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A History of WEHI Cytometry

by Dr. Frank Battye

The idea of introducing flow cytometry to the Walter and Eliza Hall Institute of Medical Research (WEHI) probably came out of visits of WEHI scientists to Stanford University and contact with the Herzenberg labs where, in the 1970s, FACS had been driven into the commercial mainstream. In particular, this idea was supported by the then director, Sir Gus Nossal, together with Ken Shortman, then head of the Biochemistry & Biophysics Unit, and Noel Warner, who was eventually to take up a senior role with Becton Dickinson. So, early in 1977 a FACS II was acquired. This was not only WEHI’s first cell sorter but also Australia’s first and indeed the first cell sorter to operate in the southern hemisphere. At the same time, Frank Battye was recruited to supervise FACS operations, having been enticed from the field of solid state physics. While the FACS II was not “high-tech” by today’s standards, it is interesting that biomedical scientists of the day saw it as being so much more technically sophisticated than their current research tools, that they were ready to look further afield for a supervisor with a background in electronic instrumentation.

The dynamic nature of the institute and of its early FACS proponents saw the technique so fervently embraced that researchers’ requirements soon moved ahead of instrument capability. This led to a series of in-house instrument modifications that were to keep the FACS II abreast of current technology for nearly two decades and in use for 25 years. The initial 2 parameter sorting capability was extended stepwise to 6 parameters with elapsed time. Fluorescence compensation was added, then a second laser and a dye laser. Thus began a program of application-driven instrument development that was to be applied to cell sorters subsequently acquired. It was the synergistic combination of this technological development with the widespread adoption of flow cytometry by all of WEHI’s research units that led to important contributions in wide ranging fields of biomedical science.

From the first, the Nossal group employed FACS in their B-cell studies, very soon coming to a confirmation of “clonal anergy” as a mechanism for B-cell tolerance [1]. Once single cell sorting became routine it enabled molecular characterisation of memory B-cells [2]. These cells were identified by 6-parameter flow cytometry and sorted singly, direct into culture, thus assuring the clonal origin of progeny arising from their proliferation. Interestingly, Paul Lalor, who participated in that study, had come to WEHI from the Herzenberg labs and Michael McHeyser-Williams was one of several to go from WEHI to a post-doctoral position in the Herzenberg labs. The B-cell research is continued by David Tarlinton, an ex-Herzenberg PhD student.

At the same time, the Shortman unit delved into the mapping of T-cell differentiation in the thymus, firstly with Roland Scollay and then with Li Wu who was later to isolate the earliest T-precursor population (and the first example of a lymphoid-restricted precursor) [3]. The Stanford connection was reiterated by the participation in this and parallel work on the isolation of hematopoietic stem cells and precursor populations by Gerry Spangrude, who came to the group from the Weissman labs. The group’s emphasis then shifted to dendritic cells (DCs), which led to the finding that DCs can be produced from lymphoid restricted precursors [4]. More recent interest of the group has been in the separation of functionally distinct DC subpopulations.

By 1978, Russell Howard had brought the parasitologists into the field with the demonstration that the stained DNA of malaria parasites could be detected from within infected red blood cells [5]. Notably, this work was supported by earlier consultations with Mac Fulwyler, an example of the tradition of sharing of expertise by the leaders in flow cytometry development that continues still.

Frank Battye operating the WEHI FACS II circa 1981

Another early FACS adopter was Nick Nicola, then of Don Metcalf’s Cancer Research Unit, now WEHI Assistant Director and head of the Cancer and Hematology Division. He had remarkable success in identifying and isolating hematopoietic progenitor and stem cells [6, 7]. At that time, before the advent of monoclonal antibodies against surface receptors characteristic of hematopoietic cells, his method relied on detecting the patterns of binding to the cells of fluorescinated lectins [8]. The ability to isolate these primitive cell populations also advanced work of the day on the colony stimulating factors (CSFs) by presenting enriched populations of target cells for testing the effects of the CSFs, which could only be produced in very small quantities. Another contribution to the CSF work was a study of mature cell activation by the CSFs in which flow cytometry not only identified the target cells but also quantitated their response [9]. The search for stem cells was also extended to the rat hematopoietic system by the visiting Irv Goldschneider [10]. It is an interesting reflection on the early years of the cytometry facility that George Kannourakis’ demonstration that sorting of single hematopoietic cells did not affect their pattern of differentiation [11] was performed on a FACS II cell sorter, with modified electronics, on which a culture plate was positioned from well to well by hand for the sorting of each individual cell.

Wally Langdon may have been the first of the molecular biologists in the Adams / Cory unit to use flow cytometry and he did so in analysis of cancer development in their lymphoma-prone myc transgenic mice, developed with Alan Harris [12]. Flow cytometry and sorting was to be used subsequently by many of that group over the intervening years in analyzing other cancer-prone mouse models and for unraveling the complex story of the bcl 2 protein family in regulating apoptosis, particularly David Vaux [13], Andreas Strasser [14, 15], Philippe Bouillet [16] and colleagues.

Continuing the theme of stem cell separation was Perry Bartlett’s neuro-immunology group that set out to isolate a neural stem cell (NSC). Although traditionally considered a static organ incapable of cell genesis, it is now clear that the adult mammalian central nervous system contains a population of endogenous NSCs and maintains homeostasis (and brain function) by continually replacing lost populations of neurons both under normal conditions and, in specific instances, following injury. However, their relative rarity and absence of definitive markers made NSCs an elusive target. Taking their cue from the earlier hematopoietic stem cell work, the group investigated the pattern of binding to brain cells of a lectin, in this case peanut agglutinin, in combination with an array of other markers. Rod Rietze and colleagues were eventually successful in identifying and sorting a population of NSCs from adult mouse brain [17].

Yet another landmark finding was made quite recently by Mark Shackleton and Francois Vaillant and colleagues in the Lindeman / Visvader breast cancer research group with the discovery of a basally-located mouse mammary cell population that is highly enriched in mammary stem cells (MaSCs) [18]. They showed that highly purified sorted single cells from this population have the ability to engraft de-epithelialized mammary glands. This has led to further discoveries on the molecular regulators of MaSC self renewal, the lack of expression of estrogen and progesterone receptors on MaSCs and the identification of mammary cell differerentiation markers. Broadly, these studies showed that the hematopoietic model of organ development from single stem cells to functional, differentiated progeny via intermediate, lineage-restricted progenitors is applicable to the mammary gland – and therefore likely other solid organ systems. It also demonstrated the usefulness of applying FACS cell separation techniques to studies of solid organ biology.

Frank Battye and Ken Shortman at the retirement of the WEHI FACS II after 25 years service, June 2002

Today the cytometry lab houses 5 cell sorters, a confocal microscope and 7 flow cytometry analysers, including the most advanced instruments of the day, and is staffed by a team of seven. Our main contribution to the field of cytometry itself is now through development of specialist computer software. This practice had its beginnings in 1978 with the addition to our cell sorter of a computer data acquisition system to replace the supplied data capture system (a Polaroid camera for photographing the oscilloscope screen). The cytometry community’s generous help and advice was available at that time from groups at NIH (Bob Romanoff) and Los Alamos (Jim Leary, Gary Salzman). Elements of the software that was developed then have migrated through half a dozen computer architectures and operating systems to manifest now as part of the “WEASEL” data analysis and display program that, along with a number of smaller utility programs, is distributed to the international community [19].

The contributions of greater importance have been the large body of work that has been enabled at WEHI by flow cytometry and cell sorting and the large number of scientists, both junior and senior, who have become cytometry-literate during their stay at WEHI and have carried away with them that important additional tool to enhance their subsequent work.

Illustrative References

  1. Clonal anergy: persistence in tolerant mice of antigen-binding B lymphocytes incapable of responding to antigen or mitogen. Nossal GJV and Pike BL. Proc Natl Acad Sci USA 1980 77: 1602-1606.
  2. Molecular characterization of single memory B cells. McHeyser-Williams MG, Nossal GJ Lalor PA. Nature 1991 350: 502.
  3. CD4 expressed on earliest T-lineage precursor cells in the adult murine thymus. Wu L, Scollay R, Egerton M, Pearse M, Spangrude GJ, Shortman K. Nature 1991 349 (6304): 71-4.
  4. Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Ardavin C, Wu L, Li CL, Shortman K. Nature 1993 362 (6422): 761-3
  5. Plasmodium-infected blood cells analyzed and sorted by flow fluorimetry with the deoxyribonucleic acid binding dye 33258 Hoechst. Howard RJ, Battye FL, Mitchell GF. J Histochem Cytochem. 1979 27 (4): 803-13.
  6. Isolation of murine fetal hematopoietic progenitor cells and selective fractionation of various erythroid precursors. Nicola NA, Metcalf D, Von Melchner H, Burgess AW. Blood 1981 58 (2): 376-386.
  7. The production of committed hematopoietic colony-forming cells from multipotential precursor cells-in vitro. Nicola NA, Johnson GR. Blood 1982 60 (4): 1019-1029.
  8. Differential expression of lectin receptors during hematopoietic differentiation – enrichment for granulocyte-macrophage progenitor cells. Nicola NA, Burgess AW, Staber FG, Johnson GR, Metcalf D, Battye FL. Journal of Cellular Physiology 1980 103 (2): 217-237.
  9. Eosinophil activation by Colony-Stimulating Factor in man – Metabolic effects and analysis by flow-cytometry. Vadas MA, Varigos G, Nicola N, Pincus S, Dessein A, Metcalf D, Battye FL. Blood 1983 61 (6): 1232-1241.
  10. Analysis of rat hematopoietic-cells on the fluorescence-activated cell sorter. 1. Isolation of pluripotent hematopoietic stem-cells and granulocyte-macrophage progenitor cells. Goldschneider I, Metcalf D, Battye F, Mandel T. Journal Of Experimental Medicine 1980 152 (2): 419-437.
  11. Clonal proliferation in vitro of individual murine and human hemopoietic cells after fluorescence-activated cell sorting. Kannourakis G, Johnson GR, Battye F. Experimental Hematology 1988 16 (5): 367-70.
  12. The c-myc oncogene perturbs B lymphocyte development in E-mu-myc transgenic mice. Langdon WY, Harris AW, Cory S, Adams JM. Cell 1986 47 (1): 11 8.
  13. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Vaux DL, Cory S, Adams JM. Nature 1988 335 (6189): 440-2.
  14. Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2. Strasser A, Harris AW, Bath ML, Cory S. Nature 1990 348 (6299): 331-3.
  15. bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Strasser A, Harris AW, Cory S. Cell 1991 67 (5): 889-99.
  16. BH3-only Bcl-2 family member Bim is required for apoptosis of autoreactive thymocytes. Bouillet P, Purton JF, Godfrey DI, Zhang LC, Coultas L, Puthalakath H, Pellegrini M, Cory S, Adams JM, Strasser A. Nature 2002 415 (6874): 922-6.
  17. Purification of a pluripotent neural stem cell from the adult mouse brain. Rietze RL, Valcanis H, Brooker GF, Thomas T, Voss AK, Bartlett PF. Nature 2001 412 (6848): 736-9.
  18. Generation of a functional mammary gland from a single stem cell. Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML, Wu L, Lindeman GJ, Visvader JE. Nature 2006 439 (7072): 84-8.
  19. http://www.wehi.edu.au/cytometry/freesoftware/index.html