Analysis And Application Of Human Thymic Tissue At Different Ages For The Establishment Of Humanized Mouse Models

Background:In the past few decades, numerous fundamental discoveries were made inthe fields of biological science as well as medical sciences, which is quiteencouraging. However, limited breakthrough were achieved for the treatment ofworld widely detrimental diseases, such as HIV, cancer, diabetes and so on. Oneof the major reason is that: due to the species disparities, the results comingfrom traditional animal mouse models, like mouse, rat etc, can not represent the"real situation in human beings. Therefore, it is quite essential to develop“advanced animal models” to simulate the complex human physiological andpathological reactions in vivo, among which, humanized mouse model is atypical example.Humanized mouse models are the mice carrying human cells, tissues, orexpressing human genes. In my thesis, humanized mouse models represent thehumanized mice with human immune systems. The field of humanized mice hasdeveloped for over two decades, in which, the humanized mouse modeldiscovered by Dr. Yong-Guang Yang at Massachusetts General Hospital inHarvard Medical School by the transplantation of human fetal thymic graft(under the renal capsule) and fetal liver CD34+cells (intravenous injection) was considered one of the best humanized mouse models to study human immunesystem in vivo as well as human immune system related diseases such as HIV,cancer, graft transplantations etc. However, one deficiency of this model is thathuman fetal tissues are hard to harvested for lots of scientists due to the ethicissues, which limiting the utilization of this model, especially in China.Accordingly, we planned to use neonatal or pediatric thymic tissues (dissecteddue to the surgery for congenital heart disease) to establish a novel humanizedmouse model with functional human immune systemwhich do not require human fetal tissues.Objective:Analyze the cellular compositions of human thymic tissues at different agesor pathological situations. Utilize allogeneic adult/pediatric human thymictissues as well as human cord blood hematopoietic stem cells to establish anovel humanized mouse model which do not require human fetal tissuestransplantations. Then, evaluate the reconstitution and function of thishumanized mouse model and explore its potential in study humanimmune system related disease.Methods:Establishment of humanized mice through human fetal thymic grafts andhuman CD34+fetal liver cells(FLCs) transplantations: Implantation of humanfetal thymic grafts (17~20weeks gestation,1mm3) under the renal capsule ofNOD/SCID mice after2Gy total body regimen, and intravenous injection of around1×105CD34+human FLCs (from the same fetus as the thymic graft) atthe same time. Evaluate the human immune system function.Analyze cellular composition of human thymic tissues at differentdifferentiation ages and pathological situations: Utilize the techniques, likemultiple color flow cytometries, H/E staining, immunohistochemistry, toanalyze the combinations as well as the distributions of human immune cells inhuman thymic tissues, such as fetal thymus, pediatric thymus, adult thymus,thymomas, Inflammatory pseudotumor of the thymus.Generate a novel humanized mouse model which do not require human fetaltissues: Transplantation of pediatric human thymic tissues as well as allogeneichuman CD34+hematopoietic stem cells into NOD/SCID mice, and inhibit theimmunological rejection at early stage by the regimens like Cyclosporine A(CsA).Results:Part-I. Establishment of humanized mice with human immune system bytransplantation of human fetal thymus as well as CD34+FLCs.1-10%of humanlympho-hematopoietic cells (human CD45+cells) in peripheral bloodmononuclear cells was detected at3weeks after transplantation. The chimerimsof human immune cells increased gradually to around70%at around12weeksin our humanized mice. Human immune system was mainly composed byhuman CD3+T cells and human CD19+B cells. Furthermore, significantreconstitution of human CD14+myeloid cells was also detected. When mice were killed at around12weeks after transplantation，human lymphoid cells canbe detected in the secondary lymphoid organs, like spleens, lymph nodes andbone marrows.Part-II.Analyze cellular composition of human thymic tissues at differentdifferentiation ages and pathological situations. According to the flowcytometric analysis, we found that human thymocyte combination in humanpediatric thymic tissues is close to the one in human fetal thymus. However,human thymocyte distribution in the tissues of thymoma, thymic hyperplasiaand the inflammatory pseudotumor are different from human thymoma with ABtype. Therefore, we use human pediatric thymus instead of human fetal thymusto establish the humanized mice. In addition, we determined a rare case ofhuman inflammatory pseudotumor of thymus by analysis of H/E staining andimmunohistochemistry.Part-III. Generate a novel humanized mouse model which do not requirehuman fetal tissues: We found that human CD19+cell chimerism was lost insome humanized mice made by the transplantation of human pediatric thymicgraft as well as allogeneic CD34+hematopoietic stem cells which imply theoccurrence of human immunological rejections. Therefore, we treat the animalswith the immunosuppressant drug, CsA, to inhibit the rejection at early stages,in which both human CD19+B cells and CD14+monocytes as well as humanCD3+T cells could be detected in the mice. In addition, we are going to use lesstoxic regimens to deplete existent allogeneic thymic precursors for our novel humanized mouse model.Summary and conclusions:First, we successfully established the humanized mice by transplantation ofhuman fetal thymus as well as human CD34+FLCs with high level of functionalhuman immune system. Second, we made a comparison of the cellularcombination of human thymic tissue at different ages and pathology settings bymultiple color flow cytomeric analysis and H/E, IHC staining. Third, weestablished a novel humanized mouse model by the transplantation of humanpediatric thymus tissues as well as allogeneic human CD34+FLCs under thehelp of CsA regimen, which could bypass the requirement of human fetaltissues.