Germ cell binding to rat Sertoli cells in vitro

The interaction between male germ cells and Sertoli cells was studied in vitro by co-incubation experiments using isolated rat germ cells and primary cultures of Sertoli cells made germ cell-free by the differential sensitivity of germ cells to hypotonic shock. The germ cell/Sertoli cell interaction was examined morphologically with phase-contrast and scanning electron microscopy and then quantified by measuring radioactivity bound to Sertoli cell cultures after co-incubation with added [3H]leucine-labeled germ cells. Germ cell binding to Sertoli cell cultures was the result of specific adhesion between these two cell types, and several features of this specific adhesion were observed. First, germ cells adhered to Sertoli cell cultures under conditions during which spleen cells and red blood cells did not. Second, germ cells had a greater affinity for Sertoli cell cultures than they had for cultures of testicular peritubular cells or cerebellar astrocytes. Third, germ cells fixed with paraformaldehyde adhered to live Sertoli cultures while similarly fixed spleen cells adhered less tightly. Neither live nor paraformaldehyde-fixed germ cells adhered to fixed Sertoli cell cultures. Fourth, germ cell binding to Sertoli cell cultures was not immediate but increased steadily and approached a maximum at 4 h of co-incubation. Saturation of germ cell binding to Sertoli cell cultures occurred when more than 4200 germ cells were added per mm2 of Sertoli cell culture surface. Finally, germ cell binding to Sertoli cell cultures was eliminated when co-incubation was performed on ice. Based on these observations, we concluded that germ cell adhesion to Sertoli cells was specific, temperature-dependent, and required a viable Sertoli cell but not necessarily a viable germ cell. These results have important implications for understanding the complex interaction between Sertoli cells and germ cells within the seminiferous tubule and in the design of future experiments probing details of this interaction.     

Cell cycle length, cell size, and proliferation rate in hydra stem cells

We have analyzed the cell cycle parameters of interstitial cells in Hydra oligactis. Three subpopulations of cells with short, medium, and long cell cycles were identified. Short-cycle cells are stem cells; medium-cycle cells are precursors to nematocyte differentiation; long-cycle cells are precursors to gamete differentiation. We have also determined the effect of different cell densities on the population doubling time, cell cycle length, and cell size of interstitial cells. Our results indicate that decreasing the interstitial cell density from 0.35 to 0.1 interstitial cells/epithelial cell (1) shortens the population doubling time from 4 to 1.8 days, (2) increases the [3H]thymidine labeling index from 0.5 to 0.75 and shifts the nuclear DNA distribution from G2 to S phase cells, and (3) decreases the length of G2 in stem cells from 6 to 3 hr. The shortened cell cycle is correlated with a significant decrease in the size of interstitial stem cells. Coincident with the shortened cell cycle and increased growth rate there is an increase in stem cell self-renewal and a decrease in stem cell differentiation.     

Subsidiary cells in the Orchidaceae: their general distribution with special reference to development in the Oncidieae

Subsidiary cell formation in leaves of the Oncidieae begins with the production of a trapezoid cell on each side of the guard cell mother cell. The trapezoid cells are formed by oblique divisions in the tiles of cells next to the tile of cells containing the guard cell mother cell. The trapezoid cell usually divides unequally to form a subsidiary cell and a derivative cell. The subsidiary cell is smaller and next to the guard cell mother cell. The derivative cell enlarges and is often indistinguishable from the other epidermal cells. Rarely, polar subsidiary cells are also formed. In very rare cases the smaller of the division products of the trapezoid cell divides to form two subsidiary cells next to each guard cell. Subsidiary cells have been found in all tribes of the epidendroid and vandoid groups, all neottoid tribes examined except the Orchideae, and the subfamily Cypripedioideae. The absence of subsidiary cells in primitive genera of the epidendroid tribes and the presence of subsidiary cells in the most advanced genera of the epidendroid and vandoid groups supports the hypothesis that the presence of subsidiary cells is an advanced condition in the Orchidaceae.     

Fusion of cell ghosts with intact cells in Dictyostelium discoideum: Differential response of opposite mating-type cells

The sexual cycle of Dictyostelium discoideum is initiated by the fusion of cells that are of opposite mating types (e.g. NC4- and HM1-type cells). Cells grown in light on agar plates are not capable of sexual cell fusion, but become capable when cultured in the dark in a liquid medium. Cells in the incapable state are called fusion-incompetent cells, and cells in the latter state, fusion-competent cells. To gain some understanding of the mechanism of cell fusion, cell ghosts prepared by freeze-thawing intact cells were incubated with intact cells. The cell ghosts killed the intact cells by directly fusing with them, the extent of fusion depending on the particular strains employed and the fusion-competency of the intact cells and of the cells from which the cell ghosts had been prepared. A detailed examination revealed that fusion-competent NC4 cells were always more easily killed by cell ghosts than fusion-incompetent NC4 cells. It also became apparent that cell ghosts prepared from fusion-competent NC4 cells killed all cell types far more efficiently than did those prepared from fusion-incompetent NC4 cells. However, fusion-competent and fusion-incompetent HM1 cells were equally sensitive to cell ghosts, and cell ghosts prepared from fusion-competent HM1 cells had the same ability to kill as those prepared from fusion-incompetent HM1 cells. From these findings, it thus appears that opposite mating-type cells have distinct membrane properties related to sexual cell fusion.     

Possible role of cell cycle-dependent morphology, geometry, and mechanical properties in tumor cell metastasis.

Studies that examine the shear- and abrasion-sensitivity of proliferating cells are important in order to understand the behavior of hybridoma cells in bioreactor culture and metastasizing cancer cells in the bloodstream. Little is known about the link between morphology, structure, and mechanical properties of a given cell line, especially with respect to variations throughout the cell cycle. In our experiments with GAP A3 hybridoma cells, distinct cell morphologies were identified and correlated with phases of the cell cycle by video microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (apparent cell viscosity) properties of single cells. As the cell cycle progressed at 37 degrees C, an increase in cell volume from 1400 microns 3 to 5700 microns 3 was accompanied by an increase in apparent cell viscosity from 430 poise to 12,000 poise, consistent with an accumulation of more cytoplasmic material in the "older" cells. Hybridomas are representative of the various leukemias derived from hemopoietic cells, and even though as a whole, they appeared to be rather shear-insensitive, the wide range of property values demonstrates that a given cell line cannot be characterized by a single value for any one property, and that properties must be related to the cell cycle when considering proliferating cells. It is interesting to see if distinct stages in the metastatic sequence of events might correlate with any of these physical features of the cell cycle, irrespective of cell type or cell line. For example, the cytokinetic doublet could represent a fragile structure that may fail and produce cell death under fluid-shear conditions that would not affect the cells at any other stage in the cell cycle. Identifying such cell cycle-dependent features in metastasizing cancer cells could lead to a better understanding of the metastatic process and to possible clinical treatments directed at making cells more shear- and abrasion-sensitive, and therefore, more likely to be killed by the natural hydrodynamic forces of the circulatory system.     

Calcium wave propagation during cell extrusion

Oncogenically transformed or apoptotic cells are removed from epithelial sheets by cell–cell communication between the transformed/apoptotic cells (extruding cells) and the nearest neighboring cells. Cell extrusion is driven by actomyosin contraction and lamellipodial crawling of the nearest neighboring cells. Recent studies have found that distal cell communication also plays a role in cell extrusion. Specifically, distal cells located 3–16 cells away from the extruding cell are coordinated by calcium waves and collectively migrate toward the extruding cell to initiate cell extrusion. Here, I describe how calcium waves are generated and contribute to the extrusion of cells in mammals and zebrafish.     

Possible role of cell cycle-dependent morphology,geometry, and mechanical properties in tumor cell metastasis

Studies that examine the shear- and abrasion-sensitivity of proliferating cells are important in order to understand the behavior of hybridoma cells in bioreactor culture and metastasizing cancer cells in the bloodstream. Little is known about the link between morphology, structure, and mechanical properties of a given cell line, especially with respect to variations throughout the cell cycle. In our experiments with GAP A3 hybridoma cells, distinct cell morphologies were identified and correlated with phases of the cell cycle by video microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (apparent cell viscosity) properties of single cells. As the cell cycle progressed at 37°C, an increase in cell volume from 1400 μm³ to 5700 μm³ was accompanied by an increase in apparent cell viscosity from 430 poise to 12,000 poise, consistent with an accumulation of more cytoplasmic material in the “older” cells. Hybridomas are representative of the various leukemias derived from hemopoietic cells, and even though as a whole, they appeared to be rather shear-insensitive, the wide range of property values demonstrates that a given cell line cannot be characterized by a single value for any one property, and that properties must be related to the cell cycle when considering proliferating cells. It is interesting to see if distinct stages in the metastatic sequence of events might correlate with any of these physical features of the cell cycle, irrespective of cell type or cell line. For example, the cytokinetic doublet could represent a fragile structure that may fail and produce cell death under fluid-shear conditions that would not affect the cells at any other stage in the cell cycle. Identifying such cell cycle-dependent features in metastasizing cancer cells could lead to a better understanding of the metastatic process and to possible clinical treatments directed at making cells more shear- and abrasion-sensitive, and therefore, more likely to be killed by the natural hydrodynamic forces of the circulatory system.     

Differentiation of the adult Leydig cell population in the postnatal testis

Five main cell types are present in the Leydig cell lineage, namely the mesenchymal precursor cells, progenitor cells, newly formed adult Leydig cells, immature Leydig cells, and mature Leydig cells. Peritubular mesenchymal cells are the precursors to Leydig cells at the onset of Leydig cell differentiation in the prepubertal rat as well as in the adult rat during repopulation of the testis interstitium after ethane dimethane sulfonate (EDS) treatment. Leydig cell differentiation cannot be viewed as a simple process with two distinct phases as previously reported, simply because precursor cell differentiation and Leydig cell mitosis occur concurrently. During development, mesenchymal and Leydig cell numbers increase linearly with an approximate ratio of 1:2, respectively. The onset of precursor cell differentiation into progenitor cells is independent of LH; however, LH is essential for the later stages in the Leydig cell lineage to induce cell proliferation, hypertrophy, and establish the full organelle complement required for the steroidogenic function. Testosterone and estrogen are inhibitory to the onset of precursor cell differentiation, and these hormones produced by the mature Leydig cells may be of importance to inhibit further differentiation of precursor cells to Leydig cells in the adult testis to maintain a constant number of Leydig cells. Once the progenitor cells are formed, androgens are essential for the progenitor cells to differentiate into mature adult Leydig cells. Although early studies have suggested that FSH is required for the differentiation of Leydig cells, more recent studies have shown that FSH is not required in this process. Anti-Müllerian hormone has been suggested as a negative regulator in Leydig cell differentiation, and this concept needs to be further explored to confirm its validity. Insulin-like growth factor I (IGF-I) induces proliferation of immature Leydig cells and is associated with the promotion of the maturation of the immature Leydig cells into mature adult Leydig cells. Transforming growth factor alpha (TGFalpha) is a mitogen for mesenchymal precursor cells. Moreover, both TGFalpha and TGFbeta (to a lesser extent than TGFalpha) stimulate mitosis in Leydig cells in the presence of LH (or hCG). Platelet-derived growth factor-A is an essential factor for the differentiation of adult Leydig cells; however, details of its participation are still not known. Some cytokines secreted by the testicular macrophages are mitogenic to Leydig cells. Moreover, retarded or absence of Leydig cell development has been observed in experimental models with impaired macrophage function. Thyroid hormone is critical to trigger the onset of mesenchymal precursor cell differentiation into Leydig progenitor cells, proliferation of mesenchymal precursors, acceleration of the differentiation of mesenchymal cells into Leydig cell progenitors, and enhance the proliferation of newly formed Leydig cells in the neonatal and EDS-treated adult rat testes.     

种子细胞——组织工程连载之三

种子细胞也是组织工程的核心研究内容,获得足够数量和质量的种子细胞是开展体外组织工程的必要基础。用于组织工程的种子细胞必须具有形成新组织结构的能力,主要来源于自体、同种异体或异种,在具体应用时各有利弊。一些成体干细胞由于不存在伦理争议以及发育分化条件相对简单等优势是重要的种子细胞,包括造血干细胞、骨髓干细胞、神经干细胞、脂肪干细胞、皮肤干细胞。人胚胎干细胞及其组织工程要真正在临床医学中得到应用,还有很长的一段路要走。其他一些细胞也可以作为组织工程种子细胞,包括内皮细胞、上皮细胞、成纤维细胞、骨细胞、成骨细胞、角质细胞、前脂肪细胞、脂肪细胞、肌腱细胞等。这些细胞已分化,分裂能力有限,但仍应用于组织工程。理想的种子细胞具有一定标准。     

ORGANIZATION OF HAEMOPOIETIC STEM CELLS: THE GENERATION-AGE HYPOTHESIS

This paper proposes that the previous division history of each stem cell is one determinant of the functional organization of the haemopoietic stem cell population. Stem cells from a lineage of stem cells which have generated many stem cells (older stem cells) are used in the animal to form blood before stem cells which have generated few stem cells (younger stem cells). The stem cell generating capacity of a lineage of stem cells is finite. After a given number of generations a stem cell is lost to the stem cell compartment by forming two committed precursors of the cell lines. Its part in blood formation is taken by the next oldest stem cell. We have called this proposal the generation-age hypothesis. Experimental evidence in support of the proposal is presented. We stripped away older stem cells from normal bone marrow and 12 day foetal liver with phase-specific drugs and revealed a younger population of stem cells whose capacity for stem cell generation was three- to four-fold greater than that of the average normal, untreated population. We aged normal stem cells by continuous irradiation and serial retransplantation and found that their stem cell generative capacity had declined eight-fold. We measured the stem cell generative capacity of stem cells in the bloodstream. It was a half, to a quarter that of normal bone marrow stem cells and we found a subpopulation of circulating stem cells whose capacity for stem cell generation was an eighth to a fortieth that of normal femoral stem cells. This subpopulation was identified by its failure to express the brain-associated antigen which was present on 75% of normal femoral stem cells but was not found on their progeny, the committed precursors of granulocytes.     

Migration of multipotent interstitial stem cells in Hydra

Stem cells in Hydra represent one of the phylogenetically most ancient stem cell systems and, therefore, provide information for reconstructing the early history of stem cell control mechanisms. Hydra's interstitial stem cells are multipotent and differentiate into both somatic cell types and germ line cells. Although it is well accepted that cells of the interstitial cell lineage are migratory, the in vivo migratory potential of multipotent interstitial stem cells has never been explored. Combining in vivo tracing of genetically labeled interstitial stem cells and tissue transplantation, we show that in contrast to precursor cells, multipotent interstitial stem cells are stationary. Only when exposed to tissue depleted of the interstitial cell lineage, interstitial stem cells start to migrate and to repopulate emptied stem cell niches. We conclude that multipotent interstitial stem cells in Hydra are static and that microenvironmental cues including signals derived from the interstitial cell lineage or from niche cells can trigger a shift in collective stem cell behavior to start migration.     

成体心脏干细胞经静脉移植后在心梗小鼠体内的分布研究

目的：观察心肌梗死小鼠静脉移植成体心脏干细胞后,细胞在小鼠体内各器官的分布情况。明确心梗后静脉移植成体心脏干 细胞在小鼠体内的分布和归巢情况。方法：分离培养小鼠心脏成体干细胞,采用流式细胞仪鉴定细胞,通过亲脂性染料CM-DiI标 记细胞后行小鼠急性心肌梗死模型建立和细胞移植,分别在细胞移植后7、14、8 天取小鼠心脏、肝脏、脑、脊髓、肺脏,行冰冻切 片,在荧光显微镜下观察移植细胞在各组织器官存活和分布情况。结果：成体心脏干细胞分离培养后呈贴壁生长,流式细胞仪检 测显示细胞纯度>80%。CM-DiI标记后荧光显微镜下观察可见标记的细胞胞浆胞核均被染成呈明亮的红色。心肌梗死后经静脉 移植成体心脏干细胞,细胞在各组织中分布呈变化过程,7 天时,在肺脏和肝脏分布较多,至14 天和28 天时,肺脏和肝脏分布减 少,心脏分布逐渐增多,表现出向心脏的" 归巢" 现象,而脑和脊髓在28 天的观察时间内分布较少。结论：采用CM-DiI标记心 脏成体干细胞,操作简单,标记效果好,可用于短期的细胞体内追踪。小鼠心肌梗死后行经静脉成体心脏干细胞移植,28 天后细胞 在心脏的分布逐渐增多,表现出向心脏的" 归巢" 现象。     

Differentiation pathways of ectodermal epithelial cells in hydra

The differentiation pathways of ectodermal epithelial cells in hydra were investigated. We found that under steady state conditions the ectodermal epithelial cells of the foot, the foot mucous cells, and the ectodermal epithelial cells of the tentacles, the battery cells, differentiate from gastric ectodermal ephithelial stem cells. From stem cell to the terminally differentiated state, a single cell cycle is required. The cells undergo a final round of DNA replication, double their genome to 4 n and become arrested in the G2-phase of the cell cycle. The ectodermal ephithelial cells of the hypostome, which like the tentacle cells are part of the head structure, can also arise from gastric ectodermal epithelial stem cells, but do so only during head regeneration and budding. They differentiate from stem cell to hypostomal cell in a single cell cycle, but in contrast to foot mucous and battery cells they remain capable of cell proliferation. Due to this self-renewal potential, they do not require recruitment from the gastric stem-cell pool in steady-state animals.     

Mouse splenic macrophage cell lines with different antigen-presenting activities for CD4+ helper T cell subsets and allogeneic CD8+ T cells.

A panel of seven mouse splenic macrophage cell lines, derived from cloned progenitors, was compared for their ability to present antigen to Th1 or Th2 helper T cell lines and hybridomas, as well as to naive T cells, and to provide accessory cell function for the synthesis of antibody from primed B cells. One of the cell lines expressed MHC class II molecules and was the only line with constitutive antigen-presenting activity for Th1 cells. It may represent a subset of splenic macrophages responsible for the activation of naive Th1 helper cells in situ. The remaining six cell lines responded to INF-gamma by up-regulating their class II expression and acquiring Th1 antigen-presenting activity. They may represent cells which, in situ, lack constitutive antigen-presenting activity but are promoted to presenting status by Th1-derived INF-gamma. Five of the cell lines provided accessory cell function to Th2 cells, as indicated by antibody synthesis in suspensions of spleen cells from primed mice depleted of their antigen-presenting cells. One of the cell lines lacking accessory cell activity had constitutive antigen-presenting activity for Th1 cells. This reciprocal expression of antigen-presenting activity supports the idea that Th1 and Th2 helper cells are activated by different antigen-presenting cells. Finally, the cell lines differed in their ability to constitutively induce an allogeneic response; a response that was limited to CD8+ T cells occurred in a CD4+ helper cell-independent manner and was unaffected by the addition of INF-gamma. The alloantigen-presenting macrophage cell lines also possessed the most efficient accessory cell activity for antibody synthesis. These cell lines, which represent a spectrum of antigen-presenting activities in the spleen afford models for defining the roles of macrophages in the induction of immune responses and for resolving issues concerning their development.     

Maximal T cell IL-13 production requires monocyte or monokine participation

The T cell-secreted lymphokine interleukin 13 (IL-13) exerts pleiotropic effects on monocytes (Mphi) and B cells. Since accessory cells, like Mphi and B cells, also act in antigen-presenting and lymphokine augmentation of T cells, Mphi and B cells may be able to effect T cell IL-13 production. Purified T cells produced slightly less IL-13 than the lower T cell numbers contained in peripheral blood mononuclear cell population, further suggesting accessory cell augmentation. Addition of 10% B cells [either unstimulated or pokeweed mitogen (PWM)-stimulated] to autologous T cells only moderately augmented T cell IL-13 levels. PWM-stimulated B cell culture supernates had even less augmenting effect on T cell IL-13 levels and unstimulated B cell culture supernates did not augment T cell IL-13 production. In contrast to the moderately augmenting effect of B cells or their stimulated culture supernates, addition of 10% Mphi, either unstimulated or muramyl dipeptide (MDP)+IFN-gamma stimulated, to autologous T cells produced a highly significant increase in T cell IL-13 production. Mphi culture supernates were equally effective in augmenting T cell IL-13 levels, suggesting both that cell-to-cell contact is not critical for Mphi augmentation of T cell IL-13 levels, and that Mphi secreted factors are pivotal. CD64(+) Mphi (or their culture supernates), which are known as poor antigen-presenting cells, also effectively augmented T cell IL-13 production, further supporting the involvement of Mphi secreted factors. Finally, experiments with exogenous addition of recombinant monokines, as well as neutralization experiments with different cytokine antibodies, suggested IL-1beta as a primary cytokine involved in the augmentation of T cell IL-13 levels by accessory cells. However, these experiments also indicated other unidentified Mphi factors as playing a significant role in producing maximal T cell IL-13 production.     

Synchronization of cell cycle of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by using a cell chip platform

Cell synchrony is a critical requirement for the study of eukaryotic cells. Although several chemical and genetic methods of cell cycle synchronization are currently available, they have certain limitations, such as unnecessary perturbations to cells. We developed a novel cell cycle synchronization method that is based on a cell chip platform. The budding yeast, Saccharomyces cerevisiae, is a simple but useful model system to study cell biology and shares many similar features with higher eukaryotic cells. Single yeast cells were individually captured in the wells of a specially designed cell chip platform. When released from the cell chip, the yeast cells were synchronized, with all cells in the G1 phase. This method is non-invasive and causes minimal chemical and biological damage to cells. The capture and release of cells using cells chips with microwells of specific dimensions allows for the isolation of cells of a particular size and shape; this enables the isolation of cells of a given phase, because the size and shape of yeast cells vary with the phase of the cell cycle. To test the viability of synchronized cells, the yeast cells captured in the cell chip platform were assessed for response to mating pheromone (α-factor). The synchronized cells isolated using the cell chip were capable of mediating the mating signaling response and exhibited a dynamic and robust response behavior. By changing the dimensions of the well of the cell chip, cells of other cell cycle phases can also be isolated.     

Asymmetry and directionality in production of new cell types during clonal growth: the switching pattern of homothallic yeast.

Homothallic Saccharomyces yeasts efficiently interconvert between two cell types, the mating types a and alpha. These interconversions have been proposed to occur by genetic rearrangement ("cassette" insertion) at the locus controlling cell type (the mating type locus). The pattern of switching from one cell type to the other during growth of a clone of homothallic cells has been followed by direct microscopic observation, and the results have been summarized as "rules" of switching. First, when a cell divides, it produces either two cells with the same mating type as the original cell or two cells that have switched to the other mating type. This observation suggests that the mating type locus is changed early in the cell cycle, in late Gl or during S. Second, the ability to produce cells that have switched mating type is restricted to cells that have previously divided ("experienced cells"). Spores and buds ("inexperienced cells") rarely if ever give rise to cells with changed mating type. A homothallic yeast cell thus exhibits asymmetric segregation of the potential for mating type interconversion--at each cell division, the mother, but not the daughter, is capable of switching cell types in its next division. Homothallic cells also exhibit directionality in switching: experienced cells switch to the opposite cell type in more than 50% of cell divisions. These results show that the process of mating type interconversion is itself controlled during growth of a clone of homothallic cells. By analogy and extension of these results, we propose that multiple cell types can be produced in a specific pattern during development of a higher eucaryote in a model involving sequential cassette insertion.     

Functional maturation of murine B lymphocyte precursors. II. Analysis of cells required from the bone marrow microenvironment

The development of mature B cells in cultures of early B cell precursors depends on the presence of a confluent adherent bone marrow (aBM) cell layer. Adherent and sIgM+ cell-depleted bone marrow (BM) from untreated or 5-fluorouracil-pretreated donors or day 12 fetal liver cells were used as precursor cell populations. When adherent cells from thymus or highly enriched BM-derived macrophages were co-cultured with precursor cells, mature B cells were not developed. Similarly, aBM cell layers generated in the presence of hydrocortisone and horse serum were unable to support aBM cell-dependent precursor differentiation, even though cortisone was removed before the addition of precursor cells. In contrast, this type of microenvironment promoted the differentiation of precursor of myeloid cell lineages. Repeated treatment of established aBM cell populations with a monoclonal anti-macrophage antibody (31.3, known to recognize a surface marker on a subset of BM macrophages) and complement abolished the capacity of otherwise functional aBM cells to sustain the development of B cell precursors. Macrophage-depleted aBM cells regained their function after supplementation with highly enriched BM-derived macrophages grown in vitro. Limiting dilution analysis of aBM cells in microcultures containing saturating numbers of early B cell progenitors also suggests the participation of more than one cell type in the BM cell population. In conclusion, differentiation of early B cell progenitors requires macrophages in addition to at least one additional cell type contained in the aBM cell population.     

Density dependent growth of corneal endothelial cells cultured in vitro

Using the cornea of macaque monkey, we demonstrated the relationship between cell density and growth of endothelial cells in vitro. Corneal endothelial cells in a cell sheet grow most actively in regions with cell density of 1000 to 1800 cells/mm2, in explant cultures and cell sheets and in concentrated inocula dissociated cells. Cell morphology was well sustained in these cultures. Cells cultured at a higher cell density retained their potential to proliferate actively, showing clear contrast to cells cultured at a density lower than 200 cells/mm2. When dissociated cells were cultured at a low density and maintained for more than 4 weeks, they gradually lost their growth potential, altered into polymorphonuclear giant cells and eventually dedifferentiated. In addition, cells with no contact with each other did not express growth potential. Density dependent growth was confirmed by measuring the mitotic index against the cell density per square mm from the center to the peripheral regions in cultured explants. It is concluded that the growth pattern of corneal endothelial cells is closely related to cell density, and that growth of these cells might be regulated through intercellular communications.     

Cell interactions influence the fate of mouse blastomeres undergoing the transition from the 16- to the 32-cell stage

Newly formed polar and apolar 1/16 blastomeres were isolated and cultured singly, or in various combinations, through division to form 32-cell blastomeres. The morphology of the resulting cell cluster appeared to depend upon the nature and composition of the cell combination used. In most polar + apolar couplets, the polar cell enveloped the apolar cell, and following division, a 4/32 cluster was thereby generated containing two trophectoderm-like external cells derived from the polar cell and two ICM-like internal cells derived from the apolar cells. A polar cell cultured in isolation divided to give either two trophectoderm-like external cells or a trophectoderm-like cell and an ICM-like cell. Two polar cells cultured together generated clusters in which the ratio of trophectoderm-like:ICM-like cells was 4:0 or 3:1. Most apolar cells cultured together in couplets polarized, and generated 4/32 clusters containing either purely trophectoderm-like or a mixture of trophectoderm- and ICM-like cells. The results are consistent with the notion that continuing interactions between polar and apolar cells are necessary to maintain their respective fates as trophectoderm and ICM, and that in the absence of these interactions polar cells can generate ICM cells by a differentiative division and apolar cells can generate trophectoderm cells by polarizing in response to asymmetric cell contacts.