Method for promoting proliferation of hematopoietic stem cells and hematopoietic progenitor cells

Abstract

This method for promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells comprises a step for co-culturing hematopoietic stem cells or hematopoietic progenitor cells, and at least one immortalized hematopoietic support cell selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages.

Description

A method for promoting proliferation of hematopoietic stem cells and hematopoietic progenitor cells.

[0001] The present invention relates to a method for promoting the proliferation of hematopoietic stem cells and hematopoietic progenitor cells.

[0002] Hematopoietic stem cell transplantation is one of the treatments used for blood cancers that are difficult to treat with conventional treatments such as anticancer drug therapy and radiation therapy. The number of patients with major blood cancers such as leukemia, malignant lymphoma, and multiple myeloma is increasing year by year, and it is known that the incidence rate increases with age. Therefore, in Japan, where the population is aging, the demand for hematopoietic stem cell transplantation is expected to increase further.

[0003] There are three main methods for collecting hematopoietic stem cells: bone marrow collection, peripheral blood stem cell collection, and umbilical cord blood collection. Bone marrow collection typically requires a three- to four-day hospital stay and pain lasts for about a week after surgery. Peripheral blood stem cell collection typically requires a five- to six-day hospital stay and is painful. Both procedures place a strain on the donor's body. For this reason, the number of transplants has been declining in recent years, likely due to the burden of hospitalization and pre- and post-hospital visits for collection, especially for those in their prime working years who find it difficult to take time off work. Meanwhile, umbilical cord blood collection, while not burdening the donor because it utilizes industrial waste generated during childbirth, presents challenges for adult recipients, including the fact that the number of hematopoietic stem cells contained in umbilical cord blood is only about half the required number, and there is a high incidence of hematopoietic stem cell engraftment failure.

[0004] To solve the above problems, various methods for providing a stable supply of hematopoietic stem cells have been developed. For example, a method for expanding hematopoietic stem cells and hematopoietic progenitor cells by ex vivo co-culturing hematopoietic support cells collected from a donor with hematopoietic stem cells and hematopoietic progenitor cells derived from umbilical cord blood (Non-Patent Document 1). Stable ex vivo production of hematopoietic stem cells can also solve the problem of poor collection in autologous peripheral blood stem cell transplantation, which uses a patient's own hematopoietic stem cells for treatment of multiple myeloma. While expansion of hematopoietic stem cells promises lifelong bone marrow reconstruction, expansion of hematopoietic progenitor cells promises to reduce transplant accidents due to delayed engraftment of platelets and leukocytes, which is a problem in cord blood transplantation and other transplants. Therefore, a stable supply of hematopoietic progenitor cells is also extremely important.

[0005] De Lima et al. Cord-Blood Engraftment with Ex Vivo Mesenchymal-Cell Culture. The New England Journal of Medicine. 2012, 367, 2305-2315.

[0006] However, the method disclosed in Non-Patent Document 1 relies on donors for the hematopoietic supporting cells used, resulting in an unstable supply and inconsistent quality, and furthermore, there is the problem of invasiveness during collection, making it insufficient as a means for providing a stable supply of hematopoietic stem cells.

[0007] Therefore, an object of the present invention is to provide a novel method for promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells in vitro.

[0008] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that co-culturing immortalized hematopoietic supporting cells with hematopoietic stem cells or hematopoietic progenitor cells promotes the proliferation of hematopoietic stem cells or hematopoietic progenitor cells, and thus completed the present invention.

[0009] That is, the present invention is as follows: (1) A method for promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells, comprising a step of co-culturing one or more types of immortalized hematopoietic supporting cells selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages, and hematopoietic stem cells or hematopoietic progenitor cells. (2) The method according to (1), in which two or more types of immortalized hematopoietic supporting cells are used. (3) The method according to (1) or (2), in which the proportion of the number of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages in the total number of the immortalized hematopoietic supporting cells is 10 to 80%, respectively. (4) The method according to any one of (1) to (3), in which the immortalized hematopoietic supporting cells comprise immortalized mesenchymal stem cells and / or immortalized vascular endothelial cells. (5) The method according to any one of (1) to (4), wherein the proportion of the number of immortalized mesenchymal stem cells and / or immortalized vascular endothelial cells in the total number of the immortalized hematopoietic supporting cells is greater than the proportion of the number of immortalized megakaryocytes and / or immortalized macrophages. (6) The method according to any one of (1) to (5), wherein the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of the immortalized hematopoietic supporting cells is 10% or more, respectively, and the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of the immortalized hematopoietic supporting cells is greater than 50%. (7) The method according to any one of (1) to (6), wherein the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of the immortalized hematopoietic supporting cells is 70% or more. (8) The method according to any one of (1) to (7), wherein the proportion of the number of immortalized mesenchymal stem cells in the total number of immortalized hematopoietic supporting cells is 10% or more. (9) The method according to any one of (1) to (8), wherein SR1, UM171, or UM729 is further added and co-culture is performed. (10) The method according to any one of (1) to (9), wherein the hematopoietic stem cells or hematopoietic progenitor cells are derived from umbilical cord blood or adult bone marrow blood. (11) A proliferation promoter for hematopoietic stem cells or hematopoietic progenitor cells, comprising one or more immortalized hematopoietic supporting cells selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages.(12) The proliferation promoter according to (11), comprising two or more types of immortalized hematopoietic supporting cells. (13) The proliferation promoter according to (11) or (12), wherein the proportion of the number of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages in the total number of the immortalized hematopoietic supporting cells is 10 to 80%, respectively. (14) The proliferation promoter according to any one of (11) to (13), wherein the immortalized hematopoietic supporting cells comprise immortalized mesenchymal stem cells and / or immortalized vascular endothelial cells. (15) The proliferation promoter according to any one of (11) to (14), wherein the proportion of the number of immortalized mesenchymal stem cells and / or immortalized vascular endothelial cells in the total number of the immortalized hematopoietic supporting cells is greater than the proportion of the number of immortalized megakaryocytes and / or immortalized macrophages in the total number of the immortalized hematopoietic supporting cells. (16) The proliferation promoter according to any one of (11) to (15), wherein the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of the immortalized hematopoietic supporting cells is 10% or more, respectively, and the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of the immortalized hematopoietic supporting cells is more than 50%. (17) The proliferation promoter according to any one of (11) to (16), wherein the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of the immortalized hematopoietic supporting cells is 70% or more. (18) The proliferation promoter according to any one of (11) to (17), wherein the proportion of the number of immortalized mesenchymal stem cells in the total number of the immortalized hematopoietic supporting cells is 10% or more. (19) The proliferation promoter according to any one of (11) to (18), further comprising SR1, UM171, or UM729. (20) The proliferation promoter according to any one of (11) to (19), wherein the hematopoietic stem cells or hematopoietic progenitor cells are derived from umbilical cord blood or adult bone marrow blood.(21) A method for promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells, comprising a step of co-culturing one or more immortalized hematopoietic supporting cells selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages, with hematopoietic stem cells or hematopoietic progenitor cells, wherein the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells is 10% or more, respectively, and the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells is more than 50%. (22) A proliferation promoter for hematopoietic stem cells or hematopoietic progenitor cells, comprising one or more immortalized hematopoietic supporting cells selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages, wherein the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells is 10% or more, respectively, and the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells is more than 50%.

[0010] The present invention makes it possible to provide a method for promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells by co-culturing immortalized hematopoietic supporting cells, such as immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages, with hematopoietic stem cells or hematopoietic progenitor cells.

[0011] Furthermore, the present invention enables a stable supply of hematopoietic stem cells and hematopoietic progenitor cells in hematopoietic stem cell transplantation therapy, and further reduces the burden on donors.

[0012]

[0033] Figures 1A to 1C show the results on day 7 of culture, and Figures 1D to 1F show the results on day 21 of culture.

[0034] Figures 1A to 1C show the results on day 7 of culture, and Figures 1D to 1F show the results on day 21 of culture.

[0035] Figures 1A to 1C show the results on day 7 of culture, and Figures 1D to 1F show the results on day 21 of culture.

[0036] Figures 1A to 1C show the results of cell frequency analysis of hematopoietic stem cells when CD34-positive cells cultured in the presence of immortalized mesenchymal stem cells and / or SR1 and UM171 were transplanted into mice.

[0037] Figures 1D to 1F show the results of chimerism analysis of human CD45+ cells when CD34-positive cells cultured in the presence of immortalized mesenchymal stem cells and / or SR1 and UM171 were transplanted into mice.

[0038] Figures 1A to 1C show the results of chimerism analysis of hematopoietic stem cells and hematopoietic progenitor cells (CD34+) when CD34-positive cells cultured in the presence of immortalized mesenchymal stem cells and / or SR1 and UM171 were transplanted into mice.

[0033] Figure 1 shows the results of chimerism analysis of early hematopoietic stem cells and hematopoietic progenitor cells (CD34+38low) when immortalized mesenchymal stem cells and / or CD34-positive cells cultured in the presence of SR1 and UM171 were transplanted into mice.

[0034] Figure 1 shows the results of hematopoietic stem cell proliferation when immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages at the respective mixing ratios shown in Table 1 were co-cultured with cord blood-derived CD34-positive cells. The proliferation results are shown as relative values ​​(fold), with the number of cells in the control group defined as 1.

[0035] Figure 1 shows the results of hematopoietic progenitor cell proliferation when immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages at the respective mixing ratios shown in Table 1 were co-cultured with cord blood-derived CD34-positive cells. The proliferation results are shown as relative values ​​(fold), with the number of cells in the control group defined as 1.

[0033] Figure 1 shows the results of hematopoietic stem cell proliferation when immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages at the respective mixing ratios shown in Table 1 were co-cultured with CD34-positive cells derived from adult bone marrow blood. The proliferation results are shown as a relative value (FOLD) with the number of cells in the control group set at 1.

[0034] Figure 1 shows the results of evaluating the cell proliferation of vascular endothelial cells.

[0035] Figure 1 shows the results of flow cytometry analysis of vascular endothelial cells.

[0036] Figure 1 shows the results of marker expression in vascular endothelial cells.

[0037] Figure 1 shows the results of PCA (principal component analysis) of dox-on immortalized vascular endothelial cells, dox-off immortalized vascular endothelial cells, human iPS cells, human aortic endothelial cells (HAEC), and human umbilical cord blood endothelial cells (HUVEC).1 shows the results of expression analysis of DLL4 and Jagged 1 in dox-on imsenescent vascular endothelial cells, dox-off imsenescent vascular endothelial cells, human iPS cells, human aortic endothelial cells (HAEC), and human umbilical cord blood endothelial cells (HUVEC).

[0013] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, an embodiment of the present invention (hereinafter referred to as "the present embodiment") will be described in detail. However, the present invention is not limited to the following embodiment.

[0014] The present embodiment provides a method for promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells, comprising the step of co-culturing one or more immortalized hematopoietic support cells selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages, with hematopoietic stem cells or hematopoietic progenitor cells. Hematopoietic support cells are generally known as one of the factors that form bone marrow, and it has been reported that mesenchymal stem cells, osteoblasts, vascular endothelial cells, Schwann cells, megakaryocytes, macrophages, etc. are hematopoietic support cells. In the present embodiment, the term "immortalized hematopoietic support cells" is used as a general term for immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages. That is, in this embodiment, the immortalized hematopoietic supporting cells may be immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages, or may include two or more types selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages.

[0015] The present embodiment provides a method comprising the step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with only immortalized mesenchymal stem cells, only immortalized vascular endothelial cells, only immortalized megakaryocytes, or only immortalized macrophages, and also provides a method comprising the step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with a mixture of two, three, or four different types of immortalized hematopoietic support cells selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages.

[0016] The present embodiment also provides a method comprising the step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with immortalized hematopoietic support cells in which the proportion of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages in the total number of immortalized hematopoietic support cells is 10% or more (e.g., 10 to 70%, 10 to 80%, 10 to 90%). For example, a mixture of immortalized hematopoietic supporting cells containing two types selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages (e.g., immortalized mesenchymal stem cells + immortalized vascular endothelial cells, immortalized mesenchymal stem cells + immortalized megakaryocytes, immortalized mesenchymal stem cells + immortalized macrophages, immortalized vascular endothelial cells + immortalized megakaryocytes, immortalized vascular endothelial cells + immortalized macrophages, immortalized megakaryocytes + immortalized macrophages), each of which accounts for 10% or more (e.g., 10-90%) of the total number of immortalized hematopoietic supporting cells, is co-cultured with hematopoietic stem cells or hematopoietic progenitor cells. Alternatively, hematopoietic stem cells or hematopoietic progenitor cells are co-cultured with a mixture of immortalized hematopoietic supporting cells containing three types selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages (e.g., immortalized mesenchymal stem cells + immortalized vascular endothelial cells + immortalized megakaryocytes, immortalized mesenchymal stem cells + immortalized vascular endothelial cells + immortalized macrophages, immortalized mesenchymal stem cells + immortalized megakaryocytes + immortalized macrophages, immortalized mesenchymal stem cells + immortalized megakaryocytes + immortalized macrophages), each of which accounts for 10% or more (e.g., 10-80%) of the total number of immortalized hematopoietic supporting cells. Alternatively, hematopoietic stem cells or hematopoietic progenitor cells are co-cultured with a mixture of immortalized hematopoietic supporting cells containing four types (immortalized mesenchymal stem cells + immortalized vascular endothelial cells + immortalized megakaryocytes + immortalized macrophages) selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages, each of which accounts for 10% or more (e.g., 10 to 70%) of the total number of immortalized hematopoietic supporting cells.In this embodiment, the method for promoting the proliferation of hematopoietic progenitor cells or hematopoietic progenitor cells preferably comprises co-culturing hematopoietic stem cells or hematopoietic progenitor cells with a mixture of immortalized hematopoietic supporting cells containing immortalized mesenchymal stem cells and immortalized vascular endothelial cells at a concentration of 10% or more (e.g., 10 to 90%) of each of the immortalized hematopoietic supporting cells relative to the total cell number of the immortalized hematopoietic supporting cells. More preferably, the method comprises co-culturing hematopoietic stem cells or hematopoietic progenitor cells with a mixture of immortalized hematopoietic supporting cells containing immortalized mesenchymal stem cells and immortalized vascular endothelial cells at a concentration of more than 50% (e.g., 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%) of the immortalized hematopoietic supporting cells relative to the total cell number of the immortalized hematopoietic supporting cells.

[0017] In this embodiment, the percentage (%) of the number of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages in the total number of immortalized hematopoietic supporting cells used in co-culture may be any percentage and is not particularly limited, but may be, for example, the percentages (%) shown below.・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 100:0:0:0 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 0:100:0:0 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 0:0:100:0 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 0:0:0:100 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 70:10:10:10・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 10:70:10:10 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 10:10:70:10 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 10:10:10:70 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 40:40:10:10 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 10:10:40:40・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 40:10:40:10 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 10:40:10:40 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 40:10:10:40 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 10:40:40:10 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 25:25:25:25 Immortalized mesenchymal stem cells: immortalized vascular endothelial cells: immortalized megakaryocytes: immortalized macrophages = 80:0:10:10 Immortalized mesenchymal stem cells: immortalized vascular endothelial cells: immortalized megakaryocytes: immortalized macrophages = 80:10:0:10・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 80:10:10:0 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 0:80:10:10 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 10:80:0:10 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 10:80:10:0 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 0:10:80:10・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 10:0:80:10 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 10:10:80:0 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 0:10:10:80 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 10:0:10:80 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 10:10:0:80・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 50:50:0:0 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 50:0:50:0 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 50:0:0:50 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 0:50:50:0 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 0:50:0:50 ・Immortalized mesenchymal stem cells:immortalized vascular endothelial cells:immortalized megakaryocytes:immortalized macrophages = 0:0:50:50 Immortalized mesenchymal stem cells: immortalized vascular endothelial cells: immortalized megakaryocytes: immortalized macrophages = 33:0:33:33 Immortalized mesenchymal stem cells: immortalized vascular endothelial cells: immortalized megakaryocytes: immortalized macrophages = 33:33:0:33 Immortalized mesenchymal stem cells: immortalized vascular endothelial cells: immortalized megakaryocytes: immortalized macrophages = 33:33:33:0 Immortalized mesenchymal stem cells: immortalized vascular endothelial cells: immortalized megakaryocytes: immortalized macrophages = 0:33:33:33

[0018] In this embodiment, when the ratio of the number of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages to the total number of immortalized hematopoietic supporting cells used in co-culture is 0, 10, 20, 25, 30, 33, 40, 50, 60, 70, 80, 90, or 100%, the effect of promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells is similar to that when the ratio of the number of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages to the total number of immortalized hematopoietic supporting cells is between 0-10%, 0-20%, 10-30, 15-35, 20-40, 23-43, 30-50, 40-60, 50-70, 60-80, 70-90, 80-100, or 90-100, respectively. In this embodiment, the total number of immortalized hematopoietic support cells used in co-culture may be any number relative to the number of hematopoietic stem cells or hematopoietic progenitor cells, and may be, for example, 1 to 50 times.

[0019] In this embodiment, the method for promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells preferably comprises a step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with immortalized hematopoietic support cells comprising immortalized vascular endothelial cells, and more preferably comprises a step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with immortalized hematopoietic support cells comprising immortalized mesenchymal stem cells and immortalized vascular endothelial cells. More preferably, the method comprises a step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with immortalized hematopoietic support cells in which the proportion of immortalized mesenchymal stem cells and / or immortalized vascular endothelial cells to the total number of immortalized hematopoietic support cells is greater than the proportion of immortalized megakaryocytes and / or immortalized macrophages. Here, the phrase "the proportion of immortalized mesenchymal stem cells and / or immortalized vascular endothelial cells is greater than the proportion of immortalized megakaryocytes and / or immortalized macrophages" refers to the following cases:・Number of immortalized mesenchymal stem cells + number of immortalized vascular endothelial cells > number of immortalized megakaryocytes + number of immortalized macrophages ・Number of immortalized mesenchymal stem cells + number of immortalized vascular endothelial cells > number of immortalized megakaryocytes ・Number of immortalized mesenchymal stem cells + number of immortalized vascular endothelial cells > number of immortalized macrophages ・Number of immortalized mesenchymal stem cells > number of immortalized megakaryocytes + number of immortalized macrophages ・Number of immortalized mesenchymal stem cells > number of immortalized megakaryocytes ・Number of immortalized mesenchymal stem cells > number of immortalized macrophages ・Number of immortalized vascular endothelial cells > number of immortalized megakaryocytes + number of immortalized macrophages ・Number of immortalized vascular endothelial cells > number of immortalized megakaryocytes The number of immortalized vascular endothelial cells is greater than the number of immortalized macrophages. Preferably, the present invention provides a method comprising the step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with immortalized hematopoietic supporting cells in which the proportion of the number of immortalized macrophages in the total number of immortalized hematopoietic supporting cells is less than 80% (e.g., less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or 0%).Preferably, the present invention also provides a method comprising the step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with immortalized hematopoietic supporting cells in which the proportion of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells is 70% or more (e.g., 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, 100%).

[0020] In this embodiment, the method for promoting the proliferation of hematopoietic progenitor cells or hematopoietic progenitor cells preferably comprises a step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with immortalized hematopoietic support cells comprising immortalized mesenchymal stem cells. Also provided is a method preferably comprising a step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with immortalized hematopoietic support cells in which the proportion of immortalized mesenchymal stem cells in the total number of immortalized hematopoietic support cells is 10% or more (e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, 100%). In this embodiment, the method for promoting the proliferation of hematopoietic progenitor cells preferably comprises a step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with immortalized hematopoietic support cells comprising immortalized mesenchymal stem cells and immortalized vascular endothelial cells. Preferably, the method provided includes a step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with immortalized hematopoietic supporting cells, in which the proportions of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells are 10% or more (e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more). More preferably, the method includes a step of co-culturing hematopoietic stem cells or hematopoietic progenitor cells with immortalized hematopoietic supporting cells, in which the proportions of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells are each 10% or more (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more), and in which the proportions of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells are greater than the proportions of the number of immortalized megakaryocytes and immortalized macrophages (i.e., the proportions of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells are greater than 50%).

[0021] Furthermore, in the method of promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells in this embodiment, when hematopoietic stem cells or hematopoietic progenitor cells are co-cultured with one or more immortalized hematopoietic support cells selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages, SR1, UM171, or UM729, which are known as stem cell-expanding compounds, may be further added to the culture.

[0022] SR1 is a compound having the following structural formula (Science. 2010 Sep 10;329(5997):1345-8):

[0023] UM171 is a compound having the following structural formula (Science. 2014 Sep 19;345(6203):1509-12)

[0024] UM729 is a compound having the following structure: Preferably, when hematopoietic stem cells or hematopoietic progenitor cells are co-cultured with immortalized hematopoietic support cells, a combination of SR1 and UM171 or a combination of SR1 and UM729 is added to the culture.

[0025] In the present embodiment, "immortalization" refers to the artificial introduction of a gene that promotes cell proliferation or a gene that suppresses cell senescence into a target cell, or the artificial suppression of the expression of a tumor suppressor gene, thereby conferring a higher level of continuous cell division and proliferation ability than would be the case without such manipulation. Specifically, without limitation, this refers to a state in which, under standard cell culture conditions, the cell can be passaged for typically 10 or more passages, e.g., 15 or more passages, 20 or more passages, 30 or more passages, or 50 or more passages. Furthermore, "immortalization" can be used interchangeably with "immortalization." In the present embodiment, the immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages may be immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages.

[0026] In this embodiment, "mesenchymal stem cells" refer to somatic stem cells that produce mesodermally derived tissues such as osteoblasts, adipocytes, and chondrocytes. Furthermore, "vascular endothelial cells" refer to cells that constitute the vascular endothelium or cells that can differentiate into such cells. Furthermore, "megakaryocytes" refer to cells that can produce platelets and are sometimes called erythroid progenitor cells (MEPs). Furthermore, "macrophages" refer to immune cells that have phagocytic activity and are sometimes called myelocytes. Furthermore, "hematopoietic stem cells" refer to somatic stem cells that can differentiate into blood cells, and "hematopoietic progenitor cells" refer to cells that can differentiate into blood cells and are intermediates in the process by which hematopoietic stem cells differentiate into blood cells. In this embodiment, the mesenchymal stem cells, vascular endothelial cells, megakaryocytes, and macrophages may be obtained by inducing differentiation in vitro from pluripotent stem cells such as iPS cells and ES cells, and the hematopoietic stem cells and hematopoietic progenitor cells in this embodiment may be isolated from, for example, bone marrow, peripheral blood, or umbilical cord blood.

[0027] The method for preparing immortalized hematopoietic supporting cells and the method for co-culturing with hematopoietic stem cells or hematopoietic progenitor cells in this embodiment will be described below.

[0028] <Method for preparing immortalized hematopoietic supporting cells> First, mesenchymal stem cells, vascular endothelial cells, megakaryocytes, and macrophages are obtained by inducing differentiation from pluripotent stem cells such as iPS cells and ES cells using methods well known to those skilled in the art.

[0029] Mesenchymal stem cells can be obtained by inducing differentiation from pluripotent stem cells such as iPS cells and ES cells using methods well known to those skilled in the art. For example, according to the report by Fukuta et al. (PlosOne, 2014, 9(12):e112291), neural crest cells (NCCs) are induced by culturing iPS cells on a Matrigel-coated culture dish in the presence of SB-431542 and CHIR99021, and the NCCs are expanded by culturing them on a fibronectin-coated culture dish in a medium containing EGF, FGF2, and SB-431542. MSCs can then be induced by culturing the NCCs on a fibronectin-coated culture dish in a medium containing 10% FBS and FGF2.

[0030] Vascular endothelial cells can be induced during the process of inducing net-like structures from ES cells or iPS cells. Cell culture conditions suitable for preparing net-like structures from human pluripotent stem cells, such as human ES cells or iPS cells, vary depending on the pluripotent stem cells used. For example, IMDM supplemented with a final concentration of 15% FBS can be used as the medium. Even in serum-free media, media supplemented with appropriate growth factors and supplements can be used. Furthermore, to efficiently form net-like structures, it is recommended to add VEGF at a concentration of 0 to 100 ng / ml, more preferably approximately 20 ng / ml. The formed net-like structures have a follicular structure, and endothelial progenitor cells and hematopoietic progenitor cells are present within them in a concentrated state. Vascular endothelial progenitor cells present within the net-like structures can be separated by physical means, such as by passing the cells through a sterilized sieve-like device (e.g., a cell strainer). The CD34+ endothelial progenitor cells are then isolated using a cell sorter and cultured in the presence of VEGF to obtain vascular endothelial cells. It is known that the Notch signaling, which is transmitted by the interaction between Notch and a Notch ligand (such as Delta-like 4 (DLL4) or Jagged 1), is important for the proliferation of hematopoietic stem cells or hematopoietic progenitor cells. The immortalized vascular endothelial cells of this embodiment (preferably, immortalized vascular endothelial cells obtained by immortalizing vascular endothelial cells induced from pluripotent stem cells such as ES cells or iPS cells) may express a Notch ligand required for Notch signaling, such as DLL4 and / or Jagged 1. In this embodiment, when the immortalized hematopoietic supporting cells include immortalized vascular endothelial cells (preferably immortalized vascular endothelial cells obtained by immortalizing vascular endothelial cells induced from pluripotent stem cells such as ES cells or iPS cells), the proliferation of hematopoietic stem cells or hematopoietic progenitor cells may be further promoted by the immortalized vascular endothelial cells expressing DLL4 and / or Jagged 1.

[0031] Megakaryocytes can be obtained, for example, by isolating hematopoietic progenitor cells contained in a net-like structure induced from pluripotent stem cells such as human ES cells or iPS cells using a cell sorter and culturing them under megakaryocyte-inducing conditions (on feeder cells in the presence of TPO, SCF, and heparin) (WO 2008 / 041370, Takayama Blood 2008, 111(11):5298-5306).

[0032] Macrophages can be obtained, for example, by isolating hematopoietic progenitor cells contained in net-like structures induced from pluripotent stem cells such as human ES cells or iPS cells using a cell sorter and culturing them under megakaryocyte-inducing conditions (for example, in the presence of SCF, M-CSF, and IL-1β).

[0033] Next, in the obtained mesenchymal stem cells, vascular endothelial cells, megakaryocytes, and macrophages, genes such as MYC family genes (e.g., c-Myc gene), BMI1 gene, and BCL-XL gene are forced to be expressed, or siRNA (or its precursor, shRNA) or antisense DNA against tumor suppressor genes such as p53 gene are forced to be expressed, thereby obtaining immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages with high proliferation potential. In one embodiment, c-Myc and BMI1 are forced to be expressed (referred to as MB). In another embodiment, c-Myc, BMI1, and BCL-XL are forced to be expressed (referred to as MBX). In one embodiment, siRNA (or its precursor, shRNA) or antisense DNA against the p53 gene is forced to be expressed (referred to as p53KD).

[0034] After proliferation with MB, MBX, or p53KD, cell proliferation can be stopped and terminal differentiation promoted by suppressing their expression. For example, immortalized hematopoietic supporting cells (immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages) can be proliferated by forced expression of MB or MBX or p53KD, and then terminal differentiation can be promoted by stopping the expression of MB or MBX, or by stopping the expression of siRNA (or its precursor, shRNA) or antisense DNA against p53. As used herein, cells obtained by stopping the proliferation of proliferated immortalized hematopoietic supporting cells (immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages) and inducing terminal differentiation are also considered to be included in the immortalized hematopoietic supporting cells (immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages). Immortalized hematopoietic support cells may be inhibited from proliferating by irradiation or treatment with mitomycin C. In the case of mesenchymal stem cells, dox-off alone does not completely stop proliferation, so irradiation or mitomycin C treatment is recommended. In the case of other cells, such as vascular endothelial cells, megakaryocytes, and macrophages, proliferation stops with dox-off alone, so irradiation is not necessary.

[0035] When a gene is forced to be expressed in a cell, any method known to those skilled in the art may be used. For example, the gene may be introduced into the cell and expressed using a gene transfer system that uses a viral vector such as a lentivirus, retrovirus, or Sendai virus, or a non-viral vector such as a plasmid vector or episomal vector. A method (e.g., the PiggyBac Transposon system) may also be used in which a target gene is non-virally integrated into the genome of a cell using a transposon, a stable-expressing cell line is established, and then the unnecessary introduced gene is removed by transposonase. Target cells may be transfected with an expression vector (e.g., a viral vector) for a desired gene (e.g., a MYC family gene and a BMI1 gene, and optionally a BCL-XL gene), or target cells may be derived from pluripotent stem cells (e.g., ES cells, iPS cells) into which an expression cassette for a desired gene (e.g., a MYC family gene and a BMI1 gene, and optionally a BCL-XL gene) has been previously incorporated, and the gene may be forced to be expressed at that stage. Alternatively, differentiation of pluripotent stem cells (e.g., ES cells, iPS cells) into target cells may be induced by forcibly expressing a desired gene (e.g., a MYC family gene and a BMI1 gene, and optionally, a BCL-XL gene) in pluripotent stem cells (e.g., ES cells, iPS cells) into which an expression cassette for the desired gene has been previously incorporated. When gene expression is performed using a gene transfer vector, the gene may be operably linked downstream of a suitable promoter, which may then be inserted into a gene transfer vector and introduced into a cell to express the gene of interest. The promoter may be an exogenous promoter. As used herein, the term "endogenous" promoter refers to a promoter that is naturally linked to the gene in the genome, while the term "exogenous" promoter refers to a promoter that is artificially positioned proximal to the gene by genetic engineering (i.e., molecular biological techniques) so that transcription of the gene is directed by the operably linked promoter. Here, "operably" linked means that the promoter and gene of interest are linked such that the gene of interest are controlled in cis by the promoter, thereby achieving the desired expression of the gene of interest.The exogenous promoter may be a constitutive promoter or a regulatable promoter. Examples of constitutive promoters include the CMV promoter, EF1 promoter, and ubiquitin promoter. A regulatable promoter refers to an inducible or derepressible promoter, which has a DNA sequence that can bind to either a repressor or an inducer and acts in concert with the promoter. When a promoter is induced or derepressed, it is in the "on state," and when it is not induced or derepressed, it is in the "off state." Examples of regulatable promoters include drug-responsive promoters such as tetracycline-responsive promoters, steroid-responsive promoters, and metallothionein promoters. A tetracycline-responsive promoter is a known regulatable promoter that is reversibly controlled by the presence or absence of tetracycline or its derivatives (e.g., doxycycline (Dox)). A tetracycline-responsive promoter is a promoter containing a tetracycline response element (TRE) and is activated (i.e., induces expression of a target protein) upon binding of a reverse tetracycline-controlled transactivator (rtTA) protein or a tetracycline-controlled transactivator (tTA) to the TRE. The rtTA protein binds to the TRE in the presence of Dox, while the tTA protein binds to the TRE in the absence of Dox, inducing expression of a target gene operably linked to a promoter downstream of the TRE sequence. When a tetracycline-responsive promoter is used, expression of the gene can be induced or suppressed in a Dox-dependent manner by culturing cells into which the gene operably linked to the tetracycline-responsive promoter and the rtTA or tTA protein have been introduced in the presence of Dox. The exogenous promoter is preferably a regulatable promoter. By using a regulatable promoter, it is also possible to induce expression of a target gene by, for example, controlling the addition of a drug.Those skilled in the art can easily select an appropriate gene expression system using such drugs to achieve the desired expression control of the MYC family gene, BMI1 gene, BCL-XL gene, etc. Commercially available kits may be used to achieve such expression. Furthermore, the target genes for expression control, the MYC family gene, BMI1 gene, and BCL-XL gene, may be inserted into separate vectors or into the same vector.

[0036] <Method for Co-Culture with Immortalized Hematopoietic Stem Cells or Hematopoietic Progenitor Cells> In the co-culture of immortalized hematopoietic support cells and hematopoietic stem cells or hematopoietic progenitor cells, the immortalized hematopoietic support cells may be first induced before proceeding to the co-culture step with hematopoietic stem cells or hematopoietic progenitor cells, or the immortalized hematopoietic support cells may be stored as frozen stocks until proceeding to the co-culture step. The order in which the immortalized hematopoietic support cells and hematopoietic stem cells or hematopoietic progenitor cells are added to the medium may be arbitrary. The immortalized hematopoietic support cells may be added first, or the hematopoietic stem cells or hematopoietic progenitor cells may be added first. The order in which the immortalized hematopoietic support cells, i.e., immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages, are added may also be arbitrary. For example, co-culture may be performed by adding one type of immortalized hematopoietic support cells, then adding hematopoietic stem cells or hematopoietic progenitor cells, and then adding two types of immortalized hematopoietic support cells. Alternatively, hematopoietic stem cells or hematopoietic progenitor cells may be added to immortalized hematopoietic support cells cultured in a medium to perform co-culture, or immortalized hematopoietic support cells may be added to hematopoietic stem cells or hematopoietic progenitor cells cultured in a medium. Co-culture may be a two-dimensional culture in a well plate or the like, or a three-dimensional culture in an environment similar to that in a living body. Culture conditions can be appropriately determined by those skilled in the art depending on the type of cells and their state. For example, the culture temperature can be about 25°C to about 35°C, about 35°C to about 42°C, about 36°C to about 40°C, or about 37°C to about 39°C, and the carbon dioxide concentration can be, for example, 5% CO ２ The oxygen concentration is, for example, 1 to 20% ２The culture medium may be a static culture or a shake culture. The shake speed in the case of shake culture is not particularly limited and may be, for example, 10 rpm to 200 rpm, 30 rpm to 150 rpm, etc. The medium may be an STF medium containing SCF, Flt3L, TPO, gentamicin, phenol red, etc., and the STF medium may contain serum. The medium may also contain serum or plasma, or may be serum-free. If necessary, the medium may also contain one or more substances, such as cytokines, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (e.g., L-glutamine), ascorbic acid, heparin, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. Examples of cytokines include stem cell factor (SCF), vascular endothelial growth factor (VEGF), thrombopoietin (TPO), various TPO-like substances, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), interleukin 3 (IL3), ITS (insulin-transferrin-selenite) supplements, and ADAM (A Disintegrin And Metalloprotease) inhibitors.

[0037] In this embodiment, the promotion of proliferation of hematopoietic stem cells or hematopoietic progenitor cells can be evaluated, for example, by measuring the cell count, cell frequency analysis, chimerism analysis, or the like. For example, when the number of hematopoietic stem cells or hematopoietic progenitor cells has increased compared to a control in which hematopoietic stem cells or hematopoietic progenitor cells are cultured with only cytokines or the like added, it can be evaluated that proliferation has been promoted.

[0038] Furthermore, in this embodiment, the proliferation of hematopoietic stem cells or hematopoietic progenitor cells may be promoted more when hematopoietic stem cells or hematopoietic progenitor cells are co-cultured with two or more different immortalized hematopoietic supportive cells selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages than when hematopoietic stem cells or hematopoietic progenitor cells are co-cultured with a single species of immortalized mesenchymal stem cells only, immortalized vascular endothelial cells only, immortalized megakaryocytes only, or immortalized macrophages only. Furthermore, co-culture of immortalized hematopoietic supportive cells with the further addition of SR1 or UM171 may further promote the proliferation of hematopoietic stem cells or hematopoietic progenitor cells than when hematopoietic stem cells or hematopoietic progenitor cells are co-cultured with immortalized hematopoietic supportive cells only.

[0039] The method of this embodiment can be carried out when it is desired to culture and proliferate hematopoietic stem cells or hematopoietic progenitor cells, thereby promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells. For example, in order to perform hematopoietic stem cell transplantation, it becomes possible to proliferate hematopoietic stem cells and hematopoietic progenitor cells collected from bone marrow, peripheral blood, etc. of a donor, or hematopoietic stem cells and hematopoietic progenitor cells derived from umbilical cord blood for transplantation.

[0040] The aspects of the method for promoting proliferation of hematopoietic stem cells or hematopoietic progenitor cells in this embodiment are also applicable to other embodiments described below.

[0041] In another embodiment, the present invention also provides a proliferation promoter for hematopoietic stem cells or hematopoietic progenitor cells, comprising one or more immortalized hematopoietic support cells selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages. The proliferation promoter can be added during the culture of hematopoietic stem cells or hematopoietic progenitor cells, or co-cultured with the cells, to promote the proliferation of the hematopoietic stem cells or hematopoietic progenitor cells. Furthermore, the presence of the proliferation promoter in the medium can promote the proliferation of the hematopoietic stem cells or hematopoietic progenitor cells. In addition to immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages, the proliferation promoter may contain any substance, such as a component contained in a culture medium, as long as it can exhibit the effect of promoting the proliferation of the hematopoietic stem cells or hematopoietic progenitor cells. For example, the proliferation promoter may contain serum or plasma, or may also contain substances such as albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (e.g., L-glutamine), ascorbic acid, heparin, non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, or cytokines.

[0042] The present invention will be described in more detail below with reference to specific examples, although the present invention is not limited thereto.

[0043] Example 1 (1) Preparation of Immortalized Hematopoietic Support Cells for Co-culture (1-1) Immortalized Mesenchymal Stem Cells Mesenchymal stem cells were induced from human iPS cells. After induction, the mesenchymal stem cells were infected with forced expression vectors for the c-MYC gene, BMI1 gene, and BCLXL gene (MBX). Immediately after infection, 1 μg / ml of docycline was added to the culture medium to activate the promoter in the forced expression viral vector (dox on), forcibly inducing expression of the c-MYC gene, BMI1 gene, and BCLXL gene, thereby inducing immortalized mesenchymal stem cells. For use in co-culture, the cells were irradiated with 40 Gy of radiation while still in the dox on state to suppress proliferation, and then frozen and stocked for use in co-culture. In some tests, immortalized mesenchymal stem cells were also induced by knocking down p53 in the induced mesenchymal stem cells using the shRNA method, and the immortalized mesenchymal stem cells were frozen and stocked for use in co-culture.

[0044] (1-2) Immortalized vascular endothelial cells Vascular endothelial cells were induced from human iPS cells. After induction of vascular endothelial cells, MBX expression was forcibly induced (dox on) to induce immortalized vascular endothelial cells in the same manner as in (1-1), and the cells were further cultured under dox off conditions for 7 days to differentiate. For use in co-culture, the cells were irradiated to suppress proliferation, frozen, and used for co-culture.

[0045] (1-3) Immortalized Megakaryocytes Megakaryocytes were induced from human iPS cells. After megakaryocyte induction, MBX expression was forcibly induced (dox on) as in (1-1) to induce immortalized megakaryocytes, which were then frozen and stocked in the dox on state and used for co-culture. Since the proliferation of immortalized megakaryocytes completely stopped in the dox off state and the co-culture was performed in the dox off state, no growth suppression treatment by irradiation was performed.

[0046] (1-4) Macrophages Macrophages were induced from human iPS cells. After macrophage induction, MBX expression was forcibly induced (dox on) as in (1-1) to induce immortalized macrophages, which were then frozen and stocked in the dox on state and used for co-culture. Since the proliferation of immortalized macrophages completely stopped in the dox off state and the co-culture was performed in the dox off state, no growth suppression treatment by irradiation was performed.

[0047] Example 2 (2) Co-culture of immortalized hematopoietic support cells with hematopoietic stem cells or hematopoietic progenitor cells (2-1) Collection of hematopoietic stem cells and hematopoietic progenitor cells from umbilical cord blood and adult bone marrow blood CD34-positive bone marrow (BM) cells (#2M-101A) purchased from Lonza were used as a source of adult bone marrow-derived hematopoietic stem cells and hematopoietic progenitor cells for co-culture. Fresh umbilical cord blood (UCB) samples were collected from healthy maternal donors provided by the Japanese Red Cross Kanto-Koshinetsu Cord Blood Bank, and mononuclear cells (MNCs) were separated by density gradient centrifugation (1500 rpm, 25 minutes, 4°C) using lymphocyte separation medium (Ficoll). The mononuclear cells were washed with phosphate-buffered saline (PBS) (Nacalai Tesque) and then incubated with immunomagnetic beads (CD34) (Miltenyi Biotec). The CD34+ fraction, containing cord blood-derived hematopoietic stem and progenitor cells, was selected using a quadroMACS® device according to the manufacturer's instructions. The CD34+ fraction was stored in liquid nitrogen until use in coculture.

[0048] (2-2) Co-culture of Immortalized Mesenchymal Stem Cells with Hematopoietic Stem Cells or Hematopoietic Progenitor Cells In Vitro Experiments: 10,000 immortalized feeder cells (immortalized mesenchymal stem cells) prepared in (1) above were seeded onto a 96-well flat-bottom plate and maintained in a specific medium for 18 to 24 hours before co-culture. Thawed CD34+ cells derived from umbilical cord blood were counted and resuspended in a medium consisting of X-VIVO10 (a serum-free medium containing gentamicin and phenol red, Lonza) supplemented with 1 / 10 volume of 1% bovine serum albumin (10% BSA / IMDM, StemCell Technologies). The medium was also supplemented with 100 ng / ml hSCF (R&D Systems, Catalog No. 255-SC), 100 ng / ml hFlt3L (PeproTech EC, Catalog No. 300-19), and 10 ng / ml hTPO (R&D Systems, Catalog No. 288-TP) (hereinafter referred to as STF medium). In some tests, SR1 and UM171 (or UM729) or their solvent (DMSO) were added to the medium. CD34-positive cells were cultured in 5% CO with partial medium changes every other day. ２ The cells were maintained at 37°C in a 5% CO2-free medium. Control groups included CD34+ cells in a suspension containing no immortalized mesenchymal stem cells and supplemented with 0.1% DMSO. The medium was removed, and the cells were detached with trypsin and incubated with CD34-APC7, CD45-PC7, CD45RA-BV421, and CD90-APC for 30 minutes at 4°C. The cells were then washed with SM (Staining Medium, PBS + 2% FBS), resuspended in SM-PI (SM containing propidium iodide (PI)), and analyzed using a FACS Canto II (Figure 1). Figures 1A-1C show results from day 7 of culture, and Figures 1D-F show results from day 21 of culture.

[0049] By co-culture with immortalized mesenchymal stem cells, proliferation of hematopoietic stem cells (CD34+CD90+CD45RA-) and hematopoietic progenitor cells (CD34+) was observed up to days 7 and 21 of culture, and proliferation was significantly promoted compared to the control group (NC) (Figure 1). Furthermore, in the group in which immortalized mesenchymal stem cells were co-cultured with SR1 and UM171, proliferation of hematopoietic stem cells (CD34+CD90+CD45RA-) and hematopoietic progenitor cells (CD34+) was significantly promoted compared to co-culture with immortalized mesenchymal stem cells alone or co-culture with SR1 and UM171 alone (Figure 1). The use of UM729 instead of UM171 (i.e., SR1+UM729) also promoted the proliferation of hematopoietic stem cells and hematopoietic progenitor cells in co-culture with immortalized mesenchymal stem cells.

[0050] Transplantation Assay in Mice For the transplantation assay, nonadherent cultured cells were collected, washed, resuspended in IMDM (SIGMA-ALDRICH, 13390-500 mL) + 1% FBS, and transplanted into nonlethally irradiated (2 Gy) NOG mice. Fresh cord blood-derived CD34+ cells were thawed, counted, and resuspended in IMDM + 1% FBS for use as a control. Cells were transplanted via intrafemoral injection. Each recipient received 1,000 CD34+ cells cultured for 7 days prior to transplantation. Twelve weeks after transplantation, mice were anesthetized with isoflurane and sacrificed. Cells were collected from the injected and uninjected femurs (including two tibiae). The resulting cell suspension was filtered through a 40 μm nylon mesh and centrifuged. The resulting cells were resuspended in IMDM and incubated with an anti-differentiation cell surface antigen antibody cocktail (GlyA-PE, CD19-PE / Cy7, CD33-APC, CD45-FITC) and an anti-hematopoietic stem / progenitor cell surface antigen antibody cocktail (CD34-APC / Cy7, CD38-PE / Cy7, CD45-FITC, CD45RA-BV421, CD90-APC). After 30 minutes, the cells were washed with PBS + 2% FBS, resuspended in PBS + 2% FBS + propidium iodide (PI), and analyzed using a FACS Canto II (BD) system (Figures 2-5). Figure 2 shows the results of cell frequency analysis of CD45+ cells, and Figures 3 to 5 show the results of chimerism analysis, with Figure 3 showing the results of chimerism analysis of CD45+ cells, Figure 4 showing the results of chimerism analysis of hematopoietic stem cells and hematopoietic progenitor cells (CD34+), and Figure 5 showing the results of chimerism analysis of early hematopoietic stem cells and hematopoietic progenitor cells (CD34+38low).

[0051] The results of cell frequency analysis (Figure 2) confirmed a 3.32-fold increase when cells co-cultured with immortalized mesenchymal stem cells were transplanted, a 7.39-fold increase when SR1 and UM171 were added, and a 19.96-fold increase when SR1 and UM171 were further added to the co-culture with immortalized mesenchymal stem cells, compared to the control group. Furthermore, the results of chimerism analysis (Figures 3-5) confirmed that the addition of SR1 and UM171 to the co-culture with immortalized mesenchymal stem cells achieved greater chimerism than the transplantation of only CD34+ cells without culture (Fresh). This suggests that the addition of SR1 and UM171 to the co-culture with immortalized mesenchymal stem cells promotes the proliferation of hematopoietic stem cells and hematopoietic progenitor cells.

[0052] (2-3) Co-culture of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages with hematopoietic stem cells or hematopoietic progenitor cells The immortalized mesenchymal stem cells and immortalized vascular endothelial cells prepared in (1) above were thawed, counted, and seeded in a desired amount in EC medium in a 96-flat-bottom well plate. ２ The cells were cultured in an incubator at 37°C. The next day, the immortalized megakaryocytes and immortalized macrophages prepared in (1) above and 1,000 cord blood-derived CD34+ cells were thawed, counted, and the corresponding amount was seeded in STF medium. The total number of feeder cells (immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages) was 10,000 or 20,000 cells / well. Next, 0.1% DMSO was added to all wells, and the cells were cultured for 7 days.

[0053] For hematopoietic stem cell and hematopoietic progenitor stem cell analysis, the medium was removed, cells were detached with trypsin, and then incubated with CD34-APC7, CD45-PC7, CD45RA-BV421, and CD90-APC for 30 minutes at 4°C. The cells were then washed with PBS + 2% FBS, resuspended in PBS + 2% FBS + propidium iodide (PI), and analyzed using a FACS Canto II (BD) (Figures 6 and 7). Figure 6 shows the results for hematopoietic stem cell proliferation, and Figure 7 shows the results for hematopoietic progenitor cell proliferation. The vertical axis shows the relative value (fold) with the number of cells in the control group set at 1, and the horizontal axis shows the mixing ratio (%) corresponding to the numbers in Table 1.

[0054]

[0055] 6 and 7 , it was revealed that the proliferation of hematopoietic stem cells and hematopoietic progenitor cells was promoted when co-cultured with immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages, compared with the control group (NC). Furthermore, it was found that in many cases, proliferation was more promoted when co-cultured with two or more types of immortalized hematopoietic supportive cells than when co-cultured with only immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages. In particular, when the proportion of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells was 10% or more, respectively, and when the proportion of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells was 50% or more, a significant promotion of proliferation of hematopoietic stem cells and hematopoietic progenitor cells was confirmed, which far exceeded the case when a single type of immortalized hematopoietic supporting cell was used (e.g., Nos. 5, 6, 9, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 29, 36, and 37 in Figures 6 and 7).

[0056] Furthermore, when a similar test was performed using hematopoietic stem cells derived from adult bone marrow blood, the results were similar to those obtained with hematopoietic stem cells and hematopoietic progenitor cells derived from umbilical cord blood (Figure 8). The vertical axis of Figure 8 indicates the absolute number of cells, and the horizontal axis is the same as in Figures 6 and 7.

[0057] Reference Example: Creation of Immortalized Vascular Endothelial Cells and Evaluation of Their Characteristics Using the techniques described in Takayama Blood 2008, 111(11): 5298-5306; Takayama JEM 2010, 207(13): 2817-2830, vascular progenitor cells were induced from human iPS cells by co-culture with 10T1 / 2 cells in the presence of 20 ng / ml VEGF. On days 10 to 11, CD34+ vascular endothelial cell progenitor cells were sorted using FACS AriaIII, infected with Dox-on c-MYC / Dox-on BMI1 / Dox-on BCL-XL lentiviral vectors, and continued to be cultured in the presence of 1 μg / ml doxycycline and 100 ng / ml VEGF, and the number of cells was counted. Compared to the control (no viral infection), cell proliferation was significantly increased in the dox-on condition (Figure 9). Furthermore, immunostaining for the cell surface marker VE-cadherin was performed on the dox-on condition and on the maturation condition after 7 days of dox-off treatment. The results showed that the dox-off cells showed increased expression of VE-cadherin, indicating differentiation into vascular endothelial cells (Figure 10).

[0058] Furthermore, qPCR was used to evaluate the gene expression of endothelial cell differentiation markers ENG (CD105), MCAM (CD146), and PECAM1 in human iPS cells, imVEC-dox on (immortalized endothelial cells treated with dox on), imVEC-dox off (immortalized endothelial cells treated with dox off), HAEC (human aortic endothelial cells), and HUVEC (human umbilical vein endothelial cells). As a result, it was confirmed that cells obtained from human imVEC treated with dox off expressed endothelial cell differentiation markers at the same level as HAEC and HUVEC (Figure 11).

[0059] Furthermore, PCA (principal component analysis) was performed on imVEC-dox on, imVEC-dox off, human iPS cells (HAECs), and HUVECs, and the results showed that the major components of the dox-off immortalized vascular endothelial cells were similar to those of HAECs and HUVECs (Figure 12).

[0060] Furthermore, we found that Delta-like 4 (DLL4) and Jagged 1 were more strongly expressed in imVEC-dox off compared with imVEC-dox on, human iPS cells, HAEC, and HUVEC (Figure 13). Since the Notch signal, which is transmitted through the interaction between Notch and Notch ligands (DLL4, Jagged 1, etc.), is known to play an important role in the proliferation of hematopoietic stem cells and hematopoietic progenitor cells, it is possible that at least part of the promotion of hematopoietic stem cell and hematopoietic progenitor cell proliferation by adding immortalized vascular endothelial cells to a portion of immortalized hematopoietic support cells is due to enhanced expression of DLL4 and Jagged 1.

Claims

1. A method for promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells, comprising a step of co-culturing one or more immortalized hematopoietic supporting cells selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages, and hematopoietic stem cells or hematopoietic progenitor cells.

2. The method of claim 1, wherein two or more types of immortalized hematopoietic support cells are used.

3. The method according to claim 2, wherein the percentage of the number of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, or immortalized macrophages in the total number of the immortalized hematopoietic supporting cells is 10 to 80%, respectively.

4. The method according to any one of claims 1 to 3, wherein the immortalized hematopoietic supporting cells comprise immortalized mesenchymal stem cells and / or immortalized vascular endothelial cells.

5. The method according to any one of claims 1 to 4, wherein the proportion of the number of immortalized mesenchymal stem cells and / or immortalized vascular endothelial cells in the total number of the immortalized hematopoietic supporting cells is greater than the proportion of the number of immortalized megakaryocytes and / or immortalized macrophages.

6. The method according to any one of claims 1 to 5, wherein the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of the immortalized hematopoietic supporting cells is 10% or more, respectively, and the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of the immortalized hematopoietic supporting cells is more than 50%.

7. A method according to any one of claims 1 to 6, wherein the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells is 70% or more.

8. The method according to any one of claims 1 to 7, wherein the proportion of the number of immortalized mesenchymal stem cells in the total number of immortalized hematopoietic supporting cells is 10% or more.

9. The method according to any one of claims 1 to 8, further comprising adding SR1, UM171 or UM729 for co-culture.

10. The method of any one of claims 1 to 9, wherein the hematopoietic stem or progenitor cells are derived from umbilical cord blood or adult bone marrow blood.

11. A proliferation promoter for hematopoietic stem cells or hematopoietic progenitor cells, comprising one or more immortalized hematopoietic supporting cells selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages.

12. A method for promoting the proliferation of hematopoietic stem cells or hematopoietic progenitor cells, comprising a step of co-culturing one or more immortalized hematopoietic supporting cells selected from the group consisting of immortalized mesenchymal stem cells, immortalized vascular endothelial cells, immortalized megakaryocytes, and immortalized macrophages, and hematopoietic stem cells or hematopoietic progenitor cells, wherein the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells is 10% or more, respectively, and the proportion of the number of immortalized mesenchymal stem cells and immortalized vascular endothelial cells in the total number of immortalized hematopoietic supporting cells is more than 50%.

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