pH-controlled recovery of placenta-derived mesenchymal stem cell sheets.

Widely used in different biomedical applications, polyelectrolyte multilayers provide inter alia an attractive way for manufacturing of bio-functionalized, stimuli responsive surface coatings to control cellular behavior. In this study a novel polyelectrolyte-based platform for the engineering and controllable detachment of human mesenchymal stem cell (MSC) sheets is presented. Thin films obtained by layer-by-layer deposition of cationic poly(allylamine hydrochloride) (PAH) and anionic poly(styrene sulfonate) (PSS) polyelectrolytes on conductive indium tin oxide (ITO) electrodes allowed for the fast formation of viable sheets from human placenta-derived mesenchymal stem cells (PD-MSCs).

Biological and physicochemical characterization of a serum- and xeno-free chemically defined cryopreservation procedure for adult human progenitor cells.

While therapeutic cell transplantations using progenitor cells are increasingly evolving towards phase I and II clinical trials and chemically defined cell culture is established, standardization in biobanking is still in the stage of infancy. In this study, the EU FP6-funded CRYSTAL (CRYo-banking of Stem cells for human Therapeutic AppLication) consortium aimed to validate novel Standard Operating Procedures (SOPs) to perform and validate xeno-free and chemically defined cryopreservation of human progenitor cells and to reduce the amount of the potentially toxic cryoprotectant additive (CPA) dimethyl sulfoxide (DMSO). To achieve this goal, three human adult progenitor and stem cell populations-umbilical cord blood (UCB)-derived erythroid cells (UCB-ECs), UCB-derived endothelial colony forming cells (UCB-ECFCs), and adipose tissue (AT)-derived mesenchymal stromal cells (AT-MSCs)-were cryopreserved in chemically defined medium supplemented with 10% or 5% DMSO.

Optimization of the culturing conditions of human umbilical cord blood-derived endothelial colony-forming cells under xeno-free conditions applying a transcriptomic approach.

Establishment of fetal bovine serum (FBS)-free cell culture conditions is essential for transplantation therapies. Blood-derived endothelial colony-forming cells (ECFCs) are potential candidates for regenerative medicine applications. ECFCs were isolated from term umbilical cord blood units and characterized by flow cytometry, capillary formation and responsiveness to cytokines.

Injectable candidate sealants for fetal membrane repair: bonding and toxicity in vitro.

Objective: This study was undertaken to test injectable surgical sealants that are biocompatible with fetal membranes and that are to be used eventually for the closure of iatrogenic membrane defects.

Study Design: Dermabond (Ethicon Inc, Norderstedt, Germany), Histoacryl (B. Braun GmbH, Tuttlingen, Germany), and Tissucol (Baxter AG, Volketwil, Switzerland) fibrin glue, and 3 types of in situ forming poly(ethylene glycol)-based polymer hydrogels were tested for acute toxicity on direct contact with fetal membranes for 24 hours.

Multipotent mesenchymal stem cells from human placenta: critical parameters for isolation and maintenance of stemness after isolation.

Objective: This study was undertaken to isolate and characterize multipotent mesenchymal stem cells from term human placenta (placenta-derived mesenchymal stem cells, PD-MSCs).

Study Design: Sequential enzymatic digestion was used to isolate PD-MSCs in which trypsin removes the trophoblast layer, followed by collagenase treatment of remaining placental tissue. Karyotype, phenotype, growth kinetics, and differentiability of PD-MSC isolates from collagenase digests were analyzed.

Stem cell-like human endothelial progenitors show enhanced colony-forming capacity after brief sevoflurane exposure: preconditioning of angiogenic cells by volatile anesthetics.

Background: Endothelial progenitor cells play a pivotal role in tissue repair, and thus are used for cell replacement therapies in "regenerative medicine." We tested whether the anesthetic sevoflurane would modulate growth or mobilization of these angiogenic cells.

Methods: In an in vitro model, mononuclear cells isolated from peripheral blood of healthy donors were preconditioned with sevoflurane (3 times 30 min at 2 vol% interspersed by 30 min of air).

Engineered polyelectrolyte multilayer substrates for adhesion, proliferation, and differentiation of human mesenchymal stem cells.

Polyelectrolyte multilayer coatings have emerged as substrates to control cellular behavior, but interactions with human multipotent mesenchymal stromal cells (MSCs) have not been studied. We looked at layer-by-layer coatings of cationic poly-L-lysine (PLL) and anionic hyaluronic acid (HA) as substrates for MSCs of placenta and adipose tissue. This system allows for modulation of thickness (number of deposition cycles), stiffness (chemical cross-linking of bulk layer), and adhesiveness (fibronectin (FN) interface).

Relation between mechanical properties and microstructure of human fetal membranes: an attempt towards a quantitative analysis.

Objective: We sought to measure the mechanical baseline behavior of fetal membranes in order to determine constitutive mechanical model parameters for fetal membranes, and to examine their relation to molecular correlates for mechanical function, i.e. collagen and elastin.

Decreased umbilical artery compliance and igf-I plasma levels in infants with intrauterine growth restriction - implications for fetal programming of hypertension.

Epidemiological studies link intrauterine growth restriction (IUGR) to arterial hypertension in adulthood. We compared umbilical arteries from IUGR (n=12, <5th weight percentile) vs. appropriate for gestational age (AGA) infants (n=12) using structural and functional analyses.

Comparative characterization of cultured human term amnion epithelial and mesenchymal stromal cells for application in cell therapy.

Emerging evidence suggests human amnion tissue as a valuable source of two distinct types of pluripotent cells, amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs), for applications in cell replacement therapy. For some approaches, it may be necessary to culture and differentiate these cells before they can be transplanted. No systematic attempt has been yet made to determine the quantity and quality of amnion cells after isolation and culture.

Bioengineering of foetal membrane repair.

Preterm premature rupture of the foetal membranes (früher vorzeitiger Blasensprung) has remained a devastating complication of pregnancy with very high risk of pregnancy loss. Several methods of sealing spontaneously ruptured membranes to stop amniotic fluid leakage and prolong pregnancy have been tested, but no one of them has achieved a clinical breakthrough. Also, needle and foetoscopic puncture of membranes for diagnostic or surgical interventions in the amniotic cavity carry a significant risk of persistent membrane leakage and subsequent rupture - thus limiting the developing field of intrauterine foetal surgery.

The dosage dependence of VEGF stimulation on scaffold neovascularisation.

Growth factors are often used in tissue regeneration to stimulate vascularisation of polymeric scaffolds, with vascular endothelial growth factor (VEGF) having been extensively studied for short-term vessel ingrowth. We have therefore evaluated the effect of different concentrations of VEGF on the vascularisation of a porous scaffold in the short-, intermediate- and long-term, by delivering 15, 150 and 1500ng VEGF/day to polyurethane scaffolds by osmotic pumps for up to 6 weeks. An increased vascularisation months after termination of VEGF delivery was only achieved with 150ng/day (46%, p<0.

The role of actively released fibrin-conjugated VEGF for VEGF receptor 2 gene activation and the enhancement of angiogenesis.

A major challenge for therapeutic delivery of angiogenic agents such as vascular endothelial growth factor (VEGF) is to achieve sustained, low dose signaling leading to durable neovessel formation. To this end, we recently created a variant of VEGF(121), TG-VEGF(121) that directly binds to fibrin and gets released locally in proteolysis-triggered manner. Here we combined noninvasive biophotonic monitoring of VEGF receptor 2 gene activation in transgenic VEGFR2-luc mice and histomorphometry to compare endothelial activation and long-term neovascularization by actively released TG-VEGF(121)versus passively released, diffusible wild-type VEGF(121) in subcutaneous fibrin implants.

Fetoscopic closure of punctured fetal membranes with acellular human amnion plugs in a rabbit model.

Objective: To explore a surgical plug formed from decellularized term human amnion membrane for fetoscopic closure of iatrogenic defects in fetal membranes in a rabbit model.

Methods: The study was performed in eight rabbit does. Punctures were created at midgestational day 23 by 14-gauge needle fetoscopy on surgically exposed rabbit amniotic sacs.

Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering.

The molecular engineering of cell-instructive artificial extracellular matrices is a powerful means to control cell behavior and enable complex processes of tissue formation and regeneration. This work reports on a novel method to produce such smart biomaterials by recapitulating the crosslinking chemistry and the biomolecular characteristics of the biopolymer fibrin in a synthetic analog. We use activated coagulation transglutaminase factor XIIIa for site-specific coupling of cell adhesion ligands and engineered growth factor proteins to multiarm poly(ethylene glycol) macromers that simultaneously form proteolytically sensitive hydrogel networks in the same enzyme-catalyzed reaction.

EphA4 signaling regulates phospholipase Cgamma1 activation, cofilin membrane association, and dendritic spine morphology.

Specialized postsynaptic structures known as dendritic spines are the primary sites of glutamatergic innervation at synapses of the CNS. Previous studies have shown that spines rapidly remodel their actin cytoskeleton to modify their shape and this has been associated with changes in synaptic physiology. However, the receptors and signaling intermediates that restructure the actin network in spines are only beginning to be identified.

Enhanced intimal thickening of expanded polytetrafluoroethylene grafts coated with fibrin or fibrin-releasing vascular endothelial growth factor in the pig carotid artery interposition model.

Objective: Intimal hyperplasia and surface thrombogenicity are major factors in the high failure rate of synthetic small-diameter bypass grafts. Vascular endothelial growth factor is a potent stimulus for endothelial growth, and its provision in a fibrin matrix coating at the luminal graft surface may hold a key to spontaneous graft endothelialization and improved graft patency.

Methods: Pigs underwent bilateral carotid artery interposition of expanded polytetrafluoroethylene grafts either impregnated with fibrin (n = 11)--engineered to locally release vascular endothelial growth factor121 (vascular endothelial growth factor-fibrin; n = 11)--or left uncoated (n = 12).

MnSOD marks cord blood late outgrowth endothelial cells and accompanies robust resistance to oxidative stress.

Cord blood is source of colony-forming progenitors to vascular endothelial cells for potential use in cell therapies. These cells-called blood late outgrowth endothelial cells (OECs)-have undergone endothelial differentiation, but appear to still possess functional properties different from mature endothelial cells. A large-scale comparative proteomics screen of cord blood OECs versus human vein endothelial cells (HUVECs) using two-dimensional gel electrophoresis and mass spectrometry identified specific expression of manganese superoxide dismutase (MnSOD), a key antioxidant enzyme expressed in the mitochondria, in OECs but not in HUVECs.

PPS-PEG surface coating to reduce thrombogenicity of small diameter ePTFE vascular grafts.

Aims: Patency failure of small vascular synthetic grafts is still a major problem for coronary and peripheral revascularization. Thus, three new surface coatings of small synthetic grafts were tested in an acute pig model to evaluate their thrombogenicity (extracorporeal arterio-venous shunt) and in a chronic rat model to evaluate the tissue reaction they induced (subcutaneous implantation).

Methods: In five domestic pigs (25-30 kg) an extracorporeal femoro-femoral arterio-venous shunt model was used.

Synergy between interstitial flow and VEGF directs capillary morphogenesis in vitro through a gradient amplification mechanism.

Cell organization is largely orchestrated by extracellular gradients of morphogenetic proteins. VEGF, an essential factor for capillary formation, is stored in the extracellular matrix, but the mechanisms by which it and other matrix-bound morphogens are mobilized to form spatial gradients are poorly understood. Here, we suggest an efficient mechanism for morphogen gradient generation by subtle biophysical forces in an in vitro model of capillary morphogenesis.

Effects of protein and gene transfer of the angiopoietin-1 fibrinogen-like receptor-binding domain on endothelial and vessel organization.

The vessel-stabilizing effect of angiopoietin-1 (Ang1)/Tie2 receptor signaling is a potential target for pro-angiogenic therapies as well as anti-angiogenic inhibition of tumor growth. We explored the endothelial and vascular specific activities of the Ang1 monomer, i.e.

Adult 'endothelial progenitor cells'. Renewing vasculature.

During embryogenesis, endothelial progenitor cells participate in the initial processes of primitive blood vessel formation (vasculogenesis). It has become evident that progenitors to vascular endothelial cells also exist in the adult. Endothelial progenitors normally reside in the adult bone marrow but may become mobilized into circulation by cytokine or angiogenic growth factor signals from the periphery, enter extravascular tissue, and promote de novo vessel formation by virtue of physically integrating into vessels and/or supplying growth factors (adult vasculogenesis).

Endothelial cell proliferation and progenitor maturation by fibrin-bound VEGF variants with differential susceptibilities to local cellular activity.

A number of vascular therapies could benefit from advanced methods for presentation of angiogenic growth factors, including growth of endothelium on small caliber vascular grafts and revascularization of ischemic tissue through induction of collateral vessels and microvessels. To explore methods to optimize the presentation and release of angiogenic factors in such applications in device integration and tissue repair, we studied three variant forms of vascular endothelial growth factor 121 (VEGF121), each with differential susceptibility to local cellular proteolytic activity, formulated within fibrin matrices. (1) The prototypic variant alpha2PI(1-8)-VEGF121 remains immobilized in fibrin matrices until its liberation by cell-associated enzymes, such as plasmin, that degrade the fibrin network [slow, cell-demanded release; J.