Publications by authors named "Zhili Rong"

43 Publications

Get ready for the CRISPR/Cas system: A beginner's guide to the engineering and design of guide RNAs.

J Gene Med 2021 Jul 16:e3377. Epub 2021 Jul 16.

Cancer Research Institute, School of Basic Medical Sciences, State Key Laboratory of Organ Failure Research, National Clinical Research Center of Kidney Disease, Key Laboratory of Organ Failure Research (Ministry of Education), Southern Medical University, Guangzhou, China.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a state-of-the-art tool for versatile genome editing that has advanced basic research dramatically, with great potential for clinic applications. The system consists of two key molecules: a CRISPR-associated (Cas) effector nuclease and a single guide RNA. The simplicity of the system has enabled the development of a wide spectrum of derivative methods. Almost any laboratory can utilize these methods, although new users may initially be confused when faced with the potentially overwhelming abundance of choices. Cas nucleases and their engineering have been systematically reviewed previously. In the present review, we discuss single guide RNA engineering and design strategies that facilitate more efficient, more specific and safer gene editing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jgm.3377DOI Listing
July 2021

Tag-seq: a convenient and scalable method for genome-wide specificity assessment of CRISPR/Cas nucleases.

Commun Biol 2021 Jul 2;4(1):830. Epub 2021 Jul 2.

Dermatology Hospital, Southern Medical University, Guangzhou, China.

Genome-wide identification of DNA double-strand breaks (DSBs) induced by CRISPR-associated protein (Cas) systems is vital for profiling the off-target events of Cas nucleases. However, current methods for off-target discovery are tedious and costly, restricting their widespread applications. Here we present an easy alternative method for CRISPR off-target detection by tracing the integrated oligonucleotide Tag using next-generation-sequencing (CRISPR-Tag-seq, or Tag-seq). Tag-seq enables rapid and convenient profiling of nuclease-induced DSBs by incorporating the optimized double-stranded oligodeoxynucleotide sequence (termed Tag), adapters, and PCR primers. Moreover, we employ a one-step procedure for library preparation in Tag-seq, which can be applied in the routine workflow of a molecular biology laboratory. We further show that Tag-seq successfully determines the cleavage specificity of SpCas9 variants and Cas12a/Cpf1 in a large-scale manner, and discover the integration sites of exogenous genes introduced by the Sleeping Beauty transposon. Our results demonstrate that Tag-seq is an efficient and scalable approach to genome-wide identification of Cas-nuclease-induced off-targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42003-021-02351-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8253812PMC
July 2021

Single-cell RNA-seq reveals fibroblast heterogeneity and increased mesenchymal fibroblasts in human fibrotic skin diseases.

Nat Commun 2021 06 17;12(1):3709. Epub 2021 Jun 17.

Dermatology Hospital, Southern Medical University, Guangzhou, China.

Fibrotic skin disease represents a major global healthcare burden, characterized by fibroblast hyperproliferation and excessive accumulation of extracellular matrix. Fibroblasts are found to be heterogeneous in multiple fibrotic diseases, but fibroblast heterogeneity in fibrotic skin diseases is not well characterized. In this study, we explore fibroblast heterogeneity in keloid, a paradigm of fibrotic skin diseases, by using single-cell RNA-seq. Our results indicate that keloid fibroblasts can be divided into 4 subpopulations: secretory-papillary, secretory-reticular, mesenchymal and pro-inflammatory. Interestingly, the percentage of mesenchymal fibroblast subpopulation is significantly increased in keloid compared to normal scar. Functional studies indicate that mesenchymal fibroblasts are crucial for collagen overexpression in keloid. Increased mesenchymal fibroblast subpopulation is also found in another fibrotic skin disease, scleroderma, suggesting this is a broad mechanism for skin fibrosis. These findings will help us better understand skin fibrotic pathogenesis, and provide potential targets for fibrotic disease therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-24110-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211847PMC
June 2021

SOX9 inactivation affects the proliferation and differentiation of human lung organoids.

Stem Cell Res Ther 2021 06 10;12(1):343. Epub 2021 Jun 10.

State Key Laboratory of Organ Failure Research, National Clinical Research Center of Kidney Disease, Key Laboratory of Organ Failure Research (Ministry of Education), Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.

Background: The regulation of the transcription factor sex-determining region Y-box transcription factor 9 (SOX9) in lung development has been described in mouse, but the same principles apply to human lung development is unknown due to a lack of appropriate experimental approaches and models.

Methods: Here, we used gene editing technology to inactivate SOX9 in human embryonic stem cells that were then induced to differentiate into lung organoids to investigate the role of SOX9 in human lung epithelium development.

Results: Complete knockout of the transactivation domain of SOX9 by gene editing resulted in indels in both alleles of SOX9. SOX9 hESCs could be induced to differentiate into lung progenitor organoids. In vitro long-term expansion showed that SOX9 inactivation did not affect the differentiation of pulmonary epithelial cells, but promoted apoptosis and reduced proliferative capacity in the organoids. When lung progenitor organoids were transplanted under the kidney capsule of immunodeficient mice, expression of the club cell marker secretoglobin family 1A member 1 (SCGB1A1) was detected in SOX9 transplants but was absent in wild-type (WT) transplants. The maturation of goblet cells was also affected by SOX9 inactivation, as evidenced by the presence of mucin 5 AC (MUC5AC) in the cytoplasm of SOX9 grafts as compared to WT grafts in which most MUC5AC was secreted into the lumen. In vivo lung orthotopic transplantations showed that SOX9 inactivation had a limited effect on the differentiation of alveolar cells and lung regeneration in injured mice.

Conclusions: SOX9 modulates the proliferative capacity of lung epithelium but is not an indispensable transcription factor in the regulation of human lung epithelium development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13287-021-02422-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8194236PMC
June 2021

The role of S-nitrosylation of PFKM in regulation of glycolysis in ovarian cancer cells.

Cell Death Dis 2021 04 15;12(4):408. Epub 2021 Apr 15.

Cancer Research Institute, Guangdong Provincial Key Laboratory of Cancer Immunotherapy, Guangzhou Key Laboratory of Tumor Immunology Research, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.

One of the malignant transformation hallmarks is metabolism reprogramming, which plays a critical role in the biosynthetic needs of unchecked proliferation, abrogating cell death programs, and immunologic escape. However, the mechanism of the metabolic switch is not fully understood. Here, we found that the S-nitrosoproteomic profile of endogenous nitrogen oxide in ovarian cancer cells targeted multiple components in metabolism processes. Phosphofructokinase (PFKM), one of the most important regulatory enzymes of glycolysis, was S-nitrosylated by nitric oxide synthase NOS1 at Cys351. S-nitrosylation at Cys351 stabilized the tetramer of PFKM, leading to resist negative feedback of downstream metabolic intermediates. The PFKM-C351S mutation decreased the proliferation rate of cultured cancer cells, and reduced tumor growth and metastasis in the mouse xenograft model. These findings indicated that S-nitrosylation at Cys351 of PFKM by NOS1 contributes to the metabolic reprogramming of ovarian cancer cells, highlighting a critical role of endogenous nitrogen oxide on metabolism regulations in tumor progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-021-03681-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050300PMC
April 2021

MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo.

Nucleic Acids Res 2021 04;49(7):4171-4185

Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China.

CRISPR-mediated gene activation (CRISPRa) is a promising therapeutic gene editing strategy without inducing DNA double-strand breaks (DSBs). However, in vivo implementation of these CRISPRa systems remains a challenge. Here, we report a compact and robust miniCas9 activator (termed miniCAFE) for in vivo activation of endogenous target genes. The system relies on recruitment of an engineered minimal nuclease-null Cas9 from Campylobacter jejuni and potent transcriptional activators to a target locus by a single guide RNA. It enables robust gene activation in human cells even with a single DNA copy and is able to promote lifespan of Caenorhabditis elegans through activation of longevity-regulating genes. As proof-of-concept, delivered within an all-in-one adeno-associated virus (AAV), miniCAFE can activate Fgf21 expression in the liver and regulate energy metabolism in adult mice. Thus, miniCAFE holds great therapeutic potential against human diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkab174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8053112PMC
April 2021

OSMRβ mutants enhance basal keratinocyte differentiation via inactivation of the STAT5/KLF7 axis in PLCA patients.

Protein Cell 2021 Aug 27;12(8):653-661. Epub 2021 Jan 27.

Dermatology Hospital, Southern Medical University, Guangzhou, 510091, China.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13238-020-00818-3DOI Listing
August 2021

A Biomimetic Drug Delivery System by Integrating Grapefruit Extracellular Vesicles and Doxorubicin-Loaded Heparin-Based Nanoparticles for Glioma Therapy.

Nano Lett 2021 02 21;21(3):1484-1492. Epub 2021 Jan 21.

Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, P.R. China.

Existing nanoparticle-mediated drug delivery systems for glioma systemic chemotherapy remain a great challenge due to poor delivery efficiency resulting from the blood brain barrier/blood-(brain tumor) barrier (BBB/BBTB) and insufficient tumor penetration. Here, we demonstrate a distinct design by patching doxorubicin-loaded heparin-based nanoparticles (DNs) onto the surface of natural grapefruit extracellular vesicles (EVs), to fabricate biomimetic EV-DNs, achieving efficient drug delivery and thus significantly enhancing antiglioma efficacy. The patching strategy allows the unprecedented 4-fold drug loading capacity compared to traditional encapsulation for EVs. The biomimetic EV-DNs are enabled to bypass BBB/BBTB and penetrate into glioma tissues by receptor-mediated transcytosis and membrane fusion, greatly promoting cellular internalization and antiproliferation ability as well as extending circulation time. We demonstrate that a high-abundance accumulation of EV-DNs can be detected at glioma tissues, enabling the maximal brain tumor uptake of EV-DNs and great antiglioma efficacy in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.nanolett.0c04753DOI Listing
February 2021

Host metabolism dysregulation and cell tropism identification in human airway and alveolar organoids upon SARS-CoV-2 infection.

Protein Cell 2020 Dec 12. Epub 2020 Dec 12.

Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.

The coronavirus disease 2019 (COVID-19) pandemic is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is spread primary via respiratory droplets and infects the lungs. Currently widely used cell lines and animals are unable to accurately mimic human physiological conditions because of the abnormal status of cell lines (transformed or cancer cells) and species differences between animals and humans. Organoids are stem cell-derived self-organized three-dimensional culture in vitro and model the physiological conditions of natural organs. Here we showed that SARS-CoV-2 infected and extensively replicated in human embryonic stem cells (hESCs)-derived lung organoids, including airway and alveolar organoids which covered the complete infection and spread route for SARS-CoV-2 within lungs. The infected cells were ciliated, club, and alveolar type 2 (AT2) cells, which were sequentially located from the proximal to the distal airway and terminal alveoli, respectively. Additionally, RNA-seq revealed early cell response to virus infection including an unexpected downregulation of the metabolic processes, especially lipid metabolism, in addition to the well-known upregulation of immune response. Further, Remdesivir and a human neutralizing antibody potently inhibited SARS-CoV-2 replication in lung organoids. Therefore, human lung organoids can serve as a pathophysiological model to investigate the underlying mechanism of SARS-CoV-2 infection and to discover and test therapeutic drugs for COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13238-020-00811-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732737PMC
December 2020

Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain.

Nucleic Acids Res 2020 10;48(18):10590-10601

Department of Nephrology, Shenzhen Hospital, Southern Medical University, Shenzhen 518110, China.

The CRISPR/Cas system is widely used for genome editing. However, robust and targeted insertion of a DNA segment remains a challenge. Here, we present a fusion nuclease (Cas9-N57) to enhance site-specific DNA integration via a fused DNA binding domain of Sleeping Beauty transposase to tether the DNA segment to the Cas9/sgRNA complex. The insertion was unidirectional and specific, and DNA fragments up to 12 kb in length were successfully integrated. As a test of the system, Cas9-N57 mediated the insertion of a CD19-specific chimeric antigen receptor (CD19-CAR) cassette into the AAVS1 locus in human T cells, and induced intrahepatic cholangiocarcinoma in mice by simultaneously mediating the insertion of oncogenic KrasG12D into the Rosa26 locus and disrupting Trp53 and Pten. Moreover, the nuclease-N57 fusion proteins based on AsCpf1 (AsCas12a) and CjCas9 exhibited similar activity. These findings demonstrate that CRISPR-associated nuclease-N57 protein fusion is a powerful tool for targeted DNA insertion and holds great potential for gene therapy applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544211PMC
October 2020

Inhibition of the Extracellular Signal-Regulated Kinase/Ribosomal S6 Kinase Cascade Limits Chlamydia trachomatis Infection.

J Invest Dermatol 2021 Apr 9;141(4):852-862.e6. Epub 2020 Sep 9.

Department of Clinical Laboratory, Dermatology Hospital, Southern Medical University, Guangzhou, China. Electronic address:

Chlamydiatrachomatis is the cause of the most common bacterial sexually transmitted infection worldwide. Azithromycin is effective in treating chlamydial infection; however, resistance to this antibiotic is increasing, and it is important that new therapeutic strategies are developed. In this study, we demonstrated that inhibitors targeting each kinase in the extracellular signal-regulated kinase/ribosomal S6 kinase cascade significantly decreased the size and number of inclusions as well as the number of infectious progeny. The suppressive effects of the inhibitors were observed across the Chlamydia serotypes D, E, F, and L1 and across HeLa, McCoy, and Vero host cells. When combined with azithromycin, all the inhibitors exerted a synergistic suppressive effect on chlamydial infection. Knockdown experiments using small interfering RNA demonstrated that extracellular signal-regulated kinase 1/2 and ribosomal S6 kinase 1 were crucial for chlamydial infection. Moreover, BVD-523, a first-in-class extracellular signal-regulated kinase 1/2 inhibitor currently undergoing a phase II clinical trial, suppressed chlamydial infection both in cell culture and in a mouse model. These observations demonstrated not only that the extracellular signal-regulated kinase/ribosomal S6 kinase pathway plays a critical role in chlamydial infection but also that these kinases have potential as targets for host-directed therapy against C. trachomatis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jid.2020.07.033DOI Listing
April 2021

Robust Hi-C Maps of Enhancer-Promoter Interactions Reveal the Function of Non-coding Genome in Neural Development and Diseases.

Mol Cell 2020 08 26;79(3):521-534.e15. Epub 2020 Jun 26.

Department of Genetics and Genome Sciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA; Department of Computer and Data Sciences, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA. Electronic address:

Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415676PMC
August 2020

Cell-cell contact-induced gene editing/activation in mammalian cells using a synNotch-CRISPR/Cas9 system.

Protein Cell 2020 04;11(4):299-303

School of Basic Medical Sciences, Cancer Research Institute, Southern Medical University, Guangzhou, 510515, China.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13238-020-00690-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093359PMC
April 2020

MicroRNA-101-3p Downregulates TLR2 Expression, Leading to Reduction in Cytokine Production by Treponema pallidum-Stimulated Macrophages.

J Invest Dermatol 2020 08 11;140(8):1566-1575.e1. Epub 2020 Jan 11.

Research Center, Dermatology Hospital, Southern Medical University, Guangzhou, China. Electronic address:

Treponema pallidum (Tp) infection-induced immune responses can cause tissue damage. However, the underlying mechanism by which Tp infection induces immune response is unclear. Recent studies suggest a regulatory role of microRNAs in host immunity. We assessed whether microRNAs also have a regulatory role in immune response to Tp infection in vitro. Our results showed that microRNA-101-3p (miR-101-3p) levels were significantly higher in peripheral blood mononuclear cells of patients with primary syphilis and those in the serofast state, whereas toll-like receptor (TLR) 2 levels were higher in patients with syphilis than in healthy controls. In vitro, stimulation of THP-1 cells with Tp increased miR-101-3p expression. Moreover, miR-101-3p reduced expression levels of TLR2 mRNA and protein in THP-1 cells via binding to the 3' untranslated region of TLR2. Likewise, miR-101-3p inhibited production of inflammatory cytokines, including IL-1β, IL-6, tumor necrosis factor-α, and IL-12, in Tp-stimulated macrophages. IL-1β and IL-6 mRNA expression levels were reduced by transfection of macrophages with a TLR2-specific small interfering RNA. Conversely, overexpression of TLR2 upregulated cytokine expression. Patients with secondary syphilis exhibited the highest levels of plasma IL-6, which were negatively correlated with miR-101-3p. In conclusion, Tp infection upregulates miR-101-3p expression, which in turn inhibits the TLR2 signaling pathway, leading to reduced cytokine production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jid.2019.12.012DOI Listing
August 2020

NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2.

J Exp Clin Cancer Res 2019 Dec 5;38(1):483. Epub 2019 Dec 5.

Cancer Research Institute, Guangdong Provincial Key Laboratory of Cancer Immunotherapy, Guangzhou key laboratory of tumor immunology research, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.

Background: The dysfunction of type I interferon (IFN) signaling is an important mechanism of immune escape and metastasis in tumors. Increased NOS1 expression has been detected in melanoma, which correlated with dysfunctional IFN signaling and poor response to immunotherapy, but the specific mechanism has not been determined. In this study, we investigated the regulation of NOS1 on the interferon response and clarified the relevant molecular mechanisms.

Methods: After stable transfection of A375 cells with NOS1 expression plasmids, the transcription and expression of IFNα-stimulated genes (ISGs) were assessed using pISRE luciferase reporter gene analysis, RT-PCR, and western blotting, respectively. The effect of NOS1 on lung metastasis was assessed in melanoma mouse models. A biotin-switch assay was performed to detect the S-nitrosylation of HDAC2 by NOS1. ChIP-qPCR was conducted to measure the binding of HDAC2, H4K16ac, H4K5ac, H3ac, and RNA polymerase II in the promoters of ISGs after IFNα stimulation. This effect was further evaluated by altering the expression level of HDAC2 or by transfecting the HDAC2-C262A/C274A site mutant plasmids into cells. The coimmunoprecipitation assay was performed to detect the interaction of HDAC2 with STAT1 and STAT2. Loss-of-function and gain-of-function approaches were used to examine the effect of HDAC2-C262A/C274A on lung metastasis. Tumor infiltrating lymphocytes were analyzed by flow cytometry.

Results: HDAC2 is recruited to the promoter of ISGs and deacetylates H4K16 for the optimal expression of ISGs in response to IFNα treatment. Overexpression of NOS1 in melanoma cells decreases IFNα-responsiveness and induces the S-nitrosylation of HDAC2-C262/C274. This modification decreases the binding of HDAC2 with STAT1, thereby reducing the recruitment of HDAC2 to the ISG promoter and the deacetylation of H4K16. Moreover, expression of a mutant form of HDAC2, which cannot be nitrosylated, reverses the inhibition of ISG expression by NOS1 in vitro and decreases NOS1-induced lung metastasis and inhibition of tumor infiltrating lymphocytes in a melanoma mouse model.

Conclusions: This study provides evidence that NOS1 induces dysfunctional IFN signaling to promote lung metastasis in melanoma, highlighting NOS1-induced S-nitrosylation of HDAC2 in the regulation of IFN signaling via histone modification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13046-019-1448-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896289PMC
December 2019

Facile in situ synthesis of ultrasmall near-infrared-emitting gold glyconanoparticles with enhanced cellular uptake and tumor targeting.

Nanoscale 2019 Sep 28;11(35):16336-16341. Epub 2019 Aug 28.

Key Laboratory of Functional Molecular Engineering of Guangdong Province, School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, China.

The simultaneous possession of high tumor-targeting efficiency, long blood circulation, and low normal-tissue retention is critical for future clinically translatable nanomedicines. Herein, we reported a facile in situ glycoconjugation strategy for the synthesis of near-infrared (NIR)-emitting gold glyconanoparticles (AuGNPs, ∼2.4 nm) using 1-thio-β-d-glucose as both the surface ligand and the reducing agent in the presence of a gold precursor. The ultrasmall AuGNPs showed similar low healthy organ retention to that of the renal-clearable ultrasmall nonglyconanoparticles, but ∼10 and 2.5 times higher in vitro and in vivo tumor-targeting efficiencies, respectively, were observed. This facile glycoconjugation strategy of ultrasmall AuGNPs was found to show activity towards glucose transporters in the cancer cells and prolonged blood circulation with both renal and hepatobiliary clearance pathways, which synergistically enhanced the tumor targeting of the ultrasmall AuGNPs. This discovery provides a smart strategy for the improvement in tumor targeting by ultrasmall NPs and further strengthens our understanding of glycoconjugation in designing future clinically translatable nanomedicines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c9nr03821cDOI Listing
September 2019

Engineering macrophages to phagocytose cancer cells by blocking the CD47/SIRPɑ axis.

Cancer Med 2019 08 11;8(9):4245-4253. Epub 2019 Jun 11.

Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

The use of immunotherapy has achieved great advances in the treatment of cancer. Macrophages play a pivotal role in the immune defense system, serving both as phagocytes (removal of pathogens and cancer cells) and as antigen-presenting cells (activation of T cells). However, research regarding tumor immunotherapy is mainly focused on the adaptive immune system. The usefulness of innate immune cells (eg, macrophages) in the treatment of cancer has not been extensively investigated. Recent advances in synthetic biology and the increasing understanding of the cluster of differentiation 47/signal regulatory protein alpha (CD47/SIRPɑ) axis may provide new opportunities for the clinical application of engineered macrophages. The CD47/SIRPɑ axis is a major known pathway, repressing phagocytosis and activation of macrophages. In this article, we summarize the currently available evidence regarding the CD47/SIRPɑ axis, and immunotherapies based on blockage. In addition, we propose cell therapy strategies based on macrophage engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cam4.2332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675709PMC
August 2019

TRAF4 Promotes Fibroblast Proliferation in Keloids by Destabilizing p53 via Interacting with the Deubiquitinase USP10.

J Invest Dermatol 2019 09 30;139(9):1925-1935.e5. Epub 2019 Mar 30.

Dermatology Hospital, Southern Medical University, Guangzhou, China; Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China. Electronic address:

Keloids represent one extreme of aberrant dermal wound healing. One of the important characteristics of keloids is uncontrolled fibroblasts proliferation. However, the mechanism of excessive proliferation of fibroblasts in keloids remains elusive. In this study, we demonstrated that TRAF4 was highly expressed in keloid fibroblasts and promoted fibroproliferation. We investigated the underlying molecular mechanism and found that TRAF4 suppressed the p53 pathway independent of its E3 ubiquitin ligase activity. Specifically, TRAF4 interacted with the deubiquitinase USP10 and blocked the access of p53 to USP10, resulting in p53 destabilization. Knockdown of p53 rescued cell proliferation in TRAF4-knockdown keloid fibroblasts, suggesting that the regulation of proliferation by TRAF4 in keloids relied on p53. Furthermore, in keloid patient samples, TRAF4 expression was inversely correlated with p53-p21 signaling activity. These findings help to elucidate the mechanisms underlying keloid development and indicate that blocking TRAF4 could represent a potential strategy for keloid therapy in the future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jid.2019.03.1136DOI Listing
September 2019

Sex differences of Chinese patients with primary localized cutaneous amyloidosis.

J Dermatol 2019 07 11;46(7):e242-e243. Epub 2019 Feb 11.

Dermatology Hospital, Southern Medical University, Guangzhou, China.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1346-8138.14800DOI Listing
July 2019

Correction to: Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems.

Protein Cell 2019 Oct;10(10):776-777

Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.

In the original publication the Supplementary Material and Fig. 2 are incorrect. The correct version is provided in this correction article. The text HBG2 appearing in the article should be read as HBG1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13238-018-0585-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776487PMC
October 2019

Long-Term Engraftment Promotes Differentiation of Alveolar Epithelial Cells from Human Embryonic Stem Cell Derived Lung Organoids.

Stem Cells Dev 2018 10 21;27(19):1339-1349. Epub 2018 Aug 21.

Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University , Guangzhou, China .

Human embryonic stem cell (hESC) derived 3D human lung organoids (HLOs) provide a promising model to study human lung development and disease. HLOs containing proximal or/and immature distal airway epithelial cells have been successfully generated in vitro, such as early staged alveolar type 2 (AT2) cells (SPC/SOX9) and immature alveolar type 1 (AT1) cells (HOPX/SOX9). When HLOs were transplanted into immunocompromised mice for further differentiation in vivo, only few distal epithelial cells could be observed. In this study, we transplanted different stages of HLOs into immunocompromised mice to assess whether HLOs could expand and mature in vivo. We found that short-term transplanted HLOs contained lung progenitor cells (NKX2.1, SOX9, and P63), but not SPC AT2 cells or AQP5 AT1 cells. Meanwhile, long-term engrafted HLOs could differentiate into lung distal bipotent progenitor cells (PDPN/SPC/SOX9), AT2 cells (SPC, SPB), and immature AT1 cells (PDPN, AQP5). However, HLOs at late in vitro stage turned into mature AT1-like cells (AQP5/SPB/SOX9) in vivo. Immunofluorescence staining and transmission electron microscopy (TEM) results revealed that transplanted HLOs contained mesenchymal cells (collagen I), vasculature (ACTA2), neuroendocrine-like cells (PGP9.5), and nerve fiber structures (myelin sheath structure). Together, these data reveal that hESC-derived HLOs would be useful for human lung development modeling, and transplanted HLOs could mimic lung organ-like structures in vivo by possessing vascular network and neuronal network.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/scd.2018.0042DOI Listing
October 2018

iKA-CRISPR hESCs for inducible and multiplex orthogonal gene knockout and activation.

FEBS Lett 2018 07 15;592(13):2238-2247. Epub 2018 Jun 15.

Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China.

Human embryonic stem cells (hESCs) have a wide range of applications in early human embryonic development mimics, disease modeling, and cell therapy. To fulfill these applications, we established hESCs for inducible and multiplex orthogonal gene knockout and activation, which we named iKA-CRISPR hESCs. In cells, when complexed with a short guide RNA containing a 14-bp target sequence (14-bp gRNA) or a long 20-bp gRNA, the doxycycline-induced Cas9-p300 protein could activate gene transcription or cleave genomic DNA, respectively. We also demonstrate using iKA-CRISPR hESCs that knockout of OCT4 promoted differentiation, and developmentally relevant microRNAs and transcription factors could be efficiently activated. Thus, iKA-CRISPR hESCs provide a convenient platform to control gene expression networks and, therefore, facilitate the applications of hESCs in basic and translational biomedical research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.13127DOI Listing
July 2018

Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems.

Protein Cell 2018 04;9(4):380-383

Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13238-017-0491-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876187PMC
April 2018

Live cell imaging of genomic loci using dCas9-SunTag system and a bright fluorescent protein.

Protein Cell 2017 11;8(11):853-855

Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13238-017-0460-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5676592PMC
November 2017

Humanized mouse model for assessing the human immune response to xenogeneic and allogeneic decellularized biomaterials.

Biomaterials 2017 06 11;129:98-110. Epub 2017 Mar 11.

Department of Bioengineering, Sanford Consortium of Regenerative Medicine, University of California San Diego, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA. Electronic address:

Current assessment of biomaterial biocompatibility is typically implemented in wild type rodent models. Unfortunately, different characteristics of the immune systems in rodents versus humans limit the capability of these models to mimic the human immune response to naturally derived biomaterials. Here we investigated the utility of humanized mice as an improved model for testing naturally derived biomaterials. Two injectable hydrogels derived from decellularized porcine or human cadaveric myocardium were compared. Three days and one week after subcutaneous injection, the hydrogels were analyzed for early and mid-phase immune responses, respectively. Immune cells in the humanized mouse model, particularly T-helper cells, responded distinctly between the xenogeneic and allogeneic biomaterials. The allogeneic extracellular matrix derived hydrogels elicited significantly reduced total, human specific, and CD4 T-helper cell infiltration in humanized mice compared to xenogeneic extracellular matrix hydrogels, which was not recapitulated in wild type mice. T-helper cells, in response to the allogeneic hydrogel material, were also less polarized towards a pro-remodeling Th2 phenotype compared to xenogeneic extracellular matrix hydrogels in humanized mice. In both models, both biomaterials induced the infiltration of macrophages polarized towards a M2 phenotype and T-helper cells polarized towards a Th2 phenotype. In conclusion, these studies showed the importance of testing naturally derived biomaterials in immune competent animals and the potential of utilizing this humanized mouse model for further studying human immune cell responses to biomaterials in an in vivo environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2017.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5434867PMC
June 2017

A Safety Checkpoint to Eliminate Cancer Risk of the Immune Evasive Cells Derived from Human Embryonic Stem Cells.

Stem Cells 2017 05 23;35(5):1154-1161. Epub 2017 Feb 23.

Cancer Research Institute, Southern Medical University, Guangzhou, Guangdong, China.

Human embryonic stem cells (hESCs) hold great promise in the regenerative therapy of many currently untreatable human diseases. One of the key bottlenecks is the immune rejection of hESC-derived allografts by the recipient. To overcome this challenge, we have established new approaches to induce immune protection of hESC-derived allografts through the coexpression of immune suppressive molecules CTLA4-Ig and PD-L1. However, this in turn raises a safety concern of cancer risk because these hESC-derived cells can evade immune surveillance. To address this safety concern, we developed a safety checkpoint so that the immune evasive hESC-derived cells in the graft can be effectively eliminated if any cellular transformation is detected. In this context, we knock-in the suicidal gene herpes simplex virus thymidine kinase (HSVTK) into the constitutive HPRT locus of CP hESCs (knock-in hESCs expressing CTLA4-Ig and PD-L1), denoted CPTK hESCs. Employing humanized mice (Hu-mice) reconstituted with human immune system, we demonstrated that the CPTK hESC-derived cells are protected from immune rejection. In addition, CPTK hESC-derived cells can be efficiently eliminated in vitro and in vivo with FDA approved TK-targeting drug ganciclovir. Therefore, this new safety checkpoint improves the feasibility to use the immune evasive hESC-derived cells for regenerative medicine. Stem Cells 2017;35:1154-1161.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.2568DOI Listing
May 2017

Humanized Mice Reveal Differential Immunogenicity of Cells Derived from Autologous Induced Pluripotent Stem Cells.

Cell Stem Cell 2015 Sep 20;17(3):353-9. Epub 2015 Aug 20.

Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 920932, USA; Cancer Research Institute, Southern Medical University, Guangzhou, Guangdong 510515, China. Electronic address:

The breakthrough of induced pluripotent stem cell (iPSC) technology has raised the possibility that patient-specific iPSCs may become a renewable source of autologous cells for cell therapy without the concern of immune rejection. However, the immunogenicity of autologous human iPSC (hiPSC)-derived cells is not well understood. Using a humanized mouse model (denoted Hu-mice) reconstituted with a functional human immune system, we demonstrate that most teratomas formed by autologous integration-free hiPSCs exhibit local infiltration of antigen-specific T cells and associated tissue necrosis, indicating immune rejection of certain hiPSC-derived cells. In this context, autologous hiPSC-derived smooth muscle cells (SMCs) appear to be highly immunogenic, while autologous hiPSC-derived retinal pigment epithelial (RPE) cells are immune tolerated even in non-ocular locations. This differential immunogenicity is due in part to abnormal expression of immunogenic antigens in hiPSC-derived SMCs, but not in hiPSC-derived RPEs. These findings support the feasibility of developing hiPSC-derived RPEs for treating macular degeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2015.07.021DOI Listing
September 2015

Gene Targeting Through Homologous Recombination in Monkey Embryonic Stem Cells Using CRISPR/Cas9 System.

Stem Cells Dev 2015 May 25;24(10):1147-9. Epub 2015 Feb 25.

1 Shenzhen Children's Hospital , Shenzhen, Guangdong, China .

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/scd.2014.0507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425224PMC
May 2015

Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template.

Protein Cell 2014 Apr;5(4):258-60

Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13238-014-0032-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3978163PMC
April 2014
-->