Existence of gradient in cell adhesiveness along the developing Xenopus hind limb bud, shown by a cellular sorting-out experiment in vitro

To examine the possibility of a difference in cell adhesiveness along the developing Xenopus hind limb bud axes, single mesenchymal cells from developing hind limb buds were cultured, allowing them to form an aggregate in a gyratory culture system. By observing the distribution of cells within aggregates, it was found that sorting-out occurred between cells from different positions and different stages. Cells derived from more distal positions tended to be situated interiorly in the aggregates. According to Steinberg's differential adhesion hypothesis, these results support the idea that there is a graded difference in cell adhesiveness along the proximo-distal axis of the developing limb, with adhesiveness increasing distally. Although similar sorting-out was observed between anterior and posterior cell populations, it could not be determined which cell populations were definitely more cohesive. These properties may be correlated with the experimentally demonstrated 'positional value', which should be different among cells located at different positions along the axes of the developing vertebrate limb bud.     

The Surface Charge of Mesodermal and Neural Cells of Triturus Embryos

The electrical surface charge of disaggregated mesodermal and neural cells of the neurula stage of Triturus vulgaris embryos is estimated by using the apparatus described in Figure 1. The results showed a significant difference between the net charges of these two cell types: the neural cells had a clearly negative charge, whereas the mesodermal cells seemed to be more or less neutral.
The difference of the surface charges may partly explain the "sorting-out" phenomenon in the mixed reaggregates of the disaggregated cells.     

Cell Sorting daring Pattern Formation in Dictyostelium

Formation of the prestalk-prespore pattern in Dictyostelium was investigated in slugs and submerged clumps of cells. Prestalk and prespore cells were identified by staining with vital dyes, which are shown to be stable cell markers. Dissociated slug cells reaggregate and form slugs that contain a prestalk-prespore pattern indistinguishable from the original pattern. The pattern forms by sorting out of stained prestalk cells from unstained prespore cells. Sorting also occurs in clumps of dissociated slug cells submerged in liquid or agar. A pattern arises in 2 h in which a central core of stained cells is surrounded by a periphery of unstained cells. Sorting appears to be due to differential chemotaxis of stained and unstained cells to cAMP since exogenous cAMP (>10⁻⁷ M) reverses the normal direction of sorting-out such that stained cells sort to the periphery of the clumps.
Isolated portions of slugs regenerate a new prestalk-prespore pattern. Posterior isolates regenerate a pattern within 2 h due to sorting of a population of vitally stained 'anterior-like' cells present in posteriors. Anterior-like cells do not sort in intact slugs due to the influence of a diffusible inhibitor secreted by the anterior region. During posterior regeneration this signal is absent and anterior-like cells rapidly acquire the ability to sort. Anterior isolates regenerate a staining pattern more slowly than posterior isolates by a process that requires conversion of stained prestalk cells to unstained prespore cells.
The results suggest that pattern formation in Dictyostelium consists of two processes: establishment of appropriate proportions of two cell types and establishment of the pattern itself by a mechanism of sorting-out.     

Morphology, differentiation and matrix production of liver cells in organoid cultures (high density cultures) of fetal rat livers.

The aim of this study was to demonstrate the morphology and matrix synthesis of embryonic rat liver cells (day 18 of gestation) in organoid cultures (high density cultures) with electron microscopic and immunomorphological techniques. For this purpose the cells of embryonic rat livers were isolated enzymatically and grown in an organoid culture (high density culture) for 3 weeks in a Trowell system. During the first 48 h a sorting-out process took place, i.e. liver and blood-forming cells met to form aggregates. In between mesenchymal cells were seen. Vessel-like cavities developed. Electron microscopic inspection of the hepatocytes did not reveal any lesions of the cell organelles after 14 days in culture. As late as after a 3-week culture period mitochondrial swellings and an increased number of autophagic vacuoles were observed. A rim of collagenous fibrils or fibrillar bundles and granular matrix structures was perceptible as early as after 7 days in culture. Immunofluorescence microscopic techniques revealed collagen types III, IV and VI as well as laminin, nidogen, heparansulfate-proteoglycan and fibronectin in these areas. Thus, the composition of the matrix in this culture system corresponds (apart from the absence of collagen type I) to the embryonic situation. Therefore, the organoid culture appears to be an appropriate technique to study the behaviour of hepatocytes in vitro. It is especially suited to demonstrate the formation of matrix components in liver cells and their extracellular occurrence.     

Selective Cellular Affinities in the Unincubated Chick Blastoderm

Cell suspensions from whole unincubated chick blastoderms (stage 1,⁴) were obtained by EDTA or trypsin dissociation. They were cultured in rotating flasks and aggregates were examined after 1 to 7 days in culture. Early aggregates were solid and consisted of a continuous phase of loosely packed cells with abundant yolk granules, among which groups of tightly packed and less yolky cells were present. Loosely packed cells became peripheral while the tightly packed cells formed continuous cords traversing the aggregate. Aggregates also developed a cavity surrounded mainly by the loosely packed cells. The latter gave rise to cells similar to those of the columnar endoderm of the yolk sac. Our data suggest that sorting-out occurs in aggregates prepared from unincubatedblastoderms, before the onset of gastrulation.     

Scanning electron microscopy of cell aggregation: cardiac and mixed retina-cardiac cell suspensions

Reaggregates of cells from 7-day embryonic chick hearts, 10-day neural retinas and respective cell mixtures were examined by scanning electron microscopy (SEM). Cardiac cell aggregates formed during the first 2 hours were composed of rounded cells arranged in linear or branched arrays. By further collection of single cells and accretion of cell clusters, the aggregates increased in size and formed large grape-like masses. By 12 hours, heart aggregates assumed a spherical shape with partial sorting-out of myogenic from non-myogenic cells; the muscle elements occupied the interior of the aggregates, whereas flattened, squamous-like cells, of a non-muscle character, covered the surface in a multilayered epithelium. Single rounded cells were still found on the surface of 12 to 24-hour aggregates; however, they were absent by 48 hours, suggesting that the collection of free cells by the cardiac aggregates ceased during the second day. Early aggregates (2 hours) of retina cells showed initial stages of axonal and dendritic outgrowth characteristic of neural tissue. Examination of heterotypic aggregates of neural retina and myocardial cells after 2 to 6 hours showed small groups of retina cells attached to the surface of the heart cell clusters. The retina cells did not appear to be randomly distributed within the early aggregates but formed small tissue specific clusters even by 2 hours in culture. These results indicate that SEM should be a valuable tool in the further analysis of homo- and heterotypic cellular aggregation.     

Human in vivo antigen-induced lymphoblastoid B cells are capable of cyclical antibody production in vitro

In vivo immunization of normal volunteers with tetanus toxoid induces the formation of a circulating B cell subset that has the capacity to secrete specific antibody in vitro without the need for T cell help or mitogen stimulation. From earlier studies it was not clear whether the spontaneous antibody-secreting lymphoblastoid (LB) B cell was at a terminal stage of differentiation or if it had the capacity to give rise to additional B cell subsets, such as memory cells or more fully mature antibody-producing cells. In this study we have shown that at least two distinct waves of spontaneous antibody secretion can occur in vitro when cultures are initiated with the lymphocytes from individuals immunized 6 days earlier. The first production of antibody was completed by 3 days of in vitro culture and the second production of antibody did not initiate until day 7 or 8 of culture and was completed by day 12. The B cells responsible for the second stage of antibody production appeared derived from a portion of the antibody-secreting cells present on day 3 in that 1) treatment of the cultures on day 0 with BuDr and light equally inhibited the first and second rounds of antibody synthesis; 2) when isolated from the blood, both B cell subsets were in the large cell fraction after 1 X G sedimentation; and 3) under conditions of limiting numbers of cells, the cells responsible for the second wave of antibody production were almost exclusively found in cultures positive for a B cell that had produced antibody on days 1 to 3. Although only a portion (10 to 30%) of the LB B cells present on day 0 had the capacity to again produce antibody on days 8 to 12, the two cells were capable of producing similar quantities of antibody on a per cell basis. These results indicate that the mature circulating LB cell induced in vivo by immunization is not terminally differentiated, but under appropriate conditions has the capacity to give rise to additional antibody-secreting cells.     

Lung organoid culture

An organoid culture system for lung cells is described in which morphogenesis of lung histotypic structures and differentiation of both pneumocytes type II and mesenchyme occur. The principle of this technique is the culture of mouse fetal lung cells at high density on a membrane filter at the medium/air interface. In the course of cultivation, cell sorting-out, epithelial cell aggregation, formation of an alveolar-like lumen in the organoids and formation of a basal lamina occur. Epithelial differentiation culminates in the production of lamellar bodies, and the mesenchyme develops into mature connective tissue. Morphogenesis and differentiation depend on the stage of fetal development from which the lung cells were derived but appear independent of the formation of a basal lamina. Various drugs have been tested for their effects on morphogenesis and differentiation in this lung organoid culture: some of them inhibit differentiation or damage the mesenchyme, others stimulate surfactant production. Due to the quite complex morphogenetic and cellular events occurring in lung organoid culture, it may be an applicable tool for alternative in vitro screening methods.     

Behavior of cells in artificially made cell aggregates and tissue fragments after grafting to developing hind limb buds in Xenopus laevis

During vertebrate limb development, the limb bud grows along the proximo-distal (P-D) direction, with the cells changing their adhesiveness. To know whether the position-related differences in cell adhesiveness are actually utilized by morphogenesis to constitute limb structures, we grafted cell aggregates made of dissociated cells derived from different positions and stages of developing hind limb buds into developing hind limb buds and observed the behavior of the cells. Cell aggregates made of dissociated mesenchymal cells from two different origins were implanted in different positions and stages of limb buds or grafted on limb stumps made by cutting. The two grafted cell populations in the aggregate always sorted out from each other, but their patterning of sorting-out was quite different according to the transplanted regions. In summary, cells in the aggregate that have closer positional identity to the transplanted site were always situated at the boundary between host and donor cells. The pattern of sorting-out seemed to be determined by the relative adhesiveness of surrounding cells to the constituent cells of the aggregates. We also transplanted fragments dissected out from different regions along the P-D axis into st. 50 limb buds. The descendants of grafted cells moved distally to the region corresponding to their positional identity and participated in the formation of more distal structures from that point. These results suggest that the difference in cell adhesiveness may probably play a role in arranging cells along the P-D axis of a developing limb bud.     

Ontogeny of macrophage subpopulations and Ia-positive dendritic cells in pulmonary tissue of the rat

Summary The development of macrophage subpopulations and dendritic cells in the rat lung was studied from day 15 of gestation until day 21 after birth by means of immunohistochemical techniques combined with acid phosphatase staining. To characterize these cell populations, monoclonal antibodies raised against rat macrophage subpopulations were used (ED1, ED2, ED7, and ED8) in addition to anti-Ia antibodies. Ia-positive cells with a dendritic morphology were found on day 16 of gestation. During ontogeny, the number of these cells gradually increased. They were always found in mesenchymal lung tissue between the epithelial tubules of future alveoli, and in perivascular or peribronchial areas. ED1-positive macrophages were found on day 17 of gestation, with a distribution different from that of Ia-positive dendritic cells. The distribution of ED1-positive cells changed during ontogeny: before birth, ED1-positive cells were present in mesenchymal areas of lung tissue, whereas after the first week of postnatal life ED1 recognized all free alveolar macrophages. No Ia-expression was found on free alveolar macrophages. This developmental pattern resembles the ontogeny of Ia-positive dendritic cells and ED1-positive macrophages in gutassociated tissue. The comparable development of these cell populations in gut and lung tissue indicates a common ontogeny in the mucosal immune system.Fellow of the Royal Netherlands Academy of Arts and Sciences     

Megasporogenesis,Microsporogenesis and Development of Gametophytes in Eleutherococcus senticosus (Araliaceae)

This paper describes megasporogenesis, microsporogenesis, and development of female and male gametophytes in Eleutherococcus senticosus. The main results are as follows: Flowers of E. senticosus are epigynous, pentamerous. Anthers are 4 -microsporangiate. An ovary has 5 loculi. Each ovary loculus has 2 ovules: the upper ovule and the lower ovule. The upper one is orthotropous and degenerates after the formation of archesporial cell, while the lower one is anatropous, unitegmic and crassinucellar, and able to continue developing. In male plants, microsporogenesis and development of male gametophytes took place in regular way, but a series of abnormal phenomena were found in megasporogenesis and development of female gametophytes. The microspore mother cells gave rise to tetrahedral tetrads by meiosis. Cytokinesis was of the simultaneous type. The mature pollen was 3-celled and shed singly. The anther wall formation belonged to the dicotyledonous type. At the stage of microspore mother cell, the anther wall consisted of four layers, i.e. epidermis, endothecium, middle layer, and tapetum. The tapetum was of glandular type and its most cells were binucleate. When microspores were at the uninucleate stage, the tapetum began to degenerate in situ. When microspores developed into 3-celled pollen grains, the tapetum had fully degenerates. In the lower ovule of male flower, the megaspore mother cell gave rise to a linear or “T” -shaped tetrad. In some cases, a new archesporial cell over the tetrad or two tetrads parallel or in a series were observed. Furthermore, the position of functional megaspore was variable; any one or two megaspores might be functional, or one megaspore gave rise to a uninucleate embryo sac, but two other megaspores also had a potentiality of developing into the embryo sac. In generally, on the day when flowers opened, female gametophytes contained only 4 cells: a central cell, two irregular synergids and one unusual egg cell. In female plants, microspore mother cells and secondary sporogenous cells were observed. But at the stage of secondary sporogenous cell, the newly differentiated tapetum took the appearance of degeneration. Later, during the whole stage of meiosis, the trace of degenerative tapetum could be seen. At last, the microsporangium degenerated and no tetrad formed. On the blossom day, all anthers shriveled without pollen grains. In female flowers, megasporogenesis and development of female gametophytes were normal: the tetrad of megaspores was linear or “T”-shaped; the chalazal megaspore was usually functional; the development of embryo sac was of the Polygonum type. On the blossom day, most embryo sacs consisted of 7 cells with 8 nuclei or 7 cells with 7 nuclei; but the egg apparatus was not fully developed. In hermaphroditic plants, microsporogenesis was normal but the development of male gametophytes was partially abnormal. When the hermaphroditic flowers blossomed, there were more or less empty pollen grains in the microsporangium and these pollen grains were quite different in size. The development of most gynoecia was normal but numerous abnormal embryo sacs could be seen. On the blossom day, female gametophytes were mainly 7-celled with 8-nuclei or with 7-nuclei or 4-celled with antipodal cells degenerated; the egg apparatus wasnot fully developed either.     

Towards the isolation of embryonal stem cell lines from the sheep

Summary Immunosurgical isolation of inner cell masses (ICMs) from sheep embryos was most efficient at the expanded, zona-intact blastocyst stage (day 7 to 8 post oestrus) before migration of endoderm cells beyond the boundary of the ICM across the blastocoelic surface of the trophectoderm. When cultured under conditions which allow the isolation of embryonal stem (ES) cell lines from mouse ICMs, sheep ICMs attached, spread and developed areas of both ES cell-like and endoderm-like cells. After prolonged culture only endoderm-like cells were evident. The implications for the isolation of ES cell lines from sheep embryos and possible species-specific requirements are discussed.     

Relationship between timing of development,morula morphology,and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast-cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast-cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro-produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997. © 1997 Wiley-Liss, Inc.     

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/- 8.1 cells, which increased to 84.4 +/- 5.7 and 125.5 +/- 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/- 6.0 and 40.3 +/- 5.0, respectively) and then doubled on day 7 (80.6 +/- 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/- 4.0 and 41.9 +/- 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/- 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.     

Primitive lymphohematopoietic precursor cell lines generated in culture from day 7 early-mid-primitive streak stage mouse embryo.

During mouse development, the first lymphohematopoietic precursor cells and myeloid or erythroid cell lineage-determined cells can be detected in the yolk sac at days 8-8.5 of gestation. The characteristics of the cells that give rise to these yolk sac primitive lymphohematopoietic cells and the molecular events controlling this process remain poorly defined. We show here that cell suspensions from day 7 early-mid-primitive streak stage embryo proper generated early immature PgP-1+ Joro 177+ Lin- hematopoietic cells and some Mac-1+ myeloid and TER 119+ erythroid cells after co-culture with the yolk sac-derived stromal cell line YS6 without addition of exogenous cytokines. Purified Lin- hematopoietic cells generated in these cultures did not express genes known to be transcribed at early stages of lymphoid, myeloid or erythroid cell differentiation and were able to give rise to T and B lymphocytes, myeloid cells and erythroid cells after appropriate further induction in vitro. Several cell lines were established in culture with a mixture of four cytokines from the PgP-1+ Joro 177+ Lin- cell population. The cell lines shared phenotypic and genotypic characteristics with the PgP-1+ Joro 177+ Lin- cell population generated in culture from day 7 embryo proper and they were able to reconstitute the lymphohematopoietic system of irradiated mice. Taken together these results support a model of lymphohematopoiesis in which cells from day 7 early-mid-primitive streak mouse embryo proper migrate and colonize the visceral yolk sac. There they generate primitive lymphohematopoietic precursor cells and the first erythroid and myeloid hematopoietic cells under the influence of yolk sac stromal cells like the YS6 cells described here.     

Ultrastructural and karyotypic examination of in vitro produced bovine embryos developed in the sheep uterus

This study examined whether development of bovine in vitro produced (IVP) blastocysts in the sheep uterus resulted in morphologically and karyotypically normal elongation stage bovine blastocysts. Seven day IVP bovine blastocysts, resulting from either in vitro maturation and fertilization, nuclear transfer (NT), or parthenogenic activation, were surgically transferred at the blastocyst stage into sheep uteri. Sheep were sacrificed after 7-9 days, and blastocysts were flushed from their uteri. One of each kind of IVP bovine blastocyst was recovered from sheep uteri for analysis by transmission electron microscopy, and nine NT blastocysts were used to establish cell cultures that were analysed for chromosome complement. TEM analysis of in vivo-derived elongation stage bovine and ovine blastocysts was done for comparative purposes. Most ultrastructural features of the 13-19 day blastocysts were similar to earlier stage blastocysts except that distinct alternative mitochondrial morphologies were found between epiblast and trophectoderm cells. Monociliated cells, presumably nodal cells, were observed in the bovine epiblast and hypoblast, and retrovirus-like particles were elaborated by cells in these same areas. Development in the sheep uterus of IVP bovine blastocysts resulted in the presence of crystalloid bodies in the trophectoderm cells, and apoptotic and necrotic cells were observed in the epiblast tissue. Thus, in vivo incubation in the sheep uterus allowed nearly normal development to the elongated blastocyst stage and may be useful for assessment of NT bovine blastocyst developmental competence. Cell cultures derived from the NT blastocysts had normal chromosome complements suggesting that activation by ionomycin and 6-dimethyl-aminopurine did not cause detrimental changes in ploidy in those blastocysts that developed.     

Superficial cell differentiation during embryonic and postnatal development of mouse urothelium

After drastic urothelial destruction around birth and around postnatal day 6, mouse urothelial renewal starts each time de novo. The differentiation of superficial cells during urothelial restoration was followed for the first time from embryonic day 15 to postnatal day 6 by the detection of differentiation markers: cytokeratins, uroplakins and apical membrane specialization. The differentiation markers of short-lived superficial cells were studied before and after urothelial destruction. Three distinctive types of superficial cells, typical for certain developmental period, were characterised: cells at low differentiation stage with microvilli and cilia, expressing CK7 and CK18, detected on embryonic day 15; cells at advanced differentiation stage with star-like arrangement of prominent membrane ridges, expressing CK7 and CK20, present between the two urothelial destruction events; highly differentiated cells with typically jagged apical surface, expressing CK7 and CK20, found twice during development. This cell type appears for the first time on embryonic day 18 as the terminal stage of embryonic differentiation. It was found again on postnatal day 6 as an initial stage of differentiation, leading toward terminally differentiated cells of the adult urothelium. Our work proves that apical membrane specialization is the most valuable differentiation marker of superficial cells.     

Cell sensitivity to transplacental mutagenesis by N-ethyl-N-nitrosourea is greatest during early gestation in the Syrian hamster.

The extremely high rate of cell division that occurs during early embryogenesis is hypothesized to predispose to high rates of mutation after chemical exposure. We tested this supposition experimentally. To probe the variation in susceptibility to mutation induction as a function of gestation stage, somatic cells of the developing Syrian hamster were isolated after transplacental treatment with N-ethyl-N-nitrosourea (ENU). Mutants were quantified using either 6-thioguanine (6-TG) or diphtheria toxin (DT) as selective agents. Several different approaches were used. In one, three litters were exposed on each gestation day and fetuses were removed on day 13. Maximum fetal sensitivity to ENU's genotoxic action was noted when treatment was at days 8 and 9, fewer mutants being obtained with earlier and later exposures. To compensate for the low numbers of target cells early in gestation, this experiment was repeated using larger numbers of litters exposed at the earlier time points, and the highest mutation frequency was now found to occur after treatment on gestation days 6 and 7. In the second approach, mutations were quantified in cells harvested 24 h after transplacental ENU exposure. Here again, embryos exposed at earlier times of gestation were more susceptible than those treated at later periods. Based on the total cell numbers in embryos and fetuses at each gestation day, we conclude that mutation frequency is maximal on day 6, corresponding to the primitive streak stage with extremely high rates of cell division.     

In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts

The in vitro development of porcine nuclear transfer embryos constructed using primary cultures from day 25 fetal fibroblasts which were either rapidly dividing (cycling) or had their cell-cycle synchronized in G0/G1 using serum starvation (serum-starved) was examined. Oocyte-karyoplast complexes were fused and activated simultaneously and then cultured in vitro for seven days to assess development. Fusion rates were not different for either cell population. The proportion of reconstructed embryos that cleaved was higher in the cycling group compared to the serum-starved group (79 vs. 56% respectively; P < 0.05). Development to the 4-cell stage was not different using either population. Both treatments supported similar rates of development to the morula (1.5 vs. 7%, cycling vs. serum-starved) and blastocyst stage (1.5 vs. 3%, cycling vs. serum-starved). The blastocyst produced using cycling cells had a total cell number of 10. Total cell numbers for the three blastocysts produced serum-starved cells were 22, 24, and 33. These blastocysts had inner cell mass numbers of 0, 15, and 4, respectively. Six hundred and thirty-five nuclear transfer embryos reconstructed using serum-starved cells were transferred to 15 temporarily mated recipients for 3-4 days. Of these, 486 were recovered (77% recovery rate) of which 106 (22%) had developed to the 4-cell stage or later. These were transferred to a total of 15 recipients which were either unmated or mated. Seven recipients farrowed a total of 51 piglets. Microsatellite analysis revealed that none of these were derived from the nuclear transfer embryos transferred.     

In situ lymphoid cells of mouse mammary tumors. IV. Comparison of functional activity of lymphoid cells separated from mammary tumors to that of spleen and lymph node cells of tumor-sensitized mice.

Lymphoid cells isolated form several types of mouse mammary tumors are capable of stimulating tumor cell growth or survival in MCT assays. Lymph node and spleen cells of mice bearing such a tumor are specifically cytotoxic to the tumor cells. Surgical removal of the tumor is followed in 4 to 7 days by the appearance of stimulatory capacity in spleens and lymph nodes. By day 10, cytotoxic cells specific for the sensitizing tumor are again detected. These reach a peak on day 13. By day 17 no reactivity is detectable. The functional distribution of tumor-reactive lymphoid cells is different between tumor masses and peripheral lymphoid organs.