CN115976228A - Cell surface marker for distinguishing new sub-group of separated lineage-biased human pluripotent progenitor cells and application of cell surface marker - Google Patents

Abstract

The invention provides a cell surface marker for distinguishing new sub-groups of separated lineage biased human pluripotent progenitor cells and application thereof, wherein the cell surface marker is CD52; dividing the new sub-population of isolated lineage biased human pluripotent progenitor cells into a MPP1 sub-population and a MPP2 sub-population using the surface marker CD52, the MPP1 sub-population being CD52 ⁺ MPP, which is predisposed to lymphoid and myeloid differentiation in hematopoietic lineage differentiation, said MPP2 subgroup CD52 ^‑ MPP, which tends to differentiate towards erythroid and megakaryoid lineages in hematopoietic lineage differentiation. The surface marker can highly enrich myeloid-lymphoid progenitor cells and erythroid-megakaryocytic progenitor cells, thereby remarkably improving the efficiency of directionally inducing cord blood cells into specific blood cell products such as erythrocytes, megakaryocytes, neutrophils, NK cells, T cells, B cells and the like, and being used for researching the lineage differentiation and clinical application of human hematopoietic stem and progenitor cellsProviding important basis.

Description

Cell surface marker for distinguishing new sub-group of separated lineage-biased human pluripotent progenitor cells and application of cell surface marker

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a cell surface marker for distinguishing a new sub-group of a separated lineage-biased human pluripotent progenitor cell and application thereof.

Background

Hematopoietic Stem Progenitor Cells (HSPCs) and the various mature blood cells (such as erythrocytes, myeloid leukocytes, lymphocytes, etc.) produced by their differentiation play an important role in maintaining the whole life process of the organism. Abnormal proliferation and differentiation of hematopoietic cells, corresponding to the normal hematopoietic differentiation process, causes various hematologic and immune system diseases. Therefore, it is of great significance to explore the differentiation pattern of hematopoietic cells and to regulate molecules.

Multipotent blood progenitors (MPPs) are differentiated from Hematopoietic Stem Cells (HSCs), and in the Hematopoietic system of mice, MPPs are divided into four subgroups, MPP1, MPP2, MPP3, and MPP4.MPP1 can differentiate into individual lineage cells, exhibiting multi-lineage reconstitution capacity for up to 4 months in the first transplantation, similar to Short term reconstitution of hematopoietic stem cells (ST-HSC); MPP2 is a megakaryocyte biased subpopulation; MPP3 is a subset of myeloid cell bias; MPP4 is a subset of stranguria-related biases. Whereas in the human hematopoietic system, there is less research on the MPP subpopulation. Previous studies have shown a number of surface markers for purified HSCs. It has been shown that CD34 may be present in human cord blood ^- The HSC of (1). To date, CD133, CD90, CD49f, rhodamine-123 and GPI80, etc. have been used in various laboratories to purify human HSC.

In the human hematopoietic system, the study of lineage bias has also been a focus of attention for researchers: CLEC9A ^hi CD34 ^lo The cells comprise long-term reconstituted multi-differentiated HSCs and are more quiescent, whereas CLEC9A ^lo CD34 ^hi Cells are restricted to myeloid-lymphoid lineageAnd the cells are more likely to enter a proliferation state in a cycle and are more preferentially in a population in an intermediate state of HSC and LMPP. The MPP population is more fully studied in mice, and the demonstration of the MPP1-4 population provides a new direction for lineage-biased HPCs. While less research has been done on human lineage bias towards MPP.

Therefore, the development of a cell surface marker for distinguishing and separating new sub-groups of lineage biased human pluripotent progenitor cells has important application value in researching lineage differentiation and clinical application of human hematopoietic stem progenitor cells. .

Disclosure of Invention

In view of the shortcomings of the prior art, the present invention aims to provide a cell surface marker for distinguishing new sub-populations of isolated lineage biased human pluripotent progenitor cells and application thereof. The present invention finds a surface marker CD52 that distinguishes a new subset of MPPs, and CD52 can divide MPPs into two lineage biased new subsets MPP1 and MPP2. The surface marker can highly enrich myeloid-lymphoid progenitor cells and erythroid-megakaryocytic progenitor cells, thereby remarkably improving the efficiency of directionally inducing cord blood cells (comprising hematopoietic stem cells, hematopoietic progenitor cells and the like) into specific blood cell products such as erythrocytes, megakaryocytes, neutrophils, NK cells, T cells, B cells and the like, providing important basis for researching lineage differentiation and clinical application of human hematopoietic stem and progenitor cells, and having important transformation application value.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a cell surface marker for differentiating between isolated lineage biased new subpopulations of human pluripotent progenitor cells, the cell surface marker being CD52;

dividing the isolated lineage biased new subpopulation of human pluripotent progenitor cells into an MPP1 subpopulation and an MPP2 subpopulation using the surface marker CD52;

the MPP1 subgroup is CD52 ⁺ MPP, which is biased towards lymphoid and myeloid differentiation in hematopoietic lineage differentiation;

the MPP2 subgroup CD52 ^- MPP, which tends to differentiate towards erythroid and megakaryoid lineages in hematopoietic lineage differentiation.

In the invention, a lineage-biased human MPP subgroup is discovered by adopting a single-cell transcriptome sequencing method, and MPP is divided into MPP1 and MPP2 by using a cell surface marker CD 52. The two MPP subgroups are proved to have great difference in transcriptome, surface marker and hematopoiesis function through in vivo and in vitro experiments. CD52 ^- MPP is erythroid and megakaryoid bias, and CD52 ⁺ MPP is the medullary and lymphatic bias, a feature that has general applicability in the MPP of bone marrow and cord blood. CD52 is a phosphatidylinositol-linked 12-amino acid leukocyte differentiation antigen that is abundantly expressed on the surface of activated lymphocytes, monocytes, macrophages, monocyte-derived dendritic cells and endothelial cells. Previous studies have shown that cord blood mononuclear cells and CD34 are treated in vitro with CD 52-alemtuzumab ⁺ After the cells, the number of CFU-GM and CFU-GEMM increases; alemtuzumab has also been shown to increase megakaryocytopoiesis. The research of the invention finds that the MPP can be divided into two groups by the CD52 (respectively, the CD 52) ^- MPP and CD52 ⁺ MPP), and CD52 ^- MPP is biased toward erythroid and megakaryoid differentiation, and CD52 ⁺ MPP differentiates towards myeloid and lymphoid lineages.

The discovery of the invention can highly enrich myeloid-lymphoid progenitor cells and erythroid-megakaryocytic progenitor cells, thereby remarkably improving the efficiency of directionally inducing cord blood cells (comprising hematopoietic stem cells, hematopoietic progenitor cells and the like) into specific blood cell products such as erythrocytes, megakaryocytes, neutrophils, NK cells, T cells, B cells and the like, providing important basis for researching lineage differentiation and clinical application of human hematopoietic stem and progenitor cells, and having important transformation application value.

In a second aspect, the present invention provides a screening method for cell surface markers for differentiating between isolated lineage biased new subpopulations of human pluripotent progenitor cells according to the first aspect, the screening method comprising the steps of:

(1) Performing single-cell transcriptome sequencing analysis on the human hematopoietic stem progenitor cells, and analyzing the sub-populations of the pluripotent progenitor cells with different hematopoietic lineage differentiation tendencies by adopting an RNA velocity analysis method;

(2) Performing gene enrichment analysis on the sub-populations of the pluripotent progenitor cells with different lineage differentiation tendencies of the hematopoietic cells, and screening out genes which are differentially expressed in the different sub-populations, wherein the corresponding proteins of the differentially expressed genes are cell surface markers for distinguishing the new sub-populations of the human pluripotent progenitor cells with the biased isolated lineages.

Preferably, in step (1), the transcriptome data used in the transcriptome sequencing analysis comprises transcriptome MLP, B-NK1, B-NK2, neu1, neu2, MD, EBM, MEP, ery and Mk.

In the invention, single-cell transcriptome sequencing is firstly carried out on human Hematopoietic Stem and Progenitor Cells (HSPC), two groups of MPPs are analyzed by adopting an RNA velocity analysis method, one group is MPP1 which is mainly differentiated towards a gonorrhea line (transcriptome MLP, B-NK1 and B-NK 2) and a medullary line (transcriptome Neu1, neu2, MD and EBM), and the other group is MPP2 which is mainly differentiated towards an erythroid line and a megakaryoid line (transcriptome MEP, group Ery and Mk).

Preferably, in step (1), the subpopulations of multipotent progenitor cells include the MPP1 subpopulation differentiated towards the gonous and myeloid lineages, and the MPP2 subpopulation differentiated towards the erythroid and megakaryocytic lineages.

In the present invention, MPP1 and MPP2 populations are analyzed for their propensity to differentiate into hematopoietic cell lineages using gene enrichment analysis software (GSEA). The results show that the MPP1 expressed gene is enriched to a higher degree in the lymphocyte and medullary cell related gene sets, while the MPP2 expressed gene is biased to the erythrocyte and megakaryocyte related gene sets.

In the invention, MPP1 and MPP2 populations are subjected to differential gene comparison, and GO biological process enrichment analysis is carried out. The results show that: the MPP1 high-expression gene is mainly related to lymphocyte activation and extracellular secretion regulation, and the MPP2 high-expression gene is mainly enriched in biological processes such as cell transition metal ion homeostasis (iron uptake and transport), hematopoietic stem cell differentiation and the like.

In the invention, the differential genes of MPP1 and MPP2 are further analyzed, in the transcriptome MPP1, the expression of the genes of CD52, MZB1 and the like related to stranguria and the characteristic gene SPINK2 of LMPP are obviously higher than that of the MPP2 population, and in the transcriptome MPP2, the expression of the genes GATA2, RNF130 and the like related to red megakaryocytes are high.

In a third aspect, the present invention provides the use of a cell surface marker as described in the first aspect for differentiating between new subpopulations of isolated lineage biased human pluripotent progenitor cells in the preparation of a product for differentiating between subpopulations of human pluripotent progenitor cells;

the subpopulations include the MPP1 subpopulation differentiated towards the gonorrhoea and the myeloid lineage, and the MPP2 subpopulation differentiated towards the erythroid and the megakaryoid lineage.

In the present invention, the surface marker CD52 is used to divide the isolated lineage biased new subset of human pluripotent progenitor cells into a MPP1 subset and a MPP2 subset. The MPP1 subgroup is CD52 ⁺ MPP, which is biased towards lymphoid and myeloid differentiation in hematopoietic lineage differentiation; said MPP2 subgroup CD52 ^- MPP, which tends to differentiate towards erythroid and megakaryoid lineages in hematopoietic lineage differentiation.

In a fourth aspect, the present invention provides a method for the compartmentalization of a subpopulation of multipotent progenitor cells, said compartmentalization method comprising the steps of:

using the cell surface markers for differentiating the new subpopulations of isolated lineage-biased human pluripotent progenitor cells of the first aspect as the subpopulation phenotype molecules, dividing the subpopulations of pluripotent progenitor cells into the MPP1 subpopulation differentiated towards the gonorrhea and the myeloid lineage and the MPP2 subpopulation differentiated towards the erythroid and the megakaryoid lineage, based on the relative expression levels of the subpopulation phenotype molecules in each subpopulation.

Preferably, the source of the human pluripotent progenitor cells comprises: umbilical cord blood-derived human pluripotent progenitor cells or bone marrow-derived human pluripotent progenitor cells.

Preferably, the partitioning method comprises flow sorting the cells.

Preferably, the strategy of the flow sorting comprises:

Lin ^- CD34 ⁺ CD38 ^- CD45RA ^- CD52 ^- defined as MPP2 subgroup;

Lin ^- CD34 ⁺ CD38 ^- CD45RA ^- CD52 ⁺ defined as MPP1 subgroup.

In the present invention, the use of CD52 can be used to distinguish MPP1 from MPP2 cells, with the surface marker CD52 highly expressed in MPP1 and less expressed in MPP2.

In a fifth aspect, the present invention provides a kit for differentiating between isolated lineage biased new subpopulations of human pluripotent progenitor cells, the kit comprising reagents for detecting the cell surface markers of the first aspect for differentiating between isolated lineage biased new subpopulations of human pluripotent progenitor cells.

In a sixth aspect, the present invention provides the MPP1 and/or MPP2 subpopulations obtained by the method for dividing a pluripotent progenitor subpopulation according to the fourth aspect, wherein the MPP1 subpopulation is a highly enriched myeloid-lymphoid progenitor cell and the MPP2 subpopulation is a highly enriched erythroid-megakaryoid progenitor cell.

The method for dividing the cell subsets can highly enrich myeloid-lymphoid progenitor cells and erythroid-megakaryocytic progenitor cells, thereby remarkably improving the efficiency of directionally inducing cord blood cells (comprising hematopoietic stem cells, hematopoietic progenitor cells and the like) into specific blood cell products such as erythrocytes, megakaryocytes, neutrophils, NK cells, T cells, B cells and the like, providing important basis for researching lineage differentiation and clinical application of human hematopoietic stem progenitor cells and having important conversion application value.

Compared with the prior art, the invention has the following beneficial effects:

(1) The cell surface markers for differentiating the new subpopulation of isolated lineage biased human pluripotent progenitor cells of the present invention can divide MPP into two populations, CD52 respectively ^- MPP and CD52 ⁺ MPP, and CD52 ^- MPP is biased towards erythromegakaryoid differentiation, whereas CD52 ⁺ MPP is biased to the myeloid lineage to differentiate. The cell surface marker provides important basis for researching lineage differentiation and clinical application of the human hematopoietic stem progenitor cells.

(2) The method for dividing the multipotential progenitor cell subpopulation can separate the cells with different differentiation tendencies, and has important application value in clinical application.

Drawings

FIG. 1 shows the results of pseudo-timing analysis of the hematopoietic differentiation pathway of HSPC in example 1.

FIG. 2A shows the difference in MPP1 and MPP2 expression on the gonorrhoeal gene set by the gene enrichment analysis software in example 1.

FIG. 2B shows the difference in MPP1 and MPP2 expression on the myeloid gene set by the gene enrichment analysis software of example 1.

FIG. 2C shows the difference in MPP1 and MPP2 expression on the erythroid gene set by the gene enrichment analysis software of example 1.

FIG. 2D shows the difference in MPP1 and MPP2 expression on the megakaryoid gene set as shown by the gene enrichment analysis software of example 1.

Figure 3 is a differential gene GO bioprocess enrichment entry for MPP1 and MPP2 in example 1.

FIG. 4 is a comparison of the relative expression levels of genes in the MPP1 and MPP2 populations of the transcriptome of example 1.

Figure 5 is a comparison of the relative expression levels of genes for the immunophenotypic MPP1 and MPP2 populations of example 1.

FIG. 6 shows the marrow-derived CD52 in example 2 ^- MPP and CD52 ⁺ MPP flow type sorting schematic diagram.

FIG. 7A is a drawing showing the selection of bone marrow-derived CD52 in example 2 ^- MPP and CD52 ⁺ MPP was subjected to in vitro cloning to form experimental strategy diagrams.

FIG. 7B shows the results of the in vitro colony formation experiment in example 2.

FIG. 7C shows CD52 of example 2 ^- Clones representing MPP group.

FIG. 7D shows CD52 of example 2 ⁺ Clones of the MPP group represent graphs.

FIG. 8A is the marrow-derived CD52 of example 2 ^- MPP and CD52 ⁺ MPP sorting was performed on the in vitro multicellular liquid culture strategy diagram.

FIG. 8B is a statistical chart of the flow assay results of the in vitro multicellular liquid culture in example 2.

FIG. 9A shows marrow-derived CD52 of example 2 ^- MPP and CD52 ⁺ MPP sorting was performed to generate in vitro single cell liquid culture strategy diagrams.

FIG. 9B is a statistical chart of the flow detection results of in vitro single cell liquid culture in example 2.

FIG. 10A shows cord blood-derived CD52 in example 3 ^- MPP and CD52 ⁺ MPP flow type sorting schematic diagram.

FIG. 10B is the cord blood-derived CD52 of example 3 ^- MPP and CD52 ⁺ MPP in vitro cloning forms a result statistical chart.

FIG. 10C shows cord blood-derived CD52 in example 3 ^- MPP and CD52 ⁺ MPP sorting is carried out to flow detection analysis results after in vitro multicellular liquid culture.

FIG. 10D is the cord blood-derived CD52 of example 3 ^- MPP and CD52 ⁺ MPP sorting is carried out on a flow type result statistical graph of in vitro single cell liquid culture.

Figure 11A is a strategy diagram of the MPP subgroup in vivo functional validation experiments in example 4.

FIG. 11B is a statistical plot of the differentiation of human hematopoietic cells in mouse bone marrow 2 weeks after transplantation of the cord blood-derived MPP subpopulation of example 4.

FIG. 11C is a statistical chart of the differentiation of human hematopoietic cells in mouse bone marrow 2 weeks after transplantation of the MPP subpopulation derived from bone marrow in example 4.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The examples do not specify particular techniques or conditions, and are to be construed in accordance with the description of the art in the literature or with the specification of the product. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 discovery of human bone marrow subgroups MPP1 and MPP2 and characterization thereof

(1) Firstly, single-cell transcriptome sequencing is carried out on human Hematopoietic Stem Progenitor Cells (HSPC), two groups of MPPs are analyzed by adopting an RNA velocity analysis method, one group is MPP1 which is mainly differentiated towards a gonorrhea line (transcriptome MLP, B-NK1 and B-NK 2) and a medullary line (Neu 1 and Neu2, MD and EBM), and the other group is MPP2 which is mainly differentiated towards a erythroid megakaryoid line (transcriptome MEP, ery and Mk). As shown in fig. 1, fig. 1 is a result of pseudo-timing analysis of hematopoietic differentiation pathways of HSPCs, showing a distinct trilineage differentiation pathway. UMAP picture shows RNA velocity display of hematopoietic differentiation pathway, MPP1 mainly differentiates to lymphoid lineage and myeloid lineage, and MPP2 mainly differentiates to erythroid megakaryoid lineage.

(2) And analyzing the MPP1 and MPP2 populations in the aspect of differentiation tendency of hematopoietic cell lineages by using gene enrichment analysis software (GSEA). The results are shown in fig. 2A-2D, fig. 2A shows that gene enrichment analysis software (GSEA) shows differences in MPP1 and MPP2 expression on the lymphoid gene set; FIG. 2B shows the difference in MPP1 and MPP2 expression on the myeloid gene set by the Gene enrichment analysis software (GSEA); FIG. 2C shows the difference in MPP1 and MPP2 expression on the erythroid gene set for the gene enrichment analysis software (GSEA); FIG. 2D shows the differences in MPP1 and MPP2 expression on the megakaryoid gene set using the Gene enrichment analysis software (GSEA). The result shows that the MPP1 expressed gene has higher enrichment degree in a lymphocyte and medullary cell related gene set, while the MPP2 expressed gene is biased to a erythrocyte and megakaryocyte related gene set.

(3) And comparing the MPP1 and MPP2 groups with the difference genes, and carrying out GO biological process enrichment analysis, wherein the difference genes GO of the MPP1 and MPP2 are enriched as shown in figure 3. The results show that: the MPP1 high-expression gene is mainly related to lymphocyte activation and extracellular secretion regulation, and the MPP2 high-expression gene is mainly enriched in biological processes such as cell transition metal ion homeostasis (iron uptake and transport), hematopoietic stem cell differentiation and the like.

(4) The differential genes of MPP1 and MPP2 were further analyzed. Figure 4 is a comparison of relative gene expression levels for the populations of transcriptomes MPP1 and MPP2. The results show that: in the transcriptome MPP1, the expression of the genes such as CD52 and MZB1 related to the stranguria system and the characteristic gene SPINK2 of LMPP is obviously higher than that of the MPP2 population, and in the transcriptome MPP2, the expression of the genes GATA2, RNF130 related to the red megakaryocytes is high.

(5) Phenotypic MPP cells were analyzed. Figure 5 is a comparison of the relative expression levels of genes in the immunophenotypic MPP1 and MPP2 populations. CD52, SPINK2, and MZB1 were also found to be more highly expressed in the phenotypic MPP1 population, while GATA2, RNF130 were more highly expressed in the phenotypic MPP2 population.

Example 2 surface markers for differentiation of MPP subpopulations of bone marrow by CD52

(1) Base ofAs a result of single-cell transcriptome sequencing analysis in example 1, the surface marker CD52 was highly expressed in MPP1 and less expressed in MPP2. Thus, CD52 was used to differentiate MPP1 and MPP2 cells. Lin adopting flow cytometry to sort adult bone marrow ^- CD34 ⁺ CD38 ^- CD45RA ^- CD90 ^- CD52 ^- And Lin ^- CD34 ⁺ CD38 ^- CD45RA ^- CD90 ^- CD52 ⁺ Two populations of cells (individually referred to as CD 52) ^- MPP and CD52 ⁺ MPP). Bone marrow-derived CD52 ^- MPP and CD52 ⁺ MPP flow sorting schematic diagram is shown in FIG. 6, from which it can be seen that CD52 ^- The MPP accounts for about 30% of the MPP, and the CD52 ⁺ The proportion of MPP to MPP is about 60%.

(2) The two sub-populations of cells selected were seeded into semi-solid medium for Colony Forming Cell (CFC) experiments:

1) Complete methylcellulose medium H4034 was supplemented with 1% penicillin/streptomycin, 50ng/mL hIL-6, 20ng/mL hFlt3-L;

2) Sorting the corresponding population of cells (70 cells/well, total 420 cells) in 300 μ L IMDM +10% fbs, added to the prepared semi-solid medium;

3) Regulating the votex to the maximum gear 10, fully and uniformly mixing the cells for at least 1min, fully and uniformly mixing the cells in a culture medium, and inoculating the cells into a 24-pore plate which is not coated with the cells at 500 mL/pore;

4)37℃、5％CO ₂ culturing in an incubator for 10-14 days.

(3) After culturing for 10-14 days, cell morphology classification clone counting is carried out under an inverted microscope, and simultaneously, a high content imager is used for photographing the clone.

Sorting bone marrow-derived CD52 ^- MPP and CD52 ⁺ FIG. 7A shows the strategy of the MPP in vitro clone formation experiment, FIG. 7B shows the result of the MPP in vitro clone formation experiment, and CD52 shows ^- The formation amount of BFU-E in MPP group is obviously higher than that of CD52 ⁺ MPP group, CD52 ⁺ The forming quantity of CFU-GM in the MPP group is more than that of CD52 ^- There are many MPP groups, while CFU-GEMM has no difference between the two groups; the clone representation is shown in FIG. 7C and FIG. 7D, wherein FIG. 7C isCD52 ^- Clone representative map of MPP group, FIG. 7D is CD52 ⁺ Clones representing MPP group.

The results of in vitro clonogenic experiments show that CD52 ^- The formation number of MPP group erythroid burst-forming units (BFU-E) is obviously higher than that of CD52 ⁺ MPP group, and CD52 ⁺ The MPP group had a higher number of granulocyte-macrophage colony-forming units (CFU-GM) than CD52 ^- MPP groups are multiple.

(4) CD52 by using HSPC in vitro multicellular culture differentiation system ^- MPP and CD52 ⁺ MPP is detected in erythroid, megakaryocyte, granulocyte, monocyte and NK cell differentiation conditions. The detection method comprises the following steps:

1) Coating a plate: 0.1% gelatin Solution (gelatin Solution) was added to a 24-well plate, 300. Mu.L/well, incubated at 37 ℃ for 1 hour, and then blotted;

2) MS-5 cell plating: MS-5 cells were digested and resuspended in medium (a-MEM +10% FBS +1% penicillin/streptomycin), counted, resuspended in medium to the appropriate concentration (3X 10) ⁴ /mL)；

3) Take 1 mL/well (3X 10) ⁴ Individual MS-5 cells) were seeded in 24-well plates;

4) A stem cell differentiation medium (StemPro-34 SFM medium, gibco, cat # 10639011) was prepared, the composition of which is shown in Table 1, and the StemPro-34 SFM medium was filtered through a 0.22 μm filter to remove precipitated crystals:

TABLE 1

Media Components	Storage concentration	Use concentration
			StemPro-34SFM	/	/
hSCF	100μg/mL	100ng/mL
			hFlt3-L	20μg/mL	20ng/mL
hTPO	100μg/mL	100ng/mL
			hIL-6	50μg/mL	50ng/mL
hIL-3	10μg/mL	10ng/mL
			hIL-11	50μg/mL	50ng/mL
hGM-CSF	20μg/mL	20ng/mL
			hIL-2	10μg/mL	10ng/mL
hIL-7	20μg/mL	20ng/mL
			hEPO	3000units/mL	3units/mL
1% of L-glutamine	200 mM	2mM

The sources of the components described in table 1 are shown in table 2:

TABLE 2

5) Sucking out the old culture medium in the 24-well plate, and adding a newly prepared stem cell differentiation culture medium for 1 mL/well;

6) Flow sorting of 100 cells in 24-well plates at 37 ℃ 5% CO ₂ After the culture for 2-3 weeks in the incubator, the cells in the 24-well plate need to be changed in liquid half a week;

7) Observing the number of positive clones under a microscope after culturing for 14-16 days, marking the antibodies in the table 3 after sucking out the positive clones, and incubating at 4 ℃ for 30min in a dark place;

TABLE 3

Surface marking	Fluorescent channel	Volume of
			Human CD45	APC-cy7	0.3μL
Human CD14	PE-cy7	0.3μL
			Human CD15	V450	0.3μL
Human CD235a	PE	0.3μL
			Human CD56	Percp-cy5.5	0.3μL
Human CD41a	APC	0.3μL

The sources of the antibodies in table 3 are shown in table 4:

TABLE 4

/>

8) Adding 1mL of PBE buffer solution to wash the antibody, centrifuging at 1500rpm for 5min, discarding the supernatant, and using a corresponding liquid to resuspend cells to perform on-machine detection;

9) The positive clone is CD45 ⁺ Or CD235a ⁺ gates is more than or equal to 30cells; the myeloid clone is [ (CD 45) ⁺ CD14 ⁺ )+(CD45 ⁺ CD15 ⁺ )]Not less than 30cells; cloning of megakaryoid into CD41a ⁺ Not less than 30cells; the erythroid clone is (CD 45) ^- CD235a ⁺ ) Not less than 30cells, NK clone is (CD 45) ⁺ CD56 ⁺ )≥30cells。

(5) Bone marrow-derived CD52 ^- MPP and CD52 ⁺ FIG. 8A is a schematic diagram of a strategy for MPP sorting in vitro multi-cell liquid culture. FIG. 8B is a flow chart showing the results of in vitro multicellular liquid culture, wherein the histogram represents CD52 ^- MPP group and CD52 ⁺ MPP group in vitro erythroblasts (CD 45) ^- CD235a ⁺ ) Megakaryocyte (CD 41) ⁺ ) Monocyte (CD 45) ⁺ CD14 ⁺ ) Granulocytes (CD 45) ⁺ CD15 ⁺ ) And NK cells (CD 45) ⁺ CD56 ⁺ ) Statistical chart of (in the figure, CD 52) ^- MPP，n＝114；CD52 ⁺ MPP, n = 114); FIG. 8B shows that CD52 ^- MPP extracellular erythroblasts (CD 45) ^- CD235a ⁺ ) And megakaryocytes (CD 41) ⁺ ) Ratio higher than CD52 ⁺ MPP group, and CD52 ⁺ MPP forms monocytes (CD 45) in vitro ⁺ CD14 ⁺ ) And NK cells (CD 45) ⁺ CD56 ⁺ ) The ratio is higher than CD52 ^- MPP, granulocytes (CD 45) ⁺ CD15 ⁺ ) The forming ability was not significantly different in vitro.

(6) Because MPP has heterogeneity, the in vitro single cell culture method is adopted to reflect the differentiation tendency capability of the MPP in vitro. Thus, individual CD52 was sorted out in vitro ^- MPP and CD52 ⁺ MPP cells are cultured in a single cell liquid. FIG. 9A is a photograph of bone marrow derived CD52 ^- MPP and CD52 ⁺ MPP sorting was performed to generate in vitro single cell liquid culture strategy diagrams. The flow analysis results of in vitro unicellular liquid culture after 14-16 days are shown in FIG. 9B, FIG. 9B is a statistical chart of flow detection results, in the chart, ns, P>0.05；*，P≤0.05；**，P≤0.01；***，P≤0.001；After 14 to 16 days, the flow analysis result shows that the CD52 ^- MPP forms single erythroid clones and the proportion of erythro/megakaryocyte clones is higher than that of CD52 ⁺ MPP group, and CD52 ⁺ The MPP group forms a single myeloid clone in a higher proportion than CD52 ^- MPP。

The results of this example, bone marrow based in vitro experiments, show that CD52 ^- MPP is more prone to erythroid megakaryocytic lineage cell differentiation, whereas CD52 ⁺ MPP are more prone to differentiation into lymphoid and myeloid cells.

Example 3 surface marker for differentiating MPP subpopulations in cord blood CD52

To further verify the universality of this surface marker for CD52, the present example was also verified in the MPP of cord blood origin.

(1) First, the embodiment of the invention uses a flow cytometer to sort the umbilical cord blood-derived Lin ^- CD34 ⁺ CD38 ^- CD45RA ^- CD52 ^- And Lin ^- CD34 ⁺ CD38 ^- CD45RA ^- CD52 ⁺ Two populations of cells (individually referred to as CD 52) ^- MPP and CD52 ⁺ MPP)，CD52 ^- MPP and CD52 ⁺ MPP accounts for 30% and 70% of MPP similar to that in bone marrow, and FIG. 10A shows cord blood-derived CD52 ^- MPP and CD52 ⁺ MPP flow type sorting schematic diagram.

(2) FIG. 10B is a CD52 derived from cord blood ^- MPP and CD52 ⁺ MPP in vitro cloning forms a result statistical chart. The results of in vitro clonogenic experiments show CD52 ^- MPP and CD52 ⁺ No significant difference in the total clone formation amounts of two MPP groups, CD52 ^- The MPP group BFU-E is formed more than CD52 ⁺ MPP group, and CFU-GM clone count is less than CD52 ⁺ MPP group, CFU ^- GEMM had no significant difference.

(3) FIG. 10C shows cord blood-derived CD52 ^- MPP and CD52 ⁺ Flow assay analysis of the results of MPP sorting after in vitro multicellular liquid culture, CD52 ^- MPP，n＝157；CD52 ⁺ MPP, n =149. The experimental result of in vitro multi-cell culture differentiation shows that the CD52 ^- MPP group red blood cell and megakaryocyte are differentiated more than CD52 ⁺ MPP group, marrowLineage cells are less differentiated than CD52 ⁺ And MPP group.

(4) FIG. 10D is cord blood-derived CD52 ^- MPP and CD52 ⁺ Flow statistics for MPP sorting in vitro single cell liquid culture (ns, P)>0.05; * P is less than or equal to 0.05; * P is less than or equal to 0.01; * P is less than or equal to 0.001), in vitro single cell culture differentiation experiment, cord blood CD52 ^- MPP forms single erythroid clones and the proportion of erythro/megakaryocyte clones is higher than that of CD52 ⁺ MPP group, and the proportion of single myeloid clones formed is lower than that of CD52 ⁺ And MPP group.

Example 4 CD52 ^- MPP and CD52 ⁺ MPP in vivo functional characteristics of cells

The results of examples 2 and 3 show that CD52 ^- MPP is biased towards erythrocyte and megakaryocyte differentiation, while CD52 ⁺ MPP is biased toward myeloid and lymphoid cell differentiation, so this example detects MPP subpopulations for transplantation in NOG mice and short-term differentiation of hematopoietic cells in vivo. Short-term in vivo differentiation antibody markers are shown in table 5.

(1) Lin from umbilical cord blood and bone marrow respectively sorted by flow cytometry ^- CD34 ⁺ CD38 ^- CD45RA ^- CD90 ^- CD52 ^- And Lin ^- CD34 ⁺ CD38 ^- CD45RA ^- CD90 ^- CD52 ⁺ Two groups of cells, respectively called CD52 ^- MPP and CD52 ⁺ MPP, injected into NOG mice via tail vein (mice were irradiated with 2Gy dose for myeloablation 6-24 h before transplantation), and 1000 cells were transplanted per mouse, wherein the experimental scheme for in vivo functional verification of MPP subset is shown in fig. 11A.

(2) Two weeks after transplantation, the mouse bone marrow was removed, labeled with the antibodies shown in Table 5, and the differentiation of each line of human hematopoietic cells in the mouse bone marrow cells was examined by flow assay. A statistical chart of the differentiation of human hematopoietic cells in mouse bone marrow 2 weeks after transplantation of MPP subpopulation derived from umbilical cord blood is shown in FIG. 11B, which indicates that CD52 derived from umbilical cord blood ^- The MPP erythroid differentiation capability is obviously higher than that of CD52 ⁺ MPP group, while the differentiation into B cells and myeloid cells is lower than that of CD52 ⁺ MPP。

TABLE 5

Surface marking	Fluorescent channel	Volume of
			Mouse CD45	Percp-cy5.5	0.5μL
Human CD45	APC-cy7	1μL
			Human CD19	PE	1μL
Human CD33	APC	1μL
			Human CD3	FITC	1μL
Human CD235a	PE	1μL
			Human CD71	APC	1μL

The sources of the antibodies in table 5 are shown in table 6:

TABLE 6

Components	Company(s)	Goods number
			Mouse CD45	BD Bioscience	Cat#550994，Clone 30-F11
Human CD45	BD Bioscience	Cat#557833，Clone 2D1
			Human CD33	BD Bioscience	Cat#340474，Clone P67.6
Human CD19	BD Bioscience	Cat#349209，Clone 4G7
			Human CD71	BD Bioscience	Cat#551374，Clone M-A712
Human CD3	BD Bioscience	Cat#555332，Clone UCHT1
			Human CD235a	Beckman Coulter	Cat#IM2211U，Clone 11E4B-7-6

(3) A statistical plot of the differentiation of human hematopoietic cells in mouse bone marrow 2 weeks after transplantation of bone marrow-derived MPP subpopulations is shown in FIG. 11C (ns, P>0.05; * P is less than or equal to 0.05; * P is less than or equal to 0.01; * P is less than or equal to 0.001). Bone marrow derived CD52 can also be seen ^- MPP has stronger erythroid differentiation capability in vivo than CD52 ⁺ Tendency of MPP, while the differentiation ability of the myeloid lineage is weaker than that of CD52 ⁺ MPP。

In summary, the cell surface markers described in the present invention for differentiating the novel subpopulations of lineage biased human pluripotent progenitor cells can divide MPP into two lineage biased novel subpopulations MPP1 and MPP2. The surface marker of the invention provides important basis for researching lineage differentiation and clinical application of the human hematopoietic stem progenitor cells, and has important application value.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Claims (10)

1. A cell surface marker for differentiating isolated lineage biased new subpopulations of human pluripotent progenitor cells, wherein the cell surface marker is CD52;

dividing the isolated lineage biased new subpopulation of human pluripotent progenitor cells into an MPP1 subpopulation and an MPP2 subpopulation using the surface marker CD52;

the above-mentionedMPP1 subgroup is CD52 ⁺ MPP, which is biased towards lymphoid and myeloid differentiation in hematopoietic lineage differentiation;

said MPP2 subgroup CD52 ^- MPP, which tends to differentiate towards erythroid and megakaryoid lineages in hematopoietic lineage differentiation.

2. A screening method according to claim 1 for differentiating cell surface markers separating new subpopulations of lineage biased human pluripotent progenitor cells, comprising the steps of:

(1) Performing single-cell transcriptome sequencing analysis on the human hematopoietic stem progenitor cells, and analyzing the sub-populations of the multipotential progenitor cells with different hematopoietic cell lineage differentiation tendencies by adopting an RNA velocity analysis method;

(2) Performing gene enrichment analysis on the sub-populations of the pluripotent progenitor cells with different lineage differentiation tendencies of the hematopoietic cells, and screening out genes which are differentially expressed in the different sub-populations, wherein the corresponding proteins of the differentially expressed genes are cell surface markers for distinguishing new sub-populations of the human pluripotent progenitor cells with the segregation lineage deviation.

3. The screening method for cell surface markers for differentiating between new subpopulations of lineage biased human pluripotent progenitor cells according to claim 2, wherein in step (1), the transcriptome data used in the transcriptome sequencing analysis comprises transcriptomes MLP, B-NK1, B-NK2, neu1, neu2, MD, EBM, MEP, ery and Mk.

4. The screening method for cell surface markers for differentiating between new subpopulations of isolated lineage-biased human pluripotent progenitor cells according to claim 2 or 3, wherein in step (1), the subpopulations of pluripotent progenitor cells include the MPP1 subpopulation differentiated to the lymphoid and myeloid lineage and the MPP2 subpopulation differentiated to the erythroid and megakaryoid lineage.

5. Use of the cell surface markers of claim 1 for differentiating between new subpopulations of lineage biased human pluripotent progenitor cells in the manufacture of a product for differentiating between subpopulations of human pluripotent progenitor cells;

the subpopulations include the MPP1 subpopulation differentiated towards the gonorrhoea and the myeloid lineage, and the MPP2 subpopulation differentiated towards the erythroid and the megakaryoid lineage.

6. A method for compartmentalization of a subpopulation of multipotent progenitor cells, said compartmentalization comprising the steps of:

using the cell surface markers of claim 1 for differentiating new subpopulations of isolated lineage-biased human pluripotent progenitor cells as subpopulation phenotypic molecules, and dividing the subpopulation of pluripotent progenitor cells into a subpopulation MPP1 differentiated towards the gonorrhea and the myeloid lineage and a subpopulation MPP2 differentiated towards the erythroid and the megakaryoid lineage, based on the relative expression levels of the subpopulation phenotypic molecules in each subpopulation.

7. The method for dividing a subpopulation of multipotent progenitor cells according to claim 6, wherein said source of human multipotent progenitor cells comprises: umbilical cord blood-derived human pluripotent progenitor cells or bone marrow-derived human pluripotent progenitor cells.

8. The method for dividing a subpopulation of multipotent progenitor cells according to claim 6 or 7, wherein said dividing method comprises flow sorting the cells;

preferably, the strategy of the flow sorting comprises:

Lin ^- CD34 ⁺ CD38 ^- CD45RA ^- CD52 ^- defining MPP2 subgroup;

Lin ^- CD34 ⁺ CD38 ^- CD45RA ^- CD52 ⁺ defined as MPP1 subgroup.

9. A kit for differentiating isolated lineage biased new subpopulations of human pluripotent progenitor cells, comprising reagents for detecting the cell surface markers of claim 1 for differentiating isolated lineage biased new subpopulations of human pluripotent progenitor cells.

10. The MPP1 and/or MPP2 subpopulations obtained by the method of dividing a pluripotent progenitor cell subpopulation according to any one of claims 6-8, wherein the MPP1 subpopulation is a highly enriched myeloid-lymphoid progenitor and the MPP2 subpopulation is a highly enriched erythroid-megakaryoid progenitor.

Images