DNA SYNTHESIS, MITOSIS, AND DIFFERENTIATION IN PANCREATIC ACINAR CELLS IN VITRO

Pieces of mouse embryonic pancreatic epithelium cultured in an inductive situation in vitro, or when examined at critical times in vivo, show a gradient of zymogen granule accumulation. Cells located internally in explants, or in central acini in vivo, show this overt differentiation first. As the epithelia age, the more peripheral cell population proceeds in a similar differentiation. Observations of autoradiograms of H³-thymidine-labeled tissues indicate that the first cells which cease incorporating the DNA-precursor are in the central regions that differentiate first. In older explants, thymidine incorporation is largely restricted to the periphery of the tissue as zymogen appears in the internal cells. Evidence suggests that cells or nuclei which have replicated DNA move inward before dividing. Some daughter cells apparently return peripherad to divide again, whereas others remain centrally where they undergo differentiation. During at least the first 24 hours of these maturational changes, mesenchyme has a stimulatory effect upon epithelial thymidine-incorporation frequencies. The presence of a post-DNA-synthetic population is seen in the form of a group of nonlabeling central cells that remains intact in the midst of a labeled epithelium for as long as 48 hours in vitro (from 72 to 120 hours). If explants are treated with 5-bromodeoxyuridine for any 24-hour segment of the 0 to 72-hour period, before the non-incorporating population arises, no subsequent overt zymogen formation occurs. If explants are treated continuously from 72 to 120 hours, on the other hand, zymogen still forms in some internal cells. Presumably, this differentiation is limited to the postmitotic population as revealed in the thymidine autoradiograms.     

PATTERNS OF MITOSIS AND DIFFERENTIATION IN CELLS DERIVED FROM PEA ROOT PROTOPLASTS

Mitotic figures of diploid, tetraploid, octaploid and 16-ploid nuclei were observed in cultures of pea root protoplasts whose initial DNA content was apparently 2C and 4C. The distribution of these mitotic figures in the different ploidy levels paralleled the distribution of mitotic figures in the culture of intact root explants and may be related to the hormonal stimulation of mitoses in these cultures. The patterns of the time course of both DNA synthesis and cell division in the protoplast cultures were similar to such patterns observed in the culture of intact root explants, although longer lag periods were observed in the protoplast cultures. Mitotic abnormalities including both chromosome breakage and spindle disfunction were observed in protoplast cultures. A large portion of the cell pairs derived from mitoses (27 % in one experiment) contained Feulgen-positive micronuclei. An accumulation of an as yet unidentified differentiation product termed dense cytoplasmic protoplast derivative was observed. Some of the conditions influencing the development of these derivatives are reported.     

骨骼肌肌丝分化与横纹肌肉瘤分化异常的研究

本工作观察了鸡胚胸肌原代培养物内完成末次分裂的有丝分裂后肌母细胞在初生0时—22小时内形态分化与肌专一蛋白表达的时、空间特征。肌母细胞的肌分化程序是(1)退出细胞周期,(2)开始合成和集累结蛋白与肌丝蛋白,(3)细胞伸长,肌丝肌节的装配,(4)细胞融合形成多核肌管。横纹肌肉瘤的细胞周期与肌终末分化关系异常,肌丝分化阻断。少数瘤细胞形成正常肌丝肌节,表明瘤细胞内仍存在正常肌分化所需的全部遗传信息。肌分化程序的某些特定基因功能性表达异常可能是增殖与分化紊乱的主要原因。     

脂肪来源细胞体外增殖规律及定向诱导分化研究

脂肪组织由整形外科吸脂术获得(19例,31．5±5．8岁)。酶消化法分离抽吸物中细胞,体外扩增至第10代．测定细胞生长曲线、累计倍增数目,明确其体外生长规律和增殖能力;通过对表面抗原CD29、CD105、CD106、CD166、CD49d、CD34、CD31、3G5等的检测分析脂肪来源细胞的群体组成：分别向软骨、骨、脂肪定向诱导,进一步明确该细胞群体定向分化能力。实验表明,每300ml脂肪抽吸物平均可获得5×10~7个有核细胞,体外扩增10代,平均每代倍增数目为1．59±0．224．累计倍增数目为15．53。流式细胞学及免疫细胞化学检测显示,干细胞相关抗原CD29、CD105、CD106、CD166等表达率均＞60%,但与造血系相关的CD34、CD31表达率也分别达到7．3%、29．2%。ADC向软骨诱导可检测到Ⅱ型胶原表达;向成骨诱导可见矿化结节形成,并可检测到AKP、Osteonectin基因表达;向脂肪诱导可检测到PPARr2、GLU-4、Leptin基因表达,细胞内有脂滴形成。脂肪来源的细胞获得量大,体外增殖能力强,并含有具有多向分化潜能细胞,有可能作为组织构建的种子细胞。     

CYTOKINE- AND NEUROPEPTIDE-MEDIATED DIFFERENTIATION IN RETINAL PIGMENT EPITHELIAL CELLS IN VITRO

To determine the mechanism of growth and differentiation of retinal pigment epithelial (RPE) cells it is important to understand the pathogenesis of several retinal diseases. Recently it has been reported that several cytokines and neuropeptides regulate the growth of RPE cells. In this study, the role of cytokines and neuropeptides in melanin synthesis, which is one indication of the RPE cell differentiation, was examined using chick RPE cells in vitro IL-1β, TNF-α, substance P, β-endorphin and methionine-enkephalin stimulated the melanin synthesis of RPE cells in a dose-dependent manner. The most effective concentrations of these agents on RPE cell melanin synthesis were not the same as that for RPE cell proliferation. These results indicate that cytokines and neuropeptides play an important role not only for the growth but also for the differentiation of RPE cells.     

IN VITRO DIFFERENTIATION OF CHICK EMBRYONIC LENS EPITHELIAL CELLS INTO FIBER CELLS

The processes of fiber-cell formation in the lens epithelium of 9-day-old chick embryo in vitro were studied.
Mitotic activity was enhanced during the first 12 hr, but with a drop at the 4th hour of cultivation. After the 24th hour, when the cells began to elongate, almost no mitotic figures or incorporation of ³H-thymidine into the nuclei were observed.
α- and δ-crystallin were contained in and synthesized by the newly isolated lens epithelium. The content and syntheses had diminished by the 12th hour.
In the earlier phase of cultivation, both fiber cell formation and crystallin synthesis were suppressed by treatment with Actinomycin D, but after the 12th hour they were resistant to the antibiotic.
The correlation between cell division and fiber-cell differentiation in the lens epithelium in vitro is discussed and compared with that reported in Wolffian lens regeneration and in developing bovine lens.     

人胚胎干细胞分化为神经干细胞诱导方法的对比研究

目的探讨人胚胎干细胞分化为神经干细胞过程中,经拟胚体（embryonic body,EB）法和直接分化法的不同效率。方法人胚胎干细胞常规培养消化后,分为两组：A组,经EB法分化;B组,添加noggin和ITSFn直接分化法。倒置相差显微镜观察细胞形态变化,RT-PCR检测细胞各阶段标志物,免疫荧光及流式细胞仪观察两组细胞Nestin阳性细胞率。神经干细胞继续分化,免疫荧光、RT-PCR法检测MAP2、GFAP表达。结果RT-PCR检测到OCT4、nestin表达。B组nestin阳性细胞率明显高于A组,差异有统计学意义（P〈0.01）,且诱导周期短于A组。神经干细胞继续分化,得到不同数量的神经元和胶质细胞,MAP2、GFAP分别阳性。结论在体外采用定向分化诱导,人胚胎干细胞不经EB,可直接定向分化为神经干细胞,且诱导效率比EB法高。因此直接分化法是一种经济实用的诱导方法。     

高尔基体在人胃腺癌细胞诱导分化过程中的变化

MGc 80-3细胞高尔基体呈发育差、结构不典型状态,但经dBcAMP诱导后,细胞内高尔基体组数增多、分布集中、体积增大,高尔基囊数目增多、排列规则,囊的膜内颗粒增多、分布较为均匀,恢复为与其相应正常细胞相似、发育良好的典型高尔基体结构。这种变化不仅抑制了胃癌细胞的恶性分泌活动,同时对细胞表面成份的变化也起着一定的调节作用。认为高尔基体结构与功能向典型方向的转??变是癌细胞恶性表型逆转的一种重要表现,对于癌细胞由恶性向正常方向的分化具有重要影响。     

COMPARISON OF THE GROWTH AND DIFFERENTIATION OF EPITHELIAL CELLS FROM NORMAL AND HYPERPLASTIC LENSES OF THE CHICK: STUDIES OF IN VITRO CELL CULTURES

Epithelial cells from hyperplastic lenses of a strain of chicks (Hy-1) selected for high growth rate were dissociated and cultured in vitro and compared with lens epithelial cells from a normal strain (N) in similar conditions. The hyperplastic lens cells showed remarkable motility and adhesiveness after dissociation and formed cell aggregates of various sizes before attaching to the substrate, giving a rather low plating efficiency. The lens structures (lentoid bodies) developed in partially confluent cultures of Hy-1 cells at least three days earlier than those in the cultures from normal control cells, in which the lens structures developed only after the cultures reached confluence. The results of culture at low cell density showed that the Hy-1 cell population consisted of at least two cell types different from each other in growth capacity. These striking differences in in vitro behaviour of dissociated cells from normal and hyperplastic lens epithelia and the results of clonal culture are discussed in relation to the possible mechanisms of abnormal morphogenesis and growth which are likely to be involved in the development of the hyperplastic lens in situ .     

EFFECTS OF CULTURE MEDIA ON THE "FOREIGN" DIFFERENTIATION OF LENS AND PIGMENT CELLS FROM NEURAL RETINA IN VITRO

Neural retinal cells of 3.5-day-old quail embryos were cultured as a monolayer to examine their potentials for differentiation in vitro. The "foreign" differentiation into lentoid and pigment cells was much affected by the choice of medium (Eagle's MEM and Ham's F–12); in Eagle's MEM, neural retinal cells differentiated extensively into lentoid bodies and pigment cells, as previously reported in cultures of chick neural retinal cells, while in Ham's F–12, though the cells proliferated as well as in Eagle's MEM, the "foreign" differentiation is inhibited. When primary cultures were transferred to secondary cultures, the occurrence of "foreign" differentiation did not depend on the medium used for the primary culturing, but wholly on the medium used for secondary cultures. This difference in differentiation in two different media was quantitatively substantiated by measuring the amounts of α-, δ-crystallins and melanins of cultured cells.     

THE IN VITRO DIFFERENTIATION OF DENSITY SUB-POPULATIONS OF COLONY-FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY-STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub-populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSF_MLCM), post-endotoxin mouse serum (CSF_ES) and from human urine (CSF_Hu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro-phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

大鼠原生殖细胞培养和分化的研究

研究大鼠胚胎原生殖细胞（primordial germ cells,PGCs)的培养及分化,取受精后11-12.5天大鼠PGCs进行原代培养,光、电镜观察PGCs及其分化细胞的微细结构,碱性磷酸酶染色检测细胞的分化程度,结果显然显示大鼠PGCs大而圆,散在分布,或多个聚集成团,胞质中含有椭圆形的线粒体和丰富的核糖体,在鼠胚成纤维细胞饲养层存在的情况下,PGCs保持未分化状态,碱性磷酸酶反应呈强阳性,在缺乏饲养层的条件下PGCs很快分化,形态不规则,有伪足,碱性磷酸酶反应减弱,进一步分化可形成具有细长突起的神经元样细胞,胞质中含有细丝束的表皮细胞,可见节律性跳动的心肌细胞,具有分泌颗粒的分泌细胞及似血管,心脏形状的管腔结构等,由PGCs分化来的细胞碱性磷酸酶反应均呈阴性,结果表明大鼠PGCs能够分化形成三个胚层的衍生物,生殖嵴来源的PGCsp是一种具有发育全能性的胚胎多能干细胞,本研究同时证明鼠胚饲养层能抑制大鼠PGCs的分化。     

体内体外培养下飞蝗雄性生殖细胞的分化

在单一TCl99或GRACE培养液中培养的四龄三天东亚飞蝗(Locusta mtgratoria mani lensis精小管,其精子发生只发育至初级精母细胞期,培养液中添加10%小牛血清或飞蝗精巢匀浆液可促使其发育至次级精母细胞期,添加10%分别取自东亚飞蝗蝗蝻、柞蚕蛹及蓖麻蚕蛹的血淋巴可促进其产生约20%的精子。 蜕皮激素及保幼激素对精子的产生无显著影响。移植培养的精小管在受体飞蝗体内不能发育产生精子,注射20μg/虫蜕皮激素可促使其产生大量精子。完整精巢无需注射蜕皮激素即可在受体飞蝗体内发育产生精子。结果表明,昆虫血淋巴内可能含有促细胞分化类因子,此(类)因子可能无种属特异性,外源蜕皮激素可能对精子发生无直接作用,但精子发生同时需要蜕皮激素和血淋巴因子,精巢本身可能有自己的蜕皮激素来源。     

MODULATION OF OSTEOGENIC DIFFERENTIATION IN HUMAN SKELETAL CELLS IN VITRO BY 5-AZACYTIDINE

Cellular differentiation is controlled by a variety of factors including gene methylation, which represses particular genes as cell fate is determined. The incorporation of 5-azacytidine (5azaC) into DNA in vitro prevents methylation and thus can alter cellular differentiation pathways. Human bone marrow fibroblasts and MG63 cells treated with 5azaC were used as models of osteogenic progenitors and of a more mature osteoblast phenotype, respectively. The capacity for differentiation of these cells following treatment with glucocorticoids was investigated. 5azaC treatment led to significant expression of the osteoblastic marker alkaline phosphatase in MG63 osteosarcoma cells, which was further augmented by glucocorticoids; however, in human marrow fibroblasts alkaline phosphatase activity was only observed in glucocorticoid-treated cultures. MG63 cells represent a phenotype late in the osteogenic lineage in which demethylation is sufficient to induce alkaline phosphatase activity. Marrow fibroblasts are at an earlier stage of differentiation and require stimulation with glucocorticoids. In contrast, the expression of osteocalcin, an osteoblastic marker, was unaffected by 5azaC treatment, suggesting that regulation of expression of the osteocalcin gene does not involve methylation. These models provide novel approaches to the study of the control of differentiation in the marrow fibroblastic system.     

SYNTHESIS OF RNA IN MAMMALIAN CELLS DURING MITOSIS AND INTERPHASE

Chinese hamster cells in the mitotic and G₁ phases of the growth cycle were incubated for 30 or 60 min in suspension tissue culture and pulse-labeled with tritiated uridine. After appropriate chases, washes, and extractions, it was found that all incorporation into the nucleic acid may be accounted for by those cells in interphase. An average of 410 counts was found for incorporation into the cell population (approximately 2.0 x 10⁵ cells) of which over 80% of the cells was initially in mitosis. The increasing number of cells leaving mitosis and entering interphase during the 30 min incubation was theoretically able to account for 470 counts. In addition, short-pulse labeling experiments have shown a consistent linear relationship between the percentage of cells in division and the incorporation of the isotope, which strongly suggests that, if 100% of the cells were in mitosis, the counts would be essentially zero. Thus, the entire label may be attributed to those cells in interphase where portions of the chromosomal material are known to be already extended.     

DNA SYNTHESIS AND MITOSIS IN ERYTHROPOIETIC CELLS

Studies of newt (Triturus or Diemictylus viridescens) erythropoietic cells showed that DNA synthesis and mitosis normally occur throughout most of the developmental process. Mitotic divisions were found in all immature precursor stages from the proerythroblast to the highly hemoglobinized reticulocyte. Mitoses were absent in mature erythrocytes. Radioautographic examination of thymidine-³H incorporation into DNA revealed that all erythroid cells except the mature erythrocyte were labeled. Microphotometric measurements of Feulgen-stained smears showed that all immature stages were undergoing DNA synthesis whereas the mature erythrocyte was inactive. The results obtained from three independent methods clearly demonstrate that (a) no loss of DNA or of chromosomes occurs during erythrocytic development and (b) highly hemoglobinized and, therefore, well-differentiated cells normally do undergo DNA synthesis and mitosis.     

THE RELATIONSHIP BETWEEN DIURNAL VARIATION OF THE NUMBER OF CELLS IN MITOSIS AND OF THE NUMBER OF CELLS SYNTHESIZING DNA IN THE EPITHELIUM OF THE HAMSTER CHEEK POUCH

1. The numbers of cells in mitosis and in DNA synthesis in the epithelium of the hamster cheek pouch have been studied at different times of the day and night. 2. By accumulation of mitotic cells using colcemid, both the rate of entry of cells into mitosis and the duration of mitosis have been estimated at two different times of day. 3. A diurnal variation has been demonstrated in both the mitotic index and in the tritiated thymidine labelling index. Although these variations are of different amplitude and timing, the experimental data fit closely to the hypothesis that the diurnal mitotic variation is the result of a partially synchronous population moving through the DNA synthetic period. No direct action on the mitotic process need be postulated. 4. From the results of mitotic accumulation, it is clear that the rate of entry of cells into mitosis depends on the time of day at which this is studied. There is also the possibility that the duration of mitosis is slightly longer when the mitotic index is high. 5. It is concluded that, at least in the epithelium of the hamster cheek pouch, the diurnal rhythm in the number of mitoses present is a reflection of the diurnal variation in the number of cells synthesizing DNA at some time earlier. Small fluctuations in the mitotic pattern imposed by this partially synchronous population moving from S into mitosis, could be caused by slight variations in the duration of mitosis.