WO2014181783A1 - Method for enucleating erythroblasts and method for maintaining enucleated erythrocytes - Google Patents

Abstract

[Problem] To provide a method for strongly induce enucleation within a short period of time and a method for improving the survival rate of enucleated erythrocytes. [Solution] The present invention was completed by finding "incubation of erythroblasts under low-oxygen conditions exhibits an effect of advancing the starting timing of enucleation, an effect of removing nuclei, an effect of promoting the induction of enucleation and an effect of improving the survival rate of enucleated erythrocytes".

Description

Enucleation method of erythroblast and maintenance method of enucleated erythrocyte

The present invention relates to a nucleated red blood cell enucleation method and a enucleated red blood cell maintenance method characterized by culturing erythroblasts under hypoxic conditions.
This application claims priority from Japanese Patent Application No. 2013-097312, which is incorporated herein by reference.

In the medical field, blood transfusion by blood donation is currently the main, but in order to secure the supply, much labor and cost are required for collection, inspection of infection and storage. In order to supply a sufficient amount of blood in the medical and research fields, a method for inducing mature erythrocytes from cultured hematopoietic stem cells in a short time and efficiently is necessary. In the future, if the technology and application of hematopoietic stem cells can be applied from totipotent or pluripotent stem cells including iPS cells and ES cells, all processes can be cultured artificially, and the risk of infection is low and stable. In particular, blood supply becomes possible and the contribution to medical care is high. In addition, a large amount of blood culture can be industrially produced, and production at a low cost and development as a medical industry can be expected.

Red blood cells are important blood components that carry oxygen, but cannot increase because they themselves do not have a nucleus. Therefore, in the living body, erythrocyte progenitor cells in the previous stage of erythrocytes proliferate and mature through “nuclear enucleation” that discharges the nucleus.
Therefore, mass production of erythrocyte progenitor cells is necessary to produce erythrocytes in large quantities, and for that purpose, establishment of a “cell line (erythroid progenitor cell line)” is necessary.

In a study on hematopoiesis, Ihle et al. Reported that “SOCS3 is essential for hematopoiesis in the fetal liver” (see Non-patent Document 1).
In addition, Nagata et al. Reported that the fetal liver of DNase II knockout mice cannot produce mature erythrocytes, causing severe anemia, and that fetal liver macrophage DNase II is important as a support for erythroid differentiation. (Reference: Non-Patent Document 2).

On the other hand, an erythrocyte progenitor cell line was established from mouse ES cells by a group of the independent administrative institution RIKEN, which enabled large-scale culture of erythrocyte progenitor cells in vitro (see Non-Patent Document 3).
A method of transfusion of erythroid progenitor cells directly into the body is also conceivable since the large-scale culture has become possible. However, since the progenitor cells are nucleated, there are great concerns regarding safety such as canceration. Actually, there is a need for an induction method for further differentiation of the progenitor cells to enucleate them to obtain mature erythrocytes that are anucleated.

Further, in the method for producing mature erythrocytes using umbilical cord blood reported by Miharada et al. (Non-Patent Document 4), “20 days of culturing is required until the production of mature erythrocytes enucleated from erythrocyte progenitor cells. Of enucleated cells is 77% ".
In the method for producing mature erythrocytes, mifepristone, which is an antiestrogenic agent, is added as a factor that induces enucleation. Enucleation is induced by differentiating erythroid progenitor cells with a differentiation-inducing factor added at the beginning of the culture, and stopping proliferation of erythroblasts with non-physiological molecules.

As described above, several methods for producing erythroid progenitor cells are already known. However, in the process, there are almost no techniques for enucleating erythroblasts in a short time and with high efficiency using only physiological conditions and physiological molecules. unknown. However, the present inventors have reported a method in which a compound derived from proopiomelanocortin (POMC), which is a factor that induces enucleation, is added to a medium (see Patent Document 1).

Patent Document 1 discloses a “nuclear erythrocyte enucleation method characterized by culturing nucleated erythrocytes in a culture medium containing a compound derived from proopiomelanocortin (POMC)”. However, the enucleation rate is about 70% at the 7th day after maturation. Further, the enucleation method had a low maintenance rate of enucleated erythrocytes.

From the above background, about 20 to 30% of erythroblasts remain in the erythrocytes obtained by the current production method, and further there is a risk of canceration when the erythrocytes are administered into the body. .
In addition, a large amount of erythroblasts can be induced from human iPS or ES cells in the future, and a method for inducing erythrocyte enucleation in a short time will be required.

International Publication WO2010 / 098079

From the above background, an object of the present invention is to provide a new method for inducing enucleation in a short time and a method for improving the survival rate of enucleated erythrocytes.

As a result of intensive research in order to solve the above problems, the present inventors have found that “the culture of erythroblasts under hypoxic conditions is the effect of promoting the enucleation start time, the effect of enucleating, and the effect of promoting enucleation. And having an effect of increasing the survival rate of enucleated erythrocytes ”, the present invention was completed.
That is, the present invention is characterized by the following as a solution to the above problems.
“1. A method and / or enucleation of erythroblasts comprising culturing hematopoietic stem cells, erythroid progenitor cells, and / or erythroblasts in a culture solution under oxygen conditions of 1.0% to 18.0% How to maintain red blood cells.
2. 2. The method according to item 1, wherein the oxygen condition is 2.0% to 10.0%.
3. 3. The method according to item 1 or 2, wherein the oxygen condition is 3.0% to 7.0%.
4). 4. The method according to any one of items 1 to 3, wherein the oxygen condition is 4.0% to 6.0%.
5. 5. The method according to any one of items 1 to 4, wherein the culture solution contains a compound derived from proopiomelanocortin (POMC).
6). 6. The method according to item 5 above, wherein the compound is selected from any one of the following.
(1) ACTH
(2) POMC
(3) MSH
(4) LPH
(5) Endorphins (6) CLIP
7). 7. The method according to 5 or 6 above, wherein the compound is ACTH.
8). 8. The method according to any one of 1 to 7 above, wherein the expression of GATA1, GATA2 and / or MXI1 is improved.
9. 9. The method according to any one of items 1 to 8, wherein expression of MC1R, MC2R and / or MC5R is improved. "

By culturing erythroblasts under hypoxic conditions, the present invention provides an erythroblast having an effect of promoting the enucleation initiation time, an effect of promoting the enucleation induction, an effect of enucleating the nucleus, and an effect of increasing the survival rate of enucleated erythrocytes. A method of enucleation and / or a method of maintaining enucleated erythrocytes could be provided.
This makes it possible to shorten the blood production period and eliminate the risk of canceration associated with erythroblast transfusion. That is, safe blood can be obtained stably in a short time, and the contribution to medical technology is very large.

Confirmation of the effect of promoting the enucleation initiation time and enucleation induction of erythrocytes under hypoxic conditions Confirmation of increased survival of enucleated erythrocytes under hypoxic conditions Confirmation of the effect of promoting nucleation of red blood cells under hypoxic conditions and addition of POMC-derived compounds, and confirmation of increased nuclear exclusion rate under hypoxic conditions Confirmation of improved expression of GATA1, GATA2 and MXI1 under hypoxic conditions Confirmation of improved expression of MC1R, MC2R and MC5R under hypoxic conditions and under POMC-derived compound addition conditions Confirmation of enucleation induction promoting effect of bone marrow-derived CD34 positive hematopoietic progenitor erythrocytes under hypoxic condition

The present invention relates to a method and / or denucleation of erythroblasts characterized by culturing hematopoietic stem cells, erythroid progenitor cells, and / or erythroblasts in a culture solution under oxygen conditions of 1.0% to 18.0%. It relates to "a method for maintaining nuclear red blood cells". Embodiments of the present invention will be described below.
The “maintenance method of enucleated erythrocytes” includes not only preventing and eliminating mutations and disappearances of enucleated erythrocytes, but also includes methods for culturing, amplifying, differentiating and maturing enucleated erythrocytes.

(Hematopoietic stem cells)
The “hematopoietic stem cell” of the present invention is a cell having the ability to differentiate into all types of blood cells and the ability to reconstruct hematopoiesis. It is mainly present in bone marrow, umbilical cord blood, spleen or liver, and is also present in peripheral blood although it is in a trace amount. “Stem cells” mean pluripotent hematopoietic stem cells and differentiated myeloid stem cells (CFU-GEMM). These cells are CD34 and CD133 positive cells.
Hematopoietic stem cells can be obtained by a method known per se. For example, it can be obtained by separating hematopoietic stem cells present in the bone marrow, umbilical cord blood, spleen, liver or peripheral blood described above from a cell separation device (flow cytometry or the like).
More specifically, hematopoietic stem cells can be isolated using commercially available antibodies that bind to hematopoietic stem cell surface antigens (eg, CD34) using methods well known to those skilled in the art. For example, the antibody is bound to magnetic beads and immunological methods are utilized to recover the desired cell type. Preferably, the hematopoietic stem cells are in the form of CD34 positive cells. Indeed, CD34 is known as a standard marker for hematopoietic stem cells.
Separation of CD34 positive cells can be achieved by many different methods. The most widely used is positive immunological selection based on binding the cells to anti-CD34 antibodies (Cellpro, Baxter, Myltenyi) immobilized on a solid support. Other sorting methods include negative sorting that isolates all cells that do not express CD34 from CD34 positive cells based on the expression of cell lineage specific cell surface antigens. In addition, the hematopoietic stem cells to be cultured can be produced ex vivo from embryonic stem cells (see: WO 01/34776, US 6,613,568).
Further, for example, human umbilical cord blood CD34 positive cells (available from Cell Bank of RIKEN) can be used.

(Erythroid progenitor cells)
The “erythroid progenitor cell” of the present invention means a cell in which blood cells of each lineage cannot be identified from hematopoietic stem cells, but can only differentiate into blood cells in one direction of the erythroid.
Specifically, platelet colony forming cells (CFU-MEG), eosinophil colony forming cells (CFU-EO), granulocyte monocyte colony forming cells (CFU-GM), erythroid cells (BFU-E, CFU-E) ), T precursor cells, B precursor cells and the like. These are all CD34 positive cells.
Erythrocyte progenitor cells can be obtained by a method known per se. For example, it can be obtained by separating erythrocyte progenitor cells present in the above-described bone marrow, umbilical cord blood, spleen, liver or peripheral blood from a cell separation device (flow cytometry or the like).
In addition, a method for inducing erythroid progenitor cells from mouse ES cells is also known (see Non-Patent Document 3).
In addition, human bone marrow-derived CD34-positive hematopoietic progenitor cells can be obtained from Lonza or the like.

In addition, in order to amplify a large amount of hematopoietic stem cells or erythrocyte progenitor cells, various methods and development to optimum culture conditions have been attempted. And there are some reports as follows.
Therefore, hematopoietic stem cells or erythroid progenitor cells used in the present invention can also be obtained using the method described below.
(1) A method of proliferating hematopoietic stem cells by co-culturing stromal cells derived from mammals and hematopoietic stem cells (see: JP-A-10-295369).
(2) A method of obtaining hematopoietic stem cells by amplifying cells having hematopoietic support ability obtained from human umbilical cord blood outside the body (see: JP-A-2002-6520).
(3) A method of culturing human hematopoietic stem cells or erythrocyte progenitor cells in the presence of stromal cells derived from human placental tissue or umbilical cord tissue (see: JP 2004-222502 A).
(4) A method for culturing and amplifying hematopoietic stem cells or erythrocyte progenitor cells using endometrial cells (see: Special Table 2007-525231).

(Red blast)
The “erythroblast” of the present invention is used to distinguish it from enucleated red blood cells (mature red blood cells). It is a cell differentiated from erythroid progenitor cells and includes all stages of basic erythroblasts, polychromatic erythroblasts, and normal erythroblasts.
Various methods for preparing erythroblasts have been reported, and methods known per se can be used. For example, a “method of adding and preferentially aggregating and sedimenting red blood cells by adding a water-soluble polymer compound to collected whole blood” described in International Publication WO2004 / 012750 can be used, but is not particularly limited.
In addition, a method for differentiating cord blood-derived hematopoietic stem cells or erythroid progenitor cells into erythroblasts is also known (see Non-Patent Document 4).

The origin of the hematopoietic stem cell, erythroid progenitor cell or erythroblast is not particularly limited as long as it is derived from a mammal. Preferred examples include humans, dogs, cats, mice, rats, rabbits, pigs, cows and horses, with humans being more preferred. In addition, the said hematopoietic stem cell, erythrocyte progenitor cell, or erythroblast derived from a human is intended to be isolated from a living body.

{Culture solution (medium)}
The “culture solution (medium)” of the present invention can maintain or survive ES cells, hematopoietic stem cells, erythrocyte progenitor cells, erythroblasts, reticulocytes, or mature erythrocytes, or ES cells, hematopoietic stem cells or erythrocyte progenitor cells Any medium (medium) can be used as long as it does not inhibit maintenance, survival, differentiation, maturation, or self-replication.
For example, it contains inorganic substances such as sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acids, vitamins, hormones, antibiotics, cytokines, fatty acids, sugars or other chemical components or biological components such as serum depending on the purpose. You can also
Examples include hematopoietic progenitor growth medium (HPGM, Lonza), DMEM, RPMI, IMDM, 10% FBS (including antibiotics for cell culture), and the like.

(Culture conditions)
“Culture conditions” of the present invention include, except for oxygen conditions, physical cells such as temperature, osmotic pressure and light, and chemical cells such as carbon dioxide, pH and redox potential, ES cells, Hematopoietic stem cells, erythroid progenitor cells, erythroblasts, reticulocytes, or mature erythrocytes can be maintained and survived, or ES cells, hematopoietic stem cells or erythroid progenitor cells are maintained, survived, differentiated, matured, and self-replicated. Any environmental condition may be used as long as it does not inhibit anything.
The temperature is 20 to 40 ° C, preferably about 37 ° C.
The osmotic pressure is specifically an osmotic pressure under physiological conditions, and preferably an osmotic pressure equal to that of physiological saline.
The light may be as dark as a dark room, or as bright as the brightness outside in sunny weather.
The general pH in the culture system is 6.0 to 8.0, preferably a pH equivalent to physiological conditions. Carbon dioxide may be used to control the pH, or any other buffer may be used.
Specifically, the concentration of carbon dioxide is the concentration of dissolved carbon dioxide in a state where the culture is in contact with the gas phase of 5%.

(Low oxygen condition)
In the “hypoxic condition” of the present invention, the oxygen concentration dissolved in the culture solution is lower than the normal aerobic culture condition (20% O ₂ ), 1.0% to 18.0%, preferably 2.0% to 10.0%, more preferably 3.0% to 7.0%, still more preferably 4.0% to 6.0%, and most preferably about 5.0%.
In particular, the time when the oxygen concentration dissolved in the culture solution is set to 1.0% to 18.0% is the differentiation phase, the first maturity phase in the amplification phase, the differentiation phase, the first mature phase and the second mature phase. Although it is the period and the second maturity period, other periods may be set.
The oxygen concentration dissolved in the culture solution can be easily set in the range of 1.0% to 18.0% by an incubator using nitrogen gas. It can also be achieved by adding an oxygen adsorbent to the culture medium.
Furthermore, the oxygen concentration dissolved in the culture medium can be easily measured by using an oxygen sensor or the like.
Expansion phase: about 7 days. This is the time to amplify CD34 positive cells and erythroid progenitor cells 8-20 times.
Differentiation phase: 3-4 days. By culturing in the presence of erythropoietin, CD34-positive cells and erythroid progenitor cells undergo cell division and differentiate into pre-erythroblasts.
Maturation Phase 1: About 3 days. Cells divide from pre-erythroblasts and differentiate into basic erythroblasts and polychromatic erythroblasts. In addition, the polychromatic erythroblast further matures to become a normal dye erythroblast.
Maturation Phase 2 (2nd maturity phase: M3 and later): Positively dyed erythroblasts are enucleated and mature into mature erythrocytes via reticulocytes.

(Denucleation method)
The “enucleation method” of the present invention can use the following method, but is not particularly limited as long as it can induce enucleation of erythrocytes.
The cell concentration (including erythroblasts) is 1.0 × 10 ² to 1.0 × 10 ¹⁰ , preferably 1.0 × 10 ³ to 1.0 × 10 ⁸ , more preferably 1.0 in 1 ml of culture solution having an oxygen concentration of 1.0% to 18.0%. It is prepared to × 10 ⁴ to 1.0 × 10 ⁷ , most preferably about 1.0 × 10 ⁵ .
Preferably, the final concentration of the POMC-derived compound in the culture medium is 0.005 nM to 50 nM, preferably 0.01 nM to 30 nM, more preferably 0.1 nM to 20 nM, and most preferably 1.0 nM to 10 nM. Add as follows. The compound may be added at the start of the culture or may be added as appropriate during the culture.
The culture temperature is preferably 33 to 38 ° C., and the CO ₂ concentration is preferably about 3 to 7%.
The culture time for enucleation induction in erythroblasts derived from rat fetuses is 2 to 24 hours, preferably 3 to 12 hours, more preferably 4 to 8 hours.
In addition, it takes 20 days from culture of hematopoietic progenitor cells derived from human umbilical cord blood to enucleated erythrocytes (see: Non-patent Document 4). Can induce more than half of enucleation.
Note that the difference in time required for enucleation between humans and rats is thought to be due to the difference in the time required for fetal development and the number of days to survive red blood cells.

The “method of culturing and / or amplifying enucleated erythrocytes” according to the present invention can use the following method, but is not particularly limited as long as it can induce enucleation of erythroblasts.
Hematopoietic stem cells or erythrocyte progenitor cells are differentiated into nucleated erythrocytes by the method described above. In the process of differentiation, various cytokines are introduced into the culture medium as necessary. Next, when nucleated red blood cells are present in the culture solution, preferably the final concentration of the POMC-derived compound in the culture solution is 0.005 nM to 50 nM, preferably 0.01 nM to 30 nM, more preferably 0.1 nM to 20 nM, most preferably 1.0 nM to 10 nM. The compound may be added at the start of the culture or may be added as appropriate during the culture.

(Cytokine)
The “cytokine” of the present invention is a proteinous factor that is released from cells and mediates cell-cell interaction, and exhibits a control action of an immune response, an antitumor action, an antiviral action, a cell growth / differentiation regulating action, etc. Means. Specifically, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 ( IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 ( IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 ( IL-15), interleukin-16 (IL-16), interferon α (IFN-α), interferon β (IFN-β), interferon γ (IFN-γ), granulocyte colony stimulating factor (G-CSF), Granulocyte-monocyte colony stimulating factor (GM-CSF) Monocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, eosinophil colony stimulating factor, platelet colony stimulating factor, stem cell factor (SCF), stem cell growth factor, flk2 / flt3-ligand, leukemia inhibitory (blocking) factor, erythropoietin (EPO), macrophage-derived inflammatory protein 1α (MIP-1α), etc., preferably interleukin-3, stem cell factor (SCF), Flt-3L, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor Flk2 / flt3-ligand, MIP-1α or erythropoietin, thrombopoietin, transferrin, vascular endothelial growth factor (VEGF), insulin and the like.

(Effect of promoting the enucleation start time)
The “denucleation start time promoting effect” of the present invention means an effect that the enucleation start time can be advanced as shown in the results of Example 2, unlike the conventional enucleation method.

(Denucleation induction promotion effect)
The “denucleation induction promoting effect” of the present invention means an effect capable of increasing the enucleation rate as shown by the results of Example 2, unlike the conventional enucleation method.

(Effect of increasing the survival rate of enucleated erythrocytes)
The “development effect of enucleated erythrocytes” of the present invention is different from the conventional enucleation method, as shown in the results of Example 3, the survival rate of enucleated erythrocytes (denucleated erythrocytes / total Cell)).

(Nuclear emission effect)
The “nuclear ejection effect” of the present invention means an effect that, unlike the conventional denuclearization method, can increase the rate at which the denucleated nucleus is detached from the cell membrane, as the results of Example 3 show.

(Improved expression of GATA1, GATA2 and / or MXI1)
“Improved expression of GATA1, GATA2 and / or MXI1” of the present invention means that GATA1 and GATA2 under hypoxic conditions as shown in the results of Example 4 as compared with culture under normal oxygen conditions. And / or MXI1 expression is increased.
Improved expression means that the expression level of GATA1, GATA2 and / or MXI1 under hypoxic conditions is about 1.2 times higher than the expression level of GATA1, GATA2 and / or MXI1 under normal oxygen conditions It means 5.0 times, 1.5 times to 4.5 times, 2.0 times to 4.0 times, 2.5 to 3.5 times.
GATA1 and GATA2 are transcription factors required for erythroid differentiation, and MXI1 is known to be a transcription factor that induces nuclear enrichment before the enucleation process.

(Improved expression of MC1R, MC2R and / or MC5R)
The “improved expression of MC1R, MC2R and / or MC5R” of the present invention is more specifically reduced under low oxygen conditions as shown in the results of Example 5 as compared with the culture under normal oxygen conditions. It means that the expression of MC1R, MC2R and / or MC5R is increased under oxygen conditions and POMC-derived compound addition conditions.
The expression improvement means that the expression level of MC1R, MC2R and / or MC5R under hypoxic conditions is about 2.0 times higher than the expression level of MC1R, MC2R and / or MC5R under normal oxygen conditions It means 10.0 times, 3.0 times to 9.0 times, 3.5 times to 8.0 times.
MC1R, MC2R and MC5R are melanocortin receptors.

(Compound derived from POMC)
The “POMC-derived compound” of the present invention includes POMC, MSH (α-, β-, γ-MSH), ACTH, LPH (γ-, β-LPH), endorphin (α, β, γ-endorphin) or CLIP. Or a protected derivative, a sugar chain-modified product, an acylated derivative, or an acetylated derivative thereof.
The protected derivative, sugar chain modified product, acylated derivative, or acetylated derivative can be obtained by a method known per se.
Details of the POMC-derived compound are described in Patent Document 1.

POMC is a precursor of melanocyte stimulating hormone (MSH), adrenocorticotropin (ACTH), lipotropin (LPH), CLIP and β-endorphin. All these hormones are produced by cleavage from just one large precursor, POMC.

ACTH is a peptide consisting of the following amino acid residues. In addition, in this specification, ACTH may be referred to as ACTH1-39 in order to distinguish from the following ACTH fragment sequences.
Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn- Val-Ala-Glu-Asn-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe (SEQ ID NO: 1)

In addition, various ACTH fragment sequences are known. These fragment sequences themselves are considered to have an enucleating action.
ACTH1-24 (sequence 1 to 24 of ACTH) is a peptide consisting of the following amino acid residues.
Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro (SEQ ID NO: 2)
ACTH1-10 (the 1st to 10th sequence of ACTH) is a peptide consisting of the following amino acid residues.
Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly (SEQ ID NO: 13)
ACTH1-14 (sequence 1 to 14 of ACTH) is a peptide consisting of the following amino acid residues.
Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly (SEQ ID NO: 14)
ACTH1-16 (sequence 1 to 16 of ACTH) is a peptide consisting of the following amino acid residues.
Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys (SEQ ID NO: 15)
ACTH1-17 (ACTH 1st to 17th sequence) is a peptide consisting of the following amino acid residues.
Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg (SEQ ID NO: 16)
ACTH4-10 (the 4th to 10th sequence of ACTH) is a peptide consisting of the following amino acid residues.
Met-Glu-His-Phe-Arg-Trp-Gly (SEQ ID NO: 17)
ACTH7-38 (the 7th to 38th sequence of ACTH) is a peptide consisting of the following amino acid residues.
Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Val-Ala-Glu-Asn-Glu-Ser- Ala-Glu-Ala-Phe-Pro-Leu-Glu (SEQ ID NO: 18)
ACTH4-9 (ACTH 4th to 9th sequence) is a peptide consisting of the following amino acid residues.
Met-Glu-His-Phe-Arg-Trp (SEQ ID NO: 19)

MSH {(melanocyte-stimulating hormone), a melanocyte-stimulating hormone} is a peptide hormone produced in the pituitary midlobe, and α-MSH, β-MSH, and γ-MSH exist.
α-MSH is a peptide consisting of the following amino acid residues containing an Acetyl group.
Acetyl- (N) -Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH ₂ (SEQ ID NO: 3)
β-MSH is a peptide consisting of the following amino acid residues.
Ala-Glu-Lys-Lys-Asp-Glu-Gly-Pro-Tyr-Arg-Met-Glu-His-Phe-Arg-Trp-Gly-Ser-Pro-Pro-Lys-Asp (SEQ ID NO: 4)
γ-MSH is a peptide consisting of the following amino acid residues.
Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly (SEQ ID NO: 5)

LPH {(lipotropin), a fat mobilizing hormone} is a single-chain polypeptide produced in the anterior pituitary and middle lobe, and includes γ-LPH and β-LPH.
γ-LPH is a peptide consisting of the following amino acid residues.
Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu (SEQ ID NO: 6)
β-LPH is a peptide consisting of the following amino acid residues.
Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys- Ser-Gln-Thr-Pro-Leu-Val-Thr (SEQ ID NO: 7)

Endorphins (endorphins) are endogenous opioid peptides existing in the mammalian brain and pituitary gland. Three types of α, β, and γ are known.
α-Endorphin is a peptide consisting of the following amino acid residues.
Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys- Ser-Gln-Thr-Pro-Leu-Val-Thr (SEQ ID NO: 8)
β-endorphin is a peptide consisting of the following amino acid residues.
Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ile-Ile-Lys-Asn- Ala-Tyr-Lys-Lys-Gly-Glu (SEQ ID NO: 9)
γ-endorphin is a peptide consisting of the following amino acid residues.
Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu (SEQ ID NO: 10)

CLIP (cardiotropin-like peptide) is a peptide consisting of the following amino acid residues.
Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala-Glu-Ala-Phe-Pro-Leu-Glu-Phe (SEQ ID NO: 11)

The above MSH (α-, β-, γ-MSH), ACTH, LPH (γ-, β-LPH), endorphins (α, β, γ-endorphin) or CLIP are some amino acids among various types. May be different.
For example, in ACTH, the whale has the same sequence as SEQ ID NO: 1, but in pigs, the 31st Ser of SEQ ID NO: 1 is Leu, and in sheep and cattle, the 33rd Glu of SEQ ID NO: 1 is Gln. It has become.
In the present invention, MSH (α-, β-, γ-MSH), ACTH, LPH (γ-, β-LPH), endorphins (α, β, γ-endorphin) and CLIP which are different among various compounds are also derived from POMC. Include as.

In addition, the present inventors have confirmed that “ACTH1-39, ACTH1-24 and CLIP have an erythrocyte enucleation-inducing action”. Therefore, it is considered that peptides having the following amino acid residues common to ACTH1-39, ACTH1-24, and CLIP are responsible for erythrocyte enucleation induction. Therefore, the following peptides are also included as POMC-derived compounds.
Arg-Pro-Val-Lys-Val-Tyr-Pro (SEQ ID NO: 12)

In addition, the present invention provides a peptide comprising any of the peptides described above, has 90% or more homology with any of the peptides described above, and has an effect of inducing nucleation of erythrocytes of substantially the same quality as the peptide. Peptides or peptides having 1 to 5 amino acid substitutions, deletions, insertions and / or additions to peptides described in any of the above and having a substantially homogeneous erythrocyte enucleation inducing action are also derived from POMC It is included as a compound.

The “peptide containing the peptide described in SEQ ID NO: 1” substantially maintains the enucleation of erythrocytes of the peptide described in SEQ ID NO: 1, and is 1-30, 1-20 at the N-terminus and / or C-terminus. , 1 to 10, 1 to 5 means a peptide added with any amino acid.

"Enucleation-inducing action substantially the same quality as the peptide of SEQ ID NO: 1" means that the peptide has a nucleoblast-denucleating action of the erythroblast, the degree of which is compared with that of the peptide. It can be strong or weak.

“Sequence homology” usually means 70% or more of the entire amino acid sequence, preferably 80%, more preferably 85% or more, still more preferably 90% or more, even more preferably 95% or more, and most preferably 98%. It is suitable that it is% or more.

As a peptide having sequence homology with the peptide represented by the amino acid sequence described in SEQ ID NO: 1, for example, in the amino acid sequence described in SEQ ID NO: 1, for example, 1 to 15, preferably 1 to 10, more preferably Amino acid sequence having 1 to 5, even more preferably 1 to 3, even more preferably 1 to 2, most preferably 1 amino acid mutation, such as a deletion, substitution, addition or insertion mutation The peptide represented by these can be illustrated. The degree of amino acid mutation and the position thereof are not particularly limited as long as the peptide having the mutation has substantially the same quality of enucleation as the peptide represented by the amino acid sequence shown in SEQ ID NO: 1. .

(Preservation method of enucleated red blood cells)
When storing enucleated erythrocytes (including a long period of time), a method known per se can be used. Examples of storage methods include cryopreservation methods. In this case, Cell Banker (C) (Nippon Zenyaku Kogyo Co., Ltd.), glycerin, ethylene glycol, dimethyl sulfoxide (DMSO), sucrose, glucose, polyvinyl as necessary. A cryoprotectant such as pyrrolidone (PVP) or trehalose may be added, slowly frozen using a program freezer, etc., and then stored in liquid nitrogen or a deep freezer at -80 ° C.

Hereinafter, the present invention will be described in detail with specific examples, but the present invention is not limited to these examples.

(Materials and methods)
The materials and methods used in the examples are as follows.

Hematopoietic progenitor growth medium (HPGM, Lonza) was used as the culture medium in this example.
(Culture conditions in each phase)
(1) Expansion phase
Cells: Cord blood-derived or bone marrow-derived CD34 positive hematopoietic progenitor cells 1 × 10 ⁵ (cells / ml) (Purity 90% or more, Lonza)
Culture days: 7 days Addition factor of culture solution: 50 ng / ml thrombopoietin (Peprotech), 50 ng / ml Flt3 ligand (Peprotech), 50 ng / ml Stem Cell Factor (SCF) (R & D)
Temperature: 37 ° C
Oxygen concentration: 2.0% to 20.0%
(2) Differentiation phase
Cells: The cells amplified in (1) above were diluted to 2 × 10 ⁵ (cells / ml).
Culture days: 3 days Factors added to the culture: 3 U / ml erythropoietin (Kyowa Hakko Kirin), 25 ng / ml SCF, 10 ng / ml interleukin-3 (IL-3) (Peprotech), 10 ng / ml interleukin-6 (IL-6) (Peprotech)
Temperature: 37 ° C
Oxygen concentration: 2.0% to 20.0%
(3) Maturation Phase 1 (first maturity)
The culture start time is designated as M0, and hereinafter referred to as M0, M1, and M2.
Cells: The cells cultured in (2) above were diluted to 1 × 10 ⁵ (cells / ml).
Culture days: 3 days (M0-M3)
Factors added to the culture medium: 3 U / ml erythropoietin Temperature: 37 ° C
Oxygen concentration: 2.0% to 20.0%
(4) Maturation Phase 2 (2nd maturity: M3 or later)
Cells: The cells cultured in (3) above were diluted to 4 × 10 ⁵ (cells / ml).
Culture days: Measure enucleation rate after M5 Addition factor to culture medium: None Temperature: 37 ° C
Oxygen concentration: 2.0% to 20.0%

(Method of measuring enucleation rate by flow cytometry)
The method for measuring the enucleation rate by flow cytometry is as follows.
(1) Cell staining reagent Nuclear staining: SYTO16 (Molecular Probes)
Red blood cell marker: APC label-glycophorin A (GPA) (Becton Dickinson)
(2) Measurement method The cells stained in (1) above were measured by flow cytometry. The enucleation rate was calculated by the following formula. In measurement by flow cytometry, only red blood cells from which nuclei have been completely ejected (away from the cell membrane) are measured as enucleated cells. Measured as (non-nucleated cells).
Datsukakuritsu (%) = 100 × {GPA + SYTO16 - / (GPA + SYTO16 - + GPA + SYTO16 +)}
Enucleated cells (mature red blood cells): GPA ⁺ SYTO16 ^-
Akablast: GPA + SYTO16 +

(Method of measuring the enucleation rate under a microscope)
The method for measuring the enucleation rate under a microscope is as follows.
(1) Cell staining reagent Nuclear staining: Hoechst 33342 (Molecular Probes)
(2) Measurement method The cells stained with the staining reagent of (1) above were photographed under 10 fields of view (phase contrast image + fluorescence image) under a fluorescence microscope, and cells with and without nuclei were counted. The enucleation rate was calculated by the following formula. In addition, the definition of the enucleated cell is defined as a cell during enucleation in which the nucleus protrudes outside the circumference of the cell membrane, or the nucleus does not exist in the cell membrane. Those having other nuclei in the cell membrane were defined as nucleated cells. More specifically, the enucleation rate under the microscope is measured as all enucleated cells as enucleated cells. Therefore, the enucleation rate in flow cytometry (the one with the nucleus attached to the cell membrane) Compared to nucleated cells). In addition, the difference between the enucleation rate in flow cytometry and the enucleation rate under the microscope is the difference between the enucleated cells (the enucleation was induced, but the nuclei were not completely discharged from the cells and adhered to the cell membrane. )).
Enucleation rate = enucleated cells / whole cells (nucleated cells + enucleated cells)

(Effects of promoting the enucleation of erythrocytes and promoting enucleation of red blood cells under hypoxic conditions)
Erythrocyte cultures under hypoxic conditions (2% to 10% O ₂ ) are more effective in promoting the enucleation initiation time and denucleation compared to erythrocyte cultures under normal aerobic culture conditions (20% O ₂ ). It was confirmed whether there was a nuclear induction promotion effect. Details are as follows.

Under the following culture conditions, the enucleation rate on the 8th day of mature culture (M8) was measured by flow cytometry.
<Culture conditions>
Cells: Cord blood-derived CD34-positive hematopoietic progenitor cells Culture medium: HPGM + EPO (without adding ACTH)
Oxygen concentration (O ₂ ): 2, 5, 10, 20%

FIG. 1 shows the enucleation rate under each oxygen concentration condition.
Hypoxic conditions (2% -10% O ₂ ) had a 4-day enucleation rate about 4 times higher than aerobic culture conditions (20% O ₂ ). That is, under the hypoxic condition (2% to 10% O ₂ ), the effect of promoting the enucleation start time is exhibited.
Furthermore, hyponuclear conditions (2-10% O ₂ ) have a very high enucleation rate on day 7 compared to aerobic culture conditions (20% O ₂ ). In particular, the enucleation rate was about 1.8 times compared to 78% under 5% O ₂ conditions and 45% under aerobic culture conditions (20% O ₂ ). This is a very high value for the enucleation rate under physiological conditions (addition of non-physiological molecules and non-physiological conditions are not used).
From the above, it was confirmed that erythrocyte culture under hypoxic conditions (2% to 10% O ₂ ) has an effect of promoting the enucleation initiation time and the effect of promoting enucleation induction.

(Confirmation of increased survival rate of enucleated erythrocytes under hypoxic conditions)
Is erythrocyte culture under hypoxic conditions (2% to 5% O ₂ ) more effective in increasing the survival rate of enucleated erythrocytes compared to erythrocytes under normal aerobic culture conditions (20% O ₂ )? I confirmed. Details are as follows.

Under the following culture conditions, the enucleation rate on the 11th day of mature culture (M11) was measured by flow cytometry.
<Culture conditions>
Cells: Cord blood-derived CD34-positive hematopoietic progenitor cells Culture medium: HPGM + EPO (without adding ACTH)
Oxygen concentration (O ₂ ): 2, 5, 20%

The survival rate of enucleated erythrocytes under each oxygen concentration condition is shown in FIG.
Under normal aerobic culture conditions (20% O ₂ ), enucleated erythrocytes rapidly denatured and disappeared after M8, and thus could not be maintained. Regarding the enucleated red blood cell / total cell ratio under normal aerobic culture conditions (20% O ₂ ), it was 43.3% on the 8th day of the mature culture, but rapidly decreased to 3.2% on the 11th day. That is, only about 7.4% (3.2 / 43.3%) of enucleated erythrocytes could be maintained.
On the other hand, under the hypoxic condition (2%, 5% O ₂ ), the survival rate on the 11th day was very high compared with the aerobic culture condition (20% O ₂ ). More specifically, the 5% O ₂ enucleated red blood cell / total cell ratio under hypoxic conditions was 75.9% on the 8th day of mature culture compared to only 41.1% on the 11th day. There wasn't. That is, approximately 54.2% (41.1 / 75.9%) of enucleated erythrocytes could be maintained.
Based on the above, culturing with 5% O ₂ under hypoxic conditions is approximately 7.3 times (54.2 / 7.4%) of detachment compared to culturing under normal aerobic culturing conditions (20% O ₂ ). It showed the effect of increasing the survival rate of nuclear red blood cells.

(Confirmation of erythrocyte enucleation induction promotion effect under hypoxic condition and POMC-derived compound addition condition and confirmation of nuclear discharge effect under hypoxic condition)
Erythrocyte culturing under hypoxic conditions (2% to 5% O ₂ ) and under the addition of POMC-derived compounds is promoted to promote enucleation compared to erythrocytes under normal aerobic culture conditions (20% O ₂ ) It was confirmed whether it was effective. Furthermore, the effect of removing the enucleated nucleus from the cell membrane (nuclear emission effect) was also confirmed. Details are as follows.

Under the following culture conditions, the enucleation rate on the 7th day of mature culture (M7) was measured by counting enucleated erythrocytes after nuclear staining with Hoechst under a fluorescence microscope.
<Culture conditions>
Cells: CD34-positive hematopoietic progenitor cells derived from cord blood Culture medium: Addition of ACTH, a compound derived from HPGM + EPO + POMC {addition time is the first day of mature culture (M0) and the third day (M3)}
Oxygen concentration (O ₂ ): 5, 20%

FIG. 3 shows the enucleation rate of erythrocytes under each oxygen concentration condition and with or without ACTH (A39 in the figure).
The enucleation rate under hypoxic conditions and under POMC-derived compound addition conditions (O ₂ 5% A39) was very high, exceeding 90%. Under low oxygen conditions, POMC-derived compound-free condition (O ₂ 5% CNT) was 81%, and the denuclearization promoting effect of POMC-derived compounds was confirmed even under low oxygen conditions.
The enucleation rate under hypoxic conditions and POMC-derived compound addition conditions is about 1.3 times that of normal aerobic culture conditions (20% O ₂ ) and POMC-derived compound addition conditions. it was high.
Thereby, it was confirmed that the enucleation rate can be increased by the low oxygen condition and the addition of the POMC-derived compound.
In addition, the value of the enucleation rate on the 7th day of O ₂ 5% as measured by the flow cytometry in FIG. 1 and the value of the enucleation rate on the 7th day of O ₂ 5% as measured under the microscope in FIG. Is 78% in the flow cytometry measurement, which is almost the same as 80% under the microscope. On the other hand, regarding the aerobic culture condition (20%), the enucleation rate value by flow cytometry was 45%, and the enucleation rate value under a microscope was 55%. These figures indicate that the nucleus is almost completely excreted in cells that have been enucleated under hypoxic conditions, but the nucleus remains in about 10% of cells that are enucleated under aerobic conditions. It was confirmed that the cells were not detached. That is, it was confirmed that low oxygen conditions have a nuclear emission effect.

(Confirmation of improved expression of GATA1, GATA2 and MXI1 in enucleated erythrocytes under hypoxic conditions)
Erythrocyte culture under hypoxic conditions (5% O ₂ ) has improved expression of GATA1, GATA2 and MXI1 in enucleated erythrocytes compared to normal erythrocyte culture conditions (20% O ₂ ) To see if it is. Details are as follows.

<Culture conditions>
Cells: Cord blood-derived CD34-positive hematopoietic progenitor cells Culture method: Culture method described in Example 1 (without adding ACTH)
Oxygen concentration (O ₂ ): Culture was carried out at 20% oxygen concentration until the start of Maturation (M0 day), and cells were divided into two groups of oxygen 5% and 20% on M0 day.
Measurement of expression levels of GATA1, GATA2 and MXI1: On day M3, cells were collected by centrifugation, RNA extraction, and real-time PCR were performed.

The expression levels of GATA1, GATA2 and MXI1 under each oxygen concentration condition are shown in FIG.
In the transcription factors GATA1 and GATA2 required for erythroblast differentiation and the transcription factor MXI1, which induces nuclear enrichment before the enucleation process, all gene expression levels under hypoxic conditions are gene expression under normal oxygen conditions There was a marked increase compared to the amount.

(Confirmation of improved expression of MC1R, MC2R and MC5R in enucleated erythrocytes under hypoxic conditions)
Erythrocyte culture under hypoxic conditions (5% O ₂ ) improves MC1R, MC2R and MC5R expression of enucleated erythrocytes compared to erythrocytes under normal aerobic culture conditions (20% O ₂ ). To see if it is. Details are as follows.

<Culture conditions>
Cell: Cord blood-derived CD34-positive hematopoietic progenitor cells Culture method: Culture method described in Example 1 (ACTH is not added)
Oxygen concentration (O ₂ ): Culture was carried out at 20% oxygen concentration until the start of Maturation (M0 day), and cells were divided into two groups of oxygen 5% and 20% on M0 day.
Measurement of expression levels of MC1R, MC2R and MC5R: On day M3, cells were collected by centrifugation, RNA extraction, and real-time PCR were performed.

The expression levels of MC1R, MC2R and MC5R under each oxygen concentration condition are shown in FIG.
The gene expression levels of all MC1R, MC2R and MC5R under hypoxic conditions were remarkably increased compared to the gene expression levels under normal oxygen conditions.
Thus, it is considered that the enucleation promoting effect by ACTH can be obtained by increasing the expression of the receptor even without addition of ACTH.

(Confirmation of the effect of promoting enucleation of bone marrow-derived CD34-positive hematopoietic progenitor erythrocytes under hypoxic conditions)
Is erythrocyte culture under hypoxic conditions (2%, 10% O ₂ ) more effective in promoting enucleation induction than erythrocyte culture under normal aerobic culture conditions (20% O ₂ )? I confirmed. Details are as follows.

Under the following culture conditions, the enucleation rate on the seventh day of mature culture (M7) was measured by flow cytometry.
<Culture conditions>
Cells: Bone marrow blood-derived CD34 positive hematopoietic progenitor cells (Lonza)
Culturing method: Culturing was carried out in the same manner as in Example 1. ACTH is not added.
Oxygen concentration (O ₂ ): 2, 10, 20%

FIG. 6 shows the enucleation rate under each oxygen concentration condition. The denuclearization rate was 24.8% at 20% oxygen, 31.5% at 10% oxygen, and 38.4% (P <0.01) at 2% oxygen.
That is, an increase in the enucleation rate was recognized depending on the oxygen concentration. In particular, the hyponuclear condition with 2% oxygen showed an effect of increasing the enucleation rate by about 1.5 times compared with the condition with 20% oxygen.
This also corresponds to the effect of umbilical cord blood-derived cells on bone marrow-derived cells (the enucleation rate under oxygen 5% condition is 1.8 times higher than the enucleation rate under oxygen 20% condition) The result to be obtained.
As described above, even in hematopoietic progenitor cells of different origins, the enucleation rate can be improved by 1.5 to 1.8 times under hypoxic conditions.

(General)
According to the results of Examples 2 to 4, culturing erythroblasts under hypoxic conditions has an effect of promoting the enucleation initiation time, the effect of promoting the enucleation induction, the effect of promoting nuclear ejection, and the effect of increasing the survival rate of enucleated erythrocytes. Show. Furthermore, the enucleation rate of the erythroblast culture under the hypoxic condition and the POMC-derived compound addition condition is higher than the enucleation rate of the erythroblast culture under the hypoxic condition.
From the results of Examples 5 to 6, the expression of GATA1, GATA2, and MXI1 is improved in the culture of erythroblasts under hypoxic conditions.
According to the results of Example 7, nucleation of erythroblasts under hypoxic conditions can improve the enucleation rate regardless of the origin of hematopoietic progenitor cells.
As described above, the erythroblast enucleation method of the present invention was able to induce and maintain the enucleation of the final stage of mammalian erythrocyte differentiation in a short time.

The present invention was able to provide a method for enucleation that can induce and maintain the enucleation of the final stage of erythroid differentiation in a short time.
As a result, the blood production period can be shortened, and the risk of canceration associated with transfusion of erythrocyte progenitor cells and erythroblasts can be eliminated. That is, safe blood can be obtained stably in a short time, and the contribution to medical technology is very large.

Claims (9)

1. A method for enucleating erythroblasts and / or maintaining enucleated erythrocytes, comprising culturing hematopoietic stem cells, erythroid progenitor cells, and / or erythroblasts in a culture solution under oxygen conditions of 1.0% to 18.0% Method.
2. The method according to claim 1, wherein the oxygen condition is 2.0% to 10.0%.
3. The method according to claim 1 or 2, wherein the oxygen condition is 3.0% to 7.0%.
4. The method according to any one of claims 1 to 3, wherein the oxygen condition is 4.0% to 6.0%.
5. The method according to any one of claims 1 to 4, wherein the culture solution contains a compound derived from proopiomelanocortin (POMC).
6. The method according to claim 5, wherein the compound is selected from any one of the following.
   (1) ACTH
   (2) POMC
   (3) MSH
   (4) LPH
   (5) Endorphins (6) CLIP
7. The method according to claim 5 or 6, wherein the compound is ACTH.
8. The method according to any one of claims 1 to 7, wherein the expression of GATA1, GATA2 and / or MXI1 is improved.
9. The method according to any one of claims 1 to 8, wherein expression of MC1R, MC2R and / or MC5R is improved.

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