In vivo differentiation assay
Sphere cultures from representative tissues were washed twice with Hank’s balanced salt solution (Gibco). Under a microscope, 2000 spheres, each containing *1000 cells, were collected using a glass pipette and placed in a 50mL tube. Hank’s balanced salt solution (20mL) was added to each tube and subsequently centrifuged at 800rpm for 3min. The supernatant was discarded and the pellet was resuspended in 50mL of DMEM with 10% fetal bovine serum. This solution was seeded onto a sheet 3x3x1mm, composed of a nonwoven mesh of polyglycolic acid fibers, 200mm in diameter, and implanted subcutaneously into the dorsal flanks of a 4-week-old NOD/SCID mice (Jackson Laboratory).
明日は釣りだ。
Four weeks later the implants were harvested, and analyzed using immunohistochemical techniques. The implants were fixed with 10% formaldehyde, embedded in paraffin, and routinely processed into 4mm thickness. Sections were stained with hematoxylin and eosin. Cartilage was confirmed using Safranin-O (Fisher; S670-25). Duct and gland-like tissues were identified using the endoderm marker, rabbit polyclonal FOXA2 (Abcam; ab40874). Epithelium-like structures were identified using mouse monoclonal [PCK-26] Pan cytokeratin antibody (Abcam; ab6401). Muscle-like structures were identified using mouse monoclonal desmin antibody (Sigma; D1033) and anti-a-smooth muscle actin antibody (N1584; Dako). Nerve-like structures were identified using mouse monoclonal beta III tubulin antibody (Promega; G7121). In negative controls, the primary antibody was replaced with IgG-negative controls of the same isotype to ensure specificity. All sections were then peroxidase stained using the LSAB 2 kit (DakoCytomation) according to the manufacturer’s protocol. The experiments were reviewed and approved by Harvard Medical Area Standing Committee in Animals.
明日は釣りだ。
Differentiation potential of cells in vivo
When implanted subcutaneously into NOD/SCID mice, spheres generated from cells procured from each of the three germ layers demonstrated the potential to form tissue-like teratoma containing cells representative of all three germ layers. The tissues generated were encapsulated and easily resected. Each explant was *25mm3 (Fig. 6A). Individual explants contained cells representative of all three germ layers. Tissue generated from spinal spheres contained nerve (ectoderm; Fig. 6Bi, Bii), muscle (mesoderm; Fig. 6Biii, Biv), and duct-like tissue (endoderm; Fig. 6Bv, Bvi). Tissue generated from myospheres contained epithelium (ectoderm; Fig. 6Ci, Cii), muscle (mesoderm; Fig. 6Ciii, Civ), and ductlike tissue (endoderm; Fig. 6Cv, Cvi). Tissue generated from pneumospheres contained epithelium (ectoderm; Fig. 6Di, Dii), cartilage (mesoderm; Fig. 6Diii, Div), and gland (ectoderm; Fig. 6Dv, Dvi). Specific tissues were identified using immunohistochemical techniques. Nerves were identified using beta III-tubulin, epithelium identified using pancytokeratin, and muscle identified using desmin and myosin. Duct-like structures and gland were identified using FOXA2.
明日は釣りだ。
Discussion
・・・
Although the spheres in this study described were capable of generating teratoma-like tissue, the transplanted cells did not form large teratomas as did ES cells, nor they did express Eras in vitro as ESCs,31 which suggests that the teratoma-like tissue they generate may be very different from true teratomas generated from ESCs. In addition, the cells studied did not express the trophectoderm marker Cdx232 or also associated with ESCs. These differences of gene expression pattern may explain the differences of the biological function between ESCs and adult stem cells in this study.
・・・
翻訳機君、頼んどくぜ。
明日は我々は早くに釣りだ。小保方さんはESのテラトーマも作ったことがあってその大きさの違いを知ってる。
12/27Harukoを切り出した時はおどろいたろうな。くひひひひひ。
>>
the transplanted cells did not form large teratomas as did ES cells
はい、はい、機械の癖に大口叩いていないで、とっとと仕事おし。
>>
Materials and Methods
In vivo differentiation assay
Sphere cultures from representative tissues were washed twice with Hank’s balanced salt solution (Gibco). Under a microscope, 2000 spheres, each containing *1000 cells, were collected using a glass pipette and placed in a 50mL tube. Hank’s balanced salt solution (20mL) was added to each tube and subsequently centrifuged at 800rpm for 3min. The supernatant was discarded and the pellet was resuspended in 50mL of DMEM with 10% fetal bovine serum. This solution was seeded onto a sheet 3x3x1mm, composed of a nonwoven mesh of polyglycolic acid fibers, 200mm in diameter, and implanted subcutaneously into the dorsal flanks of a 4-week-old NOD/SCID mice (Jackson Laboratory).
はい、翻訳機君。
>>
Four weeks later the implants were harvested, and analyzed using immunohistochemical techniques. The implants were fixed with 10% formaldehyde, embedded in paraffin, and routinely processed into 4mm thickness. Sections were stained with hematoxylin and eosin. Cartilage was confirmed using Safranin-O (Fisher; S670-25). Duct and gland-like tissues were identified using the endoderm marker, rabbit polyclonal FOXA2 (Abcam; ab40874). Epithelium-like structures were identified using mouse monoclonal [PCK-26] Pan cytokeratin antibody (Abcam; ab6401). Muscle-like structures were identified using mouse monoclonal desmin antibody (Sigma; D1033) and anti-a-smooth muscle actin antibody (N1584; Dako). Nerve-like structures were identified using mouse monoclonal beta III tubulin antibody (Promega; G7121). In negative controls, the primary antibody was replaced with IgG-negative controls of the same isotype to ensure specificity. All sections were then peroxidase stained using the LSAB 2 kit (DakoCytomation) according to the manufacturer’s protocol. The experiments were reviewed and approved by Harvard Medical Area Standing Committee in Animals.
細かいことは後にして、続きだわ。
>>
Differentiation potential of cells in vivo
When implanted subcutaneously into NOD/SCID mice, spheres generated from cells procured from each of the three germ layers demonstrated the potential to form tissue-like teratoma containing cells representative of all three germ layers. The tissues generated were encapsulated and easily resected. Each explant was *25mm3 (Fig. 6A). Individual explants contained cells representative of all three germ layers. Tissue generated from spinal spheres contained nerve (ectoderm; Fig. 6Bi, Bii), muscle (mesoderm; Fig. 6Biii, Biv), and duct-like tissue (endoderm; Fig. 6Bv, Bvi). Tissue generated from myospheres contained epithelium (ectoderm; Fig. 6Ci, Cii), muscle (mesoderm; Fig. 6Ciii, Civ), and ductlike tissue (endoderm; Fig. 6Cv, Cvi). Tissue generated from pneumospheres contained epithelium (ectoderm; Fig. 6Di, Dii), cartilage (mesoderm; Fig. 6Diii, Div), and gland (ectoderm; Fig. 6Dv, Dvi). Specific tissues were identified using immunohistochemical techniques. Nerves were identified using beta III-tubulin, epithelium identified using pancytokeratin, and muscle identified using desmin and myosin. Duct-like structures and gland were identified using FOXA2.
Although the spheres in this study described were capable of generating teratoma-like tissue, the transplanted cells did not form large teratomas as did ES cells, nor they did express Eras in vitro as ESCs,[31] which suggests that the teratoma-like tissue they generate may be very different from true teratomas generated from ESCs. In addition, the cells studied did not express the trophectoderm marker Cdx2[32 ]or also associated with ESCs. These differences of gene expression pattern may explain the differences of the biological function between ESCs and adult stem cells in this study.
参考文献の31と32は以下よ。
>>
31. Takahashi, K., Mitsui, K., and Yamanaka, S. Role of ERas in promoting tumour-like properties in mouse embryonic stem cells. Nature 423, 541, 2003.
32. Ralston, A., and Rossant, J. Genetic regulation of stem cell origins in the mouse embryo. Clin Genet 68, 106, 2005.
