Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure and heart transplantation. A portion of familial DCM is due to mutations in the LMNA gene encoding the nuclear lamina proteins lamin A and C and without adequate treatment these patients have a poor prognosis. To get better insights into pathobiology behind this disease, we focused on modeling LMNA-related DCM using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM). Primary skin fibroblasts from DCM patients carrying the most prevalent Finnish founder mutation (p.S143P) in LMNA were reprogrammed into hiPSCs and further differentiated into cardiomyocytes (CMs). The cellular structure, functionality as well as gene and protein expression were assessed in detail. While mutant hiPSC-CMs presented virtually normal sarcomere structure under normoxia, dramatic sarcomere damage and an increased sensitivity to cellular stress was observed after hypoxia. A detailed electrophysiological evaluation revealed bradyarrhythmia and increased occurrence of arrhythmias in mutant hiPSC-CMs on β-adrenergic stimulation. Mutant hiPSC-CMs also showed increased sensitivity to hypoxia on microelectrode array and altered Ca²⁺ dynamics. Taken together, p.S143P hiPSC-CM model mimics hallmarks of LMNA-related DCM and provides a useful tool to study the underlying cellular mechanisms of accelerated cardiac degeneration in this disease.

Aim: Primary human hepatocytes (PHHs) undergo dedifferentiation upon the two-dimensional (2D) culture, which particularly hinders their utility in long-term in vitro studies. Lipids, as a major class of biomolecules, play crucial roles in cellular energy storage, structure, and signaling. Here, for the first time, we mapped the alterations in the lipid profile of the dedifferentiating PHHs and studied the possible role of lipids in the loss of the phenotype of PHHs. Simultaneously, differentially expressed miRNAs associated with changes in the lipids and fatty acids (FAs) of the dedifferentiating PHHs were investigated. Methods: PHHs were cultured in monolayer and their phenotype was monitored morphologically, genetically, and biochemically for five days. The lipid and miRNA profile of the PHHs were analyzed by mass spectrometry and Agilent microarray, respectively. In addition, 24 key genes involved in the metabolism of lipids and FAs were investigated by qPCR. Results: The typical morphology of PHHs was lost from day 3 onward. Additionally, ALB and CYP genes were downregulated in the cultured PHHs. Lipidomics revealed a clear increase in the saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) containing lipids, but a decrease in the polyunsaturated fatty acids (PUFA) containing lipids during the dedifferentiation of PHHs. In line with this, FASN, SCD, ELOVL1, ELOVL3, and ELOVL7 were upregulated but ELOVL2 was downregulated in the dedifferentiated PHHs. Furthermore, differentially expressed miRNAs were identified, and the constantly upregulated miR-27a and miR-21, and downregulated miR-30 may have regulated the synthesis, accumulation and secretion of PHH lipids during the dedifferentiation. Conclusion: Our results showed major alterations in the molecular lipid species profiles, lipid-metabolizing enzyme expression as wells as miRNA profiles of the PHHs during their prolonged culture, which in concert could play important roles in the PHHs’ loss of phenotype. These findings promote the understanding from the dedifferentiation process and could help in developing optimal culture conditions, which better meet the needs of the PHHs and support their original phenotype.

In diabetic patients, high blood glucose induces alterations in retinal function and can lead to visual impairment due to diabetic retinopathy. In immortalized retinal pigment epithelial (RPE) cultures, high glucose concentrations are shown to lead to impairment in epithelial barrier properties. For the first time, the induced pluripotent stem-cell-derived retinal pigment epithelium (hiPSC-RPE) cell lines derived from type 2 diabetics and healthy control patients were utilized to assess the effects of glucose concentration on the cellular functionality. We show that both type 2 diabetic and healthy control hiPSC-RPE lines differentiate and mature well, both in high and normal glucose concentrations, express RPE specific genes, secrete pigment epithelium derived factor, and form a polarized cell layer. Here, type 2 diabetic hiPSC-RPE cells had a decreased barrier function compared to controls. Added insulin increased the epithelial cell layer tightness in normal glucose concentrations, and the effect was more evident in type 2 diabetics than in healthy control hiPSC-RPE cells. In addition, the preliminary functionality assessments showed that type 2 diabetic hiPSC-RPE cells had attenuated autophagy detected via ubiquitin-binding protein p62/Sequestosome-1 (p62/SQSTM1) accumulation, and lowered pro- matrix metalloproteinase 2 (proMMP2) as well as increased pro-MMP9 secretion. These results suggest that the cellular ability to tolerate stress is possibly decreased in type 2 diabetic RPE cells.

Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem cells (iPSCs) offer an alternative model for primary human hepatocytes to study lipid aberrations. However, the detailed lipid profile of HLCs is yet unknown. In the current study, functional HLCs were differentiated from iPSCs generated from dermal fibroblasts of three individuals by a 3-step protocol through definitive endoderm (DE) stage. In parallel, detailed lipidomic analyses as well as gene expression profiling of a set of lipid metabolism-related genes were performed during the entire differentiation process from iPSC to HLCs. Additionally, fatty acid (FA) composition of the cell culture media at different stages was determined. Our results show that major alterations in the molecular species of lipids occurring during DE and early hepatic differentiation stages mainly mirror the quality and quantity of the FAs supplied in culture medium at each stage. Polyunsaturated phospholipids and sphingolipids with a very-long FA were produced in the cells at later stage of differentiation. This work uncovers previously unknown lipid composition of iPSC-HLCs and its alterations during the differentiation in conjunction with the expression of key lipid associated genes. Together with biochemical, functional and gene expression measurements the lipidomic analyses allowed us to improve our understanding of the concerted influence of the exogenous metabolite supply and cellular biosynthesis essential for iPSC-HLCs differentiation and function. Importantly, the study describes in detail a cell model that can be applied in exploring e.g. the lipid metabolism involved in the development of fatty liver disease or atherosclerosis.

Hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells (iPSCs) provide a renewable source of cells for drug discovery, disease modelling and cell-based therapies. Here, by using GRO-Seq we provide the first genome-wide analysis of the nascent RNAs in iPSCs, HLCs and primary hepatocytes to extend our understanding of the transcriptional changes occurring during hepatic differentiation process. We demonstrate that a large fraction of hepatocyte-specific genes are regulated at transcriptional level and identify hundreds of differentially expressed non-coding RNAs (ncRNAs), including primary miRNAs (pri-miRNAs) and long non-coding RNAs (lncRNAs). Differentiation induced alternative transcription start site (TSS) usage between the cell types as evidenced for miR-221/222 and miR-3613/15a/16-1 clusters. We demonstrate that lncRNAs and coding genes are tightly co-expressed and could thus be co-regulated. Finally, we identified sets of transcriptional regulators that might drive transcriptional changes during hepatocyte differentiation. These included RARG, E2F1, SP1 and FOXH1, which were associated with the down-regulated transcripts, and hepatocyte-specific TFs such as FOXA1, FOXA2, HNF1B, HNF4A and CEBPA, as well as RXR, PPAR, AP-1, JUNB, JUND and BATF, which were associated with up-regulated transcripts. In summary, this study clarifies the role of regulatory ncRNAs and TFs in differentiation of HLCs from iPSCs.