A total of two male rhesus macaques (age, 2 years) with a body weight of 2-3 kg were used as bone marrow and adipose tissue donors. The rhesus macaques were individually caged in an animal room with a 12/12 h light/dark cycle, and provided with commercial monkey chow, sterile water, fresh fruits and vegetables ad libitum. The temperature of the animal room was controlled between 18-26˚C and with humidity from 40 to 70%. Animal studies were approved by the Institutional Animal Care and Use Committee of Kunming University of Science and Technology (approval number: LPBR20170201) and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (25).

BM-MSCs were isolated from the tibias of young rhesus macaques using the procedures described in detail in a previous study (9). AT-MSCs were isolated from the mesenteric adipose tissue of young rhesus macaques. Briefly, the adipose tissues were washed with 75% alcohol and PBS, cut into small pieces(~0.5x0.5 cm) and placed into 10-cm plastic dishes containing Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific) and 1% (v/v) penicillin/streptomycin (Gibco; Thermo Fisher Scientific) in sterile conditions. The adipose tissues were cultured in an incubator at 37˚C with a humidified atmosphere of 5% CO₂, which was the same as the BM-MSC culture conditions. The medium was refreshed every 48 h. After 10 days, the primary cell culture was passaged at 80% confluence with 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.).

BM-MSCs and AT-MSCs were resuspended in culture medium at a dilution ratio of 1:3 and expanded on a new plastic petri dish. The morphology, surface markers and differentiation potency of the MSCs were determined at passage 3.

The procedures were described in detail in our previous study (9). Briefly, 5x10⁵ MSCs were collected, washed and centrifuged (500 x g; 5 min; room temperature) in 500 µl PBS containing 3% FBS (PBSF). MSCs were then resuspended in 100 µl PBSF and incubated with 5 µl (10 µg/µl) antibody markers (Human MSC Analysis kit; cat. no. 562245; BD Biosciences) on ice for 30 min, washed and resuspended in 500 µl PBSF, and examined using flow cytometry (C6; BD Biosciences) and analyzed using using the built-in software.

These procedures were described in detail in our previous study (9). Briefly, for adipogenic differentiation, BM-MSCs and AT-MSCs were seeded into 24-well plates (8x10⁴ cells per well), cultured for 12 h and treated with adipogenic differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) for 7 days; the medium was refreshed every 3 days. Adherent cells were stained red with 60% Oil Red O (Sigma-Aldrich; Merck KGaA) for 1 min at room temperature. For osteogenic differentiation, BM-MSCs and AT-MSCs were seeded into 24-well plates (4x10⁴ cells per well), cultured for 12 h and treated with osteogenic differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) for 21 days; the medium was refreshed every 3 days. Osteogenic differentiation was confirmed by 0.2% Alizarin Red staining for 5 min at room temperature. For chondrogenic differentiation, 2x10⁵ MSCs were collected in 15-ml centrifuge tubes and cultured with chondrogenic differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) for 21 days; the medium was refreshed every 3 days. The chondroid pellets were sectioned (8 µm) with a freezing microtome. The slices were stained with 1% toluidine blue for 3 min at room temperature and were captured using a light microscope (Olympus Corporation) at a x50 magnification.

For hepatogenic differentiation, the commonly used three-step protocol was applied, which includes serum-free culture, differentiation and maturation steps (26). Firstly, passage 3 cells were seeded on a 6-well plastic plate at a density of 2x10⁴/cm² and cultured in serum-deprived medium containing 20 ng/ml epidermal growth factor (EGF; PeproTech EC Ltd.) and 10 ng/ml basic fibroblast growth factor (bFGF; PeproTech EC Ltd.) for 2 days. Hepatogenic differentiation was sustained for 7 days, and the cells were cultured in differentiation medium consisting of DMEM supplemented with 10% FBS, 10 ng/ml bFGF, 0.6 mg/ml nicotinamide (PeproTech EC Ltd.) and 20 ng/ml hepatocyte growth factor (HGF; PeproTech EC Ltd.). At the maturation step, the cells were cultured in maturation medium consisting of DMEM supplemented with 10% FBS, 20 ng/ml oncostatin M (PeproTech EC Ltd.), 1 µM dexamethasone (Beijing Solarbio Science & Technology Co., Ltd) and 50 µg/ml insulin-transferrin-selenium (ITS; PeproTech EC Ltd.) for 2 weeks. The culture medium was changed every 3 days.

Whole liver was obtained from a 2-year-old rhesus macaque following euthanasia with intravenous sodium pentobarbital at 100 mg/kg body weight, according to the Guide for the Care and Use of Laboratory Animals Primary monkey hepatocytes were isolated using a modified multipoint puncture perfusion technique (27). Briefly, the liver tissue samples were flushed with 38˚C perfusion buffer (NaCl, 9.3 g/l; KCl, 0.5 g/l; HEPES, 2.4 g/l; EGTA, 0.95 g/l) via repetitive multipoint puncture with a 10-ml sterile syringe until the liver changed to an off-white color and the perfusion buffer turned clear. The tissue was then continuously perfused with a prewarmed digestion buffer solution (perfusion buffer with 0.05% collagenase IV). After sufficient digestion, the liver tissue was mechanically disrupted using ophthalmic scissors and an operating knife. The chopped tissue was suspended in DMEM (containing 10% FBS and 1% penicillin/streptomycin) and repeatedly pipetted up and down prior to gentle shaking. The hepatocyte suspension was divided into equal aliquots, which were then filtered through a 500-µm strainer and centrifuged at 50 x g for 2 min at 4˚C. The suspension was removed, added to red blood cell lysis buffer (Beyotime Institute of Biotechnology) and incubated for 5 min at room temperature before centrifugation at 50 x g for 2 min at 4˚C. Sedimentary cells were washed with DNaseI solution (Beyotime Institute of Biotechnology) and filtered through a 250-µm nylon membrane, and the cells were harvested by low-speed centrifugation at 50 x g for 5 min at 4˚C. Finally, the hepatocytes were seeded on 6-well plates at a density of 2x10⁴ cells/cm² and cultured with high-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C and 5% CO₂. Due to their inability to proliferate, hepatocytes were cultured for 5 days before testing in the present study.

PAS staining is a typical technique used to detect hepatocyte function (28). The functional features of HLCs were assessed for glycogen deposition using a PAS staining kit (Beijing Leagene Biotech Co., Ltd). The cell culture medium was removed from the plates, and the cells were rinsed with PBS three times. Subsequently, the cells were fixed in methyl alcohol (99% purity) for 10 min at room temperature. After being washed three more times with PBS, the cells were oxidized for 15 min with 1% periodic acid and washed three times with deionized water, then stained with Schiff's reagent at room temperature for 20 min. After being washed three times with PBS, the cells were stained with Mayer's hematoxylin (room temperature) for 1 min. Finally, the dye was washed off with PBS prior to further evaluation under a light microscope (Olympus Corporation) at a magnification of x50.

The urea production test is one of the most widely used methods to detect the function of hepatocytes (28). The culture media from MSCs and HLCs were changed every 3 days normally and collected 3-day culture before the timepoints (0, 9, 16 and 23 days), and the culture media from hepatocytes were changed at 2 days and collected after a 3-day culture. All culture media were assessed for urea production using a urease method kit (Beijing Leagene Biotech Co., Ltd.; cat.no: TC1165), according to the manufacturer's protocol. Briefly, urease working solution, phenol coloring solution and urea test solution were mixed with standard urea and with the samples, which were measured at 540 nm with a 96-well microplate reader following incubation at 37˚C for 30 min. Subsequently, the concentrations of the samples were calculated. Three independent urea samples were assessed at each time point and each assay was repeated three times.

Total RNA was extracted from monkey hepatocytes and HLCs differentiated from monkey BM-MSCs and AT-MSCs using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The precipitated RNA was solubilized in sterile diethylpyrocarbonate-treated water (Sangon Biotech), and cDNA was then synthesized using a Prime-Script RT reagent kit (Takara Biotechnology Co., Ltd) at 37˚C for 5 min and 85˚C for 5 sec. Quantification of specific genes was performed using SYBR^® Premix Ex Taq^TM II kit (Takara Biotechnology Co., Ltd) and CXF real-time PCR system (Bio-Rad Laboratories, Inc.) at particular condition (Stage 1: 95˚C for 30 sec; stage 2: 95˚C for 3 sec; 60˚C for 30 sec and repeat for 40 cycles). All experiments were performed in triplicate, and the data were analyzed using the 2^-∆∆Cq method (29). The gene-specific primers were commercially synthesized (Sangon Biotech), and sequence information is shown in Table I.

All data are presented as the mean ± SD. Statistical significance among groups was analyzed using repeated measures ANOVA with a post-hoc paired t-test with Bonferroni's correction. Statistical significance within groups at time-point was analyzed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference. Statistically analyze and histograms were generated using GraphPad Prism 5 (GraphPad Software, Inc.).

BM-MSCs and AT-MSCs were obtained from bone marrow and adipose tissue. To evaluate whether the expanded cells were genuine MSCs, the characterization of BM-MSCs and AT-MSCs at passage 3 was performed. Both cell types were compared according to morphology, immunophenotyping profiles and trilineage differentiation potential. During primary culture, BM-MSCs and AT-MSCs adhered to the plastic dishes in a scattered manner and exhibited similar morphology to each other. Cells appeared fibroblast-like, elongated and spindle-shaped with a single nucleus (Fig. 1A).

The BM-MSCs and AT-MSCs could differentiate into adipocytes (Fig. 1B), osteocytes (Fig. 1C) and chondrocytes in vitro (Fig. 1D). After the induction of adipogenic differentiation, numerous neutral lipid droplets stained with Oil Red O were observed in the cytoplasm of BM-MSCs and AT-MSCs. After the induction of osteogenic differentiation, the cells presented an aggregation of micronodules or calcium deposits that were stained by Alizarin Red. The chondrogenic differentiation of both types of MSCs was observed using an Alcian Blue stain.

The immunophenotyping profiles of BM-MSCs and AT-MSCs were analyzed by flow cytometry. The results revealed that both BM-MSCs and AT-MSCs expressed high levels of the positive markers CD44, CD90, CD73 and CD105, but did not express the negative markers CD45, CD34, CD11b, CD19 and human leukocyte antigen-DR (HLA-DR; Fig. 2A and B). No differences were observed between BM-MSCs and AT-MSCs using a t-test (Fig. 2C).

During the differentiation of BM-MSCs and AT-MSCs into HLCs, BM-MSCs (Fig. 3A) gradually changed from spindle and fibroblast-like cells to round or polygonal epithelioid cells. These changes were also observed in AT-MSCs (Fig. 3C). At day 0, before hepatogenic differentiation induction, BM-MSCs and AT-MSCs exhibited similar fibroblast-like morphology. However, a greater number of flattened and polygonal cells were observed in the BM-MSC group than in the AT-MSC group on day 9. On days 16 and 23, the induced MSCs showed epithelioid and cuboidal shapes, which were similar to the morphology of the primary hepatocytes of the control group (Fig. 3E). In addition, the presence of deposited glycogen was determined by PAS staining, to further characterize the glycogen deposition function of HLCs differentiated from BM-MSCs and AT-MSCs. After 23 days of hepatogenic differentiation induction, magenta-stained glycogen was detected in the differentiated cells but not in the undifferentiated cells. The PAS intensity of HLCs differentiated from BM-MSCs (Fig. 3B) was higher than that of HLCs differentiated from AT-MSCs (Fig. 3D) on days 16 and 23. The level of staining was similar to the PAS staining of the hepatocytes derived from liver tissue that had been cultured for 5 days (Fig. 3F). These results suggested that the morphology of HLCs differentiated from BM-MSCs and AT-MSCs was similar to primary hepatocytes. Moreover, the HLCs differentiated from both BM-MSCs and AT-MSCs exhibited the hepatic function of glycogen deposition and could be stained with PAS.

Urea assays detected the urea secretion function of HLCs differentiated from BM-MSCs and AT-MSCs at various time points (days 0, 9, 16 and 23), while urea secretion in the culture medium of hepatocytes derived from liver tissue cultured for 3 days was tested as a control. The urea production of the BM-MSCs and AT-MSCs gradually increased over the culture time (days 9, 16 and 23) during the HLC differentiation process compared to the undifferentiated BM-MSCs and AT-MSCs (Fig. 4), and the urea secretion function of differentiated hepatocytes from BM-MSCs was superior to that of AT-MSCs (P<0.05). These results showed that HLCs differentiated from rhesus macaque BM-MSCs and AT-MSCs possessed the hepatic function of urea secretion.

To further investigate the differentiation potential and hepatocyte function of BM-MSCs and AT-MSCs differentiated into HLCs, the mRNA expression levels of the hepatocyte markers, albumin (ALB; Fig. 5A), keratin 18 (CK-18; Fig. 5B), hepatocyte nuclear factor-4α (HNF-4α; Fig. 5C), cytochrome P450 family 7 subfamily A member 1 (CYP7A1; Fig. 5D) and cytochrome P450 family 3 subfamily A member 4 (CYP3A4; Fig. 5F), in BM-MSCs and AT-MSCs during the HLC differentiation process were evaluated by RT-qPCR analysis. In these assays, hepatocytes derived from liver tissue cultured for 5 days were used as a positive control. During the HLC differentiation process, the expression of these differentiated hepatocyte makers in BM-MSCs and AT-MSCs increased from day 0 to day 23. In the process of BM-MSCs differentiation into HLCs, the expression of ALB, CYP7A1 and CYP3A4 was significantly different on days 9-23 compared with expression at day 0. CK-18 and HNF-4α expression was significantly different on days 16 and 23 compared with at day 0. In the process of AT-MSC differentiation, the expression of ALB was significantly different on days 9-23 compared with that on day 0; HNF-4α, CYP7A1 and CYP3A4 expression was significantly different on days 16 and 23, and CK-18 expression was significantly different on day 23 (Fig. 5).