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Reprogramming of fibroblasts into induced pluripotent stem cells by Nanog

  • 김지현 (대학원 생명공학과 동물생명공학전공)

초록/요약

Oct4-Sox2-Nanog transcriptional networks are critical for the maintenance of embryonic stem (ES) cell self-renewal and induction of pluripotency. However, in transcription factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), Nanog is initially dispensable and Oct4 remains the sole factor that could not be substituted/omitted. Here, we show that mouse fibroblasts could be reprogrammed into iPSCs by Nanog and Bmi1, which replaces Sox2, Klf4, and c-Myc, in the absence of Oct4. Furthermore, we show that in the presence of shh agonists (oxysterol and purmophamine), which replaces the function of Bmi1, a single transcription factor, Nanog is sufficient to reprogram mouse fibroblasts into iPSCs. Nanog-induced iPSCs resemble mES cells in terms of morphology, global gene expression profiles, epigenetic status and pluripotency both in vitro and in vivo. These findings support that Nanog can replace the Oct4 for the somatic cell reprogramming and underlye the mechanisms of Nanog in reprogramming process.

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CONTENTS

1. Abstract 1

2. Introduction 2

3. Materials & Methods 6
3.1. Cell culture 6
3.2. Retrovirus infection and iPS cells generation 6
3.3. AP staining and immunofluorescence staining 7
3.4. RT-PCR and qRT-PCR 8
3.5. Western blot analysis 8
3.6. FACS analysis 9
3.7. Bisulfite sequencing 10
3.8. ChIP assay 10
3.9. In vitro differentiation and teratoma formation 11
3.10. Chimera formation and germline transmission 11
3.11. Statistical Analysis 12

4. Results 16
4.1. Nanog enhances reprogramming of mouse embryonic fibroblasts into iPSCs 16
4.2. Induction of fibroblasts into iPS cells with Nanog plus Bmi1 20
4.3. Nanog, in combination with shh agonists, is sufficient to generate iPSCs 31

5. Discussion 51

6. Reference 55

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