| BackgroundStem cells are the group of undifferentiated cells owing the self-renewal ability and multiple directional differentiation potential. Stem cells are capable of unlimited, undifferentiated proliferation in vitro, meanwhile, they can differentiate into various types of cells and tissue under specific conditions. They are an ongoing source of the differentiated cells that make up the tissues and organs. According to differentiation potential, stem cells can be divided into totipotent stem cells, pluripotent stem cells and unipotent stem cells.Embryonic stem cells (ESCs) are derived from totipotent cells of the early mammalian embryo. Embryonic stem cells owing the potential to self-renew and differentiate into multiple cell types, raising exciting prospects for organ transplantation, replacement therapy and gene therapy. However, ES cells derived from embryos are genetically divergent from the patient (allogenic) and thus any resultant transplanted cell would be rejected without the continual application of immunosuppressive drugs. The shortage sources of ES cells, and the ethical questions also greatly limit the development and clinical application of ES cells. To resolve such issues, scientists are trying to induce differentiated cells reprogramming back to an undifferentiated state. So far, three ways of reprogramming are mostly adopted: nuclear transfer, cell fusion and defined factors induced reprogramming.Somatic cell nuclear transfer is a strategy for reprogramming differentiated cells by implanting a donor nucleus from a somatic (body) cell into an enucleated oocyte. It is used in both therapeutic and reproductive cloning. Dolly, the sheep became famous for being the first successful case of the reproductive cloning of a mammal. It was the first report of live mammalian offspring following nuclear transfer from an adult mammary gland. The fact that a lamb was derived from an differentiated cell confirmed that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The discovery that somatic cells could be reprogrammed to a pluripotent state has profoundly altered the landscape in which stem cell research is conducted.Cell fusion is a process in which several uninuclear cells (cells with a single nucleus) combine to form a multinuclear cell, known as a syncytium. Somatic cells can be reprogrammed to an embryonic state when they are fused with embryonic stem cells. However, there are still many challenges to be solved before implement cell fusion as a therapeutic tool. These challenges include choosing the best cells to use for the reparative fusion, determining the best way to introduce the chosen cells into the desired tissue, discovering methods to increase the incidence in cell fusion, and ensuring that the resulting fusion products will function properly.Induced pluripotent stem cells (iPS cells or iPSCs) are a type of pluripotent stem cell that generated directly from adult cells. Somatic cells could be reprogrammed to a pluripotent state by overexpression of specific transcription factors. Induced pluripotent stem cells exhibit the morphology and growth properties of ES cells, express ES cell marker genes and own stable developmental potential to form derivatives of all three embryonic germ layers. As iPS cells can give rise to every cell type in the body (such as neurons, hematopoietic progenitor cells, Islet cell, and liver cells, bone, smooth muscle, and striated muscle, ect), they represent a stable source of cells that could be used to replace those lost to damage or disease. For a prospect, iPSCs are highly promising for regenerative medicine, disease modeling and drug screening.The thymus plays a crucial role in the immune system by supporting the develo- pment of functional T cells. It is also the main organ involved in establishing immune tolerance through the elimination of autoreactive T cell subsets. Both of these critical functions are mediated by thymic epithelial cells (TECs), the main component of the thymic stroma. The thymus has been widely studied for its role in donor-specific T-cell-dependent tolerance induction because of its critical role in T-cell maturation. Previous studies have demonstrated the importance of the thymus for rapid and stable tolerance induction in an allotransplant model. It was reported that the thymus also play an important role in inducting of tolerance in xenotransplantation model. Allo-geneic bone marrow transplantation plus thymus transplantation can induce high thymopoiesis, preserving strong graft-versus-tumour (GVT) effects without severe graft-versus-host reaction (GVHR). Thymus transplantation is highly promising for the induction of transplantation tolerance, but the shortage of transplant donors limits the progress of this therapy. Given the key role of TECs in establishing self-tolerance, differentiation of a functional thymus from stem cells also has the potential to enhance engraftment of human-stem-cell-derived tissue through the induction of graft-specific immune tolerance. Meanwhile, the use of stem cells as a potential source of TECs to enhance or restore thymic function is of great therapeutic interest.Various conditions for differentiating embryonic stem cells into thymic epithelial cells (TECs) have been successful. In 2013, two groups reported the production of thymic epithelial progenitor cells (TEPCs) from ES cells in vitro. They found that the success of coaxing ESCs into functional C depends on the accurate in vitro recapi-tulation of in vivo embryonic signaling events that are permissive for complete thymus development. Importantly, the TEPLCs could further develop into the thymic epithelium in vivo, which supports T cell development. However, the in vitro genera-tion of TECs or TEPCs still needs further improvement in differentiation efficiency. In addition, the risk of tumor development due to contamination with undifferentiated pluripotent stem cells(PSCs) must be rigorously assessed before clinical use. Compared with those generated in vitro, cells that are formed in vivo by blastocyst complementation must have gone through near-normal differentiation processes with proper epigenetic changes. The tissues obtained are presumed to be fully functional and the risk of teratoma development due to contamination of undifferentiated PSCs to be negligible. Nonetheless, differentiation of induced pluripotent stem cells into TECs has not been reported.ObjectiveTo examine an in vivo method for direct differentiation of mouse induced pluri-potent stem cells (iPSCs) into thymic epithelial cells (TECs). To establish a green fluorescent protein-expressing murine iPS cell line and produce chimeras by injecting iPS cells into mouse blastocysts. The resultant chimeras produce thymus derived from mouse iPSCs in bodies. The chimeric aninals may provide a method for the derivation of various organs from iPSCs and a foundation for further study of differentiating iPSCs into specific kinds of cell lines.Methods1. Undifferentiated iPSCs were routinely expanded on mouse embryonic fibroblasts (MEF) feeder layers (MEFs inactivated with mitomycin C) in N2B27 medium supplemented with CHIR99021 and PD0325901. The cells were incubated at 37℃ with 5% CO2 in air. For micromanipulation, iPSCs were trypsinized and suspended in iPSC culture medium.2. Superovulated ICR females were mated with ICR males. Mouse blastocyst stage embryos were collected in Medium 2 from uterus of mice 3.5 days postcoitum (dpc). These embryos were cultured in Medium 2 for 1-2 hr for blastocyst injection.3. A micromanipulator was used to drill zona pellucida and trophectoderm under the microscope and 10-15 iPSCs were introduced into blastocyst cavities near the inner cell mass.4. After blastocyst injection, embryos underwent followup culture for 1-2 hr. Mouse blastocysts then were transferred into the uteri of pseudo-pregnant recipient ICR female mice (2.5 dpc).5. Chimeras were killed by cervical dislocation and the thymic structure was observed using histopathology method to detect the morphology characteristics.The sections were stained with hematoxylin and eosin photographed using bright field optics.6. Transplanted the chimeric thymus under the renal capsule of BALB/c nude mice. The spleen was cut out from the thymus-transplanted nude mice and the cells were dispersed and were analyzed by a flow cytometer 4 weeks after transplant-ation. The antibodies used were PE-labeled anti-mouse CD3, FITC-labeled anti-mouse CD4 and APC-labeledanti-mouse CD4.Results1. Generation of GFP-transgenic C57BL/6 mouse iPSC-derived thymus in chimeras. A total of 363 blastocysts were transferred into the uterine of E2.5 pseudo-pregnant ICR mice. Chimeras were born 17 days after embryo transfer and 13 live-born chimeras were obtained. Chimeras were identified by coat color.The contribution of iPSC-derived cells in the chimeras ranged from 5% to at most 90%, estimated by their coat color.2. Typical thymic epithelium structure consisted of green fluorescent protein-expressing cells in chimera. Cytokeratin 8 (K8) and cytokeratin 5 (K5) staining identify cortical and medullary TECs.3. To analyze whether the thymus formed chimera were functional, the thymus was transplanted under the renal capsule of BALB/c nude mouse lacking thymus. There were no CD4+or CD8+single-positive (with T-cell marker, CD3 positive) cells before implantation, but these cell populations appeared 4 weeks after the thymic transplantation.The iPS-derived thymus can support T cell development upon transplantation into thymus-deficient mice.ConclusionOur study provides an in vivo method to differentiation of induced pluripotent stem cells (iPSCs) into thymic epithelial cells via producing of chimera by injecting iPS cells into mouse blastocysts. The iPSCs-derived thymic epithelial cells could support the generation of new T cells and form structures resembling normal thymic architecture in the grafts. |
PDF Full Text Request