Method for producing vascular endothelial stem cells

Abstract

The present invention provides a method for artificially producing vascular endothelial stem cells from cells other than vascular endothelial stem cells. The method for producing vascular endothelial stem cells according to the present invention comprises a step for bringing vascular endothelial cells that do not have stem-cell properties into contact with a factor that is secreted from an organ of a neonatal or infant mammal, wherein the step may be (1) a step for transplanting the vascular endothelial cells that do not have stem-cell properties into an organ of a non-human mammal, or (2) a step for culturing the vascular endothelial cells that do not have stem-cell properties using a culture system containing the factor that is secreted from an organ of a neonatal or infant mammal.

Description

[Technical field]

[0001] The present invention relates to a method for producing vascular endothelial stem cells. [Background technology]

[0002] Diseases related to blood vessels are collectively called vascular diseases, and include many diseases such as malignant tumors, retinopathy, chronic inflammatory diseases, arteriosclerosis, and ischemic diseases. There has been vigorous development of treatments for these diseases that improve the condition by controlling angiogenesis. Among these, treatments that improve the condition by regenerating blood vessels are called therapeutic angiogenesis, and are a treatment method that regenerates blood vessels to improve the pathology of diseases in which the number of blood vessels or vascular function has decreased due to various causes. Examples include: 1) treatments that improve ischemia by inducing new blood vessels in myocardial ischemia, cerebral ischemia, lower limb ischemia, etc., in other words, treatments that improve blood transport, which is the original function of blood vessels; 2) vascular regeneration treatments for diseases in which the secretion of factors normally secreted from vascular endothelial cells is suppressed from vascular endothelial cells due to genetic or acquired factors, leading to the disease (e.g., hemophilia, which develops when the production of coagulation factors in vascular endothelial cells is reduced); and 3) vascular regeneration that normalizes the blood vessels of diseased organs to restore their function (e.g., vascular regeneration for liver fibrosis, liver cirrhosis, atelectasis, osteoporosis, renal dysfunction, etc.).

[0003] The effectiveness of therapeutic vascular regeneration for the above diseases has begun to be proven in basic medicine, but cell therapy and cytokine (gene) therapy have actually been applied in clinical practice. For example, one cell therapy method is to regenerate blood vessels using vascular endothelial progenitor cells present in bone marrow. However, since these precursor cells are not stem cells, it is thought that blood vessels formed by these precursor cells will not be maintained for a long period of time. There is also a method of inducing vascular regeneration by injecting mesenchymal stem cells from fat tissue, etc. This method is thought to regenerate blood vessels by proliferating vascular endothelial cells of existing blood vessels using a substance that induces angiogenesis secreted from mesenchymal stem cells. However, this method cannot be used for patients with weakened vascular regeneration ability.

[0004] In addition to cell therapy, a method of administering growth factors that cause the proliferation of vascular endothelial cells has also been performed. Vascular endothelial growth factor (VEGF) is a cytokine that can most strongly induce the proliferation of vascular endothelial cells. A treatment to regenerate blood vessels by injecting a plasmid expressing the gene encoding this VEGF into an ischemic area was performed around 2000. However, VEGF increased vascular permeability, causing edema due to the retention of a large amount of blood components in the tissue. Due to this side effect, treatment using VEGF is no longer performed. Meanwhile, a treatment for vascular regeneration using the hepatocyte growth factor (HGF) gene is also being clinically used. Data showing its effectiveness is being introduced, but no mention has been made of its long-term effects.

[0005] For example, just as hematopoietic stem cells are necessary to reconstruct bone marrow using bone marrow from another person in bone marrow transplantation, in order to maintain regenerated blood vessels for a long period of time, blood vessels must be reconstructed with cells having stem cell properties. The present inventors have found that, although they are a very small percentage, there are cells with an extremely high ability to produce vascular endothelial cells among the vascular endothelial cells of existing blood vessels (Non-Patent Documents 1 and 2). It has been revealed that these endothelial cells are different from vascular endothelial progenitor cells present in bone marrow, and are cells that can contribute as vascular endothelial cells for a long period of time. The present inventors have found that such cells with an extremely high ability to produce vascular endothelial cells are present in existing blood vessels as vascular endothelial cells with high drug excretion ability.

[0006] Furthermore, in order to analyze in detail the characteristics of vascular endothelial cells as stem cells with an extremely high ability to produce vascular endothelial cells, the present inventors isolated surface markers expressed on these vascular endothelial cells and found that CD157 and CD200 were expressed on vascular endothelial stem cells (Non-Patent Document 2, Patent Document 1). The CD200-positive CD157-positive vascular endothelial cell population maintained its undifferentiated state and divided (self-replicated), and ultimately had the ability to produce a large amount of differentiated CD200-negative CD157-negative vascular endothelial cells. In addition, it was revealed that blood vessels once regenerated with these vascular endothelial cells were maintained as blood vessels for a long period of time without retraction. Specifically, not only the blood transport function but also the long-term production function of molecules to be secreted from vascular endothelial cells (e.g., coagulation factor VIII, etc.) was maintained. Hereinafter, the vascular endothelial cells with stem cell properties discovered by the present inventors are referred to as "vascular endothelial stem cells".

[0007] When inducing vascular regeneration, vascular regeneration using vascular endothelial cells that possess such stem cell properties is necessary, and transplantation of vascular endothelial stem cells can be a treatment that can bring therapeutic effects to patients who do not have the reserve ability for vascular regeneration. However, vascular endothelial stem cells exist in extremely small quantities in the body, and it is extremely inefficient to extract vascular endothelial stem cells from each patient who undergoes vascular regeneration. In addition, the patient's vascular endothelial stem cell function may be impaired. Therefore, there is a need to develop a method to artificially produce vascular endothelial stem cells from some kind of cell. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication WO2019 / 098264 A1 [Non-patent literature]

[0009] [Non-Patent Document 1] Naito H. et al., Identification and characterization of a resident vascular stem / progenitor cell population in preexisting blood vessels, EMBO J. 2012 Feb 15;31(4):842-55. [Non-Patent Document 2] Wakabayashi T. et al., CD157 Marks Tissue-Resident Endothelial Stem Cells with Homeostatic and Regenerative Properties, Cell Stem Cell. 2018 Mar 1;22(3):384-397. Summary of the Invention [Problem to be solved by the invention]

[0010] An objective of the present invention is to provide a method for artificially producing vascular endothelial stem cells from cells other than vascular endothelial stem cells. [Means for solving the problem]

[0011] In order to solve the above problems, the present invention includes the following inventions. [1] A method for producing vascular endothelial stem cells, comprising the step of contacting vascular endothelial cells that do not have stem cell properties with factors secreted from the organs of neonatal or juvenile mammals. [2] The method according to [1] above, wherein the step is carried out by either one of the following steps (1) or (2): (1) Transplanting the vascular endothelial cells that do not have stem cell properties into an organ of a non-human mammal; or (2) The vascular endothelial cells not having stem cell properties are cultured in a culture system containing factors secreted from the organs of mammals at the neonatal or juvenile stage. [3] The method for producing a mammal in the neonatal or juvenile stage described in [1] or [2] above, wherein the mammal in the neonatal or juvenile stage is a mammal in the neonatal stage. [4] The method according to any one of [1] to [3] above, wherein the vascular endothelial cells not having stem cell properties are cells obtained from a mammal at the neonatal or juvenile stage. [5] The method according to [4] above, wherein the vascular endothelial cells that do not have stem cell properties are cells obtained from a mammal in the neonatal stage. [6] The method according to any one of [1] to [5] above, wherein the organ is the liver. [7] A vascular endothelial stem cell inducer containing a factor secreted from the organs of a mammal in the neonatal or juvenile stage. [8] The vascular endothelial stem cell inducer described in [7] above, which contains a culture supernatant of cells prepared from an organ of a neonatal or juvenile mammal containing the factor, or an extract of an organ of a neonatal or juvenile mammal containing the factor. [9] The vascular endothelial stem cell inducer according to [7] or [8] above, wherein the neonatal or juvenile mammal is a neonatal mammal.

[10] The vascular endothelial stem cell inducer according to any one of [7] to [9] above, wherein the organ is the liver. Effect of the Invention

[0012] The present invention can provide a method for artificially producing vascular endothelial stem cells from vascular endothelial cells that do not have stem cell properties. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the results of culturing vascular endothelial cells (CD31-positive CD45-negative cells) obtained from living mice by flow cytometry in a culture system using OP9 stromal cells as feeder cells. (A) shows the results for vascular endothelial cells obtained from a 15-day-old fetal mouse, and (B) shows the results for vascular endothelial cells obtained from the liver of an 8-week-old adult mouse. [Diagram 2] This figure shows the results of extracting livers from fetal mice on embryonic day 15, and from newborn / juvenile mice on postnatal days 1, 7, 14, and 21, preparing frozen sections, and immunostaining them with an anti-CD31 antibody. [Diagram 3] FIG. 1 shows the results of flow cytometry analysis of Hoechst stained vascular endothelial cells (CD31-positive CD45-negative cells) obtained by flow cytometry from the livers of fetal mice on embryonic day 15, and newborn / juvenile mice on postnatal days 7, 14, 21, and 28. (A) shows the results of flow cytometry analysis of Hoechst staining, and (B) is a graph showing the percentage of fractions with high drug efflux capacity (SP cell fraction). [Figure 4] FIG. 1 shows the results of transplanting vascular endothelial cells (CD31-positive CD45-negative cells) from a fraction with low drug efflux capacity (MP cell fraction) obtained from the liver of a 1-day-old green mouse (C57BL / 6-Tg(CAG-EGFP)) into the liver of a 7-day-old liver disorder model mouse (C57BL / 6) and analyzing the liver of the liver disorder model mouse two months later. (A) is the result of observing the open liver with a fluorescent stereomicroscope, (B) is the result of analyzing the presence of CD31-positive GFP-positive cells in the liver of (A) by flow cytometry, and (C) is the result of analyzing the presence of SP cells in the CD31-positive GFP-positive cells of (B) by flow cytometry. [Diagram 5] FIG. 1 shows the results of flow cytometry analysis of the presence of CD200-positive CD157-positive cells in vascular endothelial cells (CD31-positive CD45-negative cells) obtained by flow cytometry from the livers of fetal mice on embryonic days 13, 15, and 18, newborn mice on postnatal days 1, 4, 7, and 14, and adult mice at 8 weeks of age. [Figure 6] FIG. 1 shows the results of transplanting CD200-negative CD157-negative vascular endothelial cells (CD31-positive CD45-negative cells) obtained from the liver of a green mouse (C57BL / 6-Tg(CAG-EGFP)) on embryonic day 15 into the liver of a mouse on postnatal day 7, and analyzing the mouse liver two months later. (A) shows the result of observing the open liver with a fluorescent stereomicroscope, and (B) shows the result of flow cytometry analysis of the presence of CD200-positive CD157-positive cells among CD31-positive CD45-negative GFP-positive cells in the liver of (A). [Figure 7]FIG. 1 shows the results of co-culture of human umbilical vein endothelial cells (HUVECs) with cells prepared from the liver of 4-day-old newborn mice, and analyzing the expression of CD157 in HUVECs by flow cytometry before and after co-culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] [Method for producing vascular endothelial stem cells] The present invention provides a method for producing vascular endothelial stem cells. The method for producing vascular endothelial stem cells of the present invention (hereinafter referred to as "the production method of the present invention") is characterized by comprising a step of contacting vascular endothelial cells not having stem cell properties with a factor secreted from an organ of a mammal at the neonatal or juvenile stage. The organ of a mammal at the neonatal or juvenile stage may be an organ of a mammal at the neonatal stage.

[0015] In this specification, vascular endothelial stem cells refer to cells that have the ability to divide in an undifferentiated state (self-renewal ability) and the ability to differentiate into vascular endothelial cells. The fact that vascular endothelial stem cells have been obtained by the production method of the present invention can be confirmed by confirming the presence of vascular endothelial cells that can form colonies from single cells. In other words, vascular endothelial cells with high colony-forming ability can be determined to be vascular endothelial stem cells. In addition, it may be confirmed that the cells obtained by the production method of the present invention have at least one of the characteristics of being cells with high drug efflux ability and being CD200-positive CD157-positive cells. For example, Hoechst staining method can be used to confirm drug efflux ability. Whether or not the cells are CD200-positive CD157-positive cells can be determined by staining the cells with anti-CD200 antibody and anti-CD157 antibody, etc.

[0016] Vascular endothelial cells are flat cells lining the vascular lumen in a single layer, and can be specified as CD31-positive CD45-negative cells. Vascular endothelial cells obtained as CD31-positive CD45-negative cells by a standard method can be suitably used as vascular endothelial cells without stem cell properties to be subjected to the production method of the present invention.

[0017] Vascular endothelial cells without stem cell properties may be vascular endothelial cells of a mammal. The mammal is not particularly limited, and examples thereof include humans, monkeys, cows, pigs, sheep, goats, dogs, cats, mice, rats, rabbits, etc. When the mammal is a human, the vascular endothelial stem cells produced by the production method of the present invention can be safely used for human cell therapy, etc.

[0018] Vascular endothelial cells without stem cell potential can be prepared from any organ. The organ may be, for example, the liver, retina, brain, heart, skin, muscle (skeletal muscle), lung, kidney, placenta, umbilical cord, adipose tissue, etc. Preferably, the organ is the liver or umbilical cord. The method for preparing vascular endothelial cells without stem cell potential is not particularly limited, but may include, for example, a method in which an isolated organ is digested and dispersed with a commercially available cell dispersion reagent to prepare a cell suspension, the cell suspension is stained with an anti-CD31 antibody and an anti-CD45 antibody, and then CD31-positive CD45-negative cells are collected using a flow cytometry technique.

[0019] The vascular endothelial cells without stem cell properties may be vascular endothelial cells obtained from a mammal, and the time of acquisition is not particularly limited. The vascular endothelial cells may be obtained from a mammal in the neonatal or juvenile stage, and are preferably vascular endothelial cells obtained from a mammal in the neonatal stage. In this specification, the neonatal stage means the period from birth to weaning, and the juvenile stage means the period from weaning to the period when reproductive ability is acquired. The present inventors have confirmed that neonatal vascular endothelial cells are more easily reprogrammed into vascular endothelial stem cells than vascular endothelial cells from adult mice.

[0020] The step of contacting vascular endothelial cells not having stem cell properties with factors secreted from a mammalian organ at the neonatal or juvenile stage may be carried out, for example, by transplanting the cells into an organ of a non-human mammal at the neonatal or juvenile stage. The non-human mammal is not particularly limited, but examples thereof include monkeys, cows, pigs, sheep, goats, dogs, cats, mice, rats, rabbits, etc. Pig or sheep is preferable. The organ is not particularly limited, but examples thereof include liver, retina, brain, heart, skin, muscle (skeletal muscle), lung, kidney, placenta, adipose tissue, etc. Preferably, the liver. The transplantation can be carried out, for example, by a method of opening the abdomen of the host animal under anesthesia and inoculating the vascular endothelial cells not having stem cell properties into the target organ by injection or the like.

[0021] The step of contacting vascular endothelial cells without stem cell properties with factors secreted from a mammalian organ at the neonatal or juvenile stage may be carried out, for example, by culturing vascular endothelial cells without stem cell properties using a culture system containing factors secreted from a mammalian organ at the neonatal or juvenile stage. Specifically, cells prepared from a mammalian organ at the neonatal or juvenile stage may be co-cultured with vascular endothelial cells without stem cell properties. Preparation of cells from an organ may include, for example, a method of digesting and dispersing an isolated organ with a commercially available cell dispersion reagent. Co-culture may be performed by culturing vascular endothelial cells without stem cell properties and cells prepared from an organ in the same culture vessel. In this case, the co-culture may be performed by attaching an instrument such as a culture insert to the culture vessel so that the vascular endothelial cells without stem cell properties and the cells prepared from the organ do not come into direct contact with each other.

[0022] Alternatively, cells prepared from a mammalian organ at the neonatal or juvenile stage may be cultured in an appropriate medium, and the culture supernatant may be collected and added to the medium for vascular endothelial cells that do not have stem cell properties. Alternatively, an extract from a mammalian organ at the neonatal or juvenile stage may be prepared, and the extract may be added to the medium for vascular endothelial cells that do not have stem cell properties. As an extract from a mammalian organ at the neonatal or juvenile stage, for example, a homogenate supernatant from the organ may be preferably used.

[0023] The period for contacting vascular endothelial cells without stem cell properties with factors secreted from mammalian organs at the neonatal or juvenile stage is not particularly limited, and an appropriate period for obtaining the desired vascular endothelial stem cells may be set in a preliminary study or the like. For example, when transplanting into a non-human mammalian organ at the neonatal or juvenile stage, the period may be 1 day or more, 2 days or more, 3 days or more, or 4 days or more, and may be 100 days or less, 50 days or less, 10 days or less, or 5 days or less. For example, when co-culturing with cells prepared from a non-human mammalian organ at the neonatal or juvenile stage, the period may be 1 day or more, 2 days or more, 3 days or more, or 4 days or more, and may be 100 days or less, 50 days or less, 10 days or less, or 5 days or less.

[0024] By contacting vascular endothelial cells without stem cell properties with factors secreted from mammalian organs at the neonatal or juvenile stage for a certain period of time, the vascular endothelial cells without stem cell properties are reprogrammed and transformed into vascular endothelial stem cells. When the vascular endothelial stem cells are produced by transplanting them into the organs of non-human mammals, the transplant site is excised, a cell suspension is prepared by using a method such as digestion and dispersion with a commercially available cell dispersion reagent, and the presence of vascular endothelial cells (CD31-positive CD45-negative cells) with high colony-forming ability is confirmed, thereby judging that the vascular endothelial stem cells have been produced. When the vascular endothelial stem cells are produced by using a method using a culture system containing factors secreted from mammalian organs at the neonatal or juvenile stage, the presence of vascular endothelial cells (CD31-positive CD45-negative cells) with high colony-forming ability is confirmed in the cells after culture, thereby judging that the vascular endothelial stem cells have been produced. Furthermore, it may be confirmed that the vascular endothelial cells with high colony-forming ability are cells with high drug-excreting ability and / or CD200-positive CD157-positive cells. The obtained vascular endothelial stem cells can be recovered, for example, by isolating and growing the cells that have formed colonies. Alternatively, cells with high drug efflux capacity or CD200-positive and CD157-positive cells may be used as indicators for collection.

[0025] [Vascular endothelial stem cell inducer] The present invention provides a vascular endothelial stem cell inducer comprising a factor secreted from a mammalian organ at the neonatal or juvenile stage. The mammalian organ at the neonatal or juvenile stage may be a mammalian organ at the neonatal stage.

[0026] The mammal is not particularly limited, and examples thereof include humans, monkeys, cows, pigs, sheep, goats, dogs, cats, mice, rats, rabbits, etc. The mammal may be a human. The organ is not particularly limited, and examples thereof include liver, retina, brain, heart, skin, muscle (skeletal muscle), lung, kidney, placenta, fat, etc. The organ may be the liver.

[0027] The vascular endothelial stem cell inducer of the present invention may be in any form as long as it contains factors secreted from the organs of mammals at the neonatal or juvenile stage. The vascular endothelial stem cell inducer of the present invention may contain, for example, a culture supernatant of cells prepared from an organ of a mammal at the neonatal or juvenile stage, or may contain an organ extract of a mammal at the neonatal or juvenile stage. Such culture supernatants and organ extracts contain factors secreted from organs. The culture supernatants can be obtained, for example, by digesting and dispersing an isolated organ with a commercially available cell dispersion reagent, and culturing the obtained cells in an appropriate medium. The organ extracts can be obtained, for example, by homogenizing and centrifuging the organ.

[0028] The vascular endothelial stem cell inducer of the present invention can be added to a medium when culturing vascular endothelial cells that do not have stem cell properties. It is recommended that the amount of the agent added be appropriately determined depending on the culture conditions, etc.

[0029] The present invention includes the following inventions. A method for inducing vascular endothelial stem cells, comprising the step of culturing vascular endothelial cells that do not have stem cell properties using a medium containing factors secreted from the organs of a mammal at the neonatal or juvenile stage. The use of factors secreted from mammalian organs at the neonatal or juvenile stage for inducing vascular endothelial stem cells. Use of a culture supernatant of cells prepared from an organ of a mammal at the neonatal or juvenile stage for inducing vascular endothelial stem cells. Use of an organ extract from a mammal at the neonatal or juvenile stage for inducing vascular endothelial stem cells. Use of a factor secreted from an organ of a mammal in the neonatal or juvenile stage for the production of a vascular endothelial stem cell inducer. Use of a culture supernatant of cells prepared from an organ of a mammal at the neonatal or juvenile stage for the production of a vascular endothelial stem cell inducer. 2. Use of an organ extract from a mammal in the neonatal or juvenile stage for the production of a vascular endothelial stem cell inducer. EXAMPLES

[0030] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

[0031] Example 1: Examination of stemness in vascular endothelial cells during fetal and postnatal periods 1-1 Experimental method C57BL / 6 mice (Japan SLC) were used. The whole fetus of E15 and the liver of 8W mice were cut into pieces as finely as possible using ophthalmic scissors. The cut tissue was immersed in a mixed solution of Dispase II (Roche Applied Science), collagenase (Wako) and type II collagenase (Worthington Biochemical) and shaken at 37°C to digest the extracellular matrix. The solution containing the digested cells was passed through a 40μm pore size filter to obtain a dispersed cell suspension. Red blood cells were lysed with ACK (Ammonium-Chloride-Potassium) solution (0.15M NH4Cl, 10mM KHCO3, and 0.1mM Na2-EDTA), and the remaining cells were used for the following experiments.

[0032] The prepared cells were subjected to immunofluorescence staining and analyzed by flow cytometry. Anti-CD31 antibody (clone MEC13.3, BD Biosciences) and anti-CD45 antibody (clone 30-F11, BD Biosciences) were used as monoclonal antibodies. Propidium iodide (PI, 2 μg / mL, Sigma-Aldrich) was added to the stained cells to stain the nuclei of dead cells and remove them. SOAP FACSAria (BD Biosciences) and FlowJo Software (Treestar Software) were used for flow cytometry analysis. CD31-positive CD45-negative vascular endothelial cells obtained by flow cytometry were seeded on a 3 cm diameter culture plate at 5,000 cells each in a culture system using OP9 stromal cells (RIKEN cell bank) as feeder cells, and cultured for 7 days.

[0033] 1-2 Results The results are shown in Figure 1. (A) shows the results of culturing vascular endothelial cells obtained from fetal mice, and (B) shows the results of culturing vascular endothelial cells obtained from the liver of an adult mouse. A small number of cells were present among the vascular endothelial cells obtained from fetuses that had the ability to proliferate and form colonies. On the other hand, it was found that vascular endothelial cells recovered from adult livers contained vascular endothelial cells that formed very large colonies. These results suggested the possibility that vascular endothelial stem cells do not exist during the fetal period, but arise after birth.

[0034] Example 2: Examination of the formation of sinusoidal blood vessels in the liver 2-1 Experimental method Livers were excised from C57BL / 6 mice (Japan SLC) fetuses on day 15 of gestation, and from newborn / juvenile mice on days 1, 7, 14, and 21 after birth, and frozen sections of the liver were prepared according to standard methods. The frozen sections were immunostained using an anti-CD31 antibody (clone MEC13.3, BD Biosciences). PE-labeled anti-rat IgG (BD Biosciences) was used as the secondary antibody.

[0035] 2-2 Results The results are shown in Figure 2. In the liver of young mice on the 21st day after birth, sinusoidal vessels were formed radially from the central vein. It was found that this liver-specific vascular structure formed radially was not formed in the liver of fetal mice, but was gradually formed after birth. Since the inventors have elucidated that sinusoidal vessels, which are vascular structures specific to the liver, are induced by vascular endothelial stem cells (Wakabayashi T. et al., Cell Stem Cell. 2018 Mar 1;22(3):384-397), it was considered possible that vascular endothelial stem cells do not exist during the fetal period and are generated after birth.

[0036] Example 3: Analysis of the developmental pattern of a fraction containing vascular endothelial stem cells (SP cell fraction) that can be isolated by utilizing their high drug efflux capacity 3-1 Experimental method Livers were excised from C57BL / 6 mice (Japan SLC) fetuses on embryonic day 15 (E15), newborns and young mice on postnatal days 1 (P1), 7 (P7), 14 (P14), 21 (P21), and 28 (P28), and adult mice at 8 weeks (8W). As in Example 1, the excised liver was cut into pieces as finely as possible using ophthalmic scissors, and the cut tissue was immersed in a mixed solution of Dispase II (Roche Applied Science), collagenase (Wako), and type II collagenase (Worthington Biochemical), and shaken at 37°C to digest the extracellular matrix. The solution containing the digested cells was passed through a filter with a pore size of 40 μm to obtain a suspension of dispersed cells. The obtained cell suspension was stained with Hoechst. Hoechst staining was performed using 1×10 6The cells were suspended in 1 ml of Hoechst-containing medium (DMEM (Sigma-Aldrich) containing 2% FBS (Sigma-Aldrich), 1 mM HEPES (Gibco), 5 μg / mL Hoechst33342 (Sigma-Aldrich)) and subjected to immunofluorescence staining at 37°C for 90 minutes. The same anti-CD31 antibody and anti-CD45 antibody as in Example 1 were used for immunofluorescence staining. Propidium iodide (PI, 2 μg / mL, Sigma-Aldrich) was added to the stained cells to stain the nuclei of dead cells and remove the dead cells. Hoechst staining analysis was performed on CD31-positive CD45-negative PI-negative cells (vascular endothelial cells from which dead cells had been removed) by flow cytometry. SOAP FACSAria (BD Bioscience) and FlowJo Software (Treestar Software) were used for flow cytometry analysis.

[0037] 3-2 Results The results are shown in Figure 3. (A) is a diagram showing typical flow cytometry analysis results for fetal and neonatal to juvenile stages, and (B) is a graph showing the percentage of the SP cell fraction. No cells of the SP cell fraction containing vascular endothelial stem cells (SP cells) were observed in the vascular endothelial cells of the fetal stage, and only cells of the MP cell fraction (MP cells), which are non-vascular endothelial stem cells, were observed. SP cells gradually appeared after birth, and it was found that in 21-day-old (P21) juvenile mice, the percentage of SP cells was about 1%, which is comparable to that of 8-week-old (8W) adult mice. This result suggests that vascular endothelial stem cells develop in the internal environment after birth.

[0038] Example 4: MP cells transform into SP cells in the liver environment 4-1 Experimental method C57BL / 6 mice and C57BL / 6-Tg(CAG-EGFP) mice (hereinafter referred to as "green mice") were purchased from Japan SLC and used. Livers were excised from green mice 1 day after birth (1 day old) and 8 weeks after birth (8 weeks old), and cell suspensions were prepared in the same manner as in Example 3. Hoechst staining and immunofluorescence staining (anti-CD31 antibody, anti-CD45 antibody, PI) were performed in the same manner as in Example 3, and vascular endothelial MP cells were collected by flow cytometry. The collected MP cells (1 x 10 5 The MP cells were transplanted into liver damage model mice. The liver vascular disorder model mice were prepared by intraperitoneally administering 300 mg / kg of monocrotaline (Sigma-Aldrich) to C57BL / 6 mice aged 7 days and 8 weeks after birth, and irradiating the whole body with 30 rads / g on the same day. Two months after the MP cell transplantation, the mice were opened under anesthesia, and the liver was observed under a fluorescent stereomicroscope (Leica). The liver was then excised and a cell suspension was prepared in the same manner as in Example 3, followed by Hoechst staining and immunofluorescence staining (anti-CD31 antibody, anti-CD45 antibody, PI), and the presence of SP cells was analyzed by flow cytometry.

[0039] 4-2 Results The results of transplanting MP cells from the liver of a 1-day-old Green mouse into a 7-day-old liver injury model mouse are shown in Figure 4. (A) is the result of observing the liver of a liver injury model mouse 2 months after transplantation, (B) is the result of analyzing the presence of CD31-positive GFP-positive cells in the liver of the mouse in (A) by flow cytometry, and (C) is the result of analyzing the presence of SP cells in the CD31-positive GFP-positive cells in (B) by flow cytometry. As shown in (A), GFP-positive cells derived from the Green mouse transplanted into the liver of the liver injury model mouse were observed. In addition, as shown in (B), a large number of CD31-positive GFP-positive cells were detected, indicating that the vascular endothelial cells of the Green mouse formed blood vessels in the host liver. Furthermore, as shown in (C), it was found that 1.5% of the CD31-positive GFP-positive cells were SP cells. This result indicates that MP cells can be reprogrammed into SP cells under appropriate conditions.

[0040] Table 1 shows the results of transplanting GFP-positive MP cells from neonatal mice (1 day old) and adult mice (8 weeks old) into neonatal liver injury model mice (7 days old) and adult liver injury model mice (8 weeks old), respectively. MP cells transplanted into the livers of adult mice were not reprogrammed into SP cells, but it was revealed that MP cells transplanted into the livers of neonatal mice were reprogrammed into SP cells. In addition, it was shown that neonatal MP cells are more easily reprogrammed into SP cells than adult MP cells. This result suggests that the neonatal liver contains many factors that reprogram MP cells into SP cells, while the adult liver contains almost no such factors. It was also considered that MP cells in the neonatal liver are more susceptible to the influence of reprogramming factors and are more easily reprogrammed into SP cells than adult MP cells.

[0041] [Table 1]

[0042] Example 5: Analysis of the developmental process of CD157-positive CD200-positive vascular endothelial stem cells 5-1 Experimental method Livers were excised from C57BL / 6 mice (Japan SLC) fetal mice on embryonic day 13 (E13), 15 (E15), and 18 (E18), newborn mice on postnatal day 1 (P1), 4 (P4), 7 (P7), and 14 (P14), and adult mice at 8 weeks (8W), and cell suspensions were prepared in the same manner as in Examples 1 and 3. As monoclonal antibodies, anti-CD31 antibody (clone MEC13.3, BD Biosciences), anti-CD45 antibody (clone 30-F11, BD Biosciences), anti-CD157 antibody (clone BP3, Biolegend), and anti-CD200 antibody (clone OX90, Biolegend) were used. Propidium iodide (PI, 2 μg / mL, Sigma-Aldrich) was added to the stained cells to stain the nuclei of dead cells and remove them. Flow cytometry analysis was performed using SOAP FACSAria (BD Bioscience) and FlowJo Software (Treestar Software).

[0043] 5-2 Results The results are shown in Figure 5. CD31+CD45-cells in the liver were gated as vascular endothelial cells in the liver, and the expression of CD200 and CD157 was analyzed by flow cytometry. Up until E15, all vascular endothelial cells were CD200-CD157-negative cells, whereas a small number of CD200-CD157-negative cells appeared at E18. After birth, at P1, the CD200-CD157-negative cell population began to be clearly separated from the CD200-CD157-negative cell population, but the CD200-CD157-positive cell population was hardly present. At P4, a small number of CD200-CD157-positive cells appeared, and at P7 and P14, the number of CD200-CD157-positive cells gradually increased, and in 8W adult mice, 4-6% of the total vascular endothelial cells were CD200-CD157-positive cells. Although data is not shown, thereafter, vascular endothelial stem cells of CD200-positive CD157-positive cells were present in the liver at almost the same rate. This result was the same as that of vascular endothelial stem cells in SP cells by the Hoechst exclusion method in Example 3. In other words, it was revealed that vascular endothelial stem cells do not exist in the liver during fetal development, and that vascular endothelial progenitor cells that have some stem cell properties but are more differentiated than stem cells appear in part just before birth, and that vascular endothelial stem cells are generated in the liver after birth.

[0044] [Example 6: CD157-negative CD200-negative vascular endothelial cells transform into vascular endothelial stem cells in the neonatal liver environment] 6-1 Experimental Method The liver of a green mouse on embryonic day 15 (E15) was excised, a cell suspension was prepared in the same manner as in Example 3, and stained with the same anti-CD31 antibody, anti-CD45 antibody, anti-CD157 antibody, and anti-CD200 antibody as in Example 5, and subjected to flow cytometry, and CD200-negative CD157-negative vascular endothelial cells (CD31-positive CD45-negative cells) were collected. The collected cells were transplanted into the liver of a liver disorder model mouse (C57BL / 6) on the 7th day after birth (7 days old) in the same manner as in Example 4. The liver disorder model mouse was produced in the same manner as in Example 4. Two months after transplantation, the mouse was opened under anesthesia, and the liver was observed with a fluorescent stereomicroscope (Leica). Furthermore, the liver was excised, a cell suspension was prepared in the same manner as in Example 3, and stained with the same anti-CD31 antibody, anti-CD45 antibody, and anti-CD157 antibody as in Example 5, and flow cytometry analysis was performed.

[0045] 6-2 Results The results are shown in Figure 6. (A) shows the results of observing the liver of the host mouse two months after transplantation, and (B) shows the results of flow cytometry analysis of the presence of CD200-positive CD157-positive cells among the CD31-positive CD45-negative GFP-positive cells in the liver of the host mouse in (A). As shown in (A), a large number of GFP-positive cells derived from the Green Mouse were observed in the liver of the host mouse, and it was found that the CD200-negative CD157-negative vascular endothelial cells (CD31-positive CD45-negative GFP-positive cells) of the Green Mouse fetus acquire the ability to contribute to sinusoidal blood vessels in the neonatal liver environment. In addition, as shown in (B), it was found that a part of the transplanted CD200-negative CD157-negative vascular endothelial cells of the Green Mouse fetus was reprogrammed into CD200-positive CD157-positive vascular endothelial stem cells.

[0046] Example 7: Conversion of human vascular endothelial cells to vascular endothelial stem cells 7-1 Experimental method Human umbilical vein endothelial cells (HUVEC) (Lonza) were used as vascular endothelial cells from newborn humans. HUVEC were cultured in HuMedia-EG2 (KURABO, Osaka, Japan) culture medium. HUVEC were cultured in a 6-well plate at a density of 1.0 × 10 5 The cells were seeded at 2.0 × 10 cells / well, and a culture insert with a pore size of 1.0 μm (Corning, NY, USA) was placed in the well. The liver of a 4-day-old newborn mouse (C57BL / 6) was excised, and a cell suspension was prepared in the same manner as in Example 1. The cell suspension contained 2.0 × 10 cells. 6 The HUVECs were seeded on culture inserts and co-cultured for 5 days without direct contact with the newborn mouse cells. After co-culture, the HUVECs were harvested and stained with CD157 antibody, and then analyzed by flow cytometry.

[0047] 7-2 Results The results are shown in Figure 7. Before co-culture, HUVECs were CD157 negative, but co-culture with cells derived from neonatal mouse liver induced positive CD157 expression. These results demonstrated that cells in the neonatal liver have the ability to induce CD157 and induce differentiation of endothelial cells into endothelial stem cells, and suggested that humoral factors secreted from the liver are involved in this transdifferentiation.

[0048] The present invention is not limited to the above-mentioned embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in the different embodiments. In addition, all academic documents and patent documents described in this specification are incorporated herein by reference.

Claims

1. A method for producing vascular endothelial stem cells from vascular endothelial cells that do not have stem cell properties, the method comprising any one of the following steps (1) to (4): (1) transplanting the vascular endothelial cells not having stem cell potential into the liver of a neonatal non-human mammal; (2) co-culturing cells prepared from the liver of a neonatal non-human mammal with the vascular endothelial cells not having stem cell properties; (3) culturing the vascular endothelial cells that do not have stem cell properties using a medium containing a culture supernatant of cells prepared from the liver of a neonatal non-human mammal; or (4) culturing the vascular endothelial cells that do not have stem cell properties in a medium containing a liver extract from a neonatal non-human mammal.

2. The method according to claim 1 , wherein the vascular endothelial cells not having stem cell properties are cells obtained from a mammal in the neonatal or juvenile stage.

3. The method according to claim 2 , wherein the vascular endothelial cells not having stem cell properties are cells obtained from a mammal in the neonatal stage.

4. A vascular endothelial stem cell inducer comprising a culture supernatant of cells prepared from the liver of a neonatal non-human mammal, or an extract of the liver of a neonatal non-human mammal.

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