Tissue RNA Integrity in Visium Spatial Protocol (Fresh Frozen Samples).

The transcriptome of a tissue can be acquired both by single-cell RNAseq (scRNA-seq) and by spatial transcriptomics (ST). The dissociation step, which is mandatory in scRNA-seq methods, might lead to the loss of fragile cells and of spatial information, thus limiting the acquisition of the tissue cellular organization. Spatial transcriptomics methods moderate the above-mentioned issues and provide single-cell transcripts detection over an intact fresh frozen tissue section. Visium platform, commercialized from 10× Genomics, provides a whole transcriptome spatial transcriptomics platform, which does not require dedicated instruments, other than those available in any pathology laboratory. In spatial transcriptomics, proper tissue handling is mandatory to preserve the morphological quality of the tissue sections and the integrity of mRNA transcripts. Proper tissue handling is critical for downstream library preparation and sequencing performance. In this chapter, we describe the most critical steps of Visium protocol on fresh frozen tissues and we provide indications on how to interpret the data obtained from the quality control analysis recommended during the workflow.

Protective Effects of Human Liver Stem Cell-Derived Extracellular Vesicles in a Mouse Model of Hepatic Ischemia-Reperfusion Injury.

Hepatic ischemia-reperfusion injury (IRI) is observed in liver transplantation and hepato-biliary surgery and is associated with an inflammatory response. Human liver stem cell-derived extracellular vesicles (HLSC-EV) have been demonstrated to reduce liver damage in different experimental settings by accelerating regeneration and by modulating inflammation. The aim of the present study was to investigate whether HLSC-EV may protect liver from IRI in a mouse experimental model. Segmental IRI was obtained by selective clamping of intrahepatic pedicles for 90 min followed by 6 h of reperfusion. HLSC-EV were administered intravenously at the end of the ischemic period and histopathological and biochemical alterations were evaluated in comparison with controls injected with vehicle alone. Intra liver localization of labeled HLSC-EV was assessed by in in vivo Imaging System (IVIS) and the internalization into hepatocytes was confirmed by fluorescence analyses. As compared to the control group, administration of 3 × 109 particles (EV1 group) significantly reduced alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release, necrosis extension and cytokines expression (TNF-α, CCL-2 and CXCL-10). However, the administration of an increased dose of HLSC-EV (7.5 × 109 particles, EV2 group) showed no significant improvement in respect to controls at enzyme and histology levels, despite a significantly lower cytokine expression. In conclusion, this study demonstrated that 3 × 109 HLSC-EV were able to modulate hepatic IRI by preserving tissue integrity and by reducing transaminases release and inflammatory cytokines expression. By contrast, a higher dose was ineffective suggesting a restricted window of biological activity. Graphical abstract.

Mesenchymal Stem Cell Derived Extracellular Vesicles Ameliorate Kidney Injury in Aristolochic Acid Nephropathy.

Limitations in the current therapeutic strategies for the prevention of progression of chronic kidney disease (CKD) to end stage renal disease has been a drawback to improving patient recovery. It is therefore imperative that a solution is found to alleviate this problem and improve the health and well-being of patients overall. Aristolochic acid (AA) induced nephropathy, a type of nephrotoxic CKD is characterised by cortical tubular injury, inflammation, leading to interstitial fibrosis. Extracellular vesicles derived from human bone marrow mesenchymal stem cells (MSC-EVs) display therapeutic properties in various disease models including kidney injury. In the current study, we intended to investigate the ability of MSC-EVs on ameliorating tubular injury and interstitial fibrosis in a mouse model of aristolochic acid nephropathy (AAN). The chronic model of AAN is comprised of an intraperitoneal injection of AA in NSG mice, followed by a three-day incubation period and then inoculation of MSC-EVs intravenously. This routine was performed on a weekly basis for four consecutive weeks, accompanied by the monitoring of body weight of all mice. Blood and tissue samples were collected post sacrifice. All animals administered with AA developed kidney injury and renal fibrosis. A gradual loss of body weight was observed, together with a deterioration in kidney function. Although no significant recovery was observed in weight loss following treatment with MSC-EVs, a significant reduction in: blood creatinine and blood urea nitrogen (BUN), tubular necrosis, and interstitial fibrosis was observed. In addition, infiltration of CD45 positive immune cells, fibroblasts, and pericytes which were elevated in the interstitium post AA induced injury, were also significantly reduced by MSC-EVs. Kidneys were also subjected to molecular analyses to evaluate the regulation of pro-fibrotic genes. MSC-EVs significantly reduced AA induction of the pro-fibrotic genes α-Sma, Tgfb1 and Col1a1. A downregulation in pro-fibrotic genes was also observed in fibroblasts activated by AA injured mTECs in vitro. Furthermore, meta-analyses of miRNAs downregulated by MSC-EVs, such as miR21, revealed the regulation of multiple pathways involved in kidney injury including fibrosis, inflammation, and apoptosis. These results therefore suggest that MSC-EVs could play a regenerative and anti-fibrotic role in AAN through the transfer of biologically active cargo that regulates the disease both at a protein and genetic level.

Indocyanine green labeling for optical and photoacoustic imaging of mesenchymal stem cells after in vivo transplantation.

The transplantation of mesenchymal stem cells (MSCs) holds great promise for the treatment of a plethora of human diseases, but new noninvasive procedures are needed to monitor the cell fate in vivo. Already largely used in medical diagnostics, the fluorescent dye indocyanine green (ICG) is an established dye to track limited numbers of cells by optical imaging (OI), but it can also be visualized by photoacoustic imaging (PAI), which provides a higher spatial resolution than pure near infrared fluorescence imaging (NIRF). Because of its successful use in clinical and preclinical examinations, we chose ICG as PAI cell labeling agent. Optimal incubation conditions were defined for an efficient and clinically translatable MSC labeling protocol, such that no cytotoxicity or alterations of the phenotypic profile were observed, and a consistent intracellular uptake of the molecule was achieved. Suspensions of ICG-labeled cells were both optically and optoacoustically detected in vitro, revealing a certain variability in the photoacoustic spectra acquired by varying the excitation wavelength from 680 to 970 nm. Intramuscular engraftments of ICG-labeled MSCs were clearly visualized by both PAI and NIRF over few days after transplantation in the hindlimb of healthy mice, suggesting that the proposed technique retains a considerable potential in the field of transplantation-focused research and therapy. Stem cells were labeled with the Food and Drug Administration (FDA)-approved fluorescent dye ICG, and detected by both PAI and OI, enabling to monitor the cell fate safely, in dual modality, and with good sensitivity and improved spatial resolution.

Imaging the pH evolution of an acute kidney injury model by means of iopamidol, a MRI-CEST pH-responsive contrast agent.

PURPOSE: To investigate in vivo possible pH level alterations following an acute renal failure disease using a MRI-CEST pH responsive contrast agent. The impact of functional evolution in different renal compartments over time was also investigated. METHODS: a mouse model of acute kidney injury was obtained by glycerol-induced rhabdomyolysis. pH maps were obtained using Iopamidol (0.75 g iodine/kg b.w. corresponding to 2.0 mmol/kg) in a control group (n = 3) and in the acute kidney injury group (n = 6) at 1, 3, 7, 14, and 21 days after the damage induction at 7T. Histology assessment of renal damage and blood urea nitrogen levels were compared with pH maps. RESULTS: during the acute kidney injury, there was a robust increase of pH values, which peaked after 3 days, compared with the predamage situation. In addition, it was possible to detect changes in contrast detection between the different functional regions of the damaged kidneys. Moreover, a slow restoration of normal pH values was observed three weeks after the glycerol injection. CONCLUSIONS: pH appears to be a good parameter to assess the early detection of kidney injury as well as it acts as a reporter of the recovery toward the physiologic functionality.