Cells derived from blood vessels of human skeletal muscle can regenerate skeletal muscle, similarly to embryonic mesoangioblasts. However, adult cells do not express endothelial markers, but instead ...express markers of pericytes, such as NG2 proteoglycan and alkaline phosphatase (ALP), and can be prospectively isolated from freshly dissociated ALP(+) cells. Unlike canonical myogenic precursors (satellite cells), pericyte-derived cells express myogenic markers only in differentiated myotubes, which they form spontaneously with high efficiency. When transplanted into severe combined immune deficient-X-linked, mouse muscular dystrophy (scid-mdx) mice, pericyte-derived cells colonize host muscle and generate numerous fibres expressing human dystrophin. Similar cells isolated from Duchenne patients, and engineered to express human mini-dystrophin, also give rise to many dystrophin-positive fibres in vivo. These data show that myogenic precursors, distinct from satellite cells, are associated with microvascular walls in the human skeletal muscle, may represent a correlate of embryonic 'mesoangioblasts' present after birth and may be a promising candidate for future cell-therapy protocols in patients.
Critical limb ischemia is the most serious form of peripheral artery disease, characterized by severe functional consequences, difficult clinical management and reduced life expectancy. The goal of ...this study was to investigate the miR-210 role in the neo-angiogenic response after acute limb ischemia. Complementary approaches were used in a mouse model of hindlimb ischemia: miR-210 loss-of-function was obtained by administration of LNA-oligonucleotides anti-miR-210; for miR-210 gain-of-function, a doxycycline-inducible miR-210 transgenic mouse was used. We tested miR-210 ability to stimulate vascular regeneration following ischemia. We found that miR-210 was necessary and sufficient to stimulate blood perfusion recovery, as well as arteriolar and capillary density increase, in the ischemic muscle. To clarify the molecular events underpinning miR-210 pro-angiogenic action, the transcriptomic changes in ischemic muscles upon miR-210 blocking were analyzed. We found that miR-210 impacted the transcriptome significantly, regulating pathways and functions linked to vascular regeneration. In agreement with a pro-angiogenic role, miR-210 also improved cardiac function and left ventricular remodeling after myocardial infarction. Moreover, miR-210 blocking decreased capillary density in a Matrigel plug assay, indicating that miR-210 is necessary for angiogenesis independently of ischemia. Collectively, these data indicate that miR-210 plays a pivotal role in promoting vascular regeneration.
In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular ...products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells.
We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCR
) and permanently disrupted
,
or
genes (IR
) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCR
-IR
and IR competent (TCR
-IR
) cells were tested in short-term co-culture assays and under a chronic stimulation setting
. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models.
We show that upon chronic stimulation, TCR
-IR
cells are superior to TCR
-IR
cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge
. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells.
These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.
Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T‐cell development, leading to immunodeficiency or autoimmunity. These ...defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene‐modified postnatal murine TECs into three‐dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline‐induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4‐expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. Stem Cells Translational Medicine 2019;8:1107–1122
Transient Oct4 expression promoted thymic epithelial cells expansion without drastically changing the cell lineage identity of adult thymic epithelial cells. iOCT4 thymic epithelial cells were able to grow into three‐dimensional collagen type I scaffolds both in vitro and in vivo demonstrating that the collagen structure reproduced a three‐dimensional environment similar to the thymic extracellular matrix, perfectly recognized by thymic epithelial cells. in vivo results showed that thymic organoids transplanted subcutaneously in athymis nude mice were vascularized but failed to support thymopoiesis because of the limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of three‐dimensional biomimetic scaffolds, represents a novel approach allowing the use of postnatal thymic epithelial cells for thymic regeneration.
In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle ...development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded
(also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.
Abstract
Background
Methods for the non-invasive quantification of changes in bladder wall thickness as potential predictors of radiation cystitis in pre-clinical research would be desirable. The use ...of ultrasound for this aim seems promising, but is still relatively unexplored. A method using ultrasound for bladder wall thickness quantification in rats was developed and applied to measure early radiation-induced bladder wall thickness changes.
Methods
Two groups (n = 9 each) of female Fischer rats were treated with a single radiation dose of 25–30 and 35–40 Gy respectively, using an image-guided micro-irradiator; six untreated rats were monitored as a control group. Empty, half-filled and fully-filled bladder volumes were determined for four non-irradiated rats by measuring axes from ultrasound 3D-images and applying the ellipsoid formula. Mean bladder wall thickness was estimated for both ventral and dorsal bladder sides through the measurement of the bladder wall area along a segment of 4 mm in the central sagittal scan, in order to minimize operator-dependence on the measurement position. Ultrasound acquisitions of all fully-filled rat bladders were also acquired immediately before, and 4 and 28 days after irradiation. Mean bladder wall thickness normalized to the baseline value and corrected for filling were then used to evaluate acute bladder wall thickening and to quantify the dose–effect.
Results
The relationship between mean bladder wall thickness and volume in unirradiated rats showed that for a bladder volume > 1.5 mL the bladder wall thickness is almost constant and equal to 0.30 mm with variations within ± 15%. The average ratios between post and pre irradiation showed a dose–effect relationship. Bladder wall thickening was observed for the 25–30 Gy and 35–40 Gy groups in 2/9 (22%) and 5/9 (56%) cases at day 4 and in 4/9 (44%) and 8/9 (89%) cases at day 28, respectively. The two groups showed significantly different bladder wall thickness both relative to the control group (
p
< 0.0001) and between them (
p
= 0.022). The bladder wall thickness increment was on average 1.32 ± 0.41, and was 1.30 ± 0.21 after 25–30 Gy and 1.47 ± 0.29 and 1.90 ± 0.83 after 35–40 Gy at days 4 and 28 respectively.
Conclusions
The feasibility of using ultrasound on a preclinical rat model to detect bladder wall thickness changes after bladder irradiation was demonstrated, and a clear dose–effect relationship was quantified. Although preliminary, these results are promising in addressing the potential role of this non-invasive approach in quantifying radiation cystitis.
Background
Malignant mesothelioma (MM) is an aggressive tumor, with a poor prognosis, usually unresectable due to late diagnosis, mainly treated with chemotherapy. BoxA, a truncated form of “high ...mobility group box 1” (HMGB1), acting as an HMGB1 antagonist, might exert a defensive action against MM. We investigated the potential of BoxA for MM treatment using experimental 40-MHz ultrasound and optical imaging (OI) in a murine model.
Methods
Murine MM cells infected with a lentiviral vector expressing the luciferase gene were injected into the peritoneum of 14 BALB/c mice (7 × 10
4
AB1-B/c-LUC cells). These mice were randomized to treatment with BoxA (
n
= 7) or phosphate-buffered saline (controls,
n
= 7). The experiment was repeated with 40 mice divided into two groups (
n
= 20 + 20) and treated as above to confirm the result and achieve greater statistical power. Tumor presence was investigated by experimental ultrasound and OI; suspected peritoneal masses underwent histopathology and immunohistochemistry examination.
Results
In the first experiment, none of the 7 controls survived beyond day 27, whereas 4/7 BoxA-treated mice (57.1%) survived up to day 70. In the second experiment, 6/20 controls (30.0%) and 16/20 BoxA-treated mice (80.0%) were still alive at day 34 (
p
= 0.004). In both experiments, histology confirmed the malignant nature of masses detected using experimental ultrasound and OI.
Conclusion
In our preclinical experience on a murine model, BoxA seems to exert a protective role toward MM. Both experimental ultrasound and OI proved to be reliable techniques for detecting MM peritoneal masses.
The prolonged, gonadotoxic effect of chemotherapy can finally lead to infertility in female cancer survivors. There is controversial evidence regarding the protective role of gonadotropin-releasing ...hormone analogue (GnRH-a) on chemotherapy-induced ovarian damage. In the present study on a murine model, ultrasound (US) and contrast-enhanced US (CEUS) were firstly used to characterise ovarian glands in normal conditions to validate a preclinical model. In addition, preliminary findings were obtained on anatomical and vascular ovarian changes induced by GnRH-a based on decapeptyl administration. Ovaries were accurately assessed with US and CEUS in a murine model placed in prone position, providing quantitative and reproducible information. Ovaries were identified in 40/40 cases and CEUS analysis was successfully performed in 20/20 cases with 100% technical success. A statistically significant increase of the diameter of the dominant follicle at US and a statistically significant reduced vascularisation at CEUS in decapeptyl-treated mice compared to untreated control mice were recorded. Further studies using US and CEUS in the murine model combining GnRH-a and chemotherapeutic agents will be needed to obtain more translational information useful for clinical practice.
Axonal transport of the lysosomal enzyme arylsulfatase A (ARSA) may be an additional mechanism of enzyme distribution after in vivo brain gene transfer in an animal model of metachromatic ...leukodystrophy (MLD). Direct molecular demonstration of the movement of this lysosomal enzyme within axonal networks was missing. We generated lentiviral vectors carrying the ARSA cDNA tagged with hemagglutinin or the green fluorescent protein and examined the subcellular localization and anatomical distribution of the tagged enzymes within the MLD hippocampus after in vivo lentiviral gene transfer. The use of tagged ARSA allowed direct real-time observation and tracking of axon-dendritic transport of the enzyme after lentiviral gene therapy. Tagged ARSA was expressed in transduced pyramidal, granule, and hilar neurons within the lentiviral-injected side and was robustly contained in vesicles within ipsilateral axon-dendritic processes as well as in vesicles associated with contralateral axons and commissural axons of the ventral hippocampal commissure. Axonal transport of tagged ARSA led to the correction of hippocampal defects in long-term treated MLD mice, which was accompanied by enzyme uptake in nontransduced contralateral neurons, enzyme accumulation within the lysosomal compartment, and clearance of sulfatide storage deposits in this region of the MLD brain. These results contribute to the understanding of the mechanisms of distribution of lysosomal enzymes within the mammalian brain after direct gene therapy, demonstrating the use of neural processes for enzyme transport.
Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different ...animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne's muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan-null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan-null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.
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