Light-stimulated ultraweak photon reemission of human amnion cells and Wish cells

Photon reemission in the ultraweak intensity range that is observed after irradiation of cell suspensions with light, reveals characteristic differences between normal human amnion cells and transformed Wish cells from the same parental tissue. The reemission kinetics, approximated best by a hyperbolical process, were studied as a function of cell density, showing that: malignant Wish cells have a photon storage capacity that is not improved by increasing the cell density; and that normal amnion cells exhibit a photon storage capacity that strongly increases with increasing cell density. The interpretation of this effect and the nature of the emitter are discussed.     

Light-Induced photon emission by rat hepatocytes and hepatoma cells

We have investigated spontaneous and light-induced photon emission of suspensions of rat hepatocytes and of HTC hepatoma cells. Rat hepatocytes exhibit spontaneous biophoton emission, but from hepatoma cells this was not detectable. In contrast, after irradiation with white light, the reemission intensity was found to be lower for hepatocytes than for the tumor cell line. Induced photon emission was neither influenced by anaerobiosis nor by the intactness of the cells. Cell-fractionation studies demonstrate that the induced photon emission was caused by the nuclear fraction and by isolated chromatin. Phenol-extracted DNA, however, has lost this capacity. Our data suggest that differences in the chromatin structure may explain the cell-specific induced photon emission.     

Light-induced photon emission by rat hepatocytes and hepatoma cells.

We have investigated spontaneous and light-induced photon emission of suspensions of rat hepatocytes and of HTC hepatoma cells. Rat hepatocytes exhibit spontaneous biophoton emission, but from hepatoma cells this was not detectable. In contrast, after irradiation with white light, the reemission intensity was found to be lower for hepatocytes than for the tumor cell line. Induced photon emission was neither influenced by anaerobiosis nor by the intactness of the cells. Cell-fractionation studies demonstrate that the induced photon emission was caused by the nuclear fraction and by isolated chromatin. Phenol-extracted DNA, however, has lost this capacity. Our data suggest that differences in the chromatin structure may explain the cell-specific induced photon emission.     

Discriminant analysis on cells from developing squamous cancer of the respiratory tract

Cytologic preparations made from the tracheobronchial tree taken by the Schreiber catheter have been scanned by three color microphotometry. The digitized cell images were processed by the analytical cytodiagnostic programs of the TICAS system. Cells were sorted into two control groups and five groups of increasing atypia ranging from normal epithelium to invasive squamous cell carcinoma. Standard statistical tests, including Wilk's Lambda, Rao's V, and the Kruskal-Wallis tests are performed on these subsets of cell image features. This study demonstrates that discriminant analyses permit differentiation between normal cells and those from marked atypia or carcinoma and that the classification achieves a high degree of agreement with visual assignment.     

Theory and practice of centrifugal elutriation (CE)

Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed. Experiments carried out with human red blood cells (rbc) indicated that aselective losses of rbc from the rotor caused by the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In addition, high cell loads (5×10⁸ rbc) resulted in better separations than low cell loads (5×10⁷ rbc). Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing the rotor speed. In all cases similar results were obtained. These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly in sedimentation velocity.     

人羊膜间充质细胞分离培养及其干细胞特性研究

目的：研究人羊膜间充质细胞（Humanamnioticmesenchymalcells,HAMCs）的分离、培养及其干细胞特性,为羊膜间充质细胞在再生医学的潜在应用奠定实验基础。方法：无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片,经胰酶胶原酶序贯消化,DMEM/F12培养,倒置显微镜下观察其形态,MTT法检测其生长规律,免疫荧光的方法对细胞进行鉴定,定向诱导方法检测细胞的多向分化潜能。结果：来源于羊膜的间充质细胞,细胞免疫荧光显示SSEA-4,OCT-4阳性,具有很强的增殖能力,并且具有一定的多向分化能力,在特定条件下可分化为脂肪细胞和成骨细胞;结论：羊膜间充质细胞能够在体外分离、培养、扩增,并且具有干细胞特性。羊膜间充质细胞在再生医学和组织工程应用有很好的前景。     

人羊膜来源成体干细胞的多向分化潜能

干细胞治疗被认为是一种非常有潜力的治疗手段,其中成体干细胞由于不存在伦理问题,更为广大学者所青睐。本研究成功从人羊膜间质细胞中分离纯化出具有自我更新能力和多向分化潜能的成体干细胞。首先从羊膜间质细胞中通过极限稀释法进一步分离得到羊膜来源成体干细胞(Amnion-derived stemcells,ADSC),分析其形态、生长方式及主要的免疫表型,并在体外分别将其向脂肪、成骨、内皮、肝细胞及神经细胞诱导分化。结果发现,ADSC在适宜条件下能够向3个胚层的细胞分化,经连续传代30次,其形态及表型稳定,并仍保持多向分化潜能。证实了ADSC的干细胞特性,可能为细胞治疗及干细胞工程提供种子细胞的新来源。     

The morphometry of the Sudan-III-positive granules in the cytoplasm of the human amniotic epithelium

Seventy-nine human amnions from different gestational ages were studied by new morphometric methods. The Sudan-III-positive granules in the cytoplasm of the amniotic epithelial cells were first observed in the reflected amnion from the second trimester of normal gestation. During the third trimester of normal gestation, the number of cytoplasmic granules increased remarkably with gestational age not only in the reflected amnion but also in the placental amnion. The number of granules was generally related to the increasing rates of anucleated cells and binucleated cells in the amniotic epithelium during the third trimester of normal gestation. During the third trimester of gestation, the number of granules was greater in the distant reflected amnion with its less adequate blood supply than in the placental amnion. A vast accumulation of the granules in the entire amnion was observed in cases of fetal death, in which blood supply had ceased. It is suggested, therefore, that the progressive accumulation of Sudan-III-positive cytoplasmic granules in the human amniotic epithelium during the third trimester of normal gestation is the result of cell degeneration.     

Kinetics of photoautotrophic growth of Marchantia polymorpha cells in suspension culture

Chlorophyllous, cultured cells of Marchantia polymorpha L. (HYA-2 cell line) grow actively under photoautotrophic (lithotrophic) conditions. The maximum specific growth rate (μ_cell) was 0.64 day⁻¹ and the doubling time was 1.08 days under optimum conditions (165 μmol m⁻² s⁻¹, 1% carbon dioxide enriched atmosphere, 25^°C). The photosynthetic activity was 1.30 μmol CO₂-fixed (10⁶ cells)⁻¹ h⁻¹ [66 μmol (mg chlorophyll)⁻¹ h⁻¹] in the exponential phase. The growth course has two distinct phases, an exponential and a linear one. The exponential phase is observed as long as the population density is sufficiently low (less than 7.9 × 10⁶ cells ml⁻¹), so that practically all individual cells directly receive the full incident light. The effect of light on the specific growth rate is a linear function of photon flux density. Linear growth occurs after the population density is so high that the incident light is almost completely absorbed by the cell suspension. The growth rate is a logarithmic function of photon flux density, in contrast to the specific growth rate, and saturates at high photon flux densities. The conditions of maximum growth, however, are not wellbalanced between cell mass production and cell division. Therefore, the maximum growth does not continue for a long time.     

Fractionation of K-562 cells on the basis of their surface properties by partitioning in two-polymer aqueous-phase systems

The K-562 cell line is a culture of human leukemia stem cells originally derived from a patient with chronic myelogenous leukemia in blast crisis. We have subjected such cells, in the log phase of growth, to countercurrent distribution in a charge-sensitive dextran-polyethylene glycol aqueous-phase system, a method that fractionates cells on the basis of subtle differences in their surface properties, and found that: (1) The cell population is heterogeneous since it is composed of cells with different partition ratios. (2) There is a correlation between increasing cell partition ratios and increasing cell electrophoretic mobilities. (3) Cells under different parts of the distribution curve have dissimilar ratios of cells in different parts of the cell cycle, a phenomenon that may, at least partially, be the basis for the subfractionation of these cells. There is a clear tendency for cells in G₀+G₁+early S to decrease and for those in late S+G₂+M to increase with increasing partition ratios. (4) Sialic acid is a major surface charge component of the cells as evidenced by a dramatic drop in their partition ratios after treatment with neuraminidase.     

Morphometric analysis of B2cAMP-induced reverse transformation in synchronized CHO cells

Summary Synchronized transformed and reverse-transformed (by 10⁻³ M B₂cAMP) CHO-K1 cells, growing adherent to plastic, are characterized by means of geometric and densitometric parameters at the level of both the entire cell and of the nuclei at various time intervals after selective mitotic detachment. Transformed and reverse-transformed cells triple-stained with Feulgen, Napthol Yellow S, and periodic acid-Schiff appeared very similar in terms of integrated optical density (IOD), related to either polysaccharides, protein, or DNA amount. On the other hand, a shift from a polygonal to a spindle-shaped morphology is accompanied by a significant decrease in both form factor and average optical density (AOD) of intact cell and nuclei, which are the most conspicuous measured changes caused by B₂cAMP, in addition to a lengthening of the cell cycle duration. In both control and treated cells, important and parallel cell-cycle-dependent modulations of geometric and densitometric parameters are also observed, for both the cytoplasmic (i.e., cell morphometry) and DNA space (i.e., nuclear morphometry). Specifically, the modulation in nuclear morphometry during G1, S, G2, andM phases confirms previous findings on synchronized HeLa cells. The optical density threshold-dependence of geometric parameters shows that, while becoming fusiform, the cytoplasm of reverse-transformed cells had a particularly low optical density precisely in the polar area. Utilization of such an approach in the development of anobjective morphological classification of all cell lines grown as monolayers “in vitro” is also discussed.     

Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis

Analysis by means of two-dimensional gel electrophoresis (IEF) of [32P]orthophosphate-labeled proteins from mitotic and interphase transformed amnion cells (AMA) has shown that keratins IEF 31 (Mr = 50,000; Hela protein catalogue number), 36 (Mr = 48,500), 44 (Mr = 44,000), 46 (Mr = 43,500), as well as vimentin (IEF 26; Mr = 54,000) are phosphorylated above their interphase level during mitosis. Similar studies of normal human amnion epithelial cells (AF type) confirmed the above observations except in the case of keratin IEF 44 whose relative proportion was too low to be analyzed. Immunofluorescent staining of methanol/acetone-treated mitotic transformed amnion cells with a mouse polyclonal antibody elicited against human keratin IEF 31 showed a dotted staining (with a fibrillar background) in all of the cells in late anaphase/early telophase (characteristic "domino" pattern) and in a sizeable proportion of the cells in other stages of mitosis. Normal mitotic amnion cells on the other hand showed a fine fibrillar staining of keratins at all stages of mitosis. Similar immunofluorescent staining of normal and transformed mitotic cells with vimentin antibodies revealed a fibrillar distribution of vimentin in both cell types. Taken together the results indicate that the transformed amnion cells may contain a factor(s) that modulates the organization of keratin filaments during mitosis. This putative factor(s), however, is most likely not a protein kinase as transformed amnion cells and amnion keratins are modified to similar extents. It is suggested that in general the preferential phosphorylation of intermediate-sized filament proteins during mitosis may play a role in modulating the various proposed associations of these filaments with organelles and other cellular structures.     

Transforming growth factor-beta inhibits prostaglandin production in amnion and A431 cells

We studied the effect of transforming growth factor-beta (TGF-beta) on prostaglandin E2 (PGE2) production and mitogenesis in human amnion cells and compared the response in amnion cells with that in A431 cells. Both amnion cells and A431 cells respond to epidermal growth factor (EGF) with increased production of PGE2 whereas EGF promotes mitogenesis in amnion cells but not in A431 cells. In amnion cells, TGF-beta was not mitogenic, and did not alter the mitogenic response of cells to EGF. Treatment of amnion cells with TGF-beta did, however, cause a decrease in PGE2 production relative to untreated cells, although EGF stimulated PGE2 production was not attenuated. In A431 cells, TGF-beta acted to decrease PGE2 production relative to untreated cells and to attenuate the stimulation of PGE2 production effected by EGF. The inhibitory action of TGF-beta on PG production in amnion and A431 cells is contrary to the stimulation of PG production in mouse calvaria reported by others and is suggestive that the effect of TGF-beta on prostaglandin production, like its effect on growth, varies between different cell types. Inhibition of PG production by treatment of amnion or A431 cells with mefenamic acid did not alter thymidine incorporation into DNA in response to EGF; similarly, the addition of PGE2 or PGF2 alpha to culture media of amnion or A431 cells had no effect on mitogenesis (in the absence or presence of EGF). Based on these findings, we conclude that PG production and EGF action on proliferation (stimulation in amnion cells; inhibition in A431 cells) are dissociated.     

Normal nonmetastatic human trophoblast cells share in vitro invasive properties of malignant cells

First-trimester normal human trophoblast cells show some phenotypic similarities to malignant cells, e.g., rapid proliferation and ability to invade neighboring tissue, including basement membrane in situ, but do not have the ability for unlimited growth or metastasis. The present study examined whether the invasive ability of normal trophoblast cells is an intrinsic property of these cells, independent of the microenvironment provided by the pregnant uterus, and if so, whether they share some of the molecular mechanisms of invasion exercized by metastatic malignant cells. The ability of in vitro grown human trophoblast lines to invade an epithelium-free human amniotic membrane was measured from the temporal kinetics of retention of radioactivity within this membrane resulting from a penetration by 125I-iododeoxyuridine-labeled trophoblast cells. The magnitude of this invasion was compared to that of the highly metastatic human JAR-choriocarcinoma cell line and murine B16F10 melanoma line. Trophoblasts were found to share some of the same molecular mechanisms of invasion with the metastatic cell lines. Inhibitors of collagenase, plasmin, plasminogen, and plasminogen activators completely prevented invasion of the amnion by the trophoblast lines as well as by the metastatic JAR and B16F10 lines. Mersalyl, a compound known to activate collagenase, stimulated invasion by all cell lines tested, including under conditions in which plasmin activity was inhibited. In addition, trophoblasts produced significant levels of type IV collagenase and laminin, both of which appear to be important products of metastatic tumor cells required for basement membrane invasion. It may be concluded from these findings that the invasive property of first trimester human trophoblasts is genetically determined; that the magnitude of amnion invasion cannot differentiate between metastatic cell lines and invasive but nonmetastatic cell lines; and that invasiveness is not a sufficient prerequisite for metastatic ability.     

Adenylate cyclase activity in cultured epithelial cells

Summary The cyclic AMP metabolism of cultured epithelial cells was investigated. Epinephrine or 1-methyl, 3-isobutylxanthine (MIX) alone had no effect on cyclic AMP levels in intact cells, whereas the combination of the two agents yielded a 6- to 10-fold increase in cyclic AMP levels. Both basal and stimulated cyclic AMP levels decreased with increasing cell density. Cell-free adenylate cyclase preparations were stimulated markedly by epinephrine or isoproterenol in the absence of MIX. Since the epithelial cells were found to have a relatively small amount of cyclic nucleotide phosphodiesterase (PDE) activity, the requirement for MIX to visualize intact cell responsiveness to epinephrine could be explained only partially by its PDE inhibitory properties. This study was supported in part by Grant PDT-16B, American Cancer Society.     

The stem cells of early embryos

Cells resident in an organism that possess the dual capacity for self-renewal and differentiation into a spectrum of subtypes are referred to as stem cells. In the past decade, basic research performed on stem cells has shed light on the molecular pathways operating in vivo which can be harnessed in vitro for the establishment of cell lines mirroring the stem cells in the organism. The attractiveness of stem cells as in vitro models of organotypic differentiation and their potential application in a clinical context holds great promise and is only beginning to be exploited. Stem cells can be broadly grouped into two categories based on their origin from either the embryonic or the adult. Only the early embryo possesses truly pluripotent cells that can give rise to all the cell types present in the embryo proper and adult. The adult, on the other hand, possesses specialized, tissue- or organ-specific stem cell types, which can give rise to the differentiated cell types of that specific organ and have in some instances been shown to transdifferentiate. However, no stem cell obtained from an adult organism has yet been shown to exhibit developmental potential matching the breadth of that of stem cells obtained from embryos. This review focuses on the different types of stem cells that are resident in early stage mammalian embryos, detailing their derivation and propagation in addition to highlighting their developmental potential and opportunities for future applications.