Developmental studies for therapeutic approaches using endothelial cells derived from mouse embryonic stem cells

The lungs together with the heart are the core of the human body. These organs maintain the homeostasis and provide substantial elements for proper functioning of the organism. Any kind of damage or dysfunction to these organs, results in serious disorders and general imbalance of the organism. Most critical are defects in the endothelial barrier, which consist of the endothelial cells (ECs). The vascular system is padded with a single layer of ECs. Endothelial cell structure and functional integrity are essential for the maintenance of the vessel wall and the circulation. Endothelial cell injury, activation or dysfunction is a feature of many pathologic states, just to mention few like inflammation or effects on vascular tone. Regenerative medicine, including cell replacement therapy, is a promising alternative for disease treatments. The application of in vitro-generated (and if required) genetically amended cells could help to recover the function of damaged parts of a specific organ. One of the approaches is to establish or restore normal function of affected endothelial cells, by therapies based on embryonic stem cells (ESCs). However, generation of those cells is ethically debatable. Development of induced pluripotent stem cells (iPS) from somatic cells has emerged as a solution. This enables the generation of patient- and disease-specific iPS cells, which may produce therapeutic cell populations without immune rejection and moral dispute. Regenerative therapies seem to carry a great promise to treat endothelial dysfunction in respiratory and cardiovascular diseases. To investigate the sites of integration of ES cell-derived endothelial (progenitor) cells, new cell lines from murine ES cells using lentiviral (LV) transduction were generated. In order to achieve the aim it had been decided to test different promoter-resistance-gene combinations. Therefore, a set of vectors containing murine promoters: VE-Cadherin (vascular endothelial) or VEGFR2 (vascular endothelial growth factor) in connection with GFP (green fluorescent protein) and antibiotic resistance genes hygromycin B, neomycin and puromycin, were produced and validated. It was feasible to generate and screen a cell line within a few weeks. The newly established cell lines are suitable for monitoring endothelial differentiation and selection by means of antibiotic resistance. These experiments demonstrated that for an effective antibiotic selection of the desired cell type, further investigations are required. It was possible to produce murine iPS cell lines, containing the above-mentioned transgenes. Carefully selected and purified murine ECs subsets will be used for in vivo studies in tumor angiogenesis models in subsequent studies.Die Lunge, zusammen mit dem Herzen, bildet den Kern des menschlichen Körpers. Gemeinsam erhalten diese Organe die Homöostase und stellen wesentliche Substanzen für die einwandfreie Funktion des Organismus zur Verfügung. Störungen oder Dysfunktionen in Teilen dieser Organe oder deren Gesamtheit führen zu schwerwiegenden Erkrankungen sowie zu einem physiologischen Ungleichgewicht. Sehr kritisch sind dabei Defekte in der endothelialen Barriere, die aus Endothelzellen aufgebaut ist. Das weit verzweigte vaskuläre System ist mit einer einzelnen Schicht dieser Endothelzellen ausgekleidet. Die Endothelzellstruktur sowie die funktionale Integrität dieser Schicht sind essentiell für die Aufrechterhaltung der Blutgefäßwand und deren zirkulatorische Funktion. Verletzungen, Aktivierungen oder Dysfunktionen der Endothelzellen sind Charakteristika verschiedener pathologischer Zustände, z.B. im Zuge von inflammatorischen Prozessen oder bei krankhaften Veränderungen des vaskulären Tonus. Die Ansätze der regenerativen Medizin, die auch eine Zellaustausch-Therapie beinhalten, sind eine vielversprechende Möglichkeit, derartige pathologische Prozesse zu behandeln. Die Verwendung in vitro-generierter und falls notwendig gentechnisch veränderter Zellen könnte zur Regenerierung der Funktion der gestörten Teile des spezifischen Organs beitragen. Die Etablierung von Therapien, basierend auf embryonalen Stammzellen könnte eine Möglichkeit sein, die normale Funktion pathologisch betroffener Endothelzellen wiederherzustellen. Jedoch befindet sich die Generierung derartiger Zellen im Spannungsfeld ethischer Diskussion. Daher ist die Entwicklung induzierter pluripotenter Stammzellen (iPS) aus somatischen Zellen eine Möglichkeit, diesen Debatten entgegenzutreten. Die Etablierung von iPS-Zellen ist individuell je nach Patient und Erkrankung und könnte zur Produktion von therapeutisch verwendbaren Zellpopulationen führen, die ohne Abstoßungsreaktion nutzbar sowie ethisch unbedenklich sind. Derartige regenerative Therapien sind eine vielversprechende Entwicklung zur Behandlung endothelialer Dysfunktionen in respiratorischen und kardiovaskulären Erkrankungen. Um die Funktionalität der aus Stammzellen entwickelten endothelialen (Vorläufer) Zellen zu analysieren und räumliche Verteilung zu verfolgen,wurden unter Verwendung der lentiviralen Transduktion neue Zelllinien aus Mausstammzellen mit verschiedenen Promoter/Resistenzgen-Kombinationen generiert. Die Vektoren besitzen einen spezifischen murinen Promotor, VE-Cadherin (Vaskulär-endotheliales) oder VEGFR2 (Vascular Endothelial Growth Factor Rezeptor), der die Expression von GFP (grün fluoreszierende Protein) reguliert, und ein Gen für antibiotische Resistenz, wie Hygromycin B, Neomycin oder Puromycin. Die verschiedenen hiermit generierten Zelllinien wurden etabliert und erwiesen sich als geeignet, eine Endothelzelldifferenzierung und Selektion auf Basis der spezifischen Antibiotikaresistenzen zu verfolgen. Die experimentellen Ergebnisse zeigen jedoch, dass weitere Untersuchungen notwendig sind, um eine Selektion des gewünschten Zelltyps vornehmen zu können. Die Generierung der murinen iPS Zelllinien, die die oben genannten Transgene beinhalten, befindet sich daher in einem derzeit voranschreitenden Entwicklungsprozess. Sorgfältig selektierte und aufgereinigte murine Endothelzellpopulationen sollen im weiteren Verlauf unter Inanspruchnahme eines Tumor-Angiogenese Modells in in vivo Studien eingesetzt werden

Emerging Therapeutic Approaches for Cystic Fibrosis. From Gene Editing to Personalized Medicine

An improved understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein structure and the consequences of CFTR gene mutations have allowed the development of novel therapies targeting specific defects underlying CF. Some strategies are mutation specific and have already reached clinical development; some strategies include a read-through of the specific premature termination codons (read-through therapies, nonsense mediated decay pathway inhibitors for Class I mutations); correction of CFTR folding and trafficking to the apical plasma membrane (correctors for Class II mutations); and an increase in the function of CFTR channel (potentiators therapy for Class III mutations and any mutant with a residual function located at the membrane). Other therapies that are in preclinical development are not mutation specific and include gene therapy to edit the genome and stem cell therapy to repair the airway tissue. These strategies that are directed at the basic CF defects are now revolutionizing the treatment for patients and should positively impact their survival rates

Isolation, cultivation, and application of primary respiratory epithelial cells obtained by nasal brushing, polyp samples, or lung explants

Here, we present a standardized protocol for isolation, maintenance, and polarization of the respiratory epithelial primary cells from patient samples acquired from nasal brushing, polyp specimens, or lung explants. This protocol generates a clearly defined polarized layer of epithelial cells on filters, with a good number of ciliated cells and a thin layer of mucus. We detail the steps for samples prepared from patients with cystic fibrosis as well as from subjects without cystic fibrosis

Differential CFTR-Interactome Proximity Labeling Procedures Identify Enrichment in Multiple SLC Transporters

International audienceProteins interacting with CFTR and its mutants have been intensively studied using different experimental approaches. These studies provided information on the cellular processes leading to proper protein folding, routing to the plasma membrane, recycling, activation and degradation. Recently, new approaches have been developed based on the proximity labeling of protein partners or proteins in close vicinity and their subsequent identification by mass spectrometry. In this study, we evaluated TurboID- and APEX2-based proximity labeling of WT CFTR and compared the obtained data to those reported in databases. The CFTR-WT interactome was then compared to that of two CFTR (G551D and W1282X) mutants and the structurally unrelated potassium channel KCNK3. The two proximity labeling approaches identified both known and additional CFTR protein partners, including multiple SLC transporters. Proximity labeling approaches provided a more comprehensive picture of the CFTR interactome and improved our knowledge of the CFTR environment

Keratin 8 is a scaffolding and regulatory protein of ERAD complexes

Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild-type (WT) A1AT with a 13-fold and fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the µs heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation and co-immunoprecipitation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients' respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD

Airway surface liquid acidification initiates host defense abnormalities in Cystic Fibrosis

Abstract Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. Morbidity is mainly due to early airway infection. We hypothesized that S. aureus clearance during the first hours of infection was impaired in CF human Airway Surface Liquid (ASL) because of a lowered pH. The ASL pH of human bronchial epithelial cell lines and primary respiratory cells from healthy controls (WT) and patients with CF was measured with a pH microelectrode. The antimicrobial capacity of airway cells was studied after S. aureus apical infection by counting surviving bacteria. ASL was significantly more acidic in CF than in WT respiratory cells. This was consistent with a defect in bicarbonate secretion involving CFTR and SLC26A4 (pendrin) and a persistent proton secretion by ATP12A. ASL demonstrated a defect in S. aureus clearance which was improved by pH normalization. Pendrin inhibition in WT airways recapitulated the CF airway defect and increased S. aureus proliferation. ATP12A inhibition by ouabain decreased bacterial proliferation. Antimicrobial peptides LL-37 and hBD1 demonstrated a pH-dependent activity. Normalizing ASL pH might improve innate airway defense in newborns with CF during onset of S. aureus infection. Pendrin activation and ATP12A inhibition could represent novel therapeutic strategies to normalize pH in CF airways