Healthy Kunming mice aged 5-6 weeks (n=15; 3 pregnant and 12 non-pregnant) were purchased from Beijing HuaFuKang Biotechnology Co., LTD (Beijing, China), and maintained on a regular diurnal lighting cycle (12:12 light:dark) with ad libitum access to food and water under specific-pathogen-free conditions at the Central Animal Care Services of Shandong University. After procedure, mice were euthanized by introduction of 100% carbon dioxide into a bedding-free cage initially containing room air with the lid closed at a rate sufficient to induce rapid anesthesia, with death occurring within 2.5 minutes. This study was approved by the Shandong Provincial ENT Hospital (Shandong Provincial Western Hospital) Ethical Committee (Project No: XYK-20200701), and all procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Preparation of retinal stem cells
At the gestational age of 17 days, pregnant Kunming mice (n=3) were euthanized, and embryos were immediately excised. As described previously [17-19], both the retina and ciliary body tissue including the pigmented layer-the ciliary marginal zone (CMZ) were cut into small pieces under a dissecting microscope for RSCs isolation (Fig 1). The resulting tissues were digested using 100 U/mL collagenase (cat. no. C0130, Sigma-Aldrich, Shanghai, China) for 1 h, followed by 0.25 % trypsin (cat. no. 15050057, Invitrogen, Shanghai, China) digestion for 15 min. Single cell was obtained by passing the suspension through use of a stainless-steel filter with 50 µm porous. Subsequently, the cells were transferred to a 25 mL culture flask at an inoculum density of 5 × 104 /mL in a 1:1 nutrient mixture of Dulbecco’s modified Eagle’s medium (DMEM) and F-12 (cat. no. 11330057, Gibco, Gaithersburg, MD, USA), supplemented with 1% B-27 (cat. no. 17504044, Gibco) and 20 ng/ml bFGF (cat. no. PHG0368, Gibco). Cells were then cultured at 37°C under 5% CO2. After 8 days of the culture, the primary suspensions of the cellular clusters were passaged in a 1:2 ratio following 1 h collagenase digestion and disruption as a result of being forced through a sterile syringe for 5 times. The process was repeated every 5–7 days. The sixth-generation cells were identified for RSCs using indirect immunofluorescence staining for Nestin and Pax6.
5-Bromo-2’-deoxyuridine (BrdU) labeling of RSCs
RSCs from the sixth-generation cultures were added to Poly-L-lysine packaged sterilized culture dishes and cultured for 3 days. Thereafter, 200 µg/ml of BrdU solution (cat no. 19-160, Sigma-Aldrich) was added to the cells. The cells were cultured for 2 days in the presence of BrdU. Incorporation of BrdU was assessed using immunohistochemical staining.
Retinal injury model
Twelve male Kunming mice aged 8 months were anesthetized with 2% pentobarbital sodium by intraperitoneal injection. This allowed us to drop tetracaine solution (cat. no. 4512, Sigma Pharmaceuticals, North Liberty, IA, USA) into the left eyes for surface anesthesia, and clamp optic nerve with a micro-artery clamp for 15 s at 2 mm behind the globe. The right eyes without any treatment were used as the controls. Retinal slicing and hematoxylin and eosin (H&E) histological staining was performed 1 week after procedure.
Retinal stem cell transplantation
The standard intravitreal injection for RSCs transplantation was used as described previously . Twelve Kunming male mice with left injured retina were randomly divided into RSCs injection group (n=6) and PBS injection group (n=6) in order to investigate the role of transplanted RSCs. Mice were anesthetized with 2% pentobarbital sodium by intraperitoneal injection, and the limbs and head were fixed well to allow access to the left (operative) eyes. Under a surgical microscope, a 10 µl micro-syringe connected to a 30G needle was inserted into the vitreous cavity from the corneoscleral limbus of the left injured eye in 3 mice, and 2 µl cell suspension ofSCs 3 x 104 cells/µl) from the 6th-generation cultures was carefully injected into the vitreous cavity. All injections were successful, which was verified by without bleeding after observing for 30 seconds. The left injured retina in the other 6 mice were injected with 2 µl of sterile PBS in the same manner, serving as the controls. After two weeks, mice were thereafter euthanized, and eyeballs were removed for immunohistochemical staining. Of note, the operator was unaware of the group allocation and disclosed it until results were obtained and analyzed.
Electroretinogram (ERG) was performed to assess the electrical activity of the retina. The German RETLport visual physiological detection system was used for ERG detection. The recording electrode was a self-made acupuncture needle electrode, placed on the limbus, and the reference electrode and ground electrode were placed under the skin of the cheek and tail, respectively. Before the test, the mouse was placed in a dark room for half an hour to adapt to the dark. After the dark adaptation, the pentobarbital sodium was anesthetized intraperitoneally, the cheeks and tail hair were shaved, and the experiment was performed in an electrically shielded dark room. The passband was set at 5 - 30 Hz, the impedance between the recording electrode and the reference electrode was <5KΩ; the sampling time was 250 ms, and the waveform was superimposed and averaged for 200 times.
H&E staining was performed on the sections of paraffin-embedded tissue according to the standard methods.
The sections of paraffin-embedded tissue were initially placed into a temperature-controlled chamber at 60°C for 30 min, followed by 10 min-deparaffinization in xylene and a series of gradient ethanol solutions: 100% ethanol for 1 min, 95 % for 1 min, and 70 % for 1 min. After hydration in distilled water for 2 min, the slices were immersed into 3% hydrogen peroxide for 15 min at room temperature to block endogenous peroxidase activity. For antigen retrieval, the slices were immersed into 2 mol/L HCl for 1 h and then dried at room temperature, followed by 0.1 mol/L NaOH for 2 s and rinse with PBS for 2 min. This step was repeated three times.
Subsequently, sections were stained with mouse anti-BrdU antibody (1:100, cat. no. ab8152, Abcam, Cambridge, United Kingdom) overnight at 4 °C. After rinsed three times with PBS, sections were incubated with sheep anti-mouse IgG solution (1:50, cat. no. ab6710, Abcam) at room temperature for 30 min. 3,3 diaminobenzidine (DAB) coloration was performed under microscope, and hematoxylin was used to stain the nuclei for 3 min. The specimens were then rinsed with water, dehydrated, and sealed for observation and imaging. Representative images were taken with optical microscope (Leica DM2500, Will and International Trade Co., LTD, Hong Kong).
Indirect immunofluorescence staining
Indirect immunofluorescence staining of Nestin and Pax6 were performed on cold acetone-fixed frozen tissue sections. After 3 washes with PBS, the sections were blocked in 10% goat serum for 1 h to decrease background signal. Rabbit anti-Pax6 (1:100, cat. no. ab195045, Abcam) and mouse anti-Nestin (1:100, cat. no. ab6320, Abcam) were added and incubated overnight at 4 ℃ in a wet box. After 5 washes with PBS, Alex488 and 594 conjugated goat anti-rabbit or mouse IgG (1:2,000, cat. no. A-11034, cat. no. A-11032, Invitrogen) were applied at 37oC for 1 h in the dark room. Notably, the staining when the primary antibodies were omitted and only the secondary antibodies were applied were used as the blank controls. After 5 washes with PBS, DAPI was added and incubated in dark for 2 min. The specimens were then sealed with glycerin and immediately observed and photographed under a fluorescence microscope (BD-YG500, Shenzhen Boshida Optical Instrument Co., Ltd., Shenzhen, China).
Data are shown as mean ± standard deviation. Two tailed t test was performed through use of Graphpad Prism 8.2.1 software (GraphPad Software, Inc., San Diego, CA, USA). P < 0.05 is considered as having significant differences.