Varani Laboratory, University of Michigan Medical School, Department of Pathology, Authors: Michael Dame (5th from right); Indiradevi Veerapaneni (2nd from right); Narasimharao Bhagavathula (4th from right); Madhav Naik (1st from left); and James Varani (center).

Human Colon Tissue in Organ Culture: Calcium and Multi-mineral-induced Mucosal Differentiation

Colon cancer is the third leading cause of cancer mortality in the US and fourth in the world. Contingent upon understanding the mechanisms of this disease is the degree to which our experimental models can reconstruct the in vivo condition. Further, our models must be dynamic and manifest in vivo responses to changing conditions and variables? Our laboratory is interested in chemoprevention and in the ability of a family of minerals that potentially prevent conversion of histologically normal cells into early premalignant lesions. Central to these studies is the role of extracellular calcium in modulating the processes of growth and differentiation in the mucosal epithelium. Previously we demonstrated that intact human colon tissue could be maintained in organ culture for two days, with preservation of histological and immunohistological features unique to normal and neoplastic tissue. Now we have shown, employing this model, that a multi-mineral extract, rich in lanthanides and other trace metals, can enhance growth control properties of calcium. Within the 2-day culture period we were able to demonstrate reduced expression of epithelial cell cycling moieties and increased markers of differentiation (normal tissue). We also observed changes in the stromal element by measuring parameters of collagen turnover. We believe that stromal integrity is fundamental to the function of the epithelium. Human colon tissue in organ culture may prove to be a valuable model for the preclinical assessment of agents that regulate growth and differentiation in the colonic mucosa.

Michael K Dame, Indiradevi Veerapaneni, Narasimharao Bhagavathula, Madhav Naik, and James Varani. Human colon tissue in organ culture: calcium and multi-mineral-induced mucosal differentiation. In Vitro Cellular & Developmental Biology-Animal, 47(1):32-8, 2011.

From left to right, Drs. LeAnn Blomberg, Wesley M. Garrett, Thomas J. Caperna, and Neil C. Talbot who are members of the Agricultural Research Service’s Animal Bioscience and Biotechnology Laboratory, Beltsville, Maryland.

Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

The goal of the work with the ARS-PICM-19 pig liver stem cell line is to improve the cell line for biomedical and agricultural applications, and the presented work was initiated under a Cooperative Research and Development Agreement (CRADA) between Hepalife Technologies, Inc., and the Agricultural Research Service (ARS).  Several potential uses for the PICM-19 cell line would be enhanced or enabled by devising a cell culture system that does not require the use of so-called “feeder-cells”, and our recent report described a simple “feeder-cell-free” method for the continuous culture of the PICM-19 cell line.  By removing the feeder-layer from the culture system, better in vitro pharmacological and toxicological assays of liver function may be able to be devised.  Also, the cell line’s use as the biological component of an artificial liver device (ALD) is markedly advanced because ideally the living component of an ALD should be fully defined, in and of itself, and in its culture components.  For agricultural purposes, the feeder-independent culture of the PICM-19 cells will simplify transfection of the cells for the in vitro analysis of transgene constructs and in the cells’ use as the “nucleus donor” in the somatic cell cloning technique, i.e., animal cloning, the method whereby precise genetic changes to pigs might be made in the future.  The feeder-independent culture of the PICM-19 cells is the first step in our attempt to establish a fully defined culture system for the cells, i.e., one free of fetal bovine serum and feeder-cell-derived substances.  If achieved, this might enable the cells to be introduced into the in vivo environment of the pig without causing rapid inflammatory responses.  The cells might then be used as a reagent for “cellular augmentation”, where PICM-19 cells, genetically engineered to produce growth stimulating substances and vaccine components, would be placed into newborn pigs in order to enhance their growth, disease resistance, and well-being.

Neil C. Talbot, LeAnn Blomberg, Wesley M. Garrett, and Thomas J. Caperna. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line.  In Vitro Cellular and Developmental Biology-Animal, 46:746-57, 2010.

Julien Serret, Kheira Errouane, Mohammad Salma, Nathalie Maréchal, Philippe Chatelet, Zvjezdana Markovic, Stéphane Dussert, André Peyrière, Thierry Joet, Florent Engelmann, Isabelle Sylvestre, Left box: Emmanuel Couturon (La Réunion). Right box: Philippe Marmey (New Caledonia).

Use of biotechnologies for the conservation of plant biodiversity

The majority of the Dessitrop team is based in IRD Montpellier, France but staff are also outposted in the tropics, currently in Réunion Island and New Caledonia. The main research topic of the team concerns the study of mechanisms of desiccation tolerance of plant tissues and organs, mostly from tropical and Mediterranean species. This study is carried out through three principal themes: the research for molecular determinants of seed desiccation tolerance; the ecology of seed desiccation tolerance in disrupted tropical ecosystems; and the acquisition of desiccation tolerance of in vitro cultured tissues and organs for their cryopreservation. In vitro techniques are very useful for conserving plant biodiversity, including genetic resources of recalcitrant seed and vegetatively propagated species; rare and endangered plant species; and biotechnology products. Medium-term conservation is achieved by reducing growth of plant material, thus increasing intervals between subcultures. For long-term conservation, cryopreservation (liquid nitrogen, -196^oC) allows storing plant material without modification or alteration for extended periods, protected from contaminations and with limited maintenance. Cryopreservation is well advanced for vegetatively propagated species. Research is much less advanced for recalcitrant species. However, various technical approaches should be explored to develop cryopreservation protocols for a larger number of recalcitrant seed species. A range of analytical techniques, which allow understanding physical and biological processes taking place in explants during cryopreservation, can be efficiently used to assist in the development of cryopreservation protocols.

Florent Engelmann. Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cellular and Developmental Biology-Plant, 47: 5-16, 2011.

Biotechnological efforts for preserving and enhancing hardwood forest tree biodiversity, health, and productivity

The research focus of the Pijut laboratory at the USDA Forest Service, Northern Research Station, Hardwood Tree Improvement and Regeneration Center located at Purdue University is on plant cell, tissue, and organ culture, genetic modification, and vegetative propagation of hardwood tree species directed toward germplasm improvement. We are currently working on black walnut, black cherry, poplar, green, white, black, and pumpkin ash, and several tropical tree species. In these studies we are developing adventitious shoot regeneration, micropropagation, rooting, genetic transformation, flowering control, pest resistance, and clonal propagation protocols for tree improvement and conservation efforts. Hardwood tree species are important ecological and economical biological resources. Preserving forest tree biodiversity through the use of biotechnological approaches is integral to any forest tree improvement program. This review focuses on biotechnological tools available for conserving, characterizing, evaluating, and enhancing hardwood forest tree biodiversity. We focused mainly on species grown for lumber and wood products. We also present a brief summary of important non-wood forest products from temperate hardwood tree species, and a few case studies for preserving forest tree biodiversity.

Paula M. Pijut, Shaneka Lawson, and Charles H. Michler. Biotechnological efforts for preserving and enhancing temperate hardwood tree biodiversity, health, and productivity. In Vitro Cellular & Developmental Biology-Plant 47: 123-147, 2011.

Photograph of several authors of the manuscript (from left to right): Dr Shane Turner, Dr Eric Bunn, Dr Anja Kaczmarczyk holding a tissue culture jar, and Dr Kingsley Dixon. The picture is taken in the tissue culture lab of the Biodiversity Conservation Centre at Kings Park and Botanic Garden, Perth, Western Australia.

The second photograph is Dr Ricardo Mancera (Curtin University of Technology, Perth WA 6845, Australia) the fifth member of the research team.

Cryopreservation of threatened native Australian species - what have we learned and where to from here?

Australia and especially the southwest of Western Australia comprise a very high diversity of plant species including many endemics. As a consequence of development of lands for agriculture, mining and infrastructure, many unique plant species are now threatened or extremely rare and need urgent conservation actions. Cryopreservation entails storage at ultra low temperature (-196°C) in liquid nitrogen and is considered the best ex situ long-term conservation method for plant species that produce few or no seeds and which have to be maintained vegetatively. Depending on the plant species and explants used, different cryopreservation protocols have been successfully applied such as two-step cooling, encapsulation/dehydration, vitrification and droplet vitrification. Some native Australian species have been cryopreserved successfully using such protocols. However, not all species respond to existing cryopreservation approaches. For taxa that are not amenable to current techniques new ways of developing and optimising cryogenic storage need to be investigated. Part of our research entails comparison of biochemical components such as sugars, proteins and cell membrane composition between cryosensitive and cryotolerant species to try and explain why one species can be more easily cryopreserved than another. An additional aspect that requires consideration is the integrity of cellular membranes during cryopreservation, as membrane damage will cause harm or even destroy plant cells. Further research is necessary on cell membranes and how they interact and change when cooled to cryogenic temperatures in the presence of various cryoprotective agents. To develop these ideas our newest research project seeks to integrate experimental cryo-procedures in the laboratory with membrane modelling and biophysical paradigms to inform development of new cryopreservation protocols and to assess the robustness of theoretical models in predicting optimum cryogenic conditions.

Anja Kaczmarczyk, Shane R. Turner, Eric Bunn, Ricardo Mancera, and Kingsely W. Dixon, Cryopreservation of threatened native Australian species - what have we learned and where to from here? In Vitro Cellular & Developmental Biology-Plant, 47:17-25, 2011.