Surgical implantation of human adipose derived stem cells attenuates experimentally induced hepatic fibrosis in rats

Isolation, culture, and expansion of human adipose tissue derived stem cells

Human ADSCs were isolated from abdominal adipose tissue collected during liposuction for cosmetic purposes. The study protocol was reviewed and approved by the Ethics and Clinical Investigation Committee of Kanazawa Medical University, Ishikawa, Japan (#G129) and has been conducted according to the principles outlined in the Declaration of Helsinki (revised 2013). Written consent was obtained from each patient undergone liposuction from abdominal subcutaneous adipose tissue during cosmetic procedures. About 30 ml of lipoaspirate was collected and processed with Lipogems device (Cat# LG PK 240, Lipogems International, Milan, Italy) and obtained 10 ml of final Lipogems product as per the manufacturer's protocol (Bianchi et al. 2013).

The adipose stem cells present in the Lipogems were isolated and purified using a kit (Cat# BMK-R001, Bio Future Technologies, Tokyo, Japan), which allows isolation of adipose stem cells without enzymatic digestion. The technique utilizes the property of adipose tissue stem cells to trap inside a three-dimensional fibrous network coated with hydroxyapatite, which allows the stem cells to proliferate fast (Fig. 1A). Inside a cell culture cabinet, a 2 cm diameter non-woven fabric polyethylene-polypropylene (PE-PP) disc coated with hydroxyapatite was placed in a cell strainer (both provided in the kit, Cat# BMK-R001) and poured 1 ml of the above Lipogem suspension on top of the disc. The disc was rinsed twice using sterile PBS pouring gently on top of the disc with a pipette. Then with tweezers, the disc with adipose stem cells was transferred to an uncoated 60 mm cell culture dish containing KBM ADSC-1 medium (Cat# 16030020, Kohjin Bio, Saitama, Japan). It was incubated at 37 °C in a humidified atmosphere with 5% CO2 on air without changing the medium for one week. The medium was changed after a week and examined the disc under a phase contrast microscope to confirm the presence of fibroblast-like adipose stem cells proliferating on the fibrous network of the disc. After 2 weeks, when hADSCs were proliferated around 20% of the surface area of the disc, the culture was washed twice with serum free media. Then the adherent cells on the disc were treated with trypsin–EDTA solution (Cat# 204-16935, FUJIFILM Wako Chemicals, Tokyo, Japan) and harvested. This process yielded about 1 million cells per disc. The cells were washed with serum free media and cultured in KBM ADSC-1 medium in a 10 ml culture dish until the culture attained 80% confluence. It took about 5 days and produced around 10 million cells per dish. The cells were harvested using trypsin–EDTA solution, and tested for mycoplasma contamination using mycoplasma detection kit (Cat# LT07-118, Lonza, Walkersville, MD, USA). A portion of the cells were analyzed on a FACS machine for various markers of stem cell and human origin. The remaining cells were frozen as 1 million cells/vial and stored in liquid nitrogen.

Fig. 1figure 1

A Photographic image of non-woven fabric polyethylene-polypropylene (PE-PP) disc coated with hydroxyapatite used for isolation and culture of human adipose tissue derived stem cells (hADSCs) without enzymatic digestion. B Representative histogram and dot plot images of the flow cytometric analysis of isolated and cultured hADSCs (2nd passage) for the expression of CD73, CD90, and CD105 and a cocktail of negative markers (CD34, CD11b, CD19, CD45, and HLA-DR) after harvested using Accutase cell detachment solution and treatment with respective anti-human mAbs conjugated with various fluorescent tags. The gated cells in A +—column indicates they are positive for the respective surface marker and the cells in A– column indicates that the negative markers are not expressed in hADSCs. Practically no cells are present in A +—column with the expression of negative markers. C Graphical presentation of the flow cytometry data for the expression of surface markers in cultured hADSCs, human ADSCs from Lonza, and human dermal fibroblast cells. The data are mean ± S.D. of 5 independent experiments

Flow cytometry

Cultured hADSCs (2nd passage), human ADSCs from Lonza (Cat# PT-5006, Walkersville, MD, USA), and human dermal fibroblast cells (HDFCs) from Kurabo (Cat# KF-4109, Chuo-ku, Osaka, Japan) were treated with Accutase (Cat# 561527, BD Biosciences, San Jose, CA, USA). After centrifugation, the cell pellet was suspended in stain buffer (Cat# 554656, BD Biosciences) to a concentration of 0.5–1.0 × 107 cell/ml. After blocking with human FcR blocking reagent (Cat# 130-059-901, Miltenyi Biotec, Bergisch Gladbach, Germany), the cells were treated with various anti-human mAbs (raised in mouse) conjugated either with FITC, phycoerythrin (PE), PerCP-Cy5.5, or allophycocyanin (APC) for 30 min at 4 °C. The following mAbs are used: anti-CD73-APC, anti-CD90-FITC, anti-CD105-PerCP-Cy5.5, and negative cocktail (anti-CD34-PE, anti-CD45-PE, anti-CD11b-PE, anti-CD19-PE, and anti-human-leukocyte antigen (HLA) -DR-PE) (Cat# 562245, Human MSC analysis kit, BD Biosciences, San Jose, CA). Cells were analyzed on a Gallios flow cytometer with Kaluza software (Beckman Coulter, Brea, CA, USA). Positive and negative cells were counted and compared with the signal of corresponding immunoglobulin isotypes.

Differentiation of hADSCs into adipocytes and osteocytes

In order to evaluate the differentiation potency of purified hADSCs into adipocytes and osteocytes, we cultured 2nd passage of hADSCs in human mesenchymal stem cell adipogenic differentiation medium BulletKit (Cat# PT-3004, Lonza, Walkersville, MD, USA) or osteogenic differentiation medium BulletKit (Cat# PT-3002, Lonza). After 2–3 weeks of culture, adipogenic differentiation was identified using AdipoRed (Cat# PT-7009, Lonza, Walkersville, MD) staining for the lipid droplets and osteogenic differentiation was characterized using Alizarin Red (Cat# ARD-A1, PG Research, Kodaira, Tokyo, Japan) staining for the mineralized matrix. As control cells, human ADSCs were purchased from Lonza (Cat# PT-5006) and human dermal fibroblasts (Cat# KF-4109) were obtained from Kurabo, Chuo-ku, Osaka, Japan.

Culture of hADSCs on polyethylene disc for surgical implantation

A vial of 2nd passage of purified and characterized hADSCs stored in liquid nitrogen was removed and thawed at 37 °C water bath. A 2 cm diameter polyethylene-polypropylene (PE-PP) disc coated with hydroxyapatite (Cat# BMK-R001, Bio Future Technologies, Tokyo, Japan) was inserted into a 2 ml sterile eppendorf tube with the help of tweezers. The thawed hADSCs were gently pipetted into the eppendorf tube containing the disc and kept in a CO2 incubator at 37 °C with intermittent gentle shaking for 3 h. This process allowed adipose stem cells to trap inside the three-dimensional fibrous network and adhere to the fibers. The disc attached with the hADSCs was transferred to a cell culture dish with KBM ADSC-1 medium (Cat# 16030020, Kohjin Bio, Saitama, Japan) and incubated at 37 °C in a humidified atmosphere with 5% CO2 on air for 5–7 days. When the disc attained around 80% confluence, it was washed in serum free media followed in sterile PBS and used for surgical implantation on the dorsal surface of rat liver.

Animals and experimental procedures

Male albino rats of Wistar strain at the age of around 2 months and weighing between 200–230 g were used for the experiments. The animals were procured from Japan SLC Corporation (SLC, Hamamatsu, Shizuoka, Japan). They were housed in temperature and humidity controlled animal house under 12-h light/12-h dark cycles with commercial rat feed pellets and water available ad libitum. All the animal experiments were carried out following the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 86-23, revised 1996). The animal experimental protocol was also approved by the Animal Care and Research Committee of Kanazawa Medical University on the ethics for the care and use of laboratory animals (#2017-89).

Fifteen rats were injected with CCl4 (Wako, Tokyo, Japan) at a dose of 0.1 ml/100 g body weight (diluted to 50% in olive oil) intraperitoneally twice a week for 8 weeks. Another 5 rats received similar injections without CCl4 and served as control. At the end of 8 weeks, 5 rats from the CCl4 group were euthanized to evaluate the development of hepatic fibrosis and early cirrhosis. The remaining 10 rats were randomly divided into two groups of 5 rats each. Under isoflurane anesthesia, the abdomen was shaved and disinfected with 70% ethanol. An upper abdominal incision was made and a 2 cm diameter disc with more than 80% confluence of hADSCs (hADSC group) or a disc without hADSCs (hADSC control group) was implanted on the surface of right lateral lobe of the liver and the laparotomy was closed with suture. All the animals in both hADSC group and hADSC control group were injected with the same dose of CCl4 once a week for another 8 weeks to maintain liver fibrosis. The control group received similar dosage of olive oil. At the end of 16th week from the beginning of exposure, all the animals were anaesthetized with isoflurane and blood was collected from right jugular vein. The liver was rapidly removed and 3 mm thick tissue was cut from both right and left lateral lobes and instantly fixed in 10% phosphate-buffered formalin for histopathological studies.

Measurement of AST, ALT, total protein, and albumin in serum

Blood was allowed to clot at 37 °C and serum was separated in the conventional method. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, and albumin levels in the serum were measured using an auto-analyzer for animal samples. AST and ALT values are presented as International Units per liter (IU/liter) of serum. Total protein and albumin were depicted as g/100 ml serum.

Histopathological evaluation of liver tissue

The formalin-fixed liver tissues were processed in an automatic tissue processor optimized for liver tissue, embedded in paraffin blocks, and cut into sections of 5-μm thickness. In hADSC group, the liver tissue was processed from right lateral lobe with disc and left lateral lobe without disc. The liver sections were stained with Azan trichrome stain for connective tissue as per the standard protocol. The stained sections were examined using an Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan) attached with Olympus DP71 digital camera (Olympus Corporation, Tokyo, Japan) and photographed.

Immunohistochemical staining for TGF-β1, α-SMA, collagen type I, and type III

Immunohistochemical staining was performed for transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), collagen type I, and collagen type III on paraffin liver sections to assess the degree of hepatic fibrosis and cirrhosis. The liver sections were deparaffinized with xylene and alcohol and hydrated to water. Immunohistochemistry was performed employing a broad-spectrum histostain kit (Invitrogen, Carlsbad, CA, USA). After blocking, the liver sections were treated with primary antibodies (Abcam, Chuo-ku, Tokyo, Japan) against TGF-β (Cat# ab92486), α-SMA (Cat# ab5694), type I collagen (Cat# ab34710), and type III collagen (Cat# ab23445) separately and incubated in a moisturized slide chamber (Evergreen Scientific, Los Angeles, CA, USA) at 4 °C overnight. The sections were then washed 3–5 times in cold phosphate-buffered saline and incubated with broad-spectrum biotinylated secondary antibody for 2 h at room temperature. The slides were washed again and treated with streptavidin-peroxidase conjugate and incubated for another 1 h at room temperature. The final stain was developed by using 3% 3-amino-9-ethylcarbazole (AEC) in N, N-dimethylformamide. The stained sections were washed and counterstained with Mayer’s hematoxylin for 2 min and mounted by using aqueous-based mounting medium. The slides were allowed to dry and examined under a microscope (Olympus BX53, Tokyo, Japan) attached with a digital camera (Olympus DP71) and photographed. The staining intensity in 10 randomly selected microscopic fields was quantified by using Image-Pro Discovery software (Media Cybernetics, Silver Spring, MD, USA).

Staining for hADSC markers Lamin B1 and CD73

In order to confirm the presence of characteristic marker proteins of hADSCs at 8 weeks after implantation, immunohistochemical staining was performed for human Lamin B1 as a marker for human origin employing Lamin B1 antibody (Cat# HS-404013, Synaptic Systems, Goettingen, Germany) and CD73 as a marker for stem cell using CD73 antibody (Cat# 12231-1-AP, Proteintech, Rosemont, IL, USA). The staining protocol was essentially same as described above, except that 3,3′-diaminobenzidine (DAB) was used for the final stain development.

Statistical analysis

Arithmetic mean and standard deviation (SD) were calculated for all the data and presented as mean ± SD. All the data were analyzed and compared either by using analysis of variance or Student's t test. A value of P < 0.05 was considered statistically significant.

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