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Cyclosporine A-Mediated IL-6 Expression Promotes Neural Induction in Pluripotent Stem Cells

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Abstract

Differentiation of pluripotent stem cells (PSCs) to neural lineages has gathered huge attention in both basic research and regenerative medicine. The major hurdle lies in the efficiency of differentiation and identification of small molecules that facilitate neurogenesis would partly circumvent this limitation. The small molecule Cyclosporine A (CsA), a commonly used immunosuppressive drug, has been shown to enhance in vivo neurogenesis. To extend the information to in vitro neurogenesis, we examined the effect of CsA on neural differentiation of PSCs. We found CsA to increase the expression of neural progenitor genes during early neural differentiation. Gene silencing approach revealed CsA-mediated neural induction to be dependent on blocking the Ca2+-activated phosphatase calcineurin (Cn) signaling. Similar observation with FK506, an independent inhibitor of Cn, further strengthened the necessity of blocking Cn for enhanced neurogenesis. Surprisingly, mechanistic insight revealed Cn-inhibition dependent upregulation of IL-6 protein to be necessary for CsA-mediated neurogenesis. Together, these findings provide a comprehensive understanding of the role of CsA in neurogenesis, thus suggesting a method for obtaining large numbers of neural progenitors from PSCs for possible transplantation.

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Abbreviations

PSC:

Pluripotent stem cells

iPSC:

Induced pluripotent stem cells

miPSC:

Mouse induced pluripotent stem cells

CsA:

Cyclosporine A

Cn:

Calcineurin

NFAT:

Nuclear factor of activated T cells

ESC:

Embryonic stem cells

iMEF:

Inactivated mouse embryonic fibroblasts

LIF:

Leukemia inhibitory factor

EB:

Embryoid body

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Acknowledgements

We would like to thank AK lab members for critical review of all the data. We thank Prof. Catherine Verfaillie for the generous gift of Oct4-GFP iPSCs. This work was supported by grants from CSIR [no. 27(0294)/13/EMRII] and intramural funding from SORM, Manipal University.

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Authors and Affiliations

  1. School of Regenerative Medicine, Manipal University, Allalasandra, GKVK post, Yelahanka, Bangalore, 560065, India

    Ashwathnarayan Ashwini, Sushma S Naganur, Bhaskar Smitha, Preethi Sheshadri, Jyothi Prasanna & Anujith Kumar

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  1. Ashwathnarayan Ashwini
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  2. Sushma S Naganur
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  3. Bhaskar Smitha
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Correspondence to Anujith Kumar.

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Electronic supplementary material

Supplementary Fig. 1

Pluripotency and lineage expression analysis of ESCs treated with CsA. a Semi –quantitative PCR analysis of Src in mESCs in presence and absence of CsA b Semi-quantitative PCR showing the expression of pluripotency genes in mESCs in absence and presence of different concentrations of CsA. c Realtime PCR for lineage specific markers in mESCs treated with RA and CsA and withdrawal of LIF. d qPCR results showing lineage specific marker expression in EBs derived from mESCs with Day 3 to Day 14 CsA treatment. (GIF 302 kb).

High Resolution Image (TIFF 2142 kb).

Supplementary Fig. 2

CsA resuces OCT4-GFP expression and maintains pluripotency of miPSCs. a Flowcytometric analysis and b quantification of percent bright GFP positive miPSCs expressing OCT4-GFP. Single asterisk indicates p < 0.05 vs cells cultured in presence of LIF. (GIF 29 kb).

High Resolution Image (TIFF 3074 kb).

Supplementary Fig. 3

Differentiation of mESCs using N2B27 and RA demarcates early and late differentiation events. a Gene expression analysis and b immunofluorescence of of neural progenitor genes in the early differentiation event. c Transcript analysis and immunofluorescence analysis (d) of neurons derived from mPSCs during late differentiation Data is representative of three independent experiments and presented as mean ± SEM. Scale bar represents 100 μm. (GIF 82 kb).

High Resolution Image (TIFF 6823 kb).

Supplementary Fig. 4

CsA inhibits nuclear translocation of NFATC3. a Immunoblot depiciting NFATC3 levels in the nuclear extracts of neurons differentiated from mESCs with and without CsA; purity of nuclear extracts assessed by presence of Nulceolin and absence of ß-actin. (GIF 6 kb).

High Resolution Image (TIFF 873 kb).

Supplementary Fig. 5

KnockdownPpp3r1 enhances neural gene expression in mESCs. a Gene expression analysis of Ppp3r1 in Scrambled and Ppp3r1 shRNA in mESCs. b Neural gene expression of mESC scramble and Ppp3r1knockdown cells. Data is representative of three independent experiments and presented as mean ± SEM. *p ≤ 0.05 vs scrambled control. (GIF 12 kb).

High Resolution Image (TIFF 1482 kb).

Supplementary Fig. 6

Blocking of Cn-NFAT pathway is essential for IL-6 expression in iPSCs. a Transcript analysis of miPSCs derived embryoid bodies in the presence and absence of CsA for IL-6 expression. b mRNA expression of interleukin genes in miPSCs transduced with PPP3r1 shRNA and scrambled control and differentiated to neural lineage. Data is representative of three independent experiments and presented as mean ± SEM. *p < 0.05, **p < 0.01 vs scrambled control. c qPCR analysis of interleukin genes in miPSCs differentiated to neural lineage in the presence and absence of FK506. (GIF 28 kb).

High Resolution Image (TIFF 3925 kb).

Supplementary Table 1

(TIFF 18 kb).

Supplementary Table 2

(TIFF 12 kb).

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Ashwini, A., Naganur, S.S., Smitha, B. et al. Cyclosporine A-Mediated IL-6 Expression Promotes Neural Induction in Pluripotent Stem Cells. Mol Neurobiol 55, 4267–4279 (2018). https://doi.org/10.1007/s12035-017-0633-0

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