Int J Stem Cells.  2023 May;16(2):156-167. 10.15283/ijsc22183.

Strategic Application of Epigenetic Regulators for Efficient Neuronal Reprogramming of Human Fibroblasts

• Fernandes GS
• Singh RD
• De D
• Kim KK

• KMID: 2542829
• DOI: http://doi.org/10.15283/ijsc22183

Abstract

Background and Objectives
Cellular reprogramming in regenerative medicine holds great promise for treating patients with neurological disorders. In this regard, small molecule-mediated cellular conversion has attracted special attention because of its ease of reproducibility, applicability, and fewer safety concerns. However, currently available protocols for the direct conversion of somatic cells to neurons are limited in clinical application due of their complex nature, lengthy process, and low conversion efficiency.
Methods and Results
Here, we report a new protocol involving chemical-based direct conversion of human fibroblasts (HF) to matured neuron-like cells with a short duration and high conversion efficiency using temporal and strategic dual epigenetic regulation. In this protocol, epigenetic modulation by inhibition of histone deacetylase and bromodomain enabled to overcome “recalcitrant” nature of adult fibroblasts and shorten the duration of neuronal reprogramming. We further observed that an extended epigenetic regulation is necessary to maintain the induced neuronal program to generate a homogenous population of neuron-like cells.
Conclusions
Therefore, our study provides a new protocol to produce neurons-like cells and highlights the need of proper epigenetic resetting to establish and maintain neuronal program in HF.

Keyword

Direct reprogramming; Patient-specific; Regenerative medicine; Dual epigenetic modification; Neurons

Figure

• 

  Fig. 1 Dual HDAC/BET inhibitors for the induction of neuronal programming. (a) Schematic design of protocol 1 for the neuronal conversion of human fibroblast into neurons. (b) Bright-field images showing the morphological changes of cells over six days using neuronal induction and neuronal maturation protocol. Scale bar, 100 μm. (c) TUJ1 and DCX immunostaining of human fibroblast cells after two days of exposure to 6C. Scale bar, 100 μm. (d) Neuronal induction efficiency two days after the treatment with 6C cocktail normalized to control human fibroblast culture on day zero. (e) mRNA levels of key genes related to neuronal reprogramming as assessed by RT–qPCR on day two. (f) Neuronal purity on day six as percentage of MAP2+ cells. (g) TUJ1 and MAP2 immunostaining on day six after treatment with 6C and FCY. Scale bar, 100 μm. (h) NeuN and MAP2 immunostaining on day six after treatment with 6C and FCY. Scale bar, 100 μm. The merge image shows the overlap of neuronal markers with the nucleus stained by DAPI. Statistical differences were examined by student’s t-test, **p<0.001, and ***p<0.0001.

• 

  Fig. 2 Role of individual epigenetic modifiers in neuronal induction. (a) Representative bright-field image of induced human fibroblast cells to investigate the role of epigenetic regulators by individually removing them from 6C. Scale bar, 100 μm. (b) DCX immunostaining of the human fibroblast cells treated with complete 6C cocktails and when epigenetic modifiers are removed from 6C cocktail, Scale bar, 100 μm. The figure shows the overlap of DCX and DAPI. (c) Neuronal induction efficiency determined on day two post neuronal induction normalized to control human foreskin culture day. Statistical differences were examined by one way ANOVA with Dennett’s post hoc test, **p<0.002, and ***p<0.001.

• 

  Fig. 3 Extended BET inhibition for generating a homogenous population. (a) Schematic design of two-step protocol 2 for converting human fibroblast into neurons. (b) Bright-field images showing the morphological changes over six days of neuronal reprogramming. (c) TUJ1 and DCX immunostaining after two days of exposure to 6C. Scale bar, 100 μm. (d) Neuronal induction efficiency two days after the treatment with 6C cocktail normalized to control human culture on day zero. (e) Neuronal purity on day six as percentage of MAP2+ cells. (f) TUJ1 and MAP2 immunostaining on day six after treatment with 6C and JYC. Scale bar, 100 μm. (g) MAP2 and NeuN immunostaining on day six after treatment with 6C and JYC. Scale bar, 100 μm. The merge image shows the overlap of neuronal markers with the nucleus stained by DAPI. Statistical differences were examined by student’s t-test, ***p<0.0001.

• 

  Fig. 4 Maturation cocktail for the generation of mature neuron-like cells. (a) Schematic design of protocol 3 for the generation of mature and functional neurons. (b) Bright-field images showing the morphological changes over 10 days of the generation of MAP2+/NeuN+/vGlUT1+ neuron-like cells. Dual immunostaining of neuron-like cells at day 10 with following markers; (c) NeuN and MAP2, (d) TUJ1 and Syn1, and (e) vGLUT1 and MAP2. Scale bar, 100 μM. The merge image shows the overlap of neuronal markers with the nucleus stained by DAPI. The boxes in lower most right panels show the magnified image of the indicated regions. (f) Fluorescent images of cells loaded with Rhod 2-AM after different time intervals for 50 sec. The arrow indicates the representative region (ROI 1) in which fluorescence intensity was measured. (g) A plot of ΔF/(F0) showing the change in fluorescent intensity over time with respect to the initial fluorescence for different ROIs as the one demonstrated in panel f. ROI 1∼6 were picked on the basis of change in the fluorescence intensity over time and the neuronal morphology as stained by the dye. Selected pseudo-color frames are baseline subtracted images (ΔF) of the cell.

Reference

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