Improved efficacy and in vivo cellular properties of human embryonic stem cell derivative in a preclinical model of bladder pain syndrome

Interstitial cystitis/bladder pain syndrome (IC/BPS) is an intractable disease characterized by severe pelvic pain and urinary frequency. Mesenchymal stem cell (MSC) therapy is a promising approach to treat incurable IC/BPS. Here, we show greater therapeutic efficacy of human embryonic stem cell (hESC)-derived multipotent stem cells (M-MSCs) than adult bone-marrow (BM)-derived counterparts for treating IC/BPS and also monitor long-term safety and in vivo properties of transplanted M-MSCs in living animals. Controlled hESC differentiation and isolation procedures resulted in pure M-MSCs displaying typical MSC behavior. In a hydrochloric-acid instillation-induced IC/BPS animal model, a single local injection of M-MSCs ameliorated bladder symptoms of IC/BPS with superior efficacy compared to BM-derived MSCs in ameliorating bladder voiding function and histological injuries including urothelium denudation, mast-cell infiltration, tissue fibrosis, apoptosis, and visceral hypersensitivity. Little adverse outcomes such as abnormal growth, tumorigenesis, or immune-mediated transplant rejection were observed over 12-months post-injection. Intravital confocal fluorescence imaging tracked the persistence of the transplanted cells over 6-months in living animals. The infused M-MSCs differentiated into multiple cell types and gradually integrated into vascular-like structures. The present study provides the first evidence for improved therapeutic efficacy, long-term safety, and in vivo distribution and cellular properties of hESC derivatives in preclinical models of IC/BPS.


Results
Characterization of M-MSCs derived from hESCs. The hESC line H9 was differentiated by embryoid body (EB) formation for 2 days and the mesenchymal cells were isolated as those migrating to the lower compartment of a porous Transwell membrane (8 μm) over 5 days (Fig. 1a). Plating of migrated cells onto collagen-coated dishes naturally selected the CD140B + CD44 + M-MSCs, thus this controlled hESC differentiation resulted in greater than 99% pure M-MSCs within 7 days 24 . The M-MSCs exhibited several typical MSC features, including spindle and fibroblast-like morphology (Fig. 1b), expression of MSC (CD73 and CD105) and pericyte (PDGFRB, CD146, and NG2) surface antigens but little MHC class II (HLA-DR), hematopoietic progenitor (CD133), or endothelial (KDR and Tie-2) marker expression (Fig. 1c). They differentiated into mesodermal osteogenic and adipogenic lineages (Fig. 1e) and substantially downregulated markers of pluripotency (OCT4, NANOG, and SOX2), and upregulated expression of EMT markers such as fibronectin, consistent with MSC-like characteristics 24 . The established M-MSCs could be expanded for more than 30 passages (once per day) without identifiable chromosomal changes (Fig. 1d). Moreover, M-MSCs exhibited tube-forming capacity on Matrigel (Fig. 1e) and also formed functional junctions with blood vessel endothelial cells, as evidenced by dye-transfer assay (Fig. 1f), indicating robust pro-angiogenic potency.
by M-MSC therapy ( Fig. 4a and b). Furthermore, gene expression analysis indicated that the bladder tissues in IC-HCl rats were characterized by the increased expression of Ngf and other genes associated with visceral hypersensitivity such as tumor necrosis factor-α and tachykinin receptor-1; however, the administration of M-MSCs significantly restored their induction in bladder tissues (Fig. 4c). These results suggest that M-MSC therapy could be beneficial in controlling visceral organ crosstalk as well as the severity and frequency of abdominal pain or discomfort in IC/BPS and IBS patients.

Significance of Wnt and downstream growth factors on M-MSC therapeutic outcome.
To investigate the mechanism of action of M-MSCs therapy for IC/BPS, we examined the expression of genes related to sonic hedgehog (Shh), Wnt, and growth factors that are activated by UCB-MSC therapy to promote SCIeNtIfIC RepoRts | 7: 8872 | DOI:10.1038/s41598-017-09330-x epithelial regeneration in a HCl-IC rat model 13 . As shown in Fig. 5a, M-MSC therapy significantly upregulated Shh and Wnt family genes (e.g., Smo, Wnt5a, Wnt8a, Wnt8b, Wnt10a, and Wnt11) as well as their downstream growth factors (e.g., Igf1, and Igf2), which were characteristically downregulated in bladders of the HCl-IC group. Accordingly, the HCl-IC + M-MSC group bladders were characterized by the increased expression and nuclear localization of β-catenin protein, a surrogate marker for Wnt signaling activation ( Fig. 5b and Supplementary  Fig. 2). Importantly, the recovery of bladder voiding functions was significantly abrogated by daily injection of indomethacin 32 or Gefitinib 33 , inhibitors for Wnt and IGF-mediated signaling activity, respectively ( Fig. 5c and Supplementary Fig. 3). The treatment of Gefitinib had little effect on the engraftment of the administrated M-MSCs at the injection site in serosa of the bladder; however, little integration of injected cells was observed in urothelium (Fig. 5d). Indomethacin significantly impaired the engraftment of administrated M-MSCs. Thus, these results indicate that the Wnt and IGF signaling cascades play a crucial role in the beneficial outcomes of M-MSC in treating IC/BPS bladders.  To gain mechanistic insight into the enhanced efficacy of M-MSCs, we examined the kinetic of in vivo engraftment/retention after transplantation of the engineered M-MSCs or BM-MSCs which stably express Nano-lantern, a chimera of enhanced Renilla luciferase and Venus fluorescent protein with high efficiency of bioluminescence resonance energy transfer (BRET) 34 . To optimally monitor the bioluminescence signals of transplanted cells, we employed a murine model in which IC/BPS was induced by CYP 35 and 2 × 10 5 Nano-lantern + M-MSCs or BM-MSCs were locally injected into bladder tissues. In optical imaging system, bioluminescence intensity from the transplanted M-MSCs were observed until 8 day after transplantation (DAT) ( Supplementary Fig. 5a). After rapid decrease (~5% as compared with that when cells were injected) in 1 DAT, the bioluminescence activity from the engrafted M-MSCs sustained until 4 DAT and largely decreasing after 7 DAT (Fig. 6d). Compared to animals treated with BM-MSCs, animals transplanted with M-MSCs demonstrated significantly brighter bioluminescence throughout the whole experimental period (Fig. 6d), indicating the superior engraftment capacity of M-MSCs.
We next monitored the tumorigenicity of transplanted M-MSCs non-invasively by employing longitudinal micro-positron emission tomography/magnetic resonance imaging (μ-PET/MRI) imaging for 12-months after injection. As shown in Supplementary Fig. 5b, only background signal for uptake of 2-[ 18 F]-fluoro-2-deoxyglucose (FDG) was detected, and no other [ 18 F]-FDG uptake characteristics of tumors were observed in any HCl-IC animal injected with 1 × 10 6 M-MSCs or PBS vehicle. We confirmed this low tumorigenic potency of M-MSCs by a thorough microscopic investigation of organs in a double-blind necropsy followed by final nano-ScanPET/MRI analysis. Taken together, these results suggest that M-MSC-based therapy, with no adverse or safety issues, exerts superior therapeutic and engraftment capacities for treating IC/BPS bladder dysfunction by supporting Wnt signaling-related epithelial regeneration.
In vivo cellular properties of infused M-MSC. In immunofluorescent analysis of M-MSCs stably expressing green fluorescence protein (GFP), the majority of GFP + cells in HCl-IC animals on 7 DAT were localized at the injection site between muscle and serosa of the bladder and some GFP + cells were observed in the lamina propria and urothelium, but few were detected in the muscular layer (Fig. 7a). In contrast, the bladder tissues of rats injected with GFP + BM-MSCs showed few engrafted GFP + cells ( Supplementary Fig. 4b), in line with the bioluminescence assay results (Fig. 6d). To analyze the phenotype of engrafted M-MSCs, multichannel laser scanning confocal microscopy was performed by labeling for engrafted cells (GFP), epithelial cells in urothelium (E-cadherin), stromal cells (vimentin), and endothelial cells (CD31). The GFP + cells found in urothelium expressed the E-cadherin in membrane, indicating their differentiation into epithelial cells (Fig. 7b and c). Majority of GFP + cells found in lamina propria was dispersed as stromal cells with strong co-staining with vimentin protein. Particularly, some GFP + cells in serosa and underneath urothelium was distributed into vessel-like structures (Fig. 7a). Confocal microscopy data indicated that GFP + cells formed in vessel-like clusters little expressed CD31 endothelial cell surface protein, but they were in close contact with the CD31 + cells and some of them expressed the vimentin, a pericyte intermediate filament (Fig. 7b). The morphology, localization, and expression of tissue markers indicated that the engrafted M-MSCs hardly differentiate into endothelial cells, but they behaved as pericytes. This perivascular M-MSC phenotype is line with a preceding in vitro data that M-MSCs expressed surface antigens for pericyte (PDGFRB, CD146, and NG2) but not endothelial cells (Fig. 1c). These results demonstrate that a considerable amount of transplanted M-MSCs survive and differentiate into multiple cell types in the bladder tissue.

Longitudinal confocal imaging of infused M-MSCs in living animals.
To investigate in vivo properties of engrafted M-MSCs at the cellular level in living animals (Fig. 8a), we employed intravital fluorescence microscopy which enables the study of in vivo cellular processes such as cell trafficking, intercellular interactions, and vascular changes 36 . Using front-view endoscopic optical probes directed toward the urothelium (Fig. 8b), we performed longitudinal imaging of infused GFP + M-MSC fluorescence for 6-months. Consistent with optical imaging results, strong focal fluorescence was detected immediately after transplantation by endoscopy. Fluorescence intensity was greatly reduced by 2 DAT but was relatively stable until 21 DAT (Fig. 8c and Supplementary Movie S1). During this period, GFP fluorescence was observed within multiple cellular structures. However, it became weak and blurred beyond 28 DAT.
We next performed high resolution in vivo confocal microscopy using objective lenses focused on the outer layer of the bladder through minimal incisions on the abdominal surface (Fig. 8b). Fluorescence intensity gradually decreased until 21 DAT. However, GFP + cells with clear morphology were still observed at 28 DAT and emission was sustained until 181 DAT, whole our observation period (Fig. 8d, Supplementary Movies S2 and S3). Little auto-fluorescence was observed in animals injected with vehicle ( Supplementary Fig. 6a and b). In M-MSC-injected animals, GFP + cells with distinct shapes were detected at 28 DAT with broad distribution over the entire bladder ( Fig. 8d and Supplementary Fig. 6c). By 2-months, the engrafted cells formed discrete foci and the majority were detected as components of blood-vessel like structures. As aforementioned intravital imaging data, confocal microscopic analysis of bladder tissues indicated that the engrafted M-MSCs functionally integrated into the epithelial cells (E-cadherin + ) in urothelium ( Supplementary Fig. 6d and e) and perivascular cells (vimentin + in close proximity to CD31 + cells) at 21 DAT (Fig. 8e) and the pericyte phenotype of M-MSCs remained stable until 6-months (Fig. 8f). Thus, these in vivo imaging data support that engrafted M-MSCs may initially replenish the urothelial layer and progressively contribute to perivascular cells.

Discussion
Although several promising preclinical studies using MSCs for treating IC/BPS have been reported, limited proliferation and impaired stemness during ex vivo expansion as well as a general lack of information on the in vivo properties of transplanted cells have provoked skepticism over current MSC-based therapy. To overcome these limitations, we demonstrate that hESCs can provide a virtually unlimited source of MSCs and that injection of  these hESC-derived M-MSCs results in improved therapeutic outcome for treating IC/BPS in a model animal compared to adult tissue-derived cells, without any adverse safety issues. Furthermore, by longitudinal intravital confocal imaging in living animals, we provide precise information on the in vivo distribution and cellular properties of transplanted cells.
IC/BPS is regarded as a heterogeneous multifactorial disease with unclear pathogenesis 37 . The disease etiology is not yet fully understood, and definitive treatments have not been reported. Even though several treatment approaches including several oral medications, bladder instillation therapies, fulguration for Hunner lesion, and hydrodistention have been used, their outcomes have not been satisfactory. IC/BPS may be chronic inflammatory and fibrotic conditions in which mast cell activation, sensory nerve hyperactivation, nitric oxide or autoimmune mechanisms, and glycosaminoglycan layer defects broadly affect the entire bladder, implying that multi-modal treatment approaches could be required [38][39][40] . For this purpose, SC therapy has several advantages, and indeed MSC-based therapy has proven beneficial in preclinical studies by regenerating damaged tissues through differentiation into target cells and creating a micro-environment favorable to tissue repair [12][13][14] .
Of note, almost 40% of patients diagnosed with inflammatory bowel syndrome presented with bladder pain, while as many as 40% diagnosed with IC/BPS had symptoms that fulfill the criteria for inflammatory bowel syndrome 29,30 . Visceral organ communication and the resulting hypersensitivity may be due to the convergence of sensory neural pathways in the dorsal root ganglion, spinal cord, and brain 41 . Other evidence showed sensitization of afferent neurons at the dorsal root ganglion as well as lumbosacral neurons that become hyper-excitable following colonic inflammation 31 . Thus, the activation of afferent nerves in response to mucosal damage may be the result of increased epithelial permeability, allowing electrolytes to have direct access to the visceral sensory neurons. In this regard, the structural regeneration and attenuation of inflammation by M-MSC therapy could prevent visceral organ crosstalk. Accordingly, we found that a single administration of M-MSCs significantly moderated the anatomical interaction between mast cells and nerve fibers, a most convincing parameter in visceral hypersensitivity ( Fig. 4a and b), and also the increased expression of Ngf, which is abundantly synthesized and released by both mast cells and nerve fibers (Fig. 4c). Thus, examination of inflammation status in the intestines or pain assessment after M-MSC therapy should be investigated further.
Consistent with previous reports using UCB-MSCs 13 , a single administration of hESC-derived M-MSCs had therapeutic effects in HCl-induced IC animal models (Fig. 2). The engrafted cells were localized mainly between muscle and serosa and more sparsely in lamina propria as vimentin + stromal cells (Fig. 7a,b, and Fig. 8g), which could stimulate Wnt-related epithelial regeneration capacity (Fig. 5). Of importance, engrafted M-MSCs exerted beneficial outcomes even with 10-fold fewer injected cells compared to BM-MSC treatment of the HCl-IC animal model (Fig. 6). This superior potency could be attributed to enhanced in vivo engraftment and survival (Fig. 6d), which are critical challenges for therapies based on MSCs from adult tissues 42 . In one report, most (≥99%) intravenously injected MSCs adhered to the lungs, and a mere 2-3% were released into the circulation 43 . As similar, around 5% of locally injected M-MSCs were detected in the bladder tissue in 1 DAT (Fig. 6d). However, our intravital imaging analysis indicated that some M-MSCs were stably engrafted into damaged bladder at the injection site and survived at least 6-months ( Fig. 8d and Supplementary Fig. 6c). As a result, the engrafted GFP + cells in bladders of IC/BPS animals were frequently detected as E-cadherin + urothelium as well as vimentin + stromal cells or pericytes ( Fig. 8e and Supplementary Fig. 6d). The stable integration of transplanted cells as naive urothelium, stromal, and perivascular cells supporting proper angiogenesis may maximize therapeutic potential by protecting the bladder from urine leakage and by boosting the pro-regenerative microenvironment, respectively. Furthermore, the unlimited proliferation capacity of hESCs could provide voluminous high-quality therapeutic cells with well controlled in vitro differentiation characteristics to enhance in vivo survival, engraftment, and functionality 23 .
Despite these advantages, one major obstacle to the therapeutic application of hESC-derivatives is the risk of tumorigenesis. However, recent clinical success on eye disorders could allay this general concern of hESC-based therapeutics 44,45 . Likewise, tumors and abnormal growth of transplanted M-MSCs were not detected by long-term longitudinal nanoScanPET/MRI monitoring and thorough necropsy ( Supplementary Fig. 5b). Furthermore, we did not observe any signs of immune rejection and tissue inflammation during the experimental period. This low immunogenic potential of hESCs and differentiated derivatives 46, 47 may stem from the absence of immunologically relevant cell surface markers, including HLA-DR (Fig. 1c) and costimulatory molecules (CD40, CD40L, B7-1, and B7-2) 24 , which may enable M-MSCs to escape the immune response and to long-term engrafted as perivascular cells. Taken together, the present preclinical data demonstrate that hESC-derived M-MSCs may overcome the limitations of current MSC therapy without adverse outcomes from hESC-derivatives. Recently, several priming strategies have been developed to enforce the function of MSCs derived from adult tissues [48][49][50] . Thus, the therapeutic efficacy and expansion of M-MSCs should be carefully compared with those of primed MSCs, which could be easily accessible and expandable, and thus seem to be relevant for clinical application.
Another significant hurdle for clinical SC therapy is the paucity of direct long-term analyses of the in vivo distribution, phenotype, and functional integration of engrafted cells in injured target organs. Critical functional properties of engrafted SCs include transcriptional activity, external signal transduction, and differentiation potency, all of which are dynamically altered by the disease environment, thereby affecting therapeutic outcome 51 . Thus, longitudinal in situ analysis of engrafted cells in living animals could advance our understanding of the cellular mechanisms underlying functional improvement, better evaluate the risks of tumorigenesis and other adverse events after transplantation, and help in the development of optimal treatment protocols. In turn, such data may accelerate the successful translation of these preclinical results to clinical trials.
In the present study, we longitudinally monitored cellular processes of transplanted M-MSCs for 6 months in living animals (Fig. 8). With high resolution from objective lenses, we repeatedly visualized a variety of in vivo cellular-level processes (Fig. 8d). However, the large sizes of these objective lenses restricted application mostly to superficial tissues such as skin and surgically exposed internal surfaces. To overcome this drawback, we also employed endo-microscopy with a small-diameter graded-index (GRIN) lens probe to visualize intact tissue in a non-invasive manner (Fig. 8b). Strikingly, we observed similar patterns of fluorescence signals using both approaches. However, the endoscopic approach was able to track the engrafted cells only within 28 DAT (Fig. 8c), while they were visualized under objective lens even at 181 DAT (Fig. 8d). This discrepancy in detection period may be attributed to the limited optical penetration depth of GRIN probes, which is only about 100 μm in most soft tissues 36 . It should be noted that until 1 month post-injection, the majority of engrafted cells were broadly observed on the bladder surface with multiple cellular morphologies, but were focally distributed by 2 months after transplantation (Fig. 8d). Thus, it could be speculated that engrafted M-MSCs may initially replenish the urothelial layer and progressively contribute to establish a micro-environment favorable to tissue repair. For further mechanistic insight, multi-colored lineage tracing with tissue-specific promoters is required.
As SC research in urology is rapidly advancing, successful clinical application of SC therapy is expected in the near future 12,52,53 . To successfully translate promising pre-clinical studies into clinical practice, we suggest that hESC-derived M-MSCs are an ideal cost-effective source of therapeutic cells with improved functional potency and minimal tumorigenic and immunogenic capacities. To our knowledge, this is the first study to longitudinally characterize the in vivo properties of transplanted SCs at the cellular level in living animals. This innovative approach could advance our understanding of the therapeutic mechanism of current SC therapy. Study Design. The purpose of this study was not only to determine greater therapeutic efficacy of human ESC-derived MSCs (M-MSCs) than BM-derived counterparts for treating IC/BPS in a rodent model but also to longitudinally monitor in vivo cellular properties of the transplanted cells. In vitro, M-MSCs was characterized using morphological and karyotypic analysis, multi-potency, angiogenic potency, and expression of surface markers and stem cell genes. In vivo, M-MSCs were administrated into rat injured bladders and the effect on bladder voiding function, urothelium denudation, mast cell infiltration, tissue fibrosis, apoptosis, and tumorigenesis was assessed. Intravital confocal fluorescence imaging in living animals tracked the infused M-MSCs for 6-months after transplantation. For every experimental setting, two independent sets with five independent animals per group were performed. They were randomly allocated to treating groups, the order of injury, the order of cell transplantation or vehicle injection, and the order of cystometry. Information for type and dosage of the injected cells was masked to investigators who were involved in surgical procedures. All cystometric, histological, and gene expression assessment were carried out with investigators who were blinded to treatment groups. Any animals that died unexpectedly by bladder insults or catheter implantation were excluded from any analyses.

Differentiation and culture of hESC-derived M-MSCs and human BM-derived MSCs.
Maintenance of undifferentiated H9-hESCs and differentiation into M-MSCs (Fig. 1a) were performed as previously described 23,24 . The established M-MSCs were cultured with EGM2-MV medium (Lonza, San Diego, CA, USA) on plates coated with rat tail collagen type I (Sigma-Aldrich, St. Louis, MO, USA) in a humidified atmosphere with 5% CO 2 at 37 °C. All M-MSCs used in experiments were expanded less than ten passages to ensure multipotency. Characterization of basic features such as surface protein expression, cell proliferation, multipotency (in vitro differentiation into osteogenic, chondrogenic, or adipogenic lineages), in vitro angiogenesis assays, and karyotyping were performed as previously described 23,24 . The M-MSC line stably expressing GFP was established by infection of GFP-expressing lentivirus produced as previously described 14 . Human BM-MSCs purchased from Lonza (Basel, Switzerland) were cultured following the manufacturer's instructions. Cells expanded for fewer than seven passages were used for experiments to ensure multipotency. All cells were tested for mycoplasm content each month (Mycoplasma Hoechst Stain Kit; 3030000; MP Biomedicals, LLC, Santa Ana, CA, USA).

Animal models and transplantation of M-MSCs. A HCl instillation IC/BPS rat model was established
as described previously 13 . One week after the HCl injury, a low abdominal incision was made and the indicated dose of hESC-derived M-MSCs or PBS vehicle was directly injected into the outer layer of the anterior wall and dome of the bladder using a 500 μm syringe and a 26-gauge needle as previously reported 13,14,54 . Starting from 1 day before stem cell injection, indomethacin (PMG Pharm Co., Ltd. Ansan, Korea; every 12 h at 2.5 mg/kg) or Gefitinib (Santa Cruz Biotechnology, Santa Cruz, CA, USA; every day at 5 mg/kg) were subcutaneously injected to block Wnt or IGF-mediated signaling, respectively.

Unanesthetized and unrestrained cystometrogram acquisition (awake cystometry).
Cystometrograms were performed on unanesthetized and unrestrained rats in metabolic cages. Simultaneous catheterizations for intravesical pressure (IVP) and intra-abdominal pressure (IAP) recordings were performed 3 days prior to cystometrogram as described previously 55,56 . Briefly, following the induction of anesthesia, a polyethylene catheter (PE-50; Becton-Dickinson, Parsippany, NJ, USA) with a cuff was implanted into the dome of the bladder through an abdominal incision. To record IAP, an abdominal balloon (Latex; Daewoo Medical, Incheon, Korea) around the cuff of a catheter tip was placed proximal to the bladder and tied to another catheter with silk thread. A polyethylene catheter (PE-50) was heated in warm water, elongated ~1.5 times its original length at the tip of the inserting side and filled with heparinized saline (100 IU/mL). As the bladder catheter was implanted, the elongated catheter was inserted into the femoral vein. These catheters were then tunneled through the subcutaneous space, exited through the back of the animal and anchored to the skin of the back. Following surgery, each rat was housed individually and maintained in the same manner.
For awake cystometric analysis, the indwelling catheter to the bladder was connected to a two-way valve connected via a T-tube to a pressure transducer (Research Grade Blood Pressure Transducer; Harvard Apparatus, Holliston, MA, USA) and a microinjection pump (PHD22/2000 pump; Harvard Apparatus). Another indwelling catheter connected to a fluid-filled abdominal balloon was connected to another pressure transducer to record the IAP. The micturition volumes were recorded continuously by means of a fluid collector connected to a force displacement transducer (Research Grade Isometric Transducer; Harvard Apparatus) as sterile saline was infused into the bladder at a rate of 0.4 mL/min. The IVP, IAP, and micturition volumes were recorded continuously using an MP150 data acquisition system with Acq Knowledge 3.8.1 software (Biopac Systems, Goleta, CA, USA) at a sampling rate of 50 Hz. The values from all reproducible micturition cycles measured for 8 min from individual animals were used for evaluation.
A non-voiding contraction (NVC) was counted when the increments of IVP exceeded 15 cmH 2 O from baseline without expelled urine. BP was defined as the lowest bladder pressure during filling, MP as the maximum bladder pressure during the micturition cycle, MV as the urine volume of expelled urine, and RV as the urine volume remaining following voiding. BC was defined as MV + RV and MI as the interval between micturition contractions.
For gene expression analysis, preparation of total RNA was performed using an RNeasy Mini Kit (Qiagen Inc., Valencia, CA), reverse transcription using TaqMan Reverse Transcription Reagents (Applied Biosystems), and real-time quantitative PCR (RQ-PCR) of the indicated transcripts with the PikoReal Real-Time PCR System (Thermo Scientific) and iQ SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA) as described 57 . Three randomly chosen areas from each slide (n = 15) using five independent animals per treatment group was used to quantify the digital image. Similarly, gene expression data are from duplicate RQ-PCR assays (n = 10) from randomly selected five animals per group.
Animal μ-PET/MRI imaging. Ten HCl-based IC/BPS rats were randomly divided into two groups and injected with 1 × 10 6 M-MSC (n = 5) or PBS vehicle (n = 5). At 6, 9, and 12 months after injection, μ-MRI/PET imaging was performed using the nanoScanPET/MRI imaging system (1 T, MEDISO, Budapest Hungary). Rats were fasted for 8 hours prior to imaging. Rats were administered 19.7 ± 1.1 MBq in 0.2 mL of 2-[ 18 F]-FDG via the tail vein while the rat was under anesthesia (2% isoflurane in 100% O 2 gas) and warmed using heated air. A T1-weighted gradient-echo (GRE) 3D sequence (TR = 25 ms, TE eff = 3, FOV = 64 mm, matrix = 128 × 128) was acquired during the FDG uptake period. Static PET images were acquired over 15 min in a 1-5 coincident in a single field of view with MRI range. Body temperature was maintained by flowing heated air on the animal bed (Multicell, Mediso, Hungary) and a pressure sensitive pad was used for respiratory triggering. PET images were reconstructed using Tera-Tomo 3D in full detector mode with all the corrections on high regularization and 8 iterations.
BRET imaging of M-MSCs. M-MSCs were infected with retrovirus containing Nano-lantern construct kindly provided by Prof. Takeharu Nagai 34 . Labeled M-MSCs or BM-MSCs (2 × 10 5 in 100 μL saline) were injected into the bladders of mice in which chronic bladder inflammation was induced by intraperitoneal administration of cyclophosphamide (CYP, 100 mg/kg, Sigma-Aldrich) every two days for one week 35 . Bioluminescence imaging was performed using IVIS Spectrum Pre-clinical In vivo Imaging System and Living Imaging 4.4 software (PerkimElmer, Waltham, MA) following manufacturer's instruction and a previously published protocol 34 . Longitudinal in vivo confocal imaging using μ-endoscopy and microscopy. M-MSCs infected with GFP-expressing lentivirus (1 × 10 6 ) were directly injected into the bladder of HCl-IC rats and confocal imaging of the infused M-MSCs was performed in living animals using μ-endoscopic optical probes or objective lenses during 6 months after transplantation. A small incision (below 5-mm) was made on the overlying abdominal skin and the outer surface of the bladder was slightly exposed to all contact with the objective lens. The micro-endoscope probe was developed using triplet GRIN lenses structured for front-view imaging 36 . The fabricated endoscope was 1.2 mm in diameter and 5.5 cm in length with transverse and lateral resolutions of 1 μm and 11 μm, respectively, sufficient for resolving single cells. The designed probe was mounted on a custom-built confocal microscope system and optically aligned to the system using a precise XYZ translational stage. By operating continuous 488 nm laser excitation, the GFP emission signal was detected and 2D-fluorescent images were acquired at 30 frames/s.