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Article |
Correspondence to Erhard Bieberich: ebieberich{at}mail.mcg.edu; or Brian G. Condie: bcondie{at}mail.mcg.edu
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Abstract |
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Introduction |
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Most recently, we have found that the expression of prostate apoptosis response-4 (PAR-4), an endogenous inhibitor protein of atypical PKC, mediates ceramide or ceramide analogue-induced apoptosis in proliferating EB-derived stem cells (Bieberich et al., 2001, 2003). The apoptotic response is specific for PAR-4(+) cells because proliferating cells with low expression of PAR-4 are less sensitive to ceramide or ceramide analogue-induced apoptosis (Bieberich et al., 2003). We also observed that in cell culture, ceramide/PAR-4induced apoptosis is predominant during or before early neural progenitor formation from mouse EB-derived cells (EBCs; Bieberich et al., 2003). However, the majority of EBC-derived nestin(+) cells do not express PAR-4 and are thus resistant to ceramide-inducible apoptosis. From these observations, we conclude that there is a portion of proliferating, Oct-4(+)/PAR-4(+) stem cells in EBCs that can be eliminated due to apoptosis by incubation with ceramide or ceramide analogues. In the current work, we tested the ability of S18 (N-oleoyl serinol), a novel ceramide analogue that has recently been synthesized in our laboratory to induce apoptosis in mouse and human EBCs (Bieberich et al., 2000, 2002). We used multilineage teratoma formation in neonatal mouse brain as an assay to measure the level of pluripotent cells in mouse EBCs with or without incubation with S18 before engraftment into mouse brain. Our results show for the first time that Oct-4(+)/PAR-4(+) stem cells can be eliminated and nestin(+) neural precursors can be enriched in EBCs by incubation with novel ceramide analogues and that this enrichment prevents teratoma formation and promotes neural differentiation after engraftment into mouse brain.
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Results |
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Fig. 1 C shows the gene expression level of PAR-4, nestin, and Oct-4, a transcription factor required for the maintenance of undifferentiated and pluripotent ES cells. Our results show that at the late EB and early NP stage Oct-4, PAR-4, and nestin were coexpressed in differentiating ES cultures, indicating that pluripotent (Oct-4(+)) cells and NPs (nestin(+)) coexisted at these differentiation stages. Coexistence of pluripotent stem cells and NPs was thus concomitant with the highest degree of S18-inducible apoptosis in EBs and EBCs.
Ceramide-induced apoptosis diminishes Oct-4(+)/PAR-4(+) mouse and human stem cells in EBs
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).
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Ceramide-induced apoptosis enriches nestin-positive neural progenitors in EBCs
We defined the population of S18-sensitive and -insensitive cells using immunocytochemistry for the expression of PAR-4, Oct-4, and nestin in attached EBs. Fig. 3 C shows that in untreated EBs, Oct-4(+)/PAR-4(+) cells are mostly confined to the center of the EBs, which is consistent with the result shown in Fig. 2 B. Nestin staining was found in cells that immediately surrounded the EB and some apparently migrating cells outside of the EBs. Consistent with our previous study (Bieberich et al., 2003), the small number of cells that coexpressed PAR-4 and nestin showed asymmetric subcellular distribution of the two proteins (Fig. 3 C, arrow). TUNEL and Annexin V staining in Fig. 3 D shows that S18 induced apoptosis selectively in nestin() cells of the central EB and in single, peripheral nestin() cells, but not in nestin(+) cells that immediately surrounded the central EB. The resistance of cells that highly express filamentous nestin toward ceramide-inducible apoptosis was also found with human EBs (unpublished data), suggesting that ceramide induces apoptosis in a similar population of pluripotent Oct-4(+)/PAR-4(+) cells derived from mouse or human EBs.
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).
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The effect of S18 treatment on the neuronal differentiation of EBC-derived NPs was determined by immunofluorescence staining for the intermediate filament protein NF-66 and the neuronal marker MAP-2 or glial marker GFAP (Chan et al., 1997; Bieberich et al., 2003). Fig. 4 D shows that NF-66 was very early expressed during NP differentiation, whereas GFAP was not expressed. In terminally differentiated cells, MAP-2(+) neurons and GFAP(+) glial cells were equally formed, which was not altered by prior incubation of EBs with S18 (unpublished data). This result indicates that S18 did not impair terminal differentiation of EBCs by eliminating Oct-4(+)/PAR-4(+) cells. On the contrary, in areas with comparable cell density, S18 treatment of EBs resulted in the early assembly of numerous NF-66(+) processes 48 h after expansion of the EBC whereas only few processes were seen without treatment (Fig. 4 E). The accelerated differentiation to NF-66(+) early neurons may have resulted from the increased population of nestin(+) NPs due to S18 treatment of EBs before expansion or may have been due to ceramide analogue-promoted neuronal differentiation.
S18-treated EBCs are less likely to form teratomas and undergo neuronal differentiation after transplantation into mouse brain
Stem cells derived from dissociated EBs are the earliest source for neural precursor cells and have been tested as a source of cells for stem cell transplantation (Deacon et al., 1998; Bjorklund et al., 2002). However, previous papers reported that in some of the animals, transplantation of EBCs resulted in the formation of stem cellderived tumors (teratomas; Bjorklund et al., 2002; Barberi et al., 2003; Erdo et al., 2003). Because S18 treatment reduced the proportion of proliferating, pluripotent Oct-4(+)/PAR-4(+) cells in EBs, we hypothesized that S18-treated EBCs would be less likely to form teratomas in vivo. The EBCs from two ES cell lines (ES-J1 and ROSA-26) were labeled with Vybrant CM diI, a red fluorescent, nontoxic, permanent dye for tracking of the injected cells (Iwaguro et al., 2002). The injected cells were also identified by additional methods based on in situ hybridization to a fluorescent DNA probe for detection of the Y-chromosome in the male donor EBCs (Y-mapping), and by immunofluorescence staining for ß-galactosidase in ROSA-26 cells (Zambrowicz et al., 1997). In each experiment, we monitored survival and neural differentiation of the EBCs by further cultivating an aliquot of the cell suspension used for injection in culture.
In preliminary studies, we found that intrastriatal injection of untreated EBCs into postnatal day 10 mice gave rise to 57 tumors throughout the brain after 6 wk in 12 out of 15 animals (Table III). Fig. 5 A shows a massive tumor that emerged on the surface of the right hemisphere at the injection site of the untreated EBCs. Immunohistochemistry using antibodies against -fetoprotein, desmin, vimentin, and GFAP confirmed that the grafted EBCs had formed teratomas containing endodermal, mesodermal, and ectodermal tissues (Fig. 5 B; Vance et al., 1988; Sangruchi and Sobel, 1989). Within the teratomas, nestin expressing cells did not express PAR-4 as we would predict from previous studies of in vitro ES cell differentiation (Bieberich et al., 2003; Fig. 5 C). However, PAR-4(+)/nestin() cells in the center of nestin(+) cell clusters expressed Oct-4 (Fig. 5 D). These results indicated that EBC-derived teratomas maintained a subpopulation of pluripotent, Oct-4(+)/PAR-4(+)/nestin() stem cells.
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Discussion |
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Apoptosis mediated by PAR-4 may involve the regulation of several downstream targets, and inhibition of PKC may at least partly explain the effects observed in our paper for the following reasons: (a) the novel ceramide analogue S18 induces the formation of a protein complex between PAR-4 and PKC
; (b) inhibition of PKC
using a specific pseudosubstrate peptide is sufficient to induce apoptosis in EBCs; and (c) S18 eliminates the Oct-4(+)/PAR-4(+) cell population, whereas nestin(+)/PAR-4() cells survive, proliferate, and differentiate into neural cells. The ceramide analogue-induced elimination of Oct-4(+)/PAR-4(+) mouse and human cells suggests that a similar subpopulation of pluripotent mouse or human EBCs is specifically sensitive toward ceramide or ceramide analogues, whereas committed neural precursors are resistant. Neither the proliferation rate of nestin(+)/PAR-4() cells nor the ability of EBCs to undergo neuronal differentiation is diminished by the elimination of residual Oct-4(+)/PAR-4(+) EBCs. In cell transplantation experiments using EBCs, we found that teratomas contained residual nonapoptotic Oct-4(+)/PAR-4(+) cells whose specific elimination by S18-induced apoptosis before grafting prevented teratoma formation and enhanced integration of the treated cells into neural tissue. From these results, we conclude that treatment of EBs with S18 prevents teratoma formation from residual, pluripotent EBCs in favor of neural differentiation from enriched neural precursors.
Prevention of teratoma formation resulted from the selective elimination of Oct-4(+)/PAR-4(+) cells that were either not apoptotic or had recovered from the initial phase of apoptosis in untreated EBCs. Cells escaping from death due to reversed or interrupted apoptosis ("Zombie cells") have been reported previously (Narula et al., 2001). In the case of pluripotent stem cells, they may be a source for unwanted teratoma formation unless forced to complete programmed cell death. Selective elimination of Oct-4(+)/PAR-4(+) cells is consistent with the observation that after plating of S18-treated EBCs the ratio of nestin(+) to Oct-4(+) cells was substantially increased. The surviving S18-treated EBCs underwent rapid differentiation to neuronal precursors as shown by nestin, ß-tubulin III, and NF-66 staining, in vitro as well as in vivo after grafting. The S18-treated cells integrated into the subependymal layer and in areas below the dentate gyrus. The intensive staining for neuronal markers shows early differentiation of neuronal cells, suggesting that treatment with novel ceramide analogues not only prevents teratoma formation but also supports neuronal differentiation. It has been suggested that ceramide may be involved in apoptosis and differentiation depending on the context of cell signaling. In particular, the expression of the antiapoptotic protein Bcl-2 has been found to channel the ceramide response toward neuronal differentiation, whereas the expression of PAR-4 favors an apoptotic response (Diaz-Meco et al., 1996; Zhang et al., 1996; Sells et al., 1997; Guo et al., 1998; Suzuki and Tsutomi, 1998; Bieberich et al., 2000, 2003; Chen et al., 2001; Esdar et al., 2001; Luberto et al., 2002; Liang et al., 2003). These results are consistent with the observation that the expression of Bcl-2 is elevated in S18-treated EBCs that survive ceramide-induced apoptosis und undergo further neuronal differentiation.
Several investigators have performed neural cell transplants using mouse or human ES cellderived cell types (Deacon et al., 1998; Brustle et al., 1999; Rossant, 2001; Reubinoff et al., 2001; Bjorklund et al., 2002; Gottlieb, 2002; Carpenter et al., 2003; Hubner et al., 2003). In many of these studies teratomas were not reported. In these cases, the transplanted cells were differentiated cell types that had been derived from the ES cells using lengthy multistep procedures involving repeated passaging and exposure to growth/differentiation factors or retinoic acid (Brustle et al., 1999; Reubinoff et al., 2001; Gottlieb, 2002; Barberi et al., 2003; Carpenter et al., 2003). An alternative approach has been reported for generating functional midbrain dopaminergic neurons from mouse ES cells by transplanting cells derived from EBs after only 4 d of differentiation in suspension culture. Although these investigators injected many fewer EBCs (12 x 103 cells) compared with the number used in our injections (105 cells), teratomas still formed in 26% (5/19) of the hosts and an additional 15% (3/19) contained graft-derived nonneural cell types (Bjorklund et al., 2002). Our work suggests that treatment of EBCs with ceramide or novel ceramide analogues kills pluripotent cells, enriches for neural progenitors, and obliterates the teratoma forming potential of these cells. In future work, we will investigate the regulatory interdependence of pluripotency (as determined by Oct-4) and sensitivity toward ceramide-inducible apoptosis (as determined by PAR-4). We will also graft S18-treated EBCs into adult mice and investigate if these cells survive and differentiate in a more hostile environment as present in the adult brain. Our results suggest that exposure to novel ceramide analogues may be a useful new method for eliminating pluripotent cell types from differentiating ES cell cultures before transplantation and, subsequently, enhancing neuronal differentiation for safe stem cell therapy.
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Materials and methods |
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Methods
ES cell differentiation, apoptosis induction, and MACS
In vitro neural differentiation of mouse and human ES cells (ES-J1, ROSA 26) followed a serum deprivation protocol as described previously (Okabe et al., 1996; Hancock et al., 2000; Bieberich et al., 2003). For MACS, cells were dissociated by incubation with trypsin, washed with PBS, and incubated for 20 min at 4°C with Annexin Vconjugated magnetic beads. Annexin Vnegative (flow through) and positive (retained cells) fractions were collected by passage through a MACS column following the manufacturer's (Miltenyi Biotec) procedures.
Transplantation of mouse EBCs
The ceramide analogue-treated or untreated mouse EBs were nonenzymatically dissociated and labeled with Vybrant CM diI or diO according to the manufacturer's protocol (Molecular Probes). The dissociated EBCs were used for in vitro neural differentiation or transplantation into the striatum of 10-d-old C57CLB6 mice by intracranial injection (bregma 1 mm, right hemisphere 2 mm off suture, 2 mm deep) of 105 EBCs in 5 µl of 0.9% sterile saline solution (Yanai et al., 1995). We transplanted equal numbers of viable untreated and S18-treated cells as determined by trypan blue staining.
Immunostaining, Y-FISH, and apoptosis assays
Differentiating ES cells (EBs and EBCs) on poly-L-ornithine/laminin-coated coverslips or frozen brain sections were fixed with 4% PFA in PBS and then permeabilized by incubation with 0.2% Triton X-100 in PBS for 5 min at RT. TUNEL assays were performed before immunostaining according to a protocol provided by the supplier (Oncogene Research Products). FLICA assays for active caspases were performed with living cells before fixation following the manufacturer's protocol (Chemicon). The immunostaining of fixed cells or brain sections followed procedures described previously using a blocking solution of 3% ovalbumin/2% donkey serum in PBS and concentrations of 5 µg/ml primary or secondary antibody in 0.1% ovalbumin/PBS (Bieberich et al., 2000, 2002). Y-chromosome FISH was performed according to the supplier's instructions (Cambio) after the brain sections were immunostained and underwent a second round of fixation with 4% PFA in PBS. Cell nuclei were stained with 2 µg/ml of Hoechst 33258 in PBS for 30 min at RT. Antigen specific immunostaining was quantified by counting cells that showed signals twofold or more above background fluorescence and the cell counts statistically evaluated using ANOVA and a Chi Square test as described previously (Bieberich et al., 2003). Epifluorescence microscopy was performed with a microscope (model Axiophot; Carl Zeiss MicroImaging, Inc.) using 40x (NA 1.0, oil, plan-apochromat) and 100x (NA 1.4, oil, plan-apochromat) objectives and a Spot II CCD camera. Confocal fluorescence microscopy was performed with a confocal scanning microscope (model LSM 510; Carl Zeiss MicroImaging, Inc.; equipped with Argon-488 and He-Neon 543, 633 lasers) using 40x (NA 1.3, oil, plan-neofluor) and 63x (NA 1.4, oil, apochromat) objectives. Fluorochromes are listed in Materials and methods and the legends for the figures. Spot Software (Scientific Diagnostics) and LSM 510 Meta 3.2 software (Carl Zeiss MicroImaging, Inc.) was used for image acquisition from epifluorescence or confocal microscopy, respectively. Adobe Photoshop 7.0 software was used for background reduction, pseudo-colorizing, and overlaying of pseudo-colorized grayscale images.
RT-PCR
Total RNA was prepared from ceramide analogue-treated or untreated EBCs using the Trizol method following the manufacturer's (Life Systems) protocol. PCR was performed by applying 35 cycles with various amounts of first strand cDNA template (equivalent to 0.050.2 µg of RNA) and 20 pmoles of sense and antisense oligonucleotide primer. The following oligonucleotide primer sequences and annealing temperatures were used: PAR-4 (sense, 5'ccagcgccaggaaaggcaaag3'; antisense, 5'ctaccttgtcagctgcccaacaac3'; 61°C), Oct-4 (sense, 5'ggagaggtgaaaccgtccctagg3'; antisense, 5'agaggaggttccctctgagttgc3'; 61°C); Tert (sense, 5'ctgcgtgtgcgtgctctggac3'; antisense, 5'gacctcagcaaacagcttgttctc, 60°C); Sox-2 (sense, 5'gtggaaacttttgtccgagac3'; antisense, 5'tggagtgggaggaagaggtaac3', 53°C); Sox-1 (sense, 5'ctgctcaagaaggacaagta3'; antisense, 5'ctcatgtagccctgagagt3', 52°C); NF-66 (sense, 5'gcacgtaccattgagataga3', antisense, 5'ctggtactttcttctgtagc3' 52°C); GAPDH (sense, 5'gaaggtgaaggtcggagtcaacg3'; antisense, 5'ggtgatgggatttccattgatgacaagc3'; 58°C). The amount of template from each sample was adjusted until PCR yielded equal intensities of amplification product using GAPDH-specific primers.
Miscellaneous
The amount of protein was determined following a modified Folin phenol reagent (Lowry) assay as described previously (Wang and Smith, 1975). Protein extracted with detergent was precipitated according to the Wessel and Flugge method (Wessel and Flugge, 1984). SDS-PAGE was performed using the Laemmli method followed by immunoblotting as described previously (Laemmli, 1970). Coimmunoprecipitation assays were performed as described previously (Wang et al., 1999).
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Acknowledgments |
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This study was funded by National Institutes of Health grants R01MH064794 to B.G. Condie and R01NS046835 to E. Bieberich.
Submitted: 24 May 2004
Accepted: 13 October 2004
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