Disertasi

Direct reprogramming fibroblas primer manusia menjadi hepatocyte-like cells dengan faktor transkripsi Cebpa, Hnf4a, dan Nr1i2. = Direct reprogramming of human primary fibroblast to hepatocyte-like cells with Cebpa, Hnf4a, and Nr1i2 transcription factors.

Latar Belakang: Terapi hepatocyte-like cells (HLC) untuk fibrosis/sirosis hati belum dapat diaplikasikan karena adanya isu malignansi sel yang telah direprogram serta sumber alogenik yang dapat menyebabkan rejeksi imun. Tujuan penelitian ini melakukan transfeksi faktor transkripsi dengan sistem penghantar protein rekombinan dan teknik direct reprogramming untuk menghindari potensi malignansi pada HLC. Penggunaan fibroblas primer manusia dalam studi ini juga dapat mengungkap peluang terapi HLC autologous. Metode: Isolasi fibroblas primer manusia dilakukan dengan metode eksplan jaringan limbah biomedis. Konstruksi plasmid rekombinan faktor transkripsi Cebpa, Hnf4a, dan Nr1i2 didesain melalui studi in silico. Direct reprogramming dilakukan dengan transfeksi faktor transkripsi pada fibroblas primer manusia menggunakan peptida rekombinan ALSV, matriks HIV, dan Rev (ALMR). Optimasi rasio faktor transkripsi dianalisis dengan marker eGFP. Kultivasi sel yang telah ditransfeksi dilakukan selama 21 hari, menggunakan medium induksi hepatosit yang telah dioptimasi. Beberapa uji karakterisasi dilakukan seperti observasi morfologi epitelial, analisis ekspresi gen dengan teknik qRT-PCR (quantitave reverse transcriptase-polyemerase chain reaction), serta immunofluorescence assay (IFA). Hasil: Diperoleh dua jaringan limbah biomedis sebagai sumber fibroblas primer manusia, breast reduction (BRED) dan palatum (PAL). Analisis fluoresens menunjukkan sistem transfeksi ALMR lebih optimal pada fibroblas BRED. Penambahan lipofectamine dapat meningkatkan efisiensi transfeksi tersebut. Perubahan morfologi BRED menghasilkan efisiensi transfeksi 82%. Peningkatan ekspresi pada gen faktor transkripsi (Cebpa, Hnf4a, dan Nr1i2) dan penanda hepatosit (GLU, ALB, dan CYP) serta hasil yang positif imunofluoresens dari antibodi Cebpa dan Hnf4a menunjukkan keberhasilan proses direct reprogramming fibroblas primer manusia menjadi HLC. Kesimpulan: Fibroblas primer manusia dilaporkan dapat dikonversi langsung menjadi HLC. Penelitian selanjutnya, analisis fungsi hepatocyte-like cells perlu diperlukan sebagai pembuktian konsep penelitian ini.
Kata kunci: direct reprogramming, hepatocyte-like cells, fibroblas primer manusia, peptida ALMR, faktor transkripsi.


Background: Hepatocyte-like cells (HLC) therapy for liver fibrosis/cirrhosis cannot be applied due to the issue of reprogrammed cell malignancy and allogeneic sources that can cause immune rejection. The purpose ofthis study was to transfect transcription factors with a recombinant protein delivery system and direct reprogramming technique to avoid potential malignancies in HLC. The use of human primary fibroblasts in this study may also reveal opportunities for autologous HLC therapy. Methods: Isolation of primary human fibroblasts was performed using the biomedical tissue explant method. The construction of the recombinant transcription factor plasmid,s Cebpa, Hnf4a, and Nr1i2 was designed by in silico studies. Direct reprogramming was performed by transfection of transcription factors in human primary fibroblasts using recombinant ALSV peptide, HIV matrix and Rev (ALMR). Optimization of the transcription factor ratio was analyzed with the eGFP marker. Cultivation of transfected cells was performed for 21 days, using an optimized hepatocyte induction medium. Several characterization assays were performed including epithelial morphology observation, gene expression analysis using the qRT-PCR (quantitave reverse transcriptase-polyemerase chain reaction) technique, and immunofluorescence assay (IFA). Results: Two biomedical tissues were obtained as sources of human primary fibroblasts, breast reduction (BRED) and palate (PAL). Fluorescence analysis showed that the ALMR transfection system was more optimal in BRED fibroblasts. The addition of lipofectamine can increase the efficiency of the transfection. Changes in the morphology of BRED resulted in a transfection efficiency of 82%. Increased expression of transcription factor genes (Cebpa, Hnf4a, and Nr1i2) and hepatocyte markers (GLU, ALB, and CYP) as well as positive immunofluorescence results of Cebpa and Hnf4a antibodies indicated the success ofthe direct reprogramming ofhuman primary fibroblasts into HLC. Conclusion: Human primary fibroblasts were reported to be directly converted to HLC. For further research, analysis of the function of hepatocyte-like cells is necessary as a proofofconcept for this research.
Keywords: direct reprogramming, hepatocyte-like cells, human primary fibroblast, ALMR peptide, transcription factor.