Int J Stem Cells.  2023 Nov;16(4):438-447. 10.15283/ijsc23053.

Assessment of Risks and Benefits of Using Antibiotics Resistance Genes in Mesenchymal Stem Cell-Based Ex-Vivo Therapy

• Bashyal N
• Lee YJ
• Jung JH
• Kim MG
• Lee KW
• Hwang WS
• Kim SS
• Chang DY
• Suh-Kim H

• KMID: 2548007
• DOI: http://doi.org/10.15283/ijsc23053

Abstract

Recently, ex-vivo gene therapy has emerged as a promising approach to enhance the therapeutic potential of mesenchymal stem cells (MSCs) by introducing functional genes in vitro. Here, we explored the need of using selection markers to increase the gene delivery efficiency and evaluated the potential risks associated with their use in the manufacturing process. We used MSCs/CD that carry the cytosine deaminase gene (CD) as a therapeutic gene and a puromycin resistance gene (PuroR) as a selection marker. We evaluated the correlation between the therapeutic efficacy and the purity of therapeutic MSCs/CD by examining their anti-cancer effect on co-cultured U87/GFP cells. To simulate in vivo horizontal transfer of the PuroR gene in vivo, we generated a puromycin-resistant by introducing the PuroR gene and assessed its responsiveness to various antibiotics. We found that the anti-cancer effect of MSCs/CD was directly proportional to their purity, suggesting the crucial role of the PuroR gene in eliminating impure unmodified MSCs and enhancing the purity of MSCs/CD during the manufacturing process. Additionally, we found that clinically available antibiotics were effective in inhibiting the growth of hypothetical microorganism, E. coli/PuroR. In summary, our study highlights the potential benefits of using the PuroR gene as a selection marker to enhance the purity and efficacy of therapeutic cells in MSC-based gene therapy. Furthermore, our study suggests that the potential risk of horizontal transfer of antibiotics resistance genes in vivo can be effectively managed by clinically available antibiotics.

Keyword

5-fluorocytosine; Mesenchymal stem cell; Puromycin resistance gene; Puromycin; Cytosine deaminase; Gene therapy

Figure

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  Fig. 1 MSCs/CD purity dependent antitumor effect to U87/GFP cells in presence of 5-FC. (A) Bystander effect of MSCs/CD purity level on U87/GFP cells measured by GFP fluorescence at indicated concentration of 5-FC. Note puromycin selected MSCs/CD cells were mixed with naïve MSCs to get different purity of MSCs/CD. Statistical significance (p-values) for Fig. 1A is in Supplementary Table S1. (B) Survival percentage (%) of U87/GFP cells at indicated MSCs/CD purity in presence of 30, 100, 300 μM 5-FC. (C) IC50 values for 5-FC in MSCs/CD at various purity level. Data are mean±SEM of at least three independent experiments (*p<0.05, ***p<0.001, compared to control; one-way ANOVA test). n.s.: not significant.

• 

  Fig. 2 Identification of residual concentration of puromycin in MSCs/CD. (A) Representative image of peaks of puromycin standard solutions containing 0.05∼8 μg/ml after injected to HPLC column. (B) Standard curve generated from standard solutions’ peak areas at retention time of ∼13.5 min. (C) Schematic flow chart showing the steps of harvesting the samples to assess the residual puromycin concentration. (D) Puromycin peak area of samples I, II, III obtained from 2 and 4 μg/ml puromycin initial addition conditions. (E) Calculated puromycin concentration from 1st and 2nd trials of sample I, II, III via HPLC. Lower limit of quantification (LLOQ), Limit of detection (LOD). Data are mean±SD of at least 2∼3 independent experiments.

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  Fig. 3 Puromycin dose dependent cytotoxicity to MSCs. (A) Representative image of MTT assay at indicated concentration of puromycin showing purple colored formazan crystals after dissolving in a dimethyl sulfoxide (DMSO). (B) MTT assay indicating the cytotoxic effect of puromycin on naïve MSCs with the puromycin dose >0.016 μg/ml. Maximum tolerated dose of puromycin is indicated by arrow. Data are mean±SEM of at least 3 independent experiments (**p<0.01, ***p<0.001, compared to 0.0 μg/ml group; one-way ANOVA test). n.s.: not significant.

• 

  Fig. 4 Generation of puromycin resistant E. coli. (A) Schematic diagram of pET and pET-PuroR plasmids. (B) Flow chart representing a hypothetical horizontal transfer into E. coli (DH5α) to generate DH5α-pET and DH5α-pET-PuroR clones and followed by validation of puromycin resistance and antimicrobial susceptibility testing (AST). (C) Optical density measured at a wavelength of 600 nm (OD600) of DH5α, and DH5α-pET clones cultured in presence (+) or absence (−) of 100 μg/ml ampicillin at indicated concentration of puromycin to identify the minimum inhibitory concentration of puromycin. Both clones were cultured in 3 ml TB media for 16 h at 37℃, 220 rpm (rotation per minute). (D) OD600 of DH5α-pET and DH5α-pET-PuroR clones cultured for 9 h and 16 h in presence or absence of ampicillin or puromycin. Data are mean±SEM of at least 3 independent experiments (***p<0.001, compared to culture media only; one-way ANOVA test). n.s.: not significant.

Reference

References

1. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. 1999; Multilineage potential of adult human mesenchymal stem cells. Science. 284:143–147. DOI: 10.1126/science.284.5411.143. PMID: 10102814.

2. Lee SH, Jin KS, Bang OY, Kim BJ, Park SJ, Lee NH, Yoo KH, Koo HH, Sung KW. 2015; Differential migration of mesenchymal stem cells to ischemic regions after middle cerebral artery occlusion in rats. PLoS One. 10:e0134920. DOI: 10.1371/journal.pone.0134920. PMID: 26241653. PMCID: PMC4524688. PMID: dd3eac219e924d009c083b1c4b6b9081.

3. Xin H, Li Y, Cui Y, Yang JJ, Zhang ZG, Chopp M. 2013; Systemic administration of exosomes released from mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats. J Cereb Blood Flow Metab. 33:1711–1715. DOI: 10.1038/jcbfm.2013.152. PMID: 23963371. PMCID: PMC3824189.

4. Wang F, Yasuhara T, Shingo T, Kameda M, Tajiri N, Yuan WJ, Kondo A, Kadota T, Baba T, Tayra JT, Kikuchi Y, Miyoshi Y, Date I. 2010; Intravenous administration of mesenchymal stem cells exerts therapeutic effects on parkinsonian model of rats: focusing on neuroprotective effects of stromal cell-derived factor-1alpha. BMC Neurosci. 11:52. DOI: 10.1186/1471-2202-11-52. PMID: 20420688. PMCID: PMC2873592. PMID: 2365f86dcd934a6baf9a6ebf1b8c13cf.

5. Kan I, Ben-Zur T, Barhum Y, Levy YS, Burstein A, Charlow T, Bulvik S, Melamed E, Offen D. 2007; Dopaminergic differentiation of human mesenchymal stem cells--utilization of bioassay for tyrosine hydroxylase expression. Neurosci Lett. 419:28–33. DOI: 10.1016/j.neulet.2007.03.070. PMID: 17475405.

6. Ma T, Gong K, Ao Q, Yan Y, Song B, Huang H, Zhang X, Gong Y. 2013; Intracerebral transplantation of adipose-derived mesenchymal stem cells alternatively activates microglia and ameliorates neuropathological deficits in Alzheimer's disease mice. Cell Transplant. 22 Suppl 1:S113–S126. DOI: 10.3727/096368913X672181. PMID: 24070198. PMID: 87cd9671aacd4917925dd93b12a2aa20.

7. Yang H, Yang H, Xie Z, Wei L, Bi J. 2013; Systemic transplantation of human umbilical cord derived mesenchymal stem cells-educated T regulatory cells improved the impaired cognition in AβPPswe/PS1dE9 transgenic mice. PLoS One. 8:e69129. DOI: 10.1371/journal.pone.0069129. PMID: 23935936. PMCID: PMC3723739. PMID: faa75a0bdf7c46409b56fea300fcf352.

8. Le Blanc K, Rasmusson I, Sundberg B, Götherström C, Hassan M, Uzunel M, Ringdén O. 2004; Treatment of severe acute graft-versus-host disease with third party haploi-dentical mesenchymal stem cells. Lancet. 363:1439–1441. DOI: 10.1016/S0140-6736(04)16104-7. PMID: 15121408.

9. Huang P, Wang L, Li Q, Xu J, Xu J, Xiong Y, Chen G, Qian H, Jin C, Yu Y, Liu J, Qian L, Yang Y. 2019; Combinatorial treatment of acute myocardial infarction using stem cells and their derived exosomes resulted in improved heart performance. Stem Cell Res Ther. 10:300. DOI: 10.1186/s13287-019-1353-3. PMID: 31601262. PMCID: PMC6785902. PMID: f353fc8aac954f2a8b85666ab60e7419.

10. Unsal IO, Ginis Z, Pinarli FA, Albayrak A, Cakal E, Sahin M, Delibasi T. 2015; Comparison of therapeutic characteristics of islet cell transplantation simultaneous with pancreatic mesenchymal stem cell transplantation in rats with type 1 diabetes mellitus. Stem Cell Rev Rep. 11:526–532. DOI: 10.1007/s12015-014-9563-7. PMID: 25297071.

11. Noronha NC, Mizukami A, Caliári-Oliveira C, Cominal JG, Rocha JLM, Covas DT, Swiech K, Malmegrim KCR. 2019; Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies. Stem Cell Res Ther. 10:131. DOI: 10.1186/s13287-019-1224-y. PMID: 31046833. PMCID: PMC6498654. PMID: 3b2ec96227cd4be081df40d2750b7ce1.

12. Han Y, Yang J, Fang J, Zhou Y, Candi E, Wang J, Hua D, Shao C, Shi Y. 2022; The secretion profile of mesenchymal stem cells and potential applications in treating human diseases. Signal Transduct Target Ther. 7:92. DOI: 10.1038/s41392-022-00932-0. PMID: 35314676. PMCID: PMC8935608. PMID: 55a254d6397d4ce7a0fde36cb8e76cb0.

13. Bashyal N, Lee TY, Chang DY, Jung JH, Kim MG, Acharya R, Kim SS, Oh IH, Suh-Kim H. 2022; Improving the safety of mesenchymal stem cell-based ex vivo therapy using herpes simplex virus thymidine kinase. Mol Cells. 45:479–494. DOI: 10.14348/molcells.2022.5015. PMID: 35356894. PMCID: PMC9260133.

14. Park JS, Chang DY, Kim JH, Jung JH, Park J, Kim SH, Lee YD, Kim SS, Suh-Kim H. 2013; Retrovirus-mediated transduction of a cytosine deaminase gene preserves the stemness of mesenchymal stem cells. Exp Mol Med. 45:e10. DOI: 10.1038/emm.2013.21. PMID: 23429359. PMCID: PMC3584665.

15. Bar D. 2011; Evidence of massive horizontal gene transfer between humans and plasmodium vivax. Nat Preced. doi: 10.1038/npre.2011.5690.1. DOI: 10.1038/npre.2011.5690.1.

16. Sun D, Jeannot K, Xiao Y, Knapp CW. 2019; Editorial: horizontal gene transfer mediated bacterial antibiotic resistance. Front Microbiol. 10:1933. DOI: 10.3389/fmicb.2019.01933. PMID: 31507555. PMCID: PMC6718914. PMID: 6c5432b9ba9f4f329bd7b71faf749cd1.

17. Southern PJ, Berg P. 1982; Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1:327–341. PMID: 6286831.

18. Kimura M, Kamakura T, Tao QZ, Kaneko I, Yamaguchi I. 1994; Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae. Mol Gen Genet. 242:121–129. DOI: 10.1007/BF00391004. PMID: 8159161.

19. Kaster KR, Burgett SG, Rao RN, Ingolia TD. 1983; Analysis of a bacterial hygromycin B resistance gene by transcriptional and translational fusions and by DNA sequencing. Nucleic Acids Res. 11:6895–6911. DOI: 10.1093/nar/11.19.6895. PMID: 6314265. PMCID: PMC326422.

20. Mulsant P, Gatignol A, Dalens M, Tiraby G. 1988; Phleomycin resistance as a dominant selectable marker in CHO cells. Somat Cell Mol Genet. 14:243–252. DOI: 10.1007/BF01534585. PMID: 2453083.

21. Vara J, Malpartida F, Hopwood DA, Jiménez A. 1985; Cloning and expression of a puromycin N-acetyl transferase gene from Streptomyces alboniger in Streptomyces lividans and Escherichia coli. Gene. 33:197–206. DOI: 10.1016/0378-1119(85)90094-0. PMID: 3888783.

22. Chang DY, Yoo SW, Hong Y, Kim S, Kim SJ, Yoon SH, Cho KG, Paek SH, Lee YD, Kim SS, Suh-Kim H. 2010; The growth of brain tumors can be suppressed by multiple transplantation of mesenchymal stem cells expressing cytosine deaminase. Int J Cancer. 127:1975–1983. DOI: 10.1002/ijc.25383. PMID: 20473873.

23. Longley DB, Harkin DP, Johnston PG. 2003; 5-fluorouracil: mec-hanisms of action and clinical strategies. Nat Rev Cancer. 3:330–338. DOI: 10.1038/nrc1074. PMID: 12724731.

24. Chang DY, Jung JH, Kim AA, Marasini S, Lee YJ, Paek SH, Kim SS, Suh-Kim H. 2020; Combined effects of mesenchymal stem cells carrying cytosine deaminase gene with 5-fluorocytosine and temozolomide in orthotopic glioma model. Am J Cancer Res. 10:1429–1441. PMID: 32509389. PMCID: PMC7269785.

25. Kim SS, Choi JM, Kim JW, Ham DS, Ghil SH, Kim MK, Kim-Kwon Y, Hong SY, Ahn SC, Kim SU, Lee YD, Suh-Kim H. 2005; cAMP induces neuronal differentiation of mesenchymal stem cells via activation of extracellular signal-regulated kinase/MAPK. Neuroreport. 16:1357–1361. DOI: 10.1097/01.wnr.0000175243.12966.f5. PMID: 16056139.

26. Clinical and Laboratory Standards Institute (CLSI). 2021. M100: performance standards for antimicrobial susceptibility testing. 31st ed. CLSI;Wayne (PA):

27. Portnow J, Synold TW, Badie B, Tirughana R, Lacey SF, D'Apuzzo M, Metz MZ, Najbauer J, Bedell V, Vo T, Gutova M, Frankel P, Chen M, Aboody KS. 2017; Neural stem cell-based anticancer gene therapy: a first-in-human study in recurrent high-grade glioma patients. Clin Cancer Res. 23:2951–2960. DOI: 10.1158/1078-0432.CCR-16-1518. PMID: 27979915. PMCID: PMC8843778.

28. Wright JC, Dolgopol VB, Logan M, Prigot A, Wright LT. 1955; Clinical evaluation of puromycin in human neoplastic disease. AMA Arch Intern Med. 96:61–77. DOI: 10.1001/archinte.1955.04430010075008. PMID: 14387321.