JP2019103391A - Human monocytic precursor cell for differentiating into only monocytic lineage and method for isolating the same - Google Patents

Abstract

An object of the present invention is to provide an isolated human monocyte precursor cell which does not differentiate into cells other than the monocyte lineage and has a proliferative ability. The present invention relates to obtaining precursor cells restricted to monocyte differentiation by isolating cells of Lin-CD34 + CD38 + CD10-CD123int / -CD45RA + CD135 + CLEC12AhiCD64hi from a cord blood sample or a bone marrow sample. [Selection diagram] FIG.

Description

  The present invention relates to human monocyte precursor cells, a method of isolating the same, and a method of using the same.

  Mononuclear phagocytes (MPs), including monocytes, macrophages and dendritic cells (DCs), play a central role in tissue homeostasis and immune responses. Most of the macrophages resident in normal tissues are derived from embryonic progenitor cells, while some of the macrophages in the intestine, heart, lung, mammary gland, dermis and bone are derived from monocytes.

Furthermore, inflammation promotes the differentiation of monocytes invading tissues into monocyte-derived macrophages and dendritic cells, which are involved in homeostatic defense responses and inflammatory diseases. Mouse monocytes are classified as classical LY6C ^hi monocytes and non-classical LY6C ^lo monocytes. LY6C ^lo monocytes present only in the blood but, LY6C ^hi monocytes present in blood and other tissues, where they differentiate into macrophages and dendritic cells. Also, most of LY6C ^lo monocytes is thought to be derived from LY6C ^hi monocytes. Human monocytes are classified into classical CD14 ⁺ CD16 ⁻ monocytes, intermediate CD14 ⁺ CD16 ⁺ monocytes, and non-classical CD14 ^{lo / −} CD16 ⁺ monocytes. By functional analysis and gene expression analysis, human CD14 ⁺ CD16 ^- have been shown to be CD16 ⁺ monocytes counterparts mice each LY6C ^hi monocytes and LY6C ^lo monocytes - monocytes and ^CD14 lo /.

  Monocytes differentiate in the bone marrow (BM) from hematopoietic stem cells (HSCs) via successive intermediate progenitor cells. Common monocyte precursor cells (cMoP) having differentiation potential localized to monocytic cells have been identified in mice (Non-patent Document 1). These cMoPs are considered to be derived from monocytes-dendritic cell precursor cells (MDPs) (Non-patent Document 2). In humans, differences in the expression patterns of IL-3 receptor (CD123), Flt3 (CD135), and CD45RA are used as an indicator to determine common myeloid precursor cells (CMP), erythroid precursor cells (MEP), and granulocytes Monocyte precursor cells (GMP) have been identified. Recently, human MDP has been identified in GMP fractions (Non-patent Document 3).

Hettinger, J. et al. Origin of monocytes and macrophages in a committed progenitor. Nat. Immunol. 14, 821-830 (2013). Fogg, D. K. et al. A clonogenic bone marrow progenitor specific for macrophages and dendritic cells. Science 311, 83-87 (2006). Lee, J. et al. Restricted dendritic cell and monocyte progenitors in human cord blood and bone marrow. J. Exp. Med. 212, 385-399 (2015).

  Due to the presence of human GMP and MDP, it was thought that human cMoP, which differentiated only into cells of monocytic lineage and not differentiated into any other hematopoietic cells, was present downstream, but so far human cMoP has been isolated It was not done. On the other hand, it is known that many of the macrophages responsible for disease pathogenesis and maintenance such as metabolic syndrome and cancer are derived from bone marrow monocytes, and monocytes for development of therapeutic agents and methods for these diseases, etc. There has been a strong demand for the isolation and identification of human cMoP that differentiates only into lineages.

Conventional human granulocyte-monocyte precursor cells (hereinafter cGMP), defined as Lin ^- CD34 ⁺ CD38 ⁺ CD10 ^- CD123 ^lo CD135 ⁺ CD45RA ⁺ cells, include granulocyte-monocyte precursor cells but T cells And some other cells that can differentiate into the lymphatic system. In addition, human MDP, which is a precursor cell that differentiates only to the monocyte-DC lineage, has been identified in the cGMP fraction (Non-patent Document 3). In other words, cGMP is actually a mixed population of true GMP and other progenitor cells. Therefore, one object of the present invention is to identify true GMP, and further to provide cMoP that differentiates only to the monocytic lineage.

Aspects of the present invention relate to the following matters.
1. An isolated human monocyte precursor cell which does not differentiate into cells other than the monocytic lineage and has proliferation ability.
2. An isolated human monocyte precursor cell according to the above 1, characterized in that it expresses CLEC12A and CD64.
3. CD34 ⁺ CD38 ⁺ CD10 ^- CD123 ^{int /} -CD45RA ⁺ CD135 ⁺ CLEC12A ^hi CD64 ^hi The isolated human monocytic precursor cells according to 1 or 2, characterized in that they have a phenotype of ^hi .
4. An isolated human monocyte precursor cell according to any one of the above 1 to 3, which is a cord blood or bone marrow derived cell.
5. A method of isolating human monocyte precursor cells, comprising
Comprising the step of isolating cells of Lin ^- CD34 ⁺ CD38 ⁺ CD10 ^- CD123-CD123 ^{int /} -CD45RA ⁺ CD135 ⁺ CLEC12A ^hi CD64 ^hi from the isolated cord blood sample or bone marrow sample, and the precursor cells have a non-monocyte lineage Not differentiating into the cells of the above, and the precursor cells have the ability to proliferate.
6. Isolating mononuclear cells (MNC) from the isolated cord blood sample or bone marrow sample and / or isolating Lin ⁻ cells from the isolated mononuclear cells, The method described in 5.
7. A method according to claim 5 or 6, wherein the isolation is performed using flow cytometry.
8. A macrophage related disease comprising, as an active ingredient, a substance that kills common monocyte precursor cells (cMoP), a substance that inhibits proliferation or differentiation of common monocyte precursor cells (cMoP), or a substance that inhibits monocyte or macrophage formation Pharmaceutical composition for use in the treatment of
9. The pharmaceutical composition according to the above 8, wherein the active ingredient is a small molecule, a nucleic acid, a polypeptide or an antibody.
10. A pharmaceutical composition for use in the treatment of a macrophage related disease, which comprises an anti-CLEC12A antibody and / or an anti-CD64 antibody as an active ingredient.
11. The pharmaceutical composition according to any of the above 8 to 10, wherein the disease is selected from the group consisting of cancer, bone related diseases, arteriosclerosis, fibrosis, inflammatory bowel disease and metabolic syndrome.
12. A pharmaceutical composition for use in treatment of cancer, comprising an anti-CLEC12A antibody and / or an anti-CD64 antibody as an active ingredient.
13. The pharmaceutical composition according to the above 12 for use in combination with another agent.
14. A kit for use in labeling or isolating human common monocytic precursor cells (cMoP), comprising an anti-CLEC12A antibody and / or an anti-CD64 antibody.
15. A method of screening for a substance that inhibits the production of monocytes, comprising
A method comprising culturing human cMoP in monocyte differentiation medium containing a test substance, and evaluating whether the test substance inhibits monocyte production.
16. A method of screening for a substance that inhibits the generation of osteoclasts, comprising
A method comprising culturing human cMoP in osteoclast differentiation medium containing a test substance, and evaluating whether the test substance inhibits osteoclast formation.
17. A method for screening a substance that affects differentiation, proliferation or survival of human cMoP, comprising
A method comprising the steps of culturing human cMoP in a medium containing a test substance, and evaluating whether the test substance affects the differentiation, proliferation or survival of human cMoP.
18. A method of screening for an antibody that affects human cMoP differentiation, proliferation or survival, comprising:
Identifying a molecule expressed on the cell surface of human cMoP,
Obtaining an antibody specific to said cell surface molecule,
A method comprising the steps of culturing human cMoP in a medium containing said antibody, and evaluating whether said antibody affects the differentiation, proliferation or survival of human cMoP.
19. A method of producing a mouse having human monocytes, comprising:
Transplanting human cMoP to the bone marrow of an immunodeficient mouse.
20. The method according to the above 19, further comprising the steps of intravenously administering human Flt3L, TPO, SCF and M-CSF to a mouse, and / or implanting human tumor cells into a mouse.
21. 22. The method according to any one of 19 to 20, wherein human cMoP is genetically modified.
22. Mice with human monocytes and no human granulocytes.
23. 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more of human monocyte precursor cells having a phenotype of CD34 ⁺ CD38 ⁺ CD10 ^- CD123 ^{int /} -CD45RA ⁺ CD135 ⁺ CLEC12A ^hi CD64 ^hi An isolated cell population comprising% or more, or 99% or more.
24. A method of treating a macrophage-related disease in a human patient, comprising: a substance that kills common monocyte precursor cells (cMoP) to the human patient, a substance that inhibits proliferation or differentiation of common monocyte precursor cells (cMoP), or A method of treatment comprising the step of administering a composition comprising as an active ingredient a substance that inhibits the production of monocytes or macrophages.
25. The therapeutic method according to the above-mentioned 24, wherein the active ingredient is a small molecule, a nucleic acid, a polypeptide or an antibody.
26. A method of treating a macrophage related disease in a human patient, comprising administering an anti-CLEC12A antibody and / or an anti-CD64 antibody to said human patient.
27. The treatment method according to any of the above 24 to 26, wherein the disease is selected from the group consisting of cancer, bone related diseases, arteriosclerosis, fibrosis, inflammatory bowel disease and metabolic syndrome.
28. A method of treating cancer in a human patient, comprising administering an anti-CLEC12A antibody and / or an anti-CD64 antibody to said human patient.
29. The treatment method according to the above-mentioned 28 comprising the step of administering another agent as a concomitant drug.
30. Substance that kills common monocytic precursor cells (cMoP), substance that inhibits common monocytic precursor cells (cMoP) proliferation or differentiation, or monocytes or macrophages in the manufacture of a medicament for use in the treatment of a macrophage-related disease Use of substances that inhibit production.
31. The use according to the above 30, wherein the substance is a small molecule, a nucleic acid, a polypeptide or an antibody.
32. Use of an anti-CLEC12A antibody and / or an anti-CD64 antibody in the manufacture of a medicament for use in the treatment of a macrophage related disease.
33. The use according to any of paragraphs 30-32, wherein the disease is selected from the group consisting of cancer, bone related diseases, arteriosclerosis, fibrosis, inflammatory bowel disease and metabolic syndrome.
34. Use of an anti-CLEC12A antibody and / or an anti-CD64 antibody in the manufacture of a medicament for use in the treatment of cancer.
35. 35. The use according to 34, wherein the medicament is for use in combination with other agents.

  According to one aspect of the present invention, it is possible to provide an isolated human monocyte precursor cell (human cMoP) which does not differentiate into cells other than the monocyte lineage and has proliferation ability.

Figure 7 shows a subset of conventional human GMP (cGMP) (see <Example 1> below). It shows the results of the MNC was analyzed by FCM - (a) ^Lin from UCB human. (B) ^Lin from BM and PB - MNC shows the results of analysis by FCM a. (C) Shows the myeloid colony forming ability of each UCB subpopulation. (D) Shows the results of Diff-Quick staining of each UCB precursor cell. The ability of the cGMP subpopulation to differentiate into myeloid and lymphoid cells is shown (see <Example 2>). (A) shows monocyte and DC differentiation from R1-R5 fractions ex vivo. (B) Shows the proportion of cell types in each fraction. (C) Shows the ability to differentiate each fraction into T cells ex vivo. (D) Shows the ability to differentiate each fraction into NK cells ex vivo. The analysis results of human cMoP and rGMP at the single cell level are shown (see <Example 3>). (A) FCM profile of representative colonies derived from cMoP. (B) Shows the FCM profile of a representative colony from rGMP. (C) Represents the proportion of monocytes and / or granulocyte colonies detected by FCM. (D) Shows the results of limiting dilution analysis of cMoP. (E) Shows the results of limiting dilution analysis of rGMP. FIG. 6 shows the proliferative capacity of human cMoP and rGMP (see Example 4). (A) It is a figure which shows the proliferation ability of the cell of each fraction labeled with CSFE. (B) Indicates the number of cells recovered from each cell, cultured as in (a) without CFSE labeling. (C) FCM profile of Ki67 expression in each cell population “immediately isolated”. The evaluation results of the in vivo differentiation ability of human cMoP and rGMP are shown (see <Example 5>). (A) shows an assay system for evaluating the differentiation ability of cMoP and rGMP in vivo. (B) FCM profile of progeny cells in bone marrow. (C) Percentage of cell types after differentiation of cMoP and rGMP. (D) shows the results of Diff-Quick staining of monocytes and granulocytes derived from cMoP-derived monocytes and rGMP. The differentiation relationship of premonocytes, cMoP and rGMP is shown (see <Example 6>). (A) Numbers indicate the purity of sorted cMoP and rGMP. The developmental stages of (b) sorted rGMP and (c) cMoP were analyzed. The results of transcriptional analysis of CMP, rGMP, cMoP, pre-monocytes, cMoP-derived monocytes, and PB monocytes are shown (see <Example 7>). (A) Heat map (log ₂ ) of RNA expression of monocyte specific gene in the indicated cells. (B) Shows relative mRNA levels of transcription factors involved in monocyte differentiation in the indicated cell populations. (C) Shows relative mRNA levels of chemokine receptors involved in cell migration. (D) Shows the results of principal component analysis of normalized gene expression profiles in the indicated cell populations. (E) Euclidean distance of the indicated cell population from cMoP-Mo or PB-Mo. (F) Comparison of human blood monocyte signatures in each cell population. The results of FCM analysis of R1, R2, R3 fractions are shown (see <Example 1>). A gating strategy is shown to detect granulocytes, monocytes and subsets of dendritic cells (see <Example 2>). The state of differentiation into monocyte-derived dendritic cells arising from R1, R2 and R3 fractions is shown (see <Example 2>). It is a figure which shows the lymphoid differentiation potential of R4 and R5 fraction (refer <Example 2>). It is a figure which shows the expression pattern of the cytokine receptor of R1-R5 fraction (refer <Example 2>). It is a figure which shows the expression pattern of CLEC12A and CD64 on cMoP of mouse | mouth, and GMP (refer <Example 2>). It is a figure which shows the comparison of the phenotype of MDP and R2-R5 fraction (refer <Example 2>). Shows a heat map (log ₂₎ of RNA expression of DC-specific genes in the progenitor cells (see <Example 7>). FIG. 5 schematically shows a revised version of the human myeloid cell differentiation pathway.

First, the abbreviations used in the present specification and their meanings are described below. Further, in the case where there is no explicit description, the following tissues, cells and the like represent human.
BM: bone marrow
cDC: conventional dendritic cell
CDP: Common DC progenitor cells (common DC progenitors)
CMP: common myeloid progenitor cells
cGMP: conventional granulocyte-monocyte progenitor cells (conventional granulocyte-monocyte progenitors)
cMOP: common monocyte progenitor cell
DC: dendritic cells
E: erythrocyte
FCM: multi-color flow cytometry
G: Granulocytes
GEMM: granulocytes-erythroblasts-macrophages-megakaryocytes (granulocytes-erythrocytes-macrophages-megakaryocyte)
GM: granulocyte-macrophage (granulocyte-macrophage)
GMDP: granulocyte-monocyte-DC precursor cell (granulocyte-monocyte-DC progenitor)
MDP: Monocyte-DC precursor cell (monocyte-DC progenitor)
MLP: multi-lymphoid progenitor cells
MNC: mononuclear cell
moDC: Monocyte-derived dendritic cell
MP: mononuclear phagocytes
PB: peripheral blood
pDC: plasmacytoid dendritic cell
preMo, pre-Mono: premonocytes (pre-monocyte)
rGMP: modified GMP population (revised GMP population)
TPO: Thrombopoietin
UCB: Umbilical cord blood (umbilical-cord blood)

<Human monocytic precursor cells>
The isolated human monocyte precursor cells (also described simply as "cMoP") of the present invention do not differentiate into cells other than the monocyte lineage, and have proliferative ability. Here, “does not differentiate into cells other than monocytic lineage” means cells that do not differentiate directly into granulocytes, dendritic cells and lymphocytes, but are cells of monocytic lineage (eg, pre-monocytes, monocytes , Macrophages, and monocyte-derived dendritic cells). That is, the isolated human monocyte precursor cells of the present invention do not have the ability to differentiate into granulocytes unlike GMP. Also, "having proliferation ability" means that cMoP can perform cell division and increase the number of cells. In addition, as long as the cell number continues to increase as a whole, part of cMoP may be differentiated into pre-monocytes, monocytes, macrophages and the like. Thus, in other words, the isolated human monocyte precursor cells of the present invention are monocyte precursor cells which do not have the ability to differentiate into granulocytes and have the ability to proliferate.

As mentioned above, conventional GMP (cGMP) is defined as Lin ^- CD34 ⁺ CD38 ⁺ CD10 ^- CD123 ^lo CD135 ⁺ CD45RA ⁺ cells, which is a mixed population of true GMP and other progenitor cells it is conceivable that.

  The present inventors subdivide the human cGMP population to identify cMoP, C-type lectin family (CLEC9A, CLEC12A, DC-SIGN, etc.), cytokine receptor (CD115, CD116, etc.), chemokine receptor (chem.) Various cell surface markers expressed on human monocytes, macrophages, and DCs were screened, including CX3CR1, CXCR4 etc.) and others (eg CD64). In combination with the in vitro culture results of the subdivided population, we identified CD64 and CLEC12A as useful markers to fragment cGMP to identify the cMoP fraction in cGMP.

More specifically, we focused on the expression of FcγRIA (CD64) and C-type lectin CLEC12A expressed in monocytes and macrophages to identify human cMoP and used these markers Conventional GMP (cGMP) was subdivided and examined in detail and divided into four subpopulations. As a result of conducting detailed examination for each subpopulation, CLEC12A ^hi CD64 ^hi subpopulation (population highly expressing CLEC12A and CD64) was identified as cMoP. Also, the subset of CLEC12A ^hi CD64 ^int cells contained in cGMP was redefined as modified (true) human GMP (rGMP). As shown in the Examples below, rGMP produces granulocytes and monocytes but does not differentiate into DCs or lymphocytes. CD64 of cGMP population ^- only fraction has the ability to differentiate into DC and lymphocytes. The inventors have shown that, by analysis of expression of multiple genes using microarray data, rGMP to cMoP, pre-monocytes, and monocytes are continuously generated, but this process is probably unrelated to MDP. I made it. The human cMoP and true GMP (rGMP) identified by the inventors and their analysis provide new insights on human myeloid cell differentiation pathways.

The cMoP of the present invention has a phenotype of CD34 ⁺ CD38 ⁺ CD10 ^- CD123 ^{int /} -CD45RA ⁺ CD135 ⁺ CLEC12A ^hi CD64 ^hi . In addition, human cMoP of the present invention is present in human umbilical cord blood and bone marrow, but is not detected in peripheral blood.

  As shown in detail in the examples, the present inventors identified human cMoP and rGMP, and further investigated these characteristics etc., and reviewed the myeloid cell developmental pathway (FIG. 16). In this reviewed pathway, rGMP differentiates into granulocytes and cMoP, and cMoP generates all monocyte subsets via premonocytes.

  As mentioned above, the cMoP of the present invention is a precursor cell that differentiates only to the monocytic lineage, and has proliferative ability. Also, cMoP is present in human umbilical cord blood and bone marrow but is not detected in peripheral blood. Although recent studies using mice show that most of the macrophages that are resident in tissues and responsible for maintaining homeostasis originate from the embryonic stage (yolk sac, liver), but are responsible for the formation and maintenance of disease pathologies such as metabolic syndrome and cancer. It has been revealed that many of the macrophages are derived from bone marrow monocytes. For example, macrophages responsible for intravascular plaque formation responsible for arteriosclerosis, macrophages involved in the formation of adipose tissue, osteoclasts responsible for inflammatory bone destruction, immunosuppressive macrophages (TAM associated within cancer tissue) And so on are derived from bone marrow monocytes. Thus, the cMoP of the present invention is a precursor cell that becomes a source of monocytes, and thus can be used for the development of a therapy for the above-mentioned diseases that targets cMoP specific markers. In addition, even if targeting or removing cMoP in the cell differentiation lineage does not affect dendritic cell differentiation, concerns such as susceptibility to infection can be extremely limited.

<Method of isolating human monocyte precursor cells>
One aspect of the invention relates to a method for the isolation of human common monocytic progenitor cells (cMoP). The isolation method comprises the step of isolating cells highly expressing CLEC12A and CD64 from an isolated cord blood sample or bone marrow sample, and in particular Lin ^- CD34 ⁺ CD38 ⁺ CD10 ^- CD123-CD123 ^{int /} -CD45RA It is preferable to include the step of: ⁺ CD135 ⁺ CLEC12A ^hi CD64 ^hi cells. The meaning of symbols (+ /-/ hi / int / lo etc.) representing the presence / absence (positive / negative) and amount (intensity) of cell surface markers is known in the art, and those skilled in the art If so, an apparatus such as flow cytometry can be appropriately set to isolate a target cell. For example, CLEC12A ^hi CD64 ^hi cells are the fractions with the highest expression of CLEC 12A and CD64 as compared to the other subpopulations in cGMP, as shown in FIG. 1a. Such a fraction uses, for example, BD FACSAria (registered trademark) III cell sorter manufactured by BD Biosciences as a flow cytometer, using “Lin ⁻ CD34 ⁺ CD38 ⁺ CD10 ⁻ CD123 ^{int / −} CD45RA ⁺ CD135 as the gate condition. ^It can be isolated from the cGMP population by setting “CLEC12A ^hi CD64 ^hi ”. In addition, gate conditions can also be suitably adjusted according to the number of cells required, and purity. The isolation method further comprises the step of isolating mononuclear cells (MNC) from the isolated cord blood sample or bone marrow sample, and / or from the isolated mononuclear cells, the Lin ⁻ cells are isolated from the isolated mononuclear cells. More preferably, the step of releasing is included.

The method for isolating human monocyte precursor cells of the present invention is, as one aspect,
(Step 1): isolating mononuclear cells (MNC) from the isolated cord blood sample or bone marrow sample;
(Step 2): isolating Lin ⁻ cells from mononuclear cells isolated in step 1;
(Step 3) Step 2 in isolated Lin ^{- Lin} from cells ^{- CD34 + CD38 + CD10 - CD123} int / - out of CD45RA ⁺ CD135 ⁺ CLEC12A ^hi CD64 ^hi cells isolating the process, at least one It is preferred to include a step, more preferably two or more steps, and even more preferably all steps. Among the steps 1 to 3, at least the step 3 is preferably included.

  Cell isolation can be performed, for example, using magnetic separation or flow cytometry.

  As a method of isolating mononuclear cells (MNC) from a cord blood sample or a bone marrow sample, for example, a method by density gradient centrifugation using a lymphocyte separation solution and the like can be mentioned.

“Lin ⁻ ” means that the differentiation antigen is negative, and is expressed in known mature blood cells (T cells, B cells, NK cells, myeloid cells, erythroid cells, etc.) It does not have a specific surface antigen, that is, it does not express the surface antigen on the cell surface, and in the present invention, CD2, CD3, CD11b, CD16, CD19, CD56 and CD235ab are negative. Say Lin ^- as a method of isolating the cells, can be mentioned the method described in Example.

By isolating cells of Lin ^- CD34 ⁺ CD38 ⁺ CD10 ^- CD123 ^{int /} -CD45RA ⁺ CD135 ⁺ CLEC12A ^hi CD64 ^hi , human cMoP which does not differentiate to cells other than monocytic lineage and has proliferation ability is obtained. be able to. The human cMoP according to the present invention does not necessarily have to be isolated at a purity of 100%, and a human single having a phenotype of CD34 ⁺ CD38 ⁺ CD10 ^- CD123-CD123 ^{int /} -CD45RA ⁺ CD135 ⁺ CLEC12A ^hi CD64 ^hi. A cell population comprising at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sphere precursor cells is also included in the subject matter of the present invention.

<Method for proliferating human cMoP>
The human monocyte precursor cells (cMoP) of the present invention have proliferative ability. The method for growing human cMoP is not particularly limited, but for example, culture in a medium in the presence of a cytokine cocktail is preferable. Examples of cytokines included in the cytokine cocktail include hFlt3L, hTPO, hSCF, M-CSF and the like. The medium preferably contains, for example, methyl cellulose, serum, Iscove's modified Dulbecco's medium, RPMI-1640 and the like. seeding concentration of cMoP is in the medium is preferably from 1 × 10 ⁴ cells / mL~4 × 10 ⁴ cells / mL, and more preferably 1 × 10 ⁴ cells / mL~2 × 10 ⁴ cells / mL. The concentration of the cytokine cocktail is preferably, for example, about 25 to 100 ng / mL. The culture temperature is, for example, preferably 30 to 40 ° C, more preferably 36 to 38 ° C, and still more preferably 37 ° C. In addition, from the viewpoint of maintaining or amplifying cells more suitably, it is preferable to perform medium exchange at an appropriate time. The frequency of medium replacement is not particularly limited as long as cells can be maintained or amplified, but may be, for example, every 1 to 5 days, preferably every 3 days. In the medium exchange, all or part of the medium may be exchanged. In addition, passaging may be performed if necessary in the culture of cMoP, and the passaging frequency is not particularly limited as long as cells can be maintained or amplified, and may be appropriately performed at the timing when the cell population becomes large. For example, it can be every 4 to 12 days, preferably every 6 to 10 days.

<Method for inducing differentiation of human cMoP>
The human monocyte precursor cells (cMoP) of the present invention have the ability to differentiate into monocytic cells downstream. In inducing differentiation to monocytic cells, for example, precursor cells are cultured in Iscove's modified Dulbecco's medium containing 10% FCS, 100 U / ml penicillin / streptomycin, and a cytokine cocktail. Here, hFlt3L, hTPO, and hSCF (FTS) can be used as a cytokine cocktail. To induce monocyte-derived DCs, cells can be cultured in media supplemented with GM-CSF in addition to FTS.

<Pharmaceutical composition>
One aspect of the present invention is effective to a substance that kills common monocyte precursor cells (cMoP), a substance that inhibits proliferation or differentiation of common monocyte precursor cells (cMoP), or a substance that inhibits the production of monocytes or macrophages. The present invention relates to a pharmaceutical composition for use in the treatment of a macrophage related disease, which is contained as a component. Such pharmaceutical compositions can comprise small molecules, nucleic acids (antisense oligonucleotides, siRNAs, aptamers, ribozymes etc.), polypeptides or antibodies as active ingredients. In addition, the pharmaceutical composition can include a pharmaceutically acceptable carrier, stabilizer, or excipient, and the administration route thereof is appropriately determined according to the type of drug to be used and the subject to be administered. be able to.

  One aspect of the present invention relates to a pharmaceutical composition comprising an anti-CLEC12A antibody and / or an anti-CD64 antibody as an active ingredient. As described above, since cMoP expresses CLEC12A and CD64, a pharmaceutical composition containing these antibodies as an active ingredient can target cMoP specific markers, so it can be used to treat monocyte-derived macrophage related diseases. It can be used. Clec12A (Gene Bank accession No. NM_001207010) is a C-type lectin and binds to uric acid crystals derived from dead cells. In addition, it has an ITIM motif in cells. CD64 (Gene Bank accession No. NM — 000566, also known as FcγRIA) is a glycoprotein and binds to human IgG1 and IgG3.

  A pharmaceutical composition containing anti-CLEC12A antibody and / or anti-CD64 antibody as an active ingredient can be used as an anticancer agent. The life span of monocytes and tumor associated macrophages (TAM) is usually considered to be several days to about three weeks, and tumor associated macrophages are partially or completely removed by using the pharmaceutical composition of the present invention. It is understood by those skilled in the art that (TAM) may also be partially or completely removed. The pharmaceutical composition according to the present invention can also be used in combination with other drugs such as anti-cancer agents as a concomitant drug.

  "Antibodies" can be polyclonal or monoclonal and can be isolated from any suitable biological source, such as mice, rats, sheep and canines. The antibody may also be a digested fragment of a monoclonal antibody or a specific portion thereof. Also, the antibody or antibody fragment may contain a peptide sequence identified by screening by phage display method or the like.

  The antibody may be either a chimeric antibody, a humanized antibody or a human antibody. Methods for producing these antibodies are known to those skilled in the art. For production of human antibodies, for example, KM mice (Kyowa Hakko Kirin) and XenoMouse II (Amgen) can be used. Although the class and subclass of the antibody are not particularly limited, they are preferably of the IgG class. The antibody is preferably an antibody having a constant region of a human antibody, particularly a human Fc region.

  The above mentioned antibodies may suitably be antibodies having neutralizing activity, antibody dependent cellular cytotoxicity (ADCC) activity, and / or complement dependent cytotoxicity (CDC) activity. ADCC activity and CDC activity can be increased or decreased by modifying amino acid residues in the Fc region of an antibody. In addition, an antibody, such as an antibody drug complex (ADC), can be conjugated to an anticancer drug, radioactive substance, cytotoxic substance or the like, and one or more additional domains to provide different activities or auxiliary functions. Chimeric antibodies or fusion antibodies may be generated. The above-described antibodies may also be bispecific antibodies that recognize two types of antigens.

  In addition, one aspect of the present invention relates to a small molecule, nucleic acid (antisense oligonucleotide, siRNA, aptamer, ribozyme, etc.) or polypeptide capable of reducing or increasing expression of CLEC12A and / or CD64 as an active ingredient. It relates to the pharmaceutical composition containing. For example, a pharmaceutical composition containing an active ingredient capable of reducing the expression of CLEC12A and / or CD64 can be preferably used for the treatment of a macrophage-related disease derived from monocytes.

  As used herein, a macrophage-related disorder refers to a disorder caused (or made malignant or refractory) by the unwanted activity of macrophages. The macrophage-related disease is not particularly limited, but, for example, cancer (immunosuppression, angiogenesis, metastasis), bone-related disease, neurodegeneration (ALS, MS), Alzheimer's disease, Rett syndrome, arteriosclerosis, dyslipidemia, lung Alveolar proteinosis, asthma, fibrosis, rheumatism, lupus nephritis, psoriasis, inflammatory bowel disease, Crohn's disease, diabetes, obesity, metabolic syndrome and the like. Examples of cancer include blood cancer, breast cancer, endometrial cancer, ovarian cancer, prostate cancer, lung cancer, gastric cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, esophagus cancer, bladder cancer, melanoma, colon cancer, renal cell carcinoma And non-Hodgkin's lymphoma. Examples of bone-related diseases include osteoporosis, osteopetrosis, bone metastasis of cancer, osteoarthritis, rheumatoid arthritis, hypercalcemia, bone fracture, Behcet's disease and the like. Fibrosis includes, for example, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, myocardial fibrosis, skin fibrosis and the like. Examples of inflammatory bowel disease include Crohn's disease and colitis such as ulcerative, granulomatous, ischemic, radioactive and infectious colitis.

<Methods of treatment and use in the manufacture of medicaments>
One aspect of the invention relates to a method of treating a macrophage related disease in a human patient. Such a method inhibits the production of a substance that kills common monocyte precursor cells (cMoP), a substance that inhibits proliferation or differentiation of common monocyte precursor cells (cMoP), or generation of monocytes or macrophages in human patients And the step of administering a composition containing the substance as an active ingredient. The active ingredient may be, but is not limited to, small molecule, nucleic acid, polypeptide or antibody. The method according to the invention preferably comprises the step of administering an anti-CLEC12A antibody and / or an anti-CD64 antibody to a human patient. The macrophage related disease may be, but is not limited to cancer, bone related disease, arteriosclerosis, fibrosis, inflammatory bowel disease or metabolic syndrome.

  One aspect of the invention relates to a method of treating cancer in a human patient. Such methods may comprise the step of administering an anti-CLEC12A antibody and / or an anti-CD64 antibody to a human patient. In addition, the method according to the present invention can also include the step of administering another drug such as an anticancer drug as a concomitant drug.

  One aspect of the present invention is a substance that kills common monocytic precursor cells (cMoP), a substance that inhibits proliferation or differentiation of common monocytic precursor cells (cMoP) in the manufacture of a medicament for use in the treatment of a macrophage-related disease Or the use of substances which inhibit the production of monocytes or macrophages. The substance used may be, but is not limited to, small molecules, nucleic acids, polypeptides or antibodies. The use according to the invention is preferably the use of an anti-CLEC12A antibody and / or an anti-CD64 antibody in the manufacture of a medicament for use in the treatment of a macrophage related disease. The macrophage related disease may be, but is not limited to cancer, bone related disease, arteriosclerosis, fibrosis, inflammatory bowel disease or metabolic syndrome.

  One aspect of the invention relates to the use of an anti-CLEC12A antibody and / or an anti-CD64 antibody in the manufacture of a medicament for use in the treatment of cancer. Moreover, the manufactured drug may be for use in combination with another drug such as an anticancer drug as a concomitant drug.

<Kit>
One aspect of the present invention relates to a kit for use in labeling or isolation of human common monocyte precursor cells (cMoP), comprising an anti-CLEC12A antibody and / or an anti-CD64 antibody. The kit may further contain a label, a reagent, a reaction container, an instruction manual, and the like.

<Use of Human Monocyte Progenitor Cells>
The cMoP of the present invention can be used for screening and the like in the development of methods for treating diseases associated with monocyte-derived macrophages. About the usage method of cMoP, the following aspect is mentioned, for example. In the following screening etc., any one known to those skilled in the art is known to promote or suppress the differentiation and proliferation of cMoP, promote or suppress the formation of monocytes, inhibit the differentiation / function to tumor-associated macrophages (TAM), etc. It can be evaluated by the method of

  (A) Human cMoP is isolated and subjected to microarray analysis, cell surface molecules expressed in cMoP are extracted, and an antibody against the same molecule is prepared. The same antibody is used to identify those that inhibit the differentiation and function of cMoP, monocytes and paraneoplastic macrophages (TAM).

Specifically, for example,
Identifying a molecule expressed on the cell surface of human cMoP,
Obtaining an antibody specific to said cell surface molecule,
Affecting the differentiation, proliferation or survival of human cMoP, comprising the steps of culturing human cMoP in a medium containing said antibody, and assessing whether said antibody affects differentiation, proliferation or survival of human cMoP Methods of screening for antibodies can be mentioned.

  Here, "affecting" differentiation, proliferation or survival of human cMoP refers to promoting or suppressing differentiation, proliferation or survival of human cMoP. Promoting or suppressing the differentiation, proliferation or survival of human cMoP can be evaluated by any method known to those skilled in the art.

Also, as another aspect,
Affecting the differentiation, proliferation or survival of human cMoP, including the steps of culturing human cMoP in a medium containing the test substance, and assessing whether the test substance affects differentiation, proliferation or survival of human cMoP Also included are methods of screening for substances. As a test substance, various cytokines, antibodies, polypeptides, polynucleotides, low molecular weight compounds and the like can be used.

  (B) Human cMoP is isolated, and a culture system that induces differentiation into monocytes in vitro is used to elucidate the differentiation mechanism from cMoP to monocytes. Furthermore, test substances such as various cytokines, antibodies, polypeptides, polynucleotides, low molecular weight compounds and the like are added to the same culture system to screen for those which inhibit or promote differentiation into monocytes.

In particular,
A method of screening for a substance that inhibits monocyte production comprising the steps of culturing human cMoP in a monocyte differentiation medium containing a test substance, and evaluating whether the test substance inhibits monocyte production or not. It can be mentioned.

  (C) A test system such as various cytokines, antibodies, polypeptides, polynucleotides, low molecular weight compounds and the like is added to a culture system which isolates human cMoP and induces differentiation into osteoclasts in vitro to osteoclasts. Screening for those that inhibit or promote the differentiation of

In particular,
A substance which inhibits osteoclast formation, which comprises the steps of culturing human cMoP in an osteoclast differentiation medium containing a test substance, and evaluating whether the test substance inhibits osteoclast formation. The screening method is mentioned. Whether to inhibit the generation of osteoclasts can be evaluated by any method known to those skilled in the art.

  (D) Analysis is performed in an in vivo evaluation system using a cytokine, an antibody, a low molecular weight compound or the like selected by the screening of (b) or (c) above.

  Specifically, umbilical cord blood or human cMoP is transplanted into an immunodeficient animal (NSG mouse, NOG mouse, BRGS mouse, MISTRG mouse etc.), and the "hematopoietic system is replaced by human-derived" humanized mouse ". The above antibodies, low molecular weight compounds and the like are administered to screen for those which inhibit or promote the differentiation ability to monocytes and macrophages in vivo. Furthermore, human tumor cell lines are transplanted, and the effects of antibodies, low molecular weight compounds and the like are examined using monocytes infiltrating into the tumor, differentiation of the monocytes to TAM, and increase of the tumor as indexes.

  (E) The cytokines, antibodies, low molecular weight compounds and the like selected by the screening in the above (b) or (c) are analyzed in an in vivo evaluation system using tumor-bearing mice. The mouse cMoP, monocytes and TAM are targeted and analyzed using the above-mentioned antibody and the mouse counterpart of the low molecular weight compound.

  (F) Human cMoP is isolated and subjected to metabolic analysis using MS (mass spectrometry) or the like to identify the metabolic pathway characteristic of cMoP and to block or activate the pathway, ie, the function of cMoP Search for small molecule drugs that inhibit or promote.

  (G) Human cMoP is isolated and subjected to microarray analysis, a master regulatory gene characteristic of cMoP is identified, cMoP is derived from iPS using the gene, and applied to treatment of various diseases.

  (H) Human cMoP is isolated, screened for cytokines and low molecular weight compounds that promote differentiation to M2 macrophages in vitro, and therapeutic application to inflammatory diseases.

  (I) Human cMoP is isolated, and it is applied to search for cytokines and low molecular weight compounds that promote differentiation to monocyte-derived dendritic cells and treatment of various diseases.

  (J) Human cMoP is isolated and a specific chemokine receptor or adhesion molecule gene is deleted using genome editing technology such as CRISPR-Cas9. Since proliferated and differentiated monocytes do not express specific chemokine receptors or adhesion molecule genes, they can suppress only transfer and accumulation to specific tissues.

  (K) Also, one aspect of the present invention comprises the step of transplanting human cMoP into the bone marrow of an immunodeficient mouse, preferably a hyperimmune deficient mouse (eg, NSG mouse, NOG mouse, BRGS mouse, MISTRG mouse, etc.) The present invention relates to a method for producing a mouse having human monocytes. By this method of preparation, mice having human monocytes but not human granulocytes are obtained. Furthermore, such methods of producing mice may include the steps of administering human Flt3L, TPO, SCF and M-CSF to mice, and / or implanting human tumor cells into mice. The cMoP used in the method for producing the mouse may be genetically modified by a technique known to those skilled in the art, and in particular, it may be genetically modified by a technique such as CRISPR-Cas 9 described in the above (j) Good. Thus, a mouse having human monocytes according to the present invention may have genetically modified human monocytes and cells derived therefrom. The humanized mouse produced can be used, for example, for the in vivo evaluation system described in (d) above.

<Use of human monocyte precursor cells as a cell drug>
The isolated human cMoP may be expanded ex vivo and then transplanted into a patient in need of monocyte lineage cell replacement. At that time, cMoP can be genetically modified ex vivo and then transplanted to a patient. Thus, the present invention is also directed to pharmaceutical compositions comprising human cMoP. Specifically, for example, a method of treating cMoP from a patient, a step of optionally modifying cMoP, and an amount necessary for transplantation Culturing cMoP to obtain c.

  Hereinafter, the present embodiment will be described in detail by way of examples, but the present embodiment is not limited to the following examples.

First, samples, culture methods, analysis methods, and the like used in Examples are described below.
<Sample>
Human umbilical cord blood samples were provided from Japan Red Cross Society Kanto Koshinetsu umbilical cord blood bank, and human BM samples were obtained from AllCells.

<Cell isolation, sorting, and flow cytometric analysis>
Fresh mononuclear cells (MNC) derived from UCB were isolated by density centrifugation using a lymphocyte separation solution (d = 1.077, Nacalai Tesque). Next, the MNCs are CD2 (RPA-2.10), CD3 (UCHT1), CD11b (ICRF44), CD16 (3G8), CD19 (HIB19), CD56 (HCD56), CD235ab (HIR2) (all from Biolegend), and CD14. (RMO52) (manufactured by Beckman Coulter) was reacted with a PE-Cy5-labeled antibody (Ab) against a cell line marker, and further reacted with anti-Cy5 microbeads (Miltenyi Biotech). Cells not previously expressed with cell line markers (Lin ⁻ ) were crudely purified using the “deplete” program of autoMACS Pro Separator (Miltenyi Biotech). Lin ^- cells were CD34 (581; APC-Cy7), CD10 (HI10a; BV 412), CD123 (6H6; PerCP-Cy 5.5), CD45RA (HI100, BV510), CD135 (BV10A4H2; PE), CLEC12A (50C1; FITC) ), CD64 (10.1; APC) (all available from Biolegend), and antibodies against CD38 (HB7; PE-Cy7) (BD). In order to evaluate the expression of cytokine receptor in each precursor cell, Lin ^- cells were CD34 (APC-Cy7), CD10 (BV412), CD123 (PerCP-Cy5.5), CD45RA (BV510), CLEC12A (FITC), An antibody against CD64 (APC) and the cytokine receptor CD115 (9-4D2-1E4; PE), CD116 (4H1; PE), CD117 (104D2; PE) (all manufactured by Biolegend), or CD110 (REA 250; PE) ) (Miltenyi Biotech). In order to compare R1 to R5 with MDP, Lin ^- cells were CD34 (APC-Cy7), CD10 (BV412), CD123 (PerCP-Cy5.5), CD45RA (BV711), CLEC12A (FITC), CD64 (APC), Reacted with antibodies to CD115 (PE), CD116 (Biotin), and Streptavidin (BV510). In order to evaluate the cell cycle status of each precursor cell, Lin ^- cells are fixed by a transcription factor staining buffer set (eBioscience), and CD34 (APC-Cy7), CD10 (BV412), CD123 (PerCP-Cy5.5), Antibodies were stained for CD45RA (BV510), CLEC12A (PE), CD64 (APC) (all from Biolegend), CD38 (PE-Cy7), and Ki67 (B56; FITC) (all from BD).

<Myeloid cell colony forming ability assay>
200 sorted progenitor cells were suspended in 200 μl of Iscove's modified Dulbecco's medium and mixed with 2 ml of methylcellulose medium (Metho Cult H4435 Enriched from Stemcell Technologies) supplemented with 2 μl of 10 ng / ml hTPO (Kyowa Hakko Kirin). 1 ml of cell suspension is added to one well of a 12-well plate (double experiment), cultured for 10 days, and macrophages (M), granulocytes / macrophages (GM), granulocytes (G), granulocytes / Colonies were counted based on erythroblast / macrophage / megakaryocytes (GEMM), or erythroblast (E) classification.

<Morphological analysis>
For morphological analysis, each of 1,000 sorted precursor cells was closely adhered to a glass slide at 700 rpm for 5 minutes (Cytospin 4 manufactured by Thermo Scientific). Cells were stained using the Diff-Quik staining kit according to the manufacturer's recommended procedure (Sysmex). Images were acquired at 100 × magnification (Leica DM 4500B).

<Culture and analysis of cells>
For the myeloid cell differentiation assay, 2 × 10 ³ progenitor cells were cultured in 50 μl IMDM (Sigma) containing 10% FCS, 100 U / ml penicillin / streptomycin (Nacalai Tesque), and a cytokine cocktail . Here, in the present specification, 50 ng / ml hFlt3L (Miltenyi Biotech), 50 ng / ml hTPO (Kyowa Hakko Kirin), and 100 ng / ml hSCF (Miltenyi Biotech) (FTS conditions) are used as cytokine cocktails It can be done. Cells were cultured in medium supplemented with FTS and 50 ng / ml GM-CSF (Miltenyi Biotech) to induce monocyte-derived DC. In order to detect myeloid cells, differentiated cells are expressed as CD66b (G10F5; PerCP-Cy5.5), HLA-DR (L243; BV510), CD14 (M5E2; BV412), CD16 (3G1; PE-Cy5), CD123 (6H6; FITC), CD1c (L161; PE-Cy7), Streptavidin (APC-Cy7) (all from Biolegend), BDCA-2 (AC144; Biotin) (Miltenyi Biotech), and CD11c (B-ly6; APC) ) (BD) stained with antibodies.

  To assess T cell and B-NK cell differentiation ability, Tst4 / Dll4 and Tst4 stromal cells were treated with 10% FCS, 100 U / ml penicillin / streptomycin (Nacalai Tesque), 1% non-essential amino acids (Gibco), And 100% RPMI-1640 medium (Sigma) (complete RPMI-1640 medium) containing 1% and 1% sodium pyruvate (Gibco) in 96 well plates. Two days later, 10 cells from each progenitor cell were added to stromal cell culture plates, cultured for 4 weeks, and half of the medium was changed weekly. To the media, 5 ng / ml Flt3L and 5 ng / ml IL-7 (Miltenyi Biotech) were added to induce T cells. 100 ng / ml SCF, 20 ng / ml IL-7, 50 ng / ml TPO, 10 ng / ml IL-2 (Miltenyi Biotech) were added to induce B-NK cells. Furthermore, after 2 weeks of culture, the cells were passed through a 40 μm pore size Nitex mesh (Nippon Science and Chemical Instruments) and transferred onto fresh stromal cells in complete RPMI-1640 medium containing the appropriate cytokine cocktail. After 4 weeks of culture, cells were harvested and stained with antibodies against hCD45 and CD3 to detect T cells, or stained with antibodies against hCD45, CD33, CD19, and CD56 to detect B-NK cells .

In order to evaluate the proliferative capacity of cMoP and rGMP, 1 × 10 ^{3 of} each precursor cell is sorted, labeled with CFSE (Life Technologies), and 2 ml of methylcellulose medium (MethoCult H4435-Enriched by Stemcell Technologies) is used. It was cultured for 7 days.

  Cells were analyzed using FACSAria III or FACSCanto II (BD) and FlowJo software (TreeStar).

<Single cell analysis>
One hundred cMoP or rGMP were cultured for 5 days and 8 days, respectively, using 2 ml of methylcellulose medium (Metho Cult H 4 435-Enriched by Stemcell Technologies) supplemented with 10 ng / ml hTPO and 10 ng / ml hFlt3L. Single colonies were picked up using a P20 pipette, suspended in 200 μl PBS in round bottom 96 well plates and pipetted up and down well. The cell suspension was spun down at 2,000 rpm and reacted with the above described antibodies for detection of myeloid cells. Cells were analyzed using FACSAria III and FlowJo software (TreeStar).

<In vivo reconstitution assay>
BRGS mice were irradiated with X-rays (0.5 Gy) (Faxitron). The next day, suspend sorted cMoP or CD64 ^int GMP (5 × 10 ^{3 to} 3 × 10 ⁴ ) in 10 μl PBS and use customized Ito microsyringe (Ito Corp.) in the marrow cavity of BRGS mice Direct injection. On the same day, human recombinant proteins Flt3L (Peprotech), TPO (Kyowa Hakko Kirin), SCF (Peprotech), and M-CSF (Biolegend) (8 μg each) were intravenously administered and continued for 4 consecutive days . Cells derived from cMoP and rGMP were analyzed at 5 and 7 days after transplantation, respectively.

<Microarray analysis and bioinformatics>
CMoP, rGMP, and CMP were sorted and pooled from cord blood of at least 20 volunteers. CDNA was generated from 6.622 ng of total RNA and amplified using Ovation® Pico WTA System V2 (NuGEN Technologies). The yield of amplified cDNA was measured using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific). Cyanine-3 (Cy3) labeled cDNA is prepared from 2.0 μg of cDNA using SureTag Complete DNA Labeling Kit (Agilent), then Amicon Ultra-0.5 mL centrifugal filter (Merck for purification and concentration of DNA Filtered using Millipore). Dye uptake and cDNA yield were checked using a NanoDrop ND-2000 spectrophotometer. Adjust 2 μg of Cy3-labeled cDNA to 50 μl with 1 × Agilent blocking reagent and 1 × Agilent hybridization buffer and use SurePrint G3 Human GE 8 × 60 K Microarray Ver2.0 (Agilent in a rotating Agilent hybridization oven for 17 hours at 65 ° C.) Hybridisation). After hybridization, the microarray was washed with GE Wash Buffer 1 (Agilent) for 1 minute at room temperature and with GE Wash buffer 2 (Agilent) for 1 minute at 37 ° C., then immediately dried by short centrifugation. Slides were scanned immediately after washing on an Agilent SureScan Microarray Scanner (G2600D) using a single color scan setting for 8 x 60 k array slides (scan area: 61 x 21.6 mm, scan resolution: 3 μm, dye channel settings : Green, setting of PMT: 100%). Analyze the scanned image using Feature Extraction Software 11.5.1.1 (Agilent), using default parameters, to subtract the background and obtain the processed signal intensity with spatial tilt removed did. All procedures to extract multiple expression data were performed by Takara Bio Inc. Raw data were analyzed using Subio Platform (ver 1.18.4667, Subio Inc.). Gene set enrichment analysis (GSEA) was performed using GSEA (v. 2.2.0 Broad Institute). Human blood monocyte signatures were extracted from published microarray data (GSE 35459). Gene sets for cMoP, rGMP, and CMP were extracted from microarray data as described above.

<Example 1: Segmentation of conventional GMP (cGMP)>
As described above, cGMP is defined as Lin ^- CD34 ⁺ CD38 ⁺ CD10 ^- CD123 ^lo CD135 ⁺ CD45RA ⁺ cells, mainly including granulocyte-monocyte precursor cells, but also cells capable of differentiating into T cells. Partially included. In addition, MDP, a precursor cell localized to the monocyte-DC lineage, has recently been identified in the cGMP fraction. Thus, as a result of screening for cytokine receptors, chemokine receptors, and others, the present inventors selected C-type lectins CLEC12A and FcγR (CD64) as markers for examining human cGMP subset (FIG. 1). Both markers are highly expressed in mouse and human monocytes.

FIG. 1 a shows the results of analysis of Lin ⁻ MNC from human UCB and FIG. 1 b shows the results of analysis of human BM and PB derived Lin ⁻ MNC using a flow cytometer. The numerical values described in or near the gate area indicate the frequency of each subpopulation. cGMP, CMP, and MLP were CD34 ⁺ CD38 ⁺ CD123 ^lo CD10 ⁻ CD135 ⁺ CD45RA ⁺ , CD34 ⁺ CD38 ⁺ CD123 ^lo CD10 ⁻ CD135 ⁺ CD45 RA ⁻ , and CD34 ⁺ CD38 ⁻ CD45 RA ⁺ CD10 ⁺ , respectively.

Furthermore, based on the expression of CLEC12A and CD64, cGMP derived from human umbilical cord blood (UCB) can be expressed as CLEC12A ^hi CD64 ^hi (R2), CLEC12A ^hi CD64 ^int (R3), CD64 ⁻ using multicolor flow cytometry (FCM). CLEC12A ⁺ (R4) and CD64 ^- CLEC12A ^- (R5) subdivided into four fractions of (Figure 1a). In addition, the CLEC12A ^hi CD64 ^hi fraction in the Lin ⁻ CD34 ⁻ CD38 ⁺ CD10 ⁻ CD123 ^lo CD135 ⁺ CD45RA ⁺ fraction was also added to the definition (R1). The phenotypes of R1 and R2 were identical except that R2 cells expressed CD34 and R1 cells did not.

The detailed phenotype of the cGMP subdivided population was as follows: CD34 ^- CD38 ⁺ CD123 ^lo CD10 ^- CD64 ^hi CLEC12A ^hi CD135 ⁺ CD45RA ^hi (R1), CD34 ⁺ CD38 ⁺ CD10 ^- CD123 ^{int /- CD45RA + CD135 + CLEC12A hi CD64 hi} (R2), CD34 + CD38 + CD10 - CD123 int / - CD45RA + CD135 + CLEC12A hi CD64 int (R3), CD34 + CD38 + CD10 - CD123 int / - CD45RA + CD135 + CD64 - CLEC12A ⁺ GMP (R4), and CD34 ⁺ CD38 ⁺ CD10 ^- CD123 ^{int /} -CD45RA ⁺ CD135 ⁺ CD64 ^- CLEC12A ^- (R5).

  These five fractions (R1 to R5) were also detected in human bone marrow (BM) (FIG. 1 b top). On the other hand, they were hardly detected in human peripheral blood (PB) (FIG. 1 b lower).

Colony forming units (CFU) were assayed ex vivo in order to evaluate the myeloid-erythroblast differentiation potential of the R1 to R5 fractions according to the method of the myeloid cell colony formation assay described above. The sorted precursor cells (1 × 10 ² ) were cultured in methylcellulose medium containing a cytokine cocktail, and colonies were counted 10 days later. As a ^{control, Lin - CD34 + CD38 - CD45RA} + CD10 + multipotent lymphoid progenitors (MLP) and ^{Lin - CD34 + CD38 + CD123 lo} CD10 - CD135 + CD45RA - common myeloid progenitors (CMP) as well (See FIG. 1a). FIG. 1 c shows bone marrow colony forming ability of each UCB subpopulation. Each bar represents the average number of colonies per cultured cell (1 × 10 ² ), and the meanings of abbreviations in the figure are as follows. M: macrophage, GM: granulocyte-macrophage, G: granulocyte, E: erythroblast, GEMM: granulocyte-erythroblast-macrophage- megakaryocyte. The R3 and R4 fractions gave rise to macrophage (M), granulocyte-macrophage (GM), and granulocyte (G) colonies, but more macrophage colonies resulted from R4 fraction than R3 fraction . Interestingly, the R2 and R5 fractions only formed M colonies, and the R1 fraction slightly formed myeloid colonies. As expected from previous cGMP reports, none of the fractions formed E or GEMM colonies (FIG. 1c). FIG. 1 d shows the results of Diff-Quick staining of each UCB precursor cell. The original magnification is 100 × and the scale bar represents 10 μm.

  FIG. 8 shows the results of analysis of R1 to R3 fractions by FCM. We gated the R1-R3 fraction again for the CD34 / CD38 and cell line marker (Lin) / CD34 profiles. FIG. 8 shows the R1 to R3 fractions in three colors, where dark gray indicates R1 fraction, light gray indicates R2 fraction, and black indicates R3 fraction. Of note, the myeloid differentiation potential of the R1-R3 fractions correlated with their CD34 / CD38 expression levels, cell size (Fig. 8a-c), and morphology (Fig. 1d).

Analysis of the CD34 / CD38 and lineage (Lin) / CD34 profiles of the R1 to R3 fractions showed that the cells of the R3 fraction were CD34 ⁺ CD38 ⁺ and the cells of the R2 fraction were downregulated CD34 expression (CD34 ^int CD38 ⁺ ), R1 had almost completely lost CD34 (CD34 ⁻ CD38 ⁺ ) (FIG. 8 a, b). The volume of cytoplasm gradually increased from R3 to R1. This correlated positively and negatively with their CD64 expression levels and myeloid-based colony forming ability, respectively (FIGS. 1 b, c and 8 c). In terms of cell morphology, the R2-R5 fraction had a typical precursor cell morphology with round, dilated, and relatively small cytoplasmic volume (FIG. 1d).

  In addition, each data of FIG. 1 performed independent experiment at least 20 times about a, 4 times in B, 4 times in B, 3 times in PB, c and 3 times in c and d. Each data in FIG. 8 is based on three independent experiments.

  These results indicated that cGMP can be fractionated into four independent subpopulations with different myeloid cell differentiation potential.

Example 2: Identification of human cMoP and rGMP
As in Example 1, the R1-R5 fractions had different myeloid cell differentiation potentials, so in vitro culture to detect granulocytes, monocytes and subsets of DC using the above gating strategy The system was launched (Figure 9a). Fractions (1 × 10 ³ ) sorted from UCB were collected from Fms-like tyrosine kinase receptor 3 ligand (Flt3L) (50 ng / mL), thrombopoietin (TPO) (50 ng / mL), and stem cell factor (SCF) (100 ng / mL). The cells are cultured (RTS) for 2 days (R1), 4 days (R2) or 8 to 10 days (R3 to R5) in the presence of F. mL), granulocytes (CD66b ⁺ ), monocytes (CD14 ⁺ CD16 ⁻ , It analyzed about CD14 ^{< + >} CD16 ^{< + >} and CD14 ^{< - >} CD16 ^{<+ >} ), pDC (CD123 ^<+> BDCA ^<2+> ) and cDC (CD141 ^hi CD11 c < ^- > and CD1 c < ⁺ > CD11 c ^{< +>} ). FIG. 2a represents monocyte and DC differentiation from R1-R5 fractions ex vivo, and FIG. 2b shows the percentage of cell types in the fractions. In FIG. 2, CD14 ⁺ CD16 ⁻ monocytes are described as CD14 ⁺ mono, and CD14 ⁺ CD16 ⁺ and CD14 ⁻ CD16 ⁺ monocytes are described as CD16 ⁺ mono. The numbers in or above the gate area indicate the proportion of each fraction. The R3 and R4 fractions showed the ability to differentiate into granulocytes in line with the results of the in vitro colony formation assay (FIG. 1c), while the R1, R2 and R5 fractions showed the ability to differentiate into granulocytes Not shown (Figures 2a, 2b). It should be noted that the R1 to R3 fractions produced monocyte subsets such as classical CD14 ⁺ CD16 ⁻ monocytes, intermediate CD14 ⁺ CD16 ⁺ monocytes and non-classical CD 14 ^{lo / −} CD16 ⁺ monocytes , pDC, ^CD141 hi cDC or CD1c ⁺ cDCs DC subsets comprising did not produce (Figure 2a, 2b). The major monocyte subset derived from these progenitors is not classical CD14 ⁺ CD16 ⁻ monocytes but intermediate CD14 ⁺ CD16 ⁺ cells, which is also consistent with recent reports.

Furthermore, FIG. 10 shows the state of differentiation into monocyte-derived dendritic cells produced from R1, R2, and R3 fractions. FIG. 10a shows that culture of R1-R3 fractions with 50 ng / mL of FTSGM (GM-CSF, a representative cytokine that induces FTS and monocyte-derived DC) results in CD14 ⁺ CD1c ⁺ monocyte-derived DC. It represents. In addition, when cultured with FTSM (FTS and M-CSF), the differentiation of R1 to R3 fractions into CD14 ⁺ CD16 ⁺ and CD14 ^{lo / −} CD16 ⁺ monocytes (ie, CD16 ⁺ monocytes) is slightly enhanced. It confirmed (FIG. 10 b, c). Related to this, the peak of monocyte production of the R1 fraction was faster on day 2 than the peak of R2, which takes at least 4 days (data not shown). This finding suggests that both R1 and R2 subsets are progenitor cells specialized for monocyte differentiation and that R1 is a downstream precursor of R2. On the other hand, neither CD64 ^- R4 and R5 fraction is produced a all monocytes and DC subsets (Fig. 2a, 2b). ^CD141 hi cDCs derived from R4 and R5 fraction had expressed CLEC9A (Figure 9b). These results suggested that the R4 and R5 fractions were the main sources of DC in cGMP, as the R1 to R3 fractions showed little ability to differentiate into DCs.

Next, the lymphoid differentiation ability of the R1 to R5 fractions was examined. Figure 2c shows Tst4 stromal cells, SCF (100 ng / ml), TPO (50 ng / ml) in the presence of Dll4 ⁺ Tst4 stromal cells, IL-7 (5 ng / ml) and Flt3L (5 ng / ml). 10 cells of each fraction were cultured in 48 wells of a 96 well plate for 1 month in the presence of ml), IL-2 (10 ng / ml), and IL-7 (40 ng / ml) . Each well was analyzed for CD3 ⁺ T cells (FIG. 2c) or CD19 ⁺ B cells and CD56 ⁺ NK cells (FIG. 2d). Each bar in the figure represents the percentage of wells positive for CD3 ⁺ T cells (FIG. 2c) and CD19 ⁺ B cells and / or CD56 ⁺ NK cells (FIG. 2d), indicating that ND was not detected. FIG. 2c shows the presence of the Flt3L and IL-7 on Tst4 stromal cells expressing Delta-like ligand 4 (Dll4), which is a Notch ligand essential for T cell differentiation, to evaluate T cell differentiation ability. Below, the result of culturing 10 cells of each fraction is shown. MLP was used as a control. The R1 to R3 fractions had no ability to differentiate into T cells, while the R4 and R5 fractions produced significant numbers of T cells. FIG. 2 d shows the results of evaluating the ability to differentiate into B-NK by culturing 10 cells of each fraction in the presence of SCF, TPO, IL-2 and IL-7 on Tst4 stromal cells. Indicates As a comparison control, B-NK precursor cells defined as Lin ^- CD34 ⁺ CD38 ⁺ CD123 ^int CD10 ⁺ CD45RA ⁺ cells were used. B cells and NK cells were defined as CD33 ⁻ CD19 ⁺ and CD33 ⁻ CD56 ⁺ cells, respectively.

FIG. 11 shows the lymphoid differentiation potential of R4 and R5 fractions. FIG. 11 a shows the results of analysis by FCM of CD3 ⁺ T cells derived from MLP, R4 fraction and R5 fraction. The cells were cultured in the same manner as in FIG. 2b. FIG. 11 b shows FCM analysis results of B cells derived from B-NK precursor cells, R4 fraction or R5 fraction, B-NK cells and NK cells. The cells were cultured in the same manner as in FIG. 2c. It shows that ND was not detected.

  Interestingly, the lymphoid differentiation potential of R1-R5 subsets tended to decrease as the expression of CLEC12A increased and was completely abolished by the further acquisition of CD64 (Figures 2c, 2d and 11a, b). The lymphoid differentiation ability of R4 and R5 suggests the ability to differentiate into T cells remaining in cGMP.

  The data in FIG. 2, FIG. 10 and FIG. 11 are all from three independent experiments.

Human and mouse cGMP both differentiate into DC in addition to granulocytes and monocytes. In addition to the myeloid cell differentiation ability, these precursor cells have an apparent differentiation ability to lymphoid cells. For example, mouse cGMP expresses high levels of lymphoid genes and gives rise to both T cells and B cells in vitro and in vivo. Human UCB-derived cGMP also has the ability to differentiate into T cells. As described above, the present inventors combined and cultured Tst4 stromal cells / Dll4 and Tst4 stromal cells, which are optimized methods, with a plurality of cytokine cocktails in order to evaluate the lymphocyte differentiation potential of precursor cells. In addition, human rGMP was found to be incapable of differentiating into lymphoid cells. Furthermore, rGMP did not have differentiation ability to DC, and DC differentiation ability was conserved in CD64 ^- cGMP (R4 and R5 fractions). In addition to the ability to differentiate into monocytes and DCs, R4 and R5 also had the ability to differentiate into granulocytes and / or lymphocytes. Since R5 has pluripotency and the human MLP-like cell group lacks the ability to differentiate into erythroblast lineage, the data in the above example show that the branch to erythroblast lineage is It matches the model that occurs at the stage of Lin ⁻ CD34 ⁺ CD38 ⁻ multipotent progenitor cells.

  Furthermore, for the R1 to R5 fractions, CD110 (TPO receptor), CD115 (M-CSF receptor), CD116 (GM-CSF receptor), CD117 (SCF receptor), and CD123 (IL3 receptor α), etc. The results of examining the expression characteristics of other cytokine receptors are shown in FIG. In FIG. 12, the black parts represent the expression pattern of each receptor. The expression pattern of the cytokine receptor in the R1 fraction has been previously reported for premonocytes (Breton, G. et al. Circulating precursors of human CD1c + and CD114 + dendritic cells. J. Exp. Med. 212, 401-413 (2015).) And expressed CD115, CD116, and CD117 at detectable levels. On the other hand, the expression patterns observed in the R2 and R3 fractions have not been reported at all.

  Based on these phenotypic and developmental analyzes, it is assumed that R1 is the same pre-monocyte fraction as recently defined pre-monocytes, R2 is cMoP and R3 is true GMP (modified GMP, rGMP) It concluded (Table 1).

(Comparison between human cMoP etc. and mouse cMoP etc)
FIG. 13 shows the expression patterns of CLEC12A and CD64 on cMoP and GMP in mice. The numerical values described in or near the gate area indicate the frequency of each subpopulation. CLEC12A and CD64 were also expressed in mouse cMoP and GMP, as seen in human cMoP and rGMP.

  Mouse and human cMoP are progenitor cells that produce only monocytes at the single cell level, and both express CD64, CLEC12A and CD117. On the other hand, human cMoP expresses CD135, but mouse cMoP does not express CD135. The differentiation origin of mouse cMoP is derived from MDP, but human cMoP is derived from rGMP, and it is likely to be differentiated without MDP (because rGMP has no DC differentiation ability). This difference may simply reflect the differences in myeloid cell differentiation pathways between mouse and human. Alternatively, mouse cGMP may be considered to contain rGMP. The presence of rGMP not capable of DC differentiation is consistent with that monocyte-derived DC is derived from a differentiation source different from cDC and pDC in the differentiation pathway, and the branch of monocyte-derived DC and DC subset is rGMP. It suggests that something is happening upstream.

Recently, in cGMP in human UCB and BM, granulocyte-monocyte-DC precursor cells (GMDP), MDP, and DC common precursor cells (CDP) restricted to DC differentiation were identified. The authors compared the phenotypes of CDP, MDP, cMoP, and rGMP. FIG. 14 shows a phenotypic comparison of MDP and the R2-R5 fractions. CDP appears as the CD123 ^hi fraction (the second panel at the top of Figure 1a) and is not included in cGMP. FIG. 14a shows that expression of CLEC12A and CD64 divides MDP into CD64 ^int and CD64 ⁻ subpopulations. FIG. 14 b shows that MDP overlaps with cGMP. FIG. 14 c shows the proportion of each fraction in the MDP. MDP was identified in R3 (rGMP) and R4, but not in R2 (cMoP) and R5. This, MDP is ^{CD64 int} and CD64 ^- suggesting that a heterogeneous population of some having subpopulations indicates that may be divided into (Figure 14a-c) of, MDP granulocytes differentiation potential . CD64 ^int MDP accounted for 14.4 ± 5.0% of rGMP.

Next, the myeloid colony forming ability of CD64 ^int and CD64 ^- MDP was examined. Figures 14d and 14e show myeloid colony forming ability. GMDP was used as a control. The sorted precursor cells (1 × 10 ² ) were cultured in a methylcellulose medium containing a cytokine cocktail, and the number of colonies was counted 10 days later. FIG. 14 d shows the proportion of each cell in 1 × 10 ² cultured. FIG. 14 e shows the analysis result by FCM. The abbreviations in FIG. 14 are as follows. M: macrophage, GM: granulocyte-macrophage, G: granulocyte.

As expected from the overlapping phenotypes, CD64 ^int and CD64 ^- MDP showed myeloid colony forming ability but not erythroblast cell forming ability, just like R3 (rGMP) and R4. Monocytes and granulocytes were respectively generated in culture (FIG. 1 c, FIG. 14 d, 14 e). Only CD64 ^- MDP produced DC under FTS conditions (data not shown). From the above we conclude that MDP is a heterogeneous population and CD64 ^int MDP should be defined as rGMP.

  In addition, each data of FIGS. 12-14 are based on three independent experiments.

Example 3: Clonal analysis of human cMoP and rGMP
In addition, the properties of cMoP and rGMP cells were assessed at the single cell level. cMoP and GMP were sorted, cultured for 5 and 8 days in methylcellulose medium containing cytokine cocktail, respectively, and single colonies picked up for FCM analysis (see "Single cell analysis" above) . Representative colony cells were analyzed for granulocytes and monocytes. FIG. 3 depicts the properties of human cMoP and rGMP at the single cell level. FIG. 3a shows FCM profiles of representative colonies from 100 cMoP and FIG. 3b from 100 rGMP. The numbers in the gate area indicate the proportion of each group. FIG. 3 c represents the proportion of monocytes and / or granulocyte colonies detected by FCM. The meanings of the abbreviations in FIG. 3 are as follows. ND: No colony detected, Mono: Monocyte, Gra: Granulocyte, undiff. : Undifferentiated cell.

  Of note, all single colonies derived from cMoP consisted of 100% monocytes (Fig. 3a, 3c). Although some single colonies derived from rGMP contained only monocytes (33.2 ± 3.5%), the rest were both monocytes and granulocytes (21.7 ± 2.8%), Granulocytes alone (37.6 ± 1.9%) or undifferentiated cells (6.6 ± 2.3%) were included (FIG. 3 b, 3 c). The frequency of progenitors capable of differentiating into two cell lineages at single cell level was higher than that of human MDP (12.5%) and comparable to mouse CDP (18.0%).

  Furthermore, FIG. 3 d shows the results of limiting dilution analysis of cMoP, and FIG. 3 e shows the results of limiting dilution analysis of rGMP. Cells were cultured in methylcellulose medium containing cytokine cocktail for 7 days. In (d) and (e), the vertical axis represents the percentage of colony negative wells, the horizontal axis represents the number of cultured cells, and the dotted line represents 37% detection failure at the expected clonogenic frequency. Also, the numbers in parentheses represent the average clone frequency of (d) cMoP and (e) rGMP, respectively.

  The colony formation ability was estimated by limiting dilution analysis, and under the same culture conditions, colonies were formed at a frequency of 1 out of 1.85 cMoP and 1 out of 1.04 rGMP respectively (FIG. 3 d , 3e). This human cMoP precursor frequency is reported for human preDC (1 / 7.84), mouse CDP (1 / 7.1 to 8.6), and mouse cMoP (1 / 3.9 to 9.2). It was equivalent to These results indicate that human cMoP is a unipotent progenitor cell as reported for mouse cMoP, and also that bipartite bipotent that rGMP can generate monocytes and granulocytes at the single cell level. It has been shown to contain progenitor cells.

  The data in FIG. 3 are from three (a to c) and four (d, e) independent experiments.

<Example 4: proliferation ability of human cMoP and rGMP>
Since progenitor cells differentiate with division, the inventor evaluated the proliferative ability possessed by monocytes, premonocytes (preMo), cMoP and rGMP by CFSE dilution assay. FIG. 4a shows the results of labeling these populations with CFSE, culturing 1 × 10 ³ cells of each fraction in methylcellulose medium containing appropriate cytokine cocktail for 7 days, and analyzing differentiated cells by FCM. Show. As assessed by CFSE dilution, cell division was not confirmed in monocytes and premonocytes, but cMoP and rGMP showed a large proliferative capacity.

FIG. 4 b shows the results of culturing and collecting each cell in the same manner as FIG. 4 a without CFSE labeling. Consistent with the observation of Figure 4a, but after 10 ^three pre monocytes cultured for 7 days the cells were not at all recovered, 31,000 ± from the same culture conditions 10 ³ CMOP 3,875 Individual cells were obtained (Figure 4b).

FIG. 4 c shows the FCM profile of Ki67 expression in each of the “immediately isolated” cells. Pre-monocytes "immediately isolated" showed no proliferative capacity (Fig. 4c) but they were Ki67 ⁺ (Fig. 4a, 4b). Since cells in G1, S, G2, and M phases are all Ki67 ⁺ , this label may reflect the last G2 and M cells after proliferating as cMoP in vivo. unknown. These results show a clear difference in proliferative ability (a measure of commonly used progenitor cells) between rGMP, cMoP and premonocytes.

  Each data in FIG. 4 is based on at least three independent experiments.

Example 5 Differentiating ability of cMoP and rGMP in vivo
In order to evaluate the differentiation ability of cMoP and rGMP in vivo, the inventor developed a new assay system (Fig. 5a). The inventors have determined that B6.B has NOD-type Sirpa. Rag2 ^{− / −} Il2 rg ^{− / −} mice (BRGS mice) were irradiated with 0.5 Gy, and cMoP or rGMP (5 × 10 ^{3 to} 3 × 10 ⁴ cells) were directly transplanted into the BM of the mice. On the same day, intravenous administration of human recombinant proteins Flt3L, TPO, SCF and M-CSF to the mice was started and continued for 4 consecutive days. FIG. 5 b shows the results of analysis of cMoP or rGMP BM progeny cells transplanted on day 5 (cMoP) and day 7 (rGMP) by FCM. In FIG. 5c, cMoP produced only monocytes, which were CD14 ⁺ CD16 ⁻ monocytes (CD14 ⁺ mono, 26.3 ± 5.3%) and CD14 ⁺ CD16 ⁺ and CD14 ⁻ CD16 ⁺ monocytes (CD16). ⁺ Mono, 54.2 ± 7.07%). rGMP produces both monocytes (CD14 ⁺ mono, 30.4 ± 8.7%; CD 16 ⁺ mono, 11.4 ± 5.03%) and granulocytes (15.2 ± 2.34%) (Fig. 5b, 5c). In this connection, the cell morphology of monocytes and granulocytes derived from cMoP and rGMP was confirmed by Diff-Quick staining (FIG. 5d). In FIG. 5 d, the original magnification is 100 × and the scale bar represents 10 μm. From these results, it was shown that cMoP is a precursor cell specialized in monocyte differentiation in vivo, and rGMP is a monocyte-granulocyte precursor cell.

  The data in FIG. 5 is based on at least three independent experiments.

As mentioned above, human monocytes are subdivided into three subsets: classical CD14 ⁺ CD16 ⁻ monocytes, intermediate CD14 ⁺ CD16 ⁺ monocytes and non-classical CD 14 ^{lo / −} CD16 ⁺ monocytes Be done. From a functional analysis and transcriptome analysis, the human classical CD14 ⁺ CD16 ^- that CD16 ⁺ monocytes, probably correspond respectively to LY6C ^hi and LY6C ^lo monocytes in mice - the nonclassical ^CD14 lo / Is clear. In this context, murine LY6C ^hi monocytes, but differentiate into spontaneously LY6C ^lo monocytes in the blood, which in certain conditions, is considered to be true in the case of human monocytes. At steady state, the major fraction of human monocytes is CD14 ⁺ CD16 ⁻ . However, treatment with M-CSF increases the number of CD14 ⁺ CD16 ⁺ monocytes, and CD14 ^{lo / −} CD16 ⁺ monocytes gradually expand. This suggests a stepwise differentiation pathway ^: CD14 ⁺ CD16 ⁻ , CD14 ⁺ CD16 ⁺ , and CD14 ^{lo / −} CD16 ⁺ monocytes. When cultured in vitro, or after in vivo transplantation of cMoP and rGMP, one of the major monocyte subsets produced from them was CD14 ⁺ CD16 ⁺ monocytes. In this context, human fetal liver CD34 ⁺ progenitor cells transplanted MISTG mice and MISTRG mouse blood, spleen, lungs, and the ^liver, CD14 + CD16 ^- rather CD14 ⁺ CD16 ⁺ cells The main single than monocytes It was a sphere subset.

Example 6: Continuous Differentiation of rGMP, cMoP and Premonocytes
Next, the top-bottom relationship of the differentiation pathway between rGMP, cMoP and premonocytes was examined. Since rGMP was able to differentiate into granulocytes and monocytes, we hypothesized that rGMP would differentiate into cMoP by losing its ability to differentiate granulocytes and further into premonocytes. The rGMP was sorted at 100% purity, and the cells were cultured in vitro for 2 days under FTS conditions (FIG. 6a). Numbers in FIG. 6a indicate the purity of sorted cMoP and rGMP. FIG. 6 shows the results of culturing the sorted (b) rGMP and (c) cMoP for 24 hours under FTS conditions and analyzing their developmental stages. Progeny cells from rGMP contained a population very similar to cMoP and premonocytes (FIG. 6b), and it was found that progeny cells from cMoP were very similar to premonocytes (FIG. 6c). cMoP never produced rGMP. These results strongly suggest that rGMP continuously differentiates into cMoP and premonocytes.

  Note that the data in FIG. 6 is based on at least three independent experiments.

<Example 7: monocyte signature possessed by cMoP>
Sorted and purified rGMP, cMoP, premonocytes (preMo), to determine whether cMoP has a monocyte signature (ie, whether they express a gene suitable for monocyte differentiation). The results of a comprehensive gene expression analysis of cMoP-derived CD14 ⁺ monocytes (cMoP-Mo) and peripheral blood CD14 ⁺ monocytes (PB-Mo) are shown in FIG. 7. As a comparative control, CMP that has the ability to differentiate into multiple myeloid cells and is not confined to monocyte differentiation was included. The level of monocyte-specific gene expression gradually increases with monocyte differentiation, and among precursor cells, the monocyte-specific gene expression level is lowest in CMP, weak in rGMP, and highest in cMoP. (Figure 7a). This trend was not observed for DC specific gene expression (FIG. 15). FIG. 7 b shows relative mRNA levels of transcription factors involved in monocyte differentiation, and FIG. 7 c shows relative mRNA levels of chemokine receptors involved in migration. In Figures 7b and 7c, data are described as relative to CMP. PU, a transcription factor important for monocyte development. The expression levels of 1, IRF8, CEBPB, and KLF4 leveled off with cMoP (Fig. 7b), and the expression levels of chemokine receptors CX3CR1 and CCR2, which are required for monocyte migration, were premonocyte , CMoP-derived CD14 ⁺ monocytes, and peripheral blood CD14 ⁺ monocytes were further increased in stages (FIG. 7 c). FIG. 7 d shows the results of principal component analysis of normalized gene expression profiles in the indicated cells. The plot flow (location of points) of each population based on principal component (PC) analysis was consistent with the process of continuous monocyte differentiation and correlated with the in vitro and in vivo findings. In connection with this, Euclidean distance, which represents the distance of differentiation stage between populations, was also calculated (Fig. 7e). The distance from cMoP-Mo or PB-Mo was gradually decreasing from CMP to premonocytes. In addition, expression levels of gene sets (human monocyte signature) known to be enriched in CD14 ⁺ monocytes in human blood using human gene set enrichment analysis (GSEA) are derived from cMoP-derived CD14 ⁺ monocytes , Pre-monocytes, cMoP, rGMP, and CMP were compared (FIG. 7 f). Specifically, the human blood monocyte signature was generated from the top 200 genes of human blood CD14 ⁺ monocytes (GSE 35459), and gene set enrichment analysis (GSEA) was cMoP vs rGMP, rGMP vs CMP, cMoP vs pre monocytes, Performed to compare premonocytes versus genes enriched in cMoP-Mo. In FIG. 7 f, p represents normal p value, and NES represents gene enrichment score. Data were obtained from 20 pooled samples. The human monocyte signature is significantly increased with the degree of differentiation towards monocytes (ie, cMoP-derived monocytes> pre monocytes>cMoP>rGMP> CMP), which are monocytes in each progenitor cell. Supports the differentiation level of From these results, cMoP was identified as a precursor cell with differentiation ability limited to monocytes, and a continuous pathway from rGMP to cMoP and monocytes was strongly supported.

  While preferred embodiments of the present invention are shown herein, it will be apparent to those skilled in the art that such embodiments are provided for the purpose of illustration only and should be apparent to those skilled in the art. Various modifications, changes and substitutions may be made without departing from the invention. It should be understood that various alternative embodiments of the invention described herein may be used in practicing the invention. Also, the contents described in all the publications, including the patents and patent application documents referred to in the present specification, are incorporated by reference in the same manner as the contents specified in the present specification. It should be interpreted.

According to the present invention, human cMoP has been identified and further details of the myeloid cell differentiation pathway have been clarified. Since monocytes and monocyte-derived macrophages cause various inflammatory diseases including metabolic syndrome and tumor growth, the present invention provides methods for treating and preventing the above-mentioned diseases targeting cMoP and monocytes. Application of is possible.

Claims (23)

  An isolated human monocyte precursor cell which does not differentiate into cells other than the monocytic lineage and has proliferation ability.   The isolated human monocyte precursor cell according to claim 1, characterized in that it expresses CLEC12A and CD64. The isolated human monocytic precursor cell according to claim 1 or 2, characterized by having a phenotype of CD34 ⁺ CD38 ⁺ CD10 ^- CD123 ^{int /} -CD45RA ⁺ CD135 ⁺ CLEC12A ^hi CD64 ^hi .   The isolated human monocyte precursor cells according to any one of claims 1 to 3, which are cord blood or bone marrow derived cells. A method of isolating human monocyte precursor cells, comprising
Comprising the step of isolating cells of Lin ^- CD34 ⁺ CD38 ⁺ CD10 ^- CD123-CD123 ^{int /} -CD45RA ⁺ CD135 ⁺ CLEC12A ^hi CD64 ^hi from the isolated cord blood sample or bone marrow sample, and the precursor cells have a non-monocyte lineage Not differentiating into the cells of the above, and the precursor cells have the ability to proliferate. Claim: 1. Isolated mononuclear cells (MNC) from the isolated cord blood sample or bone marrow sample and / or isolating Lin ⁻ cells from the isolated mononuclear cells. Item 5. The method according to item 5.   7. The method of claim 5 or 6, wherein the isolation is performed using flow cytometry.   A macrophage related disease comprising, as an active ingredient, a substance that kills common monocyte precursor cells (cMoP), a substance that inhibits proliferation or differentiation of common monocyte precursor cells (cMoP), or a substance that inhibits monocyte or macrophage formation Pharmaceutical composition for use in the treatment of   The pharmaceutical composition according to claim 8, wherein the active ingredient is a small molecule, a nucleic acid, a polypeptide or an antibody.   A pharmaceutical composition for use in the treatment of a macrophage related disease, which comprises an anti-CLEC12A antibody and / or an anti-CD64 antibody as an active ingredient.   The pharmaceutical composition according to any one of claims 8 to 10, wherein the disease is selected from the group consisting of cancer, bone related diseases, arteriosclerosis, fibrosis, inflammatory bowel disease and metabolic syndrome.   A pharmaceutical composition for use in treatment of cancer, comprising an anti-CLEC12A antibody and / or an anti-CD64 antibody as an active ingredient.   A pharmaceutical composition according to claim 12 for use in combination with other agents.   A kit for use in labeling or isolating human common monocytic precursor cells (cMoP), comprising an anti-CLEC12A antibody and / or an anti-CD64 antibody. A method of screening for a substance that inhibits the production of monocytes, comprising
A method comprising culturing human cMoP in monocyte differentiation medium containing a test substance, and evaluating whether the test substance inhibits monocyte production. A method of screening for a substance that inhibits the generation of osteoclasts, comprising
A method comprising culturing human cMoP in osteoclast differentiation medium containing a test substance, and evaluating whether the test substance inhibits osteoclast formation. A method for screening a substance that affects differentiation, proliferation or survival of human cMoP, comprising
A method comprising the steps of culturing human cMoP in a medium containing a test substance, and evaluating whether the test substance affects the differentiation, proliferation or survival of human cMoP. A method of screening for an antibody that affects human cMoP differentiation, proliferation or survival, comprising:
Identifying a molecule expressed on the cell surface of human cMoP,
Obtaining an antibody specific to said cell surface molecule,
A method comprising the steps of culturing human cMoP in a medium containing said antibody, and evaluating whether said antibody affects the differentiation, proliferation or survival of human cMoP. A method of producing a mouse having human monocytes, comprising:
Transplanting human cMoP to the bone marrow of an immunodeficient mouse.   20. The method of claim 19, further comprising administering human Flt3L, TPO, SCF and M-CSF to a mouse and / or implanting human tumor cells into a mouse.   21. The method according to any one of claims 19 to 20, characterized in that human cMoP is genetically modified.   Mice with human monocytes and no human granulocytes. 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more of human monocyte precursor cells having a phenotype of CD34 ⁺ CD38 ⁺ CD10 ^- CD123 ^{int /} -CD45RA ⁺ CD135 ⁺ CLEC12A ^hi CD64 ^hi An isolated cell population comprising% or more, or 99% or more.