VGLL1 cooperates with TEAD4 to control human trophectoderm lineage specification.

In contrast to rodents, the mechanisms underlying human trophectoderm and early placenta specification are understudied due to ethical barriers and the scarcity of embryos. Recent reports have shown that human pluripotent stem cells (PSCs) can differentiate into trophectoderm (TE)-like cells (TELCs) and trophoblast stem cells (TSCs), offering a valuable in vitro model to study early placenta specification. Here, we demonstrate that the VGLL1 (vestigial-like family member 1), which is highly expressed during human and non-human primate TE specification in vivo but is negligibly expressed in mouse, is a critical regulator of cell fate determination and self-renewal in human TELCs and TSCs derived from naïve PSCs. Mechanistically, VGLL1 partners with the transcription factor TEAD4 (TEA domain transcription factor 4) to regulate chromatin accessibility at target gene loci through histone acetylation and acts in cooperation with GATA3 and TFAP2C. Our work is relevant to understand primate early embryogenesis and how it differs from other mammalian species.

Live birth of chimeric monkey with high contribution from embryonic stem cells.

A formal demonstration that mammalian pluripotent stem cells possess preimplantation embryonic cell-like (naive) pluripotency is the generation of chimeric animals through early embryo complementation with homologous cells. Whereas such naive pluripotency has been well demonstrated in rodents, poor chimerism has been achieved in other species including non-human primates due to the inability of the donor cells to match the developmental state of the host embryos. Here, we have systematically tested various culture conditions for establishing monkey naive embryonic stem cells and optimized the procedures for chimeric embryo culture. This approach generated an aborted fetus and a live chimeric monkey with high donor cell contribution. A stringent characterization pipeline demonstrated that donor cells efficiently (up to 90%) incorporated into various tissues (including the gonads and placenta) of the chimeric monkeys. Our results have major implications for the study of primate naive pluripotency and genetic engineering of non-human primates.

Premorbid sarcopenia and functional outcome after acute stroke: a meta-analysis.

BACKGROUND AND OBJECTIVES: Sarcopenia is prevalent in patients with stroke. However, the relationship between sarcopenia and poor functional outcome of patients with acute stroke remains unknown. A systematic review and meta-analysis was performed to evaluate the above association. METHODS AND STUDY DESIGN: Observational studies which evaluated the influence of sarcopenia on functional outcome in patients with acute stroke were retrieved by search the PubMed, Embase, Cochrane Library, and Web of Science databases. A poor functional outcome was defined as modified Rankin scale (mRS) of two or more points during follow-up. Two authors independently collected the data of study characteristics and outcomes. A random-effects model was used to pool the results via incorporating the influence of possible between-study heterogeneity. RESULTS: Nine datasets from seven cohort studies contributed to the meta-analysis. A total of 1774 patients with stroke were included, and 481 (27.1%) of them had sarcopenia. Compared to patients without sarcopenia, those with sarcopenia were associated with a higher risk of poor functional outcome during follow-up duration up to 6 months after stroke onset (odds ratio: 2.42, 95% confidence interval: 1.76 to 3.33, p < 0.001) with mild heterogeneity (I2 = 23%). Subgroup analyses according to study design (prospective versus retrospective), sex of the patient, type of stroke (ischemic or mixed), diagnostic methods for sarcopenia, follow-up duration and cutoff scores for mRS showed consistent results (p for subgroup analyses all > 0.05). CONCLUSIONS: Sarcopenia may be associated with poor functional outcome in patients with acute stroke.

Generation of a humanized mesonephros in pigs from induced pluripotent stem cells via embryo complementation.

Heterologous organ transplantation is an effective way of replacing organ function but is limited by severe organ shortage. Although generating human organs in other large mammals through embryo complementation would be a groundbreaking solution, it faces many challenges, especially the poor integration of human cells into the recipient tissues. To produce human cells with superior intra-niche competitiveness, we combined optimized pluripotent stem cell culture conditions with the inducible overexpression of two pro-survival genes (MYCN and BCL2). The resulting cells had substantially enhanced viability in the xeno-environment of interspecies chimeric blastocyst and successfully formed organized human-pig chimeric middle-stage kidney (mesonephros) structures up to embryonic day 28 inside nephric-defective pig embryos lacking SIX1 and SALL1. Our findings demonstrate proof of principle of the possibility of generating a humanized primordial organ in organogenesis-disabled pigs, opening an exciting avenue for regenerative medicine and an artificial window for studying human kidney development.

Rolling back human pluripotent stem cells to an eight-cell embryo-like stage.

After fertilization, the quiescent zygote experiences a burst of genome activation that initiates a short-lived totipotent state. Understanding the process of totipotency in human cells would have broad applications. However, in contrast to in mice1,2, demonstration of the time of zygotic genome activation or the eight-cell (8C) stage in in vitro cultured human cells has not yet been reported, and the study of embryos is limited by ethical and practical considerations. Here we describe a transgene-free, rapid and controllable method for producing 8C-like cells (8CLCs) from human pluripotent stem cells. Single-cell analysis identified key molecular events and gene networks associated with this conversion. Loss-of-function experiments identified fundamental roles for DPPA3, a master regulator of DNA methylation in oocytes3, and TPRX1, a eutherian totipotent cell homeobox (ETCHbox) family transcription factor that is absent in mice4. DPPA3 induces DNA demethylation throughout the 8CLC conversion process, whereas TPRX1 is a key executor of 8CLC gene networks. We further demonstrate that 8CLCs can produce embryonic and extraembryonic lineages in vitro or in vivo in the form of blastoids5 and complex teratomas. Our approach provides a resource to uncover the molecular process of early human embryogenesis.

ß-Catenin safeguards the ground state of mousepluripotency by strengthening the robustness of the transcriptional apparatus.

Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that ß-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by ß-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.

Generation of an induced pluripotent stem cell line (GIBHi004-A) from a Parkinson's disease patient with mutant DJ-1/PARK7 (p.L10P).

Mutations occurring in the gene body of PARK7 (encoding DJ-1/PARK7) cause autosomal recessive early-onset parkinsonism (AREP). These mutations produce a loss of function and have been reported to lead to dopaminergic neuron degeneration in the substantia nigra. However, the underlying mechanisms are largely unknown. Here, we report the generation of a patient-derived induced pluripotent stem cell (iPSC) line carrying mutant DJ-1 (p.L10P). This cell line is a valuable tool for in vitro Parkinson's disease (PD) modeling and mechanistic studies.

Generation of three induced pluripotent stem cell lines from a Parkinson's disease patient with mutant PARKIN (p. C253Y).

Parkinson's disease (PD) is one of the most common neurodegenerative disorders and is characterized by the progressive degeneration of dopaminergic (DA) neurons in the substantia nigra. Loss of function mutations in PARK2 cause familial PD in an autosomal recessive manner. PARK2 encodes an E3 ubiquitin ligase that is involved in regulation of mitochondrial homeostasis. However, the mechanistic links between PARK2 mutations and dopaminergic neuron degeneration are unclear. Here, we have generated three patient-derived induced pluripotent stem cell (iPSC) lines from the same donor with mutant PARKIN (p. C253Y). These well characterized cell lines will facilitate the study of PARKIN function in disease relevant cell types in vitro.

Generation of a PARK2 homozygous knockout induced pluripotent stem cell line (GIBHi002-A-1) with two common isoforms abolished.

Loss of function mutations in PARK2 (encoding PARKIN) cause autosomal recessive Parkinson's disease (PD), which often manifests at a juvenile age. Molecular and biochemical studies show that PARKIN functions as an E3 ubiquitin ligase controlling mitochondrial homeostasis. Yet, the exact mechanisms are unclear due to the use of sub-optimal models including cancer cells and fibroblasts. We have generated a PARK2 knockout (KO) isogenic cell line using a well-characterized induced pluripotent stem cell (iPSC) clone with good differentiation potential. This cell line lacks the expression of all PARKIN isoforms and is valuable for elucidating the role of PARK2 mutations in PD.

Generation of an induced pluripotent stem cell line (GIBHi003-A) from a Parkinson's disease patient with mutant PINK1 (p. I368N).

Familial Parkinson's disease (PD) can be caused by deleterious mutations in PINK1 (encoding PINK1) in an autosomal recessive manner. Functional studies suggest that PINK1 works as a regulator of mitochondrial homeostasis. However, how loss of PINK1 induces dopaminergic neuron degeneration is still unclear. Here, we have generated a patient-derived induced pluripotent stem cell (iPSC) line with mutant PINK1 (p. I368N). This cell line will facilitate PD disease modeling in vitro and can be used for generating isogenic cell lines through gene correction.

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy.

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer's disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 µm pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

VSV-G Viral Envelope Glycoprotein Prepared from Pichia pastoris Enhances Transfection of DNA into Animal Cells.

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones (Muts) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.

Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection.

The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents. A luciferase expression construct (pGL3-control) and a DsRed expression cassette (pCMV-DsRed) were used to evaluate the delivery efficiency of DNA/EG/VSV-G complexes. VSV-G proteins could be incorporated into EG in static incubation under acidic conditions as evidenced by the Western blot analysis. Condensed plasmid DNA was bound mostly to the outer surface of EG, which could be detected by electromicroscopy and measured by electrophoresis. EG/VSV-G complexes stimulated the delivery of pGL3-control into Ad293 cells significantly with the luciferase activity increased about 4-fold as compared to that of the control. The delivery of pCMV-DsRed was also enhanced with the percentage of DsRed-positive Ad293 cells increased from 55 % to about 80 %. Moreover, the transfection efficiency in 3T3, HeLa, INS-1, and bone marrow stem cell (BMSC) cells increased about 2-3-fold. Finally, confocal microscopy analysis showed that incorporation of VSV-G significantly enhanced the endocytosis of EG into target cells. In the present study, a novel type of non-viral DNA delivery vehicle consisting of EG and fusogenic VSV-G proteins was formulated, which showed superior transfection efficiency even in cells resistant to classical transfection.

Niacin receptor GPR109A inhibits insulin secretion and is down-regulated in type 2 diabetic islet beta-cells.

OBJECTIVES: We previously found niacin receptor GPR109A was expressed in murine islet beta-cells, and signaling through GPR109A inhibited glucose stimulated insulin secretion (GSIS). However, the expression of GPR109A in human islets and its functional relevance is still not known. METHODS: The expression of GPR109A was examined by antibody staining and in situ hybridization on pancreatic paraffin sections. GPR109A was cloned and expressed in INS-1 islet beta-cells. Intracellular cAMP and GSIS were determined using enzyme-linked immunosorbent assay (ELISA). RESULTS: The expression of GPR109A was confirmed in murine islet beta-cells and further detected in human counterparts by using commercially available polyclonal antibodies. In situ hybridization study detected the transcripts of GPR109A, but not that of closely related GPR109B. Furthermore, GPR109A was significantly reduced in islets from diabetic individuals and animal model of db/db mice as compared to their respective controls. Further, GPR109A levels in insulinoma were also reduced dramatically as compared to islets found in corresponding non-tumor normal tissues. Quantitative RT-PCR analysis demonstrated that GPR109A transcripts were severely down-regulated in rodent insulinoma cell lines as compared to that of freshly isolated islets from mice. Finally, human and murine GPR109A expression cassettes were transfected into INS-1 cells, which resulted in reduced accumulation of cAMP and insulin secretion after incubation with niacin. The effect could be completely abrogated by pretreatment with pertussis toxin. CONCLUSIONS: These results demonstrate that GPR109A is functionally expressed in both human and murine islet beta-cells. However, the role of GPR109A in the prevention of diabetes or insulinoma needs further study.

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.