Collagenase-isolated Cells Research Articles

Mesenchymal Stem Cells Derived from Human Limbal Niche Cells

We investigated whether human limbal niche cells generate mesenchymal stem cells. Limbal niche cells were isolated from the limbal stroma by collagenase alone or following dispase removal of the limbal epithelium (D/C), and cultured on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), or coated or three-dimensional Matrigel in embryonic stem cell medium with leukemia inhibitory factor and basic fibroblast growth factor. Expression of cell markers, colony-forming units-fibroblast, tri-lineage differentiation, and ability of supporting limbal epithelial stem/progenitor cells were compared to limbal residual stromal cells. Stromal cells expressing angiogenesis markers were found perivascularly, subjacent to limbal basal epithelial cells, and in D/C and limbal residual stromal cells. When seeded in three-dimensional Matrigel, D/C but not limbal residual stromal cells yielded spheres of angiogenesis progenitors that stabilized vascular networks. Similar to collagenase-isolated cells, D/C cells could be expanded on coated Matrigel for more than 12 passages, yielding spindle cells expressing angiogenesis and mesenchymal stem cells markers, and possessing significantly higher colony-forming units-fibroblast and more efficient tri-lineage differentiation than D/C and limbal residual stromal cells expanded on plastic in DMEM with 10% FBS, of which both lost the pericyte phenotype while limbal residual stromal cells turned into myofibroblasts. Upon reunion with limbal epithelial stem/progenitor cells to form spheres, D/C cells expanded on coated Matrigel maintained higher expression of p63α and lower expression of cytokeratin 12 than those expanded on plastic in DMEM with 10% FBS, while spheres formed with human corneal fibroblasts expressed cytokeratin 12 without p63α. In the limbal stroma, cells subjacent to limbal basal epithelial cells serve as niche cells, and generate progenitors with angiogenesis and mesenchymal stem cells potentials. They might partake in angiogenesis and regeneration during corneal wound healing.

Pharmacological evaluation of the role of cytochrome P450 in intracellular calcium signalling in rat pancreatic acinar cells.

We have investigated whether the cytochrome P450 system is involved in Ca(2+) signalling in rat pancreatic acinar cells. Intracellular free [Ca(2+)] ([Ca(2+)](i)) was measured in collagenase-isolated cells using fura-2 microspectrofluorimetry and imaging. The imidazole P450 inhibitor ketoconazole (5 - 50 microM) inhibited [Ca(2+)](i) oscillations induced by cholecystokinin octapeptide (CCK). However, ketoconazole also raised baseline [Ca(2+)](i) when applied in the absence of CCK. These effects were mimicked by 5 - 50 microM SKF96365, an imidazole widely used as an inhibitor of Ca(2+) entry. The non-imidazole P450 inhibitor proadifen (SKF525A) inhibited CCK-induced [Ca(2+)](i) oscillations at a concentration of 10 - 50 microM. Proadifen alone caused intracellular Ca(2+) release at 25 or 50 microM, but not at 10 microM. Octadecynoic acid and 1-aminobenzotriazole, structurally-unrelated non-imidazole P450 inhibitors, did not alter baseline [Ca(2+)](i) or CCK-evoked oscillations. We compared cumulative CCK dose-response relationship in control cells and in cells where P450 had been induced by prior injection of animals with beta-naphthoflavone. Only minor differences were apparent, with induced cells showing some decrease in responsiveness at moderate and higher concentration of CCK (30 pM - 3 nM). Direct assessment of depletion-activated Ca(2+) entry showed no clear differences between control and induced cells. In conclusion, we could find no compelling evidence for a role of P450 in controlling Ca(2+) signalling generally, or Ca(2+) entry in particular, in pancreatic acinar cells. Induction of P450 is therefore probably toxic to acinar cells via a Ca(2+)-independent mechanism.

Reduced proliferative activity of polyploid cells in primary hepatocellular carcinoma.

The proliferative activity of tumor cells differing in DNA content (ploidy) and nuclearity was investigated in primary hepatocellular carcinomas of the rat by flow cytometric analysis of collagenase-isolated cells immunostained after labelling with bromodeoxyuridine (BrdU) in vivo. The diploid cell fraction in these euploid tumours was higher than in normal liver, and the rate of binucleation as well as the proliferative activity of the binuclear cells was very low. The highest proliferative activity (BrdU labelling index) was found among the diploid tumour cells. The activity in mononuclear tetraploid and octoploid cells was reduced in inverse proportion to their increasing DNA content, possibly suggesting a loss of proliferative potential associated with polyploidization. There was a significant correlation between the proliferative activity of hepatocellular carcinoma cells and nonparenchymal liver cells in the different tumours, indicating that different cell types within a tumour may respond to common growth stimuli. Treatment of tumour-bearing rats with a promoting carcinogen (2-acetylaminofluorene) resulted in significant stimulation of tumour cell proliferation (all ploidy classes), whereas the proliferation of non-parenchymal (stromal) cells in the tumour was slightly inhibited.

Xenobiotic induction of P-450 PB-4 (IIB1) and P-450c (IA1) and associated monooxygenase activities in primary cultures of adult rat hepatocytes

1. The long-term maintenance of metabolism of representative drugs and steroid hormone substrates by cytochromes P-450, and their inducibility, was investigated in primary cultures of adult rat hepatocytes. Collagenase-isolated cells were seeded on collagen-coated tissue culture dishes and cultured in Chee's essential media in the presence or absence of phenobarbital (PB, 0.75 mM, 96 h or continuously) and 3-methylcholanthrene (MC, 5 microM, 48 h) for up to 45 days. 2. Hepatic P-450-dependent metabolism of diazepam to its primary oxidized metabolite was inducible by PB both in vivo (monitored in isolated liver microsomes) and in cultured cells (up to 100% and 400% increases in the formation of temazepam and nordiazepam, respectively, after 25 days in culture). Hepatocyte microsomal androstenedione 16 beta-hydroxylase activity was also induced by PB treatment of the hepatocytes (350-650% increase in 20-day-old cells). 3. Western blot analysis revealed that immunoreactive P-450 form PB-4 (IIB1), which catalysed the N-demethylation of diazepam to yield nordiazepam as well as androstenedione 16 beta-hydroxylation when assayed in a purified enzyme system, was substantially elevated following PB treatment of the cultured cells. Similarly, MC induced 7-ethoxycoumarin O-deethylase activity (up to 2000% increase from 5 to 45 days) as well as immunoreactive P-450c (IA1) in the hepatocyte cultures. 4. These studies demonstrate that cytochrome P-450 activities can be maintained, and also induced, after extended periods of time in hepatocytes cultured using a simple collagen mixture as substrate and a commercially available tissue culture media. This culture system should provide an important tool for further studies of P-450-dependent xenobiotic metabolism in a well-defined, liver-derived cellular system.

Lipogenesis and insulin sensitivity of single fat cells.

1. A method for measuring the lipogenesis from [(14)C]glucose by single fat cells is described: (i) after incubation with ;carrier-free' [U-(14)C]glucose (0.55 mu-mole/ml.), collagenase-isolated fat cells were fixed with osmium tetroxide; (ii) similarly incubated pieces of epididymal fat pads were treated with osmium tetroxide for 90 sec, whereby only the superficial cells are fixed, and the tissue was then disintegrated by shaking with collagenase. The osmium-fixed free cells were washed, sucked into a micropipette, measured under a microscope and assayed individually for (14)C-activity.2. There was a quantitative recovery of (14)C-lipid activity from osmium-fixed single cells.3. Both collagenase-isolated cells and in situ fixed surface cells were normally distributed with respect to diameters (for both cell groups from ad lib. fed rats of ca. 110 g; mean diameter, about 55 mum; S.D. about 7 mum).4. Frequency distribution curves (number of fat cells versus (14)C-lipogenesis per cell) were asymmetric and very broad (coefficient of variation about 50%) for collagenase-isolated cells incubated with insulin (10(3) mu-u./ml.). Frequency distribution curves for surface cells obtained from similarly incubated pieces of epididymal fat pads showed a coefficient of variation of the same magnitude, whereas the mean lipogenesis of these cells was only about one third of that of the isolated cells.5. Collagenase-isolated cells incubated in the presence of insulin (10(3) mu-u./ml.) showed a weak but highly significant positive correlation between fat cell diameter and (14)C-lipogenesis (eight rats, r about 0.5 and P < 0.001 for each rat). Analysis of the relationship: lipogenesis = k x diameter to the exponent of beta showed that the estimates of beta varied significantly from rat to rat (range: 1.3-2.9). Similar correlations between cell size and lipogenesis were found both for cells incubated with insulin in various submaximal concentrations and for cells incubated without insulin.6. Small and large cells from the same rat were equally sensitive to insulin.7. Statistical analysis of frequency distribution curves (number of cells versus (14)C-lipogenesis per unit surface area) representing cells from the same rat incubated with insulin 0, 2.5, 5, 10, and 10(3) mu-u./ml., respectively, suggests that insulin exerts a graded influence on the lipogenesis of each fat cell.