Characterization of dermal fibroblast-derived iPSCs from a patient with low grade steatosis

AbstractPrimary fibroblasts from a low grade steatosis patient were reprogrammed by transduction of a combination of two episomal-based plasmids OCT4,SOX2, c-MYC and KLF4. iPSCs were characterized by immunocytochemistry, embryonic body-formation, DNA-fingerprint karyotype analysis and comparative transcriptome analyses with the human embryonic stem cell line H1 revealed a Pearsons correlation of 0.9251

Activation of the Renin–Angiotensin System Disrupts the Cytoskeletal Architecture of Human Urine-Derived Podocytes

High blood pressure is one of the major public health problems that causes severe disorders in several tissues including the human kidney. One of the most important signaling pathways associated with the regulation of blood pressure is the renin–angiotensin system (RAS), with its main mediator angiotensin II (ANGII). Elevated levels of circulating and intracellular ANGII and aldosterone lead to pro-fibrotic, -inflammatory, and -hypertrophic milieu that causes remodeling and dysfunction in cardiovascular and renal tissues. Furthermore, ANGII has been recognized as a major risk factor for the induction of apoptosis in podocytes, ultimately leading to chronic kidney disease (CKD). In the past, disease modeling of kidney-associated diseases was extremely difficult, as the derivation of kidney originated cells is very challenging. Here we describe a differentiation protocol for reproducible differentiation of sine oculis homeobox homolog 2 (SIX2)-positive urine-derived renal progenitor cells (UdRPCs) into podocytes bearing typical cellular processes. The UdRPCs-derived podocytes show the activation of the renin–angiotensin system by being responsive to ANGII stimulation. Our data reveal the ANGII-dependent downregulation of nephrin (NPHS1) and synaptopodin (SYNPO), resulting in the disruption of the podocyte cytoskeletal architecture, as shown by immunofluorescence-based detection of α-Actinin. Furthermore, we show that the cytoskeletal disruption is mainly mediated through angiotensin II receptor type 1 (AGTR1) signaling and can be rescued by AGTR1 inhibition with the selective, competitive angiotensin II receptor type 1 antagonist, losartan. In the present manuscript we confirm and propose UdRPCs differentiated to podocytes as a unique cell type useful for studying nephrogenesis and associated diseases. Furthermore, the responsiveness of UdRPCs-derived podocytes to ANGII implies potential applications in nephrotoxicity studies and drug screening

Derivation of the Immortalized Cell Line UM51-PrePodo-hTERT and Its Responsiveness to Angiotensin II and Activation of the RAAS Pathway

Recent demographic studies predict there will be a considerable increase in the number of elderly people within the next few decades. Aging has been recognized as one of the main risk factors for the world’s most prevalent diseases such as neurodegenerative disorders, cancer, cardiovascular disease, and metabolic diseases. During the process of aging, a gradual loss of tissue volume and organ function is observed, which is partially caused by replicative senescence. The capacity of cellular proliferation and replicative senescence is tightly regulated by their telomere length. When telomere length is critically shortened with progressive cell division, cells become proliferatively arrested, and DNA damage response and cellular senescence are triggered, whereupon the “Hayflick limit” is attained at this stage. Podocytes are a cell type found in the kidney glomerulus where they have major roles in blood filtration. Mature podocytes are terminal differentiated cells that are unable to undergo cell division in vivo. For this reason, the establishment of primary podocyte cell cultures has been very challenging. In our present study, we present the successful immortalization of a human podocyte progenitor cell line, of which the primary cells were isolated directly from the urine of a 51-year-old male. The immortalized cell line was cultured over the course of one year (~100 passages) with high proliferation capacity, endowed with contact inhibition and P53 expression. Furthermore, by immunofluorescence-based expression and quantitative real-time PCR for the podocyte markers CD2AP, LMX1B, NPHS1, SYNPO and WT1, we confirmed the differentiation capacity of the immortalized cells. Finally, we evaluated and confirmed the responsiveness of the immortalized cells on the main mediator angiotensin II (ANGII) of the renin–angiotensin system (RAAS). In conclusion, we have shown that it is possible to bypass cellular replicative senescence (Hayflick limit) by TERT-driven immortalization of human urine-derived pre-podocyte cells from a 51-year-old African male

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

Fibroblast-derived integration-free iPSC line ISRM-NBS1 from an 18-year-old Nijmegen Breakage Syndrome patient carrying the homozygous NBN c.657_661del5 mutation

Human fibroblasts cells from a female diagnosed with Nijmegen Breakage Syndrome (NBS) carrying the homozygous NBN c.657_661del5 mutation were used to generate integration-free induced pluripotent stem cells (iPSCs) by over-expressing episomal-based plasmids harbouring OCT4, SOX2, NANOG, KLF4, c-MYC and LIN28. The derived iPSC line – ISRM-NBS1 was defined as pluripotent based on (i) expression of pluripotency-associated markers (ii) embryoid body-based differentiation into cell types representative of the three germ layers and (iii) the similarity between the transcriptome of the iPSC line and the human embryonic stem cell line H1 with a Pearson correlation of 0.955

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 51 year 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 (TGFβ, MEK and GSK3β) 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

Activation of the Renin–Angiotensin System Disrupts the Cytoskeletal Architecture of Human Urine-Derived Podocytes

High blood pressure is one of the major public health problems that causes severe disorders in several tissues including the human kidney. One of the most important signaling pathways associated with the regulation of blood pressure is the renin–angiotensin system (RAS), with its main mediator angiotensin II (ANGII). Elevated levels of circulating and intracellular ANGII and aldosterone lead to pro-fibrotic, -inflammatory, and -hypertrophic milieu that causes remodeling and dysfunction in cardiovascular and renal tissues. Furthermore, ANGII has been recognized as a major risk factor for the induction of apoptosis in podocytes, ultimately leading to chronic kidney disease (CKD). In the past, disease modeling of kidney-associated diseases was extremely difficult, as the derivation of kidney originated cells is very challenging. Here we describe a differentiation protocol for reproducible differentiation of sine oculis homeobox homolog 2 (SIX2)-positive urine-derived renal progenitor cells (UdRPCs) into podocytes bearing typical cellular processes. The UdRPCs-derived podocytes show the activation of the renin–angiotensin system by being responsive to ANGII stimulation. Our data reveal the ANGII-dependent downregulation of nephrin (NPHS1) and synaptopodin (SYNPO), resulting in the disruption of the podocyte cytoskeletal architecture, as shown by immunofluorescence-based detection of α-Actinin. Furthermore, we show that the cytoskeletal disruption is mainly mediated through angiotensin II receptor type 1 (AGTR1) signaling and can be rescued by AGTR1 inhibition with the selective, competitive angiotensin II receptor type 1 antagonist, losartan. In the present manuscript we confirm and propose UdRPCs differentiated to podocytes as a unique cell type useful for studying nephrogenesis and associated diseases. Furthermore, the responsiveness of UdRPCs-derived podocytes to ANGII implies potential applications in nephrotoxicity studies and drug screening

Lymphoblast-derived integration-free iPS cell line from a 69-year-old male

AbstractHuman lymphoblast cells were used to generate integration-free induced pluripotent stem (iPS) cells employing episomal plasmids expressing OCT4, SOX2, NANOG, LIN28, C-MYC and L-MYC. The derived iPS cells were defined as pluripotent based on (i) expression of pluripotent-associated markers, (ii) embryoid body-based differentiation into cell types representative of the three germ layers and (iii) the similarity between the transcriptomes of the iPS cell line and the human embryonic stem cell line H1 with a Pearson correlation of 0.95

Chromosomal Instability and Molecular Defects in Induced Pluripotent Stem Cells from Nijmegen Breakage Syndrome Patients

Nijmegen breakage syndrome (NBS) results from the absence of the NBS1 protein, responsible for detection of DNA double-strand breaks (DSBs). NBS is characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition. Here, we show successful reprogramming of NBS fibroblasts into induced pluripotent stem cells (NBS-iPSCs). Our data suggest a strong selection for karyotypically normal fibroblasts to go through the reprogramming process. NBS-iPSCs then acquire numerous chromosomal aberrations and show a delayed response to DSB induction. Furthermore, NBS-iPSCs display slower growth, mitotic inhibition, a reduced apoptotic response to stress, and abnormal cell-cycle-related gene expression. Importantly, NBS neural progenitor cells (NBS-NPCs) show downregulation of neural developmental genes, which seems to be mediated by P53. Our results demonstrate the importance of NBS1 in early human development, shed light on the molecular mechanisms underlying this severe syndrome, and further expand our knowledge of the genomic stress cells experience during the reprogramming process

Lymphoblast-derived integration-free iPS cell line from a 65-year-old Alzheimer's disease patient expressing the TREM2 p.R47H variant

AbstractHuman lymphoblast cells from a male patient diagnosed with Alzheimer's disease (AD) expressing the TREM2 p.R47H variant were used to generate integration-free induced pluripotent stem (iPS) cells employing episomal plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and L-MYC. The iPS cells retained the TREM2 mutation, and were defined as pluripotent based on (i) expression of pluripotent-associated markers, (ii) embryoid body-based differentiation into cell types representative of the three germ layers and (iii) the similarity between the transcriptomes of the iPS cell line and the human embryonic stem cell line H1 with a Pearson correlation of 0.966