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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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EBCs from untreated EBs form highly invasive cortical and ventricular tumors, whereas S18-treated EBCs show enhanced neuronal differentiation after engraftment. (A) A tumor developed from Vybrant CM diI-labeled ROSA-26 EBCs was immunostained for β-galactosidase (Cy2, green). The arrow indicates a residual cluster of Vybrant CM diI-labeled cells. (B) S18-treated or untreated EBCs were stained with Vybrant CM diI (red, untreated cells) or Vybrant diO (green, treated cells), mixed, and injected into the striatum of neonatal mice. The figure shows settlement of treated cells in the subependymal layer, whereas untreated EBCs form a neural tube-like tumor in the lumen of the right lateral ventricle. (C) Mouse EBCs derived from S18-treated EBs were injected into the striatum of neonatal mice and immunostained for nestin (Cy3, red). (D) After nestin staining, frozen sections were FISH-stained for Y-chromosomes (FITC, green). DNA was counterstained with Hoechst dye (blue). (E) Mouse EBs were treated with 80 μM of S18, labeled with Vybrant CM diI (red), and injected into the striatum of neonatal mice. 6 wk after engraftment, brain sections were immunostained for β-tubulin III (Cy2, green). Arrrow shows cluster of Vybrant CM diI-positive (red) cells that are double-stained for β-tubulin III (Cy2, cryosectioned, confocal).
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fig6: EBCs from untreated EBs form highly invasive cortical and ventricular tumors, whereas S18-treated EBCs show enhanced neuronal differentiation after engraftment. (A) A tumor developed from Vybrant CM diI-labeled ROSA-26 EBCs was immunostained for β-galactosidase (Cy2, green). The arrow indicates a residual cluster of Vybrant CM diI-labeled cells. (B) S18-treated or untreated EBCs were stained with Vybrant CM diI (red, untreated cells) or Vybrant diO (green, treated cells), mixed, and injected into the striatum of neonatal mice. The figure shows settlement of treated cells in the subependymal layer, whereas untreated EBCs form a neural tube-like tumor in the lumen of the right lateral ventricle. (C) Mouse EBCs derived from S18-treated EBs were injected into the striatum of neonatal mice and immunostained for nestin (Cy3, red). (D) After nestin staining, frozen sections were FISH-stained for Y-chromosomes (FITC, green). DNA was counterstained with Hoechst dye (blue). (E) Mouse EBs were treated with 80 μM of S18, labeled with Vybrant CM diI (red), and injected into the striatum of neonatal mice. 6 wk after engraftment, brain sections were immunostained for β-tubulin III (Cy2, green). Arrrow shows cluster of Vybrant CM diI-positive (red) cells that are double-stained for β-tubulin III (Cy2, cryosectioned, confocal).

Mentions: We tested if treatment of EBs with S18 eliminated the residual pluripotent Oct-4(+)/PAR-4(+) stem cells and prevented teratoma formation from EBC-derived transplants. Table III shows that the majority of untreated ROSA-26–derived EBCs formed invasive striatal/cortical and noninvasive ventricular, β-galactosidase–positive tumors (Fig. 6 A). We labeled untreated EBCs with Vybrant CM diI (red) and S18-treated EBCs with Vybrant diO (green) and injected a mixed population of these cells into the striatum of the same mouse. Untreated, diI-labeled stem cells developed ventricular and invasive, striatal and cortical tumors, whereas S18-treated, diO-labeled cells integrated into the subependymal cell layer (Fig. 6 B and Table III) and ventromedial aspects of the hippocampus.


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)

EBCs from untreated EBs form highly invasive cortical and ventricular tumors, whereas S18-treated EBCs show enhanced neuronal differentiation after engraftment. (A) A tumor developed from Vybrant CM diI-labeled ROSA-26 EBCs was immunostained for β-galactosidase (Cy2, green). The arrow indicates a residual cluster of Vybrant CM diI-labeled cells. (B) S18-treated or untreated EBCs were stained with Vybrant CM diI (red, untreated cells) or Vybrant diO (green, treated cells), mixed, and injected into the striatum of neonatal mice. The figure shows settlement of treated cells in the subependymal layer, whereas untreated EBCs form a neural tube-like tumor in the lumen of the right lateral ventricle. (C) Mouse EBCs derived from S18-treated EBs were injected into the striatum of neonatal mice and immunostained for nestin (Cy3, red). (D) After nestin staining, frozen sections were FISH-stained for Y-chromosomes (FITC, green). DNA was counterstained with Hoechst dye (blue). (E) Mouse EBs were treated with 80 μM of S18, labeled with Vybrant CM diI (red), and injected into the striatum of neonatal mice. 6 wk after engraftment, brain sections were immunostained for β-tubulin III (Cy2, green). Arrrow shows cluster of Vybrant CM diI-positive (red) cells that are double-stained for β-tubulin III (Cy2, cryosectioned, confocal).
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fig6: EBCs from untreated EBs form highly invasive cortical and ventricular tumors, whereas S18-treated EBCs show enhanced neuronal differentiation after engraftment. (A) A tumor developed from Vybrant CM diI-labeled ROSA-26 EBCs was immunostained for β-galactosidase (Cy2, green). The arrow indicates a residual cluster of Vybrant CM diI-labeled cells. (B) S18-treated or untreated EBCs were stained with Vybrant CM diI (red, untreated cells) or Vybrant diO (green, treated cells), mixed, and injected into the striatum of neonatal mice. The figure shows settlement of treated cells in the subependymal layer, whereas untreated EBCs form a neural tube-like tumor in the lumen of the right lateral ventricle. (C) Mouse EBCs derived from S18-treated EBs were injected into the striatum of neonatal mice and immunostained for nestin (Cy3, red). (D) After nestin staining, frozen sections were FISH-stained for Y-chromosomes (FITC, green). DNA was counterstained with Hoechst dye (blue). (E) Mouse EBs were treated with 80 μM of S18, labeled with Vybrant CM diI (red), and injected into the striatum of neonatal mice. 6 wk after engraftment, brain sections were immunostained for β-tubulin III (Cy2, green). Arrrow shows cluster of Vybrant CM diI-positive (red) cells that are double-stained for β-tubulin III (Cy2, cryosectioned, confocal).
Mentions: We tested if treatment of EBs with S18 eliminated the residual pluripotent Oct-4(+)/PAR-4(+) stem cells and prevented teratoma formation from EBC-derived transplants. Table III shows that the majority of untreated ROSA-26–derived EBCs formed invasive striatal/cortical and noninvasive ventricular, β-galactosidase–positive tumors (Fig. 6 A). We labeled untreated EBCs with Vybrant CM diI (red) and S18-treated EBCs with Vybrant diO (green) and injected a mixed population of these cells into the striatum of the same mouse. Untreated, diI-labeled stem cells developed ventricular and invasive, striatal and cortical tumors, whereas S18-treated, diO-labeled cells integrated into the subependymal cell layer (Fig. 6 B and Table III) and ventromedial aspects of the hippocampus.

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