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Transplanted Hematopoietic Cells Seed in Clusters in Recipient Bone Marrow In Vivo

^a Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA;
^b Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky, USA;
^c Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA;
^d Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA;
^e Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

Key Words. Hematopoietic stem cells • Bone marrow transplantation • In vivo optical imaging • Fluorescence microscopy

Nadir Askenasy, M.D., Frankel Laboratory of Bone Marrow Transplantation, Center of Pediatric Hematology Oncology, Schneider Children's Medical Center of Israel, 14 Kaplan Street, Petach Tikva 49202, Israel. Telephone: 972-3-641-1475; Fax: 972-3-641-1475; e-mail: anadir{at}012.net.il or askenasy{at}andrew.cmu.edu

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
The process of hematopoietic stem and progenitor cell (HSPC) seeding in recipient bone marrow (BM) early after transplantation is not fully characterized. In vivo tracking of HSPCs, labeled with PKH dyes, through an optical window surgically implanted on the mouse femur revealed that transplanted cells cluster in the recipient BM. Within the first day after intravenous injection, 86 ± 6% of the cells seeded in clusters (p < 0.001 versus scattered cells) in the endosteal surfaces of the epiphyses. The primary clusters were formed by concomitant seeding of 6-10 cells over an area of ~70 µm, and secondarily injected cells did not join the already existing clusters but formed new clusters. Major antigen-disparate HSPCs participated in formation of the primary clusters, and T lymphocytes were also incorporated. After 4 to 5 days, some cellular clusters were observed in the more central regions of the BM, where the brightness of PKH fluorescence decreased, indicating cellular division. These later clusters were classified as secondary, assuming that the mechanisms of migration in the BM might be different from those of primary seeding. Some clusters remained in the periphery of the BM and retained bright fluorescence, indicating cellular quiescence. The number of brightly fluorescent cells in the clusters decreased exponentially to two to three cells after 24 days (p < 0.001). The data suggest that the hematopoietic niche is a functional unit of the BM stromal microenvironment that hosts seeding of a number of transplanted cells, which form a cluster. This may be the site where auxiliary non-HSPC cells, such as T lymphocytes, act in support of HSPC engraftment.

	INTRODUCTION

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Despite extensive efforts to characterize the biology of stem cell engraftment, the events that actually take place in recipient bone marrow (BM) in the days following hematopoietic stem and progenitor cell (HSPC) transplantation remain poorly characterized. Most studies use data collected days or weeks after transplantation for a functional assessment of engraftment. However, these approaches do not allow the dissection of the early stages of homing and seeding, which are of crucial importance in the determination of the long-term outcome of the transplantation procedure. These considerations have motivated us to engage in the development of new approaches for in vivo tracking of labeled HSPCs in recipient BM using high-resolution optical techniques. Some of the results obtained using this approach have shed new light on the early stages of the engraftment process, not always corresponding to the traditional transplant dogma. For example, a prevalent concept postulates that conditioning "opens space" for engraftment of donor HSPCs [1] by killing and removing host BM cells (BMCs) from a finite number of the hematopoietic niches that exist in the marrow space [2]. This concept implies that donor and host HSPCs compete for the limited number of marrow niches [3], and antigen mismatch between donor HSPCs and host BM stroma causes a disadvantage for engraftment [4]. We have shown that neither available space in host BM nor antigen disparity restrict early seeding of donor HSPCs [5], findings consistent with the hypothesis that transplanted cells may impose functional quiescence on host HSPCs [6].

Here, we report one of the consistent patterns of seeding observed in vivo, clustering of donor cells in the recipient marrow space. Studies performed decades ago described a focal organization of the BM of normal mice, which might represent the proliferative activity of a colony-forming cell (CFC) [7, 8]. In this study, we aimed to define three aspects of the clusters. First, we wanted to determine whether the clusters reflect a real aggregation tendency of transplanted cells, because some progenitors can divide very early after transplantation [9–12]. Therefore, we used PKH membrane linkers, which are evenly distributed among daughter cells and are diluted during division [9, 11, 13, 14]. Second, we assessed whether cells of various antigenic types are incorporated in common clusters or whether aggregation of the cells is antigen specific. Clustering along the major histocompatibility complex (MHC) classes may be one of the causes of the hematopoietic cell-marrow stroma antigen restriction [4] given that the process of adhesion per se is indifferent to MHC disparity [5]. Third, we evaluated whether the clusters include both lineage-negative HSPCs and subsets of lineage-positive BMCs. Prevalence of HSPCs over mature BMCs is expected from previous studies [5, 9–14], however, inclusion of more mature cells may be relevant to the facilitating phenomenon [15–18]. We chose to focus on T cells, which promote HSPC engraftment on one hand, and also mediate detrimental graft-versus-host reactions, on the other hand [19–23]. From our experimental perspective, we were interested in determining whether or not T cells colocate with HSPCs in recipient BM early after transplantation.

	MATERIALS AND METHODS

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Animal Preparation and Conditioning
B10 (C57Bl/10SnJ, H2^b) and B10.BR (C57Bl/BR, H2^k) mice purchased from Jackson Laboratories (Bar Harbor, ME; http://www.jax.org) were housed in a barrier facility. Animals aged 8-12 weeks were anesthetized with avertin (12-17 µg/g, i.p.). During surgery, the respiration was monitored with a PowerLab piezo-electric transducer (ADInstruments; Grand Junction, CO; http://www.adinstruments.com) and additional doses of anesthetic (avertin, 6 µg/g, i.p.) were administered when the rates exceeded 0.5 Hz to prevent awakening. Motion, access to food and water, and general behavior were monitored frequently during the postoperative period and at daily intervals thereafter. Recipients were conditioned with busulfan or total body irradiation (TBI). Busulfan was dissolved in dimethyl sulfoxide at a concentration of 24 mg/ml and was diluted fivefold in water at 39°C (Sigma Chemical Co.; St. Louis, MO; http://www.sigmaaldrich.com). TBI of 950 rad was administered at a rate of 105 rad/min from a cesium source (J.L. Sheppard & Assoc.; San Fernando, CA). Cells suspended in 0.2 ml phosphate-buffered saline (PBS) were injected into the lateral tail vein after heating to enlarge the veins prior to injection.

Surgical Procedure
To visualize recipient BM microenvironment, an optical window was placed over distal femoral epiphysis, as previously reported [5]. Briefly, the femur was exposed by transection of the patellar insertion of the quadriceps muscle in aseptic conditions. Bleeding was controlled by cauterization, and the bone cortex was thinned with an electrical drill using 1-mm drill tips. Upon appearance of fissures in the eroded area, bone plates were removed with a fine-tip forceps. The area of cortex removed was about 2.5 x 4 mm, above the distal epiphysis or the diaphysis. Dental cement, NeoCryl (NeoResins; Wilmington, MA; http://www.avecia.com/neoresins), was applied on the edge of the cortex, and the exposed area was covered with a (2-3) x (4-5) mm glass window (cover slip #0). The quadriceps muscle was sutured to one of the skin flaps, the skin was closed with 4/0 silk sutures, and the limb was casted with gauze and NeoCryl in a semiflexed position.

Isolation and Purification of BMCs
BMCs were harvested from femurs and tibiae of donors and crushed in Hank's balanced salt solution ([HBSS] GIBCO Laboratories; Grand Island, NY; http://www.invitrogen.com). Cells were suspended using an 18-gauge needle, filtered with a 30 µm sterile nylon mesh, collected by centrifugation (400 g, 10 min, 4°C), and resuspended in HBSS containing 2% fetal calf serum (FCS). Erythrocytes were lysed by incubation with ammonium chloride for 4 minutes at room temperature. Nucleated cells were counted after being washed twice with excess medium (whole BMCs).

Lineage-negative cells were isolated from nucleated BMCs. Cells were gently mixed for 30 minutes at 4°C with saturating amounts of rat-anti-mouse monoclonal antibodies (mAbs) specific for CD5 (clone 53-7.3), GR-1 (clone RB6-8C5), CD45R (clone RA3-6B2), Ly-76 (clone TER119), TCR-ß (clone H57-597), and TCR- (clone GL3) (Pharmingen; San Diego, CA; http://www.pharmingen.com). Cells coated with mAbs were washed twice with PBS containing 1% bovine serum albumin and were incubated with sheep-anti-rat IgG conjugated to M-450 magnetic beads at a ratio of four beads per cell (Dynal Inc.; Lake Success, NY; http://www.dynal.no). Rosetted cells were precipitated by exposure to a magnetic field, and supernatant containing lineage-negative HSPCs was collected. The average yield of this procedure was 4%-5%, and a viability of 95% was assayed with the trypan blue exclusion test.

T lymphocytes were isolated from low-density cells collected from lymphocyte separation media (1.087 g/ml; CedarLane; Hornby, ON, Canada; http://www.cedarlanelabs.com) by centrifugation (20 min, 4°C, 800 g). Low-density cells were incubated for 30 minutes at 4°C with biotinylated anti-CD4 and anti-CD8 mAb (Pharmingen). Antibody-coated cells were mixed for 20 minutes at 4°C with magnetic beads (CELLection Biotin Binder Kit; Dynal; http://www.dynal.no). Rosetted T cells were precipitated in a magnetic field, collected, and resuspended in HBSS. Immunomagnetic beads were detached by incubation for 10 minutes at room temperature with deoxyribonuclease, according to manufacturer's instructions.

PKH Staining
Cells were suspended in Diluent C at a concentration of 2 x 10⁷ cells/ml, and freshly prepared PKH26 or PKH67 dyes were added to a final concentration of 2 µM, as previously described [5]. Samples were incubated at room temperature for 5 minutes with gentle mixing. Staining was terminated by addition of 4 volumes of HBSS containing 10% FCS. Cells were collected by centrifugation (10 min, 4°C, 400 g) and washed twice with HBSS. The average recovery of this procedure was 90% with a viability of 95%, as assayed with the trypan blue exclusion test.

In Vivo Cellular Tracking Using Fluorescence Microscopy
Each animal was anesthetized, the cast and sutures were removed, the skin and quadriceps muscle were reflected, and connective tissue was removed from the window with a scalpel blade in aseptic conditions. The animal was mounted on the microscope stage, and the hind leg was immobilized without vascular occlusion. Each recipient was sequentially monitored by in vivo microscopy two to four times, a procedure associated with a mean death rate of 12%. The most prevalent cause of death was failure to recover from anesthesia, given in high doses to minimize motion artifacts during image acquisition.

Direct observation of PKH-labeled BMCs in recipient BM was performed with Eclipse 800 (Nikon; Melville, NY; http://www.microscopyu.com) and Axiophot (C. Zeiss; Thornwood, NY; http://www.zeiss.com) upright fluorescence microscopes. Cells labeled with PKH67 and PKH26 membrane linkers were detected using standard sets of fluorescein isothiocyanate (FITC) and rhodamine filters, respectively (Chroma Technology; Brattleboro, VT; http://www.chroma.com). Bone windows were inspected at a magnification of 5x, and maps containing discrete coordinates in reference to the window frame were recorded. The BM was then optically inspected at magnifications of 10-100x for a better spatial resolution.

Image Acquisition and Analysis
Images were collected with a cooled charge-coupled device (CCD) camera (Hamamatsu Photonics KK; Hamamatsu, Japan; http://www.hamamatsu.com), controlled by QED software (QED Imaging; Pittsburgh, PA; http://www.qedimaging.com), and pseudocolored to simulate the real hues (Adobe Photoshop software). The figures were reconstituted in two ways: A) superposition of fluorescence images over brightfield color images of the BM and B) RGB reconstruction by superposition of three layers of fluorescence acquired at the same magnification and position of the stage: red—rhodamine filters for detection of PKH26; green—FITC filters for PKH67; blue—a standard set of 4'-6'-diamindino-2'phenylindole (DAPI) filters to detect UV-excited bone autofluorescence.

Statistical Analysis
Data are presented as mean ± standard deviation. Results in each experimental group were evaluated for reproducibility by linear regression of duplicate measurements, and differences among protocols were estimated with a post hoc Scheffe t test at a significance level of p < 0.05.

Experimental Procedure
The standard experimental procedure included surgery for placement of the bone window on day –4, and whenever applicable, conditioning with busulfan (i.p.) or TBI 36 and 4 hours before transplantation, respectively. On day 0, mice were heated to enlarge the veins, and donor cells labeled with membrane liners were injected into the tail vein: 10⁵-10⁷ syngeneic or allogeneic whole BMCs, 10⁴-10⁶ syngeneic or allogeneic lineage-negative HSPCs alone, 2 x 10⁶ allogeneic T lymphocytes alone, or a mixture of 5 x 10⁴ HSPCs and 5 x 10⁴ T lymphocytes.

	RESULTS

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Cellular Clustering in Recipient BM
Intravenous injection of cells into mice positioned on the microscope stage allowed visualization of the BM 3-5 minutes after transplant. At this early time, some cells started to seed in clusters in the subendosteal region of the femoral epiphysis, a site also characteristic of early adhesion of cells in femurs perfused ex vivo [5]. Detailed observation of the patterns of seeding, using high-resolution fluorescence microscopy in vivo, revealed a clustering tendency of the transplanted cells. After this initial observation, we assessed a group of 38 recipients to determine the consistence of cellular seeding in clusters.

During the first hours after transplantation, donor cells showed high motility in the marrow space. On day +1, approximately 70% of the PKH-labeled cells were adherent to the BM stroma, and all cells were adherent on day +3 (NA, personal observation). To assess the patterns of seeding and clustering, we first determined that the observed cells were immobilized and did not move with the blood pulsation in reference to the BM matrix. For this reason, measurements performed 24 hours posttransplant were easier. In addition, within 3-5 hours after injection, there were no detectable levels of labeled donor cells in the peripheral blood of the recipients [24]. The intravascular space is usually invisible to in vivo fluorescence imaging, and the observations represent cells in the marrow space (extravascular).

On day +1 after transplantation, we could define, for 86 ± 6% of the cells, that they were part of clusters (Fig. 1) based on the fact that: A) cells were located at the base of the osseous trabeculae protruding from the bone cortex into the marrow space; B) those formations that were clearly defined as early clusters usually contained 6-10 cells; C) the majority of cells clustered in a round formation (Fig. 2, encircled in red), although not in all cases (encircled in yellow), and D) cells were brightly fluorescent within the range observed in vitro before transplantation. Formation of clusters refers to the joint seeding of a group of transplanted cells, as opposed to the proliferation of a CFC. It is difficult to assess the division of individual cells in vivo by measurements of the fluorescence intensity. There is significant variability in intensity of fluorescence of cells labeled with PKH membrane linkers, even in purified cell populations sorted by flow cytometry for a range of fluorescence. The short acquisition time used in Figure 2 to prevent saturation provides more reliable information on formation of the clusters by several injected cells.

View larger version (58K): [in this window] [in a new window]   Figure 1. In vivo imaging of the cellular clusters. B10 recipients preoperated for placement of bone windows (day –4) and conditioned with busulfan (day –1.5) were injected with 2 x 106 bone marrow cells from B10.BR donors labeled with PKH67. Images were acquired with a Nikon Eclipse 800 microscope on day +3 at a magnification of 40x. The figure represents superposition of a fluorescence image (pseudocolored in green to simulate the real hues) over a color image acquired in brightfield. The image presents a cluster of bright fluorescent cells (green) and red granulation tissue (yellow arrowheads) at the edge of the eroded bone cortex (white arrowheads) under the optical window at the location shown in the inset.

View larger version (51K): [in this window] [in a new window]   Figure 2. High resolution in vivo tracking of bone marrow cells (BMCs) labeled with PKH67. B10 recipients, prepared as detailed in Figure 1, were injected with 2 x 106 syngeneic BMCs labeled with PKH67. Images were acquired with a Nikon Eclipse 800 microscope on day +3 at a magnification of 100x. The figure represents superposition of a fluorescence layer (pseudocolored in green for PKH67) over a color image acquired in brightfield. The fluorescence image was acquired with a short exposure time (1.5 seconds) to demonstrate penetration of the membrane by the PKH dyes and staining of intracellular organelles. Characteristic primary clusters (encircled in red) and a less characteristic cluster (encircled in yellow) were imaged at the location shown in the inset. The image presents the distribution of a homogeneous population of labeled cells, relative to the variability in fluorescence intensity achieved by PKH staining. It can be seen that some dimmer cells (right upper corner) are out of the plane of focus.

The location of the clusters in the epiphyses, close to the endosteal surface, and the size of the early clusters were independent of the number of donor cells injected, in the range of 10⁵-10⁷ whole BMCs and 10⁴-10⁶ lineage-negative HSPCs. Figure 2 exemplifies some clusters with the prevalent round organization (red circles), while other cells seeded in various formations according to the shape of the underlying osseous structures of the BM (yellow arrows). In addition, there were 14 ± 3% cells that could not be unequivocally assigned to a certain cluster (p < 0.001 versus clustering cells). These cells were grouped at certain sites of the BM close to the bone surface and were not individually scattered in the BM. Notably, in vivo imaging presents a two dimensional section of the sample, and focusing is used to set this plane along the third dimension. Thus, virtually all the injected cells seen in the host BM presented some form of organized seeding pattern.

Primary Clusters
Repeated observations of selected regions of the host BM revealed that clusters were initially formed by simultaneous seeding of 6-10 donor cells over a stromal area of 50-100 µm. Groups of cells adhered concomitantly, and there was no apparent increase in the number of cells in the cluster. To further corroborate this observation, a second intravenous injection of BMCs labeled with a different PKH dye than the first injection (PKH26 for syngeneic BMCs and PKH67 for allogeneic BMCs) was performed 3 hours after the first transplantation (n = 4). We observed formation of new monochromatic (either PKH26 or PKH67) clusters of cells from the secondary injection, but there was virtually no inclusion of these cells in the already existing clusters of cells from the first injection.

Secondary Clusters
Four to five days after transplantation, some cells started to be observed in the more central regions of the epiphyses and in femoral diaphyses. The bright fluorescence of these cells during initial stages of translocation suggests that some of the transplanted BMCs changed their location in the BM. Interestingly, the cells that migrated toward the central regions of the marrow also clustered, although the number of cells decreased. We hypothesized that the mechanisms of migration and colonization of the central regions of the BM (and the diaphysis) may involve mechanisms other than those responsible for initial seeding in the subendosteal epiphysis. Therefore, a distinction was made between cells that seeded in the primary clusters and cells that changed their location later to form the secondary clusters.

We also observed a decrease in fluorescence intensity of some cells that migrated toward the center of the marrow space, indicating they entered a proliferative phase. In this study, we used a membrane linker that is evenly distributed among the daughter cells of a cycling progenitor. Inclusion of bright and dimmer cells in the central secondary clusters might be attributed either to migration of proliferating CFCs toward more central areas of the marrow or initiation of the proliferative activity of HSPCs by stromal environments in the more central areas of the marrow. Some cells, however, retained a peripheral location and bright fluorescence throughout the entire period of 24 days of monitoring (Fig. 3). Therefore, according to our classification, secondary clusters in the periphery included a smaller number of brightly fluorescent cells, seemingly quiescent. The characteristics of the central secondary clusters included: A) appearance on days 4-5 after intravenous transplantation; B) location in the more central regions of femoral BM (as opposed to the subendosteal location of the primary clusters); C) usually a smaller number of brightly fluorescent cells as compared with the primary clusters (vide infra), and D) increasing variability in fluorescence intensity of the various cells in the cluster, suggesting that some cells remained quiescent while others proliferated.

View larger version (20K): [in this window] [in a new window]   Figure 3. High resolution in vivo tracking of allogeneic cells labeled with PKH26. B10 recipients, prepared as detailed in Figure 1, were injected with 107 PKH26-labeled HSPCs from B10.BR donors. Images were acquired 14 days after transplantation with a Nikon Eclipse 800 microscope at a magnification of 100x. The figure represents superposition of a fluorescence layer over a color image acquired in brightfield. The fluorescence layer was displayed in red pseudocolor to simulate the real hues. Two peripheral clusters of two to three bright cells are located in proximity to the bone surface (yellow arrowheads) at the location shown in the inset.

Size of Clusters
As described above, the early clusters were formed by simultaneous seeding of several cells, without addition of new cells to the already formed cluster. We could not reach sufficient temporal and spatial resolution to determine the order and temporal frame of cellular seeding within the clusters. However, during the first day postinjection, there were no significant variations in the number of cells in those early clusters that could be repeatedly imaged. The size of primary clusters was constant, while donor inoculum consisted of varying numbers of cells, either whole BMCs (range 10⁵-10⁷), lineage-negative HSPCs (range 10⁴-10⁶), or a mixture of 5 x 10⁴ HSPCs and T cells. Tabulation of all the experimental observations showed a significantly greater number of cells (from 6-10) in the primary (centrifugal) clusters compared with two to three in the secondary (>5 days after transplantation) clusters (p < 0.001). The lower number of cells refers to those that retained sufficient fluorescence for optical tracking through the bone window (Fig. 4). The data included in the figure represent observations in myeloablated and nonconditioned recipients injected with syngeneic and allogeneic whole BMCs or lineage-negative HSPCs.

View larger version (14K): [in this window] [in a new window]   Figure 4. The average number of cells in clusters decreased as a function of time after transplantation (y = 8.3x(exp-0.37), r2 = 0.97, p < 0.001). Each point represents the mean value of 12-20 clusters observed on days 1-24. Error bars represent standard deviations.

View larger version (42K): [in this window] [in a new window]   Figure 5. (A) Syngeneic and allogeneic lineage-negative HSPCs coreside in the same clusters. B10 recipients, prepared as detailed in Figure 1, were injected with a mixture of 104 PKH67-labeled and 5 x 104 PKH26-labeled lineage-negative HSPCs from B10 and B10.BR donors, respectively. Images were acquired 24 hours after transplantation with an Axiophot microscope (C. Zeiss) at a magnification of 63x. The figure was RGB reconstructed from three fluorescence layers, pseudocolored to simulate the real hues: red—PKH26, green—PKH67, blue—UV-excited bone autofluorescence. Several clusters containing syngeneic (green) and allogeneic (red) cells are encircled. (B) Distribution of clusters containing various ratios of syngeneic and allogeneic cells. In these experiments, the ratio of syngeneic:allogeneic cells injected was 1:5. The homing efficiency of syngeneic is tenfold higher than that of allogeneic cells, therefore a syngeneic/allogeneic ratio of 2/1 may be considered as even distribution of the cells.

In another experiment, a mixture of 5 x 10⁴ lineage-negative PKH26-labeled HSPCs and 5 x 10⁴ PKH67-labeled T lymphocytes from syngeneic (B10, n = 4) or allogeneic (B10.BR, n = 6) donors was injected into myeloablated B10 recipients. Early after injection, we observed simultaneous seeding of T cells and HSPCs in common clusters. Imaging of recipient BM 24 hours after transplantation revealed that both cell types coresided in common clusters without an apparent change in number of aggregating cells, as compared with whole BMCs and lineage-negative HSPCs. Heterogeneity of the cellular composition of the clusters further supports the notion that they are formed from several injected cells and not by the early proliferative activity of CFCs.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vivo optical imaging of the BM has yielded new insights into the early patterns and mechanisms of hematopoietic stem cell engraftment. One of the new observations was the clustering tendency of transplanted cells during primary seeding in the subendosteal regions of femoral epiphyses. These patterns are consistent with regulation of seeding by the BM stroma [33–36]. The hematopoietic niche, thought to host transplanted cells [37], appears to be a functional unit of BM microenvironment in which a group of transplanted cells form a cluster.

Primary clusters were composed of 6-10 seeding cells located at the edge of the osseous trabeculae of the BM, close to the endosteal surface. This site has been shown to contain the primitive progenitors and stem cells in normal mice [25–29] and to host early seeding of the majority of transplanted cells [5, 30–32]. Consequently, 4-5 days posttransplantation, some cells started to be seen in more central regions of the BM. The decrease in number of bright cells and the increase in heterogeneity of fluorescence intensity most likely reflect the proliferative activity of some HSPCs during transition to secondary clusters. PKH membrane linkers are equally distributed among daughter cells during division, resulting in dilution of the dye below the detection threshold of optical imaging [9, 11, 13, 14]. Some hematopoietic progenitors have been shown to divide early after transplantation [9–12]. It will be interesting to determine whether HSPCs enter the cell cycle before or after their migration. On one hand, it is possible that cycling of HSPCs induces a change in expression of cell-surface adhesion molecules, which triggers their migration. On the other hand, proliferative activity might be initiated by migration of quiescent HSPCs to the center of the BM, where they encounter stromal microenvironments favorable for division. This latter possibility would be consistent with stromal regulation of stem cell activity. In addition to the apparent division of centrally located cells (dilution of the membrane linker), some clusters of quiescent cells remained close to the bone surface and retained bright fluorescence.

The size of the cellular clusters appears to continue to decrease, and at 6 weeks after transplantation, individual quiescent cells have been observed in the BM [30]. Those cells that retain fluorescence for long periods after transplantation are nondividing cells and may represent the subpopulation with long-term reconstituting potential, which is quiescent for periods of weeks in the murine BM [11, 38–40]. Although the concentration of dye we used in this study would allow monitoring of several divisions, we did not attempt to quantify cellular proliferation. Additional possible causes of a decrease in fluorescence intensity include submersion of the transplanted cells under host BM stroma [5], limited lifetime of mature donor cells in host BM (experiments under way), and photobleaching of the fluorochromes by repeated illumination [41].

Consistent with the observation that adhesion of transplanted cells to the BM stroma is not restricted by antigenic barriers [5], we found a random distribution of syngeneic and allogeneic BMCs in the primary cellular clusters. This does not preclude the possibility that, at a later stage of engraftment, antigen disparity, such as inhibition of allogeneic cobblestone formation in culture [42], may become relevant [4]. In this study, we partially corrected for fractional killing of antigen-disparate cells in the host reticuloendothelial system by increasing the size of allogeneic donor inoculum.

Formation of patches of hematopoietic cells has been previously reported in severe combined immunodeficiency (SCID) mice injected with human cells [43] and in the brain of mice injected with murine cells [44]. However, the functional significance of cellular clustering, in particular in the host BM after transplantation, is yet unknown. It may merely reflect the distribution of adhesion ligands presented by the BM stromal microenvironment, such as fibronectin and collagen, which are abundant in the subendostium [31, 45–47]. Despite the remarkably higher affinity of lineage-negative HSPCs for seeding in recipient BM compared with mature BMCs [5, 9–11, 13, 14, 32], we have observed incorporation of T cells in the primary cellular clusters. The lifetime of donor lymphocytes in recipient BM is limited to a few days (studies of T cell apoptosis in the host BM are currently under way). Death of T cells, and possibly other mature BMCs, may be an additional cause of the decrease in cluster size over time. This observation raises the question of a possible involvement of nonstem cells, such as T lymphocytes, in facilitation of HSPC engraftment [15–18]. These cells may act either by direct support of the adjacent HSPCs, indirect effects through induction of trophic factor expression by stromal elements, or veto effects that suppress the activity of host HSPCs to free space [6]. If facilitation of HSPC engraftment is indeed a significant mechanism, our data indicate that facilitation may occur in the recipient BM at very early stages after transplantation. It is important to characterize these mechanisms, because optimization of the timing of administration of posttransplant immunosuppressive therapy may improve the yield of engraftment.

In summary, in vivo optical tracking revealed a pattern of clustering of transplanted hematopoietic cells in the host BM. The primary clusters (early) were composed of 6-10 transplanted cells labeled with PKH dyes, were located close to the bone surface, and included lineage-negative HSPCs of different antigenic types, as well as T lymphocytes. Within several days after transplantation, smaller clusters were seen both close to the bone and in the more central regions of the femoral epiphyses and the diaphysis, accompanied by an apparent proliferation of some of the cells. It remains to be determined what is the functional meaning of the clustering of transplanted cells in recipient BM. In vivo optical imaging provides an important tool for the study of the early stages of hematopoietic stem cell engraftment and its relation to the long-term outcome of transplantation.

	ACKNOWLEDGMENT

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We thank Mrs. Judy Montibeller and Lisa McGow for their excellent technical assistance, Dr. Kathy Muirhead, SciGro Co., Philadelphia, PA, for providing PKH membrane linkers, Dr. Sallie S. Boggs, University of Pittsburgh Medical Center, Pittsburgh, PA, for the stimulating discussion and critical review of the manuscript, and Dr. Suzanne T. Ildstad, Institute for Cellular Therapeutics, University of Louisville, Louisville, KY, for her outstanding support in initiating this study.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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