Lab Members (left to right): Helen O’Neill, Janice Abbey, Rebecca Hinton, Jonathan Tan, Pravin Periasamy, Kristin Griffiths, Genevieve Despars (Insert)

Heterogeneity Amongst Splenic Stromal Cell Lines Which Support Dendritic Cell Hematopoiesis

The hematopoiesis of dendritic cells (DC) supported by splenic stroma has been studied in this laboratory since the early 90s. As a PhD student in the lab, Keping Ni first developed a culture system which continuously produced cells with DC characteristics. This finding occurred well before we knew markers to distinguish this cell type, and before specific antibodies became available. Over time, the maintenance of progenitors within the stromal matrix was evident. A number of studies then showed the production of immature myeloid DC by a number of approaches, including cell surface marker analysis, immune function, antigen presenting capacity, and later gene expression profiling. The importance of stromal cells in DC hematopoiesis was also proven. Studies on stromal components were only possible after Keping Ni developed the STX3 stromal cell line from one spleen long-term culture which had by chance lost hematopoietic cells over time and cell passage. STX3 has since been used as a valuable tool in studies on the stromal cell components which influence DC hematopoiesis. For example, overlaid bone marrow cells produce DC with 14 days of coculture. These DC resemble those produced in long-term spleen cultures, with production of no other known hematopoietic cell type. STX3 is, however, a heterogeneous stromal line, and so studies were limited by the interaction effects of several different cell types. This paper now describes the results of cloning STX3, and the range of different cell types which have been isolated. Over 100 cloned lines were derived which grew continuously and maintained a consistent morphological appearance varying from cobblestone, to elongated spindle-shaped cells. Some clones also maintained mixed morphology. A subset of 13 selected representative lines was studied further. Despite morphological differences, all 13 lines showed expression of the endothelial cell genes ACVRL1/ALK1, COL18A1 and MCAM. Despite a common endothelial origin, the cloned lines however, were found to vary in hematopoietic support capacity for DC development from overlaid bone marrow. Some lines were identified as supporters and some as non-supporters. Some supporters showed development of cells in foci with release of DC into supernatant, while others showed less foci development, but nevertheless release of DC into supernatant. Of further interest was the finding that support capacity did not correlate with morphology. This paper therefore reports the development of a battery of cloned stromal lines which differ in morphology, and hematopoietic support function for DC, all of which reflect cells of the endothelial lineage. This study highlights the importance of vascular niches in hematopoiesis particularly in the context of the spleen microenvironment. It also emphasises the need to study the development and heterogeneity of cells of the endothelial lineage. Future work will embrace the se issues as well as identification of the progenitors which give rise to DC under stromal cell support.

Genevieve Despars and Helen O’Neill. Heterogeneity Amongst Splenic Stromal Cell Lines Which Support Dendritic Cell Hematopoiesis, In Vitro Cellular & Developmental Biology-Animal 42:208-215, 2006.

Hailu Aynalem

A Comparison of Visual and Image Analysis for the Storage of Micropropagated Plants

The USDA-ARS National Clonal Germplasm Repository (NCGR) stores national collections of fruit, nut and specialty crops. The primary collections are maintained as growing plants fields or greenhouses. These plants could be lost from environmental stresses and insect or diseases. As a secondary back up a subset of the field collection was established as virus-free tissue cultures stored at refrigerator temperatures. Survival of these cultures in storage can vary from 6 months to 5 years depending on the plant type. Stored plants are evaluated at 4 month intervals to determine their health status. In this study we tested four cultivated pears to determine if computer anlysis of digital photographs would be more effective for determining health status than a direct visual evaluation. Correlations between digital and visual tests do not provide a definitive evaluation technique, they will assist in the development of digital imaging as an alternative technique for evaluation of stored tissue culture plantlets.

Hailu M. Aynalem, Timothy L. Righetti, Barbara M. Reed. Nondestructive Evaluation of In Vitro-Stored Plants: A Comparison of Visual and Image Analysis, In Vitro Cellular & Developmental Biology – Plant 42:561-566, 2006.