A sorting method of a high-secretion hepatocyte growth factor mesenchymal stem cell subpopulation and a screening method of a sorting index

Abstract

The present application relates to the field of biomedical technology, and more particularly to a sorting method of a mesenchymal stem cell subpopulation secreting high hepatocyte growth factor and a screening method of a sorting index. The application of cell surface protein CD10 as an index in sorting the mesenchymal stem cell subpopulation secreting high hepatocyte growth factor. The present application successfully associates CD10 with HGF secretion function and strong repair efficiency for the first time, providing an unprecedented method capable of directly and non-destructively screening and enriching a high therapeutic potential cell subpopulation from a heterogeneous cell population. The method overcomes the core bottleneck of traditional mesenchymal stem cell products, which are not uniform in function and unstable in efficacy, making it possible to sort based on the CD10 surface marker, thereby enabling the preparation of a mesenchymal stem cell subpopulation product with higher HGF expression level, more significant repair function and better quality uniformity. It has important clinical application value and industrialization prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for sorting a subpopulation of mesenchymal stem cells that secrete high levels of hepatocyte growth factor and a method for screening sorting indicators. Background Technology

[0002] Mesenchymal stem cells (MSCs) possess excellent tissue repair, immune regulation, and paracrine regulation capabilities, making them highly valuable for clinical applications in multiple biomedical fields such as tissue damage repair, inflammatory diseases, and degenerative diseases. They are currently the core cell resource for research and translation in the field of regenerative medicine.

[0003] Despite the promising potential of MSCs therapy, existing technologies have significant limitations and drawbacks. First, treatment efficacy varies considerably among different patients and cell batches, resulting in unstable clinical results. This is largely attributed to the fact that the MSCs used are a heterogeneous mixed population, with only a subset possessing stronger tissue repair potential. Second, traditional MSC preparation and identification standards are too broad, only guaranteeing basic cell "identity" (such as surface markers) without accurately identifying and enriching truly therapeutically efficient cell subpopulations. This leads to low treatment efficiency, often requiring large-scale cell infusions to achieve any effect, increasing costs and potentially introducing risks such as cell embolism. Finally, current technologies lack effective methods for precisely linking specific cell functions (such as efficient secretion of key repair factors) with cell subpopulations for screening. This hinders significant improvements in the quality control and predictability of cell therapy products, restricting their development into standardized, "off-the-shelf" biopharmaceutical products. Summary of the Invention

[0004] To address the aforementioned shortcomings, the present invention aims to provide a method for accurately and efficiently screening and identifying cell subpopulations with strong repair functions from heterogeneous MSC populations, thereby improving the efficacy stability and quality controllability of cell therapy products. Specifically, it relates to a method for sorting mesenchymal stem cell subpopulations that highly secrete hepatocyte growth factor and a method for screening sorting indicators.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of cell surface protein CD10 as an indicator in the sorting of mesenchymal stem cell subpopulations that secrete high levels of hepatocyte growth factor.

[0006] This invention also provides the application of cell surface protein CD10 as an indicator in the sorting of mesenchymal stem cell subpopulations with strong tissue repair function.

[0007] This invention also provides a method for screening sorting indicators for a subset of mesenchymal stem cells that secrete high levels of hepatocyte growth factor, comprising the following steps: (1) Construct a mesenchymal stem cell RNA sequencing library and sequence it to obtain sequencing data; (2) Bioinformatics analysis was performed on the sequencing data to obtain cell surface proteins that are related to high-secretion hepatocyte growth factor and have similar expression levels; these cell surface proteins were used as sorting indicators. The cell surface protein is CD10.

[0008] Preferably, the sequencing is paired-end sequencing.

[0009] This invention also provides a method for sorting a subset of mesenchymal stem cells that secrete high levels of hepatocyte growth factor, comprising the following steps: Mesenchymal stem cells were mixed with fluorescently labeled anti-human CD10 monoclonal antibody and incubated for 20-30 minutes. Then, they were sorted according to fluorescence intensity, and mesenchymal stem cell subpopulations with high fluorescence intensity were selected.

[0010] Preferably, the incubation temperature is 2~6℃.

[0011] The present invention also provides a subpopulation of mesenchymal stem cells obtained by the sorting method described above, wherein the subpopulation of mesenchymal stem cells is a subpopulation of mesenchymal stem cells that highly expresses CD10.

[0012] This invention also provides the application of a subpopulation of mesenchymal stem cells that highly express CD10 in the preparation of drugs with strong tissue repair functions.

[0013] The present invention also provides the application of a subpopulation of mesenchymal stem cells that highly express CD10 in the preparation of drugs with neuroreparative capabilities.

[0014] The present invention has the following advantages: This invention, by integrating single-cell sequencing, bioinformatics analysis, and flow cytometry sorting technologies, has for the first time successfully linked the cell surface marker CD10 directly to the high hepatocyte growth factor (HGF) secretion function and strong repair efficacy of mesenchymal stem cells (MSCs). It provides an unprecedented method for directly and non-destructively screening and enriching high-potential cell subpopulations from heterogeneous cell populations. This method overcomes the core bottlenecks of heterogeneous function and unstable efficacy in traditional MSCs products, making CD10-based sorting possible. This allows for the preparation of MSC subpopulation products with higher HGF expression levels, more significant repair function, and better quality uniformity. This greatly improves the predictability of efficacy and quality control of cell therapy products, laying a solid technical foundation for developing standardized, "off-the-shelf" cell therapy formulations for various HGF-dependent repair diseases such as nerve and muscle injuries, and has significant clinical application value and industrialization prospects. Attached Figure Description

[0015] Figure 1 The distribution patterns of HGF and CD10 in umbilical cord MSCs at single-cell resolution; Figure 2 The expression of HGF in CD10+ and CD10- subsets was detected by qRT-PCR and ELISA (left figure shows qRT-PCR results, right figure shows ELISA results). Figure 3 To evaluate the repair effects of CD10+ and CD10- subsets on damaged nerve cells in an in vitro cell model (A: Results of high-throughput observation of live nerve cells using a high-content cell imaging analysis system; B: Neuron lengths of different groups). Detailed Implementation

[0016] In this invention, hepatocyte growth factor (HGF) is a key core functional factor that mediates tissue repair, inhibits cell apoptosis, and improves the microenvironment. The secretion level of HGF directly determines the clinical therapeutic effect of MSCs.

[0017] The following detailed description of the solutions provided by the present invention, in conjunction with the embodiments, should not be construed as limiting the scope of protection of the present invention.

[0018] The fresh umbilical cord tissue used in this embodiment of the invention was obtained from the Department of Obstetrics of the First Medical Center Hospital of the Chinese People's Liberation Army, and an informed consent form was signed with the family members.

[0019] Example 1

[0020] Sorting methods for mesenchymal stem cell subsets that secrete high levels of hepatocyte growth factor.

[0021] (1) Heterogeneity of mesenchymal stem cells, and differences in gene expression and potential subpopulation structure within mesenchymal stem cells.

[0022] Umbilical cord-derived mesenchymal stem cell (MSC) samples for single-cell sequencing were obtained and prepared. Specifically, primary MSCs were isolated from fresh umbilical cord tissue donated by healthy full-term newborns using the tissue block adherence method. Subsequently, the obtained primary MSCs were expanded and cultured under standard conditions (serum-free medium, in a humidified incubator at 37°C and 5% CO2) until the cells reached the logarithmic growth phase and met the basic criteria for MSCs defined by the International Society for Cell Therapy (i.e., adherent growth, expression of CD73, CD90, and CD105, but not expression of CD34, CD45, CD11b, CD19, or HLA-DR, and possessing the potential for osteogenic and adipogenic differentiation). Healthy, well-conditioned MSCs from passages 3 to 5 were gently washed with calcium- and magnesium-free phosphate-buffered saline (PBS), and cell viability was confirmed to be above 95% using a suitable cell viability dye (such as trypan blue). Then, the cells were resuspended, counted, and diluted using an approved single-cell suspension preparation kit (the reagents recommended by 10×Genomics Chromium Single Cell 3'ReagentKits) to ensure that the final cell concentration and volume for sequencing met the stringent requirements of the single-cell sequencing platform (such as the 10×Genomics Chromium system). The target capture cell number was typically set at 5,000 to 10,000 cells to obtain sufficient depth for statistical analysis.

[0023] Next, single-cell RNA sequencing library construction and sequencing were performed. High-quality single-cell suspensions were loaded into a microfluidic chip, and microdroplet encapsulation technology was used to encapsulate individual cells with gel beads containing cell barcodes and unique molecular identifiers within oil droplets. Cell lysis, mRNA capture, and reverse transcription were completed within the oil droplets to generate cDNA with cell-specific barcodes. Subsequently, through amplification, fragmentation, end repair, A-tailing, and adapter ligation, a standard single-cell sequencing library was constructed. The library was sequenced using a high-throughput sequencer (Illumina NovaSeq 6000) with paired ends, achieving a sequencing depth of an average of 50,000–100,000 reads per cell to ensure effective detection of low-abundance transcripts, including HGF and MME (CD10).

[0024] (2) Bioinformatics analysis methods to find cell surface markers that are highly correlated with HGF expression.

[0025] Bioinformatics analysis was performed on the obtained single-cell sequencing data to resolve the heterogeneity of MSCs and identify associated biomarkers. The raw sequencing data underwent standard processing procedures including cell barcoding and UMI identification, sequence alignment, and gene expression quantification to obtain the gene expression matrix for each cell. First, rigorous quality control filtering was performed to remove cells with too few genes (e.g., <200), too high a proportion of mitochondrial genes (e.g., >20%), and genes expressed in only a very small number of cells. Subsequently, unsupervised clustering of cells was performed using principal component analysis, t-distributed random neighborhood embedding or unified manifold approximation and projection dimensionality reduction, and clustering algorithms to visualize and identify different transcriptomic subpopulations within the MSC population. A key step was calculating the expression level of the HGF gene in all cells at single-cell resolution and systematically analyzing its correlation with the expression levels of thousands of other genes (especially genes encoding known cell surface proteins). Statistical methods such as Pearson correlation coefficient or Spearman rank correlation coefficient were used to calculate and rank the data, and for the first time, it was discovered and confirmed that the gene expression level encoding the cell membrane surface protein MME (i.e., CD10) showed a similar expression pattern to the gene expression level of HGF. Figure 1 This finding suggests that CD10 protein may serve as a detectable surface marker for MSCs that highly express HGF, and could be used as an indicator for sorting MSCs that highly express HGF.

[0026] The expression levels of HGF in cell subpopulations with high CD10 protein expression (CD10+) and low CD10 protein expression (CD10-) were comprehensively validated after sorting. At the gene transcription level, total RNA was extracted from both subpopulations and detected using quantitative reverse transcription polymerase chain reaction (RT-PCR). Household genes (such as GAPDH or β-actin) were used as internal controls, and amplification was performed using specific primers (SEQ ID NO. 1-2) targeting HGF mRNA. The relative HGF mRNA expression level of the CD10+ subpopulation compared to the CD10- subpopulation was calculated by comparing Ct values. The results showed that the HGF mRNA level in the CD10+ subpopulation was significantly higher (p<0.001). At the protein secretion level, equal numbers of CD10+ and CD10- cells were seeded into culture plates and cultured for 48 hours. The cell culture supernatant was then collected. The concentration of HGF protein in the supernatant was detected using a highly sensitive enzyme-linked immunosorbent assay (ELISA) kit (Elabsciences) and normalized to cell number or total protein content. The data clearly show that the HGF protein secretion level in the supernatant of CD10+ subset cells was significantly higher than that in CD10- subset, thus confirming the high HGF secretion characteristic of CD10+ subset at the protein function level. Figure 2 ).

[0027] SEQ ID NO.1, F: GCCTGAAAGATATCCCGACA; SEQ ID NO. 2, R: TTCTCCTTGACCTTGGATGC.

[0028] Figure 2 CD10 high Indicates CD10+, CD10 low It represents CD10-.

[0029] The results showed that the expression level of HGF in the CD10+ subset was significantly higher than that in the CD10- subset.

[0030] (3) Obtaining a subpopulation of mesenchymal stem cells with high expression based on CD10

[0031] A flow cytometry-based cell sorting protocol based on CD10 cell surface proteins was established and optimized. Another batch of independently cultured, unsequencing umbilical cord MSCs (also passage 3-5) was prepared into a single-cell suspension. After cell counting, an appropriate amount of cells (1×10⁶) was collected. 6 Cells were washed with pre-chilled PBS buffer. The cells were resuspended in 100 μL PBS buffer, and a fluorescently labeled anti-human CD10 monoclonal antibody (APC-labeled anti-human CD10 antibody) at the optimal working concentration determined by titration was added. An isotype control antibody group was also included as a negative control. The cells were incubated at 4°C in the dark for 25 minutes. After incubation, the cells were washed twice with PBS buffer to remove unbound antibodies and finally resuspended in buffer containing a specific cell viability dye (7-AAD) to exclude dead cells during sorting. Using a high-performance flow cytometer (BD FACS II), the viable cell population (FSC-A / SSC-A and viability dye negative) was first delineated in a scatter plot. Then, based on the comparison of CD10 antibody fluorescence signal intensity with the isotype control, a sorting threshold was set, and the cell population was physically separated into two subpopulations: CD10-positive (CD10+) and CD10-negative (CD10-). The sorting process was performed under sterile conditions, and pre-filled complete culture medium was used in the collection tubes to ensure cell viability. After sorting, immediately count the cells in both groups and assess their survival rate (>90%). Then, they can be cultured separately or used directly for functional verification experiments.

[0032] Based on the above results, the CD10+ subset was selected as a subset with high HGF expression.

[0033] The above sorting method enabled the enrichment of target subpopulations while maintaining cell viability in the precursor.

[0034] Example 2

[0035] Functional validation of the sorted cell subpopulations

[0036] Human neuroblastoma cell line SH-SY5Y (purchased from the Cell Bank of the Chinese Academy of Sciences) was cultured and treated with toxic substances (okadaic acid, OA) to induce cell damage, simulating the pathological environment of neurodegenerative skeletal tangles. Damaged SH-SY5Y cells were randomly divided into three groups: a model group (OA, with only fresh culture medium), a CD10+MSCs co-culture group (OA+CD10+MSCs), and a model group. high CD10-MSCs co-culture group (OA+CD10) low The undamaged group served as the control (CON). Using the Transwell co-culture system, damaged SH-SY5Y cells were seeded in the lower chamber, and an equal number of CD10+MSCs or CD10-MSCs were seeded in the upper chamber (pore size 0.4 μm, allowing secreted factors to pass freely but cells not to come into direct contact).

[0037] After co-culturing for 48 hours, the cell nuclei and cytoplasm were labeled with the membrane-permeable live dye Hoechst and the calcium-binding agent calcein-AM, respectively. High-throughput, high-resolution structural data of live nerve cells were then observed and acquired using a high-content cell imaging analysis system (HCA). Figure 3 Experimental results showed that the cell bodies in the control group were intact with longer axons, while the cell bodies in the OA group were round with shorter axons. Treatment with CD10-high and CD10-low expression subsets improved the morphology of damaged SH-SY5Y cells, with the CD10-high expression group showing better morphological recovery. Statistical analysis of total axon length and longest dendrite length confirmed that the CD10-high expression subset had superior neural repair capacity compared to the CD10-low expression subset. Figure 3 ).

[0038] CD10 high This refers to a subset of MSCs that highly express CD10, namely CD10+; CD10 low This refers to a subset of MSCs that express low levels of CD10, namely CD10-.

[0039] As demonstrated by the above embodiments, this invention provides a complete method for obtaining a CD10+ subpopulation from a heterogeneous umbilical cord mesenchymal stem cell population based on the CD10 surface marker discovered through single-cell sequencing, using non-destructive sorting via flow cytometry. This subpopulation is then validated at multiple levels—including gene, protein, and functional experiments—as a cell subpopulation highly expressing HGF and possessing stronger damage repair capabilities. The method features clear and interconnected steps, achieving for the first time the direct targeting and enrichment of high-potential MSCs subpopulations at the live-cell level. This provides a concrete, reliable, and reproducible technical solution for preparing uniform, efficient, and standardized MSCs therapeutic products.

[0040] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of cell surface protein CD10 as an indicator in the sorting of mesenchymal stem cell subpopulations that secrete high levels of hepatocyte growth factor.

2. Application of cell surface protein CD10 as an indicator in the sorting of mesenchymal stem cell subpopulations with strong tissue repair function.

3. A method for screening a sorting marker of a subpopulation of mesenchymal stem cells that highly secretes a hepatocyte growth factor, characterized by, Includes the following steps: (1) Construct a mesenchymal stem cell RNA sequencing library and sequence it to obtain sequencing data; (2) Bioinformatics analysis was performed on the sequencing data to obtain cell surface proteins that are related to high-secretion hepatocyte growth factor and have similar expression levels; these cell surface proteins were used as sorting indicators. The cell surface protein is CD10.

4. The screening method according to claim 3, characterized in that, The sequencing was performed using paired-end sequencing.

5. A method for sorting a subset of mesenchymal stem cells that secrete high levels of hepatocyte growth factor, characterized in that, Includes the following steps: Mesenchymal stem cells were mixed with fluorescently labeled anti-human CD10 monoclonal antibody and incubated for 20-30 minutes. Then, they were sorted according to fluorescence intensity, and mesenchymal stem cell subpopulations with high fluorescence intensity were selected.

6. The sorting method according to claim 5, characterized in that, The incubation temperature is 2~6℃.

7. The mesenchymal stem cell subpopulation obtained by the sorting method according to claim 5 or 6, characterized in that, The mesenchymal stem cell subpopulation is a subpopulation of mesenchymal stem cells that highly express CD10.

8. Application of CD10-expressing mesenchymal stem cell subsets in the preparation of drugs with strong tissue repair functions.

9. Application of CD10-expressing mesenchymal stem cell subsets in the preparation of drugs with neurorepair capabilities.

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