Complete remission of tumors in mice with neoantigen-painted exosomes and anti-PD-1 therapy.

Neoantigen-based cancer vaccines are emerging as promising tumor therapies, but enhancement of immunogenicity can further improve therapeutic outcomes. Here, we demonstrate that anchoring different peptide neoantigens on subcutaneously administered serum exosomes promote lymph node homing and dendritic cell uptake, resulting in significantly enhanced antigenicity in vitro and in vivo. Exosomes anchoring of melanoma peptide neoantigens augmented the magnitude and breadth of T cell response in vitro and in vivo, to a greater extent with CD8+ T cell responses. Simultaneous decoration of different peptide neoantigens on serum exosomes induced potent tumor suppression and neoantigen-specific immune responses in mice with melanoma and colon cancer. Complete tumor eradication and sustainable immunological memory were achieved with neoantigen-painted serum exosome vaccines in combination with programmed cell death protein 1 (PD-1) antibodies in mice with colon cancer. Importantly, human serum exosomes loaded with peptide neoantigens elicited significant tumor growth retardation and immune responses in human colon cancer 3-dimensional (3D) multicellular spheroids. Our study demonstrates that serum exosomes direct in vivo localization, increase dendritic cell uptake, and enhance the immunogenicity of antigenic peptides and thus provides a general delivery tool for peptide antigen-based personalized immunotherapy.

Functional imaging and targeted drug delivery in mice and patient tumors with a cell nucleolus-localizing and tumor-targeting peptide.

Tumor-targeting peptides have profound clinical implications in early detection and delineation of microscopic lesions for surgical resection, and also delivery of therapeutics with reduced systemic toxicity. Here, we demonstrate that a peptide (RS), evolved from a previously reported hepatocellular carcinoma (HCC)-targeting peptide P47, enables improved HCC micrometastasis discrimination and delineation from noncancerous tissues in murine orthotopic mice and patient biopsies, with up to 21-fold contrast. Importantly, RS targets non-small cell lung (NSCLC) and colon cancers in mice and patient biopsies, with higher selectivity for highly proliferative tumor nodules. Moreover, RS localizes to cell nucleoli of HCC, NSCLC, breast, colon and cervical cancer cells and induces nucleolar stress when conjugated with chemotherapeutic Oxaliplatin (OXA) (RS-OXA), demonstrating both cellular and subcellular targeting. RS-delivered OXA elicits significant tumor retardation in orthotopic HCC mice with markedly reduced systemic toxicity compared to OXA alone. Injection of fluorescence-labeled RS enables dynamic visualization of tumor growth in RS-OXA-treated subcutaneous HCC mice. Our study demonstrates that RS targets a spectrum of tumors and localizes to cell nucleolus, thus enabling functional imaging and targeted delivery of OXA in HCC mice, and consequently provides a versatile tool for tumor imaging and targeted therapeutics.

Universal immunotherapeutic strategy for hepatocellular carcinoma with exosome vaccines that engage adaptive and innate immune responses.

BACKGROUND: Personalized immunotherapy utilizing cancer vaccines tailored to the tumors of individual patients holds promise for tumors with high genetic heterogeneity, potentially enabling eradication of the tumor in its entirety. METHODS: Here, we demonstrate a general strategy for biological nanovaccines that trigger tailored tumor-specific immune responses for hepatocellular carcinoma (HCC). Dendritic cell (DC)-derived exosomes (DEX) are painted with a HCC-targeting peptide (P47-P), an α-fetoprotein epitope (AFP212-A2) and a functional domain of high mobility group nucleosome-binding protein 1 (N1ND-N), an immunoadjuvant for DC recruitment and activation, via an exosomal anchor peptide to form a "trigger" DEX vaccine (DEXP&A2&N). RESULTS: DEXP&A2&N specifically promoted recruitment, accumulation and activation of DCs in mice with orthotopic HCC tumor, resulting in enhanced cross-presentation of tumor neoantigens and de novo T cell response. DEXP&A2&N elicited significant tumor retardation and tumor-specific immune responses in HCC mice with large tumor burdens. Importantly, tumor eradication was achieved in orthotopic HCC mice when antigenic AFP peptide was replaced with the full-length AFP (A) to form DEXP&A&N. Supplementation of Fms-related tyrosine kinase 3 ligand greatly augmented the antitumor immunity of DEXP&A&N by increasing immunological memory against tumor re-challenge in orthotopic HCC mice. Depletion of T cells, cross-presenting DCs and other innate immune cells abrogated the functionality of DEXP&A&N. CONCLUSIONS: These findings demonstrate the capacity of universal DEX vaccines to induce tumor-specific immune responses by triggering an immune response tailored to the tumors of each individual, thus presenting a generalizable approach for personalized immunotherapy of HCC, by extension of other tumors, without the need to identify tumor antigens.

Exosome-mediated improvement in membrane integrity and muscle function in dystrophic mice.

Duchenne muscular dystrophy (DMD) is a devastating genetic disorder that leads to compromised cellular membranes, caused by the absence of membrane-bound dystrophin protein. Muscle membrane leakage results in disrupted intracellular homeostasis, protein degradation, and muscle wasting. Improving muscle membrane integrity may delay disease progression and extend the lifespan of DMD patients. Here, we demonstrate that exosomes, membranous extracellular vesicles, can elicit functional improvements in dystrophic mice by improving muscle membrane integrity. Systemic administration of exosomes from different sources induced phenotypic rescue and mitigated pathological progression in dystrophic mice without detectable toxicity. Improved membrane integrity conferred by exosomes inhibited intracellular calcium influx and calcium-dependent activation of calpain proteases, preventing the degradation of the destabilized dystrophin-associated protein complex. We show that exosomes, particularly myotube-derived exosomes, induced functional improvements and alleviated muscle deterioration by stabilizing damaged muscle membrane in dystrophic mice. Our findings suggest that exosomes may have therapeutic implications for DMD and other diseases with compromised membranes.

Genome-wide CRISPR knockout screens identify ADAMTSL3 and PTEN genes as suppressors of HCC proliferation and metastasis, respectively.

PURPOSE: It is important for hepatocellular carcinoma (HCC) treatment that the targets related to its progression are identified. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9)-based genetic screening is a powerful tool for identifying genes with loss-of-function mutations that are critical for tumour growth and metastasis. METHODS: We transduced the human SMMC7721 HCC cell line expressing Cas9 with a human genome-scale CRISPR-Cas9 knockout (GeCKO) lentiviral library A (hGeCKOa) of 65,383 single-guide RNAs (sgRNAs) targeting 19,050 human genes; we then subcutaneously transplanted the transduced cells into nude mice. RESULTS: The transduced cells were found to proliferate and metastasize faster than the untransduced cells. Through next-generation sequencing, the genes potentially related to HCC proliferation and metastasis were identified. The sgRNAs targeting the ADAMTSL3 and PTEN genes appeared twice on the list of genes related to HCC proliferation and metastasis, respectively. Analysis based on the data mining of Oncomine revealed that the ADAMTSL3 and PTEN genes were expressed at lower levels in HCC cells than they were in normal liver cells, indicating their tumour-suppressive roles. Downregulation of ADAMTSL3 and PTEN displayed poor overall survival (OS) and predicted poor relapse-free survival (RFS), further supporting their tumour-suppressive roles. Moreover, knocking out either the ADAMTSL3 or PTEN genes promoted either the proliferation or metastasis of HCC cells, respectively. CONCLUSIONS: Using both in vitro and in vivo approaches, we described the profound role of the ADAMTSL3 and PTEN genes. This study indicates novel candidate targets for use in HCC treatment and therapy.

Alarmin-painted exosomes elicit persistent antitumor immunity in large established tumors in mice.

Treating large established tumors is challenging for dendritic cell (DC)-based immunotherapy. DC activation with tumor cell-derived exosomes (TEXs) carrying multiple tumor-associated antigen can enhance tumor recognition. Adding a potent adjuvant, high mobility group nucleosome-binding protein 1 (HMGN1), boosts DCs' ability to activate T cells and improves vaccine efficiency. Here, we demonstrate that TEXs painted with the functional domain of HMGN1 (TEX-N1ND) via an exosomal anchor peptide potentiates DC immunogenicity. TEX-N1ND pulsed DCs (DCTEX-N1ND) elicit long-lasting antitumor immunity and tumor suppression in different syngeneic mouse models with large tumor burdens, most notably large, poorly immunogenic orthotopic hepatocellular carcinoma (HCC). DCTEX-N1ND show increased homing to lymphoid tissues and contribute to augmented memory T cells. Importantly, N1ND-painted serum exosomes from cancer patients also promote DC activation. Our study demonstrates the potency of TEX-N1ND to strengthen DC immunogenicity and to suppress large established tumors, and thus provides an avenue to improve DC-based immunotherapy.

Alarmin augments the antitumor immunity of lentiviral vaccine in ectopic, orthotopic and autochthonous hepatocellular carcinoma mice.

It is a daunting therapeutic challenge to completely eradicate hepatocellular carcinoma (HCC) from patients. Alpha-fetoprotein (AFP) -based vaccines appear promising, however the efficacy needs to be improved. Methods: Here, we explore if fusing high-mobility group nucleosome binding protein 1 (HMGN1), a potent immunoadjuvant, to AFP (lenti-HA) can augment the antitumor immunity of AFP-expressing lentiviral vector (lenti-AFP), a vehicle extensively employed for genetic immunization with high transduction efficacy and good safety profiles. The antitumor immunity of Lenti-HA was systemically assessed in ectopic, orthotopic and autochthonous HCC models. Results: Lenti-HA elicited strong anti-HCC effects in mice and amplified the antitumor immunity of lenti-AFP by reducing effective dose 6-fold. Importantly, lenti-HA induced a robust antitumor immune response with prolonged survival rate and improved the immune and tumor microenvironment in mice with carcinogen-induced autochthonous HCC. Lenti-HA localized primarily to lymphoid organs with no preference for specific immune cell types. Activated dendritic cells (DCs), particularly CD103+CD11b- DCs, were also actively recruited to lymph nodes in lenti-HA-treated HCC mice. Moreover, lenti-HA-transduced human DCs elicited stronger immune response than lenti-AFP against HCC cells in vitro. Conclusion: Our study demonstrates that HMGN1 augments the antitumor immunity of AFP-expressing lentiviral vaccines in HCC mice and human cells in vitro and thus provides a new therapeutic strategy for HCC.

Correction: Intrahepatic Tissue Implantation Represents a Favorable Approach for Establishing Orthotopic Transplantation Hepatocellular Carcinoma Mouse Models.

[This corrects the article DOI: 10.1371/journal.pone.0148263.].

Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnostics and therapy.

Exosomes are circulating nanovesicular carriers of macromolecules, increasingly used for diagnostics and therapeutics. The ability to load and target patient-derived exosomes without altering exosomal surfaces is key to unlocking their therapeutic potential. We demonstrate that a peptide (CP05) identified by phage display enables targeting, cargo loading, and capture of exosomes from diverse origins, including patient-derived exosomes, through binding to CD63-an exosomal surface protein. Systemic administration of exosomes loaded with CP05-modified, dystrophin splice-correcting phosphorodiamidate morpholino oligomer (EXOPMO) increased dystrophin protein 18-fold in quadriceps of dystrophin-deficient mdx mice compared to CP05-PMO. Loading CP05-muscle-targeting peptide on EXOPMO further increased dystrophin expression in muscle with functional improvement without any detectable toxicity. Our study demonstrates that an exosomal anchor peptide enables direct, effective functionalization and capture of exosomes, thus providing a tool for exosome engineering, probing gene function in vivo, and targeted therapeutic drug delivery.

Fluorescent peptide highlights micronodules in murine hepatocellular carcinoma models and humans in vitro.

Early detection and clear delineation of microscopic lesions during surgery are critical to the prognosis and survival of patients with hepatocellular carcinoma (HCC), a devastating malignancy without effective treatments except for resection. Tools to specifically identify and differentiate micronodules from normal tissue in HCC patients can have a positive impact on survival. Here, we discovered a peptide that preferentially binds to HCC cells through phage display. Significant accumulation of the fluorescence-labeled peptide in tumor from ectopic and orthotopic HCC mice was observed within 2 hours of systemic injection. Contrast between tumor and surrounding liver is up to 6.5-fold, and useful contrast lasts for 30 hours. Micronodules (0.03 cm in diameter) in liver and lung can clearly be distinguished from normal tissue with this fluorescence-labeled peptide in orthotopic HCC mice and HCC patients. Compared to indocyanine green, a Food and Drug Administration-approved imaging contrast agent, an up to 8.7-fold higher differentiation ratio of tumor to fibrosis is achieved with this fluorescence-labeled peptide. Importantly, this peptide enables up to 10-fold differentiation between HCC and peritumoral tissue in human tissues and the complete removal of tumor in HCC mice with surgical navigation. No abnormalities in behavior or activity are observed after systemic treatment, indicating the absence of overt toxicity. The peptide is metabolized with a half-life of approximately 4 hours in serum. CONCLUSION: Our findings demonstrate that micronodules can be specifically differentiated with high sensitivity from surrounding tissue with this molecule, opening clinical possibilities for early detection and precise surgery of HCC. (Hepatology 2018).

3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145) ameliorated dexamethasone induced hepatic gluconeogenesis through activation of Akt/FoxO1 pathway.

3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145) is identified as a selective SphK2 inhibitor. It was previously reported as an anti-tumor agent, in this study we demonstrated that K145 was able to regulate hepatic gluconeogenesis and improve glucose intolerance in mice. C57BL/6 mice treated with dexamethasone injection were used as experimental animals, which exhibited impaired glucose tolerance and increased gluconeogenetic enzymes. After K145 treatment, we found that the impairment of glucose tolerance and gluconeogenetic genes mRNA expression were improved. Besides, both in vivo and in votro studies suggested that K145 stimulated insulin dependent Akt phosphorylation and subsequently activates FoxO1 phosphorylation therefore inhibited gluconeogenetic genes expression including PEPCK and G6pase. Our study figures out a potential extent increase the value of developing K145 as therapeutic candidate for diabetes.

Dendritic cell-derived exosomes elicit tumor regression in autochthonous hepatocellular carcinoma mouse models.

BACKGROUND & AIMS: Dendritic cell (DC)-derived exosomes (DEXs) form a new class of vaccines for cancer immunotherapy. However, their potency in hepatocellular carcinoma (HCC), a life-threatening malignancy with limited treatment options in the clinic that responds poorly to immunotherapy, remains to be investigated. METHODS: Exosomes derived from α-fetoprotein (AFP)-expressing DCs (DEXAFP) were investigated in three different HCC mouse models systemically. Tumor growth and microenvironment were monitored. RESULTS: DEXAFP elicited strong antigen-specific immune responses and resulted in significant tumor growth retardation and prolonged survival rates in mice with ectopic, orthotopic and carcinogen-induced HCC tumors that displayed antigenic and pathological heterogeneity. The tumor microenvironment was improved in DEXAFP-treated HCC mice, demonstrated by significantly more γ-interferon (IFN-γ)-expressing CD8+ T lymphocytes, elevated levels of IFN-γ and interleukin-2, and fewer CD25+Foxp3+ regulatory T (Treg) cells and decreased levels of interleukin-10 and transforming growth factor-ß in tumor sites. Lack of efficacy in athymic nude mice and CD8+ T cell-depleted mice showed that T cells contribute to DEXAFP-mediated antitumor function. Dynamic examination of the antitumor efficacy and the immune microenvironment in DEXAFP-treated orthotopic HCC mice at different time-points revealed a positive correlation between tumor suppression and immune microenvironment. CONCLUSIONS: Our findings provide evidence that AFP-enriched DEXs can trigger potent antigen-specific antitumor immune responses and reshape the tumor microenvironment in HCC mice and thus provide a cell-free vaccine option for HCC immunotherapy. Lay summary: Dendritic cell (DC)-derived exosomes (DEXs) form a new class of vaccines for cancer immunotherapy. However, their potency in hepatocellular carcinoma (HCC) remains unknown. Here, we investigated exosomes from HCC antigen-expressing DCs in three different HCC mouse models and proved their feasibility and capability of treating HCC, and thus provide a cell-free vaccine for HCC immunotherapy.

Metformin sensitizes sorafenib to inhibit postoperative recurrence and metastasis of hepatocellular carcinoma in orthotopic mouse models.

BACKGROUND: Sorafenib is recognized as a standard treatment for advanced hepatocellular carcinoma (HCC). However, many patients have to adopt dose reduction or terminate the use of sorafenib because of side effects. In addition, a large number of patients are resistant to sorafenib. Thus, it is essential to investigate the underlying mechanisms of the resistance to sorafenib and seek potential strategy to enhance its efficacy. METHODS: The protein expression of hypoxia-inducible factors (HIF)-2α, 30-kDa HIV Tat-interacting protein (TIP30), E-cadherin, N-cadherin, and pAMPK was detected by Western blot. Cell viability assays were performed to study the influence of metformin and sorafenib on cell proliferation. Annexin V-FITC apoptosis assays were used to detect the influence of metformin and sorafenib on cell apoptosis. The relationship between HIF-2α and TIP30 was studied using gene silencing approach and chromatin immunoprecipitation assay. To investigate the effect of metformin and sorafenib on postoperative recurrence and lung metastasis of HCC in tumor-bearing mice, the mice were orally treated either with metformin or sorafenib once a day for continuous 37 days after the operation to remove the lobe where the tumor was implanted. CD31, Ki67, and TUNEL were examined by immunohistochemistry. RESULTS: Our study demonstrated that metformin synergized with sorafenib reduced HIF-2α expression as examined by Western blot. Gene silencing approach indicated TIP30 was upregulated after knocking-down of HIF-2α and chromatin immunoprecipitation assay revealed that HIF-2α could bind to TIP30 promoter under hypoxic condition. Cell Counting Kit-8 (CCK8) cell viability assay and Annexin V-FITC apoptosis assay showed that metformin in combination with sorafenib suppressed cell proliferation and promoted cell apoptosis. Besides, combined therapy suppressed epithelial-mesenchymal transition (EMT) process both in vitro and in vivo. Moreover, metformin in combination with sorafenib significantly minimized postoperative recurrence and lung metastasis of HCC in orthotopic mouse model. Combined therapy inhibited CD31 and Ki67 expression but promoted TUNEL expression. CONCLUSIONS: Metformin may potentially enhance the effect of sorafenib to inhibit HCC recurrence and metastasis after liver resection by regulating the expression of HIF-2α and TIP30.

Tumor-derived exosomes elicit tumor suppression in murine hepatocellular carcinoma models and humans in vitro.

UNLABELLED: Hepatocellular carcinoma (HCC) remains a global challenge due to high morbidity and mortality rates and poor response to treatment. Immunotherapy, based on introduction of dendritic cells (DCs) activated by tumor cell lysates as antigens ex vivo, shows limited response rates in HCC patients. Here, we demonstrate that tumor cell-derived exosomes (TEXs), displaying an array of HCC antigens, can elicit a stronger immune response than cell lysates in vitro and in vivo. Significant tumor growth inhibition was achieved in ectopic and orthotopic HCC mice treated with TEX-pulsed DCs. Importantly, the tumor immune microenvironment was significantly improved in orthotopic HCC mice treated by TEX-pulsed DCs, demonstrated by increased numbers of T lymphocytes, elevated levels of interferon-γ, and decreased levels of interleukin-10 and tumor growth factor-ß in tumor sites. As expected, T cells played an essential role in the TEX-pulsed DC-mediated immune response. Notably, exosomes from HCC cells not only promoted HCC-specific cytolysis but also provided cross-protective effects against pancreatic cancer cells. Moreover, HCC-specific cytolysis, elicited by DCs pulsed with human HepG2 cell-derived exosomes, was observed across different human HCC cells irrespective of human leukocyte antigen types. CONCLUSION: HCC TEXs can potently carry HCC antigens, trigger a strong DC-mediated immune response, and improve the HCC tumor microenvironment. (Hepatology 2016;64:456-472).

Intrahepatic Tissue Implantation Represents a Favorable Approach for Establishing Orthotopic Transplantation Hepatocellular Carcinoma Mouse Models.

Mouse models are commonly used for studying hepatocellular carcinoma (HCC) biology and exploring new therapeutic interventions. Currently three main modalities of HCC mouse models have been extensively employed in pre-clinical studies including chemically induced, transgenic and transplantation models. Among them, transplantation models are preferred for evaluating in vivo drug efficacy in pre-clinical settings given the short latency, uniformity in size and close resemblance to tumors in patients. However methods used for establishing orthotopic HCC transplantation mouse models are diverse and fragmentized without a comprehensive comparison. Here, we systemically evaluate four different approaches commonly used to establish HCC mice in preclinical studies, including intravenous, intrasplenic, intrahepatic inoculation of tumor cells and intrahepatic tissue implantation. Four parameters--the latency period, take rates, pathological features and metastatic rates--were evaluated side-by-side. 100% take rates were achieved in liver with intrahepatic, intrasplenic inoculation of tumor cells and intrahepatic tissue implantation. In contrast, no tumor in liver was observed with intravenous injection of tumor cells. Intrahepatic tissue implantation resulted in the shortest latency with 0.5 cm (longitudinal diameter) tumors found in liver two weeks after implantation, compared to 0.1cm for intrahepatic inoculation of tumor cells. Approximately 0.1cm tumors were only visible at 4 weeks after intrasplenic inoculation. Uniform, focal and solitary tumors were formed with intrahepatic tissue implantation whereas multinodular, dispersed and non-uniform tumors produced with intrahepatic and intrasplenic inoculation of tumor cells. Notably, metastasis became visible in liver, peritoneum and mesenterium at 3 weeks post-implantation, and lung metastasis was visible after 7 weeks. T cell infiltration was evident in tumors, resembling the situation in HCC patients. Our study demonstrated that orthotopic HCC mouse models established via intrahepatic tissue implantation authentically reflect clinical manifestations in HCC patients pathologically and immunologically, suggesting intrahepatic tissue implantation is a preferable approach for establishing orthotopic HCC mouse models.

Rif1 maintains telomere length homeostasis of ESCs by mediating heterochromatin silencing.

Telomere length homeostasis is essential for genomic stability and unlimited self-renewal of embryonic stem cells (ESCs). We show that telomere-associated protein Rif1 is required to maintain telomere length homeostasis by negatively regulating Zscan4 expression, a critical factor for telomere elongation by recombination. Depletion of Rif1 results in terminal hyperrecombination, telomere length heterogeneity, and chromosomal fusions. Reduction of Zscan4 by shRNA significantly rescues telomere recombination defects of Rif1-depleted ESCs and associated embryonic lethality. Further, Rif1 negatively modulates Zscan4 expression by maintaining H3K9me3 levels at subtelomeric regions. Mechanistically, Rif1 interacts and stabilizes H3K9 methylation complex. Thus, Rif1 regulates telomere length homeostasis of ESCs by mediating heterochromatic silencing.

Infection of common marmosets with hepatitis C virus/GB virus-B chimeras.

UNLABELLED: The development of vaccination and novel therapy for hepatitis C virus (HCV) has been hampered by the lack of suitable small-animal models. GB virus B (GBV-B), closely related to HCV, causes viral hepatitis in common marmosets (Callithrix jacchue jacchus) and might represent an attractive surrogate model for HCV infection. However, differences exist between GBV-B and HCV in spite of a short genetic distance between the two viruses. Here we report common marmosets infected with two HCV/GBV-B chimeras containing HCV structural genes coding for either whole core and envelope proteins (CE1E2p7) or full envelope proteins (E1E2p7) substituted for the counterpart elements of GBV-B. Naïve animals intrahepatically injected with chimeric RNA transcripts or intravenously injected with sera from primary infected animals produced high levels of circulating infectious chimeric viruses and they developed chronic infection. Tacrolimus-treated marmosets inoculated with a CE1E2p7 chimera had higher viral loads and long-term persistent infection. A moderate elevation of serum aspartate aminotransferase (AST) levels was observed in parallel with viral replication. Chimeras recovered from liver samples revealed 1/958 adaptive viral mutations. Histopathological changes typical of viral hepatitis were observed in liver tissues from all types of HCV chimeras-infected marmosets. HCV core and E2 proteins were detected in liver tissues from infected animals by immunohistochemical staining. Fluctuations of chimeric virus replication in marmosets with spontaneous and sporadic viral clearance might be related to specific antibody and T-cell response to HCV proteins in vivo. Replication of CE1E2p7 chimera was observed in primary hepatocyte cultures by immunofluorescent staining in vitro. CONCLUSION: Infectious HCV chimeras causing chronic hepatitis in marmosets might constitute a small primate model suitable for evaluation of virus-cell interaction, vaccination, and antiviral therapy against HCV infection.

Influences of lamin A levels on induction of pluripotent stem cells.

Lamin A is an inner nuclear membrane protein that maintains nuclear structure integrity, is involved in transcription, DNA damage response and genomic stability, and also links to cell differentiation, senescence, premature aging and associated diseases. Induced pluripotent stem (iPS) cells have been successfully generated from various types of cells and used to model human diseases. It remains unclear whether levels of lamin A influence reprogramming of somatic cells to pluripotent states during iPS induction. Consistently, lamin A is expressed more in differentiated than in relatively undifferentiated somatic cells, and increases in expression levels with age. Somatic cells with various expression levels of lamin A differ in their dynamics and efficiency during iPS cell induction. Cells with higher levels of lamin A show slower reprogramming and decreased efficiency to iPS cells. Furthermore, depletion of lamin A by transient shRNA accelerates iPS cell induction from fibroblasts. Reduced levels of lamin A are associated with increased expression of pluripotent genes Oct4 and Nanog, and telomerase genes Tert and Terc. On the contrary, overexpression of lamin A retards somatic cell reprogramming to iPS-like colony formation. Our data suggest that levels of lamin A influence reprogramming of somatic cells to pluripotent stem cells and that artificial silencing of lamin A facilitates iPS cell induction. These findings may have implications in enhancing rejuvenation of senescent or older cells by iPS technology and manipulating lamin A levels.

Molecular insights into the heterogeneity of telomere reprogramming in induced pluripotent stem cells.

Rejuvenation of telomeres with various lengths has been found in induced pluripotent stem cells (iPSCs). Mechanisms of telomere length regulation during induction and proliferation of iPSCs remain elusive. We show that telomere dynamics are variable in mouse iPSCs during reprogramming and passage, and suggest that these differences likely result from multiple potential factors, including the telomerase machinery, telomerase-independent mechanisms and clonal influences including reexpression of exogenous reprogramming factors. Using a genetic model of telomerase-deficient (Terc(-/-) and Terc(+/-)) cells for derivation and passages of iPSCs, we found that telomerase plays a critical role in reprogramming and self-renewal of iPSCs. Further, telomerase maintenance of telomeres is necessary for induction of true pluripotency while the alternative pathway of elongation and maintenance by recombination is also required, but not sufficient. Together, several aspects of telomere biology may account for the variable telomere dynamics in iPSCs. Notably, the mechanisms employed to maintain telomeres during iPSC reprogramming are very similar to those of embryonic stem cells. These findings may also relate to the cloning field where these mechanisms could be responsible for telomere heterogeneity after nuclear reprogramming by somatic cell nuclear transfer.

Quantitative proteomics analysis of parthenogenetically induced pluripotent stem cells.

Parthenogenetic embryonic stem (pES) cells isolated from parthenogenetic activation of oocytes and embryos, also called parthenogenetically induced pluripotent stem cells, exhibit pluripotency evidenced by both in vitro and in vivo differentiation potential. Differential proteomic analysis was performed using differential in-gel electrophoresis and isotope-coded affinity tag-based quantitative proteomics to investigate the molecular mechanisms underlying the developmental pluripotency of pES cells and to compare the protein expression of pES cells generated from either the in vivo-matured ovulated (IVO) oocytes or from the in vitro-matured (IVM) oocytes with that of fertilized embryonic stem (fES) cells derived from fertilized embryos. A total of 76 proteins were upregulated and 16 proteins were downregulated in the IVM pES cells, whereas 91 proteins were upregulated and 9 were downregulated in the IVO pES cells based on a minimal 1.5-fold change as the cutoff value. No distinct pathways were found in the differentially expressed proteins except for those involved in metabolism and physiological processes. Notably, no differences were found in the protein expression of imprinted genes between the pES and fES cells, suggesting that genomic imprinting can be corrected in the pES cells at least at the early passages. The germline competent IVM pES cells may be applicable for germ cell renewal in aging ovaries if oocytes are retrieved at a younger age.