Brd4::Nutm1 fusion gene initiates NUT carcinoma in vivo.

NUT carcinoma (NC) is an aggressive cancer with no effective treatment. About 70% of NUT carcinoma is associated with chromosome translocation events that lead to the formation of a BRD4::NUTM1 fusion gene. Because the BRD4::NUTM1 gene is unequivocally cytotoxic when ectopically expressed in cell lines, questions remain on whether the fusion gene can initiate NC. Here, we report the first genetically engineered mouse model for NUT carcinoma that recapitulates the human t(15;19) chromosome translocation in mice. We demonstrated that the mouse t(2;17) syntenic chromosome translocation, forming the Brd4::Nutm1 fusion gene, could induce aggressive carcinomas in mice. The tumors present histopathological and molecular features similar to human NC, with enrichment of undifferentiated cells. Similar to the reports of human NC incidence, Brd4::Nutm1 can induce NC from a broad range of tissues with a strong phenotypical variability. The consistent induction of poorly differentiated carcinoma demonstrated a strong reprogramming activity of BRD4::NUTM1. The new mouse model provided a critical preclinical model for NC that will lead to better understanding and therapy development for NC.

Mesenchymal Stromal Cells Increase the Natural Killer Resistance of Circulating Tumor Cells via Intercellular Signaling of cGAS-STING-IFNß-HLA.

Circulating tumor cells (CTCs) shed from primary tumors must overcome the cytotoxicity of immune cells, particularly natural killer (NK) cells, to cause metastasis. The tumor microenvironment (TME) protects tumor cells from the cytotoxicity of immune cells, which is partially executed by cancer-associated mesenchymal stromal cells (MSCs). However, the mechanisms by which MSCs influence the NK resistance of CTCs remain poorly understood. This study demonstrates that MSCs enhance the NK resistance of cancer cells in a gap junction-dependent manner, thereby promoting the survival and metastatic seeding of CTCs in immunocompromised mice. Tumor cells crosstalk with MSCs through an intercellular cGAS-cGAMP-STING signaling loop, leading to increased production of interferon-ß (IFNß) by MSCs. IFNß reversely enhances the type I IFN (IFN-I) signaling in tumor cells and hence the expression of human leukocyte antigen class I (HLA-I) on the cell surface, protecting the tumor cells from NK cytotoxicity. Disruption of this loop reverses NK sensitivity in tumor cells and decreases tumor metastasis. Moreover, there are positive correlations between IFN-I signaling, HLA-I expression, and NK tolerance in human tumor samples. Thus, the NK-resistant signaling loop between tumor cells and MSCs may serve as a novel therapeutic target.

Definitive Endodermal Cells Supply an in vitro Source of Mesenchymal Stem/Stromal Cells.

Mesenchymal stem/Stromal cells (MSCs) have great therapeutic potentials, and they have been isolated from various tissues and organs including definitive endoderm (DE) organs, such as the lung, liver and intestine. MSCs have been induced from human pluripotent stem cells (hPSCs) through multiple embryonic lineages, including the mesoderm, neural crest, and extraembryonic cells. However, it remains unclear whether hPSCs could give rise to MSCs in vitro through the endodermal lineage. Here, we report that hPSC-derived, SOX17+ definitive endoderm progenitors can further differentiate to cells expressing classic MSC markers, which we name definitive endoderm-derived MSCs (DE-MSCs). Single cell RNA sequencing demonstrates the stepwise emergence of DE-MSCs, while endoderm-specific gene expression can be elevated by signaling modulation. DE-MSCs display multipotency and immunomodulatory activity in vitro and possess therapeutic effects in a mouse ulcerative colitis model. This study reveals that, in addition to the other germ layers, the definitive endoderm can also contribute to MSCs and DE-MSCs could be a cell source for regenerative medicine.

Engineering of human mesenchymal stem cells resistant to multiple natural killer subtypes.

Mesenchymal stem cells (MSCs) as a therapeutic promise are often quickly cleared by innate immune cells of the host including natural killer (NK) cells. Efforts have been made to generate immune-escaping human embryonic stem cells (hESCs) where T cell immunity is evaded by defecting ß-2-microglobulin (B2M), a common unit for human leukocyte antigen (HLA) class I, and NK cells are inhibited via ectopic expression of HLA-E or -G. However, NK subtypes vary among recipients and even at different pathologic statuses. It is necessary to dissect and optimize the efficacy of the immune-escaping cells against NK subtypes. Here, we first generated B2M knockout hESCs and differentiated them to MSCs (EMSCs) and found that NK resistance occurred with B2M-/- EMSCs expressing HLA-E and -G only when they were transduced via an inducible lentiviral system in a dose-dependent manner but not when they were inserted into a safe harbor. HLA-E and -G expressed at high levels together in transduced EMSCs inhibited three major NK subtypes, including NKG2A+ /LILRB1+ , NKG2A+ /LILRB1- , and NKG2A- /LILRB1+ , which was further potentiated by IFN-γ priming. Thus, this study engineers MSCs with resistance to multiple NK subtypes and underscores that dosage matters when a transgene is used to confer a novel effect to host cells, especially for therapeutic cells to evade immune rejection.

Human ESC-derived MSCs enhance fat engraftment by promoting adipocyte reaggregation, secreting CCL2 and mobilizing macrophages.

Mesenchymal stem cells (MSCs) derived from somatic tissues have been used to promote lipotransfer, a common practice in cosmetic surgery. However, the effect of lipotransfer varies, and the mechanism of action remains vague. To address these questions, we differentiated human embryonic stem cells, a stable and unlimited source, into MSCs (EMSCs). Then we subcutaneously transplanted human fat aspirates together with EMSCs or PBS as a control into the back of nude mice. Within 24 h of transplantation, EMSCs promoted aggregation and encapsulation of injected fat tissues. Afterward, all grafts gradually shrank. However, EMSC-containing grafts were larger, heavier and had fewer dark areas on the surface than the control grafts. Histologically, more live adipocytes, vascular cells, and macrophages and less fibrosis were observed in EMSC-containing grafts than in the controls. Some EMSCs differentiated into vascular cells and adipocytes in the EMSC-containing grafts. RNA sequencing revealed that human RNA was shown to decline rapidly, while mouse RNA increased in the grafts; further, human genes related to extracellular matrix remodeling, adipogenesis, and chemokine (including CCL2) signaling were expressed at higher levels in the EMSC-containing grafts than they were in the controls. CCL2 knockout reduced macrophage migration towards EMSCs in vitro and early macrophage recruitment to the grafts and the pro-engraftment effect of EMSCs in vivo. Treating mice with a macrophage inhibitor abolished the EMSC effects and converted the grafts to heavy masses of cell debris. Together, these data demonstrate that EMSCs promote fat engraftment via enhanced tissue reconstitution and encapsulation of implanted tissues, which was followed by increased angiogenesis and adipocyte survival and reduced fibrosis, in which stimulated CCL2 signaling and mobilized macrophages play pivotal roles.

Noninvasive application of mesenchymal stem cell spheres derived from hESC accelerates wound healing in a CXCL12-CXCR4 axis-dependent manner.

Mesenchymal stem cells (MSC) derived from adult tissues effectively promote wound healing. However, MSC quality varies, and the quantity of MSC is limited, as MSC are acquired through donations. Moreover, the survival and functioning of dissociated MSC delivered to an inflammatory lesion are subject to challenges. Methods: Here, spheres (EMSCSp) generated from human embryonic stem cell-derived MSC (EMSC) were directly dropped onto excised wounds in mice; the effects of EMSCSp were compared to those of dissociated EMSC (EMSCDiss). Following transplantation, we measured the extent of wound closure, dissected the histological features of the wounds, determined transcriptomic changes in cells isolated from the treated and control wounds, and evaluated the molecular mechanism of the effects of EMSC. Results: The application of EMSCSp onto murine dermal wounds substantially increased survival and efficacy of EMSC compared to the topical application of EMSCDiss. RNA sequencing (RNA-Seq) of cells isolated from the wounds highlighted the involvement of CXCL12-CXCR4 signaling in the effects of EMSCSp, which was verified in EMSC via CXCL12 knockdown and in target cells (vascular endothelial cells, epithelial keratinocytes, and macrophages) via CXCR4 inhibition. Finally, we enhanced the biosafety of EMSCSp by engineering cells with an inducible suicide gene. Conclusions: Together, these data suggest the topical application of EMSCSp as an unlimited, quality-assured, safe, and noninvasive therapy for wound healing and the CXCL12-CXCR4 axis as a key player in this treatment.

Universal Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells for Cellularization of a Corneal Scaffold.

PURPOSE: We generated universal corneal epithelial cells (CEC) from human embryonic stem cells (hESC) by genetically removing human leukocyte antigens (HLA) class I from the cell surface. METHODS: The serum-free, growth factor-free, and defined medium E6 was used to differentiate hESC to CEC. Decellularized murine corneas were recellularized with hESC-derived CEC. Using CRISPR/Cas9, ß-2-microglobulin (B2M) was deleted in hESC to block the assembly of HLA class-I antigens on the cell surface to generate B2M -/- CEC. RESULTS: E6 alone was sufficient to allow hESC differentiation to CEC. A time-course analysis of the global gene expression of the differentiating cells indicates that the differentiation closely resembles the corneal development in vivo. The hESC-CEC were highly proliferative, and could form multilayer epithelium in decellularized murine cornea, retain its transparency, and form intact tight junctions on its surface. As reported before, B2M knockout led to the absence of HLA class-I on the cell surface of hESC and subsequently derived CEC following stimulation with inflammatory factors. Moreover, B2M -/- CEC, following transplantation into mouse eyes, caused less T-cell infiltration in the limbal region of the eye than the wild-type control. CONCLUSIONS: CEC can be derived from hESC via a novel and simple protocol free of any proteins, hESC-CEC seeded on decellularized animal cornea form tight junctions and allow light transmittance, and B2M -/- CEC are hypoimmunogenic both in vitro and in vivo. TRANSLATIONAL RELEVANCE: B2M -/- hESC-CEC can be an unlimited and universal therapy for corneal repair in patients of any HLA type.

Critical Role of Tumor Necrosis Factor Signaling in Mesenchymal Stem Cell-Based Therapy for Autoimmune and Inflammatory Diseases.

Mesenchymal stem cells (MSCs) have been broadly used as a therapy for autoimmune disease in both animal models and clinical trials. MSCs inhibit T effector cells and many other immune cells, while activating regulatory T cells, thus reducing the production of pro-inflammatory cytokines, including tumor necrosis factor (TNF), and repressing inflammation. TNF can modify the MSC effects via two TNF receptors, i.e., TNFR1 in general mediates pro-inflammatory effects and TNFR2 mediates anti-inflammatory effects. In the central nervous system, TNF signaling plays a dual role, which enhances inflammation via TNFR1 on immune cells while providing cytoprotection via TNFR2 on neural cells. In addition, the soluble form of TNFR1 and membrane-bound TNF also participate in the regulation to fine-tune the functions of target cells. Other factors that impact TNF signaling and MSC functions include the gender of the host, disease course, cytokine concentrations, and the length of treatment time. This review will introduce the fascinating progress in this aspect of research and discuss remaining questions and future perspectives.

Scalable Generation of Mesenchymal Stem Cells from Human Embryonic Stem Cells in 3D.

Human embryonic stem cell (hESC) derived mesenchymal stem cells (EMSC) are efficacious in treating a series of autoimmune, inflammatory, and degenerative diseases in animal models. However, all the EMSC derivation methods reported so far rely on two-dimensional (2D) culture systems, which are inefficient, costive and difficult for large-scale production. HESC, as an unlimited source, can be successively propagated in spheroids. Here, we demonstrate that hESC spheroids can directly differentiate into MSC spheroids (EMSCSp) within 20 days in one vessel without passaging and the system is scalable to any desired size. EMSCSp can further differentiate into osteocytes and chondrocytes in spheres or demineralized bone matrix. EMSCSp also retains immune-modulatory effects in vitro and therapeutic effects on two mouse models of colitis after dissociation. Compared to EMSC differentiated in monolayer, EMSCSp-derived cells have faster proliferation and higher yield and develop less apoptosis and slower senescence. Thus, the 3D differentiation system allows simple, cost-effective, and scalable production of high-quality EMSC and subsequently bone and cartilage tissues for therapeutic application.

Concise Review: One Stone for Multiple Birds: Generating Universally Compatible Human Embryonic Stem Cells.

With ongoing clinical trials, human embryonic stem cells (hESCs) have shown substantial potential for regenerative medicine. However, due to the mismatch of human leukocyte antigens (HLAs) between hESC-derived allografts and recipients, immunosuppressant regimens must be used to prevent immune rejection of the grafts. Considerable efforts have been devoted to overcoming this hurdle via the derivation and banking of human nuclear transfer ESCs, parthenogenetic ESCs, and induced pluripotent stem cells. However, ethical and safety concerns remain, hindering the application of these types of pluripotent cells. Other approaches have recently been explored to generate universally compatible hESCs through the silencing or deletion of HLAs or genes essential for HLA expression, including ß-2-microglobulin and class-II MHC transactivator, as well as the induction of immunosuppression via the ectopic expression of non-classical HLAs (e.g., HLA-E and -G), cytotoxic T lymphocyte antigen 4 fused with immunoglobulin, and programmed death ligand-1. In this review, we introduce developments in this line of research and discuss strategies to reduce the tumorigenic concerns regarding hESCs, especially after they acquire the capability to escape immune surveillance. Stem Cells 2016;34:2269-2275.

Incorporation of VSV-G produces fusogenic plasma membrane vesicles capable of efficient transfer of bioactive macromolecules and mitochondria.

The objective of this study was to determine if plasma membrane vesicles (PMVs) could be exploited for efficient transfer of macro-biomolecules and mitochondria. PMVs were derived from mechanical extrusion, and made fusogenic (fPMVs) by incorporating the glycoprotein G of vesicular stomatitis virus (VSV-G). Confocal microscopy examination revealed that cytoplasmic proteins and mitochondria were enclosed in PMVs as evidenced by tracing with cytoplasmically localized and mitochondria-targeted EGFP, respectively. However, no fluorescence signal was detected in PMVs from cells whose nucleus was labeled with an EGFP-tagged histone H2B. Consistently, qRT-PCR measurement showed that mRNA, miRNA and mitochondrial DNA decreased slightly; while nuclear DNA was not measureable. Further, Western blot analysis revealed that cytoplasmic and membrane-bound proteins fell inconspicuously while nuclear proteins were barely detecsle. In addition, fPMVs carrying cytoplamic DsRed proteins transduced about ~40 % of recipient cells. The transfer of protein was further confirmed by using the inducible Cre/loxP system. Mitochondria transfer was found in about 20 % recipient cells after incubation with fPMVs for 5 h. To verify the functionalities of transferred mitochondria, mitochodria-deficient HeLa cells (Rho0) were generated and cultivated with fPMVs. Cell enumeration demonstrated that adding fPMVs into culture media stimulated Rho0 cell growth by 100 % as compared to the control. Lastly, MitoTracker and JC-1 staining showed that transferred mitochondria maintained normal shape and membrane potential in Rho0 cells. This study established a time-saving and efficient approach to delivering proteins and mitochondria by using fPMVs, which would be helpful for finding a cure to mitochondria-associated diseases. Graphical abstract Schematic of the delivery of macro-biomolecules and organelles by fPMVs. VSV-G-expressing cells were extruded through a 3 µm polycarbonate membrane filter to generate fusogenic plasma membrane vesicles (fPMVs), which contain bioactive molecules and organelles but not the nucleus. fPMVs can be endocytosed by target cells, while the cargo is released due to low-pH induced membrane fusion. These nucleus-free fPMVs are efficient at delivery of cytoplasmic proteins and mitochondria, leading to recovery of mitochondrial biogenesis and proliferative ability in mitochondria-deficient cells.

Transient embolization with microspheres of polyhydroxyalkanoate renders efficient adenoviral transduction of pancreatic capillary in vivo.

BACKGROUND: Our previous study showed an efficient targeting of islets of Langerhans by adenoviral injection via the celiac trunk. Unexpectedly, none of the endothelial cells was infected given the direct contact between adenoviruses and the capillary wall. The present study intended to provide an efficient approach for adenoviral targeting of the microcapillary endothelial cells in the pancreas. METHODS: We prepared microspheres of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) with a size comparable to the diameter of capillary (5-10 µm). Scanning electron microscopy was applied to verify that adenoviruses carrying a green fluorescence protein gene were complexed with PHBHHx-microspheres after 30 min of co-incubation. The complexes were then injected into the pancreas of mice via the celiac trunk. RESULTS: Approximately 40% of endothelial cells in the pancreas were labeled 5 days after surgery. Islet cells were labeled occasionally, whereas labeling of the acinar and ductal tissues was barely detectable. Endothelium targeting was inefficient in other internal organs. Consistent with the reported superior tissue compatibility of PHBHHx, no discernable microspheres were found in all of the organs examined. Furthermore, splenocyte activation was dampened when adenoviruses were complexed with the microspheres. CONCLUSIONS: The present study has established an approach for efficient pancreatic capillary targeting by using microsphere-adenoviral complexes. This procedure could be invaluable for the treatment of capillary-related diseases.