Establishment of a Lentiviral Vector Encoding Human HGF and the Infection of Human ADSCs

The delivery of adipose-derived stem cells (ADSCs) for promoting tissue repair has become a potential new therapy, while hepatocyte growth factor (HGF) is an important growth factor with angiogenic, anti-fibrotic, and anti-inflammatory benefits. In this paper, hADSCs were separated, cultured and identified based on the expression of cell surface antigens and multiple differentiation potential. We successfully generated a lentiviral vector encoding human HGF, infected hADSCs with this vector and examined the protein expression pattern. Finally we found that the hHGF lentiviral vector was successfully generated, and the lentiviral vector was able to safely infect hADSCs with high infection efficiency, thereby producing cells that overexpressed hHGF, which may provide a new strategy for the treatment of ischemic heart disease (IHD) and other ischemic diseases.


Introduction
Ischemic heart disease (IHD) remains the leading cause of death in modern society. e pathologic mechanism of IHD is characterized by the irreversible loss of functional cardiomyocytes followed by myocardial �brosis and ventricular remodeling due to the decreased number of vessels, which diminishes the blood supply [1]. erapeutic angiogenesis is currently a popular focus of study in IHD research involving growth factor protein therapy, cell transplantation, and gene therapy [2]. With the development of vascular tissue engineering, stem cell technology has been widely used and represents the latest advances in this �eld. Mesenchymal stem cells (MSCs) are a group of heterogeneous multipotent cells that can be isolated from many tissue types. Many studies have focused on MSCs isolated from bone marrow. However, there are issues associated with the clinical use of MSCs derived from bone marrow, including pain, morbidity, and low numbers of harvested cells. In contrast, adipose tissue contains an abundance of adult stem cells (termed adipose-derived stem cells, ADSCs), and these ADSCs are easy to isolate and differentiate into osteogenic, chondrogenic, myogenic, endothelial, and neurogenic lineages [3,4]. Animal studies have shown that ADSCs display potential bene�cial effects for therapeutic angiogenesis [5,6].
HGF, which was originally identi�ed and cloned for hepatocyte, has been reported to exert mitogenic, angiogenic, antiapoptotic, and anti�brotic activity in various cell types [7]. Several studies have revealed that HGF is an endogenous cardioprotective factor, as it protects cardiomyocytes from acute ischemic death during acute myocardial infarction (AMI) and enhances the survival of cardiomyocytes exposed to oxidant stress [8,9]. Recent study showed that intramyocardial injection of HGF and microbubbles (MBs) in combination with insonation enhanced neovascularization and reduced ventricular remodeling and infarct size [10]. erefore, the angiogenic effects of ADSC transplantation combined with HGF expression directed into the infracted heart area may exert more bene�cial effects than either gene therapy or stem cell therapy alone. In the present study, we generated a lentiviral vector encoding human HGF, infected ADSCs with this vector, and examined the protein expression pattern to provide a new strategy for the treatment of IHD.

Materials and Methods
2�1� �denti�cation and �e�uencing of the pcD�A3�0�hH�� and hH�� PCR Ampli�cation Products� e pcDNA3.0 plasmid encoding human HGF (pcDNA3.0-hHGF) was a kind gi provided by Dr. Y. S. Zhou at the Chinese Academy of Military Medical Sciences. e pcDNA3.0 plasmid and hHGF open reading frame (ORF) sequences were obtained from the Invitrogen and NCBI websites. Based on the genomic sequences, we selected the Hind III, Xba I, BamH I, and Not I restriction enzyme sites, and we sequenced the DNA. e hHGF mRNA primers were designed using Primer 3 soware. e forward primer (5′-GATCCGCTAGCGCTACCGGTCGCCACC ATGTGGGTGACCAAACTCC-3′) and reverse primer (5′-TCACCATGGTGGCGACCGGTAGTGACTGTGGTACCT TATATGTTA-3′) both contained an Age I restriction enzyme site. e length of the ampli�ed segment was 2218 bp.
e PCR products were assessed via electrophoresis using a 1% agarose gel. e PCR products were isolated from the gel using a viltaLight lamp. e target gene PCR products were isolated and puri�ed using a gel extraction kit.

Cloning the hHGF Genomic Fragment into the pGC-E1
Vector. e puri�ed hHGF fragment and pGC-E1 gene plasmid were separately digested with Age I. Using an In-Fusion Kit, the hHGF fragment was ligated into the pGC-E1 expression vector, which was previously digested with Age I. e total ligation reaction volume was 20 mL (2 mL of 100 mg/mL vector DNA, 2 mL of 100 ng/mL hHGF fragment, 1 mL of 10x In-Fusion exchange enzyme buffer, 0.5 mL of In-Fusion exchange enzyme, and 13.5 mL of ddH 2 O). Two control groups, either without vector DNA or without hHGF, were included in the reaction system. e reaction was run at 23 ∘ C for 15 min followed by 42 ∘ C for 15 min, and the DNA cloning ligation system was then prepared.
Aer transforming the DNA into DH5 competent bacteria cells, which were grown in LB medium, the positive clones were identi�ed via PCR to verify the successful insertion of the hHGF fragment into the lentiviral shuttle plasmid, and the PCR products were subsequently analyzed via DNA sequencing by Bei�ing GeneChem Co. e �nal plasmid was termed pGC-E1-hHGF.

Production and Titration of the Recombinant Lentiviral
Vector. e recombinant pGC-E1-hHGF vector and two packaging components (pHelper1.0 and pHelper2.0) were extracted from the positively transformed bacteria. e plasmid DNA concentration was measured using UV A260/A280 absorption values (normally between 1.8 and 2.0). e recombinant lentiviral vector, Lenti-hHGF, was generated by cotransfecting 293T cells with 20 g of pGC-E1-hHGF, 15 g of pHelper1.0, and 10 g of pHelper2.0 in 10 cm dishes with Lipofectamine 2000 (Invitrogen, USA). e 293T cells were then cultured in DMEM (Gibco, Invitrogen) containing 10% heat-inactivated fetal bovine serum FBS (Gibco, Invitrogen). e culture supernatants were collected every 24 h for 3 days, �ltered through a 0.45 um pore size �lter, and concentrated twice via ultracentrifugation at 50,000 xg at 20 ∘ C for 120 min. e viral supernatants were concentrated 1,000 times by ultracentrifugation, resuspended in sterile phosphate-buffered saline (PBS), and then stored at −80 ∘ C until use. e virus titers were determined via a one-in-one whole dilution.

Isolation� Culture� and Identi�cation of ADSCs
2.4.1. ADSC Isolation and Culture. Human adipose tissue derived from patients undergoing selective section-assisted lipectomy was collected, aer obtaining informed consent from the patients, according to procedures approved by the Ethics Committee at Chinese Academy of Medical Sciences and Anhui Provincial Hospital of Anhui Medical University. is tissue collection procedure has been described previously [1�. Brie�y, adipose tissue was extensively washed with D-Hanks' solution (Gibco Life Technologies, Paisley, UK) to remove contaminating debris and red blood cells, cut into small pieces, and then digested with 0.2% collagenase II (Sigma, St. Louis, MO, USA) at 37 ∘ C for 30 min with gentle agitation. e collagenase was inactivated with an equal volume of DMEM/10% FBS. e cells were washed twice and plated in T-75 tissue culture �asks at a density of 2 × 10 6 cells/mL. e expansion medium contained 90% DMEM, 10% FBS, 10 ng/mL epidermal growth factor (EGF, Sigma), 10 ng/mL platelet-derived growth factor BB (PDGF-BB, Sigma), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, Invitrogen). e medium was changed 24 h later to remove the nonadherent cells. Once the adherent cells were more than 90% con�uent, the cells were separated and cultured.

e Multiple Differentiation Ability of ADSCs.
For osteogenic differentiation, cells at passage 5 were incubated in DMEM medium containing 10% FBS, 20 nM dexamethasone, 100 U/mL penicillin, 100 mg/mL streptomycin, 2.5 mg/mL amphotericin, 10 mM b-glycerophosphate, and 0.05 mM L-ascorbic acid-2-phosphate. Control cultures were fed only DMEM containing 10% FBS and antibiotics. Aer 21 days of culture, the osteogenic differentiation of stem cells was con�rmed via the positive alizarin red staining of the mineralized matrix.
For adipogenic differentiation, the cells were incubated with DMEM medium containing 3% FBS, antibiotics, 33 mM biotin, 17 mM pantothenic acid, 1 mM insulin, 1 mM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 mM rosiglitazone, and 5% rabbit serum for 3 days. e cells were then treated with inducing medium without rosiglitazone and IBMX. Aer 19 days of culture, the cells were �xed with 10% formalin and incubated for 20 min with Oil-Red O to visualize lipid droplets.

Infection of ADSCs with Lentiviral Vectors.
On the day of infection, the cells were plated at a density of 4 × 10 4 cells/well in 96-well plates along with lenti-hHGF or lenti-GFP at different multiplicities of infection (MOI) in serumfree growth medium containing 5 g/mL polybrene. Serumcontaining growth medium was added aer 4 h and replaced aer 48 h. Reporter gene expression was examined using �uorescent microscopy on day 4 or 5 aer infection. e ideal MOI for the formal experiment was selected. e infected cells were passaged, and the percentage of GFP + cells was assessed via �ow cytometry.

Detection of hHGF Protein in Target
Cells. e hHGF protein expression levels in the ADSCs were assessed via western blot analysis. Brie�y, uninfected ADSCs and ADSCs infected with either lenti-hHGF or lenti-GFP were harvested, and a speci�c volume of ice cold 2x lysis buffer was applied to the cells. e supernatant was collected, and the protein concentration was assessed using a Bradford assay. Sample buffer (2x) was then added to the samples (the volume of buffer depended on the concentration of the protein sample), and the proteins were denatured at 100 ∘ C for 5 minutes. e protein samples were then separated via 12% SDS-PAGE electrophoresis (25 g/pore). e proteins were transferred to PVDF membranes at 120 V for approximately 2 hours. Subsequently, the membrane was blocked with a 5% milk TBST solution overnight. e membranes were then incubated with primary antibody (1 : 1000 dilution) at room temperature for 2 hours, washed three times with TBST (3 × 10 min), incubated with secondary antibody (1 : 1000 dilution) at room temperature for 2 hours, and washed again. Finally, ECL reagent was added to visualize the protein bands. e �lm was exposed aer a 5-minute incubation with ECL reagent, and the �lm was then developed and �xed. Actin was used as a control to assess the relative expression levels of the proteins in each group.
To detect the expression of hHGF protein in supernatant, ELISA was used. Cell culture supernatants were collected from the culture media of uninfected ADSCs and ADSCs      Figure  1). e third clone was submitted for sequencing analysis. e base sequence of the inserted gene in the pcDNA3.0 plasmid matched the genomic sequence of hHGF provided by GenBank. As predicted, the hHGF product generated by the two primers ran at approximately 2218 bp on a gel ( Figure  2).

pGC-E1-hHGF Plasmid Construction and Sequencing.
e puri�ed hHGF fragment and the pGC-E1 gene plasmid were digested separately with Age I, and the pGC-E1-hHGF vector was generated via In-Fusion enzyme ligation. DH5 competent bacteria cells were transformed, and three positive bacterial clones containing the hHGF coding sequence were identi�ed via �CR. e product size was 537 bp (Figure 3). e recombinant pGC-E1-hHGF plasmid was con�rmed via sequencing, and the sequence was consistent with the hHGF gene sequence provided by GenBank (Figure 4).

Production and Titration of the Recombinant Lentiviral
Vector. Aer cotransfection of 293T cells with the three plasmids, GF� expression was assessed using a �uorescence microscope to ensure that the lentiviral vectors were properly generated ( Figure 5). e virus titers were determined via a one-in-one whole dilution, and the �nal virus titers were 1 × 10 8 TU/mL aer concentration.
3.4. Immunophenotype Analysis of ADSCs. e freshly isolated ADSCs displayed adherence and expansion in culture, and they assumed a �broblast-like morphology when observed using a light microscope. Aer the third passage, a FACS analysis revealed that the ADSCs were positive for CD29, CD44, and CD71, but the ADSCs assayed negative for hematopoietic and endothelial lineage markers, including CD31, CD34, CD45, CD106, and CD117, resulting in a phenotype similar to that of bone marrow MSCs. Moreover, immunogenicity markers, including CD14, CD45, CD86, and HLA-DR, were not observed ( Figure 6).

Osteogenic and Adipogenic Differentiation of ADSCs.
e osteogenic differentiation of human ADSCs at passage 5 was con�rmed via alizarin red staining (Figure 7(a)). Aer feeding the ADSCs with osteogenic-inducing media, dark red mineralized bone matrix (bone nodules) was observed within the alizarin red-stained section. e adipogenic differentiation of human ADSCs was con�rmed via Oil Red-O staining. Aer feeding the ADSCs with adipogenic-inducing media for 21 d, oil droplets were observed in the cytoplasm (Figure 7(b)).

e Infection Efficiency of ADSCs with Lentiviral Vectors.
On the day of infection, hADSCs were infected with either lenti-hHGF or lenti-GFP at an MOI of 100, or 200. GFP expression was observed via �uorescent microscopy ( Figure  8), and the infection efficiency rate was assessed via �ow cytometry. e results indicated that the efficiency rate of infection with an MOI of 200 was (53 ± 15)%, which was higher than (36 ± 9)%, the efficiency rate of infection with an MOI of 100.

hHGF Protein Expression in
ADSCs. e protein lanes corresponding to the uninfected ADSCs and the ADSCs infected with either lenti-hHGF or lenti-GFP are shown in Figure 9(a). e actin protein ran at 42 kDa, and the HGF protein ran at 83 kDa. Both the HGF gene-infected ADSCs and the uninfected ADSCs expressed HGF, but the expression level observed in the lenti-hHGF-infected group represented the highest level of expression of the three groups. ELISA found that both gene infected and uninfected ADSCs produced hHGF protein, but the concentrations were signi�cantly higher in hHGF infected group at different time points aer infection (P < 0.001). And the infected cells secreted the peak level of hHGF at 5 days aer infection (Figure 9(b)).

Discussion
Stem cells possess the ability to self-renew and differentiate into multiple cell types. Due to their reproducibility and multipotency, they have a signi�cant role in many clinical and preclinical �elds �11]. Stem cells can be harvested from various mesenchymal sources, such as bone marrow, peripheral blood, cord blood and adipose tissue. e most common source of stem cells is bone marrow. However, harvesting stem cells from bone marrow causes pain and discomfort to patients, and only a relatively small number of cells can be harvested. Currently, an increasing number of researchers are focusing on ADSCs because they are multipotent, immuneprivileged, abundantly harvested, and easily expanded ex vivo. In the present study, ADSCs were isolated from adult human adipose tissue and subse�uently ampli�ed in culture.
e cultured ADSCs were positive for CD29, CD44, and CD71 expression, but they did not express hematopoietic and endothelial lineage markers (CD31, CD34, CD45, CD106, and CD117). e phenotype of these cells was similar to that of the MSCs derived from bone marrow with the exception that MSCs express CD106. Additionally, the expression of immunogenicity markers (CD14, CD45, CD86, and HLA-DR) was not observed, which demonstrated that the human ADSCs were immune privileged. e osteogenic-and adipogenic-induced differentiation experiments con�rmed that the obtained ADSCs possessed multiple differentiation abilities.
Single-stem cell therapy is characterized by some de�ciencies, such as the instability of stem cells, low cell numbers, and low activity. erefore, stem cells combined with cytokines are more effective, and this combination has become the new therapy for IHD. HGF, which was originally identi�ed and cloned as a potent for hepatocyte, has been reported to possess mitogenic, angiogenic, antiapoptotic, and anti�brotic activity in various cells. Many studies have shown that acute myocardial infarction, ischemia reperfusion injury, and congestive heart failure induce the expression of HGF in the heart. HGF gene therapy can improve cardiac function via the induction of angiogenesis, reduction of �brosis, and recruitment of stem cells derived from bone marrow, which affects myocardial regeneration [12]. To express HGF in the target cells, we selected a lentiviral vector for the gene therapy study because lentiviral vectors have several attractive gene delivery vehicle properties as follows: (i) sustained gene delivery via stable vector integration into the host genome; (ii) the capability of infecting both dividing and nondividing cells; (iii) broad tissue tropisms, including important types of gene-and cell-targeted therapy; (iv) no viral protein expression aer vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic and introncontaining sequences; (vi) a potentially safer integration site pro�le; (vii) a relatively easy system for vector manipulation and production [13].
In this study, we constructed a HGF recombinant lentiviral expression vector. Using molecular cloning techniques, such as PCR and DNA sequencing, the human HGF lentiviral expression plasmid was successfully constructed, and high titer viral particles were obtained aer plasmid transfection into packaging cells. e HGF-GFP expression levels reached more than 60% when human ADSCs were infected. e western blot and ELISA results showed that HGF geneinfected ADSCs overexpressed HGF. ese results demonstrated that the lentiviral vector successfully delivered the human HGF gene into ADSCs and mediated high levels of HGF expression in ADSCs. In the future, ADSCs infected with the recombinant human HGF lentiviral vector will be used to study the function of the target gene in combination with stem cells in IHD and other ischemic diseases.