Ann Surg Treat Res.  2021 Dec;101(6):322-331. 10.4174/astr.2021.101.6.322.

A novel strategy to promote liver regeneration: utilization of secretome obtained from survivin-overexpressing adipose-derived stem cells

• Kim CH
• Kim OH
• Park JH
• Kim SJ

• KMID: 2522929
• DOI: http://doi.org/10.4174/astr.2021.101.6.322

Abstract

Purpose
Survivin is a typical antiapoptotic protein. It is copiously expressed during human fetal development but is infrequently present in adult tissues. In this experiment, we researched the treatment effect of the secretome that adiposederived stem cells (ASCs) transfected with survivin.
Methods
First of all, we generated survivin-overexpressing ASCs transfected with a plasmid comprising a gene encoding survivin. The secreted substances released from survivin-overexpressing ASCs (survivin-secretome) were collected, and were determined their in vitro and in vivo therapeutic potential, especially in the model of liver impairment.
Results
In vitro, the survivin-secretome significantly increased cell viability and promoted the expression of proliferationrelated markers (proliferating cell nuclear antigen [PCNA], phospho-signal transducer and activator of transcription 3 (p-STAT3), hepatocyte growth factor [HGF], vascular endothelial growth factor [VEGF]) and anti-apoptosis-related markers (myeloid cell leukemia-1 [Mcl-1] and survivin) (P < 0.05). In vivo using 70% hepatectomy mice, the survivin-secretome group exhibited the lowest serum levels of interleukin-6, tumor necrosis factor-α (P < 0.05). The serum levels of liver transaminases (alanine aminotransferase and aspartate aminotransferase) were also the lowest in the survivin-secretome group (P < 0.05). The survivin-secretome group also exhibited the highest liver regeneration on the 7th day after 70% partial hepatectomy (P < 0.05). In the subsequent liver specimen analysis, the specimens of survivin-secretome exhibited the highest expression of p-STAT3, HGF, VEGF, PCNA, and Mcl-1 and the lowest expression of bcl-2-like protein 4 (P < 0.05).
Conclusion
Taken together, secretome secreted by survivin-overexpressing ASCs could be an effective way to improve liver regeneration and repair for liver injury treatment.

Keyword

Adipose-derived stem cell; Apoptosis; Liver regeneration; Secretome; Survivin

Figure

• 

  Fig. 1 Characterization of survivin-overexpressing adipose-derived stem cells (ASCs). (A) Study design. After generation of survivin-secretome from survivin-overexpressing ASCs, the liver regenerative potential of survivin-secretome was determined using a mouse model of 70% PH. (B) Western blot analysis showing the upregulated expression of survivin in the survivin-overexpressing ASCs. (C) Real-time polymerase chain reaction comparing messenger RNA (mRNA) expression between control secretome and survivin-secretome. Values are displayed as mean ± standard deviations from 3 independent experiments. *P < 0.05. HGF, hepatocyte growth factor; PH, partial hepatectomy; VEGF, vascular endothelial growth factor.

• 

  Fig. 2 In vitro validation of survivin-secretome. (A) Cell viability test of AML12 hepatocytes treated with either control secretome or survivin-secretome. (B) Real-time polymerase chain reaction comparing messenger RNA (mRNA) expression of AML12 hepatocytes treated with either control secretome or survivin-secretome. (C) [Left] Western blot of various markers in the AML12 hepatocytes treated with either control secretome or survivin-secretome. [Right] The relative densities of markers in each group. Using ImageJ software, the relative density of individual markers was quantified and then normalized to the density of β-actin in each group. Values are displayed as mean ± standard deviations from 3 independent experiments. *P < 0.05. Ct, control; SCM, survivin conditioned media (survivin-secretome); NCM, normal conditioned media (control secretome); TAA, thioacetamide; Bax, bcl-2-like protein 4; Mcl-1, myeloid cell leukemia 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; STAT3, signal transducer and activator of transcription 3; t-STAT3, total STAT3; p-STAT3, phospho-STAT3; HGF, hepatocyte growth factor; VEGF, vascular endothelial growth factor.

• 

  Fig. 3 In vivo validation of survivin-secretome. (A) Study design of in vivo experiment. One hour after partial hepatectomy (PH), the mice were administrated with normal saline (PH-only group), control secretome (control secretome group), and survivin-secretome (survivin-secretome group) through the tail vein, respectively. Each group included 2 subgroups (continuous and interrupted groups) according to the method of acquiring samples. (B) Enzyme-linked-immunosorbent assay showing the serum concentration of systemic inflammatory markers (tumor necrosis factor [TNF]-α and interleukin [IL]-6) in each group. (C) Serologic test showing the serum concentration of transaminases (ALT and AST) in each group. (D) Size comparison of the representative liver specimens in each group. (E) Comparison of liver regeneration rates based on the weights of extracted liver specimens in each group. Values are displayed as mean ± standard deviations from 3 independent experiments. *P < 0.05. Ct, control; NS, normal saline; NCM, normal conditioned media (control secretome); SCM, survivin conditioned media (survivin-secretome).

• 

  Fig. 4 In vivo effects of survivin-secretome on the liver specimens. (A) Real-time polymerase chain reaction comparing messenger RNA (mRNA) expression in the liver specimens treated with either control secretome or survivin-secretome. (B) [Left] Western blot showing the expression of various markers in the liver specimens in each group. [Right] The relative densities of markers in each group. Using ImageJ software, the relative density of individual markers was quantified and then normalized to the density of β-actin in each group. Values are displayed as mean ± standard deviations from 3 independent experiments. *P < 0.05. PH, partial hepatectomy; Ct, control; NS, normal saline; NCM, normal conditioned media (control secretome); SCM, survivin conditioned media (survivin-secretome); HGF, hepatocyte growth factor; VEGF, vascular endothelial growth factor; Mcl-1, myeloid cell leukemia 1; Bax, bcl-2-like protein 4; HNF4α, hepatocyte nuclear factor 4 α; EpCAM; epithelial cell adhesion molecule; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; STAT3, signal transducer and activator of transcription 3; t-STAT3, total STAT3; p-STAT3, phospho-STAT3; PCNA, proliferating cell nuclear antigen.

• 

  Fig. 5 Immunohistochemical stains of the liver specimens in each group. (A) H&E and immunohistochemical of bcl-2-like protein 11 (Bim), B-cell leukemia-extra large (Bcl-xL), proliferating cell nuclear antigen (PCNA), and survivin in each group. (B) Using Imag J software, the percentages of immunoreactive areas were measured and displayed as relative values to those in normal livers. Values are displayed as mean ± standard deviations from 3 independent experiments. *P < 0.05. H&E, Hematoxylin and Eosin; Ct, control; PH, partial hepatectomy; NS, normal saline; NCM, normal conditioned media (control secretome); SCM, survivin conditioned media (survivin-secretome).

Reference

1. Farid H, O’Connell T. Hepatic resections: changing mortality and morbidity. Am Surg. 1994; 60:748–752. PMID: 7944036.

2. Liu J, Zhang Y, Zhu H, Qiu L, Guo C. Prediction of perioperative outcome after hepatic resection for pediatric patients. BMC Gastroenterol. 2019; 19:201. PMID: 31775648.

3. Itoh T, Miyajima A. Liver regeneration by stem/progenitor cells. Hepatology. 2014; 59:1617–1626. PMID: 24115180.

4. Puglisi MA, Saulnier N, Piscaglia AC, Tondi P, Agnes S, Gasbarrini A. Adipose tissue-derived mesenchymal stem cells and hepatic differentiation: old concepts and future perspectives. Eur Rev Med Pharmacol Sci. 2011; 15:355–364. PMID: 21608430.

5. Baglio SR, Pegtel DM, Baldini N. Mesenchymal stem cell secreted vesicles provide novel opportunities in (stem) cell-free therapy. Front Physiol. 2012; 3:359. PMID: 22973239.

6. Filip S, Mokrý J, English D, Vojácek J. Stem cell plasticity and issues of stem cell therapy. Folia Biol (Praha). 2005; 51:180–187. PMID: 16419613.

7. Makridakis M, Roubelakis MG, Vlahou A. Stem cells: insights into the secretome. Biochim Biophys Acta. 2013; 1834:2380–2384. PMID: 23376432.

8. Kim KH, Lee JI, Kim OH, Hong HE, Kwak BJ, Choi HJ, et al. Ameliorating liver fibrosis in an animal model using the secretome released from miR-122-transfected adipose-derived stem cells. World J Stem Cells. 2019; 11:990–1004. PMID: 31768225.

9. Lee J, Choi JH, Joo CK. TGF-β1 regulates cell fate during epithelial-mesenchymal transition by upregulating survivin. Cell Death Dis. 2013; 4:e714. PMID: 23828577.

10. Deguchi M, Shiraki K, Inoue H, Okano H, Ito T, Yamanaka T, et al. Expression of survivin during liver regeneration. Biochem Biophys Res Commun. 2002; 297:59–64. PMID: 12220508.

11. Hagemann S, Wohlschlaeger J, Bertram S, Levkau B, Musacchio A, Conway EM, et al. Loss of Survivin influences liver regeneration and is associated with impaired Aurora B function. Cell Death Differ. 2013; 20:834–844. PMID: 23519077.

12. Park JH, Kim OH, Kim KH, Hong HE, Seo H, Choi HJ, et al. Isolation of secretome with enhanced antifibrotic properties from miR-214-transfected adipose-derived stem cells. J Korean Med Sci. 2019; 34:e273. PMID: 31760709.

13. Greene AK, Puder M. Partial hepatectomy in the mouse: technique and perioperative management. J Invest Surg. 2003; 16:99–102. PMID: 12746193.

14. Park MR, Wong MS, Araúzo-Bravo MJ, Lee H, Nam D, Park SY, et al. Oct4 and Hnf4α-induced hepatic stem cells ameliorate chronic liver injury in liver fibrosis model. PLoS One. 2019; 14:e0221085. PMID: 31404112.

15. Zhao Q, Zhou H, Liu Q, Cao Y, Wang G, Hu A, et al. Prognostic value of the expression of cancer stem cell-related markers CD133 and CD44 in hepatocellular carcinoma: from patients to patient-derived tumor xenograft models. Oncotarget. 2016; 7:47431–47443. PMID: 27329727.

16. Lee TH, Park DS, Jang JY, Lee I, Kim JM, Choi GS, et al. Human placenta hydrolysate promotes liver regeneration via activation of the cytokine/growth factor-mediated pathway and anti-oxidative effect. Biol Pharm Bull. 2019; 42:607–616. PMID: 30930420.

17. Altieri DC. Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene. 2003; 22:8581–8589. PMID: 14634620.

18. O’Connor DS, Grossman D, Plescia J, Li F, Zhang H, Villa A, et al. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc Natl Acad Sci U S A. 2000; 97:13103–13107. PMID: 11069302.

19. Ebrahimiyan H, Aslani S, Rezaei N, Jamshidi A, Mahmoudi M. Survivin and autoimmunity; the ins and outs. Immunol Lett. 2018; 193:14–24. PMID: 29155234.

20. Ito T, Shiraki K, Sugimoto K, Yamanaka T, Fujikawa K, Ito M, et al. Survivin promotes cell proliferation in human hepatocellular carcinoma. Hepatology. 2000; 31:1080–1085. PMID: 10796883.

21. Deveraux QL, Reed JC. IAP family proteins: suppressors of apoptosis. Genes Dev. 1999; 13:239–252. PMID: 9990849.

22. Sun C, Cai M, Gunasekera AH, Meadows RP, Wang H, Chen J, et al. NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP. Nature. 1999; 401:818–822. PMID: 10548111.

23. Shi Y. Survivin structure: crystal unclear. Nat Struct Biol. 2000; 7:620–623. PMID: 10932241.

24. Conway EM, Pollefeyt S, Steiner-Mosonyi M, Luo W, Devriese A, Lupu F, et al. Deficiency of survivin in transgenic mice exacerbates Fas-induced apoptosis via mitochondrial pathways. Gastroenterology. 2002; 123:619–631. PMID: 12145814.

25. Suzuki A, Hayashida M, Ito T, Kawano H, Nakano T, Miura M, et al. Survivin initiates cell cycle entry by the competitive interaction with Cdk4/p16(INK4a) and Cdk2/cyclin E complex activation. Oncogene. 2000; 19:3225–3234. PMID: 10918579.

26. Wang P, Zhen H, Zhang J, Zhang W, Zhang R, Cheng X, et al. Survivin promotes glioma angiogenesis through vascular endothelial growth factor and basic fibroblast growth factor in vitro and in vivo. Mol Carcinog. 2012; 51:586–595. PMID: 21761458.

27. Botto S, Streblow DN, DeFilippis V, White L, Kreklywich CN, Smith PP, et al. IL-6 in human cytomegalovirus secretome promotes angiogenesis and survival of endothelial cells through the stimulation of survivin. Blood. 2011; 117:352–361. PMID: 20930069.

28. Tran J, Master Z, Yu JL, Rak J, Dumont DJ, Kerbel RS. A role for survivin in chemoresistance of endothelial cells mediated by VEGF. Proc Natl Acad Sci U S A. 2002; 99:4349–4354. PMID: 11917134.

29. Assy N, Spira G, Paizi M, Shenkar L, Kraizer Y, Cohen T, et al. Effect of vascular endothelial growth factor on hepatic regenerative activity following partial hepatectomy in rats. J Hepatol. 1999; 30:911–915. PMID: 10365820.

30. Taniguchi E, Sakisaka S, Matsuo K, Tanikawa K, Sata M. Expression and role of vascular endothelial growth factor in liver regeneration after partial hepatectomy in rats. J Histochem Cytochem. 2001; 49:121–130. PMID: 11118484.