Nuclei of Embryonic Stem Cells Reprogram Somatic Cells
http://www.100md.com
《干细胞学杂志》
Center for Animal Transgenesis and Germ Cell Research, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania, USA
Key Words. Oct4 ? Reprogramming ? Embryonic stem cells ? Neurosphere cells ? Fusion
Correspondence: Hans R. Sch?ler, Ph.D., Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Mendelstrasse 7, 48149 Münster, Germany. Telephone: 49-0251-980-2866; Fax: 49-0251-959-2992; e-mail: schoeler@mpi-muenster.mpg.de
ABSTRACT
During mammalian development, the genomic potential of cells is being progressively restricted, with only the earliest stages containing cells of a totipotent or pluripotent phenotype. However, the restricted potential of differentiated cells can be reversed. Mammalian cloning experiments have shown us that the program of differentiated cells can be reset to that of totipotent cells . The exchange of nuclear factors between the donor cell nucleus and the enucleated egg cytoplasm is considered to be important for this process . Somatic cells can be dedifferentiated in vitro by fusion with pluripotent cells, activating genes that are not expressed in adult stem cells . For example, the fusion of a thymic lymphocyte with an embryonic germ cell or an embryonic stem cell (ESC) has led to the activation of the Oct4 gene in the somatic cells. Clarke et al. suggested that coculture of neurosphere cells (NSCs) and ESCs in embryoid bodies induced the NSCs to transdifferentiate into myocytes due to signals from ESCs. Moreover, the differentiated state of somatic cells could also be altered by fusion with another type of somatic cell, suggesting that the dynamic interaction of proteins between the fused cells might be responsible for the plasticity of nuclear function . To date, however, little is known about how somatic cells are actually reprogrammed.
The ooplasm of an enucleated mammalian oocyte has the capacity to recondition or reset the genetic program of a fully differentiated somatic cell nucleus to the point of producing a fully developed organism. However, the experimental difficulties inherent in handling oocytes render them unfeasible for conducting analyses on the underlying mechanisms of genetic reprogramming. In contrast, ESC lines are more amenable to experimental manipulation and present an equally valid tool with which to address the molecular basis of genetic reprogramming. In previous studies, we analyzed the reactivation of Oct4 and an Oct4–green fluorescent protein (GFP) transgene—markers of pluripotency—after transfer of somatic cell nuclei into oocytes . In the present study, we examined whether enucleated cytoplasts of ESCs can also activate the Oct4-GFP transgene in somatic cells or whether nuclear components are required.
MATERIALS AND METHODS
Reprogramming of Oct4-GFP in ESC-NSC Hybrids
To distinguish between the reprogramming capacity of the cytoplasm and that of the nucleus of ESCs, we first separated the cytoplasts (cy) from the karyoplasts (ka) (cyESCs and kaESCs, respectively; Fig. 1). We then fused either kaESCs or cyESCs with NSCs. NSCs were prepared from OG2/ROSA26 heterozygous transgenic mice carrying GFP under the control of the Oct4 promoter (Oct4-GFP) and a neo/lacZ transgene that is expressed ubiquitously. The Oct4 gene is expressed in the pluripotential cells of the embryo up to the gastrulation stage and is restricted to germ cells thereafter . Because Oct4 is inactive in NSCs, Oct4-GFP can only be expressed in these cells if it becomes reactivated. The reprogramming capacity of somatic cells in ESC-NSC hybrid cells induced by PEG can be estimated by examining the GFP-positive signal under a fluorescence microscope. These ES hybrid clones were subcultured in G418-containing ES media every 2 or 3 days after fusion. Oct4-GFP expression was examined to assess reprogramming of a pluripotency marker in the ESC-NSC hybrid cells (Fig. 2). The first GFP-positive cells were observed at days 2 after PEG-induced fusion (Figs. 2A, 2B). The GFP-positive ESC-NSC fusion hybrids formed ESC-like colonies, which were, along with GFP expression, stably maintained for at least 20 passages after fusion and could be recovered from cryopreservation. The reprogramming rate (Oct4-GFP–positive/lacZ-positive colony) at passage 5 was approximately 95%, and the reprogramming rate was stable over subsequent passages (in preparation). To determine whether these were hybrid cells, we examined the karyotype of the cell fusion culture as well as the morphology of the nuclei. The fused Oct4-GFP–positive cells had tetraploid karyotypes (Fig. 2C) and enlarged nuclei with multiple nucleoli (Fig. 2D). The fusion hybrids expressed markers that characterize undifferentiated ESCs, including Oct4, Rex-1, and nanog, but did not express the ectoderm marker gene glutamate receptor 6 (GluR6) (Fig. 2E). These results suggest that the ESC-NSC hybrids have ESC-like potency and that the NSC hybrid counterparts lose neuronal characteristics.
Figure 1. Schematic illustration of the fusion combination. Fusion of ESCs with NSCs induces activation of Oct4-GFP in adult stem cells. To identify whether cytoplasmic or nuclear factors are responsible for reactivation of Oct4-GFP, we fused either karyoplasts or cytoplasts of ESCs (kaESCs and cyESCs, respectively) with NSCs. To investigate whether DNA replication and division is essential for reprogramming of the somatic cell genome, we performed fusion experiments between MMC-treated ESCs and NSCs. If reprogramming of Oct4-GFP was not observed using intact NSCs, NSCs were treated with 5-aza C before fusion to facilitate reprogramming. Abbreviations: 5-aza C, 5-azacytidine; ESC, embryonic stem cell; NSC, neurosphere cell; GFP, green fluorescent protein; MMC, mitomycin C.
Figure 2. Reactivation of Oct4-GFP in ESC-NSC hybrid cells and their characteristics. (A): Phase contrast micrograph of GFP-positive colony was observed after polyethylene glycol–induced fusion. (B): Fluorescence image of GFP-positive colony. (C): Representative tetraploid karyotype of a fused hybrid cell. (D): The Oct4-GFP–positive cells had enlarged nuclei with multiple nucleoli. (E): RT-PCR analysis of gene expression in NSCs, ESC-NSC hybrids, and E14 ESCs. RT- indicates minus RT control. Scale bars = 50 μm (A) and 25 μm (D), respectively. Abbreviations: ESC, embryonic stem cell; NSC, neurosphere cell; GFP, green fluorescent protein; RT-PCR, reverse transcription–polymerase chain reaction.
Separation of ESCs and NSCs into Cytoplasts and Karyoplasts
To obtain favorable yields of enucleated ESCs and NSCs, we established effective separation conditions (Fig. 3A). To this end, we centrifuged cells through a Ficoll gradient and collected cytoplasts from the 15% and 18% regions and karyoplasts from the bottom of the gradient (30% region). To confirm successful separation, the collected cytoplasts and karyoplasts were stained with Hoechst and examined under a fluorescence microscope (Figs. 3E, 3G). More than 95% of the cells were successfully enucleated, as determined by Hoechst staining. The size of cytoplasts was smaller than that of karyoplasts (Figs. 3D, 3F). The cell viability of cytoplasts and karyoplasts estimated by trypan blue exclusion test was 99.6% and 96.7%, respectively. A karyoplast is defined as a nucleus isolated from a eukaryotic cell that is surrounded by a very thin layer of cytoplasm enclosed within a plasma membrane. The remainder of the cell is called a cytoplast. In CFSE analysis, we found that purified karyoplasts contained only trace amounts of cytoplasm (compare Fig. 3J with Fig. 3M). These observations suggested that the cytoplasts and karyoplasts purified by centrifugation through Ficoll gradient were suitable for fusion with somatic cells.
Figure 3. Separation of karyoplasts and cytoplasts through Ficoll gradient. (A): After centrifugation through the Ficoll gradient, the cytoplasts are collected from the 15% and 20% regions, whereas the karyoplasts are collected from the bottom of the 30% portion of the gradient. Bright field morphology of intact ESCs (B), cytoplasts of ESCs (D), and karyoplasts of ESCs (F) and the respective fluorescence images after Hoechst staining (C, E, G). Although some cells were not successfully enucleated (arrowhead), more than 95% of the cells were successfully enucleated. CFSE analysis of ESC controls (H–J) and karyoplasts of ESCs (K–M) was conducted to confirm successful removal of cytoplasm. Bright field morphology (H, K) and the respective fluorescence images for nuclei (Hoechst; I, L) and cytoplasm (CFSE; J, M). Purified karyoplasts contained only trace amounts of cytoplasm compared with cytoplasts (arrow) and ESCs (J). Scale bars = 20 μm. Abbreviations: CFSE, carboxyfluorescein diacetate succinimidyl ester; ESC, embryonic stem cell.
Reprogramming Ability of ESC Karyoplasts and Cytoplasts
To determine whether ESC nuclei lacking the cytoplasm have the potential to activate a marker of pluripotency in the somatic genome, we fused kaESCs with NSCs. Oct4-GFP reactivation was observed in kaESC-NSC hybrids, again on approximately day 2 after fusion (Figs. 4A, 4B). The kaESC-NSC hybrids formed ESC-like colonies and expressed the ESC markers Oct4, Rex-1, and nanog, just like ESC-NSC hybrids and E14 ESCs (data not shown). This result indicates that karyoplasts of ESCs contain factors that are required for reprogramming.
Figure 4. Reprogramming ability of ESC karyoplasts and cytoplasts. ESC karyoplast and NSC hybrid colonies (A) and GFP-positive cells in the hybrid colonies (B). Most ESC cytoplast and NSC hybrids were dying approximately 3 days after fusion (C), and some survived a few days longer, with no Oct4-GFP expression observed (D). Scale bars = 20 μm. Abbreviations: ESC, embryonic stem cell; GFP, green fluorescent protein; NSC, neurosphere cell.
To additionally determine whether ESC cytoplasm lacking nuclei has reprogramming potential, we then conducted a fusion experiment between cyESCs with either NSCs or kaNSCs. GFP was detected neither in cyESC-NSCs nor in cyESC-kaNSC hybrids, although the fusion was tested under various conditions in five independent fusion experiments. Using larger cyESCs obtained from CB-treated ESCs or changing the epigenetic state of NSCs by treatment with 5-azacytidine (5-aza C) also did not facilitate induction of Oct4-GFP expression (data not shown). Most of the cells in the cyESC-NSC fusion mixture were dying approximately 3 days after fusion (Figs. 4C, 4D), and some survived a few days longer. Expression of GFP after fusion of ESCs or kaESCs with NSCs was detected after 2 days. In contrast, even after 3 days or more, cyESCs could not reactivate Oct4-GFP in NSCs, indicating that cyESCs either lacked factors crucial for reactivation or that these factors were present but were below a critical level.
MMC-Treated ESCs Can Reprogram NSCs Pretreated with 5-aza C
To investigate whether DNA replication is important for reprogramming of the somatic cell genome, we performed fusion experiments between NSCs and MMC-treated ESCs. MMC inhibits DNA replication and cell division but does not affect gene transcription and protein synthesis. We observed that MMC-treated ESCs did not reactivate the Oct4-GFP of intact NSCs after fusion. However, treatment of NSCs with 5-aza C to facilitate reprogramming of NSCs indeed resulted in reactivation of Oct4-GFP. Figures 5A and 5B show a hybrid cell that was attached to the feeder layer but did not form a colony, remaining as a single cell even 3 days after fusion. Many Oct4-GFP–expressing cells were identified in fusion colonies (data not shown). However, only Oct4-GFP expression in a single nondividing cell indicates that a marker of pluripotency can be reactivated in NSCs without replication. In contrast, unfused control NSCs, treated with 5-aza C and PEG, did not re-express Oct4-GFP (Figs. 5C, 5D). These results indicate that ESCs that are incapable of DNA replication and cell division still retain their reprogramming ability. These results suggest that the reprogramming of Oct4-GFP in differentiated cells requires neither DNA replication nor cell division.
Figure 5. Reactivation of Oct4-GFP in MMC-treated ESC and NSC hybrids. Bright field (A) and fluorescence images (B) of a representative GFP-positive single cell observed after fusion of MMC-treated ESCs with NSCs. Representative control NSC colony treated with 5-azacytidine and polyethylene glycol (C), which did not re-express Oct4-GFP (D). Scale bars = 25 μm. Abbreviations: ESC, embryonic stem cell; GFP, green fluorescent protein; MMC, mitomycin C; NSC, neurosphere cell.
DISCUSSION
This work was supported by the Postdoctoral Fellowship Program of Korea Science and Engineering Foundation (KOSEF); the Marion Dilley and David George Jones Funds and the Commonwealth and General Assembly of Pennsylvania; and the National Institutes of Health grant 1RO1HD420 11–01 to H.S.
REFERENCES
Surani MA. Reprogramming of genome function through epigenetic inheritance. Nature 2001;414:122–128.
Stice SL, Robl JM. Nuclear reprogramming in nuclear transplant rabbit embryos. Biol Reprod 1988;39:657–664.
Collas P, Robl JM. Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos. Biol Reprod 1991;45:455–465.
Tada M, Tada T, Lefebvre L et al. Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells. EMBO J 1997;16:6510–6520.
Tada M, Takahama Y, Abe K et al. Nuclear reprogramming of somatic cells by in vitro hybridization with ESCs. Curr Biol 2001;11:1553–1558.
Ying QL, Nichols J, Evans EP et al. Changing potency by spontaneous fusion. Nature 2002;416:545–548.
Terada N, Hamazaki T, Oka M et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002;416:542–545.
Clarke DL, Johansson CB, Wilberts J et al. Generalized potential of adult neural stem cells. Science 2000;288:1660–1663.
Blau HM, Chiu CP, Webster C. Cytoplasmic activation of human nuclear genes in stable heterokaryons. Cell 1983;32:1171–1180.
Blau HM, Blakely BT. Plasticity of cell fate: insights from heterokaryons. Semin Cell Dev Biol 1999;10:267–272.
Boiani M, Eckardt S, Sch?ler HR et al. Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes Dev 2002;16:1209–1219.
Boiani M, Eckardt S, Leu NA et al. Pluripotency deficit in clones overcome by clone-clone aggregation: epigenetic complementation? EMBO J 2003;22:5304–5312.
Szabo PE, Hubner K, Scholer H et al. Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech Dev 2002;115:157–160.
Sch?ler HR, Ciesiolka T, Gruss P. A nexus between Oct-4 and E1A: implications for gene regulation in embryonic stem cells. Cell 1991;66:291–304.
Palmieri SL, Peter W, Hess H et al. Oct4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol 1994;166:259–267.
Yeom YI, Fuhrmann G, Ovitt CE et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 1996;122:881–894.
Wakayama S, Cibelli JB, Wakayama T. Effect of timing of the removal of oocyte chromosomes before or after injection of somatic nucleus on development of NT embryos. Cloning Stem Cells 2003;5:181–189.
Wakayama T, Taeno H, Mombaerts P et al. Nuclear transfer into mouse zygotes. Nat Genet 2000;24:108–109.
Du F, Sung LY, Tian XC et al. Differential cytoplast requirement for embryonic and somatic cell nuclear transfer in cattle. Mol Reprod Dev 2002;63:183–191.
Goffin J, Eisenhauer E. DNA methyltransferase inhibitors: state of the art. Ann Oncol 2002;13:1699–1716.
Chiu CP, Blau HM. 5-Azacytidine permits gene activation in a previously non-inducible cell type. Cell 1985;40:417–424.
Jiang Y, Jahagirda BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.
Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24:372–376.
Byrne JA, Simonsson S, Western PS et al. Nuclei of adult mammalian somatic cells are directly reprogrammed to oct-4 stem cell gene expression by amphibian oocytes. Curr Biol 2003;13:1206–1213.
Johansson BM, Wiles MV. Evidence for involvement of activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoietic development. Mol Cell Biol 1995;15:141–151.
Mitsui K, Tokuzawa Y, Itoh H et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003;13:631–642.(Jeong Tae Do, Hans R. Sch)
Key Words. Oct4 ? Reprogramming ? Embryonic stem cells ? Neurosphere cells ? Fusion
Correspondence: Hans R. Sch?ler, Ph.D., Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Mendelstrasse 7, 48149 Münster, Germany. Telephone: 49-0251-980-2866; Fax: 49-0251-959-2992; e-mail: schoeler@mpi-muenster.mpg.de
ABSTRACT
During mammalian development, the genomic potential of cells is being progressively restricted, with only the earliest stages containing cells of a totipotent or pluripotent phenotype. However, the restricted potential of differentiated cells can be reversed. Mammalian cloning experiments have shown us that the program of differentiated cells can be reset to that of totipotent cells . The exchange of nuclear factors between the donor cell nucleus and the enucleated egg cytoplasm is considered to be important for this process . Somatic cells can be dedifferentiated in vitro by fusion with pluripotent cells, activating genes that are not expressed in adult stem cells . For example, the fusion of a thymic lymphocyte with an embryonic germ cell or an embryonic stem cell (ESC) has led to the activation of the Oct4 gene in the somatic cells. Clarke et al. suggested that coculture of neurosphere cells (NSCs) and ESCs in embryoid bodies induced the NSCs to transdifferentiate into myocytes due to signals from ESCs. Moreover, the differentiated state of somatic cells could also be altered by fusion with another type of somatic cell, suggesting that the dynamic interaction of proteins between the fused cells might be responsible for the plasticity of nuclear function . To date, however, little is known about how somatic cells are actually reprogrammed.
The ooplasm of an enucleated mammalian oocyte has the capacity to recondition or reset the genetic program of a fully differentiated somatic cell nucleus to the point of producing a fully developed organism. However, the experimental difficulties inherent in handling oocytes render them unfeasible for conducting analyses on the underlying mechanisms of genetic reprogramming. In contrast, ESC lines are more amenable to experimental manipulation and present an equally valid tool with which to address the molecular basis of genetic reprogramming. In previous studies, we analyzed the reactivation of Oct4 and an Oct4–green fluorescent protein (GFP) transgene—markers of pluripotency—after transfer of somatic cell nuclei into oocytes . In the present study, we examined whether enucleated cytoplasts of ESCs can also activate the Oct4-GFP transgene in somatic cells or whether nuclear components are required.
MATERIALS AND METHODS
Reprogramming of Oct4-GFP in ESC-NSC Hybrids
To distinguish between the reprogramming capacity of the cytoplasm and that of the nucleus of ESCs, we first separated the cytoplasts (cy) from the karyoplasts (ka) (cyESCs and kaESCs, respectively; Fig. 1). We then fused either kaESCs or cyESCs with NSCs. NSCs were prepared from OG2/ROSA26 heterozygous transgenic mice carrying GFP under the control of the Oct4 promoter (Oct4-GFP) and a neo/lacZ transgene that is expressed ubiquitously. The Oct4 gene is expressed in the pluripotential cells of the embryo up to the gastrulation stage and is restricted to germ cells thereafter . Because Oct4 is inactive in NSCs, Oct4-GFP can only be expressed in these cells if it becomes reactivated. The reprogramming capacity of somatic cells in ESC-NSC hybrid cells induced by PEG can be estimated by examining the GFP-positive signal under a fluorescence microscope. These ES hybrid clones were subcultured in G418-containing ES media every 2 or 3 days after fusion. Oct4-GFP expression was examined to assess reprogramming of a pluripotency marker in the ESC-NSC hybrid cells (Fig. 2). The first GFP-positive cells were observed at days 2 after PEG-induced fusion (Figs. 2A, 2B). The GFP-positive ESC-NSC fusion hybrids formed ESC-like colonies, which were, along with GFP expression, stably maintained for at least 20 passages after fusion and could be recovered from cryopreservation. The reprogramming rate (Oct4-GFP–positive/lacZ-positive colony) at passage 5 was approximately 95%, and the reprogramming rate was stable over subsequent passages (in preparation). To determine whether these were hybrid cells, we examined the karyotype of the cell fusion culture as well as the morphology of the nuclei. The fused Oct4-GFP–positive cells had tetraploid karyotypes (Fig. 2C) and enlarged nuclei with multiple nucleoli (Fig. 2D). The fusion hybrids expressed markers that characterize undifferentiated ESCs, including Oct4, Rex-1, and nanog, but did not express the ectoderm marker gene glutamate receptor 6 (GluR6) (Fig. 2E). These results suggest that the ESC-NSC hybrids have ESC-like potency and that the NSC hybrid counterparts lose neuronal characteristics.
Figure 1. Schematic illustration of the fusion combination. Fusion of ESCs with NSCs induces activation of Oct4-GFP in adult stem cells. To identify whether cytoplasmic or nuclear factors are responsible for reactivation of Oct4-GFP, we fused either karyoplasts or cytoplasts of ESCs (kaESCs and cyESCs, respectively) with NSCs. To investigate whether DNA replication and division is essential for reprogramming of the somatic cell genome, we performed fusion experiments between MMC-treated ESCs and NSCs. If reprogramming of Oct4-GFP was not observed using intact NSCs, NSCs were treated with 5-aza C before fusion to facilitate reprogramming. Abbreviations: 5-aza C, 5-azacytidine; ESC, embryonic stem cell; NSC, neurosphere cell; GFP, green fluorescent protein; MMC, mitomycin C.
Figure 2. Reactivation of Oct4-GFP in ESC-NSC hybrid cells and their characteristics. (A): Phase contrast micrograph of GFP-positive colony was observed after polyethylene glycol–induced fusion. (B): Fluorescence image of GFP-positive colony. (C): Representative tetraploid karyotype of a fused hybrid cell. (D): The Oct4-GFP–positive cells had enlarged nuclei with multiple nucleoli. (E): RT-PCR analysis of gene expression in NSCs, ESC-NSC hybrids, and E14 ESCs. RT- indicates minus RT control. Scale bars = 50 μm (A) and 25 μm (D), respectively. Abbreviations: ESC, embryonic stem cell; NSC, neurosphere cell; GFP, green fluorescent protein; RT-PCR, reverse transcription–polymerase chain reaction.
Separation of ESCs and NSCs into Cytoplasts and Karyoplasts
To obtain favorable yields of enucleated ESCs and NSCs, we established effective separation conditions (Fig. 3A). To this end, we centrifuged cells through a Ficoll gradient and collected cytoplasts from the 15% and 18% regions and karyoplasts from the bottom of the gradient (30% region). To confirm successful separation, the collected cytoplasts and karyoplasts were stained with Hoechst and examined under a fluorescence microscope (Figs. 3E, 3G). More than 95% of the cells were successfully enucleated, as determined by Hoechst staining. The size of cytoplasts was smaller than that of karyoplasts (Figs. 3D, 3F). The cell viability of cytoplasts and karyoplasts estimated by trypan blue exclusion test was 99.6% and 96.7%, respectively. A karyoplast is defined as a nucleus isolated from a eukaryotic cell that is surrounded by a very thin layer of cytoplasm enclosed within a plasma membrane. The remainder of the cell is called a cytoplast. In CFSE analysis, we found that purified karyoplasts contained only trace amounts of cytoplasm (compare Fig. 3J with Fig. 3M). These observations suggested that the cytoplasts and karyoplasts purified by centrifugation through Ficoll gradient were suitable for fusion with somatic cells.
Figure 3. Separation of karyoplasts and cytoplasts through Ficoll gradient. (A): After centrifugation through the Ficoll gradient, the cytoplasts are collected from the 15% and 20% regions, whereas the karyoplasts are collected from the bottom of the 30% portion of the gradient. Bright field morphology of intact ESCs (B), cytoplasts of ESCs (D), and karyoplasts of ESCs (F) and the respective fluorescence images after Hoechst staining (C, E, G). Although some cells were not successfully enucleated (arrowhead), more than 95% of the cells were successfully enucleated. CFSE analysis of ESC controls (H–J) and karyoplasts of ESCs (K–M) was conducted to confirm successful removal of cytoplasm. Bright field morphology (H, K) and the respective fluorescence images for nuclei (Hoechst; I, L) and cytoplasm (CFSE; J, M). Purified karyoplasts contained only trace amounts of cytoplasm compared with cytoplasts (arrow) and ESCs (J). Scale bars = 20 μm. Abbreviations: CFSE, carboxyfluorescein diacetate succinimidyl ester; ESC, embryonic stem cell.
Reprogramming Ability of ESC Karyoplasts and Cytoplasts
To determine whether ESC nuclei lacking the cytoplasm have the potential to activate a marker of pluripotency in the somatic genome, we fused kaESCs with NSCs. Oct4-GFP reactivation was observed in kaESC-NSC hybrids, again on approximately day 2 after fusion (Figs. 4A, 4B). The kaESC-NSC hybrids formed ESC-like colonies and expressed the ESC markers Oct4, Rex-1, and nanog, just like ESC-NSC hybrids and E14 ESCs (data not shown). This result indicates that karyoplasts of ESCs contain factors that are required for reprogramming.
Figure 4. Reprogramming ability of ESC karyoplasts and cytoplasts. ESC karyoplast and NSC hybrid colonies (A) and GFP-positive cells in the hybrid colonies (B). Most ESC cytoplast and NSC hybrids were dying approximately 3 days after fusion (C), and some survived a few days longer, with no Oct4-GFP expression observed (D). Scale bars = 20 μm. Abbreviations: ESC, embryonic stem cell; GFP, green fluorescent protein; NSC, neurosphere cell.
To additionally determine whether ESC cytoplasm lacking nuclei has reprogramming potential, we then conducted a fusion experiment between cyESCs with either NSCs or kaNSCs. GFP was detected neither in cyESC-NSCs nor in cyESC-kaNSC hybrids, although the fusion was tested under various conditions in five independent fusion experiments. Using larger cyESCs obtained from CB-treated ESCs or changing the epigenetic state of NSCs by treatment with 5-azacytidine (5-aza C) also did not facilitate induction of Oct4-GFP expression (data not shown). Most of the cells in the cyESC-NSC fusion mixture were dying approximately 3 days after fusion (Figs. 4C, 4D), and some survived a few days longer. Expression of GFP after fusion of ESCs or kaESCs with NSCs was detected after 2 days. In contrast, even after 3 days or more, cyESCs could not reactivate Oct4-GFP in NSCs, indicating that cyESCs either lacked factors crucial for reactivation or that these factors were present but were below a critical level.
MMC-Treated ESCs Can Reprogram NSCs Pretreated with 5-aza C
To investigate whether DNA replication is important for reprogramming of the somatic cell genome, we performed fusion experiments between NSCs and MMC-treated ESCs. MMC inhibits DNA replication and cell division but does not affect gene transcription and protein synthesis. We observed that MMC-treated ESCs did not reactivate the Oct4-GFP of intact NSCs after fusion. However, treatment of NSCs with 5-aza C to facilitate reprogramming of NSCs indeed resulted in reactivation of Oct4-GFP. Figures 5A and 5B show a hybrid cell that was attached to the feeder layer but did not form a colony, remaining as a single cell even 3 days after fusion. Many Oct4-GFP–expressing cells were identified in fusion colonies (data not shown). However, only Oct4-GFP expression in a single nondividing cell indicates that a marker of pluripotency can be reactivated in NSCs without replication. In contrast, unfused control NSCs, treated with 5-aza C and PEG, did not re-express Oct4-GFP (Figs. 5C, 5D). These results indicate that ESCs that are incapable of DNA replication and cell division still retain their reprogramming ability. These results suggest that the reprogramming of Oct4-GFP in differentiated cells requires neither DNA replication nor cell division.
Figure 5. Reactivation of Oct4-GFP in MMC-treated ESC and NSC hybrids. Bright field (A) and fluorescence images (B) of a representative GFP-positive single cell observed after fusion of MMC-treated ESCs with NSCs. Representative control NSC colony treated with 5-azacytidine and polyethylene glycol (C), which did not re-express Oct4-GFP (D). Scale bars = 25 μm. Abbreviations: ESC, embryonic stem cell; GFP, green fluorescent protein; MMC, mitomycin C; NSC, neurosphere cell.
DISCUSSION
This work was supported by the Postdoctoral Fellowship Program of Korea Science and Engineering Foundation (KOSEF); the Marion Dilley and David George Jones Funds and the Commonwealth and General Assembly of Pennsylvania; and the National Institutes of Health grant 1RO1HD420 11–01 to H.S.
REFERENCES
Surani MA. Reprogramming of genome function through epigenetic inheritance. Nature 2001;414:122–128.
Stice SL, Robl JM. Nuclear reprogramming in nuclear transplant rabbit embryos. Biol Reprod 1988;39:657–664.
Collas P, Robl JM. Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos. Biol Reprod 1991;45:455–465.
Tada M, Tada T, Lefebvre L et al. Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells. EMBO J 1997;16:6510–6520.
Tada M, Takahama Y, Abe K et al. Nuclear reprogramming of somatic cells by in vitro hybridization with ESCs. Curr Biol 2001;11:1553–1558.
Ying QL, Nichols J, Evans EP et al. Changing potency by spontaneous fusion. Nature 2002;416:545–548.
Terada N, Hamazaki T, Oka M et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002;416:542–545.
Clarke DL, Johansson CB, Wilberts J et al. Generalized potential of adult neural stem cells. Science 2000;288:1660–1663.
Blau HM, Chiu CP, Webster C. Cytoplasmic activation of human nuclear genes in stable heterokaryons. Cell 1983;32:1171–1180.
Blau HM, Blakely BT. Plasticity of cell fate: insights from heterokaryons. Semin Cell Dev Biol 1999;10:267–272.
Boiani M, Eckardt S, Sch?ler HR et al. Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes Dev 2002;16:1209–1219.
Boiani M, Eckardt S, Leu NA et al. Pluripotency deficit in clones overcome by clone-clone aggregation: epigenetic complementation? EMBO J 2003;22:5304–5312.
Szabo PE, Hubner K, Scholer H et al. Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech Dev 2002;115:157–160.
Sch?ler HR, Ciesiolka T, Gruss P. A nexus between Oct-4 and E1A: implications for gene regulation in embryonic stem cells. Cell 1991;66:291–304.
Palmieri SL, Peter W, Hess H et al. Oct4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol 1994;166:259–267.
Yeom YI, Fuhrmann G, Ovitt CE et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 1996;122:881–894.
Wakayama S, Cibelli JB, Wakayama T. Effect of timing of the removal of oocyte chromosomes before or after injection of somatic nucleus on development of NT embryos. Cloning Stem Cells 2003;5:181–189.
Wakayama T, Taeno H, Mombaerts P et al. Nuclear transfer into mouse zygotes. Nat Genet 2000;24:108–109.
Du F, Sung LY, Tian XC et al. Differential cytoplast requirement for embryonic and somatic cell nuclear transfer in cattle. Mol Reprod Dev 2002;63:183–191.
Goffin J, Eisenhauer E. DNA methyltransferase inhibitors: state of the art. Ann Oncol 2002;13:1699–1716.
Chiu CP, Blau HM. 5-Azacytidine permits gene activation in a previously non-inducible cell type. Cell 1985;40:417–424.
Jiang Y, Jahagirda BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.
Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24:372–376.
Byrne JA, Simonsson S, Western PS et al. Nuclei of adult mammalian somatic cells are directly reprogrammed to oct-4 stem cell gene expression by amphibian oocytes. Curr Biol 2003;13:1206–1213.
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