Method for allele matching of donor mesenchymal stem cells and receptors

Abstract

The invention provides a method for allele matching of donor mesenchymal stem cells and a receptor. The method comprises the following steps: S10, separating mesenchymal stem cells from perinatal tissue of a donor; s20, extracting genome DNA (deoxyribonucleic acid) of the mesenchymal stem cells, and performing HLA (human leukocyte antigen) genetic typing; s30, performing HLA (human leukocyte antigen) genetic typing on the DNA of the receptor; s40, performing allele spectrum comparison on the HLA gene of the receptor and the HLA gene of the mesenchymal stem cell, and calculating the matching degree; s50, judging the matching degree of the receptor and the mesenchymal stem cells according to the matching degree; and S60, storing the HLA genotyping result of the mesenchymal stem cells and the matching degree in a database, and establishing a matching library for matching the donor mesenchymal stem cells and the receptor. According to the method, a set of standardized system is established, compatibility evaluation of a donor and a receptor at the allele level is achieved, the immunological rejection risk caused by HLA mismatching is effectively predicted and avoided before treatment, and the in-vivo survival and implantation efficiency of MSCs is improved.

Description

Technical Field

[0001] This invention relates to the fields of regenerative medicine and biotechnology, and more specifically, to a method for allelic matching of donor mesenchymal stem cells and recipients. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cell with self-renewal capacity and multi-lineage differentiation potential. MSCs can be isolated from various tissue sources, including bone marrow, adipose tissue, and perinatal tissues such as placenta, umbilical cord, and amnion. Among these, perinatal MSCs have advantages such as high proliferation potential, low immunogenicity, and enhanced therapeutic efficacy, and are becoming an important resource for cell therapy.

[0003] Despite extensive research, clinical outcomes for MSC-based treatments remain inconsistent, largely due to differences in immunocompatibility and variability among different donor sources. In traditional solid organ transplantation or hematopoietic stem cell transplantation, high-resolution HLA typing is an essential prerequisite for successful graft implantation and long-term survival. This technology minimizes the risk of rejection by the host's immune system and is the cornerstone of successful transplantation.

[0004] However, in the field of MSC transplantation and MSC therapy, a standardized system for assessing donor-recipient compatibility at the allele level has not yet been established. This lack of a crucial step leads to significant uncertainty in treatment, making it impossible to effectively predict and avoid the risk of immune rejection caused by HLA incompatibility before treatment, potentially severely impairing the in vivo survival and engraftment efficiency of MSCs. Summary of the Invention

[0005] This invention aims to provide a method for allele matching between donor mesenchymal stem cells (MSCs) and recipients, establishing a standardized system to achieve allele compatibility assessment between donors and recipients. This system effectively predicts and avoids the risk of immune rejection caused by HLA incompatibility before treatment, improving the in vivo survival and engraftment efficiency of MSCs. Furthermore, by constructing a matching database and cell bank, it provides fundamental support for subsequent rapid retrieval and intelligent donor screening.

[0006] To address the aforementioned problems, this invention provides a method for allelic typing of donor mesenchymal stem cells and recipients. The method includes the following steps: S10: isolating mesenchymal stem cells from perinatal tissue of the donor; S20: extracting genomic DNA from the mesenchymal stem cells and performing HLA genotyping; S30: performing HLA genotyping on the recipient's DNA; S40: comparing the allelic profiles of the recipient's HLA gene and the mesenchymal stem cell's HLA gene and calculating the matching degree; S50: determining the degree of matching between the recipient and the mesenchymal stem cells based on the matching degree; S60: storing the HLA genotyping results and matching degree of the mesenchymal stem cells in a database to establish a typing library for matching donor mesenchymal stem cells and recipients.

[0007] The technical effects achieved by adopting this solution are as follows: By extracting genomic DNA from donor MSCs and performing HLA genotyping, and then comparing the allele profiles of the recipient's HLA genes with those of mesenchymal stem cells, a standardized system is established. This system aims to achieve compatibility assessment between donors and recipients at the allele level, effectively predicting and avoiding the risk of immune rejection caused by HLA incompatibility before treatment, and improving the in vivo survival and implantation efficiency of MSCs. This system enhances the safety, precision, and scalability of MSC-based cell therapy in organ transplantation, autoimmune diseases, and regenerative medicine applications, contributing to the development of precision medicine in the field of cell therapy and supporting the establishment of large-scale stem cell banks for transplantation and precision cell therapy.

[0008] Further, step S10 specifically includes the following steps: S110: Isolating mesenchymal stem cells from perinatal tissues; S120: Performing quality testing on the isolated mesenchymal stem cells to obtain qualified mesenchymal stem cells; S130: Expanding and culturing the qualified mesenchymal stem cells to obtain expanded mesenchymal stem cells.

[0009] The technical effects achieved by adopting this solution are as follows: Through the complete process of isolating perinatal tissues, conducting rigorous quality testing, and expanding in vitro, a sufficient number of mesenchymal stem cells with activity and purity meeting clinical standards can be stably obtained. This provides a high-quality, standardized cell source for subsequent precise immunocompatibility assessment and transplantation therapy, fundamentally ensuring the reliability of the assessment results and the effectiveness and safety of the final treatment.

[0010] Furthermore, the expanded mesenchymal stem cells are expanded from the 2nd to the 6th generation of mesenchymal stem cells.

[0011] The technical effects achieved by adopting this technical solution are as follows: using expanded second to sixth generation mesenchymal stem cells, the mesenchymal stem cells have been sufficiently expanded to obtain a sufficient number of cells for DNA extraction and cell cryopreservation, providing high-quality standardized cells for the entire evaluation process and potential clinical applications.

[0012] Furthermore, HLA genotyping identifies at least the HLA-A, HLA-B, and HLA-C loci.

[0013] The technical effects achieved by adopting this solution are as follows: HLA genotyping identifies at least the HLA-A, HLA-B, and HLA-C loci, and compares them with the recipient's HLA gene allele profile, ultimately achieving accurate HLA identification. This not only provides a key basis for assessing immunocompatibility and reducing the risk of rejection during allogeneic transplantation, but also lays a solid technical foundation for establishing standardized cell banks and ensuring product quality control, thereby significantly improving the safety and clinical application potential of allogeneic mesenchymal stem cell therapy.

[0014] Furthermore, HLA genotyping also confirmed the HLA-DR and HLA-DQ loci.

[0015] The technical effects achieved by adopting this solution are as follows: it expands the HLA genotyping range to include HLA-DR and HLA-DQ loci, providing comprehensive immunogenotypes of donor mesenchymal stem cells, thereby constructing a more complete HLA lineage; it provides key predictive information for assessing long-term immune rejection, significantly improves the comprehensiveness and foresight of the matching assessment system, and lays a more solid genetic foundation for screening high-quality donors with lower immunogenicity and higher survival potential after transplantation.

[0016] Furthermore, the classification criteria for matching degree are as follows: perfect match: all 12 alleles are the same; partial match: 6 to 11 alleles are the same; low match: 0 to 5 alleles are the same.

[0017] The technical effects achieved by adopting this solution are as follows: Using the aforementioned classification criteria, the HLA matching degree between the recipient and donor mesenchymal stem cells can be precisely quantified and clearly graded, thereby transforming complex gene typing data into an intuitive and actionable basis for clinical decision-making. This standard clearly defines "complete match," "partial match," and "low match," greatly facilitating rapid donor screening and prioritization. Furthermore, it directly warns of the high immune rejection risk inherent in "low-match" combinations, effectively avoiding their inclusion in unnecessary subsequent experimental and clinical processes. This significantly optimizes resource allocation and evaluation efficiency while improving transplant safety. In short, this technical solution introduces a quantitative allele matching assessment mechanism, further improving the accuracy and reproducibility of matching.

[0018] Furthermore, the method for allelic matching of donor mesenchymal stem cells and recipients further includes: S70: stimulating the mesenchymal stem cells with an immunostimulatory factor to induce HLA antigen expression; S80: detecting the HLA antigen expression level of the mesenchymal stem cells before and after stimulation culture.

[0019] The technical effects achieved by this approach are as follows: Stimulating MSCs with immunostimulatory factors can simulate the in vivo inflammatory environment and induce HLA antigen expression. The expression levels of HLA-DR antigen in MSCs before and after stimulation can be quantitatively detected by flow cytometry, thus objectively assessing the strength of the stimulation-induced effect. If HLA-DR expression is low-inducible or non-inducible after stimulation, it indicates a low risk of activation and immune response in the in vivo inflammatory environment, making these donor-derived MSCs a preferred candidate for allogeneic transplantation. Conversely, if HLA-DR antigen expression is significantly upregulated after stimulation, it suggests a high risk of immune activation in the donor MSCs, making them unsuitable for allogeneic transplantation. Furthermore, by comparing the expression changes of HLA-DR and other class II HLA molecules before and after stimulation, the risk of activation and immune response in the inflammatory microenvironment of different donor MSCs can be quantified. This inducibility detection result is also recorded in the matching library for grading and ranking the immunogenicity of donors in the library.

[0020] Furthermore, the immunostimulatory factor is selected from one or more of interferon-gamma and conditioned culture supernatant of immune cells.

[0021] The technical effects achieved by adopting this technical solution are as follows: by using gamma-interferon or immune cell culture supernatant as specific immunostimulatory factors to treat mesenchymal stem cells, it is possible to effectively simulate the inflammatory microenvironment after in vivo transplantation in vitro and actively induce them to highly express HLA antigens and co-stimulatory molecules.

[0022] Furthermore, the conditions for stimulation culture using γ-interferon are as follows: add γ-interferon at a concentration of 10-50 ng / mL to the culture medium and continue culturing for 48h-72h.

[0023] The technical effects achieved by adopting this solution are as follows: By adding the aforementioned γ-interferon to the culture medium at a concentration of 10-50 ng / mL and continuously culturing for 48-72 hours, a standardized and efficient immune activation window for mesenchymal stem cells can be provided without inducing cytotoxicity or excessive aging. This condition is sufficient to fully upregulate the expression of HLA antigens to a level that can be stably detected by serum antibodies, thereby maximally mimicking the potential immune challenge of the in vivo inflammatory environment to transplanted cells. At the same time, it avoids missed detection due to insufficient stimulation or cell function damage caused by overstimulation, ensuring the reliability, sensitivity, and reproducibility of experimental results. Furthermore, it can provide predictive data on donor immunogenicity without altering the MSCs themselves.

[0024] Furthermore, the immune cells in the conditioned culture supernatant of immune cells include one or more of T cells, natural killer cells, monocytes, macrophages, and dendritic cells.

[0025] The technical effects achieved by adopting this approach are as follows: Stimulation with conditioned culture supernatants containing one or more immune cells, such as T cells, natural killer cells, monocytes, macrophages, or dendritic cells, can more comprehensively and realistically induce mesenchymal stem cells to express HLA antigen lineages that may be upregulated after actual transplantation by simulating the complex immune microenvironment in vivo, compared to single cytokines. This significantly improves the predictive accuracy and reliability of subsequent population reactive antibody functional analysis results, providing a more sufficient basis for clinical transplantation decisions. Attached Figure Description

[0026] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a flowchart illustrating the steps of a method for allelic matching between donor mesenchymal stem cells and recipients, as provided in an embodiment of the present invention. Detailed Implementation

[0027] To make the above-mentioned objectives, features, and advantages of the present invention more apparent and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] This invention provides a method for allelic typing of donor mesenchymal stem cells and recipients. The method includes the following steps: S10: Isolating mesenchymal stem cells from perinatal tissue of the donor; S20: Extracting genomic DNA from the mesenchymal stem cells and performing HLA genotyping; S30: Performing HLA genotyping on the recipient's DNA; S40: Comparing the allelic profiles of the recipient's HLA genes and the mesenchymal stem cells' HLA genes and calculating the matching degree; S50: Determining the degree of matching between the recipient and the mesenchymal stem cells based on the matching degree; S60: Storing the HLA genotyping results and matching degree of the mesenchymal stem cells in a database to establish a typing library for matching donor mesenchymal stem cells and recipients. See details below. Figure 1 As shown.

[0029] This embodiment provides a method for assessing the compatibility of mesenchymal stem cell transplantation. By extracting genomic DNA from donor MSCs and performing HLA genotyping, and then comparing the allele profiles of the recipient's HLA genes with those of the MSCs, a standardized system is established. This system aims to assess the compatibility of donor and recipient at the allele level, effectively predicting and avoiding the risk of immune rejection caused by HLA mismatch before treatment, and improving the in vivo survival and implantation efficiency of MSCs. By comparing the matching of recipient and donor MSCs at the HLA allele level and calculating the matching degree, the traditional serologically based fuzzy typing can be elevated to a precise matching at the gene level, thereby quantitatively and objectively assessing the basic immunocompatibility of the two in vitro. This not only provides a key basis for rapidly screening candidates with the highest gene matching degree among multiple potential donors and optimizing donor selection strategies, but also identifies combinations with high rejection risk due to high frequency mismatches in advance, effectively avoiding subsequent resource waste and laying a preliminary genetic foundation for personalized precision transplantation. By establishing a complete standardized process covering MSC isolation, amplification, HLA typing, and allele compatibility testing, consistency of results and clinical applicability across donor sources are ensured. Specifically, HLA typing results and matching data can be stored in a database or biobank, allowing for retrieval when needed. In clinical applications, donors with high matching scores will be prioritized for transplantation.

[0030] Existing technologies mainly reduce immune rejection by genetically modifying MSCs (e.g., using iPSC-derived MSCs to reduce HLA expression), without introducing donor-recipient matching at the allele level. This invention introduces a quantitative allele matching assessment mechanism, replacing the traditional antibody-based population response antibody (PRA) detection, and completely improving the accuracy and reproducibility of typing.

[0031] Perinatal tissues are selected from one or more of the placenta, umbilical cord, and amnion. When mesenchymal stem cells are derived from perinatal tissues such as the placenta, umbilical cord, or amnion, it ensures that the obtained cells possess superior biological characteristics such as strong originality, high proliferative capacity, and relatively low immunogenicity. These tissues, as medical waste, are obtained ethically without controversy and are widely available, providing a stable and high-quality cell source for establishing standardized, large-scale mesenchymal stem cell banks. This fundamentally guarantees the homogeneity and accessibility of cells required for subsequent HLA typing, allele matching, and transplantation therapy. The selection criteria for donor perinatal tissues are: mother age under 40 years, no family history of autoimmune or hereditary diseases, and negative infectious disease test results.

[0032] In some embodiments of this application, step S10 specifically includes the following steps: S110: Isolating mesenchymal stem cells from perinatal tissues; S120: Performing quality testing on the isolated mesenchymal stem cells to obtain qualified mesenchymal stem cells; S130: Expanding and culturing the qualified mesenchymal stem cells to obtain expanded mesenchymal stem cells.

[0033] This embodiment utilizes a complete process of isolation from perinatal tissues, rigorous quality testing, and in vitro expansion to reliably obtain sufficient quantities of mesenchymal stem cells (MSCs) with viability and purity meeting clinical standards. This provides a high-quality, standardized cell source for subsequent precise allele matching and transplantation therapy, fundamentally ensuring the reliability of evaluation results and the effectiveness and safety of the final treatment. Specifically, MSCs are isolated and cultured under aseptic and well-regulated production conditions. Quality control measures for the expanded MSCs include microbial contamination, mycoplasma detection, immunophenotypic identification, and differentiation capacity assessment. The method complies with GMP standards and is suitable for large-scale clinical library construction applications for HLA typing of MSCs.

[0034] Mesenchymal stem cells can be isolated from the perinatal tissues of the donor using enzymatic digestion or explant culture.

[0035] In some embodiments of this application, the expanded mesenchymal stem cells (MSCs) are expanded from passage 2 to passage 6. Using expanded passage 2 to passage 6 MSCs ensures that the MSCs have been sufficiently expanded to obtain a sufficient number of cells for DNA extraction and cell cryopreservation, providing high-quality standardized cells for the entire evaluation process and potential clinical applications. Preferably, the isolated MSCs are cultured and expanded to passage 4 under GMP-compliant aseptic conditions. Genomic DNA is extracted from the cultured and expanded passage 4 MSCs, which can be done using commercial kits or automated systems.

[0036] In some embodiments of this application, HLA genotyping identifies at least HLA-A, HLA-B, and HLA-C loci, and compares them with the recipient's HLA allele profile, ultimately achieving precise HLA identification. This not only provides crucial evidence for assessing immunocompatibility and reducing rejection risk during allogeneic transplantation, but also lays a solid technical foundation for establishing standardized cell banks and ensuring product quality control, thereby significantly improving the safety and clinical application potential of allogeneic mesenchymal stem cell therapy. Next-generation sequencing (NGS) or other high-resolution methods are used for HLA loci genotyping. The alleles identified by sequencing can be cross-referenced with the IPD-IMGT / HLA database to verify their accuracy.

[0037] In some embodiments of this application, HLA genotyping also identifies the HLA-DR and HLA-DQ loci. Expanding the scope of HLA genotyping to include the HLA-DR and HLA-DQ loci provides comprehensive immunogenotypes of donor mesenchymal stem cells, thereby constructing a more complete HLA lineage. This provides crucial predictive information for assessing long-term immune rejection, significantly improving the comprehensiveness and prospectiveness of the matching assessment system, and laying a more solid genetic foundation for screening high-quality donors with lower immunogenicity and higher post-transplant survival potential.

[0038] In some embodiments of this application, the matching criteria are as follows: perfect match: all 12 alleles are identical; partial match: 6 to 11 alleles are identical; low match: 0 to 5 alleles are identical. See Table 1 for details. The HLA genotyping results and matching scores are stored in a searchable clinical database or biobank system.

[0039] Table 1. Classification criteria for allele matching between recipient and donor mesenchymal stem cells By adopting the above classification criteria, the HLA matching degree between recipient and donor mesenchymal stem cells can be accurately quantified and clearly graded, thereby transforming complex gene typing data into an intuitive and actionable basis for clinical decision-making. This standard clearly defines "perfect match," "partial match," and "low match," greatly facilitating rapid donor screening and prioritization. For example, donors with "perfect matches" or "partial matches" can be prioritized for subsequent functional cross-matching. Furthermore, it can directly warn of the high immune rejection risk inherent in "low-match" combinations, effectively avoiding their involvement in unnecessary subsequent experimental and clinical processes. This significantly optimizes resource allocation and evaluation efficiency while improving transplant safety. In other words, this technical solution introduces a quantitative allele matching assessment mechanism, further improving the accuracy and reproducibility of matching. All matching data is stored in a secure and searchable database. In clinical applications, donors with high matching scores will be prioritized for transplantation. Furthermore, HLA gene typing results and matching data are stored in a searchable database or clinical biobank.

[0040] It should be noted that MSCs characterized according to the standard procedures of this invention are cryopreserved and systematically cataloged in a biobank according to their HLA genotyping results, matching degree, and relevant donor information (such as donor age, tissue origin, and health parameters). This database can also integrate detailed maternal health records and tissue origin information to improve the accuracy of donor tracing and clinical decision-making. Through this comprehensive biobank infrastructure, highly compatible MSC donors can be efficiently retrieved and selected for clinical transplantation and regenerative therapy.

[0041] In some embodiments of this application, the method for allelic matching of donor mesenchymal stem cells (MSCs) and recipients further includes: S70: stimulating the MSCs with immunostimulatory factors to induce HLA antigen expression. Stimulating MSCs with immunostimulatory factors can simulate an in vivo inflammatory environment and induce HLA antigen expression. If HLA-DR expression is low-inducible or non-inducible after stimulation, it indicates that the donor-derived MSCs have a low risk of activation and triggering an immune response in an in vivo inflammatory environment, and therefore can be preferentially selected for allogeneic transplantation. In this step, the majority of MSCs used for treatment are not treated; only a small portion of the donor MSCs undergo the aforementioned induction assessment as quality control. Therefore, step S70 can also be performed simultaneously with HLA genotyping.

[0042] In some embodiments of this application, the immunostimulatory factor is selected from one or more of interferon-gamma and conditioned culture supernatant of immune cells. By using specific immunostimulatory factors such as interferon-gamma or conditioned culture supernatant of immune cells to treat mesenchymal stem cells, the inflammatory microenvironment after in vivo transplantation can be effectively simulated in vitro, actively inducing high expression of HLA antigens and co-stimulatory molecules.

[0043] In some embodiments of this application, the conditions for stimulation culture using interferon-gamma are as follows: interferon-gamma at a concentration of 10-50 ng / mL is added to the culture medium, and the cells are cultured continuously for 48-72 hours. These stimulation conditions provide a standardized and efficient immune activation window for mesenchymal stem cells (MSCs) without inducing cytotoxicity or excessive senescence. These conditions are sufficient to adequately upregulate HLA antigen expression to a level that can be stably detected by serum antibodies, thereby maximally mimicking the potential immune challenge of the in vivo inflammatory environment to transplanted cells. Simultaneously, they avoid missed detections due to insufficient stimulation or cell function damage caused by overstimulation, ensuring the reliability, sensitivity, and reproducibility of experimental results. Furthermore, they provide predictive data on donor immunogenicity without altering the MSCs themselves. In addition, quantitative detection of changes in HLA-DR antigen expression levels in MSCs before and after stimulation using flow cytometry clarifies the induction effect of this stimulation step on HLA expression. Simultaneously, flow cytometry can quantitatively detect changes in HLA-DR antigen expression levels in MSCs before and after stimulation, further clarifying the stimulation-induced effect.

[0044] In some embodiments of this application, the immune cells in the conditioned culture supernatant include one or more of T cells, natural killer cells, monocytes, macrophages, and dendritic cells. Stimulation with conditioned culture supernatants containing one or more of these immune cells can, by simulating the complex immune microenvironment in vivo, more comprehensively and realistically induce mesenchymal stem cells to express HLA antigen lineages that may be upregulated after actual transplantation, compared to single cytokines. This significantly improves the predictive accuracy and reliability of subsequent population reactive antibody functional analysis results, providing a more comprehensive basis for clinical transplantation decisions.

[0045] Any of the above embodiments can be used for cell therapy of autoimmune diseases, diabetes, regenerative medicine or inflammatory diseases, and can also be used as adjuvant cell therapy for organ transplantation.

[0046] This invention also provides a cryopreserved MSCs cell bank with complete HLA typing records. This MSCs bank allows for rapid donor-recipient matching, improving transplant success rates. The method for allele matching of donor mesenchymal stem cells and recipients described in this invention can be widely applied to regenerative medicine and the treatment of autoimmune diseases. This invention has higher clinical applicability and relevance, as demonstrated in the various embodiments provided herein (umbilical cord, placenta, amnion applications, biobank establishment, and clinical trials). This integrated approach significantly improves graft survival rates, reduces the incidence of immune rejection, and contributes to the advancement of precision medicine in the field of cell therapy.

[0047] The MSCs cryopreservation bank catalogs each batch of MSCs samples according to their HLA genotype, allele matching score, and donor-related information. The database integrates the donor's clinical history, tissue origin information, and maternal health data to optimize donor-recipient matching.

[0048] In addition, artificial intelligence algorithms integrate HLA genotype, matching scores, and inducibility data for comprehensive analysis to rank candidate donors and predict graft survival and immune tolerance. For example, HLA typing data and matching scores from an MSCs biobank can be input into an AI algorithm model trained based on historical transplant results. This system ranks candidate donors based on predicted compatibility and graft survival probability, significantly shortening donor selection time and improving transplant success rates. With this AI-assisted matching system, donors can be ranked based on predicted graft survival, immune tolerance, and the need to reduce immunosuppression. This AI-driven selection process enables clinicians to quickly identify the optimal donor, significantly improving transplant success rates and reducing the risk of rejection or other adverse immune responses.

[0049] Example 1 Embodiments of the present invention also provide a method for assessing the compatibility of mesenchymal stem cell transplantation, comprising the following steps: S1: MSCs were isolated from umbilical cord tissue using enzymatic digestion under sterile biological conditions; S2: The isolated MSCs were tested for microbial contamination, mycoplasma, immunophenotypic identification, and differentiation capacity to obtain qualified mesenchymal stem cells; S3: Expand qualified MSCs to the 4th generation to obtain expanded mesenchymal stem cells; S4: Genomic DNA was extracted from the amplified MSCs and HLA genotyping was performed based on sequencing to determine the loci of HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ. S5: Perform HLA genotyping on the receptor to determine the HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ loci; S6: Compare the allelic profiles of the HLA genes of the receptor and the HLA genes of MSCs, and calculate the matching percentage; S7: Determine the degree of matching between the recipient and mesenchymal stem cells based on the matching percentage; S8: Store the HLA genotyping results of mesenchymal stem cells and the matching degree in a searchable database to establish a matching library for matching the donor mesenchymal stem cells with the recipient.

[0050] Example 2 S1: MSCs were isolated from umbilical cord tissue using enzymatic digestion under sterile biological conditions; S2: The isolated MSCs were tested for microbial contamination, mycoplasma, immunophenotypic identification, and differentiation capacity to obtain qualified mesenchymal stem cells; S3: Expand qualified MSCs to the 4th generation to obtain expanded mesenchymal stem cells; S4: Genomic DNA was extracted from the amplified MSCs and HLA genotyping was performed based on sequencing to determine the loci of HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ. S5: Perform HLA genotyping on the receptor to determine the HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ loci; S6: Compare the allelic profiles of the HLA genes of the receptor and the HLA genes of MSCs, and calculate the matching percentage; S7: Mesenchymal stem cells were stimulated and cultured to induce HLA antigen expression, and the inducibility of HLA class I and II expression was assessed by flow cytometry. The stimulation culture conditions are as follows: γ-interferon at a concentration of 10-50 ng / mL is added to the culture medium and cultured continuously for 48-72 hours.

[0051] S8: Detect the expression level of HLA antigens in mesenchymal stem cells before and after stimulation; S9: Determine the degree of matching between the receptor and mesenchymal stem cells based on the matching percentage and the expression level of HLA antigens; S10: Store the HLA gene typing results of mesenchymal stem cells and the matching degree in a searchable database to establish a matching library for matching the donor mesenchymal stem cells with the recipient.

[0052] It should be noted that step S7 only performs the above-mentioned induction assessment on a small portion of donor MSCs as a quality control measure. Therefore, step S7 can also be performed concurrently with step S4.

[0053] Example 3 HLA genotyping was performed on placental MSCs, and their HLA allele profiles were compared with several potential recipients. A matching algorithm was used to calculate the matching score of donor-recipient pairs, and the results were categorized as fully matched, partially matched, or poorly matched / no match. For donors with ≥10 matching alleles, their MSCs were prioritized for clinical transplantation. This allele matching system provides an objective and quantitative assessment of donor-recipient compatibility, replacing traditional antibody-based population response antibody (PRA) testing methods.

[0054] Example 4 MSCs were isolated from amniotic tissue, cultured, amplified, and HLA genotyped. Donors with rare HLA haplotypes were prioritized during donor selection. These amniotic MSCs were then formulated into cell preparations and infused into patients with systemic lupus erythematosus (SLE) for treatment. This example highlights the value of the method of the present invention in screening optimal MSC donors for personalized autoimmune disease therapy, ensuring the lowest possible risk of immune rejection after transplantation.

[0055] Example 5 Patients with Crohn's disease were included in a clinical study. Prior to each cell infusion, the best-matched MSC donors were selected from an established HLA-generated MSCs library. The selected MSCs were infused into the patients, and the efficacy was followed up for 12 months. Results showed that, compared to MSCs therapy without HLA genotyping, MSCs selected using the method of this invention exhibited a higher engraftment success rate and fewer immune-related adverse reactions in patients. This example further demonstrates the effectiveness and value of this invention in clinical applications.

[0056] Compared with existing methods, the method of the present invention for allelic matching of donor mesenchymal stem cells and recipients has higher clinical applicability and relevance. This integrated method significantly improves the survival rate of grafts, reduces the incidence of immune rejection, and helps to promote the development of precision medicine in the field of cell therapy.

[0057] The invented method for allelic matching of donor mesenchymal stem cells and recipients can also be applied to MSCs derived from adipose tissue or bone marrow.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for allelic matching between donor mesenchymal stem cells and recipients, characterized in that, The method includes the following steps: S10: Isolate mesenchymal stem cells from the perinatal tissues of the donor; S20: Extract genomic DNA from the mesenchymal stem cells and perform HLA genotyping; S30: HLA genotyping of the recipient DNA; S40: Compare the allelic profiles of the HLA gene of the receptor with those of the HLA gene of the mesenchymal stem cells, and calculate the matching degree; S50: Determine the degree of matching between the receptor and the mesenchymal stem cells based on the matching degree. S60: Store the HLA gene typing results of the mesenchymal stem cells and the matching degree in a database to establish a matching library for matching the donor mesenchymal stem cells with the recipient.

2. The method according to claim 1, characterized in that, Step S10 specifically includes the following steps: S110: Isolate the mesenchymal stem cells from the perinatal tissue; S120: The isolated mesenchymal stem cells are subjected to quality testing to obtain qualified mesenchymal stem cells; S130: The qualified mesenchymal stem cells are expanded and cultured to obtain expanded mesenchymal stem cells.

3. The method according to claim 2, characterized in that, The expanded mesenchymal stem cells are expanded from the 2nd to the 6th generation of mesenchymal stem cells.

4. The method according to claim 1, characterized in that, The HLA genotyping identifies at least the HLA-A, HLA-B, and HLA-C loci.

5. The method according to claim 4, characterized in that, The HLA genotyping also confirmed the HLA-DR and HLA-DQ loci.

6. The method according to claim 1, characterized in that, The classification criteria for the matching degree are as follows: Perfect match: all 12 alleles are identical; Partial match: There are 6 to 11 identical alleles; Low match: There are 0 to 5 identical alleles.

7. The method according to claim 1, characterized in that, Also includes: S70: The mesenchymal stem cells are stimulated and cultured using immunostimulatory factors to induce HLA antigen expression; S80: Detect the HLA antigen expression level of the mesenchymal stem cells before and after stimulation culture.

8. The method according to claim 7, characterized in that, The immunostimulatory factor is selected from one or more of gamma interferon and conditioned culture supernatant of immune cells.

9. The method according to claim 8, characterized in that, The conditions for stimulation culture using the aforementioned γ-interferon are as follows: Add the γ-interferon at a concentration of 10-50 ng / mL to the culture medium and continue culturing for 48-72 h.

10. The method according to claim 8, characterized in that, The immune cells in the conditioned culture supernatant of the immune cells include one or more of T cells, natural killer cells, monocytes, macrophages, and dendritic cells.

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