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Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell-derived neural transplants.

Bieberich E, Silva J, Wang G, Krishnamurthy K, Condie BG - J. Cell Biol. (2004)

Bottom Line: S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of beta-tubulin III.However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm.Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Genetics, School of Medicine, Medical College of Georgia, Augusta, GA 30912, USA. ebieberich@mail.mcg.edu

ABSTRACT
The formation of stem cell-derived tumors (teratomas) is observed when engrafting undifferentiated embryonic stem (ES) cells, embryoid body-derived cells (EBCs), or mammalian embryos and is a significant obstacle to stem cell therapy. We show that in tumors formed after engraftment of EBCs into mouse brain, expression of the pluripotency marker Oct-4 colocalized with that of prostate apoptosis response-4 (PAR-4), a protein mediating ceramide-induced apoptosis during neural differentiation of ES cells. We tested the ability of the novel ceramide analogue N-oleoyl serinol (S18) to eliminate mouse and human Oct-4(+)/PAR-4(+) cells and to increase the proportion of nestin(+) neuroprogenitors in EBC-derived cell cultures and grafts. S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of beta-tubulin III. However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm. Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.

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Oct-4(+)/PAR-4(+) cells persist in teratomas from brain transplants of untreated EBCs. (A) Mouse EBs were incubated with or without 80 μM of the novel ceramide analogue S18 and 100,000 untreated (left) or 200,000 S18-treated (right) EBCs injected into the striatum of C57BL6 mice (right hemisphere, arrow shows injection site). After 6 wk, mice were killed and teratoma formation was analyzed (some mice had to be killed earlier to avoid distress to the animal). (B) The teratoma obtained with untreated EBCs (left; Fig. 1 A) was vibratome sectioned and the sections immunostained for the endodermal marker α-fetoprotein (AFP, Cy2, green), the mesodermal marker desmin (Cy3, red), the ectodermal marker vimentin (Cy5, blue, middle), and the neuro-ectodermal and glial marker GFAP (Cy5, blue, right). (C) Immunohistochemistry was also performed for nestin (Cy3, red) and PAR-4 (Cy2, green). (D) Immunostaining of nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, red) at higher magnification.
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fig5: Oct-4(+)/PAR-4(+) cells persist in teratomas from brain transplants of untreated EBCs. (A) Mouse EBs were incubated with or without 80 μM of the novel ceramide analogue S18 and 100,000 untreated (left) or 200,000 S18-treated (right) EBCs injected into the striatum of C57BL6 mice (right hemisphere, arrow shows injection site). After 6 wk, mice were killed and teratoma formation was analyzed (some mice had to be killed earlier to avoid distress to the animal). (B) The teratoma obtained with untreated EBCs (left; Fig. 1 A) was vibratome sectioned and the sections immunostained for the endodermal marker α-fetoprotein (AFP, Cy2, green), the mesodermal marker desmin (Cy3, red), the ectodermal marker vimentin (Cy5, blue, middle), and the neuro-ectodermal and glial marker GFAP (Cy5, blue, right). (C) Immunohistochemistry was also performed for nestin (Cy3, red) and PAR-4 (Cy2, green). (D) Immunostaining of nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, red) at higher magnification.

Mentions: In preliminary studies, we found that intrastriatal injection of untreated EBCs into postnatal day 10 mice gave rise to 5–7 tumors throughout the brain after 6 wk in 12 out of 15 animals (Table III). Fig. 5 A shows a massive tumor that emerged on the surface of the right hemisphere at the injection site of the untreated EBCs. Immunohistochemistry using antibodies against α-fetoprotein, desmin, vimentin, and GFAP confirmed that the grafted EBCs had formed teratomas containing endodermal, mesodermal, and ectodermal tissues (Fig. 5 B; Vance et al., 1988; Sangruchi and Sobel, 1989). Within the teratomas, nestin expressing cells did not express PAR-4 as we would predict from previous studies of in vitro ES cell differentiation (Bieberich et al., 2003; Fig. 5 C). However, PAR-4(+)/nestin(−) cells in the center of nestin(+) cell clusters expressed Oct-4 (Fig. 5 D). These results indicated that EBC-derived teratomas maintained a subpopulation of pluripotent, Oct-4(+)/PAR-4(+)/nestin(−) stem cells.


Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell-derived neural transplants.

Bieberich E, Silva J, Wang G, Krishnamurthy K, Condie BG - J. Cell Biol. (2004)

Oct-4(+)/PAR-4(+) cells persist in teratomas from brain transplants of untreated EBCs. (A) Mouse EBs were incubated with or without 80 μM of the novel ceramide analogue S18 and 100,000 untreated (left) or 200,000 S18-treated (right) EBCs injected into the striatum of C57BL6 mice (right hemisphere, arrow shows injection site). After 6 wk, mice were killed and teratoma formation was analyzed (some mice had to be killed earlier to avoid distress to the animal). (B) The teratoma obtained with untreated EBCs (left; Fig. 1 A) was vibratome sectioned and the sections immunostained for the endodermal marker α-fetoprotein (AFP, Cy2, green), the mesodermal marker desmin (Cy3, red), the ectodermal marker vimentin (Cy5, blue, middle), and the neuro-ectodermal and glial marker GFAP (Cy5, blue, right). (C) Immunohistochemistry was also performed for nestin (Cy3, red) and PAR-4 (Cy2, green). (D) Immunostaining of nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, red) at higher magnification.
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Related In: Results  -  Collection

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fig5: Oct-4(+)/PAR-4(+) cells persist in teratomas from brain transplants of untreated EBCs. (A) Mouse EBs were incubated with or without 80 μM of the novel ceramide analogue S18 and 100,000 untreated (left) or 200,000 S18-treated (right) EBCs injected into the striatum of C57BL6 mice (right hemisphere, arrow shows injection site). After 6 wk, mice were killed and teratoma formation was analyzed (some mice had to be killed earlier to avoid distress to the animal). (B) The teratoma obtained with untreated EBCs (left; Fig. 1 A) was vibratome sectioned and the sections immunostained for the endodermal marker α-fetoprotein (AFP, Cy2, green), the mesodermal marker desmin (Cy3, red), the ectodermal marker vimentin (Cy5, blue, middle), and the neuro-ectodermal and glial marker GFAP (Cy5, blue, right). (C) Immunohistochemistry was also performed for nestin (Cy3, red) and PAR-4 (Cy2, green). (D) Immunostaining of nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, red) at higher magnification.
Mentions: In preliminary studies, we found that intrastriatal injection of untreated EBCs into postnatal day 10 mice gave rise to 5–7 tumors throughout the brain after 6 wk in 12 out of 15 animals (Table III). Fig. 5 A shows a massive tumor that emerged on the surface of the right hemisphere at the injection site of the untreated EBCs. Immunohistochemistry using antibodies against α-fetoprotein, desmin, vimentin, and GFAP confirmed that the grafted EBCs had formed teratomas containing endodermal, mesodermal, and ectodermal tissues (Fig. 5 B; Vance et al., 1988; Sangruchi and Sobel, 1989). Within the teratomas, nestin expressing cells did not express PAR-4 as we would predict from previous studies of in vitro ES cell differentiation (Bieberich et al., 2003; Fig. 5 C). However, PAR-4(+)/nestin(−) cells in the center of nestin(+) cell clusters expressed Oct-4 (Fig. 5 D). These results indicated that EBC-derived teratomas maintained a subpopulation of pluripotent, Oct-4(+)/PAR-4(+)/nestin(−) stem cells.

Bottom Line: S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of beta-tubulin III.However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm.Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine and Genetics, School of Medicine, Medical College of Georgia, Augusta, GA 30912, USA. ebieberich@mail.mcg.edu

ABSTRACT
The formation of stem cell-derived tumors (teratomas) is observed when engrafting undifferentiated embryonic stem (ES) cells, embryoid body-derived cells (EBCs), or mammalian embryos and is a significant obstacle to stem cell therapy. We show that in tumors formed after engraftment of EBCs into mouse brain, expression of the pluripotency marker Oct-4 colocalized with that of prostate apoptosis response-4 (PAR-4), a protein mediating ceramide-induced apoptosis during neural differentiation of ES cells. We tested the ability of the novel ceramide analogue N-oleoyl serinol (S18) to eliminate mouse and human Oct-4(+)/PAR-4(+) cells and to increase the proportion of nestin(+) neuroprogenitors in EBC-derived cell cultures and grafts. S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of beta-tubulin III. However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm. Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.

Show MeSH
Related in: MedlinePlus