Limits...
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.

Show MeSH

Related in: MedlinePlus

Nestin(+) NPs enriched from S18-treated EBs undergo rapid neuronal differentiation. (A) Mouse EBs were incubated with or without 80 μM of S18 and then expanded for 24 h to induce differentiation into NPs. Expanded NPs were stained for nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, pink). Arrow indicates Oct-4(+)/PAR-4(+) cell. (B and C) Protein and mRNA was isolated from expanded NPs and then used for immunoblotting (B) or RT-PCR (C) for the expression of various genes and proteins (nestin, Oct-4, PAR-4, Sox-2, Sox-1, Tert, Bcl-2, and NF-66), respectively. (D) EBCs 72 h after expansion of EBs were stained for nestin (Cy3, red), NF-66 (Cy2, green), or GFAP (Cy5, pink). (E) EBCs 72 h after expansion of EBs were stained for NF-66 (Cy2, green) and DNA (Hoechst, blue). Arrows indicate cells with extensive formation of processes that stain for filamentous NF-66.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172580&req=5

fig4: Nestin(+) NPs enriched from S18-treated EBs undergo rapid neuronal differentiation. (A) Mouse EBs were incubated with or without 80 μM of S18 and then expanded for 24 h to induce differentiation into NPs. Expanded NPs were stained for nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, pink). Arrow indicates Oct-4(+)/PAR-4(+) cell. (B and C) Protein and mRNA was isolated from expanded NPs and then used for immunoblotting (B) or RT-PCR (C) for the expression of various genes and proteins (nestin, Oct-4, PAR-4, Sox-2, Sox-1, Tert, Bcl-2, and NF-66), respectively. (D) EBCs 72 h after expansion of EBs were stained for nestin (Cy3, red), NF-66 (Cy2, green), or GFAP (Cy5, pink). (E) EBCs 72 h after expansion of EBs were stained for NF-66 (Cy2, green) and DNA (Hoechst, blue). Arrows indicate cells with extensive formation of processes that stain for filamentous NF-66.

Mentions: To monitor further neural differentiation of S18-treated EBCs, we determined the expression of Oct-4, PAR-4, and nestin 24 h after dissociation and replating of untreated or S18-treated EBs. Table II and Fig. 4 A show that S18 reduced the number of Oct-4(+)/PAR-4(+) by 70%, whereas the number of nestin(+) cells was not diminished. On the contrary, the portion of nestin(+) cells from S18-treated EBs showed the same proliferation rate as obtained with untreated cells and even increased by twofold (Table II), most likely due to their resistance to S18-inducible apoptosis (Bieberich et al., 2003). In contrast to untreated cells, almost all of the Oct-4(+)/PAR-4(+) cells from S18-treated EBs were FLICA(+), indicating that they were actively undergoing apoptosis and demonstrating that S18 eliminated residual Oct-4(+) positive cells from the EBCs (Table II). These results were consistent with those from MACS sorting (Table I). They also verified that S18 treatment spared the S18-resistant, nestin(+) cell population while inducing apoptosis in nearly all of the Oct-4(+) population of EBCs (Table II).


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)

Nestin(+) NPs enriched from S18-treated EBs undergo rapid neuronal differentiation. (A) Mouse EBs were incubated with or without 80 μM of S18 and then expanded for 24 h to induce differentiation into NPs. Expanded NPs were stained for nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, pink). Arrow indicates Oct-4(+)/PAR-4(+) cell. (B and C) Protein and mRNA was isolated from expanded NPs and then used for immunoblotting (B) or RT-PCR (C) for the expression of various genes and proteins (nestin, Oct-4, PAR-4, Sox-2, Sox-1, Tert, Bcl-2, and NF-66), respectively. (D) EBCs 72 h after expansion of EBs were stained for nestin (Cy3, red), NF-66 (Cy2, green), or GFAP (Cy5, pink). (E) EBCs 72 h after expansion of EBs were stained for NF-66 (Cy2, green) and DNA (Hoechst, blue). Arrows indicate cells with extensive formation of processes that stain for filamentous NF-66.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172580&req=5

fig4: Nestin(+) NPs enriched from S18-treated EBs undergo rapid neuronal differentiation. (A) Mouse EBs were incubated with or without 80 μM of S18 and then expanded for 24 h to induce differentiation into NPs. Expanded NPs were stained for nestin (Cy3, red), PAR-4 (Cy2, green), and Oct-4 (Cy5, pink). Arrow indicates Oct-4(+)/PAR-4(+) cell. (B and C) Protein and mRNA was isolated from expanded NPs and then used for immunoblotting (B) or RT-PCR (C) for the expression of various genes and proteins (nestin, Oct-4, PAR-4, Sox-2, Sox-1, Tert, Bcl-2, and NF-66), respectively. (D) EBCs 72 h after expansion of EBs were stained for nestin (Cy3, red), NF-66 (Cy2, green), or GFAP (Cy5, pink). (E) EBCs 72 h after expansion of EBs were stained for NF-66 (Cy2, green) and DNA (Hoechst, blue). Arrows indicate cells with extensive formation of processes that stain for filamentous NF-66.
Mentions: To monitor further neural differentiation of S18-treated EBCs, we determined the expression of Oct-4, PAR-4, and nestin 24 h after dissociation and replating of untreated or S18-treated EBs. Table II and Fig. 4 A show that S18 reduced the number of Oct-4(+)/PAR-4(+) by 70%, whereas the number of nestin(+) cells was not diminished. On the contrary, the portion of nestin(+) cells from S18-treated EBs showed the same proliferation rate as obtained with untreated cells and even increased by twofold (Table II), most likely due to their resistance to S18-inducible apoptosis (Bieberich et al., 2003). In contrast to untreated cells, almost all of the Oct-4(+)/PAR-4(+) cells from S18-treated EBs were FLICA(+), indicating that they were actively undergoing apoptosis and demonstrating that S18 eliminated residual Oct-4(+) positive cells from the EBCs (Table II). These results were consistent with those from MACS sorting (Table I). They also verified that S18 treatment spared the S18-resistant, nestin(+) cell population while inducing apoptosis in nearly all of the Oct-4(+) population of EBCs (Table II).

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