Application of ADGRG1 as a biomarker in the preparation of a kit for detecting the in vitro expansion efficiency of hematopoietic stem cells

Abstract

The present invention provides the use of ADGRG1 as a biomarker in the preparation of a reagent or kit for detecting the in vitro expansion efficiency and transplantation effect of hematopoietic stem cells. The use of a drug that increases the ratio of ADGRG1-positive hematopoietic stem cells in the preparation of a drug for treating leukemia and anemic diseases, and the use of a drug that inhibits the expression of ADGRG1 in the hematopoietic stem cells of a patient in the preparation of a drug for treating leukemia and anemic diseases. The present invention discovers a novel biomarker, ADGRG1, which can effectively label functional hematopoietic stem cells under in vitro expansion culture stress conditions. On the one hand, it can detect the expansion efficiency of functional hematopoietic stem cells, and on the other hand, it can screen for related drugs that promote the ADGRG1-positive cell population for the transplantation efficiency of hematopoietic stem cells in clinical practice. In addition, by detecting the genes specifically enriched in ADGRG1-positive CD34+CD133+ cells, new regulatory factors for maintaining the stemness of human hematopoietic stem cells can be discovered and identified.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the medical field of diseases related to hematopoietic stem cell transplantation as a treatment method. Background Art

[0002] Currently, there is no effective in vitro expansion system for hematopoietic stem cells in the world. The main reason is that people do not have enough knowledge about the mechanism of maintaining the stemness of human hematopoietic stem cells under in vitro culture stress conditions. During in vitro culture, hematopoietic stem cells will undergo drastic changes from phenotype to function. If there is no marker to monitor hematopoietic stem cells under in vitro culture stress conditions, it is impossible to determine the in vitro expansion efficiency of hematopoietic stem cells.

[0003] Currently available hematopoietic stem cell markers are CD34, CD133, CD38, CD45RA, CD90, CD49f, CD201, and ITGA3 (CD49c). Antibody combinations targeting these markers can selectively label hematopoietic stem cells. + CD38 - CD45RA - CD90 + CD49f + The labeled cell population is only applicable to freshly isolated hematopoietic stem cells in a resting state, and is not applicable to the labeling of functional hematopoietic stem cells after in vitro culture and expansion. + CD133 + CD45RA - CD90 + CD201 + ITGA3 + The discovery of the cell population comes from the culture after treatment with a small molecule compound such as UM171, and cannot reflect the number or proportion of functional hematopoietic stem cells and their transplantation efficiency in the culture system without any small molecule compound treatment. In other words, the discovery of this marker combination is biased. + CD133 + CD45RA - CD90 + CD201 + ITGA3 + Cell populations, but they cannot increase the transplantation efficiency of hematopoietic stem cells in recipient mice. In summary, there is currently no reliable marker that can mark functional hematopoietic stem cells under in vitro expansion and culture stress conditions, so as to truly reflect or evaluate the expansion efficiency and transplantation effect of hematopoietic stem cells. Summary of the invention

[0004] The first object of the present invention is to provide the use of ADGRG1 as a biomarker in the preparation of a reagent or a kit for detecting the in vitro expansion efficiency and transplantation effect of hematopoietic stem cells.

[0005] The second object of the present invention is to provide a drug for increasing the ratio of ADGRG1-positive hematopoietic stem cells for use in the preparation of drugs for treating leukemia and anemia.

[0006] The third object of the present invention is to provide a drug for inhibiting the expression of ADGRG1 in hematopoietic stem cells of a patient for use in the preparation of a drug for treating leukemia and anemia.

[0007] The fourth object of the present invention is to provide a kit for detecting the in vitro expansion efficiency and transplantation effect of hematopoietic stem cells.

[0008] In order to achieve the above-mentioned purpose, the present invention discloses the use of ADGRG1 as a biomarker in the preparation of a reagent or a kit for detecting the in vitro expansion efficiency and transplantation effect of hematopoietic stem cells. The present invention discovered the first functional hematopoietic stem cell marker ADGRG1 under in vitro culture stress conditions, real-time labeling of hematopoietic stem cells under in vitro expansion culture stress conditions, and using ADGRG1 as a target to detect the in vitro expansion efficiency and transplantation effect of hematopoietic stem cells.

[0009] Furthermore, the invention discloses the use of a drug for increasing the ratio of ADGRG1-positive hematopoietic stem cells in the preparation of drugs for treating leukemia and anemia. An in vitro hematopoietic stem cell expansion culture system is established using ADGRG1 as a target, and small molecule compound drugs targeting ADGRG1 are screened for expanding hematopoietic stem cells. Using ADGRG1 as a target, hematopoietic stem cells are expanded for the treatment of leukemia, anemia and other diseases that are mainly treated by hematopoietic stem cell transplantation.

[0010] Furthermore, the invention discloses the use of a drug for inhibiting the expression of ADGRG1 in hematopoietic stem cells of a patient in the preparation of a drug for treating leukemia and anemia. The invention also discloses the treatment of leukemia by directly targeting ADGRG1.

[0011] Furthermore, a kit for detecting the in vitro expansion efficiency and transplantation effect of hematopoietic stem cells is disclosed, comprising a monoclonal antibody or a polyclonal antibody targeting ADGRG1.

[0012] The ideas and methods of the present invention are as follows:

[0013] 1. By comparing the transplantation effects of freshly isolated hematopoietic stem cells and hematopoietic stem cells expanded in vitro under stress conditions in immunodeficient mice, it was found that the transplantation efficiency (stemness) of hematopoietic CD34-positive cells decreased significantly after short-term in vitro culture.

[0014] 2. RNA-seq technology was used to detect the transcriptome of freshly isolated and cultured stressed hematopoietic CD34-positive cells. It was found that the mitochondrial oxidative phosphorylation metabolic activity, mitochondrial ROS level, mitochondrial number and membrane potential of hematopoietic CD34-positive cells were significantly increased under in vitro culture stress conditions, indicating an increase in mitochondrial oxidative stress.

[0015] 3. The hematopoietic CD34 positive cells cultured in vitro under stress conditions were stained for mitochondrial ROS, and then two cell populations with low mitochondrial ROS (mitoROS low) and high mitochondrial ROS (mitoROS high) were separated. Bone marrow transplantation experiments found that functional hematopoietic stem cells with blood reconstruction ability exist in CD34 with low mitochondrial ROS. + in cells.

[0016] 4. Identification of CD34 cells with low mitochondrial ROS and blood reconstitution capacity using single-cell sequencing scRNA-seq technology + The results showed that ADGRG1 + CD34 + CD133 + The cells significantly expressed characteristic genes of functional hematopoietic stem cells such as HLF and AVP.

[0017] 5. Using limiting dilution bone marrow transplantation experiments to prove ADGRG1 + CD34 + CD133 + The cells are real functional hematopoietic stem cells with blood reconstruction ability. ADGRG1 positive CD34 + CD133 + Cells with ADGRG1 negative CD34 + CD133 + Compared with WT cells, ADGRG1 has a significantly higher transplantation efficiency. This fully proves that ADGRG1 is a new marker of functional hematopoietic stem cells under stress conditions in vitro.

[0018] The advantage of the present invention is that it has discovered a new marker ADGRG1 that can effectively mark functional hematopoietic stem cells under in vitro expansion and culture stress conditions. On the one hand, it can detect the expansion efficiency of functional hematopoietic stem cells, and on the other hand, it can screen related drugs that promote ADGRG1-positive cell populations for clinical hematopoietic stem cell transplantation efficiency. In addition, ADGRG1-positive CD34 + CD133 + Genes specifically enriched in cells were discovered and identified as new regulatory factors for maintaining the stemness of human hematopoietic stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :Limiting dilution analysis of freshly isolated and in vitro expanded human umbilical cord blood CD34 + The frequency of functional hematopoietic stem cells in cells. a. Poisson distribution of the freshly isolated group and the in vitro cultured group shows the frequency of functional hematopoietic stem cells SRC; b. The frequency of functional hematopoietic stem cells in the freshly isolated group and the in vitro cultured group per 10 6 CD34 + The number of functional hematopoietic stem cells contained in the cells. **p<0.01.

[0020] Figure 2 : Results of GSEA gene enrichment analysis after RNA sequencing. a. GSEA analysis showed that human umbilical cord blood CD34 + The expression of mitochondrial-related genes was significantly enriched after cell culture and expansion stress in vitro; b. Gene function cluster analysis showed that human umbilical cord blood CD34 + Functional gene clusters of mitochondrial-related genes were significantly enriched after cell in vitro culture amplification stress.

[0021] Figure 3 :Seahorse mitochondrial respiratory metabolic activity test results. Analysis showed that freshly isolated human umbilical cord blood CD34 + Compared with cells, CD34 + The oxygen consumption of the cells' mitochondrial respiratory metabolism increased significantly.

[0022] Figure 4 :Results of detection of mitochondrial metabolic activity indexes in freshly isolated and in vitro expanded cultured groups. a. Mitochondrial reactive oxygen species ROS in in vitro expanded cultured group CD34 + b. Mitochondrial Mito-tracker staining showed that CD34 + The amount of mitochondria in the cells increased significantly; c. JC-1 staining showed that CD34 + The mitochondrial membrane potential in cells was significantly increased. ***p<0.001.

[0023] Figure 5 :Limiting dilution transplantation experiment was used to analyze the number of functional hematopoietic stem cells (SRCs) in two cell populations with high (mitoROS high) and low (mitoROS low) mitochondrial oxidative stress levels. a. Poisson distribution of mitoROS low group and mitoROShigh group shows the frequency of functional hematopoietic stem cells (SRCs); b. The number of functional hematopoietic stem cells (SRCs) in each of the mitoROS low group and mitoROS high group was 1.17 × 10 cells / mL.6 CD34 + The number of functional hematopoietic stem cells contained in the cells. ***p<0.001.

[0024] Figure 6 :UMAP analysis results of single-cell scRNA sequencing showed that CD34 cells of umbilical cord blood expanded in vitro + The cells can be divided into 18 cell populations. Cell populations 10, 11, 12, and 16 are mainly enriched in mitoROS low cells.

[0025] Figure 7 :Ex vivo culture and expansion of stressed CD34 + Group 11 of the cells specifically expressed the hematopoietic stem cell marker genes AVP and HLF.

[0026] Figure 8 :Protein interaction network association analysis showed the correlation of 86 specifically expressed genes in cell population 11.

[0027] Fig. 9 :Bone marrow transplantation experiments showed that ADGRG1 + CD133 + CD34 + The cells have the ability to reconstitute and transplant blood. a. hCD45 antibody staining of recipient mouse bone marrow cells 4 months after transplantation shows the proportion of human blood cells in mouse bone marrow; b. hCD33 antibody staining of recipient mouse bone marrow cells 4 months after transplantation shows the proportion of human blood cells in mouse bone marrow; c. hCD19 antibody staining of recipient mouse bone marrow cells 4 months after transplantation shows the proportion of human blood cells in mouse bone marrow; d. hCD45 antibody staining of recipient mouse bone marrow cells 4 months after transplantation shows the proportion of human blood cells in mouse peripheral blood. *p<0.05; **p<0.01.

[0028] Fig.10 : Limiting dilution analysis of ADGRG1 + and ADGRG1 - Human Cord Blood CD34 + The frequency of functional hematopoietic stem cells in cells. a.ADGRG1 + group and ADGRG1 - Poisson distribution of the group shows the frequency of functional hematopoietic stem cells SRC; b. ADGRG1 + group and ADGRG1 - Every 10 in the group 6 CD34 + CD133 + The number of functional hematopoietic stem cells contained in the cells. ***p<0.001. DETAILED DESCRIPTION

[0029] The technology of the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following specific embodiments are only used to help those skilled in the art understand the present invention, and are not intended to limit the present invention.

[0030] Example

[0031]

Human umbilical cord blood CD34 + Cell isolation and culture

[0032] First, fresh human umbilical cord blood was subjected to density gradient centrifugation with Ficoll solution (GE Healthcare, Piscataway, NJ, USA) to isolate mononuclear cells (MNCs). Then, CD34 + Cells: Resuspend mononuclear cells in MACs buffer (PBS containing 0.5% BSA, 2 mM EDTA, pH 7.2); add FcR blocking reagent (Miltenyi Biotec, #130-046-702) for blocking, and then add CD34 + Magnetic beads (Miltenyi Biotec, #130-046-702) were incubated with mononuclear cells at 4°C for 30 minutes. After incubation, MACS buffer was added and centrifuged (300 g, 10 minutes) to wash the cells. After discarding the supernatant, the cells were resuspended in 1 ml of MACs buffer and the cell suspension was passed through a magnetic bead sorting column MACS column (Miltenyi Biotec, #130-042-401) to sort the CD34 +The cells were cultured in hematopoietic stem cell medium, the formula of which was: Stem Cell Expansion Medium (Sigma, S0912) + 100 ng / ml stem cell growth factor (Stem Cell Factor, SCF) (R&D Systems, #7466-SC-010 / CF) + 100 ng / ml thrombopoietin (TPO) (R&D Systems, #288-TP-200 / CF) + 50 ng / ml FMS-like tyrosine kinase 3 ligand (Flt3L) (BioLegend, #710802) + 50 IU / ml penicillin + 50 ug / ml streptomycin. The cell culture conditions were 5% O 2 , 5% CO 2 .

[0033]

Immunofluorescence staining and flow cytometry analysis of cells

[0034] The cells were centrifuged (300g for 10 minutes), washed twice with pre-cooled PBS, resuspended in 500ul of PBS, added with fluorescent antibodies and stained at 4℃ for 30 minutes, washed twice with pre-cooled PBS, and fixed with 1% formaldehyde. The cells were analyzed by flow cytometry. Antibodies for the following surface markers were used: CD34-APC (581, BD Bioscience), CD133-BV421 (293C3, BD), ADGRG1-PE (4C3, BioLegend), CD19-PE (HIB19, BD), CD33-PEcy7 (WM53, BD) and CD45-APC (HI30, BD).

[0035]

Mitochondrial staining of cells

[0036] Umbilical cord blood CD34 + MitoSOX TMRed mitochondrial superoxide fluorescent probe reagent, MitoTracker Green FM mitochondrial green fluorescent dye and JC1 mitochondrial membrane potential fluorescent probe reagent were used for staining. The staining methods were provided by Thermofisher. Cells were first stained with surface marker antibodies, and after centrifugation and discarding the supernatant to wash the cells, Mito SOX, MitoTracker or JC-1 reagents were added for incubation (37°C, 15 minutes). Cells were centrifuged with pre-cooled PBS at 4°C (300g, 10 minutes), the supernatant was discarded, and the cells were shaken and resuspended in 500 μl of PBS, and immediately loaded on the flow cytometer LSRFortessa flow cytomete (BD Biosciences) for analysis and data acquisition.

[0037]

Seahorse Cell Energy Metabolism Test

[0038] The extracellular flux analysis system Seahorse XF Extracellular Flux Analyzer (Agilent Technologies) (Guo et al., 2018) was used to detect CD34 in umbilical cord blood. + The oxygen consumption rate (OCR) of the cells. 200ul of XF calibration solution was added to a 96-well calibration plate (utility plate) and incubated at 37°C overnight. Before use, the cell culture plate (Seahorse Bioscience, #101085-004) was incubated with cell tissue adhesive Cell-Tak solution (CORNING, #354241) at room temperature for 1 hour. 10 5 Purified CD34 + The cells were centrifuged at 1000 g for 10 min.

[0039] During this process, oligomycin (Sigma, #75351), FCCP (Sigma, C2920), rotenone (Sigma, R8875) (A, B, C) were added to the calibration plate in sequence for OCAR analysis. The calibration plate with the sensor cover was placed in the instrument tray rack for calibration. After calibration, the calibration plate was removed, the cell microwell culture plate was placed in the tray rack, and "Start" was clicked to measure the umbilical cord blood CD34 + OCAR value of cells.

[0040] [Limiting dilution method for counting bone marrow reconstitution cells]

[0041] The number of transplanted bone marrow repopulating cells (SCID) (Doulatov et al., 2012; Guo et al., 2018) was calculated using the limiting dilution method reported in 2012 and 2018. + The cells were injected into the tail vein of NSG donor mice, which had been previously irradiated with a sublethal dose. Sixteen weeks after transplantation, the mice were sacrificed and samples were collected for staining and flow cytometry to determine the expression of human CD45 + The SRC frequency of functional hematopoietic stem cells was calculated using L-Calc software (Stem Cell Technologies Inc, Vancouver, BC, Canada), and the graph was drawn using ELDA software (bioinf.wehi.edu.au / software / elda / ).

[0042] RNA Sequencing

[0043] After lysing the umbilical cord blood CD34+ cells, RNA was extracted using the RNA extraction kit RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). Sequencing services were provided by SeqWright Genomic Services, a subsidiary of GE Healthcare. RNA sequencing sample pretreatment kit TruSeq RNA Sample Prep was used to select RNA fragments with Poly A tails, and then library construction and generation of different cell type groups were performed. After RNA library construction, sequencing was performed on the machine. The sequencing length of the llumina HiSeq 2500 instrument was 2×100bp, and the read length of each sample was 2×20M.

[0044] Single-cell RNA sequencing and library construction were performed using 10X Genomics Chromium Single Cell 3'V3 Reagent Kits (10X Genomics, Pleasanton, CA) and 10X Genomics Chromium Controller Instrument. +Cells were divided into two groups according to the level of mitochondrial reactive oxygen species: mitoROS low and mitoROS high. After the cell density was adjusted to 1000 cells / μL, there were approximately 10,000 cells in each channel, forming oil-in-water droplets (Gel Bead-In-Emulsions, GEMs) with a gel magnetic bead-single cell-oil droplet structure. 8,000 single cells were collected for each sample, and the mRNA was "labeled" (barcoding).

[0045] After reverse transcription, GEMs were dissolved, cDNA with "tags" was purified and amplified, and the single end was connected to the adapter sequence (adaptors) for bridge PCR amplification. We used the high-sensitivity DNA quantitative detection reagent Qubit HighSensitivity DNA assay (Thermo Fisher Scientific) for quantification, and put the high-sensitivity DNA chip into the instrument Bioanalyzer 2200 (Agilent) for library distribution determination. All libraries were sequenced at both ends (paired-end run) with a sequencing length of 150bp, and the sequencing instrument was HiSeq Xten (Illumina, San Diego, CA).

[0046] Data analysis of RNA sequencing

[0047] The RNA-seq reads without introns were aligned with the constructed reference genome hg38 index using the alignment analysis software RNA-seq alignerSTAR (v2.5) (Dobin et al., 2013) (parameter: "--outSAMmapqUnique 60"), and then the expression of unique genes was counted using the software The feature Counts for calculating the number of fragments, and then aligned with the reference genome GENCODE 25 (parameter: "-s 2-p-Q 10"). More than two-thirds of the samples were filtered to have a CPM < 0.5 (CPM: Read Count per Million, that is, the ratio of the number of reads aligned to a gene to the total number of reads aligned to all genes multiplied by 10 6) were used to standardize the data using the TMM algorithm, so that differential expression analysis could be performed using the edgeR (v3.20.8) software package (McCarthy et al., 2012; Robinson et al., 2010). Data with a false discovery rate (FDR)-corrected P value < 0.001 and a fold change (FC) > 2 were taken, and functional analysis was performed using the gene enrichment analysis software DAVID (Dennis Jr et al., 2003; Huang et al., 2009) to determine the differentially expressed genes (DEGs). After all genes were arranged according to fold change, gene cluster enrichment analysis (GSEA) was performed (Subramanian et al., 2005).

[0048] [Data analysis of single-cell RNA sequencing]

[0049] The read sequences were depleted of adapter sequences and low-quality sequences, and the measured sequences were compared with the constructed GRCh38 gene index and annotation database GENCODE V28 annotationn using the single-cell sequencing data processing software CellRanger (version 3.1.0) to calculate the number of fragments in single-cell sequencing. + The cells were sampled and the genes with a total cell number of <0.1% or cells with less than 200 genes detected were eliminated, and then the analysis software Seurat R package (Bulter et al., 2018; Stuart et al., 2019) was used for subsequent analysis.

[0050] The distribution of the number of unique genes and mitochondria was different. We used the "LogNormalized" method to select cells with a unique gene number between 2800 and 6000 and a mitochondrial count of less than 10%, and normalized the data with a conversion factor of 10,000. The data of the low-level and high-level mitoROS groups were integrated and linearly transformed. Principal Component Analysis (PCA) was performed on all cells, and the first 19 principal components (PCs) were selected. The algorithm based on the shared nearest neighbor (SNN) module optimization was used to obtain 19 cell type groups.

[0051] In order to identify the genes highly expressed in the cell cluster 11 in the cluster analysis results, we used the "findmarker" function of the Seurat package and the Wilcoxon rank sum test to compare the expression of genes in cluster 11 and other cell type clusters. Cells with a Bonferroni-corrected p value < 0.05 and an average FC value > linear 1.2 were selected to find the marker genes specifically expressed in cluster 11. We used the protein interaction database STRING database (Szklarczyk et al., 2015) to construct the protein-protein interaction (PPI) network in which the marker genes in cluster-11 participated.

[0052]

Statistical analysis

[0053] The data in this article were analyzed using statistical software GraphPad Prism 5.0., and expressed as mean ± standard deviation (SD) or standard error of the mean (SEM). The statistical analysis of the low-level and high-level MitoROS groups was performed using a two-sided T test, and P < 0.05 was considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001).

[0054] The specific invention process is as follows: We first used NSG immunodeficient mouse bone marrow transplantation experiments to compare freshly isolated and in vitro expanded human umbilical cord blood CD34 + The results of limiting dilution analysis showed that umbilical cord blood CD34 + After short-term in vitro culture, the bone marrow transplantation ability of the cells decreased significantly (Figure 1), and the frequency of functional hematopoietic stem cells SRCs (SCIDrepopulating cells) decreased from 1:3424 to 1:271949 (Tables 1 and 2), indicating that in vitro expansion culture stress can lead to the loss of stemness of human umbilical cord blood hematopoietic stem cells.

[0055] Table 1. Statistics of transplanted mice in limiting dilution transplantation experiment

[0056]

[0057] Table 2 Human umbilical cord blood CD34 cells freshly isolated and expanded in vitro + Frequency of functional hematopoietic stem cells SRC in cells

[0058]

[0059] RNA-seq transcriptome analysis comparing freshly isolated and in vitro expanded human umbilical cord blood CD34 + The results showed that human umbilical cord blood CD34 + The expression levels of genes related to mitochondrial metabolism were significantly upregulated ( Figure 2 ). Seahorse mitochondrial metabolic activity assay shows that human umbilical cord blood CD34 + The mitochondrial respiratory metabolism of cells increased significantly ( Figure 3 ). A series of mitochondrial metabolic index tests including mitochondrial ROS, mitochondrial mass, and mitochondrial membrane potential tests showed that in vitro expanded umbilical cord blood CD34 + The level of mitochondrial oxidative stress in cells was significantly upregulated ( Figure 4 ). Next, human umbilical cord blood CD34 + The cells were divided into two groups, one with high mitochondrial oxidative stress (mitoROS high) and one with low mitochondrial oxidative stress (mitoROS low). The blood reconstitution and bone marrow transplantation abilities of the two groups were analyzed and compared using bone marrow transplantation experiments in NSG immunodeficient mice. The experimental results showed that functional hematopoietic stem cells with blood reconstitution ability were mainly concentrated in CD34 cells with low mitochondrial oxidative stress levels. + In the cell population ( Figure 5 The SRC frequency of functional hematopoietic stem cells in the mitoROS high cell population was 1:112939, while the SRC frequency of functional hematopoietic stem cells in the mitoROS low cell population was 1:8918 ( Figure 5 , Table 3).

[0060] Table 3. Human umbilical cord blood CD34 in the mitoROS low and mitoROS high groups + Frequency of functional hematopoietic stem cells SRC in cells

[0061]

[0062] Single-cell sequencing technology is a powerful tool for studying cell heterogeneity and analyzing and identifying cell populations. Next, we used single-cell sequencing transcriptome analysis scRNA-seq technology to simultaneously compare and analyze the mitoROS low cell population and the mitoROS high cell population, thereby identifying the potential functional hematopoietic stem cell population in the mitoROS low cell population. We analyzed 7663 mitoROS low cells and 12688 mitoROS high cells. UMAP analysis found that in vitro cultured CD34 +The cells can be divided into 18 cell populations in total. Among them, populations 10, 11, 12, and 16 are mainly enriched in mitoROS low cells ( Figure 6 More importantly, cell population 11 expressed the human hematopoietic stem cell marker genes AVP and HLF ( Figure 7 , Figure 8 ); At the same time, several important positive regulatory factors of hematopoietic stem cells, including HOXA9, MSI2 and MLLT3, were significantly enriched in cell population 11 ( Figure 8 ). These results indicate that cell population 11 is likely to be functional hematopoietic stem cells.

[0063] The protein interaction network analysis of 86 genes specifically enriched in cell population 11 showed that the ADGRG1 gene was closely associated with human hematopoietic stem cell surface marker proteins CD34 and CD133 (Figure 8). The ADGRG1 gene was significantly enriched in cell population 11, so we believe that the in vitro culture amplification stress human umbilical cord blood CD34 + CD34 + CD133 + ADGRG1 + The cells are functional hematopoietic stem cells.

[0064] CD34 + CD133 + ADGRG1 + Cells and CD34 + CD133 + ADGRG1 - The cells were then transplanted into NSG immunodeficient mice to analyze bone marrow engraftment and blood reconstitution abilities. + CD133 + ADGRG1 + The proportion of human blood cells in the recipient mice of the cells was significantly enriched both in the bone marrow and in the peripheral blood, while the transplanted CD34 + CD133 + ADGRG1 - The proportion of human cells in the recipient mice of the cells was relatively small ( Fig. 9 ).

[0065] The results of limiting dilution assay showed that CD34 + CD133 + ADGRG1 + The frequency of functional hematopoietic stem cells SRC in cells is 1:10077, while CD34 + CD133 + ADGRG1 -The frequency of functional hematopoietic stem cells SRC in cells was 1:135972. These results fully demonstrate that human umbilical cord blood CD34 + After stem cells were expanded and cultured in vitro, ADGRG1-labeled CD34 + CD133 + The cells are functional hematopoietic stem cells with blood reconstruction ability. ADGRG1 can be used as a marker for functional hematopoietic stem cells cultured and expanded in vitro, targeting CD34 + CD133 + ADGRG1 + Cell populations can be used to screen for activator drugs for in vitro expansion of functional hematopoietic stem cells ( Fig.10 ).

[0066] Table 4. ADGRG1 + group and ADGRG1 - Human umbilical cord blood CD34 + Frequency of functional hematopoietic stem cells SRC in cells

[0067]

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Preparation of reagents for detecting ADGRG1 and CD34 + CD133 + Application of a kit for evaluating the transplantation effect of hematopoietic stem cells after in vitro expansion and culture.

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