| Deriving functional T cells from pluripotent stem cells(PSCs)is a study-hotspot in regenerative medicine.In the first section of this thesis,T cells differentiated from mouse embryonic stem cells(mESC)were assessed for in vivo tumor-killing ability in CAR-T cell therapy.In the second section,we established a method of regenerating hematopoietic progenitor cells from mESC,which gave rise to mature myeloid cells in vivo.These results provide technical insights into regeneration of functional T cells and myeloid cells for translational research.Chimeric Antigen Receptor T cells(CAR-T)therapy has become an effective therapeutic approach for treating hematological malignancies.Generally,CAR-T cells were produced by editing the autologous T cells isolated from patients’ peripheral blood.Besides the fact that T cells in tumor-bearing patients are functionally either exhausted or abnormal,the availabitiy of T cells from certain patients is often problematic.Thus,it is necessary to explore new sources of T cells.Based on the T cell induction technology established in our lab,we isolated the induced T cells(iT)from the immunodeficient mice which were generated from the inducible Runx1-Hoxa9 pluripotent stem cells and transduced them with CD 19-CAR element by retroviral infection in vitro.In order to verify the tumor killing function of CD 19-CAR transduced iT cells(CD 19-CAR iT),we established a tumor model via transplantation of luciferase-reporting Ka539(B lymphoma cell line)cells to C57BL/6 mice.The CD 19-CAR iT cells were then transplanted into the luciferase-reporting B cell lymphoma mouse model.Flow cytometry analysis revealed that the proportion of CD19+ B cells in the peripheral blood from CD19-CAR iT cells treated mice was significantly reduced than control mice.In vivo live imaging showed that the tumor cells were gradually cleared by the CD 19-CAR iT cells within one week,however,the tumor cells were dramatically growing in the control mice.Moreover,CD 19-CAR iT cell therapy also significantly extended the life span of the treated tumor-bearing mice(>70 days).Moreover,the CD 19-CAR iT cells proliferated rapidly in vivo after stimulated by CD 19 antigen,and tremendous amount of activated T cells(CD44+CD62L-)were detected in CD 19-CAR iT treated mice.These results suggest that CD 19-CAR iT cells can effectively eliminate tumor cells in vivo,which supports the potential of iPSC-derived T cells in immunotherapy.Meanwhile,myeloid cells(especially granulocytes,monocytes,and macrophages),as essential cell compotents of innate immune system,play pivotal roles in phagocytosis,sterilization,and resistance to pathogen infections.Therefore,regeneration of myeloid cells is clinically needed for the anti-infection treatment.In the second section of this thesis,myeloid cells(including granulocytes,monocytes,and macrophages)were regenerated from the mES cell line edited to inducible expressing Runx1-Hoxa10(iR10 mESCs).First,the differentiation of iR10 mESCs were initiated by embryoid bodies(EBs)formation,followed regeneration of induced hematogenic endothelial cells(iHECs,CD31+CD41lowCD45-c-Kit+CD201hi)via the inducible expression of Runx1 and Hoxa10 and addition of hematopoietic specific cytokines.The iHECs were subsequently cocultured with OP9-DL1 stromal cells to obtain induced hematopoietic progenitor cells(iHPCs).These iHPCs were transplanted into the sub-lethally irradiated Rag1-/-mice and successfully regenerated myeloid cells.The PCR sequencing results confirmed the insertion of the Runx1 and Hoxa10 genes in the regenerative myeloid cells.In addition,regenerative myeloid cells were also detected in the bone marrow and spleen,including granulocytes(CD11b+Gr1+),monocytes(CD11b+Gr1-F4/80-)and macrophages(CD11b+F4/80+).Finally,iHPCs transplanted into sub-lethally irradiated wild type recipients also successfully regenerated myeloid cells.These prospective results provide technical insights into regeneration of engraftable human myeloid cells from pluripotent stem cells for translational study. |
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