In vivo tracking of 14C thymidine labeled mesenchymal stem cells using ultra-sensitive accelerator mass spectrometry

Despite the tremendous advancements made in cell tracking, in vivo imaging and volumetric analysis, it remains difficult to accurately quantify the number of infused cells following stem cell therapy, especially at the single cell level, mainly due to the sensitivity of cells. In this study, we demonstrate the utility of both liquid scintillator counter (LSC) and accelerator mass spectrometry (AMS) in investigating the distribution and quantification of radioisotope labeled adipocyte derived mesenchymal stem cells (AD-MSCs) at the single cell level after intravenous (IV) transplantation. We first show the incorporation of 14C-thymidine (5 nCi/ml, 24.2 ng/ml) into AD-MSCs without affecting key biological characteristics. These cells were then utilized to track and quantify the distribution of AD-MSCs delivered through the tail vein by AMS, revealing the number of AD-MSCs existing within different organs per mg and per organ at different time points. Notably, the results show that this highly sensitive approach can quantify one cell per mg which effectively means that AD-MSCs can be detected in various tissues at the single cell level. While the significance of these cells is yet to be elucidated, we show that it is possible to accurately depict the pattern of distribution and quantify AD-MSCs in living tissue. This approach can serve to incrementally build profiles of biodistribution for stem cells such as MSCs which is essential for both research and therapeutic purposes.

www.nature.com/scientificreports/ (10 -18 ) ~ zeptomole (10 -21 ) quantities of 14 C in small samples) method such as accelerator mass spectrometry (AMS) is required to quantify stem cells labeled with low 14 C thymidine radioactivity without interfering with their normal function 13 . Originally developed for the field of radiocarbon dating in the late 1970s, AMS has proven to be an effective instrument for biomedical and pharmaceutical research when trace quantities of 14 C need to be quantified in small samples 14,15 . Recently, the tracking of tumor colonization was demonstrated by AMS in a xenograft model which served to quantitatively evaluate metastasis and tumor aggressiveness 16 . While the biological mechanisms and significance behind the distribution of cells throughout time remains largely a mystery due to the limited number of studies, a wider array of data could help to identify general patterns in distribution and localization dependent on cell type and route of delivery. Herein, we demonstrate using AMS in investigating the distribution and localization of adipocyte derived mesenchymal stem cells (AD-MSCs) at the single cell level after intravenous (IV) transplantation. This study corroborates some of the findings made in previous work but it also reveals the localization of single cells in unexpected organs at early time points. While the significance of these cells is yet to be elucidated, we show that it is possible to accurately depict the pattern of distribution and localization of AD-MSCs in living tissues over time.

Results
Selection of optimal concentration for 14 C-labeling AD-MSCs. Since 14 C-thymidine incorporates into the newly replicated DNA when the cell divides, we simply rationalized that AD-MSCs exhibiting a higher proliferation rate would incorporate higher amounts of 14 C-thymidine during cultivation. Therefore, 2.5 nCi/ ml of 14 C-thymidine was added into various medium conditions (10% FBS, 20% FBS, 10% FBS + bFGF, and 20% FBS + bFGF) to determine the optimal culture group for cell proliferation and consequently, 14 C-thymidine incorporation (Fig. 1A). Cell counting was performed on day 6 which revealed the proliferative capacity of each group ranking from highest to lowest; 20% FBS + bFGF > 20% FBS > 10% FBS + bFGF > 10% FBS (Fig. 1B). Interestingly and contrary to our expectation, 14 C activity was significantly higher in the groups containing 10% FBS, especially when supplemented with bFGF, despite the higher proliferation rate observed in those containing 20% FBS (Fig. 1C).
Thus, the 10% FBS + bFGF group was selected to be best suited for both AD-MSC proliferation as well as 14 C-thymidine incorporation. Next, we sought to determine whether increasing concentrations of 14 C-thymidine would negatively impact AD-MSC proliferation and morphology by comparing cultivations that received 0 (control), 1, 2.5, 5, 10, or 25 nCi/ml of 14 C-thymidine (Supplementary Table S1). Based on observation at day 6, it was clear that concentrations of 10 nCi/ml and 25 nCi/ml reduced the proliferation rate of AD-MSCs as more vacant spaces were readily identified in both groups (Fig. 1D). This was further confirmed by cell counting (hemocytometer) and CCK-8 assay which showed a marked decrease in both the total and viable cell population, respectively (Fig. 1E,F). When the level of 14 C-thymidine uptake for each group was measured as a percentage by LSC, it revealed a decline in uptake efficiency for AD-MSCs that received concentrations of 10 and 25 nCi/ ml (Fig. 1G). As expected, higher levels of 14 C activity was detected as the concentration increased (Fig. 1H) and the 14 C-thymidine incorporated DNA was able to be extracted for all groups (Fig. 1I). Based on these results, a concentration of 5 nCi/ml was determined to be the optimal concentration for AD-MSCs because 14 C activity was higher than the 1 and 2.5 nCi/ml groups while the cell proliferation rate and uptake efficiency was maintained, unlike the 10 and 25 nCi/ml groups which exhibited adverse effects.
Characterization of 14 C-labeled AD-MSCs. Unlabeled and 14 C-labeled AD-MSCs were characterized by specific CD marker expression and multi-lineage differentiation to compare surface characters and functional potential. FACS analysis revealed that both populations displayed the hallmark MSC antigen profile of positive CD73, CD90, and CD105 complemented by negative CD34 and CD45 ( Fig. 2A). However, a noticeable decrease in CD105 expression was detected as 92.72% of the unlabeled AD-MSCs expressed CD105 which was higher than the 80.62% expressed by the labeled cells. While it remains unclear as to why a difference in marker expression emerged.
The functional characteristics of AD-MSCs and its multipotency were investigated through differentiation. Both groups were subjected to differentiation conditions for adipocytes, osteoblasts, and chondrocytes which were identified by Oil Red O, Alizarin red S, and Alcian blue staining, respectively. Both groups were capable of differentiating into all three cell types, confirming that multipotency was retained in the labeled group (Fig. 2B). In addition, cell cycle analysis was performed to further compare both populations in relation to the different phases of the cell cycle. Both groups shared a very similar profile for the sub-G1, G1, S, and G2-M phases, indicating that cell growth, genome replication, and ultimately the process of mitosis remained unchanged after 14 C-labeling (Fig. 2C). These results demonstrated that the key characteristics of AD-MSCs in regards to surface markers, multipotency, and cell division were retained in the labeled cells, suggesting that the incorporation of 14 C-thymidine does not affect the key characteristics of AD-MSCs.
Pre-treatment process and calibration the LSC and AMS methods. The detection and quantification of radioactivity by LSC and AMS were compared by first transplanting 50,000 14 C-labeled AD-MSCs into a homogenized liver sample before analysis. For LSC, the organ samples were simply solubilized (SOLV-ABLE, PerkinElmer, Waltham, MA, USA) then decolorized using H 2 O 2 before sample analysis (Fig. 3A). The LSC calibration curve was prepared using 14 C activity values (disintegration per minute, dpm) according to the number of labeled AD-MSCs (200, 500, 1000, 2500, 5000, 10,000, and 50,000 cells) and the R 2 value was 0.9982, indicating linearity (Fig. 3B). Furthermore, to generate a calibration curve for the AMS analysis, 50,000 labeled AD-MSCs were spiked into the homogenized liver sample and diluted to the indicated number of cells (1, 5, 10, 25, 50, and 100 cells). In contrast to LSC, AMS required a comparatively lengthy process that involves the www.nature.com/scientificreports/ graphitization of organ samples before analysis which is detailed in the Materials and Methods section (Fig. 3C).
The diluted 14 C-labeled AD-MSCs were measured by AMS after pretreatment. The calibration curve of the AMS was prepared using modern carbon (MC, the ratio of 14 C/ 12 C in the reference atmosphere of the 1950s), and a dynamic range of the AMS from 0.1 MC to 150 MC (0.001356 -2.034 dpm/mgC) according to the number of labeled AD-MSCs. The R 2 value was 0.9997, indicating linearity (Fig. 3D). When comparing the number of detectable AD-MSCs for each assay, it showed that LSC could not detect less than 200 AD-MSCs. However, a value of 3.4 MC in one cell was obtained from AMS analysis, which was 30 times higher than the limit of detection. These results show that LSC is inadequate in detecting a small number of cells and AMS is required for tracking single cell populations.

Quantification of 14 C-labeled AD-MSCs in nude mice by LSC and AMS analyses. In order to
investigate the distribution and localization of transplanted AD-MSCs in vivo, nude mice were subjected to IV injection (1 × 10 6 cells) and 14 C radioactivity was measured in the lung, spleen, liver, heart, kidney, and brain by both LSC and AMS at 4 h, 12 h, 24 h, 48 h, and day 7. Using the calibration curve obtained from  www.nature.com/scientificreports/ LSC was obtained using the cell number and radioactivity, Eq. (1). The cell number was calculated Eq. (2), (3). Where, L sample and L blank are the radioactivity (dpm) of the sample and pre-dose organ, respectively The measurement results of AMS were obtained by the ratio of the amount of 14 C ( 14 C cell and 14 C organ ) and 12 C ( 12 C organ ) as shown in the following equation Eq. (4). R mesu and R blank are the value of 14 C/ 12 C of the sample and pre-dose organ, respectively. 12 C organ is the amounts of carbon ( 12 C) contained in the prepared organ. R sample was calculated by subtracting R mesu from R blank , Eq. (5).
(1) www.nature.com/scientificreports/ The linear regression equation of calibration curve for quantitation of cell number using AMS was obtained using the cell number and modern carbon, Eq. (6). The cell number was calculated Eqs. (7) and (8).
Cell concentration (cell number/mg) and cell amount (cell number/organ) can be obtained from cell number, the weight of the sample (W sample ), and the weight of the organ (W organ ). Therefore cell concentration and cell amount were calculated, Eq. (9), (10).   (Fig. 4A). In contrast, AMS was able to detect a small number of cells located in all other tested organs but difficult to quantify larger numbers such as the lung's case due to over-detection. At 4 h post-transplantation, the number of AD-MSCs per mg located in the spleen and liver was 19 ± 8 and 14 ± 1, respectively. And even lesser amounts or single cells were detected in the heart and kidney (1 ± 0 infused cells number/mg) while no cells were detected in the brain. Interestingly, the number of cells increased at 12 h with exception to the lung which may indicate the occurrence of cell migration from highly populated organs such as the lung to other organs such as the liver and spleen. However, the number of cells was reduced in the liver after 24 h as a fourfold, while other organs (spleen, heart, and kidney) were observed after 12 h as a twofold reduction. Even though the decline continued at 48 h, a small number of residual cells remained within each tissue at day 7 (Fig. 4B). Based on the compiled data of both LSC and AMS, we were able to determine the number of infused cells per organ based on the average mass of each organ to better illustrate AD-MSC localization following IV transplantation (Fig. 4C). The majority of AD-MSCs were present in the lung from 4 h (619,266 ± 36,239) to 48 h (157,448 ± 40,908) and even though the number of active cells continued to decline, ~ 5000 ± AD-MSCs lingered within the organ on day 7. As for the liver, the number of cells significantly increased from 4 h (14,796 ± 1,552) to 12 h (21,941 ± 1,060) before a sharp decline at 24 h (4,761 ± 907). These values were interestingly similar to that of the lung on day 7. The spleen also exhibited a rise in population from 4 h (1,497 ± 590) to 12 h (1,993 ± 454) followed by a decline at 24 h (1,018 ± 156) which plateaued at 48 h (884 ± 593) to day 7 (908 ± 674). This pattern of distribution was similar in the heart and kidney at a much lesser extent (Supplementary Table S2).  . And similar to that of 14 C-labeled AD-MSCs, we found that the majority of cells populated the lung at 4 h followed by the liver and spleen. We also found that the cells began to clear within the first day yet a small number of residual cells remained within the specified organs on day 7. These results complements the tracking of AMS by showing that cells are engrafted visually, although accurate quantification is not possible within each organ (Fig. 5).

Discussion
This study demonstrates the successful incorporation of 14 C-thymidine (5 nCi/ml) into AD-MSCs without affecting key biological characteristics pertinent to morphology, CD marker expression, differentiation, and cell cycle. These cells were then utilized to track and quantify the distribution of AD-MSCs delivered through an intravenous route (tail vein) by AMS, revealing the number of AD-MSCs existing within different organs per mg and per organ at different time points. Notably, the results show that this highly sensitive approach can quantify one cell per mg which effectively means that AD-MSCs can be detected in various tissues at the single cell level. Even though the history of AMS is extensive such as Geoscience, Archaeology, Environmental Science, and Biomedical Science, its adaptation to the field of regenerative medicine is fairly recent as only a few studies documented its usage in the tracking and quantification of distributed stem cells in vivo. Previously reported radioisotope labeling methods such as 51 Cr, 124 I, and 99m TC often require highly radioactivity for imaging purposes; 18.5 MBq (500 µCi) for 51 Cr, 3.7 MBq/0.1 ml (100 µCi/0.1 ml) for 124 I-FIAU, and 3.7 MBq (100 µCi) for 64 Cu-PISM 17,18 . Furthermore, studies that employ a β-counter, often used concentrations of 1 µCi of 3 H-thymidine or 14 C-thymidine which has also been estimated to cause cell damage 12,[19][20][21] . In this study, we show that far lower concentrations can be used for the long term tracking of AD-MSCs following transplantation due to long halflife of 14 C (half-life = 5,370 years). Since stem cells exhibit plasticity unlike somatic cells, it is very likely that they are more prone to change upon interference thereby a highly sensitive approach is advantageous to ensure that its character is not disrupted before administration.
Another important factor for cell distribution is the route of delivery. Although our study is limited to IV, it is well known that other delivery routes, whether systematic (IV) or local (direct injection) can change the distributive outcome. For example in IV and intra-atrial (IA), cells are able to be distributed throughout several organs with most of the cells accumulating within the lung, liver, and spleen as shown in this study which is also consistent with previous reports 9,22,23 . In contrast, direct injection (DI) can target a specific tissue but prohibits interactions between MSCs and secondary signaling systems 24 . And because there is no consensus on the optimal delivery route for MSCs at this time, the approach used in this study can help to elucidate the distribution and localization of stem cells following transplantation through a selected route in order to further determine effective routes for various cell types. In sum, radioisotope labeling paired with AMS can be a very useful tool in tracking and quantifying AD-MSCs at the single cell level after transplantation. Future studies should focus on applying a similar approach to other stem cell variants of both multipotent and pluripotent origin to incrementally build profiles of biodistribution which can serve to accelerate our understanding of stem cell behavior in vivo especially in light of the increasing attention and clinical studies surrounding stem cell therapeutics.

Analysis of liquid scintillation counter (LSC).
The activity of the incorporated 14 C-thymidine into the DNA of AD-MSCs was measured using Tri-Carb 4910TR LSC instrument (Perkin Elmer, Shelton, CT, USA). 14 C labeled AD-MSC and extracted DNA in PBS (DNA concentration, 100 μl) of aliquot from each sample were added into 10 ml of LSC cocktail (Perkin Elmer, Shelton, CT, USA). Each radioactivity in mixed solution was measured through LSC for 30 min.

Characterization of 14C-labeled adipose-derived mesenchymal stem cells. Characterization
of AD-MSC was conducted according to our previously described method 25 . Cells were detached by 0.25% trypsin-EDTA at ~ 90% passage, detached cells were resuspended in FACS buffer (PBS solution including 0.5% bovine albumin (BSA) and 2 mm EDTA) and filtered using a premoistened a 40-µm cell strainer. Cells were then labeled using each antibody of MSC surface markers according to manufacturer's instructions. Types of antibody are as follows. Fluorochrome-conjugated antibodies for CD73-PE, CD90-APC, and CD105-PE (BD Biosciences, Bedford, MA, USA), along with a negative marker CD34 and CD45 conjugated to PE (BD Biosciences) were used. Also, corresponding IgG controls were prepared equally, and 30,000 labeled cells were acquired and analyzed using Becton Dickinson FACS Calibur (BD Biosciences).
Assessment of multilineage differentiation. Multilineage differentiation of AD-MSC was conducted according to our previously described method 25 . For the induction of osteoblasts, adipocytes, and chondroblasts, commercially available kits were used (Thermo Fisher Scientific, Waltham, MA, USA). Cells under differentiation conditions were maintained in 4-well plates or 12-well plates. Osteogenesis was incubated for 21 days, and adipogenic and chondrogenic lineage was induced for 14 days. All experimental procedures were performed according to the manufacturer's instructions. To evaluate each differentiation process, appropriate staining was performed: Alizarin Red S to identify calcium deposits, Oil Red O to detect intracellular lipid droplets, and Alcian blue to confirm the formation of proteoglycans. Images were analyzed using and inverted microscope.
Intravenous injection of 14 C-labeled AD-MSCs in nude mice. BALB/c nude mice (6 weeks old, male, weight 19-22 g) were supplied by Orient Bio (Seongnam, Republic of Korea). All experimental procedures were performed according to the guidelines and with the approval of the Institutional Animal Care and Use Committee (IACUC) of the Korea Institute of Science and Technology (IACUC number: KIST-2019-002). Housed at the Integrated Animal Center of the Korea Institute of Science and Technology (KIST) maintained on a 12 h interval day and night cycle. Mice were allowed free access to water and feed. In order to confirm the distribution of 14 C-labeled AD-MSCs after the transplantation of cells into mice, five different groups of mice were prepared for assessment at 4 h, 12 h, 24 h, 48 h and 7 days after intravenous (IV) injection (n = 3). Then, close to 1 × 10 6 of 14 C-labeled AD-MSCs suspended in 150 μl of PBS were injected into each mouse via the tail vein. After the injection of the 14 C-labeled AD-MSCs, the mice were sacrificed with CO 2 and organs (lung, spleen, liver, heart, kidney, brain) were extracted. In order to prevent 14 C contamination between organs during separation, each part was separated a set of forceps that underwent frequent cleaning them with PBS. The extracted organs were washed with PBS and then frozen in a cryo-tube. Samples were stored in a -80 °C until further processing. All animal experiments were approved by an ethics committee organized in KIST.
Graphitization of samples and accelerator mass spectrometry (AMS) measurement. In order to homogenize collected organ were washed 2 times in PBS prior to organ homogenization using a Bead-