Culturing and differentiating human mesenchymal stem cells for biocompatible scaffolds in regenerative medicine.

Mesenchymal stem cells from a variety of sites are a natural resource that using appropriate skills can be cultured in the laboratory, in scaffolds, to provide differentiated-cell replacement tissues, for clinical application. To perform such work with human cells, strict ethical integrity must be observed at all stages. Adipocytes, osteocytes and chrondrocytes are amongst the most desirable end-point cells. Hydrolytic degradable scaffolds allow implanted cells to synthesise their own extracellular matrix in situ after implantation, degeneration of the foreign scaffold to temporally match creation of the new innate one. For preliminary in vitro stem cell differentiation protocols, initial investigation is commonly performed with stem cells in commercially available porous collagen sponges or cell-free small intestinal submucosa. Differentiation of stem cells to a specific phenotype is achieved by culturing them in apposite culture media under precise conditions. Once the cells have differentiated, they are checked and characterised in a wide variety of systems. This chapter describes differentiation media for adipocytes, osteocytes, chondrocytes, myocytes and neural precursors and methods of observing their characteristics by microscopy using phase contrast microscopy, standard light microscopy and electron microscopy with tinctorial, immunocytochemical and electron dense stains, respectively. Cell sorting techniques are not dealt with here. Immunocytochemistry/microscopy staining for specific differentiated-cell antigens is an invaluable procedure, and the range of commercially available antibodies is wide. Precautions need to be considered for using actively proliferating cells in vivo, so that implanted cells remain controlled by the body's molecular signals and avoid development of malignancy.

Heparan sulfate-dependent ERK activation contributes to the overexpression of fibrotic proteins and enhanced contraction by scleroderma fibroblasts.

OBJECTIVE: To investigate the contribution of heparan sulfate proteoglycan and Ras/MEK/ERK to the overexpression of profibrotic proteins and the enhanced contractile ability of dermal fibroblasts from patients with systemic sclerosis (SSc; scleroderma). METHODS: The effects of the MEK/ERK inhibitor U0126, the heparan sulfate side chain formation inhibitor beta-xyloside, and soluble heparin on the overexpression of profibrotic genes were compared in fibroblasts from lesional skin of patients with diffuse SSc and fibroblasts from healthy control subjects. Identified protein expressions were compared with the contractile abilities of fibroblasts while they resided within a collagen lattice. Forces generated were measured using a culture force monitor. RESULTS: Inhibiting MEK/ERK with U0126 significantly reduced expression of a cohort of proadhesive and procontractile proteins that normally are overexpressed by scleroderma fibroblasts, including integrin alpha4 and integrin beta1. Antagonizing heparan sulfate side chain formation with beta-xyloside or the addition of soluble heparin prevented ERK activation, in addition to reducing the expression of these proadhesive/contractile proteins. Treatment with either U0126, beta-xyloside, or heparin resulted in a reduction in the overall peak contractile force generated by dermal fibroblasts. Blocking platelet-derived growth factor receptor with Gleevec (imatinib mesylate) reduced overall contractile ability and the elevated syndecan 4 expression and ERK activation in SSc fibroblasts. CONCLUSION: The results of this study suggest that heparan sulfate-dependent ERK activation contributes to the enhanced contractile ability demonstrated by dermal fibroblasts from lesional skin of patients with scleroderma. These results are consistent with the notion that the MEK/ERK procontractile pathway is dysregulated in scleroderma dermal fibroblasts. Additionally, the results suggest that antagonizing the MEK/ERK pathway is likely to modulate heparan sulfate proteoglycan activity, which in turn may have a profound effect on the fibrotic response in SSc.

Copper deprivation in rats induces islet hyperplasia and hepatic metaplasia in the pancreas.

BACKGROUND INFORMATION: Prolonged copper deprivation in rats followed by refeeding with a normal diet has previously been used to induce the appearance of hepatocyte-like cells in the pancreas, but the effects on islet size and morphology have not been determined. RESULTS: In the present study we investigated the distribution of pancreatic alpha- and beta-cells and of hepatocytes in adult rats fed a copper-deficient diet followed by refeeding with a normal diet. Immunohistochemical staining for insulin and glucagon showed that the islets of the copper-deficient group were up to 2.4 times larger in mass compared with controls. The islets were disorganized, with alpha-cells found in multiple layers at the periphery of the islet and sometimes deep in the core. Isolated alpha- and beta-cells were also found in increased numbers in the ductular system. Copper deprivation caused almost complete ablation of the acinar cells, and refeeding induced adipogenesis, acinar regeneration and hepatocyte-like cells. Ductular proliferation and nerve hyperplasia were also present. The hepatocytes tended to be associated with islets or with ducts, rather than with residual pancreatic exocrine tissue. CONCLUSIONS: These data show that copper deficiency in rats, as well as inducing the appearance of hepatocytes, is capable of causing islet hyperplasia.

New multi-cue bioreactor for tissue engineering of tubular cardiovascular samples under physiological conditions.

In the present study we have developed a multi-cue bioreactor (MCB) that is capable of delivering a range of stimuli to assist the development of a tissue-engineered construct. The MCB provides an accurate and utilizable computer-controlled pulsatile pump and strain induction mechanism and it has the capability of applying physiological conditions to samples. The device described here emulates the pressure and straining environment found at the aortic root. This function, along with an integral perfusion and sterile containment system, allows for long-term culture and whole-tissue testing capability. Aortic and pulmonary arteries were obtained from freshly isolated porcine hearts and subjected to various loading regimens (Deltapressure/flow/force). Through analyzing data acquired by the MCB transducer array it was possible to differentiate the dynamic mechanical properties of the tissue types tested. In addition, the MCB illustrates a novel concept in cardiovascular tissue engineering: being able to support long-term tissue culture of cell-seeded substrates while they are under the influence of mechanical cues. After 7 days of pulsation in the MCB cell alignment was observed. The MCB represents a versatile model that will enable the development of tissue engineering not only for cardiovascular tissue, but for all tubular tissues such as esophageal, tracheal, and bronchial systems.

Assessment of growth inhibition and morphological changes in in vitro and in vivo hepatocellular carcinoma models post treatment with dl1520 adenovirus.

BACKGROUND AND AIMS: E1B-deleted virus dl1520 (ONYX-015) has been previously used in clinical trials mainly for treatment of head and neck tumors, and has been shown to have beneficial effects independent of p53 status. The main aim of this investigation was to carry out a preclinical study for assessment of the use of dl1520 in in vitro and in vivo hepatocellular carcinoma (HCC) models with various p53 status (deleted, mutant, and wild type), and study the ultrastructural changes in the carcinoma cells during and following treatment with dl1520. METHODS: dl1520 (ONYX-015) virus was used for treatment of three HCC cell lines in culture, then for treatment of developed xenografts in SCID mice. The effects of dl1520 on HCC cell growth and accompanied morphological changes were assessed by various techniques including transmission electron microscopy. dl1520 infection was confirmed using polymerase chain reaction and immunolabeling at transmission electron microscopy level. RESULTS: dl1520 was effective in killing cells and inhibiting HCC cell growth both in vitro and in vivo. The cell killing was at higher levels in cells possessing abnormal p53. Survival rates in SCID mice treated with dl1520 were statistically significantly higher in HCC tumors with deleted and mutant p53, than in tumors with wild-type p53. CONCLUSIONS: The findings in this study suggest that dl1520 could be safely and effectively used for treatment of HCC dependent on the p53 status of the cells in vivo. Characteristic morphological changes that took place in the dl1520-treated HCC cells/tumors were distinct at transmission electron microscopy level and are the first of their kind to be reported.

Tissue engineering of biological cardiovascular system surrogates.

Cardiovascular diseases are common in ageing communities globally. This fact is most striking in the industrialised world where the aged population makes up a large proportion of society. Elderly patients are frequently treated surgically with grafts to replace damaged tissues and vessels. The number of human-donated components is insufficient and synthetic surrogates are sought. These might be wholly mechanical, wholly biological, or tissue engineered complexes of cells and their products growing in a scaffold. At present, many such composites exist with potential for use as substitutes for specific blood vessels. The challenges of producing tissue engineered heart valves are now being widely explored. Neotissues must provide an effective, durable, non-thrombogenic and non-immunogenic substitute that will fulfil the purpose of the natural tissue. The aims and scope of this paper are to review current and novel concepts in the field of tissue engineering of biological cardiovascular system surrogates. Mechanical stresses and strains on cardiovascular cells in vitro have been recognised and can be measured by a culture force monitor. Physiological stresses can be generated by a tensioning culture force monitor and applied to engineered tissue, aligning the cells and mimicking arterial wall architecture. The hydrostatic forces a vessel experiences and mechanical parameters of blood vessels can be studied in the tubular culture system of a multi-cue bioreactor.