The roles of apoptotic pathways in the low recovery rate after cryopreservation of dissociated human embryonic stem cells

Abstract

Human embryonic stem (hES) cells have enormous potential for clinical applications. However, one major challenge is to achieve high cell recovery rate after cryopreservation. Understanding how the conventional cryopreservation protocol fails to protect the cells is a prerequisite for developing efficient and successful cryopreservation methods for hES cell lines and banks. We investigated how the stimuli from cryopreservation result in apoptosis, which causes the low cell recovery rate after cryopreservation. The level of reactive oxygen species (ROS) is significantly increased, F-actin content and distribution is altered, and caspase-8 and caspase-9 are activated after cryopreservation. p53 is also activated and translocated into nucleus. During cryopreservation apoptosis is induced by activation of both caspase-8 through the extrinsic pathway and caspase-9 through the intrinsic pathway. However, exactly how the extrinsic pathway is activated is still unclear and deserves further investigation.

Figure 1

Caspase activity of the cells from feeder-independent after 2h of culture, and at day 1. hES cells after regular passage or after cryopreservation procedure were plated on matrigel-coated 96-well plates in the presence of either 10 μM Y27632, 10 μM Y-27632 (R) and 1 μM pifithrin-μ (P), or 20 μM caspase-9 inhibitor (C9), or 20 μM caspase inhibitor VI (Z), or 200 μM Bax-inhibiting peptide (Bax) or in the absence of any inhibitors on the first day of culture, at the density of 7.5 × 104 cells/well. (A) Caspase-8 activity of the cells cryopreserved by 10% DMSO after 2 h of culture. (B) Caspase-8 activity of the cells cryopreserved by 10% DMSO at day 1 of culture. (C) Caspase-9 activity of the cells cryopreserved by 10% DMSO after 2 h of culture (D) Caspase-9 activity of the cells cryopreserved by 10% at day 1 of culture. Results are expressed as the mean of three independent experiments ± the standard derivation (n = 3). Statistical analysis was performed using one-way ANOVA. *P < 0.05, compared with in the absence of any inhibitor. **P < 0.05, compared with in the presence of 10 μM Y-27632 and 1 μM pifithrin-μ.

Figure 2

Cell recovery after CPA addition and removal only, and after cryopreservation using 10% DMSO. (A) Cell number per well after 4 days of culture for the cells from feeder-independent culture after regular passage and CPA addition and removal only. (B) The ratio of cell number after cryopreservation, cultured in the absence of Y-27632, in the presence of 10 μM Y-27632, and 10 μM Y-27632 and 1 μM pifithrin-μ to the cell number after cryopreservation, cultured in the presence of Y-27632Y-27632 (+)/(−): R(+)/(−), pifithrin-μ (+)/(−) : P(+)/(−). Results are expressed as the mean of three independent experiments ± the standard derivation (n = 4). Statistical analysis was performed using one-way ANOVA. *P < 0.05, compared with regular passage. **P < 0.05, compared with CPA addition and removal only. ***P < 0.05, compared with R(−)P(−). ****P < 0.05, compared with R(+)P(−). (C) Typical colony formation from feeder-dependent culture after cryopreservation using 10% DMSO, and then recovered in the absence of Y-27632, in the presence of 10 μM Y-27632, 1 μM pifithrin-μ and 10 μM Y27632. Scale bar: 100 μm.

Figure 3

Colony formation from feeder-independent culture after cryopreservation using 10% DMSO, and then recovered in the absence of Y-27632 [R(−)P(−)], in the presence of 10 μM Y-27632 and 1 μM pifithrin-μ [R(+)P(−)], 20 μM caspase-9 inhibitor [R(−)C9(+)] , 20 μM -VAD-FMK [R(−)Z(+)], 200 μM Bax-inhibiting peptide [R(−)Bax(+)], or in the combination of Y-27632 and 20 μM caspase-9 inhibitor [R(+)C9(+)], 20 μM -VAD-FMK [R(+)Z(+)], 200 μM Bax-inhibiting peptide [R(+)Bax(+)], respectively. Scale bar: 50 μm.

Figure 4

Intracellular ROS after regular passage, CPA addition and removal only, and after cryopreservation. The cells were stained with 2′7-Dichlorofluorescein diacetate (DCFH-DA), for hydrogen peroxide, or with dihydroethidine (DHE) for superoxide anion generation. (A) Hydrogen peroxide in mitochondria. (B) Superoxide content in mitochondria. Results are expressed as the mean of three independent experiments ± the standard derivation (n = 4). Statistical analysis was performed using one-way ANOVA. *P < 0.05, compared with regular passage. **P < 0.05, compared with CPA addition and removal only.

Figure 5

Distribution and quantification of intracellular F-actin after regular passage, CPA addition only, CPA addition and removal only, and cryopreservation by 10% DMSO. To quantify F-actin, the cells after treatment were fixed in the corresponding solution containing 4% formaldehyde, permeabilised with 0.1% Triton in PBS, and stained with 0.33 μM rhodamine phalloidin. F-actin content was quantitatively determined by flow cytometry. For the detection of F-actin distribution, the cells labeled with 0.33 μM rhodamine phalloidin were transferred to slides, and visualized by fluorescence microscopy. (A) intracellular F-actin content after regular passage, CPA addition only, CPA addition and removal only, and cryopreservation by 10% DMSO. Results are expressed as the mean of three independent experiments ± the standard derivation (n = 3). Statistical analysis was performed using one-way or two factor with replication method. *P < 0.05, compared with regular passage. **P < 0.05, compared with after CPA addition, ***P < 0.05, compared with CPA addition and removal only. (B) F-actin distribution in suspension of hES after regular passage, CPA addition only, CPA addition and removal only, and cryopreservation by 10% DMSO. Scale bar: 10 μm. (C) Representative FAC analysis.

Figure 6

p53 expression of the hESs after cryopreservation using 10% DMSO. p53 was expressed in green color, and blue color was for DAPI. Scale bar: 25 μm.

Figure 7

Expression of undifferendiated marker, nuclear markers, OCT4 and Nanog, and surface marker, SSEA4, for hES cells at passage 2 after cryopreservation using 10% DMSO. The cells after cryopreservation were cultured in the presence of ROCK inhibitor, R(+)P(−), or in the presence of ROCK inhibitor and pifithrin-μ, R(+)P(+). Scale bar: 50 μm for feeder-dependent culture and 25 μm fro feeder-independent culture, respectively.

8

A) Observed mediators of apoptosis induced during cryopreservation; (B) Suggested mechanism of cell responses to cryopreservation.