WO2014051154A1 - 虚血性疾患治療に適した細胞を含む細胞群の生体外増幅方法 - Google Patents
虚血性疾患治療に適した細胞を含む細胞群の生体外増幅方法 Download PDFInfo
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Definitions
- the present invention relates to a method for amplifying and modifying a cell group comprising vascular endothelial progenitor cells suitable for treatment of ischemic disease in serum-free culture from a mononuclear cell fraction, a cell group obtained by the method, and the cell group
- the present invention relates to a method for producing an ischemic disease therapeutic agent.
- Non-patent Document 1 it has been reported that co-culture of CD3-positive CD31-positive cells called Angiogenic T cells present in the CD34-negative cell population with EPC increases the ability of EPC to regenerate blood vessels. has been suggested to contain cells that enhance the ability of EPC (Non-patent Document 1).
- Non-Patent Document 1 there is a possibility that cells having the ability to enhance the ability of EPC exist in the CD34 negative cell population. Therefore, as in Patent Document 3 described above, in order to obtain an EPC fraction, the CD34 and / or CD133 positive cells are not sorted, or the CD34 positive cells and the CD34 negative cells are mixed at a certain ratio to obtain mononuclear cells.
- the culture or group of cells obtained by this method is more inflammatory with a better ability to form vascular endothelium than the culture or group of cells obtained by culturing CD34 and / or CD133 positive cells. It has proven to be effective in the treatment of ischemic diseases because it has an angiogenic action and an excellent tissue repair ability.
- the subject of this invention is providing the cell group which simplifies the preparation process of the cell group used for ischemic disease treatment, and is more effective in the said treatment. More specifically, by simplifying the cell sorting process after collecting bone marrow, umbilical cord blood and peripheral blood, not only vascular regenerative cells: specifically differentiated EPC colony forming cells with high angiogenic ability, but also anti-inflammatory Tolerance-inducing cells: Specifically, to provide a means for amplifying cell groups including M2 macrophages converted to anti-inflammatory, T lymphocyte subsets, and regulatory T cells.
- the present inventors have differentiated these cells into a group of cells that contribute to angiogenesis without sorting out mononuclear cells in vitro or by mixing CD34-sorted and unsorted cells. ⁇ Culture conditions that allowed growth were examined. Results: (1) Stem cell factor (SCF), (2) Interleukin-6 (IL-6), (3) FMS-like tyrosine kinase 3 ligand (FMS-like tyrosine kinase 3 ligand; FL) ), (4) by culturing mononuclear cells in a serum-free medium containing a factor selected from the group consisting of thrombopoietin (TPO) and (5) vascular endothelial growth factor (VEGF).
- SCF Stem cell factor
- IL-6 Interleukin-6
- FL FMS-like tyrosine kinase 3 ligand
- TPO thrombopoietin
- VEGF vascular endothelial growth factor
- This group of cells containing EPC is particularly characterized in that it contains EPC and anti-inflammatory M2 macrophages, and also includes anti-inflammatory T lymphocyte subsets and regulatory T cells. Therefore, it was clarified that this cell group has not only angiogenesis but also an anti-inflammatory effect. Furthermore, the present inventors have clarified that cell groups having similar functions can be obtained not only from healthy individuals but also from cell groups collected from diabetic patients. In particular, in mononuclear cells used for culture, it was clarified that a cell group with remarkably high blood vessel regeneration ability can be obtained when CD34 positive cells and negative cells are cultured at a certain ratio.
- the present invention is as follows.
- Mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood are obtained by culturing in a serum-free medium containing stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial growth factor.
- Cell group [2] A cell group obtained by culturing mononuclear cells derived from bone marrow, umbilical cord blood or peripheral blood in a serum-free medium, and enriched with vascular endothelial progenitor cells and anti-inflammatory macrophages.
- [3] The cell group according to [1] above, comprising vascular endothelial progenitor cells and anti-inflammatory macrophages.
- [4] The cell group according to [2] or [3] above, wherein the vascular endothelial progenitor cells are differentiated EPC colony forming cells.
- [5] The cell group according to any one of [2] to [4], wherein the anti-inflammatory macrophage is M2 macrophage.
- a mononuclear cell derived from bone marrow, umbilical cord blood or peripheral blood is cultured in a serum-free medium containing stem cell factor, interleukin 6, FMS-like tyrosine kinase 3 ligand, thrombopoietin and vascular endothelial growth factor
- a method for producing a cell group enriched in vascular endothelial progenitor cells and / or anti-inflammatory macrophages [7] The method described in [6] above, wherein the vascular endothelial progenitor cells are differentiated EPC colony forming cells.
- the anti-inflammatory macrophage is M2 macrophage.
- a therapeutic agent for ischemic disease comprising the cell group according to any one of [1] to [5] above.
- the therapeutic agent according to [9] above, wherein the ischemic disease is a disease that is cured by angiogenesis.
- a therapeutic agent for refractory ulcer or diabetes-related disease comprising the cell group according to any one of [1] to [5] above.
- the present invention By transplanting the cell group amplified by the present invention, improvement in blood flow volume and necrosis improvement rate of ischemic disease was observed. Furthermore, the present inventors have examined these effects, and the present invention is useful in producing endothelial cells both in quality and quantity, and is a useful method of cell transplantation therapy for vascular disorders such as ischemic diseases. It came to the conclusion that it is.
- a cell group having a high vascular regenerative capacity in both quantity and quality can be obtained even in a patient with reduced vascular regenerative capacity such as a diabetic patient. It can be said that it is extremely useful as a cell transplantation therapy because its presence suppresses inflammation.
- FIG. 1 shows that peripheral blood mononuclear cells (hereinafter referred to as PBMNC) were seeded with 2 ⁇ 10 6 cells and cultured for 7 days in the serum-free medium of the present invention (Quality and Quantity Cell; hereinafter referred to as QQC). It is shown as a ratio to the number of cells. *** indicates a significant difference (P ⁇ 0.001) from the subject shown in the figure.
- FIG. 2 shows undifferentiated (small) EPC colonies on the left and differentiated EPC colonies on the right. The bar is 500 ⁇ m.
- FIG. 3 shows the number of undifferentiated EPC colonies (white frame) and differentiated EPC colonies (gray frame) after seeding with PBMNC (left bar) and QQC (right bar).
- FIG. 4 shows undifferentiated EPC after PBMNC was sorted into CD34 positive and negative cells and cultured in the serum-free medium of the present invention for 7 days in the presence of CD34 negative, CD34 positive, CD34 positive and CD34 negative cells in order from the left bar. The number of colonies (white frame) and differentiated EPC colony (dot frame) formed is shown. * And *** indicate significant differences (P ⁇ 0.05, P ⁇ 0.001), respectively, from the subject shown in the figure.
- FIG. 4 shows undifferentiated EPC after PBMNC was sorted into CD34 positive and negative cells and cultured in the serum-free medium of the present invention for 7 days in the presence of CD34 negative, CD34 positive, CD34 positive and CD34 negative cells in order from the left bar. The number of colonies (white frame) and differentiated EPC colony (dot frame) formed is shown. * And *** indicate significant differences (P ⁇ 0.05, P ⁇ 0.001), respectively, from the subject shown in the figure.
- FIG. 4 shows undifferentiated
- FIG. 5A shows the expression of each protein in QQC and PBMNC using a flow cytometer, and the expression ratio (%) (left) or the number of positive cells per 100 ml of peripheral blood (right) is shown as a ratio.
- FIG. 5B shows that each of the scatter plots of PBMNC or QQC is gated by cell size into three populations (ie, lymphocyte size, monocyte size, large cell size) and the sum of cells gated into these populations is 100 The percentage change of each marker-expressing cell in the whole cell after QQc (right) in this case is shown.
- FIG. 6 shows the content of helper lymphocyte (Th) subset inflammatory Th1, anti-inflammatory Th2 and regulatory T (T reg) in QQC and PBMNC.
- Th helper lymphocyte
- the left graph shows the ratio of the content in QQC to the content in PBMNC of each Th.
- the right graph shows the increase or decrease of the real number of each T subset content relative to the QQC PBMNC.
- N 4-6.
- FIG. 7 compares the change in the total cell number and the number of CD34 positive cells when QQc is performed between a healthy person (Healthy) and a diabetic patient (DM). * p ⁇ 0.05
- FIG. 8 is a comparison of changes in cell numbers of anti-inflammatory macrophages and inflammatory macrophages when QQc is performed between healthy individuals (Healthy) and diabetic patients (DM).
- FIG. 9 is a comparison of the number of mature EPCs and the number of cell colonies having revascularization ability when QQc is performed on healthy individuals (Healthy) and diabetic patients (DM). * p ⁇ 0.05, *** p ⁇ 0.001
- FIG. 10 shows the expression of each gene in QQC (black frame) and PBMNC (gray frame) measured by the quantitative PCR method and expressed as an expression ratio with the GAPDH gene. *, ** and *** indicate significant differences (P ⁇ 0.05, P ⁇ 0.01, P ⁇ 0.001) with respect to the subject shown in the figure.
- FIG. 10 shows the expression of each gene in QQC (black frame) and PBMNC (gray frame) measured by the quantitative PCR method and expressed as an expression ratio with the GAPDH gene. *, ** and *** indicate significant differences (P ⁇ 0.05, P ⁇ 0.01, P ⁇ 0.001) with respect to the subject shown in the figure.
- FIG. 10 shows the expression of each gene in QQC (black frame) and PBMNC (gray frame) measured
- FIG. 11 shows the measurement of blood vessel formation when human vascular endothelial cells (Human vascular endocrine cells; hereinafter referred to as HUVEC) are not co-cultured (white frame), co-cultured with PBMNC (gray frame), and co-cultured with QQC (black frame). It is shown. HPF stands for High power field. * And ** indicate significant differences (P ⁇ 0.05, P ⁇ 0.01) with respect to the subjects shown in the figure.
- FIG. 12 shows PBMNC (white frame) or QQC (black frame) labeled with acLDL-DiI co-cultured with HUVEC and the number of uptake into blood vessels was measured. *** indicates a significant difference (P ⁇ 0.001) from the subject shown in the figure.
- FIG. 12 shows PBMNC (white frame) or QQC (black frame) labeled with acLDL-DiI co-cultured with HUVEC and the number of uptake into blood vessels was measured. *** indicates a significant difference (P ⁇
- FIG. 13 shows Iscove's Modified Dulbecco's Medium (hereinafter IMDM) (white frame), PBMNC (gray frame) and QQC (black frame) transplanted into ischemic mice, and then blood was analyzed by laser dropper image analysis on the 21st day. The flow rate is measured and the ratio of blood flow to the control lower limb is shown in%. * Indicates a significant difference (P ⁇ 0.05) from the subject shown in the figure. N represents the number of measured mice.
- FIG. 14 is a bar graph showing the rim salvage (lower limb remaining) score in different stages.
- FIG. 15 shows the amplification of EPC with QQ culture days by mouse EPC-CFA. On day 8 after cell seeding. Both undifferentiated and differentiated EPC colonies (pEPC-CFU, dEPC-CFU) increased gradually according to the number of days of QQ culture. The numerical values in the graph indicate the number of each EPC colony.
- FIG. 16 shows the blood flow improvement effect by transplantation of QQC in the C57BL6 / N mouse lower limb ischemia model.
- the left graph shows the results of measurement by staining the capillarity-forming ability by transplanting QQC in C57BL6 / N mouse lower limb ischemia model with islectin B4-FITC (Vector Lab).
- the right graph shows the wall cell region lined with microvessels.
- IMDM non-cell transplanted
- PBMNC (1x10 5 / animal
- FIG. 18 shows the results of measuring capillary formation by staining with islectin B4 antibody. The results when IMDM, PBMNC and QQC are transplanted are shown from the left. *** indicates a significant difference (P ⁇ 0.001) from the subject shown in the figure.
- FIG. 19 shows the results of measuring mature angiogenesis by staining with a smooth muscle alpha actin antibody (Sigma-Aldrich). The results when IMDM, PBMNC and QQC are transplanted are shown from the left.
- FIG. 20 shows myotube formation measured by HE staining of tissue fragments. The results when IMDM, PBMNC and QQC are transplanted are shown from the left. * And ** indicate significant differences (P ⁇ 0.05, P ⁇ 0.01) with respect to the subjects shown in the figure.
- FIG. 21 shows fibrosis measured by Azan staining of tissue fragments. The results when IMDM, PBMNC and QQC are transplanted are shown from the left. ** indicates a significant difference (P ⁇ 0.01) with respect to the subject shown in the figure.
- FIG. 22 shows ischemic muscle tissue stained with iNOS antibody. It has been shown that inflammatory cells are significantly confined in the QQ-MNC transplant group.
- the present invention relates to a method for proliferating a cell group containing vascular endothelial progenitor cells in vitro, particularly a method for proliferating a cell group enriched in vascular endothelial progenitor cells and anti-inflammatory macrophages, and a cell transplant obtained by the method.
- a group of cells is provided.
- the mononuclear cell used in the present invention is a general term for cells having a circular nucleus contained in peripheral blood, bone marrow or umbilical cord blood, and includes lymphocytes, monocytes, macrophages, vascular endothelial progenitor cells, hematopoietic stem cells, and the like. . Mononuclear cells further contain CD34 and / or CD133 positive cells. Mononuclear cells can be obtained by collecting bone marrow, umbilical cord blood or peripheral blood from an animal and extracting the fraction by subjecting it to, for example, density gradient centrifugation.
- the density gradient centrifugation method is not particularly limited as long as a mononuclear cell fraction is formed, but Histopaque-1077 (Sigma-Aldrich) is preferably used.
- Histopaque-1077 Sigma-Aldrich
- the mononuclear cells used in the present invention have a great feature in that the obtained mononuclear cells can be used as they are in the cell culture described later without selecting CD34 and / or CD133 positive cells (positive selection). .
- the animal species from which the cells used in the present invention are derived means general mammals including humans to which cell transplantation therapy for diseases such as ischemic diseases is applied, but in view of the object of the present invention of clinical application, it is preferable. Is a human.
- the stem cell factor (SCF) used in the present invention is a glycoprotein consisting of 248 amino acids and having a molecular weight of about 30,000.
- a soluble type and a membrane-bound type exist by selective splicing, and the SCF used in the present invention may be any type of SCF as long as it is useful for culturing such as EPC. Preferably it is a soluble type.
- the origin of SCF is not particularly limited, but a recombinant that can be stably supplied is preferable, and a human recombinant is particularly preferable. Those that are commercially available are known.
- the concentration of SCF in the serum-free medium varies depending on the type of SCF to be used and is not particularly limited as long as it is useful for culturing EPC or the like.
- human recombinant SCF for example, 10 to 1000 ng / mL, preferably Is 50 to 500 ng / mL, more preferably about 100 ng / mL.
- Interleukin 6 (IL-6) used in the present invention is a glycoprotein having a molecular weight of 210,000, which has been isolated as a factor that induces terminal differentiation of B cells into antibody-producing cells. It is known to be involved in cell line proliferation and differentiation, acute phase reaction, and the like.
- IL-6 used in the present invention is appropriately selected. When used for culturing human EPC or the like, human IL-6 is preferable, and a recombinant that can be stably supplied is particularly preferable. Those that are commercially available are known.
- the concentration of IL-6 in the serum-free medium varies depending on the type of IL-6 used, and is not particularly limited as long as it is useful for culturing EPC or the like. In the case of human recombinant IL-6, for example, 1 ⁇ 500 ng / mL, preferably 5 to 100 ng / mL, more preferably about 20 ng / mL.
- the FMS-like tyrosine kinase 3 ligand (FL) used in the present invention is known as a receptor tyrosine kinase ligand that plays an important role in the control of early hematopoiesis.
- FL FMS-like tyrosine kinase 3 ligand
- the FL used in the present invention may be any type of FL as long as it is useful for culture such as EPC. Those that are commercially available are known.
- the concentration of FL in the serum-free medium varies depending on the type of FL used, and is not particularly limited as long as it is useful for culturing EPC or the like, but in the case of human recombinant Flt-3 ligand, for example, 10 to 1000 ng / mL, preferably 50-500 ng / mL, more preferably about 100 ng / mL.
- Thrombopoietin (TPO) used in the present invention is a kind of hematopoietic cytokine and is known to act specifically on the process of producing megakaryocytes from hematopoietic stem cells and promote the production of megakaryocytes.
- the origin of TPO used in the present invention is not particularly limited, but a recombinant that is expected to be stably supplied is preferable, and a human recombinant is particularly preferable. Those that are commercially available are known.
- the concentration of TPO in the serum-free medium varies depending on the type of TPO used, and is not particularly limited as long as it is useful for culturing EPC or the like.
- human recombinant TPO for example, 1 to 500 ng / mL, preferably Is 5 to 100 ng / mL, more preferably about 20 ng / mL.
- vascular endothelial growth factor that can be used in the present invention is a growth factor that specifically acts on EPC, and is known to be produced mainly by cells surrounding blood vessels.
- VEGF proteins of different sizes are produced by alternative splicing, and the VEGF used in the present invention may be any type of VEGF as long as it enables EPC colonization.
- VEGF165 is preferable.
- the origin of VEGF and the like are not particularly limited, but a recombinant that can be stably supplied is preferable, and a human recombinant is particularly preferable. Those that are commercially available are known.
- the concentration of VEGF in the serum-free medium varies depending on the type of VEGF used and is not particularly limited as long as it is useful for culturing EPC or the like.
- human recombinant VEGF165 for example, about 5 to 500 ng / mL, Preferably it is about 20-100 ng / mL, more preferably about 50 ng / mL.
- the various factors added to the serum-free medium of the present invention are preferably unified with factors derived from animals of the same species as the animal from which the mononuclear cells are derived.
- a cell culture suitable for allogeneic transplantation such as allogeneic transplantation can be obtained.
- a cell group containing EPC or the like can be cultured in an environment that does not contain any components derived from different animals, the resulting cell culture has the advantage of avoiding infection risk and rejection during transplantation and the like. .
- a serum-free medium for in vitro amplification of a group of cells comprising progenitor cells can be prepared.
- the serum-free medium of the present invention can be prepared. Filtration sterilization can be performed according to a method commonly practiced in this field, for example, using a 0.22 ⁇ m or 0.45 ⁇ m Millipore filter.
- serum-free medium used in the present invention, a medium usually used in this field can be used, and for example, a serum-free medium known as a medium for proliferating hematopoietic stem cells can be used.
- a serum-free medium known as a medium for proliferating hematopoietic stem cells
- the basal medium used as the serum-free medium include DMEM, MEM, IMDM and the like.
- the serum-free medium of the present invention contains one or more factors selected from the group consisting of SCF, IL-6, FL, TPO and VEGF, preferably three or more factors, more preferably all factors.
- the serum-free medium used in the culture method of the present invention is, for example, a) SCF, b) IL-6, c) FL, d) TPO, e) VEGF, f) a combination of SCF and IL-6, g) Combination of SCF and FL, h) Combination of SCF and TPO, i) Combination of SCF and VEGF, j) Combination of IL-6 and FL, k) Combination of IL-6 and TPO, l) IL- 6) a combination of VEGF, m) a combination of FL and TPO, n) a combination of FL and VEGF, o) a combination of TPO and VEGF, p) a combination of SCF, IL-6 and FL, q) SCF,
- the serum-free medium of the present invention more preferably contains SCF, IL-6, FL, TPO and VEGF. More preferably, it contains about 50 ng / mL VEGF, about 100 ng / mL SCF, about 20 ng / mL IL-6, 100 ng / mL FL, about 20 ng / mL TPO.
- the mononuclear cell culture in the serum-free medium containing the above-described factor is performed by adding a cell suspension containing the mononuclear cell to the serum-free medium containing the above-mentioned factor.
- a cell suspension a body fluid itself containing mononuclear cells (for example, bone marrow fluid, umbilical cord blood, peripheral blood) can also be used.
- the culture conditions for mononuclear cells are not particularly limited, and can be carried out under conditions that are usually performed in the art.
- the cells are cultured at 37 ° C. for 7 days or longer (for example, 10 days or longer) in a 5% CO 2 atmosphere.
- the concentration of mononuclear cells in the serum-free medium is not particularly limited as long as culture such as EPC is possible.
- the concentration is about 0.5 to 10 ⁇ 10 5 cells / ml, more preferably about 1 to 5 ⁇ 10 5.
- the cell group in the present invention is a general term for cells obtained as a result of culturing mononuclear cells in a serum-free medium containing the above-mentioned factors, and together with EPC, vascular endothelial cells, anti-inflammatory macrophages, promotion of angiogenesis and fibrosis It is a cell group containing at least one or more suppressor cells.
- vascular endothelial cells, anti-inflammatory macrophages, angiogenesis-promoting and fibrosis-suppressing cells were preferentially amplified together with the above EPC. If it is a thing, it is thought that the same effect is acquired.
- a cell group including a cell group positive for CD206 and a cell group positive for CD34 is preferable, and a cell group with high expression thereof is more preferable.
- a cell group including differentiated EPC and an anti-inflammatory macrophage described later is desirable, a cell group containing more of these cell groups, that is, a cell group enriched with these cell groups. It is more desirable.
- the cell group enriched with EPC and / or anti-inflammatory macrophage is a cell after culturing compared to the respective ratio in the whole mononuclear cell before culturing in the serum-free medium containing the above-mentioned factors.
- the ratio of each cell in the whole group increases to 2 times or more, preferably 4 times or more, more preferably 5 times or more.
- the cell group obtained by the culture method of the present invention has a significantly higher ratio of EPC and anti-inflammatory macrophages than the original mononuclear cells, and thus significantly promotes angiogenesis and suppresses inflammation. Is played.
- the EPC used in the present invention is not particularly limited as long as it is an undifferentiated cell that can be a vascular endothelial cell.
- EPC is further differentiated EPC colonies mainly containing cells with a diameter of 20-50 ⁇ m (CFU-Large cell like EC, also referred to as large EPC colonies) and undifferentiated EPC colonies mainly containing cells with a diameter of 20 ⁇ m or less depending on the degree of differentiation. It can be distinguished from two types of colonies with different sizes (CFU-small cell like EC, also called small EPC colony).
- An undifferentiated (small) EPC colony that appears at an early stage can be said to be an early differentiation stage EPC colony that is excellent in proliferative ability, and a differentiated (large) EPC colony that appears at a later stage is an late stage that is excellent in blood vessel development. It can be said that it is a differentiation stage EPC colony (for example, see Masuda H. et al., Circulation Research, 109: 20-37 (2011)).
- the EPC contained in the cell group obtained by the present invention has a marked increase in the number of differentiated EPC colony forming cells compared to the case of sorting by CD34 / CD133, while the number of undifferentiated EPC colony forming cells is almost the same. ing.
- the number of undifferentiated EPC-forming cells does not change as compared with the case of selection by CD34 / CD133, whereas the number of differentiated EPC colony-forming cells is twice or more. More preferably, the number of undifferentiated EPC colony forming cells does not change as compared with the case of selection by CD34 / CD133, but 4 times, more preferably 5 times, differentiated EPC colony forming cells are included. .
- the anti-inflammatory macrophages contained in the cell group obtained by the present invention are macrophages that are CD206 positive and anti-inflammatory and contribute to angiogenesis and repair. M2 macrophages are preferable, and CD206 positive M2 macrophages are more preferable.
- the cell group obtained by the present invention comprises this cell together with vascular endothelial progenitor cells, and the ratio of anti-inflammatory macrophages in the cell group is usually more than twice that of mononuclear cells in the case of unselected. , Preferably increased by a factor of 4 or more.
- Differentiation or amplification promotion from undifferentiated EPC colony-forming cells to differentiated EPC colony-forming cells by the unexpected method of culturing unselected mononuclear cells in a serum-free medium containing the aforementioned factors, blood system It becomes possible to promote differentiation or amplification of anti-inflammatory macrophages in cells or to suppress differentiation or amplification of inflammatory macrophages.
- Measurement of vascular endothelial progenitor cell colony forming ability includes physiologically active substances, specifically, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), preferably in addition to the above factors, 1 or 2 or more, preferably 3 or more, more preferably all selected from the group consisting of stem cell factor (SCF), interleukin 3 (IL-3), insulin growth factor (IGF) and epidermal growth factor (EGF) And a methylcellulose medium containing serum and / or heparin as necessary.
- SCF stem cell factor
- IL-3 interleukin 3
- IGF insulin growth factor
- EGF epidermal growth factor
- a methylcellulose medium containing serum and / or heparin as necessary.
- VEGF vascular endothelial progenitor cells cultured in methylcellulose medium containing about 50 ng / mL EGF and about 50 ng / mL IGF in a 5% CO 2 atmosphere at 37 ° C. for usually 10 days or longer, such as 14-18 days or longer Allow colonies to form. The formation of colonies can be visually confirmed.
- vascular endothelial precursor cells depends on the ability to take up acetylated LDL (acLDL), the ability to bind to UEA-1 lectin, This is carried out by confirming the expression of VE-cadherin, KDR, vWF (for example, by RT-PCR or fluorescent immunohistochemical method). For example, when a colony is double-stained with DiI-labeled acetylated LDL (acLDL-DiI) (Biomedical Technologies) and FITC-labeled UEA-1 lectin (UEA-1 lectin-FITC) (Vector Lab), vascular endothelial precursor If cells are stained together.
- acLDL DiI-labeled acetylated LDL
- UEA-1 lectin-FITC FITC-labeled UEA-1 lectin
- Measurement of the ability to form vascular endothelial progenitor cell colonies can also be performed using a commercially available kit.
- a kit for example, a kit (catalog No. H4236) manufactured by Stem Cell Technology is commercially available.
- EPC colony forming ability can be easily measured.
- Differentiation or amplification of anti-inflammatory macrophages or inflammatory macrophages can be measured, for example, by labeling CD206 for anti-inflammatory macrophages and CCR2 for inflammatory macrophages with antibodies that have affinity for them and by flow cytometer analysis .
- the cell group obtained by the present invention may further contain anti-inflammatory Th2 cells and regulatory T cells. Therefore, the cell group obtained by the present invention may be a cell group enriched with anti-inflammatory Th2 cells and regulatory T cells in addition to EPC and anti-inflammatory macrophages.
- the ratio of each cell to the total number of cells compared with that before culturing is usually 2 times or more, preferably 4 times or more, more preferably 5 times or more.
- the cell group obtained by the present invention preferably contains differentiated EPC colony forming cells, and more preferably contains differentiated EPC colony forming cells and anti-inflammatory macrophages (CD206 positive M2 macrophages).
- This cell group is extremely effective in that it can not only generate blood vessels but also suppress inflammation at sites of inflammation such as ulcers.
- the present invention further provides a method for preparing vascular endothelial cells, comprising differentiating vascular endothelial progenitor cells obtained by culturing unselected mononuclear cells in a serum-free medium containing the above-described factors into vascular endothelial cells.
- Differentiation of vascular endothelial progenitor cells into vascular endothelial cells can be performed by a method known per se, and examples thereof include methods using EBM-2, EGM2V Single Quots (Clonetics), autoserum and the like.
- the cells such as EPC contained in the cell group of the present invention can be isolated and / or purified as appropriate.
- CD34 and CD133 as cell surface markers for blood hemangioblasts
- KDR as a cell surface marker for EPC
- KDR as a surface marker for vascular endothelial cells
- vascular endothelial cadherin as a surface marker for anti-inflammatory macrophages
- a desired cell can be separated by subjecting to a cell separation method using a substance (for example, an antibody) having affinity for these cell surface markers. Examples of such cell separation methods include magnetic cell separation (MACS) and fluorescent cell separation (FACS).
- MCS magnetic cell separation
- FACS fluorescent cell separation
- a feature of the present invention is that EPC containing differentiated EPC colony-forming cells is obtained by culturing mononuclear cells obtained without CD34 and / or CD133 positive selection in a serum-free medium containing the above-mentioned factors, anti-inflammatory properties Macrophages, pro-angiogenesis and fibrosis-suppressing cells are to be amplified. More specifically, when the mononuclear cells selected for CD34 positive are cultured by the culture method of the present invention, only undifferentiated EPC colony-forming cells are amplified. However, when unselected mononuclear cells are used, undifferentiated EPC colony-forming cells are amplified. At the same time, differentiated EPC colony forming cells are amplified, and even anti-inflammatory macrophages are amplified. Furthermore, anti-inflammatory Th2 cells and regulatory T cells are also amplified.
- the present invention also provides a therapeutic agent for ischemic diseases.
- the therapeutic agent of the present invention can be applied to diseases that can be treated by angiogenesis. That is, the therapeutic agent of the present invention can be used for the treatment of vascular disorders by virtual cell transplantation.
- diseases that can be treated by neovascularization include ischemic diseases (for example, ischemic heart diseases such as myocardial infarction and angina pectoris, lower limb ischemia such as lower limb ischemic arteriosclerosis, Burger) ) Disease), vascular injury. It can also be used to heal wounds such as skin ulcers or to make artificial blood vessels.
- the effect after cell transplantation can be confirmed by a method known per se.
- the disease that can be treated by angiogenesis is a lower limb ischemic disease
- the therapeutic effect after transplantation can be evaluated by examining, for example, the lower limb blood flow rate and the necrosis improvement rate.
- the increase in blood flow can be measured by measuring the value of laser Doppler imaging analysis.
- the necrosis improvement rate can be measured visually as a rim salvage score.
- the cardiac function after transplantation can be evaluated by examining, for example, contractility and dilatability. Measurement of the contractility and dilatability of the heart can be performed by various methods implemented in the art.
- the measurement of contractility is a + dp / dt value calculated from the mitral regurgitation waveform (value decreases as contractility decreases) or left ventricular ejection fraction (% EF, value decreases as contractility decreases) If the diastolic capacity is measured, the -dp / dt value (the value increases as the diastolic capacity decreases), the end diastolic inner diameter (EDd), etc., which are similarly calculated from the mitral regurgitation waveform, etc. (See, for example, FASEB Journal, 18: 1392-1394 (2004)).
- the agent of the present invention may be the cell group itself of the present invention, which is an active ingredient, or may be suspended in a liquid medium.
- the liquid medium may be any liquid that can be injected into humans.
- phosphate buffer, physiological saline, or DMEM that is a serum-free medium can be used.
- the liquid medium may contain a compound preferable for cell survival such as albumin.
- serum derived from a patient is used as the compound containing albumin.
- the cell group which is an active ingredient of the agent of the present invention includes vascular endothelial progenitor cells, vascular endothelial cells, anti-inflammatory macrophages, angiogenesis promotion and fibrosis-suppressing cells.
- the present invention further provides a serum-free medium containing the above-mentioned factors, and a mononuclear cell culture reagent and kit containing the serum-free medium.
- the kit of the present invention further comprises a substance (for example, an antibody) having specific affinity for a cell surface marker such as EPC, and / or a differentiation inducer of blood hemangioblasts or differentiated cells (for example, EPC to vascular endothelium). An inducer of differentiation into cells).
- a substance for example, an antibody
- a differentiation inducer of blood hemangioblasts or differentiated cells for example, EPC to vascular endothelium.
- An inducer of differentiation into cells is suitably used for the culture method of the present invention.
- Diabetes (DM) patient EPC has decreased tissue repair / regeneration ability, and the effectiveness of autologous EPC cell transplantation therapy for wound healing in DM patients is insufficient.
- the cell group in which tissue repair / regeneration ability declines due to diabetes is released from the diabetic environment, and blood vessel regeneration ability is achieved. It can be re-educated into a cell group having an action of suppressing inflammation. That is, the use of the cell group of the present invention opens the way for treatment of intractable ulcers and diabetes-related diseases in DM patients.
- the cell group obtained by the present invention is a cell group enriched with not only vascular endothelial progenitor cells but also anti-inflammatory macrophages, and also a cell group with increased regulatory T cells. It can also be useful for the prevention and treatment of diseases (eg, diabetes itself and diabetes-related diseases) that develop as a result of working.
- diseases eg, diabetes itself and diabetes-related diseases
- diabetes related disease refers to all diseases originated from vascular disorders associated with the onset of diabetes. Examples of such diseases include diabetic ischemic diseases (heart disease, brain disease, limb ischemia, intractable disease). Ulcers, renal disorders, retinal disorders, etc.).
- GraphPad Prism5 software (GraphPad Prism5 software, Inc) was used for statistical analysis of the results. Wilcoxon's signed rank test was used to analyze PBMNC and QQC cell numbers or the ratio between them. Correlation analysis was performed by linear regression analysis. In other assays, the various parameters of these cells were analyzed by Mann-Whitney U test. Kruskal-Wallis test was used for comparison between the three groups. P ⁇ 0.05 was considered statistically significant. All values are expressed as mean ⁇ standard error.
- Example 1 EPC increase from peripheral blood-derived mononuclear cells (1) Preparation of PBMNC and CD34 positive mononuclear cells 20-100 ml of peripheral blood was collected from healthy volunteers 20-55 years old using a heparinized winged needle attached to a 50 ml syringe. The collection was performed with the approval of Tokai University School of Medicine Medical Research Committee, and the obtained peripheral blood samples were handled in accordance with biological guidelines for human samples. Isolation of PBMNC from peripheral blood was performed by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, # 10771) according to the method described in Asahara et al., Science, 275: 964-7 (1997). went.
- the isolated PBMNC was washed with PBS-EDTA and then suspended in a buffer solution to prepare a cell suspension.
- the CD34 positive rate was 0.23 ⁇ 0.03%
- the CD133 positive rate was 0.20 ⁇ 0.07%.
- the number of CD34 positive cells per 100 ml of peripheral blood was (27.8 ⁇ 4.5) ⁇ 10 4
- the number of CD133 positive cells was (23.2 ⁇ 6.9) ⁇ 10 4 .
- CD34 positive cells Purification of CD34 positive cells was performed by autoMACS separator (Miltenyi Biotec) using mouse anti-human CD34 antibody coated with magnetic beads and CD34 Cell Isolation kit (Miltenyi Biotec, # 130-046-702) according to the instructions.
- the serum-free medium (hereinafter referred to as QQ medium or QQcm) used for culture is shown in Table 1 using stemline TM II Hematopoietic Stem Cell Expansion Medium (Sigma-Aldrich, Cat No. S0192). Created based on composition. That is, each component shown in Table 1 was aseptically added to a serum-free medium so as to have a predetermined concentration.
- the obtained QQC number showed a negative correlation with the PBMNC number per 100 ml of peripheral blood in linear regression analysis. That is, an average of about 4 ⁇ 10 7 cells were obtained from 100 ml of peripheral blood regardless of the total number of PBMNCs isolated from an equal amount of peripheral blood.
- EPC-CFA EPC colony formation assay
- PBMNC had 66.4% undifferentiated colony forming cells and 33.6% differentiated colony forming cells out of all EPC colony forming cells, whereas QQC had undifferentiated colony forming cells.
- the ratio of differentiated colony forming cells was greatly increased, with 8.4% of forming cells and 91.6% of differentiated colony forming cells.
- the number of differentiated EPC colony forming cells in QQC and the total EPC per dish was correlated with the number of differentiated EPC colony forming cells of PBMNC, but the number of undifferentiated EPC colony forming cells of QQC was not correlated with the number of undifferentiated EPC colony forming cells of PBMNC.
- the number of undifferentiated EPC colony forming cells or the total number of EPC colony forming cells of QQC did not correlate with the differentiated EPC colony forming cells of PBMNC.
- the total EPC colony forming frequency of QQC was dependent on that of PBMNC.
- the frequency of differentiated EPC colony forming cells in QQC is related to the frequency of undifferentiated EPC colony forming cells in PBMNC, indicating that QQ culture differentiated undifferentiated EPC colony forming cells in PBMNC. ing. From the above, it was demonstrated that the ability to form blood vessels dramatically improved both in terms of quantity and quality of cells by QQ culture.
- Example 2 Flow cytometric analysis of cell group
- cells of blood vascular stem cells, blood cells, or vascular cells were analyzed by flow cytometry. The expression of surface markers was examined.
- Flow cytometry analysis was performed as follows. Cells suspended in MACS buffer (5x10 5 cells / 200 ⁇ l MACS buffer) were incubated for 30 minutes at 4 ° C after addition of 10 ⁇ l FC blocking reagent, and then dispensed in equal volumes into reaction tubes for subsequent staining (100 ⁇ l / tube). Each aliquot was incubated with 2 ⁇ l of each primary antibody for 20 minutes at 4 ° C. and then washed twice with 1 ml MACS buffer.
- the cell positive rate of each PBMNC or QQC was first estimated at each gate, and then the positive rate in the gated total viable cell fraction was calculated by gating and measuring each cell marker in three cell size regions ( Fig. 5A left side).
- the number of positive cells per 100 ml of peripheral blood (FIG. 5A right side) was also calculated using the total number of viable cells of PBMNC or QQC and the accumulated positive rate.
- the positive rate was calculated when the total number of cells gated into the above three populations was taken as 100% (left side of FIG. 5B), and the% change of each marker-expressing cell in the whole cells before and after QQC in this case was also calculated ( Fig. 5B right).
- the ratio and number of CD34 positive or CD133 positive stem cells increased significantly compared to PBMNC (the frequency was 5.31 times for CD34 positive cells, the number was 1.96 times; for CD133 positive cells) The frequency was 4.64 times and the number was 1.80 times), and the ratio and number of positive cells decreased for most hematopoietic cell markers (FIG. 5A).
- the positive rate of endothelial cell markers was slightly reduced by 0.92 times for CD31 and 0.77 times for vWF, and 0.56 times for VEGFR-2.
- CD105 or CD146 had an increased positive rate, probably reflecting the enrichment of undifferentiated or differentiated colony-forming EPCs in QQC.
- the parameters of anti-inflammatory M2 macrophages increased to the same extent as the increase in the stem cell population, whereas the parameters of inflammatory M1 macrophages (CCR2) were clearly decreased (CD206).
- the frequency is 4.71 times, the number of cells is 1.72 times; for CCR2-positive cells, the frequency is 0.01 times, the number of cells is 0.01 times).
- This result showed the same tendency in the positive rate when the total number of cells gated in the above three populations was taken as 100%.
- QQ-MNC The ratio of the undifferentiated EPC fraction (CD34 + or CD133 + cells) per same viable cell count was increased.
- the content rate in the whole living cells also increased slightly.
- QQ-MNC showed a decrease in inflammatory T lymphocyte subset (Th1) compared to PB-MNC, whereas anti-inflammatory T lymphocyte subset (Th2) and regulatory T lymphocytes An increase in subset (T reg) was observed (FIG. 6).
- Example 3 Comparison of QQC in healthy and diabetic patients Peripheral blood 50 cc was collected from diabetic (DM) patients and healthy volunteers, mononuclear cells were collected, and cultured in QQc for one week. EPC-Colony Forming Assay (EPC-CFA), FACS, and EPC Assay were used to examine the ability of blood vessels to regenerate before and after QQc.
- EPC-CFA EPC-Colony Forming Assay
- FACS FACS
- EPC Assay EPC-CFA
- M2 macrophages which are CD206 positive anti-inflammatory cells in FACS analysis
- M1 macrophages which are inflammatory cells of CCR positive cells
- Example 4 Real-time RT-PCR analysis of a cell group The cell group was also analyzed by a real-time RT-PCR method. Analysis was performed as follows. Total mRNA was isolated by Trizol (Invitrogen), and genomic DNA was digested by DNase I treatment. DNase I treated mRNA was purified by phenol extraction and ethanol precipitation. 500 ng of purified mRNA was used for cDNA synthesis using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). For in vivo colony analysis, cDNA was amplified using TaqMan PreAmp Master Mix (Applied Biosystems) and 45 nM forward and reverse primer mix.
- Amplification conditions used in PCR are as follows: 20 cycles of 95 ° C for 10 minutes, 95 ° C for 10 seconds, and 60 ° C for 4 minutes. Dilute the amplified cDNA 10-fold and add the diluted cDNA to TaqMan RT using EagleTaq Master Mix (Roche), forward and reverse primers for cDNA amplification (0.3 mM), and TaqMan probe (Sigma Aldrich; 0.25 mM) -Used for PCR. All primers and TaqMan probes were commercially available. For in vitro colony analysis, cDNA diluted 10-fold without an amplification step was used for TaqMan RT-PCR. All gene expression levels were normalized to GAPDH.
- VEGF-A expression showed a decreasing trend, but IGF-1 and the angiogenic cytokines leptin, IL-8, and IL-10 Gene expression was significantly enhanced (21.2 times for IGF-1, 35.9 times for leptin, 6.3 times for IL-8, 5.4 times for IL-10).
- Expression of vascular maturation factors VEGF-B and Ang-1 was also enhanced (4.2 times for VEGF-B and 2.4 times for Ang-1).
- Example 5 Evaluation of angiogenic ability of a cell group
- EPC EPC
- the mixed cell suspension was incubated at 37 ° C in a water bath and added in an amount of 100 ⁇ l each to Matrigel (BD Falcon, # 354234; 50 ⁇ l / well) pre-cultured in the wells of a 96-well plate .
- the number of closed areas formed by HUVEC was counted using Photoshop software in photographs taken with a phase contrast optical microscope (x2 HPF) (Eclipse TE300, Nikon).
- the number of labeled PBMNC or QQC incorporated into the tube was also counted using Photoshop software in photographs taken with a fluorescence microscope (IX70, Olympus). Tube counts and cell counts were performed blindly by two people.
- Example 6 Human QQMNC cell transplantation experiment into ischemic model mice
- All animal experiments were conducted in accordance with national and research institution guidelines. The experimental protocol was approved according to the guidelines of the Animal Experiment Committee of the Isehara Campus of Tokai University School of Medicine based on the Guide for the Care and Use of Laboratory Animals of the National Research Council of the United States. In order to reduce the pain and discomfort of experimental animals, we paid maximum attention to anesthesia. In the experiment, as previously reported (Sasaki K. et al., Proc. Natl. Acad. Sci.
- mice BALB / cAJcl-nu / nu mice aged 8-10 weeks (Charles River, Japan).
- the proximal part of the left femoral artery including the shallow and deep branches was sutured, and the proximal and distal parts of the saphenous artery were occluded with bipolar electrocoagulation forceps (MERA, # N3-14).
- the skin was closed with surgical staples.
- an ischemic hind limb model mouse was prepared. The next day, the cells were suspended in IMDM and the suspension was injected intramuscularly at 4 sites on the hind limb of ischemia, i.e.
- the QQC transplant group had the lowest frequency of necrosis in the lower limbs (4.3%, 15.8%, 9.5% for the QQC transplant group, control, and PBMNC transplant group, respectively)
- the overall residual frequency was the highest (21.7%, 10.5%, and 9.5% for the QQC transplant group, control group, and PBMNC transplant group, respectively) (FIG. 14).
- Example 7 Mouse QQMNC cell transplantation experiment into an ischemic model mouse (1) Evaluation of the regenerative capacity of mouse QQMNC by EPC colony assay In C57BL6 / N (male) mice of 10-12 weeks of age under pentobarbital anesthesia Intracardiac heparin blood was collected using a tuberculin needle, mouse mononuclear cells (MNC) were collected by density gradient method using Histopaque-1083, and used as mouse PBMNC for experiments. QQ culture was performed in the same manner as in human QQC in Example 1, and EPC colony production ability of cells before and after the culture was evaluated by EPC-CFA.
- MNC mouse mononuclear cells
- QQ culture solution StemLine II, mouse recombinant TPO, SCF, Flt-3 ligand, IL-6, and VEGF were used.
- EPC-CFA Stem Cell Tec's MethoCult TM SF M3236 was used, 10% FBS was added, and mouse recombinant growth factors and cytokines similar to human EPC-CFA were adjusted to the same concentration as humans.
- PBMNC and QQC cell QQ cultured for 3 days, after 5 days) were seeded into 35 mm Primaria dish one by each 1x10 5 cells. Colony counts were made 8 days after cell seeding. Both undifferentiated and differentiated EPC colonies (pEPC-CFU, dEPC-CFU) gradually increased according to the QQ culture days (FIG. 15).
- Microvessels and wall cells were observed and quantitatively evaluated with a fluorescence microscope in the same manner as in humans.
- QQ-MNC low and high
- IMDM non-cell transplanted group
- the increase in microvessel density (Fig. 17 left) and the increase in the number of lining blood vessels by mural cells (promotion of arteriogenesis) Fig. 17 (Right) was observed, confirming that the revascularization ability was enhanced by QQ culture.
- Example 8 Histochemical evaluation A tissue section was prepared from the mice subjected to the transplantation experiment in Example 6, and histochemical evaluation was performed. The protocol is as follows. (1) Preparation of sample for evaluation Three weeks after the operation, 50 ⁇ l of isolectin B4-FITC was injected into the tail vein using an insulin syringe. Mice were euthanized under sufficient anesthesia 20 minutes after injection, and immediately after that, 20 ml of PBS was perfused by cardiac puncture, and then fixed by switching to an equal volume of PBS containing 4% paraformaldehyde. The ischemic hind limbs were then excised and placed in graded concentrations of sucrose / PBS.
- the fibrosis area was significantly smaller in the QQC transplant group, 0.23 times that in the control group, and 0.33 times that in the PBMNC transplant group (fibrosis area).
- / mm 2 is QQC transplanted group 5.9 ⁇ 1.28, control group 25.2 ⁇ 7.57, PBMNC transplanted group 17.7 ⁇ 3.18) (FIG. 21). This indicates that QQC transplantation has significantly suppressed fibrosis compared to PBMNC transplantation and controls.
- Tissue sections were prepared from mice subjected to transplantation experiments in Example 7, and ischemic muscle tissue on the 14th day after ischemia was stained with iNOS antibody (Abcam) (brown stained part). Expression of iNOS is enhanced by inflammatory cells, and iNOS-stained cells are widely confirmed in non-cell transplanted groups and PB-MNC transplanted groups, which are control ischemic tissues, but inflammatory cells are significant in QQ-MNC transplanted groups It was confirmed that it was limited to.
- Non-cell transplanted group 10.6 ⁇ 1.6%
- PB-MNC transplanted group 11.6 ⁇ 1.6%
- QQCHigh transplanted group 4.6 ⁇ 0.8%.
- Example 1 StemLine II, human recombinant TPO, SCF, Flt-3 ligand, IL-6, and VEGF were used as in Example 1.
- PBMNC and 4 ⁇ 1 QQC cells were turbid in PBS 25ul and transplanted to the bottom of the ulcer. After creating the ulcer, the wound healing was compared by measuring the ulcer reduction rate with the pictures taken with Canon Cyber shot on Day 0, 3, 7, 10, and 14. Day 14 ulcers were collected, and the blood vessel density in the tissue was analyzed by Anti-CD31 staining.
- the transplantation of cells amplified by the method of the present invention improved the blood flow rate and necrosis improvement rate of ischemic disease. That is, the method of the present invention is considered useful for producing endothelial cells in both quality and quantity, and can be a useful method for cell transplantation therapy for vascular disorders such as ischemic diseases.
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Abstract
Description
一方で、CD34陰性細胞集団に存在するAngiogenic T細胞と呼ばれるCD3陽性CD31陽性細胞とEPCとを共培養することで、EPCの血管再生能力が高まることも報告されており、CD34陰性細胞集団の中にEPCの能力を高める細胞が含まれていることが示唆されている(非特許文献1)。
すなわち、本発明の課題は虚血性疾患治療に用いる細胞群の調製工程の簡略化と、より当該治療に効果を示す細胞群を提供することにある。より具体的には骨髄、臍帯血および末梢血採取後の細胞選別工程の簡略化により、血管再生性細胞:具体的には血管新生能力の高い分化型EPCコロニー形成細胞だけでなく、抗炎症・免疫寛容誘導細胞:具体的には抗炎症性に転化したM2マクロファージや、Tリンパ球サブセット、制御性T細胞を含む細胞群を増幅するための手段を提供することにある。
特に培養に用いる単核球において、CD34陽性細胞と陰性細胞とを一定の割合で培養した場合、血管再生能が著しく高い細胞群を得られることを明らかにした。
[1]骨髄、臍帯血または末梢血由来の単核球を、幹細胞因子、インターロイキン6、FMS様チロシンキナーゼ3リガンド、トロンボポエチンおよび血管内皮細胞増殖因子を含有する無血清培地中で培養して得られる、細胞群。
[2]骨髄、臍帯血または末梢血由来の単核球を、無血清培地中で培養して得られる細胞群であって、血管内皮前駆細胞および抗炎症性マクロファージが富化した細胞群。
[3]血管内皮前駆細胞および抗炎症性マクロファージを含む、上記[1]に記載の細胞群。
[4]血管内皮前駆細胞が分化型EPCコロニー形成細胞である、上記[2]または[3]に記載の細胞群。
[5]抗炎症性マクロファージがM2マクロファージである、上記[2]~[4]のいずれかに記載の細胞群。
[6]幹細胞因子、インターロイキン6、FMS様チロシンキナーゼ3リガンド、トロンボポエチンおよび血管内皮細胞増殖因子を含有する無血清培地において、骨髄、臍帯血または末梢血由来の単核球を培養することを特徴とする、血管内皮前駆細胞および/または抗炎症性マクロファージが富化した細胞群の製造方法。
[7]血管内皮前駆細胞が分化型EPCコロニー形成細胞である、上記[6]に記載の方法。
[8]抗炎症性マクロファージがM2マクロファージである、上記[6]または[7]に記載の方法。
[9]上記[1]~[5]のいずれかに記載の細胞群を含んでなる、虚血性疾患の治療剤。
[10]虚血性疾患が、血管新生により治癒する疾患である、上記[9]に記載の治療剤。
[11]上記[1]~[5]のいずれかに記載の細胞群を含んでなる、難治性潰瘍または糖尿病関連疾患の治療剤。
特に本発明によれば、糖尿病患者のような血管再生能の低下した患者であっても質・量共に高い血管再生能を有する細胞群が得られ、さらに細胞移植時においても抗炎症性細胞の存在により炎症が抑えられるため、細胞移植療法として極めて有用であるといえる。
本発明で用いられる単核球は、CD34および/またはCD133陽性細胞の選別(陽性選別)を行うことなく、取得した単核球をそのまま後述する細胞培養に用いることができる点に大きな特徴がある。
ここで、EPCおよび/または抗炎症性マクロファージを富化した細胞群とは、上述した因子を含有する無血清培地で培養する前の単核球全体におけるそれぞれの比率に比べて、培養後の細胞群全体における各細胞の比率が、2倍以上、好ましくは4倍以上、より好ましくは5倍以上に増加することをいう。本発明の培養方法により得られる細胞群は、もとの単核球に比べてEPCと抗炎症性マクロファージの比率が著しく高くなっているため、血管新生を顕著に促進すると共に、炎症を抑える効果を奏するのである。
これらの細胞群も、培養前と比較した各細胞の全細胞数に対する比率が、通常2倍以上、好ましくは4倍以上、より好ましくは5倍以上に増加している。
より詳細にはCD34陽性選別をした単核球を本発明の培養方法で培養すると未分化型EPCコロニー形成細胞しか増幅されないが、無選別単核球を使用した場合は未分化型EPCコロニー形成細胞とともに分化型EPCコロニー形成細胞が増幅するし、さらに抗炎症性マクロファージまでが増幅する。さらに抗炎症性のTh2細胞や制御性T細胞までもが増幅するのである。
3群間の比較にはクラスカル・ウォリス検定を用いた。P<0.05を統計的に有意とした。全ての値は平均±標準誤差で表している。
(1) PBMNC及びCD34陽性単核球の調製
20-55歳の健康なボランティアから50mlシリンジに装着されたヘパリン化翼状針を用いて末梢血を20-100ml採取した。採取は東海大学医学部医学調査委員会の承認の下で行い、得られた末梢血サンプルの取り扱いは、ヒトサンプルに対する生物学的ガイドラインに沿って行った。末梢血からのPBMNCの単離は、Asahara et al., Science, 275: 964-7 (1997)に記載の方法に従って、Histopaque-1077 (Sigma-Aldrich, #10771)を用いた密度勾配遠心法により行った。単離されたPBMNCをPBS-EDTAで洗浄後、緩衝液中に懸濁し、細胞懸濁液を調製した。単離されたPBMNCにおいてCD34陽性率は0.23±0.03%、CD133陽性率は0.20±0.07%であった。また、末梢血100mlあたりのCD34陽性細胞数は(27.8±4.5)x104、CD133陽性細胞数は(23.2±6.9)x104であった。
培養に用いる無血清培地(以下、QQ培地またはQQcm)は、stemlineTM II Hematopoietic Stem Cell Expansion Medium (Sigma-Aldrich, Cat No. S0192)を用いて、表1に示す組成に基づいて作成した。即ち、表1に示す各成分を、所定の濃度となるように無血清培地に無菌的に添加した。
上記方法により単離されたPBMNCを、Primaria Tissue culture plate (35-mm PrimariaTM tissue culture dish, BD Falcon, #353801)を用い、1ウェル当たり2x106細胞/2ml QQ培地の条件下、(2)で調製された無血清培地中で7日間培養した。QQ培地中の上記細胞密度は、末梢血1mlあたり約1x106 MNCに相当する。
培養の結果、培養開始前のPBMNCに対する上記培養後の細胞(以下、QQCといい、上記培養法をQQcという場合がある)の数は、全ての被験者について減少していた(平均で0.49倍; 図1)。一方、得られるQQC数は、線形回帰分析において、末梢血100mlあたりのPBMNC数と負の相関を示した。即ち、等量の末梢血から単離された全PBMNC数とは関係なく、100mlの末梢血から平均で約4x107個の細胞が得られた。
元のPBMNCおよびQQCの血管形成能を調べるため、EPCコロニー形成アッセイ(EPC-CFA)により接着性EPCコロニーを定量した。EPC-CFAは、Masuda H. et al., Circulation research, 109: 20-37 (2011)に記載の方法に準じて行った。具体的には、35mm PrimariaTM dish (BD Falcon)中、表2に示す組成に基づいて作製した半固形培地中で細胞を培養し、培養開始から16-18日後に、位相差光学顕微鏡(Eclipse TE300, Nikon)下、ディッシュあたりの接着性コロニー数をgridded scoring dish (Stem Cell Tec.)を用いて測定した。未分化型EPCコロニー(PEPC-CFU (primitive EPC colony forming unit); 図2左)と分化型EPCコロニー(DEPC-CFU (definitive EPC colony forming unit); 図2右)とを別々にカウントした。
更に、QQCにおける各EPCコロニー形成細胞の頻度とPBMNCにおける各EPCコロニー形成細胞の頻度との関係を線形回帰分析により調べたところ、1ディッシュあたりで、QQCの分化型EPCコロニー形成細胞数および総EPCコロニー形成細胞数はPBMNCの分化型EPCコロニー形成細胞数と相関していたが、QQCの未分化型EPCコロニー形成細胞数とPBMNCの未分化型EPCコロニー形成細胞数とは相関がなかった。一方、QQCの未分化型EPCコロニー形成細胞数または総EPCコロニー形成細胞数はPBMNCの分化型EPCコロニー形成細胞と相関がなかった。
要約すれば、QQCの総EPCコロニー形成細胞頻度はPBMNCのそれに依存していた。特に、QQCにおける分化型EPCコロニー形成細胞の頻度は、PBMNCにおける未分化型EPCコロニー形成細胞の頻度と関連しており、QQ培養によりPBMNC中の未分化型EPCコロニー形成細胞が分化したことを示している。以上のことから、QQ培養により、血管形成能が細胞の量的にも質的にも劇的に向上することが実証された。
実施例1で得られた細胞群の特徴をより明らかにするため、フローサイトメトリーにより、血液血管系の幹細胞、血液系細胞、または血管系細胞の細胞表面マーカーの発現を調べた。フローサイトメトリー解析は下記の通りに行った。
MACSバッファー中に懸濁した細胞(5x105細胞/200μl MACSバッファー)を10μlのFCブロッキング試薬の添加後に4℃で30分間培養し、その後の染色のために反応チューブ中に等量に分注した(100μl/チューブ)。各アリコートを2μlの各一次抗体と共に4℃で20分間培養した後、1mlのMACSバッファーで2回洗浄した。細胞をMACSバッファー中に懸濁した(200μlのMACSバッファーあたり2x105細胞)。フローメトリー解析はLSRFortessaTM cell analyzer (BD)およびFlowJoTMソフトウェア(Tomy Digital Biology)を用いて行った。抗体はいずれも市販のものを使用した。vWFの染色の際は、細胞を各一次抗体と培養後、ビオチン抱合ラット抗マウスIgG1と培養し、次いでストレプトアビジン-PE/Cy7にコンジュゲートさせた。
PBMNCまたはQQCの散布図をそれぞれ、細胞サイズにより3つの集団、即ちリンパ球サイズ、単球サイズ、および大型細胞サイズにゲートした(図5A)。PBMNCまたはQQCの各々の細胞陽性率を先ず各ゲートにおいて推定し、その後、各表面マーカーについて3つの細胞サイズ領域にゲートし測定することで、ゲートした全生存細胞画分における陽性率を計算した(図5A左側)。また、末梢血100mlあたりの陽性細胞数(図5A右側)についても、PBMNCまたはQQCの全生存細胞数と上記の累積された陽性率とを用いて算出した。
さらに、上記3集団にゲートした細胞の合計を100%とした場合の陽性率を算出し(図5B左側)、さらにこの場合のQQC前後の細胞全体における各マーカー発現細胞の%変化も算出した(図5B右側)。
注目すべきこととして、抗炎症性M2型マクロファージのパラメータ(CD206)が幹細胞集団の増加と同程度に増加し、逆に炎症性M1型マクロファージのパラメータ(CCR2)は明らかに減少していた(CD206陽性細胞について、頻度は4.71倍、細胞数は1.72倍; CCR2陽性細胞について、頻度は0.01倍、細胞数は0.01倍)。
この結果は上記3集団にゲートした細胞の合計を100%とした場合の陽性率でも同様の傾向であった。具体的には、PB-MNCに比較してQQ-MNCでは、
・未分化EPC分画(CD34+またはCD133+細胞)の同一生細胞数当たりの比率が上昇していた。また、生細胞全体における含有率も若干上昇した。これは、未分化EPC分画がQQcにより増幅されたことを意味する(図5B最上段四角内)。
・炎症性マクロファージ(CCR2+細胞)の同一生細胞数当たりの比率(左側)は減少しており、さらに生細胞全体における含有率(右側)は減少した。一方、抗炎症性マクロファージ(CD206+細胞)においては、いずれの指標も上昇した(図5B下段上側四角内)。
・また血管形成性T細胞(angiogenic T cell)の指標としてはCD3/CXCR4/CD31が挙げられるが、これについてもいずれの指標も上昇した(図5B下段下側四角内)。
以上のことから、QQcにより幹細胞集団が富化されるのと同時に、抗炎症性環境が提供されると考えられる。
糖尿病(DM)患者と健常人ボランティアから末梢血50ccを採血し、単核球を採取し、QQcにて一週間培養した。EPC-Colony Forming Assay法(EPC-CFA)、FACS、EPC AssayにてQQc前後の細胞における血管再生能力などを検討した。
FACS(CD34抗体)によれば、QQc前後で全体の細胞数は共に減少するものの(図7左側)、DM患者においても健常人と同様にCD34陽性細胞の増幅を認めた(図7右側)。
また、FACS解析においてCD206陽性の抗炎症細胞であるM2マクロファージがDM患者と健常人においてQQc後に細胞数が優位に上昇し、CCR陽性細胞の炎症性細胞であるM1マクロファージ数が優位に低下する(図8)。
なお、糖尿病患者において実施例2と同様に散布図をとり3集団にゲートし、各表面マーカーの陽性率を計算して生細胞数あたりの比率や生細胞全体における含有率をカウントしたが、糖尿病患者でも健常人と同様の傾向を示した。
リアルタイムRT-PCR法による細胞群の解析も行った。解析は以下の通りに行った。
トータルmRNAをTrizol (Invitrogen)により単離し、ゲノムDNAをDNase I処理により消化した。DNase I処理したmRNAをフェノール抽出およびエタノール沈殿により精製した。500 ngの精製したmRNAをSuperScript VILO cDNA Synthesis Kit (Invitrogen)を用いたcDNA合成のために用いた。in vivoコロニーの解析のために、TaqMan PreAmp Master Mix (Applied Biosystems)、および45nMのフォワードおよびリバースプライマーミクスチャーを用いてcDNAを増幅した。PCRで用いた増幅条件は以下の通りである:95℃で10分の後、95℃で10秒、60℃で4分を20サイクル。増幅したcDNAを10倍に希釈し、希釈したcDNAを、EagleTaq Master Mix (Roche)、cDNA増幅用のフォワードおよびリバースプライマー(0.3mM)、およびTaqManプローブ(Sigma Aldrich; 0.25mM)を用いたTaqMan RT-PCRのために使用した。全てのプライマーおよびTaqManプローブは市販のものを用いた。in vitroコロニーの解析のために、増幅工程なしで10倍に希釈されたcDNAをTaqMan RT-PCRのために使用した。遺伝子の発現レベルは全て、GAPDHに対して正規化した。
次に、本発明の方法で得られた、EPCを含む細胞群(QQC)の血管形成能を評価するために、in vitroでのマトリゲルアッセイを行った。アッセイは以下の通りに行った。
Masuda H. et al., Circulation research, 109: 20-37 (2011)に記載のように、37℃のCO2インキュベータ中で30分間、acLDL-DiI (20μg/ml; 500μl中に2-4x104)を添加したEBM-2/ 2% FBS (500μl)を含有する1.5mlチューブ中で細胞を培養した。4℃において400gで10分間遠心分離し、上清を吸引後、細胞ペレットを1ml PBSで洗浄し、EBM-2/ 2% FBS (50μl中に1.0x103細胞)で懸濁した。標識された各細胞をHUVECと共に再懸濁した(100μlのEBM-2/ 2% FBS中、EPC:HUVEC = 1x103:1.5x104細胞)。混合細胞の懸濁液を水浴中37℃で培養し、それを96ウェルプレートのウェル中に予め培養されたマトリゲル(BD Falcon, #354234; 50μl/ウェル)に対して各100μlの量で添加した。12時間の培養後、HUVECにより形成された閉じた領域の数を、位相差光学顕微鏡(x2 HPF)(Eclipse TE300, Nikon)により撮られた写真中でPhotoshopソフトウェアを用いてカウントした。更に、チューブ中に取り込まれた標識されたPBMNCまたはQQC数についても、蛍光顕微鏡(IX70, Olympus)により撮られた写真中でPhotoshopソフトウェアを用いてカウントした。チューブ数および細胞数のカウントは、盲検で2人により行った。
また、QQCは、PBMNCと比較して、チューブ内への取り込み能が顕著に高いことも分かった(x4 HPFあたりの、チューブ中に取り込まれたacLDL-DiI染色細胞数 = 38.5±8.30 (QQC), 8.72±1.89 (PBMNC))(図12)。
以上のことから、本発明の方法で得られた細胞群は、PBMNCと比較して、血管形成能や血管内取り込みの増大を示すことが実証された。
本発明の方法で得られる細胞の細胞移植療法における有用性を評価するために、虚血症モデルマウスへの細胞移植実験を行った。全ての動物実験は、国および研究機関のガイドラインに沿って行った。また、実験プロトコルは、米国National Research CouncilのGuide for the Care and Use of Laboratory Animalsに基づく東海大学医学部伊勢原キャンパスの動物実験委員会のガイドラインによる承認を得た。実験動物の痛みや不快感を軽減すべく、麻酔についても最大限の注意を払った。
実験では、既報(Sasaki K. et al., Proc. Natl. Acad. Sci. USA, 103: 14537-14541 (2006))の通り、8-10週齢のBALB/cAJcl-nu/nuマウス(Charles River, 日本)を用いた。浅枝および深枝を含む左大腿動脈の近位部を縫合し、伏在動脈の近位部および遠位部をバイポーラの電気凝固鉗子(MERA, #N3-14)で閉塞した。その部分の皮膚を外科用ステープルで閉じた。このようにして虚血症後肢モデルマウスを作製した。
翌日、細胞をIMDM中に懸濁し、虚血症後肢の4つの部位、即ち大腿部または下肢についてそれぞれ2箇所に懸濁液を筋肉内注射した(2.5x103細胞/10μl/部位; 計1x104細胞/マウス)。特に、移植細胞のin vivoでの内皮系への分化の評価の目的では、より多量の細胞を注射した(5.0x104細胞/10μl/部位; 計2x105細胞/マウス)。
その結果、虚血手術の21日後において、QQC移植動物では、コントロール(IMDM注射)またはPBMNC注射の動物と比較して、虚血症後肢における顕著な血流の回復が見られた(図13)。反対の肢に対する虚血症の肢における血流の割合は、QQC移植群においてコントロール群またはPBMNC移植群と比べて1.85倍または1.80倍であった。また、虚血症により損傷した後肢の分類の分布において、QQC移植群では最も低い下肢壊死頻度であった一方(QQC移植群、コントロール、PBMNC移植群についてそれぞれ4.3%、15.8%、9.5%)、全残存頻度は最も高かった(QQC移植群、コントロール、PBMNC移植群についてそれぞれ21.7%、10.5%、9.5%)(図14)。
以上のことから、本発明の方法で得られた細胞群を移植することにより、PBMNC移植または細胞移植なしの場合と比較して、虚血症動物に対して血流量の回復をもたらし、更に組織を壊死から守ることができることが実証された。従って、本発明の細胞群は虚血症治療に有効である。
(1)EPCコロニーアッセイによるマウスQQMNCの血管再生能の評価
10-12週齢のC57BL6/N(雄)マウスにおいて、ペントバルビタール麻酔下にツベルクリン針を用いて心腔内ヘパリン採血を実施し、Histopaque-1083を用いた密度勾配法によりマウス単核球(MNC)を採取し、マウスPBMNCとして実験に用いた。実施例1におけるヒトQQCと同様の手法でQQ培養を行い、培養前後の細胞をEPC-CFAにてEPCコロニー産生能を評価した。QQ培養液= StemLine II, マウス遺伝子組み換えTPO、SCF、Flt-3 ligand、IL-6、VEGFを用いた。また、EPC-CFAについては、Stem Cell Tec社製MethoCultTM SF M3236を用い、10%FBSを添加し、ヒトEPC-CFA と同様のマウス遺伝子組み換え成長因子、サイトカインをヒトと同様の濃度で調整した。PBMNC及びQQC細胞(QQ培養3日、5日後)を各1x105個ずつ35mm Primaria dishに播種した。コロニーの算定は、細胞播種後8日目に行った。未分化型、分化型EPCコロニー(pEPC-CFU、dEPC-CFU)はいずれもQQ培養日数に従って漸増した(図15)。
QQ培養day5の細胞をC57BL6/N(雄)マウス虚血モデル作成翌日に移植した。移植細胞数は、PB-MNC=1 x105/40 microL, QQ-MNC Low=1 x104/40 microL , QQ-MNC High=1 x105/40 microLとした。対照群は、細胞懸濁液のIMDMの培養液とした。20 microLずつを前脛骨筋及び腓腹筋に移植した。虚血作成後、14日後に、ヒトQQMNC実験例5と同様にLazer Doppler Perfusion Imagingによる血流測定を行った。対照群(IMDM)、PB-MNC移植(1x105/匹)群に比較して、QQC細胞移植群(Low= 1x104/匹、High=1x105/匹)は、各群N数が3のため、有意差が認められないものの血流改善傾向が観察された(図16)。
虚血作成14日の血流測定後、isoectinB4-FITC (Vector Lab) 40 microLを尾静脈より注入し、in vivoにて微小血管を染色した後、十分量のPentobarbitalを腹腔内に投与し、麻酔下に左心室よりPBS及び4%パラホルムアルデヒド20 mLずつ注入し還流固定を行った。OCTコンパウンドに包埋後、液体窒素にて冷却したアセトンに浸潤し凍結標本を作製した。凍結標本から切片を作成し、smooth muscle alpha actin-Cy3(Sigma-Aldrich)の蛍光免疫染色を行い、蛍光顕微鏡にてヒトと同様に、微小血管及び壁細胞を観察し定量評価した。QQ-MNC low, highともにPB-MNC、非細胞移植群(IMDM) に比較して微小血管密度の上昇(図17左)及び壁細胞による裏打ち血管数の増加(細動脈化促進)(図17右)が認められ、血管再生能がQQ培養により増強することが確認された。
実施例6において移植実験に供されたマウスから組織切片を作製し、組織化学的評価を行った。そのプロトコルは下記の通りである。
(1) 評価用試料の作製
手術から3週間後、50μlのイソレクチンB4-FITCをインスリンシリンジを用いて尾静脈に注射した。注射の20分後に十分な麻酔下でマウスを安楽死させ、その直後に心臓穿刺により、20mlのPBSをかん流した後、4%パラホルムアルデヒドを含む等量のPBSに切り替えて固定を行った。その後、虚血症後肢を切除し、段階的な濃度のショ糖/PBS中に入れた。次いで、前脛骨筋を切除し、パラフィン中に包埋し、その後の評価に用いた。
(2) 微小血管密度(MVD)および周皮細胞のリクルートメントの評価
6-8μmの組織切片試料を筋肉の組織ブロックからスライスし、微小血管密度(MVD)および周皮細胞のリクルートメントの評価に用いた。
平滑筋αアクチン(SMαアクチン)の染色のために、組織切片を脱パラフィン化し、PBSで洗浄し、室温下10%ヤギ血清で30分間ブロッキングした後、希釈したCy3抱合SMαアクチン抗体(#C6198, Sigma-Aldrich)と共に室温下で2時間培養した。PBSでの洗浄後、切片をVECTASHIELD HardSet Mounting Medium (Vector Lab, #H-1400)を用いてマウントし、蛍光顕微鏡(Biorevo, #BZ-9000, Keyence)下で観察した。一次抗体を省いた同じプロトコルをネガティブコントロールとして行った。
ソフトウェア(VH analyzer, Keyence)を用い、撮像された各群のマウスあたり2-4の横断面中、イソレクチンB4-FITCでin vivo染色された毛細血管をカウントすることによりMVDを評価した。同時に、脈管構造中への周皮細胞のリクルートメントをSMαアクチン陽性細胞の領域をカウントすることにより評価した。
(3) 筋形成および間質線維の評価
H-E染色により中央に核が見られる筋線維を顕微鏡写真機(AX80, Olympus)で撮像し、VHanalyzerにより筋芽細胞(Pesce M et al., Circ. Res., 93: e51-62 (2003))をカウントした。肢部の間質線維をAzan染色を用いて形態学的に評価し、撮像した写真をVH analyzerにより解析した(Napoli C et al., Proc Natl Acad Sci USA, 102: 17202-17206 (2005); Sica V et al., Cell Cycle, 5: 2903-2908 (2006))。評価した組織切片の数は上記(3)と同じであった。また、定量的評価を盲検で2人により行った。
(4) 移植細胞の内皮系への分化の免疫組織化学的評価
脱パラフィン化後、蒸留水で希釈(1:10)したtarget retrieval solution (Dako, #S-1699)中(98℃)で10分間、組織切片をマイクロ線照射した。次いでStreptavidin/Biotin Blocking Kit (Vector Lab, #SP-2002)で処理して内因性ビオチンをブロッキングした後、切片を室温下で30分間、5% 正常ヤギ血清/PBSと培養し、次いで、ビオチン化ヤギ抗マウスIgGと予め反応させたマウス抗ヒトCD31抗体、およびマウス血清(Rockland, #D208)と4℃で一晩培養した。最後に、ストレプトアビジン-Alexa Fluor 594コンジュゲートと室温で1時間培養した後、切片をPBSで洗浄し、DABCO (Sigma Aldrich, #D2522-25G)/グリセロール/PBS中のTOTO-3 (Invitrogen, #T3604)を用いてマウントし、レーザー走査型顕微鏡(Carl Zeiss, LSM510META)により観察した。
微小血管密度(MVD)の組織化学的評価では、QQC移植群からの切片において、PBMNC移植群およびコントロール由来の切片と比較して、顕著に高いMVDが観察された(MVD/mm2はQQC移植群400.7±37.9、コントロール群98.7±15.8、PBMNC移植群118.9±20.1; 従って、コントロール群に対して4.1倍、PBMNC移植群に対して3.4倍)(図18)。また、SMαアクチン陽性細胞で評価した周皮細胞のリクルートメントについても、QQC移植群ではコントロール群に対して2.6倍、PBMNC移植群に対して2.0倍であった(SMαアクチン陽性細胞数/mm2は、QQC移植群38.7±5.5、コントロール群15.0±2.7、PBMNC移植群19.8±4.3)(図19)。更に、筋線維の頻度においても、QQC移植群ではコントロール群に対して1.95倍、PBMNC移植群に対して1.83倍であった(筋線維/mm2は、QQC移植群112.2±16.4、コントロール群57.6±7.0、PBMNC移植群61.3±6.8)(図20)。
以上のことから、本発明の細胞群は、PBMNCやコントロールと比較して、顕著に高い血管新生能、動脈形成能、筋形成能を有することが実証された。
これは、QQCの移植は、PBMNCの移植やコントロールと比較して、線維化が有意に抑制されていることを示している。
実施例7において移植実験に供されたマウスから組織切片を作製し、虚血作成14日の虚血筋肉組織をiNOS抗体(Abcam)で染色(茶色の被染色部分)した。iNOSは炎症細胞により発現が増強されており、コントロール虚血組織である非細胞移植群およびPB-MNC移植群でiNOS染色細胞が広範に確認されるが、QQ-MNC移植群では炎症細胞が有意に限局されていることが確認できた。非細胞移植群(IMDM)=10.6±1.6%、PB-MNC移植群=11.6±1.6%、QQCLow移植群6.8±0.6% , QQCHigh移植群4.6±0.8% 。*、**、***は図に示された対象に対する有意差(*P<0.05、**P<0.01、***P<0.001)を示す。(図22)
(方法)
糖尿病患者(5例)、健常人ボランティア(4例)から50mlの末梢血を採取し、Histopaque-1077を用いた密度勾配法によりヒト単核球(MNC)を採取しヒトMNCとして実験に用いた。全てのデータはN=3(人)にて取得した。Balb/cヌードマウスの背部に6mmパンチを用いて皮膚全層欠損潰瘍を作成し、シリコンステントを潰瘍周囲に6-0ナイロンを用いて縫合し、創収縮を抑制した潰瘍モデルを作成した。
実施例1と同様の方法でヒトMNCQQ培養を行い、培養前後の細胞を本潰瘍モデルに移植した。QQ培養液としては、実施例1と同様に StemLine II,ヒト遺伝子組み換えTPO、SCF、Flt-3 ligand、IL-6、VEGFを用いた。PBMNC及びQQC細胞1x104個をPBS25ulに混濁して潰瘍底部に移植した。潰瘍作成後、Day0,3,7,10,14にてCanon Cyber shotにて撮影した写真にて潰瘍縮小率を計測し創治癒を比較した。Day14の潰瘍を採取しAnti-CD31染色にて組織内血管密度を解析した。
(結果)
糖尿病患者(DM)QQc後細胞移植群(A群)は、DMQQc前MNC細胞移植群(B群)及びPBS投与対照群(C群)に比較して有意に潰瘍の縮小を認めた(%縮小率; A群66.92±2.52vs B群84.16±3.29、C群65.61±4.19; p<0.01)。組織学的評価ではCD31染色にてDMQQc後細胞移植群は他の群に比べ高い組織内血管形成を認めた。(CD31陽性血管数; A群145±28 vs B群321±58、C群140±34; p<0.05 )
Claims (11)
- 骨髄、臍帯血または末梢血由来の単核球を、幹細胞因子、インターロイキン6、FMS様チロシンキナーゼ3リガンド、トロンボポエチンおよび血管内皮細胞増殖因子を含有する無血清培地中で培養して得られる、細胞群。
- 骨髄、臍帯血または末梢血由来の単核球を、無血清培地中で培養して得られる細胞群であって、血管内皮前駆細胞および抗炎症性マクロファージが富化した細胞群。
- 血管内皮前駆細胞および抗炎症性マクロファージを含む、請求項1に記載の細胞群。
- 血管内皮前駆細胞が分化型EPCコロニー形成細胞である、請求項2または3に記載の細胞群。
- 抗炎症性マクロファージがM2マクロファージである、請求項2~4のいずれか一項に記載の細胞群。
- 幹細胞因子、インターロイキン6、FMS様チロシンキナーゼ3リガンド、トロンボポエチンおよび血管内皮細胞増殖因子を含有する無血清培地において、骨髄、臍帯血または末梢血由来の単核球を培養することを特徴とする、血管内皮前駆細胞および/または抗炎症性マクロファージが富化した細胞群の製造方法。
- 血管内皮前駆細胞が分化型EPCコロニー形成細胞である、請求項6に記載の方法。
- 抗炎症性マクロファージがM2マクロファージである、請求項6または7に記載の方法。
- 請求項1~5のいずれか一項に記載の細胞群を含んでなる、虚血性疾患の治療剤。
- 虚血性疾患が、血管新生により治癒する疾患である、請求項9に記載の治療剤。
- 請求項1~5のいずれか一項に記載の細胞群を含んでなる、難治性潰瘍または糖尿病関連疾患の治療剤。
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CN114164174A (zh) | 2022-03-11 |
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