Thymopoiesisis regulated by the thymic microenvironment, of which epithelial cells are the major components. Both cortical and medullary thymic epithelial cells (TECs) have been shown to arise from a common progenitor cell. Here we show for the first time that mouse embryonic stem cells (mESCs) can be selectively induced in vitro to differentiate into cells that have the phenotype of thymic epithelial progenitors (TEPs). When placed in vivo, these mESC-derived TEPs self-renew, develop into TECs, and reconstitute the normal thymic architecture. Functionally, these ESC-derived TEPs enhanced thymocyte regeneration after bone marrow transplantation and increased the number of functional naive splenic T cells. In addition to providing a model to study the molecular events underlying thymic epithelial cell development, the ability to selectively induce the development of TEPs in vitro from mESCs has important implications regarding the prevention and/or treatment of primary and secondary T-cell immunodeficiencies.

BackgroundThe WNT/β-CATENIN signaling cascade is crucial for the patterning of the early lung morphogenesis in mice, but its role in the developing human lung remains to be determined. In this study, expression patterns of canonical WNT/β-CATENIN signaling components, including WNT ligands (WNT2, WNT7B), receptors ( FZD4, FZD7, LRP5, LRP6), transducers ( DVL2, DVL3, GSK-3β, β-CATENIN, APC, AXIN2), transcription factors ( TCF4, LEF1) and antagonists ( SOSTDC1) were examined in human embryonic lung at 7, 12, 17 and 21 weeks of gestation (W) by real-time qRT-PCR and in situ hybridization.ResultsqRT-PCR analysis showed that some of these components were gradually upregulated, while some were significantly downregulated from the 7 W to the 12 W. However, most components reached a high level at 17 W, with a subsequent decrease at 21 W. In situ hybridization showed that the canonical WNT ligands and receptors were predominantly located in the peripheral epithelium, whereas the canonical WNT signal transducers and transcription factors were not only detected in the respiratory epithelium, but some were also scattered at low levels in the surrounding mesenchyme in the developing human lung. Furthermore, Western blot, qRT-PCR and histological analysis demonstrated that the β-CATENIN-dependent WNT signaling in embryonic human lung was activated in vitro by CHIR 99021 stimulation.ConclusionsThis study of the expression patterns and in vitro activity of the canonical WNT/β-CATENIN pathways suggests that these components play an essential role in regulation of human lung development.

Amphiphilic brush-like block copolymers composed of polynorbonene-cholesterol/poly(ethylene glycol) (P(NBCh9-b-NBPEG)) self-assembled to form a long circulating nanostructure capable of encapsulating the anticancer drug doxorubicin (DOX) with high drug loading (22.1% w/w). The release of DOX from the DOX-loaded P(NBCh9-b-NBPEG) nanoparticles (DOX-NPs) was steady at less than 2% per day in PBS. DOX-NPs were effectively internalized by human cervical cancer cells (HeLa) and showed dose-dependent cytotoxicity, whereas blank nanoparticles were noncytotoxic. The DOX-NPs demonstrated a superior in vivo circulation time relative to that of free DOX. Tissue distribution and in vivo imaging studies showed that DOX-NPs preferentially accumulated in tumor tissue with markedly reduced accumulation in the heart and other vital organs. The DOX-NPs greatly improved survival and significantly inhibited tumor growth in tumor-bearing SCID mice compared to that for the untreated and free DOX-treated groups. The results indicated that self-assembled P(NBCh9-b-NBPEG) may be a useful carrier for improving tumor delivery of hydrophobic anticancer drugs.

Bone marrow transplantation (BMT) is often followed by a prolonged period of T cell deficiency. Therefore, the enhancement of T cell reconstitution is an important clinical goal. We have identified a novel hybrid cytokine containing IL-7 and the β-chain of hepatocyte growth factor (HGF) in the supernatant of cultured mouse BM stromal cells. We have cloned and expressed the IL-7/HGFβ gene to produce a single-chain rIL-7/HGFβ protein that stimulates the in vitro proliferation of thymocytes, early B-lineage cell, and day 12 spleen CFUs. In this study, we show that, following syngenic BMT, the in vivo administration of rIL-7/HGFβ supports the rapid and complete regeneration of the thymus and efficiently reconstitutes the pool of naive T cells having a normally diverse TCR repertoire. The rIL-7/HGFβ hybrid cytokine was significantly more effective quantitatively than was rIL-7 and differed qualitatively in its ability to cross-link c-Met and IL-7Rα and to stimulate the expansion of early thymocyte progenitors and thymic epithelial cells. It also supports the maturation and homeostatic expansion of peripheral T cells. Consequently, the in vivo administration of rIL-7/HGFβ may offer a new approach to preventing and/or correcting post-BMT T cell immune deficiency.

We have reported that mouse embryonic stem cells (mESCs) can be selectively induced in vitro to differentiate into thymic epithelial cell progenitors (TEPs). When placed in vivo, these mESC-derived TEPs differentiate into cortical and medullary thymic epithelial cells, reconstitute the normal thymic architecture, and enhance thymocyte regeneration after syngeneic BM transplantation (BMT). Here, we show that transplantation of mESCderived TEPs results in the efficient establishment of thymocyte chimerism and subsequent generation of naive T cells in both young and old recipients of allogeneic BM transplant. GVHD was not induced, whereas graft-versus-tumor activity was significantly enhanced. Importantly, the reconstituted immune system was tolerant to host, mESC, and BM transplant donor antigens. Therefore, ESCderived TEPs may offer a new approach for the rapid and durable correction of T-cell immune deficiency after BMT, and the induction of tolerance to ESC-derived tissue and organ transplants. In addition, ESC-derived TEPs may also have use as a means to reverse age-dependent thymic involution, thereby enhancing immune function and decreasing infection rates in the elderly. (Blood. 2011;118(12): 3410-3418) IntroductionBM transplantation (BMT) is widely used in the treatment of a variety of hematopoietic and nonhematopoietic diseases. However, BM transplant recipients are often subject to prolonged periods of T-cell deficiency, which is associated with high risk of common and opportunistic infections, as well as occurrence and relapse of cancers. 1-4 T-cell development in the thymus depends not only on the availability of thymocyte progenitors but also on the thymic microenvironment, of which thymic epithelial cells (TECs) are the main components. 5,6 However, TECs are vulnerable to injury from radiation, chemotherapy, immunosuppressive drugs, infection, and GVHD after BMT. 7,8 In addition, TECs undergo a qualitative and quantitative loss over time, which is believed to be one of the main factors responsible for age-dependent thymic involution. 3,9 We reported previously that mouse embryonic stem cells (mESCs) could be selectively induced to differentiate into Ep-CAM1 ϩ thymic epithelial cell progenitors (TEPs) in vitro. Furthermore, adoptive transfer of mESC-derived TEPs into syngeneic BM transplant recipients enhanced thymocyte reconstitution and increased the numbers and functions of peripheral T cells. 10 In the current study, we investigated whether mESC-derived TEPs could enhance thymocyte regeneration after allogeneic BMT (allo-BMT), a more clinically relevant model. We show here that transplantation of mESC-derived TEPs enhances thymopoiesis, leading to increased numbers of peripheral T cells in both young and old allo-BM transplant recipients. We demonstrated that the T cells in these recipients were tolerant to both host and mESC antigens. Moreover, GVHD was not induced, but graft-versustumor (GVT) activity was significantly enhanced in these recipients. Methods MiceFour-to 10-week-old C57BL/6 (B6), B...

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Thymic epithelial cells (TECs) are the major components of the thymic microenvironment for T cell development. TECs are derived from thymic epithelial progenitors (TEPs). It has been reported that human ESCs (hESCs) can be directed to differentiate into TEPs in vitro. However, the efficiency for the differentiation is low. Furthermore, transplantation of hESC-TEPs in mice only resulted in a very low level of human T cell development from co-transplanted human hematopoietic precursors. We show here that we have developed a novel protocol to efficiently induce the differentiation of hESCs into TEPs in vitro. When transplanted into mice, hESC-TEPs develop into TECs and form a thymic architecture. Most importantly, the hESC-TECs support the long-term development of functional mouse T cells or a higher level of human T cell development from co-transplanted human hematopoietic precursors. The hESC-TEPs may provide a new approach to prevent or treat patients with T cell immunodeficiency.

T cell immunodeficiency is a major complication of bone marrow (BM) transplantation (BMT). Therefore, approaches to enhance T cell reconstitution after BMT are required. We have purified a hybrid cytokine, consisting of IL-7 and the β-chain of hepatocyte growth factor (HGFβ) (IL-7/HGFβ), from a unique long-term BM culture system. We have cloned and expressed the IL-7/HGFβ gene in which the IL-7 and HGFβ genes are connected by a flexible linker to generate rIL-7/HGFβ protein. Here, we show that rIL-7/HGFβ treatment enhances thymopoiesis after allogeneic BMT. Although rIL-7 treatment also enhances the number of thymocytes, rIL-7/HGFβ hybrid cytokine was more effective than was rIL-7 and the mechanisms by which rIL-7 and rIL-7/HGFβ increase the numbers of thymocytes are different. rIL-7 enhances the survival of double negative (DN), CD4 and CD8 single positive (SP) thymocytes. In contrast, rIL-7/HGFβ enhances the proliferation of the DN, SP thymocytes, as well as the survival of CD4 and CD8 double positive (DP) thymocytes. rIL-7/HGFβ treatment also increases the numbers of early thymocyte progenitors (ETPs) and thymic epithelial cells (TECs). The enhanced thymic reconstitution in the rIL-7/HGFβ-treated allogeneic BMT recipients results in increased number and functional activities of peripheral T cells. Graft-versus-host-disease (GVHD) is not induced in the rIL-7/HGFβ-treated BMT mice. Therefore, rIL-7/HGFβ may offer a new tool for the prevention and/or treatment of T cell immunodeficiency following BMT.