Mikkel A. Rasmussen

Transient p53 Suppression Increases Reprogramming of Human Fibroblasts without Affecting Apoptosis and DNA Damage

Summary The discovery of human-induced pluripotent stem cells (iPSCs) has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently, knockout of the Tumor Protein 53 (p53) gene was reported to facilitate reprogramming but unfortunately also led to genomic instability. Here, we report that transient suppression of p53 during nonintegrative reprogramming of human fibroblasts leads to a significant increase in expression of pluripotency markers and overall number of iPSC colonies, due to downstream suppression of p21, without affecting apoptosis and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns, displayed normal karyotypes, contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion, transient p53 suppression increases reprogramming efficiency without affecting genomic stability, rendering the method suitable for in vitro mechanistic studies with the possibility for future clinical translation.

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.

Ultrastructural and molecular distinctions between the porcine inner cell mass and epiblast reveal unique pluripotent cell states.

Characterization of the pluripotent cell populations within the porcine embryo is essential for understanding pluripotency and self‐renewal regulation in the inner cell mass (ICM) and epiblast. In this study, we perform detailed ultrastructural and molecular characterization of the developing pluripotent cell population as it develops from the ICM to the late epiblast. The ultrastructural observations revealed that the outer cells of the ICM have a high nuclear:cytoplasmic ratio but are transcriptionally inactive and contain mitochondria with few cristae. In contrast, the epiblast cells have a reduced nuclear:cytoplasmic ratio, are more transcriptionally active, and contain abundant cellular organelles. This study also revealed cavitation and potential unfolding of the epiblast. As the ICM forms the epiblast, SSEA1 is lost and VIMENTIN is lost and re‐expressed. The D6 blastocyst expressed high levels of STELLA, TERF1, and GDF3, and the epiblast expressed epithelial markers, MUC1 and E‐CADHERIN, and the pluripotency markers, DNMT3B and CRIPTO. Developmental Dynamics 239:2911–2920, 2010.

Temporal Repression of Endogenous Pluripotency Genes during Reprogramming of Porcine Induced Pluripotent Stem Cells

Porcine induced pluripotent stem cells (piPSCs) have the capacity to differentiate in vitro and in vivo and form chimeras. However, the lack of transgene silencing of exogenous DNA integrated into the genome and the inability of cells to proliferate in the absence of transgene expression are underlying reported problems, suggesting that reprogramming is not complete. The aim of the present study was to evaluate reprogramming events using a partially reprogrammed piPSC-like line expressing hOCT4, hNANOG, and hcMYC under tetracycline-regulated control to investigate the effects of these particular transgenes on the expression of the porcine endogenous pluripotency machinery. Endogenous and exogenous gene expression of OCT4, NANOG, SOX2, KLF4, and cMYC was determined at passages 5, 10, 15, and 20, both in cells cultured at 1 μg/mL doxycycline or 4 μg/mL doxycycline. Our results revealed that endogenous genes are repressed by their transgene counterparts in culture and that lack of expression of the transgenes, SOX2 and KLF4 allows for expression of endogenous SOX2 and KLF4. Furthermore, we report that alternate endogenous transcripts for pNANOG, pSOX2, and pKLF4 can also be detected in the pig. Despite the ability for some endogenous genes to be expressed in these lines, the piPSC-like cells still cannot be maintained without doxycycline, indicating that the culture system of piPSCs may not be optimal or that the reprogramming factor combination used may not currently be optimal for maintaining pluripotency in the pig. This may help to explain the difficulties in producing stable piPSCs and bona fide embryonic stem cell lines in this species.

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.

Epigenetic regulation of gene expression in porcine epiblast, hypoblast, trophectoderm and epiblast-derived neural progenitor cells

After fertilization, lineage specification is governed by a complicated molecular network in which permissiveness and repression of expression of pluripotency- and differentiation-associated genes are regulated by epigenetic modifications. DNA methylation operates as a very stable repressive mark in this process. In this study, we investigated the relationship between DNA methylation and expression of pluripotency-associated genes (OCT4, NANOG and SOX2), a trophectoderm (TE)-specific gene (ELF5), and genes associated with neural differentiation (SOX2 and VIMENTIN) in porcine Day 10 (E10) epiblast, hypoblast, and TE as well as in epiblast-derived neural progenitor cells (NPCs). We found that OCT4, NANOG, and SOX2 were highly expressed in the epiblast and hypoblast, while VIMENTIN was only highly expressed in the epiblast. Moreover, low expression of OCT4, NANOG, SOX2 and VIMENTIN was noted in the TE. Most CpG sites of OCT4, NANOG, SOX2 and VIMENTIN displayed low methylation levels in the epiblast and hypoblast and, strikingly, also in the TE. Hence, the expression patterns of these genes were not directly related to levels of DNA methylation in the TE in contrast to the situation in the mouse. In contrast, ELF5 was exclusively expressed in the TE and was correspondingly hypomethylated in this tissue. In NPCs, we observed down-regulation of NANOG and OCT4 expression, which correlated with hypermethylation of their promoters, whereas VIMENTIN displayed up-regulation in accordance with hypomethylation of its promoter. In conclusion, DNA methylation is an inconsistently operating epigenetic mechanism in porcine E10 blastocysts, whereas in porcine epiblast-derived NPCs, expression of pluripotency-associated and differentiation genes appear to be regulated by this modification.

Flavobacterium sp. Strain 4221 and Pedobacter sp. Strain 4236 β-1,3-Glucanases That Are Active at Low Temperatures

ABSTRACT Secretion of β-1,3-glucanases by the arctic bacterial isolates 4221 and 4236, related to the genera Flavobacterium and Pedobacter, was discovered. Escherichia coli and Lactococcus lactis expression of β-1,3-glucanases Glc4221-1 and Glc4236-1 from the respective isolates was achieved. The enzymes hydrolyzed fungal cell walls and retained activity at low temperatures.

Patient iPSC-Derived Neurons for Disease Modeling of Frontotemporal Dementia with Mutation in CHMP2B

Summary The truncated mutant form of the charged multivesicular body protein 2B (CHMP2B) is causative for frontotemporal dementia linked to chromosome 3 (FTD3). CHMP2B is a constituent of the endosomal sorting complex required for transport (ESCRT) and, when mutated, disrupts endosome-to-lysosome trafficking and substrate degradation. To understand the underlying molecular pathology, FTD3 patient induced pluripotent stem cells (iPSCs) were differentiated into forebrain-type cortical neurons. FTD3 neurons exhibited abnormal endosomes, as previously shown in patients. Moreover, mitochondria of FTD3 neurons displayed defective cristae formation, accompanied by deficiencies in mitochondrial respiration and increased levels of reactive oxygen. In addition, we provide evidence for perturbed iron homeostasis, presenting an in vitro patient-specific model to study the effects of iron accumulation in neurodegenerative diseases. All phenotypes observed in FTD3 neurons were rescued in CRISPR/Cas9-edited isogenic controls. These findings illustrate the relevance of our patient-specific in vitro models and open up possibilities for drug target development.

Directed differentiation of porcine epiblast-derived neural progenitor cells into neurons and glia

Neural progenitor cells (NPCs) are promising candidates for cell-based therapy of neurodegenerative diseases; however, safety concerns must be addressed through transplantation studies in large animal models, such as the pig. The aim of this study was to derive NPCs from porcine blastocysts and evaluate their in-vitro differentiation potential. Epiblasts were manually isolated from expanded hatched blastocysts and cultured on MEF feeder cells. Outgrowth colonies were passaged to MS5 cells and rosettes were further passaged to Matrigel-coated dishes containing bFGF and EGF. Three NPC lines were established which showed expression of SOX2, NESTIN and VIMENTIN. One line was characterised in more detail, retaining a normal karyotype and proliferating for more than three months in culture. Following differentiation, TUJI was significantly up-regulated in protocol 2 (RA and SHH; 58% positive cells) as were NF and TH. In contrast, MBP was significantly up-regulated in protocol 3 (FGF8 and SHH; 63% positive cells), whereas, GFAP was significantly up-regulated in protocols 1-4 (33%, 25%, 43% and 22%). The present study provides the first report of a porcine blastocyst-derived NPC line capable of differentiating into both neurons and glia, which may be of paramount importance for future transplantation studies in large animal models of neurodegenerative diseases.

Insulin-like growth factor 2: A modulator of anti-apoptosis related genes (HSP70, BCL2-L1) in bovine preimplantation embryos

Intrinsic defects within the embryos, reflected by elevated cell death and low proliferative ability, are considered the most critical factors associated with bovine infertility. The identification of embryonic factors, which are responsible for successful embryo development, is thus critical in designing strategies for infertility intervention. In this experiment, the possible mechanisms involved in both blastomere proliferation and regulation of cell death were studied by analysis of relative expression patterns of IGF-II, BCL2-L1, BAK1, and HSP70 in 3 classes of morphological quality groups (e.g., excellent, good, and poor) of bovine blastocysts produced by IVF. Variation in total blastocyst cell numbers as well as their allocation to inner cell mass and trophectoderm lineages were also determined by differential CDX2 staining. Results showed that transcript levels for IGF-II, BCL2-L1, and the BCL2-L1/BAK1 ratio were higher in excellent- and good-quality blastocysts compared with low-quality blastocysts (P<0.01), whereas mRNA levels for HSP70 were higher in low-quality blastocyst compared with excellent-quality bovine blastocysts (P<0.05). In addition, excellent-quality blastocysts displayed not only greater total cell number but also greater mean inner cell mass/total cell number proportion than that of poor-quality blastocysts (P<0.01). The expression levels of IGF-II showed negative correlation with the levels of HSP70 (r=-0.70; P<0.05); however, the correlation of expression levels of IGF-II with both of BCL2-L1 (r=0.91; P<0.01) and the ratio of BCL2-L1/BAK1 (r=0.78; P<0.05) were highly positive. There was no correlation between the expression levels of IGF-II and BAK1 genes. In conclusion, these observations suggested that levels of endogenous IGF-II transcripts might be associated with the quality of IVF embryos by regulating either apoptosis-related genes or mitogenic actions in bovine preimplantation embryos.