Marker tolerant, immunocompetent animals as a new tool for regenerative medicine and long-term cell tracking

Immune response of wild-type rats after syngeneic transplantation of cells from hPLAP-tg rats

In order to test whether peripheral blood cells and bone marrow cells from transgenic donors would survive and proliferate in normal wild-type rats of the same inbred strain, we intravenously injected peripheral blood leukocytes (PBL) or bone marrow cells (BMC) from hPLAP-tg donor rats daily for 1 week into wild-type F344 rats. However, histological analysis of lung, liver, spleen, lymph nodes, and bone failed to show any evidence of hPLAP-expressing cells from transgenic donors in the wild-type recipients, suggesting that it was not possible to syngeneically transplant cells from transgenic donors into wild-type rats (Fig. 1). This finding prompted us to ask the question whether expression of the transgene, and especially membrane expression of hPLAP, would induce the formation of antibodies to hPLAP in the wild-type recipients. FACS (Fluorescence-Activated Cell Sorter) analysis revealed that almost all PBL and BMC showed membrane expression of the hPLAP enzyme in hPLAP-tg rats (Fig. 2A and 2B). Immunization phenomena against hPLAP could result in immune-mediated destruction of any hPLAP-expressing cells in wild-type recipients, because the hPLAP enzyme represents a foreign protein to the rat immune system.

To test whether wild-type recipients develop antibodies against hPLAP after injection of cells from transgenic donor rats of the same strain, we intraperitoneally or subcutaneously injected wild-type F344 rats with PBL from hPLAP-tg rats three times with an 11-day interval between the injections. Subsequently, serum from naïve wild-type F344 rats or from wild-type F344 rats that had received injections of PBL from hPLAP-tg rats was incubated with PBL from transgenic rats, and the presence of antibody-labeled cells was examined by FACS analysis. As expected, serum from naïve wild-type rats that had never been in contact with transgenic cells did not react with PBL from transgenic rats (Fig. 2C). However, as shown in Fig. 2C, repeated intraperitoneal injection of transgenic PBL induced an antibody response in wild-type rats. Identical results were obtained with subcutaneous injection (data not shown). To test a cellular immune response to hPLAP in wild-type rats, we performed mixed lymphocyte reaction protocols. When spleen cells from naïve wild-type rats were co-cultured with transgenic spleen cells, we did not observe increased cell proliferation (Fig. 2D). However, cell proliferation was induced when spleen cells from wild-type rats that had previously been exposed to hPLAP-expressing cells were co-cultured with transgenic cells. These data demonstrate that exposure to hPLAP-tg cells induces immunization phenomena in wild-type rats, explaining the abovementioned failure of the experiments aimed at the syngeneic transplantation of transgenic cells into normal wild-type recipients. To verify this hypothesis, we transplanted skin from hPLAP-tg F344 rats into naïve, 3-month-old wild-type F344 recipients (n = 3). All recipients rejected the transgenic transplants within 2 weeks.

Induction of tolerance by bone marrow transplantation

So far, our results indicated that hPLAP-tg rats could serve as a very attractive model for regenerative therapies because of ubiquitous and strong expression of the transgene, and excellent preservation of the marker enzyme in paraffin and MMA sections. However, immune-mediated rejection of hPLAP-tg cells by wild-type rats exposed to cells from transgenic donors made it impossible to use this model for cell tracking. Therefore, we thought of a way to overcome these problems. Previous studies have shown that tolerance against intracellular xenogeneic genes (for example GFP or neo, neomycin phosphotransferase) can be induced by busulfan treatment of wild-type C57BL/6 mice, and subsequent transplantation of bone marrow from GFP transgenic C57BL/6 mice [8], or by transplantation of autologous hematopoietic stem cells transduced with a neo marker gene in monkeys after total body irradiation [9]. Based on these results, our idea was to combine established methods of tolerance induction to a specific marker with subsequent transplantation of otherwise syngeneic cells labeled with the same marker in order to avoid immune-mediated rejection of the labeled cells.

To test the idea of employing marker tolerant animals as a model for regenerative therapies, we initially used lethal irradiation and transplantation of bone marrow from hPLAP-tg F344 rats into wild-type F344 rats as proof-of-principle to prevent the immune response after injection of transgenic cells into wild-type rats. Because the rats are of the same inbred strain, graft-versus-host reactions can be ruled out a priori. Fig. 3A–C shows that in irradiated wild-type rats transplanted with hPLAP-tg bone marrow [WT (hPLAP-BMT)] all hematopoietic cells are of donor origin, and stably express hPLAP, 4 weeks post-transplantation. The superb histological detection quality of hPLAP permits a clear distinction between wild-type cells of recipient origin and hPLAP-expressing cells of donor origin (Fig. 3D–F). We found that a significant portion of capillary endothelial cells, but not epithelial or mesenchymal cells such as muscle cells, were of donor origin in heart, kidney, and lung of irradiated WT (hPLAP-BMT) rats (Fig. 3D–F and data not shown).

Utility of marker tolerant rats for cell tracking in regenerative medicine

An example of the usefulness of marker tolerant animals for cell therapeutic studies is shown in Fig. 4A and 4B. In this experiment, BMC from hPLAP-tg rats were injected into the knee joint of irradiated WT (hPLAP-BMT) rats. One week post-injection, the transgenic BMC had formed a dense, 2 – 3 cells wide, layer on the intact articular cartilage surface. This finding suggests that these cells underwent proliferation on the cartilage surface earlier. After 4 weeks, the genetically labeled cells displayed a more differentiated fibroblast- or chondrocyte-like phenotype and appeared to have become integrated into the surrounding cartilage tissue of wild-type origin. This experiment underscores the potential usefulness and strengths of this technology, and clearly demonstrates that mesenchymal precursors present in native bone marrow can adhere to the articular cartilage surface and can, subsequently, differentiate into a fibroblastic or chondrocytic phenotype. Although potentially marred by immune-mediated rejection, similar findings have been reported in a goat model of osteoarthritis, in which injection of autologous mesenchymal stem cells harvested from bone marrow, expanded in culture, and transduced with GFP stimulated regeneration of meniscal tissue and retarded progressive joint destruction [11].

It is clear that the background of labeled hematopoietic cells is an important shortcoming of irradiated WT (hPLAP-BMT) rats, limiting this model to the study of non-hematopoietic tissues. To test whether neonatal exposure of wild-type F344 rats with cells from hPLAP-tg F344 rats results in a sustainable tolerance to transgenic tissue, we subcutaneously injected neonatal F344 wild-type rats (n = 6) with whole blood from hPLAP-tg rats. Four months later, skin of hPLAP-tg rats was transplanted into these neonatally tolerized rats. The transgenic skin grafts were accepted in 6 out of 6 rats, while a control littermate not having received transgenic blood promptly rejected the transgenic graft. Fig. 5A shows the margin of the transgenic skin transplant in a marker tolerant rat, 4 weeks post-transplantation. It is evident that the border between wild-type and transgenic tissue is sharply delineated, and that capillaries from transgenic donor tissue invade the surrounding wild-type skin (Fig. 5B).

These data clearly demonstrate that sustainable marker tolerance also can be achieved by neonatal exposure of wild-type F344 rats to hPLAP-expressing cells of the same rat strain. Thus, any kind of hPLAP-labeled cell or tissue from F344 rats can be transplanted and studied in these marker tolerant F344 rats in the absence of immune-mediated rejection, and also in the absence of background labeling. To examine the utility of this model for long-term cell tracking, we intravenously injected neonatally tolerized F344 rats with transgenic BMC (n = 4), and assessed the occurrence of hPLAP-labeled cells 2 months after cell administration in various organs. Two months post-injection, we found hPLAP-labeled cells in lymph nodes, but not in liver, heart, kidney, bone, or cartilage (Fig. 5C–F and data not shown), with the exception of extremely rare hPLAP-labeled endothelial cells in heart and kidney (data not shown). hPLAP-stained cells were totally absent in neonatally tolerized F344 controls which had not received transgenic BMC as evidenced by histological and FACS analysis, ruling out the possibility of a background of hPLAP-labeled cells in these rats (data not shown).

Animals

All experimental procedures were conducted in compliance with prevailing animal welfare regulations. Heterozygous male or female hPLAP-tg F344 rats were mated with wild-type F344 rats, and the resulting wild-type and heterozygous transgenic offspring were genotyped by enzyme histochemistry using a drop of tail blood as described [4]. The rats were housed in pairs at 24°C and a 12 h/12 h light/dark cycle with free access to tap water and commercial rat diets (Altromin 1324 for maintenance and 1314 for breeding, Altromin, Lage, Germany).

Histology and hPLAP detection

Because hPLAP is sensitive to fixation with formalin [2], tissues and bones were harvested and were fixed in 40% ethanol at 4°C for 48 h. Subsequently, the tissue specimens were dehydrated and embedded in paraffin or in a modified MMA embedding mixture that preserves enzyme activities and can also be used for immunohistochemistry [3]. Five-μm-thick paraffin and MMA sections were cut with a HM360 microtome (Microm, Walldorf, Germany), and were mounted on slides pre-treated with 3-aminopropyltriethoxy-silane (APES, Sigma-Aldrich, Deisenhofen, Germany).

Paraffin sections were deparaffinated using xylene, whereas MMA sections were deplasticized using 2-methoxyethylacetate as described [3]. Deparaffinated and deplasticized sections were rehydrated and heated at 65°C for 30 min in deionized water to block endogenous alkaline phosphatase activity. Sections were then incubated in TRIS buffer (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 5 mM MgCl₂) containing 0.17 mg/ml of the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) at room temperature overnight. Subsequently, sections were counterstained with nuclear fast red (Sigma), dehydrated, and mounted using Vectamount (Vector, Burlingame, CA, USA).

Flow cytometry

Peripheral whole blood was taken from a tail vein. BMC were harvested from the tibia or femur by short centrifugation of the bone, and were dispersed in PBS by repeated pipetting. For labeling of cell surface hPLAP on peripheral blood and BMC, cell suspensions (approximately 10⁶ cells each) were incubated with a monoclonal mouse anti-hPLAP antibody (supernatant of clone 8B6, Dako) diluted 1:20 for 30 min on ice. Non-immune mouse IgG₁ (MOPC-21, Sigma) and cell suspensions from wild-type rats were used as negative controls. After washing twice, the cells were incubated with rat-adsorbed, FITC-labeled goat anti-mouse IgG antibody (Sigma) for 30 min on ice. Prior to FACS analysis, erythrocytes were hemolyzed in blood samples using FACS lysing solution (Becton Dickinson, Heidelberg, Germany). The analyses were performed on a FACScan flow cytometer using CellQuest Pro software (Becton Dickinson, Heidelberg, Germany).

To examine the presence of circulating anti-hPLAP antibodies in serum of wild-type rats that had previously received cells from hPLAP-tg rats, wild-type and transgenic peripheral blood cells were incubated for 30 min on ice with 10 μl undiluted serum from naïve wild-type rats or from wild-type rats that had received transgenic PBL earlier. Using FITC-labeled goat anti-rat IgG antibody (Serotec, Harwell, UK) as secondary antibody, the samples were processed and analyzed as described above.

Transplantation of transgenic blood and bone marrow cells into wild-type recipients

In order to determine the fate of genetically labeled donor cells in wild-type recipients, we intravenously injected 36 wild-type F344 rats with PBL or BMC (n = 18 each) isolated from hPLAP-tg F344 rats. Transgenic rats were killed by exsanguination from the abdominal aorta under ketamine/xylazine anesthesia. PBL were harvested using a density gradient centrifugation kit (NycoPrep 1.077A, Axis-Shield, Oslo, Norway), washed several times and resuspended in PBS. The percentage of viable cells after the isolation procedure was > 90% (trypan blue exclusion). At the same time, BMC were harvested and washed as described above. After isolation, all cells were resuspended in PBS, and were kept on ice until use. Isolated genetically labeled PBL or BMC were injected intravenously at a dose of 25 × 10⁶ or 5 × 10⁶ cells per animal, respectively, into sex-matched wild-type recipients via a lateral tail vein. All recipients received daily injections of genetically labeled cells over 7 consecutive days (days 1 – 7). The recipient rats were killed on days 8, 15, 22, or 28 (n = 4 – 6 each for PBL and BMC recipients) by exsanguination from the abdominal aorta under ketamine/xylazine anesthesia. From all recipient animals a variety of soft tissues and bones were harvested, fixed in 40% ethanol at 4°C for 48 hours, and processed as described above.

Humoral and cellular immune response to transgenic cells

To examine the development of antibody production against transgenic cells, we repeatedly exposed wild-type rats to PBL from hPLAP-tg donors. The PBL were harvested from hPLAP-tg rats as described above, and resuspended in PBS. Subsequently, 1 × 10⁶ PBL were subcutaneously or intraperitoneally injected three times into wild-type F344 rats (1 rat each) with an 11-day interval between the injections. At baseline, and 10 days after each injection, blood was drawn from a tail vein of the wild-type recipients to obtain serum. For mixed lymphocyte reaction, spleen cells were isolated by density gradient centrifugation (NycoPrep 1.077A). The spleen cells from naïve wild-type rats or from wild-type rats previously exposed to transgenic cells (3 intraperitoneal injections at 10-day intervals) were co-cultured with transgenic spleen cells for 5 days at a total density of 5 × 10⁵ cells per well (2.5 × 10⁵ wild-type and transgenic cells each). During the last 18 hours of culture, ³H thymidine (2 Ci/mmol) was added to quantify cell proliferation. ConA (10 μg/ml) was used as a positive control.

Lethal irradiation and bone marrow transplantation

To transplant bone marrow from hPLAP-tg rats into wild-type F344 rats, wild-type animals (n = 6) were lethally irradiated with a dosage of 8.5 Gy at 0.9473 Gy/min using a cobalt-60 irradiator (Eldorado, Atomic Energy of Canada, Ottawa, Canada). Four hours after the irradiation, the rats were intravenously injected with 3 – 4 million BMC isolated from hPLAP-tg rats as described above. To rule out unsuccessful engraftments, the same injection of freshly prepared transgenic BMC was repeated 24 hours after the irradiation.

Intraarticular injection of genetically labeled cells

Next, we wanted to explore whether intraarticularly injected BMC from hPLAP-tg rats would survive, proliferate, and differentiate in the knee joint of irradiated WT (hPLAP-BMT) rats. For this experiment 6 male and female wild-type F344 rats at the age of 3 months were lethally irradiated and transplanted with BMC from hPLAP-tg F344 rats. Two weeks after the bone marrow transplantation, 5 × 10⁶ BMC isolated from hPLAP-tg rats were injected once into the cavity of the left or right knee joint under medetomidine/midazolam/fentanyl anesthesia. The rats were killed 7 days or 28 days (n = 3 each) after the intraarticular injection by exsanguination from the abdominal aorta under ketamine/xylazine anesthesia. The intact knee joints were harvested, trimmed, fixed in 40% ethanol at 4°C for 48 hours, and embedded in MMA. Frontal 5-μm-thick sections were cut as described above.

Induction of tolerance by neonatal exposure to transgenic cells

To induce tolerance to the marker gene by an alternative method, 6 neonatal wild-type F344 rats pups, within 4 hours after birth, were subcutaneously injected once with 20 μl of whole blood from hPLAP-tg F344 donors. One control litter mate did not receive transgenic cells. Four months later, all 7 rats received skin grafts (approx. 1 cm²) from sex-matched hPLAP-tg donors at the back under anesthesia with medetomidine/midazolam/fentanyl. The grafts were controlled daily. Four weeks post-transplantation, biopsies were taken under anesthesia from the margin of the skin grafts, fixed in 40% ethanol at 4°C for 48 hours, embedded in paraffin, sectioned at 5 μm thickness, and stained for hPLAP enzyme activity as described above.

Tracking of transgenic cells in neonatally tolerized rats

Tolerance to hPLAP in F344 WT hosts was induced and confirmed by skin grafts as described above. At the age of 4 months, 4 neonatally tolerized WT rats were intravenously injected once with 10⁸ BMC from hPLAP-tg donors, and were killed 2 months post-injection. Various tissues were harvested, fixed in 40% ethanol at 4°C for 48 hours, embedded in paraffin or MMA, sectioned at 5 μm thickness, and stained for hPLAP enzyme activity as described above. Neonatally tolerized control rats were killed without prior injection of labeled cells to rule out a background of labeled cells in the marker tolerant recipients.

Statistical analysis

Statistics were computed using SPSS for Windows 14.0 (SPSS, Chicago, IL). The data of the mixed lymphocytes reaction experiments were analyzed using one-way analysis of variance (ANOVA). When the analysis of variance performed over all groups indicated a significant (p < 0.05) difference among the groups, statistical differences between two groups were subsequently evaluated with Fisher's least significant difference test. P values of less than 0.05 were considered significant. The data in Figure 2D are given as means ± SEM.