Effect of hypoxia on neural induction in colonies of human parthenogenetic stem cells.

We studied neural induction and generation of neuroectoderm in the colonies of human parthenogenetic SC cultured in the presence of 5 and 19±2% oxygen. We found that neuroectoderm was more actively generated at high oxygen content. At the same time, the transcription of stem cell pluripotency genes was not completely suppressed during neural induction at low oxygen content, while the expression of endoderm and mesodermal marker genes attested to the absence of specific differentiation. These findings demonstrate more efficient neuroectoderm generation induced in the colonies of pluripotent stem cells under conditions of normoxia.

Activin A suppresses induced formation of neuroectoderm in colonies of parthenogenetic stem cells in vitro.

We studied the effect of recombinant human activin A on induced neuroectoderm formation in colonies of human parthenogenetic SC in the absence of feeder cells. It was found that pretreatment of human parthenogenetic SC with activin A suppressed subsequent neural induction. Activin A in a concentration of 10 ng/ml significantly decreased transcriptional activity of genes required for neuroectoderm formation. At the same time, activin A in a concentration of 20 ng/ml increased the expression of pluripotency genes and completely inhibited the formation of structures in vitro reproducing the neural tube of the developing embryo. These findings attest to prolonged effect of activin A as an inhibitor of neuroectodermal differentiation.

[Genomic imprinting in the epigenetics of mammals].

Genomic imprinting is one of the most remarkable and important epigenetic phenomena. A biological ban on parthenogenetic and androgenetic development of mammals is an obvious consequence of genomic imprinting. Genomic imprinting defects may cause malformations, clinical syndromes, and tumor growth in humans and to the large offspring syndrome and an increased mortality after in vitro manipulations with early embryos in mammals. Differential expression of parental alleles during ontogeny implies a mechanism of reversible, selective marking of gene alleles. These relatively stable epigenetic modifications, which do not affect the primary nucleotide sequence of DNA, may be transmitted in somatic cell lines and reproduced in the germ line. The genomic imprinting mechanism may be involved in other epigenetic processes, such as epigenetic inheritance, nonrandom allele segregation, meiotic drive, etc. Artificial modulation of genomic imprinting effects with the use of growth factors and demethylating agents permits partial "normocoping" during the development of parthenogenetic mouse embryos. Targeted changes in the transcriptional activity of imprinted genes provide prerequisites for epigenetic correction of syndromes and diseases caused by genomic imprinting defects.

[Analysis of the effects of mutant hairless genes in chimeric mice]. / Analiz éffektov mutantnogo gena hairless u khimernykh myshei.

The autosomal recessive gene hairless (hr) is responsible for the complete hairlessness in mice homozygous for this gene. Hair shedding that begins at the age of 10 days is caused by an abnormal cycle of hair follicle development disturbed at the catagen stage. This results in enhanced programmed cell death (apoptosis) and ultimately leads to the complete hair follicle destruction and shedding of all hairs by the age of three weeks. To study the phenotypic expression of the hr gene in a chimeric organism, we have obtained 12 chimeric mice hr/hr <--> +/+ by means of aggregation of early embryos hr/hr and +/+. In chimeric mice, the hair shedding has begun two days later than in the hr/hr mice. By day 23 of postnatal development, hairless areas were present on the coat of chimeric mice or the latter were completely hairless depending on the percentage of the hr/hr mutant component. In four chimeras with high content of the mutant component (68-76%), the hair shedding process was similar to that in the hr/hr mice, though it was accomplished two days later. In three chimeras with 48-51% of the mutant component, alternating hairless and hair-covered bands were observed. These data suggest that the hr gene acts in epidermal cells of a hair follicle, because epidermal cell clones in embryonic skin migrate in the lateral-ventral direction coherently and without mixing. However, some chimeras displayed a pattern which was not so clear-cut: the band borders were illegible and hairs partly covered the hairless areas. In some chimeras, the uniform thinning of the coat was observed. Analysis of the effects of the hr mutant gene in chimeric mice differing in the ratio between mutant (hr/hr) and normal (+/+) components in tissues suggests that the hr gene acts in the epidermal cells of the hair follicle. The interactions between cells have an essential effect on the mode and degree of the hr gene expression, which leads to distortion of the "ectodermal" coat pattern in chimeras.

[Effects of the mutant gene wellhaarig in chimeric mice]. / Analiz deistviia mutantnogo gena wellhaarig u khimernykh myshei.

The mutant gene wellhaaring (we) confers the waved coat in mice, which is most pronounced in homozygotes at 10 to 21 days of postnatal development. Abnormal hair growth and structure in the we/we mutant mice results from defective cell differentiation in the inner root sheath of a hair follicle. To localize the site of the we gene action, we obtained ten chimeric mice by aggregation of the early C57BL/6-2we/we and BALB/c embryos. The chimera coat was waved, shaggy, or almost normal depending on the percentage of the mutant component. In the we/we +/+ chimeric animals of the first generation (G1) aged 21 days, both mutant and normal hair phenotypes were observed, which was especially discernible in zigzag hair. Note that none of the chimeras exhibited the alternating patterns of transversely oriented stripes or patches of either mutant or normal hair; i.e., they had a mixed parental hair phenotype. We also did not observe the animals with an intermediate phenotype, which suggests a discontinuous hair formation in chimeras according to the "all or nothing" principle. The data obtained indicate that the dermal papilla cells of a hair follicle are the sites for the we gene action. During the embryonic development, dermal cells are strongly mixed, which accounts for the lack of the clear-cut transverse stripes of either mutant or normal hair. The mutant gene we is probably responsible for a disrupted induction signal from the dermal papilla towards ectodermal cells of a hair follicle.

[Different influences of genomic imprinting on the development of parthenogenetic cell clones in C57BL/6 and CBA mice]. / Razlichnoe vliianie genomnogo imprintinga na razvitie partenogeneticheskikh kletochnykh klonov myshei C57BL/6 i CBA.

Clonal analysis of parthenogenetic chimeric mouse embryos C57B1/6(PG)<-->BALB/c has shown that parthenogenetic cell clones C57BL/6 are present in the brain, liver, and kidneys of 14- and 18-day-old embryos. The content of the parthenogenetic component (PG) in these organs on day 18 was lower than on day 14, and, in some 18-day-old embryos, parthenogenetic cell clones were absent from the liver and/or kidneys. These data suggest that, during the embryogenesis of parthenogenetic chimeras, parthenogenetic cell clones of mostly endodermal and mesodermal origins were actively eliminated. Therefore, in such parthenogenetic adult chimeras, parthenogenetic clones of mostly ectodermal origins were preserved. In parthenogenetic chimeras CBA(PG)<-->BALB/c, parthenogenetic cell clones were actively eliminated at early embryonic stages, and, as a result, they were absent at the post-implantation stages. Hence, during development of parthenogenetic cell clones, the effects of genomic imprinting are expressed unequally in C57BL/6 and CBA mice.

[Use of chimeric mice for studying the effects of genomic imprinting]. / Ispol'zovanie khimernykh myshei dlia izucheniia éffektov genomnogo imprintinga.

This is a review of the data of clonal analysis of developing tissues in parthenogenetic and androgenetic chimeric mice. The time and causes of death of the parthenogenetic and androgenetic cell clones in chimeras are considered. The data obtained suggest that the development of cell clones, derivatives of the mesoderm and endoderm, is determined by the expression of alleles of the imprinted loci of paternal chromosomes, while the formation of cell clones, derivatives of the ectoderm, depends on the expression of other imprinted loci of maternal chromosomes. The death of androgenetic and parthenogenetic (gynogenetic) mammalian embryos is due to the lack of the expression of certain imprinted loci of the maternal and paternal genome, respectively.

[An analysis of parthogenetic cell clones in chimeric C57BL/6(PG)<-->BALB/c mice]. / Analiz partenogeneticheskikh kletochnykh klonov u khimernykh myshei C57BL/6(PG)<-->BALB/c.

We studied the distribution of parthenogenetic cell clones in the retinal pigment epithelium and choroid of eyes on serial sections and in the brain, kidneys, and liver by electrophoretic analysis of glucose phosphate isomerase isozymes in 12 mouse chimeras C57BL/6(PG)<-->BALB/c obtained earlier. Asymmetry was noted in the distribution of the parthenogenetic cell clones in the eye structure, just as the earlier established asymmetry in the distribution of the parthenogenetic clones of epidermal melanoblasts. A high correlation was shown between the ratio of parthenogenetic to normal cells in the retinal pigment epithelium of the right or left eyes and epidermal melanoblasts in the hair cover of the corresponding body half of the chimera. These data suggest that there is a certain relationship between the processes leading to the characteristic distribution of the ectodermal parthenogenetic clones in the retinal pigment epithelium of the right and left eyes and epidermal melanoblasts in parthenogenetic chimeras. Electrophoretic analysis did not show parthenogenetic components in the liver or kidneys of any chimera, and the parthenogenetic component was found in the brain of only two chimeras, in which a high percentage of parthenogenetic cells of ectodermal origin was noted. In these cases, asymmetry was noted in the right and left cerebral hemispheres, just as in the retinal pigment epithelium of the right and left eyes. The data obtained suggest that, during the development of the chimeras, parthenogenetic C57BL/6 cells were actively eliminated from the tissues of endodermal and mesodermal origin. In adult chimeras C57BL/6(PG)<-->BALB/c, parthenogenetic cell clones of ectodermal origin are mostly preserved.

[The distribution of parthenogenetic clones of epidermal melanoblasts in chimeric mice C57BL/6(PG) <--> BALB/c]. / Raspredelenie partenogeneticheskikh klonov épidermal'nykh melanoblastov u khimernykh myshei C57BL/6(PG) <--> BALB/c.

The death of diploid parthenogenetic mammalian embryos, specifically mouse embryos, is related to the effects of genomic imprinting. But in mouse chimeras, many parthenogenetic cell clones retain their viability for long periods of development. We obtained 12 chimeric mice by aggregating diploid parthenogenetic C57BL/6 (C/C) embryos with normal BALB/c (c/c) embryos at the 8-cell stage. Distribution of parthenogenetic clones of the melanoblasts in the hair follicles of chimeric mice was estimated by the presence of pigmented regions of the hair cover. The proportion of pigmented regions of the hair cover did not exceed 35% in any of 12 parthenogenetic chimeras C57BL/6(PG) <--> BALB/c. In most chimeras, pigmented regions were present in the anterior and posterior parts of the body. At the same time, the middle part of the body was, as a rule, not pigmented, possible due to the death of parthenogenetic melanoblasts in this area. In many chimeras, bilateral distribution of pigmented regions was disturbed, possibly due to accidental death of individual parthenogenetic clones of the melanoblasts. The absence of pigmentation of the ventral body side and distal parts of the limbs in most obtained chimeras appears to be due to decreased proliferative activity of the parthenogenetic melanoblasts, as compared with the normal situation.