Proliferation Of Pluripotent Stem Cells Research Articles

High-throughput analysis of topographical cues for the expansion of murine pluripotent stem cells

The expansion of pluripotent stem cells (PSCs)in vitroremains a critical barrier to their use in tissue engineering and regenerative medicine. Biochemical methods for PSC expansion are known to produce heterogeneous cell populations with varying states of pluripotency and are cost-intensive, hindering their clinical translation. Engineering biomaterials to physically control PSC fate offers an alternative approach. Surface or substrate topography is a promising design parameter for engineering biomaterials. Topographical cues have been shown to elicit profound effects on stem cell differentiation and proliferation. Previous reports have shown isotropic substrate topographies to be promising in expanding PSCs. However, the optimal feature to promote PSC proliferation and the pluripotent state has not yet been determined. In this work, the MultiARChitecture (MARC) plate is developed to conduct a high-throughput analysis of topographical cues in a 96-well plate format. The MARC plate is a reproducible and customizable platform for the analysis of multiple topographical patterns and features and is compatible with both microscopic assays and molecular biology techniques. The MARC plate is used to evaluate the expression of pluripotency markersOct4, Nanog, andSox2and the differentiation markerLmnAas well as the proliferation of murine embryonic stem (mES) cells. Our systematic analyses identified three topographical patterns that maintain pluripotency in mES cells after multiple passages: 1µm pillars (1µm spacing, square arrangement), 2µm wells (c-c (x, y) = 4, 4µm), and 5µm pillars (c-c (x, y) = 7.5, 7.5µm). This study represents a step towards developing a biomaterial platform for controlled murine PSC expansion.

The MORC2 p.S87L mutation reduces proliferation of pluripotent stem cells derived from a patient with the spinal muscular atrophy-like phenotype by inhibiting proliferation-related signaling pathways.

Mutations in the microrchidia CW-type zinc finger protein 2 (MORC2) gene are the causative agent of Charcot-Marie-Tooth disease type 2Z (CMT2Z), and the hotspot mutation p.S87L is associated with a more severe spinal muscular atrophy-like clinical phenotype. The aims of this study were to determine the mechanism of the severe phenotype caused by the MORC2 p.S87L mutation and to explore potential treatment strategies. Epithelial cells were isolated from urine samples from a spinal muscular atrophy (SMA)-like patient (MORC2 p.S87L), a CMT2Z patient (MORC2 p.Q400R), and a healthy control and induced to generate pluripotent stem cells, which were then differentiated into motor neuron precursor cells. Next-generation RNA sequencing followed by KEGG pathway enrichment analysis revealed that differentially expressed genes involved in the PI3K/Akt and MAPK/ERK signaling pathways were enriched in the p.S87L SMA-like patient group and were significantly downregulated in induced pluripotent stem cells. Reduced proliferation was observed in the induced pluripotent stem cells and motor neuron precursor cells derived from the p.S87L SMA-like patient group compared with the CMT2Z patient group and the healthy control. G0/G1 phase cell cycle arrest was observed in induced pluripotent stem cells derived from the p.S87L SMA-like patient. MORC2 p.S87L-specific antisense oligonucleotides (p.S87L-ASO-targeting) showed significant efficacy in improving cell proliferation and activating the PI3K/Akt and MAPK/ERK pathways in induced pluripotent stem cells. However, p.S87L-ASO-targeting did not rescue proliferation of motor neuron precursor cells. These findings suggest that downregulation of the PI3K/Akt and MAPK/ERK signaling pathways leading to reduced cell proliferation and G0/G1 phase cell cycle arrest in induced pluripotent stem cells might be the underlying mechanism of the severe p.S87L SMA-like phenotype. p.S87L-ASO-targeting treatment can alleviate disordered cell proliferation in the early stage of pluripotent stem cell induction.

Arachidonic acid modulates the cellular energetics of human pluripotent stem cells and protects the embryoid bodies from embryotoxicity effects in vitro

Arachidonic acid (AA), an ω-6 polyunsaturated fatty acid involved in signalling pathways that drive cell fate decisions, has an enhancing role in the immunomodulatory effect on mesenchymal stem cells and the vasculogenesis of embryonic stem cells. 3D embryoid bodies (EBs) from pluripotent stem cells (PSCs) have been used as in vitro models for embryotoxicity for various compounds/drugs. Valproic acid (VA), a common anti-epileptic drug, is known to be embryotoxic and cause malformations in embryos. As early embryogenesis depends on AA, we investigated the embryo protective effects of AA against the embryotoxic drug VA in this study. The effects of AA on the proliferation and cell cycle parameters of PSCs were studied. In particular, the potential of AA to abrogate VA-induced embryotoxicity in vitro was evaluated using ROS detection and antioxidant assays. In response to AA, we observed modulation in cell proliferation of induced pluripotent stem cells (iPSCs) and pluripotent NTERA-2 embryonal carcinoma (EC) cells. The present study substantiates the cytoprotective effects of AA against VA. These results imply that AA plays a critical role in the proliferation and differentiation of iPSCs and EC cells and protects the EBs from cytotoxic damage, thereby ensuring normal embryogenesis. Thus, the bioactive lipid AA may be explored for supplementation to benefit pregnant women treated with long-term anti-epileptic drugs to prevent in-utero fetal growth malformations.

A high ratio of linoleic acid (n-6 PUFA) to alpha-linolenic acid (n-3 PUFA) adversely affects early stage of human neuronal differentiation and electrophysiological activity of glutamatergic neurons in vitro.

Introduction: There is a growing interest in the possibility of dietary supplementation with polyunsaturated fatty acids (PUFAs) for treatment and prevention of neurodevelopmental and neuropsychiatric disorders. Studies have suggested that of the two important classes of polyunsaturated fatty acids, omega-6 (n-6) and omega-3 (n-3), n-3 polyunsaturated fatty acids support brain development and function, and when used as a dietary supplement may have beneficial effects for maintenance of a healthy brain. However, to date epidemiological studies and clinical trials on children and adults have been inconclusive regarding treatment length, dosage and use of specific n-3 polyunsaturated fatty acids. The aim of this study is to generate a simplified in vitro cell-based model system to test how different n-6 to n-3 polyunsaturated fatty acids ratios affect human-derived neurons activity as a cellular correlate for brain function and to probe the mechanism of their action. Methods: All experiments were performed by use of human induced pluripotent stem cells (iPSCs). In this study, we examined the effect of different ratios of linoleic acid (n-6) to alpha-linolenic acid in cell growth medium on induced pluripotent stem cell proliferation, generation of neuronal precursors and electrophysiology of cortical glutamatergic neurons by multielectrode array (MEA) analysis. Results: This study shows that at a n-6:n-3 ratio of 5:1 polyunsaturated fatty acids induce stem cell proliferation, generating a large increase in number of cells after 72h treatment; suppress generation of neuronal progenitor cells, as measured by decreased expression of FOXG1 and Nestin in neuronal precursor cells (NPC) after 20days of development; and disrupt neuronal activity in vitro, increasing spontaneous neuronal firing, reducing synchronized bursting receptor subunits. We observed no significant differences for neuronal precursor cells treated with ratios 1:3 and 3:1, in comparison to 1:1 control ratio, but higher ratios of n-6 to n-3 polyunsaturated fatty acids adversely affect early stages of neuronal differentiation. Moreover, a 5:1 ratio in cortical glutamatergic neurons induce expression of GABA receptors which may explain the observed abnormal electrophysiological activity.

The adaptation of bovine embryonic stem cells to the changes of feeder layers.

Although the feeder-free culture system has been established, the microenvironment provided by the feeder cells still possesses a unique advantage in maintaining the long-term stability and the rapid proliferation of pluripotent stem cells (PSCs). The aim of this study is to discover the adaptive ability of PSCs upon changes of feeder layers. In this study, the morphology, pluripotent marker expression, differentiation ability of bovine embryonic stem cells (bESCs) cultured on low-density, or methanol fixed mouse embryonic fibroblasts were examined by immunofluorescent staining, Western blotting, real-time reverse transcription polymerase chain reaction, and RNA-seq. The results showed that the changes of feeder layers did not induce the rapid differentiation of bESCs, while resulting in the differentiation initiation and alteration of pluripotent state of bESCs. More importantly, the expression of endogenous growth factors and extracellular matrix were increased, and the expression of cell adhesion molecules was altered, which indicated that bESCs may compensate some functions of the feeder layers upon its changes. This study shows the PSCs have the self-adaptive ability responded to the feeder layer alteration.

In vitro characterization of iridoid and phenylethanoid glycosides from Cistanche phelypaea for nutraceutical and pharmacological applications.

"Desert hyacinths" are a remarkable group of parasitic plants belonging to genus Cistanche, including more than 20 accepted species typically occurring in deserts or coastal dunes parasitizing roots of shrubs. Several Cistanche species have long been a source of traditional herbal medicine or food, being C.deserticola and C.tubulosa the most used in China. This manuscript reports the isolation and identification of some phenylethanoid and iridoid glycosides, obtained from the hydroalcoholic extract of C.phelypaea collected in Spain. The present study aims to characterize the antioxidant activity of C.phelypaea metabolites in the light of their application in nutraceutical and cosmeceutical industries and the effect of acetoside, the most abundant metabolite in C.phelypaea extract, on human keratinocyte and pluripotent stem cell proliferation and differentiation. Our study demonstrated that acetoside, besides its strong antioxidant potential, can preserve the proliferative potential of human basal keratinocytes and the stemness of mesenchymal progenitors necessary for tissue morphogenesis and renewal. Therefore, acetoside can be of practical relevance for the clinical application of human stem cell cultures in tissue engineering and regenerative medicine.

Redox Homeostasis and Regulation in Pluripotent Stem Cells: Uniqueness or Versatility?

Pluripotent stem cells (PSCs) hold great potential both in studies on developmental biology and clinical practice. Mitochondrial metabolism that encompasses pathways that generate ATP and produce ROS significantly differs between PSCs and somatic cells. Correspondingly, for quite a long time it was believed that the redox homeostasis in PSCs is also highly specific due to the hypoxic niche of their origin—within the pre-implantation blastocyst. However, recent research showed that redox parameters of cultivated PSCs have much in common with that of their differentiated progeny cells. Moreover, it has been proven that, similar to somatic cells, maintaining the physiological ROS level is critical for the regulation of PSC identity, proliferation, differentiation, and de-differentiation. In this review, we aimed to summarize the studies of redox metabolism and signaling in PSCs to compare the redox profiles of pluripotent and differentiated somatic cells. We collected evidence that PSCs possess metabolic plasticity and are able to adapt to both hypoxia and normoxia, that pluripotency is not strictly associated with anaerobic conditions, and that cellular redox homeostasis is similar in PSCs and many other somatic cells under in vitro conditions that may be explained by the high conservatism of the redox regulation system.

DNA Damage Response and Cell Cycle Regulation in Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) hold great promise in cell-based therapy because of their pluripotent property and the ability to proliferate indefinitely. Embryonic stem cells (ESCs) derived from inner cell mass (ICM) possess unique cell cycle control with shortened G1 phase. In addition, ESCs have high expression of homologous recombination (HR)-related proteins, which repair double-strand breaks (DSBs) through HR or the non-homologous end joining (NHEJ) pathway. On the other hand, the generation of induced pluripotent stem cells (iPSCs) by forced expression of transcription factors (Oct4, Sox2, Klf4, c-Myc) is accompanied by oxidative stress and DNA damage. The DNA repair mechanism of DSBs is therefore critical in determining the genomic stability and efficiency of iPSCs generation. Maintaining genomic stability in PSCs plays a pivotal role in the proliferation and pluripotency of PSCs. In terms of therapeutic application, genomic stability is the key to reducing the risks of cancer development due to abnormal cell replication. Over the years, we and other groups have identified important regulators of DNA damage response in PSCs, including FOXM1, SIRT1 and PUMA. They function through transcription regulation of downstream targets (P53, CDK1) that are involved in cell cycle regulations. Here, we review the fundamental links between the PSC-specific HR process and DNA damage response, with a focus on the roles of FOXM1 and SIRT1 on maintaining genomic integrity.

QuinoMit Q10-Fluid attenuates hydrogen peroxide-induced irregular beating in mouse pluripotent stem cell-derived cardiomyocytes

BackgroundCoenzyme Q10 (CoQ10) is a crucial component of the mitochondrial structure which is involved in producing more than 90% of cellular ATP. This study aimed to investigate the protective effects and underlying mechanisms of QuinoMit Q10-Fluid against hydrogen peroxide (H2O2)-induced arrhythmias on cardiomyocytes (CMs). MethodsUndifferentiated stem cell-derived CMs were cultured in the presence of different concentrations of QuinoMit Q10-Fluid. To investigate if CoQ10 has anti-apoptotic activity, CMs were exposed to H2O2 for up to 100 h with or without CoQ10. The expression levels of cardiac reference genes were determined by RT-PCR. The structural and functional properties of CMs were examined by immunofluorescence and the xCELLigence system. Caspase 3/7 assay was also performed for cell apoptosis study. ResultsThe study showed that QuinoMit Q10-Fluid inhibits the proliferation of pluripotent stem cells at high concentrations and had less effect on cardiomyogenesis. However, the beating rate of clusters containing CMs generated under QuinoMit Q10-Fluid (1:100) was significantly increased. This increase was accompanied by the up-regulated expression level of some important cardiac markers during differentiation. Treatment of CMs with H2O2 notably induced irregular beating and decreased the amplitude of the beating signal of CMs, concomitantly with increased caspase-3/7 activity. However, CMs pretreated with QuinoMit exhibited a protective effect against H2O2-induced arrhythmia. ConclusionOur results reveal that QuinoMit Q10-Fluid attenuates H2O2-induced irregular beating in mouse pluripotent stem cell-derived CMs, at least partly by reducing the generation of ROS, suggesting a protective effect against CM dysfunctions.

The combination of dextran sulphate and polyvinyl alcohol prevents excess aggregation and promotes proliferation of pluripotent stem cells in suspension culture.

ObjectivesFor clinical applications of cell‐based therapies, a large quantity of human pluripotent stem cells (hPSCs) produced in standardized and scalable culture processes is required. Currently, microcarrier‐free suspension culture shows potential for large‐scale expansion of hPSCs; however, hPSCs tend to aggregate during culturing leading to a negative effect on cell yield. To overcome this problem, we developed a novel protocol to effectively control the sizes of cell aggregates and enhance the cell proliferation during the expansion of hPSCs in suspension.Materials and MethodshPSCs were expanded in suspension culture supplemented with polyvinyl alcohol (PVA) and dextran sulphate (DS), and 3D suspension culture of hPSCs formed cell aggregates under static or dynamic conditions. The sizes of cell aggregates and the cell proliferation as well as the pluripotency of hPSCs after expansion were assessed using cell counting, size analysis, real‐time quantitative polymerase chain reaction, flow cytometry analysis, immunofluorescence staining, embryoid body formation, teratoma formation and transcriptome sequencing.ResultsOur results demonstrated that the addition of DS alone effectively prevented hPSC aggregation, while the addition of PVA significantly enhanced hPSC proliferation. The combination of PVA and DS not only promoted cell proliferation of hPSCs but also produced uniform and size‐controlled cell aggregates. Moreover, hPSCs treated with PVA, or DS or a combination, maintained the pluripotency and were capable of differentiating into all three germ layers. mRNA‐seq analysis demonstrated that the combination of PVA and DS significantly promoted hPSC proliferation and prevented cell aggregation through improving energy metabolism‐related processes, regulating cell growth, cell proliferation and cell division, as well as reducing the adhesion among hPSC aggregates by affecting expression of genes related to cell adhesion.ConclusionsOur results represent a significant step towards developing a simple and robust approach for the expansion of hPSCs in large scale.

Kinetics and adsorption isotherm of ammonia uptake by cation exchange resins and treatment of mixed aqueous lactate–ammonia by Mg–Al layered double oxide and the resins

The proliferation of pluripotent stem cells in mass cell culture is hindered by the release of cell metabolites such as lactate and ammonia. In this work, a porous strong-acid cation exchange resin (PK216LH) and Mg–Al layered doubled oxide (LDO) were used to take up NH4+ and lactate, respectively. Initially, PK216LH could take up NH4+ from an aqueous solution. The uptake of NH4+ by PK216LH followed pseudo-second-order kinetics and corresponded to the Langmuir adsorption isotherm, with an apparent activation energy of 28.2 kJ/mol and a maximum adsorption amount of 1.38 mmol/g. The simultaneous use of Mg–Al LDO and PK216LH could not sufficiently take up lactate and NH4+. In contrast, the individual use of Mg–Al LDO and PK216LH in two stages enabled efficient removal of lactate and NH4+.

UV/ozone surface modification combined with atmospheric pressure plasma irradiation for cell culture plastics to improve pluripotent stem cell culture

Culturing pluripotent stem cells effectively requires substrates coated with feeder cell layers or cell-adhesive matrices. It is difficult to employ pluripotent stem cells as resources for regenerative medicine due to risks of culture system contamination by animal-derived factors, or the large costs associated with the use of adhesive matrices. To enable a coating-free culture system, we focused on UV/ozone surface modification and atmospheric pressure plasma treatment for polystyrene substrates, to improve adhesion and proliferation of pluripotent stem cells. In this study, to develop a feeder- and matrix coating-free culture system for embryonic stem cells (ESCs), mouse ESCs were cultured on polystyrene substrates that were surface-modified using UV/ozone-plasma combined treatment. mESCs could be successfully cultured under feeder-free conditions upon UV/ozone-plasma combined treatment of culture substrates, without any further chemical treatments, and showed similar proliferation rates to those of cells grown on the feeder cell layer or matrix-coated substrates.

Concise Review; Effects of Antibiotics and Antimycotics on the Biological Properties of Human Pluripotent and Multipotent Stem Cells.

Human Pluripotent Stem Cells (PSCs), including Embryonic Stem Cells (ESCs) and induced Pluripotent Stem Cells (iPSCs), have the remarkable potential to self-renew and develop into various cell lineages. Human Mesenchymal Stem Cells (MSCs) or multipotent stem cells that are present in various organs can self-renew and differentiate into multiple mesenchymal lineages. Both human PSCs and MSCs hold great promise in cell-based therapies, disease modeling, drug discovery, and regenerative medicine. Human stem cells must be cultured under the optimal conditions to use them in transplantology. Therefore, researchers must ensure the sterility of human stem cell lines. Bacterial contamination is a common problem in laboratories and major precautions are required to detect the types of microorganisms, and to eliminate and prevent contamination in cell cultures. Stem cell culture media usually contain antibiotics and antimycotics such as penicillin- streptomycin (pen-strep), gentamicin, and amphotericin B (AmB) to avoid bacterial, fungal, and yeast contaminants. Numerous publications recognized the serious effect of antibiotics and antimycotics on in vitro properties of human stem cells, including proliferation, differentiation, survival, and genetic instability. This review study aimed to understand the impact of routinely used antibiotics and antimycotics such as pen-strep, gentamicin, and AmB on viability, proliferation, and functional properties (differentiation and pluripotency) of human PSCs and MSCs.

Application of the RBBP9 Serine Hydrolase Inhibitor, ML114, Decouples Human Pluripotent Stem Cell Proliferation and Differentiation.

Retinoblastoma binding protein 9 (RBBP9) is required for maintaining the expression of both pluripotency and cell cycle genes in human pluripotent stem cells (hPSCs). An siRNA-based study from our group showed it does so by influencing cell cycle progression through the RB/E2F pathway. In non-pluripotent cells, RBBP9 is also known to have serine hydrolase (SH) activity, acting on currently undefined target proteins. The role of RBBP9 SH activity in hPSCs, and during normal development, is currently unknown. To begin assessing whether RBBP9 SH activity might contribute to hPSC maintenance, hPSCs were treated with ML114—a selective chemical inhibitor of RBBP9 SH activity. Stem cells treated with ML114 showed significantly reduced population growth rate, colony size and progression through the cell cycle, with no observable change in cell morphology or decrease in pluripotency antigen expression—suggesting no initiation of hPSC differentiation. Consistent with this, hPSCs treated with ML114 retained the capacity for tri-lineage differentiation, as seen through teratoma formation. Subsequent microarray and Western blot analyses of ML114-treated hPSCs suggest the nuclear transcription factor Y subunit A (NFYA) may be a candidate effector of RBBP9 SH activity in hPSCs. These data support a role for RBBP9 in regulating hPSC proliferation independent of differentiation, whereby inhibition of RBBP9 SH activity de-couples decreased hPSC proliferation from initiation of differentiation.

Nucleosides Rescue Replication-Mediated Genome Instability of Human Pluripotent Stem Cells.

SummaryHuman pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have now found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. However, we have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth.

In Situ Expansion, Differentiation, and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid.

One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3-dimensional bioprinting. Yet, inclusion of contiguous, living muscle to support pump function has not been achieved. This is largely due to the challenge of attaining high densities of cardiomyocytes-a notoriously nonproliferative cell type. An alternative strategy is to print with human induced pluripotent stem cells, which can proliferate to high densities and fill tissue spaces, and subsequently differentiate them into cardiomyocytes in situ. To develop a bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle. We optimized a photo-crosslinkable formulation of native ECM (extracellular matrix) proteins and used this bioink to 3-dimensionally print human induced pluripotent stem cell-laden structures with 2 chambers and a vessel inlet and outlet. After human induced pluripotent stem cells proliferated to a sufficient density, we differentiated the cells within the structure and demonstrated function of the resultant human chambered muscle pump. Human chambered muscle pumps demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing. The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease. This advance represents a critical step toward generating macroscale tissues, akin to aggregate-based organoids, but with the critical advantage of harboring geometric structures essential to the pump function of cardiac muscle. Looking forward, human chambered organoids of this type might also serve as a test bed for cardiac medical devices and eventually lead to therapeutic tissue grafting.

The immobilization of fibronectin- and fibroblast growth factor 2-derived peptides on a culture plate supports the attachment and proliferation of human pluripotent stem cells.

Pluripotent stem cells (PSCs) offer a promising tool for regenerative medicine. The clinical application of PSCs inevitably requires a large-scale culture in a highly defined environment. The present study aimed to devise defined coating materials for the efficient adhesion and proliferation of human PSCs (hPSCs). We tested the activity of seven fibronectin-derived peptides and three laminin-derived peptides for the attachment and proliferation of hPSCs through their immobilization on the bottom of culture dishes by creating a fusion protein with the mussel adhesion protein. Among the extracellular matrix (ECM) mimetics tested, one fibronectin-derived peptide, PHSRN-GRGDSP, significantly promoted adhesion, enhanced alkaline phosphatase activity, and increased pluripotency-related gene expression in hPSCs compared to Matrigel. Furthermore, co-immobilization of a particular canofin peptide derived from fibroblast growth factor 2 increased pluripotency marker expression, which may offer the possibility of culture without growth factor supplementation. Our findings afford a novel defined condition for the efficient culture of hPSCs and may be utilized in future clinical applications.

Pluripotent stem cells proliferation is associated with placentation in dogs

Pluripotent stem cells have been studied as source of cells for regenerative medicine and acquire or genetic diseases, as an innovative therapy. Most tissues have stem cells populations, however in few quantities or impossible to be used during adult life, which lead to scientists look for new sources. Thus, this study aimed to analyze the presence of pluripotent cells in the uterus and placenta, following up non-pregnant, pregnant (begin, middle, and final), and postpartum periods in dogs. The uteri were obtained from social castration programs for population control in Pirassununga, Sao Paulo, Brazil. It was collected 20 uteri at different stages. The samples were fixed and processed for immunohistochemical analysis of NANOG, OCT4 and SOX2 expression, knowing as pluripotent stem cells makers. Our results showed positive expression for NANOG, OCT4 and SOX2 in all stages of gestation and nonpregnant uterus; however, we highlight some quantitative different between stages. OCT4 showed more expression in non-pregnant uterus than NANOG and SOX2, and its expression increased in pregnant uterus. In pregnant uterus there was more expression of NANOG than OCT4 and SOX2. Interesting, no difference was found between these markers in the other periods. In conclusion, it was possible to identify pluripotent stem cells in all periods in dog placenta and uterus, however during the early stage of pregnancy we observed more pluripotent stem cells than in all the others periods confirming the high plasticity and regeneration capacity of the uterine tissue.

Tissue inhibitor of metalloproteinase proteins inhibit teratoma growth in mice transplanted with pluripotent stem cells.

Pluripotent stem cells (PSCs) can serve as an unlimited cell source for transplantation therapies for treating various devastating diseases, such as cardiovascular diseases, diabetes, and Parkinson's disease. However, PSC transplantation has some associated risks, including teratoma formation from the remaining undifferentiated PSCs. Thus, for successful clinical application, it is essential to ablate the proliferative PSCs before or after transplantation. In this study, neural stem cell-derived conditioned medium (NSC-CM) inhibited the proliferation of PSCs and PSC-derived neural precursor (NP) cells without influencing the potential of PSC-NP cells to differentiate into neurons in vitro and prevented teratoma growth in vivo. Moreover, we found that the NSC-CM remarkably decreased the expression levels of Oct4 and cyclin D1 that Oct4 directly binds to and increased the cleaved-caspase 3-positive cell death through the DNA damage response in PSCs and PSC-NPs. Interestingly, we found that NSCs distinctly secreted the tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 proteins. These proteins suppressed not only the proliferation of PSCs in cell culture but also teratoma growth in mice transplanted with PSCs through inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 activity. Taken together, these results suggest that the TIMP proteins may improve the efficacy and safety of the PSC-based transplantation therapy.

Putting Stem Cells on a Low-Fat Diet Switches Their Pluripotent State.

Previous work demonstrating the importance of stem cell metabolism in regulating development ignited a debate on how intrinsic versus extrinsic conditions influence metabolic programming of epigenetics. In this issue of Cell Stem Cell, Cornacchia etal. show that exogenous lipids regulate an acetyl-CoA-dependent switch to influence the intrinsic pluripotent state of hPSCs.