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
Kaynakça
- 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.
Öz
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.
Anahtar Kelimeler
Dermal fibroblast disease modelling direct conversion neuron transcriptomics
Kaynakça
- 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.
Ayrıntılar
| Birincil Dil | İngilizce |
|---|---|
| Konular | Sağlık Kurumları Yönetimi |
| Bölüm | Dahili Tıp Bilimleri Araştırma Yazıları |
| Yazarlar | |
| Yayımlanma Tarihi | 29 Aralık 2018 |
| Kabul Tarihi | 16 Aralık 2018 |
| Yayımlandığı Sayı | Yıl 2018 |