Phenotypic shift of human amniotic epithelial cells in culture is associated with reduced osteogenic differentiation in vitro.

BACKGROUND: Amniotic membrane is a highly promising cell source for tissue engineering. Being part of the placenta, this tissue is abundantly available. It can be processed easily to yield large amounts of epithelial and mesenchymal cells that have shown broad differentiation potential. For tissue-engineering purposes, cells may be applied either directly after isolation from the tissue or after a period of in vitro expansion to obtain higher cell numbers. In order to investigate the advantages and drawbacks of these strategies we compared freshly isolated and cultivated human amniotic epithelial cells (hAEC) regarding their surface antigen (Ag) expression profile and osteogenic differentiation capacity. METHODS: Expression of surface Ag that are characteristic for mesenchymal stromal and embryonic stem cells was analyzed by flow cytometry. Different protocols for osteogenic and adipogenic differentiation were compared. RESULTS: We have demonstrated that expression of surface Ag changes dramatically during cultivation of hAEC. While not or only weakly expressed on primary isolates, the mesenchymal markers CD13, CD44, CD49e, CD54, CD90 and CD105 are strongly up-regulated during in vitro propagation. In contrast, expression of the embryonic markers TRA-1-60 and TRA-1-81, but not SSEA-4, rapidly decreases upon cultivation. This phenotypic shift is associated with a reduction in osteogenic differentiation. DISCUSSION: Our results suggest that phenotypic alterations of hAEC during in vitro cultivation might be responsible for a functional reduction of the differentiation potential, which has to be considered for the potential application of these cells in regenerative medicine.

Development of standardized cell culture conditions for tumor cells with potential clinical application.

BACKGROUND: Tumor cell lines have enormous value for the study of different aspects of cancer biology and have also recently gained great importance in autologous cell-based anti-tumor therapies. However, the use of these cells is still limited because in vitro growth is hampered by suboptimal culture conditions and current media contain fetal bovine serum (FBS), which poses serious safety concerns regarding clinical application. METHODS: To address this drawback, we aimed to develop a strategy for optimization of the culture medium for human medullary thyroid carcinoma (MTC) cell lines as a model system. We combined the general cell screening system (GCSS), which continuously measured the growth behavior of cells in a 96-well plate format, with statistically based experimental designs. RESULTS: The results obtained clearly demonstrated that, just by changing the composition of the basal medium, a significantly enhanced growth rate could be observed, and by subsequent addition of several substances a serum-free cell culture medium could be developed. This medium allowed the propagation of two MTC cell lines comparable with conventionally used serum-supplemented medium. DISCUSSION: We present a fast and easy way to screen for substances that are essential for tumor cell growth in vitro. Furthermore, these tumor cells can be adapted to culture conditions that allow the use of the cells in safe cell-based therapies. This is of utmost importance because of increasing regulatory requirements.

[Electrolyte changes during and after voluntary hyperventilation]. / Elektrolytveränderungen während und nach willkürlicher Hyperventilation.

Paresthesia and tetanic finger cramps during hyperventilation-induced respiratory alkalosis are believed to derive from a pH-dependent decrease of ionized serum calcium. In the study reported here, ionized serum calcium, total calcium and total protein were measured during a three-minute hyperventilation period in ten volunteers. During hyperventilation finger paresthesias appeared in all probands without proof of any significant change in ionized serum calcium (1.26 +/- 0.05 mmol/l at the end of the three-minute hyperventilation period). Total protein increased as a consequence of hyperventilation-induced transient hemo-concentration. Paresthesias and tetanic finger cramps during the three-minute hyperventilation could not be related to changes of ionized serum calcium; however the other electrolytes, i.e. sodium, magnesium, potassium, chloride, phosphate and bicarbonate, showed, with the exception of sodium, significant changes.

["Diabetic" proliferative retinopathy and nodular glomerulosclerosis without diabetes mellitus]. / "Diabetische" proliferative Retinopathie und noduläre Glomerulosklerose ohne Diabetes mellitus.

A 65-year-old woman of normal weight, hospitalized because of pleuritis, was found to have chronic renal failure (creatinine clearance 20 ml/min). Renal biopsy (light and electron-microscopy) revealed nodular glomerulosclerosis (Kimmerstiel-Wilson disease), described as a diabetes-specific renal change. Fundoscopy discovered bilateral proliferative retinopathy as seen in diabetes. But oral and intravenous glucose tolerance tests were normal, excluding a manifest diabetic metabolic disorder. No other cause of the glomerulosclerosis (such as amyloidosis or multiple myeloma) was found. The patient had been overweight for a time when younger, reversed by dieting. It is suggested that the "diabetic" changes in the kidneys and eyes without diabetes could be the result of a transitory disorder of glucose tolerance during the period of obesity.

[Experiences with a modified, intraocular pressure-reducing excision of the ciliary body]. / Erfahrungen mit einer modifizierten, augeninnendrucksenkenden Ziliarkörperexzision.

The treatment of hemorrhagic secondary angle-closure glaucoma following central venous occlusion is generally very difficult. Sautter's ciliary body excision is preceded by two semicircular cyclocryocoagulations performed within 7 days of each other. If IOP remains above 30 mm Hg and the subjective complaints are unchanged, oculopression lasting 10 minutes is performed after local retrobulbar anesthesia. The lowering of pressure thus achieved is sufficient to render a Flieringa ring and paracentesis unnecessary. Several illustrations are included showing the surgical procedure. In 9 cases of hemorrhagic glaucoma following central venous occlusion, there was a further deterioration in vision 8 to 23 months postoperatively. Preoperatively, IOP was between 36 and 63 mm Hg; at late postoperative follow-ups it was between 5 and 30 mm Hg. Complications worth mentioning included three slight losses of vitreous, bleeding into the anterior chamber in four cases, and one loss of the anterior chamber with caput medusae of the iris and secondary opacification of the lens. In 2 cases of aphakic secondary angle-closure glaucoma there was no deterioration in vision and a lowering of pressure to under 30 mm Hg, in spite of 3 preceding intraocular operations. The authors regard the slightly modified ciliary body excision as a possible way of preserving the bulb.