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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 formation of a PAR-4–PKCζ complex and PAR-4 dependent apoptosis. (A) EBCs (NP2 stage) were incubated overnight with or without 80 μM of S18. Cellular protein was solubilized and antigen–antibody complexes were immunoprecipitated, followed by SDS-PAGE and immunoblotting. (B) EBCs (NP2, 3, and 4 stages) were incubated with various ceramide-like apoptosis inducers and the degree of apoptosis was quantified by counting of cells with activated caspases (FLICA assay). All results were from three independent experiments showing average values and SEMs of cell counts from five areas with more than 100 cells. Treated cells show statistically significant differences to nontreated control cells as evaluated by ANOVA. C2, N-acetyl sphingosine (30 μM); C16, N-palmitoyl sphingosine (2 μM, incubated in solution with dodecanol as described in Bieberich et al., 2003); PZI, myristoylated PKCζ pseudosubstrate inhibitor peptide (30 μM). Open bars, NP2 stage (24 h after replating of EBCs); gray bars, NP3 stage (48 h after replating of EBCs); black bars, NP4 stage (72 h after replating of EBCs). (C and D) The level of Oct-4, PAR-4, and nestin gene expression was determined by RT-PCR during differentiation of EBCs from mouse. The expression level of PAR-4 protein was determined by immunoblotting using a mouse monoclonal anti–PAR-4 antibody. EB6, 7, and 8 are EBs 48, 72, and 96 h after attachment of suspension EBs; NP stages as in B.
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fig1: S18-induced formation of a PAR-4–PKCζ complex and PAR-4 dependent apoptosis. (A) EBCs (NP2 stage) were incubated overnight with or without 80 μM of S18. Cellular protein was solubilized and antigen–antibody complexes were immunoprecipitated, followed by SDS-PAGE and immunoblotting. (B) EBCs (NP2, 3, and 4 stages) were incubated with various ceramide-like apoptosis inducers and the degree of apoptosis was quantified by counting of cells with activated caspases (FLICA assay). All results were from three independent experiments showing average values and SEMs of cell counts from five areas with more than 100 cells. Treated cells show statistically significant differences to nontreated control cells as evaluated by ANOVA. C2, N-acetyl sphingosine (30 μM); C16, N-palmitoyl sphingosine (2 μM, incubated in solution with dodecanol as described in Bieberich et al., 2003); PZI, myristoylated PKCζ pseudosubstrate inhibitor peptide (30 μM). Open bars, NP2 stage (24 h after replating of EBCs); gray bars, NP3 stage (48 h after replating of EBCs); black bars, NP4 stage (72 h after replating of EBCs). (C and D) The level of Oct-4, PAR-4, and nestin gene expression was determined by RT-PCR during differentiation of EBCs from mouse. The expression level of PAR-4 protein was determined by immunoblotting using a mouse monoclonal anti–PAR-4 antibody. EB6, 7, and 8 are EBs 48, 72, and 96 h after attachment of suspension EBs; NP stages as in B.

Mentions: We have reported that apoptosis of EBCs is induced by simultaneous elevation of endogenous ceramide and PAR-4, a protein that binds and inhibits atypical PKCζ and a variety of other proteins (Sells et al., 1994, 1997; Diaz-Meco et al., 1996; Guo et al., 1998; Bieberich et al., 2003; Gurumurthy and Rangnekar, 2004). We have also shown that overexpression or antisense knockdown of PAR-4 increases or reduces the sensitivity of differentiating ES cells toward ceramide or ceramide analogue-inducible apoptosis, respectively (Bieberich et al., 2003). However, it remained to be investigated at which differentiation stage and how ceramide-inducible apoptosis is regulated by PAR-4–mediated inhibition of PKCζ. It has been reported that C2-ceramide induces formation of a protein complex between PKCζ and PAR-4 in PC12 cells before apoptosis (Wang et al., 1999). A potential S18-induced formation of a PKCζ–PAR-4 complex in EBCs was tested using coimmunoprecipitation assays with protein from cells that were treated with or without S18. Fig. 1 A shows that no precipitated protein was found when the primary antibody was omitted, which verified the specificity of the immunoprecipitation reaction. If the same primary antibody was used for immunoprecipitation and immunoblotting, the signal was not altered by prior incubation of the stem cells with S18. This result indicated that the amount of immunoprecipitated protein was not affected by S18. However, treatment with S18 was required to coimmunoprecipitate PKCζ or PAR-4 using primary antibodies against PAR-4 or PKCζ, respectively. This observation showed that S18 induced formation of a complex between PKCζ and its inhibitor protein PAR-4.


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 formation of a PAR-4–PKCζ complex and PAR-4 dependent apoptosis. (A) EBCs (NP2 stage) were incubated overnight with or without 80 μM of S18. Cellular protein was solubilized and antigen–antibody complexes were immunoprecipitated, followed by SDS-PAGE and immunoblotting. (B) EBCs (NP2, 3, and 4 stages) were incubated with various ceramide-like apoptosis inducers and the degree of apoptosis was quantified by counting of cells with activated caspases (FLICA assay). All results were from three independent experiments showing average values and SEMs of cell counts from five areas with more than 100 cells. Treated cells show statistically significant differences to nontreated control cells as evaluated by ANOVA. C2, N-acetyl sphingosine (30 μM); C16, N-palmitoyl sphingosine (2 μM, incubated in solution with dodecanol as described in Bieberich et al., 2003); PZI, myristoylated PKCζ pseudosubstrate inhibitor peptide (30 μM). Open bars, NP2 stage (24 h after replating of EBCs); gray bars, NP3 stage (48 h after replating of EBCs); black bars, NP4 stage (72 h after replating of EBCs). (C and D) The level of Oct-4, PAR-4, and nestin gene expression was determined by RT-PCR during differentiation of EBCs from mouse. The expression level of PAR-4 protein was determined by immunoblotting using a mouse monoclonal anti–PAR-4 antibody. EB6, 7, and 8 are EBs 48, 72, and 96 h after attachment of suspension EBs; NP stages as in B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172580&req=5

fig1: S18-induced formation of a PAR-4–PKCζ complex and PAR-4 dependent apoptosis. (A) EBCs (NP2 stage) were incubated overnight with or without 80 μM of S18. Cellular protein was solubilized and antigen–antibody complexes were immunoprecipitated, followed by SDS-PAGE and immunoblotting. (B) EBCs (NP2, 3, and 4 stages) were incubated with various ceramide-like apoptosis inducers and the degree of apoptosis was quantified by counting of cells with activated caspases (FLICA assay). All results were from three independent experiments showing average values and SEMs of cell counts from five areas with more than 100 cells. Treated cells show statistically significant differences to nontreated control cells as evaluated by ANOVA. C2, N-acetyl sphingosine (30 μM); C16, N-palmitoyl sphingosine (2 μM, incubated in solution with dodecanol as described in Bieberich et al., 2003); PZI, myristoylated PKCζ pseudosubstrate inhibitor peptide (30 μM). Open bars, NP2 stage (24 h after replating of EBCs); gray bars, NP3 stage (48 h after replating of EBCs); black bars, NP4 stage (72 h after replating of EBCs). (C and D) The level of Oct-4, PAR-4, and nestin gene expression was determined by RT-PCR during differentiation of EBCs from mouse. The expression level of PAR-4 protein was determined by immunoblotting using a mouse monoclonal anti–PAR-4 antibody. EB6, 7, and 8 are EBs 48, 72, and 96 h after attachment of suspension EBs; NP stages as in B.
Mentions: We have reported that apoptosis of EBCs is induced by simultaneous elevation of endogenous ceramide and PAR-4, a protein that binds and inhibits atypical PKCζ and a variety of other proteins (Sells et al., 1994, 1997; Diaz-Meco et al., 1996; Guo et al., 1998; Bieberich et al., 2003; Gurumurthy and Rangnekar, 2004). We have also shown that overexpression or antisense knockdown of PAR-4 increases or reduces the sensitivity of differentiating ES cells toward ceramide or ceramide analogue-inducible apoptosis, respectively (Bieberich et al., 2003). However, it remained to be investigated at which differentiation stage and how ceramide-inducible apoptosis is regulated by PAR-4–mediated inhibition of PKCζ. It has been reported that C2-ceramide induces formation of a protein complex between PKCζ and PAR-4 in PC12 cells before apoptosis (Wang et al., 1999). A potential S18-induced formation of a PKCζ–PAR-4 complex in EBCs was tested using coimmunoprecipitation assays with protein from cells that were treated with or without S18. Fig. 1 A shows that no precipitated protein was found when the primary antibody was omitted, which verified the specificity of the immunoprecipitation reaction. If the same primary antibody was used for immunoprecipitation and immunoblotting, the signal was not altered by prior incubation of the stem cells with S18. This result indicated that the amount of immunoprecipitated protein was not affected by S18. However, treatment with S18 was required to coimmunoprecipitate PKCζ or PAR-4 using primary antibodies against PAR-4 or PKCζ, respectively. This observation showed that S18 induced formation of a complex between PKCζ and its inhibitor protein PAR-4.

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