Lung Progenitor and Stem Cell Transplantation as a Potential Regenerative Therapy for Lung Diseases.

Chronic lung diseases are debilitating illnesses ranking among the top causes of death globally. Currently, clinically available therapeutic options capable of curing chronic lung diseases are limited to lung transplantation, which is hindered by donor organ shortage. This highlights the urgent need for alternative strategies to repair damaged lung tissues. Stem cell transplantation has emerged as a promising avenue for regenerative treatment of the lung, which involves delivery of healthy lung epithelial progenitor cells that subsequently engraft in the injured tissue and further differentiate to reconstitute the functional respiratory epithelium. These transplanted progenitor cells possess the remarkable ability to self-renew, thereby offering the potential for sustained long-term treatment effects. Notably, the transplantation of basal cells, the airway stem cells, holds the promise for rehabilitating airway injuries resulting from environmental factors or genetic conditions such as cystic fibrosis. Similarly, for diseases affecting the alveoli, alveolar type II cells have garnered interest as a viable alveolar stem cell source for restoring the lung parenchyma from genetic or environmentally induced dysfunctions. Expanding upon these advancements, the use of induced pluripotent stem cells to derive lung progenitor cells for transplantation offers advantages such as scalability and patient specificity. In this review, we comprehensively explore the progress made in lung stem cell transplantation, providing insights into the current state of the field and its future prospects.

Amide proton transfer-weighted magnetic resonance imaging for application in renal fibrosis: A radiological-pathological based analysis.

BACKGROUND: Renal fibrosis (RF) being the most important pathological change in the progression of CKD, is currently assessed by the evaluation of a biopsy. This present study aimed to apply a novel functional MRI (fMRI) protocol named amide proton transfer weighting (APTw) to evaluate RF non-invasively. METHODS: Male Sprague-Dawley (SD) rats were initially subjected to bilateral kidney ischemia/reperfusion injury (IRI), unilateral ureteral obstruction (UUO) and Sham operation, respectively. All rats underwent APT mapping on the 7th and the 14th day after operation. Besides, 26 patients undergoing renal biopsy at the Nephrology Department of Shanghai Tongji Hospital between July 2022 and May 2023. Patients underwent APT and apparent diffusion coefficient (ADC) mappings within 1 week before biopsy. MRI results of both patients and rats were calculated by comparing with gold standard histology for fibrosis assessment. RESULTS: In animal models, the cortical APT (cAPT) and medullary APT (mAPT) values were positively correlated with the degree of renal fibrosis. Compared to the sham group, IRI group showed significantly increased cAPT and mAPT values on the 7th and 14th day after surgery, but no group differences were found in ADC values. Similar results were found in human patients. Cortical/medullary APT values were significantly increased in patients with moderate-to-severe fibrosis than patients with mild fibrosis. ROC curve analysis indicated that APT value displayed a better diagnostic value for RF. Furthermore, combination of cADC and cAPT improved fibrosis detection by imaging variables alone (p<0.1). CONCLUSION: APT values had better diagnostic capability at early stage of RF compared to ADC values, and the addition of APT imaging to conventional ADC will significantly improve the diagnostic performance for predicting kidney fibrosis.

Ononin Relieves the Thyroid Cancer Progression through Targeting the Caspase 3 and CD274 Expression Levels.

Thyroid cancer (TC) is the most common malignant tumor of endocrine system and head and neck. Ononin is an isoflavone component, which exhibited great antioxidant and anti-inflammatory activities. This study was conducted to explore the functions of ononin in the TC progression. The cell counting kit-8 (CCK8) assay was applied for the cell viability determination. The cell death and apoptosis rate were analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and flow cytometry. The quantitative real-time PCR (qRT-PCR) and Western blot assays were performed for the relative expressions determination. Lactate dehydrogenase (LDH) release assay was used to assess cytotoxicity. Ononin treatment prominently inhibited the cell viability and induced the cell apoptosis of the TC cells. Besides, caspase 3 (CASP3) was down-regulated and CD274 was up-regulated in TC. Ononin treatment prominently decreased the CD274 levels and increased the CASP3 levels in the TC cells. Additionally, ononin treatment dramatically enhanced the LDH release of the cytotoxicity of T cells. What is more, CASP3 overexpression or CD274 knockdown promoted the role of ononin in TC cells. Ononin treatment induced the cell death of the TC cells through regulating the CASP3 and CD274 expressions.

A histidine-rich fusion tag enables real-time monitoring of recombinant protein expression by Pauly reaction-based colorimetric assay.

Commercially available recombinant expression systems always use fusion tags to facilitate target protein purification and SDS-PAGE analysis followed by Coomassie Brilliant Blue (CBB) staining is the classical method to validate the expression level of target protein, which is time-consuming, although not very laborious. Previously, we found that a histidine-rich elastin-like polypeptide (HRELP) tag could make its fusion proteins being quickly and specifically stained with Pauly's reagent. In this study, we designed a Pauly reaction-based colorimetric assay to real-time monitoring of the expression level of recombinant protein tagged HRELP and found that the absorption value of post-induction E. coli cells stained with Pauly's reagent correlated well with both the band intensity of the target protein from Pauly's reagent-stained and CBB-stained gels. Moreover, we found the colorimetric assay could also be helpful to roughly estimate the expression efficiency by using a poly-histidine-tagged protein, which has only 1.17% histidine residue. In our opinion, Pauly reaction-based colorimetric assay could significantly shorten the time to validate the over-expression of recombinant protein tagged with either HRELP or poly-histidine. And HRELP seemed to be an ideal fusion tag for it can not only facilitate protein purification but also simplify protein detection.

A single-cell atlas identifies pretreatment features of primary imatinib resistance in chronic myeloid leukemia.

Primary resistance to tyrosine kinase inhibitors (TKIs) is a significant barrier to optimal outcomes in chronic myeloid leukemia (CML), but factors contributing to response heterogeneity remain unclear. Using single-cell RNA (scRNA) sequencing, we identified 8 statistically significant features in pretreatment bone marrow, which correlated with either sensitivity (major molecular response or MMR) or extreme resistance to imatinib (eventual blast crisis [BC] transformation). Employing machine-learning, we identified leukemic stem cell (LSC) and natural killer (NK) cell gene expression profiles predicting imatinib response with >80% accuracy, including no false positives for predicting BC. A canonical erythroid-specifying (TAL1/KLF1/GATA1) regulon was a hallmark of LSCs from patients with MMR and was associated with erythroid progenitor [ERP] expansion in vivo (P < .05), and a 2- to 10-fold (6.3-fold in group A vs 1.09-fold in group C) erythroid over myeloid bias in vitro. Notably, ERPs demonstrated exquisite TKI sensitivity compared with myeloid progenitors (P < .001). These LSC features were lost with progressive resistance, and MYC- and IRF1-driven inflammatory regulons were evident in patients who progressed to transformation. Patients with MMR also exhibited a 56-fold expansion (P < .01) of a normally rare subset of hyperfunctional adaptive-like NK cells, which diminished with progressive resistance, whereas patients destined for BC accumulated inhibitory NKG2A+ NK cells favoring NK cell tolerance. Finally, we developed antibody panels to validate our scRNA-seq findings. These panels may be useful for prospective studies of primary resistance, and in assessing the contribution of predetermined vs acquired factors in TKI response heterogeneity.

Impact of rhG-CSF on Clinical Efficacy and Immune Cell Subsets after Initial Induction Chemotherapy in AML.

BACKGROUND: The impact of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in acute myeloid leukemia (AML) is still controversial. The purpose of this study is to explore the impact of rhG-CSF administration on clinical efficacy and immune cell subsets after initial induction chemotherapy in AML. METHODS: The clinical efficacy and immune cell subsets were compared in the newly diagnosed patients with AML according to whether rhG-CSF was used after initial induction chemotherapy. Next, rhG-CSF stimulation experi-ments on leukemia cell lines and primary leukemia blasts were performed in vitro. RESULTS: There was no statistical difference between control group and rhG-CSF therapy group in complete remission rate and relapse free survival. The duration of agranulocytosis was significantly shortened in rhG-CSF therapy group compared with control group. The percentage of circulating monocytic myeloid-derived suppressor cells (M-MDSCs) and regulatory T cells (Tregs) were significantly increased after the administration of rhG-CSF. Furthermore, it was found that rhG-CSF did not promote the proliferation of leukemia cell lines and primary leukemia blasts, but increased the proportion of M-MDSCs and Tregs in vitro. CONCLUSIONS: Administration of rhG-CSF after initial induction therapy of AML does not affect the clinical remission and relapse rate, but reduces the duration of agranulocytosis and increases the proportion of M-MDSCs and Tregs.

Nanoscale coordination polymers enabling antioxidants inhibition for enhanced chemodynamic therapy.

Reactive oxygen species (ROS) generation to induce cell death is an effective strategy for cancer therapy. In particular, chemodynamic therapy (CDT), using Fenton-type reactions to generate highly cytotoxic hydroxyl radical (•OH), is a promising treatment modality. However, the therapeutic efficacy of ROS-based cancer treatment is still limited by some critical challenges, such as overexpression of enzymatic and non-enzymatic antioxidants by tumor cells, as well as the low tumor targeting efficiency of therapeutic agents. To address those problems, biomimetic CuZn protoporphyrin IX nanoscale coordination polymers have been developed, which significantly amplify oxidative stress against tumors by simultaneously inhibiting enzymatic and non-enzymatic antioxidants and initiating the CDT. In this design, cancer cell membrane camouflaged nanoparticle exhibits an excellent homotypic targeting effect. After being endocytosed into tumor cells, the nanoparticles induce depletion of the main non-enzymatic antioxidant glutathione (GSH) by undergoing a redox reaction with GSH. Afterward, the redox reaction generated cuprous ion (Cu+) works as a CDT agent for •OH generation. Furthermore, the released Zn protoporphyrin IX strongly inhibits the activity of the typical enzymatic antioxidant heme oxygenase-1. This tetra-modal synergistic strategy endows the biomimetic nanoparticles with great capability for anticancer therapy, which has been demonstrated in both in vitro and in vivo studies.

The Efficacy of Autologous Platelet-Rich Gel and Traditional Chinese Medicine in Diabetic Foot Treatment: A Parallel Randomized Controlled Clinical Trial.

BACKGROUND: Diabetic foot (DF) is a prevalent metabolic infection. DF wounds are the basis for all cases of nontraumatic lower limb amputations in diabetes. DF care approaches include debridement of wound, pressure relief in the wounded area, proper wound, infection, and ischemia management. However, there is a need for research to develop more effective therapeutic approaches. This study investigated the effectivity and safety of autologous platelet-rich gel combined with conventional treatment and traditional Chinese medicine (TCM) in DF ulcer therapy. METHODS: Sixty DF ulcer patients were divided into treatment and control groups of 30 patients each. The treatment group involved a combination of autologous platelet-rich gel, conventional treatment, and TCM. The control group was only treated with a combination of conventional therapy and TCM. Laboratory variables, including platelets, hemoglobin (Hb), albumin, and HbA1c, were analyzed and compared between the treatment and the control groups at baseline and endpoint. Healing area, volume, and rates were compared in both groups. RESULTS: Basic patients' data and the wound conditions had no significant difference between the treatment and the control group. The treatment and control groups cure rates were 93.3% vs. 50%, respectively. The healing rate per 2 weeks was significantly higher in the treatment than in the control group (0.78 ± 0.05 vs. 0.43 ± 0.04). There was no statistically significant difference in the platelets, Hb, albumin, and HbA1c levels in the treatment and control groups. CONCLUSIONS: Autologous platelet-rich gel combined with conventional treatment and TCM is effective and safe for DF ulcer treatment.

Formation of Nano-Fibrous Patterns on Aluminum Substrates via Photolithographic Fabrication of Electrospun Photosensitive Polyimide Fibrous Membranes.

The formation of polymeric micro-patterns on various substrates via a photolithography procedure has been widely used in semiconductor fabrication. Standard polymer patterns are usually fabricated via photosensitive polymer varnishes, in which large amounts of potentially harmful solvents with weight ratios over 50 wt% have to be removed. In the current work, a novel pattern-formation methodology via solvent-free electrospun photosensitive polymeric fibrous membranes (NFMs) instead of the conventional photosensitive solutions as the starting photoresists was proposed and practiced. For this purpose, a series of preimidized negative auto-photosensitive polyimide (PSPI) resins were first prepared via the two-step chemical imidization procedure from the copolymerization reactions of 3,3',4,4'-benzophenonetetracarboxylic- dianhydride (BTDA) and two ortho-methyl-substituted aromatic diamines, including 3,3',5,5'-tetramethyl-4,4'-diaminodiphenylmethane (TMMDA) and 3,7-diamino-2,8-dimethyl- dibenzothiophene sulfone (TSN). The derived homopolymer PI-1 (BTDA-TMMDA) and the copolymers, including SPI-2~SPI-6, with the molar ratio of 5~25% for TSN in the diamine units, showed good solubility in polar solvents. Then, a series of PSPI NFMs were fabricated via standard electrospinning procedure with the developed PSPI solutions in N,N-dimethylacetamide (DMAc) with a solid content of 25 wt% as the starting materials. The derived PSPI NFMs showed good thermal stability with 5% weight loss temperatures higher than 500 °C in nitrogen. Meanwhile, the derived PSPIs showed good photosensitivity to the ultraviolet (UV) emitting wavelengths of i-line (365 nm), g-line (405 nm) and h-line (436 nm) of the high-pressure mercury lamps in both forms of transparent films and opaque NFMs. Fine micro-patterns with a line width of around 100 µm were directly obtained from the representative SPI-4 NFM via standard photolithography procedure.

Multilayered control of splicing regulatory networks by DAP3 leads to widespread alternative splicing changes in cancer.

The dynamic regulation of alternative splicing requires coordinated participation of multiple RNA binding proteins (RBPs). Aberrant splicing caused by dysregulation of splicing regulatory RBPs is implicated in numerous cancers. Here, we reveal a frequently overexpressed cancer-associated protein, DAP3, as a splicing regulatory RBP in cancer. Mechanistically, DAP3 coordinates splicing regulatory networks, not only via mediating the formation of ribonucleoprotein complexes to induce substrate-specific splicing changes, but also via modulating splicing of numerous splicing factors to cause indirect effect on splicing. A pan-cancer analysis of alternative splicing across 33 TCGA cancer types identified DAP3-modulated mis-splicing events in multiple cancers, and some of which predict poor prognosis. Functional investigation of non-productive splicing of WSB1 provides evidence for establishing a causal relationship between DAP3-modulated mis-splicing and tumorigenesis. Together, our work provides critical mechanistic insights into the splicing regulatory roles of DAP3 in cancer development.

ADARs act as potent regulators of circular transcriptome in cancer.

Circular RNAs (circRNAs) are produced by head-to-tail back-splicing which is mainly facilitated by base-pairing of reverse complementary matches (RCMs) in circRNA flanking introns. Adenosine deaminases acting on RNA (ADARs) are known to bind double-stranded RNAs for adenosine to inosine (A-to-I) RNA editing. Here we characterize ADARs as potent regulators of circular transcriptome by identifying over a thousand of circRNAs regulated by ADARs in a bidirectional manner through and beyond their editing function. We find that editing can stabilize or destabilize secondary structures formed between RCMs via correcting A:C mismatches to I(G)-C pairs or creating I(G).U wobble pairs, respectively. We provide experimental evidence that editing also favors the binding of RNA-binding proteins such as PTBP1 to regulate back-splicing. These ADARs-regulated circRNAs which are ubiquitously expressed in multiple types of cancers, demonstrate high functional relevance to cancer. Our findings support a hitherto unappreciated bidirectional regulation of circular transcriptome by ADARs and highlight the complexity of cross-talk in RNA processing and its contributions to tumorigenesis.

Engineering pro-angiogenic biomaterials via chemoselective extracellular vesicle immobilization.

Nanoscale extracellular vesicles (EVs) represent a unique cellular derivative that reflect the therapeutic potential of mesenchymal stem cells (MSCs) toward tissue engineering and injury repair without the logistical and safety concerns of utilizing living cells. However, upon systemic administration in vivo,EVs undergo rapid clearance and typically lack controlled targeted delivery, thus reducing their effectiveness in therapeutic regenerative therapies. Here, we describe a strategy that enables long-term in vivo spatial EV retention by chemoselective immobilization of metabolically incoporated azido ligand-bearing EVs (azido-EVs) within a dibenzocyclooctyne-modified collagen hydrogel. MSC-derived azido-EVs exhibit comparable morphological and functional properties as their non-labeled EV counterparts and, when immobilized within collagen hydrogel implants via click chemistry, they elicited more robust host cell infiltration, angiogenic and immunoregulatory responses including vascular ingrowth and macrophage recruitment compared to ten times the higher dose required by non-immobilized EVs. We envision this technology will enable a wide range of applications to spatially promote vascularization and host integration relevant to tissue engineering and regenerative medicine applications.

RNA editing mediates the functional switch of COPA in a novel mechanism of hepatocarcinogenesis.

BACKGROUND & AIMS: RNA editing introduces nucleotide changes in RNA sequences. Recent studies have reported that aberrant adenosine-to-inosine RNA editing is implicated in cancers. Until now, very few functionally important protein-recoding editing targets have been discovered. Here, we investigated the role of a recently discovered protein-recoding editing target COPA (coatomer subunit α) in hepatocellular carcinoma (HCC). METHODS: Clinical implication of COPA editing was studied in a cohort of 125 HCC patients. CRISPR/Cas9-mediated knockout of the editing site complementary sequence (ECS) was used to delete edited COPA transcripts endogenously. COPA editing-mediated change in its transcript or protein stability was investigated upon actinomycin D or cycloheximide treatment, respectively. Functional difference in tumourigenesis between wild-type and edited COPA (COPAWTvs. COPAI164V) and the exact mechanisms were also studied in cell models and mice. RESULTS: ADAR2 binds to double-stranded RNA formed between edited exon 6 and the ECS at intron 6 of COPA pre-mRNA, causing an isoleucine-to-valine substitution at residue 164. Reduced editing of COPA is implicated in the pathogenesis of HCC, and more importantly, it may be involved in many cancer types. Upon editing, COPAWT switches from a tumour-promoting gene to a tumour suppressor that has a dominant-negative effect. Moreover, COPAI164V may undergo protein conformational change and therefore become less stable than COPAWT. Mechanistically, COPAI164V may deactivate the PI3K/AKT/mTOR pathway through downregulation of caveolin-1 (CAV1). CONCLUSIONS: We uncover an RNA editing-associated mechanism of hepatocarcinogenesis by which downregulation of ADAR2 caused the loss of tumour suppressive COPAI164V and concurrent accumulation of tumour-promoting COPAWT in tumours; a rapid degradation of COPAI164V protein and hyper-activation of the PI3K/AKT/mTOR pathway further promote tumourigenesis. LAY SUMMARY: RNA editing is a process in which RNA is changed after it is made from DNA, resulting in an altered gene product. In this study, we found that RNA editing of a gene known as coatomer subunit α (COPA) is lower in tumour samples and discovered that this editing process changes COPA protein from a tumour-promoting form to a tumour-suppressive form. Loss of the edited COPA promotes the development of liver cancer.

Characterization of Oral Microbiome and Exploration of Potential Biomarkers in Patients with Pancreatic Cancer.

Pancreatic cancer (PC) is highly malignant and lacks an effective therapeutic schedule, hence that early diagnosis is of great importance to achieve a good prognosis. Oral bacteria have been proved to be associated with pancreatic cancer, but the specific mechanism has not been comprehensively illustrated. In our study, thirty-seven saliva samples in total were collected with ten from PC patients, seventeen from benign pancreatic disease (BPD) patients, and ten from healthy controls (HC). The oral bacterial community of HC, PC, and BPD groups was profiled by 16S rDNA high-throughput sequencing and bioinformatic methods. As shown by Simpson, Inverse Simpson, Shannon and Heip, oral microbiome diversity of HC, BPD and PC groups is in increasing order with the BPD and PC groups significantly higher than the HC group. Principal coordinate analysis (PCoA) suggested that grouping by PC, BPD and HC was statistically significant. The linear discriminant analysis effect size (LEfSe) identified high concentrations of Fusobacterium periodonticum and low concentrations of Neisseria mucosa as specific risk factors for PC. Furthermore, predicted functions showed changes such as RNA processing and modification as well as the pathway of NOD-like receptor signaling occurred in both PC and HC groups. Conclusively, our findings have confirmed the destruction of oral bacterial community balance among patients with PC and BPD and indicated the potential of Fusobacterium periodonticum and Neisseria mucosa as diagnostic biomarkers of PC.

Peptide and Aptamer Decorated Delivery System for Targeting Delivery of Cas9/sgRNA Plasmid To Mediate Antitumor Genome Editing.

A multiple-functionalized targeting delivery system was prepared by self-assembly for efficient delivery of Cas9/sgRNA plasmids to targeted tumor cell nuclei. The Cas9/sgRNA plasmids were compacted by protamine in the presence of calcium ions to form nanosized cores, which were further decorated by peptide and aptamer conjugated alginate derivatives. With the help of the nuclear location signal peptide and AS1411 aptamer with specific affinity for nucleolin in the tumor cell membrane and nuclei, the delivery vector can specifically deliver the plasmid to the nuclei of tumorous cells for knocking out the protein tyrosine kinase 2 (PTK2) gene to down-regulate focal adhesion kinase (FAK). The tumor cell apoptosis induced by genome editing is mitochondrial-dependent. In addition, FAK knockout results in negative regulation on the PI3K/AKT signaling pathway. Meanwhile, favorable modulation on various proteins involved in tumor progression can be realized by genome editing. The enhanced E-cadherin and decreased MMPs, vimentin, and VEGF imply the desirable effects of genome editing on suppression of tumor development. Wound healing and transwell assays confirm that the genome editing system can suppress tumor invasion and metastasis in edited cells efficiently. The investigation provides a facile and effective strategy to fabricate multiple-functionalized delivery vectors for genome editing.

Incidence, Risk Factors, and Outcome of Immune-Mediated Neuropathies (IMNs) following Haploidentical Hematopoietic Stem Cell Transplantation.

Immune-mediated neuropathies (IMNs) following hematopoietic stem cell transplantation have been described recently, which, excluding Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy, may present with atypical patterns. This retrospective, nested, case-control study reviewed data from 3858 patients who received haploidentical hematopoietic stem cell transplantation (haplo-HSCT) during the past 10 years at a single center, and 40 patients (1.04%) with IMN following haplo-HSCT were identified. Chronic graft-versus-host disease (cGVHD) (P = .043) and cytomegalovirus (CMV) viremia (P = .035) were recognized as independent risk factors for the development of IMN after haplo-HSCT. There were no significant differences in overall survival (P = .619), disease-free survival (P = .609), nonrelapse mortality (P = .87), or the incidence of relapse (P = .583) between patients with and without IMN after haplo-HSCT. However, patients with post-transplant IMN were at higher risk of developing cGVHD (P = .012) than patients who did not develop IMN. Twenty-four of the 40 patients with IMN (60%) attained neurologic improvement after treatments including vitamins B1 and B12 and/or immunomodulatory agents. However, 19 (47.5%) patients still had persistent motor/sensory deficits despite receiving timely treatment. More studies are needed to help develop standardized diagnostic and therapeutic strategies for patients with post-transplant IMN.

Targeting Delivery of Oligodeoxynucleotides to Macrophages by Mannosylated Cationic Albumin for Immune Stimulation in Cancer Treatment.

To efficiently deliver CpG oligodeoxynucleotides (ODNs) to macrophages for the reversal of cancer-induced immunosuppression, nanoparticles ODN@MCBSA with mannosylated cationic albumin (MCBSA) as a macrophage targeting vector were constructed. Compared with ODN@CBSA with cationic albumin (CBSA) as a vector, ODN@MCBSA exhibited significantly improved cellular uptake mediated by mannose moieties, resulting in significantly enhanced secretion of proflammatory cytokines including IL-12, IL-6, TNF-α, and iNOS. The modulation of macrophages toward the favorable M1 phenotype was confirmed by the upregulated CD80 expression after being treated by ODN delivery systems. In addition to immune cells, the effects of the ODN delivery system on cancerous HeLa cells were also investigated. The results showed that ODN@MCBSA did not affect the overall tumor cell viability. However, enhanced NF-κB, p-Akt, PIK3R3, Fas, and FasL, as well as upregulated caspases were observed in tumor cells, implying the pleiotropic effects on tumor cells. Our study provides a more in-depth understanding on the immunotherapeutic effects of CpG ODNs and highlights the importance of macrophage targeting delivery to minimize the effects on tumor cells. These results indicate that MCBSA could serve as a promising delivery vector of CpG ODNs to macrophages for cancer immunotherapy.

Biofabrication of a vascularized islet organ for type 1 diabetes.

Islet transplantation is superior to extrinsic insulin supplementation in the treating severe Type 1 diabetes. However, its efficiency and longevity are limited by substantial islet loss post-transplantation due to lack of engraftment and vascular supply. To overcome these limitations, we developed a novel approach to bio-fabricate functional, vascularized islet organs (VIOs) ex vivo. We endothelialized acellular lung matrixes to provide a biocompatible multicompartment scaffold with an intact hierarchical vascular tree as a backbone for islet engraftment. Over seven days of culture, islets anatomically and functionally integrated into the surrounding bio-engineered vasculature, generating a functional perfusable endocrine organ. When exposed to supra-physiologic arterial glucose levels in vivo and ex vivo, mature VIOs responded with a physiologic insulin release from the vein and provided more efficient reduction of hyperglycemia compared to intraportally transplanted fresh islets. In long-term transplants in diabetic mice, subcutaneously implanted VIOs achieved normoglycemia significantly faster and more efficiently compared to islets that were transplanted in deviceless fashion. We conclude that ex vivo bio-fabrication of VIOs enables islet engraftment and vascularization before transplantation, and thereby helps to overcome limited islet survival and function observed in conventional islet transplantation. Given recent progress in stem cells, this technology may enable assembly of functional personalized endocrine organs.

Multifunctional Vector for Delivery of Genome Editing Plasmid Targeting ß-Catenin to Remodulate Cancer Cell Properties.

Accurate and efficient delivery of genome editing plasmids to targeted cells is of critical importance in genome editing. Herein, we prepared a multifunctional delivery vector with a combination of ligand-mediated selectivity and peptide-mediated transmembrane function to effectively deliver plasmids to targeted cancerous cells. In the delivery system, the clustered regularly interspaced short palindromic repeat-associated Cas9 nuclease (CRISPR-Cas9) plasmid is combined with protamine with membrane and nuclear translocating activities and co-precipitated with CaCO3, which is further decorated by AS1411-functionalized carboxymethyl chitosan and cell penetrating peptide (TAT)-functionalized carboxymethyl chitosan. The AS1411-mediated tumor cell/nuclear targeting and TAT-induced enhanced endocytosis result in obviously increased cellular uptake and nuclear transport. As a result, the CRISPR-Cas9 plasmid can be efficiently delivered to cancer cell nuclei to mediate genome editing, resulting in an efficacious knockout of CTNNB1 gene encoding ß-catenin. More importantly, downregulation of ß-catenin could effectively prevent its enrichment in nuclei and then significantly downregulate the expression of proteins, such as vimentin, Snail, MMP-2, MMP-9, CD44, Nanog, and Oct4 to prevent tumor progression and metastasis. The edited cancerous cells exhibit favorable remodulated properties including inhibited growth, suppressed migration and invasion, and reduced cancer stemness.

Reversal of tumor malignization and modulation of cell behaviors through genome editing mediated by a multi-functional nanovector.

To effectively reverse tumor malignization by genome editing, a multi-functional self-assembled nanovector for the delivery of a genome editing plasmid specifically to tumor cells was developed. The nanovector core consisting of protamine and calcium carbonate entrapping the CRISPR-Cas9 plasmid is decorated by aptamer incorporated heparin. Owing to a high affinity between a MUC1 specific aptamer and mucin 1 (MUC1) overexpressed in tumor cells as well as the interaction between AS1411 and nucleolin on the tumor cell surface and cell nuclei, the nanovector can target the nuclei of tumorous cells for the knockout of focal adhesion kinase (FAK). Notably, the genome editing mediated by our delivery systems can effectively modulate cell behaviors and thus reverse tumor malignization. Up-regulated p53, p16, p21, E-cadherin, CD80, MICA, MICB and Fas, together with down-regulated MMP-9, vimentin, VEGF, TGF-ß, CD47 and CD133 in genome edited cells indicate that the genome editing system can inhibit cancerous cell growth, prevent tumor invasion and metastasis, reverse tumor-induced immune suppression, and inhibit cancer stemness. More importantly, the edited cells can maintain the modulated cellular function after succeeding subcultures.