A new model of blood cell differentiation proposed—a great discovery that may lead to the updating of textbooks
22 May 2009 (Volume 4 Issue 5)
A paper on blood cell differentiation by Hiroshi Kawamoto, team leader of the Laboratory for Lymphocyte Development at the RIKEN Research Center for Allergy and Immunology, appeared in the News & Views column of the UK science journal as an achievement that will require the revision of textbook accounts. Kawamoto says with a smile, “Both T lymphocytes and B lymphocytes have an important role in immune function, and they have previously been thought to be siblings that are produced from common progenitors. However, we demonstrated that it is not B lymphocytes, but macrophages, a type of phagocyte, that are produced from progenitors shared by T lymphocytes, and proposed a new pathway map for the differentiation of blood cells. I am very happy to have launched this groundbreaking achievement from Japan, overturning the traditional theory.” This achievement is expected to lead to the establishment of a new therapy for leukemia and cancer (Fig. 1).
Figure 1: Differentiation of blood cells.
Hematopoietic stem cells descend ‘the mountain of hierarchy of differentiation’ while proliferating and dividing. There are many bifurcations in the downhill path, where little by little their fates are restricted. One of the routes leads to the thymus. After entering the thymus, the hematopoietic progenitors pass a bifurcation toward phagocytes (macrophages) and dendritic cells, and become T lymphocytes. The monolith standing at each bifurcation symbolizes the enigma. Fountains of growth factors appear here and there.
The cells of the blood system
“Since I was a child I have been very fond of manga, both reading and creating them, and when I was a graduate student I received the Encouragement Prize at a contest for the comic magazine Big Comic Spirits. Recently I have been painting posters, for example for events sponsored by the Research Center for Allergy and Immunology (RCAI),” says Kawamoto.
Figure 1 was drawn by Kawamoto to show the process of production of the various types of cells of the blood system from hematopoietic stem cells. Hematopoietic stem cells are produced in the bone marrow and differentiate into progenitors. Some of the progenitors differentiate into erythrocytes, B lymphocytes and phagocytes in the bone marrow, and the others are transported to the thymus, where they differentiate into T lymphocytes and phagocytes. “Showing how the fates of individual cells are decided, ‘differentiation pathway maps’ provide very important basic information in biology. However, blood cells have been studied without extensive versions of differentiation pathway maps,” Kawamoto points out. “The goal of our project is to clarify the pathway of the differentiation of blood cells.”
Now the key participants in the blood cell differentiation pathway map are introduced. The cells in the blood system can be divided into the erythrocyte–platelet lineage and the leukocyte lineage (Fig. 2). Leukocytes comprise the phagocyte lineage and the lymphocyte lineage. Leukocytes are responsible for the immune mechanism, working individually to eliminate foreign substances such as pathogens. Phagocytes swallow and digest pathogens, whereas killer T lymphocytes kill pathogen-infected cells. B lymphocytes produce antibodies in response to the directions of helper T lymphocytes, thus attacking pathogens. Kawamoto says with a laugh, “My illustration here (Fig. 1) is unpopular among B lymphocyte researchers, who say that antibodies are not as dirty as saliva. But ease of understanding is also important, isn’t it?”
Figure 2: Various blood cells and functions of leukocytes.
Hematopoietic stem cells, the origin of all blood cells, are produced in the bone marrow. In the bone marrow, progenitors are produced from hematopoietic stem cells and then differentiate into erythrocytes, platelets, B lymphocytes, phagocytes, and others. Some progenitors are transferred to the thymus, which lies above the heart, and T lymphocytes and phagocytes are produced there. T lymphocytes, B lymphocytes, and phagocytes constitute the immune mechanism that protects the body against pathogens and other foreign substances.
Are T lymphocytes and B lymphocytes siblings?
“This illustration appears as a model of blood cell differentiation in textbooks on medicine or biology,” says Kawamoto, showing the upper panel in Fig. 3. Hematopoietic stem cells first differentiate into common progenitors of the phagocyte–erythrocyte lineage and the lymphocyte lineage. Phagocytes and erythrocytes differentiate from the former, and B lymphocytes and T lymphocytes from the latter. “Although this model has been central for some 30 years, it is not based on experimental data. It is a product of the speculation that ‘B lymphocytes and T lymphocytes should be related to each other because they are similar in terms of shape and function’.”
B lymphocytes and T lymphocytes are not only morphologically alike, but they also share antigen specificity for the capability of responding to particular antigens. Another common feature is that they produce receptors capable of binding to a wide variety of antigens through a mechanism known as gene reconstitution. However, Kawamoto points out, “The inference that animals with similar appearances, like cats and tigers, are closely related to each other is often correct. However, if one compares flying squirrels (a rodent) and sugar gliders (a marsupial), for example, they are taxonomically remote despite their external similarity. Their process of differentiation must be evaluated without undue speculation.”
In 1994 Kawamoto began a study to elucidate the process of blood cell differentiation in the laboratory of Yoshimoto Katsura, an immunologist at the Chest Disease Research Institute (now the Institute for Frontier Medical Sciences) at Kyoto University. In 1997, jointly with Katsura, Kawamoto developed a new analytical method called a multilineage progenitor assay. This is a groundbreaking approach that enables the cultivation of single progenitors of the blood system one by one, and examination of their potential for differentiation into T lymphocytes, B lymphocytes, or phagocytes. The progenitors are individually cultured together with a thymus tissue in the presence of a variety of cytokines (proteins that are secreted from cells and are responsible for signal transduction). The differentiated cells are sorted by means of a flow cytometer.
Figure 3: The two blood cell differentiation models.
The classical model (upper panel) assumes the presence of progenitors that are common to B lymphocytes and T lymphocytes, whereas the myeloid-based model (lower panel) proposes that progenitors retain the ability to differentiate into phagocytes (myeloid cells) until their fate is sealed by differentiating into erythrocytes, B lymphocytes, and T lymphocytes.
Kawamoto and Katsura were astonished at the results. Kawamoto explains, “This is an unusual finding. Although we had been careful not to be bound by conventional ideas, we had been thinking that our study would produce results in line with the traditional model. However, we found no progenitors that would differentiate into T lymphocytes and B lymphocytes, though there were progenitors that would differentiate into T lymphocytes and phagocytes, or into B lymphocytes and phagocytes.” Kawamoto and Katsura therefore jointly conducted demonstrative tests and held discussions, proposing a novel model of blood cell differentiation (lower panel in Fig. 3). “This is our myeloid-based model, which assumes that blood cells are capable of differentiating into phagocytes (myeloid cells) until their final differentiation into erythrocytes, B lymphocytes, and T lymphocytes. It is based on the new concept that differentiation proceeds on the basis of the myeloid lineage.”
In 1997 Kawamoto published the first finding supporting the myeloid-based model in the science journal International Immunology. Soon after that, however, Irving Weissman, the hematologist, and his colleagues at Stanford University reported to Cell that they discovered progenitors common to T lymphocytes and B lymphocytes in the adult mouse bone marrow, in support of the traditional model. Meanwhile, some researchers supported Kawamoto’s new model. However, the progenitors used by Kawamoto were collected from a fetal mouse, which limited the model’s applicability to fetuses. The old model continued to be the mainstream.
Despite this, Kawamoto did not give up. “Except for T lymphocytes, which are produced in the thymus, all blood cells are produced in different sites; in the bone marrow in adults, and in the liver in fetuses. Even so, there should be no difference in the process of cell differentiation because it has been acquired through the long history of evolution.” He adds, “Many researchers tend to avoid arguments in scientific research, but it is good for a researcher to have someone against whom they can argue, because the situation provides an appropriate feeling of tension. Even if the person you are arguing against is preeminent in the relevant field, the researcher must continue to state what he believes to be right.”
T-lymphocyte progenitors are capable of differentiating into macrophages
In 2002 Kawamoto organized his team, and one of its aims was to establish a firm basis for the myeloid-based model. One possible approach was to demonstrate that the common progenitors for T lymphocytes and B lymphocytes, reportedly discovered in the bone marrow by Weissman, are also capable of producing phagocytes. In fact, we had obtained such data. However, if the results were attributed to the use of an inappropriate method of separation, endless dispute would be unavoidable.” Kawamoto decided to demonstrate that the cells about to become T lymphocytes in the thymus (T-lymphocyte progenitors) retained the ability differentiate into phagocytes, although they were deficient in their ability to differentiate into B lymphocytes.”
Then, jointly with an ex-RIKEN Research Scientist Haruka Wada (an associate professor now at St Marianna University School of Medicine, Kanagawa), Kawamoto developed a method of culturing T-lymphocyte progenitors with stroma cells that they had prepared independently as the basic cells, and determining whether they were capable of differentiating into T lymphocytes, B lymphocytes, and macrophages. The T-lymphocyte progenitors were collected from the thymus of a mouse that had been genetically modified to produce a green fluorescent protein in all the cells in its body. This animal was chosen because its cells can be seen under a fluorescence microscope, even if only a few cells are produced during cultivation.
When they cultured 192 T-lymphocyte progenitors from the thymus at a single-cell level, 123 differentiated into T lymphocytes only, 1 differentiated into macrophages only, and 13 differentiated into both macrophages and T lymphocytes. None differentiated into B lymphocytes. The team performed further experiments and demonstrated that T-lymphocyte progenitors can differentiate into macrophages even in vivo. “T-lymphocyte progenitors in the thymus are capable of differentiating into T lymphocytes and macrophages, although they have lost their ability to differentiate into B lymphocytes. This provides evidence that the conventional model is wrong, and strongly suggests the correctness of the myeloid-based model.”
In April 2008 Kawamoto published this achievement in Nature. Many researchers have come to think that the myeloid-based model, as featured in the journal’s News & Views section, is correct and is ‘an achievement that will necessitate revision of textbook accounts.’ What, then, was Weissman’s response? “Since he wrote in a review in a journal, ‘The absence of progenitors common to lymphocytes has not been established,’ he seems not to have accepted the myeloid-based model.” We asked Kawamoto, “What can you do to prove your model beyond doubt?” He answered, “My next goal is to demonstrate that B-lymphocyte progenitors are capable of differentiating into phagocytes, although they are unable to differentiate into T lymphocytes, and we have already obtained some supportive data.”
The updating of the pathway map for blood cell differentiation is not the only outcome of the current achievement. Because differentiation reflects the process of evolution, the origins of blood cells can also be discussed. “We have proposed a model in which blood cells originated from phagocytes, and B lymphocytes and T lymphocytes have evolved in different ways: B lymphocytes have evolved directly from phagocytes, whereas T lymphocytes have evolved via killer cells from phagocytes. It is quite rare that a new theory in biology is born in Japan. In this context, our achievement is of paramount importance.”
Toward a new therapy for leukemia and cancer
The present achievement is also expected to find medical applications, including in the treatment of leukemia. A certain type of leukemia cell had been known to have binary characteristics for T lymphocytes and phagocytes, but the stage at which they became leukemia cells remained elusive. “With the myeloid-based model, the leukemia cells can be estimated to be derived from progenitors common to the phagocyte–T lymphocyte lineage. I want to proceed to develop a new therapy that targets this stage.” Kawamoto has worked as a clinician in a department of hematology. “Many of my patients with leukemia were young, so I had bitter experiences as a physician. I still have a strong desire to treat it by some means or other.”
Kawamoto says, “We are leading the world in cell culture technology.” Making use of the technology, a research project aiming at regenerative medicine for blood cells is ongoing. At present, leukemia and cancer are treated by bone marrow transplants (in which bone marrow stem cells collected from a donor are transplanted to the recipient patient) and cell therapy (in which progenitors collected from the patient are differentiated into immunocytes in vitro and then returned to the patient’s body). However, these methods pose problems, including the difficulty in finding compatible donors and the great burden on the donor for the bone marrow transplant, and the need for the frequent collection of progenitors for cell therapy. If it is possible to proliferate hematopoietic stem cells or progenitors freely, the burdens on patient and donor are lessened. However, this is quite difficult. Whereas hematopoietic stem cells and progenitors proliferate in vivo by self-replication while retaining their pluripotency for differentiation into a wide variety of cells, they also become differentiated cells. In culture, however, all of them differentiate, without self-replication, even when the factors essential for self-replication are added.
Kawamoto overcame the problem with a flip-flop notion: progenitors may be proliferated by inhibiting their differentiation rather than by promoting their self-replication. Kawamoto took note of a transcription factor known as E2A. Researcher Tomokatsu Ikawa had demonstrated that in E2A-deficient mice, progenitors proliferate by self-replication without differentiation, while retaining their pluripotency. E2A had also been known to undergo functional suppression when bound to the Id protein. If the Id protein is forcibly overexpressed, the function of E2A will be inhibited, resulting in the self-replication and proliferation of progenitors. With this in mind, Kawamoto conducted experiments and found that the progenitors proliferated at a rate much higher than expected, while retaining their multipotency. It was also found that the progenitors thus proliferated would continue to produce T lymphocytes, B lymphocytes, phagocytes, and others for a long time after being returned to the body. Kawamoto says, “I believe that this method of progenitor proliferation will be widely applicable to cell therapy and bone marrow transplantation. We are now trying to develop a technique for producing functional T lymphocytes in vitro. I want to develop a new immunotherapy that will provide radical treatment for leukemia and cancers. It’s my dream.”