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Specific activation of endogenous genes can be achieved by programmable artificial transcription factors (ATFs). In this study, we compared two artificial, programmable, clustered regularly interspaced short palindromic repeats (CRISPR)-based, ubiquitous transcription factors: deficient CRISPR-associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts). Activation potential was monitored using a luciferase reporter system and we found that both CRISPRa and SAM can efficiently activate the proximal promoter of all five genes. We also observed that the guide RNA (gRNA) target sites and number of gRNAs have a major effect on gRNA-guided activation efficiency. Furthermore, increased activation efficiency (>3-folds) could be achieved by the SAM system compared to CRISPRa. In addition, we discovered that only the SAM system could efficiently activate LIN28, OCT4, and SOX2 expression (up to 100-folds compared to coexpression with a scrambled gRNA) in primary human fibroblasts. This SAM-mediated activation of LOS can be stably maintained for over 20 days in fibroblasts cultured in either fibroblasts or stem cell medium. However, when attempting to use the SAM-LOS activation as an approach for induced pluripotent stem cells-reprogramming, no embryonic stem-like colonies could be obtained from these SAM fibroblasts. In conclusion, our study showed that CRISPR/Cas9-based ATFs are potent to activate and maintain transcription of endogenous human pluripotent genes. However, future improvements of the system are still required to improve activation efficiency and cellular reprogramming using ATFs.

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The quest for porcine pluripotent stem cells (PSCs) was initiated in the early 90s. Initially, it was the intention to benefit from these cells for production of genetically modified pigs using homologous recombination followed by derivation of chimeric offspring; a technology that has been used to produce genetically modified mice since the mid-80s. However, no convincing reports on the generation of bona fide porcine embryonic stem cells or embryonic germ cells resulted from these activities, and with the advent of somatic cell nuclear transfer during the late 90s, alternative methods for creating genetically modified pigs emerged. Over the past years, renewed interest in porcine PSCs has sparked activities in deriving in particular porcine induced pluripotent stem cells to develop the pig as a faithful model for studying the potentials and risks associated with induced pluripotent stem cell-based human therapy. Here, we review the recent data on establishment of porcine PSCs and the differences in embryonic development between pig and mouse, which may be underlying factors for the continuing challenge to culture and maintain porcine PSCs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Differential staining is an immunocytochemical staining that visualizes trophectoderm (TE) and the inner cell mass (ICM) of the blastocysts. It is used to determine the blastocyst quality, but could also be a useful tool to assess the integration site of injected cells into the early embryo. This is relevant for testing of presumed pluripotent stem cells. The gold standard for pluripotent stem cells is to test if the cells are capable of contributing to germline chimeras. Differential staining can be used to evaluate the possibility of chimeric contribution; if the cells are located in the area of the ICM they are likely to contribute to the fetus and if they are located in the area of the TE they are likely to contribute to the fetal membranes. In this article, we optimize on methods for embryo staining and mounting so that the exact location of injected stem cells within preimplantation porcine embryos can be evaluated.

The factors necessary to maintain organ-specific progenitor cells are poorly understood and yet of extreme clinical importance. Here, we identify the transcription factor SOX9 as the first specific marker and maintenance factor of multipotential progenitors during pancreas organogenesis. In the developing pancreas, SOX9 expression is restricted to a mitotically active, Notch-responsive subset of PDX1(+) pluripotent progenitors and is absent from committed endocrine precursors or differentiated cells. Similar to Notch mutations, organ-specific Sox9 inactivation in mice causes severe pancreatic hypoplasia resulting from depletion of the progenitor cell pool. We show that Sox9 maintains pancreatic progenitors by stimulating their proliferation, survival, and persistence in an undifferentiated state. Our finding that SOX9 regulates the Notch-effector HES1 suggests a Notch-dependent mechanism and establishes a possible genetic link between SOX factors and Notch. These findings will be of major significance for the development of in vitro protocols for cell replacement therapies.

Alterations in neurotransmitter homeostasis, primarily of glutamate and GABA, is strongly implicated in the pathophysiology of Alzheimer's disease (AD). Homeostasis at the synapse is maintained by neurotransmitter recycling between neurons and astrocytes. Astrocytes support neuronal transmission through glutamine synthesis, which can be derived from oxidative metabolism of GABA. However, the precise implications of astrocytic GABA metabolism in AD remains elusive. The aim of this study was to investigate astrocytic GABA metabolism in AD pathology implementing human induced pluripotent stem cells derived astrocytes. Metabolic mapping of GABA was performed with [U-13C]GABA stable isotopic labeling using gas chromatography coupled to mass spectrometry (GC–MS). Neurotransmitter and amino acid content was quantified via high performance liquid chromatography (HPLC) and protein expression was investigated by Western blot assay. Cell lines carrying mutations in either amyloid precursor protein (APP) or presenilin1 (PSEN-1) were used as AD models and were compared to a control cell line of the same genetic background. AD astrocytes displayed a reduced oxidative GABA metabolism mediated by a decreased uptake capacity of GABA, as GABA transporter 3 (GAT3) was downregulated in AD astrocytes compared to the controls. Interestingly, the carbon backbone of GABA in AD astrocytes was utilized to a larger extent to support glutamine synthesis compared to control astrocytes. The results strongly indicate alterations in GABA uptake and metabolism in AD astrocytes linked to reduced GABA transporter expression, hereby contributing further to neurotransmitter disturbances. [ABSTRACT FROM AUTHOR]

Recent studies indicate a crucial role for neuronal glycogen storage and degradation in memory formation. We have previously identified alpha‐amylase (α‐amylase), a glycogen degradation enzyme, located within synaptic‐like structures in CA1 pyramidal neurons and shown that individuals with a high copy number variation of α‐amylase perform better on the episodic memory test. We reported that neuronal α‐amylase was absent in patients with Alzheimer's disease (AD) and that this loss corresponded to increased AD pathology. In the current study, we verified these findings in a larger patient cohort and determined a similar reduction in α‐amylase immunoreactivity in the molecular layer of hippocampus in AD patients. Next, we demonstrated reduced α‐amylase concentrations in oligomer amyloid beta 42 (Aβ42) stimulated SH‐SY5Y cells and neurons derived from human‐induced pluripotent stem cells (hiPSC) with PSEN1 mutation. Reduction of α‐amylase production and activity, induced by siRNA and α‐amylase inhibitor Tendamistat, respectively, was further shown to enhance glycogen load in SH‐SY5Y cells. Both oligomer Aβ42 stimulated SH‐SY5Y cells and hiPSC neurons with PSEN1 mutation showed, however, reduced load of glycogen. Finally, we demonstrate the presence of α‐amylase within synapses of isolated primary neurons and show that inhibition of α‐amylase activity with Tendamistat alters neuronal activity measured by calcium imaging. In view of these findings, we hypothesize that α‐amylase has a glycogen degrading function within synapses, potentially important in memory formation. Hence, a loss of α‐amylase, which can be induced by Aβ pathology, may in part underlie the disrupted memory formation seen in AD patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells (hESCs) offer tremendous potential for not only treating neurological disorders but also for their ability to serve as vital reagents to model and investigate human disease. To further our understanding of a key protein involved in Alzheimer disease pathogenesis, we stably overexpressed amyloid precursor protein (APP) in hESCs. Remarkably, we found that APP overexpression in hESCs caused a rapid and robust differentiation of pluripotent stem cells toward a neural fate. Despite maintenance in standard hESC media, up to 80% of cells expressed the neural stem cell marker nestin, and 65% exhibited the more mature neural marker β-3 tubulin within just 5 days of passaging. To elucidate the mechanism underlying the effects of APP on neural differentiation, we examined the proteolysis of APP and performed both gain of function and loss of function experiments. Taken together, our results demonstrate that the N-terminal secreted soluble forms of APP (in particular sAPPβ) robustly drive neural differentiation of hESCs. Our findings not only reveal a novel and intriguing role for APP in neural lineage commitment but also identify a straightforward and rapid approach to generate large numbers of neurons from human embryonic stem cells. These novel APP-hESC lines represent a valuable tool to investigate the potential role of APP in development and neurodegeneration and allow for insights into physiological functions of this protein. [ABSTRACT FROM AUTHOR]

The rising prevalence of progressive neurodegenerative diseases coupled with increasing longevity poses an economic burden at individual and societal levels. There is currently no effective cure for the majority of neurodegenerative diseases and disease-affected tissues from patients have been difficult to obtain for research and drug discovery in pre-clinical settings. While the use of animal models has contributed invaluable mechanistic insights and potential therapeutic targets, the translational value of animal models could be further enhanced when combined with in vitro models derived from patient-specific induced pluripotent stem cells (iPSCs) and isogenic controls generated using CRISPR-Cas9 mediated genome editing. The iPSCs are self-renewable and capable of being differentiated into the cell types affected by the diseases. These in vitro models based on patient-derived iPSCs provide the opportunity to model disease development, uncover novel mechanisms and test potential therapeutics. Here we review findings from iPSC-based modeling of selected neurodegenerative diseases, including Alzheimer’s disease, frontotemporal dementia and spinocerebellar ataxia. Furthermore, we discuss the possibilities of generating three-dimensional (3D) models using the iPSCs-derived cells and compare their advantages and disadvantages to conventional two-dimensional (2D) models. [ABSTRACT FROM AUTHOR]

Alterations in the cellular metabolic machinery of the brain are associated with neurodegenerative disorders such as Alzheimer's disease. Novel human cellular disease models are essential in order to study underlying disease mechanisms. In the present study, we characterized major metabolic pathways in neurons derived from human induced pluripotent stem cells (hiPSC). With this aim, cultures of hiPSC-derived neurons were incubated with [U- 13 C]glucose, [U- 13 C]glutamate or [U- 13 C]glutamine. Isotopic labeling in metabolites was determined using gas chromatography coupled to mass spectrometry, and cellular amino acid content was quantified by high-performance liquid chromatography. Additionally, we evaluated mitochondrial function using real-time assessment of oxygen consumption via the Seahorse XF e 96 Analyzer. Moreover, in order to validate the hiPSC-derived neurons as a model system, a metabolic profiling was performed in parallel in primary neuronal cultures of mouse cerebral cortex and cerebellum. These serve as well-established models of GABAergic and glutamatergic neurons, respectively. The hiPSC-derived neurons were previously characterized as being forebrain-specific cortical glutamatergic neurons. However, a comparable preparation of predominantly mouse cortical glutamatergic neurons is not available. We found a higher glycolytic capacity in hiPSC-derived neurons compared to mouse neurons and a substantial oxidative metabolism through the mitochondrial tricarboxylic acid (TCA) cycle. This finding is supported by the extracellular acidification and oxygen consumption rates measured in the cultured human neurons. [U- 13 C]Glutamate and [U- 13 C]glutamine were found to be efficient energy substrates for the neuronal cultures originating from both mice and humans. Interestingly, isotopic labeling in metabolites from [U- 13 C]glutamate was higher than that from [U- 13 C]glutamine. Although the metabolic profile of hiPSC-derived neurons in vitro was particularly similar to the profile of mouse cortical neurons, important differences between the metabolic profile of human and mouse neurons were observed. The results of the present investigation establish hallmarks of cellular metabolism in human neurons derived from iPSC. [ABSTRACT FROM AUTHOR]

Nuclear reprogramming efficiency has been shown to be highly variable among different types of somatic cells and different individuals, yet the underlying mechanism remains largely unknown. Several studies have shown that reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) requires remodeling of mitochondria and a metabolic shift from an oxidative state to a more glycolytic state. In this study, we evaluated the nuclear reprogramming efficiency in relation to mitochondrial bioenergetic parameters of fibroblasts from seven different human individuals. Using the Seahorse extracellular energy flux analyzer, we measured oxygen consumption rate (OCR) profiles of the cells, along with their nuclear reprogramming efficiency into iPSCs. Our results showed that fibroblasts with the lowest mitochondrial spare respiratory capacity (SRC) had the highest nuclear reprogramming efficiency, opposed to fibroblasts with the highest mitochondrial SRC, which showed lowest reprogramming efficiency. Furthermore, we found that targeted fluorescent tagging of endogenous genes ( MYH6 and COL2A1) by CRISPR/Cas9-mediated homologous recombination was accompanied by an increase in the SRC level of the modified fibroblasts and impaired reprogramming efficiency. Our findings indicate a negative correlation between high mitochondrial SRC in somatic cells and low reprogramming efficiencies. This type of analysis potentially allows screening and predicting reprogramming efficiency before reprogramming, and further suggests that nuclear reprogramming might be improved by approaches that modulate the SRC. [ABSTRACT FROM AUTHOR]