Key words : Cell differentiation, Immunocytochemistry, Mammalian retina, MicroRNA mimic, Retinal degeneration disease

Introduction

Mesenchymal stem cells (MSCs) have gained signi-ficant recognition in the field of cell therapy and regenerative medicine due to their numerous advantages. Unlike embryonic stem cells, the use of MSCs does not raise ethical concerns or encounter significant constraints.¹ Mesenchymal stem cells possess the ability to evade immune reactions and, moreover, can effectively suppress immune responses, similar to regulatory T cells. The isolation process for these cells is relatively simple, and MSCs have demonstrated the potential to achieve favorable outcomes in cell therapy through various mechanisms.²

These cells can be treated under in vitro conditions with a variety of factors, such as growth factors, to guide their individual differentiation along specified pathways. Furthermore, the effectiveness of MSCs can be modulated by regulation of desired tissue-specific regulatory factors. Paracrine networks play a crucial role to regulate key features of MSCs and thereby influence the regenerative profiles of these cells. One notable characteristic of MSCs affected by paracrine networks is the production of various cytokines and growth factors.³

The immunomodulatory properties of these cells, particularly the ability to suppress T lymphocytes, have a significant effect on the regenerative process of ocular vasculopathies. This effect is achieved through signaling pathways associated with growth factors, cytokines, and interventions in angiogenesis. Recently, miRNA-mediated reprogramming techno-logy has been discovered for stem cells. MicroRNAs play a crucial role in reprogramming, maintaining pluripotency, and directing differentiation, as well as regulation of the fate of stem cells by targeting pluripotency markers and differentiation pathways.⁴

MicroRNAs represent a promising therapeutic target in the field of regenerative medicine. Inves-tigation of the regulatory activities of miRNAs will contribute to the development of stem cell-based clinical treatments. MicroRNAs offer several advan-tages as an alternative to growth factors and inhibitors for direction of cell differentiation. These advantages include small size, rapid synthesis, resistance to nucleic acid degradation, long half-life, and bioactivity. Research has shown that miRNAs play a crucial role in stem cell differentiation by binding to the 3′ untranslated region of genes involved in pluripotency factors.⁵

Moreover, miRNAs affect stem cell separation by focusing on particular translation components.⁶ MicroRNAs have apparent potential for the treatment of different diseases, including myocardial localized necrosis, neurodegenerative maladies, and retinopathies. In developing nations, severe visual deficiency is frequently caused by dysfunctional photoreceptors due to retinal degeneration.^7,8 Diverse sorts of infections with various etiologies can eventually lead to photoreceptor devastation and result in visual deficiency.

Unfortunately, the limited number of commonly used therapies are ineffective for prevention of these pathological conditions.⁹ Therefore, it is crucial to explore new strategies, particularly stem cell-based therapies, with a focus on photoreceptor recons-truction and restoration of visual function in affected individuals.¹⁰ Photoreceptor cells, as post mitotic neurons, are typically not regenerated in most mam-mals once degradation ensues, which emphasizes the importance to utilize stem cells in the treatment of various retinopathies.¹¹ In this research, we aimed to design an in vitro study to gain a better understanding of the role of the miRNA known as MIR96 in differentiation of human bone marrow-derived MSCs (hBMSCs) into photoreceptor-like cells.

Materials and Methods

The hBMSCs, prepared from the bank of BMSCs at the Cell Therapy Center of Royan Institute in Tehran, Iran, were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS) and 1% streptomycin and penicillin. The culture medium was refreshed every 3 days. The differentiation of hBMSCs into photoreceptors was initiated at the third passage.

Cell transfection
The hBMSCs were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, 1.5 × 10⁵ cells in the logarithmic growth phase were seeded in each well of a 6-well plate containing 1.5 mL of FBS-free DMEM sup-plemented with antibiotics. A transfection mixture was prepared by combining 50 nM of miRNA-96 with 10 μL of Lipofectamine in 200 μL of DMEM. The mixture was incubated at room temperature for 15 minutes and then added to the hBMSCs. The cells were incubated for 5 hours after transfection.

The transfected cells were then supplemented with medium containing FBS and antibiotics and incubated for 24 hours and 48 hours. Additionally, a scrambled sequence was used as a negative control. The efficacy of cell transfection was assessed with real-time polymerase chain reaction technique. All transfection procedures were repeated 3 times, and the cells were allowed to reach 60% to 70% confluence prior to transfection.

Detection of expression levels of MIR96 using real-time polymerase chain reaction
The analysis of miRNA expression in hBMSCs involves 2 steps: stem-loop reverse transcription (RT) reaction and real-time polymerase chain reaction detection. In the stem-loop RT reaction, reverse transcriptase was used to transcribe the primers of the stem-loop structure, which bind to the 3′ end of miRNA molecules. To reverse-transcribe the extracted RNA, a poly-A polymerase was utilized in the presence of an oligo-dT adaptor. After transfection, miRNA expression in hBMSCs was assessed using real-time polymerase chain reaction. The RNA extraction was performed after 24 hours and 48 hours of cell transfection using the miRCURY RNA isolation kit (Exiqon). Subsequently, cDNA synthesis was performed (LNA Universal cDNA synthesis kit; Exiqon).

For the real-time polymerase chain reaction analysis of miRNA, we used a nucleic acid gel stain (SYBR Green master mix kit; Exiqon), along with specific primers. The RNA U6 small nuclear 1 gene served as the internal control for normalization. The real-time polymerase chain reaction data were analyzed using the 2^-ΔΔCt formula, which enables relative quantification. The real-time polymerase chain reaction was performed using a real-time nucleic acid amplification and detection system (Rotor-Gene Q instrument; Qiagen). The thermal cycling profile consisted of an initial denaturation step at 95 °C for 2 minutes, followed by 40 amplification cycles (95 °C for 5 seconds and 60 °C for 30 seconds).

Real-time polymerase chain reaction assay for detection of gene expression level
The total RNA from the transfected cells, both cells transfected with miRNA and control cells, was extracted using an RNA extraction kit from Ambion. The extraction procedure was performed following the manufacturer’s protocol. Following the extraction, the qualitative and quantitative assessments of the RNA samples were conducted with a spectrop-hotometer (NanoDrop system; Thermo Fisher Scientific). The spectrophotometer allowed for the measurement of absorbance, which provided information about the concentration and purity of the RNA samples.

We used 2 μg of total RNA for the synthesis of cDNA using a cDNA synthesis kit (RevertAid first strand cDNA synthesis kit No. K1622; Thermo Scientific) and real-time nucleic acid amplification and detection (Rotor-Gene Q instrument; Qiagen). It should be noted that the thermal conditions included the initial stage of denaturation for 3 minutes at 95 °C, then for 40 cycles at 95 °C lasting 15 seconds, and then 72 °C for 25 seconds. The primers we used for each gene in this study are presented in (Table 1). The changes in expression level of mRNA for each gene were analyzed via the 2^-ΔΔCt method, and the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as the internal control gene.

Immunostaining
Immunocytochemical assays were performed with cultures grown on tissue culture dishes coated with fibronectin or on glass coverslips coated with poly-L-The cells were first fixed in 4% paraformaldehyde (Merck) at room temperature for 10 to 15 minutes.

To block nonspecific binding sites and perform permeabilization of the cells, we used a solution of phosphate-buffered saline containing 0.1% Triton X-Cell cultures were kept in PBT1 solution for 15 minutes; then the primary antibody (diluted in PBT1) was added and incubated to 37 °C for 3 hours at room temperature.

The primary antibodies were rabbit monoclonal antibodies against rhodopsin (ROH) and cone-rod homeobox (CRX) (Sigma; used at a 1:300 dilution), and secondary antibodies were rabbit polyclonal antibodies against ROH and CRX (Abcam; used at 1 μg/mL). For the next step, before addition and dilution of the secondary antibody, the cultures underwent a series of washes with phosphate-buffered saline at room temperature. We then stained nuclear For the final step, we observed protein expression under a fluorescence microscope (Eclipse 90i; Nikon).

Statistical analyses
All data in this study were obtained from more than 3 independent experiments and analyzed with GraphPad Prism software (version 5.0). We used the unpaired Student t test and 1-way analysis of variance or the Kruskal-Wallis test followed by the Dunnett test to compare different groups. P < .05 was considered significant.

Results

The gene expression levels of hBMSCs transfected with the mimic to miRNA-96 and gene expression levels of the control hBMSCs were investigated using real-time polymerase chain reaction after 24 and 48 hours to assess the expression of photoreceptor-like cell markers (Figure 1). The analysis of mRNA expression revealed that markers such as ortho-denticle homeobox 2 (OTX2), CRX, neural retinal leucine zipper (NRL), and protein kinase C (PKC) were upregulated in cells transfected with the mimic to miRNA-96 after 48 hours. Additionally, RHO and recoverin showed upregulation after both 24 and 48 hours ((Figure 2) and (Figure 3)).

The expression of photoreceptor cell markers was significantly higher in cells transfected with mimic to miRNA-96 compared with the control cells. In addition, transfection of hBMSCs with the mimic to miRNA-96 created favorable conditions for the differentiation of these cells into photoreceptor-like cells. We used GAPDH as the internal control gene for normalization. As depicted in (Figure 2) and (Figure 3), the gene expression levels of OTX2, CRX, NRL, RHO, recoverin, and PKC were significantly higher in cells transfected with mimic to miRNA-96 versus the control cells. However, no significant differences were observed in the expression of solute carrier family 1 member 1 (SLC1A1) between the 2 groups after the designated time points.

The real-time polymerase chain reaction analysis revealed the relative expression levels of OTX2, CRX, NRL, RHO, recoverin, SLC1A1, and PKC mRNA in hBMSCs transfected with equal doses of miRNA-negative control (scrambled) and the mimic to miRNA-96. Interestingly, overexpression of MIR96 had no significant effect on mRNA levels of OTX2, CRX, NRL, SLC1A1, and PKC after 24 hours.

We gained some valuable insight into the underlying mechanisms involved in the differ-entiation of hBMSCs into photoreceptor-like cells. To further validate the effect of increased gene expression on protein levels, we conducted immunocyto-chemistry assays. We conducted these assays to demonstrate the expression of photoreceptor marker proteins, namely, RHO and cryptochrome circadian regulator (CRY). Positive staining of these markers was observed in hBMSCs transfected with the mimic to miRNA-96 after both 24 and 48 hours, which confirmed that the increased gene expression led to corresponding changes at the protein level. After 24 and 48 hours, significant upregulation in expression of RHO was observed in hBMSCs that were infected with the miRNA-96 mimic, in comparison to their uninfected counterparts.

Although CRX expression showed better changes, the changes were less obvious. The immunocyto-chemistry results further support these findings because the presence of RHO expression in transplanted cells after 24 and 48 hours was found to be a positive marker for induced photoreceptor-expressing cells. In contrast, the control group (scrambled) did not show the presence of this marker ((Figure 4) and (Figure 5)).

Discussion

To our knowledge, in this in vitro study, the effect of MIR96 mimic on the expression of photoreceptor markers in hBMSCs was investigated for the first time. Known for pluripotency, hBMSCs have great potential for therapeutic applications. The real-time polymerase chain reaction results showed that RHO and recoverin mRNA levels increased after 24 hours in transfected cells. In addition, OTX2, CRX, NRL, RHO, recoverin, and PKC mRNA levels increased after 48 hours in transfected cells.

The immunocytochemistry results confirmed the expression of RHO and CRX in transfected cells at 24 hours and 48 hours, which indicated the success of photoreceptor-like cells. The transcription factor NRL exhibits a leucine zipper motif that acts as a biomarker for rod photoreceptors. The importance of NRL is a consequence because its absence causes the loss of all rod photoreceptors.¹²

The transcription factor OTX2 is involved in the differentiation of different types of photoreceptors.¹³ Another important transcription factor is CRX, which plays an important role in photoreceptor differentiation by regulation of the expression of several photoreceptor-specific genes.¹⁴ The CRY binding site sequences are located upstream of many photoreceptor-specific genes, including opsin genes found in many species.¹⁵ The photoreceptor-specific transcription factor CRX is important for regulation of the 16 phenotypes and function of cells.¹⁶ The SLC1A1 gene is a target gene of miRNAs 183/96/182 in photoreceptors, indicating that it is regulated by miRNAs.¹⁷

The PKC family includes a serine/threonine-specific protein kinase group. These enzymes play an important role for regulation of cellular physiology by phosphorylating serine/threonine residues in many proteins. Rhodopsin is an optical pigment abundant in many organisms. It is believed that even some single-celled organisms can use RHO photoreceptors if the pigments found are of ancient origin.¹⁸

Bone marrow-derived stem cells have been shown to be effective for improvement of the survival of photoreceptor cells. Thus, BMSCs have emerged as a promising cell therapy for the treatment of retinal degenerative diseases. The challenges of retinal repair and regeneration using autologous retinal cells have led to research into MSC-based therapies for a variety of eye diseases, including age-related macular degeneration, diabetic retinopathy, prema-ture infant retinopathy, and glaucoma.

A previously published investigation into the regenerative capacity of rat BMSCs was conducted using an animal model with damaged retinal pigment epithelium. The BMSCs were injected into the subretinal space region and demonstrated proliferation, differentiation into retinal cells, and expression of specific retinal genes such as ROH, glial fibrillary acidic protein, and pan-cytokeratin following differentiation.¹⁹

In another study on retinopathy, human BMSCs were injected into Royal College of Surgeons rats, which is a rat model characterized by severe degeneration of retinal cells. Surprisingly, the results were positive, as the BMSCs exhibited regenerative potential for improvement of the retinopathy process. Remarkably, the transplant of human BMSCs into rats did not require immunosuppression factors and showed no signs of rejection despite the inter-species aspect of the transplant (human to rat).²⁰

A crucial aspect of photoreceptor function is the change in electrophysiological activity in response to mild optical stimuli, which can subsequently modulate the expression of specific markers.²¹ The regulation of gene expression and the maintenance of photoreceptor function heavily relies on epigenetic mechanisms. Among these mechanisms, the role of MIR96 is particularly noteworthy, as its expression levels can influence the differentiation of stem cells into photoreceptors. It is worth mentioning that fully differentiated sensory neurons, including photoreceptors, typically exhibit high levels of MIR96 expression.

The findings of this research demonstrated the potential of MIR96 to induce the differentiation of hBMSCs into photoreceptor-like cells by upregulating the expression of OTX2, CRX, NRL, RHO, recoverin, and PKC genes. The expression analysis of photoreceptor markers, including OTX2, CRX, NRL, RHO, recoverin, and PKC at the mRNA level, as well as the protein levels of CRX and RHO using real-time polymerase chain reaction and immunocytochemistry, confirmed that hBMSCs transfected with the mimic to miRNA-96 could effectively promote the differentiation of hBMSCs into photoreceptor-like cells. These achievements can contribute to the development of a novel method to derive photoreceptor-like cells from hBMSCs through transfection with a mimic to miRNA-96. This approach holds promising potential for stem cell-based therapies to target retinal degenerative diseases in the future. Overall, our study suggested that hBMSCs have the capability to differentiate into cells with a photoreceptor phenotype in vitro when transfected with a mimic to miRNA-96.²²

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