Ebola and Lassa viruses require biosafety-level-4 (BSL4) containment, infect the liver, and cause deadly hemorrhagic fevers. The cellular effects of these viruses, and whether different families of hemorrhagic-fever viruses elicit similar effects, remain fundamental questions in BSL4 virology. Here, we introduce a new metabolic selection approach to create nearly-pure hepatocytes from human pluripotent stem cells, killing non-liver cells by withholding essential nutrients. Unexpectedly, Ebola and Lassa exerted starkly different effects on human hepatocytes. Ebola infection activated the integrated stress response (ISR) and WNT pathways in hepatocytes in vitro and killed them, whereas Lassa did not. Within non-human primates, Ebola likewise infected hepatocytes and activated ISR signaling in vivo . In summary, we present a single-cell transcriptional and chromatin accessibility roadmap of human hepatocyte differentiation, purification, and viral infection.

Human mesenchymal stem cells (MSCs) have been studied in over 1500 clinical trials to treat over 30 diseases. However, the understanding towards MSC stemness remains under studied. So far, little is known about how MSCs maintain undifferentiated state or commit to specific lineages under different microenvironmental cues. The lack of comprehensive understanding regarding MSC stemness greatly hampers the translation of research findings into successful clinical application due to unclear mechanism of action. Emerging evidence shows that a variety of genetic factors delicately regulate MSC self-renewal and differentiation. In this review, we summarize the role of transcriptional factors, cell cycle regulators, genomic stability genes, cellular quality control, epigenetic regulators, non-coding RNAs, mitochondrial function, growth factors and m6A modification in regulating the stemness of MSCs. Strategies to maintain MSC stemness during ex-vivo expansion are also discussed. This review will deepen understanding of MSC stemness for advancing clinical applications and provide insights into future directions for research aimed at improving MSC-based therapies.

RATIONALE:Cardiac-expressed long noncoding RNAs (lncRNAs) are important for cardiomyocyte (CM) differentiation and function. Several lncRNAs have been identified and characterized for early CM lineage commitment, however those in later CM lineage specification and maturation remain less well studied. Moreover, unique atrial / ventricular lncRNA expression has never been studied in detail. OBJECTIVES:Here, we characterized a novel ventricular myocyte-restricted lncRNA, not expressed in atrial myocytes, and conserved only in primates. METHODS AND RESULTS:First, we performed single cell RNA-seq on human pluripotent stem cell derived cardiomyocytes (hPSC-CM) at the late stages of 2, 6 and 12 weeks of differentiation. Weighted correlation network analysis identified core gene modules, including a set of lncRNAs highly abundant and predominantly expressed in the human heart. A lncRNA (we call VENTHEART, VHRT) co-expressed with cardiac maturation and ventricular-specific genes MYL2 and MYH7, and was expressed in fetal and adult human ventricles, but not atria. CRISPR-mediated deletion of the VHRT gene led to impaired CM sarcomere formation and significant disruption of the ventricular CM gene program. Indeed, a similar disruption was not observed in VHRT KO hPSC-derived atrial CM, suggesting that VHRT exhibits only ventricular myocyte subtype-specific effects. Optical recordings validated that loss of VHRT significantly prolonged action potential duration at 90 % repolarization (APD90) for ventricular-like, but not atrial-like, CMs. CONCLUSION:This reports the first lncRNA that is exclusively required for proper ventricular, and not atrial, CM specification and function.

During the development of the central nervous system, the proliferation of neural progenitors and differentiation of neurons and glia are tightly regulated by different transcription factors and signaling cascades, such as the Wnt and Shh pathways. This process takes place in cooperation with several microRNAs, some of which evolutionarily conserved in vertebrates, from teleosts to mammals. We focused our attention on miR-7, as its role in the regulation of cell signaling during neural development is still unclear. Specifically, we used human stem cell cultures and whole zebrafish embryos to study, in vitro and in vivo, the role of miR-7 in the development of dopaminergic (DA) neurons, a cell type primarily affected in Parkinson's disease. We demonstrated that the zebrafish homologue of miR-7 (miR-7a) is expressed in the forebrain during the development of DA neurons. Moreover, we identified 143 target genes downregulated by miR-7, including the neural fate markers TCF4 and TCF12, as well as the Wnt pathway effector TCF7L2. We then demonstrated that miR-7 negatively regulates the proliferation of DA-progenitors by inhibiting Wnt/β-catenin signaling in zebrafish embryos. In parallel, miR-7 positively regulates Shh signaling, thus controlling the balance between oligodendroglial and DA neuronal cell fates. In summary, this study identifies a new molecular cross-talk between Wnt and Shh signaling pathways during the development of DA-neurons. Being mediated by a microRNA, this mechanism represents a promising target in cell differentiation therapies for Parkinson's disease.

The effects of ascorbate on adult cell fate specification remain largely unknown. Using our stepwise and chemically defined system to derive lateral mesoderm progenitors from human pluripotent stem cells (hPSCs), we found that ascorbate increased the expression of mesenchymal stromal cell (MSC) markers, purity of MSCs, the long-term self-renewal and osteochondrogenic capacity of hPSC-MSCs in vitro. Moreover, ascorbate promoted MSC specification in an iron-dependent fashion, but not in a redox-dependent manner. Further studies revealed that iron synergized with ascorbate to regulate hPSC-MSC histone methylation, promote their long-term self-renewal, and increase their osteochondrogenic capacity. We found that one of the histone demethylases affected by ascorbate, KDM4B, was necessary to promote the specification of hPSC-MSCs. This mechanistic understanding led to the metabolic optimization of hPSC-MSCs with an extended lifespan in vitro and the ability to fully repair cartilage defects upon transplantation in vivo. Our results highlight the importance of ascorbate and iron metabolism in adult human cell fate specification.

Lung cancer is the world's leading cause of cancer death and shows strong ancestry disparities. By sequencing and assembling a large genomic and transcriptomic dataset of lung adenocarcinoma (LUAD) in individuals of East Asian ancestry (EAS; n = 305), we found that East Asian LUADs had more stable genomes characterized by fewer mutations and fewer copy number alterations than LUADs from individuals of European ancestry. This difference is much stronger in smokers as compared to nonsmokers. Transcriptomic clustering identified a new EAS-specific LUAD subgroup with a less complex genomic profile and upregulated immune-related genes, allowing the possibility of immunotherapy-based approaches. Integrative analysis across clinical and molecular features showed the importance of molecular phenotypes in patient prognostic stratification. EAS LUADs had better prediction accuracy than those of European ancestry, potentially due to their less complex genomic architecture. This study elucidated a comprehensive genomic landscape of EAS LUADs and highlighted important ancestry differences between the two cohorts. Genomic and transcriptomic analysis of lung adenocarcinoma (LUAD) in Asia indicates that Asian LUADs have fewer mutations, lower driver prevalence and fewer copy number alterations than European LUADs.

Tumor initiating cells (TIC) are resistant to conventional anticancer therapy and associated with metastasis and relapse in cancer. Although various TIC markers and their antibodies have been proposed, it is limited to the use of antibodies for in vivo imaging or treatment of TIC. In this study, we discovered heme oxygenase 2 (HMOX2) as a novel biomarker for TIC and developed a selective small molecule probe TiNIR (tumor initiating cell probe with near infrared). TiNIR detects and enriches the functionally active TIC in human lung tumors, and through the photoacoustic property, TiNIR also visualizes lung TIC in the patient-derived xenograft (PDX) model. Furthermore, we demonstrate that TiNIR inhibits tumor growth by blocking the function of HMOX2, resulting in significantly increased survival rates of the cancer model mice. The novel therapeutic target HMOX2 and its fluorescent ligand TiNIR will open a new path for the molecular level of lung TIC diagnosis and treatment.

In the version of this article originally published, there is an error in Fig. 5a. Originally, ‘MAT2A’ appeared between ‘Methionine’ and ‘Homocysteine’. ‘MAT2A’ should have been ‘MTR’. The error has been corrected in the PDF and HTML versions of this article.

It is not fully clear why there is a higher contribution of pluripotent stem cells (PSCs) to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells (ESCs) but also induced pluripotent stem cells (iPSCs) can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a "dose effect". Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast (EPI) lineage development while promoting trophectoderm (TE) differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A (cultured from 4-cell stage to early blastocyst at E3.5) could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improve their EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understanding of PSCs effects on early embryo development.

The identification of 101 genes upregulated or differentially expressed by mature human oocytes is provided herein. These genes and the corresponding gene products will facilitate a greater understanding of oogenesis, folliculogenesis, fertilization, and embryonic development. In addition these genes and the corresponding gene products can be used to effect dedifferentiation and/or transdifferentiation of desired somatic cells. The resultant dedifferentiated cells and somatic cells derived therefrom can be used in cell therapies such as in the treatment of cancer, autoimmunity, and other diseases wherein specific types of cells such as hematopoietic cells may be depleted because of the underlying disease or the treatment of the disease. Also, a core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This invention therefore provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes will also expand knowledge on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis. Classifications C12N15/8509 Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic View 4 more classifications WO2008030610A2 WO Application Find Prior Art Similar Other languages French Inventor Jose Bernardo Cibelli Arif Murat Kocabas Bing Lim Hasan H. Otu Original Assignee Michigan State University Priority date 2006-09-08 Family: US (1)WO (1) DateApp/Pub NumberStatus 2007-09-10PCT/US2007/019651 2008-03-13WO2008030610A2Application 2008-12-18WO2008030610A3Application Info Patent citations (1) Non-patent citations (9) Cited by (8) Legal events Similar documents Priority and Related Applications External links Espacenet Global Dossier PatentScope Discuss

EGFR-mutant lung adenocarcinomas (LUAD) display diverse clinical trajectories and are characterized by rapid but short-lived responses to EGFR tyrosine kinase inhibitors (TKIs). Through sequencing of 79 spatially distinct regions from 16 early stage tumors, we show that despite low mutation burdens, EGFR-mutant Asian LUADs unexpectedly exhibit a complex genomic landscape with frequent and early whole-genome doubling, aneuploidy, and high clonal diversity. Multiple truncal alterations, including TP53 mutations and loss of CDKN2A and RB1, converge on cell cycle dysregulation, with late sector-specific high-amplitude amplifications and deletions that potentially beget drug resistant clones. We highlight the association between genomic architecture and clinical phenotypes, such as co-occurring truncal drivers and primary TKI resistance. Through comparative analysis with published smoking-related LUAD, we postulate that the high intra-tumor heterogeneity observed in Asian EGFR-mutant LUAD may be contributed by an early dominant driver, genomic instability, and low background mutation rates.

Tumor initiating cells (TICs) have been implicated in clinical relapse and metastasis of a variety of epithelial cancers, including lung cancer. While efforts toward the development of specific probes for TIC detection and targeting are ongoing, a universal TIC probe has yet to be developed. We report the first TIC-specific fluorescent chemical probe, TiY, with identification of the molecular target as vimentin, a marker for epithelial-to-mesenchymal transition (EMT). TiY selectively stains TICs over differentiated tumor cells or normal cells, and facilitates the visualization and enrichment of functionally active TICs from patient tumors. At high concentration, TiY also shows anti-TIC activity with low toxicity to non-TICs. With the unexplored target vimentin, TiY shows potential as a first universal probe for TIC detection in different cancers.

H3.3 is a histone variant, which is deposited on genebodies and regulatory elements, by Hira, marking active transcription. Moreover, H3.3 is deposited on heterochromatin by Atrx/Daxx complex. The exact role of H3.3 in cell fate transition remains elusive. Here, we investigate the dynamic changes in the deposition of the histone variant H3.3 during cellular reprogramming. H3.3 maintains the identities of the parental cells during reprogramming as its removal at early time-point enhances the efficiency of the process. We find that H3.3 plays a similar role in transdifferentiation to hematopoietic progenitors and neuronal differentiation from embryonic stem cells. Contrastingly, H3.3 deposition on genes associated with the newly reprogrammed lineage is essential as its depletion at the later phase abolishes the process. Mechanistically, H3.3 deposition by Hira, and its K4 and K36 modifications are central to the role of H3.3 in cell fate conversion. Finally, H3.3 safeguards fibroblast lineage by regulating Mapk cascade and collagen synthesis.

How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs). Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines) are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-beta, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% +/- 7.35% TBX3(+) HNF4A(+) human liver bud progenitors and 81.5% +/- 3.2% FAH(+) hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah(-/-)Rag2(-/-)II2rg(-/-) mouse model of liver failure.

Multiple adult tissues are maintained by stem cells of restricted developmental potential which can only form a subset of lineages within the tissue. For instance, the two adult lung epithelial compartments (airways and alveoli) are separately maintained by distinct lineage-restricted stem cells. A challenge has been to obtain multipotent stem cells and/or progenitors that can generate all epithelial cell types of a given tissue. Here we show that mouse Sox9+ multipotent embryonic lung progenitors can be isolated and expanded long term in 3D culture. Cultured Sox9+ progenitors transcriptionally resemble their in vivo counterparts and generate both airway and alveolar cell types in vitro. Sox9+ progenitors that were transplanted into injured adult mouse lungs differentiated into all major airway and alveolar lineages in vivo in a region-appropriate fashion. We propose that a single expandable embryonic lung progenitor population with broader developmental competence may eventually be used as an alternative for region-restricted adult tissue stem cells in regenerative medicine.

While, the genomic landscape of early stage, treatment naïve lung adenocarcinomas (LUADs) has been described quite elaborately in recent literature, the genomic profile of late stage, drug resistant tumors remains largely unknown. Further, most of the published studies are based on smoker dominated Caucasian cohorts and EGFR-mutant LUAD remains under-represented in them. Despite response rates of upto 70% to EGFR tyrosine kinase inhibitors (TKIs), resistance ensues in most of these EGFR-mutant patients, limiting responses to a median of 10-12 months. Thus, to better understand the evolution of these tumors in context of drug resistance, we perform a comparative analysis of the mutational and copy number landscape of early stage, treatment naïve vs late stage, resistant tumors. Whole exome sequencing was performed on: (i) 100 tumor sectors from 24 early stage, treatment naïve EGFR-mutant LUAD cases. (ii) 81 biopsies from 58 late stage, TKI and chemotherapy resistant cases. Copy number analysis using SNP arrays was performed for a subset of these patients. The significantly higher mutation burden in the late stage, drug resistant tumors elucidated a driver mutation landscape beyond just recurrent TP53 mutations, which was dominated by PIK3CA (14%), RB1 (10%), NF1 (7%) and other rare mutations in EGFR (in 5/58 cases), many of which co-occurred with the T790M mutation. Functional studies validated the oncogenicity of some of these rare mutations in the PI3K/AKT1 pathway. The copy number landscape revealed pervasive, truncal genome doubling events in both cohorts (~80% cases). While comparable fraction of genome was affected by overall copy number gains or losses (copy change >=1) across the two cohorts (49.2% vs 46.3%, P=0.51), significantly higher fraction of genome was affected by amplifications (copy change >=2; 8.7% vs 5%, P=0.02) and loss of heterozygosity (LOH; 33.1% vs 20.6%, P=0.003) in the drug resistant tumors compared to the treatment naive tumors. In summary, our study reveals (i) increased mutation and driver burden with co-occurring resistance mutations and (ii) higher fraction of genome with amplifications and LOH in the drug resistant cohort, suggesting ways in which genomic landscape of EGFR-mutant LUAD evolves, potentially making them more tolerant to drug treatment.

This protocol describes the isolation and long-term culture of Sox9 + distal tip lung progenitors from mouse embryonic lung tissue.We have developed a serum-free, feeder-free 3D system to stably expand a 95% pure Sox9 + mouse embryonic lung progenitor population _in vitro_.These lung progenitors retain their multipotency and can differentiate into the full spectrum of lung epithelial lineages-i.e., airway and alveolar lineages-_in vitro_ as well as _in vivo_ upon transplantation into the injured mouse lung.Sox9 + progenitors can be isolated from mouse embryonic lungs within 4h and maintained for at least 6 months \(expanding ~10 20 fold in number).Cultured Sox9 + progenitors can be also cryopreserved and resurrected without losing their properties.The ability to expand multipotent Sox9 + lung progenitors should avail multiple future applications including the generation of lung organoids and gene editing, with rami cations for disease modeling, drug screening and regenerative medicine.

Self-renewing tumor-initiating cells (TICs) are thought to be responsible for tumor recurrence and chemo-resistance. Glycine decarboxylase, encoded by the GLDC gene, is reported to be overexpressed in TIC-enriched primary non-small-cell lung carcinoma (NSCLC). GLDC is a component of the mitochondrial glycine cleavage system, and its high expression is required for growth and tumorigenic capacity. Currently, there are no therapeutic agents against GLDC. As a therapeutic strategy, we have designed and tested splicing-modulating steric hindrance antisense oligonucleotides (shAONs) that efficiently induce exon skipping (half maximal inhibitory concentration [IC50] at 3.5-7 nM), disrupt the open reading frame (ORF) of GLDC transcript (predisposing it for nonsense-mediated decay), halt cell proliferation, and prevent colony formation in both A549 cells and TIC-enriched NSCLC tumor sphere cells (TS32). One candidate shAON causes 60% inhibition of tumor growth in mice transplanted with TS32. Thus, our shAONs candidates can effectively inhibit the expression of NSCLC-associated metabolic enzyme GLDC and may have promising therapeutic implications.

Despite decisive progress in differentiating pluripotent stem cells (PSCs) into diverse cell-types, the often-lengthy differentiation and functional immaturity of such cell-types remain pertinent issues. Here we address the first challenge of prolonged differentiation in the generation of hepatocyte-like cells from PSCs. We delineate a roadmap describing the extracellular signals controlling six sequential branching lineage choices leading from pluripotency to endoderm, foregut, and finally, liver progenitors. By blocking formation of unwanted cell-types at each lineage juncture and manipulating temporally-dynamic signals, we accelerated generation of 89.0±3.1% AFP + human liver bud progenitors and 87.3±9.4% ALBUMIN + hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. 81.5±3.2% of hepatocyte-like cells expressed metabolic enzyme FAH (as assayed by a new knock-in reporter line) and improved short-term survival in the Fah -/- Rag2 -/- Il2rg -/- mouse model of liver failure. Collectively the timed signaling interventions indicated by this developmental roadmap enable accelerated production of human liver progenitors from PSCs.