Combined transplantation of human mesenchymal stem cells and human retinal progenitor cells into the subretinal space of RCS rats

Retinitis pigmentosa (RP) is one of hereditary retinal diseases characterized by the loss of photoreceptors. Cell transplantation has been clinically applied to treat RP patients. Human retinal progenitor cells (HRPCs) and human bone marrow-derived mesenchymal stem cells (HBMSCs) are the two commonly and practically used stem cells for transplantation. Since combined transplantation could be a promising way to integrate the advantages of both stem cell types, we transplanted HRPCs and HBMSCs into the subretinal space (SRS) of Royal College of Surgeons (RCS) rats. We report that HRPCs/HBMSCs combined transplantation maintains the electroretinogram results much better than HRPCs or HBMSCs single transplantations. The thickness of outer nuclear layer also presented a better outcome in the combined transplantation. Importantly, grafted cells in the combination migrated better, both longitudinally and latitudinally, than single transplantation. The photoreceptor differentiation of grafted cells in the retina of RCS rats receiving combined transplantation also showed a higher ratio than single transplantation. Finally, activation of microglia and the gliosis of Müller cells were more effectively suppressed in combined transplantation, indicating better immunomodulatory and anti-gliosis effects. Taken together, combining the transplantation of HRPCs and HBMSCs is a more effective strategy in stem cell-based therapy for retinal degenerative diseases.


Identification of HMSCs.
HBMSCs can express CD44, CD73, CD90 and CD105 simultaneously. Using FACS, we found that the positive percentages of CD44, CD73, CD90 and CD105 in HBMSCs on passage 3 were 99.2%, 98.8%, 96.4% and 95%, respectively (Fig. 2a-d) (see supplementary methods). HBMSCs should also be able to show multi-lineage potential 38 . After being induced in osteogenic conditions for 10-12 days, HBMSCs formed sphere-or olive-matrices (Fig. 2e). With the staining of Alizarin Red S, osteogenic calcium depositions could be visualized (Fig. 2f). An adipocyte-differentiation test showed that HBMSCs were able to form vesicle-like structures. After staining with Oil Red O, lipid vesicles could be visualized (Fig. 2g). To test whether our HBMSCs had cartilage-forming ability, HBMSCs were incubated with chondrogenic medium for 4 weeks. With Alcian Blue staining, cartilage could be detected (Fig. 2h).
To exclude the possibility of contamination with other cells, CD11b, CD19, CD34, CD43 and HLA-DR expression was tested by FACS, with negative results (Fig. 2i). All these results illustrate that the isolated cells from bone marrow were HBMSCs. To perform transplantation surgery, HBMSCs were transfected with lentivirus-GFP. Using immunofluorescence and FACS, approximately 90% of HBMSCs were GFP positive cells (Fig. 2j,k).

ERG recording.
As an electrophysiology-testing method, ERG can reflect the function of the retina, especially the function of photoreceptors by measuring the amplitudes of both a and b waves. The main purpose of transplantation into RCS rats is to measure the functional improvement of injured photoreceptors. Thus, ERGs were recorded on all post-operational rats at 3, 6, 9 and 12 weeks after cell transplantations. The results showed that both combined transplantation or single transplantation displayed a significant improvement of the amplitude of a and b waves at 3, 6 and 9 weeks, compared to those in the untreated group and PBS injection group ( Fig. 3a-t,u,v). In particular, there was no significant difference among single HRPCs transplantation, single HBMSCs transplantation or combined transplantation by 3 weeks (Fig. 3a-e,u,v). However, the amplitude of both a and b waves in the combined transplantation group (a wave: 42.25 ± 4.52, b wave: 114.33 ± 25.23) increased significantly by 6 weeks, compared to those in the single HRPCs transplantation group (a wave: 29.75 ± 3.03, P = 0.000910; b wave: 71.93 ± 13.18, P = 0.006055) and single HBMSCs transplantation group (a wave: 32.78 ± 5.10, P = 0.006987; b wave: 82.55 ± 19.58, P = 0.030260) ( Fig. 3f-j,u,v). By 9 weeks, though decreased, the amplitudes of both a and b waves in the combined transplantation group (a wave: 29.95 ± 4.52; b wave: 79.40 ± 17.74) were significantly higher than that in the single HRPCs transplantation group (a wave: 16.93 ± 3.30, P = 0.000054; b wave: 54.75 ± 12.84, P = 0.013526) and single HBMSCs transplantation group (a wave: 13.00 ± 3.00, P = 0.000003; b wave: 39.25 ± 4.40, P = 0.000378) ( Fig. 3k-o,u,v). However, after 12 weeks, there was no significant difference of ERG readings among single HRPCs transplantation, single HBMSCs transplantation and PBS injection groups. Meanwhile, the amplitudes of a and b waves in the combined transplantation group (a wave: 18.63 ± 2.09; b wave: 38.23 ± 8.20) still showed significantly better effects over the single HRPCs transplantation group (a wave: 4.45 ± 1.41, P = 0.000000; b wave: 15.23 ± 4.29, P = 0.000032) and the single HBMSCs transplantation group (a wave: 4.73 ± 1.79, P = 0.000000; b wave: 11.93 ± 3.23, P = 0.000007) ( Fig. 3p-t,u,v). There showed now significant difference between untreated group and PBS injection group in all time points (Fig. 3a,b,f,g,k,l,p,q,u,v).
ONL thickness analysis. Since retinal function was profoundly improved after combined transplantation, we also investigated the morphological evidence, which supported our findings. RCS rats mainly suffered from loss of photoreceptors. Therefore, the ONL thickness was measured to assay for the protection of the ONL among different types of cell transplantations. The results showed that all cell transplantation groups presented significantly thicker ONLs than the untreated group and PBS-alone injection groups at 3, 6, 9 and 12 weeks post-operation ( Fig. 4a-p,q). The combined transplantation groups displayed a significant increase of ONL thickness (56.82 ± 6.79) over the HRPCs transplantation group (42.02 ± 4.81, P = 0.001122) by 3 weeks post-operation For Ki67 staining, the significance between P1 and P3 is P = 0.004637. The significance between P1 and P5 is P = 0.000349. The significance between P3 and P5 is P = 0.028506. For SOX2 staining, the significance between P1 and P3 is P = 0.010751. The significance between P1 and P5 is P = 0.000505. The significance between P3 and P5 is P = 0.020324. For GFAP staining, the significance between P1 and P5 is P = 0.000635. The significance between P3 and P5 is P = 0.005400. *P < 0.05; **P < 0.01; ***P < 0.001.

Longitudinal and latitudinal migration of transplanted cells.
After HRPCs, HMSCs and their combination were individually transplanted to the SRS of RCS rats, and cells were counted among three groups. Our results showed that combined transplantation presented a significant increase in the number of cells by 6, 9 and 12 weeks post-operation when compared with single transplantation groups (Fig. 5p). This result indicates that cells survived better under our combined transplantation regimen. To assess the change more specifically, the numbers of transmitted cells were measured both longitudinally and latitudinally. During the 3-week to 12-week post-transplantation period, cells gradually migrated from the SRS into the inner retina ( Fig. 5a-l). At 3 weeks post-operation, there was no obvious longitudinal migrations in all 3 groups (Fig. 5a-c). The combined transplantation group started to show significant migration by 6 weeks post-operation ( Fig. 5f,q). However, the HRPCs and HBMSCs single transplantation groups did not display migration until 9 weeks post-operation (Fig. 5d,e,g,h). At that time, the combined transplantation group showed a significant increase of migrated cells in the inner retina (Fig. 5i,q). By 12 weeks, although the number of migrated cells in the combined transplantation group decreased, it still presented significantly high trends compared to the single transplantation groups ( Fig. 5j-l,q). We also selected 6 weeks post-transplantation to measure the latitudinal migration of transplanted cells. Our results showed that rare migration occurred in the HBMSCs transplantation group (Fig. 5n,r). Although cells migrated in the HRPCs transplantation group, the number of migrated cells was significantly lower compared to that in the combined transplantation group (Fig. 5m,o,r). Taken together, our results demonstrated that cells in the combined transplantation group migrated better than in the single transplantation groups.

Photoreceptor differentiation of transplanted cells.
Since the total number of transplanted cells reached the highest level by 6 weeks post-operation (Fig. 5p), the differentiation of transplanted cells was measured at this time point. Using photoreceptor marker recoverin, we found that in the HRPC single transplantation group, there was co-expression of recoverin and h-mitochondria, indicating that the transplanted cells differentiated into photoreceptors (Fig. 6a,a'). However, this differentiation was seldom observed (Fig. 6a,a'). In the HBMSC single transplantation group, there was no co-expression of recoverin and h-mitochondria observed ( Fig. 6b,b'). In contrast, there were many more cells co-expressing recoverin and h-mitochondria in the combined transplantation group compared to those in the HRPC single transplantation group, indicating that transplanted cells differentiated into photoreceptors more often under our combined transplantation regimen (Fig. 6c,c'). To confirm the results, we also used rhodopsin. Our results were consistent with the recoverin staining assays ( Fig. 6d-f,d'-f '). The differences were that more cells co-expressed rhodopsin and h-mitochondria under HRPC single and combined transplantations, indicating that more differentiation occurred.

Inflammatory modulation of transplanted cells. Immunoreactions and inflammation can influ-
ence retinal function in many ways, and microglia cells play an important role 39,40 . We evaluated the number of Iba1-immunoreactive retinal microglia cells in the PBS injection, HRPC transplantation, HBMSC transplantation and HPRC/HBMSC combined transplantation groups. Our results showed that there were no significant differences among these four groups in the cell-grafted areas by 3 post operational weeks (Fig. 7a-d). However, there were significant differences in the para-grafted areas at this time point (Fig. 7e-h). Cell-transplanted groups all displayed significant decreases in microglia cell number. More importantly, the HBMSC transplantation group and the combined transplantation groups showed more effective inflammation suppression compared with the HRPC transplantation group (Fig. 7f-h,q). These differences were not found to be statistically significant, however (Fig. 7g,h,q). To verify this finding, a western blot analysis of Iba1 expression was performed. By 3 post Results showed that all cell transplantation groups displayed increases of the ONL thickness, which indicated the protection of photoreceptor. Combined transplantation showed longest photoreceptor-protection effect. *P < 0.05; **P < 0.01; ***P < 0.001. (n = 3).
operational weeks, our results showed that the Iba1 expression level was significantly decreased in all three groups (Fig. 7r,s). The combined transplantation groups showed significant decreased Iba1 expression levels compared to the HRPC and HBMSC transplantation group (P = 0.016298 and P = 0.000988). The HRPC transplantation group showed decreased Iba1 expression levels compared to the HBMSC transplantation group (Fig. 7r,s). By 6 post operational weeks, no significant differences could be detected among all groups in either the grafted or para-grafted areas (Fig. 7i-p). Western-blot analysis results were consistent with this finding (Fig. 7r,s). In addition, we also assayed the expression levels of inflammatory factors and neurotrophic factors with RT-PCR. Our results showed that all 3 types of cell transplantation decreased TNF and IL-1β expression levels and increased NGF and BDNF expression levels ( Supplementary Fig. 1a-d). These differences were strongest in the cell transplantation groups containing HBMSCs ( Supplementary Fig. 2a-d).

The combined cell transplantation suppressed Müller cells.
To evaluate the gliosis state after cell transplantation, GFAP counter-staining was performed in the untreated, PBS injection, HRPC transplantation, HBMSC transplantation and HPRC/HBMSC combined transplantation groups. There showed no significant difference between untreated group and PBS injection group on all time points (Fig. 8a,b,f,g,k,l,p,q,u,v). After 3 weeks, the cell-transplantation groups presented a more severe gliosis state than PBS injection group and untreated group (Fig. 8a-e,u,v). However, the gliosis state is significantly overturned in all three transplantation groups by 6 weeks post-operation. The gliosis was obviously reduced in all cell-transplantation groups but increased in the PBS injection group and untreated group (Fig. 8f-j,u,v). After 9 weeks, gliosis remained at a high level in the PBS injection group and untreated group and increased moderately in the cell-transplantation groups ( Fig. 8k-o,u,v). As time went on, gliosis in the HBMSC transplantation group increased significantly after 12 weeks, while gliosis in the HRPC and combined transplantation groups remained in a low level (Fig. 8p-t,u,v). The western blot results also confirmed the findings (Supplementary Fig. 3).

Discussion
Retinal degenerative diseases, including RP and age-related macular degeneration, are far from being fully understood and treated. Promising treatments include those focused on gene, drug and stem cell therapies 41 . There are many different types of stem cells that can be selected as candidate cells 42 . In addition, stem cell transplantation can perform a variety of functions including cell replacement, anti-hazardous substances, trophic action, neural plasticity and immune modulation 43 . Taken together, transplantation of stem cells possesses huge potential in treating degenerative disease. However, the characteristics of one particular stem cell type is fixed, which means that the effects of transplantation of a single cell type is limited. In this way, to integrate the different functions of stem cell transplantation, the combined transplantation of two or more types of cells can be implemented [32][33][34][35][36][37]44 . Ranging from mice and rats to cats and chickens, many animal models have been used to mimic the process of RP. However, the RCS rat is the classic naturally occurring inherited retinal degenerative disease model 45 . In the present study, HRPCs and HBMSCs were combined and transplanted into the SRS of RCS rats to show the effects of combined transplantation.
ERG tests showed that the visual function in all groups declined over time. Furthermore, combined transplantation did not present an advantage over single transplantation until 6 weeks post-operation. This effect lasted at least through 12 weeks post-transplantation. Single transplantation of HRPCs and HBMSCs showed no significant differences between each other, illustrating the same rescue effects of these two cells. By 12 weeks post-operation, neither HRPCs nor HBMSCs alone preserved visual function. In other HRPCs single transplantation experiments, ERG testing showed better results at 12 weeks 24 . This might be due to different states and amounts of HRPCs used for transplantation. However, though HRPCs and HBMSCs lost their rescue effect in our experiments, the combined transplantation of both cell types maintained visual function better, indicating the mutual promotion effect of these two cell types. Interestingly, though without achieving statistical significance, HBMSCs single transplantation showed a high to low rescue effect compared with HRPCs single transplantation. This result indicates the different mechanisms in preserving visual function between HRPCs and HBMSCs. In retina transplantation research, the thickness of ONL is commonly used to measure the degree of photoreceptor protection 24,46,47 . Our study showed that the HBMSCs and combined transplantation groups displayed thicker ONL at 3 weeks. However, it decreased more quickly in the HBMSCs group with time. In later time points, 9 and 12 weeks post-operation, the HRPCs group showed better photoreceptor protection than the HBMSCs group did. However, the combined transplantation group still displayed the best protection effect, especially in the late stage of transplantation. This may be due to the complementary effect of HBMSCs and HRPCs, where the former cells create better microenvironments and the latter cells play a major role in repairing the retina.
To best perform their cell replacement function, transplanted cells should migrate into the inner retina. However, in a previous HRPCs SRS transplantation study, a low migration rate was observed 48 . In the present study, increases of both longitudinal and latitudinal migration were discovered under a combined transplantation regimen. Chemokines played a significant role in helping migration of the cells. As the major role of HBMSCs in rescuing retinal damage is to secret factors and improve the microenvironment 29-31 , combined transplantation may actually coordinate the characteristics of these two cells and further facilitate HRPCs migration.
Differentiation of transplanted cells is the most important but difficult sign of cell replacement. In the photoreceptor degeneration model, differentiation of transplanted cells into photoreceptors is an exciting achievement. In single transplantation groups, HRPCs were able to differentiate into recoverin-and rhodopsin-positive photoreceptors while HBMSCs could not, illustrating the different functions of these two cells. These results are similar to what has been shown in a previous study 49 . In our combined transplantation tests, the differentiation of HRPCs increased compared to single transplantation, indicating that HBMSCs helped HRPCs to differentiate. As acquisition of HBMSCs was through a well-established clinical procedure 49 , the combined transplantation of HBMSCs and HRPCs is realistic for clinical application.
Our previous studies showed that activation of microglia may lead to a vicious cycle 39,40 and that SRS transplantation of neural stem cell was able to suppress this activation 20 . In the present study, we found that all SRS transplantations actually suppressed the microglial activation. This effect is only restricted to the para-grafted area by post-operational week 3. Additionally, we showed no significant difference between HBMSCs single transplantation and combined transplantation. Unfortunately, the immune suppressing effect of HRPCs is less than that of HBMSCs. Taken together, HBMSCs played a major role in the immune regulation of SRS transplantation 50 , which might support the vitality of HRPCs. The limited immune suppressing effect might be due to the limited number of transplanted cells or might be an irreversible effect of severe photoreceptor degeneration.
Gliosis is the adaptive response of Müller cells that may further impede the differentiation and integration of transplanted cells 51 . Our study found transplantation of HRPCs and HBMSCs singly or combined was able to decrease the gliosis levels at early stages, approximately 3-9 weeks post-transplantation. However, HBMSCs single transplantation resulted in a reduced gliosis-suppressing effect at 12 weeks post-operation, while HRPCs single and combined transplantation still inhibited gliosis well at this time point, indicating that HRPCs are better than HBMSCs at performing the gliosis-suppressing effect. A previously published study found that the transplantation of HBMSCs into the retina caused gliosis 52 , which could be the reason why GFAP levels increased again in the later stage of HBMSCs transplantation. More importantly, combined transplantation still maintained a gliosis-suppressing state, providing more strong evidence for the benefits of mutual coordination in our combined transplantation protocol.

Materials and Methods
Ethical approval on human tissue. All the experimental procedures throughout the present study involving human tissue were approved by the Human and Animal Research Ethics Committees of the Third Military Medical University. The research adhered to the tenets of the Declaration of Helsinki and written informed consent and surgical consent were obtained from all patients (WHO Trial Registration, ChiCTR-TNRC-08000193). Participants provided their written consent and approved the consent procedure for our studies.
Animal and ethics. The RCS rats were kindly donated by the Becjnab Vision Center and were raised in a specific-pathogen-free room and maintained on a 12-h light/dark cycle. Rats were supplied by the Animal Care Centre of Southwest Hospital. All tissue collection and experimental procedures were performed according to protocols approved by the Institutional Review Board of the Third Military Medical University and conformed to the National Institutes of Health (NIH) guidelines on the ethical use of animals (for more information, please see supplementary ethical statement).
Isolation and culture of HRPCs. HRPCs were harvested from human fetal eyes between 11-13 weeks of gestation. The neuroretinas were separated as previously described 1,53 . Fetal neuroretinas were cut into pieces, rinsed, and digested for 20-30 min with 1 U/ml papain at 37 °C. After a 10-second agitation, the cell suspension was centrifuged at 1000 rpm for 5 min. Any remaining undissociated tissue was processed again. Then, cell pellets were re-suspended in culture medium containing 10 ng/mL epidermal growth factor, 20 ng/mL bFGF, 2 mM L-glutamine, 1% Penicillin/Streptomycin (P/S; Invitrogen), and 5% fetal bovine serum (FBS, Invitrogen). The number of live and dead cells was counted using a trypan blue assay (Sigma-Aldrich). The isolated cells were plated onto fibronectin-coated (100 μg/mL) tissue culture flasks at a density of 9-13 × 10 3 cells/cm 2 and were incubated in a 5% CO 2 saturation-humidity atmosphere in 37 °C. Twenty-four hours after plating, FBS was removed by changing the culture medium. The viability of purified cells was determined using trypan blue staining and only cell preparations with greater than 95% viability in passage 2 were used for transplantation.
Isolation and purification of HMSCs. HMSCs were isolated from the bone marrow aspirates of donors (20-30 years old). The marrow was diluted with phosphate buffered saline (PBS) (pH 7.3) and loaded over a Ficoll solution with a density of 1.073 g/ml. After a 20 min centrifugation at 900 g, nucleated cells were collected and washed with PBS at room temperature 54 . The precipitate was re-suspended in MSC culture medium, containing DMEM/F12, 10% FBS (Hyclone). Cells were cultured up to 5 passages. At passage 3, the identification of HMSCs was tested by FACS.
Cell transplantation to RP animal model. Sub-retinal transplantation was performed as previously described 55 . In brief, HRPCs, HBMSCs or their combination were injected into the SRS of the left eyes of RCS rats at the age of postnatal 3 weeks (12/group). The identical volume of 0.01 M PBS was injected into the SRS of the right eyes of RCS rats (12/group). In the HRPCs and HBMSCs singly transplanted groups, all transplanted cells were labeled with the fluorescent marker CM-DiI (2 mg/ml, Invitrogen). Cell suspensions (5 μl/eye, total 4 × 10 5 cells/eye) were slowly injected into the left temporal SRS. Then, 5 μl 0.01 M PBS was slowly injected into the right temporal SRS. In the HRPCs and HBMSCs combined transplanted group, HRPCs were labeled with CM-DiI and HBMSCs were labeled with GFP via lentivirus infection. Cell suspension mixtures (5 μl/eye, total 4 × 10 5 cells/eye) containing HRPCs (2 × 10 5 cells/eye) and HBMSCs (2 × 10 5 cells/eye) were slowly injected into the left temporal SRS. Then, 5 μl 0.01 M PBS was slowly injected into the right temporal SRS. The pupils were dilated with 1% of tropicamide (Santen Pharmaceutical Co., Ltd. Osaka, Japan) 30 mins before the surgery. A 10 μl Hamilton syringe (30 gauge; Hamilton, Nevada, USA) containing the cell suspension was tangentially inserted into the SRS through the conjunctiva and sclera, causing a self-sealing wound tunnel. The edge of cornea was punctured to reduce intraocular pressure and limit the efflux of cells at the injection site. Fundus examinations were performed via direct ophthalmoscope viewing after the operations. ERG Recording. ERG was performed on 3, 6, 9 and 12 post-operation weeks to evaluate the retinal function changes as we previously described. In brief, RCS rats were dark adapted for at least 12 hours prior to conduct ERG 56 . Anesthetization were performed by intraperitoneal injection a solution of ketamine (120 mg/kg) and xylazine (20 mg/kg). Pupils were dilated using 1% of tropicamide. Body temperature of animals was maintained at 37 °C by a bottom heating pad to prevent hypothermia. Two active gold electrodes were placed on each cornea as the recording electrodes. The reference and ground electrodes were placed subcutaneously in the mid-frontal area of head and the tail, respectively. Light stimulations were delivered with a xenon lamp at 3.0 cd·s/m 2 . The amplitudes of a-and b-waves were recorded and processed by the RETI-Port device (Roland consult, Brandenburg, Germany). All the procedures were performed in a dark room with dim red safety light.
Quantitative RT-PCR. The retinas of RCS and rdy rats were collected at postnatal week 3 to evaluate mRNA levels using quantitative RT-PCR (qRT-PCR). Total mRNA was extracted using the RNeasy Mini kit (Qiagen, ML, USA) and cDNA synthesis was conducted using SuperScript III First-Strand Synthesis SuperMix (Invitrogen, CA, USA). The procedures of mRNA extraction and cDNA synthesis were performed according to manufacturer's instructions. Primers are listed in Supplementary Table. Reactions were performed in a 25 μl Eppendorf tube with 2 μl of cDNA, 0.3 μl of forward and reverse primer (10 pmol/μl), 10 μl of 2× Mix (full velocity SYBR green qPCR master mix, Stratagene) Taq and 7.4 μl of ddH 2 O. The procedure for real-time qRT-PCR included 4 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C (Roche LC480, Roche Applied Science). All of the qRT-PCR reactions were performed in triplicate for yielding averaged Ct values. Expression (evaluated as fold change for each target gene) was normalized to GAPDH in microglial cells following the well-established Δ-Δ method 58,59 . Data were presented as fold change over the control. A non-template control was included in the experiment to estimate DNA contamination of isolated mRNA and reagents.
Western blot. Animals were euthanized with CO2 at postoperative weeks 6 and 12. Eyeballs were enucleated and retinas were quickly isolated on ice. After being rinsed in 0.01 M PBS and drained, the retinas were lysed in ice-cold tissue lysis buffer (10% PMSF + 90% RIPA). The lysates were then cleared by centrifugation at 15,000 g for 6 min at 4 °C. Protein concentration was determined using the BCA Protein Assay (Beyotime, China) according to the manufacturer's instructions. Total proteins (10 μg per slot) were electrophoresed on a 12% sodium dodecyl sulfate polyacrylamide gel and then transferred onto polyvinylidene fluoride membranes. After blocking in 5% fat-free milk for 2 h at 37 °C, membranes were incubated with anti-Iba1 (1:500; Wako, Japan), anti-GFAP antibody (1:500, rabbit, Sigma Chemical Co) and anti-GADPH (1:1000, mouse, Proteintech Group, Chicago) antibody overnight at 4 °C. The next day, membranes were further incubated with peroxidase-conjugated immuno-globulin G (1:2000; Santa Cruz Biotechnology). Finally, membranes were scanned using the Bio-rad exploding system (Bio-rad, CA, USA) with corresponding software.