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Plastics have been part of our ecosystem for about a century and their degradation by different environmental factors produce secondary microplastics (MPs). To date, the impact of MPs on human health has not been well investigated. To understand the possible effects of polystyrene-MPs (PS-MPs) on the human brain, a 3D model of human forebrain cortical spheroids has been derived, which mimics early development of human cerebral cortex. The spheroids were exposed to 100, 50, and 5 µg/mL of 1 µm and 10 µm PS-MPs during day 4-10 and day 4-30. The short-term MP exposure showed the promoted proliferation and high gene expression of Nestin, PAX6, ATF4, HOXB4 and SOD2. For long-term exposure, reduced cell viability was observed. Moreover, changes in size and concentration of PS-MPs altered the gene expression of DNA damage and neural tissue patterning. In particular, β-tubulin III, Nestin, and TBR1/TBR2 gene expression decreased in PS-MP treated conditions compare to the untreated control. The results of this study suggest that the size- and concentration-dependent exposure to PS-MPs can adversely affect embryonic brain-like tissue development in forebrain cerebral spheroids. This study has significance in assessing environmental factors in neurotoxicity and degeneration in human., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification., (© 2021. The Author(s).)

The three peripheral sensory neuron (SN) subtypes, nociceptors, mechanoreceptors, and proprioceptors, localize to dorsal root ganglia and convey sensations such as pain, temperature, pressure, and limb movement/position. Despite previous reports, to date no protocol is available allowing the generation of all three SN subtypes at high efficiency and purity from human pluripotent stem cells (hPSCs). We describe a chemically defined differentiation protocol that generates all three SN subtypes from the same starting population, as well as methods to enrich for each individual subtype. The protocol yields high efficiency and purity cultures that are electrically active and respond to specific stimuli. We describe their molecular character and maturity stage and provide evidence for their use as an axotomy model; we show disease phenotypes in hPSCs derived from patients with familial dysautonomia. Our protocol will allow the modeling of human disorders affecting SNs, the search for treatments, and the study of human development., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

2,4,6-trichlorophenol (TCP), 2,4,6-tribromophenol (TBP) and 2,4,6-triiodophenol (TIP) are a new class of halophenolic disinfection byproducts (DBPs) which have been widely detected in drinking water. In recent years, their developmental toxicity has got increasing public attention due to their potential toxic effects on embryo development towards lower organisms. Nonetheless, the application of human embryos for embryonic toxicologic studies is rendered by ethical and moral considerations, as well as the technical barrier to sustaining normal development beyond a few days. Human extended pluripotent stem (EPS) cells (novel totipotent-like stem cells) represent a much more appropriate cellular model for studying human embryo development. In this study, we utilized human EPS cells to study the developmental toxicity of TCP, TBP and TIP, respectively. All three halophenolic DBPs showed cytotoxicity against human EPS cells in an obvious dose-dependent manner, among which TIP was the most cytotoxic one. Notably, the expression of pluripotent genes in human EPS cells significantly declined after 2,4,6-trihalophenol exposure. Meanwhile, 2,4,6-trihalophenol exposure promoted ectodermal differentiation of human EPS cells in an embryoid bodies (EBs) differentiation assay, while both endodermal and mesodermal differentiation were impaired. These results implied that phenolic halogenated DBPs have specific effects on human embryo development even in the early stage of pregnancy. In summary, we applied human EPS cells as a novel research model for human embryo developmental toxicity study of environmental pollutants, and demonstrated the toxicity of phenolic halogenated DBPs on early embryo development of human beings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The mechanisms of kidney injury and fibrosis can now be studied using kidney organoids derived from human pluripotent stem cells (hPSCs). Mature kidney organoids contain nephrons and stromal cells with fibrogenic potential, spatially organized in a manner that resembles the anatomy of the kidney. Organoid nephron damage and interstitial fibrosis can be induced under well-controlled experimental conditions in vitro, making this an ideal system for the study of tissue-intrinsic cell signaling and intercellular crosstalk mechanisms in the absence of systemic signals and immune cells that are present in vivo. Here we describe methods for the generation of kidney organoids from a widely used hPSC line, and for the induction and analysis of nephron damage and interstitial fibrosis.

Tremendous progress has been made in recent years to produce functional cells for cell therapy products. Hundreds of clinical trials of stem cell products (SCPs) have shown promising therapeutic potential worldwide, including the products derived from human pluripotent stem cells (hPSCs), adult stem cells and mesenchymal stem cells (MSC). Before starting a clinical trial, comprehensive chemistry, manufacturing and control (CMC) study is required to assure the safety and quality consistency of SCPs. The heterogeneity of stem cell products arises from the variability in the donor tissues, isolation of cells and differentiation processes, and appropriate testing approaches are needed to characterize and release SCPs. Here we summarize the regulatory considerations of CMC study in Investigational New Drug (IND) application of SCPs in China based on the current knowledge, and they will be updated in the future with the advance of stem cell biology and regulatory science., (Copyright © 2020 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Background: Enhancers are distal regulators of gene expression that shape cell identity and control cell fate transitions. In mouse embryonic stem cells (mESCs), the pluripotency network is maintained by the function of a complex network of enhancers, that are drastically altered upon differentiation. Genome-wide chromatin accessibility and histone modification assays are commonly used as a proxy for identifying putative enhancers and for describing their activity levels and dynamics., Results: Here, we applied STARR-seq, a genome-wide plasmid-based assay, as a read-out for the enhancer landscape in "ground-state" (2i+LIF; 2iL) and "metastable" (serum+LIF; SL) mESCs. This analysis reveals that active STARR-seq loci show modest overlap with enhancer locations derived from peak calling of ChIP-seq libraries for common enhancer marks. We unveil ZIC3-bound loci with significant STARR-seq activity in SL-ESCs. Knock-out of Zic3 removes STARR-seq activity only in SL-ESCs and increases their propensity to differentiate towards the endodermal fate. STARR-seq also reveals enhancers that are not accessible, masked by a repressive chromatin signature. We describe a class of dormant, p53 bound enhancers that gain H3K27ac under specific conditions, such as after treatment with Nocodazol, or transiently during reprogramming from fibroblasts to pluripotency., Conclusions: In conclusion, loci identified as active by STARR-seq often overlap with those identified by chromatin accessibility and active epigenetic marking, yet a significant fraction is epigenetically repressed or display condition-specific enhancer activity.

Chromosome structure is a crucial regulatory factor for a wide range of nuclear processes. Chromosome conformation capture (3C)-based experiments combined with computational modelling are pivotal for unveiling 3D chromosome structure. Here, we introduce TADdyn, a tool that integrates time-course 3C data, restraint-based modelling, and molecular dynamics to simulate the structural rearrangements of genomic loci in a completely data-driven way. We apply TADdyn on in situ Hi-C time-course experiments studying the reprogramming of murine B cells to pluripotent cells, and characterize the structural rearrangements that take place upon changes in the transcriptional state of 21 genomic loci of diverse expression dynamics. By measuring various structural and dynamical properties, we find that during gene activation, the transcription starting site contacts with open and active regions in 3D chromatin domains. We propose that these 3D hubs of open and active chromatin may constitute a general feature to trigger and maintain gene transcription.

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is increasingly used for detection of various macromolecules and metabolites in biological samples. Here, we present a detailed analysis of the CuAAC reaction conditions in cells and tissue sections. Using the optimized CuAAC conditions, we have devised a highly sensitive immunostaining technique, based on the tyramide signal amplification/catalyzed reporter deposition (TSA/CARD) method with a novel alkyne tyramide substrate. The described method offers improved detection threshold compared to conventional immunofluorescent staining and produces significantly lower non-specific background than TSA/CARD with fluorescent tyramides.

Metabolic remodelling has emerged as critical for stem cell pluripotency; however, the underlying mechanisms have yet to be fully elucidated. Here, we found that the glycine cleavage system (GCS) is highly activated to promote stem cell pluripotency and during somatic cell reprogramming. Mechanistically, we revealed that the expression of Gldc, a rate-limiting GCS enzyme regulated by Sox2 and Lin28A, facilitates this activation. We further found that the activated GCS catabolizes glycine to fuel H3K4me3 modification, thus promoting the expression of pluripotency genes. Moreover, the activated GCS helps to cleave excess glycine and prevents methylglyoxal accumulation, which stimulates senescence in stem cells and during reprogramming. Collectively, our results demonstrate a novel mechanism whereby GCS activation controls stem cell pluripotency by promoting H3K4me3 modification and preventing cellular senescence., (© 2019 Tian et al.)

High-throughput biological technologies (e.g. ChIP-seq, RNA-seq and single-cell RNA-seq) rapidly accelerate the accumulation of genome-wide omics data in diverse interrelated biological scenarios (e.g. cells, tissues and conditions). Integration and differential analysis are two common paradigms for exploring and analyzing such data. However, current integrative methods usually ignore the differential part, and typical differential analysis methods either fail to identify combinatorial patterns of difference or require matched dimensions of the data. Here, we propose a flexible framework CSMF to combine them into one paradigm to simultaneously reveal Common and Specific patterns via Matrix Factorization from data generated under interrelated biological scenarios. We demonstrate the effectiveness of CSMF with four representative applications including pairwise ChIP-seq data describing the chromatin modification map between K562 and Huvec cell lines; pairwise RNA-seq data representing the expression profiles of two different cancers; RNA-seq data of three breast cancer subtypes; and single-cell RNA-seq data of human embryonic stem cell differentiation at six time points. Extensive analysis yields novel insights into hidden combinatorial patterns in these multi-modal data. Results demonstrate that CSMF is a powerful tool to uncover common and specific patterns with significant biological implications from data of interrelated biological scenarios., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cohesin and CTCF are master regulators of genome topology. How these ubiquitous proteins contribute to cell-type specific genome structure is poorly understood. Here, we explore quantitative aspects of topologically associated domains (TAD) between pluripotent embryonic stem cells (ESC) and lineage-committed cells. ESCs exhibit permissive topological configurations which manifest themselves as increased inter- TAD interactions, weaker intra-TAD interactions, and a unique intra-TAD connectivity whereby one border makes pervasive interactions throughout the domain. Such 'stripe' domains are associated with both poised and active chromatin landscapes and transcription is not a key determinant of their structure. By tracking the developmental dynamics of stripe domains, we show that stripe formation is linked to the functional state of the cell through cohesin loading at lineage-specific enhancers and developmental control of CTCF binding site occupancy. We propose that the unique topological configuration of stripe domains represents a permissive landscape facilitating both productive and opportunistic gene regulation and is important for cellular identity.

Stem cell is critical to regeneration of tissue or organ of human. How to promote repair or regeneration in the tissues/organ using its pluripotency is always an important issue. Lgr5-possitive cell is one type of the stem cell-like cells capable of pluripotent differentiation in various tissues/organs of both humans and mice. Current study showed that single or small amount Lgr5-possitive stem cells can grow and form a plurality of organs in 3D culture system, and some organs can present similar biological and physiological properties with the progenitor they were derived. These studies provided new insight into future orientation, for example, Lgr5-possitive inner ear cells were confirmed as inner ear pluripotent cells population, the experiences obtained from organoid studies of Lgr5-possitive cells have certainly showed potential in the future study of inner ear stem cells. This review will focus on the recent progress associated with Lgr 5-positive stem cells forming organoids in the 3D culture.

Higher-order chromatin organization controls transcriptional programs that govern cell properties and functions. In order for pluripotent stem cells (PSCs) to appropriately respond to differentiation signals, developmental gene loci should be structurally and spatially regulated to be readily available for immediate transcription, even though these genes are hardly expressed in PSCs. Here, we show that both chromatin interaction profiles and nuclear positions at developmental gene loci differ between human somatic cells and hPSCs, and that changes in the chromatin interactions are closely related to the nuclear repositioning. Moreover, we also demonstrate that developmental gene loci, which have bivalent histone modifications, tend to colocalize in PSCs. Furthermore, this colocalization requires PRC1, PRC2, and TrxG complexes, which are essential regulatory factors for the maintenance of transcriptionally poised developmental genes. Our results indicate that higher-order chromatin regulation may be an integral part of the differentiation capacity that defines pluripotency.

A sensitive method to simultaneously quantitate chlorpyrifos, chlorpyrifos oxon and the detoxified product 3,5,6-trichloro-2-pyridinol (TCP) was developed using either liquid-liquid extraction for culture media samples, or protein precipitation for cell samples. Multiple reaction monitoring in positive ion mode was applied for the detection of chlorpyrifos and chlorpyrifos oxon, and selected ion recording in negative mode was applied to detect TCP. The method provided linear ranges from 5 to 500, 0.2-20 and 20-2000ng/mL for media samples and from 0.5-50, 0.02-2 and 2-200ng/million cells for CPF, CPO and TCP, respectively. The method was validated using selectivity, linearity, precision, accuracy, recovery, stability and dilution tests. All relative standard deviations (RSDs) and relative errors (REs) for QC samples were within 15% (except for LLOQ, within 20%). This method has been successfully applied to study the neurotoxicity and metabolism of chlorpyrifos in a human neuronal model., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8 + T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.

Endothelial cells derived from human pluripotent stem cells are a promising cell type for enhancing angiogenesis in ischemic cardiovascular tissues. However, our understanding of microenvironmental factors that modulate the process of endothelial differentiation is limited. We examined the role of combinatorial extracellular matrix (ECM) proteins on endothelial differentiation systematically using an arrayed microscale platform. Human pluripotent stem cells were differentiated on the arrayed ECM microenvironments for 5 days. Combinatorial ECMs composed of collagen IV + heparan sulfate + laminin (CHL) or collagen IV + gelatin + heparan sulfate (CGH) demonstrated significantly higher expression of CD31, compared to single-factor ECMs. These results were corroborated by fluorescence activated cell sorting showing a 48% yield of CD31 + /VE-cadherin + cells on CHL, compared to 27% on matrigel. To elucidate the signaling mechanism, a gene expression time course revealed that VE-cadherin and FLK1 were upregulated in a dynamically similar manner as integrin subunit β3 (>50 fold). To demonstrate the functional importance of integrin β3 in promoting endothelial differentiation, the addition of neutralization antibody inhibited endothelial differentiation on CHL-modified dishes by >50%. These data suggest that optimal combinatorial ECMs enhance endothelial differentiation, compared to many single-factor ECMs, in part through an integrin β3-mediated pathway.

Despite their well-known function in maintaining normal cell physiology, how inorganic elements are relevant to cellular pluripotency and differentiation in human pluripotent stem cells (hPSCs) has yet to be systematically explored. Using total reflection X-ray fluorescence (TXRF) spectrometry and inductively coupled plasma mass spectrometry (ICP-MS), we analyzed the inorganic components of human cells with isogenic backgrounds in distinct states of cellular pluripotency. The elemental profiles revealed that the potassium content of human cells significantly differs when their cellular pluripotency changes. Pharmacological treatment that alters cell membrane permeability to potassium affected the maintenance and establishment of cellular pluripotency via multiple mechanisms in bona fide hPSCs and reprogrammed cells. Collectively, we report that potassium is a pluripotency-associated inorganic element in human cells and provide novel insights into the manipulation of cellular pluripotency in hPSCs by regulating intracellular potassium.

Background: Human embryonic stem cells (hESCs) hold tremendous promise for cell replacement therapies for a range of degenerative diseases. In order to provide cost-effective treatments affordable by public health systems, HLA-matched allogeneic tissue banks of the highest quality clinical-grade hESCs will be required. However only a small number of existing hESC lines are suitable for clinical use; they are limited by moral and ethical concerns and none of them apply Good Manufacturing Practice (GMP) standards to the earliest and critical stages of gamete and embryo procurement. We thus aimed to derive new clinical grade hESC lines of highest quality from fresh surplus GMP grade human embryos., Methods: A comprehensive screen was performed for suitable combinations of culture media with supporting feeder cells or feeder-free matrix, at different stages, to support expansion of the inner cell mass and to establish new hESC lines., Results: We developed a novel two-step and sequential media system of clinical-grade hESC derivation and successfully generated seven new hESC lines of widely varying HLA type, carefully screened for genetic health, from human embryos donated under the highest ethical and moral standards under an integrated GMP system which extends from hESC banking all the way back to gamete and embryo procurement., Conclusions: The present study, for the first time, reports the successful derivation of highest-quality clinical-grade hESC lines from fresh poor-quality surplus human embryos generated in a GMP-grade IVF laboratory. The availability of hESC lines of this status represents an important step towards more widespread application of regenerative medicine therapies.

Human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, but their derivatives need to be rigorously evaluated for residual stem cells to prevent teratoma formation. Here, we report the development of novel surface-enhanced Raman scattering (SERS)-based assays that can detect trace numbers of undifferentiated hPSCs in mixed cell populations in a highly specific, ultra-sensitive, and time-efficient manner. By targeting stem cell surface markers SSEA-5 and TRA-1-60 individually or simultaneously, these SERS assays were able to identify as few as 1 stem cell in 10(6) cells, a sensitivity (0.0001%) which was ∼2000 to 15,000-fold higher than that of flow cytometry assays. Using the SERS assay, we demonstrate that the aggregation of hPSC-based cardiomyocyte differentiation cultures into 3D spheres significantly reduced SSEA-5(+) and TRA-1-60(+) cells compared with parallel 2D cultures. Thus, SERS may provide a powerful new technology for quality control of hPSC-derived products for preclinical and clinical applications., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The advent of technology has led to an established range of engineered nanoparticles that are used in diverse applications, such as cell-cell interactions, cell-material interactions, medical therapies and the target modulation of cellular processes. The exponential increase in the utilization of nanomaterials and the growing number of associated criticisms has highlighted the potential risks of nanomaterials to human health and the ecosystem. The existing in vivo and in vitro platforms show limitations, with fluctuations being observed in the results of toxicity assessments. Pluripotent stem cells (PSCs) are viable source of cells that are capable of developing into specialized cells of the human body. PSCs can be efficiently used to screen new biomaterials/drugs and are potential candidates for studying impairments of biophysical morphology at both the cellular and tissue levels during interactions with nanomaterials and for diagnosing toxicity. Three-dimensional in vitro models obtained using PSC-derived cells would provide a realistic, patient-specific platform for toxicity assessments and in drug screening applications. The current review focuses on PSCs as an alternative in vitro platform for assessing the hazardous effects of nanomaterials on health systems and highlights the importance of PSC-derived in vitro platforms. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Ovarian cancer is among the most common gynecologic cancers and unfortunately the most common cause of death from gynecologic malignancies. Due to few early symptoms and insufficient screening programs, an early diagnosis of ovarian cancer is very difficult and new biomarkers related to early ovarian carcinogenesis are needed. In the last years a growing scientific knowledge about cancer stem cells and their markers opened a new perspective on screening and early diagnosis of ovarian cancer. The transcription factor NANOG is not only a pluripotency and cancer stem cell-related marker, but also promotes cancer stem cell-like characteristics of tumor, tumor growth, dissemination, immune evasion, and resistance to conventional therapy. The recent data showed that small stem cells resembling very small embryonic-like stem cells are present in the ovarian surface epithelium of adult human ovaries. These cells expressed several genes related to primordial germ cells, germinal lineage, and pluripotency, including NANOG, therefore their involvement in the manifestation of ovarian cancer are not excluded. As majority of cancer cells within a tumor are non tumorigenic, the therapies targeting these cells cause tumor regression, but the survived cancer stem cells regenerate the tumor, so tumor relapse or reoccur. The eradication of cancer actually requires the elimination of cancer stem cells, therefore new strategies in treatment that specifically target cancer stem cells are urgently needed. Although the therapeutic efficacy of targeting NANOG as a cancer treatment method is still in experimental phase, the gene therapy with small interfering RNA or short hairpin RNA have already shown some promising therapeutic potential. The authors can conclude that NANOG represents a promising diagnostic marker and agent for target therapy of ovarian cancer.

Aims: In vitro expansion changes replication and differentiation capacity of mesenchymal stem cells (MSCs), increasing challenges and risks, while limiting the sufficient number of MSCs required for cytotherapy. Here, we characterized and compared proliferation, differentiation, telomere length and pluripotency marker expression in MSCs of various origins., Main Methods: Immunophenotyping, proliferation and differentiation assays were performed. Pluripotency marker (Nanog, Oct-4, SOX-2, SSEA-4) expression was determined by immunofluorescence. Quantitative PCR was performed for relative telomere length (RTL) analyses, while expression of relevant genes for pluripotency markers, differentiation state (Cbfa1, human placental alkaline phosphatase, peroxisome proliferator activated receptor, Sox9 and Collagen II a1), and telomerase reverse transcriptase (hTERT) was determined by semiquantitative RT-PCR., Key Findings: Peripheral blood MSCs (PB-MSCs) and umbilical cord MSCs (UC-MSCs) showed the highest, while periodontal ligament MSCs (PDL-MSCs) and adipose tissue MSCs (AT-MSCs) the lowest values of both the replication potential and RTL. Although MSCs from exfoliated deciduous teeth (SHEDs), PDL-MSCs and AT-MSCs showed higher mRNA expression of pluripotency markers, all MSCs expressed pluripotency marker proteins. SHEDs and PDL-MSCs showed prominent capacity for osteogenesis, PB-MSCs and UC-MSCs showed strengthened adipogenic differentiation potential, while AT-MSCs displayed similar differentiation into both lines., Significance: The MSCs populations derived from different sources, although displaying similar phenotype, exhibited high degree of variability regarding biological properties related to their self-renewal and differentiation capacity. These data indicate that for more accurate use in cell therapy, individualities of MSCs isolated from different tissues should be identified and taken into consideration when planning their use in clinical protocols., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A chemically defined thermoresponsive hydrogel, poly(AEtMA-Cl-co-DEAEA) cross-linked with N,N'-methylenebisacrylamide, which allows enzyme-free passaging, was used as a substrate to culture murine embryonic stem cells (mESCs) under defined and undefined conditions. Analysis of 14 stem cell markers showed that the mESCs remained in a "naïve" state of pluripotency with differentiation potential to form endoderm, mesoderm, and ectoderm derived lineages. These results validate the use of a chemically defined hydrogel for standardised and inexpensive mESC culture.

The histogenesis of Merkel cell carcinoma (MCC) has remained unresolved. Moreover, one of the questions is whether pure MCC and combined MCC represent the same histogenesis and entity. The existence of combined MCC suggests that MCC likely arise from pluripotent stem cells. Merkel cells (MC) localize within the bulge area, which is populated by hair follicle stem cells. We used hair follicle stem cell markers to investigate whether MCC share certain characteristics of these stem cells. Fourteen MCC specimens were examined histologically and immunohistochemically. There were six pure MCC and eight combined MCC. In six combined MCC, both MCC components and squamous components at least focally shared the expression of one or more of cytokeratin (CK)15, CK19 and CD200, which are hair follicle stem cell markers. On the other hand, four cases of pure MCC showed partially distinct CK19 expression, but did not show CK15 and/or CD200 expression. There was a distinct difference between pure MCC and combined MCC on the expression of hair follicle stem cell markers. The normal skin expressed CK15, CK19 and CD200 in the bulge area, whereas CK15 and CD200 were absent in the MC-rich glabrous skin and touch domes. The results led us to hypothesize that combined MCC originate from the hair follicle stem cells. We postulate that combined MCC undergo multidirectional differentiation into squamous, glandular, mesenchymal and Merkel cells. Further investigation is warranted to confirm the histogenesis of pure MCC and combined MCC., (© 2015 Japanese Dermatological Association.)

Background: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here, we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development., Methods and Results: By relying on human pluripotent stem cell differentiation models, we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs, TERMINATOR, ALIEN, and PUNISHER, specifically expressed in undifferentiated pluripotent stem cells, cardiovascular progenitors, and differentiated endothelial cells, respectively. Functional characterization, including localization studies, dynamic expression analyses, epigenetic modification monitoring, and knockdown experiments in lower vertebrates, as well as murine embryos and human cells, confirmed a critical role for each lncRNA specific for each analyzed developmental stage., Conclusions: We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development, and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development, mesodermal commitment, and cardiovascular specification., (© 2015 American Heart Association, Inc.)

Nucleosomes help structure chromosomes by compacting DNA into fibers. To gain insight into how nucleosomes are arranged in vivo, we combined quantitative super-resolution nanoscopy with computer simulations to visualize and count nucleosomes along the chromatin fiber in single nuclei. Nucleosomes assembled in heterogeneous groups of varying sizes, here termed "clutches," and these were interspersed with nucleosome-depleted regions. The median number of nucleosomes inside clutches and their compaction defined as nucleosome density were cell-type-specific. Ground-state pluripotent stem cells had, on average, less dense clutches containing fewer nucleosomes and clutch size strongly correlated with the pluripotency potential of induced pluripotent stem cells. RNA polymerase II preferentially associated with the smallest clutches while linker histone H1 and heterochromatin were enriched in the largest ones. Our results reveal how the chromatin fiber is formed at nanoscale level and link chromatin fiber architecture to stem cell state., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objectives: Cross-talk between organ-specific extracellular matrix (ECM) and stem cells is often assumed but has not been directly demonstrated. We developed a protocol for the preparation of human cardiac ECM (cECM) and studied whether cECM has effects on pluripotent stem cell differentiation that may be useful for future cardiac regeneration strategies in patients with end-stage heart failure., Methods: Of note, 0.3 mm-thick cECM slices were prepared from samples of myocardium from patients with end-stage non-ischaemic dilated cardiomyopathy, using a three-step protocol involving hypotonic lysis buffer, sodium dodecyl sulphate (SDS) and foetal bovine serum (FBS). Murine embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and mesenchymal stromal cells (MSCs) were seeded and grown in standard culture, on cECM or on non-specific ECM preparations (Matrigel® or Geltrex®). Cell attachment, apoptosis induction (Caspase 3/7 activity) and metabolic activity (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium conversion) were followed. Transcriptional activation of genes involved in pluripotency; early and late myocardial development; and endothelial, ectodermal or endodermal commitment were monitored by quantitative real-time polymerase chain reaction (rtPCR). Protein expression of selected markers was confirmed by immunohistology., Results: cECM supported the proliferation of ESCs and iPSCs, and Caspase 3/7 activity was significantly lower compared with standard culture. Cardiac lineage commitment was favoured when ESCs or iPSCs were grown on cECM, as evidenced by the significantly increased mRNA expression of cardiac alpha myosin heavy polypeptide 6 (Myh6), cardiac troponin T2 (Tnnt2) and NK2 homeobox 5 (Nkx2.5) as well as positive immunohistology for cardiac troponin T and heavy-chain cardiac myosin protein. In contrast, Matrigel or Geltrex did not induce cardiac-specific markers. MSCs showed no evidence of cardiomyocyte differentiation., Conclusions: Human cardiac ECM seems to direct differentiation of pluripotent stem cells towards a cardiomyocyte phenotype. This phenomenon supports the use of cardiac ECM preparations for guided stem cell differentiation and myocardial repair, and may ultimately increase the therapeutic efficacy of cell therapy in heart failure patients., (© The Author 2014. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Induced pluripotent stem cells (iPSCs) are generated from adult somatic cells through the induction of key transcription factors that restore the ability to become any cell type found in the body. These cells are of interest for tissue engineering due to their potential for developing patient-specific therapies. As the technology for generating iPSCs advances, it is important to concurrently investigate protocols for the efficient differentiation of these cells to desired downstream phenotypes in combination with biomaterial scaffolds as a way of engineering neural tissue. For such applications, the generation of neurons within three dimensional fibrin scaffolds has been well characterized as a cell-delivery platform for murine embryonic stem cells (ESCs) but has not yet been applied to murine iPSCs. Given that iPSCs have been reported to differentiate less effectively than ESCs, a key objective of this investigation is to maximize the proportion of iPSC-derived neurons in fibrin through the choice of differentiation protocol. To this end, this study compares two EB-mediated protocols for generating neurons from murine iPSCs and ESCs: an 8 day 4-/4+ protocol using soluble retinoic acid in the last 4 days and a 6 day 2-/4+ protocol using soluble retinoic acid and the small molecule sonic hedgehog agonist purmorphamine in the last 4 days. EBs were then seeded in fibrin scaffolds for 14 days to allow further differentiation into neurons. EBs generated by the 2-/4+ protocol yielded a higher percentage of neurons compared to those from the 4-/4+ protocol for both iPSCs and ESCs. The results demonstrate the successful translation of the fibrin-based cell-delivery platform for use with murine iPSCs and furthermore that the proportion of neurons generated from murine iPSC-derived EBs seeded in fibrin can be maximized using the 2-/4+ differentiation protocol. Together, these findings validate the further exploration of 3D fibrin-based scaffolds as a method of delivering neuronal cells derived from iPSCs - an important step toward the development of iPSC-based tissue engineering strategies for spinal cord injury repair.

Mammalian pluripotent stem cells possess properties of self-renewal and pluripotency. These abilities are maintained by the strict regulation of pluripotent stem cell-specific transcription factor network and unique properties of chromatin in the stem cells. Although these major signaling pathways robustly control the characteristics of stem cells, other regulatory factors, such as metabolic pathways, are also known to modulate stem cell proliferation and differentiation. In this study, we fractionated protein samples from mouse embryonic stem (ES) cells cultured with or without the leukemia inhibitory factor (LIF). Protein expression was quantified by 2-dimensional differential gel electrophoresis (2D-DIGE). In total, 44 proteins were identified as being differentially expressed in the pluripotent stem cells and the differentiated cells. Surprisingly, half of the identified proteins were the proteins localized in mitochondria, which supply cellular energy and regulate cell cycle, development, and cell death. Some of these identified proteins are involved in the metabolic function and the regulation of pluripotency. Further analysis of the identified proteins could provide new information for the manipulation of pluripotency in ES cells.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle "in a dish" for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

MicroRNAs (miRNAs) are noncoding, regulatory RNAs expressed dynamically during differentiation of human embryonic stem cells (hESCs) into defined lineages. Mapping developmental expression of miRNAs during transition from pluripotency to definitive endoderm (DE) should help to elucidate the mechanisms underlying lineage specification and ultimately enhance differentiation protocols. In this report, next generation sequencing was used to build upon our previous analysis of miRNA expression in human hESCs and DE. From millions of sequencing reads, 747 and 734 annotated miRNAs were identified in pluripotent and DE cells, respectively, including 77 differentially expressed miRNAs. Among these, four of the top five upregulated miRNAs were previously undetected in DE. Furthermore, the stem-loop for miR-302a, an important miRNA for both hESCs self-renewal and endoderm specification, produced several highly expressed miRNA species (isomiRs). Overall, isomiRs represented >10% of sequencing reads in >40% of all detected stem-loop arms, suggesting that the impact of these abundant miRNA species may have been overlooked in previous studies. Because of their relative abundance, the role of differential isomiR targeting was studied using the miR-302 cluster as a model system. A miRNA mimetic for miR-302a-5p, but not miR-302a-5p(+3), decreased expression of orthodenticle homeobox 2 (OTX2). Conversely, isomiR 302a-5p(+3) selectively decreased expression of tuberous sclerosis protein 1, but not OTX2, indicating nonoverlapping specificity of miRNA processing variants. Taken together, our characterization of miRNA expression, which includes novel miRNAs and isomiRs, helps establish a foundation for understanding the role of miRNAs in DE formation and selective targeting by isomiRs., (© 2014 AlphaMed Press.)

A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Despite constant advances in the field of biology and medical application of human embryonic stem cells, the molecular mechanism of pluripotency remains largely unknown. So far, definitions of pluripotent stem cells (SC) have been based on a limited number of antigenic markers and have not allowed for unambiguous determination of the homogeneity of each subpopulation. Moreover, the use of some crucial pluripotency markers such as SSEA-3 and SSEA-4 has recently been questioned due to the possibility that the pattern of surface glycans may be changed depending on the content of the cell culture medium., Aim: Quantitative analysis of amniotic SC subpopulations cultured in different media, based on the following pluripotency surface markers: SSEA-3, SSEA-4, TRA- 1-60 and TRA- 1-81 expression and co-expression., Material and Methods: Immunofluorescence and fluorescence microscopy were used to identify and localize SC within a normal human placenta at term. The number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells with co-expression of the above mentioned markers, cultured in media containing different protein supplements of animal origin, was counted by flow cytometry, Results and Conclusions: Cells with characteristics of embryonic SC were identified in the amniotic epithelium and the chorion, but not in the decidua basalis. Amniotic epithelium contained various types of SC, with SSEA-4+ as the most numerous. Disproportion in the number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells characterized by co-expression of these antigens, as well as lack of quantitative differences between SC subpopulations cultured in different media, was observed. In conclusion, the amniotic epithelium is composed of SC at different stages of the development but human amnion might become an alternative source of SSEA-4+ embryonic-like SC. The composition of the evaluated media, characterized by different content of animal-derived proteins, does not influence the number of cells identified within the SC subpopulations.

Cardiomyocyte loss via apoptosis plays a crucial role in ventricular remodeling following myocardial infarction (MI). Cell-based therapy approaches using bone marrow derived c-kit⁺ pluripotent cells may attenuate apoptosis following ischemic injury. We therefore thought to examine the early course of apoptosis following myocardial infarction - in-vivo - and non-invasively determine the effect of c-kit⁺ bone marrow cells on post-MI remodeling. We studied apoptosis in wild-type Kit(+/+) , c-kit mutant Kit(W)/Kit(W-v) and Kit(W)/Kit(W-v) mice after cell therapy with bone-marrow derived c-kit⁺ cells after ischemia-reperfusion injury. Mice were followed by hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography (FMT-XCT) at 6h, 24h and 7 days after ischemia-reperfusion injury using an Annexin V-based fluorescent nanosensor targeting phosphatidylserine. Kit(W)/Kit(W-v) mice showed increased and prolonged apoptosis compared to control Kit(+/+) mice while c-kit cell therapy was able to attenuate the altered apoptosis rates. Increased apoptosis was accompanied by severe decline in heart function, determined by cardiac Magnetic Resonance Imaging, and cell therapy was able to rescue the animals from deleterious heart failure. Post-mortem cryoslicing and immunohistochemistry localized the fluorescence signal of the Annexin V sensor within the infarcted myocardium. Flow cytometry of digested infarct specimens identified apoptotic cardiomyocytes as the major source for the in-vivo Annexin V signal. In-vivo molecular imaging using hybrid FMT-XCT reveals increased cardiomyocyte apoptosis in Kit(W)/Kit(W-v) mice and shows that c-kit⁺ cardioprotective cells are able to attenuate post-MI apoptosis and rescue mice from progressive heart failure.

Fourier transform infrared (FTIR) microspectroscopy shows potential as a benign, objective and rapid tool to screen pluripotent and multipotent stem cells for clinical use. It offers a new experimental approach that provides a holistic measurement of macromolecular composition such that a signature representing the internal cellular phenotype is obtained. The use of this technique therefore contributes information that is complementary to that acquired by conventional genetic and immunohistochemical methods.

Diabetes mellitus is the leading metabolic disease and represents a major public health concern worldwide. Whereas the transplantation of pancreas donor-derived islets significantly improves the quality of life of diabetic patients who become insulin independent for few years, it can unfortunately be provided only to few patients in an advanced stage of the disease. This situation is related to the severe shortage in pancreas donors and has prompted the hunt for alternative sources of islet cells. Beside many other strategies aiming at producing new beta cells in vitro or in vivo, a particular focus has been on the plupiropent stem cells because of their abundant availability and their extreme plasticity. Progress in understanding small vertebrates embryonic development has tremendously contributed to the design of differentiation strategies applied to pluripotent stem cells. Nowadays, definitive endoderm and pancreatic progenitors can be efficiently induced from human embryonic stem cells and from human induced pluripotent stem cells. Although we are still lacking the knowledge required for deriving functional beta cells in vitro, transplantation experiments have demonstrated that stem cell-derived pancreas progenitors further generate this phenotype in vivo. All these findings gathered during the last decade witness the closer clinical application of pluripotent stem cell progenies in diabetes cell therapy., (© 2013 médecine/sciences – Inserm.)

Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increasingly lineage restricted. We examined retinoic acid-induced changes in the cellular proteoglycan composition of the well-characterized teratocarcinoma line NCCIT. Our analysis revealed changes in the abundance of transcripts for genes encoding core proteins, enzymes that are responsible for early and late linkage region biosynthesis, as well as enzymes for GAG chain extension and modification. Transcript levels for genes encoding core proteins used as backbones for polysaccharide synthesis revealed highly significant increases in expression of lumican and decorin, 1,500-fold and 2,800-fold, respectively. Similarly, glypican 3, glypican 5, versican and glypican 6 showed increases between 5 and 70-fold. Significant decreases in biglycan, serglycin, glypican 4, aggrecan, neurocan, CD74 and glypican 1 were observed. Disaccharide analysis of the glycans in heparin/heparan sulfate and chondroitin/dermatan sulfate revealed retinoic acid-induced changes restricted to chondroitin/dermatan sulfate glycans. Our study provides the first detailed analysis of changes in the glycosaminoglycan profile of human pluripotent cells upon treatment with the retinoic acid morphogen.

Genomic integrity of human pluripotent stem cell (hPSC) lines requires routine monitoring. We report here that novel karyotyping assay, utilizing bead-bound bacterial artificial chromosome probes, provides a fast and easy tool for detection of chromosomal abnormalities in hPSC lines. The analysis can be performed from low amounts of DNA isolated from whole cell pools with simple data analysis interface. The method enables routine screening of stem cell lines in a cost-efficient high-throughput manner., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Several studies have reported the generation of spermatogonia-derived pluripotent stem cells from human testes. The initial aim of the present study was the derivation of equivalent stem cells from an established and experimentally accessible non-human primate model, the common marmoset monkey (Callithrix jacchus). However, an essential prerequisite in the absence of transgenic reporters in primates and man is the availability of validated endogenous markers for the identification of specific cell types in vitro., Methods and Results: We cultured marmoset testicular cells in a similar way to that described for human testis-derived pluripotent cells and set out to characterize these cultures under different conditions and in differentiation assays applying established marker panels. Importantly, the cells emerged as testicular multipotent stromal cells (TMSCs) instead of (pluripotent) germ cell-derived cells. TMSCs expressed many markers such as GFR-α, GPR125, THY-1 (CD90), ITGA6, SSEA4 and TRA-1-81, which were considered as spermatogonia specific and were previously used for the enrichment or characterization of spermatogonia. Proliferation of TMSCs was highly dependent on basic fibroblast growth factor, a growth factor routinely present in germ cell culture media. As reliable markers for the distinction between spermatogonia and TMSCs, we established VASA, in combination with the spermatogonia-expressed factors, MAGEA4, PLZF and SALL4., Conclusions: Marmoset monkey TMSCs and spermatogonia exhibit an overlap of markers, which may cause erroneous interpretations of experiments with testis-derived stem cells in vitro. We provide a marker panel for the unequivocal identification of spermatogonia providing a better basis for future studies on primate, including human, testis-derived stem cells.

To identify early populations of committed progenitors derived from human embryonic stem cells (hESCs), we screened self-renewing, BMP4-treated and retinoic acid-treated cultures with >400 antibodies recognizing cell-surface antigens. Sorting of >30 subpopulations followed by transcriptional analysis of developmental genes identified four distinct candidate progenitor groups. Subsets detected in self-renewing cultures, including CXCR4(+) cells, expressed primitive endoderm genes. Expression of Cxcr4 in primitive endoderm was confirmed in visceral endoderm of mouse embryos. BMP4-induced progenitors exhibited gene signatures of mesoderm, trophoblast and vascular endothelium, suggesting correspondence to gastrulation-stage primitive streak, chorion and allantois precursors, respectively. Functional studies in vitro and in vivo confirmed that ROR2(+) cells produce mesoderm progeny, APA(+) cells generate syncytiotrophoblasts and CD87(+) cells give rise to vasculature. The same progenitor classes emerged during the differentiation of human induced pluripotent stem cells (hiPSCs). These markers and progenitors provide tools for purifying human tissue-regenerating progenitors and for studying the commitment of pluripotent stem cells to lineage progenitors.

In this study, we investigated whether multipotent (human-bone-marrow-derived mesenchymal stem cells [hBM-MSCs]) and pluripotent stem cells (murine-induced pluripotent stem cells [iPSCs] and murine embryonic stem cells [ESCs]) respond to nanocomposite fibrous mats of poly(L-lactic acid) (PLLA) loaded with 1 or 8 wt % of calcium-deficient nanohydroxyapatite (d-HAp). Remarkably, the dispersion of different amounts of d-HAp to PLLA produced a set of materials (PLLA/d-HAp) with similar architectures and tunable mechanical properties. After 3 weeks of culture in the absence of soluble osteogenic factors, we observed the expression of osteogenic markers, including the deposition of bone matrix proteins, in multi/pluripotent cells only grown on PLLA/d-HAp nanocomposites, whereas the osteogenic differentiation was absent on stem-cell-neat PLLA cultures. Interestingly, this phenomenon was confined only in hBM-MSCs, murine iPSCs, and ESCs grown on direct contact with the PLLA/d-HAp mats. Altogether, these results indicate that the osteogenic differentiation effect of these electrospun PLLA/d-HAp nanocomposites was independent of the stem cell type and highlight the direct interaction of stem cell-polymeric nanocomposite and the mechanical properties acquired by the PLLA/d-HAp nanocomposites as key steps for the differentiation process.

Human embryonic stem cells have the capacity for self-renewal and pluripotency and thus are a primary candidate for tissue engineering and regenerative therapies. These cells also provide an opportunity to study the development of human tissues ex vivo. To date, numerous human embryonic stem cell lines have been derived and characterized. In this review, we will detail the strategies used to direct tissue-specific differentiation of embryonic stem cells. We also will discuss how these strategies have produced new sources of tissue-specific progenitor cells. Finally, we will describe the next generation of methods being developed to identify and select stem cell-derived tissue precursors for experimental study and clinical use.

Oct4 is a well-known transcription factor that plays fundamental roles in stem cell self-renewal, pluripotency, and somatic cell reprogramming. However, limited information is available on Oct4-associated protein complexes and their intrinsic protein-protein interactions that dictate Oct4's critical regulatory activities. Here we employed an improved affinity purification approach combined with mass spectrometry to purify Oct4 protein complexes in mouse embryonic stem cells (mESCs), and discovered many novel Oct4 partners important for self-renewal and pluripotency of mESCs. Notably, we found that Oct4 is associated with multiple chromatin-modifying complexes with documented as well as newly proved functional significance in stem cell maintenance and somatic cell reprogramming. Our study establishes a solid biochemical basis for genetic and epigenetic regulation of stem cell pluripotency and provides a framework for exploring alternative factor-based reprogramming strategies.

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes. A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo. Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM(-1) sec(-1) strength. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure. Our group has applied this agent in an "off label" use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

Since the derivation of the first human embryonic stem cell (hESC) lines by Thomson and coworkers in 1998, more than 1,200 different hESC lines have been established worldwide. Nevertheless, there is still a recognized interest in the establishment of new lines of hESC, particularly from HLA types and ethnic groups currently underrepresented among the available lines. The methodology of hESC derivation has evolved significantly since 1998, when human LIF (hLIF) was used for maintenance of pluripotency. However, there are a number of different strategies for the several steps involved in establishing a new line of hESC. Here we make a survey of the most relevant parameters used between 1998 and 2010 for the derivation of the 375 hESC lines deposited in two international stem cell registries, and able to form teratomas in immunocompromised mice. Although we identify some trends in the methodology for establishing hESC lines, our data reveal a much greater heterogeneity of strategies than what is used for derivation of murine ESC lines, indicating that optimum conditions have not been consolidated yet, and thus, hESC establishment is still an evolving field of research.

An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.

5-Hydroxymethylcytosine (hmC) was recently discovered as a new constituent of mammalian DNA. Besides 5-methylcytosine (mC), it is the only other modified base in higher organisms. The discovery is of enormous importance because it shows that the methylation of cytosines to imprint epigenetic information is not a final chemical step that leads to gene silencing but that further chemistry occurs at the methyl group that might have regulatory function. Recent progress in hmC detection--most notably LC-MS and glucosyltransferase assays--helped to decipher the precise distribution of hmC in the body. This led to the surprising finding that, in contrast to constant mC levels, the hmC levels are strongly tissue-specific. The highest values of hmC are found in the central nervous system. It was furthermore discovered that hmC is involved in regulating the pluripotency of stem cells and that it is connected to the processes of cellular development and carcinogenesis. Evidence is currently accumulating that hmC may not exclusively be an intermediate of an active demethylation process, but that it functions instead as an important epigenetic marker., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)