[논문 링크] 피부세포에서 신경줄기세포로의 직접적 리프로그래밍 / 한동욱 등

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Available online 22 March 2012

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Article

Direct Reprogramming of Fibroblasts into Neural Stem Cells by Defined Factors

• Dong Wook Han^{1, 2, 7, ,} , 
• Natalia Tapia^1, 7, 
• Andreas Hermann^3, 4, 
• Kathrin Hemmer⁵, 
• Susanne Höing¹,
• Marcos J. Araúzo-Bravo¹, 
• Holm Zaehres¹, 
• Guangming Wu¹, 
• Stefan Frank¹, 
• Sören Moritz¹, 
• Boris Greber¹,
• Ji Hun Yang¹, 
• Hoon Taek Lee², 
• Jens C. Schwamborn⁵, 
• Alexander Storch^3, 4, 
• Hans R. Schöler^1, 6, , 

• ¹ Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, 48149 Münster, Germany
• ² Department of Stem Cell Biology, School of Medicine, SMART Institute of Advanced Biomedical Science, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
• ³ Division of Neurodegenerative Diseases, Department of Neurology and Center for Regenerative Therapies Dresden (CRTD), Dresden University of Technology, 01307 Dresden, Germany
• ⁴ German Centre for Neurodegenerative Diseases (DZNE), 01307 Dresden, Germany
• ⁵ Westfälische Wilhelms-Universität Münster, ZMBE, Institute of Cell Biology, Stem Cell Biology and Regeneration Group, Von-Esmarch-Str. 56, 48149 Münster, Germany
• ⁶ Medical Faculty, University of Münster, Domagkstrasse 3, 48149 Münster, Germany

• Received 14 November 2011. Revised 27 January 2012. Accepted 24 February 2012. Available online 22 March 2012. Published online: March 22, 2012.

• http://dx.doi.org/10.1016/j.stem.2012.02.021, How to Cite or Link Using DOI
• Cited by in Scopus (0)

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Summary

Recent studies have shown that defined sets of transcription factors can directly reprogram differentiated somatic cells to a different differentiated cell type without passing through a pluripotent state, but the restricted proliferative and lineage potential of the resulting cells limits the scope of their potential applications. Here we show that a combination of transcription factors (Brn4/Pou3f4, Sox2, Klf4, c-Myc, plusE47/Tcf3) induces mouse fibroblasts to directly acquire a neural stem cell identity—which we term as induced neural stem cells (iNSCs). Direct reprogramming of fibroblasts into iNSCs is a gradual process in which the donor transcriptional program is silenced over time. iNSCs exhibit cell morphology, gene expression‎, epigenetic features, differentiation potential, and self-renewing capacity, as well as in vitro and in vivo functionality similar to those of wild-type NSCs. We conclude that differentiated cells can be reprogrammed directly into specific somatic stem cell types by defined sets of specific transcription factors.

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Graphical Abstract

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Highlights

► Direct reprogramming of differentiated cells into self-renewing somatic stem cells ► Set of four transcription factors induces neural stem cell program in fibroblasts ► Reprogramming without risk of teratomas ► Induced neural stem cells behave as their endogenous counterparts

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Figures and tables from this article:

Figure 1. Direct Reprogramming of Fibroblasts into iNSCs(A) Morphology of an early iNSC cluster generated by a combination of five factors (5F; SKMBE), as assessed by bright-field microscopy. MOCK corresponds to fibroblasts that were not transduced with the reprogramming cocktails but were maintained under NSC culture conditions.(B) Immunofluorescence microscopy images of control NSCs and iNSCs (4F and 5F), using antibodies against SSEA1 and Olig2.(C) Morphology of an early iNSC cluster generated by four factors (SKMB), as assessed by bright-field microscopy. MOCK corresponds to fibroblasts that were not transduced with the reprogramming cocktails but were maintained under NSC culture conditions.(D) RT-PCR analysis showed that both 4F and 5F iNSCs exhibit a gene expression‎ profile similar to that of control NSCs.(E) 4F iNSCs could be stably maintained for more than 130 passages.See also Figures S1 and S2 and Table S1.

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Figure 2. Characterization of iNSCs(A) Heat map from microarray data demonstrating global gene expression‎ pattern in fibroblasts, control NSCs, 4F iNSCs of early and late passages, and 5F iNSCs. The color bar at the top indicates gene expression‎ in log₂ scale. Red and blue colors represent higher and lower gene expression‎ levels, respectively.(B) Hierarchical clustering of the cell lines based on the gene expression‎ profiles in (A).(C and D) Pairwise scatter plot analysis of the global gene expression‎ profiles of 4F iNSCs of early and late passages, and 5F iNSCs versus the parental fibroblasts (C) and control NSCs (D). Black lines indicate boundaries of 2-fold difference in gene expression‎ levels. The bar to the right indicates the scattering density; the higher the scattering density, the darker the blue color. Gene expression‎ levels are depicted in log₂ scale. The number of differentially expressed genes is indicated under each scatter plot.(E) DNA methylation status on the second intron of Nestin in fibroblasts, control NSCs, and 4F and 5F iNSCs was assessed by bisulfite sequencing PCR. Open and filled circles represent unmethylated and methylated CpGs, respectively.(F) Expression‎ levels of region-specific marker genes. All data are calibrated to the control NSCs, whose expression‎ is considered to be 1 for all genes. Error bars correspond to the normalization against two housekeeping genes.(G) Expression‎ level of Olig2 in control NSCs and both 4F and 5F iNSCs as percentage of Actinexpression‎.See also Figure S3 and Table S2.

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Figure 3. In Vitro Differentiation Potential of iNSCs(A) Differentiation potential of 4F and 5F iNSCs into neurons, astrocytes, and oligodendrocytes, as determined by immunocytochemistry with antibodies against Tuj1, GFAP, and O4, respectively.(B) The efficiency of differentiation into neurons, astrocytes, and oligodendrocytes from control NSCs and iNSCs (4F and 5F) was quantified and compared via immunostaining with Tuj1, GFAP, and O4, respectively. Error bars correspond to SEM.(C) Electrophysiological properties of control NSC- and iNSC-derived neurons. Representative voltage-clamp recordings in response to increasing voltage pulses from neurons derived from control NSCs, 5F iNSCs, and 4F iNSCs after 7–16 days of differentiation. Insets represent higher magnification of sodium currents. Both single and multiple action potentials were detected in neurons derived from control NSCs, 5F iNSCs, and 4F iNSCs.(D) Characterization of neurons derived from control NSCs and iNSCs. Representative immunofluorescence images of differentiated control NCS, 4F iNSCs, and 5F NSCs after 14–21 days of differentiation. All three NSC types behaved similarly in spontaneous differentiation behavior and differentiated into all major neuronal subtypes, namely GABAergic and glutamatergic neurons, ChAT⁺ cholinergic and TH⁺ dopaminergic neurons (except that 4F iNSCs did not differentiate into TH⁺ neurons). Dotted VGlut1 expression‎ in proximity of Tuj1⁺ nerve fibers suggested morphological synapse formation. Scale bars represent 50 μm and 20 μm (lowermost panel). ChAT, choline acetyltransferase; TH, tyrosine hydroxylase; Tuj, β-tubulin class III; VGluT1, vesicular glutamate transporter type 1.See also Table S3.

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Figure 4. iNSCs Are Functionally Similar to Control NSCs(A) In vivo transplantation of iNSCs. 1.5 × 10⁵ 5F iNSCs labeled with GFP were stereotactically transplanted into the subventricular zone of an adult mouse. After transplantation, the fate of transplanted iNSCs was analyzed.(B) Injected cells migrate from the graft into the rostral migratory stream.(C) Some of the transplanted cells remained as progenitors, as indicated by the presence of the marker Nestin. Some of the injected cells also maintained the ability to proliferate, as shown by Ki67 staining.(D) Transplanted iNSCs became Sox2⁻but Mash1⁺, suggesting that the iNSCs did not remain as NSCs, but had differentiated.(E) The transplanted iNSCs showed differentiation into neurons (GFP⁺/Tuj1⁺/Dcx⁺), astrocytes (GFP⁺/GFAP⁺/NG2⁺), and oligodendrocytes (GFP⁺/S100β⁺/Olig2⁺), as determined by immunohistochemistry (E).See also Figure S4.

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Document S1. Supplemental Experimental Procedures, Figures S1–S4, and Tables S1–S3.  

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