Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells☆
Section snippets
Handling and freezing hES cells
We have studied the influence of a cryopreservation protocol routinely used for hESCs [35]. The hES cell line H9 (WiCell, Wisconsin) was grown on a mouse embryonic fibroblast (MEF) layer according to the standard protocols provided by WiCell [36] using matrigel (BD Bioscience, CA) for plate coating. The cells were infected with a lentiviral construct expressing enhanced green fluorescent protein (EGFP) under a constitutive Oct-4 promoter, and were harvested using collagenase to minimize the
Co-localization of endogenous Oct-4 immunostaining and Oct-4-EGFP reporter in hESCs
For this particular experiment we have cloned a hES cell line where over 99% of cells express an Oct-4-EGFP reporter. To ensure that the EGFP reporter accurately reflected endogenous Oct-4 expression, we performed immunostaining of Oct4-EGFP hES cells for Oct-4 compared with EGFP fluorescence. The vast majority of cells that were positive for endogenous Oct-4 were also EGFP-positive (Fig. 1). Some (∼5%) EGFP-positive cells at the periphery of the colonies did not have detectable amount of
Discussion
Several basic conclusions can be drawn from these results:
- 1.
More than half the cells did not survive cryopreservation and 3 days storage at −80 °C and did not attach. Approximately a half of those that attached, eventually died in 3 days indicated by PI fluorescent staining. Thus, the overall yield (recovery) of frozen hES cells can be estimated as 25% while the vital staining indicates that 50% of attached cell are non-vital. This is similar to other reports [8], [12], [23], [24], [40]. Moreover,
Acknowledgments
We are thankful to Pamela Itkin-Ansari and Bjorn Tyrberg (UCSD Cancer Center) for useful comments and suggestions, and to the reviewers of the original version of this paper for their constructive criticisms.
References (40)
- et al.
The effects of solvents on embryonic stem cell differentiation
Toxicol. In Vitro
(2006) - et al.
Core transcriptional regulatory circuitry in human embryonic stem cells
Cell
(2005) - et al.
Establishment of pluripotent cell lines from porcine preimplantation embryos
Theriogenology
(1999) - et al.
Low- and high-temperature vitrification as a new approach to biostabilization of reproductive and progenitor cells
Int. J. Refrig.
(2006) - et al.
Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4
Cell
(1998) - et al.
Human embryonic stem cells
Fertil. Steril.
(2001) - et al.
Improved cryopreservation of human embryonic stem cells with trehalose
Reprod. Biomed. Online
(2005) - et al.
Stem cells. Setting standards for human embryonic stem cells
Science
(2003) - et al.
Cryopreservation of stem cells using trehalose: evaluation of the method using a human hematopoietic cell line
Stem Cells Dev.
(2004) Investing in frozen assets—the world’s first stem cell bank opened in the UK this week and its closely-guarded contents could change our lives. But is the vision flawed?
Times (Lond.)
(2004)
In vivo targeting of tumor endothelial cells by systemic delivery of lentiviral vectors
Hum. Gene Ther.
A simple and efficient cryopreservation method for primate embryonic stem cells
Int. J. Dev. Biol.
Cryopreservation of human embryonic stem cells without the use of a programmable freezer
Hum. Reprod.
A proposed design for the cryopreservation of intact and adherent human embryonic stem cell colonies
In Vitro Cell. Dev. Biol. Anim.
The cryopreservation of human embryonic stem cells
Biotechnol. Appl. Biochem.
Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to apoptosis rather than cellular necrosis
J. Biomed. Sci.
Activation of transiently transfected reporter genes in 3T3 Swiss cells by the inducers of differentiation/apoptosis—dimethylsulfoxide, hexamethylene bisacetamide and trichostatin A
Eur. J. Biochem.
Cryopreservation of adherent human embryonic stem cells
Biotechnol. Bioeng.
Motility and fertilization ability of boar semen frozen and stored at −80 °C
Cryobiology
Effects of type IV collagen and laminin on the cryopreservation of human embryonic stem cells
Stem Cells
Cited by (0)
- ☆
The final stages of the work were supported by the Burnham Stem Cell Center.