Publications by authors named "Pentao Liu"

112 Publications

Understanding totipotency: A role for alternative splicing.

Dev Cell 2021 Jun;56(12):1697-1699

School of Biomedical Science, Stem Cell and Regenerative Consortium, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong; Center for Translational Stem Cell Biology, Hong Kong Science and Technology Park, Hong Kong. Electronic address:

Totipotency refers to single cells' developmental capacity to form an entire organism. Understanding how totipotent stem cells form has implications for chimera generation. In a recent Cell study, Shen et al. (2021) report that inhibition of spliceosomes resets conventional pluripotent stem cells to a cellular state with totipotency features.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2021.06.002DOI Listing
June 2021

Establishment of bovine expanded potential stem cells.

Proc Natl Acad Sci U S A 2021 Apr;118(15)

The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, Inner Mongolia University, 010070 Hohhot, China;

Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2018505118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8053967PMC
April 2021

Myeloid-derived suppressor cells promote lung cancer metastasis by CCL11 to activate ERK and AKT signaling and induce epithelial-mesenchymal transition in tumor cells.

Oncogene 2021 02 15;40(8):1476-1489. Epub 2021 Jan 15.

State Key Laboratory of Respiratory Disease, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

Myeloid-derived suppressor cells (MDSCs) suppress antitumor immune activities and facilitate cancer progression. Although the concept of immunosuppressive MDSCs is well established, the mechanism that MDSCs regulate non-small cell lung cancer (NSCLC) progression through the paracrine signals is still lacking. Here, we reported that the infiltration of MDSCs within NSCLC tissues was associated with the progression of cancer status, and was positively correlated with the Patient-derived xenograft model establishment, and poor patient prognosis. Intratumoral MDSCs directly promoted NSCLC metastasis and highly expressed chemokines that promote NSCLC cells invasion, including CCL11. CCL11 was capable of activating the AKT and ERK signaling pathways to promote NSCLC metastasis through the epithelial-mesenchymal transition (EMT) process. Moreover, high expression of CCL11 was associated with a poor prognosis in lung cancer as well as other types of cancer. Our findings underscore that MDSCs produce CCL11 to promote NSCLC metastasis via activation of ERK and AKT signaling and induction of EMT, suggesting that the MDSCs-CCL11-ERK/AKT-EMT axis contains potential targets for NSCLC metastasis treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41388-020-01605-4DOI Listing
February 2021

is a critical regulator of adult long-term hematopoietic stem cell quiescence.

Proc Natl Acad Sci U S A 2020 12 2;117(50):31945-31953. Epub 2020 Dec 2.

Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814;

Regulation of quiescence is critical for the maintenance of adult hematopoietic stem cells (HSCs). Disruption of transcription factor gene during mouse embryonic development has been shown to cause a severe loss of fetal liver HSCs; however, the underlying mechanisms and the function of in adult HSCs remain unclear. To investigate the role of in adult HSCs, we generated a novel conditional knockout mouse model and deleted in adult mouse hematopoietic system using the IFN-inducible Our results show that deletion in the adult mouse hematopoietic system has a less severe effect on HSCs, causing a gradual decline of adult HSC numbers and a concomitant increase in the multipotent progenitor (MPP) compartment. deletion in the hematopoietic system following transplantation produced the same phenotype, indicating that the defect is intrinsic to adult HSCs. This HSC loss was also exacerbated by stress induced by 5-fluorouracil injections. Annexin V staining showed no difference in apoptosis between wild-type and knockout adult HSCs. In contrast, Bromodeoxyuridine analysis revealed that loss of significantly increased cycling of long-term HSCs (LT-HSCs) with the majority of the cells found in the S to G2/M phase. Consistently, RNA sequencing analysis of mouse LT-HSCs with and without deletion showed that loss induced a significant decrease in the expression of several known cell cycle regulators of HSCs, among which and were identified as direct targets of Our results suggest that preserves the function of adult LT-HSCs by promoting their quiescence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2017626117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749346PMC
December 2020

Reprogramming Porcine Fibroblast to EPSCs.

Methods Mol Biol 2021 ;2239:199-211

Stem Cell and Regenerative Medicine Consortium, Li Ka Shing Faculty of Medicine, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong.

The development of porcine expanded potential stem cells (pEPSCs) provides an invaluable tool for investigation of porcine stem cell pluripotency and opens a venue for research in biotechnology, agriculture, and regenerative medicine. Since the derivation of pEPSC from porcine pre-implantation embryos has been demanding in resource supply and technical challenges, it is more feasible and convenient for most laboratories to derive this new type of porcine stem cells by reprogramming somatic cells. In this chapter, we describe the detailed procedures for reprogramming porcine fetal fibroblast cells to EPSC with the eight reprogramming factors cloned on the piggyBac vectors followed by a selection for pluripotent cells independent of transgene expression using the EPSC media. This technique allows the generation of pEPSCs for stem cell research, genome editing, biotechnology, and agriculture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-0716-1084-8_13DOI Listing
April 2021

IL-6 trans-signaling promotes the expansion and anti-tumor activity of CAR T cells.

Leukemia 2021 05 9;35(5):1380-1391. Epub 2020 Nov 9.

Key Laboratory of Regenerative Biology South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

Chimeric antigen receptor (CAR) T cell therapies lead to high clinical response rates in B cell malignancies, and are under investigation for treatment of solid tumors. While high systemic interleukin- (IL-) 6 levels are associated with clinical cytokine release syndrome (CRS), the role of IL-6 trans-signaling within CAR T-cells has not been reported. We generated CAR T cells that constitutively express hyper IL-6 (HIL-6), a designer cytokine that activates the trans-signaling pathway. HIL-6-expressing CAR T-cells exhibited enhanced proliferation and antitumor efficacy in vitro and in xenograft models. However, HIL-6 CAR T cells caused severe graft-versus-host disease (GVHD). Transcriptomic profiling revealed that HIL-6 stimulation of CAR T cells upregulated genes associated with T cell migration, early memory differentiation, and IL-6/GP130/STAT3 signaling. Since IL-6 trans-signaling acts via surface GP130, we generated CAR T cells expressing a constitutively-active form of GP130 and found these retained improved antitumor activity without signs of GVHD in preclinical models of B-cell leukemia and solid tumors. Taken together, these results show that IL-6 trans-signaling can enhance expansion and antitumor activity of CAR T cells via the GP130/STAT3 pathway, and suggest that expression of GP130 within CAR T cells could lead to improved antitumor efficacy without systemic IL-6 trans-signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41375-020-01085-1DOI Listing
May 2021

Expanded potential: the key to synthetic embryo?

Curr Opin Genet Dev 2020 10 9;64:72-77. Epub 2020 Jul 9.

School of Biomedical Sciences, Stem Cell and Regenerative Medicine Consortium, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 5 Sassoon Road, Pokfulam, Hong Kong. Electronic address:

How does an embryo acquire totipotency and develop into an adult is a fundamental scientific question. Stem cells derived from pre-implantation embryos or reprogrammed from somatic cells with totipotency features have been established. They have enriched molecular features, including transcription, epigenetic modification, chromatin structure and metabolism, similar to early embryos from 2 cell (2C) to morula. Functionally, they display a broader developmental potential to differentiate into cell types in the embryonic and extraembryonic tissues. The expanded developmental potential was further demonstrated by inducing these stem cells into embryo-like structures alone or aggregating with other embryo-derived stem cells. The synthetic embryo-like structures not only facilitate the dissection of key events in early embryonic development, but also serve as a model for investigating pregnancy related complications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.gde.2020.06.008DOI Listing
October 2020

BCL11A Promotes the Progression of Laryngeal Squamous Cell Carcinoma.

Front Oncol 2020 20;10:375. Epub 2020 Mar 20.

Shanghai Institute of Immunology, Shanghai Jiaotong University School of Medicine, Shanghai, China.

We report functional and clinical data uncovering the significance of B-cell lymphoma/leukemia 11A (BCL11A) in laryngeal squamous cell carcinoma (LSCC). We examined BCL11A expression in a cohort of LSCC patients and evaluated the association between BCL11A expression and clinicopathological features. We investigated the consequences of overexpressing BCL11A in the LSCC cell line on proliferation, migration, invasion, cell cycle, chemosensitivity, and growth . We explored the relationship between BCL11A and MDM2 in LSCC and tumorigenesis pathways by using the Human Cancer PathwayFinder Array. High levels of BCL11A were found in LSCC tissues and were more frequently associated with advanced lymphatic metastasis stages with poor prognoses. BCL11A overexpression enhanced LSCC proliferation and . A positive correlation between MDM2 and BCL11A expression was identified. These data uncover important functions of BCL11A in LSCC and identify BCL11A as a prognostic biomarker and potential therapeutic target in LSCC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fonc.2020.00375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098986PMC
March 2020

A Virus-Infected, Reprogrammed Somatic Cell-Derived Tumor Cell (VIReST) Vaccination Regime Can Prevent Initiation and Progression of Pancreatic Cancer.

Clin Cancer Res 2020 01 25;26(2):465-476. Epub 2019 Nov 25.

National Center for International Research in Cell and Gene Therapy, Sino-British Research Centre for Molecular Oncology, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, China.

Purpose: Pancreatic cancer remains one of the most lethal cancers, and late detection renders most tumors refractory to conventional therapies. Development of cancer prophylaxis may be the most realistic option for improving mortality associated with this disease. Here, we develop a novel individualized prophylactic and therapeutic vaccination regimen using induced pluripotent stem cells (iPSC), gene editing, and tumor-targeted replicating oncolytic viruses.

Experimental Design: We created a Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime. iPSCs from healthy cells were induced to pancreatic tumor cells using gene editing via stable provision of and tumor driver mutations. These cells were preinfected with oncolytic Adenovirus (AdV) as prime or Vaccinia virus (VV) as boost, to improve vaccine immunogenicity, prior to delivery of vaccines in a sequential regime to young KPC transgenic mice, genetically programmed to develop pancreatic cancer, to prevent and delay disease development.

Results: Tumor cells preinfected with oncolytic AdV as prime or VV as boost were the best regime to induce tumor-specific immunity. iPSC-derived tumor cells were highly related in antigen repertoire to pancreatic cancer cells of KPC transgenic mice, suggesting that an individual's stem cells can provide an antigenically matched whole tumor cell vaccine. The VIReST vaccination primed tumor-specific T-cell responses, resulting in delayed disease emergence and progression and significantly prolonged survival of KPC transgenic mice. Importantly, this regime was well-tolerated and nontoxic.

Conclusions: These results provide both proof of concept and a robust technology platform for the development of personalized prophylactic cancer vaccines to prevent pancreatic malignancies in at-risk individuals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-19-1395DOI Listing
January 2020

A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan.

EMBO Rep 2019 11 30;20(11):e47967. Epub 2019 Sep 30.

Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK.

Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embr.201947967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832011PMC
November 2019

Expanded potential stem cell media as a tool to study human developmental hematopoiesis in vitro.

Exp Hematol 2019 08 18;76:1-12.e5. Epub 2019 Jul 18.

Department of Haematology, Wellcome & MRC Cambridge Stem Cell Institute, Cambridge, UK. Electronic address:

Pluripotent stem cell (PSC) differentiation in vitro represents a powerful and tractable model to study mammalian development and an unlimited source of cells for regenerative medicine. Within hematology, in vitro PSC hematopoiesis affords novel insights into blood formation and represents an exciting potential approach to generate hematopoietic and immune cell types for transplantation and transfusion. Most studies to date have focused on in vitro hematopoiesis from mouse PSCs and human PSCs. However, differences in mouse and human PSC culture protocols have complicated the translation of discoveries between these systems. We recently developed a novel chemical media formulation, expanded potential stem cell medium (EPSCM), that maintains mouse PSCs in a unique cellular state and extraembryonic differentiation capacity. Herein, we describe how EPSCM can be directly used to stably maintain human PSCs. We further demonstrate that human PSCs maintained in EPSCM can spontaneously form embryoid bodies and undergo in vitro hematopoiesis using a simple differentiation protocol, similar to mouse PSC differentiation. EPSCM-maintained human PSCs generated at least two hematopoietic cell populations, which displayed distinct transcriptional profiles by RNA-sequencing (RNA-seq) analysis. EPSCM also supports gene targeting using homologous recombination, affording generation of an SPI1 (PU.1) reporter PSC line to study and track in vitro hematopoiesis. EPSCM therefore provides a useful tool not only to study pluripotency but also hematopoietic cell specification and developmental-lineage commitment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.exphem.2019.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859476PMC
August 2019

Establishment of porcine and human expanded potential stem cells.

Nat Cell Biol 2019 06 3;21(6):687-699. Epub 2019 Jun 3.

Key Laboratory of Regenerative Biology of Chinese Academy of Sciences, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41556-019-0333-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035105PMC
June 2019

PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice.

Nat Commun 2019 03 29;10(1):1415. Epub 2019 Mar 29.

The Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-09180-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440946PMC
March 2019

An Id2-Reporter Mouse Redefines Innate Lymphoid Cell Precursor Potentials.

Immunity 2019 04 26;50(4):1054-1068.e3. Epub 2019 Mar 26.

Innate Immunity Unit, Institut Pasteur, Paris 75724, France; Inserm U1223, Institut Pasteur, Paris 75724, France. Electronic address:

Innate lymphoid cell (ILC) development proposes that ILC precursors (ILCPs) segregate along natural killer (NK) cell versus helper cell (ILC1, ILC2, ILC3) pathways, the latter depending on expression of Id2, Zbtb16, and Gata3. We have developed an Id2-reporter strain expressing red fluorescent protein (RFP) in the context of normal Id2 expression to re-examine ILCP phenotype and function. We show that bone-marrow ILCPs were heterogeneous and harbored extensive NK-cell potential in vivo and in vitro. By multiplexing Id2 with Zbtb16 and Bcl11b strains, we made a single-cell dissection of the ILCP compartment. In contrast with the current model, we have demonstrated that Id2Zbtb16 ILCPs included multi-potent ILCPs that retained NK-cell potential. Late-stage ILC2P and ILC3P compartments could be defined by differential Zbtb16 and Bcl11b expression. We suggest a revised model for ILC differentiation that redefines the cell-fate potential of helper-ILC-restricted Zbtb16 ILCPs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2019.02.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477155PMC
April 2019

Genome-Scale CRISPRa Screen Identifies Novel Factors for Cellular Reprogramming.

Stem Cell Reports 2019 04 21;12(4):757-771. Epub 2019 Mar 21.

Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK; UK Dementia Research Institute, Department of Clinical Neuroscience, Cambridge Biomedical Campus, University of Cambridge, Cambridge CB2 0AH, UK. Electronic address:

Primed epiblast stem cells (EpiSCs) can be reverted to a pluripotent embryonic stem cell (ESC)-like state by expression of single reprogramming factor. We used CRISPR activation to perform a genome-scale, reprogramming screen in EpiSCs and identified 142 candidate genes. Our screen validated a total of 50 genes, previously not known to contribute to reprogramming, of which we chose Sall1 for further investigation. We show that Sall1 augments reprogramming of mouse EpiSCs and embryonic fibroblasts and that these induced pluripotent stem cells are indeed fully pluripotent including formation of chimeric mice. We also demonstrate that Sall1 synergizes with Nanog in reprogramming and that overexpression in ESCs delays their conversion back to EpiSCs. Lastly, using RNA sequencing, we identify and validate Klf5 and Fam189a2 as new downstream targets of Sall1 and Nanog. In summary, our work demonstrates the power of using CRISPR technology in understanding molecular mechanisms that mediate complex cellular processes such as reprogramming.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2019.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450436PMC
April 2019

Mesothelin is a target of chimeric antigen receptor T cells for treating gastric cancer.

J Hematol Oncol 2019 02 18;12(1):18. Epub 2019 Feb 18.

Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

Background: Gastric cancer (GC) is a common cancer in Asia and currently lacks a targeted therapy approach. Mesothelin (MSLN) has been reported to be expressed in GC tissue and could be targeted by chimeric antigen receptor (CAR) T cells. Mesothelin targeting CAR-T has been reported in mesothelioma, lung cancer, breast cancer, and pancreas cancer. However, the feasibility of using anti-MSLN CAR T cells to treat GC remains to be studied.

Methods: We verified MSLN expression in primary human GC tissues and GC cell lines and then redirected T cells with a CAR containing the MSLN scFv (single-chain variable fragment), CD3ζ, CD28, and DAP10 intracellular signaling domain (M28z10) to target MSLN. We evaluated the function of these CAR T cells in vitro in terms of cytotoxicity, cytokine secretion, and surface phenotype changes when they encountered MSLN+ GC cells. We also established four different xenograft GC mouse models to assess in vivo antitumor activity.

Results: M28z10 T cells exhibited strong cytotoxicity and cytokine-secreting ability against GC cells in vitro. In addition, cell surface phenotyping suggested significant activation of M28z10 T cells upon target cell stimulation. M28z10 T cells induced GC regression in different xenograft mouse models and prolonged the survival of these mice compared with GFP-transduced T cells in the intraperitoneal and pulmonary metastatic GC models. Importantly, peritumoral delivery strategy can lead to improved CAR-T cells infiltration into tumor tissue and significantly suppress the growth of GC in a subcutaneous GC model.

Conclusion: These results demonstrate that M28z10 T cells possess strong antitumor activity and represent a promising therapeutic approach to GC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13045-019-0704-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380000PMC
February 2019

In vitro establishment of expanded-potential stem cells from mouse pre-implantation embryos or embryonic stem cells.

Nat Protoc 2019 02;14(2):350-378

Wellcome Trust Sanger Institute, Hinxton, UK.

Molecular and embryology studies have demonstrated that mouse pre-implantation embryo development is a process of progressive cell fate determination. At the time of implantation, three cell lineages are present in the developing blastocyst: the trophectoderm (TE), the epiblast (Epi) and the primitive endoderm (PrE). From these early embryo cells, trophoblast stem (TS) cells, embryonic stem (ES) cells and extra-embryonic endoderm stem (XEN) cells can be derived. Recently, we derived stem cells with blastomere-like features from mouse cleavage-stage embryos, which we named expanded-potential stem cells (EPSCs). Here, we provide detailed protocols that describe how to establish EPSCs from single eight-cell-stage blastomeres or whole eight-cell pre-implantation mouse embryos, or by conversion of mouse ES cells or induced pluripotent stem (iPS) cells reprogrammed from fibroblasts. It takes 2-3 weeks to derive EPSCs from each cell source. The EPSCs derived from these protocols can differentiate into all embryonic and extra-embryonic lineages when implanted into chimeras. Furthermore, bona fide TS and XEN cell lines can be derived from EPSCs in vitro.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41596-018-0096-4DOI Listing
February 2019

DNAX-activating protein 10 co-stimulation enhances the anti-tumor efficacy of chimeric antigen receptor T cells.

Oncoimmunology 2019;8(1):e1509173. Epub 2018 Nov 2.

Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

Chimeric antigen receptor (CAR) T cell immunotherapies have shown remarkable efficacy in treating multiple types of hematological malignancies but are not sufficiently effective at treating solid tumors. NKG2D is a strong activating receptor for NK cells and a co-stimulatory receptor for T cells. NKG2D signal transduction depends on DNAX-activating protein 10 (DAP10). Here, we introduced the cytoplasmic domain of DAP10 into the second-generation CARs M28z and G28z to generate M28z10 and G28z10, which target mesothelin (MSLN) and glypican 3 (GPC3), respectively. T cells expressing M28z10 or G28z10 showed enhanced and prolonged effector function against MSLN+ lung cancer or GPC3+ hepatocellular carcinoma cell lines in culture and secreted elevated levels of cytokines, including IL-2, IFN-γ, granzyme B, and GM-CSF. In addition, M28z10 CAR-T cells showed greater anti-tumor activity than those expressing M28z in both A549 cell line xenografts and human lung cancer patient-derived xenografts (PDX). Similarly, G28z10 exhibited higher efficacy in causing tumor regression than did G28z in hepatocellular carcinoma PDX. Therefore, our results show that DAP10 signaling contributes to the function of CAR-T cells in both lung cancer and hepatocellular carcinoma and can enhance the efficacy of CAR-T cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/2162402X.2018.1509173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287795PMC
November 2018

PiggyBac Transposon-Based Insertional Mutagenesis in Mice.

Methods Mol Biol 2019 ;1907:171-183

Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.

While sequencing and array-based studies are creating catalogues of genetic alterations in cancer, discriminating cancer drivers among the large sets of epigenetically, transcriptionally or posttranslationally dysregulated genes remains a challenge. Transposon-based genetic screening in mice has proven to be a powerful approach to address this challenge. Insertional mutagenesis directly flags biologically relevant genes and, combined with the transposon's unique molecular fingerprint, facilitates the recovery of insertion sites. We have generated transgenic mouse lines harboring different versions of PiggyBac-based oncogenic transposons, which in conjunction with PiggyBac transposase mice can be used for whole-body or tissue-specific insertional mutagenesis screens. We have also developed QiSeq, a method for (semi-)quantitative transposon insertion site sequencing, which overcomes biasing limitations of previous library preparation methods. QiSeq can be used in multiplexed high-throughput formats for candidate cancer gene discovery and gives insights into the clonal distribution of insertions for the study of genetic tumor evolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-8967-6_14DOI Listing
July 2019

Xist Intron 1 Repression by Transcriptional-Activator-Like Effectors Designer Transcriptional Factor Improves Somatic Cell Reprogramming in Mice.

Stem Cells 2019 05 21;37(5):599-608. Epub 2019 Feb 21.

Research Center for Animal Genetic Resources of Mongolian Plateau, Inner Mongolia University, Hohhot, People's Republic of China.

Xist is the master regulator of X chromosome inactivation. In order to further understand the Xist locus in the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) and in somatic cell nuclear transfer (SCNT), we tested transcription-activator-like effectors-based designer transcriptional factors (dTFs), which were specific to numerous regions at the Xist locus. We report that the selected dTF repressor 6 (R6) binding the intron 1 of Xist, which caused higher H3K9me3 followed by X chromosome opening and repression of X-linked genes in mouse embryonic fibroblasts, rather than affecting Xist expression, substantially improved the iPSC generation and the SCNT preimplantation embryo development. Conversely, the dTF activator targeting the same genomic region of R6 decreased iPSC formation and blocked SCNT-embryo development. These results thus uncover the critical requirement for the Xist locus in epigenetic resetting, which is not directly related to Xist transcription. This may provide a unique route to improving the reprogramming. Stem Cells 2019;37:599-608.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.2928DOI Listing
May 2019

Establishment of peripheral blood mononuclear cell-derived humanized lung cancer mouse models for studying efficacy of PD-L1/PD-1 targeted immunotherapy.

MAbs 2018 Nov-Dec;10(8):1301-1311. Epub 2018 Oct 2.

b Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine , Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences , Guangzhou , China.

Animal models used to evaluate efficacies of immune checkpoint inhibitors are insufficient or inaccurate. We thus examined two xenograft models used for this purpose, with the aim of optimizing them. One method involves the use of peripheral blood mononuclear cells and cell line-derived xenografts (PBMCs-CDX model). For this model, we implanted human lung cancer cells into NOD-scid-IL2Rg-/- (NSI) mice, followed by injection of human PBMCs. The second method involves the use of hematopoietic stem and progenitor cells and CDX (HSPCs-CDX model). For this model, we first reconstituted the human immune system by transferring human CD34+ hematopoietic stem and progenitor cells (HSPCs-derived humanized model) and then transplanted human lung cancer cells. We found that the PBMCs-CDX model was more accurate in evaluating PD-L1/PD-1 targeted immunotherapies. In addition, it took only four weeks with the PBMCs-CDX model for efficacy evaluation, compared to 10-14 weeks with the HSPCs-CDX model. We then further established PBMCs-derived patient-derived xenografts (PDX) models, including an auto-PBMCs-PDX model using cancer and T cells from the same tumor, and applied them to assess the antitumor efficacies of anti-PD-L1 antibodies. We demonstrated that this PBMCs-derived PDX model was an invaluable tool to study the efficacies of PD-L1/PD-1 targeted cancer immunotherapies. Overall, we found our PBMCs-derived models to be excellent preclinical models for studying immune checkpoint inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/19420862.2018.1518948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284590PMC
June 2019

BCL11A interacts with SOX2 to control the expression of epigenetic regulators in lung squamous carcinoma.

Nat Commun 2018 08 20;9(1):3327. Epub 2018 Aug 20.

Department of Pharmacology, University of Cambridge, Cambridge, CB2 1PD, UK.

Patients diagnosed with lung squamous cell carcinoma (LUSC) have limited targeted therapies. We report here the identification and characterisation of BCL11A, as a LUSC oncogene. Analysis of cancer genomics datasets revealed BCL11A to be upregulated in LUSC but not in lung adenocarcinoma (LUAD). Experimentally we demonstrate that non-physiological levels of BCL11A in vitro and in vivo promote squamous-like phenotypes, while its knockdown abolishes xenograft tumour formation. At the molecular level we found that BCL11A is transcriptionally regulated by SOX2 and is required for its oncogenic functions. Furthermore, we show that BCL11A and SOX2 regulate the expression of several transcription factors, including SETD8. We demonstrate that shRNA-mediated or pharmacological inhibition of SETD8 selectively inhibits LUSC growth. Collectively, our study indicates that BCL11A is integral to LUSC pathology and highlights the disruption of the BCL11A-SOX2 transcriptional programme as a novel candidate for drug development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-05790-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102279PMC
August 2018

Functional characterization of common BCL11B gene desert variants suggests a lymphocyte-mediated association of BCL11B with aortic stiffness.

Eur J Hum Genet 2018 11 8;26(11):1648-1657. Epub 2018 Aug 8.

Division of Experimental Medicine & Immunotherapeutics (EMIT), Department of Medicine, University of Cambridge, Cambridge, UK.

The recent genome-wide analysis of carotid-femoral pulse wave velocity (PWV) identified a significant locus within the 14q32.2 gene desert. Gene regulatory elements for the transcriptional regulator B-cell CLL/lymphoma 11B (BCL11B) are within this locus and an attractive target for the gene association. We investigated the functional impact of these gene desert SNPs on BCL11B transcript in human aorta to characterize further its role in aortic stiffness. To do this, we used a large repository of aortic tissues (n = 185) from an organ transplant program and assessed ex vivo stiffness of the aortic rings. We tested association of three lead SNPs from the GWAS meta-analysis with ex vivo aortic stiffness and BCL11B aortic mRNA expression: rs1381289 and rs10782490 SNPs associated significantly with PWV and showed allele-specific differences in BCL11B mRNA. The risk alleles associated with lower BCL11B expression, suggesting a protective role for BCL11B. Despite strong association, we could not detect BCL11B protein in the human aorta. However, qPCR for CD markers showed that BCL11B transcript correlated strongly with markers for activated lymphocytes. Our data confirm the significance of the 14q32.2 region as a risk locus for aortic stiffness and an upstream regulator of BCL11B. The BCL11B transcript detected in the human aorta may reflect lymphocyte infiltration, suggesting that immune mechanisms contribute to the observed association of BCL11B with aortic stiffness.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41431-018-0226-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189060PMC
November 2018

BCL11B mutations in patients affected by a neurodevelopmental disorder with reduced type 2 innate lymphoid cells.

Brain 2018 08;141(8):2299-2311

Département de Génétique, Hôpital La Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Paris, France.

The transcription factor BCL11B is essential for development of the nervous and the immune system, and Bcl11b deficiency results in structural brain defects, reduced learning capacity, and impaired immune cell development in mice. However, the precise role of BCL11B in humans is largely unexplored, except for a single patient with a BCL11B missense mutation, affected by multisystem anomalies and profound immune deficiency. Using massively parallel sequencing we identified 13 patients bearing heterozygous germline alterations in BCL11B. Notably, all of them are affected by global developmental delay with speech impairment and intellectual disability; however, none displayed overt clinical signs of immune deficiency. Six frameshift mutations, two nonsense mutations, one missense mutation, and two chromosomal rearrangements resulting in diminished BCL11B expression, arose de novo. A further frameshift mutation was transmitted from a similarly affected mother. Interestingly, the most severely affected patient harbours a missense mutation within a zinc-finger domain of BCL11B, probably affecting the DNA-binding structural interface, similar to the recently published patient. Furthermore, the most C-terminally located premature termination codon mutation fails to rescue the progenitor cell proliferation defect in hippocampal slice cultures from Bcl11b-deficient mice. Concerning the role of BCL11B in the immune system, extensive immune phenotyping of our patients revealed alterations in the T cell compartment and lack of peripheral type 2 innate lymphoid cells (ILC2s), consistent with the findings described in Bcl11b-deficient mice. Unsupervised analysis of 102 T lymphocyte subpopulations showed that the patients clearly cluster apart from healthy children, further supporting the common aetiology of the disorder. Taken together, we show here that mutations leading either to BCL11B haploinsufficiency or to a truncated BCL11B protein clinically cause a non-syndromic neurodevelopmental delay. In addition, we suggest that missense mutations affecting specific sites within zinc-finger domains might result in distinct and more severe clinical outcomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/brain/awy173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6061686PMC
August 2018

Stability and Function of Hippocampal Mossy Fiber Synapses Depend on .

Front Mol Neurosci 2018 5;11:103. Epub 2018 Apr 5.

Institute of Molecular and Cellular Anatomy, Ulm University, Ulm, Germany.

Structural and functional plasticity of synapses are critical neuronal mechanisms underlying learning and memory. While activity-dependent regulation of synaptic strength has been extensively studied, much less is known about the transcriptional control of synapse maintenance and plasticity. Hippocampal mossy fiber (MF) synapses connect dentate granule cells to CA3 pyramidal neurons and are important for spatial memory formation and consolidation. The transcription factor is expressed in dentate granule cells and required for postnatal hippocampal development. Ablation of in the adult hippocampus results in impaired adult neurogenesis and spatial memory. The molecular mechanisms underlying the behavioral impairment remained unclear. Here we show that selective deletion of in the adult mouse hippocampus leads to a rapid loss of excitatory synapses in CA3 as well as reduced ultrastructural complexity of remaining mossy fiber boutons (MFBs). Moreover, a dramatic decline of long-term potentiation (LTP) of the dentate gyrus-CA3 (DG-CA3) projection is caused by adult loss of . Differential transcriptomics revealed the deregulation of genes associated with synaptic transmission in mutants. Together, our data suggest to regulate maintenance and function of MF synapses in the adult hippocampus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fnmol.2018.00103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895709PMC
April 2018

Bcl11b, a novel GATA3-interacting protein, suppresses Th1 while limiting Th2 cell differentiation.

J Exp Med 2018 05 7;215(5):1449-1462. Epub 2018 Mar 7.

Molecular and Cellular Immunoregulation Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD

GATA-binding protein 3 (GATA3) acts as the master transcription factor for type 2 T helper (Th2) cell differentiation and function. However, it is still elusive how GATA3 function is precisely regulated in Th2 cells. Here, we show that the transcription factor B cell lymphoma 11b (Bcl11b), a previously unknown component of GATA3 transcriptional complex, is involved in GATA3-mediated gene regulation. Bcl11b binds to GATA3 through protein-protein interaction, and they colocalize at many important cis-regulatory elements in Th2 cells. The expression of type 2 cytokines, including IL-4, IL-5, and IL-13, is up-regulated in -deficient Th2 cells both in vitro and in vivo; such up-regulation is completely GATA3 dependent. Genome-wide analyses of Bcl11b- and GATA3-regulated genes (from RNA sequencing), cobinding patterns (from chromatin immunoprecipitation sequencing), and Bcl11b-modulated epigenetic modification and gene accessibility suggest that GATA3/Bcl11b complex is involved in limiting Th2 gene expression, as well as in inhibiting non-Th2 gene expression. Thus, Bcl11b controls both GATA3-mediated gene activation and repression in Th2 cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20171127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940260PMC
May 2018

Transformation of Accessible Chromatin and 3D Nucleome Underlies Lineage Commitment of Early T Cells.

Immunity 2018 02;48(2):227-242.e8

Systems Biology Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892, USA. Electronic address:

How chromatin reorganization coordinates differentiation and lineage commitment from hematopoietic stem and progenitor cells (HSPCs) to mature immune cells has not been well understood. Here, we carried out an integrative analysis of chromatin accessibility, topologically associating domains, AB compartments, and gene expression from HSPCs to CD4CD8 T cells. We found that abrupt genome-wide changes at all three levels of chromatin organization occur during the transition from double-negative stage 2 (DN2) to DN3, accompanying the T lineage commitment. The transcription factor BCL11B, a critical regulator of T cell commitment, is associated with increased chromatin interaction, and Bcl11b deletion compromised chromatin interaction at its target genes. We propose that these large-scale and concerted changes in chromatin organization present an energy barrier to prevent the cell from reversing its fate to earlier stages or redirecting to alternatives and thus lock the cell fate into the T lineages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2018.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5847274PMC
February 2018

Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes.

Nature 2018 02 24;554(7690):62-68. Epub 2018 Jan 24.

Anthropology & Human Genomics, Department of Biology II, Ludwig-Maximilians Universität, 82152 Martinsried, Germany.

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest Kras levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous Kras in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfβ-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature25459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097607PMC
February 2018
-->