High efficient differentiation of functional hepatocytes from porcine induced pluripotent stem cells

Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs) by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM), and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP) 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications. © 2014 Ao et al

Cold-preservation of human adult hepatocytes for liver cell therapy

Hepatocyte transplantation is a promising alternative therapy for the treatment of hepatic failure, hepatocellular deficiency, and genetic metabolic disorders. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in sufficient quantity and of the quality required for biotherapy. In this study, first we assessed how cold storage in three clinically safe preservative solutions (UW, HTS-FRS, and IGL-1) affects the viability and in vitro functionality of human hepatocytes. Then we evaluated whether such cold-preserved human hepatocytes could engraft and repopulate damaged livers in a mouse model of liver failure. Human hepatocytes showed comparable viabilities after cold preservation in the three solutions. The ability of fresh and cold-stored hepatocytes to attach to a collagen substratum and to synthesize and secrete albumin, coagulation factor VII, and urea in the medium after 3 days in culture was also equally preserved. Cold-stored hepatocytes were then transplanted in the spleen of immunodeficient mice previously infected with adenoviruses containing a thymidine kinase construct and treated with a single dose of ganciclovir to induce liver injury. Engraftment and liver repopulation were monitored over time by measuring the blood level of human albumin and by assessing the expression of specific human hepatic mRNAs and proteins in the recipient livers by RT-PCR and immunohistochemistry, respectively. Our findings show that cold-stored human hepatocytes in IGL-1 and HTS-FRS preservative solutions can survive, engraft, and proliferate in a damaged mouse liver. These results demonstrate the usefulness of human hepatocyte hypothermic preservation for cell transplantation

Efficient neutralization of Tat by high avidity antibodies.

<p>(A) The HTL-Tat proteins induce the generation of high avidity antisera. Antisera diluted 500-fold were incubated in microtiter wells coated with the amino-terminal peptide (1-20 amino acids) of Tat. Percent absorbance of the NaSCN treated wells was calculated considering the absorbance of the control wells as 100%. The mean percent absorbance + SD values are shown on the y-axis and the concentration of NaSCN on the x-axis. (B) The HTL-Tat antisera block the exogenous Tat efficiently. Percent transactivation was calculated considering the amount of p24 secreted by HLM1 cells in the absence of a neutralizing antibody as 100%. *P<0.05 and **P<0.005.</p

The Grafting of Universal T-Helper Epitopes Enhances Immunogenicity of HIV-1 Tat Concurrently Improving Its Safety Profile

<div><p>Extracellular Tat (eTat) plays an important role in HIV-1 pathogenesis. The presence of anti-Tat antibodies is negatively correlated with disease progression, hence making Tat a potential vaccine candidate. The cytotoxicity and moderate immunogenicity of Tat however remain impediments for developing Tat-based vaccines. Here, we report a novel strategy to concurrently enhance the immunogenicity and safety profile of Tat. The grafting of universal helper T-lymphocyte (HTL) epitopes, Pan DR Epitope (PADRE) and Pol₇₁₁ into the cysteine rich domain (CRD) and the basic domain (BD) abolished the transactivation potential of the Tat protein. The HTL-Tat proteins elicited a significantly higher titer of antibodies as compared to the wild-type Tat in BALB/c mice. While the N-terminal epitope remained immunodominant in HTL-Tat immunizations, an additional epitope in exon-2 was recognized with comparable magnitude suggesting a broader immune recognition. Additionally, the HTL-Tat proteins induced cross-reactive antibodies of high avidity that efficiently neutralized exogenous Tat, thus blocking the activation of a Tat-defective provirus. With advantages such as presentation of multiple B-cell epitopes, enhanced antibody response and importantly, transactivation-deficient Tat protein, this approach has potential application for the generation of Tat-based HIV/AIDS vaccines.</p></div

HTL-Tat proteins are transactivation deficient.

<p>(A) Domain structures of the five Tat constructs are illustrated. WT: wild type, N-term: amino terminal region, CRD: Cystine-rich domain, CD: core domain, BD: basic domain and C-term: carboxy terminal of exon-1 (B) The HTL-Tat proteins fail to transactivate in HLM1 (left panel) or TZM-bl (right panel) cells. The data for the 72 h time point are presented. The x-axis represents the mean amount of p24 or luciferase + SD and the y-axis the Tat construct used. The experiments were performed in biological triplicates and the plotted data are representative of two independent experiments.</p

IgG1 and IgG2a profile of anti-Tat antibodies.

<p>The isotype profile of the Tat-specific antibodies was determined using IgG2a (Th1) and IgG1- (Th2) specific secondary antibodies. Each bar corresponds to the antisera used in the assay as enlisted in the figure key and are grouped into two based on the isotype-specific secondary antibody used shown on the y-axis and x-axis corresponds to antibody titers. Representative data of two independent experiments are shown. *P<0.05.</p

The HTL-Tat proteins elicit a strong immune response.

<p>The immunization protocol is represented in the inset schema. (A) Antisera collected 14 days after the final booster were used for the titer determination. X-axis corresponds to antibody titer and y-axis to the Tat protein used in immunizations. (B) Splenocytes harvested from mice 14 days after the final booster immunization were stained with CFSE and dilution of the dye in CD4+ cells was evaluated and the mean stimulation index (SI) + SD was plotted on the x-axis. SI  =  Percent CFSE-low (Peptide-stimulated cells)/Percent CFSE-low (DMSO-treated cells). The data presented are representative of two independent experiments (n = 10, * P<0.05).</p