Establishment and characterization of an immortalized epicardial cell line

Abstract Recently, the increasing significance of the epicardium in cardiac development and regeneration is beginning to be recognized. However, because of the small proportion of primary epicardial cells and the limited cell culture time, further research on the mechanism of epicardial cells is hindered. Here, we transfected simian virus 40 Large T (SV40‐LT) into primary epicardial cells to establish an immortalized cell line, named EpiSV40. We further demonstrated that EpiSV40 can be easy to culture and has the proliferation, migration and differentiation capacities comparable to primary epicardial cells. EpiSV40 can serve as an ideal in vitro model for epicardial cell research, which will booster the study of the epicardium in cardiac development and heart regeneration.

regenerative medicine, including cell transplantation therapy and tissue-engineering approaches, [9][10][11] while the underlying molecular mechanisms of epicardial cells are still largely unknown.
In vitro cellular experiment is an important strategy for investigating the molecular mechanisms of epicardium function, but the primary culture of epicardial cells from embryonic mouse heart faces challenges. The epicardial cell isolation is a laborious and timeconsuming process, which required a 14-day lead time for pregnant mice and microscopic manipulation of embryos. 12 Besides, the amount of epicardial cells is limited and cannot expand in vitro. 13,14 Hence, it is necessary to establish a cell line that could maintain the primary epicardial cell characterization for mechanism research.
Introgression of exogenous genes like Epstein-Barr virus, 15 simian virus 40 Large T 16 and telomerase reverse transcriptase is commonly used for immortalization of primary cells. 17 The simian virus SV40, a polyomavirus of rhesus macaques, was identified in 1960, which encodes two proteins, large T antigen and small t antigen. 18,19 The large T antigen of SV40 (SV40-LT) has been widely used for primary cell immortalization by regulating the p53-mediated cell cycle. 16 SV40-LT-mediated cell immortalization has been applied to melanocyte cells, glomerular podocytes, atrial myocytes, ventral mesencephalic neuronal progenitor cells, dental mesenchymal cells and retinal microglial cells. [20][21][22][23][24][25] In this study, we transfected the SV40-LT into the primary epi-

| Mice
All experiments involving animals were conducted following the Guide for the Use and Care of Laboratory Animals. All animal protocols were approved by the Institutional Animal Care and Use

| Isolation and culture of primary epicardial cells
Hearts from E11.5 embryos are isolated in a sterile environment and placed in a 24-well plate which is coated with 1% gelatin. Slowly add 250ul DMEM containing 10% FBS along the wall of the 24-well plate until the liquid just covers half of the heart (too much medium will cause the heart to float away from the centre because of the liquid surface pressure, while too little medium will cause the migrated cell death because of the loss of the medium supply). Hearts are incubated for 8 hours in a CO 2 incubator at 37°C. Then another 250 μl medium is added to the well and continue to culture for 24 hours.
After that, it can be seen that the primary epicardial cells migrated onto the well in a circular shape around the heart. Then hearts are sucked from the well with a 200ul yellow pipette tip, the medium was changed every 48 hours thereafter.

| EdU incorporation
EdU was used to label primary epicardial cells and EpiSV40 undergoing mitosis in vitro. Cells grown on gelatin-coated chamber-slides were treated with 10 μM EdU for 2 hours in culture medium and subjected to EdU immunostaining. EdU incorporation was detected using BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime catalog no. C0071S).

| Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was isolated from cells using TRIzol (Life Technologies, Cat. no. 15596-018). The PrimeScript RT Master Mix was used to convert RNA into cDNA. qRT-PCR for the analysis of expression of different genes (Table 1) was performed in triplicate using the SYBR Green qPCR Master Mix (Applied Biosystems) in a QuantStudio 5 Real-Time PCR Systems (Applied Biosystems). The results were analysed using GraphPad Prism and differences among groups were analysed using Student's t test. Significance levels are indicated by Prism Software as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

| Karyotype analysis
The karyotype analysis was performed using standard G-banding techniques. Briefly, EpiSV40 cells cultured in a T25 flask were incubated with 100 ng/ml colcemid at 37°C for 5 hours, followed by dissociation with trypsin/EDTA and centrifugation at 1000 rpm for 10 min. Cells were re-suspended in 0.05 mol/L potassium chloride hypotonic solution and incubated for 20 minutes. Following centrifugations, the cells were re-suspended in fixative and aspirated on the glass slide, followed by staining with Giemsa solution. The protein samples were mixed with 4 × SDS loading buffer and 10 × SDS loading buffer for 10 minutes at 70°C. Subsequently, 10 μg of total protein was loaded onto 10% sodium dodecyl sulphatepolyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After blocking the non-specific background staining, the membranes were incubated at 4°C overnight with the primary antibody: anti-SV40 T Ag (1:1000). The membranes were washed with Tris-buffered saline/0.1% Tween 20 (TBST) and incubated with secondary antibodies for 1 hour at room temperature.

| Western blot analysis
Signals were detected using Pierce™ ECL Western Blot Substrate (Thermo Fisher Scientific). Krt14 Reactions were performed with the following cycles: 10 minutes at 95°C, 40 cycles of 94°C for 30 s and annealing temperature for 60 s, and a final cycle of 10 minutes at 98°C. Following PCR, the droplets were read on the QX200 (Bio-Rad), set for detecting absolute levels of FAM/HEX probe fluorophores.

| Cell differentiation assay
Cells grown on gelatin-coated chamber-slides were treated with PBS or TGF-β1 (R&D Systems, 10 ng/mL) for 72 hours in the culture medium and subjected to α-SMA and β-catenin immunostaining.

| Virus packaging and infection
Lentivirus packaging and cell infection were performed as described previously. 16 Briefly, the pLVX-IRES-Puro-SV40LT lentiviral expression vector was cotransfected with psPAX2 (Addgene, #12260) and pMD2.G (Addgene, #12259) packaging plasmids into showed in Figure S1A and the detailed sequence information was added in Table S1.

| FACS analysis
FACS was performed as described previously. 28 In brief, embryonic mouse hearts from WT1 GFPCre/+ mice at E12.5 were dissected and dissociated into single cells using the Neonatal Heart Dissociation Kit (Miltenyi Bio-Tech) with a heated shaking block at 37°C and 1000 rpm for 15 minutes. Cells suspension were then centrifuged at 300 g for 5 min and re-suspended in DMEM with 1% BSA and kept on ice. FACS sorting of GFP populations was carried out by gating against a GFP-negative control littermate. Samples were analysed by flow cytometry (BD FACS Arial II, BD bioscience), and the data processing was performed using FlowJo V.10 (Tree Star, Inc).

| Statistical analysis
All results were analysed using GraphPad Prism (version 7.0, GraphPad Software). All data were expressed as the mean ± standard error of the mean (SEM). Differences between groups were evaluated using an unpaired Student's t test. Differences were considered significant when the P-value was < 0.05.

| Epicardium constitutes a low fraction of cardiac cells
We performed two typical markers of epicardium (WT1 and RALDH2) immunostaining on embryonic hearts from E9.5 to E12.5. At E9.5, WT1 expression was noted in the proepicardium (PE), which was a grape-like cell protrusion from the venous pole of the developing heart tube, while Raldh2 was not expressed in epicardial cells before E10.5 ( Figure 1A, B, E, F). After E11.5, the epicardium formed a thin cell monolayer covering the growing myocardium (Figure 1C, D, G, H). A recently reported singlecell sequencing study has shown epicardial cells account for no more than 10% of all heart cells during the development. 29 The WT1 GFPCre/+ mice line is a powerful reporter line to label the WT1positive epicardial cells with GFP expression ( Figure 1I). We confirmed the low proportion of epicardial cells by Fluorescenceactivated Cell Sorting (FACS) analyses of E12.5 embryonic hearts from WT1 GFPCre/+ mice ( Figure 1J, K). The immunofluorescence staining of WT1 further proved that the cells obtained from FACS were highly purified epicardial cells ( Figure 1L). Taken together, these data demonstrated that the complete epicardial layer was formed after E11.5 which is suitable for cell culture and epicardial cells constitute a modest fraction of the foetal cardiac cells.

| Serial passage of primary epicardial cells was limited
Tissue attachment was adopted to separate highly purified primary epicardial cells from E11.5 mouse hearts. The schematic representation of the cell isolation strategy is shown in Figure 2A. The isolated epicardial cells showed typical epithelial cell characteristics of cobblestone morphology ( Figure 2B). Immunofluorescence staining showed that these cells expressed WT1 ( Figure 2C Figure 2E). Taken together, these results revealed that the primary epicardial cells of high purity were successfully isolated by the tissue attachment method.
To expand the epicardial cell amount, we continuously passaged 3 generations from P0 to P3. We found that P0 and P1 were typical cobblestone-like cells. However, the shape of the cells became large and flattened at P2 and P3 ( Figure 3A

| Establishment of an immortal epicardial cell line EpiSV40
To establish an immortal epicardial cell model, we transfected SV40-LT into the primary epicardial cells P1 separated from E11.5 mouse hearts and obtained a cell line named EpiSV40 ( Figure 4A).
Immunostaining, qPCR and Western blot analysis showed a robust expression of SV40 T antigen in EpiSV40, which confirmed the successful infection ( Figure 4D,G-H). Karyotype analysis of EpiSV40 was performed, showing 40 chromosomes (20 pairs), consistent with normal mouse chromosomes ( Figure 4E). We further quantitatively established that on average EpiSV40 had 4.69 copies of the SV40-LT gene in the genome by ddPCR assay (Figure 4F). After ten passages, EpiSV40 still maintained typical cobblestonelike morphology like primary epicardial cells P1 ( Figure 4B). The expression of epicardial cell marker genes was not changed over passaging ( Figure 4C). Compared to primary epicardial cells, the expression of major epicardial marker gene Wt1 and EMT-related genes was not changed while epithelial genes expression decreased, indicating EpiSV40 might be more comparable to the primary epicardial cells on the EMT process ( Figure S1B). Immunostaining of EdU, pH3 and Ki67 exhibited that both of the primary epicardial cells P1 and EpiSV40 responded uniformly to serum stimulation, though EpiSV40 had a higher basal proliferation level ( Figure 5A-D). Meanwhile, the EdU, pH3 or Ki67 positive cells can be barely detected in primary epicardial cells P3, indicating the loss of proliferation capacity after serial passage ( Figure S1C).
TGFβ1 has been widely used to examine the capacity of migration and differentiation of the primary epicardial cells. [30][31][32] Scratch wound healing assay revealed that EpiSV40 migration was enhanced significantly by TGFβ1, which coincides with primary epicardial cells ( Figure 6A-D). Immunostaining showed that EpiSV40 differentiation  was induced by TGFβ1 with β-catenin decrease and up-regulation of α-SMA, a marker of the vascular smooth muscle cell (VSMC) ( Figure 6F-G). We detected the expression of EMT and VSMCrelated genes by qRT-PCR and found that EpiSV40 has a similar response to TGFβ1 stimulation compared to the primary epicardial cells ( Figure 6E), indicating EpiSV40 is suitable for EMT and VSMC differentiation research.
In conclusion, EpiSV40 had comparable proliferation, migration and differentiation capacities as primary epicardial cells, suggesting it is an adapted tool for epicardial cell in vitro study.

| D ISCUSS I ON
The epicardium plays an important role in cardiac development and heart repair. [3][4][5][6][7][8] Here, we established and characterized an immortalized mouse epicardial cell line EpiSV40, which maintained the morphology and proliferation, migration and differentiation abilities of primary epicardial cells, suggesting its utility as an ideal in vitro study model for epicardium research.
Primary culture of epicardial cells from the embryonic heart was a difficult process, and there are two methods for primary epicardial cells isolation: tissue attachment and FACS. The technical comparison of these two methods from different groups is detailed in Table 2. In brief, the tissue attachment method is gentler and has less impact on primary epicardial cells, in which cell morphology approximates the situation in vivo. 12 The FACS method requires enzymatic digestion and flow cytometry, which has a great impact on the cells, and the sorted cells are spindle-shaped mesenchymal cells. 3 Besides, this method is more laborious with additional steps, such as the non-specific labelling method of CMFDA incubation or the transgenic mice line carrying the fluorescent reporter for labelling the epicardium. 26,33,34 We obtained highly purified primary epicardial cells with cobblestone morphology using the tissue attachment method, which showed a robust expression of epicardial markers.
As shown in our study, the major constrain of the primary epicardial cells in vitro culture is low cell number and limited life span.
Immortalization of the primary mammalian cells is a simple and feasible approach to the manufacture of target cells for in vitro culture. SV40-LT is the most widely used gene for cell immortalization by inhibiting p53 functions to prolong cell cycle. 35  To sum up, we established an easy culture epicardial cell line, EpiSV40, to substitute primary epicardial cells for mechanism research. EpiSV40 has the cobblestone morphology and comparable functions to primary cells. We believe the application of EpiSV40 will facilitate the study of the epicardium in cardiac development and heart repair.

CO N FLI C T O F I NTE R E S T
The authors declare no competing conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.