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Stem Cells 2002;20:80-85 www.StemCells.com
© 2002 AlphaMed Press

RAPID COMMUNICATION

Antigen Barriers or Available Space Do Not Restrict in Situ Adhesion of Hemopoietic Cells to Bone Marrow Stroma

Nadir Askenasya,b,d, Daniel L. Farkasa,c

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 Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA;
d Present address: Frankel Laboratory of Bone Marrow Transplantation, Center of Pediatric Hematology Oncology, Schneider Children's Medical Center of Israel, Petach Tikva, Israel

Key Words. Bone marrow cells • Hemopoietic stem cells • Bone marrow • Seeding • Adhesion • Antigen barriers

Nadir Askenasy, Ph.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: 9-723-925-3669; Fax: 9-723-925-3042; e-mail: anadir{at}012.net.il


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, optical techniques were used to characterize adhesion of hematopoietic cells to bone marrow (BM) stromal microenvironment in situ. Bone marrow cells (BMC) labeled with PKH membrane linkers were infused into nonconditioned femurs and were monitored by fluorescence microscopy through an optical bone window. Repeated infusions of BMC into the femoral lumen resulted in a progressive increase in the number of adherent cells (p < 0.01), indicating that the availability of hemopoietic niches in the nonconditioned BM was not a rate-limiting factor of early BMC seeding. Adhesion of hemopoietic progenitor and stem cells (HSPC) was 30-fold higher than lineage+ BMC (p < 0.001), suggesting that adhesion molecules on the surface of HSPC have a higher propensity for adhesion. BMC antigen-matched to and disparate from BM stroma adhered at equal rates, opposing the idea of involvement of antigen barriers during early seeding. It is concluded that primary adhesion to BM stromal microenvironment is favorable for HSPC and is not restricted by antigenic barriers or availability of vacant niches.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The early stages of bone marrow cell (BMC) engraftment include homing and seeding in recipient bone marrow (BM). Recent in vivo microscopy studies showed high traffic of homing BMC in recipient femur, which stopped when BMC seeded in stromal microenvironment [submitted manuscript]. If seeding is the dominant factor in determining the efficiency of homing, then adhesion should have the characteristics of the homing process. First, an approximately 16-fold higher homing efficiency of lineage- hemopoietic stem and progenitor cells (HSPC) compared with lineage+ BMC suggested that adhesion molecules on the surface of stem cells had a higher propensity for adhesion [1–5]. Second, homing of allogeneic BMC was one order of magnitude lower than syngeneic BMC. Theoretically, antigen disparity could limit engraftment at three levels: reduction in the effective number of circulating BMC seeking the BM; restriction of migration across vascular endothelium and interaction with BM stroma across antigen barriers; and dysfunction of the seeding cells. The number of circulating allogeneic BMC that seek the BM might be lowered by their excessive entrapment in host reticuloendothelial system [6–9]. Considering that syngeneic and allogeneic BMC co-resided in common cellular clusters and adhesion appeared to improve BMC survival, it is possible that antigen disparity does not affect BMC interaction with stromal cells. Alternatively, deficient seeding of donor cells in recipient BM stroma could be restricted by antigenic barriers [10], as substantiated by inhibition of cobblestone formation in stromal cell cultures [11]. To elaborate on the characteristics and restrictions of donor BMC adhesion to recipient BM stroma, the seeding efficiency of syngeneic versus allogeneic lineage+ BMC and lineage- HSPC was quantitatively assessed in situ.

The observation that large numbers of BMC injected over several days engraft in nonconditioned recipients led to the hypothesis that donor cells must compete with host HSPC for hemopoietic niches [12–14]. According to this concept, the higher homing efficiencies reported in nonmyeloablated recipients are an apparent paradox [8,, 9], because cytoreductive conditioning is assumed to increase the number of available niches by fractional killing of host HSPC [15–18]. A more detailed analysis revealed that the higher numbers of cells in the nonconditioned BM exceed the probabilities calculated assuming competitive niche occupation or replacement of host BMC, suggesting that donor cells may repress the activity of host HSPC [19]. Aiming to determine whether availability of space is one of the limitations of BMC adhesion to BM stroma, femurs were repeatedly perfused with a large number of BMC in situ. The results suggest that the adhesion probability of antigen-matched and disparate BMC to BM stroma is equal, and increases with repeated infusions.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Preparation and Surgical Procedure
Mice used in this study were B10 (C57Bl/10, H2b) and B10.BR (C57Bl/BR, H2k) purchased from Jackson Laboratories (Bar Harbor, ME; http://www.jax.org). Animals, aged 8-12 weeks, were anesthetized with Avertin (12-17 µg/g, i.p.) and euthanized by CO2 asphyxiation. Access to food and water and general behavior were monitored frequently during the post-operative period. All procedures were approved by the Institutional Animal Care Committee.

Isolation and Staining of Cells
BMC were harvested from femurs and tibia of donors crushed in Hank' balanced salt solution (HBSS; GIBCO Laboratories; Grand Island, NY; http://www.invitrogen.com). BMC were resuspended with an 18G needle and were filtered with a 30 µm sterile nylon mesh. Cells were collected by centrifugation (400 g, 10 minutes, 4°C) and were resuspended in HBSS containing 2% fetal calf serum (FCS). RBC were lysed by incubation with ammonium chloride for 4 minutes at room temperature.

For isolation of lineage- HSPC, low-density cells were collected by centrifugation (20 minutes, 4°C, 800 g) from lymphocyte separation media (1.087 g/ml; CedarLane; Hornby, Ontario, Canada; http://www.cedarlanelabs.com) and washed twice with HBSS. Then, cells were gently mixed for 30 minutes at 4°C with saturating amounts of rat-anti-mouse monoclonal antibodies (mAb) specific for lineage markers (CD4, CD5, CD8, GR-1, Mac-1, B220, and TER119; PharMingen; San Diego, CA; http://www.pharmingen.com). mAb-coated cells were incubated with phosphate-buffered solution (PBS) containing 10% FCS, washed twice with PBS, and incubated with sheep-anti-rat immunoglobulin G conjugated to M-450 magnetic beads at a ratio of 4 beads per cell (Dynal Inc.; Lake Success, NY; http://www.dynal.no). Lineage+ BMC conjugated to beads were precipitated by exposure to a magnetic field, and lineage- HSPC were collected from the supernatant. The absence of lineage+ BMC was confirmed by flow cytometry (Coulter Elite; Miami, FL) using fluorescein isothiocyanate-labeled mAb against lineage markers. The average yield of the procedure was 4%-5%, with a viability of 95% as assayed by the trypan blue exclusion test.

Staining with PKH membrane linkers (provided by K. Muirhead, SciGro Co.; Malvern, PA; http://www.maconsultants.com/scigro) was performed by addition of 2 µM freshly prepared dye to 2x 107 cells/ml diluent C (according to manufacturer' instructions). Samples were incubated at room temperature for 5 minutes with gentle mixing. Staining was terminated by addition of four volumes of HBSS containing 10% FCS, cells were collected by centrifugation (400 g, 10 minutes, 4°C), and washed twice with HBSS. The average recovery of this procedure was 80% with a viability of 95% as assayed by the trypan blue exclusion test.

Placement of Bone Windows
To visualize the recipient BM microenvironment, an optical window was implanted over distal femoral epiphysis. The use of windowed femurs was based on two considerations. First, this preparation simulates ex vivo the conditions of microscopic observations performed through bone windows in vivo. Second, in preliminary studies we found that this preparation was superior to longitudinal sections of the femur in preservation of the viability of stromal cells. In some experiments, femurs were excised 4 days after placement of the windows, while in other experiments, the windows were placed ex vivo. After removing the debris from the excised femurs, the cortex was thinned with an electrical drill using 1 mm tip drills. Upon appearance of fissures, the eroded bone fragments were removed with a fine-tip forceps. Dental cement Neocryl (NeoResins; Wilmington, MA; http://www.avecia.com/neoresins) was applied to the bone edge, and the exposed area was covered with a 3 x 5 mm glass window (cover slip #0). The window was secured by dry cement within a few minutes. The same procedure was performed in vivo, using cauterization to control bleeding. Muscle and skin were closed with 4/0 silk sutures and the limb was casted with gauze and Neocryl in a semiflexed position.

In Situ Bone Perfusion
Epiphyseal cartilages were removed, 26G blunt needles were inserted into the femoral lumen, and the contents of the lumen were gently flushed with PBS using a peristaltic minipump (P720; Instech Lab.; Plymouth Meeting, PA; http://www.instechlabs.com). PKH-labeled BMC suspended in PBS were then infused into the femoral lumen at a rate of 0.1 ml/minute. Nonadherent cells were removed by 10 minutes of perfusion with PBS.

In Situ Staining of BM Stroma
The same femoral preparation was used for staining of BM stroma with PKH dyes. After gentle flushing of femoral contents, the bone lumen was superfused with 2 µM PKH67 in diluent C for 10 minutes. Then, the femur was consecutively perfused for 10 minutes with PBS containing 2% FCS and PBS. Viability of stromal cells was assayed by the propidium iodide (PI) exclusion test. As previously shown, the cellular lining of femoral BM was confluent and there was no significant uptake of PI up to 3 hours after excision of the femurs.

Image Acquisition and Data Analysis
Direct observation of flurochrome-labeled BMC in recipient BM was performed through the optical bone window using Nikon Eclipse 800; Melville, NY (http://www.microscopyu.com) and Axiophot (C. Zeiss; Thornwood, NY; http://www.zeiss.com) upright fluorescence microscopes. Images acquired with standard sets of filters (Chroma Technology; Brattleboro, VT; http://www.chroma.com) were recorded with a charge-coupled device (CCD) camera (Hamamatsu Photonics KK; Hamamatsu, Japan; http://www.hamamatsu.com), processed, and pseudocolored to simulate the real hues (Adobe Photoshop software). Images were RGB (color gamut R, red; G, green; B, blue) reconstructed by superposition of three fluorescence layers acquired at the same magnification and position of the stage: the red layer represents PKH26, the green layer represents PKH67, and the blue layer represents UV-excited bone autofluorescence observed with a standard set of DAPI (4'-6'-diamidino-2'phenylindole) filters. The number of cells adherent to BM stroma was calculated using a visibility coefficient of the optical window in our experimental conditions.

Adhesion of BMC to BM stroma was assessed with the aid of laser tweezers (Cell Robotics Intl.; Albuquerque, NM; http://www.cellrobotics.com). Briefly, the optical trap uses the energy of a laser light beam at 1,000 nm to trap particles under focus. Trapped cells can be translocated by deflecting the light beam using a joystick-operated set of mirrors. Preliminary studies showed that the trapping force of the laser tweezers was insufficient for detachment of adherent cells and was effective for translocation of nonadherent cells.

Data are presented as means ± standard deviation (SD) for each experimental protocol. Results in each experimental group were evaluated for reproducibility by linear regression of duplicate measurements. Differences between the experimental protocols were estimated with a post hoc Scheffe t-test and significance was considered at p < 0.05.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In Situ Imaging of BM Microenvironment
Implantation of bone windows allowed detailed inspection of femoral BM in situ (Fig. 1Go). In this experiment, BM stroma of windowed femurs was stained with PKH67 and, after washout of excess dye, 106 PKH26-BMC/ml were infused into femoral lumen at a rate of 0.1 ml/min. Then, nonadherent cells were removed by perfusion with PBS, and images of the BM were acquired through the bone window. This in situ preparation showed that clustering of hemopoietic cells is an intrinsic property of BM microenvironment.



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  		Figure 1. High-resolution in situ imaging of transplanted cells and the infrastructure of BM stroma observed through a bone window. Windowed femurs were excised and cellular lining of the bone marrow was stained with PKH67. The lumen was superfused with solution containing BMC labeled with PKH26. Fluorescence images were acquired at the location delineated in the insert with an Axiophot microscope (C. Zeiss) at a magnification of 63xand the figure was reconstructed from three layers: red = PKH26-labeled cells; green = PKH67-labeled BM stroma; blue = UV-excited bone autofluorescence visualized with a DAPI set of filters. Images were RGB reconstructed from three layers pseudocolored to simulate the real hues. A cluster of five to six cells is seen in a ridge of BM stroma (encircled in red), and adjacent clusters are demarcated by red arrows.

 
In Situ Adhesion to BM Stromal Microenvironment
In a series of controlled experiments, a visibility factor for the bone window was calculated from the ratio between visualized cells and the total number of PKH-labeled BMC harvested from that bone. This factor was found to be 23%-27% for cells labeled with PKH67 and PKH26. To determine whether antigen disparity between BMC and BM stroma affects adhesion, windowed femurs harvested from nonconditioned H2b mice were infused with 105 whole BMC/ml at a rate of 0.1 ml/minute for 10 minutes. After perfusion with PBS for 10 minutes, 96 ± 11 syngeneic (H2b) and 82 ± 9 allogeneic (H2k) adherent BMC were observed (n = 6). Using the window visibility factor, seeding efficiencies of 0.33%-0.38% were calculated for the 105 syngeneic and allogeneic BMC infused. The nonsignificant difference in seeding suggested that BMC-stroma antigen disparity did not affect adhesion per se.

Seeding of Lineage-Negative BMC
To assess the adhesion probability of lineage- versus lineage+ BMC, windowed femurs excised from B10 mice were perfused with 5 x 103 lineage- HSPC (n = 6). Visualization of 58 ± 5 syngeneic and 51 ± 6 allogeneic adherent cells corresponded to seeding efficiencies of 4.6% and 4.1%, respectively. Considering that immunomagnetic isolation yielded 5% lineage- HSPC and using adhesion probabilities of syngeneic whole and lineage- HSPC of 0.38% and 4.6%, respectively, the calculated seeding efficiency for the 95% lineage+ BMC was 0.16%. The calculated seeding efficiency of allogeneic lineage+ BMC was 0.13%. These data correspond to an adhesion affinity of HSPC 29- to 31-fold higher (p < 0.001) than lineage+ BMC (Fig. 2Go).



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  		Figure 2. Lineage- HSPC have a higher affinity of adhesion to BM microenvironment in situ, compared with lineage+ BMC. The seeding efficiency of lineage+ BMC was calculated considering that the yield of immunomagnetic isolation of lineage- BMC was 5%. Data represent mean ± SD of six mice.

 
Is Lodging Limited by Availability of Hematopoietic Niches?
To evaluate whether availability of vacant niches limits the number of lodging cells, windowed H2b femurs were repeatedly superfused (four times) in situ with 5 x 105 H2k BMC/ml PBS (20 minutes, 0.1 ml/minute). Free-floating cells were removed by superfusion of femoral lumen with PBS for 10 minutes, and the number of retained cells was quantified by fluorescence microscopy through the bone window (n = 5). The mean numbers of cells observed were: 850 ± 72, 970 ± 94, 1,172 ± 78, and 1,360 ± 121, after each one of the four repeated perfusions at 1-hour intervals, respectively. These data showed a progressive increase of 15%-25% (p < 0.01 versus constant number of cells) in number of cells retained by BM stroma during four repeated infusions. Although the efficiency of seeding is in the order of 0.33%, the use of 5 x 105 cells might be insufficient to occupy the total number of available niches in femoral BM. Therefore, the number of infused cells was increased to 108 BMC/ml, and sites within the BM were sampled for cell counting through the bone window (n = 6). Under these conditions, the femurs retained large numbers of cells, which increased progressively during four repeated infusions. Figure 3Go presents a low-magnification image that demonstrates the increase in number of seeding cells. These data suggest that availability of hemopoietic niches for engraftment is not a significant rate-limiting factor for cellular seeding in nonconditioned femurs.



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  		Figure 3. There is no limitation of niches available for engraftment. Images were acquired after two consecutive perfusions of femurs with 108 PKH67-labeled allogeneic BMC/ml at a rate of 0.1 ml/minutes. Free-floating cells were removed by perfusion with PBS. Fluorescence images were acquired through the bone window with an Axiophot microscope (C. Zeiss) at a magnification of 10 x. Images were pseudocolored to simulate the real hues.

 
Qualitative Assessment of BMC Seeding
The spatial organization of BMC seeding in the isolated femur preparation showed: A) cells lodged exclusively in femoral epiphyses, whereas the diaphysis was cell free; B) BMC seeded close to the endosteal bone surface of the femur; C) adherent BMC formed clusters (Fig. 1Go), and D) cells penetrated into the depth of stromal microenvironment (revealed by time-lapse microscopy, Fig. 4Go).



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  		Figure 4. High-resolution in situ imaging of transplanted cells interacting with recipient BM stroma. Images were acquired with an Axiophot microscope (C. Zeiss) at a magnification of 100x through the optical window and were RGB reconstructed. Repeated acquisitions at 20- to 30-minute intervals were performed with the aid of a computerized stage that automatically recovered the coordinates of individual cells. In-depth penetration of an adherent PKH26+ cell under BM stromal cells is demonstrated by the change in position and the decrease in fluorescence intensity.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study makes three contributions toward elucidation of the interactions between hemopoietic cells and BM stroma. It was demonstrated that: A) antigenic barriers do not restrict BMC adhesion; B) HSPC have a significantly higher probability of adhesion to the BM stroma compared with lineage+ BMC, and C) repeated BMC infusions into nonconditioned femurs result in an increase in number of adherent BMC.

Equal seeding efficiencies of H2-matched and disparate BMC in situ demonstrate that antigenic barriers did not restrict the adhesion process per se. It is unlikely that similar adhesion rates were caused by a megadose effect, shown to overcome major histocompatibility complex (MHC) barriers in the absence of T cells [20], because the femurs were exposed to a normalized number of BMC. In this study, 105 whole BMC and 5 x 103 HSPC were infused for a rough simulation of in vivo injection of 2-10 x 106 BMC, considering that one femur consists of sim;6% of the total hemopoietic space in the mouse [21], and an approximate 5% yield of the HSPC purification procedure. The experimental data present several characteristics of BMC seeding. Considering that cobblestone formation was inhibited in MHC class Ia-disparate stromal cell cultures [11], our findings imply that antigen disparity may affect later stages of engraftment, but not adhesion itself. In the face of MHC-unrestricted adhesion, the higher affinity of BMC to antigen-matched BM [10] may be a result of facilitation of HSPC engraftment by donor-antigen-matched osteoblasts and stromal cells [22,23]. In addition, the effective number of circulating allogeneic BMC might be reduced in vivo by excessive entrapment in host reticuloendothelial system [6–9]. This is a reasonable explanation for the fact that syngeneic and allogeneic BMC adhered at equal rates in situ whereas the seeding efficiency of allogeneic BMC was one order of magnitude lower in vivo (manuscript in preparation).

Repeated infusions of BMC into femoral lumen over a short period of time resulted in a gradual and constant increase in the number of adherent BMC. Although initial infusion of either 1 or 200 million cells resulted in retention of a larger number of BMC compared with the subsequent infusions, the cumulative profile of seeding suggests that there was no limitation in availability of stromal sites for seeding. From the experimental perspective of direct microscopic observations of recipient BM, the hemopoietic niche for seeding is composed of several stromal cells over an area of 50-100 microns that hosts initial seeding of 6-10 cells. Considering the complexity of the homing-lodging process, it will be interesting to determine experimentally whether a causal relationship exists between the number of homing/seeding cells and the levels of donor hemopoietic chimerism. Remarkably, the in situ seeding efficiency of lineage- HSPC was sim;30-fold higher than that of BMC expressing lineage markers. Superior adhesion of HSPC has been reported in previous studies [8,9]. In view of the efficient hematopoietic reconstitution of myeloablated recipients by one or a few stem cells [24,25], it is possible that the number of seeding BMC represents a statistical probability for engraftment of the one omnipotent stem cell.

In summary, repeated infusions of BMC resulted in a progressive increase in the number of adherent cells in nonconditioned femurs in situ. Although seeding appears to be the rate-limiting factor of hemopoietic cell homing to the BM, there was no apparent limitation in the number of available niches in nonconditioned femurs. Corroborating previous in vivo observations, adherent cells formed clusters in proximity to endosteal bone surface. This pattern of hemopoietic cell lodging is consistent with stromal regulation of seeding with a highly organized topology of femoral BM microenvironment. The adhesion process itself was not restricted by antigenic barriers, and stem cells presented a remarkable affinity for BM stroma, approximately 30-fold higher than lineage+ BMC. Direct observation of BMC behavior at the level of BM stroma provides important experimental tools for dissection of the complex process of hemopoietic stem cell engraftment.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Mrs. Judy Montibeller and Mrs. Lisa McGown for the excellent technical assistance. The stimulating discussions and comments of Dr. Sallie S. Boggs and Dr. Tatiana Zorina (University of Pittsburgh Medical Center, Pittsburgh, PA) are gratefully acknowledged.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received October 12, 2001; accepted for publication October 23, 2001.



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