Introduction

Various etiologies such as toxins, drugs, viral infections, immune reactions, or genetic disorders may cause acute or chronic liver injury. Once liver cirrhosis is present, patients have a high incidence of serious complications, including portal hypertension, liver failure, and liver cancer. Increasing evidence suggests that mesenchymal stem cells (MSCs) transplantation can be a promising treatment for liver disease. MSCs can be obtained from various somatic tissues [1] and show favorable characteristics, such as ease of isolation and expansion, differentiation potential, lack of teratoma formation, and low ethics controversy. MSCs can differentiate into mature hepatocytes in vitro under adequate stimuli, such as cytokines and growth factors [2–5], coculture with hepatocytes or nonparenchymal liver cells [6–8], three-dimensional (3D) culture [9, 10], and transcription factor transduction [11]. In liver injury animal models, MSCs not only differentiate into hepatocytes to provide hepatic function [12–14], but also produce a series of bioactive factors that reduce hepatocyte apoptosis and inhibit inflammatory responses [15–17]. Initial studies in humans also predict the potential clinical application of MSCs in the treatment of liver disease [18, 19].

Before broadening the use of MSCs to clinical therapy, variables like cell dose, dosing frequency, delivery route, timing of delivery, and host microenvironment should be defined to ensure safe and effective administration. To satisfy these demands, it is desirable to establish novel molecular imaging methods to longitudinally and noninvasively track cellular fate (i.e., distribution, localization, engraftment, and repopulation) in vivo following transplantation. In former studies of liver disease, MSCs were labeled with superparamagnetic iron oxide (SPIO) [20], quantum dots [21], or radionuclides [22], and their distribution was monitored using in vivo magnetic resonance imaging (MRI), fluorescence imaging (FLI), or single-photon emission computed tomography (SPECT), respectively. In another study, SPIO and luciferase reporter gene were codelivered into MSCs with polymer-based nonviral vector, and then, cells were tracked using both in vivo MRI and bioluminescence imaging (BLI) [23]. The transiently labeled contrast agents or reporter genes are diluted with each cellular division, and therefore are not able to accurately assess cell viability. In this study, MSCs were stably labeled with Luc2-mKate2 dual-fusion reporter gene by lentiviral transduction, and the optimal dose and delivery route for liver disease were determined through in vivo BLI in a mouse model.

Materials and Methods

Construction of pLenti-CMV-Luc2-mKate2 and Preparation of MSCs-Luc2-mKate2 (MSCs-R)

Lentiviral expression vector pLenti6.3/V5-DEST carrying Luciferase2 (Luc2, Promega Inc., Madison, WI, USA) and mKate2 (Evrogen Inc., Moscow, Russia) dual-fusion reporter gene was constructed according to the manufacturer’s protocol (Invitrogen Inc., Carlsbad, CA, USA). The expression vector (pLenti-CMV-Luc2-mKate2) and ViraPower™ Packaging plasmid Mix were cotransfected into 293FT cells with Lipofectamine 2000 (Invitrogen Inc.). The culture supernatants were collected, concentrated, and used as a virus stock.

The immortalized human bone marrow-derived MSCs UE7T-13 were provided by the RIKEN BioResource Center through the National Bio-Resource Project of the MEXT, Japan. These cells were maintained under standard conditions as described previously [24, 25]. MSCs were infected with pLenti-CMV-Luc2-mKate2 lentivirus with a multiplicity of infection (MOI) of 21 and 8 μg/ml polybrene for 48 h. Through flow cytometry, the percentage of mKate2-positive cells was determined, and positive cells (MSCs-R) were further purified.

Multilineage Differentiation of MSCs-R

To evaluate the effect of the Luc2-mKate2 reporter gene on the multilineage differentiation potential of MSCs, MSCs-R were induced to differentiate into adipogenic, osteogenic, and chondrogenic lineages using STEMPRO® Differentiation Kit (Invitrogen Inc.). After induction, cells were stained with oil red O for lipid droplets, alizarin red S for calcium, or alcian blue for mucopolysaccharides, respectively.

Preparation of Acute Liver Injury Model and MSCs-R Transplantation

Animal procedures were performed according to the protocol approved by the Sun Yat-sen University Institutional Animal Care and Use Committee. Male 6- to 8-week-old athymic nude mice (BALB/c nu/nu) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). To prepare acute liver injury model, mice were treated by peritoneal injection of 20 % CCl4 every other day for two times. The injection dose was 2.5 ml/kg body weight. MSCs-R transplantation was performed at 24 h after the secondary administration of CCl4.

All operations were performed under anesthesia with 10 mg/ml ketamine and 2 mg/ml xylazine in PBS. Mice underwent aseptic laparotomy. MSCs-R suspended in 0.25 ml saline were transplanted into mice via three different routes, including inferior vena cava (IVC), superior mesenteric vein (SMV) [20], and intrahepatic (IH) injection. A 29-gauge needle was used for puncture and injection of cells. The total injection time was no less than 1 min.

In Vitro, In Vivo and Ex Vivo Bioluminescence Imaging

BLI was performed using Xenogen IVIS Lumina II imaging system and analyzed using IVIS Living Imaging 4.2 software (Caliper Life Sciences, Alameda, CA). Bioluminescence images were normalized and reported as photons per second per centimeter squared per steradian (p/s/cm2/sr). For in vitro BLI, 1.0 × 103, 1.25 × 103, 2.5 × 103, 5.0 × 103, 1.0 × 104, 1.25 × 104, 2.5 × 104, 5.0 × 104, and 1.0 × 105 MSCs-R were planted on black 96-well plate. After the incubation of D-luciferin (150 μg/ml), cell bioluminescence signals were detected with an acquisition time of 5 s. For in vivo BLI of transplanted MSCs-R, mice were injected with D-luciferin intraperitoneally at a dose of 150 mg/kg body weight. Bioluminescence signals were detected with the acquisition time of 30 s. For ex vivo BLI, mice were euthanized after D-luciferin injection. The major organs were dissected and subjected to ex vivo BLI. Bioluminescence signals were detected with the acquisition time of 30 s.

Postmortem Tissue Assay

Postmortem tissue assay was performed as described previously [26–28]. Organs were dissected at 3-day posttransplantation of MSCs-R. Frozen tissue sections (5 μm thick) were fixed for 20 min at room temperature in 4 % paraformaldehyde and subsequently covered with DAPI-containing mounting medium. Images were obtained using fluorescence microscopy.

Statistical Analysis

Quantitative data are expressed as mean ± SD. Means were compared using one-way ANOVA and the Student’s t test. P values of <0.05 were considered statistically significant.

Results

Labeling MSCs with Luc2-mKate2 Dual-Fusion Reporter Gene by Lentivirus Transduction

To develop the imaging approach, MSCs were labeled with the Luc2-mKate2 dual-fusion reporter gene by lentiviral transduction. Luc2 can be used for in vivo BLI while mKate2 can be used for fluorescence assay at the cellular level in vitro and the tissue level ex vivo. Luc2 and mKate2 are joined by an 8-amino acid peptide (NRDPPVAT). The dual-fusion reporter gene was driven by a constitutive CMV promoter (Fig. 1a). The labeling efficiency was 17.5 % based on the flow cytometric analysis of mKate2-positive cells (MSCs-R) (Fig. 1b). After fluorescence-activated cell sorting, the percentage of MSCs-R was raised to 92.8 % (Fig. 1b). Confocal analysis of MSCs-R demonstrated uniform mKate2 expression within the cytoplasm (Fig. 1c). MSCs carrying Luc2-mKate2 (MSCs-R) showed similar morphology to nonlabeled MSCs (control). Like control MSCs, MSCs-R were still able to differentiate into adipogenic, osteogenic, and chondrogenic lineages (Fig. 1d).

Fig. 1.
figure 1

a Schema of lentivirus vector carrying dual-fusion reporter gene (Luc2-mKate2) driven by a constitutive CMV promoter. b Flow cytometric analysis of Luc2-mkate2 lentivirus-transduced MSCs before and after fluorescence-activated cell sorting. c Confocal analysis of MSCs-Luc2-mKate2 (MSCs-R). Scale bar, 20 μm. d Assessment of multilineage differentiation ability of MSCs-R. After induction, adipocytes, osteoblasts, and chondrocytes were stained with oil red O, alizarin red S, and alcianblue, respectively. Scale bar, 200 μm.

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To confirm that the levels of Luc2 reporter gene activities correlated with cell numbers, MSCs-R were assayed through both in vitro and in vivo BLI. In vitro BLI showed a linear relationship between cell number (1.0 × 103 to 1.0 × 105) and bioluminescence signal (R 2 = 0.997) (Fig. 2a, b). For in vivo BLI, different numbers (1.0 × 104 to 1.0 × 106) of MSCs-R were transplanted into the liver of nude mice via the SMV. The blood of SMV flows directly into the hepatic portal vein, and therefore delivers MSCs-R into the liver. BLI was performed at 3-h posttransplantation (PT). As shown in Fig. 2c, d, there is also a robust relationship between cell number and bioluminescence signal of the liver (R 2 = 0.996). The above results suggest that BLI of Luc2 is a reliable approach to monitor viable transplanted MSCs-R quantitatively.

Fig. 2.
figure 2

a In vitro bioluminescence imaging (BLI) of varying numbers of MSCs-R plated on 96-well plate. CTL, control. b Linear relationship analysis between the cell number and bioluminescence intensity of a. c In vivo BLI of varying numbers of MSCs-R transplanted into the liver of nude mice via superior mesenteric vein (SMV) at 3-h posttransplantation. d Linear relationship analysis between the cell number and bioluminescence intensity of c.

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Determination of the Optimal Delivery Dose of MSCs-R

To determine the optimal delivery dose, different numbers (1.0 × 106, 5.0 × 105, and 2.5 × 105) of MSCs-R were transplanted into the liver of nude mice via the SMV. The 1.0 × 106 group mice demonstrated the highest liver BLI signal (Fig. 2c), but also had high PT 24-h mortality (50.0 %, 3/6). Liver histology analysis in Fig. 3 showed large area of necrosis accompanied by infiltrating neutrophils, which was caused by portal vein embolization (PVE). The 5.0 × 105 group mice had lower 24-h mortality (33.3 %, 1/6) and smaller area of necrosis (Fig. 3). By contrast, no PVE-caused death occurred in the 2.5 × 105 group mice, and histology analysis showed no necrosis in the liver (Fig. 3). Given the above results, 2.5 × 105 is the optimal delivery dose and therefore chosen for further study.

Fig. 3.
figure 3

Appearance and HE staining of the liver 24 h after 1.0 × 106, 5.0 × 105, or 2.5 × 105 of MSCs-R transplanted into the liver of nude mice via SMV. Black arrows indicate necrotic areas accompanied by infiltrating neutrophils. Scale bar, 200 μm.

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Determination of the Optimal Delivery Route of MSCs-R

To determine the optimal delivery route, 2.5 × 105 MSCs-R were transplanted into acute liver injury model nude mice via three different routes, including IVC, SMV, and IH injection. The validity of the acute liver injury model was confirmed through histological assay (Supplementary Material Fig. S1). Representative in vivo BLI images and quantifications at different time points PT are shown in Fig. 4a–c. In the IVC group mice, a clear signal can be seen in the lungs at 3-h PT, demonstrating that the majority of infused MSCs-R were trapped inside the pulmonary system. The lung BLI signal declined quickly and reduced to background levels at 7-day PT. However, no BLI signal could be detected in any of the other organs. In the IH group mice, BLI signal was only localized in the injection region of the liver and decreased to background levels at 14-day PT. By contrast, MSCs-R demonstrated dispersed distribution within the liver after SMV transplantation. The liver BLI signal declined progressively with time and retreated to background levels at 10-day PT. No BLI signal could be detected in the lungs or other organs. Ex vivo BLI (Fig. 4d, e) further confirmed the in vivo BLI results.

Fig. 4.
figure 4

a In vivo BLI of acute liver injury model nude mice after transplantation of 2.5 × 105 MSCs-R via three different routes, including inferior vena cava (IVC), SMV, and intrahepatic (IH) injection. b Time-liver bioluminescence intensity curves of a. c Time-lung bioluminescence intensity curves of a. d Ex vivo BLI of major organs harvested at 3-day posttransplantation of MSCs-R. e Bioluminescence intensity quantification of major organs at 3-day posttransplantation of MSCs-R. All data in b, c, and e are presented as the mean ± standard deviation(SD).

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To clearly demonstrate MSCs-R migration direction in these three delivery routes, digital subtraction angiography (DSA) was performed on mice (Supplementary Material Fig. S2). In IVC puncture route, blood reentered the heart through the right atrium and went to the lungs through the pulmonary artery. In the SMV puncture route, blood flowed directly into the hepatic portal vein and went to the various hepatic lobes through portal venous branches (Supplementary Material Fig. S2, S3). In the IH puncture route, contrast agent was confined to the injection region of the liver and could not disperse to other regions. The above DSA results were in accordance with the in vivo BLI results.

Analysis of MSCs-R Tissue distribution

Fluorescence microscopy analysis of mKate2+ MSCs-R tissue distribution (Fig. 5) was further performed to validate the BLI measurements. In the IVC group, MSCs-R accumulated mainly in the lungs and not in the liver. In the SMV group, MSCs-R were detected to reside in the liver instead of other tissues, including the lungs, heart, kidneys, and spleen (Fig. 5, Supplementary Material Fig. S4). In the IH group, MSCs-R distributed only in the injection region of the liver (Fig. 5) but could not migrate to the noninjection region of the liver (Supplementary Material Fig. S5).

Fig. 5.
figure 5

MSCs-R distribution analysis on lung or liver sections 3-day posttransplantation via IVC, SMV, or IH, respectively. Scale bar, 100 μm.

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Discussion

Both experimental [12–17] and initial clinical [18, 19] studies have shown the therapeutic potential of MSCs in liver disease. It is of great importance to get direct evidence that transplanted MSCs do survive for enough time and engraft accurately into the injured liver in animal models before initiating clinical applications in humans. Molecular imaging enables in vivo cell tracking in a real-time, longitudinal, noninvasive way and could further facilitate assessment, optimization, and guidance of cell transplantation for clinical translation [29, 30].

For cell imaging, cells can be directly labeled with contrast agents such as magnetic particles [20], quantum dots [21], and radionuclides [22] and so on. While direct imaging techniques can be straightforwardly implemented and commonly used, it cannot specifically differentiate viable cells from dead cells because the contrast agents are diluted after every cell division and keep lingering in tissues even after cell death. An alternative approach to cell imaging is reporter gene imaging, which requires the integration of specific reporter genes. This is a favorable imaging technique for long-term assessment of cell survival since its signal generation counts on cell viability. Among various imaging modalities, BLI possesses many advantages including low background, low cost, high sensitivity, no radiation, and simplicity. Thus, it has been widely used in small animal studies. In this study, we use BLI reporter gene for MSCs imaging to determine the optimal delivery dose and route for liver disease.

In the initial study, the Luc2-mKate2 dual-fusion reporter gene was constructed. Luc2 is a BLI reporter gene, a synthetic firefly luciferase with humanized codon optimization. Compared to wild-type luciferase, Luc2 shows higher expression and reduced abnormal transcription. mKate2 is a far-red fluorescent reporter gene. The brightness of mKate2 is higher compared to any other monomeric fluorescent proteins. Its far-red fluorescence allows high signal-to-background ratio. Besides, it offers excellent pH resistance, high photostability, and low toxicity [31]. The above characteristics of mKate2 make it a superior fluorescent reporter gene. The inclusion of mKate2 is helpful for the cell sorting and tissue analysis. After lentiviral transduction, MSCs-R constitutively express Luc2-mKate2 reporter gene and still keep multileage differentiation ability (Fig. 1). Both in vitro and in vivo BLI (Fig. 2) show a linear relationship between cell number and bioluminescence signal, which suggests that BLI of Luc2 is a reliable approach to monitor viable transplanted MSCs-R quantitatively.

Delivery dose and route of MSCs are two important parameters that determine therapeutic efficacy. To identify the optimal delivery dose, different numbers (1.0 × 106, 5.0 × 105, and 2.5 × 105) of MSCs-R were transplanted into the liver of nude mice via the SMV. The blood from SMV flows directly into the hepatic portal vein, and therefore delivers MSCs-R into the liver. The 1.0 × 106 and 5.0 × 105 group mice demonstrated high liver BLI signal, but also had 50.0 and 33.3 % mortality within 24-h PT, respectively. The death was caused by PVE (Fig. 3). By contrast, no PVE and its related death occurred in the 2.5 × 105 group mice. Therefore, in order to avoid side effects like PVE, 2.5 × 105 is the optimal dose of MSCs when delivered through the SMV. Lethal pulmonary embolism was also reported after intravenous administration of high-dose MSCs (1.0–3.0 × 106/mouse) [32, 33], likely because MSCs are prone to aggregate in high concentration. One study showed that a combination of MSCs and heparin could avoid pulmonary embolism [33]. Further studies are needed to validate the efficacy of heparin on PVE.

Systemic infusion, portal vein injection, and IH injection are commonly applied MSC delivery approaches for liver disease. Therefore, these three delivery routes were quantitatively compared through in vivo BLI in mice with acute liver injury (Fig. 4). Carbon tetrachloride (CCl4), a potent hepatotoxic agent, was used to induce acute liver injury. Treatment with CCl4 can cause hepatic steatosis, inflammation, apoptosis, and necrosis through various mechanisms, such as formation of reactive free radicals, induction of hypomethylated ribosomal RNA, and alterations in calcium homeostasis [34]. Peripheral vein (such as tail vein or femoral vein) is the usually used route for systemic infusion. To avoid extravasation at the injection site and ensure the similar level of surgical trauma as the other two routes, systemic infusion of MSCs was implemented through IVC. Portal vein infusion of MSCs was carried out through SMV. After IVC infusion, MSCs-R were quickly trapped inside the lungs and eliminated within 7 days, which is consistent with previous studies [35–37]. However, no detectable homing of MSCs-R to the liver and other organs was demonstrated. Relatively large size and cell surface adhesion molecules might account for the lung entrapment of MSCs [37]. It is reported that MSCs passage through the lung barrier could be significantly increased through prior treatment with vasodilator sodium nitroprusside [35, 36], heparin saturation of MSCs [38], or pronase detachment [39]. Further studies are needed to confirm whether the application of these methods could facilitate MSCs engraftment to the injured liver after systemic infusion. By IH and SMV injection, lung entrapment is bypassed, and MSCs-R were mainly distributed in the liver. Compared to IVC route, these two routes showed higher delivery efficacy to target organ liver and less systemic engraftment, which may reduce side effects. After IH injection, MSCs-R exhibited a longer retention time in the liver than by SMV injection, but their distribution was only localized in the injection region of the liver. In liver disease, the lesion of the liver is usually diffused. In this condition, localized distributed MSCs may execute less efficacious therapy. Also, locally aggregated MSCs are predisposed to establish their own microenvironments [40]. Thus, cautions should be taken when considering direct injection of MSCs into tissues [41]. By contrast, MSCs-R were dispersedly distributed and stayed as long as 7-day PT in the liver after SMV infusion. DSA showed that after SMV puncture, blood flowed directly into the portal vein and went to the various hepatic lobes through portal venous branches (Supplementary Material Fig. S2, S3), which confirmed the in vivo BLI results. From the above results, we conclude that the SMV (i.e., portal vein) is the optimal MSCs delivery route for liver disease. In a clinical setting, portal vein puncture is performed through percutaneous transhepatic catheterization [18, 42], which is a minimally invasive intervention surgery. MSCs could also be delivered through artery route. Left ventricular or aortic arch injection is a systematic administration method. When delivered through this route, liver engraftment efficiency of MSCs (less than 10 %) is pretty low [43]. Besides, MSCs are also distributed in other organs, such as the kidney, lung, brain, and heart [43], which may cause side effects. Intra-arterial regional administration, on the other hand, can bypass nonspecific organs and deliver cells directly to the target organs. Therefore, it is often used as delivery route in various disease models [44–46]. Hepatic artery is the regional delivery route in liver disease. Actually, MSCs have been transplanted through the hepatic artery for the treatment of liver disease in clinic trials [19, 47]. Hepatic artery puncture is performed through femoral artery catheterization, which is less invasive than portal vein puncture. It is difficult to carry out hepatic artery puncture in small animals, such as mice. Therefore, the engraftment efficiency and therapy efficacy of MSCs delivered through these two routes need to be compared through large animal experiments or clinic trials in the future.

In conclusion, BLI, which could dynamically and quantitatively track cellular location and survival, is useful in determining MSCs transplantation parameters, such as delivery dose and route. However, further molecular imaging studies are needed to answer essential questions about clinical application of MSCs. BLI is limited to small animal studies due to its shallow penetration depth. The application of radionuclide reporter genes, such as herpes simplex virus type 1 thymidine kinase (HSV1-tk) or sodium iodide symporter (NIS), will facilitate large animal studies and clinical translation. Besides, the accurate cellular spatial localization information can be obtained through the incorporation of MRI reporter genes, such as Ferritin or lysine-rich protein (LRP). Former studies have confirmed the therapeutic effect of MSCs in acute liver injury [16, 23]. This study investigated MSCs delivery parameters exclusively on cell retention. Therefore, the relationship between cell retention and subsequent therapeutic efficacy needs to be further explored. MSCs are heterogeneous and contain subpopulations with different self-renewal and differentiation ability [48]. To date, little information is available to distinguish between individual subpopulations. The identification of MSCs subpopulations with favorable characteristics will facilitate specialized MSCs therapy in liver disease. Besides, some studies report tumorigenic potential of MSCs [49]. Thus, the safety of MSCs transplantation must be thoroughly observed especially when applied to premalignant liver disease. For a long time, MSCs were thought to be “immune privileged”. However, recent studies indicate that allogeneic or xenogeneic transplanted MSCs can be immunogenic. Activated NK cells [50], memory T cells [51, 52], macrophages [53, 54], and antidonor IgG [55] were reported to mediate the immune rejection of MSCs. In this study, the number of MSCs engrafted in the liver decreased progressively with time, and they were not detected at 10-day PT after SMV infusion. The liver is an organ of the immune system and contains abundant NK cells, macrophages, and lymphocytes [56, 57]. Therefore, it is crucial to elucidate the mechanism of the immune response against MSCs in the liver, which will further aid in the optimization of treatment strategy.