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Abstract

Stem cell therapy plays an important role in regenerative therapy; however, there is little information on the in vivo dynamics of transplanted stem cells and the influence of the inflammation of affected tissues or organs on these dynamics. In this study, we revealed real-time dynamics of transplanted adipose tissue–derived stem cells (ASCs) and the influence of the inflammatory states on these dynamics in acute liver failure mice. Quantum dots (QDs) labeling did not affect the cytokine profile of ASCs, and intravenously transplanted ASCs labeled with QDs could be detected in real time with high efficiency without laparotomy. Until 30 min after ASC transplantation, no marked differences in the behavior or accumulation of transplanted ASCs in the liver were observed among the three groups with different degrees of liver damage (normal, weak, and strong). However, significant differences in the engraftment rate of transplanted ASCs in the liver were observed among the three groups from 4 h after transplantation. The engraftment rate was inversely correlated with the extent of the liver damage. These data suggested that QDs are useful for in vivo real-time imaging of transplanted cells, and the inflammatory state of tissues or organs may affect the engraftment rate of transplanted cells.

Keywords

quantum dots (QDs)acute liver failure stem cells transplantation

Introduction

Stem cell transplantation plays an important role in regeneration therapy for many disorders such as lung and liver diseases because the reassembly of these tissues and organs is very difficult. Indeed, stem cell transplantation therapy with somatic stem cells, such as bone marrow stem cells (BMSCs)^1,2 and adipose tissue–derived stem cells (ASCs)^3,4, has been applied in clinical practice. In stem cell transplantation, the accumulation and engraftment of transplanted stem cells in the affected tissue or organs strongly affect the therapeutic efficacy^5,6. While the inflammatory state of the affected tissue or organs is also considered to influence the behavior, accumulation, and engraftment of transplanted stem cells^7–9, little is known on this subject. Accordingly, the in vivo real-time imaging and diagnosis of the dynamics, including the behavior, accumulation, and engraftment, of transplanted stem cells are essential for ensuring maximum therapeutic safety and efficacy with stem cell transplantation^10,11.

The presently available in vivo real-time imaging technologies for detecting transplanted stem cells are not sufficient. Some modalities, such as ultrasonic diagnostics, a roentgen diagnosis, X-ray computed tomography (CT)¹², magnetic resonance imaging (MRI)¹³, positron emission computerized-tomography (PET)¹⁴, and single-photon emission computed tomography (SPECT)¹⁵, have been used in clinical practice. However, these modalities are primarily used to diagnose tissues and organs, so the highly sensitive detection and real-time imaging of transplanted stem cells are very difficult. Fluorescence imaging was expected to contribute to the development of stem cell transplantation because fluorescence imaging can detect transplanted stem cells at a cellular level^16,17. Quantum dots (QDs) have specific fluorescence properties, such as a high quantum yield, wide range of wavelength, narrow fluorescence wavelength, and superior photostability. In particular, increasing attention is being paid to QDs with water-soluble and near-infrared region (NIR) fluorescence as useful fluorescence probes for overcoming these problems^18–20. We previously showed that QDs were useful for the fluorescence imaging of transplanted fetal hepatocytes, corneal endothelial cells, and stem cells derived from adipose tissue and exfoliated deciduous teeth^21–26.

In addition, we reported that ASC transplantation attenuated acute liver failure induced by carbon tetrachloride (CCl₄) injection in mice⁴. We then showed that ASCs could be labeled with QDs using the octa-arginine peptide (R8) with high efficiency and that R8-labeled QDs were effective for in vivo imaging of transplanted ASCs^27,28. However, the details concerning the dynamics of transplanted ASCs in mice are poorly understood. In this study, we developed in vivo real-time fluorescence imaging technology using QDs to reveal the dynamics after transplantation and then investigated whether or not the inflammatory state of acute liver failure influenced the behavior, accumulation, and engraftment of the transplanted ASCs in the mouse livers. To our knowledge, this is the first study to apply QD technology to in vivo real-time fluorescence imaging of transplanted cells just after transplantation.

Methods

Materials

Qdot ITK carboxyl QDs (QDs655 and QDs800: emission peak at 655 and 800 nm, respectively), Hank’s balanced salt solution, Hoechst 33342 solution, and enzyme-linked immunosorbent assay kits for mouse tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), and interleukin (IL)-12p70 were purchased from Thermo Fisher Scientific K.K. (Kanagawa, Japan). Fetal bovine serum (FBS) was purchased from Trace Scientific Ltd. (Melbourne, Australia). Octa-arginine peptide (R8), PKH26 Red Fluorescent Cell Linker Kit (PKH26GL-IKT), and fluorescein isothiocyanate-dextran of average molecular weight 40,000 (FD-40) were purchased from Sigma Aldrich^® Japan (Tokyo, Japan). BBx pack for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was purchased from Nittobo Medical Co., Ltd. (Tokyo, Japan). Novo-Heparin (10,000 units/10 ml for injection) was purchased from Mochida Pharmaceutical Co., Ltd. (Tokyo, Japan). Non-luminescence feed without alfalfa was purchased from SLC Japan, Inc. (Tokyo, Japan). A cell counting kit-8 (CCK-8) was purchased from DOJINDO Laboratories (Kumamoto, Japan). GSL I-B4 isolectin fluorescein isothiocyanate (FITC) conjugate was purchased from Funakoshi Co., Ltd. (Tokyo, Japan). XenoLight DiR (DiR) was purchased from Perkin Elmer Inc. (MA, USA).

Animals

C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). The mice were housed in a controlled environment (12 h light/dark cycles at 22°C) with free access to water and a standard chow diet before being killed. All conditions and handling of animals in this study were in accordance with the protocols approved by the Nagoya University Committee on Animal Use and Care (No. 20242).

Isolation and Culture of ASCs

The isolation and culture of ASCs have been reported previously²¹. In brief, 7- to 14-month-old female C57BL/6 mice were killed by cervical dislocation, and adipose tissue specimens were isolated from the inguinal groove and washed with Hank’s buffer to remove the blood cells. The adipose tissues were cut finely and digested with type II collagenase at 37°C in a shaking water bath for 90 min. Adipose tissue cells were suspended in cell culture medium [Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 20% FBS and 100 U/ml penicillin/streptomycin]. The cells were centrifuged at 1,200 × g for 5 min at room temperature to obtain pellets containing the ASCs. The cells were washed three times by suspension and centrifugation in the culture medium. The primary cells were cultured for 4 to 5 days until they reached confluence and were defined as passage “0.” Cells from passages 2 to 5 were used in all the experiments.

Transduction of QDs Into ASCs

The transduction method of QDs using R8 into ASCs has been reported previously²⁷. In brief, QDs (8.0 nM) and R8 (80 µM) were mixed in the transduction medium (DMEM/F12, 2% FBS, 100 U/ml penicillin/streptomycin) at 37°C for 15 min, and then the medium including QDs and R8 complex was added to the ASC culture flask. After 4-h incubation, the cells were washed using transduction medium. The transduction efficiency and fluorescence intensity were evaluated by flow cytometry (BD LSR Fortessa^TM X-20; Japan BD, Tokyo, Japan). QDs800 show a strong near-infrared fluorescence (about 800 nm wavelength) which can pass through the body with high efficiency; thus, QDs800 is very useful for in vivo imaging. However, near-infrared fluorescence at an 800-nm wavelength is very difficult to detect by conventional fluorescence microscopy. Therefore, QDs800 were used for in vivo imaging, and QDs655 composed of the same elements as QDs800 were used for cell evaluations.

Measurement of Inflammation Markers

ASCs (1 × 10⁵ cells) were seeded into a 24-well plate and cultured for 24 h. QDs655 (8 nM) and R8 (80 µM) were mixed in the transduction medium, and the complex was transduced into ASCs. After 4-h incubation, the medium was replaced with new cell culture medium and the ASCs were cultured for 1, 2, or 3 days. The cell culture medium was collected, and the levels of inflammation cytokines (TNF-α, IFN-γ, IL-12p70) and anti-inflammation cytokine (IL-10) were measured. In addition, these cytokine levels in serum derived from acute liver failure mice were measured via the same method.

Histological Analyses

Liver tissue specimens were resected at 4 or 24 h after CCl₄ injection and fixed in 10% formalin for 24 h. They were then embedded in paraffin, cut to 2-µm thickness, and stained with hematoxylin and eosin (HE) after deparaffinization. The stained slides were observed under a stereoscopic microscope.

Measurement of Liver Failure Markers

The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum of normal or CCl₄-treated mice were evaluated using the BBx pack. This experiment was performed in accordance with the manufacturer’s instructions. In brief, the serum (50 µl) of normal or CCl₄-treated mice was placed dropwise onto the BBx pack, and then the levels of ALT and AST were detected using the BBx device after a few minutes.

Transplantation of ASCs Labeled With QDs Into Mice With or Without Acute Liver Failure

To induce acute liver failure in 8- to 10-week-old C57BL/6 male mice, CCl₄ at 0.5 ml/kg that had been diluted 10 times with olive oil was intraperitoneally injected into mice. ASCs (1.0 × 10⁶ cells/200 µl saline) labeled with QDs655 or QDs800 using R8 (ASCs-QDs655 or QDs800) were mixed with 10 µl of heparin and then transplanted into mice with or without acute liver failure via a tail vein at 4 or 24 h after CCl₄ treatment.

In Vitro Fluorescence Imaging of ASCs-QDs in Microtubes

ASCs were labeled with QDs800 using R8 in the same manner as described above. The fluorescence images and intensities of ASCs (0.075, 0.15, 0.3, 0.6, 1.3, 2.5, and 5.0 × 10⁵ cells) labeled with QDs800 (8.0 nM) in 1.5 ml tubes were investigated using an IVIS^® Lumina K Series III (excitation filter: 720 ± 20 nm; emission filter: 790 ± 20 nm). The correlation coefficient between the number of ASCs-QDs800 and the fluorescence intensity was calculated.

In Vivo and Ex Vivo Fluorescence Imaging of ASCs-QDs in Mice

The mice transplanted with ASCs-QDs800 (1.0 × 10⁶ cells/200 µl saline) were anesthetized, and the fluorescence derived from QDs800 in the mouse bodies was monitored using an IVIS^® Lumina K Series III (excitation filter: 720 ± 20 nm; emission filter: 790 ± 20 nm), either in kinetics mode (movie mode: 1 slide/500 ms from 0 to 30 min after ASC transplantation) or in sequential mode [multiple time points, from 1 to 168 h (day 7) after ASC transplantation].

For the ex vivo fluorescence imaging analysis, the liver was harvested and the fluorescence intensity was analyzed using the IVIS^® Lumina K Series III. The measurement conditions were the same as for the in vivo imaging analysis. The region of interest (ROI) was measured under the assistance of the IVIS^® Lumina K Series III, and the data were expressed as the total radiant efficiency (TRE) in units of photons/second within the ROI: [p/s]/[µW/cm²].

Labeling of ASCs-QDs by Cell Membrane Staining Reagents

XenoLight DiR

ASCs-QDs were incubated in DiR staining solution (1 ml of Diluent C buffer containing 200 µg of XenoLight DiR) for 1 h, and then those labeled with DiR (ASCs-QDs-DiR) were washed with fresh cell culture medium three times. The labeled cells were collected and transplanted into mice.

PKH26

ASCs-QDs were incubated in PKH26 staining solution (1 ml of Diluent C buffer containing 4 µl of PKH26) for 1 h, and then those labeled with PKH26 (ASCs-QDs-PKH26) were washed with fresh cell culture medium three times. The labeled cells were collected and transplanted into mice.

Intravital and Ex Vivo Multiphoton Fluorescence Imaging of Transplanted ASCs-QDs-DiR in the Liver by a High-Speed Multiphoton Confocal Laser Microscope

Intravital

The liver of a mouse was exposed by an abdominal operation, and the mouse was set on a high-speed multiphoton confocal laser microscope, as shown in Fig. 5B. Hoechst 33342 solution (100 µl; 0.1 mg/ml) and FD-40 solution [100 µl; 1.5% (v/v)] were injected through a tail vein. ASCs-QDs-DiR (1.0 × 10⁶ cells/150 µl saline) were then transplanted into the mouse through a tail vein, and the transplanted ASCs-QDs-DiR were observed using a multiphoton excitation fluorescence microscope (A1plus; Nikon Corporation, Tokyo, Japan) equipped with an eHPDS-007 femtosecond-laser (Spectra Physics Mai Tai) and water immersion objective lens (CFI Plan Apo IR 60XW, CFI Apo Lambda S LWD 40XW). The fluorescent signals were detected using a GaAsP multi detector unit (Nikon Corporation) in kinetics mode (movie mode: 500 ms/frame, 0–10 min after ASC transplantation).

Ex vivo

The livers were harvested and incubated overnight in staining solution [1 ml of phosphate buffered saline (PBS) containing 20 µl of isolectin conjugated with FITC]. The isolectin-stained livers were then observed using a multiphoton excitation fluorescence microscope. The excitation laser was tuned at 920 nm and routed through an A1plus ×40 and ×60 water immersion objective lens (Nikon Corporation). The stained liver was observed using a bandpass emission filter 525/20 nm (for FITC) and 629/ 53 nm (for QDs655). In general, 212 × 212 µm or 322 × 322 µm areas were scanned, and 20- to 30-µm z-series were acquired using a 0.6-µm step. The fluorescent signals were detected using a GaAsP multi detector unit (Nikon Corporation). The acquired images were analyzed and reconstituted using the NIS-Elements AR software program (Nikon Corporation).

Statistical Analyses

The data are expressed as mean ± standard deviation (SD). Statistical significance was determined by Dunett’s test and a one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple-comparison test. A significant difference was defined as a P value of <0.05. All the statistical analyses were performed using the SPSS software package

Results

Levels of Inflammatory Cytokines With ASCs-QDs

As previously reported^27,28, the degree of liver failure after CCl₄ injection and the labeling efficiency and intensity of ASCs-QDs were evaluated. The highest degree of liver failure was observed 24 h after CCl₄ injection, followed in order by 4 h after CCl₄ injection and normal mice (Supplementary Fig. 1). The maintenance of labeling efficiency and fluorescence intensity of ASCs-QDs655 was evaluated; the labeling efficiency of ASCs-QDs655 was 93.9% at day 1 and 92.5% at day 7, and the mean fluorescence intensity (MFI) at day 7 remained the same at day 0 (Supplementary Fig. 2). To investigate whether or not QD labeling causes inflammation reactions in ASCs, the levels of inflammatory cytokines (TNF-α, IFN-γ, IL-12p70) and anti-inflammatory cytokine (IL-10) were measured in the culture supernatant of ASCs-QDs655 after 1, 2, and 3 days of incubation. All measurements were less than the lower limit of detection, and no significant differences between the non-labeled and labeled ASCs were observed in the degree of inflammation or anti-inflammation cytokine levels (Fig. 1A–D).

Figure 1.

The analysis of the levels of inflammatory cytokines with ASCs-QDs. (A–D) The comparison of levels of inflammatory cytokines [TNF-α (a), IFN-γ (b), IL-12p70 (c)] and anti-inflammatory cytokines [IL-10 (d)] in the cell culture supernatant of non-labeled ASCs (control) and ASCs-QDs655 on days 1, 2 and 3 after transduction of QDs655. The analysis was done in triplicate, and the data are shown as mean ± SD (n = 3). ASCs: adipose tissue–derived stem cells; QD: quantum dots; TNF-α: tumor necrosis factor-α; IFN-γ: interferon γ; IL: interleukin.

In Vitro Fluorescence Imaging of ASCs-QDs in Microtubes

To examine whether or not ASCs-QDs800 could be quantitatively detected, various numbers of labeled ASCs (5.0 × 10⁵, 2.5 × 10⁵, 1.3 × 10⁵, 0.6 × 10⁵, 0.3 × 10⁵, 0.15 × 10⁵, 0.75 × 10⁴ cells) were collected in PBS and spun down. The cell pellets were then prepared for a fluorescence analysis in microtubes (Fig. 2A). The labeled cell pellets were able to be quantitatively detected at high levels of fluorescence intensity (Fig. 2B). These data suggest that the ASCs-QDs could be detected quantitatively, and the fluorescence intensity was sufficient for cell visualization using the IVIS in vivo imaging system.

Figure 2.

The quantitative capability and in vivo real-time imaging of transplanted ASCs-QDs in kinetics mode. (A) The fluorescence images and intensities of ASCs-QDs800 (5.0, 2.5, 1.3, 0.6, 0.3, 0.15, 0.075, and 0 cells) using an in vivo imaging system. (B) The relationship between the fluorescence intensity and number of ASCs-QDs800. Representative data are shown (n = 3). (C) In vivo imaging of the mice treated without (left) and with ASCs-QDs800 transplantation (right) at 15 min after transplantation. (D) The time course of in vivo fluorescence images of transplanted ASCs-QDs800 (1 × 10⁶ cells) in normal and acute liver failure mice 4 and 24 h after CCl₄ injection. (E–G) The fluorescence intensity [total radiant efficiency (TRE)] of in vivo images of transplanted ASCs-QDs800 (1 × 10⁶ cells) in normal (E) and acute liver failure mice 4 (F) and 24 h (G) after CCl₄ injection. The approximate curves are shown from 0 to 120 s and from 120 to 900 s. Representative data are shown (n = 3). ASCs: adipose tissue–derived stem cells; QD: quantum dots.

In Vivo Fluorescence Imaging of the Transplanted ASCs-QDs in Kinetic Mode

ASCs-QDs800 (1.0 × 10⁶ cells) in a microtube were detected with high sensitivity using the IVIS^® Lumina K Series III before transplantation (Supplementary Fig. 3a). To investigate the behavior and accumulation of transplanted ASCs-QDs800 in mice, in vivo real-time fluorescence imaging was conducted in kinetics mode (movie mode) from 0 to 30 min after ASC transplantation (Fig. 2C, D). The fluorescence changes in the liver were recorded in video mode (Supplementary Fig. 3b, c). The fluorescence intensity in the liver region increased sharply in the first 3 min after transplantation and then decreased slightly with time. There were no significant differences in the fluorescence intensities among the three groups (normal, 4 h or 24 h transplantation after CCl₄ injection) (Fig. 2E–G). These data suggest that the transplanted ASCs accumulated in the liver of mice within a few minutes, before moving away from the liver slightly regardless of the degree of liver injury.

In Vivo and Ex Vivo Fluorescence Imaging of the Transplanted ASCs-QDs in Sequential Mode

Changes in the fluorescence intensities in liver were compared among the three groups up to 168 h (day 7) after ASC transplantation in sequential mode. In normal group, the fluorescence intensity in the liver decreased slightly with time, and the fluorescence signal was detected even at 168 h after ASC transplantation. However, in the CCl₄ injection group, the fluorescence intensity in the liver decreased relatively quickly in comparison with that in the normal group. Furthermore, the speed of the fluorescence intensity depended on the degree of hepatic damage at ASC transplantation (Fig. 3A–D). In addition, the fluorescence images of livers removed from mice at each time point (10 min and 1, 3, 6, 12, 24, 48, and 72 h) after ASC transplantation were observed and the fluorescence intensity was evaluated. The fluorescence derived from ASCs-QDs800 was confirmed until 48 h after transplantation (Fig. 3E). The fluorescence intensity gradually decreased in a logarithmic fashion (Fig. 3F).

Figure 3.

In vivo and ex vivo imaging of transplanted ASCs-QDs in sequential mode. (A) The time course of in vivo fluorescence images of transplanted ASCs-QDs800 (1 × 10⁶ cells) in normal and acute liver failure mice 4 and 24 h after CCl₄ injection. (B–D) The fluorescence intensity [total radiant efficiency (TRE)] of in vivo images of transplanted ASCs-QDs800 (1 × 10⁶ cells) in normal (B) and acute liver failure mice 4 (C) and 24 h (D) after CCl₄ injection. Representative data are shown (n = 3). (E, F) Ex vivo fluorescence images (E) and time course of the fluorescence intensity (F) of ASCs-QDs800 accumulated in the liver of acute liver failure mice 24 h after CCl₄ injection. ASCs: adipose tissue–derived stem cells; QD: quantum dots.

We also investigated the change in the fluorescence intensity in the liver region after injection of QDs800 alone into the normal mice using the IVIS in vivo imaging system. QDs800 alone in the microtube showed strong fluorescence (Supplementary Fig. 4a, b). The fluorescence intensity of QDs800 in the liver region was drastically lower than that of ASCs-QDs800, and the fluorescence decay curve was markedly different from that of ASCs-QDs800 (Supplementary Fig. 4c–f).

Intravital and Ex Vivo Multiphoton Fluorescence Imaging of Transplanted ASCs-QDs in the Liver of Acute Liver Failure Mice

To investigate the dynamics of ASCs just after transplantation in the liver, the intravital multiphoton fluorescence imaging of transplanted ASCs-QDs in the liver of acute liver failure mice was performed using a high-speed multiphoton confocal laser microscope (Fig. 4A). To ensure the labeling of ASCs, the surface of ASCs-QDs655 was co-labeled with DiR emitting at 780 nm. Two- and three-dimensional fluorescence images of ASCs labeled with QDs655 (inside) and DiR (cell membrane) (ASCs-QDs655-DiR) are shown in Fig. 4B. A large number of transplanted ASCs-QDs655-DiR were confirmed to arrive at the liver within about 3 min and stay in the same position until at least 10 min after transplantation (Fig. 4C). To obtain more information concerning the dynamics of ASCs just after transplantation, the intravital real-time behavior of transplanted ASCs-QDs655-DiR in the liver of acute liver failure mice was recorded in video mode (Supplementary Fig. 5). These results agreed with the data of in vivo fluorescence imaging in Fig. 2.

Figure 4.

Intravital and ex vivo multiphoton fluorescence imaging of transplanted ASCs-QDs in the liver of acute liver failure mice. (A) The fluorescence images of ASCs labeled with Hoechst 33342 (nucleus), QDs655 (inside), and DiR (membrane) (ASCs-QDs655-DiR). (B) The photograph of intravital imaging to acute liver in mice using a high-speed multiphoton confocal laser microscope. (C) The time course (0–10 min) of intravital multiphoton fluorescence images of ASCs-QDs655-DiR accumulated in the liver of acute liver failure mice 24 h after CCl₄ injection. The yellow arrows show the transplanted ASCs-QDs655-DiR (red and violet fluorescence). The blue and green fluorescence show the nuclei of hepatocytes and the blood vessels in the liver, respectively. (D) The time course (0.5, 24, 48, and 72 h after transplantation) of the ex vivo multiphoton fluorescence images of ASCs-QDs655 accumulated in the liver of normal and acute liver failure mice 4 and 24 h after CCl₄ injection. The yellow arrows show the transplanted ASCs-QDs655 (red fluorescence). The blood vessels in the liver were labeled with isolectin B4-FITC (green fluorescence). Representative data are shown (n = 3). ASCs: adipose tissue–derived stem cells; QD: quantum dots; FITC: fluorescein isothiocyanate.

To further investigate the engraftment of transplanted ASCs in livers removed from mice at 0.5, 24, 48, and 72 h after transplantation, the sacrificed liver was observed using a multiphoton excitation fluorescence microscope. In the normal group, ASCs-QDs655 were observed <72 h after ASC transplantation (Fig. 4D). The fluorescence signal derived from QDs655 was observed in liver sinusoids; therefore, almost all transplanted ASCs appeared to have been localized to and were still present in the liver sinusoids. Furthermore, the state of accumulation of ASCs-QDs655 was clearly different from that of only QDs655 (Supplementary Fig. 6). In the CCl₄-treated groups (4 and 24 h), ASCs-QDs655 were also observed in the liver sinusoids until 24 h after ASC transplantation, whereas the number of ASCs-QDs655 decreased in a time-dependent manner (Fig. 4D). To confirm the presence of ASCs, ex vivo multiphoton fluorescence imaging of transplanted ASCs-QDs655 co-labeled with PKH26 (ASCs-QDs655-PKH26) was performed. ASCs-QDs655-PKH26 were also observed in the liver sinusoids just after ASC transplantation, whereas the number of ASCs-QDs655 had markedly decreased at 72 h (Supplementary Fig. 7). These results agreed with the data of in vivo and ex vivo fluorescence imaging in Fig. 3.

Discussion

Many studies have reported the successful labeling of stem cells with QDs through various methods, such as electroporation, microinjection, and liposome-based transduction^29,30. We have already demonstrated that the labeling of QDs using cell-penetrating peptides (octa-arginine: R8) was efficient for ASCs, and that the transplanted ASCs-QDs800 could be detected in vivo with high efficiency^27,28. In this study, we confirmed that QD labeling did not affect the inflammation of ASCs, and intravenously transplanted ASCs-QDs800 were able to be detected in video mode without laparotomy using in vivo real-time fluorescence imaging technology. The number of ASCs-QDs800 was able to be analyzed quantitatively based on the fluorescence intensity of QDs800. In addition, we performed intravital multiphoton fluorescence imaging of transplanted ASCs-QDs at a single-cell level in the liver of mice.

These findings indicated that non-laparotomic processes can be used to conduct serial observations of transplanted ASCs and eliminate the influence of laparotomy on the inflammation system in mice. In acute liver failure, hepatocytes are drastically damaged, and the function of the liver is markedly decreased. Fulminant hepatitis (FH) shows particularly dramatic and extensive damage to the hepatocytes based on whether it is induced by hepatitis viruses or drugs^31,32. We used the major acute liver failure mouse model induced by CCl₄ injection in this study^33,34. We previously showed that acute liver failure in mice could be attenuated by ASC transplantation at 4 h but not 24 h after CCl₄ injection⁴. Indeed, the liver histology and levels of liver failure markers (ALT and AST) and inflammation cytokines (TNF-α) were markedly different between the two treated groups (ASC transplantation 4 and 24 h after CCl₄ injection). We assessed the influence of the inflammatory state of the affected liver on the behavior, accumulation, and engraftment of transplanted ASCs using this acute liver failure mouse model.

No marked differences in the behavior and accumulation of the transplanted ASCs was observed among the three groups (normal and 4 or 24 h transplantation after CCl₄ injection) until at least 30 min after ASC transplantation. The transplanted ASCs were found to have accumulated in the affected liver within at least 5 min, before gradually decreasing. However, significant differences in the engraftment of the transplanted ASCs in the affected liver were observed among the three groups from 1 h after ASC transplantation. In the normal group, the transplanted ASCs in the liver could be detected until at least 168 h after ASC transplantation. In the CCl₄ injection groups (4 h and 24 h), transplanted ASCs were observed in the liver sinusoids until 24 h after transplantation, but the number of transplanted ASCs decreased dramatically in a time-dependent manner. In addition, the engraftment rate of transplanted ASCs was inversely correlated with the degree of the liver damage and inflammation.

These differences in the engraftment rate of transplanted ASCs seem to have been induced by different immune states of the affected liver.^35–37 Wang et al.³⁵ showed that an inflammatory cytokine (TNF-α) antagonist promoted the survival of transplanted neural stem cells. Wei et al.³⁸ showed that CCl₄ injection induced the accumulation of natural killer (NK) cells in the affected liver and the production of TNF-α or IFN-γ. Furthermore, Scoazec and Feldmann³⁹ and Volpes et al.⁴⁰ showed that the liver damage induced changes in the expression of adhesion molecules, such as PECAM-1 (platelet-endothelial cell adhesion molecule-1), ICAM-1 (intercellular adhesion molecule-1), and E-selectin, on the surface of sinusoidal endothelial cells. In the initial phase of acute liver failure induced by CCl₄ injection, CCl₄ is converted to high trichloromethyl radical (CCl₃) by CYP2E1, and then CCl₃ activates the Kupffer cells and induces the production of inflammatory cytokines, such as TNF-α^41–43. Therefore, the production levels of inflammatory cytokines, the degree of accumulation of different immunocompetent cells, including Kupffer cells and NK cells, and the changes in the production of cell adhesion proteins of sinusoidal endothelial cells are considered to affect the engraftment of transplanted stem cells. Indeed, the influence of Kupffer cells on the transplanted ASCs was confirmed using QD technology in our latest studies (data not shown).

However, how these phenomena are associated with a decrease in the engraftment of transplanted ASCs in the affected liver is unclear. Clarifying these relationships will require the quantitative estimation of the accumulated immunocompetent cells in the affected liver and the production changes in the cell adhesion proteins of sinusoidal endothelial cells. QDs are able to label many kinds of cells and molecules; as such, in vivo fluorescence imaging using QDs may be useful for the analysis of the relationship between the transplanted ASCs and accumulated immunocompetent cells or sinusoidal endothelial cells.

In conclusion, we developed in vivo real-time fluorescence imaging technology of intravenously transplanted ASCs in video mode without the need for laparotomy and intravital multiphoton fluorescence imaging of transplanted ASCs at a single-cell level in the liver of mice. In addition, we clarified whether or not the inflammatory state of acute liver failure affects the dynamics (behavior, accumulation, and engraftment) of transplanted ASCs in the mouse liver using these QD imaging-based technologies. No marked differences in the behavior or accumulation of transplanted ASCs were observed among the three liver damage groups (normal, weak and strong) until at least 30 min after ASC transplantation. However, significant differences in the engraftment of transplanted ASCs in the affected liver were observed among the three groups from 4 h after transplantation. The engraftment rate of the transplanted ASCs was inversely correlated with the degree of the liver damage and inflammation. Our findings suggest that the inflammatory state of acute organs affects the engraftment of transplanted stem cells.

Supplemental Material

sj-docx-1-cll-10.1177_09636897231176442 – Supplemental material for In Vivo Real-Time Quantum Dots Imaging to Track Transplanted Adipose Stem Cells in Different Inflammatory States of Acute Liver Failure Mice

Supplemental material, sj-docx-1-cll-10.1177_09636897231176442 for In Vivo Real-Time Quantum Dots Imaging to Track Transplanted Adipose Stem Cells in Different Inflammatory States of Acute Liver Failure Mice by Shota Yamada, Hiroshi Yukawa, Koudai Kitamura, Toshiki Mizumaki, Yasuma Yoshizumi, Tomomi Oohara, Eri Nanizawa, Fumika Hirano, Kazuhide Sato, Ayae Sugawara-Narutaki, Tetsuya Ishikawa and Yoshinobu Baba in Cell Transplantation

Footnotes

Acknowledgements

We appreciate the help of Yoko Tsutsui (Nagoya University) for the treatment of ASCs.

Author Contributions

S.Y. and H.Y. devised the concept. S.Y., Y.Y., K.K., T.M., T.O., E.N., F.H., and K.S. carried out the experiments and analyzed the data. H.Y., K.S., A.S.-N., T.I., and Y.B. directed this project and developed the procedures. S.Y., H.Y., and T.I. wrote the manuscript. All authors contributed to the discussion of the project.

Availability of Data and Materials

The data presented in this study are available upon request to the corresponding author.

Ethical Approval

This study was approved by our institutional review board.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Research Ethics and Patient Consent

The animal study protocol was approved by the Nagoya University Committee on Animal Use and Care (protocol code: M220370-003, date of approval: March 31, 2022). Patient consent is not applicable.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was mainly supported by the Japan Agency for Medical Research and Development (AMED) through its “Research Center Network for Realization of Regenerative Medicine.” This work was partially supported by JSPS KAKENHI Grant Numbers 21H05589, 21H04663, 22H03938, 22H03924), Quantum Leap Flagship Program (Q-LEAP, JPMXS0120330644) of MEXT, Advanced Research Infrastructure for Materials and Nanotechnology in Japan (ARIM, Nagoya University) of MEXT, and SEI group CSR foundation.

ORCID iD

Shota Yamada

Supplemental Material

Supplemental material for this article is available online.

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