TY - JOUR
AU - Nicola, Nicos A.
AB - Abstract A collection of tributes and remembrances from esteemed colleagues, mentees, and friends on the life and work of “the father of hematopoietic cytokines” Open in new tabDownload slide Tribute to Donald Metcalf February 26, 1929 - December 15, 2014 Open in new tabDownload slide Tribute to Donald Metcalf February 26, 1929 - December 15, 2014 The Editors of STEM CELLS and its publisher AlphaMed Press sincerely thank the contributors to this memorial tribute to Donald Metcalf. In the following pages, we celebrate his life, his work, and his legacy. It is fitting that the Journal recognize and honor Don, who co-founded the Journal 33 years ago. Tribute to Donald Metcalf 3401 “Summon up the Blood”… In Memoriam    Ann Murphy, Martin J. Murphy 3402 In His Own Words: Introduction to “Summon up the Blood: In Dogged Pursuit of the Blood Cell Regulators” by Donald Metcalf 3404 My Tribute to a Real Master Craftsman, Don Metcalf    C. Glenn Begley 3406 Donald Metcalf: A Disciplined and Devoted Medical Biologist    E. Richard Stanley 3408 A Perspective on the Legacy of Donald Metcalf from a Former Postdoctoral Fellow    Anna Rita Migliaccio 3412 Career Lessons Learned from a Giant: Don Metcalf    Kenneth Kaushansky 3414 Professor Donald Metcalf    Ashley P. Ng, Warren S.Alexander, Douglas J. Hilton, Nicos A. Nicola 3419 Profile: Reprinted from “Blood Lines: An Introduction to Characterizing Blood Diseases of the Post-Genomic Mouse” by Donald Metcalf 3420 Photograph Collection: Reprinted from “Polyfunctionality of Hemopoietic Regulators: The Metcalf Forum” 3401 “Summon up the Blood”… In Memoriam    Ann Murphy, Martin J. Murphy 3402 In His Own Words: Introduction to “Summon up the Blood: In Dogged Pursuit of the Blood Cell Regulators” by Donald Metcalf 3404 My Tribute to a Real Master Craftsman, Don Metcalf    C. Glenn Begley 3406 Donald Metcalf: A Disciplined and Devoted Medical Biologist    E. Richard Stanley 3408 A Perspective on the Legacy of Donald Metcalf from a Former Postdoctoral Fellow    Anna Rita Migliaccio 3412 Career Lessons Learned from a Giant: Don Metcalf    Kenneth Kaushansky 3414 Professor Donald Metcalf    Ashley P. Ng, Warren S.Alexander, Douglas J. Hilton, Nicos A. Nicola 3419 Profile: Reprinted from “Blood Lines: An Introduction to Characterizing Blood Diseases of the Post-Genomic Mouse” by Donald Metcalf 3420 Photograph Collection: Reprinted from “Polyfunctionality of Hemopoietic Regulators: The Metcalf Forum” Open in new tab Open in new tabDownload slide Professor Donald Metcalf AC, BSc(Med) Sydney, MD Sydney, FRACP, HonDSc Sydney, HonMD Oslo, HonFRCPath London, FRCPA, FAA, FRS Research Professor of Cancer Biology, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia Open in new tabDownload slide Professor Donald Metcalf AC, BSc(Med) Sydney, MD Sydney, FRACP, HonDSc Sydney, HonMD Oslo, HonFRCPath London, FRCPA, FAA, FRS Research Professor of Cancer Biology, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia “Summon up the Blood”… In Memoriam Open in new tabDownload slide ANN MURPHY, PHD Managing Editor STEM CELLS Open in new tabDownload slide ANN MURPHY, PHD Managing Editor STEM CELLS Open in new tabDownload slide MARTIN J. MURPHY, DMEDSC, PHD, FASCO Executive Editor STEM CELLS Open in new tabDownload slide MARTIN J. MURPHY, DMEDSC, PHD, FASCO Executive Editor STEM CELLS Little need be written to introduce this tribute to our dear friend, colleague, and mentor, Don Metcalf. Don not only opened the era of cytokines to the world, he continued on its leading edge, designing-and personally executing-innovative experiments, always asking probing and challenging questions whilst inspiring and training generations of new scientists. Throughout his life, Don had that special blend of enthusiasm for exciting ideas and the perseverance to convert them into life-lengthening hope for patients with catastrophic disease, and he instilled these values into the lives of those blessed to be within his orbit. Those of us honored by his friendship were doubly blessed. We treasure our memories of Don as a co-founder and sustainer of the Journal STEM CELLS. We also treasure our memories of serving as the editors of his autobiography, Summon up the Blood: In dogged pursuit of the blood cell regulators published by AlphaMed Press in 2000, and of another opus, Blood Lines: An Introduction to Characterizing Blood Diseases of the Post-Genomic Mouse, published in 2004. To his beloved and devoted Josephine (Jo), his wife and life-long partner who forever supported him, to his daughters and grandchildren, we dedicate this tribute to Don, whose life truly exemplified the words of Goethe: In the realm of ideas, everything rests on enthusiasm; In the real world, all rests on perseverance. As Don often reminded us in prosecuting science with carefully executed experiments, and for all things of value… “Summon up the Blood”! Open in new tabDownload slide Open in new tabDownload slide Don's editors Open in new tabDownload slide Open in new tabDownload slide In His Own Words The following excerpt is the Introduction to “Summon up the Blood: In Dogged Pursuit of the Blood Cell Regulators” by Donald Metcalf, published in 2000. Open in new tabDownload slide Open in new tabDownload slide The 35-year saga of the discovery, development, and medical application of the blood cell regulators, the colony-stimulating factors (CSFs), has all the elements needed for an absorbing story. There was a cast of characters who shared between them a full range of human foibles, the gradual opening of an exciting new field of medical science, distrust between opposing groups, the tedium of endless purifications, the excitement of gene cloning and expression, the tensions of animal then clinical trials, opportunities lost, battles over patent rights, the aggression or indolence of pharmaceutical companies, the frustrations of licensing procedures, and always the need to push techniques and ideas beyond existing limits. Despite repeated requests, I had been reluctant to attempt to write a semipopular account of the CSFs, suspecting correctly that this might prove to be quite difficult. However, those of us who were involved from the early phases have been chagrined to realize that we have already begun to forget key elements of the story. There seemed no other option therefore than to set down as many of the details as could be recalled before too much else was forgotten. As most of the participants in the saga are still alive and remain on friendly, if sometimes wary, terms, I certainly had no inclination to write a warts-and-all exposé. I do have my own views on certain episodes where tensions arose but I am not well placed to describe how others felt or acted at these times. This book was not intended to be an ordered and fully referenced scientific account of the CSFs. Such an account has already been written by Nick Nicola and me. Rather, I wished to give a chronological description of how order slowly emerged out of confusion in this field, and to attempt this without becoming so technical that a nonexpert reader would become discouraged. My attempt to write a blow-by-blow account soon ran into difficulties. It was easy enough to write a personal account of the early days of the project when only three or four workers were involved. This became much more difficult when there were a hundred or so in various groups working in the field. It clearly became impossible to introduce any personal touches to the account when there probably were thousands involved in various aspects of the work. By default, the account became progressively transformed to a skewed description of the activities of a single research group. This then became in danger of implying that we were the only serious contributors to the field. I have tried to avoid this distortion by inserting at various stages the important contributions of others buy may not have been wholly successful in this attempt. It became increasingly more difficult to describe, in the form of a single account, a story that became composed of quite separate issues and projects, proceeding simultaneously. In the latter part of the book, this problem forced a divergence from a single chronological account to separate descriptions of four major streams of work that were really proceeding simultaneously. Try as I could, the account could not be restricted to the CSFs. Other hematopoietic regulators were being discovered as the work on the CSFs progressed and, because these often interact with the CSFs, they needed to be introduced from time to time. No account of the development of the CSFs was able to be given without some reference to the target hematopoietic cells on which they act, because the characterization of these cells was making major advances throughout the period of work on the CSFs. So, from time to time, there were necessary asides in the account to fill the reader in on these simultaneous developments. Work on the CSFs did not cease with their clinical development. Much further crucial information on these regulators needed to be discovered and indeed these studies continue today. The latter part of the account therefore is a description of what we and others felt impelled to do in the postclinical era. While most of the salient features of the CSF story have at least been mentioned, the account has ended up neither fish nor fowl. It is heavily autobiographical but without many of the expected features of an autobiography. I have tried to portray laboratory life as it happens for a group tackling complex and prolonged projects and I hope this gives some insights into how findings slowly emerge. The description omits dramas, personal frictions, and periods of angst that of course occurred and possibly can be read between the lines. What has resulted may well be a rather dull account of a subject that could have made a sustaining page-turner. It is interesting, in the development and expansion of a subject, how the intensely personal excitements for the few in the initial days do transform to a somewhat shapeless pattern as the field expands: advances go on, but in a manner that progressively appears to lose its personal aspects. For each individual this may never actually be the case, but as more and more investigators enter a field it becomes impossible to follow in detail their individual hopes and actions. I have ended up with an account that is little more than a history of a single research unit over the past 35 years. This may interest those who know the field and perhaps not many others. If this is the case, so be it. If my account does not seem to agree with accounts of the CSFs that often appear in more recent review publications, then I suggest that the readers go back to the scientific literature and establish exactly who did what and when. This may find some surprises. Donald Metcalf Melbourne, 2000 Once more unto the breach, dear friends, once more; Or close the wall up with our English dead! In peace there's nothing so becomes a man As modest stillness and humility; But when the blast of war blows in our ears, Then imitate the action of the tiger: Stiffen the sinews, summon up the blood, Disguise fair nature with hard-favour'd rage; Then lend the eye a terrible aspect. William Shakespeare King Henry V Actlll,Scene1 The first two lines of this excerpt were used by me on more than one occasion as an exhortation to my colleagues when things were going badly and spirits were sagging. Not that we were English, butwe were being sorely tried. Donald Metcalf My Tribute to a Real Master Craftsman, Don Metcalf Open in new tabDownload slide C. Glenn Begley, PhD TetraLogic Pharmaceuticals Corporation, Malvern, Pennsylvania, USA Open in new tabDownload slide C. Glenn Begley, PhD TetraLogic Pharmaceuticals Corporation, Malvern, Pennsylvania, USA How do I write a tribute to Don Metcalf? Many have talked about his outstanding scientific contribution and the direct impact he has had on the lives of millions of people, all of which is true. But for me, it is much more personal. Don took me on as his Ph.D. student—a raw clinician with no research training. I didn't know how to grow cells, I contaminated our cultures, I mistakenly used the centrifuge reserved for precious FACS sorts. However, for whatever reason, Don saw something, and as a result of working with him for 3 years, I was infected forever with the research bug. That was, in fact, the first lesson Don taught me: if someone is enthusiastic, determined, committed, then “give them a go.” At that time, I had no idea what a tremendous privilege had been afforded me. Even now, given the respect Don commanded, I am amazed and thankful that he provided me that opportunity. That is not to imply that Don wasn't demanding. He was. Very demanding. It should not be taken to mean that he didn't have high expectations. He did. He had very high expectations. But what he expected was for people to work hard, be thoughtful, work hard, be thorough, work hard, be careful, and to work hard. My second lesson began immediately upon arriving in the lab. That was one of humility. Science is humbling. We know much less than we think we know. Our models are naive and mostly wrong. We are not nearly as clever or as knowledgeable or as indispensable as we think. Despite his expansive knowledge and his breadth of experience, Don knew how humbling science could be. To help me learn this key lesson, Don had me start as the technician to his technicians, some of whom were a decade younger than me. Having just completed a successful clinical training program, this was an invaluable lesson. Don's technicians were extremely skilled. They were thorough and precise. From a technical perspective, just like Don, they were superb. Don chose them carefully and trained them well. Over 30 years later, I am acutely aware of the solid technical grounding that he provided. Open in new tabDownload slide Open in new tabDownload slide As a student, it was impossible not to notice that Don was always among the first, if not the first, into the lab every morning. That was a great time to catch him and talk. It was also impossible not to notice his extraordinary capacity for work. It was typical that at morning tea (a compulsory event that took place at 10:30 am and was never to be missed) with a sense of pride he would say, “I've already counted a thousand plates this morning; what have you done?” He could be extremely intimidating (although I suspect he probably never knew just how intimidating!).Toa new Ph.D. student, his constant example provided clear motivation. I was never able to count a thousand plates in a day, let alone before morning tea, but he had finely honed his craft. His work ethic was extraordinary. He would much rather spend time performing his own experiments than reading about someone else's. Over recent years I have increasingly realized the wisdom of his position. He eschewed the “top-tier” journals and the flashy science that they promoted. He was much more interested in being right than being first. These were very important lessons that, if anything, are true even more today than they were in the 1980s. In the same vein, when writing a paper, Don always cautioned against a long Discussion. He was clear that the results would stand the test of time: there was never any doubt in his mind as to how robust and reproducible the data should be. In contrast it was the Discussion that, in the fullness of time, would reveal the foolishness of our current thinking! Early in my research career, Don told me that mine was an apprenticeship and that his job was to teach me the trade. He told me many times that it was impossible for him to train more than two people at a time. And true to his word, he seldom if ever had more than two people for whom he was directly responsible at any time: one Ph.D. student and one postdoctoral fellow. Again, that is a lesson well remembered. He was an outstanding, remarkable teacher. It is instructive to realize that someone of his caliber and stature would take his teaching and training responsibilities so seriously. Science today is suffering because we have forgotten this crucial lesson. Don surrounded himself with people he liked, respected, and trusted. He believed in teams, and recruited people with the variety of expertise required to advance a project. He was always direct and expected others to be the same. He was open to challenge; in the 15 years I worked with him, I cannot remember a time when he shut someone down for challenging his data or its interpretation. But if someone did challenge him, they had to be able to stand up to the no-holds-barred intellectual tussle that would ensue. Equally, Don had no qualms about questioning anyone else's data. There was no greater compliment than Don returning a manuscript covered in red ink; it was clear he had gone through it line by line. The environment he nurtured was extremely rigorous. Debate, challenge, argument, and discussion were expected. There was no dogma that could not be overturned. But it was clear, above all else, that we were a team. Before members of the team showed data to the rest of the world, it would be debated, argued, tested, and corrected. It was a wonderful, healthy environment that fostered debate, criticism, and built confidence. It was never personal. As teammates, friends, and colleagues we owed to each other to be as harsh on the data as possible. We all “owned” the data, we all “owned” the presentation, and we all shared that responsibility. After that, any other presentation was a breeze! We also had a lot of fun together. It was not unusual that at the aforementioned morning tea, Don would sit there with tears running down his face as he laughed at a story or recounted the most recent irreverent episode of South Park from the night before. He was a wonderful colleague. A story that captures the Don I know occurred one day at morning tea. That day he had received a paper from Nature to review. It related to a project we had been working on for years, the cloning of G-CSF. He sat down to tell us that the day was off to a terrible start. He had opened his mail only to discover we had just been beaten (“scooped” was the word he used). When I asked, he said that he had already reviewed the paper and placed it in the immediate return mail. He had added a covering letter requesting that the paper be published as soon as possible. Truly remarkable! Too few scientists behave in that manner. When pressed, he said he was compelled to do so because now we were compromised: we were at an unfair advantage in having knowledge of the confidential information contained in that manuscript. What a superb example of scientific integrity. Don was my teacher, mentor, role model, colleague, and friend. Of all the things that are said about him, and all the contributions he made, the two lessons I value most highly are his scientific commitment and his scientific rigor. Don told me that science is an apprenticeship. It is true. I had the honour and privilege of learning from a real Master. Vale my Friend. Open in new tabDownload slide Open in new tabDownload slide Photographs are reproduced with permission from the Archives of The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia. Donald Metcalf: A Disciplined and Devoted Medical Biologist Open in new tabDownload slide E. Richard Stanley, PhD Albert Einstein College of Medicine, Bronx, New York, USA Open in new tabDownload slide E. Richard Stanley, PhD Albert Einstein College of Medicine, Bronx, New York, USA I was Don Metcalf s second Ph.D. student. As an undergraduate, I had completed my honors research year in biochemistry at the University of Western Australia. As Perth was quite isolated, I applied to work at the Walter and Eliza Hall Institute (WEHI) in Melbourne, which had just appointed the young and dynamic Gus Nossal to succeed the Nobel Prize-winning immunologist, Frank Macfarlane Burnet, as the new director. My application was accepted, and in early 1967, I was assigned to the Cancer Research Unit, on the recently added sixth floor of the Institute, under Don's mentorship. Don was unit head and deputy director of the Institute. My project was to characterize and purify the factor in mouse serum responsible for the formation of colonies of granulocytes and macrophages using the 7-day assay developed by Ray Bradley. At that time, Don's lab comprised Don's first Ph.D. student, Bill Robinson, from Colorado, two technicians, and Don's secretary. Don was away on sabbatical at the Roswell Park Cancer Institute in Buffalo, New York, but had arranged for me to collaborate with the head of the biochemistry unit, Gordon Ada, during his absence. Ray Bradley was just across the road. With Gordon's help, we were quickly able to establish that the mouse serum factor responsible for colony-stimulating activity was a protein or glycoprotein. However, I was painfully aware of the impossibility of purifying the factor from mouse serum. Ray had identified embryo cell conditioned medium, which was very active compared with serum, but it was when Bill and I discovered activity in human urine that purification seemed feasible. Upon Don's return from sabbatical, he at first seemed somewhat skeptical of the progress that had been made but, after a week, decided that I should receive technical assistance to move the project forward and even spent much of his time counting and staining colonies stimulated by the fractions from my purifications. Our first purification paper together was published in The Australian Journal of Experimental Biology and Medical Science in 1969. Named a Current Contents Citation Classic in 1981, it indicated that complete purification of the colony-stimulating factor (CSF) from human urine was possible and emphasized its possible role as a humoral regulator of hematopoiesis. The purification work and other studies progressed well during my Ph.D., and in 1969, just before my thesis was submitted, Don requested that I stay on as a postdoctoral fellow. I agreed with Don that we were in a good position to further establish our leadership in the CSF field and stayed on for 2 more years. By the time I left to set up my own laboratory in mid-1972, we had, by using 200-liter batches of preselected human urine, essentially purified CSF (CSF-1 or M-CSF), showing that it was a glycoprotein and less than 1/100,000 of the total urinary protein. In addition, several other studies were completed that benefitted future work in both of our laboratories. For example, tissue expression and fractionation studies by John Sheridan and myself, together with Don's colony counts and stained colony analyses, indicated the existence of a different type of CSF, which was subsequently named granulocyte-macrophage CSF (GM-CSF). During my time in his laboratory, Don extensively exploited the potential of the new agar culture technique, using it to assay colony-stimulating activity in mouse and human leukemic, polycythemic, and anemic sera, as well as the sera of germfree, antigen-stimulated, irradiated, or phlebotomized mice, and to assess the effect of lymphoid and myeloid cells on colony formation. He studied the in vitro development of colonies in detail and the effect of agents, such as cortisone and antigen, on both circulating CSF and hematopoietic organ colony-forming cells. With Malcolm Moore, he investigated the development of the colony-forming cells of the hematopoietic system and, via cell separation, their relationship to more primitive cells. As significant amounts of partially purified CSF-1 became available, he examined CSF half-life and hematologic effects following its administration to mice. Characteristically, Don did not direct this work remotely, but was intimately involved in its execution. He considered it a privilege to work in the laboratory as a scientist. Don spent long days at the bench or microscope, and he routinely worked on Saturday mornings. Although Don could be intimidating at times, I was a particularly independent-minded young man and not as intimidated as most. Hence, we did have differences in opinion on publications and collaborations that were painful for both of us to discuss. However, despite our differences, I appreciated his perspective of the field, his curiosity, his detailed colony analyses, and his disciplined approach. I also realized very early on that Don really appreciated my work. He encouraged me to make a long seminar tour of the United States and to attend important meetings in Europe that gave me a global perspective of the work going on in my area of interest. Furthermore, as my mentor, Don demonstrated commitment, persistence, hard work, and a focus on biological relevance that were all important lessons for me. Don suffered from major back problems, having at least one back surgery performed while I was there. Despite this, I remember him sitting bolt upright in a straight-backed chair, all day, counting colonies and enumerating cells in picked colonies (Fig. 1). He claimed the straight-backed chair was the only way to cope with the back problem. For me, a glowing example of how Don's hard work and persistence paid off was his identification of pure granulocyte colonies that led to the discovery of granulocyte CSF (G-CSF). Another area of his mentorship by example was how effectively he used his time in the laboratory, limiting distracting outside interactions and writing productively, with great self-discipline, at work and at home. In addition, he spent a considerable proportion of his time working in the laboratory. Don, as a role model, demonstrated the importance of commitment to focused, hard work in the life of the successful professional scientist. When Don was not working, he could relax. He would routinely take lunch with colleagues in the tearoom, as well as the morning and afternoon teas that were really institutions in themselves. Don was a lively participant, appreciating and contributing humorous anecdotes. I also have fond memories of dinners at his home with his wife, Jo, and their kids. He enjoyed parties, holding a memorable one at his place and attending at least one of ours. I enjoyed his extremely dry sense of humor and sarcasm. This was a great foil to the enthusiasm and exuberance of Gus Nossal in seminars, where Don would often ask Gus to call a spade a spade. However, this obviously reflected a good working relationship, because Don remained deputy director of the Institute during Gus' entire tenure as director. Open in new tabDownload slide Fig.1. Left: Don counting colonies. Right: A welcome interruption from counting colony cells (circa 1968). Open in new tabDownload slide Fig.1. Left: Don counting colonies. Right: A welcome interruption from counting colony cells (circa 1968). When I left the laboratory for a faculty position at the University of Toronto as a member of the Ontario Cancer Institute, Don informed me that from now on he regarded me as competition! In spite of this, on a return visit, after I had presented a seminar at WEHI, he was unrestrained in appreciating the work I had presented. Furthermore, he supported me as I developed my career in the Northern Hemisphere. When I heard that he was not well, I wrote to him and thanked him for his valuable mentorship during my time in his laboratory. I also mentioned our disagreements. The last paragraph of his handwritten reply was, “It has been great having you as a colleague. We certainly did not agree on too many things, but did on the ones that matter.” I guess that sums up our relationship nicely—like that of father and rebellious son! Don had an enormous impact on the field of blood cell regulators. I was fortunate enough to significantly contribute to it with him from the beginning. He is sorely missed. A Perspective on the Legacy of Donald Metcalf from a Former Postdoctoral Fellow Open in new tabDownload slide Anna Rita Migliaccio, PhD Department of Medicine, Ichan School of Medicine at Mount Sinai, New York, NY, USA; Histology and Embriol-ogy, Department of DIBIDEM, Alma Mater University, Bologna, Italy Open in new tabDownload slide Anna Rita Migliaccio, PhD Department of Medicine, Ichan School of Medicine at Mount Sinai, New York, NY, USA; Histology and Embriol-ogy, Department of DIBIDEM, Alma Mater University, Bologna, Italy It is impossible for fellows training in experimental hematology today, who are used to all the sophistications (single cell transplants, chimeric recombinant growth factors, full genome sequencing, direct reprogramming, etc.), to even imagine what it meant to be a fellow in the Seventies. There was limited instrumentation (mainly optical and, for a few lucky fellows, electron microscopes). Few animal and human experimental models were available. Most of the knowledge in humans was provided by the medical consequences of either volunteer (bombing of Hiroshima and Nagasaki, Japan, August 1945) or accidental (damage to the nuclear reactor at the Vinča Research Institute for Physical Science, Yugoslavia, October 15, 1958) atomic explosions in inhabited areas. It was during this revolutionary and unfamiliar time that approximately 20 “giants” invented experimental hematology as we know it today. With only the anatomy of hematopoietic cell compartments as their guide, these men and women embarked on a crusade to decipher the mysterious process of hematopoiesis. Together, they devised both the constructive hypothesis and the tools necessary to decipher it. Decoding the erythroid lineage was facilitated by the agricultural practice of transhumance, the seasonal migration of shepherds and their livestock from pastures on the plain to those at high altitude. The low oxygen at high altitudes increases the red cell mass of both shepherds and sheep. Erythrocytosis is also expressed by patients suffering from a rare form of kidney tumor. Based on these observations, Dr. Eugene Goldwasser, a giant of these early days, provided physical evidence for the existence of erythropoietin, a hormone produced by the kidney and present at high levels in the plasma and urine of sheep and humans experiencing erythrocytosis [1]. By contrast, there was no natural or pathological experimental model that could provide clues on how the generation of myeloid cells is controlled and how alterations of this control may determine insurgency of leukemia. To this aim, James Till and Ernest McCulloch, two other giants of the time, took a top-to-bottom approach. Being atomic physicists by training, they generated a theoretically perfect model of hematopoiesis, positing that cells for the various myeloid lineages are generated by stochastic division and commitment of a hematopoietic-specific stem cell. In its formulation, this stochastic mechanism resembles the statistical fall of an electron toward the atomic nucleus. This model is supported by experimental approaches based on cell transplantation in sublethally irradiated animals [2]. Because these approaches rely on the supportive environment of the animals, they neither need growth factors nor are suited for growth factor identification. Donald Metcalf: A Disciplined Mind The mystery of the factors that control myelopoiesis and that may determine the cause of leukemia was Don's to solve. From the only pa per he published on erythroid cells [3]—one of the first indicating the kidneys as the site of erythropoietin production—he expressed the conviction that every hematopoietic lineage is controlled by a specific growth factor. Don tested this one lineage-one growth factor hypothesis with a bottom-up approach. He first defined a quantitative assay, the colony-forming assay, suited for characterization of myeloid-specific activities [4]. He then moved on to identify sources that may promote growth of myeloid colonies in vitro, such as the plasma from polycythemic patients, in the case of the erythroid lineage [5, 6]. During this period, Don's publications were systematic analyses of the ability of culture media conditioned by any possible organ stimulated in a variety of ways to sustain the growth of myeloid colonies in vitro. His intention was to identify the source most suited for identification, purification, and finally, cloning of the growth factors that would support the growth of cells for each myeloid lineage. Becausethese first growth factors were identified on the basis of colony assays, they were first defined as colony-stimulating activities, and then, once the protein was purified, as colony-stimulating factors (CSF). The name included an adjective (macrophagic, myeloid, granulo-monocytic, etc.) indicating the specific lineage promoted. Over the years, a sequence of graduate students and postdoctoral fellows tested this theory by identifying many hematopoietic growth factors. In some cases, Don's fellows came first (granulocyte-macrophage (GM)-CSF) [7]. In other cases, his students made their original discoveries under Don's supervision and then moved on to clone the factor in their own laboratory (macrophage colony-stimulating factor, M-CSF) [8, 9]. Or, they arrived either second (granulocyte colony-stimulating factor, G-CSF) [10] or never, and Don's contribution to the field was his commentary to the discovery (thrombopoietin,TPO) [11]. A group of his fellows were also the first to discover the leukemia inhibitory factor, while not a significant megakaryocytic-promoting factor, but is indispensable for mass culture of embryonic stem cells, a stem cell source with great potential for regenerative medicine [12, 13]. Establishing Strong Bonds with His Fellows Insights into the special relationships that Don established with his fellows are provided in each of the dedicated articles included in this special issue of Stem Cells. It amazes me how Don succeeded in establishing such strong bonds even with me, a hema-tologist in training in Europe, a continent far away from the land Down Under. The first time my path crossed Don's was when I was a postdoctoral fellow in the laboratory of Dr. Cesare Peschle in Naples, Italy. I was working on a project aimed to identify the “switching” factor that suppresses hemoglobin F production at birth. Because the concentration of prostaglandin E2 (PGE2) sharply increases during labor, we hypothesized that PGE2 might represent the switching factor we were looking for. The experimental data failed to prove this hypothesis but instead identified for the first time that PGE2 stimulates proliferation of human progenitor cells by inducing macrophages to establish cell-cell contacts that favor colony formation in vitro [14]. These experiments required high numbers of macrophages; therefore, I needed a “suitable conditioned media” to expand these cells in vitro. One of the most powerful conditioned media for the growth of macrophages had been developed by Don. The observation that macrophages are often engulfed by bacteria had suggested that these cells may represent the first line of defense against bacterial infection. Don hypothesized that endotoxin, the wall of gram-positive bacteria, may trigger the body's defense mechanism, which includes production of growth factors to stimulate the proliferation of macrophages. This idea was translated into the discovery that treatment with endotoxin stimulates production of growth factors, predominantly GM-CSF but also M-CSF, in many organs but especially in the lungs [15]. Because the lungs from endotoxin-treated mice release, in culture, high levels of these growth factors, the conditioned media from these cultures may be used to stimulate macrophage formation in vitro. I still remember the great respect I had for the clarity and simplicity of Don's method for preparing lung-conditioned media. I was capable of reproducing it at my first attempt. These were years during which, even without knowing Don's face, I was humbled by his productivity and by the numerous insights coming from his laboratory. My path crossed Don's for the second time in 1982 when I did a postdoctoral fellowship in radiobiology in the laboratory of Dr. Gerard Wagemaker at the TNO institute (Netherlands Organisation for Applied Scientific Research) in Rijswijk, The Netherlands. It was a near-miss. Don had spent a sabbatical in the same lab in 1981, attracted by Gerard's identification of the stem cell-activating factor (SAF), which induces hematopoietic stem cells into cycle [16]. This factor was later identified to be identical to multi-CSF (CSF for multiple lineages), a factor being studied by Don [17], and to IL-3 [18], a factor cloned thanks to the existence of the WEHI-3 cell line established in Don's laboratory [19]. Don is recorded in the TNO Annual Reports of 1980–1981 as “Visiting Scientist, Associate Director of the Walter and Eliza Hall Institute, Melbourne, Victoria, Australia.” Don left lasting memories in the labs and co-published a paper on the factors that affect granulocyte-macrophage lineage decisions [20]. After a lunch conversation, during which I learned that I had inherited his desk, I sent Don a letter (it was not yet the e-mail era). He not only answered me, but considered me a postdoctoral colleague. Over the years, he continued to follow my research and, in personal encounters, always had comments and feedback about my papers. Since then, our paths crossed many times at ASH and at other international meetings. Of all of them, I like to remember the very first scientific meeting to which I had been invited as plenary speaker. It was the Italy–United States workshop “Stem Cell Factor and Related Cytokines in Bone Marrow Congenital Dysplasias” organized in Cattolica, Italy, March 8–9, 1993, by Dr. Martin Murphy and Dr. Gian Paolo Bagnara (Fig. 1). I remember being excited not so much for the opportunity to talk as I was to be speaking at the same meeting where Don was also speaking. And then came my last interaction with Don. It was September 2014. I had e-mailed him asking for his collaboration on a project. Even in this most critical personal moment of his life, he replied in his customary considered and logical fashion. He e-mailed me back that he would not be able to help because he had just been diagnosed with advanced pancreatic cancer. What could I do other than wait? The news arrived in the middle of December of the same year, just after ASH. Open in new tabDownload slide Cover of the STEM CELLS supplement dedicated to the meeting in Cattolica, Italy, on March 8–9,1993, organized by Dr. Martin Murphy and Dr. Gian Paolo Bagnara [21]. The inset presents a photo of Donald Metcalf, his wife Josephine (Jo), and the Bagnaras (Gian Paolo and Laura) taken during a break of the meeting. A group photograph of the workshop participants is shown on page 11. Photos courtesy of Prof. Laura Bolsi Bagnara. Open in new tabDownload slide Cover of the STEM CELLS supplement dedicated to the meeting in Cattolica, Italy, on March 8–9,1993, organized by Dr. Martin Murphy and Dr. Gian Paolo Bagnara [21]. The inset presents a photo of Donald Metcalf, his wife Josephine (Jo), and the Bagnaras (Gian Paolo and Laura) taken during a break of the meeting. A group photograph of the workshop participants is shown on page 11. Photos courtesy of Prof. Laura Bolsi Bagnara. Pearls of Don's wit from his visit to the Bagnara laboratory in 1993 Visiting a tidy laboratory where a postdoctoral fellow (Dr. Pierluigi Strippoli, now Professor in his own right) was working hard, Don passed his finger on the bench to collect the dust and then, looking at his finger, commented, “Too clean!” Dr. GianPaolo Bagnara and his wife were discouraged by the recent rejection of their latest paper. Don commented, “Do you see this briefcase? It is full of papers of mine that have been rejected. There was a time when my papers were rejected because I was nobody. Presently, they are rejected because I am too famous.” Dr. Bagnara caught Don seated on a bench looking at graphs with high standard deviations. Don commented, “Everybody is afraid to show standard deviations so high, while this is reality. Cells do not behave always the same way.” Prof. Bagnara saw Don, at the age of 70, at the bench in the la boratory. Don commented, “I want to do my cultures.” He kept going until the very last. Don's Legacy Don was a great and generous mentor! In his tribute, Dr. Begley mentioned that Don never took more than two students at a time. However most, if not all, of Don's students did great under his supervision and opened new fields in their own right. In addition, after having stimulated the original discovery, Don always left the novel field to his postdoctoral students for further development while he moved on to tackle new scientific challenges with new fellows. Every 2 years, the scientific academia all over the world had one new “scientific creature,” formed by Don, to enjoy—some names: Bill Robinson, Nick Nicola, Richard Stanley, Ashley Dunn, Doug Jilton, and last but not least, Warren Alexander. The sense of loss and gratitude experienced by numerous investigators is demonstrated by the numerous obituaries published in his honor [22-25], including this special issue of StemCells. It is ironic that the last paper by Don is one that strongly challenges the basic hypothesis of his scientific life: one lineage-one growth factor. In this paper, he demonstrated that Mpl, the receptor for thrombopoietin, the megakaryocyte-specific growth factor, may not be necessary to make platelets after all [26]. There must be another receptor out there still to be discovered. The legacy of Donald Metcalf continues to inspire new areas of investigation. Open in new tabDownload slide Workshop CNR ITALIA-U.S.A. –Cattolica, Italy, March 8-9,1993 Stem Cell Factorand Related Cytokines in Bone Marrow Congenital Dysplasias Open in new tabDownload slide Workshop CNR ITALIA-U.S.A. –Cattolica, Italy, March 8-9,1993 Stem Cell Factorand Related Cytokines in Bone Marrow Congenital Dysplasias Remembrance Gerald Spangrude, PhD I was happy to pause for a moment to recall my interactions with Donald Metcalf. My memories of Don are mostly derived from stories I was told by others during the 18 months I spent in Roland Scollay's laboratory at the Walter and Eliza Hall Institute (WEHI). At that time (1989-1990), the excitement revolved around several research topics, including the work on LIF (leukemia inhibitory factor) and the interest in the SOCS (suppressor of cytokine signaling) molecules. Greg Johnson and I were eager to test the Thy-Sca-Lin protocol I brought to the institute using the Metcalf approaches and to try to transduce these cells with vectors expressing growth factors. I also collaborated with Uli Duhrson, one of Don's fellows, in characterizing a leukemic cell line, and this work resulted in the honor of publishing a paper with Don. I can relate the memory from long-term scientists at WEHI of large flasks placed in the men's toilet to be filled with source material (urine) for M-CSF purification, a method I replicated in my own laboratory. Also interesting is the memory of Don seemingly asleep in the back of the WEHI lecture hall as a lecture presented on hematopoiesis progressed, only to be punctuated by the most insightful and piercing questions from Don at the conclusion of the lecture. Don could frequently be found peering into his microscope and provided a great example of a working scientist, in contrast to the constant travel of many of his contemporaries. Don always knew the experimental details of every project going forward in his laboratory and always provided useful and insightful suggestions for students and fellows to pursue. I often joined the Metcalf team's table during tea time at WEHI, a half hour in the morning and in the afternoon, during which all work stopped for a social moment and the conversation most frequently revolved around current events and football but occasionally involved science as well. Don was a central figure at WEHI but remained very personable and approach-ablethroughout my acquaintance with him. Career Lessons Learned from a Giant: Don Metcalf Open in new tabDownload slide Kenneth Kaushansky, MD Stony Brook Medicine, Stony Brook University, Stony Brook, New York, USA Open in new tabDownload slide Kenneth Kaushansky, MD Stony Brook Medicine, Stony Brook University, Stony Brook, New York, USA In late 1982, nearing the completion of my internal medicine residency and clinical fellowship training in hematology at the University of Washington, I sat with my soon-to-be mentor, Dr. John Adamson, and discussed research topics that might launch my career as a physician-scientist, a calling instilled into my psyche by the bedazzling academic environment created by John and his colleagues, including Bob Petersdorf, Paul Beeson, Belding Scribner, Don Thomas, Earl Davie, and Clem Finch. John shared with me the excitement of experimental hematopoiesis—and that the normal and malignant blood cells he and Clem and others had taught me to recognize on clinical marrow rounds could actually be grown and studied in the laboratory. As an introductory primer, John handed me a copy of a compact, blue and white book entitled “Hemopoietic Colonies: In Vitro Cloning of Normal and Leukemic Cells” by Donald Metcalf. John explained that in order to grow marrow and blood cells in tissue culture, “colony-stimulating activities” must be present, and that since his pioneering publication in 1966 Don Metcalf had done more to identify the cells and substances necessary to stimulate the survival, proliferation, and differentiation of blood cells in culture than anyone on the planet. I rapidly consumed Don's book (and subsequently hundreds of other papers by Don on purified progenitors and growth factors) and have been hooked on hema-topoiesis ever since. In 1984, my first full year in Earl Davie's laboratory, I began to biochemically purify granulocyte-macrophage colony-stimulating factor (GM-CSF) from a tumor cell line, using huge 4L ion exchange and gel filtration chromatography columns. That July, Don and his colleagues published in Nature their landmark paper on the molecular cloning of GM-CSF from messenger RNA collected from murine lung. My career was again hugely impacted; I absolutely had to learn how to molecularly clone, because of its incredible scientific power, and because it allowed me to trade in the 4L chromatography set up for 1-mL oligo-dT mRNA purification columns! The best I can recall, I first met Don at the Walterand Eliza Hall Institutein May1994, when I was asked to deliver a talk on a new hematopoietic growth factor we had just cloned and characterized, thrombopoietin. The night before my talk, Nic Nicola, Glenn Begley, and Doug Hilton took me to dinner at a BYOB restaurant; Nic brought 4 bottles of wine and, by bottle three, began peppering me with stories of how at my talk the next day Don will sit in the back of the auditorium, seemingly not pay attention, and then askthree impossible-to-address questions and storm out when I could not answer them. As intended, for a relatively young academic about to speak at the Mecca of hematopoietic growth factor biology, they had achieved their goal of profound intimidation. The next morning, after a fitful sleep, Don's research fellow, John Rasko, picked me up early and brought me to “the WEHI” to meet Don. Of course, Don was already at work, passionately counting colonies. Don shared with me his and John's approach to cloning thrombopoietin, which was remarkably similar to ours, mutating an interleukin-3-dependent cell line engineered to express the presumptive/putative thrombopoietin receptor, the c-MpI proto-oncogene, to become growth factor independent by virtue of secreting its own growth factor, hopefully thrombopoietin. Don shared that John had cloned IL-3125 times and nothing else; I shared that we had cloned I L-3 22 times, but twice found “something else,” thrombopoietin. By comparing notes, we concluded that retroviral mutagenesis (John's approach) loves the I L-3 locus, but chemicals (our approach) mutate other sites as well. Then, as my pulse raced, I delivered my talk. Don sat in the front row, took copious notes, was gracious in his comments and compliments, and asked seriously insightful and answerable questions. I quickly realized the alcohol, and not Nic or Glenn or Doug, was speaking the night before! A month later, our papers on the cloning of thrombopoietin appeared in Nature, accompanied by an editorial by Don; he was again very gracious in praise, although if you read between the lines, he called us lucky. That we were lucky was true, but as Pasteur said, “chance favors the prepared mind.” Don had prepared all our minds. Over the past 20 years, I met with and discussed hematopoietic science and philosophy with Don on many occasions. We also discussed surfing, remarkably, one of his many non-hematopoietic interests. Don, along with Tariq Enver, Clare Heyworth, and Mike Dexter, helped me, as Editor-in-Chief of Blood, launch a point and counterpoint feature in Blood in 1998, tackling the major questions and controversies in hematology, in that inaugural case: do growth factors direct hematopoietic lineage commitment (Don's view) or merely keep cells alive to allow the stochastic rise and fall of transcription factors that mediate cell fate determination (Tariq et al's view)? When the American Society of Hematology annual meeting was held in San Diego in December of 2004, my wife Lauren and I invited about 75 friends to our home to unwind from the rigors of the meeting. I believe the group photograph below, shared by Misaki Mori (on the far left, back row), and including (clockwise from Misaki) Nobuyoshi Kosaka, Nami Nogawa-Kosaka, Youichi Aizawa, Takashi Kato (who also cloned thrombopoietin), Don, and yours truly, captured the essence of Don: intensely focused, surrounded by students of his work of all levels, and pleased with his impact on his science. With his passing, the world of hematology has lost a giant; we will all miss him. Open in new tabDownload slide Open in new tabDownload slide Professor Donald Metcalf, AC, BSc(Med) Sydney, MD Sydney, FRACP, Hon DSc Sydney, HonMD Oslo, HonFRCPath London, FRCPA, FAA, FRS Open in new tabDownload slide Ashley P. Ng, PhDa,c Open in new tabDownload slide Ashley P. Ng, PhDa,c Open in new tabDownload slide Warren S. Alexander, PhDa,c Open in new tabDownload slide Warren S. Alexander, PhDa,c Open in new tabDownload slide Douglas J. Hilton, PhDb,c Open in new tabDownload slide Douglas J. Hilton, PhDb,c Open in new tabDownload slide Nicos A. Nicola, PhDa,c Open in new tabDownload slide Nicos A. Nicola, PhDa,c aDivision of Cancer and Haematology, bDivision of Molecular Medicine, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia, cDepartment of Medical Biology, University of Melbourne, Parkville, Victoria, Australia Donald Metcalf, Don to his friends and colleagues, was one of the world's preeminent experimental hematologists. He dedicated his life to research into blood cells and their regulation and led the back-breaking effort over several decades that identified the colony-stimulating factors (CSFs). This pioneering research heralded a new era of modern molecular hematology and not only provided deep insights into blood cell development and regulation but also led to paradigm shifts in the treatment of blood cell disorders and cancers. The use of CSFs, to support patients through bone marrow suppressing cancer treatment and for mobilization of hematopoietic stem cells into the peripheral blood for use in bone marrow transplantation, has become the standard of care in hematology units around the world. The middle child of two schoolteachers, Don was born in 1929 in the small town of Mittagong in New South Wales. His father, Scottish by descent and Presbyterian by religion, immigrated to Australia as a child and moved to New South Wales from Victoria to pursue his career. Influenced by the Great Depression, Don's parents recognized the importance of education. Thus, Don's schooling commenced not long after he could walk, the classroom evolving from place of childcare to one in which he learned to read and write at a precocious age. As his family's movements followed the promotions of his father, Don's childhood was itinerant and rather isolated. Along with his two sisters, Beryl and Rosalind, he attended schools from Lithgow to Tamworth, barely keeping himself out of mischief, while helping to run the small family farm. Studying, however, became a major focus, and although he could have completed high school at the age of 14, he was too young to enter university and was made to repeat 2 years. He duxed Tamworth High School for several years running and qualified for a scholarship to study medicine at Sydney University just after the end of the Second World War. The expectations of his parents had instilled in him a discipline and work ethic that he combined with a keen inquisitiveness. These qualities would stay with him for the rest of his life. Don was part of the first class to go through medical school after the war. It was survival of the fittest for more than 600 students, an eclectic mix of ex-servicemen and young students out of high school, taught by a department depleted of staff and resources. But it was a new research training course at Sydney University, a Bachelor in Medical Science, that made the biggest impression on Don and his future career. Don was the first student to take this opportunity, joining the Department of Bacteriology with Professor Hugh Ward, a bacteriologist who had trained at Harvard University with Hans Zinsser. Don was directly supervised by Patrick de Burgh, with whom he studied murine ectromelia and hepatitis viruses in a rather rundown and poorly equipped postwar laboratory. Rather than treating Don as one student among hundreds sitting in overcrowded lectures, Ward and de Burgh, both ardent intellectuals, took time to sit, talk, and debate with a young Don “as a human who had something worth saying.” This was an experience Don would find electrifying and provided him a lesson in the importance of mentorship that he would take to heart. It was here that Don became increasingly drawn to the field of hematology as he became familiar with the emerging literature describing viruses and their potential role in leukemia development. Don graduated in 1953 with a Bachelor of Medicine and Surgery. He undertook his residency at Royal Prince Alfred Hospital in 1954. He was determined to undertake medical research, but acutely appreciated the perspective his medical training had instilled, and seriously considered specializing in hematology if he could not undertake research. It was also during his residency that he met his future wife and life-partner, Josephine Lentaigne, a nurse, who came from the rural town of Bangalow in New South Wales, where her father, John Lentaigne, was a highly respected and skilled rural doctor. An advertisement for the Carden Fellowship from the then Anti-Cancer Council of Victoria precipitated Don's dramatic move from clinical medicine to medical research. The Anti-Cancer Council was searching for a professor to start a cancer research unit in Melbourne. Having already approached several expatriate Australians who politely declined, Don's old mentor, Hugh Ward, a wartime friend of an executive member of the Anti-Cancer Council, suggested they talk to Don. Don suggested that, given his inexperience, he would accept the fellowship if offered on half-salary (he was, by his own admission, “never this stupid again!”) and was promptly granted an interview in Melbourne. At the age of 26, in the middle of assisting in an operation, he was notified by telegram that he had been awarded the Carden Fellowship. Whether by serendipity or tremendous foresight, the Anti-Cancer Council of Victoria backed the right horse. It was this fellowship that sustained Don's research for the next 60 years and was critical in providing him the security and freedom to focus on long-term research questions through the inevitable difficult periods. In 1954, Don began work at what he regarded as the best medical research institute in the country, the Walter and Eliza Hall Institute. The Institute had been established within the infrastructure of the then Melbourne Hospital, from a bequest from the Walter and Eliza Hall Trust 4 decades earlier. Under the directorship of Charles Kellaway and subsequently his protégé, the eminent virologist and future Nobel laureate in immunology, Frank Macfarlane Burnet, the Institute had become a beacon for infectious disease research, particularly in virology. It had taken some persuading on the part of the Anti-Cancer Council to convince Burnet to admit Don to the Institute. Burnet regarded cancer as an inevitable disease and considered nascent research into cancer causes and treatments as a rather futile exercise. Indeed, this was the prevailing opinion not only of Burnet but also of other oncologists and hematologists at the time. It is perhaps telling that Don attended clinical rounds at The Royal Children's Hospital with another pioneer, pediatrician Jonathan Colebatch, who would change the face of leukemia treatment by not accepting the status quo. Burnet, however, made Don prove himself to be a “genuine scientist” by working for 2 years as a “virologist” on vaccinia virus. Don duly complied, but not one to be stifled for long, he began to “do his own thing” by investigating chicken leukemia viruses. This way, he would be able to fall within the remit of his Carden fellowship, fulfill Burnet's wishes, and begin to study blood cells. What Don didn't learn from Burnet about blood cell and leukemia development, he did in terms of research techniques and scientific strategy: mobilize your resources, focus on the area you are good at, and stick to it. Don became interested in the thymus gland, a then mysterious organ, which seemed important in the development of certain forms of leukemia. The opportunity arose from 1956-1958 for Don to undertake a postdoctoral position at Harvard University with Jacob Furth, a Hungarian-born scientist who had demonstrated that removing the thymus prevented mice from developing lymphoid leukemia. Here at last, Don could undertake formal training in a large center with a scientist devoted to the emerging field of cancer research. Furth's voluminous ideas about cancer development had a significant impact on Don. In particular, Furth's careful observations that certain endocrine tumors may be caused by an imbalance of hormonal regulators piqued Don's interest. This was an idea that defined a new research focus for him: seeking to identify regulators that would favor blood cell proliferation and, perhaps, contribute to leukemia. Open in new tabDownload slide Research scientist. Dr. Donald Metcalf, with Dr. K. Nakamura injecting a laboratory mouse during leukemia research at the Walter and Eliza Hall Institute for Medical Research, Royal Melbourne Hospital, Parkville, Victoria (1959). Open in new tabDownload slide Research scientist. Dr. Donald Metcalf, with Dr. K. Nakamura injecting a laboratory mouse during leukemia research at the Walter and Eliza Hall Institute for Medical Research, Royal Melbourne Hospital, Parkville, Victoria (1959). On his return, Don applied his new skills, particularly in animal pathology, to the next phase of his research career. His monograph, “The Thymus: Its Role in Immune Response, Leukaemia Development and Carcinogenesis” clearly demonstrated that he had become a peerless anatomist, pathologist, and cytologist. The balance sheet approach that he applied to dissection of the hematopoietic hierarchy later in his career, he had first applied in his studies of the thymus, revealing novel insights into thymus biology, including the fact that the majority of the cells that were made within it died. This observation gained wide acceptance, and was understood to be necessary for elimination of self-reactive T lymphocytes, only years later. His analysis of the AKR mouse model of thymoma was definitive, and the foreword written by Burnet to Don's monograph showed the regard and respect the former had developed for Don. Despite this groundbreaking work that aligned Don with Burnet's and the Institute's growing focus on immunology, the thymus remained a frustration to him. The in vivo systems he was using did not lend themselves easily to the precise dissection of regulators that could control cellular behavior. A scientific collaboration with Ray Bradley at the University of Melbourne, however, was about to bring about a monumental change in direction. Virtually overnight, all work on the thymus stopped. In 1964, Ray Bradley walked across the road from Melbourne University's Department of Physiology to show Don dishes of semi-solid agar. In these were growing small colonies of cells from mouse bone marrow, appearing as star-like clusters when illuminated under a microscope. This was the first time Don had seen bone marrow cells grown in vitro. Independently, Leo Sachs and Dov Pluznick at the Weizmann Institute in Israel had grown bone marrow “mast cell” colonies using a similar technique. Each colony derived from a single cell. The growth of colonies was dependent upon an appropriate underlayer of tissue fragments that “conditioned” the medium. This observation was Don's “eureka” moment. It was the system that would allow Don to systematically search for regulators that stimulated the growth of blood cells. Through further experiments, Bradley and Don believed that there were soluble “factors” in the culture medium that supported the survival and growth of these clonogenic bone marrow myeloid colonies. The game was now afoot to find them. At this time, nothing was known of any blood cell regulating factor, or if they even existed, other than the indirect observation of support of colony growth. It had to be established that CSFs were proteins and not, for example, viruses and not due to contamination of the culture medium by bacterial products that could be stimulating cellular proliferation. Serum, urine, and medium conditioned by various human and murine tissues were all surveyed for bone marrow colony-stimulating activity. It became clear that a number of these sources supported different types of colony growth. Rather disconcertingly, this implied there may be several types of CSFs. Don assembled a tight-knit team of research assistants and biochemists who would focus on purifying “active” fractions from impure source material, with Don always personally overseeing the colony assays to test for CSF activity. Remarkably, the number of staff working on this project never grew to more than ten for the first decade of work. Indeed, the initial task of purifying CSFs initially had fallen to just one Ph.D. student! There eventually turned out to be at least four distinct CSFs. Macrophage CSF (M-CSF, CSF-1), granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), and multipotential CSF (Multi-CSF, interleukin 3) were recognizable by their distinct biochemical properties and colony-stimulating potential, characteristics that Don defined in exhaustive detail. What also couldn't be known at the time was the monumental difficulty and scale of the task. At commencement, the goal of being able to obtain sufficient pure CSF to test in mice, let alone apply to human trials, far exceeded the technological capabilities of the day. Multiple challenges and frustrations needed to be overcome, including controlling the variability of the ever-important colony assay, the adhesion of CSFs to glassware and plastic, and the accidental discarding of the only pure vial of M-CSF in the world by an over-enthusiastically tidy researcher. Following a decade of work, purification of M-CSF from literally thousands of liters of volunteer human urine was achieved. Murine GM-CSF and G-CSF were purified from batches of lung conditioned medium. Initially, thousand-fold purifications using protein separation columns yielded reproducible colony-stimulating activity. This progressed to one million-fold purifications with the development of the high-performance liquid chromatography. Importantly, this new technology was able to yield protein “pure” enough for amino acid sequencing. Overcoming the challenges of infinitesimally small protein yields, a collaboration with the CSIRO (Commonwealth Scientific and Industrial Research Organization) led to the first amino acid sequence of murine GM-CSF. With the entry of molecular biologists at the Melbourne branch of the Ludwig Institute for Cancer Research to the project, Don and his team were able to isolate the genes encoding CSFs. Finally, recombinant CSFs were made and found to be biologically active, first in murine models, and then in humans. This was the final validation of the program that Don began decades ago, much to his satisfaction and relief. Never one to stop investigating the effect of the CSFs on hematopoiesis, it was Don's analysis of patient blood from clinical trials that revealed that G-CSF administration induced the appearance of stem cells in the peripheral blood, a critical observation that led to G-CSF being used for stem cell mobilization for bone marrow transplant therapy. What had begun as a simple project in a field in which few research groups had taken a particular interest, took decades to complete and culminated in intense competition with several other laboratories around the world and with pharmaceutical companies scrambling for patent rights. Open in new tabDownload slide Professor Metcalf, shown here in the 1960s, spent many laborious hours at his microscope counting blood cell colonies. Open in new tabDownload slide Professor Metcalf, shown here in the 1960s, spent many laborious hours at his microscope counting blood cell colonies. The crucial task of scoring colonies over all those years fell principally to Don. Six-to eight-hour stretches at a microscope scoring hundreds of cultures each day (in addition to other work), led to back pain, two laminectomies, and spinal fusion. It is not surprising that one of his favorite passages was from Shakespeare's Henry V (from which he borrowed the line “Summon up the Blood” for the title of his book describing the discovery of the CSFs). This passionate call to arms began “Once more unto the breach, dear friends, once more.” For Don, every colony in every culture dish on every tray counted. But, most importantly to Don, the program to identify and clone the CSFs had been successful. Profound insights into blood cell production and function ensued, and CSFs entered the clinic to ultimately benefit millions of patients. What started as an observation from a culture dish with Bradley pioneered a new field of endeavor in which Don and his team were world leaders. The accolades followed: the Wellcome Medal (1986), Bristol-Myers Award for Cancer Research (1987), Robert Koch Prize (1988), Alfred P. Sloan Award—General Motors Cancer Research Foundation (1989), Companion of the Order of Australia (1993), Albert Lasker Clinical Medical Research Award (1993), Jessie Stevenson Kovalenko Medal U.S. National Academy of Sciences (1994), Gairdner Foundation International Award (1994), Royal Medal of The Royal Society (1995), Prime Minister's Prize for Science (2001), and Grand Hamdan International Award (2008) were some of the prizes that rightly recognized his leadership in the field. Throughout the long process of CSF discovery and characterization, Don led the Cancer Research Unit at the Walter and Eliza Hall Institute. In 1966, following the appointment of Gustav Nossal as the successor to Burnet as director of the Institute, Nossal promptly also appointed Don deputy director. In the Cancer Research Unit, Don built a team of talented and trusted colleagues who shared his vision and worked in close-knit collaboration. The Cancer Research Unit grew, devoting itself to dissecting the biology of blood cell development more broadly, building on the discoveries of the colony-stimulating factors with further ground-breaking discoveries of novel cytokine receptors, signal transduction pathways, and the molecular genetics of blood cell and cancer development. Throughout, the collaborative approach established by Don in the unit's infancy became the team's defining characteristic. This was the integrated research team defining the mechanisms of blood and cancer development that Don had always aspired to develop. Don hated to be interrupted at the microscope and expected a quiet laboratory environment; disturb his concentration when he was scoring a plate on which hundreds of colonies might be present, and you would incur his wrath. This created a sometimes-intimidating aura for colleagues wanting to consult him. However, this was largely undeserved. A generous collaborator, Don often recounted that he could not recall having refused someone who asked to help. Once through his door, Don invariably would give you his time despite whatever else he was doing and whether you were a professor or a graduate student. Typically, he was collaborating on more than a dozen projects throughout the Institute. Appreciation of good science was the key, because in the end, that is what mattered to Don. Being first with a discovery was important, but not as important as the quality of what was being produced. What really mattered was that what came out of your laboratory could be reproduced in any laboratory of equal standing around the world and would stand the test of time. If Don could help his colleagues achieve this, he invariably gave his time and expertise. Open in new tabDownload slide Open in new tabDownload slide Although little time was left in Don's grueling work schedule, he found time to appreciate music and the arts. His appreciation of classical music was exemplified by his exhaustive classical CD collection and knowledge of music, of which most would be envious. He and Josephine regularly attended the opera and other classical music performances. It was a moment of mutual admiration when Jose Carreras, having benefited from G-CSF during his own leukemia therapy, visited Don's laboratory. Don's fascination with the natural world wasn't limited to biology. He was just as fascinated watching the transit of Venus as a zealous colleague projected the solar reflection on the laboratory wall or through the telescope set up on the Institute forecourt, harking back to the time when the country sky at nighttime whetted his underdeveloped interest in astronomy. Don also traveled regularly with Josephine on a yearly cruise, a treasured time when he could take a break from the microscope and see the world. Although Don “retired” in the sense that he stood down as leader of the Cancer Research Unit in the mid 1990s, there was no pause in his experiments and he still arrived at work at 6:30 in the morning, usually before anyone else, to examine what emerged from the incubator. The excitement of potentially seeing something no one else in the world had seen before and making the next discovery never waned. This continued after he was diagnosed with pancreatic cancer in mid 2014. Limited in his ability to drive to work, Don insisted on moving his trusty microscope to his house to be able to continue to pursue his research and not to let down the colleagues with whom he was collaborating. A fitting and memorable event during these last months was the tribute paid to Don at a bursting-at-the-seams Walter and Eliza Hall Institute at which his colleagues and friends emotionally acknowledged his extraordinary contribution to them personally, and to the Institute, with stories, tears, and laughter. All were reminded not only of Don's own contribution, but also of the generosity of Josephine, their daughters Katherine, Mary-Ann, Johanna, and Penelope, and their families, who shared Don with the medical research community and who were themselves so much a part of the Walter and Eliza Hall Institute family. At the time of writing, Don's office sits empty. His microscope has been returned to the desk, and the stiff-backed wooden chair (painted pink for some unknown reason) sits quietly facing a desk that is still covered with some of his papers. The space in the tearoom where he sat during his obligatory morning break, where he would enthusiastically catch up with gossip (scientific and otherwise) after a long morning counting colonies, is now occupied by others. Colleagues still hesitate, however, to occupy the seat at the back right corner of the Institute's lecture theater in which Don invariably sat during seminars. While these day-to-day imprints of Don on the memories of his colleagues will gradually fade, the legacy of his dogged determination and devotion to medical research, his extraordinary discoveries in molecular hematology and their continuing impact on clinical practice, his contribution to the training and careers of countless colleagues, and his professional generosity will permanently endure. Open in new tabDownload slide Photographs are reproduced with permission from the Archives of The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia. Open in new tabDownload slide Photographs are reproduced with permission from the Archives of The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia. The following profile of Professor Donald Metcalf first appeared in his book “Blood Lines: An Introduction to Characterizing Blood Diseases of the Post-Genomic Mouse” Open in new tabDownload slide Open in new tabDownload slide Professor Donald Metcalf was born in Mittagong, New South Wales, Australia, and was educated in various country schools. Hegraduated from Sydney University in1953 after completing a BSc (Med) in virology. After an internship at The Royal Prince Alfred Hospital, Sydney, he joined the staff of The Walter and Eliza Hall Institute of Medical Research, a position he continues to hold. From 1965 to 1996, he was Head of the Cancer Research Unit and Assistant Director of The Walter and Eliza Hall Institute, and in 1996 became Professor Emeritus of The University of Melbourne. His work at The Walter and Eliza Hall Institute has been interspersed with sabbatical years as a Visiting Scientist at Harvard Medical School, Boston; the Roswell Park Memorial Institute, Buffalo; the Swiss Institute for Experimental Cancer Research, Lausanne; the Radiobiological Institute, Rijswijk; and the University of Cambridge. Professor Metcalf has been rightfully called “the father of hematopoietic cytokines” for his pioneering work on the control of blood cell formation. In early studies, he discovered the function of the thymus gland in controlling the formation of lymphocytes, and beginning in 1965, co-developed a series of specialized culture techniques permitting the growth of various types of blood cells. These cultures led him and his team to the discovery of the “colony-stimulating factors” (CSFs), hormones that control white blood cell formation and are, therefore, responsible for one's resistance to infection. His work, with that of others, led to the successful cloning of the genes for all four mouse and human CSFs, and the mass production of these hormones by bacterial, yeast, and other cells. His work provided the pivotal demonstration that the CSFs, when injected into animals, stimulated the formation and regulated the activity of white blood cells. Exploiting this, his collaborators then documented the effectiveness of GM-CSF and G-CSF (two primary white blood cell regulators) when injected into patients. These blood cell regulators are now in extensive clinical use throughout the world as valuable drugs, which can accelerate the regrowth of blood cells following anti-cancertreatment and bone marrow or peripheral blood transplantation. The corpus of his fundamental and applied research is to be found in more than 400 peer-reviewed scientific papers, 200 other scientific papers, ad seven books. Professor Metcalf has received some of the highest honors in the world of contemporary science. Among them, he was made a Companion of the Order of Australia; he was elected a Fellow of the Australian Academy of Science, a Fellow of the Royal Society, London; and a Foreign Associate of the National Academy of Sciences of the U.S. His prizes for research include the Wellcome Prize of the Royal Society (shared), the Bristol-Myers Award for Distinguished Achievement in Cancer Research, the Hammer Prize for Cancer, the Koch Prize of the Federal Republic of Germany, and a Gairdner Foundation International Award of Canada. In addition, Professor Metcalf shared the Alfred P. Sloan Prize of the General Motors Cancer Research Foundation; received the Bertner Foundation Award of the MD Anderson Cancer Center, and the Rabbi Shai Shacknai Prize of the Hadassah University, Jerusalem. He is also a recipient of the Albert Lasker Clinical Medical Research Award, the Louisa Gross Horwitz Prize of Columbia University, the Jessie Stevenson Kovalenko Medal of the U.S. National Academy of Sciences, and was the inaugural recipient of the Kantor Family Prize for Cancer Research Excellence from the Hippie Cancer Research Center. In 1995 he received the Ernst Neumann Award, International Society for Experimental Hematology and the Royal Medal, Royal Society, London. In 1996 he shared the Amgen Australia Prize and The Warren Alpert Foundation Prize, Harvard Medical School. In 1998 he was made an Honorary Member of the Alpha Omega Alpha Medical Society, U.S. and in 2000 hewill receive the Chiron International Award, National Academy of Medicine, Italy. Unusual among scientists of almost any age, certainlythose in their seventh decade of life, he continues to work at the laboratory bench nine hours each day. The brilliant and incredibly productive life of Professor Metcalf may be viewed in this book as a magnificent painting, but one of its primary colors would be missing without recognition of the loving support of Josephine, his wife of 46 years. Mother of their four grown daughters, “Jo” (as she is affectionately called) has been a tower of strength and a source of perspective for Don, their children and grandchildren, and for all those fortunate to know and thereby to be touched by her. Therein lies another profile…and another fascinating book. The following photographs and legends were originally provided courtesy of Josephine Metcalf for publication in “Polyfunctionality of Hemopoietic Regulators: The Metcalf Forum”, edited by Martin J. Murphy, Jr., and published in 1994 by AlphaMed Press as a supplement to the Journal STEM CELLS. They are reprinted here in fond remembrance. Open in new tabDownload slide Open in new tabDownload slide Open in new tabDownload slide Open in new tabDownload slide References 1 Jacobson LO , Plazak L, Fried W et al. Plasma factor(s) influencing red cell production . Nature 1956 ; 177 : 1240 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Becker AJ , McCollouch EA, Till JE. Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells . Nature 1963 ; 197 : 452 – 454 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Metcalf D , Bradley R. The growth and erythropoietic activity of spleen grafts placed under the kidney capsule . Aust J Exp Biol Med Sci 1965 ; 43 : 229 – 236 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Bradley TR , Metcalf D. The growth of mouse bone marrow cells in vitro . Aust J Exp Biol Med Sci 1966 ; 44 : 287 – 299 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Metcalf D . A lymphocytosis stimulating factor in the plasma of chronic lymphatic leu-kaemic patients . Br J Cancer 1956 ; 10 : 169 – 178 . Google Scholar Crossref Search ADS PubMed WorldCat 6 Bradley TR , Metcalf D, Robinson W. Stimulation by leukaemic sera of colony formation in solid agar cultures by proliferation of mouse bone marrow cells . Nature 1967 ; 213 : 926 – 927 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Gough NM , Gough J, Metcalf D et al. Molecular cloning of cDNA encoding a murine haematopoietic growth regulator, granulocyte-macrophage colony stimulating factor . Nature . 1984 ; 309 ( 5971 ): 763 – 767 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Stanley ER , Hansen G, Woodcock J et al. Colony stimulating factor and the regulation of granulopoiesis and macrophage production . Fed Proc 1975 ; 34 : 2272 – 2278 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 9 Stanley ER , Chen DM, Lin HS. Induction of macrophage production and proliferation bya purified colony stimulating factor . Nature 1978 ; 274 : 168 – 170 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Gough NM , Gough J, Metcalf D et al. Molecular cloning of cDNA encoding a murine haematopoietic growth regulator, granulocyte-macrophage colony stimulating factor . Nature 1984 ; 309 : 763 – 767 . Google Scholar Crossref Search ADS PubMed WorldCat 11 Metcalf D . Blood. Thrombopoietin—At last . Nature 1994 ; 369 : 519 – 520 . Google Scholar Crossref Search ADS PubMed WorldCat 12 Gearing DP , Gough NM, King JA et al. Molecular cloning and expression of cDNA encoding a murine myeloid leukaemia inhibitory factor (LIF) . EMBO J 1987 ; 6 : 3995 – 4002 . Google Scholar Crossref Search ADS PubMed WorldCat 13 Gough NM , Gearing DP, King JA et al. Molecular cloning and expression of the human homologue of the murine gene encoding myeloid leukemia-inhibitory factor . Proc Natl Acad Sci USA 1988 ; 85 : 2623 – 2627 . Google Scholar Crossref Search ADS PubMed WorldCat 14 Rossi GB , Migliaccio AR, Migliaccio G et al. In vitro interactions of PGE and cAMP with murine and human erythroid precursors . Blood 1980 ; 56 : 74 – 79 . Google Scholar Crossref Search ADS PubMed WorldCat 15 Sheridan JW , Metcalf D. A low molecular weight factor in lung-conditioned medium stimulating granulocyte and monocyte colony formation in vitro . J Cell Physiol 1973 ; 81 : 11 – 23 . Google Scholar Crossref Search ADS PubMed WorldCat 16 Migliaccio AR , Visser JW. Proliferation of purified murine hemopoietic stem cells in serum-free cultures stimulated with purified stem-cell-activating factor . Exp Hematol 1986 ; 14 : 1043 – 1048 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 17 Hapel AJ , Fung MC, Johnson RM et al. Biologic properties of molecularly cloned and expressed murine interleukin-3 . Blood 1985 ; 65 : 1453 – 1459 . Google Scholar Crossref Search ADS PubMed WorldCat 18 Lee JC , Hapel AJ, Ihle JN. Constitutive production of a unique lymphokine (IL 3) by the WEHI-3 cell line . J Immunol 1982 ; 128 : 2393 – 2398 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 19 Warner NL , Moore MA, Metcalf D. A transplantable myelomonocytic leukemia in BALB-c mice: Cytology, karyotype, and muramidase content . J Natl Cancer Inst 1969 ; 43 : 963 – 982 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 20 Metcalf D , Merchav S, Wagemaker G. Commitment by GM-CSF of bipotential GM progenitor cells to granulocyte or macrophage formation . Experimental Hematology Today 1982 ;( suppl ); 3 – 9 . Google Scholar OpenURL Placeholder Text WorldCat 21 Stem Cell Factor and Related Cytokines in Bone Marrow Congenital Dysplasias . Stem Cells 1993 ; 11 ( suppl 2 ): 1 – 189 . Available at http://onlinelibrary.wiley.com/doi/10.1002/stem.v11:2%2B/issuetoc. Accessed July 31, 2015. OpenURL Placeholder Text WorldCat 22 Hilton DJ , Nicola NA, Alexander WS et al. Donald Metcalf (1929–2014) . Cell 2015 ; 160 : 361 – 362 . Google Scholar Crossref Search ADS PubMed WorldCat 23 Hilton D . Donald Metcalf (1929–2014) . Nature 2015 ; 517 : 554 . Google Scholar Crossref Search ADS PubMed WorldCat 24 Adams JM , Cory S. Donald Metcalf: The father of modern hematology . Proc Natl Acad Sci USA 2015 ; 112 : 2628 – 2629 . Google Scholar Crossref Search ADS PubMed WorldCat 25 Shortman K , Nicola NA, Ng AP. Professor Donald Metcalf (1929–2014) . Immunity 2015 ; 42 : 1 – 3 . Google Scholar Crossref Search ADS PubMed WorldCat 26 Ng AP , Kauppi M, Metcalf D et al. Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation . Proc Natl Acad Sci USA 2014 ; 111 : 5884 – 5889 . Google Scholar Crossref Search ADS PubMed WorldCat © 2015 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Special Issue Collection: In Memoriam
JF - Stem Cells
DO - 10.1002/stem.2237
DA - 2015-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/special-issue-collection-in-memoriam-fUaLSzeRzn
SP - 3397
EP - 3422
VL - 33
IS - 12
DP - DeepDyve
ER -