A Wide Variety of Tissues were Successfully Engrafted in Mouse Ear Pinna and Survived Long-term
To test whether different tissues can engraft in mouse ear pinna, we transplanted a variety of tissues and pieces of organs including aorta, kidney, bone marrow, spleen, lymph node, skeletal muscle, adrenal gland, ovary, lung, trachea, and thyroid gland from adult B6 CD45.1 mice into the pinnae of the ear in syngeneic recipients. The recipient mice were sacrificed at different time points after transplantation. The engrafted tissues were then harvested for histological analyses. The results are shown in
Figure 1 Histological analysis of adult tissues transplanted into ear pinnae (H&E stain, scale bar=100 µm).
- Aorta: it demonstrated a patent vascular structure resembling the “normal aortic tissue.” Since there was no vascular flow, the central lumen was not fully patent at the later date.
- Kidney: renal tissue was observed up to 8 weeks at the implantation site. Glomeruli and interstitial stromal tissue was well preserved.
- Bone marrow: it demonstrated the remodeling of the bony structures with residual cellular matrix at 8 weeks.
- Spleen: lymphoid aggregates were evident in the engrafted splenic tissue.
- Lymph node: the well-preserved lymphoid follicles were observed at the implantation site for up to 4 weeks.
- Skeletal muscle: viable skeletal muscle exemplified by the presence of the striation of the muscle fibers and the peripheral localization of the nuclei were demonstrated.
- Adrenal gland: it showed the adrenal cortical cells with clear cytoplasm. The cells were forming sheets resembling the “normal” adrenal tissue.
- Ovary: the ovary sections showed normal ovarian tissue. The germ cells appeared to be degenerated quickly.
- Lung: the bronchial and alveolar structures were well preserved up to 8 weeks in the engrafted lung tissue.
- Trachea: the tracheal tissue was exemplified by the presence of cartilage and the lining epithelium. Well preserved cilia were observed.
- Thyroid gland: the follicular structure that is characteristic for thyroid tissue could be observed in the engrafted thyroid gland up to 4 weeks after implantation.
We also used near-term fetal donors because some of the tissues could not be obtained in a sterile manner from adult donors (e.g., intestine) or the adult tissues did not function as well as fetal tissues (e.g., thymus). The histological results are shown in
Figure 2 Histological analysis of fetal tissues transplanted into ear pinnae (H&E stain, scale bar=100 µm).
- Heart: cardiac muscle could be observed up to 8 weeks after transplantation.
- Stomach: the gastric tissue could be well maintained up to 8 weeks. The parietal cells and the chief cells were noticed in the glandular compartment.
- Ileum: the properly oriented small intestinal villi were evident in the engrafted ileum. At the base of the crypts, Paneth cells were readily seen. The intestinal lumen was also patent.
- Colon: a patent colonic structure was seen in the engrafted colon. The colonic mucosa was well-preserved. The lining epithelium contained normal mucin.
- Thymus: the lymphoid tissue was seen at the implantation site, which resembled the “normal” thymic tissue.
These histological data demonstrated that a wide variety of tissues survived in this area of the ear for up to 8 weeks. Histologically the morphology of these tissues was preserved.
Survival and Function of Neonatal Heart Tissue Engrafted into Ear Pinna
One of the critical questions is whether the engrafted tissues can function. To answer this, we first verified whether cardiac muscle truly survived in the transplanted heart graft using Masson’s Trichrome stain. As shown in , the transplanted heart muscle as shown in red could be easily visualized in the space anterior to the cartilage of the ear. As expected, the transplanted fetal heart continued to beat more than 100 days after transplantation (Video S1). Moreover, an electrocardiogram (ECG) that was performed more than 60 days after transplantation demonstrated that the ectopic heart could produce an expected ventricular depolarization (). Local bipolar recordings of another transplanted heart also demonstrated intact atrial and ventricular depolarization with intermittent AV nodal conduction ().
Survival and function of near-term fetal heart tissue engrafted into ear pinna.
T Cell Reconstitution in Mice Engrafted with Thymic Tissue in the Pinna of the Ear
To further demonstrate that it is not simply the survival of the organ in this space but that the organ can actually function as would be predicted based on its origin, we transplanted a thymus from a B6 CD45.1 neonatal mouse (<48 hours old) into the pinna of a BALB/c nude animal (CD45.2). We then monitored the development of T cells. Since nude animals have competent lymphoid precursors but lack a thymus, new T cells, especially functional T cells would have to be generated from the transplanted thymus. Indeed, we were able to detect signal joint T-cell receptor rearrangement excision circles (siTREC, a marker for active thymopoiesis 
) in nude mice engrafted with thymic tissue in the pinna of the ear (). Moreover, since the T cells from the host and within the thymus differ in the CD45 isoform, we were able to determine the origin of the peripheral T cells as shown in (thymus donor: CD45.1+
, host: CD45.1−
). New T cells, especially CD4+
cells, were readily detectable in the peripheral blood beginning approximately 8 weeks after transplantation of the donor thymus (). Moreover, these T cells arose predominantly from the BALB/c marrow precursors, which were then educated in the donor thymus (). As demonstrated in , ear-thymus recipients had significantly higher numbers of CD4+
T cells in peripheral blood than the sham transplanted group 8 weeks post transplantation (P<0.05).
T cell reconstitution in mice engrafted with ear-thymus.
To determine whether transplantation of thymic tissues in the ear pinna truly restores T cell function in nude mice, we transplanted third-party skin grafts from C3H/HeJ into the animals that had received either a piece of thymic tissue or just a sham operation. As can be seen in , the animals receiving the thymic transplant were able to reject the donor graft within 60 days after skin transplantation while the animals receiving a sham thymic transplant did not reject their skin graft (P<0.0001).
Ear-tissue can be Visualized at the Cellular Level in Living Animal
The data above clearly demonstrated that not only were the tissues surviving in this resident space but also that they were able to function as would be expected. To demonstrate whether the engrafted tissues can be imaged in the ear pinna, we utilized enhanced green fluorescent protein (EGFP)- or discosoma red fluorescent protein (DsRed)-transgeneic donor mice. More than two weeks after transplantation, engrafted ear-tissues were imaged in live animals using two-photon microscopy. shows the location of the transplanted organs in the ear pinna. Images of engrafted fluorescent tissues are shown in . Both EGFP+ muscle cells and small intestinal villi engrafted in the ear pinnae could be imaged clearly (). As shown in and Video S2, EGFP+ heart muscle fiber was clearly evident in the beating heart graft. EGFP+ glomeruli could also be recognized in the ear-kidney graft ( and Video S3). In and Videos S2 and S3, rhodamin B labeled dextron (shown in red) was injected to visualize the blood vessel. In order to study cell-to-cell interaction using this model, we transplanted EGFP+ ear-thymus recipients with DsRed+ T cell depleted bone marrow cells 8 weeks after thymic transplantation. Imaging was then performed 4 weeks after bone marrow transplantation. As shown in and Video S4, single EGFP+ and DsRed+ cells could be clearly recognized as deep as 143 µm below the surface. These data clearly demonstrated that tissues engrafted in the pinna of the ear could be visualized at the single cell level in living animals.
Ear-tissue can be visualized at the cellular level in living animal.
Visualizing Radiation-induced Thymocyte Apoptosis in Ear-thymus Graft
To demonstrate whether this novel model can be used to study biological questions, we followed the fate of thymocytes in the ear-thymus graft after irradiation using intravital two-photon imaging. A nude BALB/c chimera containing DsRed+ hematopoietic cells were transplanted with a thymus from a B6 CD45.1 neonatal mouse (<48 hours old) into the ear pinna. Five weeks later, the ear pinna containing thymus graft was irradiated with a lethal dose of radiation (8.5 Gy). Cell apoptosis was then followed in the thymus graft over time by in vivo two-photon imaging after injection of carboxyfluorescein (FAM)-FLIVO™, a green fluorescent probe specific for apoptosis. In this model, almost all hematopoietic cells including thymocytes are DsRed+ (red) and the apoptotic cells are green. As demonstrated in , only 2.7% apoptotic cells were observed before irradiation. Apoptotic cells were increased to 14.5% of total thymocytes as early as 6 hours after irradiation. The numbers of apoptotic cells kept increasing over time. By 72 hours, 61.7% of thymocytes were apoptotic.
Visualizing radiation-induced thymocyte apoptosis in ear-thymus graft.
Visualizing Hematopoietic Cell Engraftment in Spleen Engrafted in the Mouse Pinna
To further demonstrate that this model is suitable for studying biological questions using in vivo imaging, we tracked hematopoietic cell engraftment in spleen engrafted in the mouse pinna. A BALB/c nude recipient of EGFP+ spleen was lethally irradiated and then transplanted with DsRed+ T-cell-depleted bone marrow (TCD BM) cells. Engraftment of hematopoietic cells (red) in ear-spleen (green) was tracked over time by two-photon microscopy. As shown in , EGFP+ lymphocytes were evident in ear-spleen graft before irradiation and transplantation. One week after irradiation and stem cell transplantation, some of the host EGFP+ lymphocytes had disappeared. By two weeks, the majority of the host EGFP+ lymphocytes had disappeared and non-hematopoietic cells survived. DsRed+ donor cells started to appear in the spleen graft starting from 5 weeks after stem cell transplantation. The numbers of DsRed+ donor cells kept increasing in the following weeks. By week 9, a large amount of DsRed+ donor cells were evident in the ear-spleen graft. By week 14, almost all lymphocytes found in the spleen graft were from the DsRed+ donor.
Visualizing hematopoietic cell engraftment in spleen engrafted in the mouse pinna.