Scientific Resources

Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However,research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly,this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose. View Publication

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent,thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes,which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages,starting with endoderm,which can develop into several organs,including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A in combination with several growth factors. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition,which can be achieved in vitro by addition of cyclopamine. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation. Although successful,this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation,which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation. The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture,herein we present our data with rat heart microvascular endothelial Cells (RHMVEC),primarily for the ease of analysis. View Publication

Nanog,Oct4,and Sox2 are the core regulators of mouse (m)ESC pluripotency. Although their basic importance in human (h)ESCs has been demonstrated,the mechanistic functions are not well defined. Here,we identify general and cell-line-specific requirements for NANOG,OCT4,and SOX2 in hESCs. We show that OCT4 regulates,and interacts with,the BMP4 pathway to specify four developmental fates. High levels of OCT4 enable self-renewal in the absence of BMP4 but specify mesendoderm in the presence of BMP4. Low levels of OCT4 induce embryonic ectoderm differentiation in the absence of BMP4 but specify extraembryonic lineages in the presence of BMP4. NANOG represses embryonic ectoderm differentiation but has little effect on other lineages,whereas SOX2 and SOX3 are redundant and repress mesendoderm differentiation. Thus,instead of being panrepressors of differentiation,each factor controls specific cell fates. Our study revises the view of how self-renewal is orchestrated in hESCs. View Publication

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development,we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm,then into replicating Nkx2.1+ lung endoderm,and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP,FGF,and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs),creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice. View Publication

A major difficulty of producing induced pluripotent stem cells (iPSCs) has been the low efficiency of reprogramming differentiated cells into pluripotent cells. We previously showed that 5% of mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs when they were transduced with a fusion gene composed of Oct4 and the transactivation domain of MyoD (called M(3)O),along with Sox2,Klf4 and c-Myc (SKM). In addition,M(3)O facilitated chromatin remodeling of pluripotency genes in the majority of transduced MEFs,including cells that did not become iPSCs. These observations suggested the possibility that more than 5% of cells had acquired the ability to become iPSCs given more favorable culture conditions. Here,we raised the efficiency of making mouse iPSCs with M(3)O-SKM to 26% by culturing transduced cells at low density in serum-free culture medium. In contrast,the efficiency increased from 0.1% to only 2% with the combination of wild-type Oct4 and SKM (OSKM) under the same culture condition. For human iPSCs,M(3)O-SKM achieved 7% efficiency under a similar serum-free culture condition,in comparison to 1% efficiency with OSKM. This study highlights the power of combining the transactivation domain of MyoD with a favorable culture environment. View Publication

In this unit,we describe a simple coculture-free method for obtaining mast cells from mouse and human embryonic stem (ES) cells. Much of our knowledge regarding the mechanisms by which mast cells are activated comes from studies of mouse bone marrow-derived mast cells. Studies of human mast cells have been hampered by the limited sources from which they can be cultured,the difficulty in introducing specific genetic changes into these cells,and differences between established cultures that reflect the unique genetic makeup of the tissue donor. Derivation of mast cells from embryonic stem cells addresses these limitations. ES-derived mast cells can be generated in numbers sufficient for studies of the pathways involved in mast cell effector functions. These ES cell-derived mast cells respond to antigens and other stimuli by releasing histamine,cytokines,lipids,and other bioactive mediators. The derivation of human mast cells from ES cells carrying mutations introduced by homologous recombination should provide a novel means of testing the function of genes in both the development and the effector functions of mast cells. View Publication

Dental pulp is particularly interesting in regenerative medicine because of the accessibility and differentiation potential of the tissue. Dental pulp has an early developmental origin with multi-lineage differentiation potential as a result of its development during childhood and adolescence. However,no study has previously identified the presence of stem cell populations with embryonic-like phenotypes in human dental pulp from the third molar. In the present work,we describe a new population of dental pulp pluripotent-like stem cells (DPPSCs) that were isolated by culture in medium containing LIF,EGF and PDGF. These cells are SSEA4(+),OCT3/4(+),NANOG(+),SOX2(+),LIN28(+),CD13(+),CD105(+),CD34(-),CD45(-),CD90(+),CD29(+),CD73(+),STRO1(+) and CD146(-),and they show genetic stability in vitro based on genomic analysis with a newly described CGH technique. Interestingly,DPPSCs were able to form both embryoid-body-like structures (EBs) in vitro and teratoma-like structures that contained tissues derived from all three embryonic germ layers when injected in nude mice. We examined the capacity of DPPSCs to differentiate in vitro into tissues that have similar characteristics to mesoderm,endoderm and ectoderm layers in both 2D and 3D cultures. We performed a comparative RT-PCR analysis of GATA4,GATA6,MIXL1,NANOG,OCT3/4,SOX1 and SOX2 to determine the degree of similarity between DPPSCs,EBs and human induced pluripotent stem cells (hIPSCs). Our analysis revealed that DPPSCs,hIPSC and EBs have the same gene expression profile. Because DPPSCs can be derived from healthy human molars from patients of different sexes and ages,they represent an easily accessible source of stem cells,which opens a range of new possibilities for regenerative medicine. View Publication

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a structurally endogenous peptide with many biological roles. Maxadilan,a 61-amino acid vasodilatory peptide,specifically activates the PACAP type I receptor (PAC1). Although PAC1 has been identified in embryonic stem cells,little is known about its presence or effects in human induced pluripotent stem (iPS) cells. In the present study,we investigated the expression of PAC1 in human iPS cells by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. To study the physiological effects mediated by PAC1,we evaluated the role of maxadilan in preventing apoptotic cell death induced by ultraviolet C (UVC). After exposure to UVC,the iPS cells showed a marked reduction in cell viability and a parallel increase of apoptotic cells,as demonstrated by WST-8 analysis,annexin V/propidium iodide (PI) analysis and the terminal transferase dUTP nick end labeling (TUNEL) assay. The addition of 30 nM of maxadilan dramatically increased iPS cell viability and reduced the percentage of apoptotic cells. The anti-apoptotic effects of maxadilan were correlated to the downregulation of caspase-3 and caspase-9. Concomitantly,immunofluorescence,western blot analysis,real-time quantitative polymerase chain reaction (RT-qPCR) analysis and in vitro differentiation results showed that maxadilan did not affect the pluripotent state of iPS cells. Moreover,karyotype analysis showed that maxadilan did not affect the karyotype of iPS cells. In summary,these results demonstrate that PAC1 is present in iPS cells and that maxadilan effectively protects iPS cells against UVC-induced apoptotic cell death while not affecting the pluripotent state or karyotype. View Publication

Transactive response DNA-binding (TDP-43) protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP). Identification of mutations in the gene encoding TDP-43 (TARDBP) in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts by using induced pluripotent stem cells (iPSCs). Here,we report the generation of iPSCs that carry the TDP-43 M337V mutation and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent-resistant TDP-43 protein,decreased survival in longitudinal studies,and increased vulnerability to antagonism of the PI3K pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell-autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening. View Publication

The subunit genes encoding human chorionic gonadotropin,CGA,and CGB,are up-regulated in human trophoblast. However,they are effectively silenced in choriocarcinoma cells by ectopically expressed POU domain class 5 transcription factor 1 (POU5F1). Here we show that POU5F1 represses activity of the CGA promoter through its interactions with ETS2,a transcription factor required for both placental development and human chorionic gonadotropin subunit gene expression,by forming a complex that precludes ETS2 from interacting with the CGA promoter. Mutation of a POU5F1 binding site proximal to the ETS2 binding site does not alter the ability of POU5F1 to act as a repressor but causes a drop in basal promoter activity due to overlap with the binding site for DLX3. DLX3 has only a modest ability to raise basal CGA promoter activity,but its coexpression with ETS2 can up-regulate it 100-fold or more. The two factors form a complex,and both must bind to the promoter for the combination to be transcriptionally effective,a synergy compromised by POU5F1. Similarly,in human embryonic stem cells,which express ETS2 but not CGA,ETS2 does not occupy its binding site on the CGA promoter but is found instead as a soluble complex with POU5F1. When human embryonic stem cells differentiate in response to bone morphogenetic protein-4 and concentrations of POU5F1 fall and hCG and DLX3 rise,ETS2 then occupies its binding site on the CGA promoter. Hence,a squelching mechanism underpins the transcriptional silencing of CGA by POU5F1 and could have general relevance to how pluripotency is maintained and how the trophoblast lineage emerges from pluripotent precursor cells. View Publication

The glycogen synthase kinase 3 (GSK3) inhibitor,6-bromoindirubin-3'-oxime (BIO),is a key regulator of many signaling pathways to maintain pluripotency of human and mouse embryonic stem cells (ESCs). However,the effect of BIO on derivation of dairy goat male germline stem cells (mGSCs) remains unclear. The objectives of this study were to investigate whether BIO influences derivation of dairy goat mGSCs. Dairy goat mGSCs were cultured in mTeSR containing BIO medium and its effects on the proliferation ability of goat mGSCs (derived from goats ≤2 mo of age) were evaluated by 5-Bromo-2-deoxyuridine (BrdU) incorporation and alkaline phosphatase (AP) staining. Furthermore,its effects on maintenance of the undifferentiated state of mGSCs in late passages of cultures,as well as the capacity of mGSCs to differentiate into embryoid bodies (EBs) were examined. The presence of BIO increased the mitosis index and the number of AP positive colonies,as well as expression of pluripotent markers,Oct4,Nanog,Sox2,C-myc,Klf4,E-cadherin,and the proliferative markers,Pcna and C-myc. In contrast,there was no significant change in expression of apoptosis markers,P53,P21 and cyclin-related genes (Cyclin A,CDK2,Cyclin D1),as determined by RT-PCR analysis. When mGSCs were cultured in mTeSR medium containing BIO,EBs were formed,which were capable of further differentiating into various cell types found in the three embryonic germ layers,as determined by immunofluorescence and/or histologic staining. In conclusion,adding BIO to cultures BIO significantly promoted establishment of goat mGSC colonies and maintained their undifferentiated state. View Publication

Human embryonic stem cells (hESCs) require specific niches for adhesion,expansion,and lineage-specific differentiation. In this study,we showed that a membrane substrate offers better tissue niches for hESC attachment,spreading,proliferation,and differentiation. The cell doubling time was shortened from 46.3±5.7 h for hESCs grown on solid substrates to 25.6±2.6 h for those on polyester (PE) membrane substrates with pore size of 0.4 μm. In addition,we observed an increase of approximately five- to ninefold of definitive endoderm marker gene expression in hESCs differentiated on PE or polyethylene terephthalate membrane substrates. Global gene expression analysis revealed upregulated expressions of a number of extracellular matrix and cell adhesion molecules in hESCs grown on membrane substrates. Further,an enhanced nuclear translocation of β-catenin was detected in these cells. These observations suggested the augmentation of Wnt signaling in hESCs grown on membrane substrates. These results also demonstrated that a membrane substrate can offer better physicochemical cues for enhancing in vitro hESC attachment,proliferation,and differentiation. View Publication