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Mesenchymal stem cells (MSCs) in their immature state express a variety of genes of the three germ layers at relatively low or moderate levels that might explain their phenomenal plasticity. Numerous recent studies have demonstrated that under the appropriate conditions in vitro and in vivo the expression of different sets of these genes can be upregulated,turning MSCs into variety of cell lineages of mesodermal,ectodermal and endodermal origin. While transdifferentiation of MSCs is still controversial,these unique properties make MSCs an ideal autologous source of easily reprogrammable cells. Recently,using the approach of cell reprogramming by biological active compounds that interfere with chromatin structure and function,as well as with specific signalling pathways that promote neural fate commitment,we have been able to generate neural-like cells from human bone marrow (BM)-derived MSCs (hMSCs). However,the efficiency of neural transformation of hMSCs induced by this approach gradually declined with passaging. To elucidate the mechanisms that underlie the higher plasticity of early-passage hMSCs,comparative analysis of the expression levels of several pluripotent and neural genes was conducted for early- and late-passage hMSCs. The results demonstrated that early-passage hMSCs expressed the majority of these genes at low and moderate levels that gradually declined at late passages. Neural induction further increased the expression of some of these genes in hMSCs,accompanied by morphological changes into neural-like cells. We concluded that low and moderate expression of several pluripotent and neural genes in early-passage hMSCs could explain their higher plasticity and pliability for neural induction. Copyright textcopyright 2012 John Wiley & Sons,Ltd. View Publication

PURPOSE: The presence of multipotent human limbal stromal cells resembling mesenchymal stromal cells (MSC) provides new insights to the characteristic of these cells and its therapeutic potential. However,little is known about the expression of stage-specific embryonic antigen 4 (SSEA-4) and the embryonic stem cell (ESC)-like properties of these cells. We studied the expression of SSEA-4 surface protein and the various ESC and MSC markers in the ex vivo cultured limbal stromal cells. The phenotypes and multipotent differentiation potential of these cells were also evaluated.backslashnbackslashnMETHODS: Limbal stromal cells were derived from corneoscleral rims. The SSEA-4(+) and SSEA-4(-) limbal stromal cells were sorted by fluorescence-activated cells sorting (FACS). Isolated cells were expanded and reanalyzed for their expression of SSEA-4. Expression of MSC and ESC markers on these cells were also analyzed by FACS. In addition,expression of limbal epithelial and corneal stromal proteins such as ATP-binding cassette sub-family G member 2 (ABCG2),tumour protein p63 (p63),paired box 6 (Pax6),cytokeratin 3 (AE5),cytokeratin 10,and keratocan sulfate were evaluated either by immunofluorecence staining or reverse transcription polymerase chain reaction. Appropriate induction medium was used to differentiate these cells into adipocytes,osteocytes,and chondrocytes.backslashnbackslashnRESULTS: Expanded limbal stromal cells expressed the majority of mesenchymal markers. These cells were negative for ABCG2,p63,Pax6,AE-5,and keratocan sulfate. After passaged,a subpopulation of these cells showed low expression of SSEA-4 but were negative for other important ESC surface markers such as Tra-1-60,Tra-1-81,and transcription factors like octamer-binding transcription factor 4 (Oct4),SRY(sex determining region Y)-box 2 (Sox2),and Nanog. Early passaged cells when induced were able to differentiate into adipocytes,osteocytes and chondrocytes.backslashnbackslashnCONCLUSIONS: The expanded limbal stromal cells showed features of multipotent MSC. Our study confirmed the expression of SSEA-4 by a subpopulation of cultured limbal stromal cells. However,despite the expression of SSEA-4,these cells did not express any other markers of ESC. Therefore,we conclude that the cells did not show properties of ESC. View Publication

Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities,caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level,CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development,we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here,we showed that the lack of functional CSB does not represent a barrier to genetic reprogramming. However,iPSCs derived from CSB patient's fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover,these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells,regulating the expression of TP53 and TXNIP and ROS production. View Publication

Human pluripotent stem cell (hPSC-) derived cardiomyocytes have potential applications in drug discovery,toxicity testing,developmental studies,and regenerative medicine. Before these cells can be reliably utilized,characterization of their functionality is required to establish their similarity to native cardiomyocytes. We tracked fluorescent beads embedded in 4.4-99.7 kPa polyacrylamide hydrogels beneath contracting neonatal rat cardiomyocytes and cardiomyocytes generated from hPSCs via growth-factor-induced directed differentiation to measure contractile output in response to changes in substrate mechanics. Contraction stress was determined using traction force microscopy,and morphology was characterized by immunocytochemistry for α-actinin and subsequent image analysis. We found that contraction stress of all types of cardiomyocytes increased with substrate stiffness. This effect was not linked to beating rate or morphology. We demonstrated that hPSC-derived cardiomyocyte contractility responded appropriately to isoprenaline and remained stable in culture over a period of 2 months. This study demonstrates that hPSC-derived cardiomyocytes have appropriate functional responses to substrate stiffness and to a pharmaceutical agent,which motivates their use in further applications such as drug evaluation and cardiac therapies. View Publication

Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. Here we show that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs under defined,growth factor-free conditions. shRNA knockdown of β-catenin during the initial stage of hPSC differentiation fully blocked cardiomyocyte specification,whereas glycogen synthase kinase 3 inhibition at this point enhanced cardiomyocyte generation. Furthermore,sequential treatment of hPSCs with glycogen synthase kinase 3 inhibitors followed by inducible expression of β-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually (up to 98%) pure functional human cardiomyocytes from multiple hPSC lines. The robust ability to generate functional cardiomyocytes under defined,growth factor-free conditions solely by genetic or chemically mediated manipulation of a single developmental pathway should facilitate scalable production of cardiac cells suitable for research and regenerative applications. 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

Functional cardiomyocytes can be efficiently derived from human pluripotent stem cells (hPSCs),which collectively include embryonic and induced pluripotent stem cells. This cellular platform presents exciting new opportunities for development of pharmacologically relevant in vitro screens to detect cardiotoxicity,validate novel drug candidates in preclinical trials and understand complex congenital cardiovascular disorders,to advance current clinical therapies. Here,we discuss the progress and impediments the field has faced in using hPSC-derived cardiomyocytes for these in vitro applications,and highlight that rigorous protocol optimisation and standardisation,scalability and automation are remaining obstacles for the generation of pure,mature and clinically relevant hPSC cardiomyocytes. View Publication

Embryonic stem cell (ESC) markers are molecules specifically expressed in ES cells. Understanding of the functions of these markers is critical for characterization and elucidation for the mechanism of ESC pluripotent maintenance and self-renewal,therefore helping to accelerate the clinical application of ES cells. Unfortunately,different cell types can share single or sometimes multiple markers; thus the main obstacle in the clinical application of ESC is to purify ES cells from other types of cells,especially tumor cells. Currently,the marker-based flow cytometry (FCM) technique and magnetic cell sorting (MACS) are the most effective cell isolating methods,and a detailed maker list will help to initially identify,as well as isolate ESCs using these methods. In the current review,we discuss a wide range of cell surface and generic molecular markers that are indicative of the undifferentiated ESCs. Other types of molecules,such as lectins and peptides,which bind to ESC via affinity and specificity,are also summarized. In addition,we review several markers that overlap with tumor stem cells (TSCs),which suggest that uncertainty still exists regarding the benefits of using these markers alone or in various combinations when identifying and isolating cells. View Publication

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. View Publication

We describe a method using atomic force microscopy (AFM) to quantify the mechanobiological properties of pluripotent,stem cell-derived cardiomyocytes,including contraction force,rate,duration,and cellular elasticity. We measured beats from cardiomyocytes derived from induced pluripotent stem cells of healthy subjects and those with dilated cardiomyopathy,and from embryonic stem cell lines. We found that our AFM method could quantitate beat forces of single cells and clusters of cardiomyocytes. We demonstrate the dose-responsive,inotropic effect of norepinephrine and beta-adrenergic blockade of metoprolol. Cardiomyocytes derived from subjects with dilated cardiomyopathy showed decreased force and decreased cellular elasticity compared to controls. This AFM-based method can serve as a screening tool for the development of cardiac-active pharmacological agents,or as a platform for studying cardiomyocyte biology. View Publication

The sodium-iodine symporter (NIS) is expressed on the cell membrane of many thyroid cancer cells,and is responsible for the radioactive iodine accumulation. However,treatment of anaplastic thyroid cancer is ineffective due to the low expression of NIS on cell membranes of these tumor cells. Human embryonic stem cells (ESCs) provide a potential vehicle to study the mechanisms of NIS expression regulation during differentiation. Human ESCs were maintained on feeder-independent culture conditions. RT-qPCR and immunocytochemistry were used to study differentiation marker expression,(125)I uptake to study NIS function. We designed a two-step protocol for human ESC differentiation into thyroid-like cells,as was previously done for mouse embryonic stem cells. First,we obtained definitive endoderm from human ESCs. Second,we directed differentiation of definitive endoderm cells into thyroid-like cells using various factors,with thyroid stimulating hormone (TSH) as the main differentiating factor. Expression of pluripotency,endoderm and thyroid markers and (125)I uptake were monitored throughout the differentiation steps. These approaches did not result in efficient induction of thyroid-like cells. We conclude that differentiation of human ESCs into thyroid cells cannot be induced by TSH media supplementation alone and most likely involves complicated developmental patterns that are yet to be understood. View Publication

Human embryonic stem cells (hESCs) can self-renew and become all three germ layers. Nodal/Activin signaling specifies developmental status in hESCs: moderate Nodal/Activin signaling maintains pluripotency,while enhancement and inhibition promote definitive endoderm (DE) and neuroectoderm (NE) development,respectively. However,how modulation of Nodal/Activin signaling influences developmental competence and commitment toward specific lineages is still unclear. Here,we showed that enhancement of Nodal/Activin signaling for 4 days was necessary and sufficient to upregulate DE markers,while it diminished the upregulation of NE markers by inhibition of Nodal/Activin signaling. This suggests that after 4 days of enhanced Nodal/Activin signaling,hESCs are committed to the DE lineage and have lost competence toward the NE lineage. In contrast,inhibition of Nodal/Activin signaling using LY364947 for 2 days was sufficient to impair competence toward the DE lineage,although cells were still able to activate LEFTY1 and NODAL,direct targets of Nodal/Activin signaling. Expression analyses indicated that the levels of pluripotency regulators NANOG and POU5F1 were significantly diminished by 2 days of LY364947 treatment,although the expression of NANOG,but not POU5F1,was restored immediately upon Activin A treatment. Thus,downregulation of POU5F1 coincided with the abrogation of DE competence caused by inhibition of Nodal/Activin signaling. View Publication