WO2023207244A1 - Molecular marker for red blood cell denucleation and use thereof - Google Patents

Abstract

The present application relates to a molecular marker for red blood cell denucleation and a use thereof. According to a method for identifying or sorting red blood cells being denucleated, by detecting markers such as CD44, CD235a or Ter119, and CD45, red blood cells being denucleated are specifically identified.

Description

Molecular markers of red blood cell denucleation and their uses

This application claims priority from the Chinese patent application (Application No. 2022104528957) submitted on April 27, 2022.

Technical field

This application involves the fields of medicine, biology, and clinical diagnosis. Specifically, it relates to molecular markers of red blood cell denucleation and their uses.

Background technique

Red blood cells are the main carriers of oxygen in the body and are essential for maintaining life. In the treatment of diseases such as hemorrhagic anemia, thalassemia, and hematopoietic dysfunction, red blood cell transfusion is the main means of saving patients' lives. However, there are currently problems such as insufficient supply and the inability to completely eliminate blood-borne viruses. Therefore, the development of new blood sources is an urgent need for clinical treatment of anemia.

Inducing erythroid differentiation and denucleation to produce mature red blood cells through an in vitro culture system can provide a continuous and safe blood source. In the in vitro hematopoietic system, hematopoietic stem/progenitor cells, embryonic stem cells, induced pluripotent stem cells, and immortalized adult erythroid progenitor cells from umbilical cord blood, adult bone marrow, and peripheral blood are often used to induce erythroid differentiation in vitro. However, the source of hematopoietic stem and progenitor cells is limited. In the in vitro culture system of embryonic stem cells, induced pluripotent stem cells, and immortalized adult erythroid progenitor cells, the low efficiency of red blood cell denucleation is one of the main reasons that hinders efficient hematopoiesis in vitro.

Hematopoiesis begins with hematopoietic stem cells, in which the proliferation rate of erythroid hematopoietic development decreases significantly after completing the massive expansion of the commitment and progenitor cell stages, and enters the terminal differentiation stage, where a large amount of erythroid-specific expression products accumulate, providing the basis for the production of morphologically and functionally mature cells. Red blood cells make final preparations. The terminal differentiation process of the erythroid system is accompanied by 3-5 cell divisions, which include: proerythroblast (hereinafter referred to as ProE), proerythroblast (Basophilic erythroblast, BasoE), intermediate erythroblast (Polychromatic erythroblast, PolyE), late erythroblast ( Orthochromatic erythroblast, OrthoE) four nucleated cell stages (Doty RT et al. Single-cell analyzes demonstrate that a heme-GATA1 feedback loop regulates red cell differentiation. Blood. 2019,133(5):457-469). During this process, the cells gradually become smaller, hemoglobin accumulates, the nucleus shrinks, and finally denucleates. Reticulocytes and nuclei (pyrenocytes) coated only by a thin layer of cytoplasm (McGrath, Kathleen E et al.Enucleation of primitive erythroid cells generates a transient population of "pyrenocytes" in the mammalian fetus.Blood.2008,111( 4):2409-17; Sankaran, Vijay G et al. Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number. Genes&development. 2012, 26(18): 2075-87). The extruded erythrocyte nuclei (pyrenocytes) are phagocytosed by central macrophages of blood islands or other macrophages in the bone marrow in a membrane phosphatidylserine-dependent manner (Chasis, Joel Anne, and Narla Mohandas. Erythroblastic islands: niches for erythropoiesis. Blood. 2008.112(3):470-8). The reticulocytes further degrade the remaining RNA and organelles, generate mature red blood cells and release them into the blood circulation to perform the function of transporting oxygen (Pasini, Erica M et al. In-depth analysis of the membrane and cytosolic proteome of red blood cells. Blood .2006, 108(3):791-801). Mature red blood cells can survive for 120 days in humans and 55 days in mice.

Red blood cell denucleation is a key step in in vitro erythroid differentiation to produce mature red blood cells, involving complex dynamic cell deformation. The use of cell immunofluorescence and flow imaging technology can help reveal cell deformation and molecular mechanisms during the denucleation process. Studies have found that erythroblasts before denucleation need to undergo chromatin condensation and nuclear condensation, exit the cell cycle, and then undergo nuclear polarization, and then the nuclei are expelled from the cytoplasm, and the nuclei separated from the nascent reticulin are Phagocytosis by macrophages. Such dramatic cellular changes involve the participation of a variety of biological processes, such as membrane protein sorting, cytoskeleton remodeling, vesicle transport, vacuole fusion, organelle clearance, etc. A variety of cytoskeletal proteins play an important role in this process, such as microtubules that help establish cell polarity; actin and myosin interact to form a CAR contractile ring between the nucleus and new reticulocytes. Affecting the remodeling of skeleton proteins will significantly hinder the denucleation process and cause morphological and functional abnormalities in the reticulum produced. Lipid raft aggregation helps separate the nucleus and form new cell membranes (Konstantinidis, Diamantis G et al. "Signaling and cytoskeletal requirements in erythroblast enucleation." Blood vol. 119, 25 (2012): 6118-27).

In addition, the denucleation process of red blood cells is accompanied by the sorting of membrane proteins. Studies on in vitro culture of human erythroblasts suggest that cytoskeletal proteins, spectrin and glycophorin, which are important for the shape and deformability of red blood cells, are sorted into the network. Histoerythrocyte membrane, while those related to cell adhesion Proteins such as Emp1 and β1 integrin are sorted into the pyrenocyte (Bell, Amanda J et al. “Protein distribution during human erythroblast enucleation in vitro.” PloS one vol. 8,4(2013):e60300 ). In a study of in vitro erythroid differentiation of mouse bone marrow, 75% _of EMP and ₇₀ % of the β1 subunit of _α4β1 and _α5β1 integrins partitioned to the plasma membrane surrounding the nucleus. This process will lead to efficient binding of macrophages at this site, thereby promoting phagocytosis (Lee, James CM et al. "Mechanism of protein sorting during erythroblast enucleation: role of cytoskeletal connectivity" Blood vol. 103,5 (2004) :1912-9).

The above-mentioned research on the regulatory mechanism of erythrocyte denucleation is often based on morphological detection. In view of this, there is still a need in the field for effective surface markers to identify and enrich red blood cells undergoing enucleation.

Contents of the invention

In response to the aforementioned needs in the art, the use of reagents targeting CD45 (especially human CD45) in preparing a detection device is provided, wherein the detection device is in the form of a kit or a chip, and the detection device is used to determine the isolation of autologous The denucleation state of erythroid cells in or from in vitro culture, the denucleation state is selected from any one of the following: denucleation preparation/not denucleation, denucleation in progress, denucleation, the reagent targeting CD45 can Determine the presence of CD45 or determine the level of CD45 and be selected from any of the following: an anti-CD45 antibody or antigen-binding fragment thereof, a primer targeting CD45, or a probe targeting CD45. In some embodiments, the denucleation being performed is selected from any of the following: early denucleation, late denucleation.

In some embodiments, when the erythroid cells are CD45 positive, the proportion of the erythroid cells that are determined to be in a denucleating state, or the erythroid cells that are determined to be in a denucleating state is statistically significantly increased.

In other embodiments, when the erythroid cells are CD45 negative, the proportion of the erythroid cells that are determined to be ready for denucleation or in a state of being denucleated is statistically significantly reduced.

In some embodiments, the detection device further includes a reagent that targets CD235a (also known as Ter119); the reagent that targets CD235a refers to a reagent that determines the presence of CD235a or determines the level of CD235a. The reagent targeting CD235a is selected from any one of the following: anti-CD235a antibody or antigen-binding fragment thereof, primer targeting CD235a, targeting Probe for CD235a.

In some embodiments, when the erythroid cells are positive for CD235a and CD45, the proportion of the erythroid cells that are determined to be in a denucleating state, or the erythroid cells that are determined to be in a denucleating state is statistically significantly increased.

In some embodiments, when CD235a and CD45 are co-localized on the surface of erythroid cells, the erythroid cells are determined to be in a denucleating state, or the proportion of the erythroid cells determined to be in a denucleating state is statistically significant significantly improved.

In some embodiments, the detection device further comprises a reagent targeting CD44; the reagent targeting CD44 refers to a reagent that determines whether CD44 is present or determines the level of CD44; the reagent targeting CD44 is selected from any of the following One item: anti-CD44 antibody or antigen-binding fragment thereof, primers targeting CD44, and probes targeting CD44.

In some embodiments, when CD44 and CD45 are co-localized on the surface of the prolapsed cell nucleus, the erythroid cell is determined to have been enucleated, or the denucleation rate of the erythroid cell is determined to be statistically significantly improved.

In some embodiments, the detection device further comprises an agent targeting any of the following, or a combination thereof: hemoglobin, ApoE, Vcam1, C1qa, C1qb, C1qc, Ly6D, Ighm, Igkc, Cd79a, CD79b, Ebf1, Pax5, Lef1, Elane, S100A8/A9, Prtn3, Cebpb, Lyz2, S100a4, Lgals3.

In some embodiments, when the erythroid cells highly express hemoglobin, ApoE, Vcam1, C1qa, C1qb, and C1qc at the RNA level, and the erythroid cells are positive for CD44 and CD45 at the protein level, the erythroid cells are determined to be in degeneration. The ratio of the state of nuclear late stage or the state of the erythroid cells being judged to be in the late stage of denucleation was statistically significantly improved.

In some embodiments, when erythroid cells highly express one of Ly6D, Ighm, Igkc, Cd79a, CD79b, Ebf1, Pax5, Lef1, Elane, S100A8/A9, Prtn3, Cebpb, Lyz2, S100a4, and Lgals3 at the RNA level or more, and when CD44, CD235a positive and CD45 negative are present at the protein level, it is determined that the denucleation potential of the erythroid cells is significantly improved.

In some embodiments, the determination is at the RNA level or at the protein level.

In some embodiments, the antibody is derived from any of the following: murine, rabbit, human, camel, canine, ovine, equine, recombinantly expressed antibody.

In some specific embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

In some specific embodiments, the antigen-binding fragment is selected from any one of: Fab, Fab', F(ab')2, Fv fragment, single chain antibody, domain antibody, multispecific antibody.

This application also provides a method for enriching erythroid cells undergoing denucleation in vitro, including the steps:

1) Providing erythroid cells in vitro, the erythroid cells isolated from the body or from in vitro culture;

2) contacting said erythroid cells with a CD45-targeting agent according to the present application,

3) Sort CD45-positive erythroid cells, preferably by flow cytometry.

In some embodiments, the CD45 refers to human CD45.

In some embodiments, the methods are not used for diagnosis or treatment of disease.

This application also provides a method for enriching erythroid cells undergoing denucleation in vitro, including the steps:

1) Provide erythroid cells in vitro,

2.1) contacting said erythroid cells with a CD45-targeting agent according to the present application,

2.2) Optionally, contacting said erythroid cells with a CD44-targeting agent according to the present application,

2.3) Optionally, contacting said erythroid cells with an agent targeting CD235a according to the present application,

3) Sorting CD45-positive (optionally CD44-positive, optionally CD235a-positive) erythroid cells;

Steps 2.1), 2.2), and 2.3) can be performed in any order or simultaneously.

This application also provides a device (kit or chip) for determining the denucleation status of erythroid cells, where the denucleation status is selected from any one of the following: denucleation preparation/not denucleation, denucleation in progress , has been denuclearized.

In some embodiments, the device comprises any one (at the protein or RNA level) selected from: an agent targeting CD45 according to the application, an agent targeting CD44 according to the application, a target according to the application Reagents for CD235a.

In some embodiments, the device further comprises an agent or combination thereof (at the protein or RNA level) that targets any of the following: hemoglobin, ApoE, Vcam1, C1qa, C1qb, C1qc, an immune signature RNA marker (e.g., Ly6D , Ighm, Igkc, Cd79a, CD79b, Ebf1, Pax5, Lef1, Elane, S100A8/A9, Prtn3, Cebpb, Lyz2, S100a4, Lgals3).

Description of drawings

Figure 1A: Circle the red blood cells undergoing denucleation through flow imaging, and show the enrichment of CD44 high-expressing cells and the corresponding cell fluorescence photos.

Figure 1B: Statistical diagram showing high expression of CD44 in red blood cells undergoing denucleation.

Figure 1C: Flow cytometry analysis and statistics of the proportion of CD44 ^hi Ter119 ⁺ cell population (defined as Nonpro) undergoing denucleation.

Figure 1D: Nonpro cell population was sorted by flow cytometry, and the smear was stained with Giemsa and benzidine.

Figure 1E: Nonpro enriches late-enucleated erythrocytes.

Figure 1F: Representative cell photographs showing the distribution of CD44 protein during red blood cell denucleation.

Figure 1G: Flow cytometry shows the positional relationship between actin and CD44 during red blood cell denucleation.

Figure 1H: Immunofluorescence 3D imaging demonstrates the dynamic distribution of CD44 and Ter119 during the denucleation process.

Figure 1I: Using multispectral immunohistochemistry technology to detect the distribution of CD44 and Ter119 on denucleated red blood cells in mouse bone marrow tissue.

Figure 2A: UMAP analysis of CD45 ^- Nonpro heterogeneity.

Figure 2B: Dotplot diagram showing the expression of erythroid cells, cytoskeleton, transcription factors and immune response-related characteristic genes.

Figure 3A Venn diagram shows functionally enriched GO entries for immune cells and immune red blood cells.

Figure 3B: Clustering heatmap demonstrating biological functions of immune red blood cell-specific enrichment.

Figure 4A: Flow cytometry analysis of the co-expression of CD44 and CD45 protein levels in mouse bone marrow nucleated erythrocytes and denucleated erythrocytes, and statistics of CD45 ⁺ %.

Figure 4B: Flow cytometry analysis of the ratio of CD45 ⁺ nucleated red blood cells to CD45 ⁻ nucleated red blood cells undergoing denucleation.

Figure 4C: Fluorescence photos of cells showing the distribution of signals in enucleated red blood cells.

Figure 4D to Figure 4E: Statistics of the proportion of CD45 ⁺ and CD45 ^- in the early and late stages of denucleation in denucleated red blood cells, and the proportion of nuclear staining intensity subpopulations.

Figure 4F: CD45 weakly positive cells are also present in some anucleated red blood cells.

Figure 4G to Figure 4H: CD45 ⁺ Ter119 ⁺ % is positively correlated with the proportion of red blood cells undergoing denucleation, and negatively correlated with the denucleation rate of red blood cells.

Figure 4I: Immunofluorescence 3D reconstruction shows CD45 ⁺ enucleating red blood cells.

Figure 4J: Immunofluorescence of mouse bone marrow tissue demonstrates the presence of CD45 ⁺ enucleating red blood cells.

Figure 4K: The myosin II inhibitor blebbistatin reverses denucleation of CD45 ⁺ erythrocytes.

Figure 5A: Single-cell sequencing of Nonpro and OrthoE in whole mouse bone marrow by flow sorting.

Figure 5B: UMAP demonstrates cellular heterogeneity in mixed samples of mouse whole bone marrow Nonpro, CD45 ^- Nonpro and Ortho (EB: Erythroblast). The upper right corner shows the UMAP of a single sample, and the lower right corner UMAP shows the RNA content of the combined sample.

Figure 5C: UMAP displays the expression of erythroid signature genes, immune cell signature genes, and Ery/ApoE ⁺ subgroup signature genes.

Figure 6A: Flow cytometry analysis and demonstration of enucleating cells in CD45 ^- Nonpro.

Figure 6B: Giemsa staining of CD45 ^- Nonpro shows mononuclear/granulocyte-like cells.

Figure 6C: Live cell imaging shows that sorted CD45 ^- Nonpro contains 10% to 15% monocyte/granulocyte-like cells.

Figure 6D: Immunofluorescence photos show that CD45 ^- Nonpro produces a large number of new reticulocytes after in vitro culture.

Figure 6E: Flow cytometry analysis after overnight culture of CD45 ^- Nonpro, showing the cell morphology of denucleation preparation (not denucleation), denucleation and denucleation, and the distribution of CD45 protein and Ter119 protein in CD45 ⁺ red blood cells.

Figure 6F: CD45 ⁺ % of each subpopulation after overnight culture with CD45 ^- Nonpro and Ortho.

Figure 7A: Gating strategy for flow imaging analysis of CD45 ⁺ proportion in human bone marrow red blood cells and the proportion of them undergoing denucleation.

Figure 7B: Fluorescence photo of cells showing the morphology of CD45 ⁺ and ^CD45- denucleating red blood cells.

Figure 7C: Statistics of the proportion of CD45 ⁺ and CD45 ^- nucleated erythrocytes undergoing denucleation in human bone marrow nucleated erythrocytes and anucleated erythrocytes.

Figure 7D and Figure 7E: Histogram showing the ratio of CD45 ⁺ and ^CD45- in early and late stages of enucleated red blood cells.

Detailed ways

the term

In the context of this application, "red blood cells" (also called erythroid cells) should be interpreted in the broadest sense, including cells at all stages of development into mature red blood cells; such as, but not limited to, erythroid progenitor cells, proerythroblasts, and proerythroblasts. , intermediate erythrocytes, late erythrocytes, reticulocytes, and mature erythrocytes.

When "red blood cells" refers specifically to cells of a certain stage, the skilled artisan will be able to determine obviously from the context.

In the context of this application, "erythroblasts" refers to nucleated erythroid cells, including the following: proerythroblasts, proerythroblasts, mesoblasts, and metablasts; the population of erythroblasts includes erythroid cells undergoing denucleation. .

In the context of this application, "immune red blood cells" refer to cells that express both immune markers (such as CD45 protein or immune cell signature RNA) and erythroid markers (such as murine Ter119, corresponding to human CD235a).

Immune red blood cells are divided into two categories: 1) immune cell characteristic RNA-positive erythroid cells, 2) CD45 protein-positive erythroid cells. As examples of immune cell signature RNAs, mention can be made of Ly6D, Ighm, Igkc, Cd79a, CD79b, Ebf1, Pax5, Lef1, Elane, S100A8/A9, Prtn3, Cebpb, Lyz2, S100a4, Lgals3. The expression of RNA during denucleation is very diverse and changes rapidly. Therefore, when identifying immune cell characteristic RNA-positive erythroid cells, a single RNA marker is not used, but the profile of the RNA transcriptome is evaluated, for example, using methods known in the art or the method used in Example 2.

In the context of this application, "denucleation" refers to the process by which erythroblasts shed their nuclei to produce mature red blood cells.

In the context of this application, "immune denucleation" means that erythroblasts express the immune marker CD45 protein or myeloid/lymphoid lineage characteristic RNA during the process of removing their nuclei to produce mature red blood cells.

In the context of this application, early denucleation: the process in which erythroid cells begin to remove their nuclei, usually showing the following characteristics: there is Ter119 (i.e., CD235a) signal in flow imaging, the center distance (Delta Centroid) of the nuclear signal is greater than 1, and Less than 4.

In the context of this application, late-stage denucleation usually shows the following characteristics: Ter119 (i.e., CD235a) signal in flow imaging, and the center-to-center distance of the nuclear signal is greater than 4 and less than 8.

In the context of this application, "enucleating" refers to the intermediate process from metablasts to reticulocytes. It usually shows the following characteristics: there is Ter119 (i.e. CD235a) signal in flow imaging, and the center distance of the nuclear signal is greater than 1 and less than 8.

In the context of this application, "completed denuclearization" or "denuclearized" are used interchangeably and refer to a core-free state. Usually, when Ter119 ⁺ and the nuclear signal is negative, it indicates that denucleation has been completed.

In the context of this application, "not denuclearized" is also called denuclearized preparation in this article, which refers to a state in which the center distance of the nuclear signal is less than 1.

In the context of this application, "denucleation potential" means that erythroid cells have activation of denucleation-related regulatory pathways at the transcriptome level.

In the context of this application, "denucleation potential score" refers to the score of denucleation-related regulatory pathways at the transcriptome level (such as but not limited to: calcium ion response, MAPK activity, binding actin, cell deformation, Vesicle transport and other parameters) are scored using the AddModuleScore of the well-known Seurat system. The higher the score, the higher the degree of activation of the pathway.

In the context of this application, "denucleation rate" refers to the percentage of anucleate red blood cells in total erythroid cells.

In the context of this application, "high expression", also expressed as the superscript hi, refers to a higher expression level, signal intensity, or amount relative to a control. From the context and in conjunction with the assay used, the skilled person will be able to understand exactly what control is being referred to. As an example, CD44 ^hi means that the CD44 signal intensity is higher than the CD44 signal intensity of promyeloid BasoE.

Targeting reagents

In this application, the target refers to the object targeted by the targeting reagent of this application; it can be a nucleic acid (gene, mRNA, etc.) or a protein (precursor, isotype). As an example, the target is an antigen (such as the CD45 protein) as the target. As another example, the target is mRNA as the target.

Reagents that target a target (such as CD45) are those that can determine whether the target is present (qualitative) or determine the level of the target (quantitative). The determination may be at the protein level or at the nucleic acid level.

In some embodiments, when determining the presence of a target or determining the level of a target at the protein level, the agent targeting the target is an antibody or antigen-binding fragment thereof against the target.

"Antigen" refers to a molecule or portion of a molecule that is specifically recognized or bound by an antigen-binding protein (eg, an antibody). An antigen can have one or more epitopes. An "epitope" refers to a region on an antigen capable of specifically binding to an antibody or antigen-binding fragment thereof. An epitope can be formed from a contiguous string of amino acids (linear epitope); or contain non-contiguous amino acids (conformational epitope).

"Capable of specifically binding", "specifically binding" or "binding" means that an antibody is capable of binding to a target antigen or epitope thereof with higher affinity than other antigens or epitopes. Typically, antibodies bind an antigen or epitope thereof with an equilibrium dissociation constant (KD) of about 1×10 ⁻⁷ M or less (eg, about 1×10 ⁻⁸ M or less). KD can be measured using known methods, for example by Measured by surface plasmon resonance assay.

"Antibody" is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies; full-length antibodies and antibody fragments, so long as they exhibit the desired Antigen binding activity is sufficient.

"Antibody fragment" or "antigen-binding fragment" refers to a molecule that is different from an intact antibody and contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds (eg, CD45, CD44, Ter119). Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'F(ab')2, single domain antibody, single chain Fab (scFab), diabody, linear antibody, scFv; and polypeptides formed from antibody fragments. specific antibodies.

Skilled persons understand that the technical effects of this application do not depend on specific antibody strains. As long as they are antibodies or antigen-binding fragments that can target targets (such as antigens), they can implement the technical solutions of this application, which can be commercially available antibodies or experimental antibodies. Antibodies prepared in chamber.

CD45 should be interpreted broadly and refers to various forms of molecules of the CD45 gene in various stages, such as but not limited to molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification of the CD45 gene. , such as cDNA, mRNA, precursor proteins, mature proteins, natural variants, modified forms, and fragments thereof. As an example, CD45 is the CD45 protein. As an example, CD45 is human CD45. Nucleic acid and amino acid sequence information for CD45 can be obtained from databases such as Genbank No. 5788 or Uniprot No. P08575.

CD44 should be interpreted broadly and refers to various forms of molecules of the CD44 gene in various stages, such as but not limited to molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification processes of the CD44 gene. , such as cDNA, mRNA, precursor proteins, mature proteins, natural variants, modified forms, and fragments thereof. As an example, CD44 is human CD44. Nucleic acid and amino acid sequence information for CD44 can be obtained from databases such as Genbank No. 960 or Uniprot No. P16070.

Ter119 should be interpreted broadly and refers to various forms of molecules of the Ter119 gene at various stages, such as but not limited to molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification processes of the Ter119 gene. , such as cDNA, mRNA, precursor proteins, mature proteins, natural variants, modified forms, and fragments thereof. As an example, Ter119 is mouse Ter119. Nucleic acid and amino acid sequence information for Ter119 can be obtained from databases, such as Genbank No. 14934 or Uniprot No. P14220. The human homolog of mouse Ter119 is GYPA (also known as CD235a). Nucleic acid and amino acid sequence information for GYPA can be obtained from databases such as Genbank No. 2993 or Uniprot No. P02724.

In some embodiments, when determining the presence or level of a target at the nucleic acid (eg, RNA) level, the reagent targeting the target is in the form of a primer (pair) or probe that recognizes and binds to a segment or all of the target nucleic acid. long sequence.

A primer refers to a molecule with a specific nucleotide sequence that promotes synthesis when nucleotide polymerization is initiated. Primers are usually two artificially synthesized nucleotide sequences. One primer is complementary to one end of the target region (or template, target sequence), and the other primer is complementary to the other end of the target region. Its function is to polymerize nucleotides. The starting point, so that the nucleic acid polymerase can start synthesizing a new nucleotide chain along its 3' end.

Primers can be DNA primers or RNA primers. In the specific examples of this application, RNA primers are preferred. It should be understood that DNA primers corresponding to RNA primers still fall within the scope of the present application. Since primers usually come in pairs, they are called primer pairs. One primer in the primer pair is specific upstream of the target sequence and serves as the forward primer; the other primer is specific downstream of the target sequence and serves as the reverse primer.

When the target sequence is given, technicians follow the principles of textbooks and nucleotide sequence complementarity (such as "Molecular Cloning Experiment Guide"2017; P450 "Designing PCR Primers Using Primer3 Plus"; Chapter 13 "Labeled DNA Probes, RNA Probes""Preparation of needles and oligonucleotide probes"), know the principles of primer amplification of target sequences, the principles of probes binding to target sequences, and the design principles of primers and probes. There are a variety of primer/probe design software in the existing technology, such as Primer Premier, Oligo7, BeaconDesigner, etc. When the skilled person knows the target sequence, he can refer to and obtain the sequence information and structural information of specific primers or probes. Therefore, the technical solutions of the present application are not limited to specific primer pairs or probe sequences.

As an example, the length of the primer/probe does not exceed 50 nt, such as but not limited to 1, 2, 3, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 46, 48, 50 nt.

Detection device

The skilled person is aware that the detection device may be embodied in any known or future form, for example in the form of a reagent (or assembled into a kit) or a chip.

When the detection device is in the form of a reagent (or kit), it contains the targeting reagent of the present application, prepared in the form of a liquid or lyophilized powder. When the detection device is in the form of a chip, the targeting reagent of the present application is bound (coated) to the solid carrier.

One or more targeting agents according to the present application may be present in the form of conjugates or labels to obtain detectable/quantifiable signals. When used with suitable labels or detectable biomolecules (or chemicals), targeting reagents are especially useful for identification, identification, differentiation, sorting, localization, diagnosis and other applications in vitro and in vivo.

Labels for immunoassays are known to those skilled in the art and include enzymes, radioisotopes, fluorescence, luminescence, particles (eg latex, magnetic particles), chromogenic substances (eg colloidal gold).

As an example of particular interest, antibodies (or fragments thereof), primers, or probes are labeled with a detectable label (e.g., enzyme, fluorescent, radioactive label, etc.) to enable visualization, quantification, sorting, and /or enrichment.

In some embodiments, a detection device includes at least one container containing each targeting agent of the present application or a combination thereof.

Enrichment method

The present application provides methods for enriching target cell populations, especially methods for enriching red blood cells undergoing denucleation.

The skilled person understands that enrichment should not be construed restrictively as purification.

In some embodiments, "enrichment" is performed such that the proportion of the target cell population in the final cell population is significantly higher than the proportion of the target cell population in the initial cell population. As an example, in the initial cell population, the initial cell population is contacted with the targeting reagent of the present application, the target cell population bound by the targeting reagent is identified, and the identified target cell population is sorted to collect the final cells. group, thereby increasing the proportion of the target cell group.

Methods of sorting are well known in the art. For example, a carrier can be used for capture. Another example is using flow cytometry to sort red blood cells that are being denucleated.

In this application, "significant" means that there is a statistically significant difference, especially at a set p-value level. For example, p-values are set to 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, or even lower. For example, two groups are considered to have a statistically significant difference when their p-values for measured expression levels are less than a specific p-value level.

Example

Example 1. Enrichment of red blood cells undergoing denucleation

1. Expression of CD44

Red blood cells have typical morphological characteristics during denucleation. As the denucleation process proceeds, the distance (Δcentroid) from the center of the nucleus to the center of the cytoplasm gradually increases, while the aspect ratio of the cell (Aspect ratio) gradually decreases. With the help of flow imaging technology, the cells undergoing denucleation (Extruding) of mouse whole bone marrow erythroblasts are circled (Figure 1A). Interestingly, the cell surface molecule CD44, which gradually declines with the terminal differentiation of the erythroid lineage, showed high expression on some denucleated cells, and its proportion was further quantified as high as 60% (Figure 1B). It is suggested that CD44 may be a potential marker for denucleated red blood cells.

2.CD44 ^hi Ter119 ⁺ (Nonpro)

In view of the high expression of CD44 on denucleating cells, the inventors further analyzed the CD44 ^hi Ter119 ⁺ cell population (named Nonpro). Flow cytometry results show that the denucleation ratio of Nonpro is 40-60%, which is much higher than the denucleation ratio of erythroblasts (EBs) (Figure 1C).

3. Morphological verification

Through flow cytometry, Nonpro cells from mouse bone marrow were collected for smear staining. Giemsa staining stained the nuclei in blue-purple, and benzidine staining stained the hemoglobin-rich cytoplasm in deep yellow. It is very easy to see the denucleated cell morphology in Nonpro, which is highly consistent with the results of flow imaging analysis (Figure 1D).

4.Distribution of CD44 in early and late stages of denucleation

The characteristics of Nonpro's enucleated cells were further explored. Through flow cytometry, denucleating red blood cells were divided into early and late denucleation stages according to Δcentroid, and Nonpro was found to be enriched in late denucleation cells (Figure 1E). Representative images showing the presence of CD44 protein in the early stages of denucleation Agglutinates and polarizes to one side of the cell, separates from nascent reticulin at a late stage and eventually surrounds the cell perinuclear (Fig. 1F).

5. Distribution of F-actin

In view of the important role of cytoskeletal proteins in denucleation, the distribution of F-actin during the denucleation process and its positional relationship with CD44 were detected in Nonpro.

The distribution of F-actin is consistent with existing research reports. In the middle and late stages of denucleation, it is concentrated between the newly formed reticulocytes and the discharged nuclei. Later, it is located in the reticulocytes rather than in the discharged nuclei, while the CD44 protein is It is separated from F-actin and distributed around the nucleus (Figure 1G).

6.Dynamic distribution of CD44 and Ter119

In order to clarify the spatial distribution of CD44 during nuclear egress, high-resolution confocal microscopy and 3D imaging were performed on sorted Nonpro cells (Figure 1H). In the early stage of denucleation, Ter119 partially aggregates and bulges out of the cell surface. In the middle stage of denucleation, the nucleus is extruded and deformed into an asymmetric dumbbell shape, and CD44 is discontinuously distributed in the narrowed parts of the deformed nucleus and on the nuclear surface. In the late stage of denucleation, CD44 and Ter119 are completely distributed to the discharged nucleus and the surface of the new reticulum, respectively.

7. CD44 ^hi Ter119 ⁺ (Nonpro) is expressed in situ in bone marrow

Using multispectral immunohistochemistry technology, adult mouse bone marrow tissue sections were co-stained for CD44 and Ter119. It can be observed that Ter119 aggregates into clusters on cells in the early stage of denucleation and is asymmetrically distributed on the nuclei with high expression of CD44. In cells at the late stage of denucleation, CD44 and Ter119 were distributed to the discharged nuclei and the surface of new reticulum, respectively (Figure 1I).

In summary, the use of CD44 ^hi Ter119 ⁺ defined Nonpro cells can enrich denucleated red blood cells in mouse bone marrow.

Example 2. Sequencing of Nonpro single cell transcriptome

To better characterize subpopulations of Nonpro, single-cell RNA sequencing was performed to identify molecular markers of ongoing denucleation.

CD44 ^hi Ter119 ⁺ Nonpro cells were isolated and collected from the bone marrow of two mice by flow cytometry. To exclude erythrocyte phagosomes, all CD45-labeled immune cells were removed, resulting in CD45 ^- Nonpro for 10× single-cell transcriptome sequencing. After strict quality control, a total of 10,361 single cells were obtained for subsequent analysis.

UMAP analysis identified 7 cell subpopulations, including a continuous spindle and two independent subpopulations (Figure 2A). Based on the expression pattern of erythroid characteristic genes, five subgroups continuously distributed on the main axis were defined as E1 to E5.

Interestingly, two independent subpopulations are highly expressed in B lymphocytes (Ly6D, Ighm, Igkc, Cd79a, CD79b, transcription factors including Ebf1, Pax5 and Lef1), neutrophils (Elane, S100A8/A9, Prtn3 and Transcription factor Cebpb) and monocyte (Lyz2, S100a4, Lgals3)-related immune genes, which were identified as immune erythrocytes Ery/B and Ery/Mono (Figure 2B).

In addition, GO enrichment analysis showed that immune red blood cells not only have immune response-related functions such as regulation of innate immune response, positive regulation of cell-cell adhesion, leukocyte migration, and phagocytosis, but also retain the function of erythroid differentiation. , and acquired the ability for actin filament polymerization and cell shape regulation (data not shown). This suggests that immune RBCs may have potential functions related to RBC denucleation.

In summary, through single-cell transcriptome sequencing, it was identified that Nonpro cells are composed of early erythroblasts (51.72%), late erythroblasts (15.84%), and immune red blood cells (32.44%).

To further explore the biological functions unique to immune red blood cells, Ery/B and Ery/Mono were compared with public single-cell RNA-seq data of mouse bone marrow immune cells. Although Ery/B and Ery/Mono have high similarity with immune cells at the transcriptome level (data not shown), the results of biological function enrichment analysis revealed differences in cellular functions between them. (Figure 3A). Clustering heatmap showing specific biological function GO entries in immune red blood cells. Denucleation-related pathways, including responses to calcium ions, regulation of MAP kinase activity, actin binding, vesicles, and vacuoles, were significantly and specifically activated in immune erythrocytes (data not shown).

The denucleation potential score of each subpopulation in CD45 ^- Nonpro was calculated based on gene expression in GO entries. Immune red blood cells (two independent subpopulations, Ery/B and Ery/Mono), have significantly higher denucleation potential scores than the five subpopulations continuously distributed on the main axis (Figure 3B).

Overall, the results indicate that immune erythrocytes in CD45 ^- Nonpro possess denucleation-related biological functions and prepare immune erythrocytes for denucleation at the transcriptional level.

Example 3. Using the immune molecule CD45 to effectively capture red blood cells undergoing denucleation in the body

In order to clarify the relationship between immune red blood cells and denucleation, we chose to compare immune red blood cells with CD44 co-expressed CD45 molecules were used as surface markers for flow cytometry imaging.

Taking into account the particularity of immune red blood cells (expressing both immune markers and erythroid markers), the experimental protocol of flow cytometry was optimized. The step of sorting CD45 ^- cells with magnetic beads was abandoned, and mouse whole bone marrow cells were used for flow cytometry. Imaging to prevent loss of immune red blood cells. Flow cytometry detected a positive correlation between CD45 and CD44 at the protein level, and the positive rate of CD45 in denucleated red blood cells reached 60%-80% (Figure 4A). Further statistics show that the proportion of CD45 ⁺ erythroblasts that are denucleating is as high as 60%-80% (Figure 4B), which is slightly higher than the proportion of Nonpro-enriched erythrocytes that are denucleating (40%-60%) (Figure 1C), and CD45 can Capturing CD44 ^low CD45 ^low early denucleated red blood cells (Figure 1C to Figure 1D) makes up for the shortcoming of CD44 that cannot capture this subpopulation of cells. Therefore, CD45 ⁺ red blood cells can enrich denucleating red blood cells more effectively than Nonpro.

Figure 4C shows representative photos of CD45 ⁺ enucleating red blood cells. As CD45 expression changes from weak to strong, CD45 protein-rich vesicles bulge out of the cell (I), and then the nucleus deforms into CD44 and CD45-positive vesicles (II). Ter119 membrane separates from CD44 and CD45 and is distributed to On the membrane surface of new reticulocytes, the prolapsed nuclei are surrounded by CD44 and CD45 proteins (III). Interestingly, the staining intensity of the detached CD44 ⁺ CD45 ⁺ nuclear DNA became weaker, suggesting possible DNA degradation.

Next, the proportion of CD45 ⁺ in the early and late stages of denucleation and the intensity of the nucleus in denucleated red blood cells were calculated. CD45 ⁺ was found to be more enriched in nucleated erythrocytes with late-stage denucleation and weak nuclear signal (Figure 4D and Figure 4E). In addition, CD45 weakly positive cells were also present in some anucleated red blood cells (Figure 4F). Photos of cells show that CD45 protein still remains in the reticulum depression, which may be because the immune signal has not completely subsided or is broken at the denucleated junction.

In order to clarify whether CD45 can be used as a molecular marker of denucleating red blood cells, correlation analysis was performed on the proportion of CD45 ⁺ Ter119 ⁺ , the proportion of denucleating red blood cells and the denucleation rate (Figure 4G and Figure 4H). The results showed that the proportion of CD45 ⁺ Ter119 ⁺ was positively correlated with the proportion of red blood cells undergoing denucleation and negatively correlated with the denucleation rate. Therefore, CD45 can effectively mark a type of denucleated red blood cells with immune characteristics.

3D reconstruction of immunofluorescence staining revealed the spatial distribution of CD45 and Ter119 proteins (Figure 4I). In erythrocytes in the late stage of denucleation, CD45 is mainly distributed on the surface of the prolapsed nucleus. An interaction between Ter119 and CD45 signals can be seen on the membrane connecting the newly formed reticulocytes and the prolapsed nucleus. This result further demonstrates that when red blood cells are denucleated, Ter119 and CD45 for protein sorting. Detection of mouse bone marrow tissue sections using tissue immunofluorescence demonstrated the presence of CD45 ⁺ denucleated red blood cells (Figure 4J).

CD45 ⁺ nucleated red blood cells were sorted for in vitro culture, and the myosin II inhibitor blebbistatin was added for live cell real-time imaging. It was observed that the denucleation process of immune red blood cells was reversed (Figure 4K).

Example 4. Expression of Apoe in late-stage immune red blood cells undergoing denucleation

In order to comprehensively and completely characterize the transcriptome dynamics during the denucleation process of immune red blood cells, the whole bone marrow Nonpro cells of mice were sorted (in Example 2, Nonpro is not from the whole bone marrow, but the immune cells are removed. In this example, the whole bone marrow Nonpro did not remove immune cells and was the same cell population as Nonpro in Example 1), and single-cell transcriptome sequencing was performed using OrthoE cells as a control (Figure 5A). After strict quality control, 2004 whole bone marrow Nonpro cells and 4379 OrthoE cells were obtained respectively.

Whole bone marrow Nonpro, two CD45 ^- Nonpro (Nonpro1, Nonpro2; Nonpro1, Nonpro2 are experimental replicates, respectively from two samples, both covering the aforementioned 7 subpopulations) and OrthoE were integrated, and UMAP analysis identified 9 subpopulations. The grouping pattern of whole bone marrow Nonpro was similar to that of CD45 ^- Nonpro, including a continuous main axis and two independent subpopulations (Fig. 5B).

In contrast to CD45 ^- Nonpro, Ery/ApoE ⁺ is specific to whole bone marrow Nonpro. Ery/ApoE ⁺ subpopulation cells highly expressed hemoglobin, Apoe, Vcam1, and complement components (C1qa, C1qb, and C1qc) at the RNA level, and were CD45 positive and CD44 strongly positive at the protein level (Figure 5C).

GO enrichment analysis showed that cells in the Ery/ApoE ⁺ subpopulation have significant roles in complement activation, response to inorganic substances, regulation of epithelial cell migration, response to lipoprotein particles, regulation of inflammatory response, production of tumor necrosis factor, and endocytosis. It plays a role in the positive regulation of action, red blood cell homeostasis, and heme binding.

These results suggest that the Ery/ApoE ⁺ subpopulation of erythrocytes acquires monocyte-like characteristics. In addition, the denucleation potential score shows that the denucleation potential of this subpopulation is significantly weaker at the transcriptional level than Ery/B and Ery/Mono, but there are still a small number of cells that obtain a higher denucleation potential score, thus suggesting that they have entered the late stage of denucleation. , which is also consistent with its morphological performance.

As an apolipoprotein, ApoE is mainly responsible for lipoprotein-mediated lipid transport, and lipoprotein metabolism Xie can affect the denucleation and maturation of red blood cells. Studies have shown that ApoE inactivation aggravates red blood cell denucleation and maturation abnormalities in hypercholesterolemic mice (Blood 2002Mar1;99(5):1817-24). Therefore, the Ery/ApoE ⁺ subpopulation discovered in this application may play an important role in erythrocyte denucleation.

Example 5. Immune denucleation of CD45 ^- Nonpro cells cultured in vitro

The next step is to determine whether immune denucleation occurs in CD45 ^- Nonpro.

First, the morphology of CD45 ^- Nonpro after strict removal of immune cells from mouse bone marrow was detected by flow imaging (Figure 6A). The proportion of denucleated cells was significantly reduced (about 4% to 8%), and among them, the number of cells that were denucleated was significantly reduced. All are in early stages, with Ter119 still surrounding the nucleus. CD45 ^- Nonpro was further sorted by flow cytometry after strict removal of immune cells from mouse bone marrow, and smears were stained. It was found that some cells showed a monocyte/granulocyte-like morphology, such as surface bubbling or protruding pseudopods. The nuclei were lobed or ring-shaped (Fig. 6B). Observation using live cell imaging fluorescence microscopy showed that approximately 10% to 15% of the cells were denucleating and had a monocyte/granulocyte-like morphology (Figure 6C). It is suggested that the denucleated cells with immune characteristics are generated after sorting.

The sorted CD45 ^- Nonpro was cultured in vitro. New reticulin production could be seen after 4 hours, and the proportion of reticulin increased after overnight culture (Figure 6D).

Next, flow cytometry was used to detect whether CD45 ^- Nonpro and OrthoE cells expressed CD45 protein after culture in vitro. Regardless of whether in unenucleated, denucleated, or denucleated erythrocytes, the CD45 positive rate of CD45 ^- Nonpro cells after overnight culture was significantly higher than that of OrthoE (Figure 6E, Figure 6F). Representative photos show that CD45 is gradually separated from Ter119 on the cell membrane before Nonpro denucleation. As denucleation progresses, it is distributed to the side of the cell nucleus that is about to denucleate, and is completely separated from Ter119. This is the same as the distribution pattern of CD45 in immune denucleation in vivo. CD45 is also expressed in some anucleated erythrocytes (Fig. 6E), and its proportion is slightly higher than in vivo. It is suggested that differences in the microenvironment may cause the slowdown of CD45 protein after in vitro immune denucleation.

Example 6. Enrichment of denucleating cells in human bone marrow

There may be species differences between human and mouse denucleation. When human red blood cells are denucleated, the nuclei do not deform, but when mice are denucleated, the cells deform into dumbbell shapes and are expelled from the red blood cells.

The biological functions of immune red blood cells are enriched in calcium ion response, MAPK kinase activity, muscle There may be species differences in immune denucleation between mice and humans (data not shown).

Bone marrow aspiration samples from healthy adults were collected and analyzed by flow imaging after red splitting (Figure 7A to Figure 7B). Compared with CD45 ⁻ erythrocytes, the proportion of human CD45 ⁺ erythrocytes undergoing denucleation was significantly increased (Fig. 7C), and late denucleated erythrocytes were relatively enriched (Fig. 7D and Fig. 7E). This is highly consistent with the expression of immune denucleation in mouse bone marrow.

Taken together, RNA and protein levels suggest that immune red blood cells in adult bone marrow are significantly enriched in cells undergoing enucleation morphology.

Claims (10)

1. Use of a CD45-targeting reagent in preparing a detection device, wherein:

   The detection device is in the form of a kit or a chip;

   The detection device is used to determine the denucleation status of erythroid cells, and the denucleation status is selected from any one of the following: denucleation preparation, denucleation in progress, and denucleation;

   The reagent targeting CD45 refers to a reagent that determines whether CD45 is present or determines the level of CD45;

   The CD45-targeting reagent is selected from any one of the following: anti-CD45 antibodies or antigen-binding fragments thereof, CD45-targeting primers, and CD45-targeting probes;

   The erythroid cells are isolated from the body or from in vitro culture;

   Preferably, the denucleation being carried out is selected from any one of the following: early denucleation, late denucleation;

   Preferably, said CD45 refers to human CD45.
2. The use according to claim 1, wherein:

   When the erythroid cells are CD45 positive, the proportion of the erythroid cells that are judged to be in a denucleated state, or the erythroid cells that are judged to be in a denucleated state is statistically significantly increased;

   When the erythroid cells are CD45 negative, the proportion of the erythroid cells that are determined to be in preparation for denucleation or in the process of denucleation is statistically significantly reduced.
3. The use according to claim 1 or 2, wherein:

   The detection device further includes a reagent targeting CD235a or Ter119;

   The reagent targeting CD235a or Ter119 refers to a reagent that determines whether CD235a or Ter119 is present or determines the level of CD235a or Ter119;

   The reagent targeting CD235a or Ter119 is selected from any one of the following: anti-CD235a or Ter119 antibodies or antigen-binding fragments thereof, primers targeting CD235a or Ter119, and probes targeting CD235a or Ter119.
4. The use according to claim 3, wherein:

   When erythroid cells are CD235a positive or Ter119 positive and CD45 positive, The erythroid cells are determined to be in a denucleated state, or the proportion of the erythroid cells determined to be in a denucleated state is statistically significantly increased; or

   When CD235a and CD45 are co-localized, or Ter119 and CD45 are co-localized on the surface of erythroid cells, it is determined that the erythroid cells are in a state of denucleation, or the proportion statistics of the determination that the erythroid cells are in a state of denucleation. significantly improved.
5. The use according to claim 1 or 3, wherein:

   The detection device further includes a reagent targeting CD44;

   The CD44-targeting reagent refers to a reagent that determines whether CD44 is present or determines the level of CD44;

   The CD44-targeting reagent is selected from any one of the following: anti-CD44 antibodies or antigen-binding fragments thereof, CD44-targeting primers, and CD44-targeting probes.
6. The use according to claim 5, wherein:

   When CD44 and CD45 are co-localized on the surface of prolapsed cell nuclei, it is determined that the erythroid cells have been denucleated, or the denucleation rate of the erythroid cells is determined to be statistically significantly improved.
7. The use according to any one of claims 1 to 6, wherein:

   The detection device also includes reagents or combinations thereof targeting any of the following: hemoglobin, ApoE, Vcam1, C1qa, C1qb, C1qc, Ly6D, Ighm, Igkc, Cd79a, CD79b, Ebf1, Pax5, Lef1, Elane, S100A8/ A9, Prtn3, Cebpb, Lyz2, S100a4, Lgals3;

   Preferably, when the erythroid cells highly express hemoglobin, ApoE, Vcam1, C1qa, C1qb, and C1qc at the RNA level, and are positive for CD44 and CD45 at the protein level, the erythroid cells are determined to be in a late stage of denucleation, or The proportion of the erythroid cells determined to be in the late stage of denucleation is statistically significantly increased;

   Preferably, when the erythroid cells highly express one or more of Ly6D, Ighm, Igkc, Cd79a, CD79b, Ebf1, Pax5, Lef1, Elane, S100A8/A9, Prtn3, Cebpb, Lyz2, S100a4, and Lgals3 at the RNA level , and when they are CD44 positive, CD235a positive or Ter119 positive and CD45 negative at the protein level, it is determined that the erythroid cells have significantly increased denucleation potential.
8. The use according to any one of claims 1 to 7, wherein:

   The determination is at the RNA level or protein level;

   The antibody is derived from any of the following: mouse, rabbit, human, camel, dog, sheep, horse, recombinantly expressed antibody;

   Preferably, the antibody is a monoclonal antibody or a polyclonal antibody;

   Preferably, the antigen-binding fragment is selected from any one of the following: Fab, Fab′, F(ab′)2, Fv fragment, single chain antibody, domain antibody, multispecific antibody.
9. A method for enriching denucleated erythroid cells in vitro, including the steps:

   1) Provide erythroid cells in vitro,

   2) contacting said erythroid cells with an agent targeting CD45,

   3) Sorting CD45-positive erythroid cells, preferably by flow cytometry;

   in:

   The reagent targeting CD45 refers to a reagent that determines whether CD45 is present or determines the level of CD45;

   The CD45-targeting reagent is selected from any one of the following: anti-CD45 antibodies or antigen-binding fragments thereof, CD45-targeting primers, and CD45-targeting probes;

   The erythroid cells are isolated from the body or from in vitro culture;

   Preferably, the CD45 refers to human CD45;

   The methods are not intended for diagnosis or treatment of disease.
10. The method of claim 9, wherein:

    1) Provide erythroid cells in vitro,

    2.1) contacting said erythroid cells with an agent targeting CD45,

    2.2) Optionally, contacting said erythroid cells with an agent targeting CD44,

    2.3) Optionally, contacting the erythroid cells with an agent targeting CD235a or Ter119,

    3) Sorting CD45-positive, optionally CD44-positive, optionally CD235a or Ter119-positive erythroid cells;

    Steps 2.1), 2.2), and 2.3) can be performed in any order or simultaneously;

    The CD44-targeting reagent refers to a reagent that determines whether CD44 is present or determines the level of CD44;

    The CD44-targeting reagent is selected from any one of the following: anti-CD44 antibodies or antigen-binding fragments thereof, CD44-targeting primers, and CD44-targeting probes;

    The reagent targeting CD235a or Ter119 refers to a reagent that determines whether CD235a or Ter119 is present or determines the level of CD235a or Ter119;

    The reagents targeting CD235a or Ter119 are selected from any one of the following: anti-CD235a or Ter119 antibodies or antigen-binding fragments thereof, primers targeting CD235a or Ter119, and probes targeting CD235a or Ter119;

    The determination is at the RNA level or protein level.