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

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

Mapping of the B-cell epitopes in Tat.

<p>The antisera collected from mice immunized with (A) WT-Tat or (B) PADRE-CRD proteins were diluted 500 and 1,000 times, respectively, and used in the pepscan analysis. Twenty-mer synthetic peptides with a 10-residue overlap and encompassing the full-length C-Tat consensus sequence were used in the assay. Peptides 1-9 represent full-length subtype C Tat protein. The gray bars (A and B) represent the amino acid sequences generated due to the grafting of the HTL in Tat. The data are presented as the mean absorbance + SD on the x-axis with corresponding peptides on the y-axis. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114155#pone.0114155.s002" target="_blank">Figure S2</a> for data pertaining to the other three HTL-Tat antisera.</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

Cross-clade reactivity of the anti-Tat antibodies.

<p>The antibody titers were determined by incubating serially diluted antisera in triplicate microtiter wells coated with recombinant Tat from subtypes A, B, C and D. The x-axis represents the serum dilution and the y-axis to mean absorbance + SD. Representative data of two or more independent experiments are shown.</p

Proof-of-Concept Gene Editing for the Murine Model of Inducible Arginase-1 Deficiency

Arginase-1 deficiency in humans is a rare genetic disorder of metabolism resulting from a loss of arginase-1, leading to impaired ureagenesis, hyperargininemia and neurological deficits. Previously, we generated a tamoxifen-inducible arginase-1 deficient mouse model harboring a deletion of Arg1 exons 7 and 8 that leads to similar biochemical defects, along with a wasting phenotype and death within two weeks. Here, we report a strategy utilizing the Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system in conjunction with piggyBac technology to target and reincorporate exons 7 and 8 at the specific Arg1 locus in attempts to restore the function of arginase-1 in induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) and macrophages in vitro. While successful gene targeted repair was achieved, minimal urea cycle function was observed in the targeted iHLCs compared to adult hepatocytes likely due to inadequate maturation of the cells. On the other hand, iPSC-derived macrophages expressed substantial amounts of "repaired" arginase. Our studies provide proof-of-concept for gene-editing at the Arg1 locus and highlight the challenges that lie ahead to restore sufficient liver-based urea cycle function in patients with urea cycle disorders