scholarly journals Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells

2010 ◽  
Vol 432 (3) ◽  
pp. 575-599 ◽  
Author(s):  
Peter Burton ◽  
David R. Adams ◽  
Achamma Abraham ◽  
Robert W. Allcock ◽  
Zhong Jiang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Large Scale ◽  
Embryonic Stem ◽  
Culture Methods ◽  
Relationship Analysis ◽  
Pou Domain ◽  
Spontaneous Differentiation ◽  
Exogenous Cytokine

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure–activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

scholarly journals Identification and characterization of small-molecule ligands that maintain pluripotency of human embryonic stem cells

2010 ◽  
Vol 38 (4) ◽  
pp. 1058-1061 ◽  
Author(s):  
Peter Burton ◽  
David R. Adams ◽  
Achamma Abraham ◽  
Robert W. Allcock ◽  
Zhong Jiang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Large Scale ◽  
Pharmaceutical Research ◽  
Embryonic Stem ◽  
Good Manufacturing Practice ◽  
Exogenous Cytokine ◽  
Small Molecule Ligands ◽  
Hesc Lines

hESCs (human embryonic stem cells) offer great potential for pharmaceutical research and development and, potentially, for therapeutic use. However, improvements in cell culture are urgently required to allow the scalable production of large numbers of cells that maintain pluripotency. Supplementing feeder-free conditions with either EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine] or readily synthesized analogues of this compound maintains hESC pluripotency in the absence of exogenous cytokines. When the hESC lines SA121 or SA461 were maintained in feeder-free conditions with EHNA they displayed no reduction in stem-cell-associated markers such as Nanog, Oct4 (octamer-binding protein 4) and SSEA4 (stage-specific embryonic antigen 4) when compared with cells maintained in full feeder-free conditions that included exogenously added bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but EHNA did not limit efficient spontaneous or directed differentiation following its removal. We conclude that EHNA or related compounds offers a viable alternative to exogenous cytokine addition in maintaining hESC cultures in a pluripotent state and might be a particularly useful replacement for bFGF for large-scale or GMP (good manufacturing practice)-compliant processes.

scholarly journals Endogenous WNT Signals Mediate BMP-Induced and Spontaneous Differentiation of Epiblast Stem Cells and Human Embryonic Stem Cells

2015 ◽  
Vol 4 (1) ◽  
pp. 114-128 ◽  
Author(s):  
Dorota Kurek ◽  
Alex Neagu ◽  
Melodi Tastemel ◽  
Nesrin Tüysüz ◽  
Johannes Lehmann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Epiblast Stem Cells ◽  
Spontaneous Differentiation

scholarly journals Human Embryonic Stem Cells Where To Draw The Line

2012 ◽  
Vol 7 (2) ◽  
pp. 40-43
Author(s):  
T Hasan
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Large Scale ◽  
Human Life ◽  
Embryonic Stem ◽  
Eternal Life ◽  
Human Embryos ◽  
Ethical Perspective ◽  
Wide Range

Introduction: Human-embryonic stem cells (hESC) are derived from very early stages of the human embryo. These cells have immense plasticity and can be conditioned to develop into any type of cell of the human body. Despite all their promising utility, hESC researches have recently been the subject of fervent debate. Objective: This paper explores the implications of hESC therapy from a bio-ethical perspective. Method: Published literature with strict inclusion and exclusion criteria was extensively reviewed through use of general and meta search engines to elucidate the applications and implications of hESC. Discussion: Studies indicate that the potential of hESC in reconstructive and regenerative medicine is undisputable but complex social and moral issues are hopelessly intertwined beneath the pleasant facade. hESC offer endless possibilities in understanding bio-molecular disease patterns, supplying readymade healthy organs, interpreting aging and organogenesis at the cellular level. The use of hESC is well established in leukemia and scientists anticipate diverse applications in a wide range of congenital and acquired medical conditions. However, many dilemmas arise in context of their biomedical usage because of the destruction of donor human embryos in producing stem cells, adverse transplant reactions, teratogenecity, phenotypic / genotypic abnormalities, nonstandardized research laws, logistic issues and the possibility of eternal life and humanoid chimeras. Conclusion: The wisdom to choose between ' mindful utilization' and 'senseless exploitation' lies with us. The large scale commercialization of human life or the killing of viable embryos cannot be justified by any means. A neutral approach with increased involvement of uncontroversial progenitors should be adopted. DOI: http://dx.doi.org/10.3329/jafmc.v7i2.10396 JAFMC 2011; 7(2): 40-43

Comparison of Three Embryo Culture Methods for Derivation of Human Embryonic Stem Cells from Discarded Embryos

2011 ◽  
Vol 13 (3) ◽  
pp. 233-239 ◽  
Author(s):  
Ying Liu ◽  
Yang Li ◽  
Andrew Hwang ◽  
Shu-yu Wang ◽  
Chan-wei Jia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryo Culture ◽  
Embryonic Stem ◽  
Culture Methods

Caspase Inhibitor Z-VAD-FMK Enhances the Freeze-Thaw Survival Rate of Human Embryonic Stem Cells

2007 ◽  
Vol 27 (4-5) ◽  
pp. 257-264 ◽  
Author(s):  
Boon Chin Heng ◽  
Marie Veronique Clement ◽  
Tong Cao
Keyword(s):  
Stem Cells ◽  
Survival Rate ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Culture Media ◽  
Embryonic Stem ◽  
Immunocytochemical Staining ◽  
Irreversible Inhibitor ◽  
Slow Cooling ◽  
Spontaneous Differentiation

Previous study demonstrated that the low survival of human embryonic stem cells (hESC) under conventional slow-cooling cryopreservation protocols is predominantly due to apoptosis rather than cellular necrosis. Hence, this study investigated whether a synthetic broad-spectrum irreversible inhibitor of caspase enzymes, Z-VAD-FMK can be used to enhance the post-thaw survival rate of hESC. About 100 mM Z-VAD-FMK was supplemented into either the freezing solution, the post-thaw culture media or both. Intact and adherent hESC colonies were cryopreserved so as to enable subsequent quantitation of the post-thaw cell survival rate through the MTT assay, which can only be performed with adherent cells. Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhance the post-thaw survival rate (10.2% vs. 9.9%, p > 0.05). However, when 100 mM Z-VAD-FMK was added to the post-thaw culture media, there was a significant enhancement in the survival rate from 9.9% to 14.4% (p < 0.05), which was further increased to 18.7% when Z-VAD-FMK was also added to the freezing solution as well (p < 0.01). Spontaneous differentiation of hESC after cryopreservation was assessed by morphological observations under bright-field microscopy, and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. The results demonstrated that exposure to Z-VAD-FMK did not significantly enhance the spontaneous differentiation of hESC within post-thaw culture.

Sign in / Sign up

Export Citation Format

Share Document