Three‐dimensional hydrogel encapsulated embryonic stem and carcinoma cells as culture platforms for cytotoxicity studies

The utility of alginate hydrogels for three‐dimensional (3‐D) culture of mouse embryonic stem cells (mESCs) and future development of 3‐D stem cell culture‐based in vitro screens of toxicity is described. Using alginate hydrogels of various stiffness, we first evaluated the impact of substrate modulus on mESC viability, proliferation, as well as expression of pluripotency and germ‐layer markers and observed that low concentration alginate hydrogels (0.5% and 1% alginate) were most suitable for long‐term culture of mESCs. These results were not unique to mESCs; long‐term viability and proliferation of mouse embryonic carcinoma cells (mECCs) was also best supported by similar conditions. Finally, we determined cytotoxic responses of alginate encapsulated cells to commercially available chemicals and interestingly observed similar responses for mESCs and mECCs, thereby suggesting that mECCs can predict stem cell responses to chemicals. These studies will facilitate future design of optimal stem cell‐based platforms of organ‐specific and developmental toxicity. © 2015 American Institute of Chemical Engineers AIChE J, 61: 3180–3184, 2015     

hiPSC-Derived Epidermal Keratinocytes from Ichthyosis Patients Show Altered Expression of Cornification Markers

Inherited ichthyoses represent a large heterogeneous group of skin disorders characterised by impaired epidermal barrier function and disturbed cornification. Current knowledge about disease mechanisms has been uncovered mainly through the use of mouse models or human skin organotypic models. However, most mouse lines suffer from severe epidermal barrier defects causing neonatal death and human keratinocytes have very limited proliferation ability in vitro. Therefore, the development of disease models based on patient derived human induced pluripotent stem cells (hiPSCs) is highly relevant. For this purpose, we have generated hiPSCs from patients with congenital ichthyosis, either non-syndromic autosomal recessive congenital ichthyosis (ARCI) or the ichthyosis syndrome trichothiodystrophy (TTD). hiPSCs were successfully differentiated into basal keratinocyte-like cells (hiPSC-bKs), with high expression of epidermal keratins. In the presence of higher calcium concentrations, terminal differentiation of hiPSC-bKs was induced and markers KRT1 and IVL expressed. TTD1 hiPSC-bKs showed reduced expression of FLG, SPRR2B and lipoxygenase genes. ARCI hiPSC-bKs showed more severe defects, with downregulation of several cornification genes. The application of hiPSC technology to TTD1 and ARCI demonstrates the successful generation of in vitro models mimicking the disease phenotypes, proving a valuable system both for further molecular investigations and drug development for ichthyosis patients.     

SMA Human iPSC-Derived Motor Neurons Show Perturbed Differentiation and Reduced miR-335-5p Expression

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by mutations in the Survival Motor Neuron 1 gene, resulting in very low levels of functional Survival of Motor Neuron (SMN) protein. SMA human induced Pluripotent Stem Cells (hiPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. MicroRNAs (miRNAs) are often reported as playing a key role in regulating neuronal differentiation and fate specification. In this study SMA hiPSCs have been differentiated towards early motor neurons and their molecular and immunocytochemical profile were compared to those of wild type cells. Cell cycle proliferation was also evaluated by fluorescence-activated cell sorting (FACS). SMA hiPSCs showed an increased proliferation rate and also higher levels of stem cell markers. Moreover; when differentiated towards early motor neurons they expressed lower levels of NCAM and MN specific markers. The expression of miR-335-5p; already identified to control self-renewal or differentiation of mouse embryonic stem cells (mESCs); resulted to be reduced during the early steps of differentiation of SMA hiPSCs compared to wild type cells. These results suggest that we should speculate a role of this miRNA both in stemness characteristic and in differentiation efficiency of these cells.     

Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs) generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF) cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1) and Rex-1 (ZFP-42, zinc finger protein 42). Using embryonic stem cells (ESCs) conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs) using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF) cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.     

Recent Developments in β-Cell Differentiation of Pluripotent Stem Cells Induced by Small and Large Molecules

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold promise as novel therapeutic tools for diabetes treatment because of their self-renewal capacity and ability to differentiate into beta (β)-cells. Small and large molecules play important roles in each stage of β-cell differentiation from both hESCs and hiPSCs. The small and large molecules that are described in this review have significantly advanced efforts to cure diabetic disease. Lately, effective protocols have been implemented to induce hESCs and human mesenchymal stem cells (hMSCs) to differentiate into functional β-cells. Several small molecules, proteins, and growth factors promote pancreatic differentiation from hESCs and hMSCs. These small molecules (e.g., cyclopamine, wortmannin, retinoic acid, and sodium butyrate) and large molecules (e.g. activin A, betacellulin, bone morphogentic protein (BMP4), epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), noggin, transforming growth factor (TGF-α), and WNT3A) are thought to contribute from the initial stages of definitive endoderm formation to the final stages of maturation of functional endocrine cells. We discuss the importance of such small and large molecules in uniquely optimized protocols of β-cell differentiation from stem cells. A global understanding of various small and large molecules and their functions will help to establish an efficient protocol for β-cell differentiation.     

Lessons Learned about Human Stem Cell Responses to Ionizing Radiation Exposures: A Long Road Still Ahead of Us

Human stem cells (hSC) possess several distinct characteristics that set them apart from other cell types. First, hSC are self-renewing, capable of undergoing both asymmetric and symmetric cell divisions. Second, these cells can be coaxed to differentiate into various specialized cell types and, as such, hold great promise for regenerative medicine. Recent progresses in hSC biology fostered the characterization of the responses of hSC to genotoxic stresses, including ionizing radiation (IR). Here, we examine how different types of hSC respond to IR, with a special emphasis on their radiosensitivity, cell cycle, signaling networks, DNA damage response (DDR) and DNA repair. We show that human embryonic stem cells (hESCs) possess unique characteristics in how they react to IR that clearly distinguish these cells from all adult hSC studied thus far. On the other hand, a manifestation of radiation injuries/toxicity in human bodies may depend to a large extent on hSC populating corresponding tissues, such as human mesenchymal stem cells (hMSC), human hematopoietic stem cells (hHSC), neural hSC, intestine hSC, etc. We discuss here that hSC responses to IR differ notably across many types of hSC which may represent the distinct roles these cells play in development, regeneration and/or maintenance of homeostasis.     

Design of polymeric materials for culturing human pluripotent stem cells: Progress toward feeder-free and xeno-free culturing

This review describes recent developments regarding the use of natural and synthetic polymers to support the propagation of human pluripotent stem cells (hPSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (hiPSCs) while maintaining pluripotency in feeder-free and xeno-free cultures. The development of methods for culturing these cells without using mouse embryonic fibroblasts (MEFs) as a feeder layer will enable more reproducible culture conditions and reduce the risk of xenogenic contaminants, thus increasing the potential clinical applications of differentiated hPSCs. Human or recombinant fibronectin, laminin-511, and vitronectin, which are components of the extracellular matrix (ECM), have been used instead of Matrigel for the feeder-free growth of undifferentiated hPSCs. Successful hPSC cultures have been described for the following conditions: on oligopeptide-immobilized surfaces derived from vitronectin, on microcarriers prepared from synthetic polymers, and encapsulated within three-dimensional (3D) hydrogels composed of alginate and other hydrophilic natural polymers. Recently, synthetic biomaterials that allow hPSCs to maintain pluripotency by secreting endogenous ECM components have been designed. The combination of human ECM proteins or cell adhesion molecules (e.g., oligopeptides and poly-d-lysine) and synthetic biomaterials with well-designed surfaces and/or structures (e.g., scaffolds, hydrogels, microcarriers, microcapsules, or microfibers) in the presence of a chemically defined medium containing recombinant growth factors would offer a xeno-free alternative to feeder cells for culturing hPSCs and maintaining their pluripotency.     

Efficient Generation of Neural Stem Cells from Embryonic Stem Cells Using a Three-Dimensional Differentiation System

Mouse embryonic stem cells (ESCs) are useful tools for studying early embryonic development and tissue formation in mammals. Since neural lineage differentiation is a major subject of organogenesis, the development of efficient techniques to induce neural stem cells (NSCs) from pluripotent stem cells must be preceded. However, the currently available NSC differentiation methods are complicated and time consuming. This study aimed to propose an efficient method for the derivation of NSCs from mouse ESCs; early neural lineage commitment was achieved using a three-dimensional (3D) culture system, followed by a two-dimensional (2D) NSC derivation. To select early neural lineage cell types during differentiation, Sox1-GFP transgenic ESCs were used. They were differentiated into early neural lineage, forming spherical aggregates, which were subsequently picked for the establishment of 2D NSCs. The latter showed a morphology similar to that of brain-derived NSCs and expressed NSC markers, Musashi, Nestin, N-cadherin, and Sox2. Moreover, the NSCs could differentiate into three subtypes of neural lineages, neurons, astrocytes, and oligodendrocytes. The results together suggested that ESCs could efficiently differentiate into tripotent NSCs through specification in 3D culture (for approximately 10 days) followed by 2D culture (for seven days).     

Polymeric design of cell culture materials that guide the differentiation of human pluripotent stem cells

Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the potential to differentiate into many cell types that originate from the three germ layers, such as dopamine-secreting cells and insulin-secreting cells for the treatment of Alzheimer's disease and diabetes, respectively. However, it is challenging to guide hPSC differentiation into desired cell lineages due to their varying differentiation ability. A reasonable strategy is to mimic the stem cell microenvironment for the differentiation of hPSCs into specific cell lineages using optimal polymeric biomaterials for hPSC culture. This review summarizes various methods for differentiating hPSCs cultured on polymeric biomaterials and discusses the optimal methods and cell culture polymeric biomaterials for hPSC differentiation into specific cell lineages. The recent trend in protocols avoids embryoid body (EB, aggregated cells) formation because EBs contain different types of cells. The combination of appropriate differentiation protocols and cell culture polymeric biomaterials for the differentiation of hPSCs into specific cell lineages will produce a large quantity of highly pure GMP-grade differentiated cells for use in translational medicine.     

Modeling and Targeting Neuroglial Interactions with Human Pluripotent Stem Cell Models

Generation of relevant and robust models for neurological disorders is of main importance for both target identification and drug discovery. The non-cell autonomous effects of glial cells on neurons have been described in a broad range of neurodegenerative and neurodevelopmental disorders, pointing to neuroglial interactions as novel alternative targets for therapeutics development. Interestingly, the recent breakthrough discovery of human induced pluripotent stem cells (hiPSCs) has opened a new road for studying neurological and neurodevelopmental disorders “in a dish”. Here, we provide an overview of the generation and modeling of both neuronal and glial cells from human iPSCs and a brief synthesis of recent work investigating neuroglial interactions using hiPSCs in a pathophysiological context.     

Epidermal Development in Mammals: Key Regulators,Signals from Beneath,and Stem Cells

Epidermis is one of the best-studied tissues in mammals that contain types of stem cells. Outstanding works in recent years have shed great light on behaviors of different epidermal stem cell populations in the homeostasis and regeneration of the epidermis as well as hair follicles. Also, the molecular mechanisms governing these stem cells are being elucidated, from genetic to epigenetic levels. Compared with the explicit knowledge about adult skin, embryonic development of the epidermis, especially the early period, still needs exploration. Furthermore, stem cells in the embryonic epidermis are largely unstudied or ambiguously depicted. In this review, we will summarize and discuss the process of embryonic epidermal development, with focuses on some key molecular regulators and the role of the sub-epidermal mesenchyme. We will also try to trace adult epidermal stem cell populations back to embryonic development. In addition, we will comment on in vitro derivation of epidermal lineages from ES cells and iPS cells.     

The Key Enzyme of the Sialic Acid Metabolism Is Involved in Embryoid Body Formation and Expression of Marker Genes of Germ Layer Formation

The bi-functional enzyme UDP-N-acetyl-2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme of the sialic acid biosynthesis. Sialic acids are negatively charged nine carbon amino sugars and are found on most glycoproteins and many glycolipids in terminal positions, where they are involved in a variety of biological important molecular interactions. Inactivation of the GNE by homologous recombination results in early embryonic lethality in mice. Here, we report that GNE-deficient embryonic stem cells express less differentiation markers compared to wild-type embryonic stem cells. As a result, GNE-deficient embryonic stem cells fail to form proper embryoid bodies (EB) within the first day of culture. However, when culturing these cells in the presence of sialic acids for three days, also GNE-deficient embryonic stem cells form normal EBs. In contrast, when culturing these cells in sialic acid reduced medium, GNE-deficient embryonic stem cells proliferate faster and form larger EBs without any change in the expression of markers of the germ layers.     

Multi-parametric profiling network based on gene expression and phenotype data: a novel approach to developmental neurotoxicity testing

The establishment of more efficient approaches for developmental neurotoxicity testing (DNT) has been an emerging issue for children's environmental health. Here we describe a systematic approach for DNT using the neuronal differentiation of mouse embryonic stem cells (mESCs) as a model of fetal programming. During embryoid body (EB) formation, mESCs were exposed to 12 chemicals for 24 h and then global gene expression profiling was performed using whole genome microarray analysis. Gene expression signatures for seven kinds of gene sets related to neuronal development and neuronal diseases were selected for further analysis. At the later stages of neuronal cell differentiation from EBs, neuronal phenotypic parameters were determined using a high-content image analyzer. Bayesian network analysis was then performed based on global gene expression and neuronal phenotypic data to generate comprehensive networks with a linkage between early events and later effects. Furthermore, the probability distribution values for the strength of the linkage between parameters in each network was calculated and then used in principal component analysis. The characterization of chemicals according to their neurotoxic potential reveals that the multi-parametric analysis based on phenotype and gene expression profiling during neuronal differentiation of mESCs can provide a useful tool to monitor fetal programming and to predict developmentally neurotoxic compounds.     

Engineered Human Induced Pluripotent Cells Enable Genetic Code Expansion in Brain Organoids

Human induced pluripotent stem cell (hiPSC) technology has revolutionized studies on human biology. A wide range of cell types and tissue models can be derived from hiPSCs to study complex human diseases. Here, we use PiggyBac-mediated transgenesis to engineer hiPSCs with an expanded genetic code. We demonstrate that genomic integration of expression cassettes for a pyrrolysyl-tRNA synthetase (PylRS), pyrrolysyl-tRNA (PylT) and the target protein of interest enables site-specific incorporation of a non-canonical amino acid (ncAA) in response to an amber stop codon. Neural stem cells, neurons and brain organoids derived from the engineered hiPSCs continue to express the amber suppression machinery and produce ncAA-bearing reporter. The incorporated ncAA can serve as a minimal bioorthogonal handle for further modifications by labeling with fluorescent dyes. Site-directed ncAA mutagenesis will open a wide range of applications to probe and manipulate proteins in brain organoids and other hiPSC-derived cell types and complex tissue models.     

Improved Establishment of Embryonic Stem (ES) Cell Lines from the Chinese Kunming Mice by Hybridization with 129 Mice

Chinese Kunming mice (Mus musculus Km), widely used as laboratory animals throughout China, remain very refractory for embryonic stem (ES) cell isolation. The present study was aimed to evaluate the effects of hybridization with 129/Sv mice, and culture media containing fetal bovine serum (FBS) or Knockout serum replacement (KSR) on ES cell isolation from Kunming mice. The results demonstrated that ES cells had been effectively isolated from the hybrid embryos of Kunming and 129/Sv mice using all three media containing 15% FBS, 15% KSR and their mixture of 14% KSR and 1% FBS, individually. These isolated ES cells had maintained in vitro undifferentiated for a long time, exhibiting all features specific for mouse ES cells. In addition, the rates of ES cell isolation in the medium containing 14% KSR and 1% FBS, was 46.67% and significantly higher than those in another two media containing only FBS or KSR (p < 0.05). Contrarily, no ES cell line had been established from Kunming mouse inbred embryos using the same protocols. These results suggested that ES cells with long-term self-renewal ability could be efficiently generated from hybrid embryos of Kunming and 129/Sv mice, and a small volume of FBS was necessary to isolate ES cells in the KSR medium when embryos and early ES cells cultured.     

Synthesis and characterization of novel biodegradable and biocompatible poly(ester-urethane) thin films prepared by homogeneous solution polymerization

Novel biodegradable and biocompatible poly(ester-urethane)s were synthesized by in situ homogeneous solution polymerization of poly(?-caprolactone) diol, dimethylolpropionic acid (DMPA), and methylene diphenyl diisocyanate in acetone followed by solvent exchange with water. The effects of the DMPA content and hard segment content on the properties of the polyurethanes were measured by DSC, TGA, and hydrolytic degradation measurements. The results showed that DMPA had a dramatic effect on the particle size; the particle size decreased rapidly with increasing DMPA content. The hydrolytic degradation test showed that the degradation rate was little affected by the DMPA content in the range investigated, but was observed to be influenced by the hard segment content. Cell toxicity analysis showed that the biodegradable poly(ester-urethane)s synthesized in this study did not exhibit any detectable toxicity to human umbilical vein endothelial cells and mouse embryonic stem cells. Both types of cells can effectively adhere to and spread on the surface of pure poly(?-caprolactone) or poly(ester-urethane)s. The present study demonstrates the feasibility of a facile synthesis of biodegradable polyurethanes and of their aqueous dispersions with prescribed properties for biomedical applications.