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.

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.

The use of low-power laser irradiation for faster vascularization of tissue transplants.

We studied the effect of low-power laser irradiation on vascularization and take of transplanted rabbit renal and pancreatic tissue in athymic nude mice. The mean size of the transplant and the number of blood vessels in it were higher in irradiated mice compared to nonirradiated controls. Moreover, the organ-specific structure of the transplants was preserved in irradiated mice, but not in the control group. These findings suggest that low-power laser irradiation can be used for promotion of vascularization and take of tissue transplants.

Stimulation of the growth of human tumor by low-power laser irradiation.

We studied the effect of low-power laser on the growth of human gastric adenocarcinoma transplanted to athymic mice. Irradiation shortened the latency of tumor growth in recipients from 4-6 months to 21-24 days. After 17 serial passages on athymic mice, the size of tumor node in irradiated recipients on day 33 after transplantation was 161.1 mm(3) (vs. 10.2 mm(3) in nonirradiated mice). These findings suggest that low-power laser irradiation can stimulate the growth of metastases in patients with a history of malignancy.

Vocal cord augmentation with cultured autologous fibroblasts.

Bovine collagen is an acceptable agent for vocal cord medialization; however, it produces only a temporary effect. As a foreign protein bovine collagen is susceptible to host collagenase and can induce immune response. Autologous collagen has become recently available, but it is less effective as a medialization agent. The study examines human skin fibroblasts growing in culture. Human skin bioptates were taken from the retroauricular area. Fibroblasts in culture were tested for scar contractility and ability to produce type I collagen (by flow cytometry with labeled antibodies). After five passages in culture the cells produced normal type I collagen, exhibited normal contractility, and did not induce no tumors in nude mice.

Safety of injectable autologous human fibroblasts.

Autologous dermal fibroblasts after propagation in cell culture were used for face soft tissue augmentation. Twenty patients aged 37-61 years with facial rhytides and atrophic scars were treated with autologous fibroblasts from cell culture. Significant sustained clinical improvement was observed. Cells of early passages (4, 5, 6) were used for injection. The study showed that cultured fibroblasts were functionally active and produced large quantities of type I collagen. In vitro studies of scar formation potency of injectable fibroblasts showed that these cells possessed normal collagen gel contraction capacity. In vivo experiments showed that cultured fibroblasts exhibited no oncogenic properties and induced no tumors in nude mice.

Glucosaminylmuramyldipeptide-induced changes in phenotype of melanoma cells result in their increased lysis by peripheral blood cells.

Flow cytometry was used to show that biologically active N-acetylglucosamine-containing muramylpeptides (GMPs) induced in vitro dose-dependent increase in the expression of tumor-associated antigens (TAAs) characteristic for colon and mammary gland carcinomas, melanoma and lung adenocarcinoma. Forty to two hundred percent enhancement in TAA-expressing cells was observed after 18-48 h incubation with GMPs. In contrast, MHC class I antigen expression was not altered. Using MTT and chromium-release assays, melanoma cells treated in vitro with GMDP were shown to be more susceptible to killing by peripheral blood cells of healthy donors than non-treated cells. Fractionation of blood cells revealed that platelets were responsible for this effect.

Changes in expression of surface and core antigens of hepatitis B virus in different mutant clones of hepatoma PLC-PRF-5 cells.

Mutant clones of human hepatocarcinoma PLC-PRF-5 cells carrying a hepatitis B virus (HBV) genome have been obtained using selection for resistance to the toxic action of a variety of preparations to induce cell differentiation. The clones differed in various features such as expression of alpha-fetoprotein (AFP) and albumin as well as in growth rates, ability to grow in semisolid media and to be cloned in agar. Expression of the surface antigen (HBsAg) was significantly increased in mutant cells exhibiting differentiation features in contrast to the parental cells. In addition, the core antigen (HBcAg), which was silent in the original cells, was detected in some clones. There was no temporal correlation between the peak of enhanced expression of HBsAg and activation of HBcAg observed at different life periods of each clone. Evidence of cell fusion in cell culture such as premature chromatin condensation and increased numbers of binucleate cells was detected in clones with differentiation features and an increased level of viral gene expression. The approach used in this study can be used to develop cell lines of the same origin with different degrees of differentiation whilst maintaining HBV expression. This model system may be useful in the study of HBV.

[Plasmid DNA transfection in fibroblasts of athymic rats]. / Issledovanie transfektsii plazmidnoi DNK v fibroblasty bestimusnoi krysy.

Nontumorigenic clone FR-7 cl 13 from fibroblasts of athymic rat was obtained from stroma of human colon carcinoma xenograft propagated on nude animals. Spontaneous transformation of this cells was absent after 40 passages in vitro and treatment with pSV2neo. But cells give rise to tumors in athymic mice after transfection with pEJ. This cell clone can be recommended as cells-targets for transfection.

[Induction of stromal cell transformation in xenografts of human colonic cancer]. / Induktsiia transformatsii stromal'nykh kletok v ksenotransplantatakh raka tolstoi kishki cheloveka.

Two transformed stromal cell lines FM-7 and FR-7 were obtained from human xenografts of colon tumor (HCT-7), propagated in nude mice and nude rats, respectively. These two cell lines were confirmed to be murine and rat's by a cytogenetic study and were tumourigenic in BALB/c nude mice with the morphology of fibrosarcoma. Human DNA sequences were not detected by hybridization with pBlur8 probe in DNA of FR-7 cell line grown in vitro. These results indicate that human cancer cells from xenograft HCT-7 can induce malignancy in adjacent normal cells of nude mice and nude rats in the absence of DNA transfer.

[The evaluation of the mutagenic activity of pertussis vaccinal preparations]. / Otsenka mutagennoi aktivnosti kokliushnykh vaktsinal'nykh preparatov.

Two vaccine preparations obtained from Bordetella pertussis, whole-cell vaccine constituting one of the components of adsorbed DPT vaccine and acellular vaccine, were tested for mutagenicity. The doses of the preparations covered the range 1-100 ED50. Ames' test and the metaphase analysis of marrow cells of C57BL/6J mice were used. The acellular preparation was also tested on thymectomized mice, taking into consideration chromosomal aberrations in marrow metaphases. Whole-cell and acellular pertussis vaccines did not induce mutations in Salmonella typhimurium and chromosomal aberrations in mouse marrow cells.

[Xenograft of human RShM32 cervical cancer and comparative DNA flow cytometry]. / Geterotransplantat raka sheiki matki cheloveka RShM 32 i sravnitel'naia tsitometriia DNK.

Xenograft CC-32 was obtained from uterine cervical carcinoma of 24 years old woman in IV stage of the disease. Morphological diagnosis of clinical material and xenograft at various passages was the same-squamous cell carcinoma. Examination tumour DNA showed HPV-16 DNA integrated in five selected points. Tumor CC-32 contained a large quantity a diploid and tetraploid cells in contrast with another cervical carcinoma xenograft.