Malte Sgodda

Optimal reprogramming factor stoichiometry increases colony numbers and affects molecular characteristics of murine induced pluripotent stem cells.

Somatic cells can be reprogrammed toward pluripotency by overexpression of a set of transcription factors, yielding induced pluripotent stem cells (iPSCs) with features similar to embryonic stem cells. Little is known to date about stoichiometric requirements of the individual reprogramming factors (RFs) for efficient reprogramming and especially about whether stoichiometry also influences the quality of derived iPSCs. To address this important issue, we chose bicistronic lentiviral vectors coexpressing fluorescent reporters (eGFP, dTomato, Cerulean, or Venus) along with the canonical RFs to transduce a bulk of murine embryonic fibroblasts (MEFs). Using a flow cytometric approach, we were able to independently and proportionally quantify all fluorophores in multiple‐infected MEFs and more importantly could sort these cells into all 16 stoichiometric combinations of high or moderate expression of the four factors. On average, we obtained about 600 alkaline phosphatase‐expressing colonies from 20,000 seeded cells. Interestingly, only seven different stoichiometric ratios gave rise to any colonies at all. The by far most colonies were obtained from those fractions, where Oct4 was in excess over the other three factors (2,386 colonies/20,000 cells), or where both Oct4 and c‐Myc were in excess over Sox2 and Klf4 (1,593 colonies/20,000 cells). Our findings suggest that increased Oct4 levels opposite to modest ones for Sox2 and Klf4 are required for satisfying reprogramming efficiencies and that these stoichiometries are also highly beneficial for achieving a stable pluripotent state independent of ectopic RF expression. Finally, the eligible Oct4high, Sox2low, and Klf4low subpopulation only resembles a small fraction of cells targeted by equal vector amounts, suggesting the necessity to address stoichiometry also in alternative approaches for iPSC generation or between different experimental systems.

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH −/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH −/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH −/− iPS cell lines, we aggregated FAH −/−-iPS cells with tetraploid embryos and obtained entirely FAH −/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH −/− mice. Then, we transduced FAH cDNA into the FAH −/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.

Sustained Knockdown of a Disease-Causing Gene in Patient-Specific Induced Pluripotent Stem Cells Using Lentiviral Vector-Based Gene Therapy

Patient‐specific induced pluripotent stem cells (iPSCs) hold great promise for studies on disease‐related developmental processes and may serve as an autologous cell source for future treatment of many hereditary diseases. New genetic engineering tools such as zinc finger nucleases and transcription activator‐like effector nuclease allow targeted correction of monogenetic disorders but are very cumbersome to establish. Aiming at studies on the knockdown of a disease‐causing gene, lentiviral vector‐mediated expression of short hairpin RNAs (shRNAs) is a valuable option, but it is limited by silencing of the knockdown construct upon epigenetic remodeling during differentiation. Here, we propose an approach for the expression of a therapeutic shRNA in disease‐specific iPSCs using third‐generation lentiviral vectors. Targeting severe α‐1‐antitrypsin (A1AT) deficiency, we overexpressed a human microRNA 30 (miR30)‐styled shRNA directed against the PiZ variant of A1AT, which is known to cause chronic liver damage in affected patients. This knockdown cassette is traceable from clonal iPSC lines to differentiated hepatic progeny via an enhanced green fluorescence protein reporter expressed from the same RNA‐polymerase II promoter. Importantly, the cytomegalovirus i/e enhancer chicken β actin (CAG) promoter‐driven expression of this construct is sustained without transgene silencing during hepatic differentiation in vitro and in vivo. At low lentiviral copy numbers per genome we confirmed a functional relevant reduction (−66%) of intracellular PiZ protein in hepatic cells after differentiation of patient‐specific iPSCs. In conclusion, we have demonstrated that lentiviral vector‐mediated expression of shRNAs can be efficiently used to knock down and functionally evaluate disease‐related genes in patient‐specific iPSCs.

MicroRNA-199a-5p inhibition enhances the liver repopulation ability of human embryonic stem cell-derived hepatic cells

BACKGROUND & AIMS Current hepatic differentiation protocols for human embryonic stem cells (ESCs) require substantial improvements. MicroRNAs (miRNAs) have been reported to regulate hepatocyte cell fate during liver development, but their utility to improve hepatocyte differentiation from ESCs remains to be investigated. Therefore, our aim was to identify and to analyse hepatogenic miRNAs for their potential to improve hepatocyte differentiation from ESCs. METHODS By miRNA profiling and in vitro screening, we identified miR-199a-5p among several potential hepatogenic miRNAs. Transplantation studies of miR-199a-5p-inhibited hepatocyte-like cells (HLCs) in the liver of immunodeficient fumarylacetoacetate hydrolase knockout mice (Fah(-/-)/Rag2(-/-)/Il2rg(-/-)) were performed to assess their in vivo liver repopulation potential. For target determination, western blot and luciferase reporter assay were carried out. RESULTS miRNA profiling revealed 20 conserved candidate hepatogenic miRNAs. By miRNA screening, only miR-199a-5p inhibition in HLCs was found to be able to enhance the in vitro hepatic differentiation of mouse as well as human ESCs. miR-199a-5p inhibition in human ESCs-derived HLCs enhanced their engraftment and repopulation capacity in the liver of Fah(-/-)/Rag2(-/-)/Il2rg(-/-) mice. Furthermore, we identified SMARCA4 and MST1 as novel targets of miR-199a-5p that may contribute to the improved hepatocyte generation and in vivo liver repopulation. CONCLUSIONS Our findings demonstrate that miR-199a-5p inhibition in ES-derived HLCs leads to improved hepatocyte differentiation. Upon transplantation, HLCs were able to engraft and repopulate the liver of Fah(-/-)/Rag2(-/-)/Il2rg(-/-) mice. Thus, our findings suggest that miRNA modulation may serve as a promising approach to generate more mature HLCs from stem cell sources for the treatment of liver diseases.

Efficient Hematopoietic Redifferentiation of Induced Pluripotent Stem Cells Derived from Primitive Murine Bone Marrow Cells

Heterogeneity among induced pluripotent stem cell (iPSC) lines with regard to their gene expression profile and differentiation potential has been described and at least partly linked to the tissue of origin. Here, we generated iPSCs from primitive [lineage negative (Lin(neg))] and nonadherent differentiated [lineage positive (Lin(pos))] bone marrow cells (BM-iPSC), and compared their differentiation potential to that of fibroblast-derived iPSCs (Fib-iPSC) and embryonic stem cells (ESC). In the undifferentiated state, individual iPSC clones but also ESCs proved remarkably similar when analyzed for alkaline phosphatase and SSEA-1 staining, endogenous expression of the pluripotency genes Nanog, Oct4, and Sox2, or global gene expression profiles. However, substantial differences between iPSC clones were observed after induction of differentiation, which became most obvious upon cytokine-mediated instruction toward the hematopoietic lineage. All 3 BM-iPSC lines derived from undifferentiated Lin(neg) cells yielded high proportions of cells expressing the hematopoietic differentiation marker CD41 and in 2 of these lines high proportions of CD41+/ CD45+ cells were detected. In contrast, little hematopoiesis-specific surface marker expression was detected in 4 Lin(pos) BM-iPSC and 3 Fib-iPSC lines. These results were corroborated by functional studies demonstrating robust colony outgrowth from hematopoietic progenitors in 2 of the Lin(neg) BM-iPSCs only. Thus, in conclusion, our data demonstrate efficient generation of iPSCs from primitive hematopoietic tissue as well as efficient hematopoietic redifferentiation for Lin(neg) BM-iPSC lines, thereby supporting the notion of an epigenetic memory in iPSCs.

Hepatic differentiation of pluripotent stem cells

Abstract In regenerative medicine pluripotent stem cells are considered to be a valuable self-renewing source for therapeutic cell transplantations, given that a functional organ-specific phenotype can be acquired by in vitro differentiation protocols. Furthermore, derivatives of pluripotent stem cells that mimic fetal progenitor stages could serve as an important tool to analyze organ development with in vitro approaches. Because of ethical issues regarding the generation of human embryonic stem (ES) cells, other sources for pluripotent stem cells are intensively studied. Like in less developed vertebrates, pluripotent stem cells can be generated from the female germline even in mammals, via parthenogenetic activation of oocytes. Recently, testis-derived pluripotent stem cells were derived from the male germline. Therefore, we compared two different hepatic differentiation approaches and analyzed the generation of definitive endoderm progenitor cells and their further maturation into a hepatic phenotype using murine parthenogenetic ES cells, germline-derived pluripotent stem cells, and ES cells. Applying quantitative RT-PCR, both germline-derived pluripotent cell lines show similar differentiation capabilities as normal murine ES cells and can be considered an alternative source for pluripotent stem cells in regenerative medicine.

Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG)

PMM2-CDG, formerly known as congenital disorder of glycosylation-Ia (CDG-Ia), is caused by mutations in the gene encoding phosphomannomutase 2 (PMM2). This disease is the most frequent form of inherited CDG-diseases affecting protein N-glycosylation in human. PMM2-CDG is a multisystemic disease with severe psychomotor and mental retardation. In order to study the pathophysiology of PMM2-CDG in a human cell culture model, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of a PMM2-CDG-patient (PMM2-iPSCs). Expression of pluripotency factors and in vitro differentiation into cell types of the three germ layers was unaffected in the analyzed clone PMM2-iPSC-C3 compared with nondiseased human pluripotent stem cells (hPSCs), revealing no broader influence of the PMM2 mutation on pluripotency in cell culture. Analysis of gene expression by deep-sequencing did not show obvious differences in the transcriptome between PMM2-iPSC-C3 and nondiseased hPSCs. By multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) we could show that PMM2-iPSC-C3 exhibit the common hPSC N-glycosylation pattern with high-mannose-type N-glycans as the predominant species. However, phosphomannomutase activity of PMM2-iPSC-C3 was 27% compared with control hPSCs and lectin staining revealed an overall reduced protein glycosylation. In addition, quantitative assessment of N-glycosylation by xCGE-LIF showed an up to 40% reduction of high-mannose-type N-glycans in PMM2-iPSC-C3, which was in concordance to the observed reduction of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide compared with control hPSCs. Thus we could model the PMM2-CDG disease phenotype of hypoglycosylation with patient derived iPSCs in vitro. Knock-down of PMM2 by shRNA in PMM2-iPSC-C3 led to a residual activity of 5% and to a further reduction of the level of N-glycosylation. Taken together we have developed human stem cell-based cell culture models with stepwise reduced levels of N-glycosylation now enabling to study the role of N-glycosylation during early human development.

Biphasic modulation of Wnt signaling supports efficient foregut endoderm formation from human pluripotent stem cells.

Pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) are of great promise in regenerative medicine, including molecular studies of disease mechanisms, if the affected cell type can be authentically generated during in vitro differentiation. Most existing protocols aim to mimic embryonic development steps by the supplementation of specific cytokines and small molecules, but the involved signaling pathways need further exploration. In this study, we investigated enhanced initial activation of Wnt signaling for definitive endoderm formation and subsequent rapid shutdown of Wnt signaling for proper foregut endoderm specification using 3 μM CHIR99021 and 0.5 μg/mL of secreted frizzled‐related protein 5 (sFRP‐5) for biphasic modulation of the Wnt pathway. The definitive endoderm and foregut endoderm differentiation capabilities of Wnt pathway‐modulated cells were determined based on the expression levels of the endodermal transcription factors SOX17 and FOXA2 and those of the transcription activator GATA4 and the α‐fetoprotein (AFP) gene, respectively. Furthermore, the resulting biphasic Wnt pathway modulation was investigated at the protein level by analyzing phosphorylation of glycogen synthase kinase 3 beta (GSK3β) and β‐catenin. Finally, Wnt target gene expression was determined using an improved lentiviral reporter construct that enabled robust T‐cell transcription factor 4 (TCF4)/lymphoid enhancer‐binding factor 1 (LEF1)‐mediated luciferase expression in differentiating pluripotent stem cells. In conclusion, we demonstrated robust, homogeneous, and efficient derivation of foregut endodermal cells by inducing a biphasic modulation of the Wnt signaling pathway.

Improved lentiviral gene transfer into human embryonic stem cells grown in co-culture with murine feeder and stroma cells.

Abstract Genetic modification of human embryonic stem cells (hESCs) using biophysical DNA transfection methods are hampered by the very low single cell survival rate and cloning efficiency of hESCs. Lentiviral gene transfer strategies are widely used to genetically modify hESCs but limited transduction efficiencies in the presence of feeder or stroma cells present problems, particularly if vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped viral particles are applied. Here, we investigated whether the recently described semen derived enhancer of virus infection (SEVI) and alternative viral envelope proteins derived from either Gibbon ape leukaemia virus (GALV) or feline leukaemia virus (RD114) are applicable for transducing hESCs during co-culture with feeder or stroma cells. Our first set of experiments demonstrates that SEVI has no toxic effect on murine or hESCs and that exposure to SEVI does not interfere with the pluripotency-associated phenotype. Focusing on hESCs, we were able to further demonstrate that SEVI increases the transduction efficiencies of GALV and RD114 pseudotyped lentiviral vectors. More importantly, aiming at targeted differentiation of hESCs into functional somatic cell types, GALV pseudotyped lentiviral particles could efficiently and exclusively transduce hESCs grown in co-culture with OP9-GFP stroma cells (which were often used to induce differentiation into haematopoietic derivatives).

Hepatic differentiation of murine disease-specific induced pluripotent stem cells allows disease modelling in vitro.

Direct reprogramming of somatic cells into pluripotent cells by retrovirus-mediated expression of OCT4, SOX2, KLF4, and C-MYC is a promising approach to derive disease-specific induced pluripotent stem cells (iPSCs). In this study, we focused on three murine models for metabolic liver disorders: the copper storage disorder Wilsons disease (toxic-milk mice), tyrosinemia type 1 (fumarylacetoacetate-hydrolase deficiency, FAH−/− mice), and alpha1-antitrypsin deficiency (PiZ mice). Colonies of iPSCs emerged 2-3 weeks after transduction of fibroblasts, prepared from each mouse strain, and were maintained as individual iPSC lines. RT-PCR and immunofluorescence analyses demonstrated the expression of endogenous pluripotency markers. Hepatic precursor cells could be derived from these disease-specific iPSCs applying an in vitro differentiation protocol and could be visualized after transduction of a lentiviral albumin-GFP reporter construct. Functional characterization of these cells allowed the recapitulation of the disease phenotype for further studies of underlying molecular mechanisms of the respective disease.