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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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S18-induced apoptosis eliminates Oct-4(+)/PAR-4(+) pluripotent stem cells and enriches for nestin(+) NPs. (A and B) MACS sorting of apoptotic EBCs. Mouse EBCs from untreated (A) or S18-treated (80 μM; B) EBs were incubated with Annexin V–conjugated magnetic beads and fractionated using MACS. Flow through cells (Annexin V(−)) and retained cells (Annexin V(+)) were precipitated on lysine-coated coverslips, incubated with FLICA substrate, and after fixation, immunostained for the expression of PAR-4 (Cy3, red) and Oct-4 (Cy5, green). FLICA-negative and -positive cells are labeled with (−) or (+), respectively. Arrows indicate Oct-4(+)/PAR-4(+) cells. Asterisks label cells in the Annexin V(+) fraction that recovered from the initial phase of apoptosis. (C) Radial expansion and neural differentiation of mouse EBs stained for the expression of nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, blue). Arrow points at cell showing coexpression and subcellular segregation of nestin and PAR-4. (D) Mouse EBs incubated overnight with 80 μM of S18 were stained for apoptotic cells (TUNEL or Annexin V, FITC, green) and nestin (Cy3, red).
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fig3: S18-induced apoptosis eliminates Oct-4(+)/PAR-4(+) pluripotent stem cells and enriches for nestin(+) NPs. (A and B) MACS sorting of apoptotic EBCs. Mouse EBCs from untreated (A) or S18-treated (80 μM; B) EBs were incubated with Annexin V–conjugated magnetic beads and fractionated using MACS. Flow through cells (Annexin V(−)) and retained cells (Annexin V(+)) were precipitated on lysine-coated coverslips, incubated with FLICA substrate, and after fixation, immunostained for the expression of PAR-4 (Cy3, red) and Oct-4 (Cy5, green). FLICA-negative and -positive cells are labeled with (−) or (+), respectively. Arrows indicate Oct-4(+)/PAR-4(+) cells. Asterisks label cells in the Annexin V(+) fraction that recovered from the initial phase of apoptosis. (C) Radial expansion and neural differentiation of mouse EBs stained for the expression of nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, blue). Arrow points at cell showing coexpression and subcellular segregation of nestin and PAR-4. (D) Mouse EBs incubated overnight with 80 μM of S18 were stained for apoptotic cells (TUNEL or Annexin V, FITC, green) and nestin (Cy3, red).

Mentions: To quantify the effect of ceramide-induced apoptosis on EBCs, mouse EBs cultivated in serum-free medium were incubated for 24 h with 80 μM of S18 or 2 μM of myriocin, a ceramide biosynthesis inhibitor, before magnetic-activated cell sorting (MACS) for Annexin V (+) EBCs and fluorochrome inhibitor of caspase (FLICA) tagging of activated caspases. Annexin V–MACS separated cells that were in the initial phase of apoptosis induction, whereas FLICA staining identified cells that executed apoptosis by caspase activation. Table I and Fig. 3 show the results of Oct-4 and PAR-4 staining in MACS-sorted Annexin (+) or (−) EBCs. With untreated control cells, the Annexin V(−) and (+) fractions contained PAR-4(+) and Oct-4(+) cells (Fig. 3 A), whereas with S18-treated cells, only the Annexin V(+) fraction contained PAR-4(+) and Oct-4(+) cells (Fig. 3 B). Almost all of the PAR-4(+) cells were also Oct-4(+) indicating that S18-induced apoptosis could efficiently eliminate Oct-4(+) cells from EBCs (Table I and Fig. 3 B). In cells not treated with S18, inhibition of endogenous ceramide biosynthesis with myriocin prevented apoptosis in PAR-4(+) cells, which implied that Oct-4(+) cells were preserved as well (Table I). FLICA staining revealed that S18 increased apoptosis from 25% in untreated cells to 55% in S18-treated cells, whereas <10% of the myriocin-treated cells were FLICA positive.


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)

S18-induced apoptosis eliminates Oct-4(+)/PAR-4(+) pluripotent stem cells and enriches for nestin(+) NPs. (A and B) MACS sorting of apoptotic EBCs. Mouse EBCs from untreated (A) or S18-treated (80 μM; B) EBs were incubated with Annexin V–conjugated magnetic beads and fractionated using MACS. Flow through cells (Annexin V(−)) and retained cells (Annexin V(+)) were precipitated on lysine-coated coverslips, incubated with FLICA substrate, and after fixation, immunostained for the expression of PAR-4 (Cy3, red) and Oct-4 (Cy5, green). FLICA-negative and -positive cells are labeled with (−) or (+), respectively. Arrows indicate Oct-4(+)/PAR-4(+) cells. Asterisks label cells in the Annexin V(+) fraction that recovered from the initial phase of apoptosis. (C) Radial expansion and neural differentiation of mouse EBs stained for the expression of nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, blue). Arrow points at cell showing coexpression and subcellular segregation of nestin and PAR-4. (D) Mouse EBs incubated overnight with 80 μM of S18 were stained for apoptotic cells (TUNEL or Annexin V, FITC, green) and nestin (Cy3, red).
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fig3: S18-induced apoptosis eliminates Oct-4(+)/PAR-4(+) pluripotent stem cells and enriches for nestin(+) NPs. (A and B) MACS sorting of apoptotic EBCs. Mouse EBCs from untreated (A) or S18-treated (80 μM; B) EBs were incubated with Annexin V–conjugated magnetic beads and fractionated using MACS. Flow through cells (Annexin V(−)) and retained cells (Annexin V(+)) were precipitated on lysine-coated coverslips, incubated with FLICA substrate, and after fixation, immunostained for the expression of PAR-4 (Cy3, red) and Oct-4 (Cy5, green). FLICA-negative and -positive cells are labeled with (−) or (+), respectively. Arrows indicate Oct-4(+)/PAR-4(+) cells. Asterisks label cells in the Annexin V(+) fraction that recovered from the initial phase of apoptosis. (C) Radial expansion and neural differentiation of mouse EBs stained for the expression of nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, blue). Arrow points at cell showing coexpression and subcellular segregation of nestin and PAR-4. (D) Mouse EBs incubated overnight with 80 μM of S18 were stained for apoptotic cells (TUNEL or Annexin V, FITC, green) and nestin (Cy3, red).
Mentions: To quantify the effect of ceramide-induced apoptosis on EBCs, mouse EBs cultivated in serum-free medium were incubated for 24 h with 80 μM of S18 or 2 μM of myriocin, a ceramide biosynthesis inhibitor, before magnetic-activated cell sorting (MACS) for Annexin V (+) EBCs and fluorochrome inhibitor of caspase (FLICA) tagging of activated caspases. Annexin V–MACS separated cells that were in the initial phase of apoptosis induction, whereas FLICA staining identified cells that executed apoptosis by caspase activation. Table I and Fig. 3 show the results of Oct-4 and PAR-4 staining in MACS-sorted Annexin (+) or (−) EBCs. With untreated control cells, the Annexin V(−) and (+) fractions contained PAR-4(+) and Oct-4(+) cells (Fig. 3 A), whereas with S18-treated cells, only the Annexin V(+) fraction contained PAR-4(+) and Oct-4(+) cells (Fig. 3 B). Almost all of the PAR-4(+) cells were also Oct-4(+) indicating that S18-induced apoptosis could efficiently eliminate Oct-4(+) cells from EBCs (Table I and Fig. 3 B). In cells not treated with S18, inhibition of endogenous ceramide biosynthesis with myriocin prevented apoptosis in PAR-4(+) cells, which implied that Oct-4(+) cells were preserved as well (Table I). FLICA staining revealed that S18 increased apoptosis from 25% in untreated cells to 55% in S18-treated cells, whereas <10% of the myriocin-treated cells were FLICA positive.

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