Stem cells from hyaluronic acid-rich node and catheter systems, methods of isolation and uses thereof
阅读说明：本技术 来源于透明质酸丰富的节点和导管系统的干细胞、其分离方法及用途 (Stem cells from hyaluronic acid-rich node and catheter systems, methods of isolation and uses thereof ) 是由 权炳世 于 2015-04-28 设计创作，主要内容包括：本发明涉及来源于透明质酸丰富的节点和导管系统(Hyaluronic acid-rich node and duct system,HAR-NDS)的干细胞、其分离方法及用途,更详细地,涉及作为具有分化为来源于透明质酸丰富的节点和导管系统的神经细胞的能力的来源于透明质酸丰富的节点和导管系统的成体干细胞(Node and ductal stem cells,NDSCs)及具有分化为造血细胞的能力的造血干细胞。本发明可将很难从骨髓、外周血及脐带血(脐血)获取的作为成体干细胞的来源于透明质酸丰富的节点和导管系统的成体干细胞及造血干细胞作为替代来源从来源于透明质酸丰富的节点和导管系统分离,从而可有效地适用于脑部疾病、神经疾病、慢性感染疾病、癌、自体免疫性疾病、脏器再生及各种难治性疾病的治疗。(The present invention relates to stem cells derived from a Hyaluronic acid-rich Node and conduit system (HAR-NDS), a method for isolating the same, and a use thereof, and more particularly, to adult stem cells (NDSCs) derived from a Hyaluronic acid-rich Node and conduit system having the ability to differentiate into nerve cells derived from a Hyaluronic acid-rich Node and conduit system, and hematopoietic stem cells having the ability to differentiate into hematopoietic cells. The present invention can isolate adult stem cells and hematopoietic stem cells derived from a node and a catheter system rich in hyaluronic acid, which are hardly obtained from bone marrow, peripheral blood and umbilical cord blood (cord blood), as alternative sources from the node and the catheter system rich in hyaluronic acid, and thus can be effectively used for the treatment of brain diseases, neurological diseases, chronic infectious diseases, cancer, autoimmune diseases, organ regeneration and various intractable diseases.)
1. A method for obtaining stem cells derived from HAR-NDS, comprising:
(a) obtaining a sample of HAR-NDS from a mammal;
(b) isolating hematopoietic stem cells from the sample of HAR-NDS in (a);
(c) culturing the hematopoietic stem cells of (b) in a methylcellulose medium comprising serum and cytokines to form Colony Forming Cells (CFCs); and
(d) isolating cells from the Colony Forming Cells (CFC) of (c), wherein the isolated cells comprise cells selected from the group consisting of Lin-，Sca-1+，c-kit+，CD135-And CD34-One or more immunological features of (a).
2. The method of claim 1, wherein the sample of HAR-NDS is obtained after staining HAR-NDS with a dye, wherein the dye is alcian blue, methylene blue, or janus green b (jgb).
3. The method of claim 2, wherein staining the HAR-NDS comprises injecting a dye into the lymphatic system of the subject.
4. The method of claim 1, wherein the sample of HAR-NDS of step (a) is obtained by Fluorescence Activated Cell Sorting (FACS).
5. The method of claim 1, wherein the methylcellulose medium of (c) is colony forming units-granulocyte erythroid macrophages, megakaryocytes (CFU-GEMM) methylcellulose.
6. The method of claim 1, wherein the cytokine of (c) is selected from erythropoietin, Stem Cell Factor (SCF), granulocyte macrophage colony stimulating factor (GM-CSF), IL-3/-7, flt3/flt2ligand (FL), Leukemia Inhibitory Factor (LIF), or Thrombopoietin (TPO).
7. The method of claim 1, further comprising identifying the colony type for the CFCs in (c) as CFU-GEMM (colony forming unit-granulocyte erythroid macrophage, megakaryocyte), CFU-GM (colony forming unit-granulocyte, macrophage), BFU-E (burst forming unit erythroid colony) or MCP (mast cell progenitor).
8. The method of claim 7 wherein the CFC is stained with a dye prior to determining the colony type and the CFC dye is toluidine blue or Wright-Giesma.
9. A method for differentiating stem cells derived from HAR-NDS, comprising:
(a) co-culturing the HAR-NDS-derived hematopoietic stem cells of claim 6 with hematopoietic supporting cells in serum and cytokine-containing medium to form Cobblestone Area Forming Cells (CAFCs);
(b) expression of CD45 from (a)-And FLK-1+Isolating hematopoietic stem cells in the immunologically-characterized CAFC of (a); and
(c) subculturing the HAR-NDS-derived hematopoietic stem cells isolated in (b) in CFC methylcellulose medium to form mature hematopoietic cells.
10. The method of claim 9, wherein the hematopoietic support cells in (a) are OP9 or OP9-DL1 cells.
11. The method of claim 9, wherein the cytokine in the cytokine-containing medium of (a) is selected from erythropoietin, Stem Cell Factor (SCF), granulocyte macrophage colony stimulating factor (GM-CSF), IL-3/-7, flt3/flt2ligand (FL), Leukemia Inhibitory Factor (LIF), or Thrombopoietin (TPO).
12. The method of claim 9, wherein the medium in (a) is CFU-GEMM methylcellulose.
13. The method of claim 9, further comprising determining the size and differentiation morphology of the cells forming the CFC in (c).
14. The method of claim 13, wherein the CFC is stained with a dye prior to determining the size and differentiation morphology of the cells forming the CFC in (c), and the dye for the CFC is toluidine blue or Wright-Giesma.
15. The method of any one of claims 1 to 14, wherein HAR-NDS is a network structure consisting of nodes and conduits on the surface of organs, in blood vessels, lymphatic vessels and skin.
Technical Field
The present invention relates to stem cells derived from a Hyaluronic acid-rich Node and conduit system (HAR-NDS), a method for isolating the same, and a use thereof, and more particularly, to Node and conduit stem cells (NDSCs) as adult stem cells having the ability to differentiate into nerve cells derived from a Hyaluronic acid-rich Node and conduit system, and hematopoietic stem cells having the ability to differentiate into hematopoietic cells.
Background
Nodes and ductal systems abundant in hyaluronic acid after meridians and acupoints are structures found in 1960 as the third circulatory System, which have alternative names of the phoenix han's tube (Bonghan) or the Primo Vascular System (Primo Vascular System). Nodes and ductal systems rich in hyaluronic acid are formed of nodes (nodes) and ducts (ducts), and the entire body is networked along the Organ surface (Organ surface), the intravascular (Organ blood vessel), the intralymphatic (Organ lymphatic), the skin and the nervous system (neural system) (Kim BH [ non-patent document 1 ]; Soh KS [ non-patent document 2 ]; Lee et al [ patent document 1 ]). Furthermore, nodes rich in hyaluronic acid and nodes in the ductal system are filled with innate immune cells, and particularly, mast cells, eosinophils, basophils, neutrophils, and macrophages (histocytes) are abundant (Kwon BS et al [ non-patent document 3 ]).
Stem cells (stem cells) are cells having an ability to self-regenerate and proliferate and an ability to differentiate into various tissue cells, and can be classified into totipotent stem cells (totipotent stem cells), pluripotent stem cells (pluripotent stem cells), and pluripotent stem cells (multipotent stem cells).
For culturing optimized stem cells, an appropriate combination of growth factors and cytokines (cytokines) is essential. Typically, these functions are performed by growth factors and cytokines such as Stem Cell Factor (SCF) (Broudy et al [ non-patent document 4]), FL (flt3/flt2ligand), Interleukin (IL), Leukemia Inhibitory Factor (LIF), Thrombopoietin (TPO), Basic fibroblast growth factor (Basic FGF), and the like, which are required for the survival, proliferation, and maturation (differentiation) of stem cells. For example, when growth factors or cytokines are stimulated by co-culturing hematopoietic stem cells and hematopoietic support cells, hematopoietic progenitor cells (hematopoietic cells) that form Cobblestone-area-forming cells (CAFCs) can be confirmed. The presence or absence, proliferation and differentiation of hematopoietic stem cells can be confirmed by the above-mentioned method (Nakahata et al [ non-patent document 5], Eaves et al [ non-patent document 6 ]).
Adult non-hematopoietic stem cells (non-hematopoietic stem cells) present in bone marrow include very small embryonic-like stem cells (VSELs), multipotent adult stem cells (multipotent adult stem cells), multipotent adult progenitor cells (multipotent adult cells), bone marrow-derived adult multi-inducible cells (marrowseamlessage cells), mesenchymal stem cells (mesenchymal stem cells), and endothelial progenitor cells (endothelial progenitor cells) (Zuba-Surma EK et al [ non-patent document 7 ]; Beltrami AP et al [ non-patent document 8 ]; Jiang Y et al [ non-patent document 9], Pittenger SC [ non-patent
In particular, VSELs are negative for lineage (linkage) and CD45 as small embryonic-like stem cells rarely present in rodent and human bone marrow, express stem cell markers, and can be differentiated into 3 germ layers such as ectoderm, mesoderm, and endoderm in vitro (Kucia M et al [ non-patent document 11 ]).
In particular, it is known that genes (Notch, Delta, neurogenin, OCT, Presentin, etc.) and growth factors (NGF, broad-derived neurotrophic factor) associated with the development of nerve cells are known, and when such growth factors are administered, it is possible to observe the phenomenon that non-hematopoietic stem cells having differentiation ability differentiate into neurons (neurones), astrocytes (astrocytes) and oligodendrocytes (oligodendrocytes), and whether or not the differentiation into neurons (neurones, neurocytes, neurons) occurs by nerve cell markers (GFAP, NeuN, β III-tubulins, neuroamelions,
The best known areas of neuronal differentiation (neuropoiesis) are the basal thin cell layer located on the lateral ventricular surface and the Dentate Gyrus (DG) of the hippocampus and the sub-granular gyrus (SGZ) which is part of the hippocampus horn (CA), called the subventricular zone (SVZ) present in the brain. Type b1cells are reacted with neural stem cells at SVZ to form neural precursor cells (type ccels) or neural blasts (type a neuroblasts), and after passing through a mouth side migration stream (RMS), they move to an olfactory bulb (olfactory bulb) and mature into interneurons (interneuron). The DG of the SGZ differentiates Radial type 1cells and
As described above, although adult stem cells or hematopoietic stem cells derived from bone marrow have been identified and methods for their in vitro production and differentiation have been found, there is still a limitation in the fundamental supply of adult stem cells and hematopoietic stem cells.
Therefore, as a result of the present inventors' efforts to develop a new source (source) of adult stem cells and hematopoietic stem cells, the adult stem cells and hematopoietic stem cells can be isolated from hyaluronic acid-rich nodes and ductal systems, and the isolated cells can be expanded in vitro. In the case of adult stem cells, the ability to differentiate into nerve cells is excellent, and hematopoietic stem cells have excellent ability to differentiate into hematopoietic cells, thereby completing the present invention.
Disclosure of Invention
Technical problem
The present invention aims to provide adult stem cells derived from a hyaluronic acid-rich node and conduit system and having the ability to differentiate into nerve cells, and a method for isolating the same.
It is still another object of the present invention to provide a method for preparing neural cells by differentiating the above-mentioned adult stem cells derived from nodes and conduit systems rich in hyaluronic acid.
It is still another object of the present invention to provide a therapeutic agent for treating a disease requiring organ regeneration associated with a nerve disease, cancer, an autoimmune disease, a chronic infectious disease, a refractory atopic disease, and tissue damage as a cell therapeutic agent treatment comprising the above-mentioned adult stem cell derived from a hyaluronic acid-rich node and a catheter system.
Another object of the present invention is to provide a method for isolating hematopoietic stem cells having the ability to differentiate into hematopoietic cells as cells derived from nodes and ductal systems rich in hyaluronic acid.
It is still another object of the present invention to provide a method for producing mature hematopoietic cells so as to differentiate hematopoietic stem cells derived from the node and ductal system rich in hyaluronic acid.
It is still another object of the present invention to provide a therapeutic agent for treating a disease requiring replacement of hematopoietic function derived from bone marrow and spleen, a disease mediated by mast cells (mast cells) and eosinophils (eosinophils), or a disease caused by reduction and increase of immune function due to bone marrow, which comprises the above-mentioned hematopoietic stem cells derived from a node and a ductal system rich in hyaluronic acid as an active ingredient.
Means for solving the problems
In order to achieve the above object, the present invention provides adult stem cells derived from a hyaluronic acid-rich node and conduit system having the ability to differentiate into nerve cells.
Also, the present invention provides a method for isolating adult stem cells derived from a hyaluronic acid-rich node and conduit system, comprising: the method comprises the following steps that (a) the node and the catheter system rich in hyaluronic acid are dyed by using a dyeing sample to obtain a node and catheter system sample rich in hyaluronic acid; and a step (b) of separating Sca-1 from the hyaluronic acid-rich node and catheter system sample obtained in the step (a)+、Lin-And CD45-Adult stem cells derived from hyaluronic acid-rich node and conduit systems.
Also, the present invention provides a method for differentiating adult stem cells derived from a hyaluronic acid-rich node and conduit system, comprising: a step (a) of forming spheres (spheres) by co-culturing adult stem cells derived from hyaluronic acid-rich node and conduit systems with supporting cells in a neural cell differentiation medium; and (b) dissociating the formed spheres with single cells (single cells), and then treating the growth factors that are transformed into nerve cells to differentiate them into nerve cells.
The present invention also provides a therapeutic agent for treating a neurological disease or disorder, which is a cell therapeutic agent containing, as an active ingredient, adult stem cells derived from a hyaluronic acid-rich node and a catheter system.
The present invention also provides a therapeutic agent for treating a disease requiring organ regeneration associated with tissue injury, which comprises, as an active ingredient, adult stem cells derived from a node and a catheter system rich in hyaluronic acid.
Also, the present invention provides a therapeutic agent for treating cancer, autoimmune diseases, chronic infectious diseases, and intractable atopic diseases, which contains, as an active ingredient, adult stem cells derived from a hyaluronic acid-rich node and a catheter system.
Furthermore, the present invention provides hematopoietic stem cells derived from hyaluronic acid-rich node and conduit systems.
Also, the present invention provides a method for isolating hematopoietic stem cells derived from a hyaluronic acid-rich node and conduit system, comprising: a step (a) of staining the hyaluronic acid-rich node and catheter system with a staining sample using the hyaluronic acid-rich node and catheter system, and extracting the stained hyaluronic acid-rich node and catheter system to isolate the hematopoietic stem cells derived from the hyaluronic acid-rich node and catheter system; and (b) forming colony forming cells by culturing the hyaluronic acid-rich node and duct system tissue cells isolated in the step (a) with a methylcellulose medium in Colony Forming Cells (CFCs) containing serum and cytokines.
Also, the present invention provides a method for differentiating hematopoietic stem cells derived from a hyaluronic acid-rich node and ductal system, comprising the steps of (a) forming pebble zone-forming cells by co-culturing hemangioblast (hemangioblast) -derived cells and hematopoietic support cells in a serum-and cytokine-containing medium derived from a hyaluronic acid-rich node and ductal system-derived hematopoietic stem cells; and (b) forming mature hematopoietic cells (hematopoietic cells) by subculturing the colony forming cells with methylcellulose medium, the pebble zone forming cells formed in the above step (a).
Also, the present invention provides a therapeutic agent for treating a disease requiring replacement of hematopoietic function derived from bone marrow or spleen, comprising hematopoietic stem cells derived from a node and a ductal system rich in hyaluronic acid as an active ingredient.
The present invention also provides a therapeutic agent for treating diseases mediated by mast cells and eosinophils, which comprises, as an active ingredient, hematopoietic stem cells derived from nodes and ductal systems rich in hyaluronic acid.
The present invention also provides a therapeutic agent for treating a disease caused by a decrease or increase in immune function due to bone marrow, which comprises, as an active ingredient, hematopoietic stem cells derived from a node or duct system rich in hyaluronic acid.
ADVANTAGEOUS EFFECTS OF INVENTION
The present invention can separate adult stem cells and hematopoietic stem cells derived from a hyaluronic acid-rich node and catheter system, which are hardly obtained from bone marrow, peripheral blood and umbilical cord blood (cord blood), as adult stem cells, from the hyaluronic acid-rich node and catheter system as alternative sources, and thus can be effectively used for the treatment of brain diseases, neurological diseases, chronic infectious diseases, cancer, autoimmune diseases, organ regeneration and various intractable diseases.
Drawings
FIGS. 1A, 1B, 1C, 1D are diagrams showing hyaluronic acid-rich node and conduit systems present on the surface of the mouse intestinal tract, inside veins and lymphatic vessels (lymphatics), FIG. 1A is a graph showing the results of staining hyaluronic acid-rich nodes and ductal systems present in the interior of veins and lymph vessels with 1% alcian blue after anesthesia of C57BL/6 mice (5-6 weeks old), FIG. 1B is a photograph showing observation of hyaluronic acid-rich nodes and catheter systems by Scanning Electron Microscope (SEM), FIG. 1C is a photograph showing observation of the HAR-node by a Transmission Electron Microscope (TEM), FIG. 1D is a graph comparing Hyaluronic Acid (HA) concentrations in hyaluronic acid-rich nodes and ductal systems, serum, urine, Peritoneal Fluid (PF), and Lymphatic Vessels (LV).
Fig. 2 is a view showing hyaluronic acid-rich nodes and ductal systems (staining lines) in the center of lymphatic vessels after injecting 1% alcian blue, which is one of staining methods of HAR-NDS, into the muscle of the tail of a mouse by intramuscular injection (it is known that separated hyaluronic acid-rich nodes and ductal systems are generally curled up). Further, when 1% alcian blue was injected by intravenous injection and then the blood in the venous tube was withdrawn, the node and catheter system rich in hyaluronic acid and shown by a stain line were confirmed.
Fig. 3A, 3B, 3C, and 3D are diagrams illustrating a method of isolating bone marrow-derived VSELs or adult stem cells derived from a hyaluronic acid-rich node and conduit system. FIG. 3a is a diagram showing the isolation of Sca-1 in a manner that allows for flow cytometric analysis of VSELs or adult stem cells derived from hyaluronic acid rich node and conduit systems+Lin-CD45-Graph of cells by flow cytometry sorter (FACS sorter) method. FIG. 3b is Sca-1 showing the presence of bone marrow or hyaluronic acid rich nodal and ductal systems in percent by flow cytometry analysis+Lin-CD45-Graph of the amount of cells present. Fig. 3c is a graph showing the number of cells separated from bone marrow or hyaluronic acid rich nodes and conduit systems using a flow cytometric sorter. FIG. 3d is a graph showing apoptosis (apoptosis) in isolated cells by immunological staining with 7-AAD and Annexin V.
Fig. 4 is a photograph showing SEM and TEM of adult stem cells derived from hyaluronic acid-rich node and conduit systems. Fig. 4, section a, is an SEM photograph of the cellular appearance and cell diameter of adult stem cells derived from hyaluronic acid-rich nodes and conduit systems isolated by a flow cytometric sorter, and fig. 4, section B, is a TEM photograph showing intracellular small organs of adult stem cells derived from hyaluronic acid-rich nodes and conduit systems isolated by a flow cytometric sorter.
Fig. 5 is a graph showing sphere formation (sphere formation) formed by bone marrow-derived VSELs or adult stem cells derived from a hyaluronic acid-rich node and conduit system. Section A of figure 5 is a diagram showing the separation of VSELs or adult stem cells derived from hyaluronic acid rich node and conduit systems with C2C12The sphere formation pattern was achieved in a manner that supported cell (feeder) co-culture. Part B of fig. 5 is a graph showing the results of the color development of spheres derived from isolated VSELs-or adult stem cells derived from a hyaluronic acid-rich node and conduit system-on Alkaline Phosphatase (AP), part C of fig. 5 is a graph showing the efficiency of sphere formation, and part D of fig. 5 is a graph showing the proliferation of cells having the differentiation ability of VSELs-or adult stem cells derived from a hyaluronic acid-rich node and conduit system.
Fig. 6A, 6B are graphs showing expression of totipotent stem cell markers in adult stem cell spheres (spheres) derived from hyaluronic acid-rich node and catheter systems. FIG. 6A is a diagram showing analysis of expression patterns of Oct-4, Sox-2 and Nanog as totipotent stem cell markers by reverse transcription-polymerase chain reaction (RT-PCR) (a) and Western blot (b) using cDNA and cell lysate derived from VSELs-and adult stem cell-derived spheres of a hyaluronic acid-rich node and conduit system, and using deoxyribonucleic acid (cDNA) and cell lysate of embryonic stem cells (ES) as positive controls, C2C12The cDNA and cell lysate of the support cells were used as negative controls. FIG. 6B is a graph showing the results of immunostaining spheres derived from adult stem cells-and VSELs-derived from hyaluronic acid-rich node and conduit systems for the expression of totipotent stem cell markers (scale: 100X).
Fig. 7A, 7B, and 7C are graphs showing differentiation of VSELs in vitro and adult stem cells derived from hyaluronic acid-rich node and conduit systems into neural cells. FIG. 7A is a graph showing the results of culturing a medium for neural cell differentiation treated with a neural cell differentiation inducer for 25 days after mechanically dissociating spheres derived from VSELs (part a of FIG. 7A) and from adult stem cells derived from a hyaluronic acid-rich node and conduit system (part b of FIG. 7A) into single cells, and showing the results of immunostaining the differentiated cells with NeuN and MAP-2 as neural cell markers (the cytoplasm of the neural cell expresses NeuN positive, the neural cell expresses MAP-2 positive (ratio:. times.100)). Fig. 7B is a diagram showing analysis of expression forms of VSELs derived from undifferentiated single cells and neural markers of adult stem cells derived from a hyaluronic acid-rich node and conduit system by RT-PCR and western blotting, and fig. 7C is a diagram showing analysis of expression forms of VSELs derived from differentiation induction and neural markers of adult stem cells derived from a hyaluronic acid-rich node and conduit system by RT-PCR and western blotting (cDNA and cell lysate of embryonic stem cells (ES) were used as a negative control group, and cDNA and cell lysate of mouse brain were used as a positive control group).
Fig. 8A, 8B, and 8C are graphs showing differentiation of nerve cells in vivo after transplantation of adult stem cells derived from hyaluronic acid-rich nodes and catheter systems in a hypoxic-ischemic brain injury model. Fig. 8A is a graph showing the results of inducing hypoxic ischemic brain injury in mice, implanting CM-Dil-labeled adult stem cells derived from hyaluronic acid-rich nodes and ductal systems, isolating the brain, confirming the damaged part of the brain in the isolated brain by TTC staining (part a of fig. 8A), and detecting the ratio of the damaged part of the brain (part b of fig. 8A). FIG. 8B is a graph showing confirmation of nerve cell differentiation of transplanted CM-Dil-labeled adult stem cells derived from a hyaluronic acid-rich node and catheter system at the DG site of the hippocampus. Fig. 8C is a photograph showing confirmation of nerve cell differentiation of transplanted CM-Dil-labeled adult stem cells derived from hyaluronic acid-rich nodes and ductal systems at CA1 and CA3 sites of the hippocampus (NeuN immunostaining was used as a marker of nerve cell differentiation).
Fig. 9 is a graph showing hematopoietic progenitor cells found in hyaluronic acid-rich nodes and ductal systems. Part a of fig. 9 is a diagram showing colonies of hematopoietic progenitor cells, and part B of fig. 9 is a photograph showing cells derived from each colony stained by Wright-Giemsa. In addition, fig. 9, section C, is a graph showing the total number of Mast Cell Progenitors (MCPs) present in BM and hyaluronic acid-rich nodes and the catheter system, and fig. 9, section D, is a graph showing flow cytometry analysis of the phenotype of cells present in MCPs colonies.
Fig. 10A, 10B, 10C, 10D, and 10E are diagrams showing hematopoietic stem cells found in hyaluronic acid-rich nodes and catheter systems. FIG. 10A is a photograph (left side: magnification,. times.50; right side: enlargement of single colony,. times.100) of pebble zone forming cells cultured in vitro (invitro), FIG. 10B is a graph showing flow cytometric analysis of the presence or absence of angioblasts under co-culture of a hyaluronic acid-rich node and catheter system/OP 9, and FIG. 10C is a graph showing Hematopoietic Stem Cells (HSC) whose presence or absence has been confirmed under co-culture of a hyaluronic acid-rich node and catheter system/OP 9. And, FIG. 10D is a diagram illustrating a method for Lin-Pebble zone forming cells and Lin marked with each cell-CD45+Graph of relative percentage of pebble zone forming cells, and figure 10E is a graph showing the differentiation of pebble zone forming cells into bone marrow (myeloid), B-cell lines and T-cell lines under co-culture of a hyaluronic acid rich node and a catheter system/OP 9.
Fig. 11 is a graph showing characteristics of colonies formed in such a manner as to induce hematopoietic stem cells, part a of fig. 11 is a graph showing a pebble zone-forming cell formed by co-culture of a hyaluronic acid-rich node and a catheter system/OP 9, and part B of fig. 11 is a graph showing flow cytometric analysis of a phenotype of colony-forming cells. Furthermore, part C of FIG. 11 is a graph showing the proliferation potency of colony forming cells, and part D of FIG. 11 is a photograph showing colony forming cells stained with Wright-Giemsa or toluidine blue (scale bar: 10 μm; ratio: X400).
FIG. 12 is a graph showing the recombinant morphology of IFN-. gamma.involved in the appearance of MCPs derived from bone marrow, spleen and hyaluronic acid-rich node and ductal systems, a part A of FIG. 12 is a graph showing the frequency of appearance of MCPs colonies under hyaluronic acid-rich node and ductal systems in normal mice and various gene-deficient mice, and a part B of FIG. 12 is a graph comparing the number of hematopoietic progenitor cells formed from the spleen in normal mice with IFN-. gamma. -KO mice. And, part C of FIG. 12 is a diagram showing that the present in C-kitW-sh/W-shAnd c-kitW-sh/+And a plot of frequency of appearance of MCPs colonies from the catheter system.
Fig. 13 is a diagram showing a morphology of moving bone marrow cells to a hyaluronic acid-rich node and catheter system.
Detailed Description
The present invention relates to stem cells derived from a hyaluronic acid-rich node and conduit system, and preferably, to adult stem cells having the ability to differentiate into nerve cells derived from a hyaluronic acid-rich node and conduit system and hematopoietic stem cells derived from a hyaluronic acid-rich node and conduit system.
First, adult stem cells derived from hyaluronic acid-rich node and conduit systems having the ability to differentiate into nerve cells will be described.
Adult stem cells derived from a hyaluronic acid-rich node and duct system can be extracted from a network structure formed by nodes and ducts existing on the surface of an organ, in a blood vessel, in a lymph vessel, and in the skin, and particularly, in the case of a human, can be extracted from a placenta (placenta). The above extraction method is a theoretically acceptable method, and can also be a rapid and effective method for extracting adult stem cells without imposing a biomedical burden on researchers or patients.
In order to separate the hyaluronic acid-rich node and catheter system from the body, it is preferable that the hyaluronic acid-rich node and catheter system be obtained by injecting the staining reagent into the body at an appropriate concentration using Methylene blue (Methylene blue), Janus Green B (JGB), Alcian blue (Alcian blue), and the like, which are samples for selectively staining the hyaluronic acid-rich node and catheter system.
In the present invention, 4 methods for determining adult stem cells derived from hyaluronic acid-rich node and conduit systems include: 1) flow cytometric analysis for identifying the type of a cell by using a cell-specific phenotype Marker (Marker) as an object; 2) a method for detecting replication ability (proliferation ability) by plate efficiency (platinggeffeciency) and morphology of cell colonies; 3) a method of using immunostaining as a specific phenotype marker for pluripotent stem cells; and 4) a differentiation potency assay method using co-culture with a differentiation inducer and supporting cells in vitro and a method of testing differentiation potency by transplanting cells in vivo. The above-described method can be implemented by the contents described in the present specification or known in the art.
To isolate adult stem cells derived from hyaluronic acid-rich node and catheter systems, Flow cytometry (Flow cytometry) and Flow cytometric methods can be used. That is, an antibody that specifically recognizes a marker (antigen) expressed on the surface of a cell can be labeled by flow cytometry (FACS), the presence or absence of the antigen is analyzed by a fluorescent substance attached to the antibody, and a desired cell is obtained using FACS sorter, alone or in combination. Among the fluorescent substances that can be used are Fluorescein Isothiocyanate (FITC), Phycoerythrin (PE), Allophycocyanin (APC), Texas Red (Texas Red, TR), Cy3, Cy5, CyChrome, Red613, Red670, Tri-Color, Quantum Red, Alexa Fluor647, and the like.
Common immunological phenotype markers for undifferentiated adult stem cells are distinguished individually and also in the differentiation step. For example, in the case of hematopoietic stem cells, long-term hematopoietic stem cells (long-term HSCs, LT-HSCs) have c-kithigh、Sca-1high、Thy1.1low、IL-17R-、CD150+、Flt3-、Enderlin+、Rhodaminelow，CD34-Short-term hematopoietic Stem cells (Short-term HSC, ST-HSC) have c-kit+、Sca-1+、Lin-、IL-17R-、Flt3+、Thy1.1low、CD11blow、CD34+. And, in the case of a pluripotent precursor cell (multipotent proliferocell) differentiated into hematopoietic stem cells, has c-kit+、Sca-1+、Lin-、IL-17R-、Flt3+、Thy1.1-、CD11blow、CD34+. In the case of human hematopoietic stem cells, CD34 is present+、CD59+、Thy1+、CD38low/-、C-kit-/lowOr Lin-(Baumetal [ non-patent document 16]])。
Depending on the degree of differentiation, the phenotypic markers of stem cells have different expression profiles. Immunological markers that manifest as phenotype of stem cells and transcellular cells include: 1) oct4, Sox2, Nanog, SSEA-1, SSEA-4, TRA-1-60, TRA1-81 of embryonic stem cells having a full differentiation ability; 2) immunological markers include Type 1 (GFAP) according to the degree of differentiation of nerve cells in the hippocampal region of the brain+、Nestin+、BLBP+、Sox2+)、Type 2a(GFAP+/-、Nestin+、BLBP+、Sox2+)、Type 2b(DCX+、NeuroD+、Prox1+、Nestin+、Ki67/PCNA+)、Type 3(DCX+、NeuroD+、Prox1+、PSA-NCAM+、GAD65+、βIII-tubulin+、MAP2ab+、Ki67/PCNA+) And (Calretinin)+、NeuN+、NeuroD+、Prox1+、Calbindin+、βIII-tubulin+、MAP2ab+) And the like.
In the present invention, the Medium used in the process of stem cell proliferation includes, as a Basal Medium (Basal Medium), Dulbecco's Modified Eagle Medium (DMEM), DMEM-F12, NeuroCult Basal Medium, and the like, and any Medium can be used as long as it is used in the field to which the present invention pertains.
In the present invention, as a commonly used marker, a lipophilic (lipophilic) fluorescent marker capable of staining transplanted adult stem cells in a state of survival and differentiation includes CM-DiI [ Ex553/Em570 ]]、SP-DiOC18(3)[Ex497/Em513]、FM-Dil[Ex553/570]、DiIC18(3)-DS[Ex555/Em570]、SPDiIC18(3)[Ex556/Em573]And DiIC18(5)-DS[Ex650/Em670]And the like, but not limited thereto (abbreviation: Ex is activation (Excitation) and Em is Emission (Emission)).
In the present invention, an ischemic disease refers to dysfunction, tissue degeneration or necrosis caused by reduction or interruption of blood supply to a tissue, and specifically includes ischemic heart diseases such as myocardial infarction and angina pectoris, and wounds and fractures accompanied by ischemia of limbs, injury and interruption of blood flow. That is, in the present invention, ischemic diseases include not only ischemic conditions but also ischemic states caused by injury or injury.
In the present invention, a neurological disease refers to a case where there is an abnormality in a neurite (neuron) protruding (projection) from a cell body of a nerve cell, and representative diseases may include depression (depression), epilepsy (epilepsy), multiple sclerosis (multiple sclerosis), xerosis (mania), and the like, in addition to alzheimer's disease and parkinson's disease.
In the present invention, the brain diseases are roughly expressed in 2 forms. The first form is a cerebral disease of the vascular system, and includes diseases caused by cerebral ischemia, reperfusion brain injury, cerebral infarction, stroke, traumatic brain injury (trauma), hypoxic brain injury (hypoxic brain damage). The ischemic brain diseases include stroke, cerebral hemorrhage, cerebral infarction, head injury, Alzheimer's disease, vascular dementia, Creutzfeldt-Jakob disease, lethargy, and shocked brain injury, but are not limited thereto. When transient cerebral ischemia occurs in the brain, ATP production and edema (edema) are reduced in nerve cells due to termination of oxygen and glucose supply, resulting in extensive damage to the brain. After a considerable time following cerebral ischemia, apoptosis of nerve cells occurs, which is called delayed neuronal damage (Kirino T et al [ non-patent document 16 ]).
The second form is a degenerative brain disease, which is accompanied by degenerative changes in nerve cells of the central nervous system. Degenerative brain diseases include, but are not limited to, alzheimer's disease, mild cognitive impairment, stroke, vascular dementia, frontotemporal dementia, dementia with lewy bodies, creutzfeldt-jakob disease, traumatic head injury, syphilis, acquired immunodeficiency syndrome, other viral infections, brain abscesses, brain tumors, multiple sclerosis, dementia due to metabolic disease, hypoxemia, parkinson's disease, muscle atrophy, huntington's disease, pick's disease, amyotrophic lateral sclerosis, epilepsy, ischemia, stroke, attention deficit hyperactivity disorder, schizophrenia, melancholia, manic depression, post-traumatic stress disorder, spinal cord injury, myelitis, and the like.
Unless otherwise indicated, the term "treating" or "treatment" refers to reversing or alleviating the diseases and disorders or one or more symptoms of the diseases and disorders indicated above or inhibiting or preventing the progression of the diseases and disorders. As used herein, the term "treatment" refers to the act of performing a treatment, where "for treatment" is defined as described above. Thus, "treatment" or "treatment" of a disease in a mammal includes: (1) inhibiting the growth of the corresponding disease; (2) preventing the spread of the disease; (3) alleviating the disease; (4) preventing the recurrence of the disease; and (5) alleviating the symptoms of the disease (palliating).
For the treatment of ischemic diseases, the composition of the present invention is administered in a pharmacologically effective dose. "pharmacologically effective dose" refers to an amount of a compound administered that reduces to some extent one or more symptoms of a disorder being treated. Therefore, the pharmacologically effective agent is used in an amount including: (1) the effect of reversing the rate of progression of the disease; (2) the effect of prohibiting further progression of the disease to some extent; and (3) the effect of reducing, preferably eliminating, to some extent one or more symptoms associated with the disease.
The cell therapeutic agent of the present invention may be a composition containing a pharmaceutically acceptable carrier (transporter) and/or an additive or the like. For example, it may include sterile water, physiological saline, a conventional buffer (phosphoric acid, citric acid, other organic acids, etc.), a stabilizer, a salt, an antioxidant (ascorbic acid, etc.), a surfactant, a suspending agent, an isotonic agent, an antistaling agent, etc. For topical administration, it is preferably combined with an organic substance such as a biopolymer, an inorganic substance such as hydroxyapatite, and the like, specifically, a collagen matrix, a polylactic acid polymer or copolymer, a polyethylene glycol polymer or copolymer, a chemical derivative thereof, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19)thed.,1995)。
For example, a quantitative dose of 1.0X 10 can be administered to 1 site or a plurality of sites in a living organism (skeletal muscle, cardiac muscle, etc.) in the vicinity of an ischemic site5～1.0×108Cells/kg (body weight), more preferably, 1.0X 106～1.0×107Cells/kg (body weight). However, the dose may vary depending on the body weight, age, sex, symptoms of the patient, the form of the composition to be administered, the method of administration, and the like, and can be appropriately adjusted by one of ordinary skill in the art to which the present invention pertains. The number of administration is1 or a plurality of administrations are carried out within the range of clinically allowable side effects, and administration may be carried out at 1 site or a plurality of sites with respect to the administration site. Animals other than humans may also be administered the same amount of the drug as humans per kg. As the subject animal of the present invention, a human and a mammal for other purposes can be included, specifically, a human, a monkey, a mouse, a rabbit, a sheep, a cow, a dog, a horse, a pig, etc.
The therapeutic agent for ischemic diseases of the present invention is preferably administered parenterally including intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, local administration, etc., more preferably, subcutaneous administration or local administration is used, and administration is mainly carried out by a method of directly injecting into the site of injury.
The cell therapeutic agent can be filled in the form of a syringe or a final injection form contained in an apparatus (device), a form of a cryovial (cryovial) or a pyrogen-free glass bottle containing a liquid drug, a rubber stopper, or an aluminum cap. The device may be in the form of a syringe, a multifunctional syringe, or the like, and in the case of ischemic diseases of limbs, it is preferable to use a material that does not affect the viability of cells in consideration of the depth of a part or muscle to which cells are administered in the range of 20Guage to 31Guage, using an injection needle that can prevent the cells from being damaged by shearing (shear) during administration of the cells and minimize pain of a patient.
In one aspect of the present invention, it relates to adult stem cells derived from hyaluronic acid-rich node and conduit systems having the ability to differentiate into nerve cells.
The present invention is characterized in that the mammal having the hyaluronic acid-rich node and the catheter system is selected from the group consisting of mice, rabbits, sheep, cows, dogs, horses, pigs, monkeys, and humans. The present invention is characterized in that the hyaluronic acid-rich node and catheter system is a network structure formed by nodes and catheters present on the surface of an organ, in a blood vessel, in a lymph vessel, and on the skin.
The present invention is characterized in that the adult stem cells derived from the hyaluronic acid-rich node and conduit system have a structure selected from the group consisting of Sca-1+、Lin-And CD45-Immunological characterization of VSELs (Very small implantable stem cells) of the group. And, the above-mentioned adult stem cells derived from hyaluronic acid-rich node and duct system have a structure selected from the group consisting of Oct4+、Sox2+、Nanog+And SSEA-1+Immunological properties of the embryonic stem cells of the group.
In still another aspect of the present invention, there is provided a method for isolating adult stem cells derived from a hyaluronic acid-rich node and conduit system,namely, comprising: obtaining samples of the hyaluronic acid-rich nodes and catheter systems by dyeing the hyaluronic acid-rich nodes and catheter systems with a dyeing sample; and (b) separating Sca-1 from the hyaluronic acid-rich node and catheter system sample obtained in the step (a)+、Lin-And CD45-Adult stem cells derived from hyaluronic acid-rich node and duct systems are formed.
The present invention is characterized in that the hyaluronic acid-rich node and catheter system is a network structure formed by nodes and catheters present on the surface of an organ, in a blood vessel, in a lymph vessel, and on the skin. The present invention is characterized in that the stained sample for a hyaluronic acid-rich node and catheter system is selected from the group consisting of alcian blue, methylene blue and janus green B.
The present invention is characterized in that the adult stem cells derived from the hyaluronic acid-rich node and conduit system have a structure selected from the group consisting of Sca-1+、Lin-And CD45-Immunological properties of VSELs selected from the group consisting of Oct4+、Sox2+、Nanog+And SSEA-1+Immunological properties of the embryonic stem cells of the group.
In still another aspect, the present invention relates to a method for differentiating adult stem cells derived from a hyaluronic acid-rich node and conduit system, the method comprising: a step (a) of forming spheres in such a manner that adult stem cells derived from a hyaluronic acid-rich node and conduit system are co-cultured with supporting cells in a neural cell differentiation medium; and (b) dissociating the formed sphere with a single cell, and then treating the growth factor that is transformed into the neural cell, thereby differentiating into the neural cell.
The present invention is characterized in that the supporting cell is C2C12(Mouse myoblast cell line). The present invention is characterized in that the spheres express markers specific to embryonic stem cells expressing Alkaline Phosphatase (AP), Oct4, Sox2, Nanog and SSEA-1。
The present invention is characterized in that the growth factor that differentiates into nerve cells is selected from the group consisting of rhEGF, FGF-2 and NGF, and the nerve cells express a nerve cell-specific marker that expresses NeuN, MAP2, glial acidic protein (GFAP), nestin (nestin) and β III tubulin (tubulin).
The present invention is characterized in that the nerve cells have a therapeutic effect on a brain disease, a disease of the nervous system, and a disease model in vivo.
In another aspect, the present invention relates to a therapeutic agent for treating neurological diseases and disorders, which is a cell therapeutic agent containing, as an active ingredient, adult stem cells derived from a hyaluronic acid-rich node and a catheter system.
In another aspect, the present invention relates to a therapeutic agent for treating a disease requiring organ regeneration associated with tissue injury, which comprises, as an active ingredient, adult stem cells derived from a node and a catheter system rich in hyaluronic acid.
In another aspect, the present invention relates to a therapeutic agent for treating cancer, autoimmune diseases, chronic infectious diseases, and allergic intractable diseases, which contains, as an active ingredient, adult stem cells derived from hyaluronic acid-rich nodes and catheter systems.
In one embodiment of the present invention, morphology and intracellular structure of adult stem cells derived from a hyaluronic acid-rich node and conduit system are observed by an electron microscope. As a result, adult stem cells derived from hyaluronic acid-rich node and conduit systems may have characteristics of undifferentiated cells and form spheres.
In still another embodiment of the present invention, the characteristics of spheres formed from adult stem cells derived from hyaluronic acid-rich node and conduit systems are studied based on proliferative capacity and phenotype, as a result, adult stem cells derived from hyaluronic acid-rich node and conduit systems have higher sphere-formation rate and plate efficiency than bone marrow-derived VSELs, it was confirmed that the adult stem cells derived from the hyaluronic acid-rich node and duct system had a vigorous proliferation ability, the expression of totipotent stem cell markers is also closer to that of embryonic stem cells than bone marrow stem cells, so that the adult stem cells derived from a hyaluronic acid-rich node and duct system have higher differentiation capacity.
In still another embodiment of the present invention, as a result of comparing characteristics before and after differentiation into nerve cells of the adult stem cells derived from the hyaluronic acid-rich node and conduit system, the spheres of the adult stem cells derived from the hyaluronic acid-rich node and conduit system having the characteristics of the stem cells are differentiated into the nerve cells in vitro by the medium specific for differentiation of the growth factors (rhEGF, FGF-2 and NGF) and the nerve cells, and the nerve cells are labeled to express GFAP, nestin, β III tubulin, NeuN and MAP-2, thereby confirming the adult stem cells having the differentiation ability of the adult stem cells derived from the hyaluronic acid-rich node and conduit system.
In another example of the present invention, it was confirmed that adult stem cells derived from a hyaluronic acid-rich node and conduit system decreased the volume of an infarct site where brain injury was generated and differentiated into nerve cells in such a manner as to distribute DG, CA1 and CA3 in the hippocampus, thereby showing an anatomically restored morphology of the brain, as a result of observing the therapeutic effect in a manner that adult stem cells derived from a hyaluronic acid-rich node and conduit system were transplanted in a mouse induced with an hypoxic ischemic brain disease.
Next, hematopoietic stem cells derived from hyaluronic acid-rich nodes and ductal systems will be described.
"hematopoietic cell" refers to any cell from which the hematopoietic pathway originates. The cells express allowable morphological characteristics and phenotypic (immunological) markers that are characteristic of the hematopoietic system. Such hematopoietic cells include hematopoietic progenitor cells, colony forming cells, and fully differentiated cells. "hemangioblast (precursor)", "hematopoietic progenitor cell", or "hematopoietic stem cell" is a cell that has the ability to produce fully differentiated hematopoietic cells and to self-replicate.
The precursor cells described in the present invention refer to Hematopoietic cells having all intermediate self-replicating and differentiating abilities, including from undifferentiated cells to fully differentiated hemangioblasts and Hematopoietic progenitor cells (HSCs). Hematopoietic stem cells derived from hemangioblasts are differentiated into hematopoietic cells such as megakaryocytes, red blood cells, mast cells, basophils, neutrophils, eosinophils, monocytes (macrophages) or natural killer cells of the lymphatic (lymphoid) system, T-lymphocytes, and B-lymphocytes by various cytokines.
Hematopoietic stem cells derived from a hyaluronic acid-rich node and duct system can be extracted from a network structure formed by nodes and ducts existing on the surface of an organ, in a blood vessel, in a lymph vessel, and in the skin, and particularly, in the case of a human, can be extracted from the placenta. The above extraction method is a theoretically acceptable method, and can also be a rapid and effective method for extracting adult stem cells without imposing a biomedical burden on researchers or patients.
In order to separate the hyaluronic acid-rich node and catheter system from the body, it is preferable to use methylene blue, janus green B, alcian blue, and the like as a sample for selectively staining the hyaluronic acid-rich node and catheter system, and specifically, to inject the staining reagent described above into the body at an appropriate concentration to smoothly visualize the acquisition of the hyaluronic acid-rich node and catheter system.
In the present invention, 3 methods for determining hematopoietic stem cells include: 1) flow cytometric analysis in which the cell-specific phenotype marker is used as a target to identify the type of cell; 2) a method for detecting the replication ability (proliferation ability) based on the plate efficiency and the morphology of cell colonies; and 3) a differentiation potency assay method in which the cells are co-cultured with a differentiation inducer and a supporting cell. The above-described method can be implemented by the contents described in the present specification or known in the art.
To isolate adult stem cells derived from hyaluronic acid-rich node and conduit systems, flow cytometry analysis may be used. That is, an antibody that specifically recognizes a marker (antigen) expressed on the surface of a cell can be labeled by FACS (Fluorescence-activated cell priming) alone or in combination, the presence or absence of the antigen is analyzed by a fluorescent substance attached to the antibody, and desired cells are isolated and obtained. Among them, usable fluorescent substances are FITC, PE, APC, TR, Cy3, Cy5, Cychrome, Red613, Red670, Tri-Color, Quantum Red, Alexa Fluor647, and the like.
Common immunological phenotype markers for undifferentiated hematopoietic stem cells differ between mouse, which has CD34, and humanlow/-、SCA-1+、Thy1+/low、CD38+、C-kit+And Lin-In the case of humans, with CD34+、CD59+、Thy1+、CD38low/-、C-kit-/lowAnd Lin-(Baum et al [ non-patent document 16]])。
CD135(FLK2, FLT3, STK1) is a marker that is not expressed in hematopoietic stem cells, but not in lymphoid progenitor cells of pluripotent stem cells.
Common immunological phenotype markers of hemangioblasts are CD31(PECAM-1), CD34, ECadrein (CD324), Endoglin (CD105), EphB4, Tie2(CD202b), VE-Cadherin (CD144), VEGFR2(Flk1), etc.
In the present invention, the basic Medium used for the growth of hematopoietic stem cells includes Minimum Essential Medium (MEM), DMEM-F12, rpmi (roswell park memorial institute), K-sfm (keratinocyte Serum Free Medium), NeuroCult basal Medium, and the like, and any other Medium may be used as long as it is used in the technical field to which the present invention belongs.
In a further aspect the invention relates to hematopoietic stem cells derived from hyaluronic acid rich node and conduit systems.
The animal having the hyaluronic acid-rich node and the catheter system is a vertebrate, and the vertebrate is preferably a mouse, a rabbit, a sheep, a cow, a dog, a horse, a pig, a monkey, or a human, but is not limited thereto.
The present invention is characterized in that the hyaluronic acid-rich node and catheter system is a network structure formed by nodes and catheters present on the surface of an organ, in a blood vessel, in a lymph vessel, and on the skin.
The invention is characterized in that the hematopoietic stem cells derived from the hyaluronic acid-rich node and conduit system originate from a source with CD45-、B220-And FLK-1+Hemangioblasts of a cell population of immunological character.
The present invention is characterized in that hematopoietic stem cells derived from a node and duct system rich in hyaluronic acid include cells having Sca-1 derived from the above-mentioned angioblasts+、CD59+、Lin-、CD45+、B220+、c-kit+、CD34-And CD135-Hematopoietic progenitor cells of an immunologically competent cell population.
The present invention is characterized in that the hematopoietic cells derived from the node and ductal system rich in hyaluronic acid are selected from the group consisting of megakaryocytes, erythrocytes, mast cells, basophils, neutrophils, eosinophils, and monocytes (macrophages) of the bone marrow system differentiated from the above hematopoietic progenitor cells, and cells differentiated into natural killer cells, T-lymphocytes, and B-lymphocytes of the lymphatic system.
In another aspect, the present invention relates to a method for isolating hematopoietic stem cells derived from a hyaluronic acid-rich node and conduit system, comprising: a step (a) of staining the hyaluronic acid-rich node and the catheter system with a staining sample through the hyaluronic acid-rich node and the catheter system, and separating cells of the hyaluronic acid-rich node and the catheter system in an extraction manner; and (b) culturing the tissue cells of the hyaluronic acid-rich node and duct system isolated in the step (a) in a methylcellulose medium to form colony forming cells comprising serum and cytokines.
The present invention is characterized in that the hyaluronic acid-rich node and catheter system is a network structure formed by nodes and catheters present on the surface of an organ, in a blood vessel, in a lymph vessel, and on the skin.
The present invention is characterized in that the stained sample for hyaluronic acid-rich node and catheter systems is selected from the group consisting of alcian blue, methylene blue and janus green B.
The present invention is characterized in that the cytokine is selected from the group consisting of erythropoietin (erythropoetin), SCF, Granulocyte-macrophage colony stimulating factor (GM-CSF), IL-3/-7, FL ((flt3/flt2ligand), LIF and TPO.
The present invention is characterized in that the methyl cellulose medium for colony-forming cells is granulocyte, erythrocyte, monocyte and megakaryocyte colony-forming unit (CFU-GEMM), methyl cellulose (methylcellulose).
The present invention is characterized by further comprising a step of staining the colony-forming cells with a staining sample to thereby classify the types of colonies into CFU-GEMM, granulocyte-macrophage colony-forming unit (CFU-GM), erythroid burst-forming unit (BFUE), and Mast Cell Progenitors (MCPs).
The present invention is characterized in that the staining sample for colony-forming cells is toluidine blue (or a complex staining solution of Rui-Giemsa).
In another aspect, the present invention relates to a method for differentiating hematopoietic stem cells derived from a hyaluronic acid-rich node and conduit system, comprising: culturing cells originated from angioblasts in hematopoietic stem cells derived from a hyaluronic acid-rich node and ductal system in a medium containing serum and cytokines together with hematopoietic supporting cells, and forming pebble zone-forming cells; and (b) subculturing the cells formed in the pebble zone in a methylcellulose medium to form mature hematopoietic cells.
The present invention is characterized in that the hematopoietic progenitor cells are OP9 or OP9-
The present invention is characterized in that the cytokine is selected from the group consisting of erythropoietin, SCF, GM-CSF, IL-3/-7, FL ((flt3/flt2ligand), LIF and TPO.
The present invention is characterized in that the culture medium for colony-forming cells is CFU-GEMM methylcellulose.
The present invention is characterized by further comprising a step of differentiating the size and differentiation morphology of colony-forming cells so that the mature hematopoietic cells are stained with a staining sample by the colony-forming cells.
The present invention is characterized in that the stained sample for colony forming cells is toluidine blue or Wright-Giemsa.
In another aspect, the present invention relates to a therapeutic agent for treating a disease requiring replacement of hematopoietic function derived from bone marrow or spleen, which comprises, as an active ingredient, hematopoietic stem cells derived from a node and a ductal system rich in hyaluronic acid. The present invention is characterized in that the disease is selected from the group consisting of paralysis of hematopoietic function due to bone marrow disorders, ischemic diseases, and disorders due to bone marrow destruction during organ transplantation.
In another aspect, the present invention relates to a therapeutic agent for treating a disease mediated by mast cells and eosinophils, which comprises, as an active ingredient, hematopoietic stem cells derived from a node and a duct system rich in hyaluronic acid. The invention is characterised in that the disease is selected from the group consisting of local or systemic allergy (allergy), Asthma (Asthma), cancer and parasitic infections.
In another aspect, the present invention relates to a therapeutic agent for treating a disease caused by a decrease or increase in immune function due to bone marrow, which comprises, as an active ingredient, hematopoietic stem cells derived from a node and a ductal system rich in hyaluronic acid. The present invention is characterized in that the disease is selected from the group consisting of autoimmunity, cancer, viral infection, and bacterial infection.
In another example of the present invention, in order to analyze characteristics of hematopoietic cells present in hyaluronic acid-rich nodes and ductal system tissues and hyaluronic acid-rich nodes and ductal systems, it was confirmed that a plurality of hematopoietic cells and immune cells are distributed in a system called hyaluronic acid-rich nodes and ductal system that is heterogeneous to bone marrow, peripheral blood, and umbilical cord blood (cord blood) that are sources of hematopoietic cells by separating hyaluronic acid-rich nodes and ductal systems present on the surface of a mouse organ and inside veins and lymph vessels (fig. 1A, 1B, 1C, and 1D).
In still another embodiment of the present invention, as a result of analyzing characteristics of colonies (cell colonies) derived from nodes and ductal systems rich in hyaluronic acid, it was confirmed that hematopoietic progenitor cells exist in the nodes and ductal systems rich in hyaluronic acid, and when cells derived from the nodes and ductal systems rich in hyaluronic acid are cultured under in vitro conditions, various types of hematopoietic colonies can be formed, and the presence of angioblast-like cells in the nodes and ductal systems rich in hyaluronic acid means that hematopoietic phenomenon occurs (fig. 9), but is not limited thereto.
In another embodiment of the present invention, in order to find out the characteristics of hematopoietic stem cells obtained from a node and ductal system rich in hyaluronic acid, when a suitable cytokine is added to cells derived from a node and ductal system rich in hyaluronic acid and cultured on hematopoietic progenitor cells, totipotent stem cells can be differentiated from hematopoietic stem cells into blood cells by using hemangioblasts (fig. 10A, 10B, 10C, 10D, and 10E), but the present invention is not limited thereto.
In another embodiment of the present invention, as a result of observing the characteristics of colonies formed in such a manner as to induce hematopoietic progenitor cells derived from a node and ductal system rich in hyaluronic acid, after differentiating into angioblast-like cells by means of totipotent stem cells derived from a node and ductal system rich in hyaluronic acid, it is possible to additionally differentiate into hematopoietic progenitor cells, and further, it is possible to produce various hematopoietic cells (fig. 11 and table 2), but the present invention is not limited thereto.
The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely illustrative of the present invention and the scope of the present invention is not limited to these examples.
EXAMPLE 1 hyaluronic acid-Rich node and ductal System tissue (tissue) and hematopoietic cell characteristics derived from hyaluronic acid-Rich node and ductal System
1-1: hyaluronic acid rich node and catheter system acquisition process
Intramuscular injection of IFN-gamma with wild type-/-Or IFN-gamma+/-C57BL/6 mice (Orient, Korea) were injected with Sulatai (Zoletil) (2.5mg/kg) and Loppon (Rompun) (0.5mg/kg) to complete anesthesia. Then, the hyaluronic acid-rich node and catheter system was obtained using a stereomicroscope (Zeiss Stereo discovery. v20) in the following manner.
1) In order to obtain the hyaluronic acid-rich node and catheter system existing on the surface of the small intestine (or liver), a cut was made along the white line (linear alba) of the abdomen, and the abdominal wall was carefully lifted, and the hyaluronic acid-rich node and catheter system between the anterior wall (antrorwall) and the small intestine (or liver) and on the surface of the visceral organ were obtained during the laparotomy.
2) To obtain a hyaluronic acid-rich node and catheter system of the vein, about 0.5ml of 1% alcian blue was injected into the iliac veins (iliac veins), after fixing the upper and lower lumbar veins (lumbar vein) with clamps, blood was discharged in a cut blood vessel manner, and a hyaluronic acid-rich node and catheter system forming a staining line was obtained from the inside of the vein.
3) To obtain hyaluronic acid rich node and catheter systems inside the lymphatic vessels (Intra-lymphatic), 0.5ml of 1% alcian blue was injected in the form of Subcutaneous injections (SC) towards the lateral caudal base, and after completion of the injections towards the 1cm end of the rectum and in the middle of the caudal vein, hyaluronic acid rich node and catheter systems were obtained.
As a result, as shown in fig. 1A, when C57BL/6 mice (5 to 6 weeks old) were anesthetized and the hyaluronic acid-rich node and ductal system present in the vein and the lymph vessel were stained with 1% alcian blue, 3 ducts (fig. 1A, part a) connecting the Large Intestine (LI), the Small Intestine (SI), and the Abdominal Wall (AW) around the node and the ductal system rich in hyaluronic acid (star) were found. Nodes and ductal systems (triangular arrows) rich in hyaluronic acid are observed inside the lumbar veins (dotted arrows) (part b of fig. 1A, showing the boundaries of the veins in wavy lines) and in the center of lymphatic vessels (part c of fig. 1A, arrows). In the enlarged photograph of fig. 1A, the boundary of the Lymphatic Vessel (LV) (section d of fig. 1A) is shown in wavy lines, and several branches originating from the node (star) (section b of fig. 1A) are also confirmed.
That is, when the nodes and the duct system rich in hyaluronic acid inside the lymphatic vessels (intralymphatic) are stained by injecting 1% alcian blue into the right and left tail bases, a staining line (fig. 1A, part a, fig. 1A, part b) is formed inside the lymphatic vessels of the lumbar arteries, ischials, and/or coccyx, and the stained nodes and duct system rich in hyaluronic acid are obtained inside the lymphatic vessels by fixing the ends of the duct. When the hyaluronic acid-rich node and the ductal system have been detached from the inside of the lymphatic vessels, a phenomenon of roll-up (portion c of fig. 1A) due to elastic force is observed.
As a result of injecting 1% alcian blue to the left tail vein in order to separate the hyaluronic acid-rich node and catheter system inside the vein, a stained line (portion a of fig. 1B) or blue knot (portion c of fig. 1B) is formed inside the waist vein, the ends of both veins are fixed, and blood is discharged in a manner of being longitudinally incised along the vein (portion B of fig. 1B), so that the hyaluronic acid-rich node and catheter system, which is completely stained, can be obtained.
As a result, when the node and the catheter system rich in hyaluronic acid inside the lymphatic vessels and veins were stained in such a manner that 1% alcian blue was injected into the mice by intramuscular and intravenous injections, the node and the catheter system rich in hyaluronic acid could be easily obtained.
1-2: electron microscopy of hyaluronic acid-rich node and catheter systems
The obtained hyaluronic acid-rich nodes and catheter systems derived from the organ surfaces were fixed with Karnovsky fixing solution (2% paraformaldehyde, 2% glutaraldehyde, 0.05M sodium cacodylate buffer (ph7.2) at a temperature of 4 ℃ for about 2 hours, the hyaluronic acid-rich nodes and catheter systems were finally fixed with 1% osmium tetroxide (EMs, Washington) at a temperature of 4 ℃ for about 2 hours, in the case of transmission electron microscope (transmission EM), dehydrated with ethanol, inserted into SURR resin (ERL, DER, NSA, and DMAE mixture) (EMs, Washington (Washington)), and polymerized overnight at a temperature of 70 ℃, cut into sections of 1 to 5 μ M with a diamond-knife (diamond-on, Switzerland (Switzerland)) using an ultra-thin microtome (RMC MTX, USA), after 20 minutes of staining with uranyl acetate (EMS, Washington), lead citrate (leader citrate) was treated for 10 minutes. The sections were analyzed using a transmission electron microscope (JEM1010, JEOL, Japan (JAPAN)) operating at an acceleration voltage of 80-kV.
After the hyaluronic acid-rich node and catheter system were fixed in a Karnovsky fixing solution using a Scanning Electron Microscope (SEM), the node and catheter system were washed 3 times with 0.05M sodium cacodylate buffer (pH7.2, 4 ℃) for 10 minutes each. Then, the hyaluronic acid-rich node and catheter system were finally fixed with 1% osmium tetroxide in 0.05M sodium cacodylate buffer (pH7.2), and then washed 2 times with distilled water at room temperature. The dehydration process of the hyaluronic acid-rich node and catheter system was carried out with ethanol at room temperature for 10 minutes. The hyaluronic acid rich node and conduit system was solidified 2 times at normal temperature in 10 minutes each using 100% isoamyl acetate and dried at critical point (critical point) with liquid carbon dioxide. The dried hyaluronic acid-rich node and catheter system was mounted on a metal base (stubs), coated with gold using a spray etcher (Sputter Coater), and observed using a Field Emission scanning electron microscope (Field-Emission SEM; Carl Zeiss SUPRA 55VP, Germany).
As a result, as shown in fig. 1B, the surface of the node rich in hyaluronic acid and the node of the catheter system (part a in fig. 1B), the inside of the node (part B in fig. 1B), and the catheters (parts c and d in fig. 1B) can be observed by a scanning electron microscope, and the node, the inside of the node, and the catheters (parts c and d in fig. 1B) are formed of
1-3: quantitative analysis of Hyaluronic acid (Hyaluronic acid)
After acquiring and quantifying a hyaluronic acid-rich node and catheter system present on the surface of an organ of a mouse, the mouse was immersed in Phosphate Buffered Saline (PBS) and rapidly frozen with liquid nitrogen. The frozen hyaluronic acid-rich nodes and the catheter system were homogenized by a disintegrator, and the supernatant was obtained after centrifugation (20 minutes, 4 ℃, 2000 xg). The hyaluronic acid content present in the supernatant was determined using a mouse hyaluronic acid enzyme-linked immunosorbent assay (ELISA) kit (SunRed, Shanghai Shanred Biological Technology) using the manufacturer's (SunRed) protocol. And, the hyaluronic acid of serum, urine, peritoneal fluid and lymphatic vessel is compared and quantified.
As a result, as shown in fig. 1D, when serum, urine, abdominal fluid, and lymphatic vessels were compared, hyaluronic acid was detected at the highest concentration in the node and the duct system where hyaluronic acid was abundant.
1-4: statistical analysis
All data were analyzed as a statistical program prism5.0 graphic pad (San Diego, CA) using student's t-test to confirm statistical significance between groups (. P <0.01,. P < 0.05). The same applies to the results of fig. 1A to 13.
From the above results, it was confirmed that the node and the catheter system rich in hyaluronic acid formed a spider-web-shaped node and catheter on the surface of the mouse organ (part a in fig. 1A). in order to easily obtain the node and the catheter system rich in hyaluronic acid, alcian blue staining was required, and as a result, the inside of the vein (part b in fig. 1A) and the lymphatic vessels (parts c and d in fig. 1A) were stained, and it was found that the HAR-catheter branched from the node (asterisk (★) in parts a and b in fig. 1A).
Referring to the SEM results of the hyaluronic acid-rich node and the catheter system using an electron microscope, HAR-nodes on the organ surface were found to have an elliptical pocket shape and have elongated catheters along both side ends (part a in fig. 1B). Furthermore, it was found that the HAR-knot was filled with cells (part B of fig. 1B), and the HAR-duct was formed of 3 small ducts (parts c and d of fig. 1B).
And, according to TEM results, 3 canals (arrows) of HAR-junctions function as a pathway of 3 ducts and are filled with cells (part a of fig. 1C), some of which are mast cells (part b of fig. 1C), polymorphonuclear leukocytes (part C of fig. 1C), monocytes (part d of fig. 1C), eosinophils (part e of fig. 1C) and small immature cells (parts f, g of fig. 1C) having a relatively small ratio of cytoplasm with respect to the macrocore.
Furthermore, the hyaluronic acid-rich nodes and ductal system, lymphatic vessels, serum, urine and peritoneal fluid had the highest concentration of hyaluronic acid (fig. 1D).
As a result, tissues (tissue) having high concentrations of hyaluronic acid, which are heterogeneous to bone marrow, peripheral blood, and umbilical cord blood (cord blood) that are production sources of hematopoietic cells, called hyaluronic acid-rich nodal and ductal systems, contain a variety of hematopoietic cells and immune cells.
Example 2 isolation of adult Stem cells derived from hyaluronic acid-rich node and conduit System
In order to obtain adult stem cells derived from a hyaluronic acid-rich node and conduit system (control group: bone marrow-derived VSELs) derived from the hyaluronic acid-rich node and conduit system, experiments were conducted in such a manner that the adult stem cells derived from the hyaluronic acid-rich node and conduit system were separated from a cell suspension containing mononuclear cells (monouchear cells) separated from the hyaluronic acid-rich node and conduit system using a phenotypic marker antibody, a flow cytometry, and a flow cell sorter. Mononuclear cells from hyaluronic acid-rich nodal and ductal systems were suspended in PBS (pH7.4, Ca/Mg) containing 1% Fetal Bovine Serum (FBS) (Gibco, Carlsbad, CA), 1mM Ethylene Diamine Tetraacetic Acid (EDTA) and 25mM hydroxyethylpiperazine ethanesulfonic acid (HEPES)++free), the cells were stained with anti-Ly-6A/E (Sca-1) -PE (clone E13-161.7), anti-CD 45-pecy5 (clone 30-F11) and biotinylated lineage (linkage) protease inhibitor, anti-CD 45R/B220-Biotin (clone RA-3H57-597), anti-Gr-1-biotin (clone RB6-8C5), anti-TCR αβ -biotin (clone H57-597), anti-TCR γ δ -biotin (clone GL-3), anti-CD 11 b-biotin (clone M1/70) and anti-Ter-119-biotin (clone Ter-119), 2 antibodies were isolated using Streptavidin (Streptavidin) -FITC that specifically binds to the
All single antibodies were added to the separated cell suspension, incubated in ice water (incubation) for 30 minutes, washed 2 times with PBS, and separated by suspension in a flow cytometer and separation medium. All single antibodies used in the above experiments were purchased from BD Pharmingen (San Diego, CA) of jacobo biotechnology limited, shanghai. Cell separation was performed using FACSAria (BD Biosciences, san jose, CA) and cell analysis was performed using FACSCalibur (BDBiosciences, san jose, CA).
As a result, Sca-1 having a size of 2 to 5 μm can be separated from the hyaluronic acid-rich node and the catheter system+Lin-CD45-The adult stem cells derived from hyaluronic acid-rich node and ductal system isolated from the immunologically labeled cells were negative for 7AAD and annexiv, and were confirmed to have no apoptosis phenomenon (fig. 3A, 3B, 3C, and 3D).
As a result, the adult stem cells derived from the hyaluronic acid-rich node and conduit system obtained by the VSELs separation method are similar in VSELs and size or morphology, and the flow cytometry analysis results that the adult stem cells derived from the hyaluronic acid-rich node and conduit system have a specific gravity of 100 times greater than that of the bone marrow-derived VSELs. That is, the hyaluronic acid-rich node and conduit system has many number of adult stem cells derived from the hyaluronic acid-rich node and conduit system as the adult stem cells.
EXAMPLE 3 cellular characteristics of adult Stem cells derived from hyaluronic acid-rich node and conduit System
In order to analyze morphology and internal cell structure of adult stem cells derived from a hyaluronic acid-rich node and conduit system by SEM and TEM, adult stem cells derived from a hyaluronic acid-rich node and conduit system obtained by a flow cytometer and a separator were prepared and observed under an electron microscope.
Electron microscopy of hyaluronic acid-rich node and catheter systems
Adult stem cells derived from hyaluronic acid-rich node and catheter systems obtained by flow cytometry and isolation were fixed with Karnovsky fixing solution (2% paraformaldehyde, 2% glutaraldehyde, 0.05M sodium cacodylate buffer solution (ph7.2)) at a temperature of 4 ℃ for about 2 hours. In the case of using a transmission electron microscope, a hyaluronic acid-rich node and catheter system were finally fixed using 1% osmium tetroxide in a manner of being left at a temperature of 4 ℃ for about 2 hours, seeded on SURR resin (ERL, DER, NSA and DMAE mixture) (EMS, Washington) after a dehydration process was performed according to an ethanol concentration, and polymerized overnight at a temperature of 70 ℃. After ultrathin (0.5 to 1.0 μm) sections were cut with a diamond knife (Switzerland) of an microtome (RMC MTX, USA), staining was performed for 20 minutes with uranyl acetate (EMS, Washington), and then lead citrate was treated for 10 minutes. Sections were analyzed using a transmission electron microscope (JEM1010, JEOL, JAPAN) operating at an acceleration voltage of 80-kV.
After fixation of the hyaluronic acid-rich node and catheter system with a Karnovsky fixative using a scanning electron microscope, the cells were washed 3 times with 0.05M sodium cacodylate buffer (pH7.2, 4 ℃) for 10 minutes each. After the fixed adult stem cells derived from the hyaluronic acid-rich node and catheter system were finally fixed with 1% osmium tetroxide in 0.05M sodium cacodylate buffer (pH7.2), they were washed 2 times with distilled water at room temperature. And, adult stem cells derived from hyaluronic acid-rich node and duct systems were treated in terms of ethanol concentration, and a dehydration process was performed at normal temperature for 10 minutes in each step. The hyaluronic acid rich node and duct system was solidified 2 times at normal temperature in 10 minutes each using 100% isoamyl acetate and dried at the appropriate time with liquid carbonic acid. The dried hyaluronic acid-rich node and catheter system described above was mounted on a metal base (stubs), coated with gold using a spray etcher, and then the adult stem cells derived from the hyaluronic acid-rich node and catheter system were observed using a field emission scanning electron microscope (Carl Zeiss SUPRA 55VP, Germany).
As a result, NDSC under SEM and TEM is a circular cell having a diameter of about 3.5 to 4.5. mu.m, has a nuclear membrane and nucleosomes, and is formed of a huge nucleus and a slightly smaller cytoplasm. Further, mitochondria (mitochondria) which is a small organ of a cell can be observed. Vacuoles (vacuoles) and endoplasmic reticulum (endoplasmic reticulum) of cells, and the like. Thus, NDSCs were determined to have characteristics of immature cells (fig. 4, part a and fig. 4, part B).
Example 4 Properties of spheres formed from adult Stem cells derived from hyaluronic acid-rich node and conduit System and bone marrow-derived VSELs derived from hyaluronic acid-rich node and conduit System
4-1 in vitro proliferation of adult stem cells derived from hyaluronic acid-rich node and catheter systems
C at the completion of radiation irradiation (at 40Gy)2C12Split Central pillars (1X 10) of adult Stem cells derived from hyaluronic acid-rich node and duct System (control group: VSELs) isolated on murine myoblast (myoblast) supporting cells3cells/well) and cultured in a manner of co-culture with 20% knock-out serum (KSR, Invitrogen, Carlsbad, CA), 2mM L-glutamine (Invitrogen), 100. mu.M MEMNEAA (Invitrogen), 100. mu.M β -mercaptoethanol (Sigma-Aldrich), and 4ng/ml human basic (basic) FGF (bFGF, Sigma-Aldrich) in supplemented DMEM-F12 medium (Sigma-Aldrich, StLouis, MO) for 7 days to form spheres2C12The support cells were again cultured in separate columns and in a co-culture manner to observe the spheres. To confirm the characteristics of the stem cells, after the spheres were fixed with 4% paraformaldehyde for 15 minutes, they were washed 2 times with TBST (0.15M NaCl, 0.05% Tween-20 in 20mM tris-HCl, pH7.4) and stained with an alkaline phosphatase confirmation kit (Millipore, Billerica, Mass.).
4-2: immunofluorescence staining
Mouse brains or cells were fixed on slides with 4% paraformaldehyde for 20 minutes. In order to prevent the generation of non-specific binding sites, PBS (pH7.4) containing 2% BSA was treated for 30 minutes, and in order to stain intracellular proteins and nuclei, 2% BSA and 0.1% triton X-100 were used for 30 minutes. Then, the sample was treated at 4 ℃ overnight as 1-time antibody. The following day, removal was completed by 3 washes of 1 antibody in PBST (PBS supplemented with 0.05% (v/v) Tween 20) for 5 minutes each. Anti-rabbits were used as 2-fold antibodies
4-3: reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis
VSELs, adult stem cell spheres derived from hyaluronic acid-rich node and catheter systems, differentiated neural cells, ES-D3, C were extracted using a tr-zol agent (Invitrogen, LaJolla, Calif.)2C12Total (total) ribonucleic acid (RNA) of the supporting cells or mouse brain. The synthesis was performed using total RNA with cDNA of 2. mu.g, oligo (dT) primer (Promega, Madison, Wis.), 20 units (units) ribonuclease (RNase) inhibitor (Ambion Inc, Austin, TX) and M-MLV reverse transcriptase by the manufacturer (Promega). PCR amplification was performed using PyrothtatTartTaq (BioneerInc., Korea) and 10pmoles for each oligonucleotide primer (Table 1) in a thermal cycler (thermocycler). Specifically, c-DNA was denatured (denaturation) at 94 ℃ as a hot start for 5 minutes, amplified in 25 to 40 cycles (cycles) of "94 ℃ to 30 seconds, 52 ℃ to 62 ℃ to 30 seconds, and 72 ℃ to 30 seconds", and finally elongated (final extension) at 72 ℃ for 10 minutes. PCR products amplified in the above reaction were analyzed by 1% agarose gel electrophoresis.
For Western blot analysis, cells were lysed with a RIPA solution (50mM tris-HCl [ pH7.2], 150mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 1mM PMSF, 25mM MgCl2) to which a phosphatase inhibitor was added, the Cell eluate was resolved by 8-12% SDS-polyacrylamide gel electrophoresis (protein electrophoresis), and after the separated proteins were moved to NCMEMbrane, immunoblotting (immunoblotting) was performed using the following antibodies as1 time antibody and anti-Oct 2(Abcam, 18976), anti-Sox 2(Abcam, ab59776), anti-Nanog (Abcam, 80892), anti-GFAP (monoclonal Technology, 12389), anti-Sox protein (Abcam, 27952) or anti-36III protein (Abcam, Ab-GFAP (Invacling) was performed using the above-mentioned primers, Invitrogen probe (Invitrogen) and the above-mouse protein electrophoresis (Invitrogen) was performed using the above-probe (Invitrogen probe) to confirm whether the concentration of the above-IgG-binding protein was used (Invitrogen probe, Invitrogen probe was determined using the above.
TABLE 1
At C2C12As a result of co-culturing VSELs and adult stem cells derived from a hyaluronic acid-rich node and ductal system in murine myoblast (myoblast) -supporting cells, spheroids similar to embryoid bodies (fig. 5 a) were formed, and as a result of staining with alkaline phosphatase, it was confirmed that the cells had the characteristics of totipotent stem cells by a positive reaction (fig. 5D). As a result of comparing the efficiency of forming spheres of VSELs and adult stem cells derived from a hyaluronic acid-rich node and conduit system (sphere formation efficiency), when 1000 VSELs and NDSC cells were cultured in separate central columns together with supporting cells, the adult stem cells derived from the hyaluronic acid-rich node and conduit system were formed into-176 spheres and the VSELs were formed into-14 spheres. From the above results, it is understood that hyaluronic acid-rich node-and duct-derived systems are obtained relative to bone marrow-derived VSELsThe adult stem cells derived from the hyaluronic acid-rich node and duct system had a sphere-forming efficiency about 12.5 times higher (fig. 5, section C). Subculture experiments were also performed to investigate the proliferation potential of VSELs and adult stem cells derived from hyaluronic acid-rich nodal and ductal systems. After the spheres were dissociated into single cells, pairs of C were administered every 7 days2C12As a result of observing the number of alkaline phosphatase-positive reaction spheres after the support cells were subcultured, the number of alkaline phosphatase-positive reaction spheres increased as the adult stem cells derived from hyaluronic acid-rich node and conduit systems were subcultured, whereas the number of alkaline phosphatase-positive reaction spheres was hardly changed regardless of subculture of VSELs (D-a, -b of FIG. 5).
It was analyzed by RT-PCR, Western blotting and immunofluorescence staining whether VSELs or adult stem cells derived from hyaluronic acid-rich node and catheter systems expressed Oct4, Sox2, Nanog and SSEA-1 as totipotent stem cell markers. Adult stem cells derived from hyaluronic acid-rich node and conduit systems expressed mRNA and protein of Oct4, Sox2, and Nanog to a similar degree to murine embryonic stem cell line (ES-D3) as a control group, whereas VSELs expressed Oct4 and Nanog but not Sox2 (fig. 6A, parts a and b). When Oct4, Sox2, Nanog, and SSEA-1 were stained with VSELs and adult stem cell spheres derived from a hyaluronic acid-rich node and catheter system by immunofluorescence staining, the adult stem cell spheres derived from a hyaluronic acid-rich node and catheter system reacted positively with Oct4, Sox2, Nanog, and SSEA-1, while the VSELs spheres reacted positively with Oct4, Nanog, and SSEA-1 and negatively with Sox2 (FIG. 6B).
According to the above results, adult stem cells derived from hyaluronic acid-rich node and conduit systems show higher sphere-formation rate and plate efficiency through subculture, and thus have higher proliferative capacity, as compared to bone marrow-derived VSELs. In addition, adult stem cells derived from a node and duct system rich in hyaluronic acid also have characteristics closer to embryonic stem cells in terms of expression of totipotent stem cell markers, as compared to bone marrow-derived VSELs.
EXAMPLE 5 ability of adult stem cells derived from hyaluronic acid-rich node and conduit system and bone marrow-derived VSELs to differentiate into neural cells
Whether an adult stem cell sphere derived from a hyaluronic acid-rich node and a catheter system, which is expressed by a totipotent stem cell marker in a neural cell differentiation condition medium, is differentiated into neural cells is investigated. First, in order to generate lysogenic cells (neurons, oligodendrocytes and glial cells) of nerve cells, 10 VSELs or adult Stem cell spheres derived from a hyaluronic acid-rich node and ductal system were prepared and dissociated into single cells, which were then cultured in NeuroCult cult basic medium (Stem cell Technologies, Vancouver, BC, Canada) supplemented with 10ng/ml rhEGF, 20ng/ml FGF-2 and 20ng/ml NGF. The above culture was performed on an 8-well culture slide (SPL Life Science, Korea), and whether the cultured cells differentiated into nerve cells was observed 21 to 25 days later. Growth factors (R & DSystem, Minneapolis, MN) were re-added every 24 hours and the medium was replaced every 3 days.
As a result, both adult stem cells and VSELs derived from the hyaluronic acid-rich node and ductal system, which were cultured for 25 days under the neural cell differentiation condition, showed NeuN-positive reaction and differentiated into neural cells (parts a and B of FIG. 7A) as MAP-2 positive by using an immunofluorescence staining method, as for the expression of GFAP, nestin and β III tubulin as neural cell markers, the same expression as ES-D3 cells was performed in single cells and VSELs that were free spheres, and mRNA was expressed from the VSELs or the node and ductal system-derived adult stem cells at the level of differentiation of the hyaluronic acid-rich node and ductal system was induced by mRNA (FIG. 7B) at the level of mRNA, as well as in FIG. 7C).
From the above results, it was found that when nerve cell growth factors (rhEGF, FGF-2 and NGF) were added to a single cell released from a sphere of adult stem cells derived from a hyaluronic acid-rich node and a ductal system having characteristics of stem cells, the cells were differentiated into nerve cells in vitro and markers (NeuN, MAP-2, GFAP, nestin and β III tubulin) specifically expressed in the nerve cells were expressed, and from the above results, it was found that the adult stem cells derived from the hyaluronic acid-rich node and the ductal system have characteristics of adult stem cells.
EXAMPLE 6 therapeutic Effect of mice with hypoxic ischemic brain diseases Using adult Stem cells derived from hyaluronic acid-rich nodes and ductal System
Adult stem cells derived from hyaluronic acid-rich nodes and ductal systems were transplanted in a model for inducing hypoxic-ischemic brain injury in mice, and experiments for neural cell differentiation and treatment effect of brain diseases were performed in vivo in the following experimental procedures. First, 7-week-old male ICR mice were anesthetized and a small incision was made on the right side of the neck. The right carotid artery was exposed and double-ligation (double-ligation) was performed with 7-0 wire suture (Ethicon LCC, SanLorenzo, Puerto Rico). The cut sections were sutured with 5-0 nylon suture (Ethicon LCC, San Lorenzo, Puerto Rico). The mice sutured in the cage were allowed to recover for about 2 hours, then at 8% O2/balance N2Systemic hypoxia (systemic hypoxia) was induced in the global province by exposure for 20 minutes. The following day, the mice were maintained at a body temperature of 38 ℃ for about 5 hours, and the above procedure was repeated every 2 days. The mouse model is a model in which the contralateral (collateral) and ipsilateral (ipsilateral) cerebral hemispheres are injured by inducing hypoxia-ischemic brain injury in mice by inducing unilateral (unilateral) carotid artery ligation and Heat failure (Heat stop) in miceAnd (4) modeling. 7 days after the induction of hypoxic-ischemic brain injury, adult stem cells (5X 10) derived from the hyaluronic acid-rich node and catheter system labeled with CM-DiI derived from the hyaluronic acid-rich node and catheter system were injected by tail vein injection3cells). Animals in the control group were injected with the same volume of 1 × PBS in tail vein.
35 days after injection of adult stem cells derived from hyaluronic acid-rich node and catheter systems, mice were sacrificed and brains were isolated, and the isolated brains were treated in a 2% TTC (2, 3, 5-triphenyltetrazolium chloride); Sigma-Aldrich, St, Louis, Mo.) solution at a temperature of 37 ℃ for 30 minutes and photographed under a dissecting microscope. To confirm the infarct site, the contralateral and ipsilateral cerebral hemisphere and infarct brain site were analyzed by NIH Image J software (NIHImage, version 1.47). Infarct volume was calculated as the percentage of the area (white area) damaged relative to the total area of the left and right hemispheres. Infarct volume (%) × 100 [ lesion area (ipsilateral area + contralateral area)/(total ipsilateral area + contralateral area) ]. The sacrificed ischemic mice brains were finally fixed using immunohistochemical staining and seeded in paraffin. The brain was subjected to coronal sections (Coronalsections) to form sections (thickness: 10 μm), and mounted on a microscope slide. The above slide glass was subjected to an experiment using an immunofluorescence staining method.
As a result, when cerebral hypoxemia ischemia is induced by inducing unilateral carotid artery ligation and heat failure in mice, apoptosis occurs in the cortex, striatum (striatum) and hippocampus of the contralateral and ipsilateral hemispheres.
As described above, adult stem cells derived from hyaluronic acid-rich nodes and ductal systems, which are marked as CM-DiI, were transplanted into a brain disease-inducing mouse, and after 5 weeks, the brain of the mouse was removed and confirmed by staining the infarct site with TTC (fig. 8A, part a). In the control group of mice injected with PBS, the volume of the infarct site was 47.5%, and in the mice transplanted with adult stem cells derived from the hyaluronic acid-rich node and ductal system, the volume of the infarct site was 15.8%, and thus, it was found that the infarct volume was reduced (fig. 8A, part b).
Cells marked as CM-DiI (red) were found in a plurality of parts of the hippocampus DG by brain section, and it was found that these cells were NeuN positive cells as a marker of nerve cells (fig. 8B, parts a and B). Furthermore, it was found that CM-DiI positive cells were found in the hippocampal horn 1(
According to the above experimental results, when adult stem cells derived from a hyaluronic acid-rich node and conduit system are transplanted into a hypoxic-ischemic brain disease mouse, the volume of an infarct site showing brain damage is reduced, and the transplanted adult stem cells derived from a hyaluronic acid-rich node and conduit system are differentiated into nerve cells so as to move to DG, CA1, and CA3 of the hippocampus. That is, the stem cell function of adult stem cells derived from hyaluronic acid-rich nodes and ductal systems in vivo is demonstrated by showing an anatomically restored morphology of a hypoxic-ischemic brain disease.
EXAMPLE 7 characteristics of hematopoietic colonies (cell colonies) derived from hyaluronic acid-rich node and vessel systems
Clonogenic assay (Clonogenic assay)
2-1: bone marrow mononuclear cells (BM-MNCs) were obtained by washing the marrow cavity of the neck and thigh bones of the mice with PBS (pH7.4) by 25G needle injection. Obtaining a single cell suspension of the hyaluronic acid-rich node and the catheter system in such a manner that the obtained hyaluronic acid-rich node and the catheter system are cell-dissociated by the cell filter.
As a result, as shown in part a of fig. 9, colonies of hematopoietic progenitor cells derived from hyaluronic acid-rich nodes and ductal systems present in the inside of veins (intravein), the inside of lymphatic vessels (intralymphatic), and the surface of organs were detected, and 4 types of colonies such as CFU-GEMM (part a of fig. 9), CFU-GM (part b of fig. 9), BFU-E (part c of fig. 9), and MCPs (part d of fig. 9) were formed in vitro.
2-2: the clonogenic capacity in methylcellulose was used to examine the formation of hematopoietic progenitor cells present in the hyaluronic acid-rich node and ductal system and bone marrow. To confirm cell morphology, single colonies were suspended in PBS and fixed in 10% neutral buffered formalin (nbf (neutral buffered formalin), ph 7.4). Then, the cells of the above colonies were centrifuged, resuspended in PBS, and mounted on a slide glass. The cells were stained with Wright-Giemsa or toluidine blue and observed under an optical microscope (Leica DMD 108).
As a result of the staining of the cells derived from the hyaluronic acid-rich node and duct system with Wright-Giemsa (part B of FIG. 9), the CFU-GEMM colonies contained basophils, megakaryocytes, eosinophils, and red blood cells, the CFU-GM colonies contained neutrophils and macrophages, the BFU-E colonies contained erythroblasts/erythroblasts, and the MCPs colonies contained mast cells.
2-3 colonies formed from mast cell progenitors that inoculated the hyaluronic acid-rich node and conduit system were obtained and cultured in 10% FBS, 2mM L-glutamine (Gibco), 0.1mM NEAA (Gibco), 50uM 2-ME and 100U/mL RPMI1640 medium containing penicillin/streptomycin supplemented with 10ng/mL mice and rIL-3(STEMCELL Technologies) and 10ng/m mice and rSCF (STEMCELL Technologies). The cells were cultured for 2-4 weeks at 37 ℃ and 5% CO2 and subjected to flow cytometry analysis.
Antibodies used in flow cytometry analysis include: 1) sca-1(E13-161.7) which binds phycoerythrin fluorescence; 2) c-kit (2B8), IgE (R35-72), Gr-1(RB3-8C5), CD11B (M1/70), CD8(53-6.7), Flk-1(AVAS12) which bind fluorescein isothiocyanate fluorescence; 3) phycoerythrin-
As a result, hyaluronic acid-rich nodes and catheter systems can be obtained from the surface of the small intestine (or liver), inside the veins and inside the lymphatic vessels. The above-mentioned cells of the hyaluronic acid-rich node and conduit system are isolated and adapted to the step of analyzing the characteristics of hematopoietic progenitor cells, enabling the formation of CFU-GM, BFU-E and CFU-GEMM colonies (a, b, c of section A of FIG. 9) in a manner that the cells of the hyaluronic acid-rich node and conduit system are cultured in vitro. The hyaluronic acid-rich nodes and ductal cells derived from the surface of the organ form colonies of the above 3 hematopoietic progenitor cells, the hyaluronic acid-rich nodes and ductal cells in the vein form CFU-GEMM and BFU-E, and the hyaluronic acid-rich nodes and ductal cells in the lymph vessel form CFU-GM only. Moreover, most of the colonies grown under the CFU-GM condition were formed from mast cells (d in part A of FIG. 9).
Based on the results of Wright-Giemsa staining, it was confirmed that various kinds of hematopoietic progenitor cells were formed from individual colonies of the precursor. That is, basophils/megakaryocytes/eosinophils (part B of FIG. 9) of CFU-GEMM colonies (part a of FIG. 9) were confirmed; macrophages/neutrophils from CFU-GM colonies (B of fig. 9, panel a) (panel B of fig. 9); erythroblasts/red blood cells of the BFU-E colony (c of part a of fig. 9) (part B of fig. 9); and mast cells (part B of fig. 9) of MCPs colonies (part d of fig. 9).
CFU-GEMM, CFU-GM and BFU-E colonies derived from the hyaluronic acid-rich node and conduit system appeared much less frequently than BM (Table 2), and CFU-GM and BFU-E colonies located in the spleen appeared much more frequently than the hyaluronic acid-rich node and conduit system, but CFU-GEMM colonies were similar in the spleen and hyaluronic acid-rich node and conduit system (Table 2).
On the other hand, the number of MCPs derived from hyaluronic acid-rich node and duct systems was approximately 5 times higher relative to BM and 100 times higher relative to spleen according to unit cell level (aper-cell basis) (table 2 and fig. 9C). In this case, the rate-limiting table 2 compares the frequency of appearance of hematopoietic progenitor cells in bone marrow, spleen, and hyaluronic acid-rich nodes and ductal systems.
TABLE 2
To confirm whether colonies of MCPs could develop into mast cells, cells were isolated from MCPs and cultured in a medium containing IL-3 and rSCF for 14 days. As a result, cells derived from MCPs showed Lin-Sca-1+c-kit + FceRI + immunological properties (phenotype), and thus confirmed to be mast cells (FIG. 9, part D; both sides).
For the analysis of immature cells, hyaluronic acid-rich nodes and catheters were obtained from the surface of the organ (part a in FIG. 1A)After the system, flow cytometry was performed. Based on phenotype analysis, approximately 2% of the hyaluronic acid-rich nodal and ductal system cells had a linkage-, Sca-1+C-kit + and CD34-Has the immunological properties of hyaluronic acid, thereby having a small number of hematopoietic stem cells among hyaluronic acid-rich nodes and ductal system cells. Then, it was confirmed whether the hyaluronic acid-rich node and conduit system had adult multipotent stem cells that could continuously generate hematopoietic progenitor cells. That is, in order to confirm whether or not the hemangioblasts, which are precursors of immature blood cells, can be induced from the hyaluronic acid-rich node and duct system cells, the entire cell structure derived from the hyaluronic acid-rich node and duct system was co-cultured with OP9 cells.
As a result, CD45 was detected-Or Flk-1 (which can differentiate into all kinds of blood cells and endothelial cells).
As a result, when cells derived from hyaluronic acid-rich nodes and ductal systems are cultured under in vitro conditions, various kinds of hematopoietic colonies (cell colonies) can be formed. In particular, the induction of angioblast-like cells from hyaluronic acid-rich nodes and ductal system cells means that hematopoiesis occurs in hyaluronic acid-rich nodes and ductal system.
EXAMPLE 8 characteristics of totipotent Stem cells derived from hyaluronic acid-rich node and catheter systems
After 1 × 105 cells among hyaluronic acid-rich nodal and ductal system cells harvested from the surface of the small intestine (or liver) were split into central columns on OP9 cells, 20% FBS, antibiotics, cytokine recombinant mouse SCF (50ng/ml, peproTech), recombinant mouse Flt3L (5ng/ml, Prospec), and recombinant mouse IL-7(5ng/ml, Prospec) were CO-cultured in α -MEM (37 ℃, 5% CO 2). OP9 cells were suitable for the process of inducing B-system and bone marrow cell line, and 9-DL 1cells were suitable for the process of inducing T-cell line.
Flow cytometric analysis was performed by culturing the pebble-forming cells on OP9 with or without mSCF addition and staining the angioblast-like cells formed with CD45(PE-Cy5) and Flk-1(FITC) antibodies. Bone marrow and B-cell lines induced to cytokines were flow cytometrically analyzed using Gr-1/CD11B (FITC), CD45(PE-Cy5) and B220(PE-Cy5) antibodies, respectively, and T-cell lines were flow cytometrically analyzed using CD4(PECy5) and CD8(FITC) antibodies.
As a result, when nodes rich in hyaluronic acid and ductal system cells were co-cultured for 6 days on OP9 cells, pebble zone-forming cells were produced (fig. 10A). After 6 days, all the cells forming the pebble zone were harvested and subjected to flow cytometry, and as a result, about 2.3% of the cells were CD45-Flk-1+Approximately 12.4% of the cells were CD45+Flk-1-(parts a, B of FIG. 10B). That is, when hyaluronic acid-rich node and ductal system cells were co-cultured with OP9 hematopoietic progenitor cells, angioblast-like cells (CD 45) were produced-Flk-1+ cells).
Immunological properties (phenotype) of the pebble zone-forming cells formed during co-culture of cells derived from hyaluronic acid-rich node and catheter systems and OP9(NDS/OP9) were also analyzed. When NDS/OP9 co-culture was carried out for 10 days, most of the cells were CD45+Flk 1-cells (part a of FIG. 10C), if adding rSCF, increase CD45+Flk-cell number. When NDS/OP9 was cultured and analyzed on
As a result, when cells derived from nodes and ductal systems rich in hyaluronic acid are cultured in hematopoietic progenitor cells with addition of appropriate cytokines, the cells can be differentiated into hemangioblast-like cells derived from totipotent stem cells having differentiation ability and various kinds of mature hematopoietic cells.
EXAMPLE 9 differentiation characteristics of Hematopoietic Stem/progenitor cells (HSPC) derived from hyaluronic acid-rich node and catheter systems
To induce hematopoietic progenitor cells from hematopoietic stem cells derived from hyaluronic acid-rich node and conduit systems, CAFC of example 8 was subjected to split-type pericycle in CFU-GEMMM medium based on 1% methylcellulose, colonies were obtained every 10 days, and 1 × 10 colonies at the same concentration in CFU-GEMM medium5cells/mL were split into cylinders. Hematopoietic stem/progenitor cells (HSPCs) that differentiate into multiple hematopoietic cells were observed under a light microscope after Wright-Giemsa or toluidine blue staining.
As a result, when cells were formed in the pebble region in the CFU-GEMM methylcellulose culture medium by 10 days of polyculture, uniform and small cells (CFC) were formed (part A in FIG. 11), and existed in the cellsThe cells of the colony (diameter:. about.5 μm) had Lin-Sca-1+c-kit+CD34-CD135-Immunological properties and maintenance of the differentiation state of hematopoietic stem/progenitor cells (fig. 11, part B). As a result of splitting the center column every 10 days, the number of cells increased during the 50-day culture period (fig. 11, part C), and the appearance of Red Blood Cells (RBC), immature megakaryocytes, mast cells, and monocytes (fig. 11, part D) was confirmed as a result of staining the cells obtained by the 5-passage culture with Wright-Giemsa toluidine blue.
As a result, hemangioblast-like cells originating from totipotent stem cells derived from hyaluronic acid-rich node and ductal systems can also differentiate into hematopoietic stem/progenitor cells, thereby allowing the production of various hematopoietic cells.
Example 10 comparison of the regulatory mechanisms of MCPs derived from hyaluronic acid-rich nodes and ductal system-, bone marrow-and blood (spleen)
Since MCPs of hyaluronic acid-rich nodes and ductal systems occur at a high frequency when compared with bone marrow or spleen, in order to investigate whether the production of MCPs in hyaluronic acid-rich nodes and ductal systems is regulated in a manner different from that of bone marrow or spleen, a variety of types of mutant mice lacking specific genes clearly showing the difference in production of MCPs between bone marrow, spleen, and hyaluronic acid-rich nodes and ductal systems were used. In particular, IFN-. gamma.plays a role in regulating the development and function of mast cells in vitro and in vivo, and thus a mutant mouse lacking the above gene is used.
As a result, IFN-. gamma.-/-In the hyaluronic acid-rich node and conduit system of the mouse, the production of MCPs was drastically reduced, which means that IFN- γ was closely related to the production of the hyaluronic acid-rich node and conduit system MCPs (part a of fig. 12). In contrast, the production of MCPs in the spleen was independent of IFN- γ (fig. 12, part B, table 3). C-kit when compared with W-sash heterozygote (heterozygate) miceW-sh/W-shThe hyaluronic acid-rich node and ductal MCPs production was significantly reduced (FIG. 12, part C), and it is understood that the formation of MCPs was associated with bone marrow or spleen hypertrophySimilarly, c-kitlocus plays an important role in the development of hyaluronic acid-rich nodal and ductal system mast cells. In this case, the frequency of colonies was compared between the control group (B6) and IFN-. gamma.deficient mutant mice in Table 3 below.
TABLE 3
As a result, in the results carried out as a list, IFN-. gamma.-/-Mice have a dramatic decrease in the production of MCPs at hyaluronic acid-rich nodes and ductal systems, and therefore rely on the IFN- γ signaling system. That is, it is known that the regulation of hematopoiesis is different in the production of MCPs in the bone marrow, blood, and nodes and ductal system where hyaluronic acid is abundant.
Example 11 compatibility of hematopoietic cells derived from hyaluronic acid-rich node and ductal System-and bone marrow
In order to investigate the Hematopoietic cell engraftment potential of Hematopoietic stem cells derived from hyaluronic acid-rich node and catheter systems, Hematopoietic cell engraftment analysis (Hematopoietic engraftment assay) was performed in 2 ways.
That is, in the case of competitive recombinant hematopoietic stem cell analysis (competitive repopulation HSC assay), 8-week-old C57Bl/6F1 mice (CD 45.1) were severely irradiated with radiation (absorption dose of 1100cGy)+/CD45.2+) As recipient, B6(CD 45.2)+) Derived from hyaluronic acid-rich node and catheter systems of
In the non-competitive implantation analysis (analysis), a homologous gene (genetic) mouse (thy 1.1) irradiated with a severe radiation (1100cGy) was subjected to+) By intravenous injection (i)Iv) into a hyaluronic acid rich node and catheter system (thy 1.2)+)
In the above-mentioned transplantation assay of competitive or non-competitive hematopoietic cell recombination, host (host) mice or the survival (rate) of cells based on the injection thereof are analyzed 1 month or 3 months after transplantation.
As a result, hyaluronic acid-rich node and duct system cells (CD 45.2) were analyzed by competitive recombinant (repopulation) HSC+) F1(CD 45.1) not implanted and irradiated seriously+/CD45.2+) Mouse, but BM-MNC (CD 45.1)+) Implantation can be achieved. In non-competitive experimental analysis, syngeneic (syngeneic) mice heavily irradiated with radiation were transplanted with EGFP at various concentrations (doses)+Node and ductal system cells rich in hyaluronic acid up to
On the other hand, it was investigated whether bone marrow cells (BM-MNC) were implanted in a mobile manner into hyaluronic acid-rich node and catheter systems. To examine the migration pattern of bone marrow cells into hyaluronic acid-rich nodes and ductal systems, 2 × 10 cells were irradiated to heavily irradiated (1100cGy) B6 mice6cells/mouse concentration intravenous EGFP+Homologous BM-MNC. Observation under a fluorescent inverted microscope (Fluoronceinverted microsco PE; Observer Z1, Zeiss) to EGFP+BM-Hyaluronic acid-rich nodes of MNC cells and movement of the catheter system.
As a result, EGFP, which is a bone marrow cell having green fluorescence, was injected into a syngeneic B6 mouse irradiated with severe radiation+BM-MNC(2×106cells/mouse), EGFP could be detected at hyaluronic acid-rich nodes and vessels (b of part a of fig. 13) and nodes (c of part a of fig. 13) of the
As a result, in terms of enabling the migration and implantation of hematopoietic cells between bone marrow and hyaluronic acid-rich nodes and ductal systems, which are sites of hematopoiesis with fundamental differences in the anatomy, this means that the hyaluronic acid-rich nodes and ductal systems are independent systems and play a role in perfecting the bone marrow and blood systems during hematopoiesis.
While certain portions of the present disclosure have been described in detail, it is needless to say that those skilled in the art will recognize that these specific techniques are merely preferred embodiments and that the scope of the present disclosure is not limited to these techniques. Accordingly, the true scope of the present invention should be defined only by the following claims and their equivalents.
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