Interactions between diploid embryonal carcinoma cells and early embryonic cells

Two diploid embryonal carcinoma (EC) cell lines, P10 and P19, differ in their response to the embryonic environment. P10 produces mostly normal chimeras following injection into blastocysts, whereas P19 produces mostly abnormal chimeras. In this study, P10 cells were aggregated with morulae, and all resulting fetuses were chimeric with very large contributions from the EC cells. However, all embryos were abnormal. Following aggregation of P19 cells with morulae, very few embryos were recovered and they were all non-chimeric. Both P10 and P19 were capable of forming functional gap junctions with morula cells and with the ICM of the blastocyst but not with trophoblast, showing that differences in the ability to make junctional contact with the embryo cannot explain the differences between the two cell lines.     

Developmental potency of teratocarcinoma cells in chimeric embryos

The developmental potential of various teratocarcinomas of different origins was examined by making chimeras with mouse embryos. Of 7 teratocarcinoma lines examined, only 2 were found to contain stem cells having the ability to form live-born chimeras; one was experimentally induced OTTBALB-2 and the other was spontaneously occurring STT-3. The latter showed remarkable ability to colonize mid-gestational fetuses and adults. This result not only demonstrates that EC cells of male primordial germ cells have the ability to form viable chimeras but also suggest that this kind of tumor is a useful source of EC cells to make chimeras. The advantage of using pluripotent cell lines such as EC cells or embryonic stem (ES) cells as a vector for introducing foreign genes into mouse embryos was discussed in relation to the study on gene function and on gene regulation during development.     

Efficacious Production of Viable Germ-Line Chimeras between Embryonic Stem (ES) Cells and 8-Cell Stage Embryos

Many embryonic stem (ES) cell lines have been isolated from various mouse strains, but production of germ-line chimeras has been achieved with only strain 129. This report describes the isolation of a new ES cell line, F1/1, from a mouse blastocyst with the C57BL/6 X CBA male genotype and tests on its ability to produce germ-line chimeras by two techniques, blastocyst injection and 8-cell embryo injection. Chimera production using CD-1 blastocysts as a host was low (20%), as reported by others. But by the 8-cell embryo injection method, in which F1/1 cells were injected into the perivitelline space through a slit in the zona pellucida of 8-cell embryos, chimeric mice with extremely high chimerism were obtained at a rate of 80%. Breeding tests showed that 89% of the fertile males were germ-line chimeras and in most case, the majority of the sperms in their testes were derived from F1/1 cells. This F1/1 cell line with a different genotype from the 129 strain shows high ability to produce functional germ cells, moreover, the 8-cell embryo injection method using F1/1 cells seems to be an efficient way to produce viable germ-line chimeras.     

Easy assessment of ES cell clone potency for chimeric development and germ-line competency by an optimized aggregation method.

Production of germ-line competent chimeric mice from embryonic stem (ES) cells is an inevitable step in establishing gene-manipulated mouse lineages. A common method used for creating chimeric mice is the injection of ES cells into the blastocoelic cavity (blastocyst injection). The aggregation method is an alternative way to introduce ES cells to the host embryo which is less difficult than blastocyst injection. Here we re-examined the condition of embryo-ES cell coculture on the aggregation method and found improvement of germ-line competent chimeric production by a simple modification of the coculture medium. Moreover, R1 ES cell and its 10 gene-manipulated subclones were tested by this method. Although all ES cell clones showed good morphology and a normal karyotype, the efficiency of chimeric development and germ-line transmission varied among clones and were classified into three grades according to germ-line competency. In the first group (class A), both the incidence of chimera with high ES cell contribution and the rate of germ-line transmission were fairly high. Germ-line competent chimeras were obtained but with rather low efficiency in the second group (class B), while another group (class C) showed an absence of high ES cell-contributed chimeras and no germ-line transmission. These results suggest the usefulness of this modified aggregation method to predict the potency of ES cell clones for germ-line competency.     

The stem cells of a primordial germ cell-derived teratocarcinoma have the ability to form viable mouse chimeras

A euploid testicular teratocarcinoma line, STT-3, has been established from a tumor spontaneously occurring in the testis of a 129/Sv-ter male. Developmental ability of the STT-3 stem cells was tested by injecting these cells into mouse blastocysts. The frequency and the extent of chimerism were examined in mid-gestational fetuses and in live-born mice. STT-3 stem cells form viable chimeras at a high rate and differentiate into normal tissues. This is the first reported testicular teratocarcinoma-derived stem line with a proven capacity to form viable chimeric mice upon injection into the blastocysts.     

Comparison of male chimeric mice generated from microinjection of JM8.N4 embryonic stem cells into C57BL/6J and C57BL/6NTac blastocysts

To identify ways to improve the efficiency of generating chimeric mice via microinjection of blastocysts with ES cells, we compared production and performance of ES-cell derived chimeric mice using blastocysts from two closely related and commonly used sub-strains of C57BL/6. Chimeras were produced by injection of the same JM8.N4 (C57BL/6NTac) derived ES cell line into blastocysts of mixed sex from either C57BL/6J (B6J) or C57BL/6NTac (B6NTac) mice. Similar efficiency of production and sex-conversion of chimeric animals was observed with each strain of blastocyst. However, B6J chimeric males had fewer developmental abnormalities involving urogenital and reproductive tissues (1/12, 8?%) compared with B6NTac chimeric males (7/9, 78?%). The low sample size did not permit determination of statistical significance for many parameters. However, in each category analyzed the B6J-derived chimeric males performed as well, or better, than their B6NTac counterparts. Twelve of 14 (86?%) B6J male chimeras were fertile compared with 6 of 11 (55?%) B6NTac male chimeras. Ten of 12 (83?%) B6J chimeric males sired more than 1 litter compared with only 3 of 6 (50?%) B6NTac chimeras. B6J male chimeras produced more litters per productive mating (3.42?±?1.73, n?=?12) compared to B6NTac chimeras (2.17?±?1.33, n?=?6). Finally, a greater ratio of germline transmitting chimeric males was obtained using B6J blastocysts (9/14; 64?%) compared with chimeras produced using B6NTac blastocysts (4/11; 36?%). Use of B6J host blastocysts for microinjection of ES cells may offer improvements over blastocysts from B6NTac and possibly other sub-strains of C57BL/6 mice.     

Short-term rescue by RNA injection of a mitotic arrest mutation that affects the preimplantation mouse embryo

The mutation oligosyndactyly results in syndactyly, abnormal fusion and insertion of certain limb muscles, and diabetes insipidus in heterozygous mice. When homozygous the mutation is lethal; beginning at the blastocyst stage, the homozygous cells arrest in metaphase with intact spindles. The mutant phenotype cannot be corrected by forming aggregation chimeras with wild-type cells, suggesting that the mutation results in a cell autonomous lethal condition. Short-term rescue of the homozygous-induced mitotic arrest can be achieved, however, by cytoplasmic injection of polyadenylated RNA obtained from a rapidly dividing embryo-derived stem cell line.     

Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines

Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation. Using morula aggregation to produce chimeras, five newly derived 129/SvEv and two C57BL/6 ES cell lines tested at early passages were found to contribute extensively to chimeras and produce germline-transmitting male chimeras. Furthermore, the two 129S/vEv ES cell lines that were tested and one of the C57BL/6 ES cell lines were able to maintain these characteristics after many passages in vitro. Our results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages. C56BL/6-derived ES cell lines, however, have a greater tendency than 129-derived ES cell lines to lose their ability to colonize the germline.     

B cells are required for generation of protective effector and memory CD4 cells in response to Pneumocystis lung infection

B cell-deficient mice are susceptible to infection by Pneumocystis carinii f. sp. muris (PC). To determine whether this susceptibility is due to a requirement for B cells to prime T cells, we compared CD4 T cell responses to PC in bone marrow chimeric mice that express MHC class II (MHCII) on all APCs (wild-type (WT) chimeras) and in bone marrow chimeric mice that express MHCII on all APCs except B cells (MHCII(-/-) chimeras). Although PC was rapidly cleared by WT chimeric mice, PC levels remained high in chimeric mice that lacked MHCII on B cells. In addition, although T cells were primed in the draining lymph nodes of MHCII(-/-) chimeric mice, the number of activated CD4 T cells infiltrating the lungs of these mice was reduced relative to the number in the lungs of WT chimeras. We also adoptively transferred purified CD4 T cells from the draining lymph nodes of PC-infected normal or B cell-deficient mice into SCID mice. Mice that received CD4 cells from normal mice were able to mount a response to infection in the lungs and clear PC. However, mice that received CD4 cells from B cell-deficient mice had a delayed T cell response in the lungs and failed to control the infection. These data indicate that B cells play a vital role in generation of CD4(+) memory T cells in response to PC infection in the lungs.     

Comparison of aggregation and injection techniques in producing chimeras with embryonic stem cells in mice

We analyzed embryonic stem cell lines for their capacity to produce aggregation chimeras with diploid or developmentally compromised tetraploid embryos. Descendants of embryonic stem cells which contributed to midgestation fetuses at high levels were capable of supporting fetal development also with tetraploid partners. Different numbers of embryonic stem cells were introduced into diploid and tetraploid morulae as well as into blastocysts by microinjection. There were no differences in the frequency of embryonic stem cell-containing fetuses when comparing aggregation or injection into morulae versus blastocysts. However, the distribution pattern of embryonic stem cell derivatives in chimeric fetuses suggested that pre-compaction embryos are more suitable for generating fetuses with high embryonic stem cell contribution. Injection of embryonic stem cells into tetraploid embryos showed that completely embryonic stem cell-derived fetuses can also be produced by this technique. Totally embryonic stem cell derived fetuses were observed in each group, when embryonic stem cells were injected into diploid embryos. However, the rate of chimeras and chimerism was lower when 1 or 3 embryonic stem cells were used versus 8 or 15 cells. This suggests that the number of embryonic stem cells introduced might play a role in the colonization ability.     

Antiviral immune responses of fully allogeneic irradiation bone marrow mouse chimeras

In (B10.BR----B10) chimeras infected with lymphocytic choriomeningitis (LCM) virus higher titers were attained in spleens and livers than in organs of the mice used for their construction, and the subsequent elimination was retarded, but eventually the virus was cleared. The numbers of LCM virus-specific CTL and their precursors as quantitated with chromium-release assay and limiting dilution method, respectively, were lower in chimeras than in B10.BR or C57BL/10J mice, and fewer were restricted for the haplotypes of the donors than of the recipients. The same was true with regard to antiviral effector cells, which were determined by adoptive immunization. The numbers of spleen cells releasing IgM and IgG antiviral antibodies were virtually as high in chimeras as they were in C57BL/10J and B10.BR mice. Transfer of immune splenocytes from either B10.BR or C57BL/10J mice resulted in incomplete virus elimination from the spleens of infected chimeras, whereas injection of a mixture of the two types of immune cells led to efficient clearance. We conclude that in the chimeras cells of both donor and recipient haplotypes participate in the infection, which is terminated by H-2k- and H-2b-restricted T lymphocytes that these animals are capable of generating. We conclude, furthermore, that clearance of the LCM virus from the tissues requires contact between effector and target cells.     

NK cell tolerance in mixed allogeneic chimeras

Alterations in inhibitory receptor expression on NK cells have been detected in mixed allogeneic chimeras and in mosaic MHC class I-expressing transgenic mice. However, it is not known whether or not NK cells are tolerant to host and donor Ags in mixed chimeras. In vitro studies have shown a lack of mutual tolerance of separated donor and host NK cells obtained from mixed chimeras. Using BALB/c-->B6 fully MHC-mismatched mixed chimeras, we have now investigated this question in vivo. Neither donor nor host NK cells in mixed chimeras showed evidence for activation, as indicated by expression of B220 and Thy-1.2 on NK cells in chimeric mice at levels similar to those in nonchimeric control mice. Lethally irradiated, established mixed BALB/c--> B6 chimeras rejected a low dose of beta(2)-microglobulin-deficient bone marrow cells (BMC) efficiently but did not reject BALB/c or B6 BMCs. In contrast, similarly conditioned B6 mice rejected both BALB/c and beta(2)-microglobulin-deficient BMCs. Thus, NK cells were specifically tolerant to the donor and the host in mixed allogeneic chimeras. The similar growth of RMA lymphoma cells in both chimeric and control B6 mice further supports the conclusion that donor BALB/c NK cells are tolerant to B6 Ags in chimeras. Administration of a high dose of exogenous IL-2 could not break NK cell tolerance in chimeric mice, suggesting that NK cell tolerance in chimeras is not due to a lack of activating cytokine. No reduction in the level of expression of the activating receptor Ly-49D, recognizing a donor MHC molecule, was detected among recipient NK cells in mixed chimeras. Thus, the present studies demonstrate that NK cells in mixed chimeras are stably tolerant to both donor and host Ags, by mechanisms that are as yet unexplained.     

Analysis of a nontumorigenic embryonal carcinoma cell line

Embryonal carcinoma (EC) cells have proven to be of particular value in studies of both oncogenesis and mammalian development as well as in evaluating the relationship between these two phenomena. We have infected EC cells with a retrovirus in an effort to obtain by insertional mutagenesis cell lines defective in either differentiative or oncogenic potentials. One such cell line, identified originally by its unique morphological phenotype, is abnormal with respect to both parameters. These cells do not differentiate along typical EC cell lineages, possibly having lost their ability to elaborate endodermal derivatives. They do, however, retain certain cell surface markers characteristic of EC cells and lose these markers after exposure to retinoic acid. Most significantly, they also fail to form tumors in vivo in syngeneic mice, although they grow as well as the parental cells in vitro. Southern blot analysis indicates that this variant cell line has a single viral insert and the original cell was probably hemizygous for the insertion site, suggesting that a single gene may regulate both the tumorigenic and differentiative capacities of the cell.     

HDC细胞与MESPU—13细胞形成嵌合鼠能力的比较研究

ＥＳ细胞嵌合能力的强弱是人们利用ＥＳ细胞获得转基因小鼠时十分关心的问题。本言语通过囊胚显微注射法将１５个左右ＥＳ细胞注入Ｃ５７ＢＬ／６Ｊ品系小鼠３．５天囊胚的囊胚腔中观察嵌合鼠毛色嵌合情况。统计嵌合鼠的出生率;以及用葡萄糖磷酸异构酶（ＧＰＩ）电泳地检测ＥＳ细胞在嵌合鼠体内各种组织和器官的嵌合情况,对于ＨＰＲＴ缺陷（ＨＤＣ）细胞和ＭＥＳＰＵ－１３细胞的嵌合能力我们作了较详细的研究,结果表明ＭＥＳＰＵ     

Development to term of mouse androgenetic aggregation chimeras.

Diploid androgenetic eggs contain two sperm-derived genomes, and only rarely develop to the early somite stage. Also, previous studies have indicated that androgenetic eggs cannot be rescued in aggregation chimeras beyond embryonic stages. Paradoxically, in blastocyst injection chimeras made with androgenetic embryonic stem (ES) cells of the 129/Sv strain, we previously obtained considerable improvement in developmental potential. Although considerable death occurred in utero, overtly normal chimeric fetuses and occasional postnatal chimeras that developed skeletal abnormalities were observed. Consequently, we have re-evaluated the developmental potential of androgenetic aggregation chimeras utilizing androgenetic eggs of the 129/Sv strain, and of the BALB/c and CD-1 strains for comparison. Regardless of strain, androgenetic aggregation chimeras were generally more inviable than previously observed with androgenetic ES cell chimeras, and often the embryoproper was abnormal even when an androgenetic contribution was detected only in the extra-embryonic membranes. This is at least a partial explanation of the greater viability of androgenetic ES cell chimeras, as ES cells do not colonize significantly certain extra-embryonic tissues. Nevertheless, in the 129/Sv strain, occasional development of chimeras to term was obtained, and one chimera that survived postnatally developed identical skeletal abnormalities to those observed previously in androgenetic ES cell chimeras. This result demonstrates that at least one example of paternal imprinting is faithfully conserved in androgenetic ES cells. Also, the postnatal chimerism shows that androgenetic eggs can give rise to terminally differentiated cell types, and are therefore pluripotent. In contrast, only possibly one BALB/c and no CD-1 androgenetic aggregation chimeras developed to term. Therefore, the developmental potential of androgenetic aggregation chimeras is to some extent dependent on mouse strain.     

源于129S1品系的小鼠胚胎干细胞系的建立及其生物学特性分析

从129S1小鼠早期胚胎的内细胞团分离、培养类胚胎样细胞,经反复传代,成功地建立了129S1小鼠胚胎干细胞系,命名为NM-2细胞系。形态学鉴定具有胚胎干细胞的典型形态特征,正常核型率为80％;呈碱性磷酸酶阳性、表达胚胎干细胞特异性转录因子OCT-4;体内分化后可形成源于三胚层的组织结构;经囊胚腔显微注射后所获得的子代个体中79％具有毛色嵌合表型;雄性嵌合个体中31％发生生殖腺嵌合;同时,通过育种观察到所有生殖腺嵌合体的子代小鼠表型正常。以上结果证实NM-2细胞系为一株具高生殖腺嵌合能力的小鼠胚胎干细胞系。     

高转移潜力食管癌细胞株的建立及其生物学特征

目的建立具有高转移潜力食管癌细胞株并研究其生物学特征。方法将食管癌细胞系EC109细胞悬液异位移植到SCID小鼠胃壁,约3个月后或动物濒临死亡时处死,行病理学解剖,将肉眼可见的纵隔淋巴结转移瘤块接种于SCID鼠皮下扩增,然后取小鼠皮下瘤组织块进行细胞培养,得到性状稳定的细胞株NMC109后,用MTT法分析细胞生长曲线,Western bloting法检测与细胞分裂增殖能力密切相关的TopoⅡα表达,酶谱法检测MMP-2和MMP-9的活性,划痕实验和Transwell体外移动实验检测细胞的移动能力。结果与母本细胞EC109相比,所获得的细胞株NMC109其增殖能力和TopoⅡα表达明显增强,MMP-9的活性明显升高,移动能力明显增强。结论获得了具有高转移潜力的食管癌细胞株。     

Adaptive differentiation of H-2- and Igh-restricted B lymphocyte in tetraparental bone marrow chimera

Immunization of BALB/c mice with MOPC-104E myeloma protein induced idiotype-specific enhancing B cells that acted on anti-dextran antibody producing B cells. The enhancing cells have the surface phenotype of B cells. With the use of several H-2 or Igh congenic mice, it was found that the cooperation among B cells was controlled by both the major histocompatibility complex (MHC) and Igh. The capability to generate enhancing B cell activity was analyzed by using tetraparental bone marrow chimeras. (C57BL/6 X BALB/c)F1 mice, for example, were lethally irradiated and were reconstituted with C57BL/6 and BALB/c bone marrow cells. Nine to 12 wk after the reconstitution, the chimeras were immunized with the myeloma protein and were tested for their enhancing B cell activity. After the removal of C57BL/6 origin cells by treatment with anti-H-2b + complement, residual cells exhibited enhancing B cell activity on BALB.B, as well as BALB/c antidextran antibody response. This indicates that the generation of H-2-restricted, idiotype-specific enhancing B cell activity differentiated adaptively so as to recognize foreign MHC as self under chimeric conditions. On the other hand, splenic B cells treated with anti-H-2d + complement did not enhance the responses of BALB/c or BALB.B. Even in a chimeric environment, the B cells of C57BL/6 origin could not obtain the ability to generate enhancing B cell activity upon immunization of the idiotype. The results described here, taken in conjunction with our previous studies, suggest that the Ig heavy chain gene(s) predominantly control the Igh restriction properties of enhancing B cells, and the capability of MHC recognition by B cells is selected under chimeric conditions.     

Chimeric primates: embryonic stem cells need not apply

In this issue, Tachibana et?al. report the generation of the first chimeras from a nonhuman primate, the rhesus monkey. Unlike mice, rhesus chimeras fail to form when embryonic stem cells are injected into blastocysts. Instead, chimera formation is achieved by aggregation of several four-cell embryos.     

Simple and efficient production of mice derived from embryonic stem cells aggregated with tetraploid embryos

Six newly derived hybrid mouse embryonic stem (ES) cell lines and two inbred ES cell lines were tested for their ability to produce completely ES cell-derived mice by aggregation of ES cells with tetraploid embryos. Forty-five ES cell-tetraploid pups were generated from six hybrid ES cell lines and no pups from two inbred ES cell lines. These pups were found to have increased embryonic and placental weights than control mice. Twenty-two pups survived to adulthood and produced normal offsprings, and the other 23 pups died of several reasons including respiratory distress, abdomen ulcer-like symptoms, and foster failure. The 22 adult ES cell-tetraploid mice were completely ES cell-derived as judged by coat color and germline transmission, only two of them was found to have tetraploid component in liver, blood, and lung as analyzed by microsatellite loci. Our data suggested that genetic heterozygosity is a crucial factor for postnatal survival of ES cell-tetraploid mice, and tetraploid embryo aggregation using hybrid ES cells is a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.