[Show abstract][Hide abstract] ABSTRACT: CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR) destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.
[Show abstract][Hide abstract] ABSTRACT: Wnt/beta-catenin signaling plays a central role in guiding the differentiation of the posterior parts of the primitive gut tube into intestinal structures in vivo and some studies suggest that FGF4 is another crucial factor for intestinal development. The aim of this study was to define the effects of Wnt and FGF4 on intestinal commitment in vitro by establishing conditions for differentiation of human pluripotent stem cells (hPSC) into posterior endoderm (hindgut) and further to self-renewing intestinal-like organoids. The most prominent induction of the well-established intestinal marker gene CDX2 was achieved when hPSC-derived definitive endoderm cells were treated with Wnt agonist molecule CHIR99021 during differentiation to hindgut. FGF4 was found to be dispensable during intestinal commitment, but it had an early role in repressing development towards the hepatic lineage. When hindgut stage cells were further cultured in 3D, they formed self-renewing organoid structures containing all major intestinal cell types even without exogenous R-spondin1 (RSPO1), a crucial factor for the culture of epithelial organoids derived from adult intestine. This may be explained by the presence of a mesenchymal compartment in the hPSC-derived organoids. Addition of WNT3A increased the expression of the Paneth cell marker Lysozyme in hPSC-derived organoid cultures, whereas FGF4 inhibited both the formation and maturation of intestinal-like organoids. Similar hindgut and organoid cultures were established from human induced pluripotent stem cells, implying that this approach can be used to create patient-specific intestinal tissue models for disease modeling in vitro.
PLoS ONE 08/2015; 10(7):e0134551. DOI:10.1371/journal.pone.0134551 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transgenic E1-DN mice express a kinase-negative epidermal growth factor receptor in their pancreatic islets and are diabetic from two weeks of age due to impaired postnatal growth of β-cell mass. Here, we characterize the development of hyperglycaemia-induced renal injury in the E1-DN mice. Homozygous mice showed increased albumin excretion rate (AER) at the age of 10 weeks; the albuminuria increased over time and correlated with blood glucose. Morphometric analysis of PAS-stained histological sections and electron microscopy images revealed mesangial expansion in homozygous E1-DN mice, and glomerular sclerosis was observed in the most hyperglycaemic mice. The albuminuric homozygous mice developed also other structural changes in the glomeruli, including thickening of the glomerular basement membrane and widening of podocyte foot processes that are typical for diabetic nephropathy. Increased apoptosis of podocytes was identified as one mechanism contributing to glomerular injury. In addition, nephrin expression was reduced in the podocytes of albuminuric homozygous E1-DN mice. Tubular changes included altered epithelial cell morphology and increased proliferation. In conclusion, hyperglycaemic E1-DN mice develop albuminuria and glomerular and tubular injury typical of human diabetic nephropathy and can serve as a new model to study the mechanisms leading to the development of diabetic nephropathy.
[Show abstract][Hide abstract] ABSTRACT: Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative devel-opment- related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs) and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs.
PLoS ONE 03/2015; 10(3):e0116668. DOI:10.1371/journal.pone.0116668 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 1. Background and utility of this document In 2009 the International Stem Cell Banking Initiative (ISCBI) contributors and the Ethics Working Party of the International Stem Cell Forum published a consensus on principles of best practice for the procurement, cell banking, testing and distribution of human embryonic stem cell (hESC) lines for research purposes , which was broadly also applicable to human induced pluripotent stem cell (hiPSC) lines. Here, we revisit this guidance to consider what the requirements would be for delivery of the early seed stocks of stem cell lines intended for clinical applications. The term 'seed stock' is used here to describe those cryopreserved stocks of cells established early in the passage history of a pluripotent stem cell line in the lab that derived the line or a stem cell bank, hereafter called the 'repository'. The seed stocks should provide cells with suitable documentation and provenance that would enable them to be taken forward for development in human therapeutic applications. WHO recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biologicals and for the characterization of cell banks were updated in 2010 and provide a number of definitions and guiding principles that may apply to stem cells. The term 'cell bank' is used to describe a stock of vials or other containers of cells with consistent composition aliquoted from a single pool of cells of the same culture history (for other specific definitions see PAS 84  and WHO ). Three important assumptions have been made in the preparation of this document. First, that seed stocks of hPSCs are used as starting materials to make cell banks for use in clinical trials. The cell banks made within a clinical trial would need to be established according to Good Manufacturing Practice (GMP) in a facility with a relevant product manufacturing license. These banks would need additional risk assessment focused on the new banking process/reagents and the specific intended clinical application. Second, it has been assumed that undifferenti-ated pluripotent stem cells would not be inocu-lated into patients. Third, where feeder cells are used to culture hPSC lines, their cellular nature and intimate contact with the therapeutic cells means that they should be subject to similar risk assessment and banking procedures as applied to the hPSC cells. It is important to note that responsibility for establishing and updating national regulations for medicinal products relies on National Regulatory Authorities. Therefore, national requirements for cell therapy may vary considerably. Accordingly, it is not intended that this international consensus provides comprehensive guidance that will ensure compliance with requirements in any given jurisdiction. Rather, it is designed to aid the development of clinical grade materials by providing points to consider in the preparation of seed stocks of stem cell lines for use in cell therapy. It may arise that there are circumstances where it is not reasonably possible to meet specific procedures presented in this document. Where this is the case any alternative procedures should be justified and mitigate against any adverse consequences. Finally, this document could also serve as a useful reference to assist in the evaluation of potential sources of candidate cell lines for the development of cell-based medicines, and provide the links necessary to identify some of the key differences in re gulatory requirements between countries. 2. Governance and ethics
Regenerative Medicine 03/2015; DOI:10.2217/RME.14.93 · 2.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human genomic variations, including single nucleotide polymorphisms (SNPs) and copy number variations (CNVs), are associated with several phenotypic traits varying from mild features to hereditary diseases. Several genome-wide studies have reported genomic variants that correlate with gene expression levels in various tissue and cell types.
We studied human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) measuring the SNPs and CNVs with Affymetrix SNP 6 microarrays and expression values with Affymetrix Exon microarrays. We computed the linear relationships between SNPs and expression levels of exons, transcripts and genes, and the associations between gene CNVs and gene expression levels. Further, for a few of the resulted genes, the expression value was associated with both CNVs and SNPs. Our results revealed altogether 217 genes and 584 SNPs whose genomic alterations affect the transcriptome in the same cells. We analyzed the enriched pathways and gene ontologies within these groups of genes, and found out that the terms related to alternative splicing and development were enriched.
Our results revealed that in the human pluripotent stem cells, the expression values of several genes, transcripts and exons were affected due to the genomic variation.
[Show abstract][Hide abstract] ABSTRACT: Growing evidence supports an association between diabetes or abnormal insulin signalling and cancer [1–4]; however, because of their rare occurrence, there is no established epidemiological evidence to support the relationship between neonatal diabetes and congenital hyperinsulinism and cancer occurrence.This article is protected by copyright. All rights reserved.
Diabetic Medicine 12/2014; 32(5). DOI:10.1111/dme.12670 · 3.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Generation of validated human induced pluripotent stem cells (iPSCs) for biobanking is essential for exploring the full potential of iPSCs in disease modeling and drug discovery. Peripheral blood mononuclear cells (PBMCs) are attractive targets for reprogramming, because blood is collected by a routine clinical procedure and is a commonly stored material in biobanks. Generation of iPSCs from blood cells has previously been reported using integrative retroviruses, episomal Sendai viruses, and DNA plasmids. However, most of the published protocols require expansion and/or activation of a specific cell population from PBMCs. We have recently collected a PBMC cohort from the Finnish population containing more than 2,000 subjects. Here we report efficient generation of iPSCs directly from PBMCs in feeder-free conditions in approximately 2 weeks. The produced iPSC clones are pluripotent and transgene-free. Together, these properties make this novel method a powerful tool for large-scale reprogramming of PBMCs and for iPSC biobanking.
[Show abstract][Hide abstract] ABSTRACT: Retinopathy is an important manifestation of trifunctional protein (TFP) deficiencies but not of other defects of fatty acid oxidation. The common homozygous mutation in the TFP α-subunit gene HADHA (hydroxyacyl-CoA dehydrogenase), c.1528G>C, affects the long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity of TFP and blindness in infancy. The pathogenesis of the retinopathy is unknown. This study aimed to utilize human induced pluripotent stem cell (hiPSC) technology to create a disease model for the disorder, and to derive clues for retinopathy pathogenesis.
We implemented hiPSC technology to generate LCHAD deficiency (LCHADD) patient-specific retinal pigment epithelial (RPE) monolayers. These patient and control RPEs were extensively characterized for function and structure, as well as for lipid composition by mass spectrometry.
The hiPSC-derived RPE monolayers of patients and controls were functional, as they both were able to phagocytose the photoreceptor outer segments in vitro. Interestingly, the patient RPEs had intense cytoplasmic neutral lipid accumulation, and lipidomic analysis revealed an increased triglyceride accumulation. Further, patient RPEs were small and irregular in shape, and their tight junctions were disorganized. Their ultratructure showed decreased pigmentation, few melanosomes, and more melanolysosomes.
We demonstrate that the RPE cell model reveals novel early pathogenic changes in LCHADD retinopathy, with robust lipid accumulation, inefficient pigmentation that is evident soon after differentiation, and a defect in forming tight junctions inducing apoptosis. We propose that LCHADD-RPEs are an important model for mitochondrial TFP retinopathy, and that their early pathogenic changes contribute to infantile blindness of LCHADD.
[Show abstract][Hide abstract] ABSTRACT: Pluripotent stem cells are capable of differentiating into cells of any tissue. The fact that iPS cell lines can be produced from skin cells or blood cells and directed to differentiate into a desired direction makes it possible to investigate e.g. myocardial or nerve cells having a disease-associated genotype. This will enable the development of experimental models of disease mechanisms and also apply them to drug screening, which may allow the development of novel types of treatment. In the future it may become possible to replace injured cells of a patient with autologous iPS cell derived transplants.
[Show abstract][Hide abstract] ABSTRACT: All forms of diabetes mellitus (DM) are characterized by the loss of functional pancreatic β cell mass, leading to insufficient insulin secretion. Thus, identification of novel approaches to protect and restore β cells is essential for the development of DM therapies. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-inducible protein, but its physiological role in mammals has remained obscure. We generated MANF-deficient mice that strikingly develop severe diabetes due to progressive postnatal reduction of β cell mass, caused by decreased proliferation and increased apoptosis. Additionally, we show that lack of MANF in vivo in mouse leads to chronic unfolded protein response (UPR) activation in pancreatic islets. Importantly, MANF protein enhanced β cell proliferation in vitro and overexpression of MANF in the pancreas of diabetic mice enhanced β cell regeneration. We demonstrate that MANF specifically promotes β cell proliferation and survival, thereby constituting a therapeutic candidate for β cell protection and regeneration.
[Show abstract][Hide abstract] ABSTRACT: Placental lactogen (PL) induced serotonergic signaling is essential for gestational β-cell mass expansion. We have previously shown that intact Epidermal growth factor -receptor (EGFR) function is a crucial component of this pathway. We now explored more specifically the link between EGFR and pregnancy-induced β-cell mass compensation. Islets were isolated from wild-type and β-cell-specific EGFR-dominant negative mice (E1-DN), stimulated with PL and analyzed for β-cell proliferation and expression of genes involved in gestational β-cell growth. β-cell mass dynamics were analyzed both with traditional morphometrical methods and three-dimensional optical projection tomography (OPT) of whole-mount insulin-stained pancreata. Insulin-positive volume analyzed with OPT increased 1.4-fold at gestational day 18.5 (GD18.5) when compared to non-pregnant mice. Number of islets peaked by GD13.5 (680 vs 1134 islets per pancreas, non-pregnant vs. GD13.5). PL stimulated beta cell proliferation in the wild-type islets, whereas the proliferative response was absent in the E1-DN mouse islets. Serotonin synthesizing enzymes were upregulated similarly in both the wild-type and E1-DN mice. However, while survivin (Birc5) mRNA was upregulated 5.5-fold during pregnancy in the wild-type islets, no change was seen in the E1-DN pregnant islets. PL induced survivin expression also in isolated islets and this was blocked by EGFR inhibitor gefitinib, mTOR inhibitor rapamycin and MEK inhibitor PD0325901. Our 3D-volumetric analysis of β-cell mass expansion during murine pregnancy revealed that islet number increases during pregnancy. In addition, our results suggest that EGFR signaling is required for lactogen-induced survivin expression via MAPK and mTOR pathways.
PLoS ONE 04/2014; 9(4):e93651. DOI:10.1371/journal.pone.0093651 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neural crest (NC) cells are specified at the border of neural plate and epiderm. They are capable of differentiating into various somatic cell types, including craniofacial and peripheral nerve tissues. Notch signaling plays significant roles during neurogenesis; however, its function during human NC development is poorly understood. Here, we generated self-renewing premigratory NC-like cells (pNCCs) from human pluripotent stem cells and investigated the roles of Notch signaling during the NC differentiation. pNCCs expressed various NC specifier genes, including SLUG, SOX10 and TWIST1, and were able to differentiate into most NC derivatives. Blocking Notch signaling during the pNCC differentiation suppressed the expression of NC specifier genes. In contrast, ectopic expression of activated Notch1 intracellular domain (NICD1) augmented the expression of NC specifier genes, and NICD1 was found to bind at their promoter regions. Notch activity was also required for the maintenance of premigratory NC state, and suppression of Notch led to generation of NC-derived neurons. Taken together, we provide a protocol for the generation of pNCCs, and show that Notch signaling regulates the formation, migration and differentiation of NC from hPSCs.