Influence of physical factors on the growth of insect cells in vitro

Summary The tolerances of a cell line (IMC-HZ-1) from a moth,Heliothis zea, for the monovalent cations Na⁺ and K⁺ were defined. Cells shifted to media containing more than 70mm of K⁺ showed decreased growth rates. No evidence was obtained for Na⁺ toxicity. The osmotic pressure tolerances were influenced by the K⁺ concentration of the medium. The richer the medium was in K⁺, the narrower was the spectrum of osmotic pressure tolerance. Once the limit of K⁺ tolerance was exceeded, the rate of decline of growth was linear with respect to further increases in K⁺. This rate of decline was independent of osmotic pressure. The initial responses of cells during one subculture (2 to 4 population doublings) in media differing from the standard medium (used to maintain the cell line) were not reliable indicators of the growth potential of the cells. Continued subculture in such media resulted in an upward trend in population growth rates in most cases. This investigation was supported by U. S. Public Health Service Research Grant no. AI 09914 from the National Institute of Allergy and Infectious Diseases. This is Paper no. 8637, Scientific Journal Series, Minnesota Agricultural Experiment Station. The material is part of the dissertation of T. J. K. presented for the Ph.D. degree at the University of Minnesota.     

Influence of physical factors on the growth of insect cells in vitro. II. Sodium and potassium as osmotic pressure regulators of moth cell growth.

The tolerance of a cell line (IMC-HZ-1) from a moth, Heliothis zea, for the monovalent cations Na+ and K+ were defined. Cells shifted to media containing more than 70 mM of K+ showed decreased growth rates. No evidence was obtained for Na+ toxicity. The osmotic pressure tolerances were influenced by the K+ concentration of the medium. The richer the medium was in K+, the narrower was the spectrum of osmotic pressure tolerance. Once the limit of K+ tolerance was exceeded, the rate of decline of growth was linear with respect to further increases in K+. This rate of decline was independent of osmotic pressure. The initial responses of cells during one subculture (2 to 4 population doublings) in media differing from the standard medium (used to maintain the cell line) were not reliable indicators of the growth potential of the cells. Continued subculture in such media resulted in an upward trend in population growth rates in most cases.     

Two polypeptide factors that promote differentiation of insect midgut stem cells in vitro

Isolated stem cells from the midguts of Manduca sexta and Heliothis virescens can be induced to differentiate in vitro by either of two polypeptide factors. One of the peptides was isolated from culture medium conditioned by differentiating mixed midgut cells; we used high performance liquid chromatographic separation and Edman degradation of the most prominent active peak. It is a polypeptide with 30 amino acid residues (3,244 Da), with the sequence HVGKTPIVGQPSIPGGPVRLCPGRIRYFKI, and is identical to the C-terminal peptide of bovine fetuin. A portion of this molecule (HVGKTPIVGQPSIPGGPVRLCPGRIR) was synthesized and was found to be very active in inducing differentiation of H. virescens midgut stem cells. It was designated Midgut Differentiation Factor 1 (MDF1). Proteolysis of bovine fetuin with chymotrypsin allowed isolation of a pentamer, Midgut Differentiation Factor 2 (MDF2) with the sequence HRAHY corresponding to a portion of the fetuin molecule near MDF1. Synthetic MDF2 was also biologically active in midgut stem cell bioassays. Dose response curves indicate activity in physiological ranges from 10(-14) to 10(-9) M for MDF1 and 10(-15) to 10(-5) M for MDF2.     

Effects of growth factors and feeder cells on porcine primordial germ cells in vitro

As embryonic stem (ES) cells are not available in swine, embryonic germ (EG) cells derived from primordial germ cells (PGCs) are an alternate source of pluripotent embryonic cells for genetic modification through homologous recombination. Although morphological and biochemical characteristics are similar between ES and EG cells, culture conditions are quite different. To optimize the culture condition for the establishment of porcine EG cells, porcine PGCs were cultured in vitro with various combinations of growth factors (leukemia inhibitory factor [LIF], stem cell factor [SCF], and basic fibroblast growth factor [bFGF]) and on different kinds of feeder cells (STO, TM(4), Sl/Sl(4) m220, porcine embryonic fibroblasts, and COS-7 cells). Optimal results were obtained when all three growth factors (LIF, SCF, and bFGF) were present in the media. Also, feeder cells expressing membrane-bound SCF are required for survival and establishment of porcine EG cells. Therefore, a combination of growth factors and proper feeder cells are critical for the establishment of undifferentiated porcine EG cells.     

Mitogenic effects of fibroblast growth factors on chicken granulosa and theca cells in vitro

We have investigated the role that fibroblast growth factors (FGFs) may play in the rapid growth of preovulatory ovarian follicles in chickens. Granulosa and theca cells, dissected from the follicles of laying hens, were cultured in vitro and treated with FGF-1, FGF-2, FGF-5, and FGF-7. The synthesis of DNA by cultured cells was measured by incorporation of [(3)H]thymidine, which was added to the cultures. FGF-1 and -2 increased the synthesis of DNA in a dose-dependent manner in both cell types; however, FGF-5 and -7 had no effect in this respect. When genistein, a tyrosine kinase inhibitor, was added to these cultures, the synthesis of DNA due to FGF-2 was abolished. Treatment of cells with the glycosaminoglycans heparan sulphate and chondroitin sulphate had no effect on FGF-2-induced mitogenesis, while heparin inhibited it. Addition of a glycosaminoglycan antagonist, hexadimethrine bromide, to FGF-2-treated cultures inhibited DNA synthesis due to FGF-2, although not completely. Our data show that FGF-1 and FGF-2 are mitogenic for chicken granulosa and theca cells, and indicate that the actions of FGF-2 may be mediated via both tyrosine-kinase-type and glycosaminoglycan-type receptors on the surface of these cells.     

Effect of hormones and growth factors on the proliferation of adult cricket neural progenitor cells in vitro

In the adult cricket brain, a cluster of neuroblasts produces new interneurons that integrate into the mushroom body (MB), the main associative structure for multisensory information of the insect brain. In previous study we showed the antagonist role of the two morphogenetic hormones, juvenile hormone (JH) and ecdysone, on the regulation of adult MB neurogenesis in vivo. In order to examine whether these hormones act directly on neural progenitor cells, we developed an organotypic culture of MB cortices. Cell proliferation was assessed by 5-bromo, 2'-deoxyuridine (BrdU) incorporation. We showed that JH increased mushroom body neuroblast (MBNb) proliferation, confirming the mitogenic effect of JH observed in vivo. By contrast, ecdysone did not affect the amount of BrdU-labeled nuclei, suggesting that the inhibitory effect observed in vivo probably proceeded from an indirect pathway. We then examined the role of growth factors known to stimulate neural stem cell/progenitor cell proliferation in vertebrates. As shown by calcium imaging, MBNb only expressed functional receptors for insulin whereas mature interneurons responded to IGF-I and bFGF. Both insulin (10 microg/ml) and IGF-I (10 ng/ml) enhanced MB progenitor cell proliferation in culture, although the insulin effect was more pronounced. This effect was abolished when an inhibitor of polyamine biosynthesis was present in the medium, suggesting a link between polyamines and the insulin signaling pathway. By contrast, bFGF (20-200 ng/ml) failed to stimulate MBNb proliferation. Our results point to conserved and divergent mechanisms between vertebrates and invertebrates in the regulation of adult neural progenitor cell proliferation.     

生长因子对骨髓间充质干细胞增殖、分化的影响

骨髓间充质干细胞(bone mesenchymal stemcell,BMSC)是骨髓基质细胞的重要组成部分,由于其不但能与其他细胞一起支持造血干细胞造血,而且还具有较强的增殖功能及多向分化潜能,在一定诱导因素下可定向分化成骨细胞、软骨细胞和脂肪细胞等,近年来已成为生物学和医学的研究热点。本文简要介绍了不同生长因子如血管内皮生长因子、碱性成纤维细胞生长因子、转化生长因子-β等对BMSC增殖、分化的影响。     

Influence of growth factors on neuronal differentiation

With so many neutrophins and receptors now known, how is our picture of neurotrophism changing? Recent studies on knockout mice have confirmed our expectations of neurotrophin action in neuronal development. A notable exception is the activation of TrkB, on motor neurons, by an unknown ligand. It is also clear that some neurotrophins have diverse activities and influence early developmental stages. There are interesting new data concerning the role of p75, the low affinity neurotrophin receptor, as a modulator of neurotrophin activity. Even more exciting are new studies on glia-derived neurotrophic factor (GDNF) which demonstrate that this growth factor acts as a potential protector of motor neurons and striatal dopaminergic neurons.     

Bubble-column design for growth of fragile insect cells

A mathematical model for the design of bubble-columns for growth of shear-sensitive insect cells is presented. The model is based on two assumptions. First, the loss of cell viability as a result of aeration is a first-order process. Second, a hypothetical volume X, in which all viable cells are killed, is associated with each air bubble during its lifetime. The model merely consists of an equation for k _d, the first-order death-rate constant, and A _min, the minimum specific surface area of the air bubbles to supply sufficient oxygen. In addition to X, the equation for k _d contains the air flow F, the air-bubble diameter d _b, the diameter D and the height H of the bubble column. This equation has been experimentally validated. Comparison of the equations for k _d and A _min shows that especially H is the key parameter to manipulate in bubble-column design in order to meet the demands set by A _min and k _dg, the first-order growth-rate constant. It is concluded that net growth of cells is enhanced as size and height of the bubble column increase.     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

Modulation of the epidermal growth factor receptor of mouse embryonic palatal mesenchyme cells in vitro by growth factors.

A single class of high-affinity receptors for EGF were detected on mouse embryonic palatal mesenchyme (MEPM) cells cultured in vitro. The degree of confluence of the cultured cells did not affect the number or affinity of the binding sites. Culture of MEPM cells in the presence of bFGF, IGF-II or TGF-beta 1 induced changes in 125I-EGF binding. TGF-beta 1 caused a marked reduction in binding to 40% of control levels. This reduction was achieved after 2 h and persisted for 24 h after addition of the growth factor. IGF-II induced a similar reduction but this effect was transitory; after a 12 h pretreatment with IGF-II, binding was restored to control levels. The effects of bFGF were biphasic. Initially, a short pre-treatment period (3-5 h) with bFGF caused a small reduction in 125I-EGF binding; longer periods of pre-incubation (24 h) resulted in a large increase in receptor number. Pre-incubation in medium containing both bFGF and TGF-beta 1 resulted in a decrease in EGF binding. Thus, TGF-beta 1 negated the large increase in receptor number induced by bFGF alone. Changes in receptor number were usually, but not always, directly related to changes in the biological activity of EGF, as assessed by a thymidine incorporation assay. This study highlights the possible interactive role of growth factors known to be present in the developing palate.     

Hormonal control of growth and differentiation of insect tissues cultured in vitro

Summary This paper reviews the effects of insect hormones on lepidopteran imaginal discs cultured in vitro.β-ecdysone stimulated both evagination and cuticle deposition of wing discs ofPlodia interpunctella (Hübner). However, evagination required a shorter exposure to ecdysone than did cuticle deposition. Cuticle deposition was obtained under the following conditions: (a) a 24-hr pulse ofβ-ecdysone (0.5–5.0μg/ml); (b) continuous treatment with 0.2μg/mlβ-ecdysone; or (c) continuous treatment with 0.5 to 50.0μg/mlβ-ecdysone in medium conditioned with larval fat body. Investigations of some biochemical effects of ecdysone showed that RNA and protein synthesis was required for evagination and cuticle deposition. In particular, studies with actinomycin D and cycloheximide (at nontoxic levels) showed that RNA and protein synthesis during the ecdysone-dependent period was essential for subsequent development. These findings support the hypothesis that stimulation of macromolecular synthesis is fundamental to the action of ecdysone on imaginal discs. The influence of beta-ecdysone on chitin synthesis was also examined.β-ecdysone stimulated uptake and incorporation of tritiated-glucosamine by culturedP. interpunctella wing discs. Addition of hexosamines to the culture medium had no influence on ecdysone-induced cuticle deposition, but inhibition of glucose-uptake by cytochalasin B prevented the formation of cuticle. The action of ecdysone on particular enzymes in the chitin pathway remains to be elucidated. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Influence of genetic factors on the fertilization of mouse ova in vitro

In-vitro fertilization was studied in F1 hybrids, 4 inbred and outbred strains of mice. Experiments were performed with intact ova (Series I) and denuded ova (Series II). In Series I the highest percentages of fertilization were obtained with gametes of F1 hybrids (86.7%) and of the CBA strain (87.5%). The KP, KE, and particularly the C57 strains, which in vivo give less than 100% fertilization, also gave low indices in vitro (42.9, 26.0 and 8.6% respectively). Testing gametes of these inbred strains with F1 gametes showed that spermatozoa and ova are responsible for the low percentages of fertilization. The rate of fertilization was mainly dependent on the genotype of the spermatozoon. In Series II, high percentages of fertilization (97-100%) were regularly obtained in all groups, indicating that differences between strains pertained mainly to binding of spermatozoa with the zona pellucida. The incidence of polyspermy, which did not exceed 5% in Series I and reached 58% in Series II, was dependent on the genotype of both gametes.     

Neural stem cell-derived factors inhibit the growth and invasion of U87 stem-like cells in vitro

Glioma is one of the most common and aggressive tumors in the brain. Significant attention has been paid to the potential use of neural stem/progenitor cells (NSCs/NPCs) as delivery vehicles to cure gliomas. However, whether the NSCs/NPCs or the factors they produced could make a contribution still remains to be seen. In this study, we focused on the inhibitory effects of the factors produced by NSCs/NPCs on the biological behavior of the glioma stem-like cell in vitro. The human glioma cell line U87 was selected and the U87 stem-like cells were addressed. After being cultured in the NSC condition medium (NSC-CM), the viability and proliferation of U87 stem-like cells were significantly reduced. The invasion of U87 stem-like cells and the migration of U87 cells were also significantly decreased. However, no significant change was observed in regard to the astrocytic differentiation of U87 stem-like cells. These indicated that NSCs/NPCs produced some factors and had an inhibitory effect on the growth and invasion but not the terminal differentiation of U87 stem-like cells. It is worth paying attention to NSCs/NPCs as a high-potential candidate for glioma treatment.     

Recombinant scorpion insect excitatory toxin BmK IT accelerates the growth of insect Spodoptera frugiperda 9 cells

Several scorpion insect toxins are selectively active on the lepidopterous and dipterous insects. The gene encoding insect excitatory neurotoxin (BmK IT) from the scorpion Buthus martensii Karsch was expressed in Escherichia coli BL21(DE3) at a high level of 3 mg/0.5 L using the prokaryotic expression system pTWIN1. Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), whole-cell patch-clamp technique and immunofluorescence assays were used to evaluate the toxicity of rBmK IT to insect Spodoptera frugiperda 9 (Sf9) cells and to analyze the potential mechanism of this toxicity. rBmK IT accelerated the growth of Sf9 cells in a dose-dependent manner. Voltage-gating sodium channel activity could not be detected in Sf9 cells using a whole-cell patch-clamp technique. However, immunofluorescence analysis clearly showed co-localization of tetrodotoxin (TTX) and rBmK IT on the Sf9 cell membrane, which demonstrated that rBmK IT could bind to and act on the voltage-gated sodium channels on the Sf9 cells by the high affinity action power. The findings presented in this study are essential for further study of this peptide.     

Influence of hormone and growth factor interactions on the proliferative potential of normal rat mammary epithelial cells in vitro

These experiments were aimed at using a recently developed serum-free culture system for growth of normal rat mammary epithelial (RME) cells in vitro to examine the interactions of specific hormones and growth factors on the proliferative potential of these cells. RME cells were obtained by enzymatic dissociation of mammary tissues of Lewis rats. Primary cultures were started by plating 2 X 10(5) RME cells per 60-mm type I collagen-coated tissue culture dish. Cultures were maintained in a basal medium that consisted of Ham's F-12 medium supplemented with bovine serum albumin (BSA), ethanolamine (EA), and transferrin (Tf), which, by itself, did not support RME cell proliferation. Insulin (I), hydrocortisone (HC), and epidermal growth factor (EGF), when added to the basal medium interacted synergistically to stimulate RME cell proliferation, but this effect was dependent on the additional presence of cholera toxin (CT). Under these conditions a greater-than-tenfold increase in cell number over a 10-day culture period was obtained. Insulin could be replaced by physiological levels of insulin-like growth factor-I (IGF-I). CT could be replaced by other agents that elevate intracellular levels of cyclic adenosine 3':5' monophosphate (cAMP) such as dibutyryl-cAMP (db-cAMP), prostaglandin E1 (PGE-1), and/or isobutylmethylxanthine (IBMX). Prolactin (M) or progesterone (P) potentiated the effect of I, HC, EGF, and CT, resulting in an additional twofold increase in cell number over that found in their absence. However, addition of both hormones was no more effective than either one alone. Furthermore, addition of M or P in the absence of EGF had no effect on RME cell proliferation. Addition of 17-B-estradiol (E2) to the I-, HC-, EGF-, and CT-containing medium also resulted in enhanced RME cell proliferation. These results point to a number of hormone and growth factor interactions that influence the proliferation of normal RME cells in vitro.