Manufacturing, quality control, and GLP-grade preclinical study of nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles.

BACKGROUND: Human adipose stromal cells-derived extracellular vesicles (haMSC-EVs) have been shown to alleviate inflammation in acute lung injury (ALI) animal models. However, there are few systemic studies on clinical-grade haMSC-EVs. Our study aimed to investigate the manufacturing, quality control (QC) and preclinical safety of clinical-grade haMSC-EVs. METHODS: haMSC-EVs were isolated from the conditioned medium of human adipose MSCs incubated in 2D containers. Purification was performed by PEG precipitation and differential centrifugation. Characterizations were conducted by nanoparticle tracking analysis, transmission electron microscopy (TEM), Western blotting, nanoflow cytometry analysis, and the TNF-α inhibition ratio of macrophage [after stimulated by lipopolysaccharide (LPS)]. RNA-seq and proteomic analysis with liquid chromatography tandem mass spectrometry (LC-MS/MS) were used to inspect the lot-to-lot consistency of the EV products. Repeated toxicity was evaluated in rats after administration using trace liquid endotracheal nebulizers for 28 days, and respiratory toxicity was evaluated 24 h after the first administration. In vivo therapeutic effects were assessed in an LPS-induced ALI/ acute respiratory distress syndrome (ARDS) rat model. RESULTS: The quality criteria have been standardized. In a stability study, haMSC-EVs were found to remain stable after 6 months of storage at - 80°C, 3 months at - 20 °C, and 6 h at room temperature. The microRNA profile and proteome of haMSC-EVs demonstrated suitable lot-to-lot consistency, further suggesting the stability of the production processes. Intratracheally administered 1.5 × 108 particles/rat/day for four weeks elicited no significant toxicity in rats. In LPS-induced ALI/ARDS model rats, intratracheally administered haMSC-EVs alleviated lung injury, possibly by reducing the serum level of inflammatory factors. CONCLUSION: haMSC-EVs, as an off-shelf drug, have suitable stability and lot-to-lot consistency. Intratracheally administered haMSC-EVs demonstrated excellent safety at the tested dosages in systematic preclinical toxicity studies. Intratracheally administered haMSC-EVs improved the lung function and exerted anti-inflammatory effects on LPS-induced ALI/ARDS model rats.

Release of O-GlcNAc transferase inhibitor promotes neuronal differentiation of neural stem cells in 3D bioprinted supramolecular hydrogel scaffold for spinal cord injury repair.

Precise fabrication of biomimetic three-dimensional (3D) structure and effective neuronal differentiation under the pathological environment are the key to neural stem cell (NSC)-based spinal cord injury (SCI) therapy. In this study, we have developed a spinal cord-like bioprinted scaffold loading with OSMI-4, a small molecule O-GlcNAc transferase (OGT) inhibitor, to induce and guide the neuron differentiation of NSCs for efficient SCI repair. To achieve this, we developed a supramolecular bioink (SM bioink) consisting of methacrylated gelatin and acrylated ß-cyclodextrins to load NSCs and OSMI-4. This bioink showed fast gelation and stable mechanical properties, facilitating bioprinting of functional neural scaffolds. Moreover, the weak host-guest cross-linking of the SM scaffolds significantly improved the cell-matrix interaction for the infiltration and migration of NSCs. What's more, the sustained delivery of OSMI-4 remarkably enhanced the intrinsic neuronal differentiation of the encapsulated NSCs in vitro by inhibiting Notch signaling pathway. In vivo experiment further revealed that the functional bioprinted scaffolds promoted the neuronal regeneration and axonal growth, leading to significant locomotor recovery of the SCI model rats. Together, the NSC-laden bioprinted SM scaffolds in combination with sustained release of the therapeutic agent OSMI-4 largely induced neuronal differentiation of NSCs and thus leading to efficient SCI repair. STATEMENT OF SIGNIFICANCE: Efficient neuronal differentiation of neural stem cells (NSCs) under the complex pathological microenvironment of spinal cord injury (SCI) is a major challenge of neural regeneration. By the use of a supramolecular bioink, we bioprinted a spinal cord-like scaffold loaded with NSCs and a small molecule drug OSMI-4 to significantly induce neuronal differentiation of NSCs for efficient SCI repair in vivo. The scaffolds with spinal cord-like structure can support the interaction and neuronal differentiation of NSCs by providing a dynamic matrix and a source of molecular release of OSMI-4. The influences of OSMI-4 on NSCs and its molecular mechanism were investigated for the first time in this study. Altogether, three-dimensional bioprinting fabrication of NSC- and small molecule drug-laden biomimetic construct may represent a promising therapeutic strategy for SCI repair.

CT/bioluminescence dual-modal imaging tracking of stem cells labeled with Au@PEI@PEG nanotracers and RfLuc in nintedanib-assisted pulmonary fibrosis therapy.

Mesenchymal stem cells (MSCs) are promising in idiopathic pulmonary fibrosis (IPF) therapy. However, low survival rate and ambiguous behavior of MSCs after transplantation impede their clinical translation. To this end, we have developed a new strategy to improve the survival rate and monitor the behavior of the transplanted MSCs simultaneously. In our strategy, nintedanib, a tyrosine kinase inhibitor, is employed to protect the human MSCs (hMSCs) from excessive oxidative stress responses and inflammatory environment in the damaged lung. Moreover, by labeling of the transplanted hMSCs with a computed tomography (CT) nanotracer, Au nanoparticles functionalized with polyethylenimine (PEI) and polyethylene glycol (PEG) (Au@PEI@PEG), in combination with red-emitting firefly luciferase (RfLuc), in vivo CT/bioluminescence (BL) dual-modal imaging tracking of the location, distribution, and survival of the transplanted hMSCs in presence of nintedanib were achieved, which facilitates the profound understanding of the role the stem cells play in IPF therapy.

Facile engineering of ECM-mimetic injectable dual crosslinking hydrogels with excellent mechanical resilience, tissue adhesion, and biocompatibility.

Injectable hydrogels have aroused ever-increasing interest for their cell/biomaterial delivery ability through minimally invasive procedures. Nevertheless, it is still a challenge to simply fabricate natural biopolymer-based injectable hydrogels possessing satisfactory mechanical properties, bioadhesion, and cell delivery ability. Herein, we describe a facile dual crosslinking (DC) strategy for preparing extracellular matrix (ECM) mimetic hydrogels with desirable comprehensive performance. The chondroitin sulfate (CS)- and gelatin (Gel)-based single crosslinked (SC) hydrogels were first developed via reversible borate ester bonds, and further strengthened through the Michael-addition crosslinking reaction or visible-light initiated photopolymerization with thiol-containing polyethylene glycol (PEG) crosslinkers. The dynamic SC hydrogels showed good injectability, pH-sensitive gel-sol transformation, and self-adhesion ability to various biological tissues such as skin, liver, and intervertebral disc. The mechanically tough DC hydrogels displayed tunable stiffness, and resilience to compression load (up to 90% strain) owing to the effective energy dissipation mechanism. The formed DC hydrogels after subcutaneous injection well integrated with surrounding tissues and exhibited fast self-recovery properties. Moreover, the photoencapsulation of human mesenchymal stem cells (hMSCs) within the developed DC hydrogels was achieved and has been proved to be biocompatible, highlighting the great potential of the photopolymerized DC hydrogels in cell delivery and three-dimensional (3D) cell culture. This biomimetic, mechanically resilient, adhesive, and cytocompatible injectable DC hydrogel could serve as a promising candidate for tissue engineering.

pH-Triggered Aggregation of Gold Nanoparticles for Enhanced Labeling and Long-Term CT Imaging Tracking of Stem Cells in Pulmonary Fibrosis Treatment.

Gold nanoparticles (AuNPs) pose a great challenge in the development of nanotracers that can self-adaptively alter their properties in response to certain cellular environments for long-term stem cell tracking. Herein, pH-sensitive Au nanotracers (CPP-PSD@Au) are fabricated by sequential coupling of AuNPs with sulfonamide-based polymer (PSD) and cell-penetrating peptide (CPP), which can be efficiently internalized by mesenchymal stem cells (MSCs) and undergo pH-induced self-assembly in endosomes, facilitating long-term computed tomography (CT) imaging tracking MSCs in a murine model of idiopathic pulmonary fibrosis (IPF). Using the CPP-PSD@Au, the transplanted MSCs for the first time can be monitored with CT imaging for up to 35 days after transplantation into the lung of IPF mice, clearly elucidating the migration process of MSCs in vivo. Moreover, we preliminarily explored the mechanism of the CPP-PSD@Au labeled MSCs in the alleviation of IPF, including recovery of alveolar integrity, decrease of collagen deposition, as well as down-regulation of relevant cytokine level. This work facilitates our understanding of the behavior and effect of MSCs in the therapy of IPF, thereby providing an important insight into the stem cell-based treatment of lung diseases.

3D bioprinted neural tissue constructs for spinal cord injury repair.

Three-dimensional (3D) bioprinting has emerged as a promising approach to fabricate living neural constructs with anatomically accurate complex geometries and spatial distributions of neural stem cells (NSCs) for spinal cord injury (SCI) repair. The NSC-laden 3D bioprinting, however, still faces some big challenges, such as cumbersome printing process, poor cell viability, and minimal cell-material interaction. To address these issues, we have fabricated NSC-laden scaffolds by 3D bioprinting and explore for the first time their application for in vivo SCI repair. In our strategy, we have developed a novel biocompatible bioink consisting of functional chitosan, hyaluronic acid derivatives, and matrigel. This bioink shows fast gelation (within 20 s) and spontaneous covalent crosslinking capability, facilitating convenient one-step bioprinting of spinal cord-like constructs. Thus-fabricated scaffolds maintain high NSC viability (about 95%), and offer a benign microenvironment that facilitates cell-material interactions and neuronal differentiation for optimal formation of neural network. The in vivo experiment has further demonstrated that the bioprinted scaffolds promoted the axon regeneration and decreased glial scar deposition, leading to significant locomotor recovery of the SCI model rats, which may represent a general and versatile strategy for precise engineering of central nervous system and other neural organs/tissues for regenerative medicine application.

Enhanced and long-term CT imaging tracking of transplanted stem cells labeled with temperature-responsive gold nanoparticles.

Gold nanoparticles (AuNPs) have been extensively employed for computed tomography (CT) imaging in cell labeling and tracking because of their strong X-ray attenuation coefficient and excellent biocompatibility. However, the design and synthesis of stimuli-responsive AuNPs to modulate their endocytosis and exocytosis for optimal cell labeling and tracking are promising but challenging. Herein, we report an innovative labeling strategy based on temperature-responsive AuNPs (TRAuNPs) with high cell labeling efficiency and extended intracellular retention duration. We have manifested that the TRAuNP labeling imposes a negligible adverse effect on the function of human mesenchymal stem cells (hMSCs). Further experiment with idiopathic pulmonary fibrosis (IPF) model mice has demonstrated the feasibility of TRAuNP labeling for long time CT imaging tracking of transplanted hMSCs. What's more, the survival of transplanted hMSCs could also be monitored simultaneously using bioluminescence imaging after the expression of luciferase reporter genes. Therefore, we believe that this dual-modal labeling and tracking strategy enables visualization of the transplanted hMSCs in vivo, which may provide an important insight into the role of stem cells in the IPF therapy.

CT/MR Dual-Modality Imaging Tracking of Mesenchymal Stem Cells Labeled with a Au/GdNC@SiO2 Nanotracer in Pulmonary Fibrosis.

Mesenchymal stem cells (MSCs) have shown potential as an innovative treatment for pulmonary fibrosis (PF), due to their capability to ameliorate the inflammation and moderate the deterioration of PF. The fate of the stem cells transplanted into the lung, including survival, migration, homing, and functions, however, has not been fully understood yet. In this paper, we report the development of a computed tomography/magnetic resonance (CT/MR) dual-modal nanotracer, gold/gadolinium nanoclusters overcoated with a silica shell (Au/GdNC@SiO2), for noninvasive labeling and tracking of the transplanted human MSCs (hMSCs) in a PF model. The Au/GdNC@SiO2 nanotracer exhibits good colloidal and chemical stability, high biocompatibility, enhanced longitudinal MR relaxivity, and superior X-ray attenuation property. The hMSCs can be effectively labeled with Au/GdNC@SiO2, resulting in a significantly increased cellular CT/MR imaging contrast, without any obvious adverse effect on the function, including proliferation and differentiation of the labeled stem cells. Moreover, by using the Au/GdNC@SiO2 nanotracer, the hMSCs transplanted in the lung can be tracked for 7 d via in vivo CT/MR dual-modality imaging. This work may provide an insight into the role the transplanted hMSCs play in PF therapy, thus promoting the stem cell-based regenerative medicine.

CT/Bioluminescence Dual-Modal Imaging Tracking of Mesenchymal Stem Cells in Pulmonary Fibrosis.

Human mesenchymal stem cells (hMSCs), due to their immune regulation and collateral secretion effects, are currently explored for potential therapy of idiopathic pulmonary fibrosis (IPF). Understanding the migration, homing, functions, and survival of transplanted hMSCs in vivo is critical to successful IPF treatment. Therefore, it is highly desired to develop noninvasive and effective imaging technologies to track the transplanted hMSCs, providing experimental basis for improving the efficacy of hMSCs in the treatment of IPF. The rational design and development of a dual-labeling strategy are reported by integrating gold nanoparticle (AuNP)-based computed tomography (CT) nanotracers and red-emitting firefly luciferase (RfLuc)-based bioluminescence (BL) tags for CT/BL multimodal imaging tracking of the transplanted hMSCs in a murine model of IPF. In this approach, the CT nanotracer is prepared by sequential coupling of AuNPs with polyethylene glycol and trans-activator of transcription (TAT) peptide (Au@TAT), and employed it to monitor the location and distribution of the transplanted hMSCs in vivo by CT imaging, while RfLuc is used to monitor hMSCs viability by BLI. This facile strategy allows for visualization of the transplanted hMSCs in vivo, thereby enabling profound understanding of the role of hMSCs in the IPF treatment, and advancing stem cell-based regenerative medicine.

WS1 gene mutation analysis of Wolfram syndrome in a Chinese patient and a systematic review of literatures.

Wolfram syndrome is a rare hereditary disease characterized by diabetes mellitus and optic atrophy. The outcome of this disease is always poor. WFS1 gene mutation is the main cause of this disease. A patient with diabetes mellitus, diabetes insipidus, renal tract disorder, psychiatric abnormality, and cataract was diagnosed with Wolfram syndrome. Mutations in open reading frame (ORF) of WFS1 gene was analyzed by sequencing. Mutations in WFS1 gene was also summarized by a systematic review in Pubmed and Chinese biological and medical database. Sequencing of WFS1 gene in this patient showed a new mutation, 1962G>A, and two other non-sense mutations, 2433A>G and 2565G>A. Systematic review included 219 patients in total and identified 172 WFS1 gene mutations, most of which were located in Exon 8. These mutations in WFS1 gene might be useful in prenatal diagnosis of Wolfram syndrome.