Stem Cell Factor Gene Overexpression Enhances the Fusion Potential of Rat Bone Marrow Mesenchymal Stem Cells with Cardiomyocytes

Kanwal Haneef1,2, Rakhshinda Habib1,3, Nadia Naeem1,3 and Asmat Salim1,* 1Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 2Dr. Zafar H. Zaidi Center for Proteomics, University of Karachi, Karachi-75270 3Dow Research Institute of Biotechnology and Biomedical Sciences, Dow University of Health Sciences, Ojha Campus, Gulzar-e-Hijri, SUPARCO Road, KDA Scheme-33, Karachi Article Information Received 23 August 2020 Revised 03 September 2020 Accepted 21 September 2020 Available online 03 December 2020


INTRODUCTION
H eart diseases are one of the main causes of morbidity and mortality all over the world particularly in developing countries (Lachtermacher et al., 2010;Herrmann et al., 2011;Abdelwahid et al., 2016). The pathological mechanisms aiding in cardiac arrest consists of cardiomyocyte death, necrosis, cardiomyocyte hypertrophy, reduced contractility, inflammation and oxidative stress (Burchfield and Dimmeler, 2008;Steven et al., 2019). The discovery of differentiation potential of stem cells, which are unspecialized cells and capable of differentiating into specialized cell type, has opened new horizons in the field of regenerative medicine (Ioannidou, 2006;Keshtkar et al., 2018). However, as the injured myocardium is commonly ischemic with the scar formation, stem cell transplantation to the infarcted heart encountered many challenges and majority of the engrafted cells die due to lack of support and inaccessibility of nutrient and oxygen. So far several strategies have been adopted to improve the survival of engrafted cells and their resistance to oxidative stress (Abdelwahid et al., 2016).
Mesenchymal stem cells (MSCs) are nonhematopoietic cells and are adherent heterogeneous multipotent stem cell originated from mesoderm. Characteristically, these cells have perivascular residence where they contribute in the maintenance of other cell types and tissues (Psaltis et al., 2008). MSCs seem to reduce cardiac injury, as having potential to regenerate myocardium (Vertesaljai et al., 2008). MSCs engrafted into the injured heart may possibly acquire the cardiac cell characteristics which suggest that MSCs differentiation perhaps is associated with ischemic microenvironment therefore cardiac microenvironment might be a key factor in mesenchymal stem cell differentiation (Shim et al., 2004;Xie et al., 2006). Paracrine factors, for instance, cytokines and growth factors are generally produced by cardiac endogenous cells in response to stress and injury. These soluble molecules possibly function through paracrine signaling to facilitate stem cell mobilization from bone marrow or tissue to the place of damage, in this way promoting tissue regeneration (Witman et al., 2019).

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MSCs are capable of regulating different types of cells in their vicinity by means of direct cell to cell contact or communication and also via secretion of a broad range of soluble growth factors and cytokines (Fan et al., 2020). Stem cell factor (SCF), also known as Steel factor or c-kit ligand, aids in the mobilization of stem cells towards the site of injury (Xu et al., 2007;Xiang et al., 2009). c-kit is a tyrosine kinase receptor expressed in stem cells and progenitor cells including stromal cells (Smith et al.,200l). SCF plays pivotal role in proliferation, survival and migration of stem cells through p38 MAPK pathway (Kuang et al., 2008;Mazzoldi et al., 2019). Functional cardiac regeneration and repair has been observed after myocardial infarction following implantation of MSCs, which is further enhanced by SCF (Fazel et al., 2005). It has been reported that SCF-c-kit pathway provides protection against cardiovascular diseases (Wigren et al., 2016). In a recent study, the role of SCF and its c-kit receptor for neovascularization has been explored. It was observed that hypoxic treatment of human endothelial cells results in increased expression of c-kit receptor in endothelial cells (Kim et al., 2019).
In the present study, we analyzed the myogenic potential of bone marrow MSCs virally transduced with SCF after co-culturing these cells with cardiomyocytes (CMs) at different time periods. Co-culture studies were performed to observe the fusion potential of SCF transduced MSCs and CMs. This study is an attempt to provide insight into designing of improved cell based therapeutic approach for cardiac diseases.

Animals
International guidelines for the care and use of laboratory animals were followed for all animal procedures after the approval from the local ethical committee. Throughout the study, Sprague Dawley (SD) rats of both sexes weighing 200-300 g were used.

Cell culture
MSCs were isolated from SD rats. Tibia and femur were aseptically removed. DMEM supplemented with 10% fetal bovine serum (FBS), 100 unit/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate and 4 mM L-glutamine was flushed down the cavities of the bones. Culture was maintained at 37°C in a humidified atmosphere containing 5% CO 2 . After 4 to 5 days, non adherent hematopoietic cells were removed. MSCs were isolated by their ability to adhere to the plastic surface. Fresh medium was provided after every 3 days. Cells were subjected to sub-culture when they reached approximately 70% confluency. MSCs of passages 1-2 (P1 or P2) were used throughout the study.

Transfection of MSCs with SCF gene
Retroviral vector construction PLXIN retroviral vectors containing SCF cDNA were custom synthesized by Clonetex systems Inc, USA. The 5' LTR promoter controls the expression of SCF and neomycin resistant genes of PLXIN-NeoR/SCF retroviral vectors derived from Moloney murine leukemia virus (MoMuLV).

Packaging of recombinant retrovirus
Retrovirus packaging cell line PT67 (CRL-12284) was purchased from American Type Culture Collection (ATCC), USA. Prior to transfection, PT67 cell line was seeded in 75 cm 2 flask in DMEM supplemented with 10% FBS, 1 mM sodium pyuvate, 4 mM L-glutamine, 100 U/ mL penicillin and 100 μg/mL streptomycin. When cells reached 50-60% confluence, they were transfected with PLXIN-NeoR/SCF retroviral vector by using calcium phosphate kit (Stratagene, USA) according to the manufacturer's instructions. Cells were incubated for 17 h at 37°C using air jacketed CO 2 incubator. Medium was aspirated and cells were washed twice with 5-10 mL of Ca ++ and Mg ++ free PBS. Cells were provided with 10 mL of antibiotic-free DMEM supplemented with 10% FBS O n l i n e

F i r s t A r t i c l e
and incubated for 48 h at 37°C in CO 2 incubator. Medium was transferred to a 15 mL falcon tube and centrifuged at 4500 x g for 10 min. Viral supernatant was collected and filtered through 0.45 µm filter and stored at -80°C or used immediately for infection.

Infection of mesenchymal stem cells
MSCs were infected twice with viral supernatant diluted at a ratio of 1:1 with FBS-free and antibiotics-free DMEM. 10 mL of diluted viral supernatant was added into a 75 cm 2 flask and incubated for 7 h at 37°C in CO 2 incubator. Viral supernatant was removed and replaced with 10 mL of antibiotic-free DMEM supplemented with 10% FBS to stop transfection and incubated for further 24 h at 37°C in CO 2 incubator. Medium was removed and MSCs were re-infected with viral supernatant and incubated for 4 h under same conditions. Viral supernatant was removed and replaced with 10 mL of antibiotic-free DMEM supplemented with 10% FBS and incubated for 48 h, 1 week, 2 weeks and 3 weeks under same conditions. Fresh Medium was provided after every 3 days.

O n l i n e F i r s t A r t i c l e
RT-PCR Isolation of total RNA (treated and control groups) was carried out by using RNeasy Mini kit (74904, Qiagen) according to the manufacturer's protocol. One (1) µg of RNA was utilized for reverse transcription using the Superscript RT kit (18080-051, Invitrogen) and then amplified using oligonucleotide primer corresponding to stem cell factor (SCF) gene. Rat GAPDH gene was used as an internal control. Both primer sequences along with the expected product sizes and annealing temperatures are listed in Table I. Reversed transcribed products were denatured for 1 min at 94°C, followed by 35 cycles of amplification i.e. denaturation at 94°C (1 min), annealing at 56-60°C (1 min), and extension at 72°C (1 min), and a final extension at 72°C for 10 min. PCR product was subjected to be resolved on 1% agarose gel through gel electrophoresis. The integrated band density of SCF gene was normalized with the corresponding GAPDH band density and compared with the control group using gel documentation system (Alpha Innotech, FluorChemTM, USA).

Co-culture studies
Co-culture of SCF transfected MSCs and CMs were performed for the analysis of fusion potential of transfected MSCs with normal CMs. 48 h transfected MSCs were used in all experiments. Prior to co-culture, cells were treated with cell labeling dyes, PKH26 and PKH67 to label transfected MSCs and CMs, respectively according to the manufacturer's instructions. Cells were co-cultured for 15 days and analyzed by fluorescence microscope and flow cytometer.

Statistical analysis
Statistical analysis was performed by using one way ANOVA and bonferroni's post hoc test for multiple comparisons. Data is presented as means ± standard error of mean (SEM). p<0.05 was considered statistically significant.

Characterization of isolated MSCs and CMs
MSCs at passage 0 (P0) showed heterogonous population of cells, while at passage 1 (P1), MSCs predominately showed homogeneous population of cells with fibroblast-like morphology (Fig. 1A). P0 CMs showed heterogonous population of cells, while P1 CMs showed adherent spindle shaped population of cells (Fig. 1A). Immunocytochemistry analysis of rat bone marrow MSCs showed the presence of mesenchymal markers, CD44, CD90 and CD117 cell surface markers, as shown in our previous study (Haneef et al., 2014) (Fig. 1B). CMs showed positive expression of cardiac specific proteins, actin and cardiac troponin T (Fig. 1B). Results are expressed as Mean ± SEM (n = 3). Differences between groups with P < 0.05 is considered statistically significant where *P < 0.05, **P < 0.01, and ***P < 0.001.

Transfection of MSCs with SCF viral vectors
Before transfection, MSCs predominately showed homogenous population of cells with fibroblast-like morphology. After transfection with retrovirus, cells were slightly shrunken in the beginning but when normal condition was restored, they regained normal morphology ( Fig. 2A). Gene expression analysis of SCF in transfected MSCs by RT-PCR showed that the expression level was maximum (P < 0.01) after 48 h of transfection. The expression started to increase till 48 h and then decreased subsequently after 1 week, 2 weeks and 3 weeks of transfection ( Fig. 2B-C). The 15 day co-culture of SCF transfected MSCs and CMs have shown an increase in the number of fused cells as compared to control. Merged images of both cell types showed yellow fluorescence of fused cells (Fig. 3). Quantification of double labeled cells in co-culture was performed through flow cytometry. Results have shown a gradual increase in the number of fused cells as compared to control. Significantly higher number of fused cells is seen in case of SCF transfected MSCs and CMs as compared to control with levels of significance p < 0.05 (Fig. 4).  Differences between groups with P < 0.05 is considered statistically significant where *P < 0.05, **P < 0.01, and ***P < 0.001.

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K. Haneef et al.

DISCUSSION
Present study successfully set up a co-culturing system of stem cell factor (SCF) transfected mesenchymal stem cells (MSCs) with cardiomyocytes (CMs) to simulate the cell-cell cross talk that may appear beneficial following stem cell transplantation for heart diseases. This in turn may improve differentiation potential MSCs into cardiomyocytes.
Growth factors and cytokines are predominant factors required for trans-differentiation of stem cells into cardiac cells (Psaltis et al., 2008). They are released by cells and play critical role in repair and regeneration of damaged myocardium (Takahashi et al., 2006). Earlier, we have reported the analyses of the mRNA expressions for SCF in DNP-treated and untreated MSCs and CMs. In this study, SCF was upregulated in MSCs following hypoxic stress (Haneef et al., 2014). The response of growth factors varies with the extent of hypoxia and time of re-oxygenation and if crucially monitored, they can be beneficially used against many ischaemic conditions in stem cell-based cellular therapy.
Stem cell factor or c-kit ligand (SCF) binds to its receptor and plays pivotal role in proliferation, survival and migration of stem cells. SCF is important for cardiomyocyte survival after cardiac injury (Kuang et al., 2008). In this study, we assessed the effect of SCF transfected MSCs in terms of their transdifferentiation potential. We analyzed the overexpression of SCF mRNA in control and transfected MSCs. The expression pattern of SCF showed that the expression reached maximum after 48 h of transfection, and decreased subsequently after 1 week, 2 weeks and 3 weeks of transfection. On the basis of our gene expression result, we selected 48 h transfected MSCs for further studies.
Large number of data has shown that cytokines and growth factors alone are insufficient to induce transdifferentiation of MSCs into cardiomyocytes and direct cell to cell cross talk is obligatory for differentiation (Perán et al., 2011). To investigate the cell to cell cross talk, we set up a co-culture system between SCF transfected MSCs and CMs. Cell fusion may result in transdifferentiation that can occur in vivo when stem cells are introduced in the infarcted myocardium. We found several double labeled cells after different days in co-culture through fluorescent microscopy and flow cytometry, which is indicative of cell fusion. Some elongated cell projections protruding from different cells indicate that the cells are interacting with each other. This most probably would result in direct cell to cell connections in due course of time. Co-culture studies have shown improved fusion of SCF transfected MSCs with CMs as compared to control, reflected by increase in the number of double labeled cells which appeared in upper right quadrant.
Extrapolating our results, it can be hypothesized that the engineered MSCs stimulate a higher SCF growth factor production, which would induce fusion capabilities between transplanted MSCs and recipient CMs and ultimately induce efficient differentiation of the transplanted MSCs. The exact molecular mechanism involved in the cross talk of two types of cells is not well understood. The fused cells may proliferate and contribute to the regeneration of damaged myocardium and would have the potential to replace damaged heart cells. It is important to determine the contribution of various factors in the fusion process so that a rational basis for the use of these cells for the therapy of cardiac diseases can be determined.

CONCLUSION
Diseases involving terminally differentiated tissues e.g. coronary artery diseases are a major challenge all over the world as cardiomyocytes have very low proliferation capacity. We conclude from our study that the stem cell factor or c-kit ligand (SCF) enhances the fusogenic abilities of MSCs. SCF plays significant role in proliferation, survival and migration of stem cells. Hence, SCF promotes fusion or transdifferentiation of MSCs into cardiomyocytes. This study emphasizes the role of SCF in MSCs transdifferentiaion and would aid in designing a therapeutic strategy in which SCF transfected MSCs can be used for the treatment of ischemic diseases.