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. 2020 Mar 30;13(1):80-92.
doi: 10.15283/ijsc19097.

Robust and Reproducible Generation of Induced Neural Stem Cells From Human Somatic Cells by Defined Factors

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Free PMC article

Robust and Reproducible Generation of Induced Neural Stem Cells From Human Somatic Cells by Defined Factors

Tae Hwan Kwak et al. Int J Stem Cells. .
Free PMC article

Abstract

Background and objectives: Recent studies have described direct reprogramming of mouse and human somatic cells into induced neural stem cells (iNSCs) using various combinations of transcription factors. Although iNSC technology holds a great potential for clinical applications, the low conversion efficiency and limited reproducibility of iNSC generation hinder its further translation into the clinic, strongly suggesting the necessity of highly reproducible method for human iNSCs (hiNSCs). Thus, in orderto develop a highly efficient and reproducible protocol for hiNSC generation, we revisited the reprogramming potentials of previously reported hiNSC reprogramming cocktails by comparing the reprogramming efficiency of distinct factor combinations including ours.

Methods: We introduced distinct factor combinations, OSKM (OCT4+SOX2+KLF4+C-MYC), OCT4 alone, SOX2 alone, SOX2+HMGA2, BRN4+SKM+SV40LT (BSKMLT), SKLT, SMLT, and SKMLT and performed comparative analysis of reprogramming potentials of distinct factor combinations in hiNSC generation.

Results: Here we show that ectopic expression of five reprogramming factors, BSKMLT leads the robust hiNSC generation (>80 folds enhanced efficiency) from human somatic cells compared with previously described factor combinations. With our combination, we were able to observe hiNSC conversion within 7 days of transduction. Throughout further optimization steps, we found that both BRN4 and KLF4 are not essential for hiNSC conversion.

Conclusions: Our factor combination could robustly and reproducibly generate hiNSCs from human somatic cells with distinct origins. Therefore, our novel reprogramming strategy might serve as a useful tool for hiNSC-based clinical application.

Keywords: Defined factors; Direct conversion; Human induced neural stem cells; Robust and reproducible generation.

Conflict of interest statement

Potential Conflict of Interest

The authors have no conflicting financial interest.

Figures

Fig. 1
Fig. 1
Generation of hiNSCs from hFFs using BSKM with SV40LT. (A) Schematic illustration of the reprogramming procedure for generating hiNSCs. Morphological changes during the reprogramming period are shown. Scale bars, 100 μm. (B) Morphology of hiNSCs after 2 weeks of transduction. Scale bars, 100 μm. (C) Morphology of the established hiNSCs at different passages. Scale bars, 100 μm. (D) Expression pattern of NSC- and fibroblast-specific markers were analyzed by RT-PCR in early and later passages of hiNSCs. GAPDH was used as a positive control.
Fig. 2
Fig. 2
Comparative analysis of reprogramming potentials of distinct factor combinations. (A) The schematic illustration depicting the strategy for comparing the reprogramming efficiency of distinct factor combinations. (B) Time-course immunofluorescence analysis for comparing reprogramming potentials of distinct factor combinations. Scale bars, 100 μm. (C) Morphology of hiNSC clusters at 2 weeks after transduction. Scale bars, 100 μm. (D) The number of BLBP/MSI1 colonies were counted in a time-course manner. Data are presented as mean±SD from six independent experiments. *p<0.05, *p<0.01, ***p<0.001.
Fig. 2
Fig. 2
Comparative analysis of reprogramming potentials of distinct factor combinations. (A) The schematic illustration depicting the strategy for comparing the reprogramming efficiency of distinct factor combinations. (B) Time-course immunofluorescence analysis for comparing reprogramming potentials of distinct factor combinations. Scale bars, 100 μm. (C) Morphology of hiNSC clusters at 2 weeks after transduction. Scale bars, 100 μm. (D) The number of BLBP/MSI1 colonies were counted in a time-course manner. Data are presented as mean±SD from six independent experiments. *p<0.05, *p<0.01, ***p<0.001.
Fig. 3
Fig. 3
Expression pattern of NSC-specific markers during hiNSC generation. Expression pattern of NSC markers in hFFs transduced with distinct factor combinations was analyzed by qPCR in a time-course manner. All the values were normalized to those of non-transduced hFFs. Data are presented as mean±SD of triplicate values. *p<0.05, **p<0.01, ***p<0.001.
Fig. 4
Fig. 4
Characterization of SKMLT hiNSCs. (A) Immunofluorescence images of hESC-derived NSCs and SKMLT hiNSCs using antibodies against BRN2, BLBP, MSI1, and MSI2. Scale bars, 100 μm. (B) A heat map representing expression profile of genes with more than two-fold expression level difference between hFFs and hESC-derived NSCs. The color represents z-score for gene expression level in log2 scale. Clusters with red and blue bar on the left represent genes with lower and higher expression in hFFs compared to hESC-derived NSCs, respectively. (C, D) Differentiation potential of SKMLT hiNSCs into astrocytes (C) and neurons (D) as determined by immunocytochemistry with antibodies against GFAP and TUJ1, respectively, Scale bars, 100 μm. (E, F) The efficiency of differentiation into astrocytes (E) and neurons (F) from hESC-derived NSCs and SKMLT hiNSCs was quantified and compared via immunostaining with GFAP and TUJ1, respectively. Data are presented as mean±SD from eight independent experiments. N.S.: not significant.
Fig. 4
Fig. 4
Characterization of SKMLT hiNSCs. (A) Immunofluorescence images of hESC-derived NSCs and SKMLT hiNSCs using antibodies against BRN2, BLBP, MSI1, and MSI2. Scale bars, 100 μm. (B) A heat map representing expression profile of genes with more than two-fold expression level difference between hFFs and hESC-derived NSCs. The color represents z-score for gene expression level in log2 scale. Clusters with red and blue bar on the left represent genes with lower and higher expression in hFFs compared to hESC-derived NSCs, respectively. (C, D) Differentiation potential of SKMLT hiNSCs into astrocytes (C) and neurons (D) as determined by immunocytochemistry with antibodies against GFAP and TUJ1, respectively, Scale bars, 100 μm. (E, F) The efficiency of differentiation into astrocytes (E) and neurons (F) from hESC-derived NSCs and SKMLT hiNSCs was quantified and compared via immunostaining with GFAP and TUJ1, respectively. Data are presented as mean±SD from eight independent experiments. N.S.: not significant.

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