Sustained knockdown of a disease-causing gene in patient-specific induced pluripotent stem cells using lentiviral vector-based gene therapy.

Patient-specific induced pluripotent stem cells (iPSCs) hold great promise for studies on disease-related developmental processes and may serve as an autologous cell source for future treatment of many hereditary diseases. New genetic engineering tools such as zinc finger nucleases and transcription activator-like effector nuclease allow targeted correction of monogenetic disorders but are very cumbersome to establish. Aiming at studies on the knockdown of a disease-causing gene, lentiviral vector-mediated expression of short hairpin RNAs (shRNAs) is a valuable option, but it is limited by silencing of the knockdown construct upon epigenetic remodeling during differentiation. Here, we propose an approach for the expression of a therapeutic shRNA in disease-specific iPSCs using third-generation lentiviral vectors. Targeting severe α-1-antitrypsin (A1AT) deficiency, we overexpressed a human microRNA 30 (miR30)-styled shRNA directed against the PiZ variant of A1AT, which is known to cause chronic liver damage in affected patients. This knockdown cassette is traceable from clonal iPSC lines to differentiated hepatic progeny via an enhanced green fluorescence protein reporter expressed from the same RNA-polymerase II promoter. Importantly, the cytomegalovirus i/e enhancer chicken ß actin (CAG) promoter-driven expression of this construct is sustained without transgene silencing during hepatic differentiation in vitro and in vivo. At low lentiviral copy numbers per genome we confirmed a functional relevant reduction (-66%) of intracellular PiZ protein in hepatic cells after differentiation of patient-specific iPSCs. In conclusion, we have demonstrated that lentiviral vector-mediated expression of shRNAs can be efficiently used to knock down and functionally evaluate disease-related genes in patient-specific iPSCs.

MicroRNA regulation of endothelial homeostasis and commitment-implications for vascular regeneration strategies using stem cell therapies.

Human embryonic (hESC) and induced pluripotent (hiPSC) stem cells have broad therapeutic potential in the treatment of a range of diseases, including those of the vascular system. Both hESCs and hiPSCs have the capacity for indefinite self-renewal, in addition to their ability to differentiate into any adult cell type. These cells could provide a potentially unlimited source of cells for transplantation and, therefore, provide novel treatments, e.g. in the production of endothelial cells for vascular regeneration. MicroRNAs are short, noncoding RNAs that act posttranscriptionally to control gene expression and thereby exert influence over a wide range of cellular processes, including maintenance of pluripotency and differentiation. Expression patterns of these small RNAs are tissue specific, and changes in microRNA levels have often been associated with disease states in humans, including vascular pathologies. Here, we review the roles of microRNAs in endothelial cell function and vascular disease, as well as their role in the differentiation of pluripotent stem cells to the vascular endothelial lineage. Furthermore, we discuss the therapeutic potential of stem cells and how knowledge and manipulation of microRNAs in stem cells may enhance their capacity for vascular regeneration.

MicroRNA-221 overexpression accelerates hepatocyte proliferation during liver regeneration.

UNLABELLED: The tightly controlled replication of hepatocytes in liver regeneration and uncontrolled proliferation of tumor cells in hepatocellular carcinoma (HCC) are often modulated by common regulatory pathways. Several microRNAs (miRNAs) are involved in HCC progression by modulating posttranscriptional expression of multiple target genes. miR-221, which is frequently up-regulated in HCCs, delays fulminant liver failure in mice by inhibiting apoptosis, indicating a pleiotropic role of miR-221 in hepatocytes. Here, we hypothesize that modulation of miR-221 targets in primary hepatocytes enhances proliferation, providing novel clues for enhanced liver regeneration. We demonstrate that miR-221 enhances proliferation of in vitro cultivated primary hepatocytes. Furthermore, applying two-thirds partial hepatectomy as a surgically induced liver regeneration model we show that adeno-associated virus-mediated overexpression of miR-221 in the mouse liver also accelerates hepatocyte proliferation in vivo. miR-221 overexpression leads to rapid S-phase entry of hepatocytes during liver regeneration. In addition to the known targets p27 and p57, we identify Aryl hydrocarbon nuclear translocator (Arnt) messenger RNA (mRNA) as a novel target of miR-221, which contributes to the pro-proliferative activity of miR-221. CONCLUSION: miR-221 overexpression accelerates hepatocyte proliferation. Pharmacological intervention targeting miR-221 may thus be therapeutically beneficial in liver failure by preventing apoptosis and by inducing liver regeneration.

Increased reprogramming capacity of mouse liver progenitor cells, compared with differentiated liver cells, requires the BAF complex.

BACKGROUND & AIMS: Ectopic expression of certain transcription factors can reprogram somatic cells to a pluripotent state. Hematopoietic and muscle stem cells can be more efficiently reprogrammed than differentiated blood or muscle cells, yet similar findings have not been shown in other primary organ systems. Moreover, molecular characteristics of the cellular hierarchy of tissues that influence reprogramming capacities need to be delineated. We analyzed the effect of differentiation stage of freshly isolated, mouse liver cells on the reprogramming efficiency. METHODS: Liver progenitor cell (LPC)-enriched cell fractions were isolated from adult (6-8 wk) and fetal (embryonic day 14.5) livers of mice and reprogrammed to become induced pluripotent stem (iPS) cells. Different transcription factors were expressed in liver cells, and markers of pluripotency were examined, along with the ability of iPS cells to differentiate, in vitro and in vivo, into different germ layers. RESULTS: Fetal and adult LPCs had significantly greater reprogramming efficiency after transduction with 3 or 4 reprogramming factors. Transduction efficiency-corrected reprogramming rates of fetal LPCs were 275-fold higher, compared with unsorted fetal liver cells, when 3 reprogramming factors were transduced. The increased reprogramming efficiency of LPCs, compared with differentiated liver cells, occurred independently of proliferation rates, but was associated with endogenous expression of reprogramming factors (Klf4 and c-Myc) and BAF (Brg1/Brm associated factor)-complex members Baf155 and Brg1, which mediate epigenetic changes during reprogramming. Knockdown of BAF complex members negated the increased reprogramming efficiency of LPCs, compared with non-LPCs. CONCLUSIONS: LPCs have intrinsic, cell proliferation-independent characteristics resulting in an increased reprogramming capacity compared to differentiated liver cells.

Hepatic differentiation of murine disease-specific induced pluripotent stem cells allows disease modelling in vitro.

Direct reprogramming of somatic cells into pluripotent cells by retrovirus-mediated expression of OCT4, SOX2, KLF4, and C-MYC is a promising approach to derive disease-specific induced pluripotent stem cells (iPSCs). In this study, we focused on three murine models for metabolic liver disorders: the copper storage disorder Wilson's disease (toxic-milk mice), tyrosinemia type 1 (fumarylacetoacetate-hydrolase deficiency, FAH(-/-) mice), and alpha1-antitrypsin deficiency (PiZ mice). Colonies of iPSCs emerged 2-3 weeks after transduction of fibroblasts, prepared from each mouse strain, and were maintained as individual iPSC lines. RT-PCR and immunofluorescence analyses demonstrated the expression of endogenous pluripotency markers. Hepatic precursor cells could be derived from these disease-specific iPSCs applying an in vitro differentiation protocol and could be visualized after transduction of a lentiviral albumin-GFP reporter construct. Functional characterization of these cells allowed the recapitulation of the disease phenotype for further studies of underlying molecular mechanisms of the respective disease.

Hepatic differentiation of pluripotent stem cells.

In regenerative medicine pluripotent stem cells are considered to be a valuable self-renewing source for therapeutic cell transplantations, given that a functional organ-specific phenotype can be acquired by in vitro differentiation protocols. Furthermore, derivatives of pluripotent stem cells that mimic fetal progenitor stages could serve as an important tool to analyze organ development with in vitro approaches. Because of ethical issues regarding the generation of human embryonic stem (ES) cells, other sources for pluripotent stem cells are intensively studied. Like in less developed vertebrates, pluripotent stem cells can be generated from the female germline even in mammals, via parthenogenetic activation of oocytes. Recently, testis-derived pluripotent stem cells were derived from the male germline. Therefore, we compared two different hepatic differentiation approaches and analyzed the generation of definitive endoderm progenitor cells and their further maturation into a hepatic phenotype using murine parthenogenetic ES cells, germline-derived pluripotent stem cells, and ES cells. Applying quantitative RT-PCR, both germline-derived pluripotent cell lines show similar differentiation capabilities as normal murine ES cells and can be considered an alternative source for pluripotent stem cells in regenerative medicine.

Pretreatment with statin attenuates the cardiotoxicity of Doxorubicin in mice.

Cardiotoxicity, which may result from intense cardiac oxidative stress and inflammation, is the main limiting factor of the anticancer therapy using doxorubicin. Because statins might exert beneficial pleiotropic cardiovascular effects, among other things, by anti-inflammatory and antioxidative mechanisms, we investigated whether or not fluvastatin pretreatment can attenuate doxorubicin-induced cardiotoxicity. Five days after a single injection of doxorubicin (20 mg/kg; i.p.), left ventricular (LV) function was measured in fluvastatin-treated (DoxStatin; 100 mg/kg/day, p.o.) and saline-treated (doxorubicin) mice (n = 8 per group) by a micro conductance catheter. Untreated mice served as controls (placebo; n = 8 per group). After measurement of cardiac function, LV tissues were analyzed by molecular biological and immunohistologic methods. Injection resulted in significantly impaired LV function (LV pressure, -29%; dp/dtmax, -45%; cardiac output, -68%; P < 0.05) when compared with placebo. This was associated with a significant increase in cardiac oxidative stress, inflammation and apoptotic mechanisms, as indicated by significant increased cardiac lipid peroxidation activity, protein expression of nitrotyrosine, tumor necrosis factor alpha and Bax (P < 0.05). In contrast, DoxStatin mice showed improved LV function (LV pressure, +24%; dp/dtmax, +87%; cardiac output, +87%; P < 0.05) when compared with untreated doxorubicin mice. This was associated with reduced cardiac expression of nitrotyrosine, enhanced expression of the mitochondrial located antioxidative SOD 2, attenuated mitochondrial apoptotic pathways, and reduced cardiac inflammatory response. Statin pretreatment attenuates doxorubicin-induced cardiotoxicity via antioxidative and anti-inflammatory effects.