Improved Metabolic Pathways of Glycolysis, Glycogen Synthesis, the Urea Cycle, and Cytochrome Peroxidase Oxidative Reabsorption in a Miniature Bioreactor.

BACKGROUND/AIMS: Bioreactor-based bioartificial liver support systems have had limited success in a translational setting and at preclinical stages. None of the existing systems monitor the metabolic pathways of glycolysis, glycogen synthesis, the urea cycle, and cytochrome peroxidase oxidative reabsorption. Herein, we designed a bioreactor that mimics the human liver microenvironment in vivo and monitors different hepatic metabolic pathways in order to help establish in vitro culture conditions for improved glycolysis, glycogen synthesis, the urea cycle, cytochrome peroxidase oxidative reabsorption and improved hepatic functions in a miniature bioartificial liver. An abnormality in such pathways negatively influences survivability and hepatic functions, including spontaneous liver regeneration. METHODS: We investigated the metabolic functions of primary mouse adult hepatocytes cultured in a three-dimensional configuration under direct oxygenation conditions (5%, 10%, 20%, and 40% O2) for 14 days in the bioreactor. We analyzed the expression of the genes of hepatic metabolic pathways, such as glycolysis (glucokinase, phosphofructokinase, and pyruvate kinase), glycogen synthesis (glycogen synthetase, UTP glucose-1-phosphate uridylylisomerase, phosphoglucomutase, and glycogen phosphorylase), the urea cycle (arginase, ornithine carbomoyltransferase, fumarate hydratase), oxidative reabsorption (peroxidase), and cytochrome peroxides (catalase and superoxide dismutase), and compared it with the level in vivo. The metabolic mini-map was used to represent the above-mentioned metabolic genes. RESULTS: Increased urea secretion under normoxia and hyperoxia conditions (20% and 40% O2, respectively) was observed, while albumin secretion was decreased in hyperoxic cultures. Lactate formation was up to 15 mg/L-g/h-h/106 cells, 2 mg/L-g/h-h/106 cells, and 0.2 mg/L-c/h-h/106 cells in 5%, 20%, and 40% O2 conditions, respectively while glucose consumption was enhanced under hypoxic conditions (5% and 10% O2). Cellular membrane integrity was estimated by lactate dehydrogenase assay and was found to be negligible in only 20% and 40% O2 conditions. The expression of the phase II enzyme UDP-glucuronosyltransferase was only upregulated in 20% oxygenation. CONCLUSION: Taken together, 20% O2 was found to be an optimal condition for the long-term culture (up to 14 days) of hepatocytes that promoted the expression of genes in metabolic pathways such as glycolysis, glycogen synthesis, the urea cycle, and cytochrome peroxidase oxidative reabsorption, and improved hepatic functions in a miniature bioreactor for bioartificial liver construction.

Isolation and Characterization of CD271⁺ Stem Cells Derived from Sheep Dermal Skin.

BACKGROUND: Mesenchymal stem cells (MSCs) have great promise in the field of regenerative medicine due to their differentiation potential into several lineages. Besides the bone marrow, MSCs can be obtained from the dermis, which represents a large stem cell reservoir in the skin. Sheep provide an appropriate large animal model for preclinical studies. In this study, we focused on the isolation and characterization of MSCs from sheep dermis as an alternative to bone marrow MSCs (bmMSCs). METHODS: Primary ovine cells were obtained from the dermis for comparison with bone marrow. CD271(+)/45(-) dermal MSCs (CD271-dMSCs), which were sorted by flow cytometry, and plastic-adherent bmMSCs were examined for morphology, proliferation and senescence-associated ß-galactosidase activity in both low and high oxygen conditions. CD271 expression on cultured cells was assessed by flow cytometry. Adipogenic and osteogenic potentials of CD271-dMSCs were evaluated by oil red O and von Kossa staining. Chondrogenic capacity of CD271-dMSCs and CD271(+)/CD45(-) bone marrow cells (CD271-bmMSCs) was detected using immunohistochemistry and measurement of sulfated glycosaminoglycans. RESULTS: The cell proliferation assay demonstrated no significant difference between CD271-dMSCs and bmMSCs under low oxygen conditions. Cultured CD271-dMSCs revealed much more CD271 expression compared to CD271-bmMSCs. CD271-dMSCs and CD271-bmMSCs showed basically similar expression of the cartilage-specific proteins aggrecan and collagen type II, although with a stronger staining in CD271-bmMSC-derived cultures. Remarkably, there was co-expression of CD271 and aggrecan during chondrogenic differentiation, suggesting an involvement of CD271 in chondrogenesis. CONCLUSION: Based on these findings, CD271-dMSCs might serve as an appropriate alternative cell source in preclinical research.

Stem-cell-based, tissue engineered tracheal replacement in a child: a 2-year follow-up study.

BACKGROUND: Stem-cell-based, tissue engineered transplants might offer new therapeutic options for patients, including children, with failing organs. The reported replacement of an adult airway using stem cells on a biological scaffold with good results at 6 months supports this view. We describe the case of a child who received a stem-cell-based tracheal replacement and report findings after 2 years of follow-up. METHODS: A 12-year-old boy was born with long-segment congenital tracheal stenosis and pulmonary sling. His airway had been maintained by metal stents, but, after failure, a cadaveric donor tracheal scaffold was decellularised. After a short course of granulocyte colony stimulating factor, bone marrow mesenchymal stem cells were retrieved preoperatively and seeded onto the scaffold, with patches of autologous epithelium. Topical human recombinant erythropoietin was applied to encourage angiogenesis, and transforming growth factor ß to support chondrogenesis. Intravenous human recombinant erythropoietin was continued postoperatively. Outcomes were survival, morbidity, endoscopic appearance, cytology and proteomics of brushings, and peripheral blood counts. FINDINGS: The graft revascularised within 1 week after surgery. A strong neutrophil response was noted locally for the first 8 weeks after surgery, which generated luminal DNA neutrophil extracellular traps. Cytological evidence of restoration of the epithelium was not evident until 1 year. The graft did not have biomechanical strength focally until 18 months, but the patient has not needed any medical intervention since then. 18 months after surgery, he had a normal chest CT scan and ventilation-perfusion scan and had grown 11 cm in height since the operation. At 2 years follow-up, he had a functional airway and had returned to school. INTERPRETATION: Follow-up of the first paediatric, stem-cell-based, tissue-engineered transplant shows potential for this technology but also highlights the need for further research. FUNDING: Great Ormond Street Hospital NHS Trust, The Royal Free Hampstead NHS Trust, University College Hospital NHS Foundation Trust, and Region of Tuscany.

Three dimensional cultures of rat liver cells using a natural self-assembling nanoscaffold in a clinically relevant bioreactor for bioartificial liver construction.

Till date, no bioartificial liver (BAL) procedure has obtained FDA approval or widespread clinical acceptance, mainly because of multifactorial limitations such as the use of microscale or undefined biomaterials, indirect and lower oxygenation levels in liver cells, short-term undesirable functions, and a lack of 3D interaction of growth factor/cytokine signaling in liver cells. To overcome preclinical limitations, primary rat liver cells were cultured on a naturally self-assembling peptide nanoscaffold (SAPN) in a clinically relevant bioreactor for up to 35 days, under 3D interaction with suitable growth factors and cytokine signaling agents, alone or combination (e.g., Group I: EPO, Group II: Activin A, Group III: IL-6, Group IV: BMP-4, Group V: BMP4 + EPO, Group VI: EPO + IL-6, Group VII: BMP4 + IL-6, Group VIII: Activin A + EPO, Group IX: IL-6 + Activin A, Group X: Activin A + BMP4, Group XI: EPO + Activin A + BMP-4 + IL-6 + HGF, and Group XII: Control). Major liver specific functions such as albumin secretion, urea metabolism, ammonia detoxification, phase contrast microscopy, immunofluorescence of liver specific markers (Albumin and CYP3A1), mitochondrial status, glutamic oxaloacetic transaminase (GOT) activity, glutamic pyruvic transaminase (GPT) activity, and cell membrane stability by the lactate dehydrogenase (LDH) test were also examined and compared with the control over time. In addition, we examined the drug biotransformation potential of a diazepam drug in a two-compartment model (cell matrix phase and supernatant), which is clinically important. This present study demonstrates an optimized 3D signaling/scaffolding in a preclinical BAL model, as well as preclinical drug screening for better drug development.

Relation Between Gender and Concomitant Medications With Erythropoietin-Treatment on Wound Healing in Burn Patients. Post Hoc Subgroup-Analysis of the Randomized, Placebo-Controlled Clinical Trial "EPO in Burns".

Burns are leading causes of mortality and morbidity, including prolonged hospitalization, disfigurement, and disability. Erythropoietin (EPO) is a well-known hormone causing erythropoiesis. However, EPO may play a role in healing acute and chronic wounds due to its anti-inflammatory and pro-regenerative effects. Therefore, the large, prospective, placebo-controlled, randomized, double-blind, multi-center clinical trial "EPO in Burns" was initiated to investigate the effects of EPO versus placebo treatment in severely burned patients. The primary endpoint of "EPO in Burns" was defined as the time elapsed until complete re-epithelialization of a defined split skin graft donor site. Additional analyses of post hoc defined subgroups were performed in view of the primary endpoint. The verum (n 45) and control (n 39) groups were compared with regard to the time it took for study wounds (a predefined split skin graft donor site) to reach the three stages of wound healing (re-epithelialization levels). In addition, the effects of gender (females n 18) and concomitant medications insulin (n 36), non-steroidal anti-inflammatory drugs (NSAIDs) (n 41), and vasopressor agents (n 43) were tested. Life tables were used to compare study groups (EPO vs. placebo) within subgroups. The Cox regression model was applied to evaluate interactions between the study drug (EPO) and concomitant medications for each re-epithelialization level. Using our post hoc defined subgroups, we observed a lower chance of wound healing for women compared to men (in terms of hazard ratio: hr100%: 5.984 [95%-CI: (0.805-44.490), p = 0.080]) in our study population, regardless of the study medication. In addition, results indicated an earlier onset of re-epithelialization in the first days of EPO treatment (EPO: 10% vs. Placebo: 3%). Moreover, the interpretation of the hazard ratio suggested EPO might have a positive, synergistic effect on early stages of re-epithelialization when combined with insulin [hr50%: 1.307 (p = 0.568); hr75%: 1,199 (p = 0.715)], as well as a stabilizing effect on critically ill patients [reduced need for vasopressors in the EPO group (EPO: 44% vs. Placebo 59%)]. However, additional high-quality data from clinical trials designed to address these endpoints are required to gain further insight into these effects.

Determination of pore size distribution at the cell-hydrogel interface.

BACKGROUND: Analyses of the pore size distribution in 3D matrices such as the cell-hydrogel interface are very useful when studying changes and modifications produced as a result of cellular growth and proliferation within the matrix, as pore size distribution plays an important role in the signaling and microenvironment stimuli imparted to the cells. However, the majority of the methods for the assessment of the porosity in biomaterials are not suitable to give quantitative information about the textural properties of these nano-interfaces. FINDINGS: Here, we report a methodology for determining pore size distribution at the cell-hydrogel interface, and the depth of the matrix modified by cell growth by entrapped HepG(2) cells in microcapsules made of 0.8% and 1.4% w/v alginate. The method is based on the estimation of the shortest distance between two points of the fibril-like network hydrogel structures using image analysis of TEM pictures. Values of pore size distribution determined using the presented method and those obtained by nitrogen physisorption measurements were compared, showing good agreement. A combination of these methodologies and a study of the cell-hydrogel interface at various cell culture times showed that after three days of culture, HepG(2) cells growing in hydrogels composed of 0.8% w/v alginate had more coarse of pores at depths up to 40 nm inwards (a phenomenon most notable in the first 20 nm from the interface). This coarsening phenomenon was weakly observed in the case of cells cultured in hydrogels composed of 1.4% w/v alginate. CONCLUSIONS: The method purposed in this paper allows us to obtain information about the radial deformation of the hydrogel matrix due to cell growth, and the consequent modification of the pore size distribution pattern surrounding the cells, which are extremely important for a wide spectrum of biotechnological, pharmaceutical and biomedical applications.

Moving towards in situ tracheal regeneration: the bionic tissue engineered transplantation approach.

In June 2008, the world's first whole tissue-engineered organ - the windpipe - was successfully transplanted into a 31-year-old lady, and about 18 months following surgery she is leading a near normal life without immunosuppression. This outcome has been achieved by employing three groundbreaking technologies of regenerative medicine: (i) a donor trachea first decellularized using a detergent (without denaturing the collagenous matrix), (ii) the two main autologous tracheal cells, namely mesenchymal stem cell derived cartilage-like cells and epithelial respiratory cells and (iii) a specifically designed bioreactor that reseed, before implantation, the in vitro pre-expanded and pre-differentiated autologous cells on the desired surfaces of the decellularized matrix. Given the long-term safety, efficacy and efforts using such a conventional approach and the potential advantages of regenerative implants to make them available for anyone, we have investigated a novel alternative concept how to fully avoid in vitro cell replication, expansion and differentiation, use the human native site as micro-niche, potentiate the human body's site-specific response by adding boosting, permissive and recruitment impulses in full respect of sociological and regulatory prerequisites. This tissue-engineered approach and ongoing research in airway transplantation is reviewed and presented here.

Telomerase activity and hepatic functions of rat embryonic liver progenitor cell in nanoscaffold-coated model bioreactor.

Presently, there is growing interest on telomerase activity in all cells (somatic cells, stem cells, cancerous cells and others) since this activity is associated with cellular changes such as proliferation, differentiation, immortalization, cell injury and ageing. Telomerase activity is absent in most of the somatic cells but present in over 90% of cancerous cells and other immortalized cell lines. In our present study, we cultured a rat embryonal liver progenitor cell line RLC-18 in a self-assembly nanostructured scaffold-coated bioreactor (NCB), collagen-coated plates (CCP) and uncoated plates (UP), and evaluated changes of telomerase activity by non radioactive techniques (Telo TAGGG Telomerase PCR ELISA, cell proliferation based on mitochondria number by MTT assay and hepatic functions such as albumin secretion, urea metabolism, Cytochrome P450 activity like ethoxyresorufin-O-deethylase (EROD) activity. We found less telomerase activity and less cell proliferation, but more hepatic functions on the NCB than on the CCP and UP. Our data support the concept that cell-scaffold interaction may play a significant in controlling the telomerase activity as well as enhanced hepatic functions. Although our present study does not focus on the exact mechanism of telomerase regulation, our result may provide basic clues on cell differentiation whereby telomerase activity inhibits differentiation of cells as in the rat embryonic liver cell line, may be regulated by cell-scaffold interaction and where there is less proliferation, cells perform enhanced hepatic functions, thereby implying that bioartificial liver support may be possible.

Cryopreservation of primary porcine liver cells in an organotypical sandwich model in a clinically relevant flat membrane bioreactor.

To overcome the logistical difficulties of continuously supplying freshly-isolated, primary porcine liver cells to bioartificial liver support bioreactors, we developed a cryopreservation method using an organotypical sandwich model in a flat membrane bioreactor (FMB). We measured albumin secretion rate, urea synthesis rate and 7-ethoxy coumarin (ECOD) in long-term cultures of cryopreserved cells (up to 14 days). The albumin secretion rate was 62% that of non-cryopreserved cells at days 11 and 14. The ECOD activity was 54% that of fresh, control cells initially and increased up to 79% by the 14th day. The urea synthesis rate was stable at 60% that of the control. This study showed that cryopreserved cells can recover liver-specific functions. This result has the potential to dramatically expand the clinical application of bioartificial liver supports.

Determination of the decay rate constant for hepatocytes immobilized in alginate microcapsules.

Primary mouse hepatocytes (between 10-250 cells per capsule) were immobilized within 1.0% w/v alginate microbeads. The textural properties of the alginate matrix were characterized and a full protocol based upon the measurement of the initial rate of Resazurin reduction was studied and standardized. Using this method, the decay rate constant (K(d) = 0.45 +/- 0.01 days(-1)) and the time in which the cell viability decreases in half (VI(50) = 37 +/- 0.7 h) have been measured. The method was compared with the analysis of cell vitality using Calcein A/M and Ethidium Homodimer I. Differences between the two methods were found in the viability profile due to the significant presence of double stained cells along the culture time. According to the author's knowledge, this is the first report of a systematic study and determination of the K(d) value for immobilized hepatocytes, incorporating a wide range of cell concentrations within the alginate matrix.

Functionalized self-assembling peptide hydrogel enhance maintenance of hepatocyte activity in vitro.

There is a major challenge in maintaining functional hepatocytes in vivo as these cells rapidly lose their metabolic properties in culture. In this work we have developed a bioengineered platform that replaces the use of the collagen I--in the traditional culture sandwich technique--by a defined extracellular matrix analogue, the self-assembling peptide hydrogel RAD16-I functionalized with biologically active motifs. Thus, after examining side by side the two culture systems we have found that in both cases hepatocytes acquired similar parenchymal morphology, presence of functional bile canaliculi structures, CYP3A2 induction by dexamethasone, urea production, secretion of proteins such as apolipoprotein (class A1, E, J), alpha(1)-microglobulin, alpha(1)-macroglobulin, retinol binding protein, fibronectin, alpha(1)-inhibitor III and biotin-dependent carboxylases. Interestingly, by assessing in more detail some other hepatic markers, one of the functionalized matrix analogues--carrying the 67 kD laminin receptor ligand--enhanced the gene expression of albumin, HNF4-alpha, MDR2 and tyrosine aminotransferase. We conclude that the use of a synthetic culture system with designed matrix functionalization has the advantage in controlling specific cellular responses.

Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity.

This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 muM which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 muM. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to beta-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.

Two compartment model of diazepam biotransformation in an organotypical culture of primary human hepatocytes.

Drug biotransformation is one of the most important parameters of preclinical screening tests for the registration of new drug candidates. Conventional existing tests rely on nonhuman models which deliver an incomplete metabolic profile of drugs due to the lack of proper CYP450 expression as seen in human liver in vivo. In order to overcome this limitation, we used an organotypical model of human primary hepatocytes for the biotransformation of the drug diazepam with special reference to metabolites in both the cell matrix phase and supernatant and its interaction of three inducers (phenobarbital, dexamethasone, aroclor 1254) in different time responses (1, 2, 4, 8, 24 h). Phenobarbital showed the strongest inducing effect in generating desmethyldiazepam and induced up to a 150 fold increase in oxazepam-content which correlates with the increased availability of the precursor metabolites (temazepam and desmethyldiazepam). Aroclor 1254 and dexamethasone had the strongest inducing effect on temazepam and the second strongest on oxazepam. The strong and overlapping inductive role of phenobarbital strengthens the participation of CYP2B6 and CYP3A in diazepam N-demethylation and CYP3A in temazepam formation. Aroclor 1254 preferentially generated temazepam due to the interaction with CYP3A and potentially CYP2C19. In parallel we represented these data in the form of a mathematical model with two compartments explaining the dynamics of diazepam metabolism with the effect of these other inducers in human primary hepatocytes. The model consists of ten differential equations, with one for each concentration c(i,j) (i=diazepam, temazepam, desmethyldiazepam, oxazepam, other metabolites) and one for each compartment (j= cell matrix phase, supernatant), respectively. The parameters p(k) (k=1, 2, 3, 4, 13) are rate constants describing the biotransformation of diazepam and its metabolites and the other parameters (k=5, 6, 7, 8, 9, 10, 11, 12, 14, 15) explain the concentration changes in the two compartments.

Enrichment of human beta 1 bri/alpha 6 bri/CD71 dim keratinocytes after culture in defined media.

Stem cell-like keratinocytes are responsible for the high regenerative potential of the skin. For clinical applications using keratinocytes in artificial skin constructs, it is suitable to work with serum-free medium under defined conditions. This is also true for the preceding expansion of the stem cell-like keratinocyte population. Therefore, we analyzed the effect of a serum-free medium on the population distribution in comparison to an established serum-containing standard medium for keratinocyte culture. We quantified the freshly isolated as well as cultured primary human keratinocytes by their expression of the beta(1) integrin (CD29) in combination with the expression of the alpha(6) integrin (CD49f) and the transferrin receptor (CD71) by flow cytometric methods. We were able to show that cultivation with serum-free medium induces a switch of the cell population to higher expression of the beta(1) integrin. In addition, the proportion of the alpha(6)(bri)/ CD71(dim)-expressing keratinocyte cell population was enhanced about 35.4 +/- 6.56% after cultivation with serum-free medium. Culture in serum-containing medium increased this proportion of the keratinocyte cell population only about 17.3 +/- 8.06%, when compared to the alpha(6)(bri)/ CD71(dim)-expressing keratinocyte cell population measured directly after isolation. Our data show that the applied culture conditions already have an enormous impact on the development of a stem cell-like phenotype of keratinocytes. This work demonstrates that the serum-free medium significantly increases the proportion of beta(1)(bri)/alpha(6)(bri)/CD71(dim)-expressing keratinocytes. In conclusion, these findings implicate new applications in keratinocyte stem cell research and regenerative medicine.

Porcine endogenous retrovirus released by a bioartificial liver infects primary human cells.

BACKGROUND: Porcine endogenous retrovirus (PERV) remains a safety risk in pig-to-human xenotransplantation. There is no evidence of in vivo productive infection in humans because PERV is inactivated by human serum. However, PERV can infect human cell lines and human primary cells in vitro and inhibit human immune functions. AIMS: We investigated the potential of primary porcine liver cells to transmit PERV to primary human cells in a bioreactor-based bioartificial liver (BAL). METHODS: Primary human hepatocytes, endothelial cells and the human cell line HEK 293 were exposed to supernatants from BAL or from the porcine cell line PK-15. PERV polymerase-specific reverse-transcriptase polymerase chain reaction (RT-PCR) and PCR were used to investigate PERV transmission to human cells. An assay of RT activity was used to detect the presence of retrovirus in the supernatants of BAL, primary human hepatocytes and endothelial cells. RESULTS: Primary human hepatocytes (hHep), endothelial cells and HEK 293 cells were reproducibly infected by PERV, originating from primary porcine liver cells within the BAL and from PK-15 cells. Infected cells were positive for PERV-specific DNA and RNA after 8-10 days on an average, and RT activity was detectable in the supernatants of infected hHep and HEK 293 cells. CONCLUSION: A risk of PERV infection in human cells is documented in this study, indicating that short-term contact of primary porcine liver cell supernatants with primary human cells could result in PERV transmission.

Timing and targeting of cell-based VEGF165 gene expression in ischemic tissue.

BACKGROUND: Therapeutic angiogenesis has become a key technology in experimental and clinical medicine. Only few data are available on the effects of timing and targeting of therapeutic proteins after cell-based gene transfer. This work investigates such effects after temporary expression of vascular endothelial growth factor 165 (VEGF(165)), the most commonly used angiogenic protein for therapeutic purposes. METHODS: We established a cell-based gene-transfer model using fibroblasts to temporarily produce VEGF(165). Cells were implanted into 40 rats. Protein expression and angiogenic effects were measured by PCR, immunohistology, and microangiography. To determine an improvement for survival of ischemically challenged tissue, cells were implanted in an ischemic flap model at different locations and time points. RESULTS: After implantation of modified cells, a temporary increase was found in the target tissue for VEGF(165), endothelial cell counts, and capillary network formations. Four wk later, histological alterations in the target tissue area were not different from controls. Implantation of modified cells into flap plus wound margin 1 wk before surgery showed significant improvement of tissue survival demonstrated by planimetric measurements and blood vessels counting in the target tissue. CONCLUSION: In our model, temporary expression of VEGF(165) induces therapeutically relevant angiogenesis and improves blood supply only if applied 1 wk before ischemia. It is essential to include the surrounding area for induction of angiogenesis in this model. In contrast, the angiogenic effects are not effective in the target area and its surrounding tissue, if therapeutic gene expression is started during onset of ischemia or 2 wk before ischemia in this model.

Therapeutic potential of endogenous stem cells and cellular factors for scar-free skin regeneration.

Injured human skin fails to regenerate, resulting in scar formation. Annually, 100 million new skin-scarring incidents, occurring as a result of surgery, disease, burns, or sports-related damage, remain untreated. Here, we review knowledge gained from scar-free experimental animal models that have natural regenerative mechanisms for scar-free skin recovery. We also focus on the unique role of endogenous stem cells and other cellular and molecular factors, including the balance of the transforming growth factor-beta (TGF-ß) pathway in the context of human skin regeneration. This new strategy opens a new window in drug development for scar-free skin regeneration treatments in both the clinical and cosmetic practice settings.

The regenerative potential of skin and the immune system.

Skin has the natural ability to heal and replace dead cells regulated by a network of complex immune processes. This ability is conferred by the population of resident immune cells that act in coordination with other players to provide a homeostatic environment under constant challenge. Other than providing structure and integrity, the epidermis and dermis also house distinct immune properties. The dermal part is represented by fibroblasts and endothelial cells followed by an array of immune cells which includes dendritic cells (DCs), macrophages, mast cells, NK-cells, neutrophils, basophils, eosinophils, αß T lymphocytes, B-cells and platelets. On the other hand, the functionally active immune cells in the epidermis comprise keratinocytes, DCs, NKT-cells, γδ T cells and αß T cells (CD4+ and CD8+). Keratinocytes create a unique microenvironment for the cells of the immune system by promoting immune recognition and cellular differentiation. T lymphocytes exhibit tissue-specific tropism toward the epidermis and the lymphatic drainage system important for their function in immune regulation. This diversity in immune regulators makes the skin a unique organ to overcome pathogenic or foreign invasion. In addition, the highly coordinated molecular events make the skin an attractive model to understand and explore its regenerative potential.

The effect of a mesenchymal stem cell conditioned medium fraction on morphological characteristics of hepatocytes in acetaminophen-induced acute liver failure: a preliminary study.

Background: In our studies, it was shown that the effectiveness of the conditioned medium obtained by cultivating mesenchymal stem cells depends on the microenvironment conditions used to cultivate the cells. It was demonstrated that the conditioned medium obtained by culturing cells with low oxygen content (10%) has a much more pronounced protective effect. Methods: Protein compositions obtained from MSCs cultured under hypoxic (10% O2 hc-MSC) and normal (21% O2 nc-MSC) conditions were used to treat acute liver failure (ALF) induced in mice by acetaminophen injection. Thus, we obtained fractions normalized by volume, which predominantly contained proteins with masses > 50, 50-30, 30-10, and 10-3 kDa. Results: The data from biochemical studies have shown that only fractions from 10 to 30 kDa (hcMSC and ncMSC) significantly reduced the level of liver enzymes in the beginning of the acute period after acetaminophen administration. Mass spectrometry analysis of the proteins contained in the isolated fractions showed a sharp increase in the protein levels in the 10-30 kDa hcMSC fraction as compared with that in 10-30 kDa ncMSCs. The composition obtained from MSCs cultured at lower O2 level (fraction 10-30 kDa hcMSC) was shown to be more potent than the composition prepared from normoxic cells. Conclusion: The results have shown that a composition obtained by culturing the cells under a reduced content of O2 (10%), significantly improves the biochemical parameters, and histological arrester reduces the degree of inflammation and stimulates regenerative processes in liver, compared to both the control group and group treated with the composition that was obtained by culturing the cells under normal oxygen content.

Multilineage differentiation potential of human dermal skin-derived fibroblasts.

Dermal skin-derived fibroblasts from rodent and human have been found to exhibit mesenchymal surface antigen immunophenotype and differentiation potential along the three main mesenchymal-derived tissues: bone, cartilage and fat. Human dermal skin-derived mesenchymal stem cells constitute a promising cell source in clinical applications. Therefore, we isolated fibroblastic mesenchymal stem-cell-like cells from human dermis derived from juvenile foreskins, which share a mesenchymal stem cell phenotype and multi-lineage differentiation potential. We could show similar expression patterns for CD14(-), CD29(+), CD31(-), CD34(-), CD44(+), CD45(-), CD71(+), CD73/SH3-SH4(+), CD90/Thy-1(+), CD105/SH2(+), CD133(-) and CD166/ALCAM(+) in well-established adipose tissue derived-stem cells and fibroblastic mesenchymal stem-cell-like cells by flow cytometry. Immunostainings showed that fibroblastic mesenchymal stem-cell-like cells expressed vimentin, fibronectin and collagen; they were less positive for alpha-smooth muscle actin and nestin, while they were negative for epithelial cytokeratins. When cultured under appropriate inducible conditions, both cell types could differentiate along the adipogenic and osteogenic lineages. Additionally, fibroblastic mesenchymal stem-cell-like cells demonstrated a high proliferation potential. These findings are of particular importance, because skin or adipose tissues are easily accessible for autologous cell transplantations in regenerative medicine. In summary, these data indicate that dermal fibroblasts with multilineage differentiation potential are present in human dermis and they might play a key role in cutaneous wound healing.