Functional calcium-responsive parathyroid glands generated using single-step blastocyst complementation.

Patients with permanent hypoparathyroidism require lifelong replacement therapy to avoid life-threatening complications, The benefits of conventional treatment are limited, however. Transplanting a functional parathyroid gland (PTG) would yield better results. Parathyroid gland cells generated from pluripotent stem cells in vitro to date cannot mimic the physiological responses to extracellular calcium that are essential for calcium homeostasis. We thus hypothesized that blastocyst complementation (BC) could be a better strategy for generating functional PTG cells and compensating loss of parathyroid function. We here describe generation of fully functional PTGs from mouse embryonic stem cells (mESCs) with single-step BC. Using CRISPR-Cas9 knockout of Glial cells missing2 (Gcm2), we efficiently produced aparathyroid embryos for BC. In these embryos, mESCs differentiated into endocrinologically mature PTGs that rescued Gcm2-/- mice from neonatal death. The mESC-derived PTGs responded to extracellular calcium, restoring calcium homeostasis on transplantation into mice surgically rendered hypoparathyroid. We also successfully generated functional interspecies PTGs in Gcm2-/- rat neonates, an accomplishment with potential for future human PTG therapy using xenogeneic animal BC. Our results demonstrate that BC can produce functional endocrine organs and constitute a concept in treatment of hypoparathyroidism.

TX99 Is a Neutralizing Monoclonal Antibody Against Mouse TIGIT.

T cell immunoglobulin and ITIM domains (TIGIT) is an inhibitory immunoreceptor expressed on NK cells, effector and memory T cells, and regulatory T cells (Tregs). The ligands for TIGIT are CD155 (PVR) and CD112 (PVRL2, nectin-2), which are broadly expressed on hematopoietic cells and nonhematopoietic cells. TIGIT negatively regulates antitumor responses, but promotes autoimmune reaction. Although neutralizing anti-human TIGIT mAbs are under clinical trials for cancers, how the blockade of TIGIT interaction with the ligands shows tumor immunity still remains unclear. Although analyses of mouse tumor model using a neutralizing anti-mouse TIGIT (mTIGIT) mAbs should be useful to address this issue, there are limitations to this type of studies due to unavailability of neutralizing anti-mTIGIT mAbs. In this study, we generated five clones of anti-mTIGIT mAbs, designated TX99, TX100, TX103, TX104, and TX105. We show that TX99 and TX100 showed the strongest binding to TIGIT. We also show that TX99 interfered with the interaction between TIGIT and CD155 and increased NK cell-mediated cytotoxicity against CD155-expressing RMA-S cells. Thus, TX99 is a unique neutralizing mAb that can be used for studies of mTIGIT functions.

Development and Characterization of Novel Monoclonal Antibodies Against Human DNAM-1.

DNAM-1 (CD226) is an activating immunoreceptor expressed on lymphocytes and myeloid cells. CD155 and CD112 are the ligands for DNAM-1. DNAM-1 plays an important role in tumor immunity mediated by CD8+ T cells and NK cells. Moreover, the interaction of DNAM-1 with the ligands contributed to the development of acute graft versus host disease (GVHD) and treatment with anti-DNAM-1 monoclonal antibodies (mAb) dramatically improved acute GVHD in a mouse model, suggesting that DNAM-1 may be a good molecular target for therapy to acute GVHD in human. In this study, we generated and characterized five novel clones of anti-human DNAM-1 mAbs, named TX94, TX95, TX96, TX107, and TX108. Among these mAbs, TX94 is a unique neutralizing mAb that most efficiently blocked the interaction between DNAM-1 and CD155. Furthermore, TX94 inhibited NK cell-mediated cytotoxicity against a tumor cell line and suppressed CD8+ T cell proliferation mediated by allogeneic mixed lymphocyte reaction. Thus, TX94 may be useful for molecular therapy targeting DNAM-1.

Soluble DNAM-1, as a Predictive Biomarker for Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (aGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Because diagnosis of aGVHD is exclusively based on clinical symptoms and pathological findings, reliable and noninvasive laboratory tests for accurate diagnosis are required. An activating immunoreceptor, DNAM-1 (CD226), is expressed on T cells and natural killer cells and is involved in the development of aGVHD. Here, we identified a soluble form of DNAM-1 (sDNAM-1) in human sera. In retrospective univariate and multivariate analyses of allo-HSCT patients (n = 71) at a single center, cumulative incidences of all grade (grade I-IV) and sgrade II-IV aGVHD in patients with high maximal serum levels of sDNAM-1 (≥30 pM) in the 7 days before allo-HSCT were significantly higher than those in patients with low maximal serum levels of sDNAM-1 (<30 pM) in the same period. However, sDNAM-1 was not associated with other known allo-HSCT complications. Our data suggest that sDNAM-1 is potentially a unique candidate as a predictive biomarker for the development of aGVHD.

Increased Soluble CD155 in the Serum of Cancer Patients.

Emerging evidence suggests that DNAM-1 (CD226) play an important role in the recognition of tumor cells and their lysis by cytotoxic T lymphocytes (CTL) and NK cells. Although the DNAM-1 ligand CD155 is ubiquitously expressed in various tissues, many human tumors significantly upregulate the expression of CD155; DNAM-1 on CTL and NK cells may be involved in tumor immunity. However, unlike those in mice, human tissues also express soluble isoforms of CD155 (sCD155) that lack the transmembrane region. Here, we show that sCD155 levels were significantly higher in the sera of 262 patients with lung, gastrointestinal, breast, and gynecologic cancers than in sera from healthy donors. In addition, the sCD155 levels were significantly higher in patients with early stage (stages 1 and 2) gastric cancer than in healthy donors, and were significantly higher in patients with advanced stage (stages 3 and 4) disease than in patients in those with early stage disease and healthy donors. Moreover, the sCD155 levels were significantly decreased after surgical resection of cancers. Thus, sCD155 level in serum may be potentially useful as a biomarker for cancer development and progression.

Establishment and characterization of a novel anti-DNAM-1 monoclonal antibody.

DNAM-1 (CD226) is expressed on the majority of NK cells, CD8(+) T cells, and CD4(+) T cells and mediates an activating signal in these cells upon binding to the ligands CD155 or CD112 expressed on target cells or antigen-presenting cells. DNAM-1 plays an important role in tumor immunity mediated by NK cells and CD8(+) T cells and the development of graft-versus-host disease (GVHD) in mice. We previously generated a monoclonal antibody against mouse DNAM-1, TX42, which inhibited DNAM-1 binding to its ligands CD155 and CD112 and inhibited activation of NK cells and CD8(+) T cells in vitro. Injection of mice with TX42 ameliorated the development of GVHD in mice. Here, we generated a new clone of anti-DNAM-1 MAb, TX92. TX92 similarly stained primary spleen cells, including CD8(+) and CD4(+) T cells and NK cells. TX92 as well as TX42 interfered with the interaction between DNAM-1 and ligands CD155 and CD112. However, TX92 recognizes a different epitope and, unlike TX42 partially, but not completely, depleted peripheral blood (PB) CD8(+) T cells in vivo. Thus, TX92 is a unique MAb that is characterized not only by inhibitory function of DNAM-1 binding to the ligands but also by function of partial depletion of PB CD8(+) T cells.

In-vitro reconstitution of hepatic tissue architectures with neonatal mouse liver cells using three-dimensional culture.

Stem cells have the potential to differentiate into multiple lineages, and the capabilities to self-renew, and reconstitute tissues following transplantation. Thus, stem cells are expected to be useful for regenerative medicine; however, the mechanisms that regulate the reconstitution of three-dimensional (3-D) tissues remain to be elucidated. To study such mechanisms, we have established a novel procedure of 3-D culture that supports the formation of tissues from isolated cells, including hepatic stem/progenitor cells in vitro. We cultured neonatal mouse liver cell populations, including hepatic stem/progenitor cells, in a simulated microgravity environment produced by a Rotating Wall Vessel bioreactor. After 8 days in culture, we obtained a 3-D tissue architecture. Histological analysis showed that bile duct structures secreting mucin formed complicated tubular branches in the peripheral region. In the non-bile duct structure region, we observed mature hepatocytes that were capable of producing albumin and storing glycogen. Thus, we were able to establish a novel 3-D culture system that is able to reconstitute functional hepatic tissue architecture from isolated neonatal mouse liver cells.

Chondrogenic differentiation of human mesenchymal stem cells cultured in a cobweb-like biodegradable scaffold.

Human mesenchymal stem cells (MSCs) were cultured in vitro in a cobweb-like biodegradable polymer scaffold: a poly(dl-lactic-co-glycolic acid)-collagen hybrid mesh in serum-free DMEM containing TGF-beta3 for 1-10 weeks. The cells adhered to the hybrid mesh, distributed evenly, and proliferated to fill the spaces in the scaffold. The ability of the cells to express gene encoding type I collagen decreased, whereas its ability to express type II collagen and aggrecan increased. Histological examination by HE staining indicated that the cells showed fibroblast morphology at the early stage and became round after culture for 4 weeks. The cartilaginous matrices were positively stained by safranin O and toluidine blue. Immunostaining with anti-type II collagen and anti-cartilage proteoglycan showed that type II collagen and cartilage proteoglycan were detected around the cells. In addition, a homogeneous distribution of cartilaginous extracellular matrices was detected around the cells. These results suggest the chondrogenic differentiation of the mesenchymal stem cells in the hybrid mesh. The PLGA-collagen hybrid mesh enabled the aggregation of mesenchymal stem cells and provided a promotive microenvironment for the chondrogenic differentiation of the MSCs.

The use of a novel PLGA fiber/collagen composite web as a scaffold for engineering of articular cartilage tissue with adjustable thickness.

It has been a great challenge to make the thickness of engineered cartilage adjustable to cover the range of both partial-thickness and full-thickness articular cartilage defects. We developed a novel kind of composite web scaffold that could be used for tissue enginnering of articular cartilage with the thickness adjustable between 200 microm and 8 mm. The composite web showed a unique structure having web-like collagen microsponges formed in the openings of a mechanically strong knitted mesh of poly(lactic-co-glycolic acid). The knitted mesh served as a skeleton reinforcing the composite web, while the web-like collagen microsponges facilitated cell seeding, cell distribution, and tissue formation. Bovine chondrocytes cultured in the composite web showed a spatially even distribution, maintained their natural morphology, and produced cartilaginous extracellular matrices such as type II collagen and aggrecan. The thickness of the implant can be simply adjusted by laminating or rolling the web sheets. Not only did the histological structure of the engineered cartilage patches match the bovine native articular cartilage, but also their dynamic complex modulus, structural stiffness, and phase lag reached 37.8, 57.0, and 86.3% of those of native bovine articular cartilage, respectively. The composite web could be an important scaffold for tissue engineering.

Redifferentiation of dedifferentiated bovine chondrocytes when cultured in vitro in a PLGA-collagen hybrid mesh.

Bovine articular chondrocytes dedifferentiated and lost their ability to express articular cartilage-specific extracellular matrices such as type II collagen and aggrecan when cultured in a culture flask during in vitro multiplication. A poly(DL-lactic-co-glycolic acid) (PLGA)-collagen hybrid mesh was prepared and used to redifferentiate the dedifferentiated cells. The two passaged dedifferentiated chondrocytes were seeded in a PLGA-collagen hybrid mesh and cultured in vitro in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The cells adhered to the hybrid mesh, distributed evenly, and proliferated to fill the spaces in the scaffold. The gene expression of type I collagen, type II collagen, and aggrecan was analyzed after the cells were cultured in the hybrid mesh for 2-12 weeks. The expression of the gene encoding type I collagen was downregulated, whereas those of type II collagen and aggrecan were upregulated. Histological examination by hematoxylin-eosin and safranin O/fast green staining indicates that the cells regained their original round morphology. In addition, a homogeneous distribution of articular cartilage extracellular matrices was detected around the cells. These results suggest redifferentiation of the differentiated chondrocytes in the hybrid mesh. The hybrid mesh, which facilitated the redifferentiation of the dedifferentiated multiplied chondrocytes, would be an effective scaffold for the assembly of cells to regenerate three-dimensional cartilaginous tissue.