Intravital imaging longitudinally monitors the in vivo distributions and properties of the grafted cells for targeted stem cell therapy. Submitted to the Graduate School of the University of Ulsan, in partial fulfillment of the requirements for the degree of. However, little direct analysis of in vivo properties of the transplanted cells hampers the accurate understanding of their underlying therapeutic mechanisms after transplantation and the development of optimal transplantation protocols.
Long-term engraftment ensured the effective and long-lasting therapeutic effects of M-MSCs in improving the defective bladder emptying function and repairing the IC/BPS bladder pathological characteristics. Thus, the current study longitudinally monitors the in vivo properties of the long-term engrafted hESC-derived M-MSCs in living animals as well as demonstrates their improved efficacy for.
LIST OF ABBREVIATIONS
INTRODUCTION
However, important questions remain about the functional integration of MSCs into existing tissue and the reasons for poor in vivo engraftment. Importantly, the details of the therapeutic mechanisms remain controversial, and therefore the use of MSC therapy as standard therapy is questionable. By using a combination of a high-resolution confocal optical imaging system and endoscopic optical probes, dynamic changes of the infused stem cells can be repeatedly visualized longitudinally at a single cell level.
In this study, we have extended this novel imaging approach to a lipopolysaccharide (LPS) instillation IC/BPS rat model, which represents more realistic chronic bladder injury, to reveal the in vivo distribution of M-MSCs after transplantation into the IC/BPS bladder . We demonstrate the implantation and migration and migration of M-MSCs from the serosa to the damaged urothelium by a novel combination of cystoscopy and intravital confocal imaging over a period of 40 days and follow up with ex vivo imaging of immunostained tissue to demonstrate mechanisms for differentiation of the M-MSCs into epithelial, stromal and endothelial cells, as well as functional integration of MSC into pre-existing perivascular cell clusters.
MATERIALS AND METHODS
For confocal microscopy, a 5 mm incision was made in the abdomen and widened to expose the bladder and allow imaging using 40 inch objective lenses. The transverse and lateral resolutions were 1 and 11 mm in air, respectively, which was sufficient to monitor single-cell resolution M-MSCs in the bladder mucosa. Consistent with previous research, MI was defined as the interval between micturition contractions and BC was defined as MV + RV.
GFP+ M-MSCs injected into the bladder were tracked by immunofluorescent staining with specific rabbit polyclonal antibody (ab290; Abcam, Cambridge, MA). Data are reported as mean ± standard error of the mean (SEM) and were analyzed by GraphPad Prism 6.0 software (GraphPad Software, La Jolla, CA).
RESULTS
Time-lapse microendoscopy imaging of grafted M-MSCs in the bladders of LPS-IC rats (three independent animals) 3-42 days post-transplantation (DAT). Time-lapse confocal microscopy of grafted M-MSCs in the bladders of LPS-IC rats (three independent animals) 3-42 days post-transplantation (DAT). Time-lapse images of the bladders of LPS-IC rats were acquired using an anterior view GRIN optical probe inserted endoscopically into the bladder (left, magnification × 40) or an objective lens (center; magnification × 40, right). ; magnification x 100). ).
Representative confocal micrographs of bladder sections from LPS-IC + M-MSC rats stained for hB2M (red) and vimentin at 7 DAT (upper panel) and 30 DAT (lower panel) in vascular-like structures (magnification × 1000, scale bar = 10mm). Given the confirmation of long-term implantation of more than 1 month, we next evaluated the in vivo efficacy of M-MSC therapy by a single administration of 1´106M-MSC directly into the bladders of LPS-IC animals. In particular, animals in the LPS-IC group characteristically showed increased contraction frequency during the non-emptying period (non-emptying contraction, NVC), even at low doses of M-MSC therapies.
However, little tissue fibrosis was detected in the bladder of LPS-IC group rats at these lower doses. Next, we investigated whether the LPS-IC animals could show chronic symptoms like IC/BPS patients. In particular, normal NVC, the most common factor for evaluating bladder emptying function in clinical care, was observed for 4 weeks in the LPS-IC animals transplanted with M-MSCs (Figure 14).
Representative awake cystometry results at 2 weeks (left) and 4 weeks (right) after injection of 1×106 M-MSC (LPS-IC + M-MSC) or PBS (LPS-IC) into LPS-IC mice. In the chronic IC/BPS model, gene expression analysis revealed that bladder tissues of LPS-IC animals characteristically upregulate Smoothened (Smo), a transducer of Shh signaling and WNT family genes (eg, Wnt2b, Wnt4, Wnt5a, Wnt8a, Wnt8b and Wnt10a), as well as their downstream growth factors (eg, Igf2, Fgf9 and Notch1) (Figure 18A). The activation of WNT pathways in the LPS-IC + M-MSC group was confirmed by the increased expression and nuclear localization of b-catenin protein, a surrogate marker for the activation of WNT signaling (Figure 18B and Figure 19A, 19B).
Both inhibitors significantly abrogated the therapeutic effects of M-MSC to recover bladder emptying functions in LPS-IC animals (Figure 18C). -PCR analyzes the expression levels of Shh, WNT and downstream growth factors in bladder tissues of LPS-IC mice at 1 week after injection of 1×106 M-MSC or PBS. Quantitative data of bladder emptying in LPS-IC mice (eight independent animals per group) 1 week after injection of 1×106M-MSC in the absence or presence of indomethacin or Gefitinib, which inhibit WNT- and IGF-mediated signaling, respectively . data are presented as mean ± SEM.
Nuclei were stained with DAPI (blue). B) Bladder sections of LPS-IC + M-MSC rats were stained with mouse and rabbit control IgG antibodies as a negative control v.
DISCUSSION
Through a combination of immunostaining and intravital imaging, we were able to verify that the beneficial results of M-MSC are due to WNT and IGF signaling cascades, and therefore it can be speculated that the grafted M-MSCs supplement the urothelial layer of the bladder against further self-induction . -inflicted damage to the environment while contributing to a microenvironment favorable to tissue repair. Indeed, M-MSCs showed the superior therapeutic potency over BM-MSCs in the LPS-IC animal model (Figure 20). The defocusing and dimming of observed cells after 21 DAT are likely due to limitations in the focal depth of the GRIN probe and to the limited optical penetration depth of visible light-based endoscopy down to ~100 mm in most soft tissues, respectively.
Due to limitations of the observation area of the GRIN probe and confocal objective, it was not possible to verify distribution of injected stem cells over the entire bladder. However, the chronic model of IC/BPS was not targeted to specific bladder regions and has an equal impact on the entire urothelium; cystometry revealed improvement in bladder regulation, and outcomes that were not limited to the single point of MSC injection. We observed migration of M-MSCs from the bladder serosa, where the MSCs were injected, to the highly damaged urothelium over a period of three days, which is the first confirmed observation of stem cell migration that supports therapeutic treatment in living bladder models of IC/BPS, to the best knowledge of the authors.
After tracking the migration of transplanted M-MSC from the bladder serosa to the mucosa over a seven-day period, in vivo cellular differentiation was observed in the urothelium, lamina propria and serosa, as well as subsequent differentiation of M-MSC cells into endothelial and epithelial over a 30 day period, which correlated with improvement of IC/BPS symptoms including bladder denudation and NVC in the animal in the LPS-IC + M-MSC group. Of importance, the transplanted M-MSCs clearly integrated into functional compartments in the bladder, including E-cadherin+urothelial cells, CD31+endothelial cells, and vimentin+stromal or pericytes. In particular, the engrafted cells in the lamina propria and partly in the urothelium stimulate WNT-related epithelial regeneration capacity.
Different tissue microenvironments during acute (HCl instillation) and chronic (LPS instillation) IC injuries could determine the engraftment and integration potency of transplanted M-MSCs. Although no adverse outcomes related to immune rejection and tissue inflammation were observed during our experimental period, the different immune responses between the two animal models could contribute to the different in vivo biological dynamics of xenogeneic transplanted cells. Despite the low immunogenicity of M-MSCs, the impact of the immune response to grafted M-MSCs in pathological bladders should be carefully investigated in a further study.
CONCLUSION
Terminal differentiation is not a major determinant of the success of stem cell therapy – crosstalk between muscle-derived stem cells and host cells. Improved efficacy and in vivo cellular properties of human embryonic stem cell derivative in a preclinical model of bladder pain syndrome. Hunner-type (classic) interstitial cystitis: a distinct inflammatory disorder characterized by pancystitis, with frequent clonal B-cell expansion and epithelial denudation.
쥐의 화학적 유발 방광염에 대한 험 및 배아줄기세포 유래 다능성 중간엽줄기세포의 치료 효과. 간질성 방광염/방광통증증후군을 표적으로 하는 줄기세포치료를 위한 이식세포의 생체내 분포와 특성을 모니터링합니다. 간질성 방광염(IC)은 결절화 및 Hunner 궤양이 특징입니다.
방광경 검사 소견과 염증 유발 비만세포 클러스터의 조직학적 소견이 있는 방. 광병성 통증 증후군은 심한 방광염 반응과 요로 상피 기능 장애를 특징으로 하는 질병입니다. 간질성방광염 환자가 뚜렷한 치료법이 없어 평생 동안 겪는 극심한 통증.
이전 논문에서 현재 연구 그룹은 HCl 주입으로 유발된 간질성 방광염 모델에서 MSC를 보고했습니다. 줄기세포치료제 연구와 메커니즘이 규명됐다. PS는 IC 환자의 중요한 병인학적 요인으로 받아들여졌습니다.
