2.1. Cell Culture. Human Embryonic Kidney 293T (HEK293T) and breast cancer cell line 4T1 were purchased from Mede Bioeconomy Company, Iran, and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Darmstadt, Germany). The cells were supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), penicillin (100 units/ml)/streptomycin (100 mg/ml) (Invitrogen, Carlsbad, CA, USA). Finally, the cells were incubated in a humidified atmosphere with 5% CO2 at 37°C.
2.2. Gene Design. The coding sequences (CDS) of EPO (Accession#: XM_001468996.1) were retrieved from GeneBank, NCBI. The sequences were synthesized by Genscript, USA, and were incorporated into pUC57 plasmid. In this experimental study, a dual promoter lentiviral vector, pCDH-513B, was purchased from System Bio, USA. The first promoter is the cytomegalovirus (CMV) promoter with a downstream multiple cloning site (MCS) used for gene cloning. The second promoter is the EF1a promoter which regulates the expression of CopA-GFP (copepod green fluorescent protein) (cGFP) and puromycin resistance genes.
The Plasmid LOX-GFP-IRES-TK was obtained from Mede Bioeconomy Company, Iran. Then, EPO and herpes simplex virus type 1 thymidine kinase (HSV1-TK) fragments were sub-cloned into pCDH-513B under a CMV promoter with SpeI-EcoRI restriction enzymes (NEB, USA). To confirm the cloning procedure, the recombinant pCDH-EPO and pCDH-TK were digested using XbaI-ApaI andSpeI –EcoRI, respectively, and then sequenced.
2.3. hWJMSCs Isolation and Characterization. Human healthy umbilical cord (UCs) Wharton’s jelly tissue (n= 1) was collected from full-term newborn in Vali-e-Asr Central Hospital and processed after obtaining the mother's informed consent and approved by the Ethics Committee of Fasa University of Medical Sciences (IR.FUMS.REC.1397.177). It was washed in phosphate-buffered saline (PBS) to eliminate the blood clots, disinfected, and cut into 1-2 mm lengths, and then were expanded on culture dishes with a minimum of Dulbecco’s Modified Eagle Medium containing Nutrient Mixture F-12 (DMEM/F12) medium (Gibco, USA), supplemented with 30-40% FBS. Then, it was incubated at 37°C with 5% CO2 which routinely monitored. After one week, solid umbilical cord pieces were removed, and cell migration was evaluated under the invert light microscope. Upon reaching 90% cell confluence, in the second changing medium, the adherent cells detached by the addition of 0.25% Trypsin-Ethylene Diamine and re-plated, usually at 4_6 days’ intervals[25]. For adipogenic, osteogenic, and chondrogenic differentiation, a six-well plate was cultured with 1× 105 cells per well. In the third passage, after reaching a 40-45% cell confluence, an adipogenic differentiation medium (Gibco, USA), osteogenesis supplement (Gibco, USA), and chondrogenic supplement (Gibco, USA) were added to the basic medium, respectively. After about 20 days, lipid droplets were visualized using Oil Red O (Sigma-Aldrich, USA) staining. Successful osteogenic differentiation was verified by Alizarin Red (Sigma-Aldrich, USA) staining. Safranin-O (Sigma-Aldrich, USA) staining was used to determine the presence of proteoglycans (PGs). In the control group, hWJMSCs were grown in the culture medium without adipogenic, osteogenesis, and chondrogenic supplement. Passage 3 WJMSCs were used in all experiments. A small number of undifferentiated hWJMSCs (passage 3, 105 cells) were analyzed using BD FACSCalibur flow cytometry (BD Bioscience, USA) to evaluate surface markers expressed by hWJMSCs. After adding specific antibodies at the recommended concentrations, the tubes were incubated in the dark at room temperature for 30-60 minutes. Then, flow cytometry analysis was performed to study two positive markers _CD105 and CD90_ and two negative markers _ CD45 and CD34_ and data were analyzed using FlowJo (version 7.6.1) software.
2.4. Lentivirus Production. HEK293T was used to produce two recombinant lentivirus rLV-GFP, rLV-EPO, and rLV-TK using transfer vectors pCDH, pCDH-EPO, and pCDH-TK, respectively. To do this, due to Prof. Trono lab protocol, CaPO4 transfection of HEK293T cells was performed with some modifications using the following amounts of DNA: 21 μg transfer/control vector, 10.5 μg pMD2.G vector, 15 μg pMDLg/pRRE vector, and 13 μg pRSV-Rev vector which all were dissolved in HEPES buffered water to reach 921μl. Then, 33μl Tris-EDTA (TE) buffer was added, and the mixture was strongly mixed and kept at room temperature for 3 minutes. Then, 105 μl CaCl2 2.5 M was added, and the mixture was strongly vortexed and left for 3 min for making DNA-CaCl2 interaction. Then, 1050 μl HEPES 2X was added while the mixture was being vortexed. HEK293T cells (2×106 cells) medium was changed 2 h before transfection. Early-passage HEK293T cells (passages under 15) with 80 % confluency was co-transfected by plasmids in a way that 2100 μl transfection master mix was added per 10 cm HEK293T cells as a droplet to all areas of the plate. Then, they were incubated at 37 °C in 5 % CO2 for 16 h.
After 24 hours, the transfection efficiency was assessed by GFP expression and visualized by an inverted fluorescent microscope (Leica, German). We selected five fields randomly under a fluorescent microscope, and the percentage of GFP-positive cell numbers determined the transfection efficiency to total cell number. Mediums containing the virus were collected after 24, 48, and 72 h following transfection which were passed through 0.25μm pore filters to remove cellular debris. Recombinant lentivirus concentration was measured by the addition of polyethylene glycol (PEG) 600 50%, NaCl4 M, and PBS to recombinant viruses inside polypropylene bottles which stored at 4°C for 1.5 hours. Then, the tubes were centrifuged at 15000 rpm for 15 minutes at 4°C. To determine the titration of recombinant lentiviral, the number of GFP positive cells were counted using flow cytometry according to the equation “TU (Transduction Unite/ml) = [F × C/V] × D” in which F is the frequency of GFP-positive cells, C is the total number of cells in the well at the time of transduction, V is the volume of inoculum in mL, and D is the lentivirus dilution factor. Fresh recombinant titrated viruses at the volumes of 1000, 500, 100, 50, 20, and 0 μl were used for transducing hWJMSCs and 4T1 cells.
2.5. Transduction and Viability Assay. The second passage of hWJMSCs and 4T1 cells were cultured at low confluency of 30-40% in a 6-well dish and incubated at 37 °C, 5 % CO2 overnight. Then, they were transduced by rLV-GFP, rLV-EPO, and rLV-TK to generate rhWJMSCs, rhWJMSCs-EPO, and r4T1-TK respectively. Cell transduction was evaluated using a fluorescent microscope, 72 hours after the transduction, and was compared with non-transduced MSCs and 4T1. Puromycin (1.5 μg/ml) selection started 72 hours after the transduction at passage 3 for the next 5 days. Then, we analyzed them morphologically by phase-contrast microscopy to confirm that transduction with lentiviral vectors has not changed the morphology of WJMSCs.
For MTT assay, we cultured 5×103 cells from transduced and normal hWJMCSs and 4T1 cells in 96-wells plates. After 1 day, we added MTT reagents and incubated them for 4 hours. Also, to measure r4T1-TK cells' sensitivity to GCV, we add 20 μg/ml GCV to the r4T1-TK cells culture medium. Then, we added the dimethyl sulfoxide (DMSO) to terminate the reaction and the plate was read at 570 nm wavelength using BioTek Instruments (Vermont, United States) microplate reader.
2.6. Western Blot Analysis. EPO and TK gene expression was confirmed after the transduction by western blotting assay. rhWJMSCs-EPO and r4T1-TK supernatants were collected 72 hours after the transduction. Evaluating the protein concentrations was examined using a BCA Protein Assay Kit (Thermo Fisher, USA). Equivalent amounts of proteins (30 µg/lane) were loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes (Bio-Rad, USA). The membranes were blocked using 5% non-fat milk and immunoblotting was performed using antibodies against EPO and TK (Santa Cruz, USA). Proteins of interest were detected using HRP-conjugated sheep anti-mouse IgG antibody (Abcam, ab6785). Finally, the protein bands were visualized using chemiluminescence (ECL) reagent, and the integrated optical density (IOD) of each protein band was measured. The internal standard β-actin adjusted IOD values.
2.7. CRA Mice Model. The mice were obtained from the laboratory animal center of Pasteur Institute of Iran. Male and female BALB/c mice (6- to 8 weeks old, n=60; weight, 18-20 g; n=10 mice/group) were housed and treated in a pathogen-free environment with the access to autoclaved food and water ad libitum according to national rules on animal experiments. Also, the mice were maintained on an iron-sufficient diet for 2 weeks before the injection of tumor cells or saline, which continued until the end of the study[26]. 5 ×105 recombinant 4T1-TK cells diluted in 100 μl PBS were injected into the right fourth mammary gland of mice using a 25-gauge needle[27]. The tumor was palpably detected after one week of injection in all 60 mice; thereafter, twice a week routinely, the tumor volume was measured until the end of the study. Because r4T1-TK tumors were metastatic within 2–3 weeks after injection, to evaluate the consequences of tumor progression on erythropoiesis compare to the control group, peripheral blood samples were obtained from the tail vein at week 3 and were analyzed using a Sysmex XT-2000i automated hematology analyzer (Sysmex Corp., Hyogo, Japan) for the measurements of red blood cell (RBC) count, reticulocyte numbers, Hct, and Hb concentration to confirm anemia in mice. Those r4T1-TK-bearing BALB/c mice qualified for developing the anemia were randomly divided into 6 groups (n= 10 mice/group). In this study, taking into account our published findings in cancer-free and cancerous mice, three groups of animals were treated intraperitoneal (IP) injection with Ganciclovir (GCV, 100 mL) at 75 mg/kg twice daily for 10 continuous days[28]. The other 3 groups of animals received PBS for the same period. The experimental animal protocols were performed based on the specific National Ethical Guideline for Biomedical Research issued by the Research and Technology Deputy of Ministry of Health and Medicinal Education (MOHME) of Iran (2005).
2.8. Implantation of rhWJMSCs-EPO. We evaluated our anemia treatment protocol in six groups of animals, all of which had anemia (3 cancer-free groups and 3 cancerous groups) according to the following instructions: A: cancer-free mice that received a moderate dose of rhWJMSCs-EPO to evaluate the effects of treatment during cancer suppression. B: control cancer-free mice that received control rhWJMSCs. C: control cancer-free mice that received PBS. D: control cancerous mice that received rhWJMSCs-EPO to evaluate treatment effects during the active phase of cancer. E: cancerous control mice which received rhWJMSCs. F: control cancerous mice which received PBS. Based on recent studies, 10×106 MSCs expressing EPO is considered a high dose of treatment which can develop polycythemia and 4.5×106 MSCs expressing EPO as a low dose of treatment has shown no correction of anemia[29]. So, to avoid polycythemia and also to achieve a better therapeutic response, we used a moderate dose of rhWJMSCs-EPO (~7×106 ) implanted into mice's skeletal muscle with a 29-G insulin syringe[29].
For laboratory measurements, the whole peripheral blood samples were collected from the tail vein once per week which were immediately analyzed for the measurements of Hct and Hb plasma levels. Moreover, the changes in plasma EPO levels in all CRA mice were detected by ELISA (Quantikine ELISA kit, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.
2.9. Statistical Analysis. All data are present as mean ± standard error of the mean (S.E.M.). The statistical analysis of data was performed using the Student’s t-test and analyzed by Prism software (GraphPad, San Diego, CA). P-value < 0.05 were considered statistically significant.