lox66-71 mice were treated with equivalent doses of tamoxifen, which resulted in the generation of control CreERT2 c-Kit +/+ , CreERT2 c-Kit +/DGFP , CreERT2 c-Kit +/W (controls), and c-Kit-deficient CreERT2 c-Kit W/ DGFP mice (Fig. 1C). After day 10 of tamoxifen injection, c-Kit null phenotype on Lin 2 Sca-1 + cells was confirmed by flow cytometric analysis in the BM of c-Kit W/DGFP mice ( Fig. 1C-D). Furthermore, c-Kit::GFP was expressed in both c-Kit-deficient c-Kit W/DGFP and heterozygous c-Kit +/DGFP mice, after tamoxifen treatment of c-Kit W/ lox66-71 and c-Kit +/lox66-71 mice, respectively (Fig. 1D). The c-Kit expression level on LSK cells from the heterozygous c-Kit +/DGFP mice was reduced proportionally to that from c-Kit +/W heterozygoutes (Fig. 1D), while all of the pre-treated mice, c-Kit +/+ , c-Kit +/ lox66-71 , c-Kit +/W , and c-Kit W/lox66-71 mice showed normal c-Kit cell surface expression (Fig. S1). These data strongly support the notion that the targeting strategy illustrated in Fig. 1A is efficient to delete the c-Kit gene and to switch on GFP expression driven by the c-Kit native promoter.

Adult c-Kit deficiency alters hematopoiesis
The majority of mice in the series of naturally-occurring W/c-Kit mutants have macrocytic anemia [5]. Therefore, we measured complete blood counts (CBC) of the c-Kit-deficient c-Kit W/DGFP mice to investigate the effect of c-Kit deficiency on blood cell productions at steady state conditions. Although prior to tamoxifen treatment all of the CBC were normal, white and red blood cell counts as well as hemoglobin and hematocrit values in peripheral blood in the c-Kit-deficient c-Kit W/DGFP mice were significantly lower in number in comparison to those in control mice ( Fig. 2A). Notably, platelet counts were normal in the c-Kitdeficient c-Kit W/DGFP mice ( Fig. 2A). Furthermore, BMs of c-Kitdeficient c-Kit W/DGFP mice were hypocellular with disruption of the normal architecture ( Fig. 2B-C), and there was a profound decrease in the spleen size and reduced number of splenocytes compared to those in controls ( Fig. 2D-E). These data suggest that c-Kit is required for the maintenance of the steady state hematopoiesis and spleen mass in adult mice. c-Kit is required for maintenance of adult hematopoietic stem and progenitor cells Next, we investigated whether the homeostasis of the phenotypically marked primitive hematopoietic cells was altered by c-Kit deficiency at the steady state conditions. Since the total cellularity of BM cells of c-Kit-deficient c-Kit WD/GFP mice was reduced in number and the c-Kit signaling is known to regulate apoptotic pathway in some cell types [13,14,15], we examined apoptotic status of c-Kit-deficient Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + cells. Flow cytometric analysis revealed that no apoptotic cells were detected in LSK and Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + cells from the BM of control and c-Kit-deficient c-Kit W/GFP mice, respectively (Fig. S2). The number of the stem cell subsets as measured by the proportions of the CD34 2 Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + and Thy1.1 low Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + cells in the BM of c-Kitdeficient c-Kit W/DGFP mice were significantly decreased as compared to control mice ( Fig. 3A) due to the reduction of BM cellularity in the c-Kit-deficient c-Kit W/DGFP mice (Fig. 2C) although the frequencies of these cell subsets in the BM were comparable between the control and c-Kit-deficient c-Kit W/DGFP mice (data not shown). Moreover, the total number of colonyforming cells in the BM of c-Kit-deficient c-Kit W/DGFP mice was significantly decreased as compared to controls (Fig. 3B), suggesting that at steady state conditions, c-Kit is essential for the maintenance of phenotypically marked HSPCs.
To assess the hematopoietic reconstitution potential of c-Kitdeficient HSPCs, we performed competitive reconstitution assays using immunodeficient NOD/SCID mice lacking CD3 + T cells.

LSK cells isolated from BM mononuclear cells (BMMNCs) of c-
Kit +/+ mice, Lin 2 Sca-1 + c-Kit dim c-Kit::GFP + purified from BMMNCs of heterozygous c-Kit +/DGFP mice or c-Kit-deficient Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + cells isolated from BMMNCs of c-Kit-deficient c-Kit W/DGFP mice, were transplanted with BMMNCs from naïve NOD/SCID mice into irradiated-recipient NOD/ SCID mice (Fig. 3C). Quantification CBC in the recipient mice on day 34 after transplantation revealed that the numbers of white blood cells and lymphocytes were increased in peripheral blood of the recipient mice transplanted with either LSK or Lin 2 Sca-1 + c-Kit dim c-Kit::GFP + cells (Fig. 3D). This suggests that the transplanted HSPCs engrafted in the BM of the recipient mice and gave rise to lymphocyte cell subsets, which lacked in NOD/SCID mice. However, those numbers in the recipient mice transplanted with c-Kit-deficient Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + cells were not increased compared to controls (Fig. 3D). Additionally, the flow cytometric analyses revealed that CD3 + T cells were reconstituted in the recipient NOD/SCID mice transplanted with LSK or Lin 2 Sca-1 + c-Kit dim c-Kit::GFP + cells, while no CD3 + T cells were detected in the recipient NOD/SCID mice transplanted with c-Kit-deficient Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + cells (Fig. 3E). Notably, c-Kit::GFP + cells were reconstituted in the BM of the recipient NOD/SCID mice transplanted with Lin 2 Sca-1 + c-Kit dim c-Kit::GFP + HSPCs, but not in NOD/SCID recipients transplanted with c-Kit-deficient Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + HSPCs (Fig. 3E). These results indicate that c-Kit-deficient LSK cells fail to reconstitute hematopoiesis in the irradiated recipient mice, suggesting that c-Kit activation is essential for hematopoietic reconstitution.

c-Kit mediates interaction of HSPCs with niche cells
Cellular adhesion of HSPCs with prototypical BM niche cells, such as endothelial and stromal cells could play a major role in maintenance of hematopoiesis. Therefore, we employed in vitro adhesion and migration assays to determine whether c-Kit activation is essential for proper interaction of the HSPCs with the niche cells. To this end, lineage-depleted hematopoietic cells (Lin 2 cells) isolated from c-Kit-deficient c-Kit W/DGFP and control mice were used for Boyden chamber assay and adhesion assay to laminin, an extracellular matrix component that is deposited by niche cells. Lin 2 cells derived from c-Kit-deficient c-Kit W/DGFP mice manifested severe defects in migration and adhesion as compared to Lin 2 cells derived from c-Kit +/+ mice (Fig. 4A). These data indicate that c-Kit is required for migration and adhesion of HSPCs to niche cells.
To define the mechanism by which c-Kit deficiency results in the impairment of hematopoiesis, we interrogated the role of c-Kit in mediating interaction of HSPCs with BM niche cells [4,16,17,18,19,20,21]. Specifically, we hypothesized that c-Kit might regulate adhesion and/or migration of HSPCs to BM endothelial and stromal cells. To test this hypothesis, we determined the effect of c-Kit deficiency on adhesion and migration activities of HSPCs using c-Kit-deficient cells purified from BMs of c-Kit W/DGFP mice, in a coculture system with niche cells [22]. Exogenous sKL stimulated adhesion of Lin 2 cells derived from the control mice onto both primary endothelial cells (ECs) and BM-derived stromal cells, cocultured in serum-and cytokine-free condition. The cocultured hematopoietic cells formed typical cobblestone colonies on endothelial and stromal cells, while c-Kit-deficient Lin 2 cells from BMs of c-Kit W/DGFP mice detached from endothelial and stromal cells and failed to form adherent or migrating colonies (Fig. 4B). In addition, some cells were observed to tightly adhere to ECs and transmigrated underneath the ECs. The ratio of c-Kit-deficient Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + HSPCs that interact with niche cells were much lower than those of wild-type LSK HSPCs (Fig. 4C). These data suggest that c-Kit signaling stimulates tight interactions of HSPCs with niche cells [21,23].
Integrin molecules expressed on cell surface are known to provide tight adhesion between cells. Unexpectedly, there were no significant difference in cell surface expressions of a4, a5, a6, and b1 integrins on between LSK and c-Kit-deficient Lin 2 Sca1 + c-Kit 2 c-Kit::GFP + cells (Fig. S3). However, as membrane KL (mKL) supports interaction of mast cells with stromal cells [11,24], we speculated that mKL expressed on niche cells might also support interactions of c-Kit-expressing cells to the various spliced isoforms of KLs expressed on niche cells. After undergoing splicing KLs generate the membrane isoforms, mKL-1 and mKL-2, which lacks the metalloproteinase-cleavage site (Fig. S4) [25,26]. Each KL isoform supported the tight adhesion of wild-type Lin 2 BMMNCs onto ECs (Fig. 4D-E), suggesting that the HSPC-niche cell interaction is enhanced by the KL-c-Kit signaling. To investigate whether the c-Kit-mediated interaction was important for maintenance and expansion of HSPCs, we extended the cocultivation period. There was an incremental expansion of LSK cells purified from BMs of c-Kit +/+ mice over a 10 day culture period (Fig. 4F-G). By contrast, c-Kit-deficient Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + cells failed to form colonies or proliferate in coculture with endothelial and stromal cells (Fig. 4F-G). In addition, mKL-1 or mKL-2 expressed on ECs supported expansion of large numbers of LSK cells (Fig. 4H-I). These findings indicate that the KL-c-Kit interaction accelerates motility and adhesion of HSPCs to the ECs and stromal cells thereby supporting their long-term expansion.

c-Kit is essential in regeneration of adult hematopoiesis
To determine whether c-Kit-mediated interaction of HSPCs with the BM microenvironment is essential for hematopoietic recovery and conferring HSPCs resistance to BM suppression [16] in adult mice, we assessed the capacity of the c-Kit conditional KO GFP-reporter mice to recover from high dose 5-FU mediated BM suppression [27]. Although there was a complete recovery of CBC in the control mice, in the c-Kit-deficient c-Kit W/DGFP mice there was a severe impairment in the hematopoietic recovery resulting in the death of the mice due to persistent pancytopenia by day 14 after 5-FU treatment (Fig. 5A). There was no major obvious organ  toxicity observed in the vital organs of the 5-FU-treated mice (data not shown). Under BM suppressive conditions, c-Kit-deficient c-Kit W/DGFP mice did not reveal the rebound extramedullary splenomegaly, which was observed in control mice (Fig. 5B). The spleen size was significantly reduced and atrophied in c-Kitdeficient c-Kit W/DGFP mice as compared to even the control untreated mice. Furthermore, on day 14 after BM suppression, c-Kit-deficient Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + HSPCs were decreased in number (data not shown). Notably, in the BM of c-Kit-deficient c-Kit W/DGFP mice the GFP expression was enhanced compared to that in heterozygous c-Kit +/DGFP mice on day 14 after the 5-FU injection (Fig. 5C). These data suggest that c-Kit activation is essential for hematopoietic reconstitution after BM myeloablation.

c-Kit regulates interactions of HSPCs to KL + VEGFR3 + sinusoidal ECs (SECs) in the BM
The advantage of the lox66-lox71 Cre-recombinase conditional KO GFP-reporter strategy allows us to precisely localize the c-Kit-deficient c-Kit::GFP + cells in the BM during the recovery from BM suppression. Indeed, c-Kit-deficient c-Kit::GFP + cells were detected in BMs of the 5-FU-treated c-Kit-deficient c-Kit W/DGFP mice (Fig. 6). Using VEGFR3 as a marker for SECs in the BM [27], confocal analysis demonstrated that c-Kit-deficient c-Kit::GFP + cells were close cellular apposition of but detached from VEGFR3 + SECs (Fig. 6B). Although at steady state conditions the expression of mKL was low on VEGFR3 + SECs (Fig. 6A), after BM suppression there was a significant upregulation of mKL expression on VEGFR3 + SECs in the BM (Fig. 6B). These data suggest that interaction of HSPCs with the SECs enhanced by KL-c-Kit signaling could be important for hematopoietic recovery.

Discussion
We have employed a lox66-lox71 Cre-recombinase conditional KO approach to dissect c-Kit functions in adult mice. This strategy not only results in c-Kit deletion but also by genetic labeling of the c-Kit-deficient cells with GFP, facilitates isolation of these cells for in vitro studies and imaging of the c-Kit-deficient cells in vivo. We demonstrate for the first time that c-Kit expression is required for adult hematopoiesis within the BM and spleen at the steady state conditions and hematopoietic recovery from BM depletion. Furthermore, we show that c-Kit signaling enhances the interaction of HSPCs with niche cells, allowing for proper functional positioning of the repopulating HSPCs within the BM microenvironment.
Among the known RTKs, expression of Tie2, VEGFR2, VEGFR1, and FGF-receptors have been implicated in modulating the homeostasis of HSPCs during the development. However, none of these RTKs have been shown to play a major role in mediating the homeostasis of HSPCs in adult mice at steady state conditions. Therefore, c-Kit is one of the critical RTKs that modulates the steady state and long-term maintenance of HSPCs. Here, we demonstrate that both at steady state and after BM depletion, KL enhances the interaction of c-Kit-expressing cells with KL + niche cells. The mKL-c-Kit pair not only performs as a survival pathway but also allows for functional positioning of HSPCs to the various BM niches to maintain HSPCs and accelerate hematopoietic recovery. Notably, both at steady state conditions and after 5-FU mediated BM depletion in the c-Kitdeficient mice there is a profound decrease in the size of spleen, with a major defect in physiological increase in the spleen size after BM depletion. It is conceivable that during hematopoietic recovery the atrophy in the size of spleen might be due to impaired mobilization of HSPCs from the BM to the spleen. Alternatively, c-Kit activation may be essential for expansion and maintenance of HSPCs within the spleen. Mutations of the juxtamembrane domain tyrosines in c-Kit RTK results in splenic hypertrophy but in normal hematocrit values in adult mice due to the loss-of-docking sites for their particular target molecules and reduced kinase activity [28]. Thus, at steady state conditions, proper c-Kit signaling is essential to maintain the spleen mass supporting the homeostasis of HSPCs.
We have taken advantage of lox66-lox71 conditional knock down of the c-Kit in adult mice, enabling simultaneous tracking of HSPCs, which lack c-Kit in the BM during the steady state and after 5-FU hematopoietic suppression. The finding that c-Kit expression regulates hematopoiesis and spleen size in adult mice even at the steady state has major clinical implications, setting

Targeting construct
The fragment containing pGK-HSV-TK was excised from pDEL-BOY (a gift from D. J. Rossi, Stanford) and inserted between EcoRI and

Mice
The targeting vector was linearized by AhdI and then transfected by electroporation into C2J ES cells derived from C57BL/6-Tyr c-2J / J mice. Colonies doubly resistant for G418 and gancyclovir were screened by Southern blot analysis for homologous recombination with XbaI digestion using 39-flanking probe. Subsequently, the Neo was excised from the targeted allele in the homologous recombinant ES clones by transient expression of Flippase-puromycin plasmid. After screening of Neo-excised ES clones by Southern blot hybridization with BglII digestion using 39-flanking probe, the screened ES clones were used for microinjections with C57BL/6 blastocysts. Generated chimeric mice were bred with C57BL/6-Tyr c-2J /J female mice, and their albino-looking offspring were genotyped to confirm germline transmission of the c-Kit lox66-71 allele. The ROSA-CreERT2 mice (CD1 back ground) were obtained from T. N. Sato at Weill Cornell Medical College (WCMC), NY. C57BL/6-Tyr c-2J /J, NOD/SCID, and C57BL/ 6-background c-Kit +/W mice were purchased from Jackson Laboratory (Bar Harbor, ME).

c-Kit conditional KO GFP-reporter mice
For generation of c-Kit W/lox66-71 mice, the c-Kit +/lox66-71 mice were crossed with heterozygous c-Kit +/W mice carrying hemizygous ROSA CreERT2 transgene. The mice hemizygously carrying the ROSA CreERT2 transgene were treated with tamoxifen at a dose of 450 mg/Kg body i.p. for three days. The treated mice on day 10-14 post tamoxifen were used for the experiments. All mice were maintained in specific pathogen-free conditions in the Animal Facility of WCMC. Reaction conditions were as described above. Cycling conditions were 94uC for 5 minutes, 35 cycles of 94uC for 30 seconds/58uC for 30 seconds/72uC for 2 minutes, followed by 72uC for 10 minutes.

CBC
Retro-orbital peripheral blood was collected into microhematocrit capillary tubes (Fisher Scientific, Pittsburgh, PA). CBC were counted by Bayer Advia 120 Multi-species Hematology Analyzer with multispecies software (Bayer HealthCare, Tarrytown, NY).

Methylcellulose culture
The colony forming was assayed using MethoCult M3434 (Stem Cell Technologies, Vancouver, Canada).

Organ histology and immunofluorescence
All organs were fixed in 4% paraformaldehyde (PFA). Femurs were decalcified in 10% EDTA in PBS containing Ca and Mg and then transferred into either 30% ethanol for preparation of paraffin sections or 30% sucrose for preparation of frozen sections. The treated tissues were embedded in OCT medium and frozen in liquid nitrogen. Hematoxylin and eosin (H&E) stains were done by Histoserv (Germantown, MD). For IF, the frozen sections (10 mm) were blocked with 10% normal donkey serum/0.05% Tween 20 in PBS containing Ca and Mg for 2 hr at RT followed by primary antibody reactions for over night at 4uC. On the next day, the stained sections were reacted with proper secondary antibodies for 1 hr at RT and then stained with DAPI (Invitrogen) for 20 min at RT for nuclei counter staining. Fluorescent images were obtained using Carl Zeiss LSM710 system (Carl Zeiss, Thornwood, NY). The primary and fluorophore-conjugated secondary antibodies (Abs) were anti-KL (Santa Cruz, Santa Cruz, CA), anti-VEGFR3 (mF4-31c1, a gift from ImClone Systems, New York, NY), anti-GFP conjugated with biotin (Invitrogen), and fluorophoreconjugated Abs (Jackson ImmunoResearch Laboratories, West Grove, PA).
Migration assay 1610 5 lineage-depleted BMMNCs in 100 mL 0.1% BSA IMDM (Invitrogen) were placed in the top compartments and 600 mL medium containing 50 ng/mL mouse recombinant SDF-1a (Pepro Tech Inc., Rocky Hill, NJ) in the bottom chambers of the 5.0 mm transwell plates (Corning, Corning, NY). After 6 hr at 37uC in a 5% CO 2 incubator, the migrated cells in the bottom chambers were harvested and then measured in number using Cell Titer-Glo Luminescent Cell Viability Assay (Promega, Madison, WI).

Adhesion and expansion assays
For adhesion and expansion assays of HSPCs to ECs and BM stromal cells, 2.5610 5 Lin 2 BMMNCs were placed onto either ECs or BM-derived stromal cells in non-serum X-VIVO 20 medium (Lonza, Walkersville, MD) with or without 20 ng/mL mouse recombinant KL (Pepro Tech Inc.) in 12-well plates. After 18 hr or 10 day culture, adhered cells were harvested by mild treatment of trypsin and then counted in cell numbers. The number of LSK or c-Kit-deficient Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + cells were calculated by frequencies of these cells quantified with a flow cytometry LSRII and the counts of BMMNCs with hemocytometer. In-put LSK or c-Kit-deficient Lin 2 Sca-1 + c-Kit 2 c-Kit::GFP + cells were also calculated by the same way, and the % adhesion was determined by the number of the adhered cells/the number of the in-put cells. For adhesion assay to laminin, 1610 5 Lin 2 BMMNCs in 100 mL 0.1% BSA IMDM containing 10 ng/mL mouse recombinant KL were placed in laminin-coated wells of 96-well plates (BD Bioscience). After 1 hr incubation at 37uC in a 5% CO 2 incubator, non-adherent cells were washed out two times by PBS. The adherent cells were quantified in number using Cell Titer-Glo Luminescent Cell Viability Assay. The % adhesion was calculated by the number of adherent cells/in-put cell numbers.