Since derived pedigreed non-human primate ESCs were injected into mice to identify their pluripotency by ability to form teratomas, we could utilize them for more detailed analysis and comparison of tissue morphology and distribution within each teratoma and between teratomas using histopathology and MRM.
Overall, no statistical differences were found for number of blocks needed (crude estimate of size) when comparing all teratomas between families and when comparing all teratomas by gender. Eight teratomas were derived out of 12 injections (67% success) from three lines within one family, compared to 19 teratomas derived from 22 injections (86% success) of 4 lines in the other family. Two injections (106-A1, A2) resulted in a moderate to poorly differentiated tumor (see below) without evidence of teratoma formation. Two injections from another line (306-C1, C2) produced a dermoid cyst on one side (left testis) and no detectable lesion from the other injection (right testis) (not shown). Two injections did not produce a detectable lesion and one produced a lesion with only two germ layers represented. shows characteristics of the three teratomas that also underwent MRM.
Demographic and germ layer scores for teratomas derived from pedigreed primate ESCs for which MRM was performed
We next compared in a semi-quantitative fashion, the relative contributions of each germ layer to the composition of each teratoma using the scale provided in the Methods. also demonstrates the median contributions of each germ layer for each teratoma for which MRM was performed. For these teratomas, wide discrepancies were found in tissue type quantity with wide variations in ectodermal versus mesodermal derivatives. However, in the overall analysis of the teratomas, no differences were noted between families for median contribution of germ layers or between sexes either within families or between families.
Overall, approximately 23 different tissue types were identified as derivatives of ectoderm (), mesoderm (2F-2O), and endoderm (2P-2T). Specific tissues seen infrequently are shown in .
Figure 2 Collage of images of hematoxylin and eosin stained tissue sections representing the range of tissues present in teratomas from pedigreed nhpESC lines. A-E represent ectodermal derivatives, F-O-mesodermal derivatives, P-T-endodermal derivatives, and U-Y-interesting (more ...)
Ectodermal derivatives included immature neuroglial tissue represented by either neuroepithelial tissue composed of stratified elongated cells with hyperchromatic oval nuclei and pink cytoplasm arranged in ribbons and rosettes (Flexner-Wintersteiner and Homer Wright) (A) or adjacent cellular neuroglial tissue composed notably of small cells with scant cytoplasm resembling neuroblasts (B). Cells with granular brown pigment resembling retinal cells arranged in rosettes were seen sporadically and in low quantity in some teratomas associated with immature neural areas (B). Mature neuroglial tissue was defined as tissue containing mature neurons, glial cells, and background mature neuropil resembling disorganized brain or areas with well-developed ganglion (C, D). Skin was also present and in most cases demonstrated a well-developed epidermis and adnexal structures including in some cases hair follicles (E).
Mesodermal derivatives comprised the largest percentage of tissues overall in these teratomas and consisted of muscle (skeletal and smooth), fat, mesenchyme, cartilage, and bone (F-O). Smooth muscle showed a consistent morphology typical of mature smooth muscle found in the muscle layers of the gastrointestinal tract. Well-defined and recognizable smooth muscle was associated with glandular epithelium in most cases and rarely seen in isolation arising from the mesenchymal stroma. Often smooth muscle bundles were found in layers recapitulating the two distinct layers seen in the gastrointestinal tract of non-human primates, humans, and other mammals. In other areas where more primitive glandular epithelium was present, the surrounding stroma had the appearance of immature smooth muscle (G). Immature skeletal muscle (H) was delineated by multinucleated myotubes, immature myofibers, and single rhabdomyoblastic cells interspersed in cellular mesenchymal stroma. Mature skeletal muscle was apparent by long eosinophilic myofibers showing striations (I). Skeletal muscle comprised a large portion of the mesodermal component of teratoma from the 3106 line (J). Immature and mature adipose tissue was seen (K, L). The majority of adipose tissue was composed of lobules of mature adipocytes with focal areas of immature fat characterized by variation in size of adipocytes and the presence of lipoblasts. Undifferentiated stroma demonstrated several morphological variants including cellular spindle cell stroma with early collagenization (M), fibromuscular (N), mixed collagenous/myxoid stroma, and densely fibrotic (not shown). Within cellular areas numerous mitotic figures could be readily identified although atypical nuclear structures or other malignant features were not seen. Bone and cartilage were frequently present in many teratomas (O). Cartilage displayed a range of morphology from cellular immature cartilage generally associated with neuroglial regions to more mature well-formed lobules of cartilage some forming structures resembling tracheal rings (see ).
Endodermal derivatives exhibited no specific morphology, but had epithelium that generally lined cystic or glandular spaces (structures having a lumen, not neuroepithelial) (P-T). The epithelium varied considerably and included immature foveolar/intestinal-type (P), primitive glandular (Q, R), stratified (S), and primitive endodermal (yolk sac) type (T). Ciliated and well-developed intestinal type epithelium was also present in some teratomas. Conspicuously missing from all teratomas examined was evidence of well-developed thyroid, liver, kidney, or pancreas.
Both injections of line 106 (A1, A2) resulted in tumors with similar morphology best characterized as moderately to poorly differentiated tumor () shown here to illustrate one of the non-teratomatous lesions resulting from ESC injections. These tumors exhibited both focal areas of solid growth and areas of glandular differentiation. Interestingly, most of the tumor cells exhibited a mixed epithelial and mesenchymal phenotype by expression of cytokeratin and vimentin ( respectively). Scattered periglandular cells demonstrated an immunophenotype reminiscent of myoepithelial cells including expression of smooth muscle actin, all muscle actin, and S100 respectively (3F, 3G, 3H). These periglandular cells were also negative for p63, a marker of basal cells in the sinonasal tract, breast, and prostate. Tumor cells were negative for PGP9.5, myogenin, and alpha-fetoprotein making neuroblastic, skeletal muscle, or endodermal differentiation less likely. Oct-4 (pluripotency marker) and p63 (epithelial progenitor marker and marker of basal cells) did not show characteristic nuclear staining in any tumor cells. Three lines from 306 also formed undifferentiated/poorly differentiated tumors, but exhibited abnormal karyotypes in the derived lines.
Figure 3 Tumor derived from 106-A1 and A2 lines. A-C are hematoxylin and eosin stained images of the tumor. In panel A, many areas showed glandular profiles composed of cells with basally oriented nuclei and abundant amphophilic cytoplasm (200X). Focal areas showed (more ...)
As histopathological and immunohistochemistry analyses require removal of the teratoma from the mouse, we examined if we could utilize a non-invasive technique, i.e., MRM, for teratoma analysis. We performed MRM on two teratomas from line 3006 and an additional teratoma from line 3106. shows volumetric measurements and 3D volume renderings of each teratoma made from MRM images. Corresponding numerical measurements are shown in Supplemental Table 2
. All three teratomas had measured volumes between 3.0 cc and 6.2 cc. A cystic volume fraction, ranging between 1% and 3%, was found only in teratomas from line 3006. In agreement with previous T2
-weighted MRI of teratomas, all cysts were high-intensity and solid tissue had heterogeneous contrast [14
]. shows a histological section co-registered with a 2D MRM slice. Structures and tissues that could be identified from MRM images with little or no ambiguity were adipose tissue, cyst, cartilage, and epidermal lining. A series of co-registrations similar to that shown in were used to determine which teratoma tissue types and structures were identifiable using MRM. About 30% of attempted slice registrations were successful, while the rest lacked compelling and unambiguous correlation between the two imaging modalities since few landmark features were present in disorganized teratomas. Successful registrations, especially when aided by fiduciary markers (, See Supplemental Movies for 3D rendering and orthogonal slices), allowed homogeneous tissue patches in histology to be correlated with pixel intensity values in T2
-weighted images. shows the results of this series of cross-teratoma correlations. Tissues with low intensity in MR images, defined as 3-22% of maximum (cyst) intensity, included adipose and necrotic tissues, mature bone/cartilage, and neuroectoderm. Tissues with medium intensity (22-52% of maximum intensity) included neuroectoderm, immature bone/cartilage, skin, muscle, connective, gastrointestinal, and necrotic tissues. High-intensity tissues (53-100% of maximum intensity) were limited to cystic lining/fluid and gastrointestinal lining. Notably, because these MR image intensity categories (i.e. Low, Medium, and High) were found to represent multiple tissue types in histology, MR images were not able to unambiguously label any tissue types except for adipose tissue, cyst, and calcified bone/cartilage. These tissue types were unambiguous due to their texture features. For example, although adipose tissue deposits and mature (calcified) bone/cartilage are both low-intensity in MR images, adipose deposits tended to have poorly delineated boundaries, while bone/cartilage exhibited shard-like patterns. High-intensity cysts had bulging shapes with well-delineated linings.
Figure 4 Volumetric teratoma data compiled from 3D MRM segmentation of three teratomas (rows A, B, C). From left to right: Gross Volume represents teratoma volume including cysts and mouse tissues; Solid Tissue (gray) represents teratoma volume after removing (more ...)
Figure 5 Histological section (left) co-registered with 2D MRM slice (right). Tissues and structures that are clearly differentiated in both histology and MRM are outlined in color. These outlines represent adipose tissue (cyan), cartilage (orange), cystic regions (more ...)
Regional correlation of teratoma tissue types with T2-weighted MRI image pixel intensities.
Previously, clinical T2
-weighted MR images also identified adipose tissue as low-intensity, cyst as high-intensity, and calcification (which increases with bone/cartilage maturity) as low-intensity with shard-like textures [14
]. High resolution MRM was critical for enabling correlation between MR image intensity with tissue type (); lower resolution images would not have sufficed. However, these higher resolutions did not allow us to uniquely identify any additional tissue types directly from MR images, when compared with previous clinical imaging. Yet with further analysis, MRM may allow direct, non-invasive identification of additional tissue types, because they revealed textured patterns not visible in low-resolution MRI. For example, in the three teratomas shown in , unidentified textures included radial patterns with alternating dark-bright spokes, faint stacks of filamentous parallel lines, dark finger-like projections, faint concentric lines against bright backgrounds, and dark patches with interior textures such as bright speckles or membrane-like lines. Correlating these textures and other regional patterns with the tissues they represent in histology, especially aided by computational statistical methods, could identify additional tissue types with varying levels of confidence, directly from MRM of teratomas.