From left to right: Luc Decombel, Guy Smagghe and Luc Tirry

Insecticides in Insect Cell Cultures and Larvae of Spodoptera

In a first series of experiments, the biological response of a continuous cell line of the beet armyworm, Spodoptera exigua, was tested with different groups of insecticides with different modes of actions: acetylcholine esterase inhibitors, acetycholine receptor agonists, inhibitors and uncouplers of oxidative phosphorylation, site I electron transport inhibitors, GABA receptor inhibitors, chitin synthesis inhibitors and juvenile hormone analogs. From the concentration response curves, IC50's were calculated. The most active compound in vitro was pyridaben with an IC50 of 0.0083 ppm. In a second series of experiments, the toxicity of these insecticide groups was determined on third-instar larvae of S. exigua, and LC50's were used in the evaluation of their in vivo biological activity. Toxicity bioassays showed that lufenuron was the most toxic (LC50=0.098 ppm). To explain the discrepancies in biological responses in vitro with insect cells compared to in vivo conditions with whole third instar larvae, the significance of different detoxifying enzyme systems was tested. P450 mono-oxygenases, esterases and glutathion S-transferases were measured in third-instar larvae and cells of S. exigua. Data are discussed in terms of the usefulness of insect cell cultures as tools in the screening for novel insecticide actions. Luc Decombel, Guy Smagghe, and Luc Terry. Action of Major Insecticide Groups on Insect Cell Lines of the Beet Armyworm, Spodoptera exigua, Compared with Larvidical Toxicity, In Vitro Cellular and Developmental Biology - Animal, 43-51, 2004.

Mammary Tumors in Transgenic Mice

We have established an explant-cell culture system for mammary gland tumors from c-Myc oncogene-expressing transgenic mice and potentially other transgenic strains. By coating culture dish surfaces with fetal bovine serum and using culture media supplemented with low serum and growth factors, the mammary tumor specimens could be maintained in culture for over three months. Throughout the culture period, the explants produced abundant outgrowths of epithelial cells. As the outgrowths of epithelial cells filled the dishes, the explants were serially transferred from one dish to another - a process that could be repeated at least 6 times, thus providing a continuous supply of primary tumor cells. This culture system provides a useful tool for studying the biology of mouse mammary gland tumors and possibly tumors form other organ sites. Xu Fang Pei, Marcia S. Noble, Maria Antonietta Davoli, Edward Rosfjord, Maddalena T. Tilli, Priscilla A. Furth, Robert Russell, Michael D. Johnson, and Robert B. Dickson. Explant-cell Culture of Primary Mammary Tumors from MMTV-c-Myc Transgenic Mice, In Vitro Cellular and Developmental Biology -Animal, 14-21, 2004.

From left to right: Patricia Hicks, Pi-Ling Chang, and Gong Feng

Differential Gene Expression

Although expression vectors using viral and mammalian promoters constitutively express genes of interest in adherent cells, few studies have examined whether the function of these vectors in suspended cells, such as in over-agar or soft agar assay (an in vitro cell transformation assay) is as robust as when they are in adherent cells. The selection of appropriate expression vector to optimally express genes in suspended cells would be useful in determining whether these genes play critical role in maintaining colony formation or cell transformation. To compare promoter driven expression vector function in adherent versus suspension cells, we performed transient transfection assays using viral (simian virus 40 [SV40] and cytomegalovirus [CMV]) and mammalian ( -actin) promoters fused to luciferase or -galactosidase reporter gene. Over-agar assay was used to suspend cells on top of agar, which allowed for cell retrieval and analysis. We found that -actin and SV40 promoter exhibited suppressed gene expression of 70 and 56%, respectively in cells suspended on agar compared to those attached on plates. The suppressed response by the exogenous -actin promoter in suspension was consistent with the response of the endogenous -actin promoter activity, since the steady-state level of -actin messenger ribonucleic acid in suspended cells was significantly reduced by 50% relative to those expressed in attached cells. In contrast to SV40 promoter, CMV promoter activity was not decreased in cells suspended in over-agar when compared to adherent cells. These studies show that regardless of mammalian or viral vectors, one cannot assume that all expression vectors behave similarly in both suspension and adherent state. Gong Feng, Patricia Hicks, and Pi-Ling Chang. Differential Expression of Mammalian or Viral Promotoer-driven Gene in Adherent Versus Suspension Cells, In Vitro Cellular and Developmental Biology - Animal, 420-423, 2003.

From left to right: Amit Gal-On, Victor Gaba, and Aaron Zelcer

Curcurbit Biotechnology

The cucurbit family includes a number of valuable crop species (melon, cucumber, squash/pumpkin, watermelon). Much of this review is concerned with transgenic resistance to viruses, shown to be the major application of biotechnology in the cucurbit family. Progress made with the production of transgenic cucurbit crops is discussed. Published data on field tests of transgenic cucurbits is reviewed, showing that much progress has been made with multiple virus resistant cucurbit crops which can be productive without chemical control of insect virus vectors. Modes of virus resistance in transgenic cucurbits are discussed, as is the bio-safety of such crops. For the first time a detailed analysis has been made of world wide and US field test applications for cucurbit crops. World wide most field test applications were for melon (54%), followed by squash (32%). World wide most field test applications were for virus resistance (84%), and most applications (77%) were in the USA. Two transgenic multiple virus resistant squash crops have been deregulated (released for sale). Additionally, the analysis shows that there are transgenic multiple virus resistant crops in all major cucurbit species already available, for which several different companies have applied for field tests. This would imply that such crops are ready to be marketed should conditions permit, which would have an impact world wide in reduction of ecological damage due to chemical control of the insect viral vectors. Victor Gaba, Aaron Zelcer, and Amit Gal-On. Cucurbit Biotechnology - The Importance of Virus Resistance, In Vitro Cellular and Developmental Biology - Plant, 346 -358, 2004.

From left to right: front row: M. Noguchi, J. Kano, N. Nakamura; back row: T. Iijima, Y. Morishita, T. Ishiyama

Hepatic Stem-like Cell Lines

The existence, origin, and bipotency of the hepatic stem cell (HeSC) have been investigated. However, the isolation and culture of HeSCs from adult liver tissue is not yet well established, and the mechanism by which HeSCs differentiate into mature cells remains unclear. On the other hand, the development of HeSC-isolating and -culturing methods and the in vitro clonal analysis of their mechanism of differentiation are required to enable clinical applications of regenerative medicine in the liver. For the purpose of providing HeSCs for these studies, we attempted to establish an HeSC line from a normal adult porcine liver using a unique culture system, a poly-D-lysine-coated culture dish with NAIR-1 medium (the PDL-NAIR-1 culture system). Moreover, we examined the differentiating capacity of HeSCs in vitro. We demonstrated that it was possible in the culture system that immature epithelial cells capable of proliferating grew selectively into aggregates and that two hepatic stem-like cell lines, PHeSC-A1 and PHeSC-A2, were established. The results from our data suggest that these hepatic stem-like cell lines were capable of self-renewing and differentiating into hepatocytes or biliary epithelial cells and show that the PDL-NAIR-1 culture system offers the immense advantage of isolating and culturing HeSCs from a normal adult liver. Furthermore, because of the ability to use a clonal analysis in vitro, these cell lines are useful for the investigation of various mechanisms in which HeSCs seem to participate and their application in the study of regenerative medicine in the liver. Junko Kano, Tadashi Ishiyama, Naoko Nakamura, Tatsuo Iijima, Yukio Morishita, and Masayuki Noguchi. Establishment of Hepatic Stem-like Cell Lines from Normal Adult Porcine Liver in a Poly-D-lysine-coated Dish with Nair-1 Medium, In Vitro Cellular and Developmental Biology - Animal, 440-448, 2003.

From left to right: Scott Merkle, Jianliang Dai (lower), and Wagner Vendrame (upper)

Yellow Poplar and Sweetgum Embryogenesis

High-frequency embryogenesis systems were established for hybrid yellow-poplar (Liriodendron tulipifera × L. chinense) and hybrid sweetgum (Liquidambar styraciflua × L. formosana) by modifying a medium originally developed for embryogenic yellow-poplar cultures. Embryogenic cultures of both hybrids, consisting of proembryogenic masses (PEMs), were initiated from immature hybrid seeds on an induction-maintenance medium (IMM) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA) and casein hydrolysate (CH). For hybrid yellow-poplar, as many as 2100 germinable somatic embryos/4000 cells or cell clumps were produced when PEMs were grown in liquid IMM lacking CH, at a pH that varied with genotype (3.5 or 5.6), followed by size fractionation and plating on semisolid embryo development medium (DM; IMM lacking 2,4-D and BA) without CH, but supplemented with 4.0 mg/L (15 µM) abscisic acid (ABA). For hybrid sweetgum, up to 1650 germinable somatic embryos/4000 cells or cell clumps were produced when PEMs were grown in liquid IMM without CH, but with 550 mg/L L-glutamine, 510 mg/L asparagine and 170 mg/L arginine at pH 5.6. Somatic embryos developed from cell clumps on DM without any plant growth regulators (PGRs) or other supplements. Hundreds of somatic embryos of both hybrids were germinated on DM without CH, transferred to potting mix and hardened off in a humidifying chamber for transfer to the greenhouse. Jianlian Dai, Wagner A. Vendrame, and Scott A. Merkle. Enhancing the Productivity of Hybrid, Yellow-poplar and Hybrid Sweetgum Embrogenic Cultures, In Vitro Cellular and Developmental Biology - Plant, 376-383, 2004.

From left to right: Philip Woost, Ulrich Hopfer, Phyllis Frisa, Martin Resnick, and David Orosz

Growth of Adult Human Proximal Tubule Cells

Human proximal tubule epithelial cell lines are potentially useful models to elucidate the complex cellular and molecular details of water and electrolyte homeostasis in the kidney. Samples of normal adult human kidney tissue were obtained from surgical specimens, and S1 segments of proximal convoluted tubules were microdissected, placed on collagen-coated, culture plate inserts, and co-cultured with lethally irradiated 3T3 fibroblasts. Primary cultures of proximal tubule epithelial cells were infected with a replication-defective retroviral construct encoding either wild-type or temperature-sensitive SV40 large T-antigen. Cells forming electrically resistive monolayers were selected and expanded in culture. Three cell lines (HPCT-03-ts, HPCT-05-wt, and HPCT-06-wt) were characterized for proximal tubule phenotype by electron microscopy, electrophysiology, immunofluorescence, Southern hybridization, and RT-PCR. Each of the three formed polarized, resistive epithelial monolayers with apical microvilli, tight junctional complexes, numerous mitochondria, well-developed Golgi complexes, extensive endoplasmic reticulum, convolutions of the basolateral plasma membrane, and a primary cilium. Each exhibited succinate, phosphate, and Na,K-ATPase transport activity, as well as acidic dipeptide- and ATP-regulated mechanisms of ion transport. Transcripts for Na+-bicarbonate cotransporter, Na+-H+ exchanger (NHE3), Na,K-ATPase, PTH receptor, EGF receptor, and vasopressin V2 receptor were identified. Furthermore, immunoreactive sodium phosphate cotransporter type II (NPT2), vasopressin receptor V1a, and CLIC-1 (NCC27) were also identified. These well differentiated, transport-competent cell lines demonstrated the growth, immortalization, and differentiation potential of normal, adult, human proximal tubule cells, and consequently have wide applicability in cell biology and renal physiology. David E. Orosz, Philip G. Woost, Robert J. Kolb, Margaret B. Finesilver, Wenwu Jin, Phyllis S. Frisa, Chee-Keong Choo, Chung-Fai Yau, Kwok-Wah Chan, Martin I. Resnick, Janice G. Douglas, John C. Edwards, James W. Jacobberger, and Ulrich Hopfer. Growth, Immortalization, and Differentiation Potential of Normal Adult Human Proximal Tubule Cells, In Vitro Cellular and Developmental Biology - Animal, 22 -34, 2004.

Rida A. Shibli, Nabila S. Karam, Asmara D. Moges

Cryopreservation of African Violet Shoot Tips

Cryopreservation of African violet via encapsulation-dehydration, vitrification, and encapsulation-vitrification of shoot tips was evaluated. Encapsulation-dehydration, pretreatment of shoot tips with 0.3 M sucrose for 2 d followed by air dehydration for 2 and 4 h resulted in complete survival and 75% regrowth, respectively. Dehydration of encapsulated shoot tips with silica gel for 1 h resulted in 80% survival but only 30% regrowth. Higher viability of shoot tips was obtained when using a step-wise dehydration of the material rather than direct exposure to 100% plant vitrification solution (PVS2). Complete survival and 90% regrowth were achieved with a four-step dehydration with PVS2 at 25° C for 20 min prior to freezing. The use of 2 M glycerol plus 0.4 M sucrose or 10% dimethyl sulfoxide (DMSO) plus 0.5 M sucrose as a cryoprotectant resulted in 55% survival of shoots. The greatest survival (80-100%) and regrowth (80%) was obtained when shoot tips were cryoprotected with 10% DMSO plus 0.5 M sucrose or 5% DMSO plus 0.75 M sucrose followed by dehydration with 100% PVS2. Shoot tips cryoprotected with 2 M glycerol plus 0.4 M sucrose for 20 min exhibited complete survival (100%) and the highest regrowth (55%). In encapsulation-vitrification, dehydration of encapsulated and cryoprotected shoot tips with 100% PVS2 at 25° C for 5 min resulted in 85% survival and 80% regrowth. Asmara D. Moges, Rida A. Shibli, and Nabila S. Karam. Cryopreservation of African Violet (Saintpaulia ionantha Wendl.) Shoot Tips,In Vitro Cellular and Developmental Biology - Plant, 389-395, 2004.

Arazdordi Toumadje	Ken-Ichi Kusumoto

In Vitro Differentiation of Embryonic Stem Cells

Although the ES-D3 murine embryonic stem cell line was one of the first derived, little information exists on the in vitro differentiation potential of these cells. We have used immunocytochemical and flow cytometric methods to monitor ES-D3 embryoid body differentiation in vitro over a 21-d period. Spontaneous differentiation of embryoid body cells was induced by leukemia inhibitory factor withdrawal in the absence of feeder cells. The pluripotent stem cell markers Oct-3/4, SSEA-1 and EMA-1 were found to persist for at least 7 d, whereas the primitive endoderm marker cytokeratin endo-A was expressed at increasing levels from day 6. The localization of these antigens within the embryoid bodies suggested that embryonic ectoderm- and primitive endoderm-derived tissues were segregated. Localized expression of class III beta-tubulin and sarcomeric myosin also was detected, indicating that representatives of all three embryonic germ layers were present after induction of differentiation in vitro. Arazdordi Toumadje, Ken-Ichi Kusumoto, Angela Parton, Patricia Mericko, Lori Dowell, Guozhong Ma, Luping Chen, David W. Barnes, and J. Denry Sato. Pluripotent Differentiation In Vitro of Murine ES-D3 Embryonic Stem Cells, In Vitro Cellular and Developmental Biology - Animal, 449-453, 2003.