Sylvia Merkert

Generation of Induced Pluripotent Stem Cells from Human Cord Blood

Induced pluripotent stem cells (iPSCs) may represent an ideal cell source for future regenerative therapies. A critical issue concerning the clinical use of patient-specific iPSCs is the accumulation of mutations in somatic (stem) cells over an organisms lifetime. Acquired somatic mutations are passed onto iPSCs during reprogramming and may be associated with loss of cellular functions and cancer formation. Here we report the generation of human iPSCs from cord blood (CB) as a juvenescent cell source. CBiPSCs show characteristics typical of embryonic stem cells and can be differentiated into derivatives of all three germ layers, including functional cardiomyocytes. For future therapeutic production of autologous and allogeneic iPSC derivatives, CB could be routinely harvested for public and commercial CB banks without any donor risk. CB could readily become available for pediatric patients and, in particular, for newborns with genetic diseases or congenital malformations.

Long term expansion of undifferentiated human iPS and ES cells in suspension culture using a defined medium.

Therapeutic application of stem cell derivatives requires large quantities of cells produced in defined media that cannot be produced via conventional adherent culture. We have applied human induced pluripotent stem (hiPS) cells expressing eGFP under control of the OCT4 promoter to establish the expansion of undifferentiated human embryonic stem (hES) and hiPS cells in suspension culture. A defined culture medium has been identified that results in up to six-fold increase in cell numbers within four days. Our culture system is based on initial single cell dissociation which is critical for standardized process inoculation. HES / hiPS cells were expanded for up to 17 passages. The cells maintained a stable karyotype, their expression of pluripotency markers and their potential to differentiate into derivatives of all three germ layers. The ability to expand HES / hiPS cells in a scalable suspension culture represents a critical step towards standardized production in stirred bioreactors.

Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.

Gene Correction of Human Induced Pluripotent Stem Cells Repairs the Cellular Phenotype in Pulmonary Alveolar Proteinosis

RATIONALE Hereditary pulmonary alveolar proteinosis (hPAP) caused by granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α-chain (CSF2RA) deficiency is a rare, life-threatening lung disease characterized by accumulation of proteins and phospholipids in the alveolar spaces. The disease is caused by a functional insufficiency of alveolar macrophages, which require GM-CSF signaling for terminal differentiation and effective degradation of alveolar proteins and phospholipids. Therapeutic options are extremely limited, and the pathophysiology underlying the defective protein degradation in hPAP alveolar macrophages remains poorly understood. OBJECTIVES To further elucidate the cellular mechanisms underlying hPAP and evaluate novel therapeutic strategies, we here investigated the potential of hPAP patient-derived induced pluripotent stem cell (PAP-iPSCs) derived monocytes and macrophages. METHODS Patient-specific PAP-iPSCs were generated from CD34(+) bone marrow cells of a CSF2RA-deficient patient with PAP. We assessed pluripotency, chromosomal integrity, and genetic correction of established iPSC lines. On hematopoietic differentiation, genetically corrected or noncorrected monocytes and macrophages were investigated in GM-CSF-dependent assays. MEASUREMENTS AND MAIN RESULTS Although monocytes and macrophages differentiated from noncorrected PAP-iPSCs exhibited distinct defects in GM-CSF-dependent functions, such as perturbed CD11b activation, phagocytic activity, and STAT5 phosphorylation after GM-CSF exposure and lack of GM-CSF uptake, these defects were fully repaired on lentiviral gene transfer of a codon-optimized CSF2RA-cDNA. CONCLUSIONS These data establish PAP-iPSC-derived monocytes and macrophages as a valid in vitro disease model of CSF2RA-deficient PAP, and introduce gene-corrected iPSC-derived monocytes and macrophages as a potential autologous cell source for innovative therapeutic strategies. Transplantation of such cells to patients with hPAP could serve as a paradigmatic proof for the potential of iPSC-derived cells in clinical gene therapy.

Derivation and Characterization of Sleeping Beauty Transposon-Mediated Porcine Induced Pluripotent Stem Cells

The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (iPS) cells showed long-term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives of the 3 germ layers. Upon injection of putative iPS cells under the skin of immunodeficient mice, we observed teratomas in 3 of 6 cases. These results form the basis for in-depth studies toward the derivation of porcine iPS cells, which hold great promise for preclinical testing of novel cell therapies in the pig model.

Primate iPS cells as tools for evolutionary analyses

Induced pluripotent stem cells (iPSCs) are regarded as a central tool to understand human biology in health and disease. Similarly, iPSCs from non-human primates should be a central tool to understand human evolution, in particular for assessing the conservation of regulatory networks in iPSC models. Here, we have generated human, gorilla, bonobo and cynomolgus monkey iPSCs and assess their usefulness in such a framework. We show that these cells are well comparable in their differentiation potential and are generally similar to human, cynomolgus and rhesus monkey embryonic stem cells (ESCs). RNA sequencing reveals that expression differences among clones, individuals and stem cell type are all of very similar magnitude within a species. In contrast, expression differences between closely related primate species are three times larger and most genes show significant expression differences among the analyzed species. However, pseudogenes differ more than twice as much, suggesting that evolution of expression levels in primate stem cells is rapid, but constrained. These patterns in pluripotent stem cells are comparable to those found in other tissues except testis. Hence, primate iPSCs reveal insights into general primate gene expression evolution and should provide a rich source to identify conserved and species-specific gene expression patterns for cellular phenotypes.

Efficient Designer Nuclease-Based Homologous Recombination Enables Direct PCR Screening for Footprintless Targeted Human Pluripotent Stem Cells

Summary Genetic engineering of human induced pluripotent stem cells (hiPSCs) via customized designer nucleases has been shown to be significantly more efficient than conventional gene targeting, but still typically depends on the introduction of additional genetic selection elements. In our study, we demonstrate the efficient nonviral and selection-independent gene targeting in human pluripotent stem cells (hPSCs). Our high efficiencies of up to 1.6% of gene-targeted hiPSCs, accompanied by a low background of randomly inserted transgenes, eliminated the need for antibiotic or fluorescence-activated cell sorting selection, and allowed the use of short donor oligonucleotides for footprintless gene editing. Gene-targeted hiPSC clones were established simply by direct PCR screening. This optimized approach allows targeted transgene integration into safe harbor sites for more predictable and robust expression and enables the straightforward generation of disease-corrected, patient-derived iPSC lines for research purposes and, ultimately, for future clinical applications.

Fast and Efficient Multitransgenic Modification of Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a prime cell source for pharmacological research and regenerative therapies because of their extensive expansion potential and their ability to differentiate into essentially all somatic lineages in vitro. Improved methods to stably introduce multiple transgenes into hPSCs will promote, for example, their preclinical testing by facilitating lineage differentiation and purification in vitro and the subsequent in vivo monitoring of respective progenies after their transplantation into relevant animal models. To date, the establishment of stable transgenic hPSC lines is still laborious and time-consuming. Current limitations include the low transfection efficiency of hPSCs via nonviral methods, the inefficient recovery of genetically engineered clones, and the silencing of transgene expression. Here we describe a fast, electroporation-based method for the generation of multitransgenic hPSC lines by overcoming the need for any preadaptation of conventional hPSC cultures to feeder-free conditions before genetic manipulation. We further show that the selection for a single antibiotic resistance marker encoded on one plasmid allowed for the stable genomic (co-)integration of up to two additional, independent expression plasmids. The method thereby enables the straightforward, nonviral generation of valuable multitransgenic hPSC lines in a single step. Practical applicability of the method is demonstrated for antibiotic-based lineage enrichment in vitro and for sodium iodide symporter transgene-based in situ cell imaging after intramyocardial cell infusion into explanted pig hearts.

Targeted genome engineering using designer nucleases: State of the art and practical guidance for application in human pluripotent stem cells

Within the last years numerous publications successfully applied sequence specific designer nucleases for genome editing in human PSCs. However, despite this abundance of reports together with the rapid development and improvement accomplished with the technology, it is still difficult to choose the optimal methodology for a specific application of interest. With focus on the most suitable approach for specific applications, we present a practical guidance for successful gene editing in human PSCs using designer nucleases. We discuss experimental considerations, limitations and critical aspects which will guide the investigator for successful implementation of this technology.

Induction of Pluripotent Stem Cells from a Cynomolgus Monkey Using a Polycistronic Simian Immunodeficiency Virus–Based Vector, Differentiation Toward Functional Cardiomyocytes, and Generation of Stably Expressing Reporter Lines

Induced pluripotent stem cells (iPSCs) represent a novel cell source for regenerative therapies. Many emerging iPSC-based therapeutic concepts will require preclinical evaluation in suitable large animal models. Among the large animal species frequently used in preclinical efficacy and safety studies, macaques show the highest similarities to humans at physiological, cellular, and molecular levels. We have generated iPSCs from cynomolgus monkeys (Macaca fascicularis) as a segue to regenerative therapy model development in this species. Because typical human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors show poor transduction of simian cells, a simian immunodeficiency virus (SIV)-based vector was chosen for efficient transduction of cynomolgus skin fibroblasts. A corresponding polycistronic vector with codon-optimized reprogramming factors was constructed for reprogramming. Growth characteristics as well as cell and colony morphology of the resulting cynomolgus iPSCs (cyiPSCs) were demonstrated to be almost identical to cynomolgus embryonic stem cells (cyESCs), and cyiPSCs expressed typical pluripotency markers including OCT4, SOX2, and NANOG. Furthermore, differentiation in vivo and in vitro into derivatives of all three germ layers, as well as generation of functional cardiomyocytes, could be demonstrated. Finally, a highly efficient technique for generation of transgenic cyiPSC clones with stable reporter expression in undifferentiated cells as well as differentiated transgenic cyiPSC progeny was developed to enable cell tracking in recipient animals. In conclusion, our data indicate that cyiPSCs represent a valuable cell source for establishment of macaque-based allogeneic and autologous preclinical cell transplantation models for various fields of regenerative medicine.