Identification of a Novel Subset of Human Airway Epithelial Basal Stem Cells.

The basal cell maintains the airway's respiratory epithelium as the putative resident stem cell. Basal cells are known to self-renew and differentiate into airway ciliated and secretory cells. However, it is not clear if every basal cell functions as a stem cell.

Complete correction of murine phenylketonuria by selection-enhanced hepatocyte transplantation.

Background And Aims: Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, the low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by selectively expanding transplanted donor cells until they replace enough of the liver mass to achieve therapeutic benefit.

In vivo tracing of the Cytokeratin 14 lineages using self-cleaving guide RNAs and CRISPR/Cas9.

The current gold-standard for genetic lineage tracing in transgenic mice is based on cell-type specific expression of Cre recombinase. As an alternative, we developed a cell-type specific CRISPR/spCas9 system for lineage tracing. This method relies on RNA polymerase II promoter driven self-cleaving guide RNAs (scgRNA) to achieve tissue-specificity.

Complete correction of murine phenylketonuria by selection-enhanced hepatocyte transplantation.

Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by selectively expanding transplanted donor cells until they replace enough of the liver mass to achieve therapeutic benefit.

Cell networks in the mouse liver during partial hepatectomy.

In solid tissues homeostasis and regeneration after injury involve a complex interplay between many different cell types. The mammalian liver harbors numerous epithelial and non-epithelial cells and little is known about the global signaling networks that govern their interactions. To better understand the hepatic cell network, we isolated and purified 10 different cell populations from normal and regenerative mouse livers.

Postsynaptic density radiation signature following space irradiation.

The response of the brain to space radiation is an important concern for astronauts during space missions. Therefore, we assessed the response of the brain to Si ion irradiation (600 MeV/n), a heavy ion present in the space environment, on cognitive performance and whether the response is associated with altered DNA methylation in the hippocampus, a brain area important for cognitive performance. We determined the effects of Si ion irradiation on object recognition, 6-month-old mice irradiated with Si ions (600 MeV/n, 0.

Strong ubiquitous micro-promoters for recombinant adeno-associated viral vectors.

Significant progress has been made in developing recombinant adeno-associated virus (rAAV) for clinical gene therapy. While rAAV is a versatile gene delivery platform, its packaging limit of 4.7 kb limits the diseases it can target.

Replication stress increases mitochondrial metabolism and mitophagy in FANCD2 deficient fetal liver hematopoietic stem cells.

Fanconi Anemia (FA) is an inherited bone marrow (BM) failure disorder commonly diagnosed during school age. However, in murine models, disrupted function of FA genes leads to a much earlier decline in fetal liver hematopoietic stem cell (FL HSC) number that is associated with increased replication stress (RS). Recent reports have shown mitochondrial metabolism and clearance are essential for long-term BM HSC function.

Micelle-Based Nanocarriers for Targeted Delivery of Cargo to Pancreas.

Innovations in the field of amphiphilic block copolymers have led to the development of a series of attractive polymer-based drug and gene delivery micellar formulations. The amphiphilic block copolymers' low critical micelle concentration (CMC) results in highly stable nanoscale micelles possessing favorable in vivo safety profiles and biocompatibility, making them an excellent carrier choice for the systemic administration of various poorly soluble drugs. These micelles can also be used as an actively targeted drug delivery system.

Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo.

Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci.

Targeted delivery of harmine to xenografted human pancreatic islets promotes robust cell proliferation.

Type 1 diabetes (T1D) occurs as a consequence of the autoimmune destruction of insulin-producing pancreatic beta (β) cells and commonly presents with insulin deficiency and unregulated glycemic control. Despite improvements in the medical management of T1D, life-threatening complications are still common. Beta-cell replication to replace lost cells may be achieved by using small-molecule mitogenic drugs, like harmine.

Human hepatocyte PNPLA3-148M exacerbates rapid non-alcoholic fatty liver disease development in chimeric mice.

Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of broadly immunodeficient chimeric mice respond to hypercaloric diets. As early as four weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatosis with ballooning degeneration selectively in the human graft, followed by pericellular fibrosis after eight weeks of hypercaloric feeding.

Metformin for treatment of cytopenias in children and young adults with Fanconi anemia.

Fanconi anemia (FA), a genetic DNA repair disorder characterized by marrow failure and cancer susceptibility. In FA mice, metformin improves blood counts and delays tumor development. We conducted a single institution study of metformin in nondiabetic patients with FA to determine feasibility and tolerability of metformin treatment and to assess for improvement in blood counts.

Development of a scalable method to isolate subsets of stem cell-derived pancreatic islet cells.

Cell replacement therapy using β cells derived from stem cells is a promising alternative to conventional diabetes treatment options. Although current differentiation methods produce glucose-responsive β cells, they can also yield populations of undesired endocrine progenitors and other proliferating cell types that might interfere with long-term islet function and safety of transplanted cells. Here, we describe the generation of an array of monoclonal antibodies against cell surface markers that selectively label stem cell-derived islet cells.

Proteomic analysis distinguishes extracellular vesicles produced by cancerous versus healthy pancreatic organoids.

Extracellular vesicles (EVs) are produced and released by both healthy and malignant cells and bear markers indicative of ongoing biological processes. In the present study we utilized high resolution flow cytometry to detect EVs in the plasma of patients with pancreatic ductal adenocarcinoma (PDAC) and in the supernatants of PDAC and healthy control (HC) pancreatic organoid cultures. Using ultrafiltration and size exclusion chromatography, PDAC and HC pancreatic organoid EVs were isolated for mass spectrometry analysis.

Cancer stem cells: advances in biology and clinical translation-a Keystone Symposia report.

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control.

Generation of functional ciliated cholangiocytes from human pluripotent stem cells.

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) provides a model for studying the pathogenesis of cholangiopathies and for developing therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of cystic fibrosis transmembrane conductance regulator (CFTR) and the presence of primary cilia capable of sensing flow.

Therapeutic liver repopulation by transient acetaminophen selection of gene-modified hepatocytes.

Gene therapy by integrating vectors is promising for monogenic liver diseases, especially in children where episomal vectors remain transient. However, reaching the therapeutic threshold with genome-integrating vectors is challenging. Therefore, we developed a method to expand hepatocytes bearing therapeutic transgenes.

Is Essential in Proliferating Ductal Cell-Mediated Development of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is frequently driven by aberrant activation and develops in the liver with chronic inflammation. Although the Notch signaling pathway is critically involved in ICC development, detailed mechanisms of Notch-driven ICC development are still unknown. Here, we use mice whose Notch signaling is genetically engineered to show that the Notch signaling pathway, specifically the Notch/Hes1 axis, plays an essential role in expanding ductular cells in the liver with chronic inflammation or oncogenic activation.

Induced Liver Regeneration Enhances CRISPR/Cas9-Mediated Gene Repair in Tyrosinemia Type 1.

The efficiency of gene repair by homologous recombination in the liver is enhanced by CRISP/Cas9 incision near the mutation. In this study, we explored interventions designed to further enhance hepatocyte gene repair in a model of hereditary tyrosinemia. A two-AAV system was employed: one virus carried a Staphylococcus pyogenes Cas9 (SpCas9) expression cassette and the other harbored a U6 promoter-driven sgRNA and a fragment of fumarylacetoacetate hydrolase () genomic DNA as the homologous recombination donor.

Leptin stimulates synaptogenesis in hippocampal neurons via KLF4 and SOCS3 inhibition of STAT3 signaling.

Normal development of neuronal connections in the hippocampus requires neurotrophic signals, including the cytokine leptin. During neonatal development, leptin induces formation and maturation of dendritic spines, the main sites of glutamatergic synapses in the hippocampal neurons. However, the molecular mechanisms for leptin-induced synaptogenesis are not entirely understood.

Insights From Liver-Humanized Mice on Cholesterol Lipoprotein Metabolism and LXR-Agonist Pharmacodynamics in Humans.

Background And Aims: Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver-humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism.