Differential basal-to-apical accessibility of lamin A/C epitopes in the nuclear lamina regulated by changes in cytoskeletal tension.

Nuclear lamins play central roles at the intersection between cytoplasmic signalling and nuclear events. Here, we show that at least two N- and C-terminal lamin epitopes are not accessible at the basal side of the nuclear envelope under environmental conditions known to upregulate cell contractility. The conformational epitope on the Ig-domain of A-type lamins is more buried in the basal than apical nuclear envelope of human mesenchymal stem cells undergoing osteogenesis (but not adipogenesis), and in fibroblasts adhering to rigid (but not soft) polyacrylamide hydrogels. This structural polarization of the lamina is promoted by compressive forces, emerges during cell spreading, and requires lamin A/C multimerization, intact nucleoskeleton-cytoskeleton linkages (LINC), and apical-actin stress-fibre assembly. Notably, the identified Ig-epitope overlaps with emerin, DNA and histone binding sites, and comprises various laminopathy mutation sites. Our findings should help decipher how the physical properties of cellular microenvironments regulate nuclear events.

Stretched extracellular matrix proteins turn fouling and are functionally rescued by the chaperones albumin and casein.

While evidence is mounting that cells exploit protein unfolding for mechanochemical signal conversion (mechanotransduction), what mechanisms are in place to deal with the unwanted consequences of exposing hydrophobic residues upon force-induced protein unfolding? Here, we show that mechanical chaperones exist that can transiently bind to hydrophobic residues that are freshly exposed by mechanical force. The stretch-upregulated binding of albumin or casein to fibronectin fibers is reversible and does not inhibit fiber contraction once the tension is released.

Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes.

BACKGROUND: A clonal cell line that combines both stable hepatic function and proliferation capacity is desirable for in vitro applications that depend on hepatic function, such as pharmacological or toxicological assays and bioartificial liver systems. Here we describe the generation and characterization of a clonal human cell line for in vitro hepatocyte applications. RESULTS: Cell clones derived from human fetal liver cells were immortalized by over-expression of telomerase reverse transcriptase. The resulting cell line, cBAL111, displayed hepatic functionality similar to the parental cells prior to immortalization, and did not grow in soft agar. Cell line cBAL111 expressed markers of immature hepatocytes, like glutathione S transferase and cytokeratin 19, as well as progenitor cell marker CD146 and was negative for lidocaine elimination. On the other hand, the cBAL111 cells produced urea, albumin and cytokeratin 18 and eliminated galactose. In contrast to hepatic cell lines NKNT-3 and HepG2, all hepatic functions were expressed in cBAL111, although there was considerable variation in their levels compared with primary mature hepatocytes. When transplanted in the spleen of immunodeficient mice, cBAL111 engrafted into the liver and partly differentiated into hepatocytes showing expression of human albumin and carbamoylphosphate synthetase without signs of cell fusion. CONCLUSION: This novel liver cell line has the potential to differentiate into mature hepatocytes to be used for in vitro hepatocyte applications.

Isolation of primary human hepatocytes after partial hepatectomy: criteria for identification of the most promising liver specimen.

Demands for primary human hepatocytes are continuously increasing, while supply is insufficient due to limited cell sources. To improve cell availability, the present study investigates the influence of donor liver characteristics on the outcome of hepatocyte isolation from surgically removed liver tissue (n = 50). Hepatocytes were isolated from liver specimens using a standardized two-step collagenase perfusion technique. The patient's sex, previous chemotherapy, or histopathology have shown no influence. Donor age significantly affected the isolation outcome, but was not found suitable for predicting cell yields. Preoperative blood parameters did not correlate with cell yield, although cell function was affected: total protein, albumin synthesis, and cell viability were significantly decreased for serum gamma-glutamyl-transferase (GGT) levels >60 U/L. Specimens from patients with benign diseases gave significantly higher cell yields than tissue removed due to secondary and primary tumors, respectively. The indication for surgery is a valuable basis for identifying the most yielding specimens. Hepatocytes from donors with high GGT levels appear to show reduced functional properties.

Isolation and culture of different epidermal and dermal cell types from human scalp suitable for the development of a therapeutical cell spray.

BACKGROUND: Previous studies demonstrated, that cultured epithelial autografts (CEA) can be isolated and skin cell sprays can be produced for application on different types of wounds. The purpose of the present study was to determine which cell types can be isolated from the human scalp and whether these cells can be used for spray transplantation. METHODS: Outer root sheath cells (ORS), keratinocytes, melanocytes, dermal papilla cells (DP), and dermal sheath cells (DSC) were isolated from human scalp tissue. Isolated cells were characterized, expanded and sprayed in an in vitro model. Growth behaviour, morphology and cell counts were compared with non-sprayed cells. RESULTS: With acceptable time, equipment and laboratory personnel a sufficient amount of keratinocytes, ORS, melanocytes, DP cells and DSC cells could be achieved. The cells are sufficient for application as a cell spray. Cells, positive for Integrin alpha6, Cytokeratin 19, CD73 and CD105 were identified within the cultures. CONCLUSIONS: Human scalp is suitable to gain epidermal and dermal cells for the development of therapeutic cell spray transplantation. Further studies have to determine, whether these cells can be combined to produce wound specific skin substitutes.

Hypothermia suppresses inflammation via ERK signaling pathway in stimulated microglial cells.

Hypothermic perfusion is a standard method for neuroprotection during cardiac surgery in children. However, the cellular responses underlying these mechanisms have not been clearly elucidated. In the present study we demonstrated that the inflammatory response of stimulated microglial cells is significantly reduced after moderate hypothermia. Continuous hypothermia caused a diminished NO release. Moderate hypothermia and rewarming caused a downregulation of phosphorylated MEK, ERK and iNOS-expression, diminished cytokine release and reduced CD-11a and ICAM-1 expression. Thus, neuroprotection offered by hypothermia could be attributed to reduced cytotoxic products released from stimulated microglial cells mediated by the MEK/ERK signal transduction pathway.

Continuously microscopically observed and process-controlled cell culture within the SlideReactor: proof of a new concept for cell characterization.

Certain cell types, especially primary human cells, favor a well-defined culture environment offering continuous supply of nutrients and oxygen and waste product removal. Several bioreactors based on special matrices or hollow fibers have been developed that provide such conditions. However, characterization of matrix re-organization or growth of tissue within these systems is possible only after culture termination. Evaluation of the influence of certain medium additives or culture conditions (e.g., temperature, oxygenation) on cell viability, expansion, and differentiation within these systems remains a challenging task. The SlideReactor, a miniaturized hollow fiber-based bioreactor, was developed to enable the observation of cells during culture. An operation concept offering predefined conditions for various cell types has been designed. For proof of concept, primary human cells (hepatocytes, fibroblasts, keratinocytes) and cell lines (HepG2, HuH7, C3A, WiDr, SkHep1) were cultured and observed. A series of experiments (n=40) showed the feasibility of the set-up; determination of process parameters and continuous observation is possible. The SlideReactor may serve as a simple and cost-efficient tool for cell characterization and optimization of cell-culture conditions.

Safety and efficacy of cardiopoietic stem cells in the treatment of post-infarction left-ventricular dysfunction - From cardioprotection to functional repair in a translational pig infarction model.

To date, clinical success of cardiac cell-therapies remains limited. To enhance the cardioreparative properties of stem cells, the concept of lineage-specification through cardiopoietic-guidance has been recently suggested. However, so far, only results from murine studies and from a clinical pilot-trial in chronic heart-failure (CHF) are available, while systematic evidence of its therapeutic-efficacy is still lacking. Importantly, also no data from large animals or for other indications are available. Therefore, we here investigate the therapeutic-efficacy of human cardiopoietic stem cells in the treatment of post-infarction LV-dysfunction using a translational pig-model. Using growth-factor priming, lineage-specification of human bone-marrow derived MSCs was achieved to generate cardiopoietic stem cells according to GMP-compliant protocols. Thereafter, pigs with post-infarction LV-dysfunction (sub-acute phase;1-month) were randomized to either receive transcatheter NOGA 3D electromechanical-mapping guided intramyocardial transplantation of cardiopoietic cells or saline (control). After 30days, cardiac MRI (cMRI) was performed for functional evaluation and in-vivo cell-tracking. This approach was coupled with a comprehensive post-mortem cell-fate and mode-of-repair analysis. Cardiopoietic cell therapy was safe and ejection-fraction was significantly higher when compared to controls (p = 0.012). It further prevented maladaptive LV-remodeling and revealed a significantly lower relative and total infarct-size (p = 0.043 and p = 0.012). As in-vivo tracking and post-mortem analysis displayed only limited intramyocardial cardiopoietic cell-integration, the significant induction of neo-angiogenesis (∼40% higher; p = 0.003) and recruitment of endogenous progenitors (∼2.5x higher; p = 0.008) to the infarct border-zone appeared to be the major modes-of-repair. This is the first report using a pre-clinical large animal-model to demonstrate the safety and efficacy of cardiopoietic stem cells for the treatment of post-infarction LV-dysfunction to prevent negative LV-remodeling and subsequent CHF. It further provides insight into post-delivery cardiopoietic cell-fate and suggests the mechanisms of cardiopoietic cell-induced cardiac-repair. The adoption of GMP-/GLP-compliant methodologies may accelerate the translation into a phase-I clinical-trial in patients with post-ischemic LV-dysfunction broadening the current indication of this interesting cell-type.

The ultrastructure of fibronectin fibers pulled from a protein monolayer at the air-liquid interface and the mechanism of the sheet-to-fiber transition.

Fibronectin is a globular protein that circulates in the blood and undergoes fibrillogenesis if stretched or under other partially denaturing conditions, even in the absence of cells. Stretch assays made by pulling fibers from droplets of solutions containing high concentrations of fibronectin have previously been introduced in mechanobiology, particularly to ask how bacteria and cells exploit the stretching of fibronectin fibers within extracellular matrix to mechano-regulate its chemical display. Our electron microscopy analysis of their ultrastructure now reveals that the manually pulled fibronectin fibers are composed of densely packed lamellar spirals, whose interlamellar distances are dictated by ion-tunable electrostatic interactions. Our findings suggest that fibrillogenesis proceeds via an irreversible sheet-to-fiber transition as the fibronectin sheet formed at the air-liquid interface of the droplet is pulled off by a sharp tip. This far from equilibrium process is driven by the externally applied force, interfacial surface tension, shear-induced fibronectin self-association, and capillary force-induced buffer drainage. The ultrastructural characterization is then contrasted with previous FRET studies that characterized the molecular strain within these manually pulled fibers. Particularly relevant for stretch-dependent binding studies is the finding that the interior fiber surfaces are accessible to nanoparticles smaller than 10 nm. In summary, our study discovers the underpinning mechanism by which highly hierarchically structured fibers can be generated with unique mechanical and mechano-chemical properties, a concept that might be extended to other bio- or biomimetic polymers.

Effect of matrine on primary human hepatocytes in vitro.

Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO2 (-) induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.