Method for evaluating transplantation compatibility of mesenchymal stem cells
By performing HLA genotyping and immune stimulation culture on donor mesenchymal stem cells, combined with recipient serum analysis, the problem of unpredictable immune responses in mesenchymal stem cell transplantation has been solved, achieving efficient pre-transplantation assessment and improved safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG TIANYUAN BIOTECHNOLOGY CO LTD
- Filing Date
- 2025-12-03
- Publication Date
- 2026-04-17
AI Technical Summary
In existing technologies, high-resolution HLA typing is not performed before mesenchymal stem cell transplantation, leading to unpredictable immune responses, which reduces implantation efficiency and long-term treatment efficacy. Furthermore, traditional methods cannot completely avoid the risk of immune rejection.
Mesenchymal stem cells were isolated from donor perinatal tissues, HLA genotyping and immunostimulation culture were performed, HLA antigen expression was induced by simulating the in vivo inflammatory environment, and high-resolution immunocompatibility assessment was carried out by combining the functional analysis of population reactive antibodies in recipient serum.
This approach enables a comprehensive assessment of the transplant compatibility of donor MSCs, significantly reduces the risk of immune rejection, improves the safety and efficacy of treatment, ensures high quality and standardization of cell sources, and provides a reliable basis for pre-transplant screening.
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Figure CN121874331A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical materials technology, and more specifically, to a method for evaluating the compatibility of mesenchymal stem cell transplantation. 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. They are widely studied globally and are considered a promising cell therapy tool for the treatment of various chronic diseases, autoimmune diseases, and tissue damage.
[0003] In traditional solid organ transplantation or bone marrow transplantation, HLA matching is an essential prerequisite for successful graft implantation and long-term survival. High-resolution HLA matching can minimize the risk of rejection of the graft by the host's immune system and is the cornerstone of transplant success.
[0004] In contrast, in the field of MSC transplantation, neglecting pre-transplant assessment of MSC immunogenicity can lead to unpredictable immune responses, resulting in low engraftment efficiency and rapid immune clearance of MSCs, ultimately weakening or even eliminating their long-term therapeutic effects. Currently, in allogeneic MSC transplantation practice, due to the widespread belief that MSCs have low immunogenicity, HLA typing pretreatment is often not performed clinically, or only a limited partial typing strategy is adopted. However, this unmatched transplantation method cannot completely eliminate the risk of immune rejection; studies have reported that unmatched MSC transplantation can still trigger recipient immune responses. For example, pre-existing anti-HLA antibodies in recipient serum are considered one of the main causes of rejection. Traditionally, flow cytometry cross-matching (such as PRA-based functional cross-matching) is used to pre-screen for such immune risks, but this strategy has not yet been widely adopted in MSC transplantation.
[0005] Therefore, there is an urgent need to develop a reliable and comprehensive method to assess the immunocompatibility between allogeneic MSCs and recipients before transplantation, in order to screen for the best MSCs donors, thereby avoiding the risk of immune rejection and improving the efficiency of MSCs implantation and the overall success rate of clinical treatment. Summary of the Invention
[0006] The present invention aims to provide a method for assessing the compatibility of mesenchymal stem cell transplantation, which is used to evaluate the immunocompatibility between allogeneic MSCs and recipients before transplantation, so as to screen the best MSCs donors, thereby avoiding the risk of immune rejection and improving the implantation efficiency of MSCs and the overall success rate of clinical treatment.
[0007] To address the aforementioned problems, this invention provides a method for assessing the compatibility of mesenchymal stem cell transplantation. The method includes the following steps: S10: isolating mesenchymal stem cells from perinatal tissue of a donor; S20: extracting genomic DNA from the mesenchymal stem cells and performing HLA genotyping; S30: stimulating the mesenchymal stem cells with immunostimulatory factors to induce HLA antigen expression; S40: performing functional analysis based on population reactive antibodies on recipient serum and the stimulated mesenchymal stem cells; S50: determining transplant compatibility based on the HLA genotyping results and the functional analysis results of the population reactive antibodies.
[0008] The technical effects achieved by this approach are as follows: Genomic DNA is extracted from donor MSCs and HLA genotyping is performed. Based on this, MSCs are stimulated and cultured using immunostimulatory factors to simulate an in vivo inflammatory environment, inducing upregulation of HLA antigen expression and placing the cells in a state more easily recognized by the immune system. Subsequent functional cross-matching of recipient serum based on population reactive antibodies (PRA) is performed. Flow cytometry is used to detect the binding degree between anti-HLA antibodies in recipient serum and HLA antigens induced by donor MSCs. This allows for a prospective assessment of the risk of antibody-mediated immune rejection before transplantation, achieving a comprehensive evaluation of donor MSC transplant compatibility. This provides a reliable basis for clinical selection of high-quality MSC donors with low immunogenicity and avoidance of transplant rejection, significantly improving the safety and efficacy of treatment. Compared with existing technologies, this invention, through high-resolution HLA genotyping and PRA cross-matching assessment of donor MSCs before transplantation, achieves prospective screening of donor-recipient immunocompatibility, significantly reducing the risk of immune rejection, demonstrating its novelty and clinical value.
[0009] 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.
[0010] 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.
[0011] Furthermore, the expanded mesenchymal stem cells are expanded from the 2nd to the 6th generation of mesenchymal stem cells.
[0012] The technical effects achieved by adopting this solution are as follows: Mesenchymal stem cells from the 2nd to 6th generation are expanded. At this time, the mesenchymal stem cells have been sufficiently expanded to obtain a sufficient number of cells for DNA extraction and cell cryopreservation, thereby ensuring the consistency of the cell population used in subsequent HLA typing and PRA-based functional crossmatch analysis, and providing high-quality standardized cell materials for the entire evaluation process and potential clinical applications.
[0013] Further, step S20 specifically includes the following steps: S210: extracting genomic DNA from mesenchymal stem cells; S220: performing HLA genotyping based on sequencing to determine at least HLA-A, HLA-B, and HLA-C loci; S230: comparing the loci with a database to determine allele allocation.
[0014] The technical effects achieved by adopting this solution are as follows: High-resolution genotyping of mesenchymal stem cells at least at HLA-A, HLA-B, and HLA-C loci based on sequencing, and accurate allele allocation through database comparison, ultimately achieving precise 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.
[0015] Furthermore, in step S220, HLA genotyping also confirmed the HLA-DR and HLA-DQ loci.
[0016] The technical effects achieved by adopting this solution are as follows: In step S220, the HLA genotyping range is expanded to cover the 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.
[0017] Furthermore, in step S30, the immunostimulatory factor is selected from one of interferon-gamma and the supernatant of conditioned medium obtained from immune cells.
[0018] The technical effects achieved by adopting this approach are as follows: By treating mesenchymal stem cells with specific immunostimulatory factors such as interferon-gamma or conditioned medium supernatant obtained from immune cells, the inflammatory microenvironment after transplantation can be effectively simulated in vitro, actively inducing high expression of HLA antigens and co-stimulatory molecules. This functionally activated state enables subsequent cross-matching experiments with recipient serum to more realistically and sensitively detect the risk of pre-existing antibody-mediated immune rejection, thereby overcoming the limitations of insufficient assessment of immunogenicity in resting cells and significantly improving the accuracy and clinical predictive value of pre-transplant compatibility prediction.
[0019] Furthermore, the conditions for stimulation culture using 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 48h-72h.
[0020] 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 (MSCs) can be provided 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 to transplanted cells from the in vivo inflammatory environment. Simultaneously, it avoids missed detections due to insufficient stimulation or cell function damage caused by overstimulation, ensuring the reliability, sensitivity, and reproducibility of subsequent PRA-based functional crossmatching experiments. This provides crucial and reliable functional experimental data for clinical prediction of antibody-mediated rejection risk. Furthermore, it can provide predictive data on donor immunogenicity without altering the MSCs themselves.
[0021] Furthermore, the immune cells in the conditioned medium supernatant obtained from the immune cells include one or more of T cells, natural killer cells, monocytes, macrophages, and dendritic cells.
[0022] The technical effects achieved by adopting this approach are as follows: Stimulation with conditioned medium supernatants obtained from 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. 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.
[0023] Furthermore, in step S40, the functional analysis of the population reactive antibody is performed by using flow cytometry to detect the binding level of anti-HLA antibodies in the recipient serum to HLA antigens on the surface of the stimulated mesenchymal stem cells.
[0024] The technical effects achieved by adopting this technical solution are as follows: By using flow cytometry to detect the binding level of anti-HLA antibodies in recipient serum to HLA antigens on the surface of stimulated cultured mesenchymal stem cells, direct and quantitative analysis of the specific binding reaction between antibodies and cells can be achieved. This method not only has high sensitivity and multi-parameter detection, but also can simultaneously assess the immunoreactivity of multiple HLA antigens while maintaining cell integrity.
[0025] Furthermore, in step S50, the functional analysis results of the population reactive antibodies are expressed as a percentage of population reactive antibodies, which is used to assess transplant compatibility; wherein, the percentage is less than 20% and indicates a low risk of immune rejection; the percentage is 20% to 50% and indicates a moderate risk of immune rejection; the percentage is greater than 50% and indicates a high risk of immune rejection.
[0026] The technical effects achieved by adopting this solution are as follows: risk stratification of donor MSC transplant compatibility based on the quantitative standard of the percentage of population reactive antibodies, which enables clinicians to determine the level of transplant rejection risk based on a clear threshold, thereby providing an intuitive and reliable reference for donor selection and transplantation decisions, and further improving transplantation safety.
[0027] Furthermore, the perinatal tissues are selected from one or more of the placenta, umbilical cord, and amnion.
[0028] The technical effects achieved by adopting this solution are as follows: Mesenchymal stem cells are derived from perinatal tissues such as the placenta, umbilical cord, or amnion, ensuring that the obtained cells have superior biological characteristics such as strong originality, high proliferative capacity, and relatively low immunogenicity. As these tissues are medical waste, their acquisition process is ethically uncontroversial and their sources are widespread, providing a stable and high-quality cell source for establishing a standardized and large-scale mesenchymal stem cell bank. This fundamentally guarantees the homogeneity and accessibility of cell materials required for subsequent HLA typing, crossmatching, and transplantation therapy.
[0029] Furthermore, based on the HLA genotyping results and the functional analysis results of population reactive antibodies, donors with rare HLA haplotypes and / or low population reactive antibody response rates are given priority.
[0030] The technical effects achieved by adopting this approach are as follows: By prioritizing donors with rare HLA haplotypes and / or low PRA reactivity based on HLA genotyping and population reactive antibody functional analysis, the probability of cross-reactivity between recipient pre-existing anti-HLA antibodies and donor MSCs can be significantly reduced, thereby decreasing the incidence of antibody-mediated rejection and improving transplant compatibility. This strategy enables a shift from traditional donor-recipient matching to refined screening based on immunogenicity risk stratification, which helps reduce the burden of immunosuppression, improve long-term graft survival, and provides a foundation for building an MSC cell bank centered on low-immunogenic donors and personalized clinical dosing regimens. Attached Figure Description
[0031] 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 A flowchart illustrating the steps of a method for evaluating mesenchymal stem cells for transplantation donors, provided in an embodiment of the present invention. Detailed Implementation
[0032] 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.
[0033] This invention provides a method for assessing the compatibility of mesenchymal stem cell transplantation. The method includes the following steps: S10: Isolating mesenchymal stem cells from perinatal tissue of a donor; S20: Extracting genomic DNA from the mesenchymal stem cells and performing HLA genotyping; S30: Stimulating the mesenchymal stem cells with immunostimulatory factors to induce HLA antigen expression; S40: Performing functional cross-matching analysis based on population reactive antibodies between recipient serum and the stimulated mesenchymal stem cells; S50: Determining transplant compatibility based on the HLA genotyping results and the functional analysis results of the population reactive antibodies. See details below. Figure 1 As shown.
[0034] This embodiment provides a method for assessing the compatibility of mesenchymal stem cell transplantation. Genomic DNA is extracted from donor MSCs and HLA genotyping is performed. Based on this, MSCs are stimulated and cultured using immunostimulatory factors to simulate an in vivo inflammatory environment, inducing upregulation of HLA antigen expression and placing the cells in a state more easily recognized by the immune system. Subsequent PRA-based functional crossmatching analysis of recipient serum, using flow cytometry to detect the binding degree between anti-HLA antibodies in recipient serum and HLA antigens induced by donor MSCs, allows for a prospective assessment of the risk of antibody-mediated immune rejection before transplantation. This comprehensive assessment of donor MSC transplantation compatibility provides a reliable basis for clinical selection of high-quality MSC donors with low immunogenicity and avoidance of transplant rejection, significantly improving the safety and effectiveness of treatment.
[0035] The selection criteria for donor perinatal tissues are: mothers under 40 years of age, no history of autoimmune or hereditary diseases, and negative results for infectious diseases.
[0036] 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.
[0037] This embodiment provides a method for assessing the compatibility of mesenchymal stem cell (MSC) transplantation. Through a complete process of isolation from perinatal tissues, rigorous quality testing, and in vitro expansion, it is possible to stably obtain sufficient quantities of MSCs with viability and purity meeting clinical standards. 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. Specifically, MSCs are isolated and cultured under aseptic and well-manufactured conditions. The quality of the expanded MSCs is assessed, including quality control tests for microbial contamination, mycoplasma detection, immunophenotypic identification, and differentiation capacity evaluation. Enzymatic digestion is preferably used to isolate MSCs.
[0038] In some embodiments of this application, the expanded mesenchymal stem cells are expanded generation 2 to 6 mesenchymal stem cells. Preferably, the expanded mesenchymal stem cells are generation 4 mesenchymal stem cells.
[0039] This embodiment provides a method for assessing the compatibility of mesenchymal stem cell transplantation using expanded MSCs from passage 2 to passage 6. At this stage, the MSCs have been sufficiently expanded to obtain a sufficient number of cells for DNA extraction and cell cryopreservation, ensuring the consistency of the cell population used for subsequent HLA typing and PRA-based functional crossmatching analysis. This provides high-quality, standardized cell material for the entire assessment process and potential clinical applications. Genomic DNA is extracted from the expanded passage 4 MSCs, preferably using commercial kits or automated extraction systems to ensure consistent DNA yield and purity.
[0040] In some embodiments of this application, step S20 specifically includes the following steps: S210: extracting genomic DNA from mesenchymal stem cells; S220: performing HLA genotyping based on sequencing to determine at least HLA-A, HLA-B, and HLA-C loci; S230: comparing the loci with a database to determine allele allocation.
[0041] This embodiment provides a method for assessing the compatibility of mesenchymal stem cell transplantation. High-resolution genotyping of at least HLA-A, HLA-B, and HLA-C loci in mesenchymal stem cells is performed based on sequencing, and precise allele allocation is achieved through database comparison. This ultimately enables accurate HLA identification, providing crucial evidence for assessing immunocompatibility and reducing rejection risk during allogeneic transplantation. It also lays a solid technical foundation for establishing standardized cell banks and ensuring product quality control, thus significantly improving the safety and clinical application potential of allogeneic mesenchymal stem cell therapy. This embodiment uniquely combines sequencing-based genotyping with PRA testing to ensure transplant compatibility assessment. Next-generation sequencing (NGS) or other high-resolution methods are used for HLA locus genotyping.
[0042] This method involves extracting genomic DNA and performing sequencing-based genotyping at HLA-A, HLA-B, and HLA-C loci. Allele results were compared with international HLA reference databases. The practical application of this method in generating HLA profiles for umbilical cord-derived MSCs used in allogeneic transplantation for autoimmune diseases is demonstrated. International reference databases include IPD-IMGT / HLA databases.
[0043] In some embodiments of this application, in step S220, HLA genotyping further confirms the HLA-DR and HLA-DQ loci.
[0044] In some embodiments of this application, the HLA genotyping range is expanded to include HLA-DR and HLA-DQ loci in step S220, providing 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 survival potential after transplantation.
[0045] In some embodiments of this application, in step S30, the immunostimulatory factor is selected from one of interferon-gamma and conditioned medium supernatant obtained from immune cells. By using specific immunostimulatory factors such as interferon-gamma or conditioned medium supernatant obtained from 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. This functional activation state enables subsequent cross-matching experiments with recipient serum to more realistically and sensitively detect the risk of pre-existing antibody-mediated immune rejection, thereby overcoming the limitations of insufficient assessment of immunogenicity of resting cells and significantly improving the accuracy and clinical predictive value of pre-transplant compatibility prediction.
[0046] In some embodiments of this application, the conditions for stimulation culture using interferon-gamma are as follows: interferon-gamma is added to the culture medium at a concentration of 10-50 ng / mL, and cultured continuously for 48-72 hours. The inducibility of HLA-DR expression is quantified by flow cytometry as a fold change relative to baseline, wherein donors with low or non-inducible HLA-DR expression are preferred to reduce the risk of immune rejection.
[0047] By employing a stimulation culture condition involving the addition of 10-50 ng / mL of the aforementioned interferon-gamma to the culture medium and continuous culturing for 48-72 hours, a standardized and efficient immune activation window for mesenchymal stem cells (MSCs) can be provided without inducing cytotoxicity or excessive senescence. This condition is sufficient to adequately upregulate HLA antigen expression to a level stably detectable by serum antibodies, thereby maximally mimicking the potential immune challenge posed by the in vivo inflammatory environment to transplanted cells. Simultaneously, it avoids missed detections due to insufficient stimulation or cell function damage caused by overstimulation, ensuring the reliability, sensitivity, and reproducibility of subsequent crossmatching experiments. This provides crucial and reliable functional experimental data for clinical prediction of antibody-mediated rejection risk. Furthermore, it can provide predictive data on donor immunogenicity without altering the MSCs themselves.
[0048] In some embodiments of this application, the immune cells in the conditioned medium supernatant obtained from immune cells include one or more of T cells, natural killer cells, monocytes, macrophages, and dendritic cells. Stimulation with the conditioned medium supernatant obtained from 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.
[0049] In some embodiments of this application, in step S40, the functional analysis of the population-reactive antibody involves detecting the binding level of anti-HLA antibodies in the recipient serum to HLA antigens on the surface of stimulated cultured mesenchymal stem cells using flow cytometry. Detecting the binding level of anti-HLA antibodies in the recipient serum to HLA antigens on the surface of stimulated cultured mesenchymal stem cells using flow cytometry enables direct and quantitative analysis of the specific binding reaction between antibodies and cells. This method not only has high sensitivity and multi-parameter detection but also allows for the simultaneous assessment of the immunoreactivity of multiple HLA antigens while maintaining cell integrity.
[0050] In some embodiments of this application, in step S50, the functional analysis results of the population reactive antibodies are expressed as a percentage of population reactive antibodies, which is used to assess transplant compatibility. A percentage below 20% indicates a low risk of immune rejection; a percentage between 20% and 50% indicates a moderate risk of immune rejection; and a percentage above 50% indicates a high risk of immune rejection. This quantitative standard of population reactive antibody percentage allows for risk stratification of donor MSC transplant compatibility, enabling clinicians to determine the level of transplant rejection risk based on clear thresholds. This provides an intuitive and reliable reference for donor selection and transplantation decisions, further improving transplantation safety.
[0051] Using PRA analysis as a crossmatching method, a pre-screened spectrophotometer containing dozens of common HLA antigens can systematically detect the presence of a wide range of anti-HLA antibodies in recipient serum. This allows for a single assessment of the overall risk of "pre-sensitization" to donor mesenchymal stem cells (MSCs). This not only identifies single antibodies against specific donor HLA antigens but also efficiently screens for "high-risk" recipients highly sensitized due to previous pregnancy, transfusion, or transplantation. It provides a comprehensive view of the recipient's immune background for clinical decision-making, significantly enhancing the comprehensiveness and efficiency of pre-transplant risk assessment. The crossmatching results quantify immunocompatibility through the percentage of binding between donor antigens and recipient antibodies (PRA value). A low PRA percentage (e.g., below 20%) indicates low immune risk, meaning fewer pre-existing antibodies against donor HLA antigens in the recipient serum; while a high PRA percentage (e.g., above 50%) indicates the presence of a wide range of anti-donor HLA antibodies, a high risk of immune rejection, and suggests against using the donor MSCs for that recipient. Patient serum is tested using a PRA test targeting MSCs. The results showed reduced immunogenicity compared to the unmatched control group. This example illustrates how the present invention can be used to pre-screen donor MSCs for compatibility in solid organ transplantation (such as kidney transplantation).
[0052] In some embodiments of this application, perinatal tissues are selected from one or more of the placenta, umbilical cord, and amnion. Mesenchymal stem cells derived from perinatal tissues such as the placenta, umbilical cord, or amnion ensure that the obtained cells possess superior biological characteristics, including high primacy, high proliferative capacity, and relatively low immunogenicity. These tissues, as medical waste, are ethically uncontroversial to obtain 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 cell materials required for subsequent HLA typing, matching, and transplantation therapy. The ethical acquisition of perinatal tissues refers to the ethically compliant acquisition and processing of perinatal tissues such as the placenta, umbilical cord, and amnion, with informed consent.
[0053] In some embodiments of this application, based on HLA genotyping results and functional analysis results of population reactive antibodies, by preferentially selecting donors with rare HLA haplotypes and / or low PRA reactivity rates, the probability of cross-reaction between recipient pre-existing anti-HLA antibodies and donor MSCs can be significantly reduced, thereby reducing the incidence of antibody-mediated rejection and improving transplant compatibility.
[0054] 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 safety conditions; S2: The isolated MSCs were tested for quality by microbial contamination, mycoplasma detection, immunophenotypic identification, and differentiation capacity assessment; qualified mesenchymal stem cells were obtained. 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 at least the HLA-A, HLA-B, and HLA-C loci; the results were compared with international reference databases to confirm allele assignments. S5: Stimulate and culture mesenchymal stem cells to induce HLA antigen expression, and assess the inducibility of HLA class I and II expression by flow cytometry; The conditions for stimulation culture are as follows: γ-interferon at a concentration of 10-50 ng / mL is added to the culture medium and cultured continuously for 48h-72h. S6: Perform PRA-based functional analysis on recipient serum and stimulated cultured MSCs; S7: Determine transplant compatibility based on HLA genotyping results and functional analysis of population reactive antibodies.
[0055] Finally, donors with rare HLA haplotypes and low PRA response rates can be selected.
[0056] Example 2 MSCs were isolated from amniotic tissue, amplified, and HLA genotyped. Donors with rare HLA haplotypes were selected, and these MSCs were prepared for therapeutic infusion in patients with systemic lupus erythematosus (SLE). This method plays a role in selecting optimal donors and ensuring minimal immune rejection for personalized treatment of autoimmune diseases.
[0057] Of course, a method for assessing mesenchymal stem cell transplant compatibility can be based on a standardized protocol designed from MSCs derived from different perinatal tissues, such as placenta, umbilical cord, and amnion, ensuring reproducibility and clinical applicability across multiple donor sources. This means using a standardized protocol to process multiple perinatal tissue donors, amplifying and HLA-genotyping the MSCs from each donor, and recording the results in a searchable database. The MSC bank is organized according to HLA profiles for rapid donor-patient matching. This method demonstrates the scalability of the invention in clinical cell banks and precision transplantation programs. This embodiment exhibits higher clinical relevance; this integrated approach improves graft survival, reduces immune rejection, and supports advancements in precision medicine within cell therapy.
[0058] Example 3 Patients with Crohn's disease were recruited for a clinical study. Perinatal MSCs were selected from an HLA typing database prior to infusion to achieve optimal donor-recipient compatibility. PRA testing was performed to confirm immunological safety. The selected MSCs were infused into patients, and treatment outcomes were monitored over 12 months. Results showed improved engraftment and fewer adverse immune responses compared to untyped MSC therapy. This demonstrates the clinical applicability and therapeutic value of the invention.
[0059] It should be noted that MSCs characterized according to the standard procedures of this invention are cryopreserved and systematically cataloged according to their HLA genotyping results, PRA reactivity, and inducibility profiles, and stored in a searchable database for clinical use. This database also integrates donor clinical history, maternal health records, and tissue origin information to optimize donor-recipient selection. Therefore, this invention also provides a cryopreserved MSC library capable of establishing complete HLA genotyping profiles. Based on this MSC library, efficient donor-recipient matching can be achieved, significantly improving graft survival rates and supporting large-scale applications in regenerative medicine and the treatment of autoimmune diseases.
[0060] Of course, this method can also integrate artificial intelligence (AI) algorithms into the donor selection process: HLA genotyping results, PRA reactivity data, and induced expression profiles from the MSCs biobank are uploaded to a secure database. A machine learning model trained based on historical transplant results predicts the compatibility score of each donor-recipient combination, and candidate donors are ranked according to predicted graft survival rate, immune tolerance, and required immunosuppression intensity. With this AI-assisted selection, clinicians can quickly identify the optimal donor, significantly improving transplant success rates and reducing the risk of rejection or other adverse immune reactions. The entire workflow is designed to comply with Good Manufacturing Practice (GMP) standards and can be used to establish a large-scale HLA-generated MSCs clinical cell bank. Furthermore, the standardized HLA genotyping strategy of this invention can also be extended to MSCs derived from adipose tissue or bone marrow and can be integrated with AI donor matching software to further improve the efficiency of donor-recipient compatibility prediction.
[0061] 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 assessing the compatibility of mesenchymal stem cell transplantation, 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: The mesenchymal stem cells are stimulated and cultured using immunostimulatory factors to induce HLA antigen expression; S40: Functional analysis of population-reactive antibodies in recipient serum and the stimulated cultured mesenchymal stem cells; S50: Determine transplant compatibility based on the HLA genotyping results and the functional analysis results of the population reactive antibodies.
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 S20 step specifically includes the following steps: S210: Extract genomic DNA from the mesenchymal stem cells; S220: Perform HLA genotyping based on sequencing to determine at least the HLA-A, HLA-B, and HLA-C loci; S230: Compare the locus with the database to determine allele allocation.
5. The method according to claim 4, characterized in that, In step S220, the HLA genotyping also identifies the HLA-DR and HLA-DQ loci.
6. The method according to claim 1, characterized in that, In step S30, the immunostimulatory factor is selected from one of interferon-gamma and the supernatant of conditioned medium obtained from immune cells.
7. The method according to claim 6, 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.
8. The method according to claim 6, characterized in that, The immune cells in the conditioned medium supernatant obtained from the immune cells include one or more of T cells, natural killer cells, monocytes, macrophages, and dendritic cells.
9. The method according to claim 1, characterized in that, In step S40, the functional analysis of the population reactive antibody is as follows: The binding levels of anti-HLA antibodies in the recipient serum to HLA antigens on the surface of the stimulated and cultured mesenchymal stem cells were detected by flow cytometry.
10. The method according to claim 1, characterized in that, In step S50, the functional analysis results of the population reactive antibodies are expressed as a percentage of population reactive antibodies, which is used to assess transplant compatibility; wherein, a percentage below 20% indicates a low risk of immune rejection; a percentage between 20% and 50% indicates a moderate risk of immune rejection; and a percentage above 50% indicates a high risk of immune rejection.
11. The method according to claim 1, characterized in that, The perinatal tissues are selected from one or more of the placenta, umbilical cord, and amnion.
12. The method according to any one of claims 1-11, characterized in that, Based on the HLA genotyping results and the functional analysis results of the population reactive antibodies, donors with rare HLA haplotypes and / or low population reactive antibody response rates are preferentially selected.