Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4.
The potential of induced pluripotent stem (iPS) cell technology is enormous, but comprehension of the molecular mechanisms that underlie reprogramming is meager, largely because the procedure is still very inefficient. Recently, a number of groups have demonstrated that the inactivation of p53 markedly increases the efficiency of iPS cell generation 1, 2, 3, 4, 5, 6. Furthermore, by reducing the expression of both p16Ink4a and p19Arf (both of which are encoded by alternative reading frames of the Ink4a/Arf locus, also known as the Cdkn2a locus), iPS cell formation was increased relative to that achieved by reducing the expression of p19Arf alone.
Bmi1 was first identified as a proto-oncogene that cooperates with c-Myc to promote the formation of B- and T-cell lymphomas 7, 8 by inhibiting c-Myc-induced apoptosis through repression of the Ink4a/Arf locus 9, 10. Bmi1 is also required for the self-renewal of stem cells and the Ink4a/Arf locus is the main target of Bmi1 stem cell proliferation activity 11, 12, 13. Furthermore, activation of the sonic hedgehog (Shh) signaling pathway induces Bmi1, Sox2, and N-Myc expression, resulting in increased proliferation of neural precursors 14, 15, 16. In this study, we hypothesized that cells with reduced expression of p16Ink4a and p19Arf as well as increased N-Myc, Klf4, and Sox2 expression 13, 16 mediated by Bmi1 overexpression could be efficiently converted into iPS cells with either two factors (Oct4 and Sox2: hereafter designated as 2F-Bmi1-iPS cells (OSB)) or only one factor (Oct4: hereafter designated as BO-iPS cells). Furthermore, we also asked whether Shh or activation of the Shh signaling pathway by oxysterol or purmorphamine can regulate the downstream target genes of Bmi1 in the generation of BO-iPS cells. Our study demonstrates that Bmi1 has dual effects on iPS cell generation from fibroblasts by both suppressing p16 Ink4a and p19 Arf and augmenting Sox2 and N-Myc. By inducing Bmi1 with some chemicals, we have also shown that only one transcription factor (Oct4) is required to reprogram fibroblasts into pluripotent cells, which have the ability to differentiate into all three germ layer cell types and are capable of germline transmission.
Bmi1 replaces the function of Klf4 and c-Myc, and increases reprogramming efficiency
Considering that Bmi1 is essential for the self-renewal of stem cells through repression of the p53 and Rb pathways, we investigated whether Bmi1 could enhance the reprogramming of fibroblasts into iPS cells. To answer this question, we first determined the expression levels of reprogramming-related genes (p16Ink4a, p19Arf, Sox2, N-Myc, p53, and Klf4) in parental mouse embryonic fibroblasts (MEFs) and Bmi1-transduced cells. p16Ink4a, p19Arf, and p53 were significantly repressed in Bmi1-transduced cells compared to MEFs; however, consistent with a previous report 16, Sox2, N-Myc, and Klf4 were abundantly expressed (Figure 1A and 1B). Next, we tested whether Bmi1 could replace Oct4, Sox2, Klf4, or C-Myc. The four transcription factors (Oct4, Sox2, Klf4, and C-Myc; OSKM) or a combination of three of the transcription factors with Bmi1 (OSKB, OSBM, OBKM, and BSKM) were introduced into MEFs. Bmi1 was able to replace Sox2, Klf4, or C-Myc in inducing Nanog-positive colonies that resemble embryonic stem (ES) cells (Figure 1C and Supplementary information, Figure S1). However, we found that in the absence of Oct4, Nanog positive colonies were not formed (data not shown), indicating that Bmi1 is not able to replace Oct4 for reprogramming MEFs. We also tested whether Bmi1 was able to replace both Klf4 and C-Myc (Figure 1A). We reprogrammed mouse fibroblasts with Oct4 and Sox2 or with the two factors plus Bmi1 (2F-Bmi1-iPS (OSB)). Overexpression of Bmi1 in these two-factor experiments increased the number of Nanog-positive colonies, consistent with a role of Bmi1 as a replacement for Klf4 and C-Myc, as well as a limited role in regulating the p53 and Rb pathways during reprogramming. The OSB colonies were very similar to those comprised of mouse ES cells and expressed pluripotency-associated transcription factors as well as pluripotent cell surface markers (Figure 1D and Supplementary information, Figure S1). Taken together, these data suggest that Bmi1-mediated regulation of N-Myc, Klf4, p16Ink4a, and p19Arf activity markedly increases reprogramming efficiency.
Induction of fibroblasts into iPS cells with Bmi1 plus Oct4
Methods designed to reduce the number of factors necessary for reprogramming have taken advantage of endogenously expressed reprogramming factors such as Sox2 17. For example, studies show that adult mouse neural stem cells (NSCs), which exhibit endogenous Sox2 expression, can be reprogrammed by Oct4 alone 17, 18. However, methods reprogramming somatic cells that lack endogenous Sox2 expression to pluripotency with Oct4 alone need to be explored. Based on previous results 16, we hypothesized that cells transduced by Bmi1 could be transdifferentiated into NSC-like cells and then converted into iPS cells by Oct4 (Figure 2A left panel). We first determined whether Bmi1 could transdifferentiate MEFs into NSC-like cells. Bmi1-transduced MEFs, but not empty vector-transduced MEFs, formed colonies exhibiting an NSC-like morphology within 3-7 days in NSC culture (Figure 2A and 2B). Of the 45 colonies generated, four were selected and grown using standard mouse NSC culturing methods. All four selected colonies expressed genes and cell surface markers characteristic of mouse NSCs, including Nestin and Sox2, as well as AP activity (Figure 2C). In addition, Bmi1-transduced spheres gave rise to neurons, oligodendrocytes, and astrocytes (Figure 2D). Next, we investigated whether Bmi1-transduced NSC-like cells could be reprogrammed into iPS cells by transduction with Oct4 alone. Indeed, we succeeded in generating ES-like colonies within 10-14 days. We refer to these reprogrammed cells as transdifferentiated BO-iPS cells (dBO-iPS cells). These cells were generated from MEFs that were first transdifferentiated into NSC-like cells and then further reprogrammed into iPS cells (Figure 2E). We also determined whether Bmi1 could replace Sox2, Klf4, and C-Myc and, in combination with Oct4, reprogram MEFs into iPS cells (Figure 2A right panel). We introduced Bmi1 and Oct4 into MEFs and were able to induce the formation of ES-like cells, hereafter designated as BO-iPS cells. The estimated reprogramming efficiency was calculated at 0.01% and 0.17% for dBO- and BO-iPS cells, respectively. The reprogramming efficiency of dBO-iPS cells was approximately 6% of that obtained by BO-iPS cells. The reason for the lower reprogramming efficiency of dBO-iPS than BO-iPS cells is not clear; however, it may be due to the poor transduction efficiency of Oct4 into the NSC-like spheres. To further characterize the BO-iPS cells, six colonies that were morphologically indistinguishable from mouse ES (mES) cell colonies were selected and grown under standard conditions (Figure 2E). BO-iPS cells expressed marker genes at levels typical of mES cells (Figure 2F and Supplementary information, Figures S2A, B, and S3A, B). Furthermore, scatter plots of the global gene expression profiles showed that the BO-iPS cell profile was similar to mES cells and was different from MEFs (Figure 2G). To further compare BO-iPS cells with mES cells, the methylation state of CpG dinucleotides in the Oct4 and Nanog promoter regions was analyzed. Bisulfite genomic sequencing analyses showed that the Oct4 and Nanog promoter regions were demethylated in BO-iPS cells relative to the parental fibroblasts and showed a similar pattern to that of mES cells (Figure 2H). Chromatin immunoprecipitation (ChIP) analyses showed that the Oct4, Sox2, and Nanog promoters had increased acetylation of histone H3 (AcH3) and decreased dimethylation of lysine 9 of histone H3 (diMeK9H3) (Supplementary information, Figure S2C), consistent with the epigenetic remodeling that occurs during reprogramming. Oct4 mRNA was transcribed from the endogenous Oct4 locus, which can be distinguished from the virally expressed human Oct4. Genomic integration of the Bmi1 and Oct4 transgenes was confirmed (Supplementary information, Figure S3C), and the expression of both transgenes was efficiently silenced in all iPS cell lines examined (Supplementary information, Figure S3D) 19.
We next investigated the differentiation potential of BO-iPS cells in vitro with the embryoid body (EB) assay. EBs derived from BO-iPS cells expressed markers of the three germ layers, including the endoderm marker GATA4, the mesoderm markers smooth muscle actin and Brachyury, and the ectoderm marker Nestin (Figure 3A). To investigate the differentiation potential of BO-iPS cells in vivo, we analyzed teratomas that formed after subcutaneous injection of BO-iPS cells into nude mice. These teratomas contained derivatives of the three embryonic germ layers, including neural epithelium, muscle, cartilage, and various glandular structures (Figure 3B and Supplementary information, Figure S3E). To assess their developmental potential, we tested whether BO-iPS cells could generate chimeric mice after injection into blastocysts. According to coat color, BO-iPS clones generated chimeric mice with germline transmission (Figure 3C). Taken together, these results demonstrate that Bmi1 can either transdifferentiate MEFs into NSC-like cells and/or replace Sox2, Klf4, and C-Myc during reprogramming of MEFs into iPS cells in the presence of Oct4. In addition, our results suggest that chemically induced activation of Bmi1 may be a useful strategy for enhancing reprogramming efficiency without genetic manipulation of the potential oncogenic Bmi1.
Shh induces Bmi1 and stimulates the generation of iPS cells from fibroblasts by transduction with Oct4 alone
Shh, the most prominent member of the Hedgehog family, plays an essential role during development. The Shh signaling pathway involves the activation of Gli transcription factors, which regulate the transcription of target genes including Gli1 and Ptch1. Furthermore, Bmi1, Sox2, and N-Myc expression was upregulated in response to Shh treatment and parallels the expression of Gli1, suppressor of fused (Sufu), and cyclin D2, which is indicative of the activation of the Shh pathway and induction of proliferation 14, 15, 16, 20, 21. Moreover, overexpression of Gli1 induces Bmi1 expression, suggesting that Bmi1 is a downstream target in the Shh pathway 15.
Therefore, we tested whether Shh could replace Bmi1 in the generation of dBO- and BO-iPS cells. Bmi1, Sox2, N-Myc, Klf4, and Gli1 mRNAs were upregulated (in contrast to p16Ink4a and p19Arf mRNAs, which were suppressed) in response to Shh treatment as early as 72 h after incubation, indicating the activation of the Shh pathway (Figure 4A and 4B). Moreover, cells developed into colonies exhibiting an NSC-like morphology within 3-7 days of Shh treatment in standard NSC culture conditions (Figure 4C). These NSC-like cells expressed genes and cell surface markers characteristic of mouse NSCs, including Sox2, Nestin, and SSEA1, as well as AP activity (Figure 4D). Shh-treated NSC-like cells were then transduced with Oct4 to reprogram them into iPS cells (1F combination of Shh and Oct4, hereafter designated as ShO-iPS cells). ShO-iPS colonies obtained within 14 days in culture were further analyzed by the same tests described above for BO-iPS cells to confirm reprogramming to pluripotency (Figure 4E-F and Supplementary information, Figure S4). Taken together, these results demonstrate that Shh can induce Bmi1, and together with Oct4, can reprogram MEFs into iPS cells that are very similar to mES cells (Supplementary information, Figure S4I).
Recently, it was demonstrated that specific oxysterol and purmorphamine not only stimulate the Shh pathway but also activate Shh target gene transcription through the protein Smo 22, 23. Similar to ShO-iPS cells, treatment of MEFs with either oxysterol or purmorphamine activated the Shh pathway, reprogramming MEFs into NSC-like cells that exhibited gene expression profiles characteristic of NSCs (Supplementary information, Figure S5A-S5C). Furthermore, the treatment of MEFs with oxysterol and/or purmorphamine enhanced the reprogramming of MEFs to pluripotency by the forced expression of Oct4 (1F combinations of oxysterol and/or purmorphamine and Oct4, hereafter designated as OxyO-iPS, PO-iPS, or POxyO-iPS cells) (Supplementary information, Figure S5D). Again, the tests described above were successfully conducted with PO-iPS and OxyO-iPS cells (Supplementary information, Figures S5E-M and S6A-H). Furthermore, PO-iPS cells were germline competent, as demonstrated by the generation of albino offspring from crossing chimeric mice with wild-type mice (Supplementary information, Figure S6I). These results demonstrate that MEFs can be reprogrammed to pluripotency by Oct4 alone when the Shh pathway is activated.
Given that Bmi1 is an important regulator of reprogramming-related genes (Figure 1A and 1B) 13, the transdifferentiation of MEFs into NSC-like cells, and the generation of iPS cells with Oct4, we studied whether knocking down Bmi1 expression would blunt neural sphere formation. Transdifferentiation was performed in the presence of oxysterol and/or purmorphamine to induce the transdifferentiation of MEFs into NSC-like cells. The formation of neurospheres and reprogramming-related gene expression were significantly altered by the knock down of Bmi1 (Figure 5A-5C). Moreover, when cells treated with both oxysterol and purmorphamine were transduced with Oct4 in the presence of Bmi1 siRNA, the formation of Nanog-positive colonies was significantly decreased compared to control (Figure 5D).
We next tested the combination of oxysterol and Oct4 on the reprogramming of tail-tip fibroblasts (TTFs) from 10-week-old male mice (hereafter designated as OxyO-iPS-TTF cells) and established two independent iPS cell lines. These cells were very similar to ES cells with an identical differentiation potential (Supplementary information, Figure S7). Growth rates and hierarchial clustering analysis of the global gene expression profiles showed that 1F (Oct4) and 2F (Oct4 and Bmi1) iPS cells clustered close to mES cells and were distinct from the parental MEFs and Bmi1-transduced neurospheres (Supplementary information, Tables S1-S2 and Supplementary information, Figure S8). The chromosomal stability of 1F and 2F iPS cells was confirmed by metaphase spread (Supplementary information, Figure S9). Taken together, these results show that Bmi1 promotes iPS cell generation from fibroblasts; Bmi1 likely has dual effects on iPS cell generation from fibroblasts, by both suppressing p16Ink4a and p19Arf and augmenting Sox2 and N-Myc (Figure 5E).
In summary, our data support two main conclusions. First, Bmi1 can replace Sox2, Klf4, and C-Myc during reprogramming and enhances the reprogramming efficiency of mouse fibroblasts infected with Oct4 and Sox2. This suggests that not only p16Ink4a and p19Arf 1, 2, 3, 4, 6, but also Sox2 and C-Myc 17, 18, 19, 24 are rate-limiting determinants in the reprogramming process. The second conclusion relates to the number of factors applied to somatic cells for iPS cell generation. We demonstrated previously that mouse and human NSCs, which express endogenous Sox2, can be reprogrammed with Oct4 alone 17, 18. Here, we expand upon these results by showing that Oct4, together with Bmi1 or activators of the Shh signaling pathway (Shh, oxysterol, and purmorphamine in this study), is sufficient for the generation of iPS cells from mouse embryonic and adult fibroblasts. This is especially crucial because adult fibroblasts do not express Sox2. This study, together with others is an important step forward in defining the critical determinants for the generation of iPS cells from differentiated fibroblasts 25, 26, 27. Future studies will determine if we can combine our direct reprogramming procedure with small molecule compounds to activate endogenous expression of Oct4, knock down Oct4-specific suppressor(s), or achieve reprogramming with Oct4 recombinant protein alone.
Materials and Methods
Generation of 4F and 2F iPS cells
pBabe-based retroviral vectors encoding Oct4, Sox2, Klf4, or C-Myc (from Gou Young Koh), and Bmi1 (from Goberdhan P. Dimri) were transfected into PT67 amphotropic packaging cells (Clontech) using Turbofect (Fermentas) according to the manufacturer's protocol. The cells were subjected to drug selection with 3 μg/ml puromycin (Bmi1, Klf4 and C-Myc) for 4 days or 1 000 ng/ml G418 (Sox2 and Oct4) for 14 days. The viruses were collected after 24 h and filtered through a 0.45-μm filter before transduction. Induction of iPS cells was performed as described previously 24 with some modification. In brief, MEF cells (2 × 105) were seeded in one well of a six-well plate and 2 ml of each retroviral supernatant (Bmi1 and Oct4) was added to the cells in the presence of 6 μg/ml of polybrene (Sigma). Two days after the initial transduction, cells were subcultured on mitomycin C-treated CF1 mouse feeder layers and maintained in mES cell medium consisting of DMEM supplemented with 15% FBS (Hyclone), β-mercaptoethanol, 1% penicillin-streptomycin, and 1 000 units/ml leukemia inhibitory factor (LIF; Millipore). ES and iPS cells were passaged every 3-5 days using 0.05% trypsin-EDTA and seeded at 2 × 105 cells/well in a six-well plate. The induction of dBO-iPS cells was performed in two steps. Generation of NSC-like cells was achieved with MEFs infected with retroviruses containing Bmi1, as described previously 16, and these NSC-like cells were then further infected with retroviruses containing Oct4, as above. Two days after Oct4-transduction, cells were grown in mES-culture conditions as described above. For the quantification of transduction efficiency, we introduced GFP to MEF as above and more than 70% of cells expressed GFP. To quantify efficiency of iPS generation, total number of Nanog-positive colonies were counted and calculated as percentage of infected cell numbers, which was calculated as GFP+ cells 28.
Generation of 1F iPS cells
To induce ShO-, PO-, OxyO-iPS, and OxyO-iPS-TTF cells, cells were cultured in NSC medium 16 with either Shh (500 ng/ml; R&D systems), purmorphamine (0.5, 1 μM; Calbiochem), or 25-hydroxycholesterol (oxysterol; 0.1, 0.5 μM; Sigma) for 1 day. Next, retroviral supernatants (Oct4) in NSC medium containing 6 μg/ml polybrene were added to the cells with Shh, purmorphamine, or oxysterol. A third round of transduction was performed and the cells were incubated for 2 days. Two days after the initial transduction, cells were transferred and grown on mitomycin C-treated MEF layers in mES cell medium. For siRNA experiments, cells were transfected with lipofectamine RNAiMAX (Invitrogen). Bmi1 siRNAs and control siRNAs were purchased from Bioneer.
Total RNA from MEF, mES, and iPS cells was labeled with Cy3. Labeled RNA was hybridized to the Agilent mouse whole-genome array (G4122F) according to the manufacturer's instructions. Arrays were scanned with the G2565BA Microarray Scanner System (Agilent Technologies). All data normalization and gene selection was performed using GeneSpring GX 7.3 (Agilent Technologies) 24.
Bisulfite sequencing analysis
DNA from MEF, mES, and iPS cells was isolated using the Genomic DNA Purification Kit (Promega). DNA was prepared for bisulfite sequencing with the EpiTect Bisulfite Kit (QIAGEN). Treated DNA was used to amplify the sequences of interest. The primers used for promoter fragment amplification are listed in Supplementary information, Table S3. The resulting fragments were cloned using the pGEM-T Easy Vector (Promega) for sequencing and sequenced with T7 forward and SP6 reverse primers.
The ChIP assay was performed on MEFs, ES cells, and iPS cells using the EZ ChIP Kit (Millipore) according to the manufacturer's instructions. Anti-acetyl H3 and anti-dimethyl K9 H3 antibodies were used in this experiment. PCR primers used for real-time PCR are listed in Supplementary information, Table S3.
Differentiation of iPS cells
iPS cells were examined by the in-vitro differentiation of EBs. iPS cells were trypsinized and single cells were cultured in suspension with EB medium without LIF for 7 days, and EBs were then replated onto 0.1% gelatin-coated plates. Spontaneous differentiation was examined by immunostaining and RT-PCR for representative lineage-specific markers with the indicated antibodies and primers at various timepoints (5-7 days).
iPS cells (1 × 106/mouse) were injected under the kidney capsule or subcutaneously into the dorsal flank of nude mice. Eight to ten weeks later, the mice developed teratomas, which were removed, immediately rinsed with PBS, fixed in 10% formalin, and embedded in paraffin. Tissue sections 5-6 μm thick were cut and processed for hematoxylin-eosin staining.
Chimera formation and germline transmission
Four- to five-week-old female mice (C57BL/6) were induced to superovulate by intraperitoneal injection of 7.5 IU PMSG followed 48 h later by intraperitoneal injection of 7.5 IU hCG and mated with a stud male mouse (C57BL/6). Blastocysts were collected 3.5 days after vaginal plug check and flushing in H-CZB medium. Approximately 10 iPS cells then were expelled from the injection pipette against the inner cell mass of the blastocyst. Injected blastocysts were transferred into the uterine horn of 2.5 days post coitum (dpc) pseudopregnant CD1 female mice that had been mated with vasectomized male mice. Chimerism was ascertained by the contribution rate of albino coat color (from iPS cells) in black host pups. High-contribution chimeras were mated with C57BL/6 mice to test for germline transmission.
Data were analyzed by analysis of variants using the general linear model procedures of the Statistical Analysis System (SAS, 9.13 PACKAGE). Data were expressed as the means ± SD. P < 0.05 was considered significant.
Gene Expression Omnibus (GEO): GSE24208
Further details and other methods can be found in the supplementary information, Data S1.
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We are grateful to Drs Goberdhan P Dimri (NorthShore University HealthSystem Research Institute, USA)and Gou Young Koh (Korea Advanced Institute of Science and Technology, Republic of Korea) for kindly providing the pBabe-Bmi1, pMX-Oct4, pMX-Sox2, pMX-Klf4, and pMX-C-Myc constructs. We thank Jihyun Kim, Jihye Hwang, and Suhyun Kwon for technical support. This research was supported by a grant (SC-5150) from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology, Republic of Korea, a grant (09172KFDA653) from the Korea Food and Drug Administration, and a grant (2010-0020347) from National Research Foundation (NRF) funded by the Korea government (MEST). Work in Germany was supported by the Max Planck Society and the Federal Ministry of Education and Research (BMBF) on Cell-Based Regenerative Medicine (Grant 01GN0539).
( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Phase-contrast photographs of cell morphology and immunofluoresence of reprogrammed iPS cells. (PDF 176 kb)
Characterization of BO-iPS cells. (PDF 135 kb)
Generation and characterization of BO-iPS clones (1–6) from established BO-iPS cells by single cell culture. (PDF 189 kb)
Characterization of ShO-iPS generated from MEFs by treatment of Shh and retroviral transduction with Oct4. (PDF 157 kb)
Generation and characterization of PO-iPS cells and OxyO-iPS cells. (PDF 222 kb)
Generation and characterization of 1F PO-iPS cells. (PDF 203 kb)
Generation and characterization of 1F OxyO-iPS-TTF cells. (PDF 189 kb)
Global gene expression analyses by DNA microarray (PDF 113 kb)
Karyotype analysis of iPS cells. (PDF 105 kb)
Population doubling time (PDF 35 kb)
Genes belonging to groups in Supplementary information, Figure S8 (PDF 99 kb)
Primers used in this study (PDF 134 kb)
Supplemental Methods (PDF 63 kb)
Antibodies and dilutions used in this study (PDF 48 kb)
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Moon, JH., Heo, J., Kim, J. et al. Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1. Cell Res 21, 1305–1315 (2011). https://doi.org/10.1038/cr.2011.107
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