Surface morphology and ultrastructure of normal and cyclic hematopoietic canine bone marrow in long-term liquid cultures

Long-term liquid cultures of normal and cyclic hematopoietic (CH) dog bone marrow produce committed granulocyte-macrophage progenitor cells (CFU-GM) and differentiated granulocytes for several weeks. Analysis of in situ fixed cultures or of cells harvested from the culture supernatants revealed that the cells had ultrastructure and surface morphology characteristic of immature and mature myeloid cells. The surface morphologies of adherent cells from both normal and CH dogs were similar. The characteristic abnormalities previously reported in neutrophils obtained from CH dogs were not observed in neutrophils obtained from long-term marrow cultures of CH dogs. These results indicate that the cellular abnormalities in the neutrophils of CH dogs may be secondary manifestations of the disease and are not inherent to the pathogenesis of the hematopoietic cells.     

Cell cycle status of stromal cells in long-term haematopoietic cultures

This study was performed to further define the mechanism by which the stromal micro-environment regulates haematopoiesis. In long-term marrow cultures the interactions between stromal cells and haematopoietic cells can be investigated at the cellular level. Long-term marrow cultures from hamsters do not require repopulation or addition of hydrocortisone and are suitable for investigation of cell kinetics. The cellular kinetics of haematopoietic and stromal cells, as studied by tritiated thymidine ([3H]dT) incorporation, revealed that DNA synthesis occurred in both the non-adherent and the adherent cells. In established cultures the adherent stromal cells were predominantly in a quiescent non-cycling state: less than 2% adherent cells incorporated [3H]dT within 5 h. Removal of the supernatant cells did not affect the labelling index of adherent cells, since the labelling indices at the 50-75 h time point were 14.3% and 12.5% in the presence and absence of supernatant cells respectively. An apparent stimulus for stromal cells to incorporate [3H]dT was attachment or adhesion. Following replating of supernatant cells of long-term marrow cultures, 23.3% of the reformed adherent layer cells were labelled compared with 12-14% in cultures with previously formed unmobilized adherent cells (P less than 0.01). The data indicate that adherent cells are not required to synthesize DNA for maintenance of haematopoiesis in established long-term marrow cultures, and that recruitment into the cell cycle has an independent mechanism that is not influenced by feed-back from the supernatant cells.     

Proteins in the amniotic and allantoic fluids of fetal pigs

Seven proteins were identified in the amniotic and allantoic fluids of fetal pigs (Sus scrofa domesticus) using crossed immunoelectrophoresis: albumin, fetuin, transferrin, alpha-fetoprotein (AFP), alpha 1-acid glycoprotein, alpha 1-antitrypsin and alpha 2-macroglobulin. The total protein concentrations were determined by the method of Bradford and individual protein concentrations by radial immunodiffusion or rocket immunoelectrophoresis. Transferrin and fetuin were the major proteins in amniotic fluid during the second trimester of gestation and together with AFP and albumin accounted for the majority of the total protein in amniotic, but not allantoic fluid.     

Cell viability and proliferation capability of long-term human dental pulp stem cell cultures

Background aimsEvaluation of cell viability is one of the most important steps of the quality control process for therapeutic use of cells. The aim of this study was to evaluate the long-term cell viability profile of human dental pulp stem cell (hDPSC) subcultures (beyond 10 passages) to determine which of these passages are suitable for clinical use and to identify the cell death processes that may occur in the last passages.MethodsFour different cell viability assays were combined to determine the average cell viability levels at each cell passage: trypan blue exclusion test, water-soluble tetrazolium 1 (WST-1), LIVE/DEAD Viability/Cytotoxicity Kit and electron probe x-ray microanalysis (EPXMA). Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and caspase 4 and BCL7C Western blotting, and cell proliferation was analyzed by WST-1 and proliferating cell nuclear antigen protein detection.ResultshDPSCs showed high average cell viability levels from passages 11–14, with adequate cytoplasmic and mitochondrial functionality at these subcultures. A non-significant trend to decreased cell proliferation was found from passages 16–20. EPXMA and TUNEL analyses suggested that a pre-apoptotic process could be activated from passages 15–20 (P < 0.001), with a correlation with caspase 4 and BCL7C expression.ConclusionshDPSCs corresponding to passages 11–14 show adequate cell function, proliferation and viability. These cells could be considered as potentially useful for clinical applications.     

Chromosome lesions in amniotic fluid cell cultures

Summary The frequencies of chromosome lesions were determined on 3537 mitoses in samples of varying sizes from cultures of 25 amniotic fluid specimens taken from patients at cytogenetic risk. The average percentage values of aberrant cells, including and excluding gaps, were 12.5 and 4.9, respectively. The corresponding values for fibroblasts and peripheral blood lymphocytes from normal adult donors, calculated under the same laboratory conditions, were 5.0 (including gaps) and 2.4 (excluding gaps) and 2.4 (including gaps) and 1.0 (excluding gaps), respectively. The hypothesis of a correlation between the increased incidence of chromosome lesions and the occurrence of abnormal karyotypes in amniotic fluid cell cultures is discussed.     

Chromosome analysis of BALB/c mouse spermatozoa with normal and abnormal head morphology.

To assess the relationship between mouse sperm head morphology and karyotype, sperm heads with either a normal or an abnormal morphology were injected individually into enucleated mouse oocytes that were karyotyped at the metaphase of the first cleavage. BALB/c male mice that produce an unusually high proportion of morphologically abnormal spermatozoa were used as sperm donors. Abnormal karyotypes were found in a significantly higher proportion of eggs injected with severely misshapen sperm heads (36-38%) as compared to those injected with normal and quasi-normal heads (15-21%) (p < 0.01). Most karyotype abnormalities were structural rather than numerical, the most common being breaks and exchanges of chromosome type in both normal and abnormal spermatozoa.     

Chromosomal mosaicism in amniotic fluid cell cultures.

Over the past 6 years, using in situ processing methods, we have identified 32 cases of mosaicism in amniotic fluid cell cultures prepared from 1,100 samples. Two of these (45,X/46,XX and 46,XX/47,XX, + 21) were called true mosaics because multiple colonies demonstrated the same abnormal chromosome complement, and on subsequent evaluation of the newborn blood or fetal tissues, mosaicism was confirmed. Of the remaining cases, 29 were designated as pseudomosaics because only single or partial colonies exhibited an aberrant chromosome complement, 12 having a trisomy 2 line. In the final case, a double trisomy was demonstrated in only one of eight colonies in the first culture, but in the culture from a repeat sample an additional two colonies showed the same double trisomy. Since no abnormal cells were observed in infant blood, it was postulated that the mosaicism may only have been present in the extraembryonic tissues. It is our conviction that the use of these cloning methods should diminish the danger of misdiagnosis in genetic amniocentesis.     

Characteristics of long-term human epithelial cell cultures derived from normal human fetal kidney

Summary Epithelial cell cultures were prepared from normal human fetal kidney and established in long-term culture. The growth characteristics and production of keratin, and alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGT) activities were compared in a modified minimal essential medium (mMEM),d-valine-containing modified alpha-MEM (mALPHA) andl-valine mALPHA. The mean number of cumulative population doublings (CPDL) was significantly (P<0.001) enhanced with thel-valine mALPHA (40.8 CPDL) over that achievable in mMEM (14.2 CPDL) ord-valine mALPHA (18.3 CPDL) media. In all three media, greater than 95% of the cells in culture produced keratin throughout the life span of these cultures. Surface-associated fibronectin was absent in these cell cultures. AP and GGT activities increased as a function of subpassage and time in culture, with the greatest activity in thel-valine mALPHA. The expression of these renal cell-associated functions suggests that these cells in culture are proximal tubule epithelial cells. The conditions and procedures described in this paper can provide a human kidney epithelial cell culture system for studying human renal function, metabolism, cytotoxicity, genotoxicity, and transformation. Research was supported by a NIEHS (ES 3101) grant to S. M. D’Ambrosio and a NCI grant (CA21104) to J. E. Trosko.     

Cell culture density as a modulator of collagenase expression in normal human fibroblast cultures

The effect of various stages of normal cell growth on human fibroblast collagenase found in the culture medium was studied, so that the regulatory mechanisms of synthesis, secretion and activity of the enzyme could be established. Specific activity of collagenase increased 6- to 10-fold shortly after confluence was reached when compared with low density levels and decreased in post-confluent cultures, suggesting that synthesis and/or release of the enzyme changes with culture density. To assess this possibility, culture medium was examined for immunoreactive collagenase protein by radioimmunoassay. After confluence was reached, immunoreactive collagenase had increased approx. 2-fold, indicating greater secretion, and probably synthesis, of the enzyme. However, the increase in specific activity of the enzyme observed shortly after confluence was greater than could be accounted for by an increase in immunoreactive enzyme protein. As a result of the disproportionate increase in collagenase activity, the collagenase activity per unit immunoreactive protein was also found to be greatest shortly after confluence and decreased in post-confluent cultures. This density-associated modulation of collagenase expression could be reproduced by initiating the cultures at high density after subculture. Expression of collagenase activity was dependent upon intact protein synthetic mechanisms, since cultures maintained in the presence of cycloheximide failed to secrete collagenase into the culture medium.     

Preservation of human chorionic gonadotropin and placental lactogen secretion and normal morphology following long-term culture of normal human placental cells

In order to study the regulation of hCG and hPL secretion during gestation, a system for the preservation of the functional integrity of normal placental cells in long-term culture was established. Normal term placental cells were dispersed with 0.25% trypsin-500 units DNAse I and cultured in a monolayer in Dulbecco's modified Eagle medium with 10% fetal bovine serum. Normal cell morphology, basal hCG and hPL production and hCG responses to dibutyryl cAMP were preserved till 54 days of culture. This model may be useful for the study of long-term regulation of normal placental hCG and hPL synthesis and secretion.     

Suppression of lymphocyte reactivity in vitro by porcine allantoic and amniotic fluids

Porcine allantoic and amniotic fluids from early, mid and late pregnancy were analyzed for protein constituents and ability to suppress phytohemagglutinin (PHA)-induced lymphocyte transformation. All fetal fluids contained alpha-fetoprotein as evaluated by polyacrylamide gel electrophoresis (PAGE), with the highest concentrations appearing in mid-pregnancy amniotic fluids. In addition, allantoic fluids from mid-pregnancy contained proteins assumed to be secreted by the uterus. Both allantoic and amniotic fluids from mid-pregnancy were suppressive to PHA-induced lymphocyte transformation (P<.01). Thus it was concluded that substances, potentially able to suppress the immune response locally, exist within porcine fetal fluids, but it was not determined whether this material is of solely fetal or maternal origin, or a combination.