J Korean Soc Transplant.  2016 Mar;30(1):13-23. 10.4285/jkstn.2016.30.1.13.

Liver Regenerating Potential of the Secretome Obtained from Adipose-derived Stem Cells Cultured under the Hypoxic Environment

• Jeong HJ
• Lee SC
• Kim OH
• Jeong WJ
• Kim SJ

• KMID: 2202623
• DOI: http://doi.org/10.4285/jkstn.2016.30.1.13

Abstract

BACKGROUND
The stem cell-derived secretome has received considerable attention as an alternative to stem cells for therapeutic applications. However, establishing optimal culture conditions is key to obtaining appropriate secretome contents. Here, the optimal culturing environment for achieving a high-efficiency secretome was determined via hypoxic preconditioning of human adipose-derived stem cells (ASC).
METHODS
Normoxic conditioned media (NCM) and hypoxic conditioned media (HCM) were obtained after culturing human ASCs under normoxia (20% O2) or hypoxia (1% O2), respectively. Subsequently, both normal and thioacetamide-induced hepatotoxic hepatocytes were treated with NCM or HCM. In addition, partially hepatectomized mice were infused with control saline, NCM, and HCM. The effects on liver regeneration and serum transaminases levels were then compared.
RESULTS
Hypoxic preconditioning significantly increased mRNA expression of proinflammatory cytokines (interleukin-6 and tumor necrosis factor-α) and growth factors (hepatocyte growth factor and vascular endothelial growth factor). In both normal and thioacetamide-induced hepatotoxic hepatocyte (alpha mouse liver 12 [AML12]) cell lines, HCM treatment resulted in the highest cell viability (122% and 95%, respectively), followed by NCM (111% and 78%, respectively). In addition, intravenous administration of HCM to partially hepatectomized mice resulted in substantially enhanced liver regeneration compared with the NCM group (P<0.05).
CONCLUSIONS
Taken together, the secretome obtained from ASC with hypoxic preconditioning showed potential to alleviate liver damage both in vitro and in vivo. Hypoxic culture of ASC is expected to play an important role in regenerative medicine by inducing secretome production that is beneficial for improving liver regeneration.

Keyword

Adipose-derived stem cell; Conditioned culture media; Hypoxic reconditioning; Liver regeneration; Paracrine effect; Secretome

MeSH Terms

Administration, Intravenous
Animals
Anoxia
Cell Line
Cell Survival
Culture Media, Conditioned
Cytokines
Hepatocytes
Humans
Intercellular Signaling Peptides and Proteins
Liver Regeneration
Liver*
Mice
Necrosis
Regenerative Medicine
RNA, Messenger
Stem Cells*
Transaminases
Culture Media, Conditioned
Cytokines
Intercellular Signaling Peptides and Proteins
RNA, Messenger
Transaminases

Figure

• 

  Fig. 1. Effect of hypoxia on the expression of cytokines and growth factor in adipose-derived stem cells (ASCs). (A) Real-time quantitative polymerase chain reaction analysis of hypoxia preconditioning ASCs. (B) Western blotting of ASCs under normoxia and hypoxia. (C) Enzyme-linked immunosorbent assay analysis for measure secreted interleukin-6 (IL-6) levels in normoxic and hypoxic condition of ASC. Values represent mean±SD of three independent experiments. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TNF-, tumor necrosis factor ; HGF, hepatocyte growth factor; VEGF, vascular endothelial growth factor; p-Erk, phosphorylated extracellular signal-regulated kinase; Erk, extracellular signal-regulated kinase; p-STAT3, phosphorylated signal transducer and activator of transcription 3; STAT3, signal transducer and activator of transcription 3. aP＜0.05.

• 

  Fig. 2. Effect of hypoxic conditioned media (HCM) on mouse hepatocyte (alpha mouse liver 12 [AML12]) cells. (A) Cell viability assay showing dose-dependent effects of the hepatotoxic molecule thioacetamide (TAA) on mouse hepatocyte AML12 proliferation. (B) Western blotting of AML12 cells under series concentration of TAA. (C) Relative densities of proliferating cell nuclear antigen (PCNA) according to TAA concentrations. (D) Effect of HCM on TAA-treated or untreated AML12 cells viabilities. (E) Effect of HCM on TAA-treated AML12 cells. (F) Relative densities of PCNA in each group. Values represent mean±SD of three independent experiments. Abbreviation: NCM, normoxic conditioned media. aP＜0.05.

• 

  Fig. 3. Hypoxic conditioned media (HCM) effects on inflammation in the liver of partially hepatectomized mice. (A) Enzyme-linked immunosorbent assay (ELISA) analysis for the serum levels of interleukin-6 (IL-6) in each group at 1, 2, 3 days after infusion. (B) ELISA analysis for the serum levels of tumor necrosis factor  (TNF-) in each group at 1, 2, 3 days after infusion. In the experiment presented in (A) and (B), each group included 20 mice at each time point (80 mice in total). Values represent mean±SD. Abbreviation: NCM, normoxic conditioned media. aP＜0.05.

• 

  Fig. 4. Hypoxic conditioned media (HCM) effects on liver regeneration in partially hepatectomized mice. (A, upper) Immunohistochemical stain of Ki67 (regeneration marker) in the liver specimens of each group at 1 day after infusion. (A, under) Percentage of Ki67 positive cells in each group at 1, 2, 3, and 7 days after infusion. (B, upper) Immunohistochemical stain of bromodeoxyuridine (BrdU; regeneration marker) in the liver specimens of each group at 1day after infusion. (B, under) Percentage of BrdU positive cells in each group at 1, 2, 3, and 7 days after infusion. (C, upper) Western blotting analysis for expression of proliferating cell nuclear antigen (PCNA; liver regeneration marker) and phosphorylation of signal transducer and activator of transcription 3 (STAT3) at 2 days after infusion. (C, under) Relative densities of these markers in each group. (D) Comparison of liver weight to body weight of each group at 7 days after infusion. In the experiment presented in (A) to (D), each group included 20 mice at each time point (80 mice in total). Values represent mean±SD. Abbreviations: NCM, normoxic conditioned media; p-STAT3, phosphorylated signal transducer and activator of transcription 3. aP＜0.05.

• 

  Fig. 5. Hypoxic conditioned media (HCM) effects on hepatic function in partially hepatectomized mice. (A) Serology tests of aspartate transaminase (AST) in each group. (B) Serology tests of alanine transaminase (ALT) in each group. In the experiment presented in (A) and (B), in the serologic tests, each group included 5 mice (20 mice in total). Values represent mean±SD. Abbreviation: NCM, normoxic conditioned media. aP＜0.05.

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