[Comparative characteristics of mesenchymal stem cell lines derived from bone marrow and muscle of limb of early human embryo].

In this work, we have carried out a comparative analysis of the characteristics of mesenchymal stem cell lines isolated from different tissues of 5-6-weeks homan embryo: bone marrow (line FetMSC) and muscle of limb (line M-FetMSC). The basic characteristics of these lines were obtained at the 6th passage. Average population doubling time was 33.0 ± 1.4 h (FetMSC) and 25.0 ± 0.1 h (M-FetMSC). Growth curves also indicated active proliferation of cells of both lines. Numerical and structural karyotypic analysis showed that both lines have a normal karyotype: 46, XY. In order to determine the status of the lines, cell surface markers were analyzed by flow cytometry. The analysis revealed the presence of surface antigens specific for human MSCs, CD44, CD73, CD90, CD105, HLA-ABC, vimentin, and the lack of CD34 and HLA-DR, in both lines. The ability to differentiate into osteogenic, chondrogenic and adipogenic directions has been also shown for both lines. Im- munofluorescence and flow cytometry analysis has detected no expression of the surface antigen TRA-1-60 in both lines, but has revealed high expression of the surface antigen SSEA-4 and low expression of transcription factor Oct-4 characteristic of human embryonic stem cells. In these lines, immunofluorescence analysis has shown the presence of the markers of early differentiation in the derivates of three germ layers characteristic of human embryonic stem cells, which provides significant opportunities for MSC to be useful, in corresponding microenvironments, for repair of tissue injures. Dispite confirming MSC status for FetMSC and M-FetMSC lines, a number of interlinear differences related to growth characteristics and differentiation potential were revealed. Adipogenic differentiatiation potential of M-FetMSC line was reduced compared with FetMSC line. Immunofluorescence analysis showed that, in the process of skeletal-muscle differentiation, Z-disks were revealed only in sarcomeres of M-FetMSC line. These findings suggest the possible influence of different microenvironments in which the cells are in the body before their transfer in vitro.

[Developing of a new feeder-free system and characterization of human embryonic stem cell sublines derived in this system under autogenic and allogenic culturing].

A new feeder-free culture system for human embryonic stem cells (hESC) was developed. It consist of extracellular matrix proteins synthesized by feeder cells--mesenchymal stem cell line SC5-MSC, which was derived from initial hESC line SC5. The major ECM proteins--fibronectin and laminin--that maintain hESC growth in feeder-free system were identified. An essential component of this system is a SC5-MSC-conditioned medium. Two hESC sublines were derived. The subline SC5-FF was cultured in autogenic and subline SC7-FF in allogenic system. Sublines SC5-FF and SC7-FF passed through more than 300 and 115 cell population doublings, retained normal diploid karyotype and an ability of in vitro differentiation into derivates of three germ layers. These sublines express markers of undifferentiated hESC: alkaline phosphatase, Oct-4, SSEA-4, TRA-1-81 and multidrug resistance transporter--ABCG2. The RT-PCR analysis revealed that undifferentiated cells SC5-FF subline, like cells of initial feeder-maintained hESC line SC5, expressed genes OCT4 and NANOG, and germ line specific genes such as DPPA3/STELLA and DAZL. An expression of OCT4, NANOG, DPPA3/STELLA ans DAZL was down-regulated during embryonic bodies differentiation, whereas expression of somatic lineages specific genes like GATA4 and AFP (extra embryonic and embryonic endoderm), PAX6 (neuroectoderm) and BRY (mesoderm) was up-regulated. The comparative analysis of some typical features (karyotype structure, the average population doubling time and the number of undifferentiated cells in populations) did not reveal essential differences between initial SC5 and SC7 lines and their sublines SC5-FF and SC7-FF. This shows that feeder-free culture systems, which are much more stable than any feeder systems, do not break main hESC features during long cultivation and can be recommended for fundamental, biomedicine and pharmacological investigations, using hESCs.

[Comparative characteristics of new mesenchymal stem cell lines derived from human embryonic stem cells, bone marrow and foreskin].

New nonimmortalized fibroblast-like cell lines SC5-MSC and SC3a-MSC, FetMSC, FRSN were obtained from human embryonic stem cells (ESC), bone marrow of a 5-6-days embryo and foreskin of a 3-years-old boy, respectively. All the lines are successfully used as the feeder at human ESC cultivation. It is determined that the average cell population doublings time varies from 25.5 h for ISC5-MSC to 38.8 h for SC3a-MSC. Active proliferation of all the lines is also shown by the corresponding growth curves. Numerical and structural karyotypic analysis showed that these lines had normal karyotype: 46,XX (SC5-MSC and SC3a-MSC) and 46,XY (FetMSC and FRSN). To determine the status of the lines, their cell surface markers were analyzed by flow cytometry. This analysis revealed the presence of surface antigens CD44, CD73, CD90, CD105 and HLA-ABC, characteristic of human MSC, and the absence of CD34 and HLA-DR. Different lines were found to express CD117(c-kit) to a different level. Immunofluorescence and flow cytometry analysis did not detect TRA-1-60 and Oct-4, characteristic of human embryonic stem cells, and revealed interlinear variations in the level of SSEA, which did not depend on the cell origin. It is not clear yet whether these interlinear variations affect functional MSC status. In all the lines, immunofluorescence analysis showed the presence of the markers of early differentiation in the derivates of three germ layers which may allow MSC to be useful, in corresponding microenvironments, for reparation of tissue injures. Adipogenic and osteogenic differentiatiation of all cell lines has been shown.

[Comparative characteristics of new human embryonic stem cell lines SC5, SC6, SC7, and SC3a].

Numerous human embryonic stem cell lines with different genetic background are widely used as cell models for fundamental, biomedical and pharmacological research. New hES cell lines SC5, SC6, SC7, and SC3a are derived from the blastocysts and maintained on mitotically inactivated human feeder cells. All derived hES cell lines passed through more than 120 cell population doublings, retained normal diploid karyotype and ability of in vitro differentiation in the derivates of three germ layers. These lines express the markers of undifferentiated hES cells: Oct-4, Nanog, SSEA-4, TRA-1-60, and alkaline phosphatase. Moreover, undifferentiated cells of SC5, SC6, and SC7 lines expressed germ line specific genes DPPA3/STELLA and DAZL and did not express somatic lineages specific genes. In contrast, undifferentiated cells of SC3a line did not express DPPA3/STELLA and DAZL but expressed extra embryonic endoderm cell markers GATA4 and AFP. Double staining of SC5 and SC3a colonies by antibodies against transcription factors Oct-4 and GATA4 has demonstrated that most SC3a cells in colonies were positive for both factors. Furthermore, the cells of SC5, SC6, SC7 lines but not of SC3a line formed teratomas containing the derivates of the three germ layers. These results indicate that, in contrast to the other cell lines, the cells in the SC3a colonies represent an early committed cell population. Moreover, expression of the multidrug resistance transporter gene ABCG2 was detected in undifferentiated cells and differentiating embryonic bodies during 10 days of all lines by immunofluorescent and RT-PCR analyses, whereas RT-PCR analysis has revealed up-regulation of the ABCB1 transporter gene expression in differentiating embryoid bodies of SC5, SC6, and SC7 cells only. Thus, these findings demonstrate different characteristics and differentiation potential of SC5, SC6, SC7, and SC3a hES cell lines which were derived in different conditions.

[Delayed biological consequences induced by gamma-radiation in human tumorigenic diploid line SK-UT-1B]. / Otdalennye biologicheskie posledstviia gamma-radiatsii v chelovecheskoi opukholevoi diploidnoi linii SK-UT-1B.

The progeny of SK-UT-1B cells that survived gamma-irradiation with 4 Gy up to the 80th passage was examined. Descendants of irradiated cells lost p53 transactivation properties. Simultaneously, in the presence of nocodazole coordination between M and S phases was disrupted. Meanwhile, descendants of irradiated cells maintained the accurate spindle assembly checkpoint. These data suggest that p53 transactivation function may be required for coordination of M and S phases, rather than for spindle assembly checkpoint. Since it is known that p53 regulates both these processes on the basis of data obtained, we suggest that functions of p53 required for coordination of M and S-phases and for spindle assembly checkpoint are separated. Besides, the data obtained indicate that radiation-induced chromosomal rearrangements are associated with activation of DNA recombination process.

[Comparison of cytogenic response of human lymphocytes to in vivo and in vitro exposure to low doses of gamma-rays. Unstable chromosomal exchanges detected by FPG techniques]. / Sravnenie tsitogeneticheskoi reaktsii limfotsitov cheloveka na obluchenie v malykh dozakh gamma-izlucheniia in vivo i in vitro. Nestabil'nye khromosomnye obmeny, opredeliaemye FPG-metodom.

On peripheral lymphocytes of eight cancer patients undergone whole-body therapeutic irradiation (at daily dose of 10 cGy up to total dose of 50 cGy of 60Co gamma-rays) the dose-response of unstable chromosome exchanges (dicentrics and centric rings) was studied. This dose response fitted well linear function. The lower slope of dose-response curve was found for in vivo irradiated lymphocytes as compared to the dose response curve obtained for in vitro irradiated lymphocytes of the same patients. This finding seems to provide evidence that in case of protracted irradiation of individuals an absorbed dose could be underestimated if for biological dosimetry an in vitro dose response curve for unstable chromosome aberrations is used as referent one.

[Cytogenetics. Science and people]. / Tsitogenetika. Nauka i liudi.

Some little-known facts are presented about foundation of the Institute of Cytology, Academy of Sciences of the USSR (now of Russia). The advancement of main lines investigation in the field of cytogenetics, carried out at the Institute from its inception up to now, has been traced, with special attention being called to organization of a scientific school of cytogenetics.

[Chromosomal restructurings and the distribution of chromosome fragile sites in the peripheral blood lymphocytes in a breast cancer patient after cytostatic therapy]. / Khromosomnye perestroiki i raspredelenie saitov lomkosti khromosom v limfotsitakh perifericheskoi krovi bol'noi rakom molochnoi zhelezy posle tsitostaticheskoi terapii.

Cytogenetic analysis was performed repeatedly on a breast cancer patient since the beginning of the antitumor treatment. Double minute chromosomes (DMS, 2-10 per cell) were found in less than 2% of peripheral blood lymphocytes besides other chromosomal abnormalities after radiation therapy and 8 months after chemotherapy. The level of structural chromosomal aberrations two years after the therapeutic treatment was 0.13-0.14 aberrations per cell, but DMS were not observed. Estimation of the fragile site (FS) frequency and distribution at this time revealed a significant expression of the common FS FRAGF (9q1.2) after the treatment of blood culture with 5-bromo-2-deoxyuridine at dose levels of 7 and 50 g/l and enhanced fragility in chromosome band 1p35-36.1 (FRA1A) in folate-deprived conditions. Rare FS were not found. The presented data are discussed.

[The cytogenetic and hematotoxic effect of cesium-137 incorporated in the rat body]. / Tsitogeneticheskii i gemotoksicheskii éffekt tseziia-137, inkorporirovannogo v organizme krys.

The influence of incorporated 137Cs on peripheral blood cells was studied at different times after a single per os administration to rats. Moderate lymphopenia occurred in 26 days. A 30-70% increase in the number of aberrant lymphocytes was revealed throughout the entire period of observation (up to 547 days). Rats are suggested to develop a pronounced immune depression and chronic radiation sickness.

[Chromosomal damage in peripheral blood lymphocytes of rats in nitrosomethylurea-induced carcinogenesis]. / Khromosomnye narusheniia v limfotsitakh perifericheskoi krovi krys pri kantserogeneze, indutsirovannom nitrozometilmochevinoi.

Chromosomal damages and their dynamics in blood lymphocytes of mongrel white L10 rats treated (once) intravenously with nitrosomethyl urea (NMU) at a dose of 50 mg/kg have been analyzed. A direct correlation is revealed between carcinogenesis, chromosome aberration level in somatic cells, polyploid and hyperaneuploid cell frequency 24 hs after the NMU treatment and in the precancerous period. An increase of hyperaneuploid and polyploid cells in the organism can serve as a prognostic factor of carcinogenesis.

[Comparative evaluation of the cytogenetic effect of cyclophosphamide on lymphosarcoma cells and the bone marrow of intact and irradiated rats]. / Sravnitel'naia otsenka tsitogeneticheskogo deistviia tsiklofosfana na limfosarkomu i kostnyi mozg intaktnykh i obluchennykh krys.

The paper deals with a comparative evaluation of cytogenetic action of cyclophosphamide on tumor and bone marrow cells in intact and radiation--exposed Pliss' lymphosarcoma-bearing mice. In both study groups, the level of cells with drug--induced chromosome damage was shown to be higher in tumor. No significant difference was established between the 2 groups in the effect of cyclophosphamide on similar tissues.

[Factors responsible for increasing the cytogenetic effectiveness of cyclophosphamide administered to irradiated animals. Cytogenetic effect of mutagens not requiring metabolic activation on bone marrow cells of irradiated rats]. / Izuchenie prichin, obuslovlivaiushchikh usilenie tsitogeneticheskoi éffektivnosti tsiklofosfana pri vvedenii ego obluchennym zhivotnym. Issledovanie tsitogeneticheskogo deistviia mutagenov, ne trebuiushchikh metabolicheskoi aktivatsii, na kletki kostnogo mozga obluchennykh krys.

The cytogenetic action of mutagens, that do not require metabolic activation (embichin and sarcolysine), and X-radiation on rat bone marrow was studied 1 month after preliminary irradiation with the dose of 4.25 Gy. The frequency of chromosome aberrations induced by the above-mentioned effects was the same as that in nonirradiated rats.

[Factors responsible for increasing the cytogenetic effectiveness of cyclophosphamide administered to irradiated animals. Mutagenic effect of cyclophosphamide on irradiated and intact cells in culture]. / Izuchenie prichin, obuslovlivaiushchikh usilenie tsitogeneticheskoi éffektivnosti tsiklofosfana pri vvedenii ego obluchennym zhivotnym. Mutagenno deistvie tsiklofosfana na obluchennye i intaktnye kletki v kul'ture.

The frequency of cyclophosphamide-induced chromosome aberrations in preirradiated cultures of L-cells and embryonal rat fibroblasts has been estimated. The mutagen was subjected to nonenzymatic activation in solution at 37 degrees C. In contrast to the results previously obtained on animals we failed to observe any influence of preirradiation on the cytogenetic effect of cyclophosphamide.

[Factors responsible for increasing the cytogenetic effectiveness of cyclophosphamide administered to irradiated animals. Frequency of chromosome aberrations in locally irradiated rat bone marrow cells]. / Izuchenie prichin, obuslovlivaiushchikh usilenie tsitogeneticheskoi éffektivnosti tsiklofosfana pri vvedenii ego obluchennym zhivotnym. Issledovanie chastoty indutsirovannykh khromosomnykh aberratsii v kletkakh kostnogo mozga krys pri lokal'nom obluchenii.

A study was made of the frequency of chromosome aberrations induced by cyclophosphamide (CP) in the bone marrow of preirradiated rats (exposure of crus with the body shielded, and vice versa). A reliable evidence was obtained in favor of a change in the metabolism of CP in the irradiated body which was the cause of the enhancement of its cytogenetic action.

[Activation of cyclophosphane when stored as a solution]. / Aktivatisiia tsiklofosfana pri khranenii ego rastvora.

There was found an effect of nonenzymic activation of cyclophasphane when keeping its solution during 24 hours at 37 degrees C. The products, formed as a result of its hydrolysis, induce chromosome rearrangement in the L-fibroblast culture in vitro while cyclophosphane dissolved immediately before the injection into the cell culture does not show a marked cytogenetic effect.

[Ultraviolet fluorescence of irradiated cultured cells]. / Izuchenie ul'trafioletovoi fluorestsentsii obluchennykh kletok v kul'ture.

Using cytofluorometry in the UV region of spectrum, a dose-dependent increase in ultra-violet fluorescence (UVF) intensity of L-fibroblasts was shown after X-irradiation at dose range of 80 to 500 rad, with maximum effect at 400 rad. The time dynamic observation of fluorescence intensity changes after irradiation at does 400 rad has demonstrated the highest values of UVF on the first days (1--3 days) with the following decrease within a month, and returning to the control level by the end of the study.

[Mutagenic effect of cyclophosphan on bone marrow cells of irradiated rats]. / Mutagennoe deistvie tsiklofosfana na kletki kostnogo mozga obluchennykh krys.

The rate of chromosome aberrations in bone marrow cells of male rats was investigated in 24 hours after the cyclophosphan intraperitoneal injection (25 mg/kg). Cyclophosphan was given to rats exposed earlier (15 days, 1, 3, 4, 6 or 9 months before) to X- and gamma-irradiation (400 rads). It was found that preliminary irradiation led to the increase in the mutagenic effect of cyclophosphan as compared to that obtained for intact rats. This effect was demonstrated during 4 months after acute X-irradiation at a dose rate of 70 rads/min and during 1 month after chronic gamma-irradiation at a dose rate of 100 rads/day. Later the effect was shown to disappear in both cases. Chronic irradiation was found to be less efficient in the stimulation of chromosome damages caused by chemical mutagens. The increase of the mutagenic effect of cyclophosphan resulted in the increase of both the number of cells carrying chromosome breaks and the severity of a damage per cell. Different ways of the irradiation effect on the mutagenic action of chemicals are discussed.