Expression of fetal antigens in fetal and adult cells during long-term culture

Summary Expression of fetal antigens in early and late passages of tissue culture cells derived from C3H/HeN and C57BL/KaLw mouse fetal cells and from lung tissue of young C57BL/6N mice was investigated by the isotopic antiglobulin technique. The late passage lines of fetal cells had undergone “spontaneous” neoplastic transformation in culture. The antisera were produced by syngeneic immunization with 5000 R x-irradiated tissues from C3H/HeN and C57BL/6N fetuses of 1 to 2 weeks gestation. Fetal antigens were found to be retained even after 5 years in cell lines derived from fetal tissues In these lines no consistent change in fetal antigen expression could be correlated with neoplastic transformation. In contrast, the early passages of adult cells did not have detectable amounts of fetal antigens. However, fetal antigen(s) was demonstrated in cells of the late passages, and cells of both lines grew as sarcomas when next assayed 55 days later. In addition, fetal antigens were also present in established tumor lines in culture.     

Expression of fetal antigens by normal human skin cells grown in tissue culture.

Normal human sera are capable of causing complement-mediated lysis of normal human skin cells grown in tissue culture. This lytic reactivity can be completely removed by absorption with first trimester fetal tissue. Absorption with a variety of normal adult human tissues including lymphocytes, decidua, skin, and muscle are incapable of absorbing reactivity. Absorption of reactivity by fetal tissue is specific and not due to the introduction of anti-complementary or other nonspecific factors, as evidenced by the inability of simultaneous fetal absorption to remove reactivity from antisera with specificity for HLA antigens. Similarly, absorption of lytic sera with fetal calf serum proteins was incapable of removing reactivity against normal cells in tissue culture. It thus appears that normal human cells in tissue culture express antigens shared by the first trimester human fetus, but not present on a variety of adult human tissues. This "neoantigen" present on normal human cells when grown in tissue culture is a potential source of confusion and must be accounted for in searching for human tumor-specific antigens utilizing tissue culture cells.     

Lack of glucose-induced functional maturation during long-term culture of human fetal islet cells

To investigate the long-term effects of glucose on the function of human fetal islets we cultured islet-like cell clusters (ICC) obtained from 12 human fetuses with a mean age of 16.1 weeks in media containing 2.8, 11.1 or 16.7 mM glucose. On the 8th day of culture, the ICC that had been maintained in 16.7 mM glucose contained 60% less insulin than the ICC cultured in 2.8 mM glucose. However, insulin release was similar in both groups, and was not affected by a 24-h incubation in high vs. low glucose. Also (pro) insulin biosynthesis was not significantly affected. During a 24-day culture period, the total release of insulin and glucagon was similar in all glucose concentrations. The ICC released about 75% of their insulin content but only 15% of their glucagon content during the last 48 h of the 24-day culture period, again regardless of glucose concentration in media. Insulin release was insensitive to acute glucose and leucine challenges in perifusion experiments after culture for 1, 5, 8 or 16 days in 11.1 mM glucose, whereas glucagon was always a potent stimulus. In conclusion, the function of cultured young human fetal islet cells is remarkably independent of glucose, even during prolonged exposure. Moreover, the primary role of glucagon in fetal life may be that of a paracrine stimulator of beta-cell function.     

DNA synthesis of adult mammalian cardiac muscle cells in long-term culture

Adult rat cardiac ventricular muscle cells were isolated and cultured in monolayer for 30-45 days. Most of the cardiac muscle cells undergo external and internal structural alterations, resembling embryonic/neonatal cardiac muscle cells in culture (Nag and Cheng, 1981; Nag et al., 1983). These cultured cells underwent DNA synthesis and mitosis as revealed by autoradiography studies that involved the exposure of the cells to [3H]-thymidine for 24 hr prior to the termination of the culture at selected intervals. During the first week of culture, cardiac muscle cells showed less than 5% labeled cells. The labeling index of myocytes attained a peak in the second week of culture, exhibiting approximately 23% labeled cells. The labeling indices of cardiac muscle cells declined over the period of 30 days of culture. During the end of the incubation period, approximately 4% of the myocytes were labeled. When the extent of the total cell population involved in DNA synthesis was examined by exposing the cells to [3H]-thymidine continuously for long periods of time, it was observed that approximately 26% of the cardiac muscle cells regained the capacity for DNA synthesis during 1-10 days of culture. From day 1 to day 14, approximately 29% of the total muscle cell population was labeled. When the cells were exposed to the radioactive isotope continuously for 30 days, approximately 31% of the cells incorporated radioactive isotope, showing their capacity for DNA synthesis. Approximately 90% of the cardiac muscle cells in long-term culture contained more than one nucleus. The nuclei were often observed in multiples of two. Labeled mitotic apparatus was observed in cardiac myocytes, indicating the replication of DNA, followed by karyokinesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation,characterization, and long-term culture of fetal bovine tracheal epithelial cells

Summary Epithelial cells were isolated from fetal bovine trachea by exposing and stripping the mucosal epithelium from the adjacent connective tissue. The tissue was minced and enzymically dissociated in Ca-Mg-free medium containing dispase and dithiothreitol. The stripping procedure and selective trypsinization produced epithelial cell cultures free of fibroblasts. Seeded on plastic, the plating efficiency was 21.5% with a doubling time of 24 h. Dome formation, evidence of occluding junctions and active ion transport characteristic of epithelial cells, was common. Growth of the cells on glass, collagen, and Engelbreth-Holm-Swarm (EHS) substrate demonstrated a striking difference in morphology. Cells grown on EHS presented a more distinctly three-dimensional growth pattern and many more microvilli when compared to cells grown on glass or collagen. The cells retained their epithelioid characteristics through more than 30 passages as shown by the presence of distinct apical and basolateral membranes, tight junctions, and positive keratin staining. This study was supported in part by grants BRSG S07 RR05408-25, Biomedical Research Support Grant Program, Division of Research Resources, by ES 00159, Center Grant, National Institutes of Environmental Health Sciences, by R23-HL37621, New Investigator Award, National Heart, Lung and Blood Institutes, National Institutes of Health, and by the Health Effects Institute, an organization jointly funded by the U.S. Environmental Protection Agency (Assistance Agreement X-8120059) and automotive manufactures. The contents do not necessarily reflect the views of policies of HEI, EPA, or automotive manufacturers.     

Marsupial cells in long-term culture

Cell lines have been developed from several species of Australian marsupials and studied during long-term growth. Cell lines developed from macropodid skin or heart tissues all had reproducible finite life-spans. However, cell lines developed from dasyurids showed bariable behavior in culture: lines developed from Antechinus stuartii and Dasyurus viverrinus had finite life-spans, while lines developed from Sminthopsis crassicaudata had indefinite life-spans. S. crassicaudata lines usually became heteroplloid, but one was still diploid after 150 population doublings, while another contained a proportion (10%) of haploid cells. Other lines were developed from the peramelid, Perameles nasuta, and the phanlngerid, Trichosurus vulpecula.     

Marsupial cells in long-term culture

Summary Cell lines have been developed from several species of Australian marsupials and studied during long-term growth. Cell lines developed from macropodid skin or heart tissues all had reproducible finite life-spans. However, cell lines developed from dasyurids showed variable behavior in culture: lines developed fromAntechinus stuartii andDasyurus viverrinus had finite life-spans, while lines developed fromSminthopsis crassicaudata had indefinite life-spans.S. crassicaudata lines usually became heteroploid, but one was still diploid after 150 population doublings, while another contained a proportion (10%) of haploid cells. Other lines were developed from the peramelid,Perameles nasuta, and the phalangerid,Trichosurus vulpecula.     

Human oral epithelial cell culture II. Keratin expression in fetal and adult gingival cells

Summary Epithelial cells from human fetal and adult gingiva were cultured in keratinocyte growth medium (KGM), a serum-free medium. The expression of keratin proteins in these cells was evaluated using immunohistochemistry and SDS-PAGE-immunoblot analysis and compared with expression in the tissue. Keratins 5, 6, 14, 16, and 19 were identified in cells cultured from both fetal and adult tissues. K19 was localized in basal cells of fetal oral tissue but was not seen in adult gingiva (except for scattered Merkel cells). K1 and K10 were expressed in tissue, but not in cultured cells. The keratin profiles of cultured epithelial cells from several adult donors were similar and were identical in cultures from primary through Passage 5. K13, a differentiation-specific keratin, was expressed in all suprabasal cells of fetal oral epithelium, but shows only spotty expression in adult gingival tissue. K13 was expressed in cultures of fetal cells, but very weakly or not at all in cultures of adult cells. K13 expression was greater in cultures grown with physiologic calcium concentrations (1.2 mM) than in those grown at 0.15 mM or less. Our findings are consistent with basal-like characters of these cells in 0.15 mM calcium growth conditions. Differentiation of fetal oral cells in culture to the suprabasal basal cell stage in 1.2 mM Ca²⁺ is shown by the expressionof K13. This work was supported by Biomedical Research grant RR05346, National Institutes of Health grant DE04660, University of Washington Graduate Fund and Hack Foundation Fund, Department of Periodontology, University of Washington.     

Isolation,long-term culture,and ultrastructural characterization of adult cardiomyopathic cardiac muscle cells

Summary A long-term cell culture system for adult cardiomyopathic hamster cardiac muscle cells has been established. The diseased and control hearts were dissociated into single cell suspension with the modifications of our previous technique using collagenase and hyaluronidase as applied to the dissociation of the adult rat heart. The postperfusion of the diseased heart with Krebs-Ringer phosphate buffer and bovine serum albumin was very helpful in obtaining greater yield of viable diseased muscle cells; the cells were cultured for 4 wk. Approximately 60% of the myocytes from the diseased heart and 85% of the myocytes from the normal heart attached to the substrates and survived throughout the culture period. Approximately 60 to 70% of the cardiac myocytes from the diseased and control hearts were bi- or multinucleated; 30% of the diseased and 80% of the normal myocytes showed rhythmic contractility. Electron microscopy revealed the presence of two kinds of cardiac muscle cells in the diseased cell culture on the basis of their myofibril content: one with scanty myofibrils and another with abundant myofibrils. Myocytes with sparse myofibrils showed certain characteristic features that included autophagic vacuoles, amorphous matrix of fine filamentous texture, scattered strips of myofibrils, and abnormal organization of the Z-line. Cardiac muscle cells with abundant myofibrillar content contained unorganized myofibrils in certain sarcomeres. These studies demonstrate the feasibility of maintaining diseased cardiac muscle cells from adult cardiomyopathic hamsters for at least 4 wk in monolayer culture. This study was supported by a grant from the American Heart Association of Michigan, National Institutes of Health grant HL-25482, and by an Oakland University Biomedical Research Support Grant.     

Isolated tumor endothelial cells maintain specific character during long-term culture

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells.In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.     

Genetic and epigenetic properties of mouse male germline stem cells during long-term culture

Although stem cells are believed to divide infinitely by self-renewal division, there is little evidence that demonstrates their infinite replicative potential. Spermatogonial stem cells are the founder cell population for spermatogenesis. Recently, in vitro culture of spermatogonial stem cells was described. Spermatogonial stem cells can be expanded in vitro in the presence of glial cell line-derived neurotrophic factor (GDNF), maintaining the capacity to produce spermatogenesis after transplantation into testis. Here, we examined the stability and proliferative capacity of spermatogonial stem cells using cultured cells. Spermatogonial stem cells were cultured over 2 years and achieved approximately 10(85)-fold expansion. Unlike other germline cells that often acquire genetic and epigenetic changes in vitro, spermatogonial stem cells retained the euploid karyotype and androgenetic imprint during the 2-year experimental period, and produced normal spermatogenesis and fertile offspring. However, the telomeres in spermatogonial stem cells gradually shortened during culture, suggesting that they are not immortal. Nevertheless, the remarkable stability and proliferative potential of spermatogonial stem cells suggest that they have a unique machinery to prevent transmission of genetic and epigenetic damages to the offspring, and these characteristics make them an attractive target for germline modification.     

Protective potential of hematopoietic cells of the embryonic liver during long-term culture

The influence of the duration of the organ cultivation on the protective potential of mouse fetal liver hemopoietic cells was investigated. The protective potential was evaluated according to a 3-week viability of the lethally irradiated (mean LD88/21) mice following syngeneic transplantation. During 20 days of cultivation the protective potential (calculated per number of injected cells) remained at the initial level. With more prolonged cultivation (24-62 days) the protective potential was retained only in part of cultures and the mean effect was reduced. No parallelism has been revealed between the totipotent hemopoietic precursors capable of replacing the hemopoietic cells of an irradiated recipient and the CFUs in long-cultivated fetal liver cultures.     

Murine adult hematopoietic cells produce adult erythrocytes in fetal recipients

Bone marrow cells from normal adult mice were introduced by microinjection via the placenta into W/W^v genetically anemic fetuses of 11 days' gestation. After birth, erythrocytes were fractionated by fluorescence-activated cell sorting on the basis of antibody binding to a fetal-specific antigen (Ft). Lysates of Ft-positive, i.e., fetal, erythrocytes did not detectably contain hemoglobin of the donor type, as judged from electrophoresis of strain-specific hemoglobin variants. Thus, adult hematopoietic bone marrow cells did not resume fetal differentiation despite their post-transplant maturation in a fetal environment.     

Expression of bone morphogenetic proteins in stromal cells from human bone marrow long-term culture.

Highly purified primitive hemopoietic stem cells express BMP receptors but do not synthesize bone morphogenetic proteins (BMPs). However, exogenously added BMPs regulate their proliferation, differentiation, and survival. To further explore the mechanism by which BMPs might be involved in hemopoietic differentiation, we tested whether stromal cells from long-term culture (LTC) of normal human bone marrow produce BMPs, BMP receptors, and SMAD signaling molecules. Stromal cells were immunohistochemically characterized by the presence of lyzozyme, CD 31, factor VIII, CD 68, S100, alkaline phosphatase, and vimentin. Gene expression was analyzed by RT-PCR and the presence of BMP protein was confirmed by immunohistochemistry (IHC). The supportive role of the stromal cell layer in hemopoiesis in vitro was confirmed by a colony assay of clonogenic progenitors. Bone marrow stromal cells express mRNA and protein for BMP-3, -4, and -7 but not for BMP-2, -5, and -6 from the first to the eighth week of culture. Furthermore, stromal cells express the BMP type I receptors, activin-like kinase-3 (ALK-3), ALK-6, and the downstream transducers SMAD-1, -4, and -5. Thus, human bone marrow stromal cells synthesize BMPs, which might exert their effects on hemopoietic stem cells in a paracrine manner through specific BMP receptors.     

Expression of adult fast pattern of acetylcholinesterase molecular forms by mouse satellite cells in culture

The pattern of acetylcholinesterase (AChE) molecular forms, obtained by sucrose gradient sedimentation, was studied at different in vitro developmental stages of myogenic cells isolated from adult mouse skeletal muscle. Only the globular forms were present in rapidly dividing satellite cells during the first days in culture. After myotube formation, a pattern similar to that described in mammalian fast-twitch skeletal muscle was observed. This pattern did not change during the following period in culture (up to 1 month) nor could it be modified by co-culturing with spinal cord motoneurons or by addition of brain-derived extracts. The internal-external localization of AChE molecular forms has been determined by the use of echothiophate iodide, a membrane-impermeant irreversible inhibitor of AChE. Echothiophate-treated cultures showed about 40% of both asymmetric and globular forms localized on the sarcolemma, with their active sites oriented outward. Analysis of culture medium from untreated cultures revealed the presence of both asymmetric and globular forms. When the same analysis was repeated on cultures of myoblasts derived from 16-day-old mouse embryos, the pattern of AChE forms was different. The myotubes derived from these cells exhibit a very small proportion of asymmetric form, which was not released into the medium. This pattern was not further modified during the following days of culture, nor by co-cultures with spinal cord motoneurons or by incubations with brain-derived extracts. Thus, the myotubes derived from myoblasts express in culture a clear phenotypic difference when compared to the corresponding myotubes from satellite cells, supporting the view that these two myogenic cells are endowed with different developmental programs.     

DNA methylation in bovine adult and fetal fibroblast cells

The aim of this project was to develop a simple screening tool to measure the DNA methylation of fibroblast cells, and to determine if differences in DNA methylation could be detected in adult and fetal fibroblast cells after serum starvation (SS). Four adult and four fetal tissue explants were collected to produce presumptive fibroblast cell cultures for this experiment. All cell lines underwent three repetitions of serum starvation for 0 (control), 2, 5, or 7 days. The DNA was extracted from the cells and analyzed for DNA methylation content using methylation sensitive restriction enzyme digestion, gel electrophoresis and image analysis. There was no difference (p = 0.11) between the DNA methylation of the adult and fetal nonclonal cell lines. A cubic trend (p = 0.09) of increased DNA methylation at 2 days of serum starvation followed by periods of decreasing DNA methylation at 5 and 7 days were observed for the adult nonclonal cell lines. A significant interaction (p = 0.03) was observed between fetal cell line and day. This simple, rapid DNA methylation assay may be beneficial when evaluating cells' DNA methylation content.     

Expression of the potassium channel ROMK in adult and fetal human kidney

The renal potassium channel ROMK is a crucial element of K⁺ recycling and secretion in the distal tubule and the collecting duct system. Mutations in the ROMK gene (KCNJ1) lead to hyperprostaglandin E syndrome/antenatal Bartter syndrome, a life-threatening hypokalemic disorder of the newborn. The localization of ROMK channel protein, however, remains unknown in humans. We generated an affinity-purified specific polyclonal anti-ROMK antibody raised against a C-terminal peptide of human ROMK. Immunoblotting revealed a 45 kDa protein band in both rat and human kidney tissue. In human kidney sections, the antibody showed intense staining of epithelial cells in the cortical and medullary thick ascending limb (TAL), the connecting tubule, and the collecting duct. Moreover, a strong expression of ROMK protein was detected in cells of the macula densa. In epithelial cells of the TAL expression of ROMK protein was mainly restricted to the apical membrane. In human fetal kidney expression of ROMK protein was detected mainly in distal tubules of mature nephrons but not or only marginally in the collecting system. No expression was found in early developmental stages such as comma or S shapes, indicating a differentiation-dependent expression of ROMK protein. In summary, these findings support the proposed role of ROMK channels in potassium recycling and in the regulation of K⁺ secretion and present a rationale for the phenotype observed in patients with ROMK deficiency.