This article is mentioned in:

Introduction

Mesenchymal stem cells (MSCs) have self-renewal capabilities and multi-directional differentiation potential, and are induced by different conditions to differentiate into various different tissues such as bone, cartilage, adipose, muscle, endothelial, epithelial and nerve tissue (1,2). MSCs may be used in transplantation for the treatment of systemic diseases, including leukemia, autoimmune disease and β-cell defects in diabetes (3). In vivo imaging technology may be used to dynamically observe the short-term distribution, homing and long-term survival of transplanted MSCs (4,5). The intravital tracer technique means that in stem cell transplantation, cells are able to be dynamically monitored in vivo to assess their migration and survival non-invasively (6–8). To obtain high definition images with high sensitivity, tracer technology for stem cells should be combined with multi-mode imaging (9). It is therefore of interest to establish a stem cell line with multi-mode imaging function. Generally, stem cell engineering studies require genetic modification of stem cells. At present, the instruments typically used to modify genes in mammalian cells include plasmid vectors, adenovirus vectors, retroviral vectors and lentiviral vectors (10). Lentiviral vectors are vectors for gene modification developed based on human immunodeficiency virus (HIV) (11). They are able to infect dividing and non-dividing cells, and integrate the target gene into the chromosomes of primary cells, stem cells and practically all cell types. Furthermore, the use of lentiviral vectors has few safety concerns and they are able to be expressed in vivo for a long time (12). These characteristics make the lentivirus vector an ideal instrument for gene modification (13,14). In the present study, multiple labeling was performed for human umbilical cord mesenchymal stem cells (hUCMSCs) to enable them to be displayed using isotopic imaging, magnetic resonance imaging (MRI) and in vivo fluorescence imaging in order to establish effective in vivo tracer technologies, provide the basis for in-depth studies on hUCMSCs and enhance their homing ability.

Materials and methods

Extraction of hUCMSCs

hUCMSCs were obtained from umbilical cords harvested from 3 patients recruited to the Department of Obstetrics and Gynecology, the First Affiliated Hospital of Jinan University (Guangzhou, China) April to May 2013. The present study was approved by the Ethics Committee of the First Affiliated Hospital of Jinan University, and informed written consent was obtained from all patients from whom tissue was collected. Blood was removed from the blood vessels of the umbilical cord, and blood on the surface was washed off. The outer membrane of the umbilical cord was opened to excise veins and arteries, and hUCMSCs were harvested under aseptic conditions within 4–6 h of sample collection. Umbilical cord amniotic epithelium were removed to obtain Wharton's jelly, which were further dissected into ~1 mm3 sections. The sections were incubated with 0.1% collagenase IV and digested at 37°C for 24 h. The mixture was centrifuged at room temperature at 1,000 × g for 5 min. The pellet was resuspended with 0.1% trypsin and further digested for 30 min at 37°C, and then was filtered through a 74 µm cell strainer. The filtrate was centrifuged at 1,500 rpm for 10 min. The cells were then completely resuspended and cultured in a 6-well plate at a density of 5×104 cells/well in DMEM/F12 containing 2 ng/ml bFGF and 10% fetal bovine serum (all Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C under 5% CO2 and relative humidity (100%). The culture solution was replaced 2 days later and the red blood cells and parenchyma cells that did not adhere to wall in the culture solution were removed. The culture solution was added again and became clear. Cellular morphology of hUCMSCs were observed every day under an inverted light microscope (Olympus CKX41; Olympus Corporation, Tokyo, Japan) at a magnification of ×100 under normal culturing conditions.

hUCMSC identification and Induction of adipogenic and osteogenic differentiation

To confirm that the isolated and purified hUCMSCs were differentiated successfully, the following surface markers were selected: Cluster of differentiation (CD)73, CD90, CD105, hematopoietic stem/progenitor cell marker CD34, hematopoietic cell marker CD45. The expression of these markers was examined by flow cytometry. Briefly, a 50 µl cell suspension was seeded at a concentration of 2×107 cells/ml in L-15 medium (Sigma-Aldrich; Merck MgaA). A total of 50 µl CD73 (cat. no. ab175396), CD105 (cat. no. ab49228), CD90 (cat. no. ab11153), CD34 (cat. no. ab185732), CD45 (cat. no. ab38436; all 1:500; all Abcam, Cambridge, UK) antibodies was added to the cells and incubated at 4°C in dark for 30 min. The cells were then incubated with goat anti-rabbit immunoglobulin G (cat. no. ab6795; Abcam) as secondary antibodies at room temperature for 1 h. Wash cells by adding 2 ml L-15 and performing centrifugation for 5 min at 300 × g and room temperature. The stained cell pellet was resuspended in 400 ul L-15 at 4°C and blocked by 5% skimmed milk at room temperature for 15 min. Flow cytometry analysis was conducted by CellQuest Software Pro 5.1 and a BD FACSVerse™ flow cytometer (both BD Biosciences, Franklin Lakes, NJ, USA). CD73, CD90, CD105 were highly expressed on the differentiated hUCMSCs, while CD34 and CD45 were barely detected on these cells.

The hUCMSC cells (5×103 cells) were seeded on 3 cm dishes in DMEM/F12 supplemented with 2 ng/ml bFGF and 10% FBS for 2 days. The cells were incubated with osteogenic or adipogenic differentiation media for 4 weeks. The adipogenic differentiation medium was composed of DMEM/F12 supplemented with 10% FBS, 100 nM dexamethasone, 50 µg/ml ascorbic acid and 50 µg/ml indomethacin. The osteogenic differentiation medium was composed of DMEM/F12 supplemented with 10% FBS, 10 nM dexamethasone, 10 mM β-glycerophosphate and 50 µg/ml ascorbic acid. As a negative control, cells were cultured in DMEM/F12 supplemented with 2 ng/ml bFGF and 10% FBS. Adipogenic differentiation was detected by Oil Red ‘O’ staining while osteogenic differentiation was detected by Alizarin Red-S staining as previously described (15) and digitalized for analysis using a Leica light microscope DMI3009B (Leica Microsystems GmbH, Wetzlar, Germany).

Construction of lentiviral vector

Obtainment of target gene human sodium/iodide symporter (hNIS)

Expression plasmid pCMV-Tag2-hNIS preserved by the Central Laboratory at the First Affiliated Hospital of Jinan University was used. Primers were designed using Oligo 5.0 software (Molecular Biology Insights, Inc., Cascade, CO, USA) with reference to the hNIS-cDNA sequence registered in GeneBank (NM_000453.2). Upstream primer for NIS forward, 5′-GAATTCGCCACCATGGAGGCCGTGGAGAC-3′ (EcoR I) and reverse, 5′-GCGGATCCTCAGAGGTTTGTCTCCTGCTGGTCTC-3′ (BamH I), were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Purification of plasmid

A total of 4 plasmids pSico-enhanced green fluorescent protein (EGFP), pMDLg-pRRE, pRSV-REV and pMD2G were transformed. Briefly, 1 µg plasmid and 50 µl E. coli competent cells (Takara Biotechnology Co., Ltd., Beijing, China) were mixed in a 1.5 ml tube for 30 min at room temperature, then the tube was incubated at 42°C for 90 sec. The tube was placed immediately on ice for 2 min. A total of 500 µl LB medium (Thermo Fisher Scientific. Inc.) was added to each tube, which was incubated for 1 h at 37°C. Subsequently, 100 µl of the suspension was placed on a LB agar plate and incubated at 37°C for 12–16 h. A fresh toothpick was placed on a colony, which was selected by adding streptomycin 50 mg/ml, kanamycin 50 mg/ml and ampicillin 60 mg/ml; the toothpick was then dipped into a 15 ml tube containing 3–5 ml super optimal broth (SOB) medium (Spectrum Laboratory Products, Inc., Shanghai, China). The tube was vigorously shaken at 37°C (speed, 90 × g) for 8 h. The plasmids DNA used for transfections was prepared with the Endofree Plasmid Maxi kit (cat. no. 12362, Qiagen, Hilden, Germany) according to manufacturer's protocol.

Lentiviral titer determination

The 293T cells (Thermo Fisher Scientific. Inc.) were used for virus titer determination. Cells were inoculated in 96-well plates at a density of ~5×104 cells/well within 100 µl medium of DMEM containing 10% fetal bovine serum (FBS) at a 37°C incubator with 5% CO2. A total of 10 sterile eppendorf (EP) tubes containing 90 µl fresh medium of DMEM containing 10% FBS were used; 10 µl virus stock was added into the first EP tube and mixed well, and 10 µl solution was transferred from the first EP tube into the second. This process was repeated for the remaining tubes until the virus stock was diluted 10 times. A total 9 µl medium was removed from 10-well in the 96-well plate and 9 µl virus solutions from each of the prepared 10 tubes were added to each well, thus there were 5,000 cells in each well, and the concentration of the virus/well were 1, 10−1 and 10−2 µl, respectively. The cell culture plate was incubated at 37°C in an atmosphere containing 5% CO2 for 48 h. A total of 100 µl fresh medium was added into each well and incubation was resumed for 96 h. GFP fluorescence expression was subsequently observed under a fluorescence microscope and the number of fluorescing cells in the last two wells were counted.

Superparamagnetic iron oxide (SPIO)-labeling of hNIS-EGFP-hUCMSCs

hNIS-EGFP-hUCMSCs were incubated with SPIOs (50 µg; Biopal, Inc., Worcester, MA, USA). SPIO solutions were prepared at a 2× concentration in DMEM/F12 for 24 h. A total of 5 ml of 2× SPIO solutions were added to 5 ml of a hUCMSCs suspension in a 10 cm dish (5×104). The cells were plated on a 96-well plate, 100 µl for each, then 10 µl MTT was added to per well and incubated for 4 h, finally 100 µl DMSO solution was added per well until the formazan crystals dissolved, which was detected with 490 nm. Growth medium without SPIOs was added to sister cultures of hUCMSCs, these cells were used as the controls. Following incubation with SPIO, the cells were collected and washed twice in PBS; labeling efficiency was determined by Prussian Blue staining at room temperature for 30 min. The cellular morphology of hUCMSCs labeled with SPIO was observed under the inverted light microscope (Olympus CKX41, ×400) for 8 days.

In vitro study of iodine uptake in transfected hUCMSCs

The experimental groups (SPIO-hNIS-EGFP-hUCMSCs) transfected with the lentivirus-hNIS-EGFP using the same kit and protocol as the 293T transfection. The control group (hUCMSCs) were untransfected cells. All groups were conventionally cultured and incubated in a 24-well plate at a density of 5×104 cells/well at 37°C in an atmosphere containing 5% CO2 overnight. When cells reached 80% confluence, the medium (DMEM/F12 containing 2 ng/ml bFGF and 10% FBS) was discarded and the cells were washed twice with Hank's Balanced Salt Solution (HBSS; Invitrogen; Thermo Fisher Scientific, Inc.). The 125I uptake method was as previously described (16). Briefly, 1 ml HBSS (containing 0.1 µl of 100 uM sodium citrate 125I) was added to each well and incubated at 37°C for 30 min. The NaClO4 inhibition test was conducted by incubation the cells with 300 µmol/l NaClO4 for 60 min. This medium was discarded, cells were washed twice with cold HBSS, then cold 95% ethanol was then added to the cells and they were incubated at room temperature for 20 min. The concentrated iodide in the cytolysate was determined with a γ-counter (2480 WIZARD2™ gamma counter; PerkinElmer, Inc., Waltham, MA, USA). The experimental and control groups each consisted of 6 independent wells in the 24-well plate.

Amplification of the hNIS gene

mRNA was extracted from the hUCMSCs using TRIzol and hNIS mRNA was amplified with one-step reverse transcription polymerase chain reaction (RT-PCR). The RT-PCR system solution was as follows: 20 µl of RNase Free dH2O, 25 µl of 2×1 Step Buffer, 2 µl of PrimeScript 1 Step Enzyme Mix (Takara Biotechnology Co., Ltd.), 2 µl of upstream primers, 3 µl of downstream primers and 1 µl RNA samples. The primers were as follows: hNIS forward, 5′-CACCATGGAGGCCGTGGAG-3′ and reverse, 5′-GAGGTTTGTCTCCTGCTGGTCTC-3′; β-actin forward, 5′-CTCCATCCTGGCCTCGCTGT-3′ and reverse, 5′-GCTGTCACCTTCACCGTTCC-3′. The thermocycling conditions were as follows: 50°C for 30 min, 94°C for 2 min, 30 cycles of 94°C for 30 sec, 50–65°C for 30 sec and 72°C for 1 min, and 72°C for 10 min. The enzyme digestion system was: 1 µl 10X NEB-buffer, 0.5 µl EcorI, 0.5 µl xhol1, 5 µl PCR products/P3.1 vector (Hangzhou Xiaoyong Biotechnology Co., Ltd., Hangzhou, China) and 3 µl ddH2O. The cells were then incubated with the digestion in 37°C for 2 h prior to the PCR product was identified by 1% agarose gel electrophoresis, which was determined to have the same size as the target gene.

Colony PCR for recombinant plasmid

NIS gene segment and P3.1 vector were linked using the following protocol: 1 ul 10X buffer, 2 ul NIS gene segment, 4 ul P3.1 vector, 1 ul T4 ligase (Thermo Fisher Scientific, Inc.) and 2 ul ddH2O were mixed overnight at 4°C. The NIS gene segment and P3.1 vector was then transformed in complete medium (DMEM and 10% FBS) for 48 h at room temperature and coated on a LB medium plate without any antibiotics. Two monoclones were selected from the resistant plate with clear colony growth and amplified by PCR with Pn1 and Pn2 as the primers. The PCR product was identified by electrophoresis using a 1% agarose gel with ethidium bromide. The two monoclones revealed 1,900 bp gene segments, which was consistent with the theoretical value. It was preliminarily proven that these two colonies contained the correctly linked recombinant plasmids.

Screening of cell line stably expressing hNIS by puromycin

Determination of puromycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) screening concentration revealed that hUCMSCs were all killed following 7 days of exposure to 8 µl/ml puromycin. At concentrations <8 µl/ml, some hUCMSCs were not apoptosed; therefore 8 µl/ml was selected as the optimal puromycin screening concentration. hUCMSCs transfected with lentivirus were subsequently sub-cultured at a dilution of 1:10. When cell attachment occurred, 8 µl/ml puromycin was added to the medium. At 7 days following screening, positive cell colonies of hUCMSCs stably expressing hNIS and EGFP were obtained by screening with 8 µg/ml puromycin, and these were named as hNIS-EGFP-hUCMSCs and transferred to a 6-well plate for multiplication culture.

Identification of hNIS and EGFP expression in hUCMSCs using western blotting

Protein was extracted from 2×105 cells using RIPA buffer (Thermo Fisher Scientific, Inc.) containing protease inhibitors (Roche Diagnostics, Basel, Switzerland). Protein concentration in the supernatants was measured using a bicinchoninic acid assay protein assay kit (cat. no. 23222; Thermo Fisher Scientific, Inc.). A total of 60 µg protein was transferred into an EP tube with 4X loading buffer and mixed well. The mixture was boiled for 10 min and centrifuged at 2,000 × g for 5 min. Proteins (10 µg/lane) were subsequently separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Membranes were washed with Tris-buffered saline with 0.1% Tween-20 and blocked at room temperature in PBS with 0.1% Tween-20 (PBST) containing 5% skim milk powder for 1 h. Membranes were subsequently incubated at room temperature for 1 h with anti-hNIS (cat. no. ab83816; Abcam, Cambridge, UK), anti-EGFP antibodies (cat. no. 2956; Cell Signaling Technology, Inc., Danvers, MA, USA; both 1:3,000) and β-actin (cat. no. BM3501-01, 1:5,000; Biomiga, Inc., San Diego, USA) and washed with PBST. Membranes were incubated at room temperature for 2 h with the secondary horseradish peroxidase-labeled anti-Rabbit IgG (cat. no. ab191866; 1:10,000; Abcam), washed with PBST, then treated with SuperSignal West Pico Chemiluminescent Substrate (cat. no. 34077; Thermo Fisher Scientific, Inc.) for 1 h; the exposure time of X-ray film was 30 min. The membrane was washed with 0.5 mol/l NaOH for 15 min.

Statistical analysis

SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) was used to perform statistic analysis. Experimental data are expressed as the mean ± standard deviation. Independent-samples t-tests were used to compare differences between groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Morphological observation of hUCMSCs in vitro

Following 12 h of culture, the majority of hUCMSCs had adhered to the bottom of the plate and spread out gradually, with flat and spindle-like shapes and fibroblast-like growth. After five days of cell proliferation, confluence reached 80–90%, with cells distributed in clusters growing in a whirl or fingerprint-like manner (Fig. 1A). The sub-cultured hUCMSCs exhibited increased proliferative abilities and spindle-like growth (Fig. 1B). The cellular morphology of hUCMSCs after the third generation was uniform and cell growth entered plateau phase.

Figure 1. Cellular morphology of hUCMSCs. (A) Following 5 days of cell proliferation, hUCMSCs exhibited whirl or fingerprint-like growth (magnification, ×100). (B) Sub-cultured hUCMSCs exhibited spindle-like growth (magnification, ×200). hUCMSCs, human umbilical cord mesenchymal stem cells.

Expression of surface antigen of hUCMSCs by flow cytometry

Third generation hUCMSCs were identified by flow cytometry and they were found to be positive for CD73 (93.30%), CD90 (99.61%) and CD105 (99.06%); however, cells were negative for CD34 (0.80%) and CD45 (0.79%; Fig. 2), which was consistent with the phenotypic features of hUCMSCs.

Figure 2. Expression of surface antigen of human umbilical cord mesenchymal stem cells determined by flow cytometry. PE, phycoerythrin; FITC, fluorescein isothyanicite.

Adipogenic induction for hUCMSCs

Cellular morphology

hUCMSCs were cultured with the adipogenic inducer and their appearance changed from spindle shaped to irregularly circular or polygonal, indicating clustering growth. At 4 days following the induction, intracellular circular lipid droplets were observed under the inverted microscope. Following continuous differentiation, the number of lipid droplets markedly increased and some fused to form large liquid droplets.

Oil red ‘O’ staining

At 28 days following adipogenic differentiation, third generation hUCMSCs were stained with oil red ‘O’. Lipid droplets were observed to possess a red color in >80% of the cell with sharp contrast (Fig. 3A).

Figure 3. (A) At 28 days following adipogenic differentiation, the third generation hUCMSCs were stained with oil red ‘O’ (magnification, ×200). (B) At 4 weeks following osteogenic induction, the third generation hUCMSCs were stained with alizarin red (magnification, ×200). hUCMSCs, human umbilical cord mesenchymal stem cells.

Osteogenic induction for hUCMSCs

Cellular morphology

Following osteogenic induction, the cellular morphology changed. On the fifth day of osteogenic induction, cell volume was observed to have increased and hUCMSCs exhibited short spindle-like or irregular polygonal shapes. Following continuous differentiation, hUCMSCs clustered and cascaded to form colonies. Calcified nodules were observed in the central regions and increased gradually.

Alizarin red staining

Following 4 weeks of osteogenic induction, third generation hUCMSCs were stained with alizarin red. Multiple black calcium nodules were observed at the central region of the cell colonies, which were stained black by reduced metallic silver (Fig. 3B).

Positive expression of hNIS-EGFP observed using fluorescence microscopy

Following transfection with EGFP and hNIS, the expression of hNIS in cells was assessed via fluorescence microscopy. At 12 h following transient transfection, a small number of hUCMSCs were observed to express green fluorescence with weak intensity. Following another 24 h, the number of fluorescing cells and the intensity of fluorescence increased, with 4–10/field in each field (magnification, ×10). Over the subsequent 48 h, the number of human amniotic epithelial cells with green fluorescence increased continuously, with >10 in each field. No obvious differences were observed in the number of hUCMSCs with green fluorescence at 72 and 48 h. The majority of hNIS-EGFP-hUCMSCs fluoresced green under a fluorescence microscope (Fig. 4). It was observed that the virus infection efficiency for the NIS gene was >95%.

Figure 4. hNIS-EGFP-hUCMSCs with puromycin. (A) and (B) Day 3 following screening of hNIS-EGFP-hUCMSCs with puromycin (magnification, ×100). (C) and (D) Day 7 following screening of hNIS-EGFP-hUCMSCs with puromycin (magnification, ×200). hNIS, human sodium/iodide symporter; EGFP, enhanced green fluorescent protein; hUCMSCs, human umbilical cord mesenchymal stem cells.

Identification of hNIS and EGFP expression in hUCMSCs by western blot

The cell lysate of the experimental (hNIS-EGFP-hUCMSCs) and control groups (hUCMSCs) underwent western blot analysis. Western blotting revealed positive bands at 90 kD (NIS) and 57 kD (eGFP) in the experimental group. Western blotting results for the control group did not reveal any specific bands (Fig. 5A).

Figure 5. Western blot analysis and Prussian blue staining. (A) Identification of hNIS and EGFP expression in hUCMSCs by western blot analysis. In vitro study of 125I uptake of hNIS-EGFP-hUCMSCs. (B) Prussian blue staining of superparamagnetic iron oxide-labeled hNIS-EGFP-hUCMSCs at (Ba and Bb) 0 h, (Bc and Bd) 24 h and (Be, Bf) 48 h. (magnification, ×100). hUCMSCs, human umbilical cord mesenchymal stem cells; hNIS, human sodium/iodide symporter; EGFP, enhanced green fluorescent protein.

SPIO-labeled hNIS-EGFP-hUCMSCs

hNIS-EGFP-hUCMSCs were co-incubated with SPIO again. It was confirmed by Prussian blue staining that SPIO transfection efficiency was >98%. The results are presented in Fig. 5B.

Study on cell biology of SPIO-labeled hNIS-EGFP-hUCMSCs Cell growth curve of hNIS-EGFP-hUCMSCs following labeling with SPIO

SPIO, hNIS and EGFP co-labeled hUCMSCs were sub-cultured. On days 1 and 2, hUCMSCs were latent and exhibited no marked proliferation. On days 3–7, hUCMSCs entered the logarithmic phase, and on days 7–8, hUCMSCs entered the plateau phase. Two double periods of cell growth were observed, the first on days 3–4 and the second on days 4–7. Compared with the control group (hUCMSCs), there was no significant difference in cell growth at any time point (Fig. 6A).

Figure 6. Cell growth curve and adipogenic and osteogenic differentiation. (A) Cell growth curve of hNIS-EGFP-hUCMSCs following labeling with SPIO. (Ba) Adipogenic and (Bb) osteogenic differentiation of hUCMSCs prior to labeling. (Bc) Adipogenic and (Bd) osteogenic differentiation of hUCMSCs following labeling (magnification, ×200). hUCMSCs, human umbilical cord mesenchymal stem cells; hNIS, human sodium/iodide symporter; EGFP, enhanced green fluorescent protein; SPIO, superparamagnetic iron oxide.

Adipogenic and osteogenic differentiation of hNIS-EGFP-hUCMSCs following labeling with SPIO

Experimental results demonstrated that modification with hNIS and EGFP and labeling with SPIO had no effect on adipogenic and osteogenic differentiation (Fig. 6B).

Phenotype identification of hNIS-EGFP-hUCMSCs following labeling with SPIO determined by flow cytometry

The adipogenic and osteogenic differentiation of normal MSCs and MSCs transfected with EGFP and SPIO were identified. The results revealed that the stemness of MSCs was not significantly decreased. Following transfection with EGFP and SPIO, and they were determined to be positive for CD73 (99.89%), CD90 (94.03%) and CD105 (91.94%) and negative for CD34 (0.13%) and CD45 (0.58%). There was no significant difference between the control and experimental groups (Fig. 7).

Figure 7. Phenotype identification of human sodium/iodide symporter-enhanced green fluorescent protein-labeled human umbilical cord mesenchymal stem cells following labeling with superparamagnetic iron oxide determined by flow cytometry. (A) Prior to and (B) following labeling. PE, phycoerythrin; FITC, fluorescein isothyanicite.

In vitro study on 125I uptake of SPIO-labeled hNIS-EGFP-hUCMSCs

Uptake of 125I was determined as previously described (17,18). 125I uptake activity significantly increased by 16.43±2.30 times in the experimental group compared with the control group (P<0.05; Table I; Fig. 8A), accumulation was faster in SPIO-hNIS-EGFP-hUCMSCs than in hUCMSCs, reaching maximal levels within 30 min (Fig. 8B). The efflux of 125I from SPIO-hNIS-EGFP-hUCMSCs was rapid, with half-maximal activity levels reached after 7.95 min (Fig. 8C).

Figure 8. Dynamic process of 125I. 125I uptake activity of SPIO-labeled hNIS-EGFP-hUCMSCs. (A) 125I uptake activity in the experimental and with the control group with and without NaClO4. The (B) accumulation and (C) efflux of 125I in the experimental and with the control group. *P<0.05 and **P<0.01. hUCMSCs, human umbilical cord mesenchymal stem cells; hNIS, human sodium/iodide symporter; EGFP, enhanced green fluorescent protein; SPIO, superparamagnetic iron oxide.

Table I. 125I uptake activity of superparamagnetic iron oxide-labeled human sodium/iodide symporter-enhanced green fluorescent protein-human umbilical cord mesenchymal stem cells.

Table I.

125I uptake activity of superparamagnetic iron oxide-labeled human sodium/iodide symporter-enhanced green fluorescent protein-human umbilical cord mesenchymal stem cells.

Group	Without NaClO4 (cpm)	With NaClO4 (cpm)
Experimental	2005.2±52.0a,b	673.8.2±31.8
Control	122.7±20.9	98.3±10.5

a P<0.05 vs. the control group without NaClO₄

b P<0.05 vs. experimental group with NaClO₄.

Discussion

Compared with bone marrow mesenchymal stem cells (BMSCs), dental pulp stem cells (DPSCs), adipose derived stem cells (ADSCs) and embryonic stem cells (ESCs), hUCMSCs possess more primitive properties, strong plasticity and differentiation potential (19). Furthermore, hUCMSCs express the specific markers (CD73, CD90 and CD105) of stem cells (20). Compared with BMSCs, UMSCs have a greater proliferative ability, making them more suitable for clinical applications (21). Compared with ADSCs, hUCMSCs exhibit lower immunogenicity and no oncogenicity (22). hUCMSCs do not express human leukocyte antigen-antigen D related, which is a major causative factor of the immune response, suggesting that hUCMSCs may be suitable for transplantation between different individuals (23). Compared with other stem cells, the proliferative activity and differentiation ability of hUCMSCs does not decrease with sub-culture. Due to the aforementioned features, hUCMSCs were selected for use in the present study. Isotopic imaging, MRI and in vivo fluorescence imaging were compared or combined in order to identify effective in vivo tracer technologies, provide the basis for in-depth studies on hUCMSCs and enhance their homing ability.

hNIS is an endogenous gene derived from human thyrocytes, and its encoding product is a physiological protein that has no immunogenicity (24,25). The product of the hNIS reporter gene is expressed on the cell membrane, making it easier to capture the reporter probe compared with herpes simplex virus type 1 thymidine kinase and other reporter genes of intracellular enzymes (26). Endogenous hNIS is only expressed in certain tissues and organs, and commonly used radionuclides, such as 123I and 99mTc may be used as reporter probes of hNIS; as such, there is no labeling requirement, which reduces the cost and increases accessibility (27). hNIS may be used for single-photon emission computed tomography (SPECT) or positron emission tomography and, as a reporter gene, hNIS is more stable than stomatostatin receptor 2 and human dopamine 2 receptor (28,29). However, the hNIS reporter gene lso has some deficiencies. As it is an endogenous gene, it is expressed in different degrees by several organs and tissues, such as the thyroid, gastric mucosa, mammary glands and salivary glands, which affects the monitoring of hNIS gene expression (30). In addition, in non-thyroid tissues and cells, iodine is released quickly because of a lack of thyroid specific proteins that hold iodine, therefore radioactive iodine does not accumulate (31–33). Although this reduces radiation damages to tissues, the signal detection is also affected.

For molecular biological techniques, the transfection of reporter genes into quiescent cells, in particular hUCMSCs, is difficult. The methods for transfecting target genes into target cells primarily include the transfection of eukaryotic expression plasmids and the transfection of viral vector-mediated genes (34). Studies on lentiviral vector (35) demonstrate that these vectors are able to effectively integrate exogenous genes into host chromosome to establish persistent expression (36). Lentiviral vectors are also able to effectively transfect neuronal, hepatic, myocardial, tumor, endothelial and stem cells (37). For cells that are difficult to transfect, such as primary, stem and non-dividing cells, the lentiviral vector increases the transduction efficiency of target genes greatly, allowing for the incorporation of RNAi, cDNA and study reporter genes (38). The lentiviral packaging system used in the present study is a four-plasmid system, consisting of pRsc-REV, pMD1g-pRRE, pMD2G and interference plasmid. The interference plasmid is able to express EGFP, pRsv-REV, pMDlg-pRRE and pMD2G, and contains the necessary elements for virus packaging (39). Eukaryotic plasmids containing the target gene, hNIS, were established and transfected with the aforementioned four-plasmids system to obtain hNIS-EGFP-hUCMSCs. Western blot analysis revealed that hNIS expression was normal. 125I uptake activity of the experimental group (hNIS-EGFP-hUCMSCs) increased by 16.43±2.30 times in comparison with that of the control group (hUCMSCs). 125I influx and efflux experiments indicated that the function of hUCMSCs was normal following the expression of hNIS, which indicates that it may be an effective reporter for SPECT in vivo cell tracing.

SPIO is a novel MR contrast, with a 20–200 nm diameter core of Fe2O3 coated with glucan (40). SPIO has very small crystal structures; under an applied magnetic field, SPIO exhibits single magnetic moment along the magnetic field (41). Even in a weak magnetic field, SPIO is highly magnetic (42). When the applied magnetic field is removed, this magnetism quickly subsides, and this is called superparamagnetism (43). Uneven distribution of SPIO in tissues results in an uneven local magnetic field (44), which shortens the transverse relaxation time (T2) and longitudinal relaxation time (T1) of tissues. The shortening of T2 is more marked, manifesting as the decrease in T2 signals (45). SPIO is negatively charged, and so in the present study, SPIO was coated with positively charged polylysine to reduce electrostatic interaction (29,32,33,46,47). Thus, SPIO was able to bind with cell surface receptors via electrostatic interaction and enter into cells via endocytosis. hNIS-EGFP-hUCMSCs are labeled with SPIO using the aforementioned technique, allowing for the successful establishment of SPIO, hNIS and EGFP co-labeled hUCMSCs. Staining with Prussian blue confirmed that SPIO entered cells, with a labeling rate of 98%.

The purpose of establishing SPIO, hNIS and EGFP co-labeled hUCMCs was to horizontally compare the advantages and disadvantages of several imaging technologies in in vivo stem cell transplantation. A series of tests were performed to determine whether the biological activity, stemness, proliferative activity or differentiation ability of hUCMSCs were affected following experimental interventions. On days 1 and 2 of sub-culture, hUCMSCs were at a latent phase with no obvious proliferation; on days 3–7, hUCMSCs entered the logarithmic phase, and on days 7–8, hUCMSCs entered the plateau phase. Cell growth had two double periods, the first on days 3–4 and the second on days 4–7. Compared with the control group (hUCMSCs), there were no significant differences at each time point. Adipogenic and osteogenic differentiation were further assessed prior to and following experimental treatments, and the experimental results suggest that the stemness of hNIS-EGFP-hUCMSCs was slightly reduced; however, the overall characteristics of stem cells remained unchanged. The results for adipogenic and osteogenic differentiation indicate that there are no significant differences between hNIS-EGFP-hUCMSCs and normal primary hUCMSCs. Western blotting also demonstrated that hNIS expression was good, and 125I influx and 125I efflux experiments indicated that the function of hUCMSCs was good following transfection with hNIS. These results suggest that hNIS-EGFP-hUCMSCs may be effective reporters for SPECT in vivo cell tracing. hNIS-EGFP-hUCMSCs were labeled with SPIO under the mediation of poly-L-lysine, and SPIO, hNIS and EGFP co-labeled hUCMSCs were successfully established. Staining with Prussian blue confirmed that SPIO successfully entered the cells, with a labeling rate of 98%. There were no significant differences in biological activity, stemness, proliferative activity or differentiation ability between SPIO, hNIS and EGFP co-labeled hUCMSCs and primary hUCMSCs. Therefore, the results of the present study suggest that, SPIO, hNIS and EGFP co-labeled hUCMSCs may be an effective treatment cell for animal models as it may be appropriate for stem cell tracing by SPECT, MRI and in vivo fluorescence imaging of animals.

Acknowledgements

Not applicable.

Funding

The present study was supported by grants from the Science and Technology Planning Project of Guangdong Province, China (grant nos. 2016A040403054 and 2013B021400002), Important Guangdong Province Science and Technology Specific Projects (grant nos. 2003A3080501 and 2015B010106008) and Important Industry-Academia-Research, Collaboration and Innovation Specific Projects of Guangzhou City (grant no. 201508020101).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

YM, HL and NB designed the experiment, established the lentivirus infection system, performed the statistical analysis and wrote the manuscript. JG processed and analyzed the single-photon emission computed tomography imaging. XZ provided plasmid of sodium/iodide co-rotor, designed the plasmid experiment and in vitro transfection. CH and WL performed magnetic resonance imaging scans and pathological research. HX guided the experiment, assisted with experiments where issues arose and reviewed the whole manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References