Neuronal Conversion of Dermal Fibroblasts as a Disease Model

Ekim Zihni Taşkıran; Beren Karaosmanoğlu

doi:10.7197/223.vi.473259

TR EN

Hastalık Modeli Olarak Dermal Fibroblasttan Dönüştürülmüş Nöron

Öz

Giriş: Kişiselleştirilmiş tıp çalışmaları için hastalık modeli oluşturmak önem kazanmıştır. Hastalardan bazı hücre türlerinin alınamıyor olması, hastalık modeli için somatik hücre farklılaştırılmasını gerektirmiştir. Hücrelerin yeniden programlanması için pek çok yöntem bulunmaktadır. İndüklenmiş pluripotent kök hücre (iPKH) ve doğrudan dönüştürme en temel iki yöntemdir. Ancak, iPKH’nin pahalılık, yüksek mutasyon oranı ve farklılaşma hasarları gibi dezavantajlarından dolayı, pek çok araştırmacı doğrudan dönüştürmeyi seçmektedir. Amaç: Bu çalışmada, dermal fibroblastların doğrudan dönüştürme ile nöronlara farklılaştırılarak kişiye özel hastalık modeli uygulamalarında kullanılabileceği amaçlanmıştır. Gereç ve yöntem: Ticari olarak satın alınmış dermal fibroblastlar lamel üzerine ekilerek küçük kimyasallar içeren besiyeri ile 24 saat süresinde indüklenmiştir. Hücreler taramalı elektron mikroskobu ile görüntülenmiş ve yeni nesil RNA dizileme ile transkriptom analizi gerçekleştirilmiştir. Bulgu: 24 saatlik indükleme sonucunda nöral hücre morfolojisi görülmüştür. Transkriptom sonuçlarında nöral genlerin artmış olduğu tespit edilmiştir. Sonuç: Dermal fibroblastlar küçük moleküller kullanılarak doğrudan dönüştürme ile başarılı olarak indüklenmiştir.

Anahtar Kelimeler

Dermal fibroblast,hastalık modeli,doğrudan dönüştürme,nöron,transkriptomik

Neuronal Conversion of Dermal Fibroblasts as a Disease Model

Abstract

Introduction: Disease modelling applications are gaining importance especially for the need of patient-specific studies. Because some cell types cannot be obtained from patients, somatic cell differentiation is needed for disease modelling. There are many methods for somatic cell reprogramming. Induced Pluripotent stem cells (iPSCs) and direct conversion are the main techniques. However, due to the disadvantages of iPSCs, such as expense, high mutation rate and differentiation defects, many researches choose direct conversion for reprogramming. Aim: In this study, we aimed to reprogram dermal fibroblasts into neurons with direct conversion in order them to be potentially used for patient-specific disease modelling applications. Materials and methods: Commercially purchased dermal fibroblasts were seeded on coverslips and then induced with a medium containing small chemicals for 24 hours. Cells were visualized by scanning electron microscopy and transcriptome analysis was done by next generation RNA sequencing. Results: Neuronal cell morphology was observed after 24 hour induction. According to the transcriptomic data, neuronal genes were upregulated. Conclusion: Dermal fibroblasts were successfully induced into neurons by direct conversion with using small chemicals.

Keywords

Dermal fibroblast,disease modelling,direct conversion,neuron,transcriptomics

References

1. Tsunemoto RK, Eade KT, Blanchard JW, Baldwin KK. Forward engineering neuronal diversity using direct reprogramming. EMBO J 2015; 34:1445-1455.
2. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663-676.
3. Gopalakrishnan S, Hor P, Ichida JK. New approaches for direct conversion of patient fibroblasts into neural cells. Brain Res 2017; 1656:2-13.
4. Yoshihara M, Araki R, Kasama Y et al. Hotspots of De Novo Point Mutations in Induced Pluripotent Stem Cells. Cell Rep 2017; 21:308-315.
5. Ghaffari LT, Starr A, Nelson AT, Sattler R. Representing Diversity in the Dish: Using Patient-Derived in Vitro Models to Recreate the Heterogeneity of Neurological Disease. Front Neurosci 2018; 12:56.
6. Koyanagi-Aoi M, Ohnuki M, Takahashi K et al. Differentiation-defective phenotypes revealed by large-scale analyses of human pluripotent stem cells. Proc Natl Acad Sci U S A 2013; 110:20569-20574.
7. Hu W, Qiu B, Guan W et al. Direct Conversion of Normal and Alzheimer's Disease Human Fibroblasts into Neuronal Cells by Small Molecules. Cell Stem Cell 2015 17:204-212.
8. Alyass A, Turcotte M, Meyre D. From big data analysis to personalized medicine for all: challenges and opportunities. BMC Medical Genomics 2015; 8:33.

9. Casamassimi A, Federico A, Rienzo M, Esposito S, Ciccodicola A. Transcriptome Profiling in Human Diseases: New Advances and Perspectives. Int J Mol Sci 2017; 18.
10. Rees MI, Harvey K, Ward H et al. Isoform heterogeneity of the human gephyrin gene (GPHN), binding domains to the glycine receptor, and mutation analysis in hyperekplexia. J Biol Chem 2003; 278:24688-24696.
11. Frischknecht R, Seidenbecher CI. Brevican: a key proteoglycan in the perisynaptic extracellular matrix of the brain. Int J Biochem Cell Biol 2012; 44:1051-1054.
12. Sato S, Omori Y, Katoh K et al. Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation. Nat Neurosci 2008; 11:923-931.
13. Fogli A, Schiffmann R, Hugendubler L et al. Decreased guanine nucleotide exchange factor activity in eIF2B-mutated patients. Eur J Hum Genet 2004; 12:561-566.
14. Fernández-Fernández D, Rosenbrock H, Kroker KS. Inhibition of PDE2A, but not PDE9A, modulates presynaptic short-term plasticity measured by paired-pulse facilitation in the CA1 region of the hippocampus. Synapse 2015; 69:484-496.
15. Cheadle L, Biederer T. The novel synaptogenic protein Farp1 links postsynaptic cytoskeletal dynamics and transsynaptic organization. J Cell Biol 2012; 199:985-1001.
16. Jorge BS, Campbell CM, Miller AR et al. Voltage-gated potassium channel KCNV2 (Kv8.2) contributes to epilepsy susceptibility. Proc Natl Acad Sci U S A 2011; 108:5443-5448.
17. Irwin N, Li YM, O'Toole JE, Benowitz LI. Mst3b, a purine-sensitive Ste20-like protein kinase, regulates axon outgrowth. Proc Natl Acad Sci U S A 2006; 103:18320-18325.
18. Fu MM, Holzbaur EL. MAPK8IP1/JIP1 regulates the trafficking of autophagosomes in neurons. Autophagy 2014; 10:2079-2081.
19. Ito H, Mizuno M, Noguchi K et al. Expression analyses of Phactr1 (phosphatase and actin regulator 1) during mouse brain development. Neurosci Res 2018; 128:50-57.

Details

Primary Language

English

Subjects

Health Care Administration

Journal Section

Research Article

Authors

Ekim Zihni Taşkıran ^*
Türkiye

Beren Karaosmanoğlu
0000-0001-5564-4813
Türkiye

Publication Date

December 29, 2018

Submission Date

October 22, 2018

Acceptance Date

December 16, 2018

Published in Issue

Year 2018 Volume: 40 Number: 4

DOI

https://doi.org/10.7197/223.vi.473259

IZ

https://izlik.org/JA74PH55DB

Cite

RIS / Bibtex

AMA

1.Taşkıran EZ, Karaosmanoğlu B. Neuronal Conversion of Dermal Fibroblasts as a Disease Model. CMJ. 2018;40(4):392-399. doi:10.7197/223.vi.473259