KR20130055313A - A method for in-vitro expansion of erythroid cells using high-density culture - Google Patents

Abstract

PURPOSE: In vitro extension method of erythron using high density culture is provided to reveal adhesion-related gene such as DCL-1 by cultivating in high-density to directly and physically connect erythrons, and enhance production rate of erythron by increasing active signal exchange between cells. CONSTITUTION: In vitro extension method of erythron cultivates erythrons in high density to directly and physically connect cells. High-density cultivation is composed of 100~150% of confluence. Erythrons which directly and physically connect cells reveal DLC-1. Composition for marking erythrocyte series cell differentiation includes DLC-1. The DLC-1 is composed of amino acid sequence represented as sequence number 1. The DLC-1 ejects adhesion-related signals by combining with VLA-4. The composition additionally contains ICAM-4. The increasing method of erythron maturation is through revelation of DLC-1, VLA-4 and ICAM-4. Maturation of erythron comprises the step of enucleating. In vitro extension method of erythron uses to increase maturation of erythron through revelation or activity stimulation of DLC-1, VLA-4, and ICAM-4.

Description

A method for in-vitro expansion of erythroid cells using high-density culture

The present invention relates to an in vitro expansion method of erythroid cells. More specifically, the present invention relates to a method for obtaining concentrated red blood cells, which is characterized by culturing erythroid cells in a high density such that the cells are in direct physical contact with each other. In particular, the expression of the adhesion-related genes DLC-1, ICAM-4, and VLA-4 increases the intercellular signal exchange activity, thereby increasing the production rate of red blood cells.

The use of red blood cells for transfusion has increased due to the aging of the population and the increase of medical demand due to the development of science and technology. In addition, transfusion infection, which can occur due to blood transfusion, causes serious problems in the use of red blood cells. Such transfusion infections include viral and bacterial contamination such as human immunodeficiency virus (HIV), hepatitis B virus. Emerging pathogens, such as blood-borne tropical diseases and variant Creutzfeldt-Jacob disease, are also problematic because of increased travel abroad.

These factors increased the need for artificially produced red blood cells (Greenwalt, TJ; Zehner Sostok, C .; Dumaswala, UJ Studies in red blood cell preservation. 1. Effect of the other formed elements.Vox Sang. 58: 85-89; 1990; Olsson, ML; Clausen, H. Modifying the red cell surface: towards an ABO-universal blood supply.Br. J. Haematol. 140: 3-12; 2008).

Currently, there is a shortage of blood required for transfusions worldwide, which is disrupting the surgery and treatment of patients. As a result, many researchers have recently tried to mass produce erythrocytes using stem cells, but most of them have only grown a small amount of cells in laboratory-level small culture wells.

As such, according to a previously published report (Mountford et al. Prospects for the manufacture of red cells for transfusion.Br J Haematol. 2010, 149, 22-34), increasing cell concentration is an important problem to be solved for mass production. did. In addition, arithmetic calculations required two tennis courts to produce a pack of red blood cells (Timmins et al. Blood cell manufacture: current methods and future challenges.Trends in Biotechnology 2009, 27, 415- 422), in fact, mass production is known to be difficult.

To date, most of the research has focused on the amplification of early stem and progenitor cells. However, little is known about the reasons and solutions for the late stage cell maturation, low denucleation rate and low cell viability, which are the most important stages.

Accordingly, the present inventors overcome the problems of the prior art described above, and in order to produce concentrated red blood cells that may be clinically useful even in a small space, the DLC-1 and ICAM by culturing at high density so as to be in direct physical contact between erythroid cells. It was confirmed that expression of adhesion-related genes such as -4 and the increase in intercellular signal exchange activity thereby increased the production rate of erythroid cells, thereby completing the present invention.

In order to solve the above problems, the present invention provides an in vitro expansion method of erythroid cells, in which the erythroid cells are cultured at high density so as to be in direct physical contact between the cells.

At this time, the high-density culture is characterized in that the confluence (confluence) of 100 ~ 150%, for example, 100% confluence is a density of about 5.0x106 cells / 2ml, 150% confluence This test includes seeding incubated at a density of approximately 7.5x106 cells / 3ml. Preferably at about 100% confluence.

The erythroid cells include cells that are in the process of maturation from erythroid progenitor cells, particularly preferably the erythroid cells that have almost completed maturation, more preferably after the final maturation step. It includes. In this final maturation stage, the enucleation process of red blood cells takes place.

In the present invention, the erythroid cells which are in direct physical contact between the cells are characterized by expressing an adhesion-related gene of DLC-1, ICAM-4 and / or VLA-4. Such adhesion-related gene expression increases the intercellular signal exchange activity, thereby increasing the production rate of erythroid cells.

Therefore, as another specific method of the present invention, instead of culturing erythroid cells at a high density, the erythroid cell culture solution may be cultured by adding DLC-1, ICAM-4, and VLA-4 proteins.

In particular, for the first time the relationship between the DLC-1 gene and the differentiation and maturation process of erythroid cells was identified. That is, it is also meaningful that the first DLC-1 has been known to play a role in the process of proliferation and differentiation of normal cells, compared to the conventional known only for cancer cells.

The amount of DLC-1 expression increases until denuclearization as the erythroid cells mature. Therefore, the present invention provides a composition for red blood cell differentiation markers including DLC-1 as another embodiment.

DLC-1 may be composed of the amino acid sequence represented by SEQ ID NO: 1, but may be appropriately modified as long as it maintains its function.

At this time, the DLC-1 emits adhesion-related signals in association with VLA-4 and ICAM-4, and the erythroid cell production rate is increased by increasing signal exchange activity between erythroid cells.

In a similar aspect, the present invention provides, as another embodiment, a method of increasing the maturation of erythroid cells, particularly terminal maturation, by promoting the expression or activity of DLC-1. In addition, such methods of promoting the expression or activity of DLC-1 may ultimately expand red blood cells in vitro enough to be clinically useful.

In this case, the final maturation of the red blood cells includes an enucleation step, and this denuclearization process is activated by promoting expression or activity of DLC-1, VLA-4 and ICAM-4. At this time, the ICAM-4 gene may be expressed as a secreted or membrane-bound protein.

That is, the present invention relates to a method of increasing the expression of DLC-1, VLA-4, and ICAM-4 between polychromatophilic or orthochromatic erythroblasts, and thereby increasing the final maturation of red blood cells. .

In addition, to implement expression or activity promotion of DLC-1, VLA-4 and ICAM-4, a method of introducing DLC-1, VLA-4 and ICAM-4 into erythroid cells by various known methods may be used. Can be.

The methods and compositions according to the present invention have the advantage of being applicable to "mass production (expansion, enrichment, amplification)" of human erythrocytes through bioreactor systems.

The present invention expresses adhesion-related genes, such as DCL-1, by culturing at high density so as to be in direct physical contact between erythroid cells, and accordingly, the increase in intercellular signal exchange activity results in the production rate of erythroid cells. It can increase red blood cells enough to be clinically useful in confined spaces.

Therefore, the present invention requires two tennis courts to make a pack of red blood cells, which completely reverses the conventional report known to be difficult to mass-produce, making it possible to produce a pack of concentrated red blood cells in an area of 260.8 / 1.

1 is a schematic diagram of embodiments of the present invention for culturing red blood cells under various density conditions.
Figure 2 shows the results of staining, denucleation rate, and apoptosis when cultured at 50%, 100%, and 150% confluence at the final maturation stage of red blood cells (arrows represent reticulocytes). ).
FIG. 3 shows the results of flow cytometry and RT-PCR analysis when cultured at 50%, 100%, and 150% confluence at the final maturation stage of red blood cells.
4 shows the results of biochemical analysis of the medium used.
5 is a result of expression analysis of adhesion-related genes DCL-1 and VLA-4.
6 shows the expression of DLC-1 in K562 cells.
FIG. 7 shows the result that DLC-1 and ICAM-4 bind to each other in K562 cells.
8 shows the results of expression analysis of secretory form ICAM-4 (secretory ICAM-4) present in cells.
9 is an analysis result in which the expression of DLC-1 increases with density.
10 is a diagram comparing the case of the conventional method and the method of the present invention with respect to the cell culture area required to form one pack of concentrated red blood cells.

The terms used in the present invention are defined as follows.

"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role in protein coding or transcription or in the regulation of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

"Primer" refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA-targeted polynucleotide and serves as a starting point for the stepwise synthesis of a polynucleotide from a mononucleotide by the action of, for example, a nucleotidyltransferase that occurs in the polymerase chain reaction .

'Progenitor cells' are undifferentiated cells with self-replicating ability and differentiation potency, but are ultimately differentiated cells in which the type of cells to differentiate is already determined. Progenitor cells have a predetermined differentiation pathway, but generally do not express markers of mature fully differentiated cells or function as mature fully differentiated cells. Thus, progenitor cells differentiate into related cell types but cannot form a wide variety of cell types under normal conditions. In the present invention, red blood cell progenitor cells are used.

'Differentiation' refers to a phenomenon in which structures or functions are specialized during the division and proliferation of cells, that is, the shape or function of a living cell or tissue is changed to perform a given task. In general, a relatively simple system is separated into two or more qualitatively different systems. For example, there may be qualitative or quantitative differences between parts of a biological system that were initially homogeneous, such as head or trunk distinction between eggs that were initially homogeneous in origin, or distinctions of cells such as muscle cells or neurons The state in which there is a difference or as a result is divided into qualitative or inferior parts or partial systems is called differentiation.

The term 'expansion or concentration' refers to an increase in the number of cells in the body of a multicellular organism, usually caused by the division of cells into homogeneous ones. When the number of cells proliferates (amplifies) and reaches a certain point, the trait is usually changed (differentiated) and controlled at the same time. When cells increase in the body and when the cytoplasm becomes new within the cell, they are often distinguished by growth. However, in view of the increase in the number of cells biologically, the time when differentiation does not occur in the generation of multicellular organisms is justified as proliferation (expansion, amplification). In the present invention, the three terms are used interchangeably.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present inventors have endeavored to develop a method for mass production (expansion, enrichment) of red blood cells in vitro to the extent that it can be industrially used, resulting in high density of red blood cells so that the cells are in direct physical contact with each other when expanded. It was confirmed that the production of a pack of concentrated red blood cells in a small size in vitro scale by incubating at a density of about 10 times or more than conventionally known cases.

This overturns the report that an area of about two tennis courts is required to make a pack of conventional red blood cells, and can produce one unit of concentrated red blood cells (U) even at 260.8-1 incubator cross-section or 2 m3 incubator volume. Demonstrating the presence of the product, it is suggested that mass production through factory manufacturing is possible.

In one aspect, the present invention relates to an in vitro expansion method of denuclearized erythrocytes, which is characterized by culturing red blood cells in high density such that the cells are in direct physical contact with each other.

Herein, erythroid cells include all cells that are in the process of maturation from erythroid progenitor cells, and in particular, cells that have almost completed maturation from erythroid progenitor cells. It is preferred, and most preferably, those of the basic chromophilic erythroblast and the orthochromatic erythroblast stage, which are terminal maturation stages. In this final maturation stage, the enucleation process of red blood cells takes place.

Erythrocyte progenitor cells can be obtained from a variety of sources, such as peripheral blood, umbilical cord blood, or bone marrow. CD34 + cells as erythroid progenitors can be isolated according to various cell isolation methods known in the art, such as immunomagnetic-bead separation methods using CD34 + antibodies, and in accordance with a preferred embodiment of the invention CD34 + cells are derived from umbilical cord blood. May be a cell.

Erythrocyte progenitor cells differentiate into mature erythrocytes through erythropoiesis, which consists of the following steps: (a) differentiating from hematopoietic stem cells to proerythroblasts; (b) differentiating from progenitor cells to basophilic erythroblasts; (c) differentiating into a polychromatophilic erythroblast in an alkaline basic cell; (d) differentiating from multibasic erythrocytes to orthochromatic erythroblasts; (e) differentiating into polychromatic erythrocytes in infectious erythrocytes; And (f) differentiating from erythrocytes to erythrocytes.

Therefore, the present invention preferably includes cells in the erythrocyte stage (d, e, f stage) of the maturation stage.

That is, the present invention can be produced in large quantities in an amount of erythrocyte pack units that are clinically available by expanding and / or concentrating mature erythrocyte cells to an industrially useful level. It is about how.

The present invention is characterized in that the erythrocyte cells are cultured at high density to the extent that the intercellular interaction can occur by physically contacting each other directly. Alternatively, in order to cause such a situation, the proteins may be put in a culture medium to induce protein-to-protein binding to reproduce a high density culture environment.

The high density culture includes, but is not limited to, exhibiting 100-150% confluence, preferably including 100% confluence. This corresponds to a density size that is about 10 times the cell density in conventional red blood cell cultures.

For example, 100% confluence includes seeding at a density of about 5.0x10 ⁶ cells / 2ml and 150% confluence at a density of about 7.5x10 ⁶ cells / 3ml.

As described above, in the present invention, by culturing the red blood cells at a high density, physical direct contact is made between the red blood cells, thereby causing interaction between the red blood cells.

In particular, the red blood cells that are in direct physical contact between the cells are characterized by expressing adhesion-related genes such as DCL-1, ICAM-4, and VLA-4.

Increasing red blood cell density in accordance with the method of the present invention results in increased DCL-1, ICAM-4 and VLA-4 expression of adhesion-related genes, thereby increasing intercellular signal exchange activity. In addition, the increase in signal exchange activity between cells enables to increase the production rate of erythroid cells.

Therefore, as another specific method of the present invention using this mechanism, in addition to the method of increasing the production rate of erythroid cells by expressing DLC-1, ICAM-4 and VLA-4 by culturing the erythroid cells at high density, DLC-1, ICAM-4, and VLA-4 proteins can be added to the erythroid cell culture to create an environment similar to that of the high density culture. Such a protein addition method can be easily used for erythroid cell culture using a bioreactor or the like.

VLA-4 is an essential gene for erythrocyte differentiation and expansion in vivo and in vitro, and is known to be highly expressed during red blood cell production (Dormer et al., Et al.), But it has been reported that DLC-1 is expressed in erythroid cells. There is no bar. The present inventors confirmed for the first time that DLC-1 is also expressed in K562 cells, which are cell lines capable of inducing differentiation into erythrocytes, and also expressed in erythroid cells. In particular, it was confirmed that DLC-1 is involved in the differentiation of erythroid cells, in particular the maturation process. In addition, expression of secretory form ICAM-4 in K562 and human erythroid cells was also confirmed for the first time. This secreted ICAM-4 expression affects the activity of intercellular signal exchange.

In addition, the expression of DLC-1 increases further during the denuclearization process, which further activates this enucleation and final maturation stage by binding to VLA-4 to release adhesion-related signals. Let's do it. Accordingly, the DLC-1 may function as a differentiation-maturation (maturation) marker of erythrocytes by increasing its expression as the differentiation-specific, ie maturation stage of erythrocytes progresses.

Therefore, the present invention relates to a composition for red blood cell differentiation markers containing DLC-1 from another aspect. For example, the composition can be used to determine whether the red blood cells are in the final stage of maturation.

In addition, by the above-described mechanism, the marker composition including the DLC-1 of the present invention may further include one or more genes of VLA-4 and ICAM-4. Preferably, it is preferable to further include both genes.

In a similar aspect, the present invention relates to a method of increasing the final maturation of red blood cells by promoting the expression or activity of the DLC-1. As described above, the terminal maturation of the red blood cells includes an enucleation step.

In addition, the expression or activity promotion of the DLC-1 may be accompanied with the promotion of the expression or activity of the VLA-4 and / or ICAM-4 gene.

At this time, the expression or activity promotion of the DLC-1, VLA-4, and ICAM-4 may be made by a method of introducing DLC-1 into red blood cells. "Introducing (transforming) DLC-1, VLA-4, and ICAM-4" refers to introducing a nucleic acid encoding the genes into erythroid cells.

DNA-calcium precipitation, liposomes, polyamines, electroporation, retroviruses, adenoviruses, etc. Intracellular transduction technology methods known in the art can be used. In one embodiment, the nucleic acid encoding DLC-1 is inserted into a separate expression vector or a single expression vector, and then introduced into red blood cells.

Each nucleic acid encoding DLC-1, VLA-4, and ICAM-4 may be used without limitation as long as it has a nucleotide sequence encoding DLC-1, VLA-4, and ICAM-4 known in the art.

In addition, the nucleic acid may have a nucleotide sequence encoding each functional equivalent of DLC-1, VLA-4, and ICAM-4. The functional equivalent means at least 70%, preferably 80%, more preferably 90% or more of each amino acid sequence represented by SEQ ID NOs: 1, 2 and 3 as a result of the addition, substitution or deletion of amino acids. It refers to a polypeptide having substantially the same biological activity as the DLC-1 of the present invention as having the same sex. 'Equivalent physiological activity' refers to an activity that further activates the denucleation and final maturation stages of red blood cells by releasing adhesion-related signals in the expansion of red blood cells.

Nucleic acids encoding the DLC-1, VLA-4, and / or ICAM-4 may also be prepared by genetic recombination methods known in the art (Sambrook, Fritsch and Maniatis, 'Molecular Cloning, A laboratory Manual, Cold Spring Harbor laboratory press, 1989; Short Protocols in Molecular Biology, John Wiley and Sons, 1992). For example, there are techniques for producing PCR amplification, chemical synthesis or cDNA sequences to amplify nucleic acids from the genome. The present invention includes methods for use in culture of recombinant DLC-1, VLA-4, and / or ICAM-4 proteins synthesized according to this technique.

Erythroid cells according to the present invention can be proliferated and expanded by any suitable method known in the art or by modifying the same.

In one embodiment, mineral (stroma-free), serum-free and / or plasma-free medium can be used. In this case, a cell culture minimum medium (CCMM) containing only a carbon source, a nitrogen source, and a trace element component may be used, for example, DMEM (Dulbecco's Modified Eagle's Medium), EDM (Endothelial differentiation medium), or MEM. (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and Iscove's Modified Dulbecco's Medium It can be used by using a commercially prepared medium of or artificially synthesized.

In addition, the medium of the present invention may include vitamin C to maintain a stable state from oxidative stress in vitro, and if necessary, antibiotics such as penicillin, streptomycin, or gentamicin may be added. Stem cell factor, interleukin-1, interleukin-3, interleukin-4, interleukin-5, interleukin-11, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, granulocyte colony stimulating factor or eryth It may further comprise at least one of the ropoietin.

In addition, in the method of the present invention, culturing the erythroid cells may be performed using a conventional culture vessel, that is, a general culture vessel (for example, a CO ₂ incubator) or a bioreactor.

The present invention increases the intercellular signal exchange activity by culturing red blood cells in high density so that the cells are in direct physical contact with each other, thereby producing a higher concentration of red blood cells. Two tennis courts are used to make a pack of red blood cells used in clinical practice. Overturning the previous announcement that the area is required, in fact, difficult to mass-produce, and proved that it can be produced in a very small bioreactor with a floor area of 1m2 and a height of 2m, suggesting that the red blood cell production can be factoryd (FIG. 10).

In other words, it is possible to produce a pack of concentrated red blood cells in an area of 260.8 / 1 of the previous report.

On the other hand, the concentrated red blood cells obtained by the method of the present invention may have various therapeutic applications.

In certain embodiments, the applied red blood cells can be used for transfusion. The ability to produce large amounts of cells for transfusion can alleviate the chronic shortage of blood experienced in blood banks and hospitals nationwide. The method of the present invention allows the production of universal cells for transfusion.

Certain aspects of the present invention are directed to expanding human erythrocyte cells to reach commercial amounts.

Human red blood cells are produced on a large scale, stored when needed, and supplied to hospitals, clinicians or other healthcare facilities. Once a patient exhibits indications such as ischemia or vascular injury, or needs hematopoietic reconstitution, human erythrocytes can be ordered and supplied in a timely manner. Accordingly, the present invention provides methods for producing and expanding human erythrocytes to reach a commercial scale, as well as cell preparations comprising human erythrocytes derived from the method, as well as providing human erythrocytes to hospitals and clinicians (ie, Produce, optionally store and sell).

In addition, another particular aspect of the present invention relates to a method for producing, storing, and distributing erythrocyte cells expanded by the methods described herein. After producing and expanding human erythrocytes in vitro, human erythrocytes can be harvested, purified and optionally stored prior to treatment of the patient. Thus, in a particular embodiment, the present invention provides a method for supplying red blood cells to hospitals, health care centers, and clinicians, whereby red blood cells produced by the methods described herein are stored, and hospital, health It is administered to patients in need of erythrocyte cell therapy, ordered at a management center, or as required by a clinician.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  1: Production of red blood cells and confirmation of various characteristics according to the present invention

1. Cell culture and yield enumeration )

CD34 + was isolated from cord blood of healthy donors using an immunomagnetic microbead selection method and the EasySep CD34 Separation Kit (StemCell Technologies, Vancouver, Canada).

CD34 + cells were stored frozen until available, and upon freezing, these cells were incubated at a density of 3 × 10 ⁵ cells / ml in the absence of substrate and serum for 17-18 days.

Several cytokines were added for differentiation of CD34 + cells. From day 0 to day 7, cells were treated with 10 μm hydrocortisone (Sigma), 100 ng / ml stem cell factor (Minneapolis, Minneapolis, SCF; R & D Systems), 10 ng / ml Interleukin (IL) -3 (R & D Systems) And in medium supplemented with 6 IU / ml erythropoietin (USA, California, La Jolla, EPO; Calbiochem).

Expanded erythrocytes from day 7 to day 13 were cultured at 50 ng / ml SCF, 10 ng / ml IL-3, and 3 IU / ml EPO. From day 13 to day 17, 50 ng / ml SCF and 2 IU / ml EPO were added. Culture medium was changed every two days. Cells were cultured in a CO 2 incubator (Sanyo, Japan) at 37 ° C. Trypan blue staining was used to use the number of cells and only remaining live cells were included in the analysis of cell expansion.

2. Cell Culture for Density Effect

From day 13 of incubation, cells were maintained at low cell density. At this stage, the cells expanded too rapidly to maintain a constant density, but the cells remained vigorous and their viability remained high regardless of cell density.

On day 17 of culture, orthochromatic erythroblasts were placed on a well culture plate in 50% (2.5x10 ⁶ cells / ml), 100% (5.0x10 ⁶ cells / 2 ml), and 150% confluence. ) (7.5x10 ⁶ cells / 3ml) (Figures 1A-C).

During incubation, the confluence of the cells gathered in the center of the plate was checked daily. Medium was supplemented daily and confluence adjusted to maintain the same culture. Cell confluence was observed under a phase contrast microscope and cell integrity was confirmed using Wright-giemsa staining.

3. Imaging of Red Blood Cells

Cell morphology was analyzed by cytospin method using cell centrifuge (Cellspin, Hanil Science Industrial, Seoul, Korea) followed by Wright-Giemsa staining (Sigma-Aldrich).

To measure the rate of nuclear clearance, 400-450 cells were counted to determine the total number at each density state and time point. Pictures of the stained cells were taken with a digital camera (Eclipse TE2000-U, Nikon, Japan) and used the Nikon ACT-1 program.

4. Determination of Cell Viability

Cell viability was measured by flow cytometry using trypan blue staining and Annexin V analysis. Cells were stained for 15 minutes at room temperature with Annexin V-PE and propidium iodide (New Jersey, Franklin Lakes, BD PharMingen). The stained cells were washed twice with wash buffer and analyzed using flow cytometry.

5. Of red blood cell detection Flow cytometry  analysis

To measure phenotypic changes during red blood cell growth, the following anti-human antibodies were attached to the cells on days 13, 17 and 21: (i) Glycoporin A (GPA) -FITC (BD PharMingen, Franklin Lakes, NJ). ) (ii) CD71-FITC (eBioscience, San Diego, CA). Immunoglobulin G1 (IgG1) -FITC and IgG1-PE (Beckman Coulter, Miami, FL) were used for homogeneous control staining. For immunophenotype, cells were washed in phosphate buffered saline (PBS) and incubated with each monoclonal antibody for 15 min at 4 ° C. After washing, the cells were suspended in phosphate buffered saline (PBS) and analyzed using a flow cytometer.

Intracellular expression of hemoglobin (Hb) was analyzed using FITC conjugated anti-fetal hemoglobin (HbF) and PerCP conjugated anti-dollar hemoglobin (Hbβ, BD PharMingen). Briefly, cells are resuspended in cold 0.05% glutaraldehyde for 10 minutes at room temperature, washed in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin, and at room temperature. It was suspended for 5 minutes in 0.1% Triton X-100. After washing, antibody was added to the cells for 15 minutes at room temperature and washed. Stained cells were analyzed using flow cytometer BD FACSCalibur ™ (BD Biosciences, San Jose, Calif.) And provided software.

6. Biochemical Analysis

On day 19 and day 21, culture medium was collected and glucose concentration was determined using an automated analyzer. pH and potassium ions (K +) were analyzed using a RapidSystems blood gas analyzer (Siemens, USA). Lactate levels were measured using an enzyme method with Cobas Integra 800 (Roce Diagnostics; Rotkreuz, Switzerland) within 2 days of collection.

7. RT - PCR

Total RNA was isolated using Trizol (Invitrogen) and reverse transcribed the total RNA using SuperScript III reverse transcription enzyme (Invitrogen). RT-PCR was performed on triplicates (LightCycler 480 II; Roche Diagnostics) using SYBR Premix ExTaq (Takara-Bio, Shiga, Japan). Relative quantitation was achieved by normalization to endogenous β-actin.

The primer set used was designed using Primer3 software and subjected to BLAST analysis to verify the properties of the primers.

The sequence of the primer set used is as follows (forward / reverse):

E-cadherin: 5'-CGGGAATGCAGTTGAGGATC-3 '(SEQ ID NO: 4)

            5'-AGGATGGTGTAAGCGATGGC-3 '(SEQ ID NO: 5);

VLA-4: 5'-AGGATGGTGTAAGCGATGGC-3 '(SEQ ID NO: 6)

            5'-TGCTGAAGAATTGGCTGAAGTGGTGG-3 '(SEQ ID NO: 7);

DLC-1: 5'-AGTGCGTGCAACAAGCGGGT-3 '(SEQ ID NO: 8)

            5'-TCCGGGTAGCTCTCGCGGTT-3 '(SEQ ID NO: 9);

ICAM-4: 5'-CCGGACTAAGCGGGCGCAAA-3 '(SEQ ID NO: 10)

5'-AGCCACGAACTCCGGGCTCA-3 '(SEQ ID NO: 11).

ELISA ( Enzyme - Linked ImmunoSpecific Assay )

The first antibody (ICAM-4, santa cruze, USA) was added to a 96 well plate coated with immuno coating and left at 4 ° C. for 20 hours. After washing three times using wash buffer (0.05% Tween-20 containing PBS), the PBS containing 10% FBS was left to stand. After 2 hours, wash again three times using wash buffer, add the supernatant obtained by culturing the same number of cells, and leave it at 4 ℃ for 18 hours, wash 5 times using wash buffer, and then remove the second antibody (HRP- conjugated secondary Ab, Jackson ImmunoResearch Antibody, USA) was added and allowed to stand at room temperature. After 1 hour, washed 7 times using wash buffer, and then added a detection solution (Bio legend, USA) and 30 minutes after reading through the ELISA reader (Applied Biosystems, USA).

9. Western  Blot Western blot )

The total protein was separated through lysis solution and then quantitated to 50 ㎍. After electrophoresis on SDS-PAGE, it was transferred to Hybond-ELC nitrocellulose membrane. Membranes from which proteins were transferred were placed in 5% skin milk (in TBST: Tris buffer saline with Tween; 25 mM Tris, 140 mM NaCl, 0.05% Tween-20, pH8.0) at room temperature for 1 hour. After 1 hour, the primary antibody (DLC-1, santa cruze, USA) was put at 4 ℃ and stored for more than 12 hours. After washing three times with TBST for 10 minutes, HRP-conjugated secondary antibody (anti-rabbit, Jackson ImmunoResearch Antibody, USA) was added and left at room temperature for 1 hour, and then washed again with TBST three times for 10 minutes. Thereafter, detection reagent (ECL solution) was added and then exposed to X-ray film to confirm the expression of each protein.

10. Statistical analysis

Statistical analysis was performed using Prism software (Prism, Version 5.0, GraphPad, San Diego, CA). Results are expressed as means ± standard error of mean (sem). Statistical significance was set at p values of 0.05 or less. Paired t-tests were used for comparison of sample groups.

Example  2: expanded red blood cell test results according to the present invention

(1) Effects of allogeneic red blood cell interactions

In vitro red blood cell production was done using CD34 + cells under serum and substrate free conditions. CD34 + cells were cultured in differentiation medium of erythrocytes including EPO, IL-3, and SCF. From day 17 of cultivation, when most of the cells were polychromatic and orthochromatic, the cells were cultured at various densities (Fig. 1). As in the previous study, the amount of media per well remained constant, while the cell numbers per range varied between wells.

Cell morphology was assessed for the effect of culture density during final maturation using Wright-Giemsa staining. As a result, cell staining confirmed poor morphology for cultures at low densities (50% confluence; FIG. 2 A), which is consistent with the results of viability tests using trypan blue (FIG. 2 B). ). In contrast, cell viability was significantly higher in cultures of 100% and 150% confluence.

As erythrocytes exited the polychromatic stage, cell proliferation declined and cell death increased. High levels in cultures of 100% and 150% confluence (25.5% and 20.6%, respectively) as compared to the erythrocyte cells of 50% confluence (18.4%) (p <0.05) (FIG. 2C). Removal (denuclearization) of was observed.

Compared to the number of RBCs produced, 50% of confluence cultures are 100% and 150% confluence when expressed as increased fold-expansion at the rate of nucleation (denucleation) at the corresponding cell density. It was significantly different from the culture of Earns (paired t-tests, p <0.05) (Fig. 2D). At 100% confluence, 8.6x105 reticulocytes were obtained with denucleation rates up to 40%.

And at the final stage of erythrocyte maturation, cell suicide rates increased dramatically. In apoptosis assays with caspase-3 activation and Annexin V, in cells cultured at 100% and 150% confluence compared to cells at 50% confluence Apoptosis was found to be significantly lower (FIGS. 2E and F).

Increasing cell viability revealed the highest expansion rate at 100% confluence. This means that at increased cell density, ie at high density, cell-to-cell contact can prevent cell death.

(2) form evaluation and Flow cell  Analysis of Cell Maturation Through Analysis

The maturation from basophilic erythroblast to polychromatophiic erythroblast and / or orthochromatic erythroblast was confirmed using Wright-Giemsa staining (data not shown).

First, on the 17th day of the density experiment, flow cytometry was used to confirm the expression of CD71 (> 53.7%) and GPA (> 87.3%) corresponding to the polyinflammatory and saline phases, respectively. (FIG. 3).

Then, on day 21, the last day of the density experiment, phenotypic changes were measured using flow cytometry. As a result, according to the culture density, CD71 and GPA expression did not appear significantly different, and the maturation markers GATA-1 and Hbβ expression also did not appear significantly different.

That is, erythrocyte maturation occurred at similar levels in all three culture densities (FIG. 3).

(3) Analysis of cultured medium

In order to minimize the potential effect of metabolic regulation, the culture density of the experiment was kept constant. To assess the culture conditions, we analyzed glucose consumption, pH, and dissolved ion concentration.

The average rate of glucose consumption during the entire incubation period did not differ significantly between the days of low density and high density culture. The pH and potassium concentrations of the medium were similar at all density conditions (FIG. 4).

(4) contact-related changes in gene expression

To investigate the role of adhesion molecules in final erythrocyte maturation, microarray analysis was used to identify differently expressed genes upon differentiation. The candidate genes included ICAM-4, VLA-4, DLC-1 and E-cadherin. The expression of each of these genes in cells cultured at different densities was evaluated.

As shown in FIG. 5, the adhesion-related signaling genes VLA-4 and DLC-1 genes were both overexpressed at medium and high levels of density (100% and 150% confluence) (VLA-4, 4.4-fold and 2.8x; DLC-1, 5.1x and 3.4x). At this time, in the case of 100% confluence, the expression levels of the two genes were slightly higher.

Through this, it can be seen that physical contact between erythrocyte cells induces autonomous adhesion-related signals by expression of adhesion-related signal genes. In other words, red blood cells cultured at high density secrete soluble signaling factors that affect the final maturation of cells.

(5) cell lines K562 In DLC -One, ICAM -4 analysis

We first identified the expression of DLC-1 and secretory form ICAM-4 in K562 cells, a cell line capable of inducing erythroid differentiation. This suggests that DLC-1 and ICAM-4 can be expressed in erythroid cells and further play a role in intercellular signal exchange. In particular, it is a new report that DLC-1, which has been reported to be found only in cancer cells, is involved in cell contact, especially between erythroid cells deeply related to blood.

6 shows that DLC-1 is expressed in K562 cells.

Furthermore, the present inventors confirmed the binding of the two genes to confirm the relationship between the two genes based on this point.

This is illustrated in FIG. 7. In other words, it was confirmed through the binding of the protein produced by the gene of ICAM-4 and DLC-1 in K562 cells. These results show that the association of two genes, which are expected to play an important role in intercellular signal exchange, is related to the contact of two genes in differentiation.

To date, it has not been reported to exist in the secretory form of ICAM-4 in mature erythrocytes. In the present invention, the presence and secretion of secretory form ICAM-4 in K562 and mature erythrocytes were confirmed.

As a result, as shown in FIG. 8, when converted to 100% of untreated control (cell-free control media), about 10% of secretory form ICAM-4 was confirmed in mature erythrocytes, and 40 in K562 cells. As much as% secretory form ICAM-4 was expressed.

In addition to the regulation of erythropoiesis (erythroid cell differentiation) process through the intercellular membrane binding through direct cell binding, soluble proteins such as secretory form ICAM-4 may play a direct or indirect role in cell differentiation. Showed.

(6) DLC -1, marker for red blood cell differentiation

The present inventors have found for the first time that DLC-1 expression appears in erythroid cells based on studies on cells capable of inducing erythroid differentiation.

Microarray analysis found that DLC-1 expression increased with maturation of erythroid cells. In particular, expression of DLC-1 was found to be affected by density. As the density was increased, the expression of DLC-1 was also increased (FIG. 9). As a result, it was confirmed that the increase in density was accompanied by an increase in the denucleation rate.

In particular, the increase in DLC-1 expression was accompanied by an increase in the rate of denucleation in medium and high density cultures. These findings indicate that DLC-1 is a differentiation marker in erythroid cells.

<110> HANYANG UNIVERSITY INDUSTRY COOPERATION FOUNDATION <120> A Method for in-vitro Expansion of Erythroid cells Using          high-density culture <130> 2011-P-079 <160> 11 <170> Kopatentin 1.71 <210> 1 <211> 3850 <212> DNA <213> Homo sapiens <400> 1 tatgggctcg agcggccgcc cgggcaggtg cccgagcgag ggcgcttcgc tcccagccag 60 gacatggccg cacctctccg catcaggagc gccggctcac ggacttctcg cccaactccc 120 tgagcgctcc ctcgtttcga tctttagaaa accccgcttt ctttctgggg ccgtgacgag 180 gggcagggag cggcgagcaa ggatgcgttg aggaccgcga gggcgcgcgt ctcgggtgcc 240 gccgtgggtc ccgacgcgga agccgagccg cctccgcctg cctcgacttc cccacagcgc 300 ttccgccgcc gcctgccgtg cttgatgtgc agaaagaagc cggacaccat gatcctaaca 360 caaattgaag ccaaggaagc ttgtgattgg ctacgggcaa ctggtttccc ccagtatgca 420 cagctttatg aagatttcct gttccccatc gatatttcct tggtcaagag agagcatgat 480 tttttggaca gagatgccat tgaggctcta tgcaggcgtc taaatacttt aaacaaatgt 540 gcggtgatga agctagaaat tagtcctcat cggaaacgaa gtgacgattc agacgaggat 600 gagccttgtg ccatcagtgg caaatggact ttccaaaggg acagcaagag gtggtcccgg 660 cttgaagagt ttgatgtctt ttctccaaaa caagacctgg tccctgggtc cccagacgac 720 tcccacccga aggacggccc cagccccgga ggcacgctga tggacctcag cgagcgccag 780 gaggtgtctt ccgtccgcag cctcagcagc actggcagcc tccccagcca cgcgcccccc 840 agcgaggatg ctgccacccc ccggactaac tccgtcatca gcgtttgctc ctccagcaac 900 ttggcaggca atgacgactc tttcggcagc ctgccctctc ccaaggaact gtccagcttc 960 agcttcagca tgaaaggcca cgaaaaaact gccaagtcca agacgcgcag tctgctgaaa 1020 cggatggaga gcctgaagct caagagctcc catcacagca agcacaaagc gccctcaaag 1080 ctggggttga tcatcagcgg gcccatcttg caagagggga tggatgagga gaagctgaag 1140 cagctcagct gcgtggagat ctccgccctc aatggcaacc gcatcaacgt ccccatggta 1200 cgaaagagga gcgtttccaa ctccacgcag accagcagca gcagcagcca gtcggagacc 1260 agcagcgcgg tcagcacgcc cagccctgtt acgaggaccc ggagcctcag tgcgtgcaac 1320 aagcgggtgg gcatgtactt agagggcttc gatcctttca atcagtcaac atttaacaac 1380 gtggtggagc agaactttaa gaaccgcgag agctacccag aggacacggt gttctacatc 1440 cctgaagatc acaagcctgg cactttcccc aaagctctca ccaatggcag tttctccccc 1500 tcggggaata acggctctgt gaactggagg acgggaagct tccacggccc tggccacatc 1560 agcctcagga gggaaaacag tagcgacagc cccaaggaac tgaagagacg caattcttcc 1620 agctccatga gcagccgcct gagcatctac gacaacgtgc cgggctccat cctctactcc 1680 agttcagggg acctggcgga tctggagaac gaggacatct tccccgagct ggacgacatc 1740 ctctaccacg tgaaggggat gcagcggata gtcaatcagt ggtcggagaa gttttctgat 1800 gagggagatt cggactcagc cctggactcg gtctctccct gcccgtcctc tccaaaacag 1860 atacacctgg atgtggacaa cgaccgaacc acacccagcg acctggacag cacaggcaac 1920 tccctgaatg aaccggaaga gccctccgag atcccggaaa gaagggattc tggggttggg 1980 gcttccctaa ccaggtccaa caggcaccga ctgagatggc acagtttcca gagctcacat 2040 cggccaagcc tcaactctgt atcactacag attaactgcc agtctgtggc ccagatgaac 2100 ctgctgcaga aatactcact cctaaagcta acggccctgc tggagaaata cacaccttct 2160 aacaagcatg gttttagctg ggccgtgccc aagttcatga agaggatcaa ggttccagac 2220 tacaaggacc ggagtgtgtt tggggtccca ctgacggtca acgtgcagcg cacaggacaa 2280 ccgttgcctc agagcatcca gcaggccatg cgatacctcc ggaaccattg tttggatcag 2340 gttgggctct tcaaaaaatc gggggtcaag tcccggattc aggctctgcg ccagatgaat 2400 gaaggtgcca tagactgtgt caactacgaa ggacagtctg cttatgacgt ggcagacatg 2460 ctgaagcagt attttcgaga tcttcctgag ccactaatga cgaacaaact ctcggaaacc 2520 tttctacaga tctaccaata tgtgcccaag gaccagcgcc tgcaggccat caaggctgcc 2580 atcatgctgc tgcctgacga gaaccgggtg gttctgcaga ccctgcttta tttcctgtgc 2640 gatgtcacag cagccgtaaa agaaaaccag atgaccccaa ccaacctggc cgtgtgctta 2700 gcgccttccc tcttccatct caacaccctg aagagagaga attcctctcc cagggtaatg 2760 caaagaaaac aaagtttggg caaaccagat cagaaagatt tgaatgaaaa cctagctgcc 2820 actcaagggc tggcccatat gatcgccgag tgcaagaagc ttttccaggt tcccgaggaa 2880 atgagccgat gtcgtaattc ctataccgaa caagagctga agcccctcac tctggaagca 2940 ctcgggcacc tgggtaatga tgactcagct gactaccaac acttcctcca ggactgtgtg 3000 gatggcctgt ttaaagaagt caaagagaag tttaaaggct gggtcagcta ctccacttcg 3060 gagcaggctg agctgtccta taagaaggtg agcgaaggac cccgtctgag gctttggagg 3120 tcagtcattg aagtccctgc tgtgccagag gaaatcttaa agcgcctact taaagaacag 3180 cacctctggg atgtagacct gttggattca aaagtgatcg aaattctgga cagccaaact 3240 gaaatttacc agtatgtcca aaacagtatg gcacctcatc ctgctcgaga ctacgttgtt 3300 ttaagaacct ggaggactaa tttacccaaa ggagcctgtg cccttttact aacctctgtg 3360 gatcacgatc gcgcacctgt ggtgggtgtg agggttaatg tgctcttgtc caggtatttg 3420 attgaaccct gtgggccagg aaaatccaaa ctcacctaca tgtgcagagt tgacttaagg 3480 ggccacatgc cagaatggta cacaaaatct tttggacatt tgtgtgcagc tgaagttgta 3540 aagatccggg attccttcag taaccagaac actgaaacca aagacaccaa atctaggtga 3600 tcactgaagc aacgcaaccg cttccaccac catggtgttt gtttttagaa gttttgccag 3660 tccttgaaga atgggttctg tgtgtaatcc tgaaacaaag aaaactacaa gctggagtgt 3720 aggaattgac tatagcaatt tgatacattt ttaaagctgc ttcctgtttg ttgagggtct 3780 gtattcatag accttgactg gaatatgtaa gactgtgcga aaaaaaaaaaaaaaaaaaa 3840 aaaaaaaaaa 3850 <210> 2 <211> 6082 <212> DNA <213> Homo sapiens <400> 2 ataacgtctt tgtcactaaa atgttcccca ggggccttcg gcgagtcttt ttgtttggtt 60 ttttgttttt aatctgtggc tcttgataat ttatctagtg gttgcctaca cctgaaaaac 120 aagacacagt gtttaactat caacgaaaga actggacggc tccccgccgc agtcccactc 180 cccgagtttg tggctggcat ttgggccacg ccgggctggg cggtcacagc gaggggcgcg 240 cagtttgggg tcacacagct ccgcttctag gccccaacca ccgttaaaag gggaagcccg 300 tgccccatca ggtccgctct tgctgagccc agagccatcc cgcgctctgc gggctgggag 360 gcccgggcca ggacgcgagt cctgcgcagc cgaggttccc cagcgccccc tgcagccgcg 420 cgtaggcaga gacggagccc ggccctgcgc ctccgcacca cgcccgggac cccacccagc 480 ggcccgtacc cggagaagca gcgcgagcac ccgaagctcc cggctggcgg cagaaaccgg 540 gagtggggcc gggcgagtgc gcggcatccc aggccggccc gaacgctccg cccgcggtgg 600 gccgacttcc cctcctcttc cctctctcct tcctttagcc cgctggcgcc ggacacgctg 660 cgcctcatct cttggggcgt tcttccccgt tggccaaccg tcgcatcccg tgcaactttg 720 gggtagtggc cgtttagtgt tgaatgttcc ccaccgagag cgcatggctt gggaagcgag 780 gcgcgaaccc ggcccccgaa gggccgccgt ccgggagacg gtgatgctgt tgctgtgcct 840 gggggtcccg accggccgcc cctacaacgt ggacactgag agcgcgctgc tttaccaggg 900 cccccacaac acgctgttcg gctactcggt cgtgctgcac agccacgggg cgaaccgatg 960 gctcctagtg ggtgcgccca ctgccaactg gctcgccaac gcttcagtga tcaatcccgg 1020 ggcgatttac agatgcagga tcggaaagaa tcccggccag acgtgcgaac agctccagct 1080 gggtagccct aatggagaac cttgtggaaa gacttgtttg gaagagagag acaatcagtg 1140 gttgggggtc acactttcca gacagccagg agaaaatgga tccatcgtga cttgtgggca 1200 tagatggaaa aatatatttt acataaagaa tgaaaataag ctccccactg gtggttgcta 1260 tggagtgccc cctgatttac gaacagaact gagtaaaaga atagctccgt gttatcaaga 1320 ttatgtgaaa aaatttggag aaaattttgc atcatgtcaa gctggaatat ccagttttta 1380 cacaaaggat ttaattgtga tgggggcccc aggatcatct tactggactg gctctctttt 1440 tgtctacaat ataactacaa ataaatacaa ggctttttta gacaaacaaa atcaagtaaa 1500 atttggaagt tatttaggat attcagtcgg agctggtcat tttcggagcc agcatactac 1560 cgaagtagtc ggaggagctc ctcaacatga gcagattggt aaggcatata tattcagcat 1620 tgatgaaaaa gaactaaata tcttacatga aatgaaaggt aaaaagcttg gatcgtactt 1680 tggagcttct gtctgtgctg tggacctcaa tgcagatggc ttctcagatc tgctcgtggg 1740 agcacccatg cagagcacca tcagagagga aggaagagtg tttgtgtaca tcaactctgg 1800 ctcgggagca gtaatgaatg caatggaaac aaacctcgtt ggaagtgaca aatatgctgc 1860 aagatttggg gaatctatag ttaatcttgg cgacattgac aatgatggct ttgaagatgt 1920 tgctatcgga gctccacaag aagatgactt gcaaggtgct atttatattt acaatggccg 1980 tgcagatggg atctcgtcaa ccttctcaca gagaattgaa ggacttcaga tcagcaaatc 2040 gttaagtatg tttggacagt ctatatcagg acaaattgat gcagataata atggctatgt 2100 agatgtagca gttggtgctt ttcggtctga ttctgctgtc ttgctaagga caagacctgt 2160 agtaattgtt gacgcttctt taagccaccc tgagtcagta aatagaacga aatttgactg 2220 tgttgaaaat ggatggcctt ctgtgtgcat agatctaaca ctttgtttct catataaggg 2280 caaggaagtt ccaggttaca ttgttttgtt ttataacatg agtttggatg tgaacagaaa 2340 ggcagagtct ccaccaagat tctatttctc ttctaatgga acttctgacg tgattacagg 2400 aagcatacag gtgtccagca gagaagctaa ctgtagaaca catcaagcat ttatgcggaa 2460 agatgtgcgg gacatcctca ccccaattca gattgaagct gcttaccacc ttggtcctca 2520 tgtcatcagt aaacgaagta cagaggaatt cccaccactt cagccaattc ttcagcagaa 2580 gaaagaaaaa gacataatga aaaaaacaat aaactttgca aggttttgtg cccatgaaaa 2640 ttgttctgct gatttacagg tttctgcaaa gattgggttt ttgaagcccc atgaaaataa 2700 aacatatctt gctgttggga gtatgaagac attgatgttg aatgtgtcct tgtttaatgc 2760 tggagatgat gcatatgaaa cgactctaca tgtcaaacta cccgtgggtc tttatttcat 2820 taagatttta gagctggaag agaagcaaat aaactgtgaa gtcacagata actctggcgt 2880 ggtacaactt gactgcagta ttggctatat atatgtagat catctctcaa ggatagatat 2940 tagctttctc ctggatgtga gctcactcag cagagcggaa gaggacctca gtatcacagt 3000 gcatgctacc tgtgaaaatg aagaggaaat ggacaatcta aagcacagca gagtgactgt 3060 agcaatacct ttaaaatatg aggttaagct gactgttcat gggtttgtaa acccaacttc 3120 atttgtgtat ggatcaaatg atgaaaatga gcctgaaacg tgcatggtgg agaaaatgaa 3180 cttaactttc catgttatca acactggcaa tagtatggct cccaatgtta gtgtggaaat 3240 aatggtacca aattctttta gcccccaaac tgataagctg ttcaacattt tggatgtcca 3300 gactactact ggagaatgcc actttgaaaa ttatcaaaga gtgtgtgcat tagagcagca 3360 aaagagtgca atgcagacct tgaaaggcat agtccggttc ttgtccaaga ctgataagag 3420 gctattgtac tgcataaaag ctgatccaca ttgtttaaat ttcttgtgta attttgggaa 3480 aatggaaagt ggaaaagaag ccagtgttca tatccaactg gaaggccggc catccatttt 3540 agaaatggat gagacttcag cactcaagtt tgaaataaga gcaacaggtt ttccagagcc 3600 aaatccaaga gtaattgaac taaacaagga tgagaatgtt gcgcatgttc tactggaagg 3660 actacatcat caaagaccca aacgttattt caccatagtg attatttcaa gtagcttgct 3720 acttggactt attgtacttc tgttgatctc atatgttatg tggaaggctg gcttctttaa 3780 aagacaatac aaatctatcc tacaagaaga aaacagaaga gacagttgga gttatatcaa 3840 cagtaaaagc aatgatgatt aaggacttct ttcaaattga gagaatggaa aacagactca 3900 ggttgtagta aagaaattta aaagacactg tttacaagaa aaaatgaatt ttgtttggac 3960 ttcttttact catgatcttg tgacatatta tgtcttcatg caaggggaaa atctcagcaa 4020 tgattactct ttgagataga agaactgcaa aggtaataat acagccaaag ataatctctc 4080 agcttttaaa tgggtagaga aacactaaag cattcaattt attcaagaaa agtaagccct 4140 tgaagatatc ttgaaatgaa agtataactg agttaaatta tactggagaa gtcttagact 4200 tgaaatacta cttaccatat gtgcttgcct cagtaaaatg aaccccactg ggtgggcaga 4260 ggttcatttc aaatacatct ttgatacttg ttcaaaatat gttctttaaa aatataattt 4320 tttagagagc tgttcccaaa ttttctaacg agtggaccat tatcacttta aagcccttta 4380 tttataatac atttcctacg ggctgtgttc caacaaccat tttttttcag cagactatga 4440 atattatagt attataggcc aaactggcaa acttcagact gaacatgtac actggtttga 4500 gcttagtgaa attacttctg gataattatt tttttataat tatggatttc accatctttc 4560 tttctgtata tatacatgtg tttttatgta ggtatatatt taccattctt cctatctatt 4620 cttcctataa cacaccttta tcaagcatac ccaggagtaa tcttcaaatc ttttgttata 4680 ttctgaaaca aaagattgtg agtgttgcac tttacctgat acacgctgat ttagaaaata 4740 cagaaaccat acctcactaa taactttaaa atcaaagctg tgcaaagact agggggccta 4800 tacttcatat gtattatgta ctatgtaaaa tattgactat cacacaacta tttccttgga 4860 tgtaattctt tgttaccctt tacaagtata agtgttacct tacatggaaa cgaagaaaca 4920 aaattcataa atttaaattc ataaatttag ctgaaagata ctgattcaat ttgtatacag 4980 tgaatataaa tgagacgaca gcaaaatttt catgaaatgt aaaatatttt tatagtttgt 5040 tcatactata tgaggttcta ttttaaatga ctttctggat tttaaaaaat ttctttaaat 5100 acaatcattt ttgtaatatt tattttatgc ttatgatcta gataattgca gaatatcatt 5160 ttatctgact ctgccttcat aagagagctg tggccgaatt ttgaacatct gttataggga 5220 gtgatcaaat tagaaggcaa tgtggaaaaa caattctggg aaagatttct ttatatgaag 5280 tccctgccac tagccagcca tcctaattga tgaaagttat ctgttcacag gcctgcagtg 5340 atggtgagga atgttctgag atttgcgaag gcatttgagt agtgaaatgt aagcacaaaa 5400 cctcctgaac ccagagtgtg tatacacagg aataaacttt atgacattta tgtattttta 5460 aaaaactttg tatcgttata aaaaggctag tcattctttc aggagaacat ctaggatcat 5520 agatgaaaaa tcaagccccg atttagaact gtcttctcca ggatggtctc taaggaaatt 5580 tacatttggt tctttcctac tcagaactac tcagaaacaa ctatatattt caggttatct 5640 gagcacagtg aaagcagagt actatggttg tccaacacag gcctctcaga tacaagggga 5700 acacaattac atattgggct agattttgcc cagttcaaaa tagtatttgt tatcaactta 5760 ctttgttact tgtatcatga attttaaaac cctaccactt taagaagaca gggatgggtt 5820 attctttttt ggcaggtagg ctatataact atgtgatttt gaaatttaac tgctctggat 5880 tagggagcag tgaatcaagg cagacttatg aaatctgtat tatatttgta acagaatata 5940 ggaaatttaa cataattgat gagctcaaat cctgaaaaat gaaagaatcc aaattatttc 6000 agaattatct aggttaaata ttgatgtatt atgatggttg caaagttttt ttgtgtgtcc 6060 aataaacaca ttgtaaaaaa aa 6082 <210> 3 <211> 3448 <212> DNA <213> Homo sapiens <400> 3 cggggtctcg caacattgcc cagacttcct ttgtgttagt taataaagct ttctcaactg 60 cctcagcctt gtgtgagttg aggggaggtg tcacatccag ctggagtcct ttctaagcag 120 ccacagcctg atcctcccac ttcctccccc aagaaaacat tgtgggttga tggccatacc 180 ctgaggttct ggtccaaatc ggactttcta tgaccttctg ggtctctagt gaaaactaaa 240 gactcctctc cagaaaaaaa catttggttt ctaatgaggc ctggaatctt attcttgacc 300 tggggagcgg aatccctttt tgcagtactc ccgggccctc tgttggggcc tccccttcct 360 ctccagggtg gagtcgagga ggcggggctg cgggcctcct tatctctaga gccggccctg 420 gctctctggc gcggggcccc ttagtccggg ctttttgcca tggggtctct gttccctctg 480 tcgctgctgt tttttttggc ggccgcctac ccgggagttg ggagcgcgct gggacgccgg 540 actaagcggg cgcaaagccc caagggtagc cctctcgcgc cctccgggac ctcagtgccc 600 ttctgggtgc gcatgagccc ggagttcgtg gctgtgcagc cggggaagtc agtgcagctc 660 aattgcagca acagctgtcc ccagccgcag aattccagcc tccgcacccc gctgcggcaa 720 ggcaagacgc tcagagggcc gggttgggtg tcttaccagc tgctcgacgt gagggcctgg 780 agctccctcg cgcactgcct cgtgacctgc gcaggaaaaa cacgctgggc cacctccagg 840 atcaccgcct acagtgaggg acaggggctc ggtcccggct ggggtgaggg gagggggctg 900 gaagaggtgg gggaagggta gttgacagtc gctctatagg gagcgcccgc ggacctcact 960 cagaggctcc cccttgcctt agaaccgccc cacagcgtga ttttggagcc tccggtctta 1020 aagggcagga aatacacttt gcgctgccac gtgacgcagg tgttcccggt gggctacttg 1080 gtggtgaccc tgaggcatgg aagccgggtc atctattccg aaagcctgga gcgcttcacc 1140 ggcctggatc tggccaacgt gaccttgacc tacgagtttg ctgctggacc ccgcgacttc 1200 tggcagcccg tgatctgcca cgcgcgcctc aatctcgacg gcctggtggt ccgcaacagc 1260 tcggcaccca ttacactgat gctcggtgag gcacccctgt aaccctgggg actaggagga 1320 agggggcaga gagagttatg accccgagag ggcgcacaga ccaagcgtga gctccacgcg 1380 ggtcgacaga cctccctgtg ttccgttcct aattctcgcc ttctgctccc agcttggagc 1440 cccgcgccca cagctttggc ctccggttcc atcgctgccc ttgtagggat cctcctcact 1500 gtgggcgctg cgtacctatg caagtgccta gctatgaagt cccaggcgta aagggggatg 1560 ttctatgccg gctgagcgag aaaaagagga atatgaaaca atctggggaa atggccatac 1620 atggtggctg acgcctgtaa tcccagcact ttgggaggcc gaggcaggag aatcgcttga 1680 gcccaggagt tcgagaccag cctggacaac atagtgagac cccgtctatg caaaaaatac 1740 acaaattagc ctggtgtggt ggcccgcacc tgtggtccca gctacccggg aggctgagtt 1800 gggaggatcc tttgagccct gaaagtcgag gttgcagtga gccttgatcg tgccactgca 1860 ctccagcctg ggggacagag cacgaccctg tctccaaaaa taaaataaaa ataaaaataa 1920 atattggcgg gggaaccctc tggaatcaat aaaggcttcc ttaaccagcc tctgtcctgt 1980 gacctaaggg tccgcattac tgcccttctt cggaggaact ggtttgtttt tgttgttgtt 2040 gttgtttttg cgatcacttt ctccaagttc cttgtctccc tgagggcacc tgaggtttcc 2100 tcactcaggg cccacctggg gtcccgaagc cccagactct gtgtatcccc agcgggtgtc 2160 acagaaacct ctccttctgc tggccttatc gagtgggatc agcgcgggcc ggggagagcc 2220 acgggcaggg gcggggtggg gttcatggta tggctttcct gattggcgcc gccgccacca 2280 cgcggcagct ctgattggat gttaagtttc ctatcccagc cccaccttca gaccctgtgc 2340 tttcctggag gccaaacaac tgtggagcga gaactcatct ccaaaataac ttaccacgct 2400 ggagtgagac cacgaatggt ggggagggga gggtcccacg gacatattga gggacgtgga 2460 tacgcagaag aggtatccat gtggtggcag ccgggaaggg gtgatcagat ggtccacagg 2520 gaatatcaca aactcgaatt ctgacgatgt tctggtagtc acccagccag atgagcgcat 2580 ggagttgggg gtggggggtg tcaaagcttg gggcccggaa gcggagtcaa aagcatcacc 2640 ctcggtccct tgttctcgcg tggatgtcag ggcccccacc caccgagcag aaggcggact 2700 caggggcgct ccagggtggc tcgagctcac acacgctgag tagacacgtg cccgctgcac 2760 cctgggtaaa tacagacccg gagccgagcg gattctaatt tagacgcccg cgaacgctgc 2820 gcgcacgcac acgtgtcctc ggctcgctgg cactttcgtc ccgccccctc cgtcgcgtgc 2880 cggagctgac ccggaggggt gcttagaggt atggctccgc ggggtcaaaa ggagaaggat 2940 cagtgagaga ggcatcccca caccctcccc tagaactgtc ctttccccat ccagtgcctc 3000 ccaaatctct cttagtcccc aaatgtatcc ccgccctaag gggcgctggt gggaggagct 3060 aaatgtgggg gcggagctcg gagtccagct tattatcatg gcatctcagc cagggctggg 3120 gtaggggttt gggaagggca acccagcatc ccccgatccc agagtcgcgg ccggggatga 3180 cgcgagagag cgtggtcgcc cccagaaggc cctgggccat catgccggcc tccacgtaga 3240 ccccaggggt cgctcactcc tgccagctcg ccttcaccaa ggccaggagc ttagcgcacg 3300 ctcgcctccc gcccccccgc cgcctctgcc gccgccccct ccttggaaac caagttacca 3360 acgttaaacc aatccccaag cgcaactctg cctcccccac accccacccg ccgcgccgcg 3420 cggagccgtc ctctagccca gctcctcg 3448 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for E-cadherin <400> 4 cgggaatgca gttgaggatc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for E-cadherin <400> 5 aggatggtgt aagcgatggc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for VLA-4 <400> 6 aggatggtgt aagcgatggc 20 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for VLA-4 <400> 7 tgctgaagaa ttggctgaag tggtgg 26 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for DLC-1 <400> 8 agtgcgtgca acaagcgggt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for DLC-1 <400> 9 tccgggtagc tctcgcggtt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for ICAM-4 <400> 10 ccggactaag cgggcgcaaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for ICAM-4 <400> 11 agccacgaac tccgggctca 20

Claims (16)

An in vitro expansion method of erythroid cells, characterized in that the erythroid cells are cultured at high density so as to be in direct physical contact between the cells.
According to claim 1, wherein the high-density culture is characterized in that the confluence (confluence) of 100 to 150%, in vitro expansion of erythroid cells.
The in vitro expansion method of erythroid cells according to claim 2, wherein the high-density culture is 100% confluence.
2. The method of claim 1, wherein the erythroid cells are cells after a terminal maturation step.
The in vitro expansion method of red blood cells according to claim 1, wherein the red blood cells in direct physical contact between the cells express DCL-1. 6. The method according to claim 5, wherein the red blood cells that are in direct physical contact between the cells further express one or more genes of ICAM-4 and VLA-4.
The in vitro expansion method of erythroid cells according to claim 5 or 6, wherein the intercellular signal exchange activity is increased by the gene expression to increase the production rate of erythroid cells.
An in vitro expansion method of erythroid cells, comprising adding DLC-1, ICAM-4, and VLA-4 proteins to erythroid cell culture.
A composition for erythropoietic cell differentiation markers comprising DLC-1.
The composition for erythroid cell differentiation marker according to claim 9, wherein the DLC-1 consists of an amino acid sequence represented by SEQ ID NO: 1. The composition for red blood cell differentiation marker according to claim 9, wherein the DLC-1 emits adhesion-related signals in combination with VLA-4.
The composition for erythroid cell differentiation marker according to claim 9, wherein the composition further contains ICAM-4.
A method of increasing the maturation of erythroid cells through promoting expression or activity of DLC-1, VLA-4 and ICAM-4.
The method of claim 10, wherein the maturation of the erythroid cells comprises an enucleation step.
The method according to claim 10, wherein the expression or activity promotion of the DLC-1, VLA-4 and ICAM-4 may be achieved by the method of introducing the DLC-1, VLA-4 and ICAM-4 genes to erythroid cells. How to. The method of in vitro expansion of erythroid cells using a method of increasing the maturation of erythroid cells through the expression or promotion of DLC-1, VLA-4 and ICAM-4 of claim 13.

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