Enhanced differentiation of human embryonic stem cells towards definitive endoderm on ultrahigh aspect ratio nanopillars

Differentiation of human embryonic stem cells is widely studied as a potential unlimited source for cell replacement therapy to treat degenerative diseases such as diabetes. The directed differentiation of human embryonic stem cells relies mainly on soluble factors. Although, some studies have highlighted that the properties of the physical environment, such as substrate stiffness, affect cellular behavior. Here, mass-produced, injection molded polycarbonate nanopillars are presented, where the surface mechanical properties, i.e., stiffness, can be controlled by the geometric design of the ultrahigh aspect ratio nanopillars (stiffness can be reduced by 25.0003). It is found that tall nanopillars, yielding softer surfaces, significantly enhance the induction of definitive endoderm cells from pluripotent human embryonic stem cells, resulting in more consistent differentiation of a pure population compared to planar control. By contrast, further differentiation toward the pancreatic ­endoderm is less successful on “soft” pillars when compared to “stiff” pillars or control, indicating differential cues during the different stages of differentiation. To accompany the mechanical properties of the nanopillars, the concept of surface shear modulus is introduced to describe the characteristics of engineered elastic surfaces through micro or nanopatterning. This provides a framework whereby comparisons can be drawn between such materials and bulk elastomeric materials

Autophagy in Human Embryonic Stem Cells

Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC

Human embryonic stem cells: preclinical perspectives

Human embryonic stem cells (hESCs) have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic

Correlated random walks of human embryonic stem cells in vitro

We perform a detailed analysis of the migratory motion of human embryonic stem cells in two-dimensions, both when isolated and in close proximity to another cell, recorded with time-lapse microscopic imaging. We show that isolated cells tend to perform an unusual locally anisotropic walk, moving backwards and forwards along a preferred local direction correlated over a timescale of around 50 min and aligned with the axis of the cell elongation. Increasing elongation of the cell shape is associated with increased instantaneous migration speed. We also show that two cells in close proximity tend to move in the same direction, with the average separation of m or less and the correlation length of around 25 μm, a typical cell diameter. These results can be used as a basis for the mathematical modelling of the formation of clonal hESC colonies

Generation of a constitutively expressing Tetracycline repressor (TetR) human embryonic stem cell line BJNhem20-TetR

AbstractHuman embryonic stem cell line BJNhem20-TetR was generated using non-viral method. The construct pCAG-TetRnls was transfected using microporation procedure. BJNhem20-TetR can subsequently be transfected with any vector harbouring a TetO (Tet operator) sequence to generate doxycycline based inducible line. For example, in human embryonic stem cells, the pSuperior based TetO system has been transfected into a TetR containing line to generate OCT4 knockdown cell line (Zafarana et al., 2009). Thus BJNhem20-TetR can be used as a tool to perturb gene expression in human embryonic stem cells

Stem Cell Therapeutics: Exploring Newer Alternatives to Human Embryonic Stem Cells

Stem cells therapeutics has come a long way since stem cells and their potential was discovered for the first time. Intense research into cellular biology of stem cells has revealed that they possess immense potential for curing many human diseases. Research done in last couple of decades revealed that a particular class of stem cells called, “Human embryonic stem cells (HESCs)” possessed exceptional self-renewal and pluripotency properties. There ability to differentiate into specialized cell lineages of all three embryonic germ layers contributed further towards their popularity. However, in recent times HESCs have come under the cross-hairs of critics, politicians and religious groups due to certain technical and ethical concerns related to them. Such problems with HESCs research have forced stem cell researchers to start exploring the prospects of using alternatives to HESCs for regenerative medicine and therapeutics. In the present review, various sources of stem cells have been described, which in near future, have the potential to replace HESCs in regenerative medicine

MPSS profiling of human embryonic stem cells

BACKGROUND: Pooled human embryonic stem cells (hESC) cell lines were profiled to obtain a comprehensive list of genes common to undifferentiated human embryonic stem cells. RESULTS: Pooled hESC lines were profiled to obtain a comprehensive list of genes common to human ES cells. Massively parallel signature sequencing (MPSS) of approximately three million signature tags (signatures) identified close to eleven thousand unique transcripts, of which approximately 25% were uncharacterised or novel genes. Expression of previously identified ES cell markers was confirmed and multiple genes not known to be expressed by ES cells were identified by comparing with public SAGE databases, EST libraries and parallel analysis by microarray and RT-PCR. Chromosomal mapping of expressed genes failed to identify major hotspots and confirmed expression of genes that map to the X and Y chromosome. Comparison with published data sets confirmed the validity of the analysis and the depth and power of MPSS. CONCLUSIONS: Overall, our analysis provides a molecular signature of genes expressed by undifferentiated ES cells that can be used to monitor the state of ES cells isolated by different laboratories using independent methods and maintained under differing culture condition

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage