Cell surface markers for distinguishing new subsets of separated lineage-biased human pluripotent progenitor cells and their applications

Abstract

The present invention provides a cell surface marker for differentiating a new subset of lineage-biased human multipotent progenitor cells and its application. The cell surface marker is CD52. The new subset of lineage-biased human multipotent progenitor cells is divided into MPP1 subset and MPP2 subset by using the surface marker CD52. The MPP1 subset is CD52+MPP, which tends to differentiate into the lymphoid and myeloid lineages during hematopoietic cell lineage differentiation. The MPP2 subset is CD52−MPP, which tends to differentiate into the erythroid and megakaryocytic lineages during hematopoietic cell lineage differentiation. The surface marker can highly enrich myeloid-lymphoid progenitor cells and erythroid-megakaryocytic progenitor cells, thereby significantly improving the efficiency of directed induction of umbilical cord blood cells into specific blood cell products such as red blood cells, megakaryocytes, neutrophils, NK cells, T cells, B cells, etc., providing an important basis for studying the lineage differentiation of human hematopoietic stem and progenitor cells and clinical applications.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a cell surface marker for distinguishing a new subpopulation of human multipotent progenitor cells with lineage bias and an application thereof. Background Art

[0002] Hematopoietic stem progenitor cells (HSPCs) and the various mature blood cells (such as red blood cells, myeloid leukocytes and lymphocytes) that they differentiate into play an important role in maintaining the entire life process of the body. Corresponding to the normal hematopoietic differentiation process, the abnormal proliferation and differentiation of hematopoietic cells can cause various blood system diseases and immune system diseases. Therefore, it is of great significance to explore the differentiation pattern and regulatory molecules of hematopoietic cells.

[0003] Multipotent blood progenitors (MPP) are differentiated from hematopoietic stem cells (HSC). In the mouse hematopoietic system, MPP is divided into four subgroups: MPP1, MPP2, MPP3, and MPP4. MPP1 can differentiate into cells of various lineages and exhibits multi-lineage reconstruction capabilities for up to 4 months in the first transplant, similar to short-term reconstitution of hematopoietic stem cells (ST-HSC); MPP2 is a subgroup biased towards megakaryocytes; MPP3 is a subgroup biased towards myeloid cells; and MPP4 is a subgroup biased towards lymphoid cells. In the human hematopoietic system, there are few studies on MPP subgroups. Previous studies focused on purified HSC surface markers. Studies have shown that CD34 may be present in human umbilical cord blood. - So far, CD133, CD90, CD49f, Rhodamine-123 and GPI80 have been used in different laboratories to purify human HSC.

[0004] In the human hematopoietic system, the study of lineage bias has always been a hot topic for researchers: CLEC9A hi CD34 lo The cells contain long-term reconstituted multilineage differentiated HSCs and are relatively quiescent, whereas CLEC9A lo CD34 hi The cells are limited to myeloid-lymphoid differentiation, are more likely to enter the cycle and proliferate, and are more inclined to the intermediate state between HSC and LMPP. The MPP population has been studied more comprehensively in mice, and the demonstration of the MPP1-4 population provides a new direction for lineage-biased HPC. However, there are few studies on human lineage-biased MPP.

[0005] Therefore, developing cell surface markers for distinguishing new subsets of lineage-biased human multipotent progenitor cells has important application value in the research of human hematopoietic stem and progenitor cell lineage differentiation and clinical applications. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a cell surface marker for distinguishing new subsets of lineage-biased human multipotent progenitor cells and its application. The present invention has found the surface marker CD52 for distinguishing new subsets of MPP, and CD52 can divide MPP into two new subsets with lineage bias, MPP1 and MPP2. The surface markers of the present invention can highly enrich myeloid-lymphoid progenitor cells and erythroid-megakaryocytic progenitor cells, thereby significantly improving the efficiency of umbilical cord blood cells (including hematopoietic stem cells and hematopoietic progenitor cells, etc.) being directionally induced into specific blood cell products such as red blood cells, megakaryocytes, neutrophils, NK cells, T cells, B cells, etc., providing an important basis for the research of human hematopoietic stem and progenitor cell lineage differentiation and clinical applications, and having important translational application value.

[0007] To achieve the purpose of this invention, the following technical solutions are adopted by the present invention:

[0008] In the first aspect, the present invention provides a cell surface marker for distinguishing new subsets of lineage-biased human multipotent progenitor cells, and the cell surface marker is CD52;

[0009] Using the surface marker CD52 to divide the new subsets of lineage-biased human multipotent progenitor cells into MPP1 subset and MPP2 subset;

[0010] The MPP1 subset is CD52 + MPP, which tends to differentiate towards the lymphoid and myeloid directions in hematopoietic cell lineage differentiation;

[0011] The MPP2 subset is CD52 - MPP, which tends to differentiate towards the erythroid and megakaryocytic directions in hematopoietic cell lineage differentiation.

[0012] In the present invention, the method of single-cell transcriptome sequencing was used to discover lineage-biased human MPP subsets, and MPP was divided into MPP1 and MPP2 using the cell surface marker CD52. In vivo and in vitro experiments have proved that these two MPP subsets have great differences in transcriptome, surface markers and hematopoietic function. CD52 - MPP is biased towards the erythroid and megakaryocytic lineages, while CD52 +MPP is myeloid and lymphoid biased, and this feature is universal in bone marrow and umbilical cord blood MPP. CD52 is a phosphatidylinositol-linked 12-amino acid leukocyte differentiation antigen that is highly expressed on the surface of activated lymphocytes, monocytes, macrophages, monocyte-derived dendritic cells, and endothelial cells. Previous studies have shown that in vitro treatment of umbilical cord blood mononuclear cells and CD34 + cells with CD52 monoclonal antibody - alemtuzumab increases the numbers of CFU-GM and CFU-GEMM; there are also studies showing that alemtuzumab can increase megakaryocyte production. The present invention finds that CD52 can divide MPP into two populations (CD52 - MPP and CD52 + MPP), and CD52 - MPP is biased towards erythroid and megakaryocytic differentiation, while CD52 + MPP is biased towards myeloid and lymphoid differentiation.

[0013] The findings of the present invention can highly enrich myeloid-lymphoid progenitor cells and erythroid-megakaryocytic progenitor cells, thereby significantly improving the efficiency of directed induction of umbilical cord blood cells (including hematopoietic stem cells and hematopoietic progenitor cells, etc.) into specific blood cell products such as red blood cells, megakaryocytes, neutrophils, NK cells, T cells, B cells, etc., providing an important basis for studying the lineage differentiation of human hematopoietic stem and progenitor cells and clinical applications, and having important translational application value.

[0014] In a second aspect, the present invention provides a screening method for cell surface markers for distinguishing and isolating new subsets of human pluripotent progenitor cells with lineage bias as described in the first aspect, and the screening method comprises the following steps:

[0015] (1) Perform single-cell transcriptome sequencing analysis on human hematopoietic stem and progenitor cells, and use the RNA velocity analysis method to resolve subsets of pluripotent progenitor cells with different hematopoietic cell lineage differentiation tendencies;

[0016] (2) Perform gene enrichment analysis on subsets of pluripotent progenitor cells with different hematopoietic cell lineage differentiation tendencies, and screen out genes that are differentially expressed in different subsets, and the proteins corresponding to the differentially expressed genes are cell surface markers for distinguishing and isolating new subsets of human pluripotent progenitor cells with lineage bias.

[0017] Preferably, in step (1), the transcriptome data used in the transcriptome sequencing analysis includes transcriptome MLP, B-NK1, B-NK2, Neu1, Neu2, MD, EBM, MEP, Ery, and Mk.

[0018] In the present invention, single-cell transcriptome sequencing is first performed on human hematopoietic stem and progenitor cells (HSPCs), and two populations of MPPs are resolved by using the RNA velocity analysis method. One population is MPP1, which mainly differentiates towards the lymphoid lineage (transcriptomes MLP, B-NK1, and B-NK2) and the myeloid lineage (transcriptomes Neu1, Neu2, MD, and EBM), and the other population is MPP2, which mainly differentiates towards the erythroid and megakaryocytic lineages (transcriptomes MEP, Ery, and Mk).

[0019] Preferably, in step (1), the subsets of the multipotent progenitor cells include the MPP1 subset that differentiates towards the lymphoid and myeloid lineages, and the MPP2 subset that differentiates towards the erythroid and megakaryocytic lineages.

[0020] In the present invention, gene set enrichment analysis software (GSEA) is used to analyze the hematopoietic cell lineage differentiation tendencies of the MPP1 and MPP2 populations. The results show that the genes expressed by MPP1 are more enriched in the gene sets related to lymphocytes and myeloid cells, while the genes expressed by MPP2 tend to be in the gene sets related to erythrocytes and megakaryocytes.

[0021] In the present invention, the MPP1 and MPP2 populations are compared for differential genes, and GO biological process enrichment analysis is performed. The results show that the genes highly expressed by MPP1 are mainly related to lymphocyte activation and regulation of extracellular secretion, while the genes highly expressed by MPP2 are mainly enriched in biological processes such as cellular transition metal ion homeostasis (iron uptake and transport) and hematopoietic stem cell differentiation.

[0022] In the present invention, further analysis is performed on the differential genes of MPP1 and MPP2. In the transcriptome of MPP1, genes related to the lymphoid lineage such as CD52 and MZB1, and the characteristic gene SPINK2 of LMPP are significantly more highly expressed than in the MPP2 population. In the transcriptome of MPP2, genes related to erythro-megakaryocytes such as GATA2 and RNF130 are highly expressed.

[0023] In a third aspect, the present invention provides the use of the cell surface markers for distinguishing novel subsets of human multipotent progenitor cells with lineage bias in the first aspect in the preparation of products for distinguishing subsets of human multipotent progenitor cells;

[0024] The subsets include the MPP1 subset that differentiates towards the lymphoid and myeloid lineages, and the MPP2 subset that differentiates towards the erythroid and megakaryocytic lineages.

[0025] In the present invention, the novel subsets of human multipotent progenitor cells with lineage bias are divided into the MPP1 subset and the MPP2 subset by using the surface marker CD52. The MPP1 subset is CD52 + MPP, which tends to differentiate towards the lymphoid and myeloid lineages in hematopoietic cell lineage differentiation; the MPP2 subset is CD52- MPP tends to differentiate into the erythroid and megakaryocytic lineages during hematopoietic cell lineage differentiation.

[0026] In a fourth aspect, the present invention provides a method for dividing subsets of multipotent progenitor cells, the division method comprising the following steps:

[0027] Using the cell surface markers for distinguishing and isolating new subsets of human multipotent progenitor cells with lineage bias described in the first aspect as subset phenotypic molecules, according to the relative expression levels of the subset phenotypic molecules in each subset, the subsets of multipotent progenitor cells are divided into an MPP1 subset that differentiates into the lymphoid and myeloid lineages, and an MPP2 subset that differentiates into the erythroid and megakaryocytic lineages.

[0028] Preferably, the sources of the human multipotent progenitor cells include: human multipotent progenitor cells derived from umbilical cord blood or human multipotent progenitor cells derived from bone marrow.

[0029] Preferably, the division method includes flow sorting of the cells.

[0030] Preferably, the flow sorting strategy includes:

[0031] Lin - CD34 + CD38 - CD45RA - CD52 - is defined as the MPP2 subset;

[0032] Lin - CD34 + CD38 - CD45RA - CD52 + is defined as the MPP1 subset.

[0033] In the present invention, CD52 can be used to distinguish MPP1 and MPP2 cells, and the cell surface marker CD52 is highly expressed in MPP1 and lowly expressed in MPP2.

[0034] In a fifth aspect, the present invention provides a kit for distinguishing and isolating new subsets of human multipotent progenitor cells with lineage bias, the kit comprising reagents for detecting the cell surface markers for distinguishing and isolating new subsets of human multipotent progenitor cells with lineage bias described in the first aspect.

[0035] In a sixth aspect, the present invention provides the MPP1 subset and / or MPP2 subset obtained by the method for dividing subsets of multipotent progenitor cells described in the fourth aspect, wherein the MPP1 subset is a highly enriched myeloid-lymphoid progenitor cell, and the MPP2 subset is a highly enriched erythroid-megakaryocytic progenitor cell.

[0036] The method for dividing cell subsets of the present invention can highly enrich myeloid-lymphoid progenitor cells and erythroid-megakaryocytic progenitor cells, thereby significantly improving the efficiency of directed induction of umbilical cord blood cells (including hematopoietic stem cells and hematopoietic progenitor cells, etc.) into specific blood cell products such as red blood cells, megakaryocytes, neutrophils, NK cells, T cells, B cells, etc., providing an important basis for studying the lineage differentiation of human hematopoietic stem and progenitor cells and clinical applications, and having important translational application value.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The cell surface markers for distinguishing and separating new subsets of human pluripotent progenitor cells with lineage bias of the present invention can divide MPP into two populations, namely CD52 - MPP and CD52 + MPP, and CD52 - MPP is biased towards erythroid-megakaryocytic differentiation, while CD52 + MPP is biased towards myeloid-lymphoid differentiation. The cell surface markers provide an important basis for studying the lineage differentiation of human hematopoietic stem and progenitor cells and clinical applications.

[0039] (2) The method for dividing subsets of pluripotent progenitor cells in the present invention can isolate cells with different differentiation tendencies and has important application value in clinical applications. Description of the Drawings

[0040] Figure 1 It is the result of the pseudotime analysis of the hematopoietic differentiation pathway of HSPC in Example 1.

[0041] Figure 2A It is the expression difference of MPP1 and MPP2 in the lymphoid gene set shown by the gene enrichment analysis software in Example 1.

[0042] Figure 2B It is the expression difference of MPP1 and MPP2 in the myeloid gene set shown by the gene enrichment analysis software in Example 1.

[0043] Figure 2C It is the expression difference of MPP1 and MPP2 in the erythroid gene set shown by the gene enrichment analysis software in Example 1.

[0044] Figure 2D It is the expression difference of MPP1 and MPP2 in the megakaryocytic gene set shown by the gene enrichment analysis software in Example 1.

[0045] Figure 3 It is the GO biological process enrichment entries of the differential genes of MPP1 and MPP2 in Example 1.

[0046] Figure 4Comparison of relative gene expression levels of the transcriptome MPP1 and MPP2 populations in Example 1.

[0047] Figure 5 Comparison of relative gene expression levels of the immunophenotype MPP1 and MPP2 populations in Example 1.

[0048] Figure 6 For CD52 derived from bone marrow in Example 2 - MPP and CD52 + Schematic diagram of flow sorting of MPP.

[0049] Figure 7A For sorting CD52 derived from bone marrow in Example 2 - MPP and CD52 + Experimental strategy diagram of in vitro colony formation for MPP.

[0050] Figure 7B Results of in vitro colony formation experiment in Example 2.

[0051] Figure 7C For CD52 in Example 2 - Colony representative diagram of the CD52 MPP group.

[0052] Figure 7D For CD52 in Example 2 + Colony representative diagram of the CD52 MPP group.

[0053] Figure 8A For CD52 derived from bone marrow in Example 2 - MPP and CD52 + Experimental strategy diagram of in vitro multicellular liquid culture for MPP sorting.

[0054] Figure 8B Statistical chart of flow cytometry detection results of in vitro multicellular liquid culture in Example 2.

[0055] Figure 9A For CD52 derived from bone marrow in Example 2 - MPP and CD52 + Experimental strategy diagram of in vitro single-cell liquid culture for MPP sorting.

[0056] Figure 9B Statistical chart of flow cytometry detection results of in vitro single-cell liquid culture in Example 2.

[0057] Figure 10A For CD52 derived from umbilical cord blood in Example 3 - MPP and CD52 + Schematic diagram of flow sorting of MPP.

[0058] Figure 10BFor CD52 derived from umbilical cord blood in Example 3 - MPP and CD52 + Statistical chart of in vitro colony formation results of MPP

[0059] Figure 10C For CD52 derived from umbilical cord blood in Example 3 - MPP and CD52 + Flow cytometry analysis results after in vitro multicellular liquid culture of sorted MPP

[0060] Figure 10D For CD52 derived from umbilical cord blood in Example 3 - MPP and CD52 + Statistical chart of flow cytometry results of in vitro single-cell liquid culture of sorted MPP

[0061] Figure 11A Experimental strategy diagram for functional verification within MPP subpopulations in Example 4

[0062] Figure 11B Statistical chart of the differentiation of human hematopoietic cells in the bone marrow of mice 2 weeks after transplantation of MPP subpopulations derived from umbilical cord blood in Example 4

[0063] Figure 11C Statistical chart of the differentiation of human hematopoietic cells in the bone marrow of mice 2 weeks after transplantation of MPP subpopulations derived from bone marrow in Example 4 Detailed implementation manners

[0064] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0065] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. For reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular commercial channels.

[0066] Example 1 Discovery and characteristics of MPP1 and MPP2 subpopulations in human bone marrow

[0067] (1) First, single-cell transcriptome sequencing was performed on human hematopoietic stem and progenitor cells (HSPC), and two populations of MPP were resolved by the RNA velocity analysis method. One population is MPP1, which mainly differentiates into the lymphoid lineage (transcriptome MLP and B-NK1 and B-NK2) and the myeloid lineage (Neu1 and Neu2, MD and EBM), and the other population is MPP2, which mainly differentiates into the erythroid-megakaryocytic lineage (transcriptome MEP, Ery and Mk). As Figure 1 shown, Figure 1The results of the pseudotime analysis of the hematopoietic differentiation pathway of HSPC show an obvious trilineage differentiation pathway. The UMAP plot shows the display of RNA velocity for the hematopoietic differentiation pathway. MPP1 mainly differentiates into the lymphoid and myeloid lineages, while MPP2 mainly differentiates into the erythroid and megakaryocytic lineages.

[0068] (2) The gene set enrichment analysis software (GSEA) was used to analyze the hematopoietic cell lineage differentiation tendency of the MPP1 and MPP2 populations. The results are as Figures 2A - 2D shown. Figure 2A shows the differential expression of MPP1 and MPP2 in the lymphoid gene set by the gene set enrichment analysis software (GSEA); Figure 2B shows the differential expression of MPP1 and MPP2 in the myeloid gene set by the gene set enrichment analysis software (GSEA); Figure 2C shows the differential expression of MPP1 and MPP2 in the erythroid gene set by the gene set enrichment analysis software (GSEA); Figure 2D shows the differential expression of MPP1 and MPP2 in the megakaryocytic gene set by the gene set enrichment analysis software (GSEA). The results show that the genes expressed by MPP1 are more enriched in the gene sets related to lymphocytes and myeloid cells, while the genes expressed by MPP2 tend to be in the gene sets related to erythrocytes and megakaryocytes.

[0069] (3) The differential genes of the MPP1 and MPP2 populations were compared, and GO biological process enrichment analysis was performed. The GO biological process enrichment entries of the differential genes of MPP1 and MPP2 are as Figure 3 shown. The results show that the genes highly expressed by MPP1 are mainly related to lymphocyte activation and regulation of extracellular secretion, while the genes highly expressed by MPP2 are mainly enriched in biological processes such as cellular transition metal ion homeostasis (iron uptake and transport) and hematopoietic stem cell differentiation.

[0070] (4) Further analysis was performed on the differential genes of MPP1 and MPP2. Figure 4 shows the comparison of the relative gene expression levels of the transcriptome MPP1 and MPP2 populations. The results show that in the transcriptome MPP1, genes related to the lymphoid lineage such as CD52 and MZB1 and the characteristic gene SPINK2 of LMPP are significantly more highly expressed than in the MPP2 population, while in the transcriptome MPP2, genes related to erythroid and megakaryocytic cells such as GATA2 and RNF130 are highly expressed.

[0071] (5) Analysis was performed on the phenotypic MPP cells. Figure 5 shows the comparison of the relative gene expression levels of the immunophenotypic MPP1 and MPP2 populations. It was also found that CD52, SPINK2, and MZB1 were more highly expressed in the phenotypic MPP1 population, while GATA2 and RNF130 were higher in the phenotypic MPP2 population.

[0072] Example 2 CD52 is a surface marker for distinguishing bone marrow MPP subsets

[0073] (1) Based on the single-cell transcriptome sequencing analysis results in Example 1, the surface marker CD52 is highly expressed in MPP1 and lowly expressed in MPP2. Therefore, CD52 was used to distinguish MPP1 and MPP2 cells. Flow cytometry was used to sort Lin - CD34 + CD38 - CD45RA - CD90 - CD52 - and Lin - CD34 + CD38 - CD45RA - CD90 - CD52 + two populations of cells (referred to as CD52 - MPP and CD52 + MPP). The schematic diagram of flow cytometry sorting of bone marrow-derived CD52 - MPP and CD52 + MPP is as shown in Figure 6 . It can be seen from the flow cytometry diagram that CD52 - MPP accounts for about 30% of MPP, while CD52 + MPP accounts for about 60% of MPP.

[0074] (2) The cells of the two sorted subpopulations were inoculated into semi-solid medium for colony-forming cell (CFC) assay:

[0075] 1) 1% penicillin / streptomycin, 50 ng / mL hIL-6, and 20 ng / mL hFlt3-L were added to the complete methylcellulose medium H4034;

[0076] 2) The cells of the corresponding population (70 cells / well, a total of 420 cells) were sorted into 300 μL IMDM + 10% FBS and added to the prepared semi-solid medium;

[0077] 3) The vortex was adjusted to the maximum gear 10, and the cells were fully mixed for at least 1 min to fully mix the cells in the medium, and then inoculated into uncoated 24-well plates at 500 μL / well;

[0078] 4) Cultured in a 37 °C, 5% CO2 incubator for 10 - 14 days.

[0079] (3) After culturing for 10 - 14 days, cell morphological classification and cloning counting were performed under an inverted microscope, and at the same time, the clones were photographed using a high-content imager.

[0080] Sort bone marrow-derived CD52 - MPP and CD52 + The experimental strategy diagram for in vitro colony formation assay of MPP is shown as Figure 7A shown, and the results of the in vitro colony formation assay are shown as Figure 7B shown, CD52 - The number of BFU-E formed in the CD52 + MPP group was significantly higher than that in the CD52 + MPP group. The number of CFU-GM formed in the CD52 - MPP group was more than that in the CD52 Figure 7C and Figure 7D shown. Among them, Figure 7C is the clone representative diagram of the CD52 - MPP group, Figure 7D is the clone representative diagram of the CD52 + MPP group.

[0081] The results of the in vitro colony formation assay showed that the number of burst-forming unit-erythroid (BFU-E) formed in the CD52 - MPP group was significantly higher than that in the CD52 + MPP group, while the number of colony-forming unit-granulocyte / macrophage (CFU-GM) formed in the CD52 + MPP group was more than that in the CD52 - MPP group.

[0082] (4) The HSPC in vitro multi-cell culture and differentiation system was used to detect the differentiation of CD52 - MPP and CD52 + MPP into erythroid, megakaryocyte, granulocyte, monocyte and NK cells. The detection method is as follows:

[0083] 1) Coating the plate: Add 0.1% Gelatin Solution to a 24-well plate, 300 μL / well, incubate in a 37 °C incubator for 1 hour and then aspirate clean;

[0084] 2) Plating MS-5 cells: After digesting MS-5 cells, resuspend them in the culture medium (α-MEM + 10% FBS + 1% penicillin / streptomycin), count, and resuspend with the culture medium to an appropriate concentration (3×10 4 / mL);

[0085] 3) Take 1 mL / well (3×10 4(An MS-5 cell) was inoculated into a 24-well plate;

[0086] 4) Prepare a stem cell differentiation medium (StemPro-34 SFM medium, Gibco, Cat#10639011). The components of the stem cell differentiation medium are shown in Table 1. Before use, the StemPro-34 SFM medium was filtered through a 0.22-μm filter to remove the precipitated crystals:

[0087] Table 1

[0088] Culture Medium Components Stock Concentration Working Concentration StemPro-34SFM / / hSCF 100 μg / mL 100 ng / mL hFlt3-L 20 μg / mL 20 ng / mL hTPO 100 μg / mL 100 ng / mL hIL-6 50 μg / mL 50 ng / mL hIL-3 10 μg / mL 10 ng / mL hIL-11 50 μg / mL 50 ng / mL hGM-CSF 20 μg / mL 20 ng / mL hIL-2 10 μg / mL 10 ng / mL hIL-7 20 μg / mL 20 ng / mL hEPO 3000 units / mL 3 units / mL 1% L-Glutamine 200 mM 2 mM

[0089] The sources of the components described in Table 1 are shown in Table 2:

[0090] Table 2

[0091]

[0092]

[0093] 5) Aspirate the old medium in the 24-well plate and add the newly prepared stem cell differentiation medium, 1 mL / well;

[0094] 6) Sort 100 cells by flow cytometry into a 24-well plate and culture them in an incubator at 37 °C and 5% CO2 for 2-3 weeks. The cells in the 24-well plate need to be replaced with half of the medium every week;

[0095] 7) Observe the number of positive clones under a microscope after culturing for 14-16 days. After aspirating the positive clones, label them with the antibodies in Table 3 and incubate them in the dark at 4 °C for 30 min;

[0096] Table 3

[0097] Surface Markers Fluorescent Channels Volume Human CD45 APC-cy7 0.3 μL Human CD14 PE-cy7 0.3 μL Human CD15 V450 0.3 μL Human CD235a PE 0.3 μL Human CD56 Percp-cy5.5 0.3 μL Human CD41a APC 0.3 μL

[0098] The sources of the antibodies in Table 3 are shown in Table 4:

[0099] Table 4

[0100]

[0101]

[0102] 8) Add 1 mL of PBE buffer to wash the antibodies. After centrifuging at 1500 rpm for 5 min and discarding the supernatant, resuspend the cells with the corresponding liquid and detect them on the machine;

[0103] 9) The positive clones are CD45 + or CD235a + gates≥30 cells; the myeloid clones are [(CD45 + CD14+ ) + (CD45 + CD15 + )] ≥ 30 cells; the megakaryocytic clone is CD41a + ≥ 30 cells; the erythroid clone is (CD45 - CD235a + ) ≥ 30 cells, the NK clone is (CD45 + CD56 + ) ≥ 30 cells.

[0104] (5) CD52 derived from bone marrow - MPP and CD52 + The strategy diagram of in vitro multicellular liquid culture for MPP sorting is as shown Figure 8A in the figure. The statistical chart of the flow cytometry detection results of in vitro multicellular liquid culture is as shown Figure 8B in the figure. The bar chart represents CD52 - MPP group and CD52 + MPP group forming red blood cells (CD45 - CD235a + ) megakaryocytes (CD41 + ), monocytes (CD45 + CD14 + ), granulocytes (CD45 + CD15 + ) and NK cells (CD45 + CD56 + ) statistical chart (in the figure, CD52 - MPP, n = 114; CD52 + MPP, n = 114); Figure 8B It shows that the proportion of CD52 - MPP cells forming red blood cells (CD45 - CD235a + ) and megakaryocytes (CD41 + ) is higher than that of the CD52 + MPP group, while the proportion of CD52 + MPP forming monocytes (CD45 + CD14 + ) and NK cells (CD45 + CD56 + ) is higher than that of CD52 - MPP, and there is no significant difference in the forming ability of granulocytes (CD45 + CD15 + ) in vitro.

[0105] (6) Due to the heterogeneity of MPP, an in vitro single-cell culture method was adopted to reflect the in vitro differentiation tendency ability of MPP. Therefore, single CD52 - MPP and CD52 + MPP cells were cultured in single-cell liquid. Figure 9A CD52 - MPP and CD52 + MPP sorting for in vitro single-cell liquid culture strategy diagram. Flow cytometry analysis results of in vitro single-cell liquid culture after 14 - 16 days were as Figure 9B shown, Figure 9B For the statistical chart of flow cytometry detection results, in the figure, ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; Flow cytometry analysis results after 14 - 16 days showed that the proportion of CD52 - MPP forming single erythroid clones and erythroid / megakaryocyte clones was higher than that of the CD52 + MPP group, while the proportion of the CD52 + MPP group forming single myeloid clones was higher than that of the CD52 - MPP.

[0106] The in vitro experimental results based on bone marrow in this example showed that CD52 - MPP was more inclined to differentiate into erythroid and megakaryocytic cells, while CD52 + MPP was more inclined to differentiate into lymphoid and myeloid cells.

[0107] Example 3 CD52 is a surface marker for distinguishing umbilical cord blood MPP subsets

[0108] To further verify the universality of this surface marker CD52, this example was also verified in MPP from umbilical cord blood.

[0109] (1) First, this example used a flow cytometer to sort Lin - CD34 + CD38 - CD45RA - CD52 - and Lin - CD34 + CD38 - CD45RA - CD52 + two populations of cells (referred to as CD52 - MPP and CD52 + MPP), CD52 - MPP and CD52 +The proportion of MPP in MPP is similar to that in the bone marrow, about 30% and 70% respectively. Figure 10A CD52 derived from umbilical cord blood - MPP and CD52 + Schematic diagram of flow sorting of MPP.

[0110] (2) Figure 10B CD52 derived from umbilical cord blood - MPP and CD52 + Statistical chart of the results of in vitro colony formation of MPP. The results of the in vitro colony formation experiment showed that CD52 - MPP and CD52 + There was no significant difference in the total number of colony formations between the two groups of MPP, and CD52 - The number of BFU-E formations in the MPP group was more than that in CD52 + MPP group, while the number of CFU-GM clones was less than that in CD52 + MPP group, CFU - There was no significant difference in GEMM.

[0111] (3) Figure 10C CD52 derived from umbilical cord blood - MPP and CD52 + Results of flow cytometry analysis after in vitro multicellular liquid culture of MPP sorting, CD52 - MPP, n = 157; CD52 + MPP, n = 149. The results of the in vitro multicellular culture and differentiation experiment showed that CD52 - The differentiation of red blood cells and megakaryocytes in the MPP group was more than that in CD52 + MPP group, and the differentiation of myeloid cells was less than that in CD52 + MPP group.

[0112] (4) Figure 10D CD52 derived from umbilical cord blood - MPP and CD52 + Statistical chart of the flow cytometry results of in vitro single-cell liquid culture of MPP sorting (ns, P>0.05; *, P≤0.05; **, P≤0.01; ***, P≤0.001). In the in vitro single-cell culture and differentiation experiment, CD52 in umbilical cord blood - The proportion of MPP forming single erythroid clones and erythroid / megakaryocyte clones was higher than that in CD52 + MPP group, while the proportion of forming single myeloid clones was lower than that in CD52 + MPP group.

[0113] Example 4 CD52 - MPP and CD52 +In vivo functional characteristics of MPP cells

[0114] The results of Examples 2 and 3 showed that CD52 - MPP tended to differentiate into erythrocytes and megakaryocytes, while CD52 + MPP tended to differentiate into myeloid cells and lymphocytes. Therefore, in this example, transplantation of MPP subsets in NOG mice and short-term in vivo differentiation of hematopoietic cells were detected. The antibody markers for short-term in vivo differentiation are shown in Table 5.

[0115] (1) Two populations of cells, Lin - CD34 + CD38 - CD45RA - CD90 - CD52 - and Lin - CD34 + CD38 - CD45RA - CD90 - CD52 + were sorted by flow cytometry from umbilical cord blood and bone marrow, and were respectively called CD52 - MPP and CD52 + MPP. They were injected into NOG mice via the tail vein (the mice were irradiated with 2 Gy before transplantation for 6 - 24 h for myeloablation), and 1000 cells were transplanted into each mouse. Among them, the experimental strategy diagram for in vivo function verification of MPP subsets is as shown in Figure 11A .

[0116] (2) Two weeks after transplantation, the bone marrow of the mice was taken out, labeled with the antibodies in Table 5, and the differentiation of each lineage of human hematopoietic cells in the bone marrow cells of the mice was detected by flow cytometry. The statistical chart of the differentiation of human hematopoietic cells in the bone marrow of mice 2 weeks after transplantation of the MPP subset from umbilical cord blood is as shown in Figure 11B . The results showed that the ability of CD52 - MPP cells from umbilical cord blood to differentiate into erythroid cells was significantly higher than that of the CD52 + MPP group, while the ability to differentiate into B cells and myeloid cells was lower than that of CD52 + MPP.

[0117] Table 5

[0118] Surface Markers Fluorescent Channels Volume Mouse CD45 Percp-cy5.5 0.5 μL Human CD45 APC-cy7 1 μL Human CD19 PE 1 μL Human CD33 APC 1 μL Human CD3 FITC 1 μL Human CD235a PE 1 μL Human CD71 APC 1 μL

[0119] The sources of the antibodies in Table 5 are shown in Table 6:

[0120] Table 6

[0121] Components Company Catalog 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

[0122] (3) Statistical chart of the differentiation of human hematopoietic cells in the bone marrow of mice after transplantation of the MPP subset derived from bone marrow for 2 weeks is shown as Figure 11C follows (ns, P>0.05; *, P≤0.05; **, P≤0.01; ***, P≤0.001). It can also be seen that the erythroid differentiation ability of CD52 - MPP derived from bone marrow in vivo is stronger than that of CD52 + MPP, while the myeloid differentiation ability is weaker than that of CD52 + MPP.

[0123] In summary, the cell surface markers for distinguishing and separating the new subsets of human pluripotent progenitor cells with lineage bias in the present invention can divide MPP into two new subsets with lineage bias, MPP1 and MPP2. The surface markers described in the present invention provide an important basis for studying the lineage differentiation and clinical application of human hematopoietic stem and progenitor cells, and have important application value.

[0124] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. Use of a reagent for detecting cell surface marker CD52, which is used to distinguish new subsets of human multipotent progenitor cells with lineage-biased differentiation, in the preparation of a product for distinguishing subsets of human multipotent progenitor cells; The subsets of human multipotent progenitor cells are the MPP1 subset that differentiates into the lymphoid and myeloid lineages, and the MPP2 subset that differentiates into the erythroid and megakaryocytic lineages; Among them, Lin - CD34 + CD38 - CD45RA - CD52 + is defined as the MPP1 subset; Lin - CD34 + CD38 - CD45RA - CD52 - is defined as the MPP2 subset.

2. A method for dividing a subpopulation of human multipotent progenitor cells, characterized in that, The classification method includes the following steps: Using the cell surface marker CD52, which is used to distinguish new subsets of human multipotent progenitor cells with lineage-biased differentiation, as a subset phenotypic molecule, and classifying the subsets of human multipotent progenitor cells into the MPP1 subset that differentiates into the lymphoid and myeloid lineages and the MPP2 subset that differentiates into the erythroid and megakaryocytic lineages according to the relative expression levels of the subset phenotypic molecule in each subset; Among them, Lin - CD34 + CD38 - CD45RA - CD52 + is defined as the MPP1 subgroup; Lin - CD34 + CD38 - CD45RA - CD52 - is defined as the MPP2 subset.

3. The method for dividing the human pluripotent progenitor cell subsets according to claim 2, characterized in that, The human multipotent progenitor cells are: human multipotent progenitor cells derived from umbilical cord blood or human multipotent progenitor cells derived from bone marrow.

4. The method for dividing the human pluripotent progenitor cell subsets according to claim 2, characterized in that, The classification method includes flow sorting of cells; The strategy for flow sorting includes: Lin - CD34 + CD38 - CD45RA - CD52 - is defined as the MPP2 subset; Lin - CD34 + CD38 - CD45RA - CD52 + is defined as the MPP1 subset.

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