Biological characterization and pluripotent identification of ovine amniotic fluid stem cells

Mesenchymal stem cells derived from amniotic fluid have become one of the most potential stem cell source for cell-based therapy for the reason they can be harvested at low cost and without ethical problems. Here, we obtained amniotic fluid stem cells (AFSCs) from ovine amniotic fluid and studied the expansion capacity, cell markers expression, karyotype, and multilineage differentiation ability. In our work, AFSCs were subcultured to passage 62. The cell markers, CD29, CD44, CD73 and OCT4 which analyzed by RT-PCR were positive; CD44, CD73, CD90, CD105, NANOG, OCT4 analyzed by immunofluorescence and flow cytometry were also positive. The growth curves of different passages were all typically sigmoidal. The different passages cells took on a normal karyotype. In addition, AFSCs were successfully induced to differentiate into adipocytes, osteoblasts and chondrocytes. The results suggested that the AFSCs isolated from ovine maintained normal biological characteristics and their multilineage differentiation potential provides many potential applications in cell-based therapies and tissue engineering.     

Effect of centrifugation on amniotic fluid phospholipid composition.

The first step in the determination of phospholipid in amniotic fluid is generally the removal of cells and debris from the fluid by centrifugation. Low-speed centrifugation of the supernatant is reported to have the same phospholipidic profile and L/S, PG/S and PI/S ratios similar to those of the uncentrifuged amniotic fluid sample. With high-speed centrifugation almost all the pulmonary surfactant seems to be recovered in pellet with the same characteristics as the uncentrifuged amniotic fluid.     

Chemical dilution and clearance studies to estimate amniotic fluid volume and amniotic fluid ingestion in normal baboon pregnancies

Amniotic fluid volume (AFV) and amniotic fluid ingestion rate (or fetal swallowing rate, FSR) were estimated by inulin and para-aminohippurate (PAH) dilution in 14 normal baboon pregnancies. Mean (± SE) AFV was significantly lower at 137–140 days of pregnancy (preterm) than at 173–178 days (term) (inulin: 326 ± 22.9 ml vs 483 ± 55.9 ml, P = 0.014; PHA:269 ± 39.4 ml vs 471 ± 39.4 ml, P = 0.002). In proportion to fetal weight, however, mean AFV was similar throughout the third trimester of pregnancy (inulin: 582 ± 40.9 ml/kg; PAH: 541 ± 39.8 ml/kg). Mean FSR was lower in preterm than in term animals when estimated by inulin dilution (587 ± 55.5 ml/day vs 784 ± 55.0 ml/day, P = 0.030) but not when estimated by PAH dilution (753 ± 65.7 ml/day vs 625 ± 50.6 ml/day). In proportion to their weights, however, preterm fetuses swallowed amniotic fluid more rapidly than term fetuses (inulin: 1,216 ± 117.6 ml/kg/day vs 840 ± 67.5 ml/kg/day, P = 0.025; PAH: 1,561 ± 142.9 ml/kg/day vs 682 ± 62.7 ml/kg/day, P < 0.001). Furthermore, our data suggest that the commonly accepted technique for estimating AFV may be based on inaccurate premises, that insulin may be a better marker than PAH to estimate AFV and FSR, and that needle aspiration of amniotic fluid does not appear to be an adequate technique to validate chemical dilution methods. Our data, however, provide estimates which indicate that the baboon is an appropriate animal model in which to seek refinements and validation of our techniques.     

Function curve of the membranes that regulate amniotic fluid volume in sheep

Seven singleton 120-day fetal lambs were prepared with a shunt from the lung to the gastric end of the esophagus, a bladder catheter, and multiple amniotic fluid and vascular catheters. The urachus was ligated. Beginning 7 days later, amniotic fluid volumes were determined by drainage, followed by replacement with 1 liter of lactated Ringer (LR) solution. Urine flow into the amnion was measured continuously. In 14 of 27 experiments, amniotic fluid volumes were determined again 2 days after the inflow into the amnion had consisted of urine only and in 13 experiments after the inflow of urine had been supplemented by an intraamniotic infusion of LR solution. Intramembranous absorption was calculated from the inflows and the changes in volume between the beginning and end of each experiment. The relations between absorption rate and amniotic fluid volume, the "function curves," were highly individual. Urine production during the infusion of LR solution did not decrease, fetal plasma renin activity decreased (P < 0.001), and amniotic fluid volume increased by 140% [SE (27%), P < 0.005], but the increase in the amniochorionic absorption rate of 411% [SE (48%), P < 0.001] was greater (P < 0.005) than the increase in volume. Each of the seven fetuses was proven capable of an average intramembranous absorption rate that exceeded 4.5 liters of amniotic fluid per day. During the infusion of LR solution, the increase in the rate of absorption matched the rate of infusion (both in ml/h), with a regression coefficient of 0.75 (P < 0.001). Thus, even for large amniotic fluid volumes, volume is not limited by the absorptive capacity of the amniochorion, and, at least in these preparations, the position of the function curve and not the natural rate of inflow was the major determinant of resting amniotic fluid volume.     

The effect of maternal fluid intake on the volume and composition of fetal urine

The effects on fetal renal function of restricting maternal water intake to 1 l/day for 6 days was investigated in 7 chronically-catheterized fetuses (gestation age 118-131 days). Restriction of water intake caused a significant decrease in maternal urine flow rate and significant increases in maternal plasma and urinary osmolality. Fetal renal function was investigated on the third and sixth days of the period of restricted maternal intake of water. Urine flow rate from the fetus was depressed significantly, and urinary osmolality increased significantly. The glomerular filtration rate remained unchanged, and free water clearance was decreased. These changes indicate increased water reabsorption in the distal parts of the nephron, probably consequent upon increased circulating levels of antidiuretic hormone. In 3 fetuses whose mothers subsequently had free access to water, these changes in urine flow rate and free water clearance that occurred during water restriction were reversed. There was an inverse correlation between maternal plasma osmolality and fetal free water clearance corrected for glomerular filtration rate. It is concluded that when water intake by a pregnant animal is restricted, the availability of water to the fetus is reduced and fetal sheep respond by producing a concentrated urine.     

Maternal, amniotic fluid and cord blood metabolic profile in normal pregnant and gestational diabetics during recurrent withholding of food

In order to advise regarding the religious practice of withholding food, we studied the metabolic changes after successive 15 days of recurrent fasting of 13 hours every day in maternal plasma and liquor amnii of obese normal gravids and gestational diabetics in their third trimester. There were no significant differences between those who fasted that period for one day prior to elective cesarean section (CS) and those who fasted the same period repeatedly for 15 days. The fasted gravids had significant rises in glycerol, beta-hydroxybutyrate (BOHB) and nonesterified fatty acids (NEFA) (P less than 0.0001, P less than 0.005 and P less than 0.01, respectively) in maternal plasma, compared to unfasted gravid groups and ungravid fasted group. No significant metabolic difference was found in the liquor amnii withdrawn from fasted and unfasted groups. The influence of such short term of starvation on the fetal metabolic profile was studied in the cord blood during cesarean section (CS). Glucose, glycerol and NEFA were significantly lower in arterial than in venous cord plasma (P less than 0.05, P less than 0.01 and P less than 0.01, respectively) indicating that the fetus could utilize these substrates. Positive correlation was found between the levels of BOHB in the mother and venous cord plasma on the one hand and their levels in the arterial cord plasma and liquor amnii on the other hand implying that this substrate passes unutilized through the fetus to the liquor amnii. A pregnant woman in the third trimester should not withhold food for long periods.     

Human amniotic fluid stem cells: a new perspective

The discovery of amniotic fluid stem cells initiated a new and very promising field in stem cell research. In the last four years amniotic fluid stem cells have been shown to express markers specific to pluripotent stem cells, such as Oct-4. Due to their high proliferation potential, amniotic fluid stem cell lineages can be established. Meanwhile, they have been shown to harbor the potential to differentiate into cells of all three embryonic germ layers. It will be a major aim for the future to define the potential of this new source of stem cells for therapies related to specific diseases.     

Desiccation-tolerant Sporobolus stapfianus: lipid composition and cellular ultrastructure during dehydration and rehydration

The desiccation-tolerant plant Sporobolus stapfianus was subjectedto slow dehydration and to rehydration either as a silica gel-drieddetached leaf or as an airdried plant. In detached leaves dehydrationresulted in a lower relative water content in comparison withleaves dried on the plant. Water loss caused a reduction inchlorophyll, carotenoid and lipid contents and an increase inconjugated dienes. In detached leaves, ultrastructure was alsoaffected by dehydration, showing damaged cells with alteredchloroplasts which retained large quantities of starch and lipid-likeinclusions in the stroma. Upon rehydration a continuous degradationof the chemical composition and cell organization was observedwith a further increase in peroxidation. Leaves dehydrated onthe plant showed degradation of chlorophyll and lipids, whereascarotenoids increased and conjugated dienes decreased. Desiccationcaused a vacuolar fragmentation and a decline in starch, whereaschloroplasts underwent slight alterations. Following rewateringa full recovery of chlorophyll and lipids occurred, while carotenoidsand dienes remained constant. Starch increased in the chloroplastsand there was complete recovery of the ordered cell arrangementand chloroplast organization. Of the chloroplast polar lipids,in both sets of leaves desiccation caused a reduction only inmonogalactosyldiacylglycerol, while phospholipids showed anopposite pattern, increasing in air-dried leaves and decreasingin detached leaves. Rewatering of leaves desiccated on the plantled to a complete recovery of the lipid composition, whereasdetached leaves suffered a complete lipid degradation with theloss of polyunsaturated fatty acids. Key words: Desiccation tolerance, lipids, resurrection plants, Sporobolus stapfianus, ultrastructure