Elongation factor 1A1 regulates metabolic substrate preference in mammalian cells.

Eukaryotic elongation factor 1A1 (EEF1A1) is canonically involved in protein synthesis but also has noncanonical functions in diverse cellular processes. Previously, we identified EEF1A1 as a mediator of lipotoxicity and demonstrated that chemical inhibition of EEF1A1 activity reduced mouse liver lipid accumulation. These findings suggested a link between EEF1A1 and metabolism. Therefore, we investigated its role in regulating metabolic substrate preference. EEF1A1-deficient Chinese hamster ovary (2E2) cells displayed reduced media lactate accumulation. These effects were also observed with EEF1A1 knockdown in human hepatocyte-like HepG2 cells and in WT Chinese hamster ovary and HepG2 cells treated with selective EEF1A inhibitors, didemnin B, or plitidepsin. Extracellular flux analyses revealed decreased glycolytic ATP production and increased mitochondrial-to-glycolytic ATP production ratio in 2E2 cells, suggesting a more oxidative metabolic phenotype. Correspondingly, fatty acid oxidation was increased in 2E2 cells. Both 2E2 cells and HepG2 cells treated with didemnin B exhibited increased neutral lipid content, which may be required to support elevated oxidative metabolism. RNA-seq revealed a >90-fold downregulation of a rate-limiting glycolytic enzyme, hexokinase 2, which we confirmed through immunoblotting and enzyme activity assays. Pathway enrichment analysis identified downregulations in TNFA signaling via NFKB and MYC targets. Correspondingly, nuclear abundances of RELB and MYC were reduced in 2E2 cells. Thus, EEF1A1 deficiency may perturb glycolysis by limiting NFKB- and MYC-mediated gene expression, leading to decreased hexokinase expression and activity. This is the first evidence of a role for a translation elongation factor, EEF1A1, in regulating metabolic substrate utilization in mammalian cells.

Deletion of p66Shc Dysregulates ERK and STAT3 Activity in Mouse Embryonic Stem Cells, Enhancing Their Naive-Like Self-Renewal in the Presence of Leukemia Inhibitory Factor.

The ShcA adapter protein is necessary for early embryonic development. The role of ShcA in development is primarily attributed to its 52 and 46 kDa isoforms that transduce receptor tyrosine kinase signaling through the extracellular signal regulated kinase (ERK). During embryogenesis, ERK acts as the primary signaling effector, driving fate acquisition and germ layer specification. P66Shc, the largest of the ShcA isoforms, has been observed to antagonize ERK in several contexts; however, its role during embryonic development remains poorly understood. We hypothesized that p66Shc could act as a negative regulator of ERK activity during embryonic development, antagonizing early lineage commitment. To explore the role of p66Shc in stem cell self-renewal and differentiation, we created a p66Shc knockout murine embryonic stem cell (mESC) line. Deletion of p66Shc enhanced basal ERK activity, but surprisingly, instead of inducing mESC differentiation, loss of p66Shc enhanced the expression of core and naive pluripotency markers. Using pharmacologic inhibitors to interrogate potential signaling mechanisms, we discovered that p66Shc deletion permits the self-renewal of naive mESCs in the absence of conventional growth factors, by increasing their responsiveness to leukemia inhibitory factor (LIF). We discovered that loss of p66Shc enhanced not only increased ERK phosphorylation but also increased phosphorylation of Signal transducer and activator of transcription in mESCs, which may be acting to stabilize their naive-like identity, desensitizing them to ERK-mediated differentiation cues. These findings identify p66Shc as a regulator of both LIF-mediated ESC pluripotency and of signaling cascades that initiate postimplantation embryonic development and ESC commitment.

Differential localization patterns of pyruvate kinase isoforms in murine naïve, formative, and primed pluripotent states.

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of the pluripotency continuum, referred to as naïve and primed pluripotent states, respectively. These divergent pluripotent states differ in several ways, including growth factor requirements, transcription factor expression, DNA methylation patterns, and metabolic profiles. Naïve cells employ both glycolysis and oxidative phosphorylation (OXPHOS), whereas primed cells preferentially utilize aerobic glycolysis, a trait shared with cancer cells referred to as the Warburg Effect. Until recently, metabolism has been regarded as a by-product of cell fate, however, evidence now supports metabolism as being a driver of stem cell state and fate decisions. Pyruvate kinase muscle isoforms (PKM1 and PKM2) are important for generating and maintaining pluripotent stem cells (PSCs) and mediating the Warburg Effect. Both isoforms catalyze the final, rate limiting step of glycolysis, generating adenosine triphosphate and pyruvate, however, the precise role(s) of PKM1/2 in naïve and primed pluripotency is not well understood. The primary objective of this study was to characterize the cellular expression and localization patterns of PKM1 and PKM2 in mESCs, chemically transitioned epiblast-like cells (mEpiLCs) representing formative pluripotency, and mEpiSCs using immunoblotting and confocal microscopy. The results indicate that PKM1 and PKM2 are not only localized to the cytoplasm, but also accumulate in differential subnuclear regions of mESC, mEpiLCs, and mEpiSCs as determined by a quantitative confocal microscopy employing orthogonal projections and airyscan processing. Importantly, we discovered that the subnuclear localization of PKM1/2 changes during the transition from mESCs, mEpiLCs, and mEpiSCs. Finally, we have comprehensively validated the appropriateness and power of the Pearson's correlation coefficient and Manders's overlap coefficient for assessing nuclear and cytoplasmic protein colocalization in PSCs by immunofluorescence confocal microscopy. We propose that nuclear PKM1/2 may assist with distinct pluripotency state maintenance and lineage priming by non-canonical mechanisms. These results advance our understanding of the overall mechanisms controlling naïve, formative, and primed pluripotency.

Analysis of TERT Isoforms across TCGA, GTEx and CCLE Datasets.

Reactivation of the multi-subunit ribonucleoprotein telomerase is the primary telomere maintenance mechanism in cancer, but it is rate-limited by the enzymatic component, telomerase reverse transcriptase (TERT). While regulatory in nature, TERT alternative splice variant/isoform regulation and functions are not fully elucidated and are further complicated by their highly diverse expression and nature. Our primary objective was to characterize TERT isoform expression across 7887 neoplastic and 2099 normal tissue samples using The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression Project (GTEx), respectively. We confirmed the global overexpression and splicing shift towards full-length TERT in neoplastic tissue. Stratifying by tissue type we found uncharacteristic TERT expression in normal brain tissue subtypes. Stratifying by tumor-specific subtypes, we detailed TERT expression differences potentially regulated by subtype-specific molecular characteristics. Focusing on ß-deletion splicing regulation, we found the NOVA1 trans-acting factor to mediate alternative splicing in a cancer-dependent manner. Of relevance to future tissue-specific studies, we clustered cancer cell lines with tumors from related origin based on TERT isoform expression patterns. Taken together, our work has reinforced the need for tissue and tumour-specific TERT investigations, provided avenues to do so, and brought to light the current technical limitations of bioinformatic analyses of TERT isoform expression.

Pannexin 1 binds ß-catenin to modulate melanoma cell growth and metabolism.

Melanoma is the most aggressive skin malignancy with increasing incidence worldwide. Pannexin1 (PANX1), a member of the pannexin family of channel-forming glycoproteins, regulates cellular processes in melanoma cells including proliferation, migration, and invasion/metastasis. However, the mechanisms responsible for coordinating and regulating PANX1 function remain unclear. Here, we demonstrated a direct interaction between the C-terminal region of PANX1 and the N-terminal portion of ß-catenin, a key transcription factor in the Wnt pathway. At the protein level, ß-catenin was significantly decreased when PANX1 was either knocked down or inhibited by two PANX1 blockers, Probenecid and Spironolactone. Immunofluorescence imaging showed a disrupted pattern of ß-catenin localization at the cell membrane in PANX1-deficient cells, and transcription of several Wnt target genes, including MITF, was suppressed. In addition, a mitochondrial stress test revealed that the metabolism of PANX1-deficient cells was impaired, indicating a role for PANX1 in the regulation of the melanoma cell metabolic profile. Taken together, our data show that PANX1 directly interacts with ß-catenin to modulate growth and metabolism in melanoma cells. These findings provide mechanistic insight into PANX1-mediated melanoma progression and may be applicable to other contexts where PANX1 and ß-catenin interact as a potential new component of the Wnt signaling pathway.

Lactate preconditioning promotes a HIF-1α-mediated metabolic shift from OXPHOS to glycolysis in normal human diploid fibroblasts.

Recent evidence has emerged that cancer cells can use various metabolites as fuel sources. Restricting cultured cancer cells to sole metabolite fuel sources can promote metabolic changes leading to enhanced glycolysis or mitochondrial OXPHOS. However, the effect of metabolite-restriction on non-transformed cells remains largely unexplored. Here we examined the effect of restricting media fuel sources, including glucose, pyruvate or lactate, on the metabolic state of cultured human dermal fibroblasts. Fibroblasts cultured in lactate-only medium exhibited reduced PDH phosphorylation, indicative of OXPHOS, and a concurrent elevation of ROS. Lactate exposure primed fibroblasts to switch to glycolysis by increasing transcript abundance of genes encoding glycolytic enzymes and, upon exposure to glucose, increasing glycolytic enzyme levels. Furthermore, lactate treatment stabilized HIF-1α, a master regulator of glycolysis, in a manner attenuated by antioxidant exposure. Our findings indicate that lactate preconditioning primes fibroblasts to switch from OXPHOS to glycolysis metabolism, in part, through ROS-mediated HIF-1α stabilization. Interestingly, we found that lactate preconditioning results in increased transcript abundance of MYC and SNAI1, key facilitators of early somatic cell reprogramming. Defined metabolite treatment may represent a novel approach to increasing somatic cell reprogramming efficiency by amplifying a critical metabolic switch that occurs during iPSC generation.

p66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity.

A key pathological feature of Alzheimer's disease (AD) is the accumulation of the neurotoxic amyloid beta (Aß) peptide within the brains of affected individuals. Previous studies have shown that neuronal cells selected for resistance to Aß toxicity display a metabolic shift from mitochondrial-dependent oxidative phosphorylation (OXPHOS) to aerobic glycolysis to meet their energy needs. The Src homology/collagen (Shc) adaptor protein p66Shc is a key regulator of mitochondrial function, ROS production and aging. Moreover, increased expression and activation of p66Shc promotes a shift in the cellular metabolic state from aerobic glycolysis to OXPHOS in cancer cells. Here we evaluated the hypothesis that activation of p66Shc in CNS cells promotes both increased OXPHOS and enhanced sensitivity to Aß toxicity. The effect of altered p66Shc expression on metabolic activity was assessed in rodent HT22 and B12 cell lines of neuronal and glial origin respectively. Overexpression of p66Shc repressed glycolytic enzyme expression and increased both mitochondrial electron transport chain activity and ROS levels in HT22 cells. The opposite effect was observed when endogenous p66Shc expression was knocked down in B12 cells. Moreover, p66Shc activation in both cell lines increased their sensitivity to Aß toxicity. Our findings indicate that expression and activation of p66Shc renders CNS cells more sensitive to Aß toxicity by promoting mitochondrial OXPHOS and ROS production while repressing aerobic glycolysis. Thus, p66Shc may represent a potential therapeutically relevant target for the treatment of AD.

Treatment with AICAR inhibits blastocyst development, trophectoderm differentiation and tight junction formation and function in mice.

STUDY QUESTION: What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function? SUMMARY ANSWER: AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur. WHAT IS KNOWN ALREADY: AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK. STUDY DESIGN, SIZE, DURATION: Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0-2000 µM) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 µM AICAR. PARTICIPANTS/MATERIALS, SETTING, METHODS: Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR. MAIN RESULTS AND THE ROLE OF CHANCE: Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin. WIDER IMPLICATIONS OF THE FINDINGS: Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles. STUDY FUNDING AND COMPETING INTEREST(S): Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests.

Connexin43 Mutant Patient-Derived Induced Pluripotent Stem Cells Exhibit Altered Differentiation Potential.

We present for the first time the generation of induced pluripotent stem cells (iPSCs) from a patient with a connexin-linked disease. The importance of gap junctional intercellular communication in bone homeostasis is exemplified by the autosomal dominant developmental disorder oculodentodigital dysplasia (ODDD), which is linked to mutations in the GJA1 (Cx43) gene. ODDD is characterized by craniofacial malformations, ophthalmic deficits, enamel hypoplasia, and syndactyly. In addition to harboring a Cx43 p.V216L mutation, ODDD iPSCs exhibit reduced Cx43 mRNA and protein abundance when compared to control iPSCs and display impaired channel function. Osteogenic differentiation involved an early, and dramatic downregulation of Cx43 followed by a slight upregulation during the final stages of differentiation. Interestingly, osteoblast differentiation was delayed in ODDD iPSCs. Moreover, Cx43 subcellular localization was altered during chondrogenic differentiation of ODDD iPSCs compared to controls and this may have contributed to the more compact cartilage pellet morphology found in differentiated ODDD iPSCs. These studies highlight the importance of Cx43 expression and function during osteoblast and chondrocyte differentiation, and establish a potential mechanism for how ODDD-associated Cx43 mutations may have altered cell lineages involved in bone and cartilage development. © 2017 American Society for Bone and Mineral Research.

Characterization and Immunomodulatory Effects of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells.

BACKGROUND: Mesenchymal stromal cells (MSC) hold promise for both cell replacement and immune modulation strategies owing to their progenitor and non-progenitor functions, respectively. Characterization of MSC from different sources is an important and necessary step before clinical use of these cells is widely adopted. Little is known about the biology and function of canine MSC compared to their mouse or human counterparts. This knowledge-gap impedes development of canine evidence-based MSC technologies. HYPOTHESIS AND OBJECTIVES: We hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC (derived from the same dogs) will have similar differentiation and immune modulatory profiles. Our objectives were to evaluate progenitor and non-progenitor functions as well as other characteristics of AT- and BM-MSC including 1) proliferation rate, 2) cell surface marker expression, 3) DNA methylation levels, 4) potential for trilineage differentiation towards osteogenic, adipogenic, and chondrogenic cell fates, and 5) immunomodulatory potency in vitro. RESULTS: 1) AT-MSC proliferated at more than double the rate of BM-MSC (population doubling times in days) for passage (P) 2, AT: 1.69, BM: 3.81; P3, AT: 1.80, BM: 4.06; P4, AT: 2.37, BM: 5.34; P5, AT: 3.20, BM: 7.21). 2) Canine MSC, regardless of source, strongly expressed cell surface markers MHC I, CD29, CD44, and CD90, and were negative for MHC II and CD45. They also showed moderate expression of CD8 and CD73 and mild expression of CD14. Minor differences were found in expression of CD4 and CD34. 3) Global DNA methylation levels were significantly lower in BM-MSC compared to AT-MSC. 4) Little difference was found between AT- and BM-MSC in their potential for adipogenesis and osteogenesis. Chondrogenesis was poor to absent for both sources in spite of adding varying levels of bone-morphogenic protein to our standard transforming growth factor (TGF-ß3)-based induction medium. 5) Immunomodulatory capacity was equal regardless of cell source when tested in mitogen-stimulated lymphocyte reactions. Priming of MSC with pro-inflammatory factors interferon-gamma and/or tumour necrosis factor did not increase the lymphocyte suppressive properties of the MSC compared to untreated MSC. CONCLUSIONS/SIGNIFICANCE: No significant differences were found between AT- and BM-MSC with regard to their immunophenotype, progenitor, and non-progenitor functions. Both MSC populations showed strong adipogenic and osteogenic potential and poor chondrogenic potential. Both significantly suppressed stimulated peripheral blood mononuclear cells. The most significant differences found were the higher isolation success and proliferation rate of AT-MSC, which could be realized as notable benefits of their use over BM-MSC.

Small-Molecule Induction of Canine Embryonic Stem Cells Toward Naïve Pluripotency.

Naïve and primed pluripotent stem cells (PSCs) reflect discrete pluripotent states that approximate the inner cell mass or the progressively lineage-restricted perigastrulation epiblast, respectively. Cells that occupy primed pluripotency have distinct epigenetic landscapes, transcriptional circuitry, and trophic requirements compared with their naïve counterparts. The existence of multiple pluripotent states has not been explored in dogs, which show promise as outbred biomedical models with more than 300 inherited diseases that also afflict humans. However, our understanding of canine embryogenesis and embryo-derived stem cells is limited. Herein, we converted leukemia inhibitory factor (LIF)-dependent and fibroblast growth factor 2 (FGF2)-dependent canine embryonic stem cells (cESCs) resembling primed PSCs toward a naïve pluripotent state using LIF and inhibitors of glycogen synthase kinase 3ß and mitogen-activated protein kinase kinase 1/2 [called 2i and LIF (2iL)]. cESCs propagated in 2iL exhibited significant induction of genes associated with the naïve pluripotent state (eg, REX1, TBX3) and downregulation of primed pluripotency markers (eg, OTX2, FGF5) (P < 0.05). Differential phosphorylation of signal transducer and activator of transcription 3 (STAT3) and cell fate decisions on exposure to bone morphogenetic protein 4 (BMP4) suggested that a novel pluripotent identity has been established with 2iL. Accordingly, cESCs cultured with 2iL formed colonies at a greater efficiency than LIF-FGF2 cESCs following single-cell dissociation. Total genomic DNA methylation and histone H3 lysine 27 trimethylation signals were reduced in 2iL-treated cESCs. Our data suggest that 2iL culture conditions promote the conversion of cESCs toward an epigenetically distinct pluripotent state resembling naïve PSCs.

The role of telomeres and telomerase reverse transcriptase isoforms in pluripotency induction and maintenance.

Telomeres are linear guanine-rich DNA structures at the ends of chromosomes. The length of telomeric DNA is actively regulated by a number of mechanisms in highly proliferative cells such as germ cells, cancer cells, and pluripotent stem cells. Telomeric DNA is synthesized by way of the ribonucleoprotein called telomerase containing a reverse transcriptase (TERT) subunit and RNA component (TERC). TERT is highly conserved across species and ubiquitously present in their respective pluripotent cells. Recent studies have uncovered intricate associations between telomeres and the self-renewal and differentiation properties of pluripotent stem cells. Interestingly, the past decade's work indicates that the TERT subunit also has the capacity to modulate mitochondrial function, to remodel chromatin structure, and to participate in key signaling pathways such as the Wnt/ß-catenin pathway. Many of these non-canonical functions do not require TERT's catalytic activity, which hints at possible functions for the extensive number of alternatively spliced TERT isoforms that are highly expressed in pluripotent stem cells. In this review, some of the established and potential routes of pluripotency induction and maintenance are highlighted from the perspectives of telomere maintenance, known TERT isoform functions and their complex regulation.

Canine Pluripotent Stem Cells: Are They Ready for Clinical Applications?

The derivation of canine embryonic stem cells and generation of canine-induced pluripotent stem cells are significant achievements that have unlocked the potential for developing novel cell-based disease models, drug discovery platforms, and transplantation therapies in the dog. A progression from concept to cure in this clinically relevant companion animal will not only help our canine patients but also help advance human regenerative medicine. Nevertheless, many issues remain to be resolved before pluripotent cells can be used clinically in a safe and reproducible manner.

Mass spectrometry-based proteomic analysis of the matrix microenvironment in pluripotent stem cell culture.

The cellular microenvironment comprises soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to regulate cellular behavior. Pluripotent stem cells utilize interactions between support cells and soluble factors in the microenvironment to assist in the maintenance of self-renewal and the process of differentiation. However, the ECM also plays a significant role in shaping the behavior of human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells. Moreover, it has recently been observed that deposited factors in a hESC-conditioned matrix have the potential to contribute to the reprogramming of metastatic melanoma cells. Therefore, the ECM component of the pluripotent stem cell microenvironment necessitates further analysis. In this study we first compared the self-renewal and differentiation properties of hESCs grown on Matrigel™ pre-conditioned by hESCs to those on unconditioned Matrigel™. We determined that culture on conditioned Matrigel™ prevents differentiation when supportive growth factors are removed from the culture medium. To investigate and identify factors potentially responsible for this beneficial effect, we performed a defined SILAC MS-based proteomics screen of hESC-conditioned Matrigel™. From this proteomics screen, we identified over 80 extracellular proteins in matrix conditioned by hESCs and induced pluripotent stem cells. These included matrix-associated factors that participate in key stem cell pluripotency regulatory pathways, such as Nodal/Activin and canonical Wnt signaling. This work represents the first investigation of stem-cell-derived matrices from human pluripotent stem cells using a defined SILAC MS-based proteomics approach.

Long telomeres bypass the requirement for telomere maintenance in human tumorigenesis.

Despite the importance of telomere maintenance in cancer cell survival via the elongation of telomeres by telomerase reverse transcriptase (TERT) or alternative lengthening of telomeres (ALT), it had not been tested directly whether telomere maintenance is dispensable for human tumorigenesis. We engineered human tumor cells containing loxP-flanked hTERT to enable extensive telomere elongation prior to complete hTERT excision. Despite unabated telomere erosion, hTERT-excised cells formed tumors in mice and proliferated in vitro for up to 1 year. Telomerase reactivation or ALT was not observed, and the eventual loss of telomeric signal coincided with loss of tumorigenic potential and cell viability. Crisis was averted via the reintroduction of active but not inactive hTERT. Thus, telomere maintenance is dispensable for human tumorigenesis when telomere reserves are long. Yet, despite telomere instability and the presence of oncogenic RAS, human tumors remain susceptible to crisis induced by critically short telomeres.

Synaptically-competent neurons derived from canine embryonic stem cells by lineage selection with EGF and Noggin.

Pluripotent stem cell lines have been generated in several domestic animal species; however, these lines traditionally show poor self-renewal and differentiation. Using canine embryonic stem cell (cESC) lines previously shown to have sufficient self-renewal capacity and potency, we generated and compared canine neural stem cell (cNSC) lines derived by lineage selection with epidermal growth factor (EGF) or Noggin along the neural default differentiation pathway, or by directed differentiation with retinoic acid (RA)-induced floating sphere assay. Lineage selection produced large populations of SOX2+ neural stem/progenitor cell populations and neuronal derivatives while directed differentiation produced few and improper neuronal derivatives. Primary canine neural lines were generated from fetal tissue and used as a positive control for differentiation and electrophysiology. Differentiation of EGF- and Noggin-directed cNSC lines in N2B27 with low-dose growth factors (BDNF/NT-3 or PDGFαα) produced phenotypes equivalent to primary canine neural cells including 3CB2+ radial progenitors, MOSP+ glia restricted precursors, VIM+/GFAP+ astrocytes, and TUBB3+/MAP2+/NFH+/SYN+ neurons. Conversely, induction with RA and neuronal differentiation produced inadequate putative neurons for further study, even though appropriate neuronal gene expression profiles were observed by RT-PCR (including Nestin, TUBB3, PSD95, STX1A, SYNPR, MAP2). Co-culture of cESC-derived neurons with primary canine fetal cells on canine astrocytes was used to test functional maturity of putative neurons. Canine ESC-derived neurons received functional GABA(A)- and AMPA-receptor mediated synaptic input, but only when co-cultured with primary neurons. This study presents established neural stem/progenitor cell populations and functional neural derivatives in the dog, providing the proof-of-concept required to translate stem cell transplantation strategies into a clinically relevant animal model.

Viable iPSC mice: a step closer to therapeutic applications in humans?

Rapid advancements have occurred in induced pluripotent stem cell research within the 3 years since Yamanaka and colleagues first reprogrammed adult mouse fibroblasts to an embryonic stem cell-like state by the forced expression of a small cohort of transcription factors. Progress has been made in overcoming various technical obstacles, including oncogenic threat, that hinder the application of iPS cell technology as a therapeutic strategy in humans. Remaining hurdles include the low efficiency of iPS cell induction and the demonstration of complete developmental potential. This latter impediment now appears to have been overcome simultaneously by two groups (Kristen Baldwin and colleagues and Qi Zhou and colleagues), who have generated viable adult mice from tetraploid complementation assays using iPS donor cells. The generation of sufficiently reprogrammed iPS cells and mice will allow for adequate genomic and functional testing to evaluate their utility in research applications and patient-specific cell replacement therapies, which may include infertility.

Improved isolation protocol for equine cord blood-derived mesenchymal stromal cells.

BACKGROUND AIMS: A robust methodology for the isolation of cord blood-derived multipotent mesenchymal stromal cells (CB-MSCs) from fresh umbilical cord blood has not been reported in any species. The objective of this study was to improve the isolation procedure for equine CB-MSCs. METHODS: Pre-culture separation of red and white blood cells was done using either PrepaCyte?-EQ medium or Ficoll-Paque? PREMIUM density medium. Regular FBS and MSC-qualified FBS were compared for their ability to support the establishment of putative primary MSC colonies. RESULTS AND CONCLUSIONS: Our results indicate that PrepaCyte-EQ medium is superior to Ficoll-Paque PREMIUM density medium for the isolation of putative equine CB MSC and that MSC-qualified FBS may improve the isolation efficiency.

Current and future regenerative medicine - principles, concepts, and therapeutic use of stem cell therapy and tissue engineering in equine medicine.

This paper provides a bird's-eye perspective of the general principles of stem-cell therapy and tissue engineering; it relates comparative knowledge in this area to the current and future status of equine regenerative medicine.The understanding of equine stem cell biology, biofactors, and scaffolds, and their potential therapeutic use in horses are rudimentary at present. Mesenchymal stem cell isolation has been proclaimed from several equine tissues in the past few years. Based on the criteria of the International Society for Cellular Therapy, most of these cells are more correctly referred to as multipotent mesenchymal stromal cells, unless there is proof that they exhibit the fundamental in vivo characteristics of pluripotency and the ability to self-renew. That said, these cells from various tissues hold great promise for therapeutic use in horses. The 3 components of tissue engineering - cells, biological factors, and biomaterials - are increasingly being applied in equine medicine, fuelled by better scaffolds and increased understanding of individual biofactors and cell sources.The effectiveness of stem cell-based therapies and most tissue engineering concepts has not been demonstrated sufficiently in controlled clinical trials in equine patients to be regarded as evidence-based medicine. In the meantime, the medical mantra "do no harm" should prevail, and the application of stem cell-based therapies in the horse should be done critically and cautiously, and treatment outcomes (good and bad) should be recorded and reported.Stem cell and tissue engineering research in the horse has exciting comparative and equine specific perspectives that most likely will benefit the health of horses and humans. Controlled, well-designed studies are needed to move this new equine research field forward.

Characterization of canine embryonic stem cell lines derived from different niche microenvironments.

Embryo-derived stem cells hold enormous potential for producing cell-based transplantation therapies, allowing high-throughput drug screening and delineating early embryonic development. However, potential clinical applications must first be tested for safety and efficacy in preclinical animal models. Due to physiological and genetic parity to humans, the domestic dog is widely used as a clinically relevant animal model for cardiovascular, neurodegenerative, orthopedic, and oncologic diseases. Therefore, we established numerous putative canine embryonic stem cell (cESC) lines by immunodissection of the inner cell mass (ICM), which we termed OVC.ID.1-23, and by explant outgrowths from whole canine blastocysts, named OVC.EX.1-16. All characterized lines were immunopositive for OCT4, SOX2, NANOG, SSEA-3, and SSEA-4; displayed high telomerase and alkaline phosphatase (ALP) activities; and were maintained in this state up to 37 passages ( approximately 160 days). Colonies from OVC.EX lines showed classic domed hESC-like morphology surrounded by a ring of fibroblast-like cells, whereas all OVC.ID lines exhibited a mixed cell colony of tightly packed cESCs surrounded by a GATA6+/CDX2- hypoblast-derived support layer. Spontaneous serum-only differentiation without feeder layers demonstrated a strong lineage selection associated with the colony niche type, and not the isolation method. Upon differentiation, cESC lines formed embryoid bodies (EB) comprised of cells representative of all germinal layers, and differentiated into cell types of each layer. Canine ESC lines such as these have the potential to identify differences between embryonic stem cell line derivations, and to develop or to test cell-based transplantation therapies in the dog before attempting human clinical trials.