Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells

Introduction: Gender-specific phenotypes of the heart were reported with respect to both physiology and pathology. While most differences were associated with the sex hormones, differential expression of genes received special attention, particularly X-Y chromosomes’ genes. Methods: Here, we compared cardiogenesis by gene expression analysis of lineage specific markers and X-Y chromosomes’ genes, during in vitro differentiation of XY and XX human embryonic stem cells (hESC), in a hormone-free setup. Results: Downregulation of pluripotency marker (NANOG) and upregulation of cardiac mesoderm and progenitor markers (GATA4, TBX5, NKX2.5, ISL1) was remained temporally similar in differentiating XY and XX hESCs. Isoproterenol treatment of XY and XX hESC-derived cardiomyocytes (hESCCM) induced hypertrophy in a sex-specific manner, with female cardiomyocytes showing response at higher isoproterenol concentration and a later time point of differentiation. Interestingly, KDM5C as an X-linked gene, was markedly upregulated in both hypertrophied male and female cardiomyocytes. Conclusion: Collectively, our results indicated a temporally identical cardiogenesis, but more susceptibility of XY hESC-CM to hypertrophic stimulus in a hormone-free condition.

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High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells

Fertility and Sterility ◽

10.1016/j.fertnstert.2008.10.017 ◽

2010 ◽

Vol 93 (3) ◽

pp. 976-985 ◽

Cited By ~ 15

Author(s):

Ji Yeon Lee ◽

Jeoung Eun Lee ◽

Dong Ku Kim ◽

Tae Ki Yoon ◽

Hyung Min Chung ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Large Scale ◽

Embryonic Stem ◽

Efficient Technique ◽

High Concentration ◽

Slow Cooling ◽

Synthetic Serum

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

Bioscience Reports ◽

10.1007/s10540-007-9051-2 ◽

2007 ◽

Vol 27 (4-5) ◽

pp. 257-264 ◽

Cited By ~ 35

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.

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