Efficient stem cell isolation from under vacuum preserved tissue samples

Different approaches for the isolation of stem/progenitor cells have been reported, including stem cell selection in stringent culture conditions. We evaluated the possibility of isolating human progenitor cells from surgical specimens preserved by under vacuum sealing and cooling, a clinical practice approached by several hospitals as alternative to formalin. Renal tissue samples (n = 20) maintained under vacuum from 6 to 48 h at 4°C were used to isolate human renal CD133(+) progenitor cells. We obtained CD133(+) progenitors from unsorted cells derived from disaggregated tissues from each sample. Phenotypic characterization as well as in vitro and in vivo differentiation of the obtained CD133(+) lines showed results comparable with sorted CD133(+) cells obtained from fresh tissue. These results indicate that the process of sealing under vacuum and cooling appears as a suitable tissue treatment to isolate hypoxia resistant cells, such as human stem/progenitor cells, and that this procedure can be exploited to render the extraction of stem cells from human samples more practical and feasible

Protective effect and localization by optical imaging of human renal CD133+ progenitor cells in an acute kidney injury model.

Recent approaches of regenerative medicine can offer a therapeutic option for patients undergoing acute kidney injury. In particular, mesenchymal stem cells were shown to ameliorate renal function and recovery after acute damage. We here evaluated the protective effect and localization of CD133(+) renal progenitors from the human inner medulla in a model of glycerol‐induced acute tubular damage and we compared the results with those obtained with bone marrow‐derived mesenchymal stem cells. We found that CD133(+) progenitor cells promoted the recovery of renal function, preventing tubular cell necrosis and stimulating resident cell proliferation and survival, similar to mesenchymal stem cells. In addition, by optical imaging analysis, CD133(+) progenitor cells accumulated within the renal tissue, and a reduced entrapment in lung, spleen, and liver was observed. Mesenchymal stem cells were detectable at similar levels in the renal tissue, but a higher signal was present in extrarenal organs. Both cell types produced several cytokines/growth factors, suggesting that a combination of different mediators is involved in their biological action. These results indicate that human CD133(+) progenitor cells are renotropic and able to improve renal regeneration in acute kidney injury

The effects of glomerular and tubular renal progenitors and derived extracellular vesicles on recovery from acute kidney injury

BACKGROUND: Mesenchymal stromal cells (MSCs) and renal stem/progenitors improve the recovery of acute kidney injury (AKI) mainly through the release of paracrine mediators including the extracellular vesicles (EVs). Several studies have reported the existence of a resident population of MSCs within the glomeruli (Gl-MSCs). However, their contribution towards kidney repair still remains to be elucidated. The aim of the present study was to evaluate whether Gl-MSCs and Gl-MSC-EVs promote the recovery of AKI induced by ischemia-reperfusion injury (IRI) in SCID mice. Moreover, the effects of Gl-MSCs and Gl-MSC-EVs were compared with those of CD133(+) progenitor cells isolated from human tubules of the renal cortical tissue (T-CD133(+) cells) and their EVs (T-CD133(+)-EVs). METHODS: IRI was performed in mice by clamping the left renal pedicle for 35 minutes together with a right nephrectomy. Immediately after reperfusion, the animals were divided in different groups to be treated with: Gl-MSCs, T-CD133(+) cells, Gl-MSC-EVs, T-CD133(+)-EVs or vehicle. To assess the role of vesicular RNA, EVs were either isolated by floating to avoid contamination of non-vesicles-associated RNA or treated with a high dose of RNase. Mice were sacrificed 48 hours after surgery. RESULTS: Gl-MSCs, and Gl-MSC-EVs both ameliorate kidney function and reduce the ischemic damage post IRI by activating tubular epithelial cell proliferation. Furthermore, T-CD133(+) cells, but not their EVs, also significantly contributed to the renal recovery after IRI compared to the controls. Floating EVs were effective while RNase-inactivated EVs were ineffective. Analysis of the EV miRnome revealed that Gl-MSC-EVs selectively expressed a group of miRNAs, compared to EVs derived from fibroblasts, which were biologically ineffective in IRI. CONCLUSIONS: In this study, we demonstrate that Gl-MSCs may contribute in the recovery of mice with AKI induced by IRI primarily through the release of EVs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13287-017-0478-5) contains supplementary material, which is available to authorized users

Acute mental stress drives vascular inflammation and promotes plaque destabilization in mouse atherosclerosis

Publisher Copyright: © 2021 The Author(s). Published by Oxford University Press on behalf of the European Society of Cardiology.Aims: Mental stress substantially contributes to the initiation and progression of human disease, including cardiovascular conditions. We aim to investigate the underlying mechanisms of these contributions since they remain largely unclear. Methods and results: Here, we show in humans and mice that leucocytes deplete rapidly from the blood after a single episode of acute mental stress. Using cell-tracking experiments in animal models of acute mental stress, we found that stress exposure leads to prompt uptake of inflammatory leucocytes from the blood to distinct tissues including heart, lung, skin, and, if present, atherosclerotic plaques. Mechanistically, we found that acute stress enhances leucocyte influx into mouse atherosclerotic plaques by modulating endothelial cells. Specifically, acute stress increases adhesion molecule expression and chemokine release through locally derived norepinephrine. Either chemical or surgical disruption of norepinephrine signalling diminished stress-induced leucocyte migration into mouse atherosclerotic plaques. Conclusion: Our data show that acute mental stress rapidly amplifies inflammatory leucocyte expansion inside mouse atherosclerotic lesions and promotes plaque vulnerability.publishersversionPeer reviewe

Molecular anatomy of adult mouse leptomeninges.

Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we identify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arachnoid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology

Leukocytes carrying Clonal Hematopoiesis of Indeterminate Potential (CHIP) Mutations invade Human Atherosclerotic Plaques.

BACKGROUND: Leukocyte progenitors derived from clonal hematopoiesis of undetermined potential (CHIP) are associated with increased cardiovascular events. However, the prevalence and functional relevance of CHIP in coronary artery disease (CAD) are unclear, and cells affected by CHIP have not been detected in human atherosclerotic plaques. METHODS: CHIP mutations in blood and tissues were identified by targeted deep-DNA-sequencing (DNAseq: coverage >3,000) and whole-genome-sequencing (WGS: coverage >35). CHIP-mutated leukocytes were visualized in human atherosclerotic plaques by mutaFISH ™. Functional relevance of CHIP mutations was studied by RNAseq. RESULTS: DNAseq of whole blood from 540 deceased CAD patients of the Munich cardIovaScular StudIes biObaNk (MISSION) identified 253 (46.9%) CHIP mutation carriers (mean age 78.3 years). DNAseq on myocardium, atherosclerotic coronary and carotid arteries detected identical CHIP mutations in 18 out of 25 mutation carriers in tissue DNA. MutaFISH ™ visualized individual macrophages carrying DNMT3A CHIP mutations in human atherosclerotic plaques. Studying monocyte-derived macrophages from Stockholm-Tartu Atherosclerosis Reverse Networks Engineering Task (STARNET; n=941) by WGS revealed CHIP mutations in 14.2% (mean age 67.1 years). RNAseq of these macrophages revealed that expression patterns in CHIP mutation carriers differed substantially from those of non-carriers. Moreover, patterns were different depending on the underlying mutations, e.g. those carrying TET2 mutations predominantly displayed upregulated inflammatory signaling whereas ASXL1 mutations showed stronger effects on metabolic pathways. CONCLUSIONS: Deep-DNA-sequencing reveals a high prevalence of CHIP mutations in whole blood of CAD patients. CHIP-affected leukocytes invade plaques in human coronary arteries. RNAseq data obtained from macrophages of CHIP-affected patients suggest that pro-atherosclerotic signaling differs depending on the underlying mutations. Further studies are necessary to understand whether specific pathways affected by CHIP mutations may be targeted for personalized treatment