技术资料

In an attempt to bring pluripotent stem cell biology closer to reaching its full potential,many groups have focused on improving reprogramming protocols over the past several years. The episomal modified Sendai virus-based vector has emerged as one of the most practical ones. Here we describe reprogramming of mesenchymal stromal/stem cells (MSC) derived from umbilical cord Wharton's Jelly into induced pluripotent stem cells (iPSC) using genome non-integrating Sendai virus-based vectors. The detailed protocols of iPSC colony cryopreservation (vitrification) and adaption to feeder-free culture conditions are also included. View Publication

In this study,because excessive polycythemia is a predominant trait in some high-altitude dwellers (chronic mountain sickness [CMS] or Monge's disease) but not others living at the same altitude in the Andes,we took advantage of this human experiment of nature and used a combination of induced pluripotent stem cell technology,genomics,and molecular biology in this unique population to understand the molecular basis for hypoxia-induced excessive polycythemia. As compared with sea-level controls and non-CMS subjects who responded to hypoxia by increasing their RBCs modestly or not at all,respectively,CMS cells increased theirs remarkably (up to 60-fold). Although there was a switch from fetal to adult HgbA0 in all populations and a concomitant shift in oxygen binding,we found that CMS cells matured faster and had a higher efficiency and proliferative potential than non-CMS cells. We also established that SENP1 plays a critical role in the differential erythropoietic response of CMS and non-CMS subjects: we can convert the CMS phenotype into that of non-CMS and vice versa by altering SENP1 levels. We also demonstrated that GATA1 is an essential downstream target of SENP1 and that the differential expression and response of GATA1 and Bcl-xL are a key mechanism underlying CMS pathology. View Publication

We have previously demonstrated that the Src family kinase Yes,the Yes-associated protein (YAP) and TEA domain TEAD2 transcription factor pathway are activated by leukemia inhibitory factor (LIF) and contribute to mouse embryonic stem (mES) cell maintenance of pluripotency and self-renewal. In addition,we have shown that fetal bovine serum (FBS) induces Yes auto-phosphorylation and activation. In the present study we confirm that serum also activates TEAD-dependent transcription in a time- and dose-dependent manner and we identify Inter-α-inhibitor (IαI) as a component in serum capable of activating the Yes/YAP/TEAD pathway by inducing Yes auto-phosphorylation,YAP nuclear localization and TEAD-dependent transcription. The cleaved heavy chain 2 (HC2) sub-component of IαI,is demonstrated to be responsible for this effect. Moreover,IαI is also shown to efficiently increase expression of TEAD-downstream target genes including well-known stem cell factors Nanog and Oct 3/4. IαI is not produced by the ES cells per se but is added to the cells via the cell culture medium containing serum or serum-derived components such as bovine serum albumin (BSA). In conclusion,we describe a novel function of IαI in activating key pluripotency pathways associated with ES cell maintenance and self-renewal. View Publication

It has been known for over 20 years that foetal calf serum can induce hypertrophy in cultured cardiomyocytes but this is rarely considered when examining cardiomyocytes derived from pluripotent stem cells (PSC). Here,we determined how serum affected cardiomyocytes from human embryonic- (hESC) and induced pluripotent stem cells (hiPSC) and hiPSC from patients with hypertrophic cardiomyopathy linked to a mutation in the MYBPC3 gene. We first confirmed previously published hypertrophic effects of serum on cultured neonatal rat cardiomyocytes demonstrated as increased cell surface area and beating frequency. We then found that serum increased the cell surface area of hESC- and hiPSC-derived cardiomyocytes and their spontaneous contraction rate. Phenylephrine,which normally induces cardiac hypertrophy,had no additional effects under serum conditions. Likewise,hiPSC-derived cardiomyocytes from three MYBPC3 patients which had a greater surface area than controls in the absence of serum as predicted by their genotype,did not show this difference in the presence of serum. Serum can thus alter the phenotype of human PSC derived cardiomyocytes under otherwise defined conditions such that the effects of hypertrophic drugs and gene mutations are underestimated. It is therefore pertinent to examine cardiac phenotypes in culture media without or in low concentrations of serum. View Publication

Human embryonic stem cells (hESCs) are pluripotent cells derived from the embryo at the blastocyst stage. Their embryonic origin confers upon them the capacity to proliferate indefinitely in vitro while maintaining the capacity to differentiate into a large variety of cell types. Based on these properties of self-renewal and pluripotency,hESCs represent a unique source to generate a large quantity of certain specialized cell types with clinical interest for transplantation-based therapy. However,hESCs are usually grown in culture conditions using fetal bovine serum and mouse embryonic fibroblasts,two components that are not compatible with clinical applications. Consequently,the possibility to expand hESCs in serum-free and in feeder-free culture conditions is becoming a major challenge to deliver the clinical promises of hESCs. Here,we describe the basic principles of growing hESCs in a chemically defined medium (CDM) devoid of serum and feeders. View Publication

The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation. View Publication

The anisotropic alignment of cardiomyocytes in native myocardium tissue is a functional feature that is absent in traditional in vitro cardiac cell culture. Microenvironmental factors cue structural organization of the myocardium,which promotes the mechanical contractile properties and electrophysiological patterns seen in mature cardiomyocytes. Current nano- and microfabrication techniques,such as photolithography,generate simplified cell culture topographies that are not truly representative of the multifaceted and multi-scale fibrils of the cardiac extracellular matrix. In addition,such technologies are costly and require a clean room for fabrication. This chapter offers an easy,fast,robust,and inexpensive fabrication of biomimetic multi-scale wrinkled surfaces through the process of plasma treating and shrinking prestressed thermoplastic. Additionally,this chapter includes techniques for culturing stem cells and their cardiac derivatives on these substrates. Importantly,this wrinkled cell culture platform is compatible with both fluorescence and bright-field imaging; real-time physiological monitoring of CM action potential propagation and contraction properties can elucidate cardiotoxicity drug effects. View Publication

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a leading monogenic neurodegenerative disorder affecting premutation carriers of the fragile X (FMR1) gene. To investigate the underlying cellular neuropathology,we produced induced pluripotent stem cell-derived neurons from isogenic subclones of primary fibroblasts of a female premutation carrier,with each subclone bearing exclusively either the normal or the expanded (premutation) form of the FMR1 gene as the active allele. We show that neurons harboring the stably-active,expanded allele (EX-Xa) have reduced postsynaptic density protein 95 protein expression,reduced synaptic puncta density and reduced neurite length. Importantly,such neurons are also functionally abnormal,with calcium transients of higher amplitude and increased frequency than for neurons harboring the normal-active allele. Moreover,a sustained calcium elevation was found in the EX-Xa neurons after glutamate application. By excluding the individual genetic background variation,we have demonstrated neuronal phenotypes directly linked to the FMR1 premutation. Our approach represents a unique isogenic,X-chromosomal epigenetic model to aid the development of targeted therapeutics for FXTAS,and more broadly as a model for the study of common neurodevelopmental (e.g. autism) and neurodegenerative (e.g. Parkinsonism,dementias) disorders. View Publication

The use of nanoparticles in medicine is ever increasing,and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here,we show that this indirect DNA damage depends on the thickness of the cellular barrier,and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling,including cytokine release,occurred only across bilayer and multilayer barriers,but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers,our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches. View Publication

The fate decisions of human pluripotent stem (hPS) cells are governed by soluble and insoluble signals from the microenvironment. Many hPS cell differentiation protocols use Matrigel,a complex and undefined substrate that engages multiple adhesion and signaling receptors. Using defined surfaces programmed to engage specific cell-surface ligands (i.e.,glycosaminoglycans and integrins),the contribution of specific matrix signals can be dissected. For ectoderm and motor neuron differentiation,peptide-modified surfaces that can engage both glycosaminoglycans and integrins are effective. In contrast,surfaces that interact selectively with glycosaminoglycans are superior to Matrigel in promoting hPS cell differentiation to definitive endoderm and mesoderm. The modular surfaces were used to elucidate the signaling pathways underlying these differences. Matrigel promotes integrin signaling,which in turn inhibits mesendoderm differentiation. The data indicate that integrin-activating surfaces stimulate Akt signaling via integrin-linked kinase (ILK),which is antagonistic to endoderm differentiation. The ability to attribute cellular responses to specific interactions between the cell and the substrate offers new opportunities for revealing and controlling the pathways governing cell fate. View Publication

Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus,they are increasingly employed as models for cancer and drug research,as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures,and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore,SBR preserves extracellular matrix-like materials and cellular proteins. These findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells,providing a pathway to enable our understanding of morphogenesis in 3D cultures. View Publication