Transcriptomic analysis of marine endophytic fungi extract identifies highly enriched anti-fungal fractions targeting cancer pathways in HepG2 cell lines.

BACKGROUND: Marine endophytic fungi (MEF) are good sources of structurally unique and biologically active secondary metabolites. Due to the increase in antimicrobial resistance, the secondary metabolites from MEF ought to be fully explored to identify candidates which could serve as lead compounds for novel drug development. These secondary metabolites might also be useful for development of new cancer drugs. In this study, ethyl acetate extracts from marine endophytic fungal cultures were tested for their antifungal activity and anticancer properties against C. albicans and the human liver cancer cell line HepG2, respectively. The highly enriched fractions were also analyzed by high performance liquid chromatography coupled with high resolution mass spectrometry (HPLC-HRMS) and their effect on the HepG2 cells was assessed via transcriptomics and with a proliferation assay. RESULTS: We demonstrated that the fractions could reduce proliferation in HepG2 cells. The detailed transcriptome analysis revealed regulation of several cancer- and metabolism-related pathways and gene ontologies. The down-regulated pathways included, cell cycle, p53 signaling, DNA replication, sphingolipid metabolism and drug metabolism by cytochrome P450. The upregulated pathways included HIF-1 signaling, focal adhesion, necroptosis and transcriptional mis-regulation of cancer. Furthermore, a protein interaction network was constructed based on the 26 proteins distinguishing the three treatment conditions from the untreated cells. This network was composed of central functional components associated with metabolism and cancer such as TNF, MAPK, TRIM21 and one component contained APP. CONCLUSIONS: The purified fractions from MEF investigated in this study showed antifungal activity against C. albicans and S. cerevisiae alone or both and reduced proliferation of the human liver cancer cell line HepG2 implicating regulation of several cancer- and metabolism-related pathways. The data from this study could be instrumental in identifying new pathways associated with liver cancer anti-proliferative processes which can be used for the development of novel antifungal and anti-cancer drugs.

Generation of two Alpha-I antitrypsin deficiency patient-derived induced pluripotent stem cell lines ISRM-AATD-iPSC-1 (HHUUKDi011-A) and ISRM-AATD-iPSC-2 (HHUUKDi012-A).

SIX2-positive urine derived renal progenitor cells were isolated from a male and female alpha1-antitrypsin deficiency (AATD) patients both harboring the homozygous PiZZ genotype. The cells were reprogrammed to generate two integration-free induced pluripotent stem cell (iPSC) lines by transfecting episomal-based plasmids expressing OCT4, SOX2, NANOG, c-MYC, KLF4 and LIN28. Pluripotency was confirmed by immunocytochemistry for associated markers and embryoid body-based differentiation into the three germ layers. The iPSC lines carried the parental PiZZ genotype. Comparative transcriptome analyses with human embryonic stem cell line H9 revealed a Pearson correlation of 0.945 for ISRM-AATD-iPSC-1 and 0.939 for ISRM-AATD-iPSC-2 respectively.

A stem cell based in vitro model of NAFLD enables the analysis of patient specific individual metabolic adaptations in response to a high fat diet and AdipoRon interference.

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial disease. Its development and progression depend on genetically predisposed susceptibility of the patient towards several 'hits' that induce fat storage first and later inflammation and fibrosis. Here, we differentiated induced pluripotent stem cells (iPSCs) derived from four distinct donors with varying disease stages into hepatocyte like cells (HLCs) and determined fat storage as well as metabolic adaptations after stimulations with oleic acid. We could recapitulate the complex networks that control lipid and glucose metabolism and we identified distinct gene expression profiles related to the steatosis phenotype of the donor. In an attempt to reverse the steatotic phenotype, cells were treated with the small molecule AdipoRon, a synthetic analogue of adiponectin. Although the responses varied between cells lines, they suggest a general influence of AdipoRon on metabolism, transport, immune system, cell stress and signalling.

The FGF, TGFß and WNT axis Modulate Self-renewal of Human SIX2+ Urine Derived Renal Progenitor Cells.

Human urine is a non-invasive source of renal stem cells with regeneration potential. Urine-derived renal progenitor cells were isolated from 10 individuals of both genders and distinct ages. These renal progenitors express pluripotency-associated proteins- TRA-1-60, TRA-1-81, SSEA4, C-KIT and CD133, as well as the renal stem cell markers -SIX2, CITED1, WT1, CD24 and CD106. The transcriptomes of all SIX2+ renal progenitors clustered together, and distinct from the human kidney biopsy-derived epithelial proximal cells (hREPCs). Stimulation of the urine-derived renal progenitor cells (UdRPCs) with the GSK3ß-inhibitor (CHIR99021) induced differentiation. Transcriptome and KEGG pathway analysis revealed upregulation of WNT-associated genes- AXIN2, JUN and NKD1. Protein interaction network identified JUN- a downstream target of the WNT pathway in association with STAT3, ATF2 and MAPK1 as a putative negative regulator of self-renewal. Furthermore, like pluripotent stem cells, self-renewal is maintained by FGF2-driven TGFß-SMAD2/3 pathway. The urine-derived renal progenitor cells and the data presented should lay the foundation for studying nephrogenesis in human.

Cell fate decisions of human iPSC-derived bipotential hepatoblasts depend on cell density.

During embryonic development bipotential hepatoblasts differentiate into hepatocytes and cholangiocytes- the two main cell types within the liver. Cell fate decision depends on elaborate interactions between distinct signalling pathways, namely Notch, WNT, TGFß, and Hedgehog. Several in vitro protocols have been established to differentiate human pluripotent stem cells into either hepatocyte or cholangiocyte like cells (HLC/CLC) to enable disease modelling or drug screening. During HLC differentiation we observed the occurrence of epithelial cells with a phenotype divergent from the typical hepatic polygonal shape- we refer to these as endoderm derived epithelial cells (EDECs). These cells do not express the mature hepatocyte marker ALB or the progenitor marker AFP. However they express the cholangiocyte markers SOX9, OPN, CFTR as well as HNF4α, CK18 and CK19. Interestingly, they express both E Cadherin and Vimentin, two markers that are mutually exclusive, except for cancer cells. EDECs grow spontaneously under low density cell culture conditions and their occurrence was unaffected by interfering with the above mentioned signalling pathways.

Establishment and characterization of an iPSC line from a 35 years old high grade patient with nonalcoholic fatty liver disease (30-40% steatosis) with homozygous wildtype PNPLA3 genotype.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome and its prevalence increases continuously. Here, we reprogrammed fibroblasts of a high grade NAFLD patient with homozygous wildtype PNPLA3 genotype. The induced pluripotent stem cells (iPSCs) were characterized by immunocytochemistry, flow cytometry, embryoid body formation, pluritest, DNA-fingerprinting and karyotype analysis.

Establishment and characterization of an iPSC line from a 58 years old high grade patient with nonalcoholic fatty liver disease (70% steatosis) with homozygous wildtype PNPLA3 genotype.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome and its prevalence increases continuously. Here, we reprogrammed fibroblasts of a high grade NAFLD patient with homozygous wildtype PNPLA3 genotype. We characterized the induced pluripotent stem cells (iPSCs) by immunocytochemistry, flow cytometry, embryoid body formation, pluritest DNA-fingerprinting, and karyotype analysis.

The presence of human mesenchymal stem cells of renal origin in amniotic fluid increases with gestational time.

BACKGROUND: Established therapies for managing kidney dysfunction such as kidney dialysis and transplantation are limited due to the shortage of compatible donated organs and high costs. Stem cell-based therapies are currently under investigation as an alternative treatment option. As amniotic fluid is composed of fetal urine harboring mesenchymal stem cells (AF-MSCs), we hypothesized that third-trimester amniotic fluid could be a novel source of renal progenitor and differentiated cells. METHODS: Human third-trimester amniotic fluid cells (AFCs) were isolated and cultured in distinct media. These cells were characterized as renal progenitor cells with respect to cell morphology, cell surface marker expression, transcriptome and differentiation into chondrocytes, osteoblasts and adipocytes. To test for renal function, a comparative albumin endocytosis assay was performed using AF-MSCs and commercially available renal cells derived from kidney biopsies. Comparative transcriptome analyses of first, second and third trimester-derived AF-MSCs were conducted to monitor expression of renal-related genes. RESULTS: Regardless of the media used, AFCs showed expression of pluripotency-associated markers such as SSEA4, TRA-1-60, TRA-1-81 and C-Kit. They also express the mesenchymal marker Vimentin. Immunophenotyping confirmed that third-trimester AFCs are bona fide MSCs. AF-MSCs expressed the master renal progenitor markers SIX2 and CITED1, in addition to typical renal proteins such as PODXL, LHX1, BRN1 and PAX8. Albumin endocytosis assays demonstrated the functionality of AF-MSCs as renal cells. Additionally, upregulated expression of BMP7 and downregulation of WT1, CD133, SIX2 and C-Kit were observed upon activation of WNT signaling by treatment with the GSK-3 inhibitor CHIR99201. Transcriptome analysis and semiquantitative PCR revealed increasing expression levels of renal-specific genes (e.g., SALL1, HNF4B, SIX2) with gestational time. Moreover, AF-MSCs shared more genes with human kidney cells than with native MSCs and gene ontology terms revealed involvement of biological processes associated with kidney morphogenesis. CONCLUSIONS: Third-trimester amniotic fluid contains AF-MSCs of renal origin and this novel source of kidney progenitors may have enormous future potentials for disease modeling, renal repair and drug screening.

Derivation and characterization of integration-free iPSC line ISRM-UM51 derived from SIX2-positive renal cells isolated from urine of an African male expressing the CYP2D6 *4/*17 variant which confers intermediate drug metabolizing activity.

SIX2-positive renal cells isolated from urine from a 51year old male of African origin bearing the CYP2D6 *4/*17 variant were reprogrammed by nucleofection of a combination of two episomal-based plasmids omitting pathway (TGFß, MEK and GSK3ß) inhibition. The induced pluripotent stem cells (iPSCs) were characterized by immunocytochemistry, embryoid body formation, DNA-fingerprinting and karyotype analysis. Comparative transcriptome analyses with human embryonic stem cell lines H1 and H9 revealed a Pearson correlation of 0.9243 and 0.9619 respectively.

Isolation and Molecular Characterization of Amniotic Fluid-Derived Mesenchymal Stem Cells Obtained from Caesarean Sections.

Human amniotic fluid cells are immune-privileged with low immunogenicity and anti-inflammatory properties. They are able to self-renew, are highly proliferative, and have a broad differentiation potential, making them amenable for cell-based therapies. Amniotic fluid (AF) is routinely obtained via amniocentesis and contains heterogeneous populations of foetal-derived progenitor cells including mesenchymal stem cells (MSCs). In this study, we isolated human MSCs from AF (AF-MSCs) obtained during Caesarean sections (C-sections) and characterized them. These AF-MSCs showed typical MSC characteristics such as morphology, in vitro differentiation potential, surface marker expression, and secreted factors. Besides vimentin and the stem cell marker CD133, subpopulations of AF-MSCs expressed pluripotency-associated markers such as SSEA4, c-Kit, TRA-1-60, and TRA-1-81. The secretome and related gene ontology (GO) terms underline their immune modulatory properties. Furthermore, transcriptome analyses revealed similarities with native foetal bone marrow-derived MSCs. Significant KEGG pathways as well as GO terms are mostly related to immune function, embryonic skeletal system, and TGFß-signalling. An AF-MSC-enriched gene set included putative AF-MSC markers PSG5, EMX-2, and EVR-3. In essence, C-section-derived AF-MSCs can be routinely obtained and are amenable for personalized cell therapies and disease modelling.

Modeling Nonalcoholic Fatty Liver Disease with Human Pluripotent Stem Cell-Derived Immature Hepatocyte-Like Cells Reveals Activation of PLIN2 and Confirms Regulatory Functions of Peroxisome Proliferator-Activated Receptor Alpha.

Nonalcoholic fatty liver disease (NAFLD/steatosis) is a metabolic disease characterized by the incorporation of fat into hepatocytes. In this study, we developed an in vitro model for NAFLD based on hepatocyte-like cells (HLCs) differentiated from human pluripotent stem cells. We induced fat storage in these HLCs and detected major expression changes of metabolism-associated genes, as well as an overall reduction of liver-related microRNAs. We observed an upregulation of the lipid droplet coating protein Perilipin 2 (PLIN2), as well as of numerous genes of the peroxisome proliferator-activated receptor (PPAR) pathway, which constitutes a regulatory hub for metabolic processes. Interference with PLIN2 and PPARα resulted in major alterations in gene expression, especially affecting lipid, glucose, and purine metabolism. Our model recapitulates many metabolic changes that are characteristic for NAFLD. It permits the dissection of disease-promoting molecular pathways and allows us to investigate the influences of distinct genetic backgrounds on disease progression.