Regulatory networks in embryo-derived pluripotent stem cells

Mammalian development requires the specification of over 200 cell types from a single totipotent cell. Investigation of the regulatory networks that are responsible for pluripotency in embryo-derived stem cells is fundamental to understanding mammalian development and realizing therapeutic potential. Extracellular signals and second messengers modulate cell-autonomous regulators such as OCT4, SOX2 and Nanog in a combinatorial complexity. Knowledge of this circuitry might reveal how to achieve phenotypic changes without the genetic manipulation of Oct4, Nanog and other toti/pluripotency-associated genes.     

Differentiation of serum-free embryoid bodies from human induced pluripotent stem cells into networks

Three-dimensional aggregation cultures allow for complex development of differentiated human induced pluripotent stem cells. However, this approach is not easily amenable to live-cell imaging and electrophysiological applications due to the thickness and the geometry of the tissue. Here, we present an improvement on the traditional aggregation method by combining the use of cell culture inserts with serum-free embryoid bodies (SFEBs). The use of this technique allows the structures to maintain their three-dimensional structure while thinning substantially. We demonstrate that this technique can be used for electrophysiological recodings as well as live-cell calcium imaging combined with electrical stimulation, akin to organotypic slice preparations. This provides an important experimental tool that can be used to bridge 3-D structures with traditional monolayer approaches used in stem cell applications.     

Glycomics of human embryonic stem cells and human induced pluripotent stem cells

Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.     

Single cell heterogeneity in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) derived from blastocysts and human induced pluripotent stem cells (hiPSCs) generated from somatic cell reprogramming. Due to their self-renewal ability and pluripotent differentiation potential, hPSCs serve as an excellent experimental platform for human development, disease modeling, drug screening, and cell therapy. Traditionally, hPSCs were considered to form a homogenous population. However, recent advances in single cell technologies revealed a high degree of variability between individual cells within a hPSC population. Different types of heterogeneity can arise by genetic and epigenetic abnormalities associated with long-term in vitro culture and somatic cell reprogramming. These variations initially appear in a rare population of cells. However, some cancer-related variations can confer growth advantages to the affected cells and alter cellular phenotypes, which raises significant concerns in hPSC applications. In contrast, other types of heterogeneity are related to intrinsic features of hPSCs such as asynchronous cell cycle and spatial asymmetry in cell adhesion. A growing body of evidence suggests that hPSCs exploit the intrinsic heterogeneity to produce multiple lineages during differentiation. This idea offers a new concept of pluripotency with single cell heterogeneity as an integral element. Collectively, single cell heterogeneity is Janus-faced in hPSC function and application. Harmful heterogeneity has to be minimized by improving culture conditions and screening methods. However, other heterogeneity that is integral for pluripotency can be utilized to control hPSC proliferation and differentiation.     

High-throughput karyotyping of human pluripotent stem cells

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.     

Biobanking of human pluripotent stem cells in China

In recent years, significant progress has been made internationally in the development of human pluripotent stem cell (hPSC)‐derived products for serious and widespread disorders. Biobanking of the cellular starting materials is a crucial component in the delivery of safe and regulatory compliant cell therapies. In China, key players in these developments have been the recently launched National Stem Cell Resource Center (NSCRC) and its partner organizations in Guangzhou and Shanghai who together, have more than 600 hPSC lines formally recorded in the Chinese Ministry of Science and Technology''s stem cell registry. In addition, 47 of these hPSCs have also been registered with the hPSCreg project which means they are independently certified for use in European Commission funded research projects. The NSCRC are currently using their own cell lines to manufacture eight different cell types qualified for clinical use, that are being used in nine clinical studies for different indications. The Institute of Zoology at the Chinese Academy of Sciences (IOZ‐CAS) has worked with NSCRC to establish Chinese and international standards in stem cell research. IOZ‐CAS was also a founding partner in the International Stem Cell Banking Initiative which brings together key stem cell banks to agree minimum standards for the provision of pluripotent stem cells for research and clinical use. Here, we describe recent developments in China in the establishment of hPSCs for use in the manufacture of cell therapies and the significant national and international coordination which has now been established to promote the translation of Chinese hPSC‐based products into clinical use according to national and international standards.     

Cardiac differentiation of human pluripotent stem cells

Due to the extremely limited proliferative capacity of adult cardiomyocytes, human embryonic (pluripotent) stem cell derived cardiomyocytes (hESC-CMs) are currently almost the only reliable source of human heart cells which are suited to large-scale production. These cells have the potential for wide-scale application in drug discovery, heart disease research and cell-based heart repair. Embryonic atrial-, ventricular- and nodal-like cardiomyocytes can be obtained from differentiated human embryonic stem cells (hESCs). In recent years, several highly efficient cardiac differentiation protocols have been developed. Significant progress has also been made on understanding cardiac subtype specification, which is the key to reducing the heterogeneity of hESC-CMs, a major obstacle to the utilization of these cells in medical research and future cell-based replacement therapies. Herein we review recent progress in cardiac differentiation of hESCs and cardiac subtype specification, and discuss potential applications in drug screening and cell-based heart regeneration.     

Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks

Efficient derivation of human cerebral neocortical neural stem cells (NSCs) and functional neurons from pluripotent stem cells (PSCs) facilitates functional studies of human cerebral cortex development, disease modeling and drug discovery. Here we provide a detailed protocol for directing the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to all classes of cortical projection neurons. We demonstrate an 80-d, three-stage process that recapitulates cortical development, in which human PSCs (hPSCs) first differentiate to cortical stem and progenitor cells that then generate cortical projection neurons in a stereotypical temporal order before maturing to actively fire action potentials, undergo synaptogenesis and form neural circuits in vitro. Methods to characterize cortical neuron identity and synapse formation are described.     

Differences in the microrheology of human embryonic stem cells and human induced pluripotent stem cells

Embryonic and adult fibroblasts can be returned to pluripotency by the expression of reprogramming genes. Multiple lines of evidence suggest that these human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells are behaviorally, karyotypically, and morphologically similar. Here we sought to determine whether the physical properties of hiPS cells, including their micromechanical properties, are different from those of hES cells. To this end, we use the method of particle tracking microrheology to compare the viscoelastic properties of the cytoplasm of hES cells, hiPS cells, and the terminally differentiated parental human fibroblasts from which our hiPS cells are derived. Our results indicate that although the cytoplasm of parental fibroblasts is both viscous and elastic, the cytoplasm of hiPS cells does not exhibit any measurable elasticity and is purely viscous over a wide range of timescales. The viscous phenotype of hiPS cells is recapitulated in parental cells with disassembled actin filament network. The cytoplasm of hES cells is predominantly viscous but contains subcellular regions that are also elastic. This study supports the hypothesis that intracellular elasticity correlates with the degree of cellular differentiation and reveals significant differences in the mechanical properties of hiPS cells and hES cells. Because mechanical stimuli have been shown to mediate the precise fate of differentiating stem cells, our results support the concept that stem cell “softness” is a key feature of force-mediated differentiation of stem cells and suggest there may be subtle functional differences between force-mediated differentiation of hiPS cells and hES cells.     

Engineering human cells and tissues through pluripotent stem cells

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DNA methylation dynamics in human induced pluripotent stem cells

Indeed human induced pluripotent stem cells (hiPSCs) are considered to be powerful tools in regenerative medicine. To enable the use of hiPSCs in the field of regenerative medicine, it is necessary to understand the mechanisms of reprogramming during the transformation of somatic cells into hiPSCs. Genome-wide epigenetic modification constitutes a critical event in the generation of iPSCs. In other words, to analyze epigenetic changes in iPSCs means to elucidate reprogramming processes. We have established a large number of hiPSCs derived from various human tissues and have obtained their DNA methylation profiles. Comparison analyses indicated that the epigenetic patterns of various hiPSCs, irrespective of their source tissue, were very similar to one another and were similar to those of human embryonic stem cells (hESCs). However, the profiles of hiPSCs and hESCs exhibited epigenetic differences, which were caused by random aberrant hypermethylation at early passages. Interestingly, continuous passaging of the hiPSCs diminished the differences between DNA methylation profiles of hiPSCs and hESCs. The number of aberrant DNA methylation regions may thus represent a useful epigenetic index for evaluating hiPSCs in the context of therapeutic applications.     

Modelling familial dysautonomia in human induced pluripotent stem cells

Induced pluripotent stem (iPS) cells have considerable promise as a novel tool for modelling human disease and for drug discovery. While the generation of disease-specific iPS cells has become routine, realizing the potential of iPS cells in disease modelling poses challenges at multiple fronts. Such challenges include selecting a suitable disease target, directing the fate of iPS cells into symptom-relevant cell populations, identifying disease-related phenotypes and showing reversibility of such phenotypes using genetic or pharmacological approaches. Finally, the system needs to be scalable for use in modern drug discovery. Here, we will discuss these points in the context of modelling familial dysautonomia (FD, Riley-Day syndrome, hereditary sensory and autonomic neuropathy III (HSAN-III)), a rare genetic disorder in the peripheral nervous system. We have demonstrated three disease-specific phenotypes in FD-iPS-derived cells that can be partially rescued by treating cells with the plant hormone kinetin. Here, we will discuss how to use FD-iPS cells further in high throughput drug discovery assays, in modelling disease severity and in performing mechanistic studies aimed at understanding disease pathogenesis. FD is a rare disease but represents an important testing ground for exploring the potential of iPS cell technology in modelling and treating human disease.     

Induced pluripotent stem cells for modelling human diseases

Research into the pathophysiological mechanisms of human disease and the development of targeted therapies have been hindered by a lack of predictive disease models that can be experimentally manipulated in vitro. This review describes the current state of modelling human diseases with the use of human induced pluripotent stem (iPS) cell lines. To date, a variety of neurodegenerative diseases, haematopoietic disorders, metabolic conditions and cardiovascular pathologies have been captured in a Petri dish through reprogramming of patient cells into iPS cells followed by directed differentiation of disease-relevant cells and tissues. However, realizing the true promise of iPS cells for advancing our basic understanding of disease and ultimately providing novel cell-based therapies will require more refined protocols for generating the highly specialized cells affected by disease, coupled with strategies for drug discovery and cell transplantation.