Identification and removal of unexpected proliferative off-target cells emerging after iPSC-derived pancreatic islet cell implantation.

Differentiation of pancreatic endocrine cells from human pluripotent stem cells (PSCs) has been thoroughly investigated for application in cell therapy against diabetes. In the context of induced pancreatic endocrine cell implantation, previous studies have reported graft enlargement resulting from off-target pancreatic lineage cells. However, there is currently no documented evidence of proliferative off-target cells beyond the pancreatic lineage in existing studies. Here, we show that the implantation of seven-stage induced PSC-derived pancreatic islet cells (s7-iPICs) leads to the emergence of unexpected off-target cells with proliferative capacity via in vivo maturation. These cells display characteristics of both mesenchymal stem cells (MSCs) and smooth muscle cells (SMCs), termed proliferative MSC- and SMC-like cells (PMSCs). The frequency of PMSC emergence was found to be high when 108 s7-iPICs were used. Given that clinical applications involve the use of a greater number of induced cells than 108, it is challenging to ensure the safety of clinical applications unless PMSCs are adequately addressed. Accordingly, we developed a detection system and removal methods for PMSCs. To detect PMSCs without implantation, we implemented a 4-wk-extended culture system and demonstrated that putative PMSCs could be reduced by compound treatment, particularly with the taxane docetaxel. When docetaxel-treated s7-iPICs were implanted, the PMSCs were no longer observed. This study provides useful insights into the identification and resolution of safety issues, which are particularly important in the field of cell-based medicine using PSCs.

CD151 expression marks atrial- and ventricular- differentiation from human induced pluripotent stem cells.

Current differentiation protocols for human induced pluripotent stem cells (hiPSCs) produce heterogeneous cardiomyocytes (CMs). Although chamber-specific CM selection using cell surface antigens enhances biomedical applications, a cell surface marker that accurately distinguishes between hiPSC-derived atrial CMs (ACMs) and ventricular CMs (VCMs) has not yet been identified. We have developed an approach for obtaining functional hiPSC-ACMs and -VCMs based on CD151 expression. For ACM differentiation, we found that ACMs are enriched in the CD151low population and that CD151 expression is correlated with the expression of Notch4 and its ligands. Furthermore, Notch signaling inhibition followed by selecting the CD151low population during atrial differentiation leads to the highly efficient generation of ACMs as evidenced by gene expression and electrophysiology. In contrast, for VCM differentiation, VCMs exhibiting a ventricular-related gene signature and uniform action potentials are enriched in the CD151high population. Our findings enable the production of high-quality ACMs and VCMs appropriate for hiPSC-derived chamber-specific disease models and other applications.

ERRγ enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes.

One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.

Early Chimerism After Liver Transplantation Reflects the Clinical Course of Recurrent Hepatitis C.

BACKGROUND Human leukocyte antigen (HLA) mismatch is a characteristic feature of post-orthotopic liver transplantation (OLT) hepatitis C. To investigate the importance of donor HLA-restricted immune cells in post-OLT hepatitis C recurrence, we analyzed the frequency of donor chimerism and the clinical course of post-OLT hepatitis C. MATERIAL AND METHODS We analyzed peripheral blood chimerism in 11 HCV-reinfected patients with post-HLA mismatched OLT. Patients were divided into 2 groups: the OLT chronic hepatitis C (CHC) group (n=8), exhibiting active hepatitis C recurrence; and the OLT-persistently normal ALT (PNALT) group (n=3), without active hepatitis. Chimerism was analyzed by flow cytometry using donor-specific anti-HLA antibodies in peripheral blood mononuclear cells from 1-100 days after OLT. Kidney (n=7) and lung (n=7) transplant recipients were also analyzed for comparison. As immune cells from the donor liver might contribute to post-OLT chimerism, the characteristics of perfusates from donor livers (n=10) were analyzed and defined. RESULTS Donor-derived cells were frequently observed in liver and lung transplant recipients. The frequency of donor-derived cells from the B cell subset was significantly higher in peripheral blood from OLT-CHC group than in that of the OLT-PNALT group. B cells, however, were not the predominant subset in the perfusates, indicating that inflow of donor-derived cells alone did not cause the chimerism. CONCLUSIONS Chimerism of B cells is frequent in liver transplant patients with early recurrence of hepatitis C. We propose that monitoring of early chimerism could facilitate early detection of chronic hepatitis C recurrence, although we need more cases to investigate.

Effective treatment against severe graft-versus-host disease with allele-specific anti-HLA monoclonal antibody in a humanized mouse model.

Graft-versus-host disease (GVHD), mediated by donor-derived alloreactive T cells, is a major cause of nonrelapse mortality in allogeneic hematopoietic stem cell transplantation. Its therapy is not well-defined. We established allele-specific anti-human leukocyte antigen (HLA) monoclonal antibodies (ASHmAbs) that specifically target HLA molecules, with steady death of target-expressing cells. One such ASHmAb, against HLA-A*02:01 (A2-kASHmAb), was examined in a xenogeneic GVHD mouse model. To induce fatal GVHD, non-irradiated NOD/Shi-scid/IL-2Rγ(null) mice were injected with healthy donor human peripheral blood mononuclear cells, some expressing HLA-A*02:01, some not. Administration of A2-kASHmAb promoted the survival of mice injected with HLA-A*02:01-expressing peripheral blood mononuclear cells (p < 0.0001) and, in humanized NOD/Shi-scid/IL-2Rγ(null) mice, immediately cleared HLA-A*02:01-expressing human blood cells from mouse peripheral blood. Human peripheral blood mononuclear cells were again detectable in mouse blood 2 to 4 weeks after A2-kASHmAb administration, suggesting that kASHmAb may be safely administered to GVHD patients without permanently ablating the graft. This approach, different from those in existing GVHD pharmacotherapy, may open a new door for treatment of GVHD in HLA-mismatched allogeneic hematopoietic stem cell transplantation.

Frequency of regulatory T-cell and hepatitis C viral antigen-specific immune response in recurrent hepatitis C after liver transplantation.

INTRODUCTION: Regulatory T (Treg) and type 1 regulatory T (Tr1) cells facilitate hepatitis C virus (HCV) recurrence after orthotopic liver transplantation (OLT). However, their frequencies and effects on HCV-specific immune responses have not been well investigated. METHODS: We determined Treg and Tr1 frequencies in OLT patients with hepatitis C and assessed their associations with HCV-specific T cell responses. These patients comprised the following groups: an early post-transplantation group (n=14); an OLT-chronic active hepatitis C group (n=14) with active hepatitis C (alanine aminotransferase of>upper limit of normal/positive for HCV-RNA); an OLT-persistently normal alanine aminotransferase group (n=12) without active hepatitis C (not interferon/positive for HCV-RNA); and an OLT-sustained viral response group (n=6) with sustained viral responses using interferon treatment (negative for HCV-RNA). The frequencies of HCV-specific CD4+ T cells that secreted interferon-γ were determined by enzyme-linked immunosorbent spot assay (except for the OLT early group). RESULTS: Treg and Tr1 frequencies were low during the early post-transplantation period. OLT patients with sustained viral responses had lower Treg frequencies than those with chronic hepatitis C, whereas Tr1 frequencies were significantly reduced in OLT patients with persistently normal alanine aminotransferase levels compared to those with chronic hepatitis C (p<0.05). Treg frequencies positively correlated with HCV NS3 antigen-specific interferon-γ responses, which corresponded to HCV clearance. CONCLUSIONS: Increased Treg frequencies and reduced HCV-NS3 antigen-specific responses recovered after viral eradication in post-OLT chronic hepatitis C patients. Reduced Tr1 frequencies were associated with hepatitis activity control, which may facilitate controlling chronic hepatitis C in patients after OLT.