Foxp3 regulatory T cells maintain the bone marrow microenvironment for B cell lymphopoiesis.

Foxp3 regulatory T cells (Treg cells) modulate the immune system and maintain self-tolerance, but whether they affect haematopoiesis or haematopoietic stem cell (HSC)-mediated reconstitution after transplantation is unclear. Here we show that B-cell lymphopoiesis is impaired in Treg-depleted mice, yet this reduced B-cell lymphopoiesis is rescued by adoptive transfer of affected HSCs or bone marrow cells into Treg-competent recipients. B-cell reconstitution is abrogated in both syngeneic and allogeneic transplantation using Treg-depleted mice as recipients.

DR3 signaling modulates the function of Foxp3+ regulatory T cells and the severity of acute graft-versus-host disease.

CD4Foxp3 regulatory T cells (Treg) are a subpopulation of T cells, which regulate the immune system and enhance immune tolerance after transplantation. Donor-derived Treg prevent the development of lethal acute graft-versus-host disease (GVHD) in murine models of allogeneic hematopoietic stem cell transplantation. We recently demonstrated that a single treatment of the agonistic antibody to DR3 (death receptor 3, αDR3) to donor mice resulted in the expansion of donor-derived Treg and prevented acute GVHD, although the precise role of DR3 signaling in GVHD has not been elucidated.

TNF-α priming enhances CD4+FoxP3+ regulatory T-cell suppressive function in murine GVHD prevention and treatment.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) have been shown to effectively prevent graft-versus-host disease (GVHD) when adoptively transferred in murine models of hematopoietic cell transplantation and in phase 1/2 clinical trials. Critical limitations to Treg clinical application are the paucity of cells and limited knowledge of the mechanisms of in vivo function. We hypothesized that inflammatory conditions in GVHD modify Treg characteristics and activity.

Human Embryonic Stem Cell Lines with Lesions in FOXP3 and NF1.

Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of blastocyst staged embryos. Spare blastocyst staged embryos were obtained by in vitro fertilization (IVF) and donated for research purposes. hESCs carrying specific mutations can be used as a powerful cell system in modeling human genetic disorders.

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease.

The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs.

Treatment with agonistic DR3 antibody results in expansion of donor Tregs and reduced graft-versus-host disease.

The paucity of regulatory T cells (Tregs) limits clinical translation to control aberrant immune reactions including graft-versus-host disease (GVHD). Recent studies showed that the agonistic antibody to DR3 (αDR3) expanded CD4(+)FoxP3(+) Tregs in vivo. We investigated whether treating donor mice with a single dose of αDR3 could alleviate acute GVHD in a MHC-mismatched bone marrow transplantation model.

Donor Requirements for Regulatory T Cell Suppression of Murine Graft-versus-Host Disease.

Adoptive transfer of freshly isolated natural occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) prevents graft-versus-host disease (GVHD) in several animal models and following hematopoietic cell transplantation (HCT) in clinical trials. Donor-derived Treg have been mainly used, as they share the same MHC with CD4(+) and CD8(+) conventional T cells (Tcon) that are primarily responsible for GVHD. Third party-derived Treg are a promising alternative for cellular therapy, as they can be prepared in advance, screened for pathogens and activity, and banked.

Engraftment of embryonic stem cells and differentiated progeny by host conditioning with total lymphoid irradiation and regulatory T cells.

Embryonic stem cells (ESCs) hold promise for the treatment of many medical conditions; however, their utility is limited by immune rejection. The objective of our study is to establish tolerance or promote engraftment of transplanted ESCs as well as mature cell populations derived from ESCs. Luciferase (luc(+))-expressing ESCs were utilized to monitor the survival of the ESCs and differentiated progeny in living recipients.

CD4+ invariant natural killer T cells protect from murine GVHD lethality through expansion of donor CD4+CD25+FoxP3+ regulatory T cells.

Dysregulated donor T cells lead to destruction of host tissues resulting in graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). We investigated the impact of highly purified (>95%) donor CD4(+) invariant natural killer T (iNKT) cells on GVHD in a murine model of allogeneic HCT. We found that low doses of adoptively transferred donor CD4(+) iNKT cells protect from GVHD morbidity and mortality through an expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs).

Mast cells suppress murine GVHD in a mechanism independent of CD4+CD25+ regulatory T cells.

To investigate the role of mast cells in hematopoietic cell transplantation, we assessed graft-versus-host disease (GVHD) in C57BL/6-Kit(W-sh/W-sh) recipients, which virtually lack mast cells, compared with C57BL/6 WT recipients. GVHD was severely exacerbated in C57BL/6-Kit(W-sh/W-sh) mice (median survival time = 13 vs 60 days in wild-type [WT] mice; P < .0001).

A new FACS approach isolates hESC derived endoderm using transcription factors.

We show that high quality microarray gene expression profiles can be obtained following FACS sorting of cells using combinations of transcription factors. We use this transcription factor FACS (tfFACS) methodology to perform a genomic analysis of hESC-derived endodermal lineages marked by combinations of SOX17, GATA4, and CXCR4, and find that triple positive cells have a much stronger definitive endoderm signature than other combinations of these markers. Additionally, SOX17(+) GATA4(+) cells can be obtained at a much earlier stage of differentiation, prior to expression of CXCR4(+) cells, providing an important new tool to isolate this earlier definitive endoderm subtype.

Generation of three-dimensional hepatocyte/gelatin structures with rapid prototyping system.

Using rapid prototyping technology, three-dimensional (3D) structures composed of hepatocytes and gelatin hydrogel have been formed. This technique employs a highly accurate 3D micropositioning system with a pressure-controlled syringe to deposit cell/biomaterial structures with a lateral resolution of 10 microm. The pressure-activated micro-syringe is equipped with a fine-bore exit needle for which a wide variety of 3D patterns with different arrays of channels (through-holes) were created.

Fabrication of viable tissue-engineered constructs with 3D cell-assembly technique.

We have recently developed an organ manufacturing technique that enables us to form cell/biomaterial complex three-dimensional (3D) architectures in designed patterns. This technique employs a highly accurate 3D micropositioning system with a pressue-controlled syringe to deposit cell/biomaterial structures with a lateral resolution of 10 microm. The pressure-activated micro-syringe is equipped with a fine-bore exit needle using which a wide variety of 3D patterns with different arrays of channels (through-holes) were created.

In vitro neuronal differentiation of cultured human embryonic germ cells.

Human embryonic germ (hEG) cells, which have been advanced as one of the most important sources of pluripotent stem cells [the other one being human embryonic stem cells], can be propagated in vitro indefinitely in the primitive undifferentiated state while being capable of developing into all three germ layer derivatives, hence have become anticipated developing novel strategies of tissue regeneration and transplantation in the treatment of degenerative diseases. In the experiments here, we derived hEG cells from cultured human primordial germ cells (PGCs) of 6- to 9-week-post-fertilization embryos. They satisfied the criteria previously used to define hEG cells, including the expression of markers characteristic of pluripotent cells-abundant alkaline phosphatase (AP) activity, stage specific embryonic antigen (SSEA)-1(+), SSEA-3(-), SSEA-4(+), TRA-1-60(+), TRA-1-81(+), Oct-4(+), and hTERT(+), the retention of normal karyotypes, and possessing pluripotency by forming embryoid bodies (EBs) in vitro.

Human embryonic germ cells isolation from early stages of post-implantation embryos.

Human embryonic germ (hEG) cell is a very important alternative pluripotent stem cell resource. We describe the derivation of hEG cells from human embryonic fetal gonads over 6-8 weeks postconception. A large number of EG-like cell clumps were obtained at passage 1 and thus facilitated the following routine culture when the donor tissues were trypsinized with gentle pipetting and plated on feeder layer cells in the initial culture.

Characterization of stage-specific embryonic antigen-1 expression during early stages of human embryogenesis.

Extensive expression of stage-specific embryonic antigen-1 (SSEA-1) has been documented in some animal species, but not in human embryos. In this study, SSEA-1 was detected during human embryogenesis by whole-mount immunohistochemistry. Alkaline phosphatase (Ap) activity was detected to identify human primordial germ cells.

Cartilage-derived morphogenetic protein-1 promotes the differentiation of mesenchymal stem cells into chondrocytes.

Mesenchymal stem cells (MSCs) are able to differentiate into many types of cells including chondrocytes. Transforming growth factor beta1 (TGF-beta1) is very important in the regulation of chondrogenesis. Since cartilage-derived morphogenetic protein-1 (CDMP-1) belongs to the TGF-beta superfamily, we tested whether CDMP-1 plays any role in the regulation of the differentiation of MSCs into chondrocytes using a high density pellet culture system.