Donor-Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte-Like Cells.

Drug-induced liver injury is the greatest cause of post-marketing drug withdrawal; therefore, substantial resources are directed toward triaging potentially dangerous new compounds at all stages of drug development. One of the major factors preventing effective screening of new compounds is the lack of a predictive in vitro model of hepatotoxicity. Primary human hepatocytes offer a metabolically relevant model for which the molecular initiating events of hepatotoxicity can be examined; however, these cells vary greatly between donors and dedifferentiate rapidly in culture. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) offer a reproducible, physiologically relevant and genotypically normal model cell; however, current differentiation protocols produce HLCs with a relatively immature phenotype. During the reprogramming of somatic cells, the epigenome undergoes dramatic changes; however, this "resetting" is a gradual process, resulting in an altered differentiation propensity, skewed toward the lineage of origin, particularly in early passage cultures. We, therefore, performed a comparison of human hepatocyte- and dermal fibroblast-derived iPSCs, assessing the impact of epigenetic memory at all stages of HLC differentiation. These results provide the first isogenic assessment of the starting cell type in human iPSC-derived HLCs. Despite a trend toward improvement in hepatic phenotype in albumin secretion and gene expression, few significant differences in hepatic differentiation capacity were found between hepatocyte and fibroblast-derived iPSCs. We conclude that the donor and inter-clonal differences have a greater influence on the hepatocyte phenotypic maturity than the starting cell type. Therefore, it is not necessary to use human hepatocytes for generating iPSC-derived HLCs. Stem Cells Translational Medicine 2017;6:1321-1331.

Multiplex reverse transcription-PCR for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group A rotavirus.

A novel multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) that can detect porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine group A rotavirus (GAR) was developed. The 3 viruses (PEDV, TGEV, and porcine GAR) are major agents in viral enteric diseases of piglets. As the clinical signs of these diseases are similar, including watery diarrhea, differential detection is required for etiologic diagnosis. A mixture of 3 pairs of published primers was used for amplification of viral nucleic acids, yielding 3 different amplicons with sizes of 859 bp, 651 bp, and 309 bp for TGEV, PEDV, and porcine GAR, respectively. A total of 157 specimens (78 fecal and 79 intestinal samples) from piglets with acute gastroenteritis were collected in Korea between January 2004 and May 2005. They were tested for the presence of 3 viruses by multiplex RT-PCR. Coinfections with PEDV and porcine GAR were identified in 16 farms (43.2%). PEDV, porcine GAR, and TGEV infection were 26.3%, 13.2%, and 2.7% respectively. The relative sensitivity and specificity of multiplex RT-PCR were evaluated, with results suggesting that this assay is equal in quality to conventional single-agent RT-PCR assays (sensitivity:100%, 92.9%, 100% for TGEV, PEDV, GARs; specificity: 100% for all 3 viruses). This multiplex RT-PCR is a simple assay and may be a potentially useful for rapid, sensitive, and cost-effective etiological diagnostic tool for acute viral gastroenteritis in piglets.

Detection of porcine circovirus type 2 in feces of pigs with or without enteric disease by polymerase chain reaction.

To establish the sensitive polymerase chain reaction(PCR) method and detect porcine circovirus type 2 (PCV2) from intestines and feces of commercial swine herds with or without enteric disease, intestinal samples from 68 pigs and 29 fecal samples from commercial swine farms were collected. A primer set, forward primer 5'-GAAGAATGGAAGAAGCGG-3' and reverse primer 5'-CTCACAGCAGTAGACAGGT-3', could detect the virus at a concentration as low as 2 infectious virions per milliliter under controlled conditions using PK-15 cell-adapted PCV2. The genomic nucleotide sequences of open reading frame 1 (ORF1) PCR products from fecal samples were found to have complete homology with other PCV2s deposited in the GenBank database. Transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) as the other enteric pathogens were also investigated by performing duplex reverse transcription-PCR (RT-PCR). Among 63 pigs with clinical enteric disease, 18 PCV2s (14 from intestines and 4 from feces), 7 TGEVs from intestines, and 18 PEDVs (14 from intestines and 4 from feces) were detected by PCR and the duplex RT-PCR. In 34 pigs (14 from intestines and 20 from feces) without clinical enteric disease, only PCV2 was detected in 19 pigs (3 from intestines and 16 from feces). Both PEDV and PCV2 were found in 6 pigs with clinical enteric disease. Among 15 PCV2 samples that were PCR-positive, 4 were culture-positive at passage level 3 in PK-15 cells. These results reveal that PCV2 is shed through the feces of pigs without clinical enteric disease, which suggests the potentiality of the fecal-oral transmission of PCV2 in feces.

Isolation of H3N2 swine influenza virus in South Korea.

Swine influenza is a significant respiratory disease causing occasional reproductive problems in naïve swine herds. Although different subtypes of swine influenza virus (SIV) have been implicated in clinical outbreaks of swine influenza in Asian countries, no virus isolation has been made to identify SIV of subtypes other than the H1N1 subtype in the Korean swine population. In December 1998, an outbreak of acute respiratory disease was identified in a commercial swine farm located in the Kyunggi province of South Korea. A causative agent, which agglutinated rooster red blood cells, was detected from the lungs of 3 piglets from the index herd and was determined to be type A influenza virus using a commercial influenza virus typing kit. Hemagglutination activity (HA) of the isolates was completely inhibited by a swine antiserum against a recent US H3N2 SIV isolate (A/Sw/IA/41305/1998) but not by H1N1 swine antiserum (A/Sw/IA/1979). Reverse transcription-polymerase chain reaction (RT-PCR) revealed all 3 isolates were H3 SIV subtypes. Sequence analysis of hemagglutinin gene PCR products supported the belief that the Korean H3 SIV isolates were genetically similar to the known mammalian H3 influenza viruses. This is the first report on a clinical outbreak of swine influenza caused by the H3N2 virus in Korea.