HLA homozygous stem cell lines derived from human parthenogenetic blastocysts

Individual HLA homozygous parthenogenetic human stem cell (hpSC-Hhom) lines have the potential for cell-based therapy in a significant number of individuals, provided the HLA haplotype is prevalent. We report the successful derivation of four stable hpSC-Hhom lines from both HLA homozygous and HLA heterozygous donors. Of these, the hpSC-Hhom-4 line carries the HLA haplotype found most commonly within the U.S. population, and is shared by different racial groups. These hpSC-Hhom lines demonstrate typical human embryonic stem cell morphology, expressing appropriate stem cell markers and possessing high levels of alkaline phosphatase and telomerase activity. Additionally, injection of these cell lines into immunodeficient animals leads to teratoma formation. G-banded karyotyping demonstrates a normal 46,XX karyotype in lines hpSC-Hhom-1 and hpSC-Hhom-4, and chromosomal anomalies in lines hpSC-Hhom-2 and hpSC-Hhom-3, both derived from the same donor. HLA genotyping of all four hpSC-Hhom lines demonstrates that they are HLA homozygous. Furthermore, in the case of HLA heterozygous donors, the hpSC-Hhom lines inherit the haplotype from only one of the donor's parents. Single-nucleotide polymorphism (SNP) data analysis suggests that hpSC-Hhom lines derived from HLA heterozygous oocyte donors are homozygous throughout the genome as assessed by SNP analysis. The protocol used for deriving these HLA homozygous stem cell lines minimizes the use of animal-derived components, which makes them more appealing for potential clinical application.     

QTL mapping under truncation selection in homozygous lines derived from biparental crosses

In plant breeding, a large number of progenies that will be discarded later in the breeding process must be phenotyped and marker genotyped for conducting QTL analysis. In many cases, phenotypic preselection of lines could be useful. However, in QTL analyses even moderate preselection can have a significant effect on the power of QTL detection and estimation of effects of the target traits. In this study, we provide exact formulas for quantifying the change of allele frequencies within marker classes, expectations of marker contrasts and the variance of the marker contrasts under truncation selection, for the general case of two QTL affecting the target trait and a correlated trait. We focused on homozygous lines derived at random from biparental crosses. The effects of linkage between the marker and the QTL under selection as well as the effect of selection on a correlated trait can be quantified with the given formulas. Theoretical results clearly show that depending on the magnitude of QTL effects, high selection intensities can lead to a dramatic reduction in power of QTL detection and that approximations based on the infinitesimal model deviate substantially from exact solutions. The presented formulas are valuable for choosing appropriate selection intensity when performing QTL mapping experiments on the data on phenotypically preselected traits and enable the calculation and bias correction of the effects of QTL under selection. Application of our theory to experimental data revealed that selection-induced bias of QTL effects can be successfully corrected.     

Paraquat-resistant cell lines derived from Chinese hamster ovary cells

Two paraquat-resistant clones, PR-1 and PR-2, were selected from CHO K1 cells pretreated with ethyl methanesulfonate. PR-1 and PR-2, routinely cultured in a normal medium without paraquat, were six fold more resistant to paraquat than the parental CHO K1 cells. There was no difference in the uptake of [3H]paraquat among PR-1, PR-2, and CHO K1 cells. Both PR-1 and PR-2 cells showed no cross resistance to free radical generating agents and no increase in total activity of superoxide dismutase. The activities of paraquat-dependent NADPH oxidase and glucose-6-phosphate dehydrogenase were significantly reduced in PR-1 and PR-2 cells, hence the rate of paraquat radical formation will be limited. In addition, an elevation of glutathione levels in PR-1 cells or an increase in glutathione S-transferase activity in PR-2 cells may also play a certain role in protective mechanisms against the toxicity of paraquat.     

Fish cell lines: Two new cell lines derived from explants of trunk musculature ofCynoscion arenarius

Summary Two cell lines were established from explants of trunk musculature of healthy, males sand seatrout,Cynoscion arenarius. One of the lines, designated CyA-1, has been carried through 150 subcultures during 6 yr. The other, designated CyA-2, has been carried through 100 subcultures during 2 yr. Both lines grow well in L15 medium adjusted to 0.150M NaCl and supplemented with 10% fetal bovine serum. Optimal growth occurs at temperatures between 24 and 30°C. The species of origin of both lines was confirmed by a cytotoxic antibody dye exclusion test. The karyotype of CyA-1 has not yet stabilized, showing a modal chromosome number of 120 at Passage 9, 89 at Passage 63, and 79 at Passage 100. The karyotype of CyA-2 is rather stable, with a modal chromosome number of 47 at Passage 1 and 49 at Passage 100. Chromosome morphology of CyA-2 is homogeneous (small, acrocentric), whereas the chromosomes of CyA-1 show considerable size variation (with small chromosomes possibly formed from fragmentation of original structures). Both lines were found to be free of bacterial or fungal contamination. Both lines supported replication of lymphocystis virus strains isolated fromBairdiella chrysura (the silver perch) and fromMicropogon undulatus (the Atlantic croaker) but were refractory to 11 other viruses (4 from fish, 1 from amphibians, and 6 from mammals). This study was supported in part by the National Oceanographic and Atmospheric Administration, Office of Sea Grant No. 04-3-158-58.     

Patient-specific stem cell lines derived from human parthenogenetic blastocysts

Parthenogenetic activation of human oocytes may be one way to produce histocompatible cells for cell-based therapy. We report the successful derivation of six pluripotent human embryonic stem cell (hESC) lines from blastocysts of parthenogenetic origin. The parthenogenetic human embryonic stem cells (phESC) demonstrate typical hESC morphology, express appropriate markers, and possess high levels of alkaline phosphatase and telomerase activity. The phESC lines have a normal 46, XX karyotype, except one cell line, and have been cultured from between 21 to 35 passages. The phESC lines form embryoid bodies in suspension culture and teratomas after injection to immunodeficient animals and give differentiated derivatives of all three embryonic germ layers. DNA profiling of all six phESC lines demonstrates that they are MHC matched with the oocyte donors. The study of imprinted genes demonstrated further evidence of the parthenogenetic origin of the phESC lines. Our research has resulted in a protocol for the production of human parthenogenetic embryos and the derivation of stem cell lines from them, which minimizes the presence of animal-derived components, making the derived phESC lines more suitable for potential clinical use.     

Hodgkin's disease derived cell lines: a review.

A number of so-called "HD cell lines" have been established over the last 10-15 years (Table 1). Or those 15 cell lines we studied, only the cell lines CO, DEV, HD-70, HDLM, KM-H2, L-428, L-540 and SUP-HD1 can be regarded to represent true HD cell lines. According to the immunostaining results and molecular genetic data, these 8 cell lines can be assigned either to the T-cell lineage (CO, HDLM, L-540) or B-cell lineage (DEV, HD-70, KM-H2, SUP-HD1). With the data currently available, the cell lineage origin of L-428 cannot be unequivocally determined, but appears to be lymphoid. All but one of these eight HD cell lines have been established from patients with the nodular sclerosis subtype. Therefore, the conclusions drawn from the in vitro studies are limited to this histological subtype of HD. It is conceivable that culture conditions select for a particular type of cell that will survive. The state of differentiation of these HD cell lines remains unclear due to the incomplete expression of T- or B-cell antigens. The in vitro cells and the in vivo H-RS cells share, however, the expression of the unique activation markers CD15, CD25, CD30, CD71 and HLA-DR. Recently published data indicate that the HD cell lines express and produce a large number of cytokines. Multiple non-random chromosomal abnormalities and the expression of various proto-oncogenes are also new and exciting findings and certainly deserve further study. In summary, although the cultured cells are not unequivocally proven to be the direct progeny of in vivo H-RS cells, several continuous HD cell lines have been established that display a variety of phenotypical features identical or similar to those of their presumed in vivo counterparts. Surface marker, molecular genetic and other features suggest a T- or B-cell derivation. An extrapolation of these conclusions would point to a lymphoid origin of H-RS cells. Whether H-RS cells can originate from other cell types such as monocytes/macrophages or reticulum cells, cannot be answered with the currently available HD cell lines.     

Macrophage cell lines derived from major histocompatibility complex II-negative mice

Summary Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II-negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-γ, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphylococcal enterotoxins A or B. C2Dt cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.     

Establishment of cell lines derived from the rat suprachiasmatic nucleus

Physiological and behavioral circadian rhythms in mammals are orchestrated by a central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Photic input entrains the phase of the central clock, and many peripheral clocks are regulated by neural or hormonal output from the SCN. We established cell lines derived from the rat embryonic SCN to examine the molecular network of the central clock. An established cell line exhibited the stable circadian expression of clock genes. The circadian oscillation was abruptly phase-shifted by forskolin, and abolished by siBmal1. These results are compatible with in vivo studies of the SCN.     

Characterization of myogenic cell lines derived by 5-azacytidine treatment

Three myogenic clonal cell lines were isolated from C3H 10T1/2 C18 cells (10T1/2) treated with 5-azacytidine (5-aza-CR). These lines reproducibly underwent fusion at confluence into functional myotubes capable of contracting in response to acetylcholine. The degree of fusion could be increased two- to threefold if the cells were grown on gelatin-coated dishes. All of the cell lines lost some of their myogenic potential after repeated passaging and the percentage of colonies capable of forming muscle was not increased by permissive media containing 2% horse serum. The 10T1/2 cells expressed only the BB form of creatine phosphokinase but all of the myogenic clones expressed additionally the MM and MB forms of the isozyme after fusion. The overall genomic level of 5-methylcytosine was decreased in some but not all of the cell clones tested. Comparisons between the 10T1/2 cells which never form muscle without 5-aza-CR treatment and clonal derivatives of committed cell types might be of value in understanding the molecular basis of the commitment process.     

Gene expression profiling of cell lines derived from T-cell malignancies

Alterations in the pentose ring of ATP have a major impact on cystic fibrosis transmembrane conductance regulator (CFTR) function. Both 2'- and 3'-deoxy-ATP (dATP) accelerate ion channel openings and stabilize open channel structure better than ATP. Purified wild-type CFTR hydrolyzes dATP. The apparent first-order rate constants for hydrolysis at low substrate concentration are the same for dATP and ATP. This suggests that product release and/or relaxation of the enzyme structure to the initial ligand free state is the rate-limiting step in the CFTR hydrolytic cycle. Circumvention of the normal requirement for protein kinase A phosphorylation of the R-domain for channel activation implies that the impact of the deoxyribonucleotide interaction with the nucleotide binding domains is transmitted to the channel-forming elements of the protein more readily than that of the ribonucleotide.     

Immunocytochemical analysis of cell lines derived from solid tumors.

Antibodies recognizing tissue-specific antigens are widely used to identify the histological origin of tumors. Here we tested the fidelity of selected tissue markers on all 167 solid tumor-derived continuous cell lines in the DSMZ cell lines bank. Most lines had an intermediate filament content consistent with the tumor type from which they were derived. Thus, 93% of all carcinoma cell lines expressed keratin filaments. With certain antibodies, some subclassification was possible. For example, the CK7 keratin 7 antibody can differentiate between colon and pancreas-derived carcinoma cell lines. Cell lines derived from non-carcinomas, in general, did not express keratin but were vimentin-positive. Four of 10 glioma/astrocytoma cell lines expressed GFAP, five of six neuroblastoma cell lines expressed neurofilaments, and the TE-671 rhabdomyosarcoma cell line expressed desmin. When other tissue markers were tested, 12/16 melanoma-derived cell lines expressed HMB-45, while PSA, CA125, and thyroglobulin were less useful. These results demonstrate that cell lines retain some but not all markers typical of the original tumor type and identify certain markers useful in characterizing the histological origin of cell lines. Our data question the identity of some cell lines submitted to the bank in the past. The immunoprofiles of 167 solid tumor-derived and 131 hematopoetic cell lines can be found at www.dsmz.de.     

Characterization of established,cell lines derived from mutagen-sensitiveDrosophila strains

Summary Six established cell lines have been generated from embryos ofDrosophila melanogaster homozygous for different X-linked mutations. Four of these mutants, confer hypersensitivity to chemical mutagens in larvae. The cell lines derived from the two mutageninsensitive stocks, serve as controls in the analyses of DNA metabolism. One cell line (UCD-Dm-mei-9-2) is uniquely identified by a strong hypersensitivity to ultraviolet radiation. Another (UCD-Dm-mus104-1) expresses an enzyme variant not found in the other lines. The population doubling time for these cultures varies between 24 and 47 h. Labeling indices of 24.4 to 37.5% were found. The duration of the S phase in one of the control cell lines is estimated to be about 9 h. Karyotype stability was monitored for five lines over a period of about 1 y. In general these cultures each, became hypotetraploid with a preferential loss of the Y and fourth chromosomes. DNA synthesis in two of the lines fails to exhibit the pattern of sensitivity to mutagens or caffeine that is observed in the corresponding primary cultures. In primary cultures three classes of cells can be identified by autoradiography. About 50% of the cells label at a moderate rate, 20% do not label within the first 1.5 d of culture, and the remaining cells exhibit a burst of labeling shortly after the cultures are initiated. This research was supported by NIH Grants GM16298 and GM22221 and by DOE Contract AT(04-3)-34 PA 210.     

Human embryonic stem cell lines derived from the Chinese population

Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.     

Alkaline phosphatase expression in human cell lines derived from primary hepatomas

Isozyme patterns of alkaline phosphatase (ALP) were electrophoretically examined in human cell lines derived from one hepatoblastoma, five hepatocellular carcinomas (HCCs) and two cholangiocellular carcinomas. Most of the cell lines tested had a liver-type ALP isozyme. In addition, an abnormal ALP isozyme, which was similar to variant ALP, was detected in one hepatoblastoma and two HCC cell lines. One HCC cell line of these variant-like ALP-positive cell lines was alpha-fetoprotein (AFP)-negative. These findings suggest that variant-like ALP may be useful for the identification of human hepatoma cell lines, especially in AFP or albumin-negative cell lines.     

Cytokeratin expression in human cell lines derived from liver tumors.

Immunohistochemical staining of cell lines derived from human liver tumours showed that five cell lines derived from hepatocellular carcinoma (HCC) and hepatoblastoma were stained positively with monoclonal keratin antibodies, CK-5 (Ker-18-specific) and KL-1 (broad specificity), but not with CK-7 (Ker-7-specific). On the other hand, four carcinoma cell lines derived from the biliary system were stained positively with not only CK-5 and KL-1, but also CK-7.     

Embryonic stem cell lines derived from blastocysts by a simplified technique

We have isolated euploid, pluripotent stem cell lines directly from mouse blastocysts by a simple culture technique. Our method permits cell lines to be derived from individual embryos, without the use of ovariectomy, immunosurgery and conditioned medium. We cultured individual intact blastocysts in MicroTest plates on top of a feeder layer of lethally irradiated STO mouse fibroblasts in a 10-microliters volume of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). The cell lines maintain a stable complement of 40 chromosomes, form embryoid bodies and differentiate both in culture and in solid tumors.