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

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 adipose-derived 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 proliferation-related 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.


INTRODUCTION
Restoring a sufficient liver volume is essential for patient survival and quality of life after surgical resection. The peak liver regeneration after surgical resection occurs on an average within 7 days. A substantial proportion of the patients undergoing liver resection often face liver dysfunction, such as liver fibrosis or cirrhosis [1], increasing patient morbidity and mortality resulting from the reduced regenerative capacity of the liver. Therefore, maximizing liver regeneration after surgical resection is a prerequisite for preventing complications and tracking morbidity and mortality [2].
Recent studies have shown the potential of stem cells to enhance liver regeneration [3]. Among various stem cell sources, adipose-derived stem cells (ASCs) have distinct advantages, including the ease of attainment and processing, long-term in vitro maintenance, and high proliferative capacity [4]. However, despite the numerous benefits of stem cells, their clinical application is still challenging due to several limitations, such as their short in vivo viability and potential for malignant transformation [5]. Recently, the use of stem cell secretome instead of stem cells has been actively studied in an attempt to overcome these limitations [6] secretome refers to substances secreted by the stem cells into the surrounding extracellular space [7]. The use of the secretome is supported by evidence that they mediate the main mechanism of action of stem cells [8]. Conditioned media (CM) obtained from MSCs were found to contain high levels of paracrine cytokines and growth factors that promote tissue repair in numerous in vitro and in vivo experimental models [6].
Survivin is a member of the inhibitor of apoptosis (IAP) protein family and is one of the representative antiapoptotic proteins [9]. It is copiously expressed during human fetal development but is infrequently present in adult tissues. Survivin inhibits cell apoptosis by inhibiting caspase activity [10] and is highly expressed in various malignancies. However, it is also expressed in various differentiated adult tissues, particularly during proliferation and regeneration [11]. We aimed to investigate whether the regenerative capacity of the liver was enhanced by the secretome obtained from survivin-overexpressing ASCs. To achieve this, we generated the secretome from survivintransfected ASCs and attempted to determine the in vitro and in vivo regenerative capacity of the secretome.
The experiments protocol was approved by the Institutional Animal Care and Use Committee of School of Medicine, The Catholic University of Korea (No. 2020-0303-01).

Serology test
Blood was collected for each mouse. Then, centrifuged for 9,500 ×g in 10 minutes serum was collected. We performed liver injury concentrations using ALT and AST with the VetTest Chemistry Analyzer (IDEXX Laboratories Inc., Westbrook, ME, USA) [8].

Statistical analysis
Analysis of all data was made by SPSS ver. 11.0 (SPSS Inc., Chicago, IL, USA). Data were presented as mean ± standard deviation. Using the Kruskal-Wallis test, groups were compared. The P-values of <0.05 were regarded as statistically significant.

Characterization of survivin-overexpressing adipose-derived stem cells
Survivin-overexpressing ASCs were generated by transfecting ASCs with survivin gene-conveying plasmids (Fig. 1A). The secretome obtained from the CM after transfecting ASCs with the survivin gene-conveying plasmid was termed the survivinsecretome. Western blot analysis showed an upregulated expression of survivin in survivin-overexpressing ASCs (Fig. 1B).
Cho-Hee Kim, et al: Survivin-overexpressing adipose-derived stem cells  In real-time PCR, survivin-overexpressing ASCs showed increased HGF and VEGF messenger RNA (mRNA) expression as well as survivin mRNA compared to the control ASCs (P < 0.05) (Fig. 1C).

In vitro characterization of the survivin-secretome
The survivin-secretome was obtained from the CM of survivin-overexpressing ASCs after processing at a 25-fold concentration using ultrafiltration units with a 3-kDa cutoff. Subsequently, thioacetamide (TAA), a hepatotoxin, was administered to AML12 hepatocytes, and the cell viability between the groups treated with either control secretome or survivin-secretome was compared. The survivin-secretome group showed significantly increased cell viability compared to the control group (P < 0.05) ( Fig. 2A). Real-time PCR showed that the survivin-secretome group significantly decreased the expression of a proapoptotic marker (Bax) and increased the expression of an antiapoptotic marker (Mcl-1) (P < 0.05) (Fig.  2B). Next, the effects of survivin-secretome were determined by western blot analysis. It showed that the survivin-secretome group significantly increased the expression of p-STAT3, HGF, VEGF, Mcl-1, and survivin compared to the control secretome group (P < 0.05) (Fig. 2C).

In vivo validation of survivin-secretome
A mouse model of 70% PH was generated by surgical resection of the hepatic lobules [13]. One hour after PH, the mice were administered normal saline (PH-only group), control secretome (control secretome group), and survivin-secretome (survivinsecretome group) through the tail vein. Each group included 2 subgroups (continuous and interrupted groups) according to the ways of sample acquisition (Fig. 3A). The expression levels of IL-6 and TNF-α (systemic inflammatory markers) in the sera of each mouse group were compared (Fig. 3B). The treatment groups (control secretome and survivin-secretome groups) showed significantly decreased serum levels of these markers on PH 2 and 3 days compared to the PH-only group (P < 0.05). Of the 2 secretome-administered groups, the survivinsecretome group showed the lowest levels of these markers (P < 0.05). Subsequently, the serum levels of liver transaminases (ALT and AST) were compared (Fig. 3C). Likewise, the survivinsecretome group showed the lowest levels of ALT and AST on PH 2 days (P < 0.05). Figure 3D shows that the size of the extracted liver was increased in the treatment group (control secretome and survivin-secretome) than in the PH-only group. Liver regeneration rate (percentage) was calculated as the ratio of the extracted liver weight to 0.7 times the excised liver weight [16]. The 2 treatment groups showed a significantly higher liver regeneration rate than the PH-only group (P < 0.05). Of the 2 treatment groups, the survivin-secretome group showed the highest liver regeneration on PH 3 and 7 days (P < 0.05) (Fig. 3E).

In vivo effects of survivin-secretome on the liver specimens
After euthanizing the mice 2 on PH 2 days, we harvested liver specimens and examined the expression of various markers related to liver regeneration at the RNA and DNA levels. Real-time PCR revealed that the survivin-secretome group showed the highest RNA expression levels of HGF, VEGF, Mcl-1, HNF4α, and albumin in all the mouse groups with PH (P < 0.05) (Fig. 4A). Also, the survivin-secretome group showed the lowest RNA expression of Bax and EpCAM (P < 0.05). Subsequently, western blot analysis was performed to compare the expression of various proteins. The survivin-secretome group showed the highest expression levels of p-STAT3, HGF, VEGF, PCNA, and Mcl-1 in all mouse groups with PH (P < 0.05) (Fig. 4B). The survivin-secretome group also showed the lowest expression of Bax (P < 0.05). Taken together, it appears that the intravenous injection of survivin-secretome cumulates in higher antiapoptotic, proliferative, and angiogenic effects than the injection of control secretome in 70% hepatectomized mice.

In vivo effects of survivin-secretome on the histology of the liver
H&E and IHC staining were performed to determine the effects of survivin-secretome on the liver of partially hepatectomized mice (Fig. 5). H&E staining showed that the survivin-secretome group exhibited the lowest expression of Bcl-xL (P < 0.05). Survivin IHC staining demonstrated that the survivin-secretome group exhibited the highest level of survivin in all groups (P < 0.05). The survivin-secretome group showed the highest expression of a proliferation marker (PCNA) (P < 0.05), suggestive of the proproliferative effects of the survivin-secretome. The survivin-secretome group also showed the highest expression of an antiapoptotic marker (Bcl-xL) and the lowest expression of a proapoptotic marker (Bim), demonstrating the antiapoptotic effect of survivin-secretome (P < 0.05). Taken together, the results of various IHC stains show that the survivin-secretome promotes liver regeneration and prevents cell apoptosis higher than control secretome.

DISCUSSION
This study focused on whether the secretome obtained from the survivin-overexpressing ASCs enhanced the proproliferative potential in the liver compared to the control secretome. In the experiments, the survivin-secretome significantly increased the cell viability. The survivin-secretome also increased the protein expression levels of markers related to proliferation (HGF, VEGF, and p-STAT3) and antiapoptosis (Mcl-1) and reduced the expression of the proapoptotic marker, Bax, in TAA-treated AML12 hepatocytes. In the experiments using hepatectomized mice, the survivin-secretome group showed the lowest serum levels of proinflammatory cytokines, such as TNF-α and IL-6. The serum levels of liver transaminases (ALT and AST) were also the lowest in the survivin-secretome group. The survivinsecretome group also exhibited the highest liver regeneration based on the liver weight on PH 3 and 7 days. 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 Bax, suggestive of the enhanced proproliferative and antiapoptotic potential of survivin-secretome. Taken together, it appears that the survivinsecretome has an enhanced potential for proproliferation and antiapoptosis compared to the control secretome in both in vitro and in vivo models of liver hepatectomy.
Survivin is the smallest member of the IAP family of proteins and is encoded by the BIRC5 gene. The survivin gene spans 15 kb of DNA and is located on chromosome 17 at band q25. Structurally, survivin includes a single baculovirus IAP repeat (BIR), a B70-amino-acid zinc-finger fold that is the hallmark of all IAPs, and an extended -COOH terminus α-helical coiledcoil, which are intimately linked with its function as an IAP [17]. A mutation in the BIR domain leads to an abrogation of its ability to inhibit apoptosis [18]. Survivin plays important roles in various cellular mechanisms, including cell cycle regulation, proliferation, apoptosis, and angiogenesis [13]. Survivin is present in the cytoplasm, nucleus, and mitochondria [19]. Survivin is highly exhibited in most human tumors and fetal tissues; however, it is entirely absent in differentiated cells. This study showed that the survivin-secretome has the potential to enhance regeneration of the damaged liver. The survivin-secretome increased the protein expression levels of the markers related to proliferation (HGF, VEGF, and p-STAT3) in both in vitro and in vivo models of liver hepatectomy. Moreover, the injection of survivin-secretome into the partially hepatectomized mice induced the highest liver regeneration on PH 3 and 7 days. In the analysis of both hepatocellular carcinoma (HCC) cell lines and HCC tissues, Ito et al. [20] found that survivin expression levels were strongly correlated with the proliferation index in HCC tissues. The overexpression of survivin resulted in a decreased G0/G1 phase and an increased S phase in all HCC cell lines. They also showed that survivin promoted cell proliferation by interacting with cyclindependent kinase 4 (Cdk4) and releasing p21 from Cdk4. Our study suggests that survivin-secretome contains higher levels of survivin, thus exerting similar proproliferative properties as survivin.
The survivin-secretome also exerted profound antiapoptotic effects in both in vitro and in vivo models of liver hepatectomy. It increased the antiapoptotic marker (Mcl-1) and decreased the pro-apoptotic marker (Bax) in TAA-treated AML12 hepatocytes. Subsequently, the liver specimens of the survivin-secretomeinjected mice exhibited the highest expression of Mcl-1 and the lowest expression of Bax. Several IAP family members have been shown to suppress apoptosis by directly inhibiting caspases via the BIR domains [21]. The structure of survivin has been compared to that of another IAP family member, XIAP, which includes 3 BIR domains. XIAP inhibits caspase-3 and caspase-7 via a linker region between the first 2 BIR domains, and also binds to and inhibits caspase-9 through its third BIR domain (BIR3) [22]. The BIR domain of survivin appears closely related to the three-dimensional structure of the BIR3 domain of XIAP, suggesting the possibility that survivin binds to caspase-9 [23]. Fas receptor (CD95, TNF receptor superfamily member 6) is a death receptor localized on the surface of various cells, which triggers a signal transduction pathway leading to apoptosis. Interestingly, Fas-induced apoptosis is associated with the upregulation of survivin in the mitochondria, nucleus, and cytosol [24]. Upon Fas stimulation in cell culture, survivin interacts with Cdk4, which leads to release p21 from its complex with Cdk4 [25] making it possible for p21 to complex with caspase-3, the initial step in inactivating caspase-3 in the mitochondria. However, further research is required to determine how survivin inhibits apoptosis by suppressing caspase-3 and other caspases. Our experiments also displayed that the survivin-secretome increased the expression of VEGF in both in vitro and in vivo models of liver hepatectomy. VEGF is a key transcription factor that regulates the angiogenesis of endothelial cells. Wang et al. [26] discovered that survivin expression in glioma cells prompted the production and secretion of VEGF and basic fibroblast growth factor, implying that survivin plays a vital role in transmitting angiogenic stimuli to the associated vasculature in glioma cells. Besides, survivin exerts favorable effects on angiogenesis by promoting the survival and proliferation of endothelial cells [27,28]. In addition, VEGF participates in the process of liver regeneration. Injection of VEGF promoted the proliferative activity of hepatocytes, demonstrating the significant role of VEGF in liver regeneration [29]. Taniguchi et al. [30] showed that VEGF promoted the proliferation of sinusoid endothelial cells during liver regeneration, which leads to the enhanced proliferation of hepatocytes. Therefore, the survivin-secretome appears to promote liver regeneration and angiogenesis, in part, by increasing VEGF levels.
In conclusion, this study purposed to determine the role of the secretome obtained from survivin-overexpressing ASCs. In the in vitro experiments, survivin-secretome enhanced cell viability and promoted the expression of proproliferative and antiapoptotic markers in TAA-treated AML12 hepatocytes. Subsequent intravenous injection of survivin-secretome into the partially hepatectomized mice enhanced cell proliferation, anti-apoptosis, and VEGF expression, all of which are beneficial for liver regeneration and tissue repair. Therefore, the use of secretome secreted by survivin-overexpressing ASCs could be an effective way to enhance liver regeneration and repair for the treatment of liver injury.