Ohmine, Seiga

Abstract Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient-derived iPSC-generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV-iPSCs) led to the formation of invasive teratocarcinoma-like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV-iPSC progeny-transplanted hosts generated secondary and metastatic tumors. Combined transgene-free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor-free transplantation, ultimately enabling regeneration of type 1 diabetes-specific human islet structures in vivo. The incidence of tumor formation in TGF-iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor-free transplantation. Thus, transgene-free cMYC-independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo. Significance Pluripotent stem cell therapy for diabetes relies on the safety as well as the quality of derived insulin-producing cells. Data from this study highlight prominent tumorigenic risks of induced pluripotent stem cell (iPSC) products, especially when reprogrammed with integrating vectors. Two major underlying mechanisms in iPSC tumorigenicity are residual pluripotent cells and cMYC overload by vector integration. This study also demonstrated that combined transgene-free reprogramming and enzymatic dissociation allows teratoma-free transplantation of iPSC progeny in the mouse model in testing the tumorigenicity of iPSC products. Further safety assessment and improvement in iPSC specification into a mature β cell phenotype would lead to safe islet replacement therapy for diabetes.

Abstract The induced pluripotent stem cell (iPSC) technology enables derivation of patient-specific pluripotent stem cells from adult somatic cells without using an embryonic cell source. Redifferentiation of iPSCs from diabetic patients into pancreatic islets will allow patient-specific disease modeling and autologous cell replacement therapy for failing islets. To date, diabetes-specific iPSCs have been generated from patients with type 1 diabetes using integrating retroviral vectors. However, vector integration into the host genome could compromise the biosafety and differentiation propensities of derived iPSCs. Although various integration-free reprogramming systems have been described, their utility to reprogram somatic cells from patients remains largely undetermined. Here, we used nonintegrating Sendai viral vectors to reprogram cells from patients with type 1 and type 2 diabetes (T2D). Sendai vector infection led to reproducible generation of genomic modification-free iPSCs (SV-iPSCs) from patients with diabetes, including an 85-year-old individual with T2D. SV-iPSCs lost the Sendai viral genome and antigens within 8–12 passages while maintaining pluripotency. Genome-wide transcriptome analysis of SV-iPSCs revealed induction of endogenous pluripotency genes and downregulation of genes involved in the oxidative stress response and the INK4/ARF pathways, including p16INK4a, p15INK4b, and p21CIP1. SV-iPSCs and iPSCs made with integrating lentiviral vectors demonstrated remarkable similarities in global gene expression profiles. Thus, the Sendai vector system facilitates reliable reprogramming of patient cells into transgene-free iPSCs, providing a pluripotent platform for personalized diagnostic and therapeutic approaches for diabetes and diabetes-associated complications.

BACKGROUND B-type natriuretic peptide (BNP), a key cardiac hormone in cardiorenal homeostasis, is produced as a 108 amino acid prohormone, proBNP1-108, which is converted to a biologically active peptide BNP1-32 and an inactive N-terminal (NT)-proBNP1-76. The widely accepted model is that the normal heart releases a proteolytically processed BNP1-32 and NT-proBNP, whereas the diseased heart secretes high amounts of unprocessed/glycosylated proBNP1-108 or inappropriately processed BNPs. In contrast, circulating proBNP1-108 has recently been identified in healthy individuals, indicating that the normal heart also secretes unprocessed proBNP1-108. However, the mechanism of proBNP1-108 secretion from the normal heart remains elusive. Our goal was to determine the molecular mechanisms underlying proBNP1-108 intracellular trafficking and secretion from the normal heart. METHODS We expressed preproBNP in cardiomyocytes, and determined the subcellular localization and dominant intracellular and extracellular forms of BNP. RESULTS Intracellular immunoreactive BNPs were first accumulated in the Golgi apparatus, and then distributed throughout the cytoplasm as secretory vesicles. The predominant intracellular form of BNP was nonglycosylated proBNP1-108, rather than BNP1-32. Glycosylated proBNP1-108, but not nonglycosylated proBNP1-108, was detected as the major extracellular form in the culture supernatants of preproBNP-expressing cell lines and primary human cardiomyocytes. Ablation of O-glycosylation of proBNP1-108 at T71 residue, near the convertase recognition site, reduced the extracellular proBNP1-108 and increased extracellular BNP1-32. CONCLUSIONS Intracellular proBNP trafficking occurs through a conventional Golgi–endoplasmic reticulum pathway. Glycosylation of proBNP1-108 controls the stability and processing of extracellular proBNP1-108. Our data establish a new BNP secretion model in which the normal cardiac cells secrete glycosylated proBNP1-108.

ABSTRACTA novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), has been identified in patients with prostate cancer and in patients with chronic fatigue syndromes. StandardMus musculuslaboratory mice lack a functional XPR1 receptor for XMRV and are therefore not a suitable model for the virus. In contrast, Gairdner's shrew-mice (Mus pahari) do express functional XPR1. To determine whetherMus paharicould serve as a model for XMRV, primaryMus paharifibroblasts and mice were infected with cell-free XMRV. Infection of cellsin vitroresulted in XMRV Gag expression and the production of XMRV virions. After intraperitoneal injection of XMRV intoMus paharimice, XMRV proviral DNA could be detected in spleen, blood, and brain. Intravenous administration of a green fluorescent protein (GFP) vector pseudotyped with XMRV produced GFP+CD4+T cells and CD19+B cells. Mice mounted adaptive immune responses against XMRV, as evidenced by the production of neutralizing and Env- and Gag-specific antibodies. Prominent G-to-A hypermutations were also found in viral genomes isolated from the spleen, suggesting intracellular restriction of XMRV infection by APOBEC3in vivo. These data demonstrate infection ofMus pahariby XMRV, potential cell tropism of the virus, and immunological and intracellular restriction of virus infectionin vivo. These data support the use ofMus paharias a model for XMRV pathogenesis and as a platform for vaccine and drug development against this potential human pathogen.