PCMs and BMMSCs were isolated according to previous reports (5,10) under the approval of the Research Ethics Committee of the School of Medicine, Ningbo University (Ningbo, China) and adhered to the Declaration of Helsinki for the use of animals in research. Briefly, male Sprague Dawley rats (n=5; age, 6–8 weeks; weight, 200–250 g) were purchased from the School of Medicine, Ningbo University, and housed separately at 20–25°C under a 12-h light/dark cycle and fed ad libitum with a normal diet. Rats were sacrificed by cervical dislocation and their eyes were harvested in oxygenated artificial cerebral spinal fluid (124 mM NaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃ and 10 mM D-glucose). The whole ciliary margin was isolated and treated with dispase for 10 min at 37°C. Tissue was subsequently dissected and placed in a trypsin solution at 37°C for 10 min. Following this, cells were centrifuged at 150 × g for 5 min at room temperature and the enzyme solution was removed and replaced with serum-free media containing trypsin inhibitor (1 mg/ml ovomucoid; Roche Diagnostics, Basel, Switzerland), for 30 min at room temperature. Dissociated cells were plated in serum-free Dulbecco's Modified Eagle's medium/F12 (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with N-2 MAX (Thermo Fisher Scientific, Waltham, MA, USA) and cultured at 37°C, 5% CO₂ in a humidified atmosphere. PCM sphere colonies were formed following ~1 week of incubation. Meanwhile, the limbs of rats were separated and bone marrow was flushed out by douching with α-minimal essential medium (α-MEM; HyClone; GE Healthcare Life Sciences) into the bone marrow cavity with a sterilized syringe. Aspirates were centrifuged at 150 × g for 5 min at room temperature, resuspended and cultured in α-MEM with 10% fetal bovine serum and 1% penicillin/streptomycin (all obtained from HyClone; GE Healthcare Life Sciences). Media was changed every 3 days and cells from passages 3–5 were used. For co-culture experiments, primary PCMs were pre-cultured in six-well plates at a density of ~3×10⁵ cells/well in serum-free DMEM/F12 + N-2 MAX for 24 h and then ~2.5×10⁵ BMMSCs were added. PCMs and BMMSCs were incubated in low serum (1%) DMEM/F12 + N-2 MAX for further analysis. Following 3 days of incubation, the growth of cells was observed by inverted microscope (Olympus Corporation, Tokyo, Japan).

The cell viability was monitored daily by MTT (MP Biomedicals, LLC, Santa Ana, CA, USA) as previously described (11). Briefly, cells were plated into 96-well plates at a concentration of 1,000 cells/well in 100 µl medium. To assess viability, the medium was exchanged for an MTT working solution (5 mg/ml in cell culture medium) and incubated for 4 h at 37°C. The solution was gently removed and the precipitation was dissolved in 100 µl dimethylsulfoxide (MP Biomedicals, LLC), and measured at 490 nm with a spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). The proliferation curve was plotted as optical density vs. culture time.

To further investigate cell division, the cell cycle was analyzed by flow cytometry, using a BD Accuri™ C6 (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, following 3 days of culture, cells were detached by trypsin and washed with PBS to obtain a single cell suspension. Cells in the suspension were fixed with ice-cold dehydrated ethanol (absolute ethanol: PBS ratio, 2:3) overnight at 4°C. The fixed cells were washed twice with PBS and stained by 100 mg/ml propidium iodide (PI; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 4°C for 30 min. A total of 1×10⁵ cells were collected for each sample and the PI-elicited fluorescence of individual cells was measured by flow cytometry. The amount of cells in the G0/G1 phase, S phase and G2/M phase was determined. Proliferation index was calculated as the percentage of cells in S + G2/M.

Following expansion for 7 days in growth medium, retinal differentiation was performed using induction medium, which contained DMEM/F12 supplemented with N-2 MAX, 1% FBS, 10 ng/ml fibroblast growth factor 2 (Prospec-Tany Technogene, Ltd. East Brunswick, NJ, USA) and 2 µg/ml heparin (Hebei Changshan Biochemical Pharmaceutical Co., Ltd., Hebei, China). Cells were cultured at 37°C and 5% CO₂ in a humidified atmosphere. Following 21 days of induction, cells were rinsed with PBS and total RNA was extracted by TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1 µg) was reverse transcribed into cDNA by using the qScript cDNA SuperMix kit (Quanta Biosciences, Beverly, MA, USA) according to the manufacturer's protocol. This was performed on Bio-Rad CFX (Bio-Rad Laboratories, Inc., Hercules, CA. USA) with Platinum^® SYBR^® Green qPCR SuperMix-UDG (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The relative mRNA levels were calculated based on housekeeping gene β-actin and normalized to BMMSCs according to their ∆∆Cq values (12). The following primers were used: Crx, 5′-TCACTATT-3′ (forward) and 5′-CCTCACGTGCATACACATCC-3′ (reverse); and β-actin, 5′-TGGCACCCAGCACAATGAA-3′ (forward) and 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′ (reverse). Primers were obtained from Takara Biotechnology Co., Ltd. (Dalian, China).

Total protein was extracted with radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.) containing 1:100 protease inhibitor cocktail. The protein concentration in the extracted lysates was measured using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Haimen, China). Proteins (50 µg per sample) were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc.). Following blocking with 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) for 1 h at room temperature, the membranes were incubated with primary antibodies against Crx (1:500; sc-30150; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (1:1,000; NB100-56875; Novus Biologicals, Ltd., Cambridge, UK) overnight at 4°C. The immune complexes were incubated for 30 min at room temperature with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2,000; BA1055; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Detection was performed using the Western-Light Chemiluminescent Detection system (Bio-Rad Laboratories, Inc.). Blots were analyzed by ImageJ 1.48v (https://imagej.nih.gov/ij/) and band gray scale was calculated. Intensity was calculated relative to GAPDH.

The PCMs and BMMSCs were either separately cultured or co-cultured on coverslips for 7 days in growth medium, followed by differentiation in induction medium for 21 days, as aforementioned. Cells were rinsed in PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were permeabilized by 0.5% Triton X-100 (in PBS) for 20 min at room temperature and then blocked in 5% normal goat serum (NBP2-23475; Novus Biologicals, Ltd.) for 30 min at room temperature. Primary rat monoclonal antibodies, including anti-rod photoreceptor rhodopsin (1:200; NBP2-25159; Novus Biologicals, Ltd.), anti-bipolar neurons visual system homebox 2 (CHX10; 1:500; NBP1-84476; Novus Biologicals, Ltd.) and anti-Müller glia heparin sulfate (1:300; MAB2040; EMD Millipore, Billerica, MA, USA) were diluted and incubated with cells overnight at 4°C. The secondary antibodies (fluorescein isothiocyanate-conjugated) were diluted 1:32 (BA1101 and BA1105; Wuhan Boster Biological Technology, Ltd.) and incubated for 30 min at room temperature in the dark. Following washing thoroughly with PBS, the coverslip was observed by an inverted fluorescence microscope (Olympus Corporation). The positive rate was determined as the percentage of green cells by ImageJ 1.48v (https://imagej.nih.gov/ij/).

All experiments were performed in triplicate and data was presented as the mean ± standard deviation. One-way analysis of variance followed by the Student-Newman-Keuls test was performed using GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA) to compare different groups. P<0.05 was considered to indicate a statistically significant difference.

PCMs formed numerous colonies under inverted microscope observation (Fig. 1A). BMMSCs exhibited a conventional spindle-like shape (Fig. 1B). When co-cultured together, numerous elongated cells were observed and the cell margin was blurred (Fig. 1C).

Generally, all groups exhibited a continuous increase in the optical density with the incubation time (Fig. 2). PCMs had the lowest cell viability throughout the 7 days, while BMMSCs had the highest (Fig. 2). PCM levels were statistically lower compared with the other 2 groups apart from on the first 2 days (P<0.05; Fig. 2). Co-cultured cells had optical density values marginally lower compared with BMMSCs, however, this was not significant (P>0.05; Fig. 2).

Cell cycle analysis was performed 3 days post-cell culture. The number of cells at G1 phase in BMMSCs was higher compared with PCMs (Fig. 3A and B). In co-cultured cells, G1 phase number was between that of the PCM and BMMSC groups (Fig. 3C). The proliferation index of PCMs was significantly lower compared with BMMSC and BMMSC + PCM groups (P<0.05; Fig. 3D), and no statistical difference was observed between BMMSC and BMMSC + PCM groups (P>0.05; Fig. 3D).

The gene expression of photoreceptor-specific homeobox gene, Crx, was measured by RT-qPCR after induction for 21 days. The PCM group expressed significantly higher levels (~10 fold higher) of Crx compared with the BMMSC group (P<0.05; Fig. 4A). Notably, Crx expression in co-cultured cells was significantly upregulated (~2 fold higher) compared with the PCM group (P<0.05; Fig. 4A). Western blot analysis revealed similar results; Crx protein expression in PCM was significantly higher compared with the BMMSC group and expression in co-cultured cells was significantly higher compared with the PCM group (P<0.05; Fig. 4B).

To further confirm the retina differentiation, RSC-associated markers were stained. All markers that were investigated were expressed in the observed cells with different positive ratios (Fig. 5A). Specifically, as illustrated in Fig. 5B, the percentage of rhodopsin-positive cells in the groups were 5.63±3.52% (BMMSC), 16.80±6.66% (PCM) and 35.50±7.66% (BMMSC + PCM). The percentage of CHX10-positive cells in different groups were 8.72±5.58% (BMMSC), 10.12±6.20% (PCM) and 23.60±4.66% (BMMSC + PCM). The percentage of heparin sulfate-positive cells in different groups were 19.30±6.58% (BMMSC), 45.50±9.83% (PCM) and 83.60±10.12% (BMMSC + PCM).