Eighteen-Year Cryopreservation Does Not Negatively Affect the Pluripotency of Human Embryos: Evidence from Embryonic Stem Cell Derivation

Abstract

Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research.

The ROCK Inhibitor Y-26732 Enhances the Survival and Proliferation of Human Embryonic Stem Cell-Derived Neural Progenitor Cells upon Dissociation

“…When the BG01 cell line was cultured on a human foreskin fibroblast feeder layer, cells displayed the typical hESC morphology and expressed pluripotent markers ( fig. 2 a) being similar to in-house-derived hESC lines reported previously by our group [Pruksananonda et al, 2012]. Undifferentiated colonies ( fig.…”

Original article. Human dental pulp stem cells as a potential feeder layer for human embryonic stem cell culture

“…Commercial human foreskin-derived fibroblasts (hFFs; CRL-2429, American Type Culture Collection, Manassas, VA, USA) were cultured and maintained according to standard protocols as previously described [37]. To use hFFs and hDPSCs as the feeder layer, confluent hFFs and hDPSCs were inactivated with 10 g/mL mitomycin C (SigmaAldrich, St. Louis, MO, USA) for 3 hours, dissociated with 0.05% trypsin-EDTA (Invitrogen, Carlsbad, CA, USA) and plated on a 0.1% gelatin coated-dish (SigmaAldrich) at a density of 5 × 10 4 cells/cm 2 .…”

“…Chula2 and Chula5 hES cell lines [37] were cultured on either HFFs or hDPSC feeders in the hES cell culture medium consisting of knockout DMEM supplemented with 20% knockout serum replacement (KSR), 1% Glutamax, 1% nonessential amino acids, 1% penicillin-streptomycin, 0.1 mM 2-mercaptoethanol (all from Invitrogen), and 8 ng/mL basic fibroblast growth factor (bFGF; R&D Systems).…”

“…Then, we evaluated the undifferentiated growth of two hES cell lines, Chula2 and Chula5 [37] on hDPSCs feeder layers. The results showed that hDPSCs can support the undifferentiated growth of hES cells in prolonged culture.…”

Cryopreservation and Cell Banking for Autologous Mesenchymal Stem Cell-Based Therapies

“…[16][17][18] Cryopreservation is based on the principle that chemical, biological, Activation of MSC's into final cell therapy product; (11) Shipping of final product in optimized, approved delivery device to medical center at 4 °C in temperature controlled packaging with temperature recorder; (12) injection of the cell therapy product into patient. notes: *In the event that a cGmP compliant cell therapy processing unit exists within the medical center, frozen mSCs could theoretically be shipped to the medical center for thawing and further processing at that unit before delivery to the patient.…”