Nuclear reprogramming of somatic tissue enables derivation of induced pluripotent stem (iPS) cells from an autologous, non-embryonic origin. The purpose of the current study was to establish efficient protocols for lineage-specification of human iPS cells into functional glucose-responsive, insulin-producing progeny. We generated human iPS cells, which were then guided with recombinant growth factors that mimic the essential signaling for pancreatic development. Reprogrammed with four stemness factors, human fibroblasts were here converted into authentic iPS cells. Under feeder-free conditions, fate-specification was initiated with activin A and Wnt3a that triggered engagement into definitive endoderm, followed by priming with FGF10 and KAAD-cyclopamine. Addition of retinoic acid, boosted by the pancreatic endoderm inducer indolactam V (ILV), yielded pancreatic progenitors expressing PDX1, NGN3 and NEUROD1 markers. Further guidance, under IGF-1, HGF and DAPT, was enhanced by glucagon like peptide-1 (GLP-1) to generate islet-like cells that expressed pancreas-specific markers including insulin and glucagon. Derived progeny demonstrated sustained expression of PDX1, and functional responsiveness to glucose challenge secreting up to 230 pM of C-peptide. A pancreatogenic cocktail enriched with ILV/GLP-1 offers a proficient means to specify human iPS cells into glucose-responsive hormone-producing progeny, refining the development of a personalized platform for islet-like cell generation.

Nuclear reprogramming of adult somatic tissue enables embryo-independent generation of autologous, patient-specific induced pluripotent stem (iPS) cells. Exploiting this emergent regenerative platform for individualized medicine applications requires the establishment of bioequivalence criteria across derived pluripotent lines and lineage-specified derivatives. Here, from individual patients with type 1 diabetes (T1D) multiple human iPS clones were produced and prospectively screened using a battery of developmental markers to assess respective differentiation propensity and proficiency in yielding functional insulin (INS)-producing progeny. Global gene expression profiles, pluripotency expression patterns, and the capacity to differentiate into SOX17- and FOXA2-positive definitive endoderm (DE)-like cells were comparable among individual iPS clones. However, notable intrapatient variation was evident upon further guided differentiation into HNF4α- and HNF1β-expressing primitive gut tube, and INS- and glucagon (GCG)-expressing islet-like cells. Differential dynamics of pluripotency-associated genes and pancreatic lineage-specifying genes underlined clonal variance. Successful generation of glucose-responsive INS-producing cells required silencing of stemness programs as well as the induction of stage-specific pancreatic transcription factors. Thus, comprehensive fingerprinting of individual clones is mandatory to secure homogenous pools amenable for diagnostic and therapeutic applications of iPS cells from patients with T1D.

Nuclear reprogramming enables patient-specific derivation of induced pluripotent stem (iPS) cells from adult tissue. Yet, iPS generation from patients with type 2 diabetes (T2D) has not been demonstrated. Here, we report reproducible iPS derivation of epidermal keratinocytes (HK) from elderly T2D patients. Transduced with human OCT4, SOX2, KLF4 and c-MYC stemness factors under serum-free and feeder-free conditions, reprogrammed cells underwent dedifferentiation with mitochondrial restructuring, induction of endogenous pluripotency genes - including NANOG, LIN28, and TERT, and down-regulation of cytoskeletal, MHC class I- and apoptosis-related genes. Notably, derived iPS clones acquired a rejuvenated state, characterized by elongated telomeres and suppressed senescence-related p15INK4b/p16INK4a gene expression and oxidative stress signaling. Stepwise guidance with lineage-specifying factors, including Indolactam V and GLP-1, redifferentiated HK-derived iPS clones into insulin-producing islet-like progeny. Thus, in elderly T2D patients, reprogramming of keratinocytes ensures a senescence-privileged status yielding iPS cells proficient for regenerative applications.

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus originally identified in human prostate cancers (33). Small numbers of XMRV-infected cells have been observed in prostatic stromal cells but not in prostate carcinoma (33). Another study identified XMRV proviral DNA in 6 and 23% of prostate tumors when analyzed by real-time PCR and immunostaining, respectively (27). While initial studies associated XMRV almost exclusively in men who were homozygous for a variant of RNase L (R462Q), which is known to have reduced antiviral activity (33), more recent work failed to link XMRV infection and RNase L mutation (4). XMRV has also been reported in patients with chronic fatigue syndrome (CFS) (17). A total of 67% of CFS patients were positive for XMRV proviral DNA, whereas only 3.7% of healthy subjects were positive for XMRV. Subsequent testing by several other groups found no evidence of infection with XMRV in CFS patients or in healthy controls (30). In Europe, no XMRV was detected in 139 prostate cancer patients in an Irish cohort (4), while no or very few XMRV-specific DNA, RNA, or antibodies were detected in Germany or the United Kingdom cohort of CFS (7, 10, 34).These conflicting data make it unclear to what degree XMRV infects humans and whether it plays a role in human diseases. If an etiological link is confirmed, detection and prevention of XMRV would provide novel intervention strategies for early diagnosis and treatment of both diseases. Moreover, since XMRV or XMRV-specific antibodies were detected in apparently healthy subjects, it would be critical to monitor XMRV contamination in clinical products for transfusion and transplantation.For a better understanding of XMRV transmission, tissue tropism, and pathogenicity, studies of XMRV infection in animal models are crucial. Laboratory mice have provided important small animal model systems for many human diseases, due to their availability, size, low cost, ease of handling, and fast reproduction rate, and extensive studies have been carried out in mice to study the pathogenesis of closely related murine leukemia viruses (MLVs) (5,11,20,23,32). However, studies of XMRV pathogenesis in a mouse model have been hampered by the lack of functional receptor for XMRV in standard laboratory mice derived from Mus musculus species.XMRV is closely related to xenotropic MLVs (X-MLVs) (33). The X-MLVs and polytropic MLVs (P-MLV) use Xpr1 as a receptor for cell entry (1, 31, 37), and so does XMRV (6, 13, 36). Xpr1 has four known variant receptor alleles in mice, Xpr1 n , Xpr1 sxv , Xpr1 c , and Xpr1 p , and each has a different susceptibility for P-MLV and/or X-MLV (35). P-MLV uses Xpr1 n as receptor and most cells from Mus musculus laboratory mice express this receptor (35). Wild mice of the Eurasian genus Mus, such as Mus dunni, express the Xpr1 sxv allele and are susceptible to both P-MLV and X-MLV, whereas the Asian mouse species Mus castaneus expresses Xpr1 c and is susceptible only to X-MLV (19). Mus pahari is another Asian wild mouse species. T...

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. Re-differentiation 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 employed non-integrating 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 to 12 passages, while maintaining the pluripotency. Genome-wide transcriptome analysis of SV-iPSCs revealed induction of endogenous pluripotency genes and down-regulation of genes involved in the oxidative stress response and the INK4/ARF pathways, including p16INK4a, p15INK4b and p21CIP1. Although SV-iPSC-specific gene expression signatures were identified including ATP5C1, SERPINE1, RGS5 and DUSP6 genes, 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 pro-hormone proBNP1-108. proBNP1-108 is converted to a biologically active peptide BNP1-32 and an inactive NT-proBNP1-76. The widely accepted model is that the normal heart releases a proteolytically processed BNP1-32 and NT-proBNP, while 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 normal subjects, indicating that the normal heart also secretes unprocessed proBNP1-108. However, the mechanism of proBNP1-108 secretion from normal heart remains elusive. Our goal is to determine the molecular mechanisms underlying proBNP1-108 intracellular trafficking and secretion from normal heart. Methods We expressed pre-proBNP in cardiomyocytes, and determined the subcellular localization, dominant intracellular and extracellular forms of BNP. Results Intracellular immunoreactive BNPs accumulated in the Golgi apparatus, which were distributed throughout the cytoplasm as secretory vesicles. The predominant intracellular form of BNP was non-glycosylated proBNP1-108, rather than BNP1-32. Glycosylated proBNP1-108, but not non-glycosylated proBNP1-108, was detected as the major extracellular form in the culture supernatants of pre-proBNP-expressing cell lines or 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-ER pathway. Glycosylation of proBNP1-108 controls the stability and processing of extracellular proBNP1-108. Our data establish a new B-type natriuretic peptide secretion model where the normal cardiac cells secrete glycosylated proBNP1-108.

BackgroundXenotropic murine leukemia virus (MLV)-related virus (XMRV) was initially identified in prostate cancer (PCa) tissue, particularly in the prostatic stromal fibroblasts, of patients homozygous for the RNASEL R462Q mutation. A subsequent study reported XMRV antigens in malignant prostatic epithelium and association of XMRV infection with PCa, especially higher-grade tumors, independently of the RNASEL polymorphism. Further studies showed high prevalence of XMRV or related MLV sequences in chronic fatigue syndrome patients (CFS), while others found no, or low, prevalence of XMRV in a variety of diseases including PCa or CFS. Thus, the etiological link between XMRV and human disease remains elusive. To address the association between XMRV infection and PCa, we have tested prostate tissues and human sera for the presence of viral DNA, viral antigens and anti-XMRV antibodies.ResultsReal-time PCR analysis of 110 PCa (Gleason scores >4) and 40 benign and normal prostate tissues identified six positive samples (5 PCa and 1 non-PCa). No statistical link was observed between the presence of proviral DNA and PCa, PCa grades, and the RNASEL R462Q mutation. The amplified viral sequences were distantly related to XMRV, but nearly identical to endogenous MLV sequences in mice. The PCR positive samples were also positive for mouse mitochondrial DNA by nested PCR, suggesting contamination of the samples with mouse DNA. Immuno-histochemistry (IHC) with an anti-XMRV antibody, but not an anti-MLV antibody that recognizes XMRV, sporadically identified antigen-positive cells in prostatic epithelium, irrespectively of the status of viral DNA detection. No serum (159 PCa and 201 age-matched controls) showed strong neutralization of XMRV infection at 1:10 dilution.ConclusionThe lack of XMRV sequences or strong anti-XMRV neutralizing antibodies indicates no or very low prevalence of XMRV in our cohorts. We conclude that real-time PCR- and IHC-positive samples were due to laboratory contamination and non-specific immune reactions, respectively.

IntroductionEnd-stage renal disease (ESRD) is a major public health problem. Although kidney transplantation is a viable therapeutic option, this therapy is associated with significant limitations, including a shortage of donor organs. Induced pluripotent stem (iPS) cell technology, which allows derivation of patient-specific pluripotent stem cells, could provide a possible alternative modality for kidney replacement therapy for patients with ESRD.MethodsThe feasibility of iPS cell generation from patients with a history of ESRD was investigated using lentiviral vectors expressing pluripotency-associated factors.ResultsIn the present article we report, for the first time, generation of iPS cells from kidney transplant recipients with a history of autosomal-dominant polycystic kidney disease (ADPKD), systemic lupus erythematosus, or Wilms tumor and ESRD. Lentiviral transduction of OCT4, SOX2, KLF4 and c-MYC, under feeder-free conditions, resulted in reprogramming of skin-derived keratinocytes. Keratinocyte-derived iPS cells exhibited properties of human embryonic stem cells, including morphology, growth properties, expression of pluripotency genes and surface markers, spontaneous differentiation and teratoma formation. All iPS cell clones from the ADPKD patient retained the conserved W3842X mutation in exon 41 of the PKD1 gene.ConclusionsOur results demonstrate successful iPS cell generation from patients with a history of ESRD, PKD1 gene mutation, or chronic immunosuppression. iPS cells from autosomal kidney diseases, such as ADPKD, would provide unique opportunities to study patient-specific disease pathogenesis in vitro.