CD90 + Use of human amnion epithelial cells in the treatment of graft versus host disease

Abstract

A method for treating subjects with graft-versus-host disease (GvHD) or at risk of developing GvHD, using CD90 with stronger immunomodulatory capabilities. + hAEC cell populations were used as the primary therapeutic cells. Studies on the related mechanism of action revealed that CD90... + hAECs showed significantly higher levels of stem cell pluripotency markers SSEA4, OCT4, and NANOG than typical hAECs, and also exhibited better immunomodulatory functions; therefore, CD90 was used as a marker. + hAECs cells can be used as a treatment to achieve better clinical outcomes.

Description

BACKGROUND

[0001] Hematopoietic stem cell transplantation (HSCT) was first introduced into clinical practice by Professor Thomas in the United States in the 1950s. After decades of development, HSCT is used to treat a large number of malignant hematological diseases and metabolic congenital defects, and is the only existing radical treatment for hematological tumors.

[0002] In hematopoietic transplantation, graft-versus-leukaemia (GVL) or graft-versus-tumour (GVT) is the main mechanism for treating cancer. On the contrary, graft-versus-host disease (GVHD) is the main complication after hematopoietic stem cell transplantation, which is the main cause of death after transplantation. According to statistics, about 50% of patients receiving hematopoietic transplantation will have GVHD symptoms, thus severely limiting the widespread development of this important therapy.

[0003] GVHD is a type of immune disorder affecting multiple organ systems, including the gastrointestinal tract, liver, skin, kidneys, and lungs, which seriously threatens the quality of life and survival time of patients after transplantation. Generally, GVHD can be divided into two categories according to the length of time of clinical symptoms: acute GVHD (aGVHD) and chronic GVHD (cGVHD).

[0004] The cause of GVHD is the immune response caused by the donor T cells responding to the genetically determined proteins (mainly HLA human leukocyte antigens, histocompatibility antigens) on the recipient cells, so the incidence of universal aGVHD is directly related to the level of HLA mismatch. The ideal donor and recipient are full-matched for HLA-A / B / C / DRB1.

[0005] The onset of GVHD can be summarized into three processes: one, the activation of antigen-presenting cells (APCs); two, the activation, proliferation, differentiation, and migration of donor T cells causing an inflammatory storm; three, the damage of target organs.

[0006] From animal experiments, T cells play a core role in the onset of GVHD. There are three existing strategies for removing T cells: removing T cells in vitro before transplantation; in vitro sorting CD34 +Stem cell approaches; in vivo by antibody specific removal of T cells. These strategies have shown good efficacy against GVHD (both aGVHD and cGVHD), however, unfortunately these treatments come at the cost of high transplant failure rates, post-transplant infections, and high relapse rates. In addition, other major approaches to prevent GVHD are non-targeted immunosuppressive treatments of the recipient, such as cyclosporin-A (CsA), mycophenolate mofetil (MMF), steroids, etc., but these regimens also simultaneously attenuate the GVL effect, resulting in increased cancer relapse probability.

[0007] In recent years, there have been reports of using cell therapy regimens such as T regulatory cells (Tregs), regulatory γδ T cells (γδTregs), mesenchymal stem cells (MSCs), etc. in experimental animals, showing certain efficacy. However, in clinical trials, MSCs have been reported to have the risk of promoting tumor recurrence. Tregs are limited in their source and difficult to expand in vitro, and cannot be widely used in clinical applications.

[0008] CD4 + T cells have been widely reported to be closely related to GVHD, and the balance between the four main subgroups Th1, Th2, Th17 and Treg has a great influence on the incidence and severity of GVHD. In acute GVHD as an inflammatory process, it is found that CD4+ is polarized to Th1. In recent years, a new class of CD4+ cell subgroups that can produce IL-17 has been found, namely Thl7. Subsequent mouse disease models and clinical studies have found that they play an important role in the initiation of inflammation and tissue damage in acute GVHD. Studies have shown that Th2 is associated with lung damage and chronic GVHD in GVHD. Among the four main subgroups of CD4+, only Treg has been widely recognized since its identification to its association with GVHD, and is believed to have inhibitory and improving effects on GVHD, while still retaining the GVL effect. In summary, the study of CD4+ subgroup polarization remodeling is particularly important.

[0009] Human amniotic epithelial cells (hAECs) are isolated from the amnion of the placenta, which is the most fetal side of the placenta. The placenta is a waste product after the birth of a baby, so the cells from this source do not have ethical problems. The placenta is anatomically divided into three layers from the inside out: the amniotic epithelial layer, the chorion, and the decidua, and the origin of each layer is completely different. The decidua comes from the mother, the chorion comes from the trophoblast, and the amniotic epithelial layer comes from the epiblast eight days after fertilization, i.e. like embryonic stem cells (ESCs), hAECs are derived from the inner cell mass. Miki et al. confirmed that hAECs can express some characteristic markers of pluripotent stem cells (such as embryonic stem cells), such as Oct4, Sox2, Nanog, SSEA-3, SSEA-4, etc., indicating that it may have the potential to differentiate into three germ layers like embryonic stem cells. This inference is confirmed by in vitro differentiation experiments. However, unlike ESCs, hAECs are negative in vivo teratoma experiments, mainly because they lack telomerase activity, so hAECs used for cell therapy have no risk of tumorigenesis (including benign tumors, sarcomas and carcinomas).

[0010] In addition, hAECs almost do not express MHC class II molecules on the cell surface, so they will not cause inflammation, allergy and immune response, and the requirement for transplantation matching is also reduced. The hAECs isolation process is relatively simple, and there are almost no other types of cell contamination except a small amount of blood cell mass on the amnion after scraping and washing, and blood cells are suspended cells under non-stimulated conditions, so they can be removed by changing the liquid after cell culture. According to our and foreign scientists' experimental statistics, there are about 800 million to 300 million hAECs cells in each human amnion. In the presence of EGF, hAECs have strong proliferative capacity, about 36 hours can proliferate a generation, and can maintain vigorous proliferative capacity within the previous generation (about 10 generations). These advantages ensure that we can obtain a sufficient number of high-purity cell products to meet the requirements of clinical treatment.

[0011] In addition to the stemness, the hAECs also have an important property, i.e. immunomodulatory property. As early as in the early 20th century, researchers began to use amniotic membrane as a transplant material for repairing skin damage of patients, and achieved good effects. Further studies found that the amniotic membrane had anti-rejection and inhibition of bacterial growth in wounds, suggesting that the amniotic membrane might have certain immunomodulatory property. Ueta et al. found that the amniotic membrane had the function of inhibiting mixed lymphocyte reaction. Li et al. further found that the main functional cells of the amniotic membrane for immunomodulation were hAECs, which could inhibit the chemotaxis of neutrophils and macrophages, and inhibit the proliferation of T and B cells stimulated by mitogens. Scientists have also reported that the amniotic membrane can induce apoptosis of IFNγ-activated macrophages. Interestingly, through a series of in vitro experiments, it was found that the amniotic membrane could inhibit the proliferation of various solid tumor cell lines. Kang et al. found that hAECs could inhibit the proliferation of breast cancer cell lines in vitro, and could inhibit the growth of breast cancer tumor in nude mice in vivo and prolong the survival time of mice. Regarding the inhibition of tumors, it is now generally believed that hAECs act by inhibiting tumor angiogenesis and promoting tumor cell apoptosis.

[0012] The present inventors further found that in the hAECs obtained according to the above separation and culture method, there is a group of CD90 + hAECs, which have significantly higher expression of stem cell pluripotency markers SSEA4, OCT4 and NANOG than other CD90 - hAECs, and have better immunomodulatory function, so as to obtain better clinical effects by using the cells as a treatment means.

[0013] SUMMARY

[0014] The present application provides a treatment method for a subject suffering from or at risk of developing graft-versus-host disease (GvHD), generally comprising administering to the subject human amniotic epithelial cells (hAECs) effective to improve at least one symptom or clinical sign of graft-versus-host disease compared to a suitable control subject. To further improve the clinical treatment effect, CD90 + hAECs cell subgroups with strong immunomodulatory ability are selected as the main treatment cells.

[0015] In another embodiment of the application, the present application relates to the use of human amniotic epithelial cells or cell preparations thereof for the preparation of a medicament for the treatment and / or amelioration of graft-versus-host disease, wherein the human amniotic epithelial cells (hAECs) are CD90 + hAECs cell subgroups.

[0016] In another embodiment of the application, the present application relates to the use of an effective amount of CD90+ human amnion epithelial cells or cell preparations thereof for the treatment and / or amelioration of graft versus host disease, alone or in combination with other drugs.

[0017] In another embodiment of the application, the cell preparation comprises human amnion epithelial cells and a pharmaceutically acceptable carrier.

[0018] In another embodiment of the application, the present application relates to a method for isolating amnion epithelial cells from amnion tissue, the method comprising the steps of:

[0019] (1) obtaining amnion from placental tissue by mechanical separation;

[0020] (2) washing the amnion and digesting it with a digestive enzyme, and centrifuging the digested liquid to obtain human amnion epithelial cells;

[0021] (3) sorting CD90-positive human amnion epithelial cells (hAECs).

[0022] The present application studies CD90 + The potential of human amnion epithelial cells in the treatment of graft versus host disease and the treatment mechanism thereof. The results show that CD90 + The treatment group of hAECs co-transplanted with PBMCs and the treatment group of unsorted hAECs have a good inhibitory effect on aGVHD, significantly improve the clinical and pathological phenotypes of mice, and significantly improve the survival rate of mice. The animal model of the present application proves that this method can effectively reduce the infiltration of inflammatory cells in target organs caused by aGVHD, and also significantly reduces the lesions of target organs. It is also found that hAECs have a pro-apoptotic effect on multiple leukemia cell lines, and can be used for the treatment of graft versus host disease, and will have a wide prospect in clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The proportion of CD3 and CD45 in each group is shown in the graph. CD3 is a type of antigen on the surface of T lymphocytes, and CD3+ is a mature T lymphocyte; CD45 molecules are expressed on all white blood cells, referred to as leukocyte common antigen. The results show that the FACS results indicate that after 2 weeks of PBMC implantation, human CD3 and human CD45 molecules are expressed on all white blood cells in the aGVHD model mice and the chimerism rate is as high as 50% or more. However, the implantation of CD90 + hAECs and the implantation of unsorted hAECs do not significantly affect the implantation of PBMCs. In addition, we found that all CD45+ cells implanted almost co-expressed CD3. The humanized aGVHD model was successfully constructed and was not affected by CD90 + hAECs.

[0024] Figure 2 CD90 + Isolation of hAECs and their immunomodulatory properties. Flow cytometry detection of CD29, CD166 and CD90 in isolated hAECs. Immunomodulatory properties of hAECs and CD90 + Functional identification of hAECs. Unsorted hAECs or sorted CD90 + and CD90 - hAECs were co-cultured with PHA-activated PBMCs for 3 days. TNFa, IL2, IL17 and IL10 in the culture medium were detected by ELISA. (A) Representative flow cytometry gating of hAECs. (B-E) ELISA detection of cytokines TNFa, IL10, IL17 and IL2 in the culture medium of hAECs. + and CD90 - Representative flow cytometry gating of hAEC sorting. (B-E) ELISA detection of cytokines TNFa, IL10, IL17 and IL2 in the culture medium of hAECs.

[0025] Figure 3 Appearance of mice in each group. After two weeks of PBMC implantation, mice implanted with PBMCs alone began to show a series of clinical manifestations typical of aGVHD: weight loss, decreased activity, listlessness, severe kyphosis, alopecia, diarrhea, etc. The hAECs treatment group without screening was better than the hAECs treatment group with CD90 + hAECs treatment groups can improve the clinical symptoms of aGVHD mice.

[0026] Figure 4 Body weight of mice in each group over time. In addition to improving the quality of life of diseased mice, the implantation of hAECs more importantly prolonged the survival time of aGVHD mice and significantly improved their survival rate, and the hAECs treatment group without screening was better than the hAECs treatment group with CD90 + The effect of the hAECs treatment group without screening was better than the hAECs treatment group with CD90

[0027] Figure 5 Survival rate of mice in each group over time. The survival rate of mice in each group over time. All mice in the model group died, and the hAECs treatment group without screening was better than the hAECs treatment group with CD90 + The treatment of hAECs cells was better, and the mortality rate was lower.

[0028] Figure 6 Flow cytometry showing the proportion of Th1, Th2 and Treg in each group of mice. Figure 6-1 Representative flow cytometry and statistics showing the proportion of human Th1 (CD4+IFNy+), Th2 (CD4+IL4+) and Treg (CD4+CD25+FOXP3+) to CD4+ cells in mice two weeks after transplantation (n=5). The results show that the hAECs treatment group without screening was better than the hAECs treatment group with CD90+ hAECs cells have better immunomodulatory and anti-inflammatory effects. Figure 6-2 CD90+ hAECs were shown to modulate T cell subsets + hAECs shift T cell subsets in vivo and affect CD4+ T cell activation, with results showing aGVHD disease group has higher Thl and lower Treg. In the CD90 + hAECs treatment group showed a clear decrease in Thl and importantly we detected a clear increase in Treg of nearly 4-fold, while in the unsorted hAECs treatment group we saw a decrease in Thl compared to CD90 + hAECs treatment group has a more pronounced suppression. In addition, we also found that regardless of CD90 + hAECs treatment group or unsorted hAECs treatment group, there was no clear change in Th2 subset. The above results show that CD90 + hAECs ameliorate aGVHD by shifting T cell subsets.

[0029] Figure 7 Figure 6 shows the expression of endothelial adhesion molecules in each group. The results showed that compared with the negative control group (i.e. PBS injection group), the expression of adhesion molecules I-CAMl and V-CAMl on the vascular endothelium of the aGVHD group implanted with PBMCs was significantly increased, and as we expected, the CD90 + hAECs co-transplanted with PBMCs significantly reduced the expression of endothelial I-CAMl and V-CAMl.

[0030] Figure 8 Figure 7 shows the HE staining of target organ sections of mice in each group. The aGVHD target organs of diseased mice (12 days after PBMC injection) were taken: liver, lung, small intestine, kidney and sectioned. HE staining showed that the aGVHD model group of mice had large areas of endothelial inflammation lesions at the portal vein of the liver. In the lungs, alveolar inflammation cell infiltration and necrotic nodules around the endothelium were observed. The kidneys showed local edema. The small intestine villi were found to be blunt. In the group of PBMCs co-transplanted with CD90+ hAECs, we found that the liver endothelial inflammation lesions almost disappeared. The alveolar inflammation cell infiltration was significantly reduced, and the necrotic area around the endothelium was significantly reduced. No significant changes were found in the kidneys and small intestines. In the unsorted hAECs treatment group, we found that the therapeutic effect on liver and lung lesions was not as good as the CD90+ hAECs group, but compared with the disease group, the degree of lesion was still significantly reduced. However, the unsorted hAECs treatment group had no significant effect on the pathological changes of the kidneys; the improvement of the small intestine was as obvious as the CD90+ hAECs group.

[0031] Figure 9Figures showing organ pathology score of mice in each group.

[0032] Figure 10 Figures showing lung masson staining of mice in each group. The results show that CD90 + hAECs treatment group is significantly better than non-screened hAECs treatment group.

[0033] Figure 11 shows hAECs lentivirus infection. Photographs of hAECs infected with GFP-labeled lentivirus under bright field (11a) and green fluorescence microscope (11b) show high infection rate.

[0034] Figure 12 Figure showing hAECs localization in mice in vivo. The results show that CD90 + hAECs mainly localize in kidney, lung, liver. DETAILED DESCRIPTION

[0036] The present application provides a method of treatment for a subject suffering from or at risk of developing graft versus host disease (GvHD), generally comprising administering to the subject human amnion epithelial cells (hAECs) effective to improve at least one symptom or clinical sign of graft versus host disease compared to a suitable control subject. To further improve the clinical treatment effect, CD90 + hAECs cell subpopulation as the main therapeutic cell.

[0037] In one aspect, the present application discloses the use of human amnion epithelial cells or cell preparations thereof in the treatment and / or amelioration of graft versus host disease, wherein the human amnion epithelial cells (hAECs) are CD90 + hAECs cell subpopulation. An effective dose of CD90 + Human amnion epithelial cells or cell preparations thereof can be used alone or in combination with other drugs for the treatment and / or amelioration of graft versus host disease. An effective dose refers to an amount sufficient to ameliorate or prevent symptoms or conditions of a medical disease. The effective amount for a particular subject can vary depending on a variety of factors, such as the disease to be treated, the overall health status of the patient, the method route and dosage of administration, and the severity of side effects. The effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxic effects.

[0038] In another embodiment of the present application, CD90 + hAECs shift T cell subpopulations in vivo and affect CD4+ T cell activation, the results show that aGVHD disease groups have higher proportions of Th1, Th17 and lower proportions of Treg. In the co-transplantation of CD90 +The therapeutic group of hAECs showed a more pronounced decrease in the proportion of Th1 cells, and importantly we detected a nearly 4-fold increase in the proportion of Tregs, while in the CD90 + In the therapeutic group of hAECs, we saw a more pronounced suppression of the proportion of Th1 cells compared to the non-selected hAECs therapeutic group. Furthermore, we also found that the proportion of Th2 subpopulation did not change significantly in either the hAECs therapeutic group or the non-selected hAECs therapeutic group. These results show that CD90 + In the therapeutic group of hAECs, we saw a more pronounced suppression of the proportion of Th1 cells compared to the non-selected hAECs therapeutic group. Furthermore, we also found that the proportion of Th2 subpopulation did not change significantly in either the hAECs therapeutic group or the non-selected hAECs therapeutic group. These results show that CD90 + hAECs improve aGVHD by shifting the proportion of T cell subpopulations and are more therapeutically valuable than non-selected hAECs cells.

[0039] In another embodiment of the present application, the animal suffering from graft versus host disease is a mammal. In a more preferred embodiment, the animal is a bovine, equine, ovine, simian, canine, rat, mouse, rabbit or human. In the most preferred embodiment, the animal suffering from graft versus host disease is a human. In one embodiment of the present application, the CD90 + In addition to improving the quality of life of the diseased mice, the engraftment of hAECs more importantly prolonged the survival time and significantly increased the survival rate of the aGVHD mice, again CD90 + The therapeutic effect of the hAECs was better than that of the non-selected hAECs.

[0040] In another embodiment of the present application, the cell preparation comprises CD90 + human amnion epithelial cells and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier of the present application refers to a substance suitable for use in humans and / or animals, with no excessive adverse side effects (such as toxicity, irritation and allergic reactions) and with a suitable benefit / risk ratio, such as a pharmaceutically acceptable solvent, suspending agent or excipient, which is beneficial to cell survival and can deliver the prepared cells to humans or animals. The carrier is selected according to the appropriate planned administration method. The carrier of the present application includes but is not limited to various physiological buffers, such as physiological saline, phosphate buffer, artificial cerebrospinal fluid or whole serum, umbilical cord serum, etc.

[0041] CD90 + The amnion epithelial cells are administered to the patient, for example, by intravenous injection or intrathecal injection, etc. Usually these cells are contained in a pharmaceutically acceptable liquid medium. The cell administration can be repeated or continuous (for example, by continuous infusion into the cerebrospinal fluid). Generally speaking, multiple administration methods are usually used at least 7-10 days apart. Another method is to plant the cells in a bioabsorbable material such as gelatin sponge, and use surgery to implant the cell-seeded bioabsorbable material into the desired site. The two methods can be combined to achieve better therapeutic effect.

[0042] CD90 + The appropriate dosage of amniotic epithelial cells will vary depending on the age, sex, weight, and general condition of the patient, as well as other factors. Generally, the dosage administered per administration will range from about 10 3 -10 9 cells, typically about 10 6 -10 8 cells.

[0043] In some embodiments of the present application, the present application discloses the use of human amniotic epithelial cells or cell preparations thereof for the preparation of a medicament for the treatment and / or amelioration of graft versus host disease, wherein the human amniotic epithelial cells (hAECs) are CD90 + hAECs cell subpopulation.

[0044] In another embodiment of the present application, the present application discloses the use of CD90 + human amniotic epithelial cells or cell preparations thereof in combination with other drugs for the treatment and / or amelioration of graft versus host disease. The CD90 + Amniotic epithelial cells are administered to patients together with one or more drugs selected from one or a combination of methylprednisolone, cyclosporine, tacrolimus, mycophenolate mofetil, methotrexate, glucocorticoids, azathioprine, thalidomide, anti-T cell monoclonal antibodies (anti-CD3 mAb), antibodies against interleukin-2 receptor, and the like.

[0045] In another embodiment of the present application, there is provided a method for isolating amniotic epithelial cells from CD90 + amniotic tissue, the method comprising the steps of:

[0046] (1) obtaining amnion from placental tissue by mechanical separation;

[0047] (2) washing the amnion and digesting it with a digestive enzyme, and centrifuging the digested liquid to obtain human amniotic epithelial cells;

[0048] (3) sorting out CD90 positive human amniotic epithelial cells (CD90 + hAECs).

[0049] In another embodiment of the present application, the amnion epithelial cells described herein are derived from humans. The amnion can be isolated from an ex vivo human placenta, rinsed with a physiological buffer to remove blood cells, and mechanically removed of residual chorion and blood vessels. Isolation refers to the removal of cells from a tissue sample and separation from other tissues. Single cells are isolated from the intact human amnion epithelial layer tissue using any conventional technique or method, including mechanical forces (chopping forces or shearing forces), enzymatic digestion with one or a combination of proteases such as collagenase, trypsin, lipase, liberase, and pepsin, or a combination of mechanical and enzymatic methods.

[0050] In another embodiment of the present application, the screening process for CD90 positive human amnion epithelial cells is by staining the hAECs with CD90 primary antibody, followed by incubation with a fluorescently conjugated secondary antibody, and flow cytometry analysis to sort out CD90 positive human amnion epithelial cells. A more preferred screening process is by staining the hAECs with CD90 (1 :20, Millipore) primary antibody, followed by incubation with a fluorescently conjugated secondary antibody. Flow cytometry analysis is performed using a FACS Calibur instrument (Becton Dickinson, Franklin Lakes, NJ, USA) to sort out CD90 positive human amnion epithelial cells.

[0051] In a preferred embodiment of the present application, the human amnion is obtained from the placental tissue of a healthy woman after caesarean section, with the consent of the woman, by mechanical separation to obtain the whole amnion.

[0052] In another preferred embodiment of the present application, the CD90 positive human amnion epithelial cells obtained in step 3 are further purified by flow cytometry analysis to sort out CD90 positive human amnion epithelial cells. + The human amnion epithelial cells are further cultured, preferably under the following conditions: the cells are seeded at a density of 1 x 105cells / cm2in a culture dish and incubated in a carbon dioxide incubator, and the culture medium is changed after the cells adhere to the dish, and the cells are digested and frozen when the cells grow to cover the dish. 6 -1 x 105cells / cm2in a culture dish and incubated in a carbon dioxide incubator, and the culture medium is changed after the cells adhere to the dish, and the cells are digested and frozen when the cells grow to cover the dish. 8 The human amnion epithelial cells are further cultured, preferably under the following conditions: the cells are seeded at a density of 1 x 105cells / cm2in a culture dish and incubated in a carbon dioxide incubator, and the culture medium is changed after the cells adhere to the dish, and the cells are digested and frozen when the cells grow to cover the dish. + The human amnion epithelial cells are further cultured, preferably under the following conditions: the cells are seeded at a density of 1 x 105cells / cm2in a culture dish and incubated in a carbon dioxide incubator, and the culture medium is changed after the cells adhere to the dish, and the cells are digested and frozen when the cells grow to cover the dish.

[0053] The skilled person can use other methods known in the art to concentrate the active cell population. These post-processing washing / concentration steps can be performed separately or simultaneously. In addition to the methods described above, the active cell population can be further purified or enriched, and the number of contaminating and dead cells reduced, after washing or after culturing the cells. The cells in suspension can be separated by techniques such as buoyant density centrifugation, differential adhesion to and elution from a solid phase, immunomagnetic beads, fluorescence-activated cell sorting (FACS), or other techniques. Examples of these different techniques and devices for performing these techniques can be found in the prior art and in commercially available products.

[0054] The type of the basic medium used in the present application is not limited as long as it is a medium that can be used for cell culture. Preferred media include DMEM medium and NPBM medium. The type of other components that can be contained in the above-mentioned basic medium is not limited, and preferred components include F-12, FCS, and nerve survival factor, etc.

[0055] In another preferred embodiment of the present application, bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) is added to the above-mentioned basic medium. In this case, one or both can be added. An example concentration of the above-mentioned bFGF or EGF is 1 ng / ml to 100 ng / ml, and a preferred concentration is 10 ng / ml. The time and method of addition are not limited. Preferably, the reagent is added every day when the above-mentioned amniotic epithelial cells are cultured in the basic medium.

[0056] The present application uses an effective dose of CD90 + Amniotic epithelial cells or CD90 + Amniotic epithelial cells are used alone or in combination with other drugs for treatment and / or improvement of graft-versus-host disease. The present application studies CD90 + The potential of human amniotic epithelial cells (hAECs) in the treatment of graft-versus-host disease and explores its therapeutic mechanism. The results show that CD90 + CD90 + The hAECs treatment group and the unselected hAECs treatment group have a better inhibitory effect on aGVHD, significantly improve the clinical and pathological phenotypes of mice compared with the disease group, and significantly improve the survival rate of mice. The animal model of the present application proves that this method can effectively reduce the infiltration of inflammatory cells in target organs caused by aGVHD, and also significantly reduces the lesions of target organs. It is also found that hAECs have a pro-apoptotic effect on multiple leukemia cell lines, can be used for the treatment of graft-versus-host disease, and will have a wide prospect in clinical application.

[0057] The present application uses CD90 + Human amniotic epithelial cells are used for the treatment of graft-versus-host disease, which fully plays the advantages of human amniotic epithelial cells. The human amniotic epithelial cells mainly have the following advantages:

[0058] (1) Can maintain pluripotency for a long time and has the potential to differentiate into tissues of three germ layers, which is unique to embryonic stem cells;

[0059] (2) The cell surface almost does not express MHC type II molecules, thus does not cause inflammation, allergy and immune response, and the transplantation matching requirement is correspondingly reduced;

[0060] (3) Has the ability to regulate immune response in vivo and in vitro, and can secrete various immune regulatory factors, anti-angiogenic proteins or anti-inflammatory factor related proteins when cultured in vitro;

[0061] (4) Has low immunogenicity, can be regarded as immune exempt cells, has no antigen presentation function, and can reduce the source of immune cells after transplantation to avoid the occurrence of immune rejection;

[0062] (5) Does not express telomerase reverse transcriptase and has no tumorigenicity (including benign tumors, sarcomas and carcinomas);

[0063] (6) Has strong proliferation ability and can maintain vigorous proliferation ability within about 10 generations;

[0064] (7) The source is wide, and the material is easy to obtain, without application restrictions and ethical problems. DETAILED DESCRIPTION

[0065] Example 1: Isolation and culture of primary amniotic epithelial cells

[0066] 1. Source of human amniotic membrane

[0067] In order to avoid contamination of the birth canal microorganisms, we selected cesarean section fetal placenta. Due to the stimulation of labor signal after full-term, the amnion will undergo apoptosis, so it is appropriate to use preterm fetal placenta (before 38 weeks). After the consent of the authorized person, the placental tissue of a healthy woman (HIV, syphilis, hepatitis A, hepatitis B, hepatitis C, etc. Serological reactions showed negative) after cesarean section was taken, and the placenta was cut with a cross-shaped knife. The whole amnion was obtained by mechanical separation.

[0068] 2. CD90 + Isolation and sorting of hAECs (sterile operation is required throughout)

[0069] Get the placenta of a baby born by cesarean section before 38 weeks, peel the amnion from the inner surface of the placenta, and immerse it in a centrifuge tube containing F12 / DMEM (containing 1X penicillin-streptomycin and amphotericin B) basic medium. 4℃ cold chain transportation to the laboratory cell.

[0070] Take out the amnion, and wash each amnion in 40ml CMF-HBSS (containing 1X penicillin-streptomycin and amphotericin B) to remove mucus, and use forceps to scrape off the mesenchymal layer close to the chorionic layer and mucus, repeat 3 times, each time change a new container and new HBSS solution.

[0071] Place washed amnion into a new container, add 10 ml of 0.05% Trypsin / EDTA, invert for 30 s, discard solution.

[0072] Place amnion into a new container, add 20 ml of 0.05% Trypsin / EDTA, incubate at 37°C for 10 min, discard solution.

[0073] Place amnion into a new container, add 25 ml of Trypsin / EDTA, incubate at 37°C for 40 min, save the digestion solution.

[0074] Place amnion into a new container, add 25 ml of Trypsin / EDTA, incubate at 37°C for 40 min, save the digestion solution.

[0075] Add equal volume of digestion stop solution (F12 / DMEM with 5% FBS, lx L-glutamine, lx beta mercaptoethanol, lx pyruvic acid), centrifuge at 400g for 10 min. Discard solution, resuspend pellet with complete culture medium: F12 / DMEM with 5% KSR (KnockOut Serum Replacement), lx L-glutamine, lx beta mercaptoethanol, lx pyruvic acid, IX ps (Penicillin-Streptomycin), 10 ng / ml hEGF.

[0076] Add equal volume of digestion stop solution, centrifuge at 400g for 10 min. Discard solution, resuspend pellet with complete culture medium.

[0077] Pass through 100 um mesh, count.

[0078] Wash with PBS.

[0079] Flow cytometry analysis was performed using a FACS Calibur instrument (Becton Dickinson, Franklin Lakes, NJ, USA). hAECs were stained with fluorescent conjugated antibodies targeting CD34, CD45, CD31, CD29, CD166, CD90, HLA-DR and HLA-DQ (1 :20, all from BioLegend) using standard protocols. For SSEA4 and TRA-1-60, hAECs were stained with SSEA4 (1 :20, Millipore) and TRA-1-60 (1 :20, Millipore) primary antibodies, followed by incubation with fluorescent conjugated secondary antibodies. Cell cycle distribution was measured using propidium iodide (PI) staining. Isotype controls were used in each experiment.

[0080] A FACS Calibur instrument program (Becton Dickinson, Franklin Lakes, NJ, USA) was set up for flow cytometry sorting of CD90 positive human amniotic epithelial stem cells. The flow rate and sample concentration were adjusted so that the concentration of target cells passing through the sample chamber was 100-300 cells per second, and CD90 positive human amniotic epithelial stem cells were screened and collected.

[0081] Seeding at 10^5 cells / cm2 to culture dishes or cryopreservation in liquid nitrogen in cryopreservation solution (90% FBS, 10% DMSO) for later use.

[0082] 3. Seeding culture and cryopreservation of hAECs

[0083] Cell counting culture: Seeding at 1 x 10 7 Cells after adhering to the plate, the culture medium was changed, and the culture medium was changed every three days thereafter.

[0084] After the cells covered the plate, the cells were digested and cryopreserved: 5 ml trypsin was added to a 15 cm dish, and after 10 minutes of observation under a microscope, the cells were rounded and the flat plate was shaken, and when the cells were completely in a suspended state, an equal amount of digestion termination solution was added to terminate the digestion. The cells on the culture dish were blown off in the same direction using a micropipette, transferred to a 15 ml centrifuge tube, and centrifuged at 300 g for 3 minutes to collect the cells, which were then counted. Cryopreservation solution was added to the cryopreservation tube, and the cryopreservation date, batch, and cell quantity were marked before the cells were placed in the cryopreservation tube. The cryopreservation tube was then immediately placed in a cryopreservation box, which was placed in a -80°C freezer. After 12 hours, the cryopreservation box was removed, and the cells were transferred to a liquid nitrogen tank for storage.

[0085] Example 2. Isolation, culture and cryopreservation of human peripheral blood mononuclear cells

[0086] The donor's peripheral blood was aseptically extracted into a collection belt containing an anticoagulant.

[0087] 5 ml of Ficoll solution was added to a 50 ml centrifuge tube, and the blood was gently added to the Ficoll solution using a sterile Pasteur pipette.

[0088] Without centrifugal acceleration, centrifuge at 400 g for 30 min at room temperature until the blood is clearly layered.

[0089] The white membrane layer in the layered portion was carefully aspirated with a new Pasteur pipette and added to 10 ml of DPBS (containing 1x PS) and mixed evenly.

[0090] Centrifuge at 300 g for 5 min at room temperature, and discard the liquid.

[0091] Resuspend the precipitate with 10 ml of DPBS (containing 1x PS), centrifuge at 300 g for 5 min at room temperature, and discard the liquid.

[0092] Resuspend in complete medium (1640, 10% FBS, PS) or in cryopreservation solution and store in liquid nitrogen.

[0093] Example 3: Establishment of mouse model and aGVHD mouse scoring criteria

[0094] 1. Establishment of NCG mouse acute GVHD model

[0095] NCG mice aged 6-8 weeks were purchased from the company and transferred to the animal room for 1 week to adapt to the environment. Drinking water was added with gentamicin (32 x 10^4 U / L) and erythromycin (250 mg / L).

[0096] The experimental mice were divided into GVHD model group, CD90 + hAECs treatment group, unselected hAECs treatment group and control group, n = 5. 10^7 human peripheral blood mononuclear cells (PBMCs) per mouse were injected into the GVHD model group, CD90 + hAECs treatment group, unselected hAECs treatment group mice were injected with 10^7 human peripheral blood mononuclear cells (PBMCs) per mouse, and after one week, CD90 + hAECs treatment group mice were injected with CD90 + hAECs 2X10^6 per mouse; unselected hAECs treatment group mice were injected with unselected hAECs 2X10^6 per mouse. The control group was injected with PBS of corresponding volume.

[0097] 2. aGVHD mouse scoring criteria

[0098] aGVHD mouse clinical phenotype scoring criteria (based on KR. Cook scoring system, a total of 10 points, observed every other day.)

[0099] Weight loss: mice with less than 10% weight loss were considered normal and scored 0; 1 point for weight loss between 10% and 25%; more than 25% weight loss was scored 2.

[0100] Posture change: mice with no abnormal posture were considered normal and scored 0; 1 point for only arching back when resting; severe arching back affecting normal activity was scored 2.

[0101] Hair texture change: mice with no abnormal hair texture were considered normal and scored 0; 1 point for slight wrinkles; severe wrinkles were scored 2.

[0102] Activity: mice with normal activity were scored 0; 1 point for slightly weakened activity; no activity without stimulation was scored 2.

[0103] Skin integrity: Normal mouse skin was scored as 0; skin peeling on the tail or footpads was scored as 1; obvious peeling area on the skin was scored as 2

[0104] aGVHD mouse organ pathology scoring criteria (total 4 points)

[0105] Lung: Normal mouse without disease was scored as 0; a small amount of leukocyte aggregation around the blood vessels was scored as 0.5; leukocyte aggregation around the blood vessels was 1-2 cell thick (involving 15% of the blood vessels), scored as 1; leukocyte aggregation around the blood vessels was 1-2 cell thick (involving 15% of the blood vessels) and infiltrated into the parenchymal tissue, scored as 1.5; leukocyte aggregation around the blood vessels was 2-3 cell thick (involving 15%-50% of the blood vessels) and infiltrated into the parenchymal tissue, scored as 2; leukocyte aggregation around the blood vessels was 2-3 cell thick (involving 25%-50% of the blood vessels) and infiltrated into the parenchymal tissue, scored as 2.5; leukocyte aggregation around the blood vessels was 4-5 cell thick (involving 25%-50% of the blood vessels) and infiltrated into the parenchymal tissue, scored as 3; leukocyte aggregation around the blood vessels was 6-7 cell thick (involving 50% of the blood vessels) and infiltrated into the parenchymal tissue, severely destroying the normal structure of the tissue, scored as 3.5; leukocyte aggregation around the blood vessels was 6-7 cell thick (involving more than 50% of the blood vessels) and infiltrated into the parenchymal tissue, severely destroying the normal structure of the tissue, scored as 4.

[0106] Intestine: Normal mouse without disease was scored as 0; occasionally, crypt cell necrosis and a small amount of inflammatory cell infiltration into the submucosal layer and small intestinal villi were scored as 0.5; crypt cell necrosis reached 15% and inflammatory cell infiltration reached 20% of the submucosal layer and small intestinal villi were scored as 1; crypt cell necrosis reached 15% and inflammatory cell infiltration reached 1 / 3 of the submucosal layer and small intestinal villi were scored as 1.5; crypt cell necrosis reached 25% and inflammatory cell infiltration reached 1 / 3 of the submucosal layer and small intestinal villi were scored as 2; crypt cell necrosis reached 25%-50% and inflammatory cell infiltration reached 1 / 3 of the submucosal layer and small intestinal villi were scored as 2.5; crypt cell necrosis was greater than 50% and inflammatory cell infiltration reached 1 / 3 of the submucosal layer and small intestinal villi were scored as 3; crypt cell necrosis was greater than 50% and inflammatory cell infiltration reached 50% of the submucosal layer and small intestinal villi were scored as 3.5; crypt cell necrosis was greater than 50% and inflammatory cell infiltration reached more than 50% of the submucosal layer and small intestinal villi were scored as 4.

[0107] Spleen: normal no disease is considered normal 0 points; each square millimeter of tissue has 10 necrosis or apoptosis cells 1 point; each square millimeter of tissue has 10 necrosis or apoptosis cells and occasionally hemolysis, 1.5 points; each square millimeter of tissue has 10-20 necrosis or apoptosis cells, occasionally hemolysis and accompanied by tissue structure damage, 2 points; each square millimeter of tissue has 10-20 necrosis or apoptosis cells, hemolysis and accompanied by 25% or less tissue structure damage, 2.5 points; each square millimeter of tissue has 20-40 necrosis or apoptosis cells, hemolysis and accompanied by 25%-50% tissue structure damage, and less than 25% tissue fibrosis, 3 points; each square millimeter of tissue has 20-40 necrosis or apoptosis cells, hemolysis and accompanied by 50% tissue structure damage, and 25%-50% tissue fibrosis, 3.5 points; large area (more than 50%) of tissue necrosis or apoptosis cells, hemolysis and accompanied by more than 50% tissue structure damage, and more than 50% tissue fibrosis, 4 points.

[0108] Liver: normal no disease is considered normal 0 points; 1-2 single nucleus cells per 0.5 cm of tissue are accumulated in the lesion area 1 point; one endothelial inflammatory blood vessel exists per 0.5 cm of tissue, and at least 2 inflammatory cells infiltrate under the endothelium of each blood vessel 2 points; three endothelial inflammatory blood vessels exist per 0.5 cm of tissue, and at least 3 inflammatory cells infiltrate under the endothelium of each blood vessel 3 points; almost all blood vessels have endothelial inflammation, and at least 3 inflammatory cells infiltrate under the endothelium of each blood vessel 4 points.

[0109] Example 4aGVHD mouse model experiment grouping

[0110] NCG mice without mature T cells, B cells, and NK cells in vivo were used as model mice, and a humanized aGVHD mouse model was established by implanting human peripheral blood mononuclear cells (PBMC). We divided the experiment into four groups: 1. The group of PBMC transplantation alone was used as the aGVHD disease model group; 2. The CD90 + hAECs and PBMCs were transplanted at the same time as the aGVHD disease CD90 + hAECs treatment group; 3. Unscreened hAECs and PBMCs were transplanted at the same time as the aGVHD disease unscreened hAECs treatment group; 4. The corresponding volume of culture solution was injected as a blank control group.

[0111]

[0112] The results show that after 2 weeks of PBMC implantation, FACS results show that in the aGVHD model mice, CD3 and CD45 (CD45 molecules are expressed on all white blood cells and the chimerism rate is as high as 50% or more Figure 1 ). While CD90 +hAECs and unsorted hAECs did not significantly affect the engraftment of PBMCs Figure 1 ). In addition, we found that almost all of the engrafted CD45+ cells co-expressed CD3 Figure 1 ).

[0113] Two weeks after PBMCs engraftment, we observed that mice engrafted with PBMCs alone started to develop a series of clinical manifestations typical of aGVHD: weight loss, decreased mobility, listlessness, severe hunched back, alopecia, diarrhea, etc. while mice co-transplanted with CD90 + hAECs (i.e. CD90 + hAECs treatment group) showed very significant improvements in all of these disease manifestations. While the unsorted hAECs treatment group did not perform as well as the CD90 + hAECs treatment group, it still showed more significant improvements than the disease group. However, the unsorted hAECs treatment group did not differ much from the CD90 + hAECs treatment group in terms of hunched back, diarrhea (which is consistent with the histology staining later) and mobility ( Figure 2 , Figure 3 ). We scored the clinical manifestations and found that the CD90 + hAECs treatment group and the unsorted hAECs group both showed significant differences from the disease group.

[0114] In addition to improving the quality of life of the disease mice, the engraftment of CD90 + hAECs more importantly prolonged the survival time and significantly improved the survival rate of the aGVHD mice. Again, the CD90 + hAECs treatment group performed better than the unsorted hAECs treatment group ( Figure 4 ) in terms of survival rate.

[0115] Example 5 Flow cytometry

[0116] 1. Cell surface marker staining

[0117] If the sample is tissue or blood cells, red blood cells need to be lysed. Add 3ml red blood cell lysis solution (ACK) to the cells in the tube to resuspend the cells, and incubate on ice for 5-20min.

[0118] Add 10ml cell staining solution (2% FBS in PBS) to the tube to stop ACK lysis. Centrifuge at 350g for 5min at room temperature, and discard the supernatant. If necessary, repeat the lysis with ACK.

[0119] Repeat step 2 to wash the cells once.

[0120] After counting, adjust the cell concentration to 5-10 x 106cells / ml with Cell Staining Buffer. Aliquot 100 ul of the cell suspension into flow tubes.

[0121] Add the appropriate amount of fluorescently labeled primary antibody to the prepared cell suspension. Incubate for 20-30 minutes on ice in the dark.

[0122] Wash twice with 2 ml of Cell Staining Buffer, centrifuge at 350 g for 5 minutes at room temperature, and discard the supernatant.

[0123] Resuspend the cells in 0.5 ml of Cell Staining Buffer and analyze by flow cytometry.

[0124] 2. Intracellular factor staining

[0125] Collect the stimulated cells, which can be tissues stimulated in vivo or cells stimulated in vitro. Add protein transport inhibitors, such as brefeldin A or monensin, to inhibit the secretion of cytokines into the extracellular space during the last 4-6 hours of stimulation. Stain the cells in separate tubes as needed.

[0126] Perform surface staining on live cells before fixing and permeabilizing the cells for intracellular staining.

[0127] Fix the cells with Fixation Buffer before intracellular staining. Add 0.5 ml of Fixation Buffer per tube, and incubate the cells at room temperature in the dark for 20 minutes.

[0128] Centrifuge at 350 g for 5 minutes, and discard the supernatant.

[0129] If necessary, pause the experiment at this step for short-term storage at 4°C. Wash the cells with Cell Staining Buffer, centrifuge at 350 g for 5 minutes, and discard the supernatant. Resuspend the cells in Cell Staining Buffer for short-term storage at 4°C. Alternatively, store the cells in 90% FCS / 10% DMSO at -80°C for long-term storage.

[0130] Resuspend the fixed cells in Permeabilization Wash Buffer, centrifuge at 350 g for 5-10 minutes, and discard the supernatant.

[0131] Repeat Step 6.

[0132] Resuspend the fixed and permeabilized cells in 100 ul of Permeabilization Wash Buffer, and add the specific fluorescently labeled antibody (add the antibody according to the instructions). Incubate at room temperature in the dark for 20 minutes.

[0133] Wash cells twice with 2 ml Permeabilization Wash Buffer. 350g for 5 minutes, discard supernatant.

[0134] Resuspend fixed and stained cells with 0.5 ml Cell Staining Buffer, and analyze by flow cytometry.

[0135] Investigate CD90 by flow cytometry + hAECs shift T cell subsets in vivo and affect CD4+T cell activation, and the results show that aGVHD disease group has a higher proportion of Th1, Th17 and a lower proportion of Treg (Figure 6). In the CD90 + The treatment group of hAECs shows a clear decrease in the proportion of Th1, and importantly we detect a clear increase in the proportion of Treg of nearly 4-fold (Figure 6), and in the CD90+hAECs treatment group we see a more pronounced inhibition of Th1 proportion compared to the unsorted hAECs treatment group (Figure 6). In addition, we also find that in both the CD90 + hAECs treatment group and the unsorted hAECs treatment group, the proportion of Th2 subsets does not change significantly (Figure 6). The above results show that CD90 + hAECs ameliorate aGVHD by shifting the proportion of each T cell subset.

[0136] Example 6 Immunofluorescence

[0137] 1. Cell immunofluorescence

[0138] Aspirate the media from each well (24-well plate), add 1 ml PBS, shake and discard.

[0139] Add 1 ml 4% paraformaldehyde (diluted in PBS), and incubate at room temperature for 30 min.

[0140] Discard the solution, and shake twice with PBS, then discard.

[0141] Add 500 ul 0.2% Triton X-100 (diluted in PBS), and incubate at room temperature for 15 min (if the antigen is a membrane protein, skip this step).

[0142] Discard the solution, and shake twice with PBS, then discard.

[0143] Add 10% FBS (or 3% goat serum or 2% horse serum) blocking solution, and incubate for 1-2 h.

[0144] Discard the solution, add PBS, and place on a horizontal shaker at 60 rpm, and wash at room temperature for 5 min, repeat 3 times

[0145] 10% FBS or 3% horse serum dilution of primary antibody, 250ul per well. Incubate at room temperature for 1h (or 4°C overnight).

[0146] Discard the solution, add 500ul wash buffer, and place it in a horizontal shaker at 60rpm for 5min at room temperature, and wash for a total of 3 times.

[0147] 10% FBS or 3% horse serum dilution of fluorescently conjugated secondary antibody, 250ul per well. Incubate at room temperature for 1h in the dark. (All subsequent steps after this step are performed in the dark.)

[0148] Discard the solution, add 500ul wash buffer, and place it in a horizontal shaker at 60rpm for 5min at room temperature, and wash for a total of 3 times.

[0149] Add PBS-diluted DAPI, 250ul per well, and incubate at room temperature for 5min.

[0150] Discard the solution, add 500ul wash buffer, and place it in a horizontal shaker at 60rpm for 5min at room temperature, and wash for a total of 2 times.

[0151] Discard the solution, and add 250ul PBS per well before microscopic examination. If it is a cell crawl sheet, add a drop of mounting solution to the cover glass, remove the crawl sheet and place it with the cells facing the cover glass. This step is mainly to try not to generate bubbles. If bubbles are generated, carefully squeeze them out with tweezers, and then examine them under a microscope.

[0152] 2、Tissue immunofluorescence

[0153] After the tissue is removed from the body, it is quickly placed in PBS to remove blood stains, and the tissue is carefully cut into the appropriate size (note that the tissue should not be squeezed). The tissue is placed in an embedding box containing a freezing section embedding agent (OCT). The embedding box is placed in dry ice-precooled isopentane until the OCT and tissue are completely frozen.

[0154] The embedded tissue is fixed on a freezing microtome, cut into 0.5um sections, and the sections are attached to an adhesive glass slide.

[0155] The sections need to be placed at room temperature for 30min before staining to allow the sections to adhere to the glass slide.

[0156] Place the sample in acetone at -20°C for 10min (if the tissue has been fixed before embedding, proceed to the next step directly).

[0157] Remove the sections and wash them with PBS for 3 times, 5min each time.

[0158] First, circle the tissue with an oil-based pen, then add the primary antibody (diluted with 5% HBS + 1% BSA in PBS) to the tissue, and incubate overnight in a humidified chamber at 4°C.

[0159] Remove the sections and wash them three times with PBS, 5 minutes each time.

[0160] Remove moisture from the slide surface, add fluorescently conjugated secondary antibody (diluted with PBS containing 5% HBS and 1% BSA), and incubate in a humidified chamber at room temperature in the dark for 1 hour. (All subsequent operations from this step onwards should be performed in the dark.)

[0161] Remove the slices and wash them three times with RBS, 5 minutes each time.

[0162] After diluting DAPI with PBS, add it to the slide and incubate at room temperature for 3 minutes. Then wash twice with PBS for 5 minutes each time.

[0163] Mount the slide with mounting solution and examine it under a fluorescence microscope.

[0164] We collected thoracic aortas from mice in both experimental and control groups, and performed frozen section analysis and immunofluorescence staining for adhesion molecules. The results showed that compared to the negative control group (i.e., the PBS-injected group), the expression of adhesion molecules I-CAM1 and V-CAM1 on the vascular endothelium was significantly increased in the aGVHD group implanted with PBMCs, while, as expected, CD90... + Co-transplantation of hAECs and PBMCs significantly reduced the expression of endothelial I-CAM1 and V-CAM1. Figure 7 Therefore, we believe that CD90 + hAECs can inhibit the expression of endothelial adhesion molecules, thereby alleviating vascular GVHD.

[0165] Example 7: Tissue paraffin embedding and sectioning

[0166] After the tissue was removed from the body, it was quickly rinsed in 10% formalin to remove blood. This was repeated three times.

[0167] Tissues were fixed at room temperature in 10% formalin or 4% PFA.

[0168] The tissue was removed, washed with PBS, and then dehydrated with 70% ethanol at room temperature.

[0169] After being dehydrated by an ethanol gradient, it is permeated in xylene.

[0170] The well-permeable tissue was embedded in paraffin and then solidified at 4°C.

[0171] Place the paraffin block in a microtome and cut it into sections of 5-10 μm, depending on the experimental requirements.

[0172] The steps of spreading, fetching, drying, and obtaining the paraffin section product are completed.

[0173] Example 8 HE staining

[0174] The paraffin section is placed in an environment of 65°C for 1 h to deparaffinize.

[0175] The section is deparaffinized with xylene solution for 10 min, 10 min, and 5 min.

[0176] The section is taken out and hydrated with 100% ethanol for 5 min, 100% ethanol for 5 min, and 95% ethanol for 2 min. Then, it is rinsed with running water for 5 min.

[0177] The section is stained with hematoxylin for 5 min, and then rinsed with running water for 5 min to remove excess dye.

[0178] The section is placed in hydrochloric acid ethanol solution for 2 s for differentiation, and then rinsed with running water for 5 min.

[0179] It is soaked in dilute ammonia water for 6 s, rinsed with running water for 5 min, and placed in 95% ethanol for 4 min.

[0180] The section is stained with eosin for 40 s.

[0181] It is passed through 95% ethanol for 2 min, 95% ethanol for 2 min, 100% ethanol for 4 min, and 100% ethanol for 4 min.

[0182] It is passed through xylene solution for 5 min, 5 min, and 5 min, respectively.

[0183] After mounting with neutral resin, it is observed under a microscope.

[0184] The results show that in the aGVHD model group, there are large areas of endothelial inflammation lesions at the portal vein of the liver. The alveoli are infiltrated with a large number of inflammatory cells, and necrotic nodules appear around the endothelium. The kidney shows local edema. The small intestinal villi are found to be blunt. In the treatment group in which PBMCs and CD90 + hAECs are transplanted at the same time, we find that the liver endothelial inflammation lesions almost disappear. The inflammatory cell infiltration of the alveoli is significantly reduced, and the necrotic area around the endothelium is significantly reduced. No obvious lesions are found in the kidney and small intestine. In the treatment group in which PBMCs and unsorted hAECs are transplanted at the same time, we find that the treatment of liver and lung lesions is not as good as that of the CD90 + hAECs group, but the lesion degree is still significantly reduced compared with the disease group, and there is no obvious effect on the pathological changes of the kidney; and the improvement of the small intestine is as obvious as that of the CD90 + hAECs group, which is consistent with the observation in the clinic that the body weight change of the treatment group is small and there is no diarrhea symptom. Figure 8 Figure 9 ​).

[0185] Example 9 Masson staining

[0186] Paraffin sections were placed in a 65 °C environment for 1 h to deparaffinize.

[0187] Sections were deparaffinized with three xylene solutions, 10 min, 10 min, 5 min, respectively.

[0188] Sections were removed and hydrated with 100% ethanol for 5 min, 100% ethanol for 5 min, and 95% ethanol for 2 min. Then they were rinsed with running water for 5 min.

[0189] Nuclei were stained with Reagent A for 5 min and rinsed with running water for 5 min.

[0190] Sections were placed in a 65 °C oven until the water on the slide surface evaporated and the section surface whitened.

[0191] Sections were placed horizontally in a wet box and Reagent B was added to cover the specimen on the sections and stained at room temperature for 10-20 min.

[0192] Reagent C was added to the sections and incubated for 3 min, the liquid was discarded, and the process was repeated twice.

[0193] The water on the section surface was removed and Reagent D solution was added and incubated for 3-5 min.

[0194] Reagent D was removed and Reagent E was added and incubated for 5-15 s.

[0195] Reagent C aqueous solution was added, the liquid was discarded, and the staining was clear.

[0196] Sections were examined after dehydration with an ethanol gradient, xylene permeation, and mounting with neutral balsam.

[0197] The results showed that a large number of positive staining areas (blue) appeared in the aGVHD model group, which suggested that fibrosis lesions may have occurred in the lungs of the disease group, while the CD90 + The positive staining of the hAECs treatment group was significantly reduced. The results showed that the CD90 + The hAECs treatment group was significantly better than the unselected hAECs treatment group. Figure 10 ).

[0198] Example 10 Immunohistochemical staining

[0199] Paraffin sections were placed in a 65 °C environment for 1 h to deparaffinize.

[0200] Sections were deparaffinized with three xylene solutions, 10 min, 10 min, 5 min, respectively.

[0201] The sections were hydrated by 100% ethanol for 5 min, 100% ethanol for 5 min and 95% ethanol for 2 min. Then washed by running water for 5 min.

[0202] The sections were placed in preheated antigen retrieval solution at 98°C and incubated at 98°C for 30 min.

[0203] Cool down to room temperature, wash the sections in PBS for 5 min at room temperature.

[0204] Add 3% hydrogen peroxide to the sections and incubate for 10 min.

[0205] Wash in PBS for 2 min x 3 times.

[0206] Spin down the sections, add primary antibody and incubate at 4°C overnight. Wash in PBS for 2 min x 3 times.

[0207] Add reagent 1 and incubate at room temperature for 20 min. Wash in PBS for 2 min x 3 times.

[0208] Add reagent 2 and incubate at room temperature for 20 min. Wash in PBS for 2 min x 3 times.

[0209] Add DAB and develop for 5-20 min.

[0210] Examine under microscope after washing in running water for 5 min, counterstaining with hematoxylin, dehydration with ethanol gradient, clearing with xylene and mounting with neutral resin.

[0211] Example 11 MACS sorting

[0212] Resuspend PBMCs to 10^7 cells per 100 ul in media and load into sterile tubes. Add 10 ul of antibody per 100 ul and incubate on ice for 15 min.

[0213] Resuspend the magnetic beads and mix well. Add 10 ul of magnetic beads per 100 ul and incubate on ice for 15 min.

[0214] Add 3 ml of MojoSort TM Buffer to the cells and mix well.

[0215] Place the cells on a magnet for 5 min.

[0216] Pour off the liquid into a collection tube and mix with the same volume of media. Spin at 350 g for 5 min. (If higher recovery is needed, repeat steps 3-5)

[0217] Discard the liquid and resuspend the cells in complete media for culture or freezing.

[0218] Example 12 CFSE proliferation assay

[0219] 1 ml CFDA SE labeling solution resuspend 10^6-5x10^6 cells.

[0220] Dilute CFDA SE stock solution (1000X) to 2X with CFDA SE labeling solution.

[0221] Add 1 ml CFDA SE stock solution (2X) to 1, mix gently, incubate at 37°C for 10 min.

[0222] Add complete medium (with serum) to stop the reaction, invert to mix.

[0223] 300g for 5 min, discard the supernatant.

[0224] Resuspend with 10 ml complete medium.

[0225] 300g for 5 min, discard the supernatant.

[0226] Resuspend with 10 ml complete medium, incubate at 37°C for 5 min to facilitate CFDA SE retention in cells and unreacted CFDA SE to enter complete cell culture medium.

[0227] 300g for 5 min, discard the supernatant.

[0228] Culture the labeled cells as needed.

[0229] Harvest the cells, and detect the mean fluorescence intensity in cells by FACS.

[0230] Example 13 Lentivirus infection and CD90 + Localization of MECs in target organs

[0231] Virus-related plasmids and vector plasmids were amplified by DH5α, and then plasmid extraction was performed using an endotoxin-free plasmid extraction kit.

[0232] Inoculate 293T into a 10 cm dish to about 80%.

[0233] Mix the virus plasmids uniformly, mix with water and calcium transfection reagent, and incubate at room temperature for 2 min.

[0234] Shake the two solutions uniformly.

[0235] Incubate overnight at 37°C, change the medium, and collect the virus at the appropriate time.

[0236] Mix the virus with an appropriate amount of polybrene, add it to the target cells, and detect the expression of the tag protein after 48-72 h of infection to determine the infection efficiency.

[0237] CD90 infected with virus+ hAECs were morphologically normal and green fluorescence microscopy pictures indicated high infection efficiency (Figure 11). CD90 + hAECs were transplanted into NCG mice alone or with PBMCs, and one week later, organs were taken from each mouse, mRNA was extracted and reverse transcribed into cDNA, and GFP expression in each tissue was detected by RT-PCR to indicate CD90 + hAECs localization in each tissue. The results showed that GFP was highly expressed in the kidney, lung, and liver, while it was very low in the small intestine, spleen, and lymph node, and almost no expression was found in the bone marrow. The results showed that CD90 + hAECs were mainly localized in the kidney, lung, and liver Figure 12 ).

[0238] The present application studies the potential ability of CD90 + The potential ability of human amniotic epithelial cells (hAECs) in the treatment of graft-versus-host disease (GvHD) and explores the treatment mechanism. The results showed that CD90 + hAECs with PBMCs co-transplantation of CD90 + The hAECs group and the unselected hAECs treatment group had better inhibition effect on aGVHD, which significantly improved the clinical and pathological phenotypes of the mice and significantly improved the survival rate of the mice compared with the disease group.

Claims

1. Use of human amnion epithelial cells or a cell preparation thereof for the preparation of a medicament for the treatment and / or amelioration of graft versus host disease, wherein the human amnion epithelial cells (hAECs) are CD90 + hAECs cell subpopulations.

2. Use according to claim 1, characterized in that: Use of an effective amount of CD90 + Human amnion epithelial cells or cell preparations thereof, alone or in combination with other drugs selected from the group consisting of cyclosporin, tacrolimus, mycophenolate mofetil, methotrexate, glucocorticoids, azathioprine, thalidomide, anti-CD3 mAb, anti-interleukin-2 receptor antibodies, or combinations thereof, for the treatment and / or amelioration of graft-versus-host disease.

3. Use according to claim 2, characterized in that: The glucocorticoid is methylprednisolone.

4. Use according to claim 1 or 2, characterized in that: The subject having graft versus host disease is a mammal.

5. Use according to claim 4, characterized in that: The mammal is a cow, a horse, a sheep, a monkey, a dog, a rat, a mouse, or a rabbit.

6. Use according to claim 1 or 2, characterized in that: The subject having graft versus host disease is a human.

7. Use according to claim 1 or 2, characterized in that: The cell preparation comprises CD90 + Human amniotic epithelial cells and a pharmaceutically acceptable carrier.

8. Use according to claim 7, characterized in that: The carrier includes physiological saline, phosphate buffer, artificial cerebrospinal fluid, or whole serum.

9. Use according to claim 1 or 2, characterized in that: CD90 is administered by intravenous injection or intrathecal injection + Human amnion epithelial cells are administered to the patient.

10. Use according to claim 9, characterized in that: CD90 + Human amnion epithelial cell administration is performed repeatedly or continuously.

11. Use according to claim 9, characterized in that: CD90 + Human amniotic epithelial cells are seeded into bioabsorbable material and the seeded bioabsorbable material is surgically implanted into the desired site.

12. Use according to claim 1 or 2, characterized in that: CD90 + The appropriate amount of human amniotic epithelial cells will vary depending on the age, sex, weight, or health of the patient.

13. Use according to claim 12, characterized in that: CD90 + Human amnion epithelial cells were administered at a dose range of 10 3 -10 9 cells per administration.

14. Use according to claim 13, characterized in that: CD90 + The dose range for each administration of human amniotic epithelial cells is 10 6 -10 8 cells.

Images