Heterogeneity of engrafted bone-lining cells after systemic and local transplantation

Abstract

The outcome of various osteoprogenitor-cell transplantation protocols was assessed using Col1a1-GFP reporter transgenic mice. The model requires the recipient mice to undergo lethal total body irradiation (TBI) followed by rescue with whole bone marrow. When the mice are rescued with total bone marrow from a Col1a1-GFP transgenic mouse, green fluorescence protein (GFP)-positive donor cells can be observed on most endosteal and trabecular bone surfaces. Although the cells express an osteoblast-restricted GFP, they fail to progress to osteocytes, do not form a mineralized matrix, and do not generate bone nodules in vitro. However when calvarial progenitor cells derived from the same transgenic mice are injected into the bone marrow space, osteogenesis by the donor cells is observed. Using different GFP colors that distinguish the donor and recipient osteoblasts, commingling of the 2 cells types is observed along the mineralizing osteoblast surface as well as within the osteocyte population of the endosteal bone. Despite the ability of the injected progenitor cells to produce bone within the injected bone, they lack the ability to form mineralized bone nodules when explanted to primary osteoblast culture. These reagents and imaging protocols will be useful in evaluating other cells having a better progenitor potential than calvarial-derived stromal cells.

Figure 1.

Patterns of GFP expression in the bones of donor mice. (A) Within trabecular bone, 3.6-green is strongly expressed on the bone surface and can extend into regions of recently formed bone. The pink cellular regions surrounded by the GFP-positive cells are bone marrow. (B) The 2.3-green is strongly expressed in bone surface cells and in the osteocytes throughout the bone. (C) The 3.6-blue expression resembles 3.6-green. (D) Nondecalcified trabecular bone of trabecular from a 3.6-green donor mouse labeled with XO 2 days before it was killed. The red label is closely associated with the majority of the GFP-positive cells although there are regions of XO labeling without overlying GFP-positive cells (arrows). (A-D) Original magnification, × 200.

Figure 2.

Engraftment of bone lining cells after transplantation with TBM from 3.6-blue donor mice. (A-B) An image of frozen decalcified bone (A, femur; B, tibia) from a 3.6-green recipient rescued with systemic TBM from a 3.6-blue donor. The 3.6-blue cells (arrows) are present on the bone surface admixed with 3.6-green cells. Note that the 3.6-blue cells do not extend into the osteocyte layer. (C-D) An image of the bone from a 2.3-green host that received an intramedullary injection of TBM from a 3.6-blue mouse. The 3.6-blue cells line the bone surface whereas the host cells express 2.3-green on the bone surface and within osteocytes. (C) Injected tibia. (D) The contralateral femur also demonstrates 3.6-blue cells, indicating engraftment by a systemic route. (A-D) Original magnification, × 200.

Figure 3.

Engraftment of bone lining cells after transplantation with TBM from 3.6-green donor mice. (A-B) Engraftment of 3.6-green donor-derived TBM cells. The recipient was a 3.6-blue mouse. (A) Injected tibia. (B) Ipsilateral femur. Now 3.6-green cells (arrows) line the bone but do not extend into the bone matrix. (C-D) Frozen nondecalcified section of bone from a nontransgenic recipient rescued by an intramedullary injection of TBM from a 3.6-green mouse. The recipient mouse was injected with XO 2 days prior to being killed. The injected femur (C) and the contralateral tibia (D) show bone surface 3.6-green cells but no associated XO labeling. (A-D) Original magnification, × 200.

Figure 4.

Temporal sequence of engraftment of donor-derived 3.6-blue bone lining cells in a 2.3-green recipient. Mice from the same TBI/rescue experiment were examined at days 7, 14, 21, 28, 56, and 78 after systemic transplantation. Shown here are representative images at day 14 (A, when 3.6-blue cells first appear, arrows), day 28 (B), and day 78 (C). Digital thresholding and counting of the number of 3.6-blue cells in each image was (A) 28, (B) 37, and (C) 48. (A-C) Original magnification, × 200.

Figure 5.

Fluorescent images of marrow stromal-cell culture grown in a 6-well plate. Each panel is formed from × 25 to × 2.5 original magnification images that are tiled together, and represents approximately 60% of the surface area of a cell-culture well. The same well is recorded at increasing days of culture (days 10-28) and the fluorescent spots that develop over time are produced by the strong GFP expression within the bone nodule. At day 21 the nodules that have mineralized are visualized by the XO staining that colocalized with the GFP nodules (Aiv,Biv). (A) Profile of developing bone nodules from an irradiated 3.6-blue transgenic mouse to illustrate the bone nodule-forming potential of the osteoprogenitor population in a bone marrow stromal culture. Images from days 14 (Ai), 18 (Aii), and 21 (Aiii) show nodule progression and the extent of mineralization at day 21 is shown by XO staining (Aiv). (B) Stromal-cell culture derived from a 2.3-green recipient mouse that was rescued by WBMT from a 3.6-blue donor. Although the number of host-derived 2.3-green bone nodules is diminished relative to nonirradiated controls (not shown), only host-derived nodules are observed. Images from days 14 (Bi), 18 (Bii), and 21 (Biii) show nodule progression and the extent of mineralization at day 21 is shown by XO staining (Biv). (C) Fluorescent image (original magnification, × 50) of a 2.3-green nodule as seen in the × 25 (original magnification) tiled image in panel B. The 2.3-green nodule (Cii) shows strong mineralization (Ciii). Panel Ci shows the cluster of 3.6-blue donor-derived cells (arrowheads) that happened to form adjacent to this 2.3-green nodule. It has a distinctly different morphology than the bone nodule (BN) that in this image is blue in color because the 2.3-green signal seen in panel Cii spills through the GFPcyan filter. Panel Civ shows the composite image of the 3.6-blue cell cluster and 2.3-green/XO bone nodule. (D) Fluorescent and phase-contrast image (original magnification, × 100) of 3.6-blue cells that develop in a stromal-cell culture derived from a recipient mouse rescued with TBM from a 3.6-blue donor mouse. The expanding cluster of cells does not become a multilayered nodule and does not stain with XO. Images from days 10 (Di), 21 (Dii), and 28 (Diii) show the cellular progression. The overlay (Div) with the phase-contrast image shows the 3.6-blue cells relative to the nonfluorescent cells in the field of view.

Figure 6.

High-power images of bone from nontransgenic recipients of an intramedullary injection of calvarial stromal cells. (A-B) Calvarial progenitor cells from a 2.3-green donor mouse injected into a nontransgenic recipient and rescued with TMB from a 3.6-blue donor. Selected images (original magnification, × 200) show engraftment of diaphyseal bone (A) and formation of new trabecular bone (B) and illustrate the association of XO label with 2.3-green cells. Because the 2.3-green marker gene remains active in osteocytes, marked donor cells become incorporated into the host bone and within the newly formed trabecular bone. In contrast, the 3.6-blue cells (arrows) remain on the bone surface and are not associated with the XO label. These images are obtained from the scan shown in Figure 7A. (C-D) Calvarial progenitor cell from a 3.6-green donor mouse injected into a nontransgenic recipient that was rescued TBM from a 3.6-blue donor. Selected images (original magnification, × 200) show engraftment of pre-existing bone (C) and formation of new trabecular bone (D) that appears to be derived from the fibroblastic tract. Both structures exhibit donor-derived osteoblasts (strong GFP signal) in association with the XO label. The fibroblastic-shaped cells along the injection tract are not associated with bone mineralization and run between regions that have produced trabecular bone. These images are obtained from the scan shown in Figure 7B. (E-F) Nondecalcified sections of a femur showing engraftment of 3.6-blue donor-derived calvarial cells after injection into a 3.6-green host that was rescued with nontransgenic TBM. (E) Trabecular region. (F) Cortical region. In both images there is commingling of 3.6-blue donor and 3.6-green host cells on the bone surface that is associated with the XO label.

Figure 7.

Histology from a nontransgenic recipient mouse that received a transplant via intramedullary injection of TBM from a 3.6-blue donor and expanded calvarial progenitor cells from a 3.6-green or 2.3-green donor. The recipient mice were injected with XO 2 days prior to being killed. (A) Full-length section of bone transplanted with calvarial progenitor cells from a 2.3-green donor mouse and TBM from a 3.6-blue donor. The needle tract (NT) is less obvious with the 2.3-green marker gene because it only visualizes mature osteoblasts. Distal engraftment is apparent in the femoral neck (FN) region. (B) Full-length section of bone transplanted with calvarial progenitor cells from a 3.6-green donor mouse and TBM from a 3.6-blue donor. The needle tract (NT) is composed of newly formed trabeculae and a core of fibroblastic-shaped progenitor cells. Bone lining cells of donor origin are present at sites distal to the injected cells such as the femoral neck region.

Figure 8.

Primary osteoblast cultures derived from bone chips of a 2.3-green or 3.6-green recipient mouse that received a transplant of calvarial progenitor cells from a 3.6-blue donor. This scenario is as illustrated in Figure 6E-F. (A) Bone chips after 7 days in primary culture and in early secondary culture showing the 2 colors of cells embedded within and growing from the bone fragments. The 3.6-green chips (Ai) show 3.6-green cells on the plate surface. The 3.6-blue cells that grow out from these chips can be appreciated at higher power in the secondary culture (Aii). The 2.3-green chips (Aiii) show the 2 colors of GFP within the bone. No 2.3-green cells are seen in the early outgrowth cells (Aiii-iv) because the transgene is not activated prior to bone nodule formation. At high power in the early secondary culture the 3.6-blue cells are evident (Aiv). (B) Mineralizing bone nodule (Biii) derived from the 3.6-green recipient mouse (Bii) that is adjacent to a cluster of 3.6-blue cells (Bi). An overlay of the 3 images (Biv) shows XO staining of the 3.6-green cells but not the 3.6-blue cells. (C) Fate of a donor-derived 3.6-blue cell cluster (Ci) that is surrounded by host-derived 2.3-green cells (Cii) that are producing a mineralized matrix (Ciii). The overlay image (Civ) shows faint XO staining over the 3.6-blue cells relative to the strong staining over the 2.3-green cells. (A-C) Original magnification, × 100.