Csilla Nemes

Generation of induced pluripotent stem cells from human foetal fibroblasts using the Sleeping Beauty transposon gene delivery system

Transposon gene delivery systems offer an alternative, non-viral-based approach to generate induced pluripotent stem cells (iPSCs). Here we used the Sleeping Beauty (SB) transposon to generate four human iPSC lines from foetal fibroblasts. In contrast to other gene delivery systems, the SB transposon does not exhibit an integration bias towards particular genetic elements, thereby reducing the risk of insertional mutagenesis. Furthermore, unlike the alternative transposon piggyBac, SB has no SB-like elements within the human genome, minimising the possibility of mobilising endogenous transposon elements. All iPSC lines exhibited the expected characteristics of pluripotent human cells, including the ability to differentiate to derivatives of all three germ layers in vitro. Re-expression of the SB transposase in the iPSCs after reprogramming resulted in the mobilisation of some of the transposons. These results indicate that the SB transposon system is a useful addition to methods for generating human iPSCs, both for basic and applied biomedical research, and in the context of future therapeutic application.

Neurosphere based differentiation of human IPSC improves astrocyte differentiation

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers  GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes.

Generation of rabbit pluripotent stem cell lines.

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.

Cloning and characterization of rabbit POU5F1, SOX2, KLF4, C-MYC and NANOG pluripotency-associated genes

While the rabbit (Oryctolagus cuniculus) is an important research model for aspects of human development and disease that cannot be studied in rodents, the lack of data on the genetic regulation of rabbit preimplantation development is a limitation. To assist in the understanding of this process, our aim was to isolate and characterize genes necessary for the induction and maintenance of cellular pluripotency. We are the first to report the isolation of complete coding regions of rabbit SOX2, KLF4, C-MYC and NANOG, which encode transcription factors that play crucial regulatory roles during early mammalian embryonic development. We determined the exon-intron boundaries and chromosomal localization of these genes using computational analysis. The sequences of mRNA and translated protein of the newly identified genes and those of POU5F1 were aligned to their mammalian orthologs to determine the degree of evolutionary conservation. Furthermore, the expression of these genes in embryonic and adult cells was studied at the mRNA and protein levels. We found the sequences and the expression pattern of these pluripotency-associated genes to be highly conserved between human and rabbit, indicating that the rabbit would be a valuable model for human preimplantation development. Implementing the newly identified genes either as biomarkers or as reprogramming factors might also pave the way towards the creation of stable pluripotent rabbit cell lines.

Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system

One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo.

Grafted murine induced pluripotent stem cells prevent death of injured rat motoneurons otherwise destined to die

Human plexus injuries often include the avulsion of one or more ventral roots, resulting in debilitating conditions. In this study the effects of undifferentiated murine iPSCs on damaged motoneurons were investigated following avulsion of the lumbar 4 (L4) ventral root, an injury known to induce the death of the majority of the affected motoneurons. Avulsion and reimplantation of the L4 ventral root (AR procedure) were accompanied by the transplantation of murine iPSCs into the injured spinal cord segment in rats. Control animals underwent ventral root avulsion and reimplantation, but did not receive iPSCs. The grafted iPSCs induced an improved reinnervation of the reimplanted ventral root by the host motoneurons as compared with the controls (number of retrogradely labeled motoneurons: 503 ± 38 [AR+iPSCs group] vs 48 ± 6 [controls, AR group]). Morphological reinnervation resulted in a functional recovery, i.e. the grafted animals exhibited more motor units in their reinnervated hind limb muscles, which produced a greater force than that in the controls (50 ± 2.1% vs 11.9 ± 4.2% maximal tetanic tension [% ratio of operated/intact side]). Grafting of undifferentiated iPSCs downregulated the astroglial activation within the L4 segment. The grafted cells differentiated into neurons and astrocytes in the injured cord. The grafted iPSCs, host neurons and glia were found to produce the cytokines and neurotrophic factors MIP-1a, IL-10, GDNF and NT-4. These findings suggest that, following ventral root avulsion injury, iPSCs are able to induce motoneuron survival and regeneration through combined neurotrophic and cytokine modulatory effects.

Establishment of induced pluripotent stem cell (iPSC) line from a 63-year old patient with late onset Alzheimer's disease (LOAD).

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically characterised 63-year old woman with late onset Alzheimers disease (LOAD). The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The transgene-free iPSC showed pluripotency verified by immunocytochemistry for pluripotency markers and differentiated spontaneously towards the 3 germ layers in vitro. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to further study the pathomechanism of sporadic AD, to identify early biomarkers and also for drug testing and gene therapy studies.

Establishment of induced pluripotent stem cell (iPSC) line from a 57-year old patient with sporadic Alzheimer's disease

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically characterised 57-year old woman with sporadic Alzheimers disease. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The transgene-free iPSC showed pluripotency verified by immunocytochemistry for pluripotency markers and differentiated spontaneously towards the 3 germ layers in vitro. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to further study the pathomechanism of sporadic AD, to identify early biomarkers and also for drug testing and gene therapy studies.

Establishment of EHMT1 mutant induced pluripotent stem cell (iPSC) line from a 11-year-old Kleefstra syndrome (KS) patient with autism and normal intellectual performance

Peripheral blood was collected from a clinically characterized female Kleefstra syndrome patient with a heterozygous, de novo, premature termination codon (PTC) mutation (NM_024757.4(EHMT1):c.3413G>A; p.Trp1138Ter). Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the human OSKM transcription factors using the Sendai-virus (SeV) delivery system. The pluripotency of transgene-free iPSC line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of Kleefstra syndrome, also for drug testing, early biomarker discovery and gene therapy studies.

Generation of human induced pluripotent stem cell (iPSC) line from an unaffected female carrier of Mucopolysaccharidosis type II (MPS II) disorder

Peripheral blood was collected from a 39-year-old unaffected female carrier of an X-linked recessive mutation of Iduronate 2-sulfatase gene (NM_000202.7(IDS):c.85C>T) causing MPS II (OMIM 309900). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC showed normal karyotype. The line offers a good platform to study MPS II pathophysiology, for drug testing, early biomarker discovery and gene therapy studies.