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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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Treatment with ceramide analogues eliminates Oct-4(+)/PAR-4(+) cells in EBs derived from mouse or human ES cells. (A) Mouse EBs were incubated for 24 (middle) or 48 h (right) with 80 μM of S18 showing ongoing cell death in the peripheral and central region of the EB. (B) The center of S18-treated mouse EBs shows intensive costaining of Annexin V (FITC, green)–, PAR-4 (Cy3, red)–, and Oct-4 (Cy5, blue)–positive cells. (C and D) S18-treated human EBs were stained for apoptotic cells using TUNEL assays (FITC, green) and confocal immunofluorescence microscopy was used to detect PAR-4 (Cy3, red)– and Oct-4 (Cy5, pink)–positive cells. Arrows show apoptotic Oct-4(+)/PAR-4(+) (arrowhead 2) or Oct-4(−)/PAR-4(+) (arrowhead 1) cells.
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fig2: Treatment with ceramide analogues eliminates Oct-4(+)/PAR-4(+) cells in EBs derived from mouse or human ES cells. (A) Mouse EBs were incubated for 24 (middle) or 48 h (right) with 80 μM of S18 showing ongoing cell death in the peripheral and central region of the EB. (B) The center of S18-treated mouse EBs shows intensive costaining of Annexin V (FITC, green)–, PAR-4 (Cy3, red)–, and Oct-4 (Cy5, blue)–positive cells. (C and D) S18-treated human EBs were stained for apoptotic cells using TUNEL assays (FITC, green) and confocal immunofluorescence microscopy was used to detect PAR-4 (Cy3, red)– and Oct-4 (Cy5, pink)–positive cells. Arrows show apoptotic Oct-4(+)/PAR-4(+) (arrowhead 2) or Oct-4(−)/PAR-4(+) (arrowhead 1) cells.

Mentions: Recently, we have reported that ceramide or S18 rapidly induces apoptosis in proliferating EBCs that express a high level of PAR-4, but little or no nestin (Bieberich et al., 2003). To determine whether or not ceramide-sensitive PAR-4(+) cells are pluripotent EBCs, we analyzed the expression of PAR-4 and Oct-4 by immunofluorescence microscopy. Fig. 2 A shows the effect of S18 on mouse EBs resulting in apoptosis and, eventually, loss of cells in the center of the EBs, whereas cells immediately surrounding the EBs were resistant toward S18. The apoptotic cells were identified as Oct-4 and PAR-4 double positive, indicating that residual Oct-4(+)/PAR-4(+) cells within the EBs maintained their sensitivity toward ceramide or ceramide analogues (Fig. 2 B). A similar subpopulation of apoptotic cells was found when EBs from human ES cells were incubated with S18 (Fig. 2 C). Confocal immunofluorescence microscopy confirmed that TUNEL(+) human cells within the EBs also stained for PAR-4 (Fig. 2 D, arrow 1) or Oct-4 and PAR-4 (Fig. 2 D, arrow 2).


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)

Treatment with ceramide analogues eliminates Oct-4(+)/PAR-4(+) cells in EBs derived from mouse or human ES cells. (A) Mouse EBs were incubated for 24 (middle) or 48 h (right) with 80 μM of S18 showing ongoing cell death in the peripheral and central region of the EB. (B) The center of S18-treated mouse EBs shows intensive costaining of Annexin V (FITC, green)–, PAR-4 (Cy3, red)–, and Oct-4 (Cy5, blue)–positive cells. (C and D) S18-treated human EBs were stained for apoptotic cells using TUNEL assays (FITC, green) and confocal immunofluorescence microscopy was used to detect PAR-4 (Cy3, red)– and Oct-4 (Cy5, pink)–positive cells. Arrows show apoptotic Oct-4(+)/PAR-4(+) (arrowhead 2) or Oct-4(−)/PAR-4(+) (arrowhead 1) cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Treatment with ceramide analogues eliminates Oct-4(+)/PAR-4(+) cells in EBs derived from mouse or human ES cells. (A) Mouse EBs were incubated for 24 (middle) or 48 h (right) with 80 μM of S18 showing ongoing cell death in the peripheral and central region of the EB. (B) The center of S18-treated mouse EBs shows intensive costaining of Annexin V (FITC, green)–, PAR-4 (Cy3, red)–, and Oct-4 (Cy5, blue)–positive cells. (C and D) S18-treated human EBs were stained for apoptotic cells using TUNEL assays (FITC, green) and confocal immunofluorescence microscopy was used to detect PAR-4 (Cy3, red)– and Oct-4 (Cy5, pink)–positive cells. Arrows show apoptotic Oct-4(+)/PAR-4(+) (arrowhead 2) or Oct-4(−)/PAR-4(+) (arrowhead 1) cells.
Mentions: Recently, we have reported that ceramide or S18 rapidly induces apoptosis in proliferating EBCs that express a high level of PAR-4, but little or no nestin (Bieberich et al., 2003). To determine whether or not ceramide-sensitive PAR-4(+) cells are pluripotent EBCs, we analyzed the expression of PAR-4 and Oct-4 by immunofluorescence microscopy. Fig. 2 A shows the effect of S18 on mouse EBs resulting in apoptosis and, eventually, loss of cells in the center of the EBs, whereas cells immediately surrounding the EBs were resistant toward S18. The apoptotic cells were identified as Oct-4 and PAR-4 double positive, indicating that residual Oct-4(+)/PAR-4(+) cells within the EBs maintained their sensitivity toward ceramide or ceramide analogues (Fig. 2 B). A similar subpopulation of apoptotic cells was found when EBs from human ES cells were incubated with S18 (Fig. 2 C). Confocal immunofluorescence microscopy confirmed that TUNEL(+) human cells within the EBs also stained for PAR-4 (Fig. 2 D, arrow 1) or Oct-4 and PAR-4 (Fig. 2 D, arrow 2).

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