The Use of Cell-penetrating Peptide for Delivery of Recombinant Transcription Factor DNA into Primary Human Fibroblast
Abstract
Background: Reprogrammed cell therapy has not been applied for clinical purposes due to the malignancy issue. The aim of this study was to design the recombinant vector of the transcription factors and analyze the effectiveness of cell-penetrating peptide delivering system for human primary fibroblast transfection to avoid the malignancy issue.
Materials and methods: The constructions of CCAT/enhancer binding protein alpha (CEBPA), hepatocyte nuclear factor 4 alpha (HNF4A), nuclear receptor subfamily 1 group I member 2 (NR1I2) were confirmed with DNA digestion and sequencing. Breast reduction (BRED) and palate (PAL) tissue were used as human primary fibroblast sources. The transcription factors were delivered into BRED and PAL with recombination of avian leukosis sarcoma virus (ALSV), human immunodeficiency virus (HIV) matrix, and regulator of expression of virion proteins (Rev) (ALMR), tagged with enhanced green fluorescence protein (eGFP). Post-transfection cells were then cultivated with optimized medium. Gene expression was measured with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).
Results: Gene expression levels of CEBPA, HNF4A, NR1I2, glutamate-ammonia ligase (GLUL), albumin (ALB), and cytochrome P450 (CYP) were increased. Transfection with ALMR, which were more efficient in BRED than PAL fibroblasts may have the advantage in autologous cell therapy for elderly patients.
Conclusion: Transfection of transcription factors to human primary fibroblast may be performed by using constructions of plasmid as designed in this study.
Keywords: recombinant plasmid, hepatocyte-like cells, primary fibroblasts, recombinant peptide, cell reprogramming, autologous cells therapy
References
Meiliana A, Wijaya A. Progress and future challenges of human induced pluripotents stem cell in regenerative medicine. Indones Biomed J. 2011; 3(2): 76-92, CrossRef.
Orge ID, Gadd VL, Barouh JL, Rossi EA, Carvalho RH, Smith I, et al. Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells. Stem Cell Res Ther. 2020; 11(1): 154, CrossRef.
Afshari A, Shamdani S, Uzan G, Naserian S, Azarpira N. Different approaches for transformation of mesenchymal stem cells into hepatocyte-like cells. Stem Cell Res Ther. 2020; 11(1): 54, CrossRef.
Song G, Pacher M, Balakrishnan A, Yuan Q, Tsay HC, Yang D, et al. Direct reprogramming of hepatic myofibroblasts into hepatocytes in vivo attenuates liver fibrosis. Cell Stem Cell. 2016; 18(6): 797-808, CrossRef.
Guo Y, Chen B, Chen LJ, Zhang CF, Xiang C. Current status and future prospects of mesenchymal stem cell therapy for liver fibrosis. J Zhejiang Univ Sci B. 2016; 17(11): 831-41, CrossRef.
Yoon S, Kang K, Cho YD, Kim Y, Buisson EM, Yim JH, et al. Nonintegrating direct conversion using mRNA into hepatocyte-like cells. Biomed Res Int. 2018; 2018: 8240567, CrossRef.
Rombaut M, Boeckmans J, Rodrigues RM, van Grunsven LA, Vanhaecke T, De Kock J. Direct reprogramming of somatic cells into induced hepatocytes: Cracking the enigma code. J Hepatol. 2021; 75(3): 690-705, CrossRef.
Vangipuram M, Ting D, Kim S, Diaz R, Schüle B. Skin punch biopsy explant culture for derivation of primary human fibroblasts. J Vis Exp. 2013; (77): e3779, CrossRef.
Baranyi U, Winter B, Gugerell A, Hegedus B, Brostjan C, Laufer G, et al. Primary human fibroblasts in culture switch to a myofibroblast-like phenotype independently of TGF beta. Cells. 2019; 8(7): 721, CrossRef.
Widyaningtyas ST, Soebandrio A, Ibrahim F, Bela B. Design, synthesis, and functional testing of recombinant cell penetrating peptides. Journal of Physics: Conference Series. 2017; 884: 012030, CrossRef.
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5): 861-72, CrossRef.
Tendean M, Oktaviono YH, Sandra F. Cardiomyocyte reprogramming: A potential strategy for cardiac regeneration. Mol Cell Biomed Sci. 2017; 1(1): 1-5, CrossRef.
Pournasr B, Asghari-Vostikolaee MH, Baharvand H. Transcription factor-mediated reprograming of fibroblasts to hepatocyte-like cells. Eur J Cell Biol. 2015; 94(12): 603-10, CrossRef.
Guo Z, Peng H, Kang J, Sun D. Cell-penetrating peptides: Possible transduction mechanisms and therapeutic applications. Biomed Rep. 2016; 4(5): 528-34, CrossRef.
Uchida M, Li XW, Mertens P, Alpar HO. Transfection by particle bombardment: Delivery of plasmid DNA into mammalian cells using gene gun. Biochim Biophys Acta. 2009; 1790(8): 754-64, CrossRef.
Lai Y, Drobinskaya I, Kolossov E, Chen C, Linn T. Genetic modification of cells for transplantation. Adv Drug Deliv Rev. 2008; 60(2): 146-59, CrossRef.
Kogiso T, Nagahara H, Otsuka M, Shiratori K, Dowdy SF. Transdifferentiation of human fibroblasts into hepatocyte-like cells by defined transcriptional factors. Hepatol Int. 2013; 7(3): 937-44, CrossRef.
Christian JE, Widyaningtyas ST, Bela B. Development of recombinant immunoblot assay diagnostic test based on HIV-1 in Indonesia. Mol Cell Biomed Sci. 2022; 6(2): 77-84, CrossRef.
Zaric M, Nikolic I, Zelen I, Mitrovic M, Milosavljevic Z. Enhancement of dermal fibroblast isolation. Ser J Exp Clin Res. 2015; 16(1): 65–9, CrossRef.
Siengdee P, Klinhom S, Thitaram C, Nganvongpanit K. Isolation and culture of primary adult skin fibroblasts from the Asian elephant (Elephas maximus). PeerJ. 2018; 6: e4302, CrossRef.
Norouzzadeh M, Kalikias Y, Mohamadpur Z, Sharifi L, Mahmoudi M. Determining population doubling time and the appropriate number of HepG2 cells for culturing in 6-well plate. Int Res Bas Sci. 2016; 10(3): 299–303, article.
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4): 663-76, CrossRef.
Ballester M, Bolonio M, Santamaria R, Castell JV, Ribes-Koninckx C, Bort R. Direct conversion of human fibroblast to hepatocytes using a single inducible polycistronic vector. Stem Cell Res Ther. 2019; 10(1): 317, CrossRef.
Godoy P, Schmidt-Heck W, Natarajan K, Lucendo-Villarin B, Szkolnicka D, Asplund A, et al. Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells. J Hepatol. 2015; 63(4): 934-42, CrossRef.
Nakamori D, Akamine H, Takayama K, Sakurai F, Mizuguchi H. Direct conversion of human fibroblasts into hepatocyte-like cells by ATF5, PROX1, FOXA2, FOXA3, and HNF4A transduction. Sci Rep. 2017; 7(1): 16675, CrossRef.
Abbey D, Elwyn S, Hand NJ, Musunuru K, Rader DJ. Self-organizing human induced pluripotent stem cell hepatocyte 3D organoids inform the biology of the pleiotropic TRIB1 gene. Hepatol Commun. 2020; 4(9): 1316-31, CrossRef.
Sugiono E, Wijaya A, Santoso A, Sandra F, Patellongi IJ, Yusuf I. Frequencies of CYP1A2 single nucleotide polymorphism in Indonesian and its effect on blood pressure. Indones Biomed J. 2018; 10(3): 297-302, CrossRef.
Ramadhani N, Soeroso NN, Tarigan SP, Eyanoer PC, Hidayat H. Correlation between genetic polymorphism of CYP2A13 genotype and lung cancer in female passive smokers. Mol Cell Biomed Sci. 2022; 6(2): 89-95, CrossRef.
Okada A, Kodaira A, Hanyu S, Izume S, Ohya K, Fukushi H. Intracellular localization of Equine herpesvirus type 1 tegument protein VP22. Virus Res. 2014; 192: 103-13, CrossRef.
Dey C, Narayan G, Krishna Kumar H, Borgohain MP, Lenka N, Thummer RP. Cell-penetrating peptides as a tool to deliver biologically active recombinant proteins to generate transgene-free induced pluripotent stem cells. Stud Stem Cells Res Ther. 2017; 3(1): 6–15, CrossRef.
Blackford SJI, Ng SS, Segal JM, King AJF, Austin AL, Kent D, et al. Validation of current good manufacturing practice compliant human pluripotent stem cell-derived hepatocytes for cell-based therapy. Stem Cells Transl Med. 2019; 8(2): 124-37, CrossRef.
Ang LT, Tan AKY, Autio MI, Goh SH, Choo SH, Lee KL, et al. A roadmap for human liver differentiation from pluripotent stem cells. Cell Rep. 2018; 22(8): 2190-205, CrossRef.
Wang H, Yang Y, Liu J, Qian L. Direct cell reprogramming: Approaches, mechanisms and progress. Nat Rev Mol Cell Biol. 2021; 22(6): 410-24, CrossRef.
DOI: https://doi.org/10.21705/mcbs.v7i1.279
Copyright (c) 2023 Cell and BioPharmaceutical Institute
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Indexed by:
Cell and BioPharmaceutical Institute
