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Knockout of beta-2 microglobulin reduces stem cell-induced immune rejection and enhances ischaemic hindlimb repair via exosome/miR-24/Bim pathway.

ABSTRACT: Generating universal human umbilical mesenchymal stem cells (UMSCs) without immune rejection is desirable for clinical application. Here we developed an innovative strategy using CRISPR/Cas9 to generate B2M^- UMSCs in which human leucocyte antigen (HLA) light chain β2-microglobulin (B2M) was deleted. The therapeutic potential of B2M^- UMSCs was examined in a mouse ischaemic hindlimb model. We show that B2M^- UMSCs facilitated perfusion recovery and enhanced running capability, without inducing immune rejection. The beneficial effect was mediated by exosomes. Mechanistically, microRNA (miR) sequencing identified miR-24 as a major component of the exosomes originating from B2M^- UMSCs. We identified Bim as a potential target of miR-24 through bioinformatics analysis, which was further confirmed by loss-of-function and gain-of-function approaches. Taken together, our data revealed that knockout of B2M is a convenient and efficient strategy to prevent UMSCs-induced immune rejection, and it provides a universal clinical-scale cell source for tissue repair and regeneration without the need for HLA matching in the future.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC6933348 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Knockout of beta-2 microglobulin reduces stem cell-induced immune rejection and enhances ischaemic hindlimb repair via exosome/miR-24/Bim pathway.

Zhang Yuqing Y Wang Yanli Y Shao Lianbo L Pan Xiangbin X Liang Chun C Liu Bin B Zhang Yu Y Xie Wenping W Yan Bing B Liu Feng F Yu Xi-Yong XY Li Yangxin Y

Journal of cellular and molecular medicine 20191115 1

Generating universal human umbilical mesenchymal stem cells (UMSCs) without immune rejection is desirable for clinical application. Here we developed an innovative strategy using CRISPR/Cas9 to generate B2M- UMSCs in which human leucocyte antigen (HLA) light chain β2-microglobulin (B2M) was deleted. The therapeutic potential of B2M- UMSCs was examined in a mouse ischaemic hindlimb model. We show that B2M- UMSCs facilitated perfusion recovery and enhanced running ...[more]

PMID: 31729180

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Project description:BackgroundUrine exosomes are small vesicles exocytosed into the urine by all renal epithelial cell types under normal physiologic and disease states. Urine exosomal proteins may mirror disease specific proteome perturbations in kidney injury. Analysis methodologies for the exosomal fraction of the urinary proteome were developed for comparing the urinary exosomal fraction versus unfractionated proteome for biomarker discovery.MethodsUrine exosomes were isolated by centrifugal filtration of urine samples collected from kidney transplant patients with and without acute rejection (AR), which were biopsy matched. The proteomes of unfractionated whole urine (Uw) and urine exosomes (Ue) underwent mass spectroscopy-based quantitative proteomics analysis. The proteome data were analyzed for significant differential protein abundances in AR.ResultsA total of 1018 proteins were identified in Uw and 349 proteins in Ue. Two hundred seventy-nine overlapped between the two urinary compartments and 70 proteins were unique to the Ue compartment. Of 349 exosomal proteins identified from transplant patients, 220 had not been previously identified in the normal Ue fraction. Eleven Ue proteins, functionally involved in an inflammatory and stress response, were more abundant in urine samples from patients with AR, three of which are exclusive to the Ue fraction. Ue AR-specific biomarkers (1) were also detected in Uw, but since they were observed at significantly lower abundances in Uw, they were not significant for AR in Uw.ConclusionA rapid urinary exosome isolation method and quantitative measurement of enriched Ue proteins was applied. Perturbed proteins in the exosomal compartment of urine collected from kidney transplant patients were specific to inflammatory responses, and were not observed in the Ue fraction from normal healthy subjects. Ue-specific protein alterations in renal disease provide potential mechanistic insights and offer a unique panel of sensitive biomarkers for monitoring AR.

Project description:BackgroundAngiogenesis is critical for forming new blood vessels from antedating vascular vessels. The endothelium is essential for angiogenesis, vascular remodelling and minimisation of functional deficits following ischaemia. The insulin-like growth factor (IGF) family is crucial for angiogenesis. Insulin-like growth factor-binding protein 5 (IGFBP5), a binding protein of the IGF family, may have places in angiogenesis, but the mechanisms are not yet completely understood. We sought to probe whether IGFBP5 is involved in pathological angiogenesis and uncover the molecular mechanisms behind it.Methods and resultsIGFBP5 expression was elevated in the vascular endothelium of gastrocnemius muscle from critical limb ischaemia patients and hindlimb ischaemic (HLI) mice and hypoxic human umbilical vein endothelial cells (HUVECs). In vivo, loss of endothelial IGFBP5 (IGFBP5EKO) facilitated the recovery of blood vessel function and limb necrosis in HLI mice. Moreover, skin damage healing and aortic ring sprouting were faster in IGFBP5EKO mice than in control mice. In vitro, the genetic inhibition of IGFBP5 in HUVECs significantly promoted tube formation, cell proliferation and migration by mediating the phosphorylation of IGF1R, Erk1/2 and Akt. Intriguingly, pharmacological treatment of HUVECs with recombinant human IGFBP5 ensued a contrasting effect on angiogenesis by inhibiting the IGF1 or IGF2 function. Genetic inhibition of IGFBP5 promoted cellular oxygen consumption and extracellular acidification rates via IGF1R-mediated glycolytic adenosine triphosphate (ATP) metabolism. Mechanistically, IGFBP5 exerted its role via E3 ubiquitin ligase Von Hippel-Lindau (VHL)-regulated HIF1α stability. Furthermore, the knockdown of the endothelial IGF1R partially abolished the reformative effect of IGFBP5EKO mice post-HLI.ConclusionOur findings demonstrate that IGFBP5 ablation enhances angiogenesis by promoting ATP metabolism and stabilising HIF1α, implying IGFBP5 is a novel therapeutic target for treating abnormal angiogenesis-related conditions.

Project description:BackgroundAn oligopeptide hepcidin is encoded by the human HAMP gene (Hamp in mice). Its deficiency can result in iron overload, while excess may lead to iron deficiency. Hepcidin knockout mice exhibited iron accumulation in multiple tissues, accompanied by degeneration of bone microarchitecture and reduced biomechanical properties. Astronauts who are exposed to weightlessness during prolonged spaceflight experience bone loss. After space missions, an interrelation exists between iron stores and bone mineral density (BMD). Bone loss in mice due to unloading is linked to iron excess and involves hepcidin. The potential role of hepcidin in unloading-induced bone loss remains unclear.MethodsOur study conducted relevant experiments using hepcidin knockout mice and their primary osteoblasts as the research subjects. We used the hindlimb unloading (HLU) model and the random positioning machine (RPM) system to simulate weightlessness in vivo and in vitro.ResultsHLU mice exhibited reduced hepcidin levels in the serum and liver. Hepcidin knockout further diminished BMD and bone mineral content (BMC) in the femurs of HLU mice. Similarly, the bone volume fraction (BV/TV) and connectivity density (Conn.Dn) followed this downward trend, whereas trabecular separation (Tb.Sp) showed an inverse pattern. Moreover, hepcidin knockout decreased the ultimate load and elastic modulus in the tibias of HLU mice. Hepcidin knockout decreased PINP levels in the serum, a commonly used marker for bone formation, alongside elevated iron levels in the serum, liver, and bone of HLU mice. We also found higher serum MDA and SOD levels in these mice. In vitro, experimental data indicated that hepcidin knockout suppresses the osteoblastic differentiation capacity under RPM conditions. Additionally, this condition upregulates SOST protein levels and downregulates LRP6 and β-catenin protein levels in osteoblasts.ConclusionHepcidin knockout exacerbates bone loss in HLU mice, most likely due to reduced osteoblastic activity.

Dataset Information

Knockout of beta-2 microglobulin reduces stem cell-induced immune rejection and enhances ischaemic hindlimb repair via exosome/miR-24/Bim pathway.

Publications

Knockout of beta-2 microglobulin reduces stem cell-induced immune rejection and enhances ischaemic hindlimb repair via exosome/miR-24/Bim pathway.

Similar Datasets

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