In this study, all experiments involving human participants were approved by the institutional review board of the Chinese Academy of Medical Science and Medical School of Shanghai Jiaotong University (Shanghai, China). All subjects provided written informed consent for participation in the study and approved publication of their case details.

This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Shanghai Jiaotong University and the protocol was approved by the Committee on the Ethics of Animal Experiments of Shanghai Jiaotong University. All surgical procedures on animals were performed under sodium pentobarbital anesthesia (P3767; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and all efforts were made to minimize suffering.

With parental consent, human umbilical cords were aseptically obtained from 12 full-term newborn male infants delivered by cesarean section at Shanghai Jiaotong University School of Medicine affiliated with Renji Hospital. Umbilical cords were preserved in cold low glucose Dulbecco's modified Eagle's medium (DMEM-LG; Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA), and cellular isolation began within 3 h after delivery. HUMSC isolation was performed using the tissue block culture attachment method (18). The blood vessels were removed, and the cord was sheared into 2-3-mm³ pieces, which were transferred to petri dishes and cultured in a 37°C incubator with 5% CO₂ in DMEM-LG containing 10% fetal bovine serum (FBS; Invitrogen), 1% penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The medium was changed every 2 days after planting and 10 days later, the tissue blocks were removed. When the cells reached 70–80% confluence, they were harvested and cultured at a density of 1×10⁴ cells/cm². Only cells from passages 3–5 were used for cell differentiation. The surface markers of HUMSCs were analyzed by flow cytometry.

Third passage cells were collected and washed twice in phosphate-buffered saline (PBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA). Cells were harvested using 0.25% trypsin (Invitrogen), washed in PBS and incubated for 30 min at 4°C in the dark with the following anti-human antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE) or allophycocyanin (APC): Anti-CD31-PE (FAB3567P), -CD45-PE (FAB1430P), -CD34-PE (FAB72271P) and-CD105-APC (FAB10971A) from R&D Systems, Inc. (Minneapolis, MN, USA), and anti-CD44-FITC (ab27285) and -CD90-FITC (ab11155) from Abcam (Cambridge, UK). PE-conjugated IgG1 (IC002P; R&D Systems, Inc.) and FITC-conjugated IgG1 (sc-2078; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used as isotype controls. After washing the cells with PBS, the expression of the markers on the cells was analyzed using flow cytometry, as previously described (16).

The osteogenic, adipogenic and chondrogenic differentiation capacity of isolated HUMSCs were assessed using third passage MSCs.

For osteogenic differentiation, cells were plated at a density of 2×10⁴ cells/cm² and cultured in DMEM-LG supplemented with 10% FBS until reaching 80–90% confluence. The DMEM-LG was then removed and Human Umbilical Cord MSC Osteogenic Differentiation Medium (Cyagen Biosciences, Inc., Santa Clara, CA, USA) was added and cells were cultured for 28 days. To detect osteogenesis, Alizarin Red S staining (Cyagen Biosciences, Inc.) was performed after 28 days of culture. The cells were then washed with PBS and fixed with 4% formaldehyde solution for 30 min. The wells were rinsed twice with 1X PBS and cells were then stained with Alizarin Red working solution for 3–5 min.

For adipogenic differentiation, cells were plated at a density of 2×10⁴ cells/cm² and cultured in DMEM-LG supplemented with 10% FBS until reaching 100–120% confluence. After the DMEM-LG was removed, Human Umbilical Cord MSC Adipogenic Differentiation Medium A and Medium B (Cyagen Biosciences, Inc.) were used to culture cells successively for 28 days. Cells were processed for Oil Red O staining (Cyagen Biosciences, Inc.) to detect adipogenesis. The staining process was similar to that used for detection of osteogenesis.

For chondrogenic differentiation, 2.5×10⁵ cells were seeded into 15 ml polypropylene culture tubes. The cells were centrifuged at 150 × g for 5 min at room temperature. The caps of the tubes were loosened by one half turn to allow gas exchange, and the tubes were incubated at 37°C, in a humidified atmosphere of 5% CO₂. After 24 h, freshly prepared Complete Chondrogenic Medium (Cyagen Biosciences, Inc.) was to each tube. After 28 days culture, the chondrogenic pellets were harvested, formalin fixed and paraffin embedded for Alcian blue staining (Cyagen Biosciences, Inc.).

Testes were obtained from 8-week-old mice, which were sacrificed by cervical dislocation. Leydig cells were isolated as described previously (19) digested by combining testes with 0.03% collagenase NB4 (SERVA Electrophoresis GmbH, Heidelberg, Germany) in a centrifuge tube. The supernatant was collected after digestion for 15 min in a 37°C shaker at 150 rpm. Cells in the supernatant were centrifuged and cultured in DMEM/F12 (Invitrogen) with 10% FBS. After 4 days, cells were harvested with 0.25% trypsin treatment. First passage cells were preserved at −80°C. The surface makers of Leydig cells were detected by immunofluorescence staining (20).

Second passage Leydig cells were plated on a Petri dish at a density of 1×10⁴ cells/cm². Leydig cells were cultured in DMEM/F12 containing 10% FBS, 1% penicillin and streptomycin. The medium was collected daily and replaced with fresh medium every 3 days. The medium was centrifuged at 500 × g for 5 min, and the supernatant was filtered with a 0.22 µm filter. The filtered medium and normal DMEM/F12 medium constitute the LC-CM, which was prepared by diluting the collected conditioned medium with an equal volume of DMEM/F12 containing 10% FBS.

DIM (16) was prepared by adding several hormones to phenol red-free DMEM/F12 as follows: 5% FBS, 1 ng/ml luteinizing hormone (LH; American Research Products, Inc., Waltham, MA, USA), 1 nM thyroid hormone (Gibco), 70 ng/ml insulin-like growth factor 1 (IGF-1), 10 ng platelet-derived factor (PDGF)-BB (American Research Products, Inc.) and insulin-transferrin-selenium cell culture supplement containing 5 mg/l insulin, 5 mg/l transferrin and 5 µg/l selenium (17-838Z; Lonza Walkersville, Inc., Walkersville, MD, USA).

For differentiation into steroidogenic cells, three experimental groups of HUMSCs were established and cultured initially at 2,000–4,000 cells/cm² in DMEM-LG with 10% FBS for 24 h. After 24 h, media from the three groups were replaced with LC-CM, DIM or DMEM/F12 with 10% FBS (control group). The medium was changed daily.

After 14 days of exposure to LC-CM or DIM, the HUMSCs were fixed with 4% polyoxymethylene for 20 min at room temperature and permeabilized with 0.25% Triton X-100 (Sigma-Aldrich) and blocked with 0.1% bovine serum albumin (Roche, Basel, Switzerland). Subsequently, cells were incubated overnight at 4°C with the following primary antibodies: Cholesterol side-chain cleavage enzyme CYP17A1 (1:200; ABC392; Merck Millipore, Merck KGaA), CYP11A1 (1:200; ab75497; Abcam) and 3β-hydroxysteroid dehydrogenase (3β-HSD; 1:200; ab154385; Abcam). After washing three times with PBS, the cells were reacted with the appropriate fluorescent dye-conjugated secondary antibody for 30 min at 37°C. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) and visualized by fluorescence microscopy (Leica Camera AG, Wetzlar, Germany). The untreated HUMSCs were used as negative controls. All antibodies were diluted in PBS.

Total cellular RNA was extracted from LC-CM and DIM induced HUMSCs at days 3, 7 and 10 using TRIzol reagent (Invitrogen) according to the manufacturer's recommendations. Phase Lock Gel (Tiangen Biotech Co., Ltd., Beijing, China) was used to remove genomic DNA according to the manufacturer's protocol. The concentration of RNA was measured by using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) and then 900 ng RNA was reversely transcribed using a reverse transcriptase kit (Takara Bio, Inc., Shiga, Japan) for 15 min at 42°C and 5 sec at 85°C. The cDNA was amplified using a PCR kit (Takara Bio, Inc.) with the following primers:: Luteinizing hormone receptor (LHR), forward 5′-GTGGCTGGGACTATGAATATGGTTT-3′ and reverse 5′-CTAGAGTGATGACGGTGAGGGTGTA-3′; Steroidogenic acute regulatory protein (StAR), forward 5′-ATGGGTGGGGTTCGTGTTTAGA-3′ and reverse 5′-TGGGGTGCTGCTTGTTCTGTG-3′; HSD3B2, forward 5′-TCCAAGCCAGTGTGCCAGTCT-3′ and reverse 5′-CTTTGGTGAGGCGTGTCATCTG-3′; CYP17A1, forward 5′-GCAAGAGATAACACAAAGTCAAGGT-3′ and reverse 5′-TGCCCATACGAACCGAATAGAT-3′; CYP11A1, forward 5′-CCAACCCAGAACGATTCCTCAT-3′ and reverse 5′-CACTACTTCCTCCAGCATCCCCT-3′; and β-actin, forward 5′-CGGGAAATCGTGCGTGACAT-3′ and reverse 5′-CGGACTCGTCATACTCCTGCTTG-3′. PCR assays were conducted by denaturation at 95°C for 30 sec followed by 30 cycles of annealing at 58°C for 30 sec and extension at 72°C for 60 sec. Finally, PCR products were separated by 2% agarose gel electrophoresis and observed under ultraviolet illumination. The expected product sizes from these primers were 454, 334, 408, 332, 387 and 481 bp for HSD3B2, CYP17A1, LHR, CYP11A1, StAR and β-actin, respectively.

After 3 weeks of exposure to LC-CM or DIM, cells were washed twice with cold PBS and harvested by trypsinization. For total protein isolation, cells were suspended in cell lysis buffer (Invitrogen) and placed on ice for 30 min. The suspension was collected after centrifugation at 15,000 × g for 30 min at 4°C. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's instruction. Proteins (20 µg) were denatured by boiling at 100°C for 5 min after the addition of a loading buffer (CWbio, Beijing, China). Equivalent quantities of protein were loaded and electrophoresed in 6–15% SDS-PAGE gels. After electrophoresis, gels were transferred to nitrocellulose (NC; Merck Millipore) membranes, and membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature. Membranes were incubated with primary antibodies targeting CYP17A1 (1:1,000; ABC392), CYP11A1 (1:1,000; ab75497), 3β-HSD (1:1,000; ab154385) or β-actin (1:1,000; ab8227; Abcam) overnight at 4°C, then with IRDye 800-conjugated goat anti-rabbit IgG secondary antibody (1:20,000; A50984P; Rockland Immunochemicals, Inc., Pottstown, PA, USA) for 2 h at room temperature. Immunoreactive bands were visualized using a LI-COR Odyssey (LI-COR Biosciences, Lincoln, NE, USA).

After three days in culture, the cells isolated from the human umbilical cord blocks were scarce and scattered, principally exhibiting a spindle-shaped morphology (Fig. 1A). After 10 days, the number of cells increased, and they grew in a parallel arrangement (Fig. 1B). Once passaged, the cells expanded rapidly with no significant changes in morphology (Fig. 1C).

After two passages, the cells exhibited positive expression of the MSC surface markers CD44, CD90 and CD105. However, the cells were negative for hematopoietic lineage markers (CD34, CD45 and CD31). These results indicated that the isolated cells in this study were HUMSCs (Fig. 2A). After 28 days of osteogenic induction, the deposition of calcium of cells could be visualized by Alizarin Red S staining (Fig. 2B). When cells were cultured in adipogenic medium for 28 days, numerous lipid droplets were observed following staining with Oil Red O solution, which used to identify adipocytes (Fig. 2C). Blue staining indicates synthesis of proteoglycans by chondrocytes after 28 days of induction (Fig. 2D).

Leydig cells predominantly exhibited round shapes with large lipid drops and tended to be arranged in clusters (Fig. 3), as previously reported (21). Immunofluorescence staining of Leydig cells for CYP17A1 and 3β-HSD was positive (Fig. 4).

To detect the Leydig cell phenotype in induced HUMSCs, immunofluorescence staining for CYP11A1, CYP17A1 and 3β-HSD was performed on cells from all three groups following 14 days of culture. Positive staining for CYP11A1, CYP17A1 and 3β-HSD was observed in the HUMSCs treated with either LC-CM or DIM. However, no positive staining was observed in the control cells (Fig. 5).

To assess the possibility of Leydig cell differentiation, HUMSCs were evaluated by RT-PCR analysis of specific testosterone-producing genes. After 7 days of culture, the results demonstrated that HUMSCs induced with LC-CM or DIM positively expressed Leydig cell lineage marker genes (Fig. 6).

Next, whether whether Leydig cell lineage enzymes can be expressed after induction was examined. 3β-HSD, CYP11A1 and CYP17A1 were induced 3 weeks after LC-CM or DIM treatment. In the testes, the production of testosterone is dependent on 3β-HSD activity (22). Additionally, 3β-HSD enzymes are essential for the production of progesterone, and CYP17A1 is required for the production of testosterone (23). These results demonstrate that HUMSCs have properties indicating their differentiation into steroidogenic cells, which are similar to those of Leydig cells (Fig. 7).