Differentiation of Mesenchymal Stem Cells into Photoreceptor-like Cells under the Inuence of a Temporary Increase in miRNA-182, -183 Expression

It is found that the death of retinal photoreceptors is the main cause of retinal degeneration, while there is not an effective treatment protocol. Data of preclinical and clinical trials indicates that the stem cell therapy is a useful way of treating retinal degeneration problems. On the other hand, previous works found that miRNA-182, -183 signicantly affected the photoreceptor maturation and maintenance in animal models. The present study aimed to investigate the impact of a temporary increase in miRNA-182, -183 expression on the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into photoreceptor-like cells. To this end, miRNA-182, -183 was transfected into hBMSCs; then, qRT-PCR was performed to measure the expression levels of miRNA-182, -183 and some retina-specic genes such as OTX2, NRL, PKCα, and recoverin. CRX and rhodopsin (RHO) levels were also measured through qRT-PCR and immunocytochemistry We indicated that the transfection of hBMSCs with miRNA-182, -183 using the Lipofectamine induce differentiation and progenitor’s genes expression consisted of CRX, OTX2, PKC, Recoverin, NRL and RHO. Moreover, the upregulation expression of transcription factors, CRX and RHO, indicated that miRNA-182, -183 could serve as crucial functions in the differentiation of hBMSCs into photoreceptor-like cells. The ndings may provide a new strategy to improve the usage of hBMSCs as a treatment for the retinal dysfunction. The show their photoreceptors. A large amount of information has been recently obtained on pivotal functions miRNAs in the vertebrate retina. Global and specic miRNA loss-of-function studies play key roles in the miRNA development, physiology and diseases of retina 11–13 . New technologies for measuring the expression of miRNA genes allow the accurate evaluation of results of removing specic miRNAs. These technologies also provide the opportunity to evaluate the interaction of miRNA with the target mRNA in a specic tissue or cell 14 . Various studies have found that the P347S rhodopsin transgenic mice, human retinitis pigmentosa (RP) model, and three other types of photoreceptor-linked mouse models express the miR-96/182/183 cluster. These results conrm the association between the reduction of their gene expression and retinal disease in humans 15 . All these studies aim to help stem cell-based therapies among a large number of people who are affected by disorders that severely challenge the of themselves and their acquaintances. The present study evaluated the differentiation of Human bone marrow-derived mesenchymal stem cells (hBMSCs) that highly expressed miRNA-182, -183 towards photoreceptor like cells.


Introduction
There are about 37 million blind people around the world; and most popular conditions are macular degeneration, diabetic retinopathy, and glaucoma 1 . Despite the fact that advances in laser and surgical procedure and drug discovery have decreased the vision loss due to these conditions, much of the vision loss, which is associated with degenerative retinal conditions, is still inalterable and advances over time 2,3 . In recent years, the stem cell therapy has been considered as a promising method of treatment of retinal diseases. Stem cells have the self-renewal ability and also can be developed to numerous cell types. Retinal anatomy has some advantages for stem cell therapy such as small space, local delivery of cells to the retina, and limited number of required stem cells. The ideal stem cell for cell substitution in eyes with retinal disorders is a cell that can be easily expanded and give rise to multiple cells 4 . Among numerous stem cell types, which have been tested for treatment of retinal diseases, the mesenchymal stem cells (MSCs) are attractive candidates as they can be easily obtained from bone marrow or adipose tissue, and be manipulated and used for autologous stem cell transplantation in clinics 5,6 . MicroRNAs (miRNAs), which are small-noncoding RNAs, play effective roles as key modulators of many physiological and pathophysiological processes often by acting as regulators of gene expression events 7 . An important family of miRNAs, which are known as sensory organen-riched miRNA clusters, is the microRNA-183 cluster consisting of miR-183, miR-96 and miR-182. It is found that these miRs are highly expressed in adult retina and other sensory organs. Moreover, their expression is necessary and su cient for the photoreceptor maturation and maintenance 8, 9 . The miR-96/182/183 cluster is necessary for the maintenance of Cone Photoreceptor Outer Segments in vitro and visual function in vivo 10 . Therefore, key roles of miRNAs cannot be ignored in photoreceptors. In this regard, miR-183 is also known as an important epigenetic regulator which plays role in the physiological function and is investigated in different pathologic conditions by various studies. The miR-182 knockout rats show serious structural defects in their photoreceptors. A large amount of information has been recently obtained on pivotal functions of miRNAs in the vertebrate retina. Global and speci c miRNA loss-of-function studies play key roles in the miRNA development, physiology and diseases of retina [11][12][13] . New technologies for measuring the expression of miRNA genes allow the accurate evaluation of results of removing speci c miRNAs. These technologies also provide the opportunity to evaluate the interaction of miRNA with the target mRNA in a speci c tissue or cell 14 . Various studies have found that the P347S rhodopsin transgenic mice, human retinitis pigmentosa (RP) model, and three other types of photoreceptor-linked mouse models express the miR-96/182/183 cluster. These results con rm the association between the reduction of their gene expression and retinal disease in humans 15 . All these studies aim to help stem cell-based therapies among a large number of people who are affected by disorders that severely MicroRNA was isolated by miRNeasy isolation kit (Qiagen) according to the manufacturer's instructions. miRCURY LNA miRNA Mimics include two short, LNA-enhanced complimentary strands which prevent any miRNA-like activity associated with passenger strands; hence, they can be sure that observed phenotypes using these mimics are due to the increased activity of the mimicked miRNA. Total RNA was extracted from cultured cells (transfected with miRNA and control cells) with TRIzol (Ambion) according to the protocol. The quality of RNA samples was then measured by NanoDrop (Thermo Fisher Scienti c).

Real Time PCR
Approximately 2 micrograms of RNA were used for the synthesis of cDNA using the thermo scienti c-K1622 kit and rotor-gene Q (Qiagen, USA). The thermal conditions included the initial stage of denaturation at 95 °C for 3 minutes, and then for 40 cycles at 95 °C and 15 seconds and 72 °C for 25 seconds. Table 1 presents the applied primers for each studied gene. The mRNA relative expression level was also calculated using the 2-ΔΔCt method. mRNA levels were normalized using the GAPDH as a housekeeping gene. Cells, which were seeded onto precoated chamber slides, were xed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized using 0.1% Triton-X100 in PBS, and blocked with 1% BSA. The cells were then immunostained by anti-CRX and anti-RHO antibody (1:500) (Santa Cruz Biotechnology) and conjugated secondary antibodies in PBS (1:500; Santa Cruz Biotechnology). After washing, cell nuclei were counterstained with DAPI. Negative control samples were processed in parallel, but without any primary antibody. Fluorescence images were captured with a uorescence's microscope (Olympus Corp., Tokyo, Japan).

Statistical Analysis
Results are presented as mean ± SD. Kruskal-Wallis test and Mann-Whitney U test were respectively used to compare gene expression levels in groups and also determine signi cance levels of groups. P-values lower than 0.05 were also considered signi cant. Statistical analysis and graph generation for gene and protein expression were conducted using GraphPad Prism (Version 7). Other data was analyzed using the SPSS 16.00.

Results
Expression of miRNA-182, -183 The real time PCR was performed 24 and 48 hours after the transfection to assess the expression in hBMSCs that were transfected by miRNA-182, -183 and scramble. Our results indicated that levels of miRNA-182, -183 expression increased in miRNA-182, -183 transfected cells in both times compared with scramble transfected cells (Fig. 1).

Expression of progenitor and differentiation genes
The real time PCR was performed 24 and 48 hours after the transfection in order to examine the effects of transfection with miRNA-182, -183 on progenitor and differentiation genes that included CRX, OTX2, NRL, SLC1A1, PKC, RHO and Recoverin. Figure 2 shows that the expression of CRX, OTX2 and RHO genes is signi cantly upregulated after 24 hours compared with the control. CRX RHO and PKC gene expression also signi cantly increased after 48 hours compared with the control in transfected cells with miR182 ( Figure 3).    Change in the expression of CRX, OTX2, PKC, NRL, SLC1A1, RHO and Recoverin genes compared with GAPDH gene after 24 hours in transfected cells. Results are expressed as fold change relative to the control group. Data is expressed as mean ± SD.

Figure 3
Changes in the expression of CRX, OTX2, PKC, NRL, SLC1A1, RHO and Recoverin genes compared with GAPDH gene after 48 hours in transfected cells. Results are expressed as fold change relative to the control group. Data is expressed as mean ± SD.

Figure 4
Changes in the expression of OTX2 gene compared with GAPDH gene after 24 and 48 hours in transfected cells. Results are expressed as fold change relative to the control group. Data is expressed as mean ± SD.   Immuno uorescence staining results of RHO 24 and 48 hours' after the transfection miR-182. Immuno uorescence staining results of CRX 24 and 48 hours' after the transfection miR-182 Figure 10 Immuno uorescence staining results of CRX 24 and 48 hours' after the transfection miR-183