Laetitia Pidial

Bone marrow‐derived mesenchymal stem cell‐loaded fibrin patches act as a reservoir of paracrine factors in chronic myocardial infarction

The combination of mesenchymal stem cells and tissue‐engineered fibrin patches improves the therapeutic efficacy of stem cells. In vivo cardiac magnetic resonance (4.7 Tesla) and ex vivo high‐spatial resolution CMR were used to track the fate of human bone marrow‐derived mesenchymal stem cell (BMSC) delivered on an epicardial scaffold and more specifically assess their potential intramyocardial migration. Fifty‐seven nude rats underwent permanent coronary artery ligation. Two months later, those with a left ventricular ejection fraction ≤55% were randomly allocated to receive a patch loaded with human BMSC (BMSC‐P, n = 10), a patch loaded with BMSCs labelled with iron oxide nanoparticles (BMSC*‐P, n = 12), an acellular patch (A‐P, n = 8) or to serve as sham‐operated animals (SHAM, n = 7). BMSC secretion of cytokines and growth factors was evaluated with flow‐cytometry. Cardiac functional parameters of cell‐treated groups (BMSC*‐P and BMSC‐P) yielded significantly better outcomes than the SHAM group (p = 0.044 and p = 0.026, respectively, for ejection fraction). Angiogenesis was higher in the cell‐patch than in control groups (e.g. BMSC*P vs. SHAM: p = 0.007). No BMSCs were identified into the myocardium on cardiac magnetic resonance or histological sections, although persisting BMSCs were identified on the epicardial surface 21 days post‐transplantation in 10% of rats hearts (Lamin A/C and CD90 positive). Cytokine and growth factor profiling demonstrated an increase in their release by cells seeded in patches. The absence of stem cell migration into the myocardium and the persistence of stem cells on the epicardial surface suggest that fibrin patches are likely to act predominantly as reservoirs of paracrine factors. Copyright

Cell Sheet Transplantation for Esophageal Stricture Prevention after Endoscopic Submucosal Dissection in a Porcine Model

Background & Aims Extended esophageal endoscopic submucosal dissection (ESD) is highly responsible for esophageal stricture. We conducted a comparative study in a porcine model to evaluate the effectiveness of adipose tissue-derived stromal cell (ADSC) double cell sheet transplantation. Methods Twelve female pigs were treated with 5 cm long hemi-circumferential ESD and randomized in two groups. ADSC group (n = 6) received 4 double cell sheets of allogenic ADSC on a paper support membrane and control group (n = 6) received 4 paper support membranes. ADSC were labelled with PKH-67 fluorophore to allow probe-based confocal laser endomicroscopie (pCLE) monitoring. After 28 days follow-up, animals were sacrificed. At days 3, 14 and 28, endoscopic evaluation with pCLE and esophagography were performed. Results One animal from the control group was excluded (anesthetic complication). Animals from ADSC group showed less frequent alimentary trouble (17% vs 80%; P = 0.08) and higher gain weight on day 28. pCLE demonstrated a compatible cell signal in 4 animals of the ADSC group at day 3. In ADSC group, endoscopy showed that 1 out of 6(17%) animals developed a severe esophageal stricture comparatively to 100% (5/5) in the control group; P = 0.015. Esophagography demonstrated a decreased degree of stricture in the ADSC group on day 14 (44% vs 81%; P = 0.017) and day 28 (46% vs 90%; P = 0.035). Histological analysis showed a decreased fibrosis development in the ADSC group, in terms of surface (9.7 vs 26.1 mm²; P = 0.017) and maximal depth (1.6 vs 3.2 mm; P = 0.052). Conclusion In this model, transplantation of allogenic ADSC organized in double cell sheets after extended esophegeal ESD is strongly associated with a lower esophageal stricture’s rate.

Designing 3D Mesenchymal Stem Cell Sheets Merging Magnetic and Fluorescent Features: When Cell Sheet Technology Meets Image-Guided Cell Therapy

Cell sheet technology opens new perspectives in tissue regeneration therapy by providing readily implantable, scaffold-free 3D tissue constructs. Many studies have focused on the therapeutic effects of cell sheet implantation while relatively little attention has concerned the fate of the implanted cells in vivo. The aim of the present study was to track longitudinally the cells implanted in the cell sheets in vivo in target tissues. To this end we (i) endowed bone marrow-derived mesenchymal stem cells (BMMSCs) with imaging properties by double labeling with fluorescent and magnetic tracers, (ii) applied BMMSC cell sheets to a digestive fistula model in mice, (iii) tracked the BMMSC fate in vivo by MRI and probe-based confocal laser endomicroscopy (pCLE), and (iv) quantified healing of the fistula. We show that image-guided longitudinal follow-up can document both the fate of the cell sheet-derived BMMSCs and their healing capacity. Moreover, our theranostic approach informs on the mechanism of action, either directly by integration of cell sheet-derived BMMSCs into the host tissue or indirectly through the release of signaling molecules in the host tissue. Multimodal imaging and clinical evaluation converged to attest that cell sheet grafting resulted in minimal clinical inflammation, improved fistula healing, reduced tissue fibrosis and enhanced microvasculature density. At the molecular level, cell sheet transplantation induced an increase in the expression of anti-inflammatory cytokines (TGF-ß2 and IL-10) and host intestinal growth factors involved in tissue repair (EGF and VEGF). Multimodal imaging is useful for tracking cell sheets and for noninvasive follow-up of their regenerative properties.

Pyruvate cellular uptake and enzymatic conversion probed by dissolution DNP-NMR: the impact of overexpressed membrane transporters

Pyruvate membrane crossing and its lactate dehydrogenase‐mediated conversion to lactate in cells featuring different levels of expression of membrane monocarboxylate transporters (MCT4) were probed by dissolution dynamic nuclear polarization‐enhanced NMR. Hyperpolarized 13C‐1‐labeled pyruvate was transferred to suspensions of rodent tumor cell carcinoma, cell line 39. The pyruvate‐to‐lactate conversion rate monitored by dissolution dynamic nuclear polarization‐NMR in carcinoma cells featuring native MCT4 expression level was lower than the rate observed for cells in which the human MCT4 gene was overexpressed. The enzymatic activity of lactate dehydrogenase was also assessed in buffer solutions, following the real‐time pyruvate‐to‐lactate conversion speeds at different enzyme concentrations. Copyright

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR.

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

Evaluation of Rat Heart Microvasculature with High-Spatial-Resolution Susceptibility-weighted MR Imaging

PURPOSE To demonstrate that strandlike hypointense signals seen in the myocardium of normal rat hearts correspond to myocardial microvessels with high-spatial-resolution susceptibility-weighted (SW) magnetic resonance (MR) imaging without injection of contrast medium. MATERIALS AND METHODS Animal experiments were performed with institutional animal care committee approval. Ex vivo cardiac MR imaging was performed in 10 normal Wistar rats with a 4.7-T imager and a cryogenic probe. The hypothesis that thin tubular hypointense signals in the myocardium of rat hearts at SW MR imaging sequences (group 1, n = 6; in-plane resolution, 39 μm) represent intramyocardial microvessels was tested. A superparamagnetic intravascular contrast agent (ferumoxsil; Lumirem) was used to explore the distribution of the intramyocardial microvessels (group 2, n = 4; three-dimensional fast imaging with steady-state free precession sequences). Nonparametric Mann-Whitney tests were performed to compare groups 1 and 2 both for microvascular densities (MVD) on histologic sections and for MR imaging signal intensities (SIs). Wilcoxon signed rank tests were used for paired comparison of subepicardial and subendocardial MVD and SI within groups. RESULTS Ferumoxsil opacified the coronary microvasculature (group 2) on MR-matched histologic sections. No statistically significant difference was found between groups 1 and 2 for either MVD or MR imaging SI expressed as ratios between subendocardium and subepicardium (P = .40 and P = .46, respectively). The comparison of mean subendocardial and subepicardial SI within groups revealed significantly more microvessels in the subepicardium with MR (group 1: P = .01; group 2: P = .004). CONCLUSION Myocardial microvessels appear as strandlike structures on high-spatial-resolution SW MR images without the aid of contrast medium injection.

ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection

In past years, the cell-sheet construct has spurred wide interest in regenerative medicine, especially for reconstructive surgery procedures. The development of diversified technologies combining adipose tissue-derived stromal cells (ADSCs) with various biomaterials has led to the construction of numerous types of tissue-engineered substitutes, such as bone, cartilage, and adipose tissues from rodent, porcine, or human ADSCs. Extended esophageal endoscopic submucosal dissection (ESD) is responsible for esophageal stricture formation. Stricture prevention remains challenging, with no efficient treatments available. Previous studies reported the effectiveness of mucosal cell-sheet transplantation in a canine model and in humans. ADSCs are attributed anti-inflammatory properties, local immune modulating effects, neovascularization induction, and differentiation abilities into mesenchymal and non-mesenchymal lineages. This original study describes the endoscopic transplantation of an ADSC tissue-engineered construct to prevent esophageal stricture in a swine model. The ADSC construct was composed of two allogenic ADSC sheets layered upon each other on a paper support membrane. The ADSCs were labeled with the PKH67 fluorophore to allow probe-based confocal laser endomicroscopy (pCLE) monitoring. On the day of transplantation, a 5-cm and hemi-circumferential ESD known to induce esophageal stricture was performed. Animals were immediately endoscopically transplanted with 4 ADSC constructs. The complete adhesion of the ADSC constructs was obtained after 10 min of gentle application. Animals were sacrificed on day 28. All animals were successfully transplanted. Transplantation was confirmed on day 3 with a positive pCLE evaluation. Compared to transplanted animals, control animals developed severe strictures, with major fibrotic tissue development, more frequent alimentary trouble, and reduced weight gain. In our model, the transplantation of allogenic ADSCs, organized in double cell sheets, after extended ESD was successful and strongly associated with a lower esophageal stricture rate.

Multiparametric optical and MR imaging demonstrate inhibition of tumor angiogenesis natural history by mural cell therapy.

To determine whether functional imaging using MRI and fibered confocal fluorescence microscopy (FCFM) could be used to monitor cell therapy by mural progenitor cells (MPC).

Assessment of Placental Perfusion in the Preeclampsia L-NAME Rat Model with High-Field Dynamic Contrast-Enhanced MRI

Purpose: To evaluate placental function and perfusion in a rat model of preeclampsia infused with L-nitro-arginine methyl ester (L-NAME) by dynamic contrast-enhanced (DCE) MRI using gadolinium chelates. Methods: Pregnant female Sprague-Dawley rats were fitted on embryonic day 16 (E16) with subcutaneous osmotic minipumps loaded to deliver, continuously, L-NAME (50 mg/day per rat; case group) or saline solution (control group). DCE MRI was performed on E19 using gadolinium chelates and a 4.7-T MRI apparatus for small animals. Quantitative analysis was performed using an image software program: placental blood flow (perfusion in mL/min/100 mL of placenta) and fractional volume of the maternal vascular placental compartment (ratio between the placental blood volume and the placental volume, Vb in %) were calculated by compartmental analysis. Results: A total of 176 placentas (27 rats) were analyzed by DCE MRI (97 cases and 79 controls). The model was effective, inducing intrauterine growth retardation, as there was a significant difference between the two groups for placental weight (p < 0.01), fetal weight (p = 0.019), and fetal length (p < 0.01). There was no significant difference in placental perfusion between the L-NAME and control groups (140.1 ± 74.1 vs. 148.9 ± 97.4, respectively; p = 0.496). There was a significant difference between the L-NAME and control groups for Vb (53 ± 12.9 vs. 46.7 ± 9%, respectively; p < 0.01). Conclusion: In the L-NAME preeclampsia model, placental perfusion is normal and the fractional blood volume is increased, suggesting that preeclampsia is not always expressed as a result of decreased placental perfusion. This highlights the usefulness of MRI for investigating the physiopathology of preeclampsia.

Thermoresponsive Gel Embedded with Adipose Stem-Cell-Derived Extracellular Vesicles Promotes Esophageal Fistula Healing in a Thermo-Actuated Delivery Strategy

Extracellular vesicles (EVs) are increasingly envisioned as the next generation of biological pro-regenerative nanotherapeutic agents, as has already been demonstrated for heart, kidney, liver, and brain tissues; lung injury repair; and skin regeneration. Herein, we explore another potential EV therapeutic application, fistula healing, together with a local minimally invasive delivery strategy. Allogenic extracellular vesicles (EVs) from adipose tissue-derived stromal cells (ASCs) are administered in a porcine fistula model through a thermoresponsive Pluronic F-127 (PF-127) gel, injected locally at 4 °C and gelling at body temperature to retain EVs in the entire fistula tract. Complete fistula healing is reported to be 100% for the gel plus EVs group, 67% for the gel group, and 0% for the control, supporting the therapeutic use of Pluronic F-127 gel alone or combined with EVs. However, only the combination of gel and EVs results in a statistically significant (i) reduction of fibrosis, (ii) decline of inflammatory response, (iii) decrease in the density of myofibroblasts, and (iv) increase of angiogenesis. Overall, we demonstrate that ASC-EV delivery into a PF-127 gel represents a successful local minimally invasive strategy to induce a therapeutic effect in a swine fistula model. Our study presents prospects for EV administration strategies and for the management of post-operative fistulas.