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Morphology of the blastocysts developed from long-term cryopreserved embryos. Six days after thawing and culture, the surviving embryos developed to the blastocyst stage. Different morphologies of the blastocysts, including fully expanded blastocysts with a clear and tightly packed ICM (white arrow; A), non-fully expanded blastocysts with thick zonae pellucidae and small ICMs (B–D), and broken zona pellucida during the freezing–thawing process, were observed (black arrow; D). All of the images were obtained using a 40× objective phase-contrast microscope. ICM, inner cell mass.
Source publication
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, culture...
Citations
... Thus, DMSO-free or DMSO-diminished cryoprotective agents might be the better alternatives for ENSdN. Therefore we additionally used a DMSO free method (StemCellKeep ™ ) based on a procedure called vitrification (Baharvand et al., 2010;Li et al., 2010;Hunt, 2011;Wagh et al., 2011;Pruksananonda et al., 2012;Beier et al., 2013), that has been successfully used for the storage of human ES/iPS cells (Matsumura et al., 2011). Carboxylated ε-poly-L-lysine (CPLL) is used as a cryoprotectant instead of DMSO, which is used widely as a food additive and is less cytotoxic than DMSO (Shima et al., 1984). ...
Introduction: Impairment of both the central and peripheral nervous system is a major cause of mortality and disability. It varies from an affection of the brain to various types of enteric dysganglionosis. Congenital enteric dysganglionosis is characterized by the local absence of intrinsic innervation due to deficits in either migration, proliferation or differentiation of neural stem cells. Despite surgery, children’s quality of life is reduced. Neural stem cell transplantation seems a promising therapeutic approach, requiring huge amounts of cells and multiple approaches to fully colonize the diseased areas completely. A combination of successful expansion and storage of neural stem cells is needed until a sufficient amount of cells is generated. This must be combined with suitable cell transplantation strategies, that cover all the area affected. Cryopreservation provides the possibility to store cells for long time, unfortunately with side effects, i.e., upon vitality.
Methods: In this study we investigate the impact of different freezing and thawing protocols (M1-M4) upon enteric neural stem cell survival, protein and gene expression, and cell function.
Results: Freezing enteric nervous system derived neurospheres (ENSdN) following slow-freezing protocols (M1-3) resulted in higher survival rates than flash-freezing (M4). RNA expression profiles were least affected by freezing protocols M1/2, whereas the protein expression of ENSdN remained unchanged after treatment with protocol M1 only. Cells treated with the most promising freezing protocol (M1, slow freezing in fetal calf serum plus 10% DMSO) were subsequently investigated using single-cell calcium imaging. Freezing of ENSdN did not alter the increase in intracellular calcium in response to a specific set of stimuli. Single cells could be assigned to functional subgroups according to response patterns and a significant shift towards cells responding to nicotine was observed after freezing.
Discussion: The results demonstrate that cryopreservation of ENSdN is possible with reduced viability, only slight changes in protein/gene expression patterns and without an impact on the neuronal function of different enteric nervous system cell subtypes, with the exception of a subtle upregulation of cells expressing nicotinergic acetylcholine receptors. In summary, cryopreservation presents a good method to store sufficient amounts of enteric neural stem cells without neuronal impairment, in order to enable subsequent transplantation of cells into compromised tissues.
... We can also speculate that other factors such as temperature fluctuations due to frequently opening the cryotank may have an impact. Nevertheless, many case reports have been published across the years showing successful pregnancies and healthy live births from either oocytes or embryos stored 3-14 years before [33][34][35][36][37]. Indeed, human embryos cryopreserved for 18 years were shown to maintain the same pluripotency as fresh embryos [38]. ...
Purpose
Few studies explored whether prolonged cryo-storage after vitrification affects embryo competence and perinatal outcomes. This systematic review and meta-analysis aims at highlighting any putative impact of cryo-storage duration on cryo-survival, miscarriage, live birth and major malformations.
Methods
A systematic review was performed using MEDLINE (PubMed), ISI Web of Knowledge, Scopus and Embase databases up to June 2021. Data were combined to obtain a pooled OR, and meta-analysis was conducted using a random effects model. Out of 1,389 screened abstracts, 22 papers were assessed for eligibility, and 5 studies were included (N = 18,047 embryos). Prolonged cryo-storage was defined as > 12 months (N = 3389 embryos). Subgroup analysis was performed for untested vitrified cleavage stage embryos (N = 1739 embryos) and for untested and euploid vitrified blastocysts (N = 13,596 and 2712 embryos, respectively).
Results
Survival rate, miscarriage, live birth and major malformation rates were all similar in the two groups.
Conclusion
These data further support the safety of long-term cryo-storage of human embryos beyond 12 months. This is reassuring for good prognosis patients with surplus embryos, couples seeking a second child from supernumerary embryos and women postponing the transfer for clinical or personal reasons.
... Our group has previously reported on live births achieved with a cohort of human embryos which were cryopreserved for more than 12 years [11]. Another study showed that human embryos cryopreserved for 18 years maintained their pluripotency similar to fresh embryos and were not adversely affected by the long duration of cryopreservation [20]. ...
Purpose Cryopreservation techniques have become an essential part of assisted reproduction technology. Embryos may be cryopreserved for several years before transfer, and the safety of long-term cryopreservation needs to be considered. This dose-response meta-analysis was conducted to evaluate whether there were dose-response relationships between the storage time of cryopreserved embryos and pregnancy outcomes such as survival rate, implantation rate, miscarriage rate, clinical pregnancy rate, and congenital malformation rate.
Methods After searching the databases PubMed, Embase, MEDLINE, CCRT and related reviews up until June 4, 2020, seven studies were included for analysis. Two reviewers extracted the relevant information and independently assessed the study quality using the Newcastle-Ottawa scale. Potential linear or non-linear dose-response relationships were assessed with a random-effect dose-response meta-analysis.
Results No dose-response association was found between duration of embryo cryostorage and survival rate, implantation rate, miscarriage rate, clinical pregnancy rate or congenital malformation rate.
Conclusion The interval between the start of embryo cryopreservation and frozen/thawed embryo transfer does not influence pregnancy outcomes.
... Moreover, there are reports of survival of ESC for 18 years, while there is no similar report for iPSC. It must be considered that iPSC were generated for the first time 13 years ago (Pruksananonda et al. 2012). However, patientderived iPSC is not suitable for age-related diseases since the somatic cells of the elderly probably have a considerable amount of genomic mutagens and hazardous epigenetic maps. ...
The first isolation of human embryonic stem cells (hESC) reported in the late 90s opened a new window to promising possibilities in the fields of human developmental biology and regenerative medicine. Subsequently, the differentiation of hESC lines into different precursor cells showed their potential in treating different incurable diseases. However, this promising field has consistently had remarkable ethical and experimental limitations. This paper is a review of clinical trial studies dealing with hESC and their advantages, limitations, and other specific concerns. Some of the hESC limitations have been solved, and several clinical trial studies are ongoing so that recent clinical trials have strived to improve the clinical applications of hESC, especially in macular degeneration and neurodegenerative diseases. However, regarding hESC-based therapy, several important issues need more research and discussion. Despite considerable studies to Date, hESC-based therapy is not available for conventional clinical applications, and more studies and data are needed to overcome current clinical and ethical limitations. When all the limitations of Embryonic stem cells (ESC) are wholly resolved, perhaps hESC can become superior to the existing stem cell sources. This overview will be beneficial for understanding the standard and promising applications of cell and tissue-based therapeutic approaches and for developing novel therapeutic applications of hESC.
... combined with difficulty of quantitatively detecting loss of function, we are led to the general perception that time nearly stops during cryopreservation (e.g. Pruksananonda et al. 2012). However, this perception must be balanced with the knowledge that inability to detect change initially is a hallmark of co-operative kinetics that occur in solid materials (Figs. ...
This study explores temperature dependency of aging rate in dry cells over a broad temperature range encompassing the fluid to solid transition (Tg) and well below. Spores from diverse species of eight families of ferns were stored at temperatures ranging from +45oC to c. -176oC (vapor phase above liquid nitrogen), and viability was monitored periodically for up to 4300 days (ca. 12 years). Accompanying measurements using differential scanning calorimetry (DSC) provide insights into structural changes that occur such as Tg, between +45 and -20oC (depending on moisture), and triacylglycerol (TAG) crystallization, between -5 and -35oC (depending on species). We detected aging even at cryogenic temperatures, which we consider analogous to unscheduled degradation of pharmaceuticals stored well below Tg caused by a shift in the nature of molecular motions that dominate chemical reactivity. We occasionally observed faster aging of spores stored at -18oC (conventional freezer) compared to 5oC (refrigerator), and linked this with mobility and crystallization within TAG, which likely influences molecular motion of dried cytoplasm in a narrow temperature range. Temperature dependency of longevity was remarkably similar among diverse fern spores, despite widely disparate aging rates; this provides a powerful tool to predict deterioration of germplasm preserved in the solid state. Future work will increase our understanding of molecular organization and composition contributing to differences in longevity.
... [29] and normal human ES cell (hESC) line (Chula2.hES) [30] were cultured in mTeSR1 medium (Stem Cell Technologies, Vancouver, Canada) for 3 days before transduction. The lentiviral vector was added to the cells at MOI of 20 with 8 μg/ml of polybrene (Sigma-Aldrich, St. Louis, MO) for 24 h. ...
Lentiviral vectors have emerged as the most efficient system to stably transfer and insert genes into cells.By adding a tetracycline-inducible promotor, transgene expression delivered by a lentiviral vector can be expressed whenever needed and halted when necessary.Here we have constructed a doxycycline (Dox)-inducible lentiviral vector which efficiently introduces a designed zinc finger protein, 2LTRZFP, into hematopoietic cell lines and evaluated its expression in pluripotent stem cells. As a result this lentiviral inducible system can regulate 2LTRZFP expression in the SupT1 T cell line and in pluripotent stem cells. Using this vector, no basal expression was detected in the T cell line and its induction was achieved with low Dox concentrations.Remarkably, the intracellular regulatory expression of 2LTRZFP significantly inhibited HIV-1 integration and replication in HIV-inoculated SupT1 cells.This approach could provide the potential tool for gene therapy applications which efficiently control and reduce the side effect of therapeutic genes expression.
... Established iPSC lines and hESCs, namely Chula.2-hESCs (Pruksananonda et al. 2012), were maintained on Matrigel (BD Biosciences, USA)-coated plates with mTeSR 1 medium (Stemcell technologies, Canada) and were subcultured every 4 days by using dispase (Stemcell technologies). ...
The use of induced pluripotent stem cells (iPSCs) as a source of cells for cell-based therapy in regenerative medicine is hampered by the limited efficiency and safety of the reprogramming procedure and the low efficiency of iPSC differentiation to specialized cell types. Evidence suggests that iPSCs retain an epigenetic memory of their parental cells with a possible influence on their differentiation capacity in vitro. We reprogramme three cell types, namely human umbilical cord vein endothelial cells (HUVECs), endothelial progenitor cells (EPCs) and human dermal fibroblasts (HDFs), to iPSCs and compare their hematoendothelial differentiation capacity. HUVECs and EPCs were at least two-fold more efficient in iPSC reprogramming than HDFs. Both HUVEC- and EPC-derived iPSCs exhibited high potentiality toward endothelial cell differentiation compared with HDF-derived iPSCs. However, only HUVEC-derived iPSCs showed efficient differentiation to hematopoietic stem/progenitor cells. Examination of DNA methylation at promoters of hematopoietic and endothelial genes revealed evidence for the existence of epigenetic memory at the endothelial genes but not the hematopoietic genes in iPSCs derived from HUVECs and EPCs indicating that epigenetic memory involves an endothelial differentiation bias. Our findings suggest that endothelial cells and EPCs are better sources for iPSC derivation regarding their reprogramming efficiency and that the somatic cell type used for iPSC generation toward specific cell lineage differentiation is of importance.
... Human embryonic stem cell line (Chula2.hES), derived from frozen-thawed embryo (Pruksananonda et al. 2012) was used as a control cell line. ...
Transgene-free human HS5-SV.hiPS line was generated from human cesarean scar-derived fibroblasts using temperature-sensitive Sendai virus vectors carrying Oct4, Sox2, cMyc and Klf4 exogenous transcriptional factors. The viral constructs were eliminated from HS5-SV.hiPS line through heat treatment. Transgene-free HS5-SV.hiPS cells expressed pluripotent associated transcription factors Oct4, Nanog, Sox2, Rex1 and surface markers SSEA-4, TRA-1-60 and OCT4. HS5-SV.hiPS cells formed embryoid bodies and differentiated into three embryonic germ layers in vivo. HS5-SV.hiPS cells maintained their normal karyotype (46, XX) after culture for extended period. HS5-SV.hiPS displayed the similar pattern of DNA fingerprinting to the parenteral scar-derived fibroblasts.
... MEFs play important roles in the regulation of hPSC pluripotency through the secretion of essential cytokines or molecules and extracellular matrices [21,22]. Due to concerns about animal pathogens and immunogens using MEFs, several reports recommended the use of human fibroblasts, including human neonatal foreskin fibroblasts (HFFs) instead of MEFs [23][24][25]. The results demonstrated that HFFs showed the characteristics of supportive feeder cells for hPSCs. ...
... hUCS has previously been demonstrated to have beneficial effects on the isolation, proliferation, and differentiation of human amniotic fluid stem cells (hAFS) and Wharton's jelly mesenchymal stromal cells by our collaborators [16,17]. The commercial human foreskin fibroblasts (HFFs) used in the present study have been demonstrated as supportive feeder cells for the derivation of hESCs and hiPSCs [23][24][25]. Therefore, the aim of the present study was to examine the feasibility of using hUCS for culturing HFFs prior to the preparation of feeder cells and the subsequent use of inactivated HFFs for coculture with hPSCs. ...
... Moreover, the quantified results of SSEA-4 expression, determined through flow cytometry, demonstrated that inactivated HFF-hUCS effectively support the pluripotency of hPSCs in a manner similar to inactivated HFF-FBS. In addition, hPSC lines differentiate into three embryonic germ layers in vitro and in vivo and maintain a normal karyotype, confirming the pluripotency of these cells [1,2,25]. ...
Although human pluripotent stem cells (hPSCs) can proliferate robustly on the feeder-free culture system, genetic instability of hPSCs has been reported in such environment. Alternatively, feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS) prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. Recently, human cord blood-derived serum (hUCS) showed a positive effect on culture of mesenchymal stem cells. It is interesting to test whether hUCS can be used for culture of feeder cells of hPSCs. This study was aimed to replace FBS with hUCS for culturing the human foreskin fibroblasts (HFFs) prior to feeder cell preparation. The results showed that HFFs cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and TGF
β
1, which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit of hUCS for hPSCs culture system.
... hESCs [Pruksananonda et al., 2012] and hiPSCs [Nishishita et al., 2012] were grown on mitotically inactivated human foreskinderived fibroblasts plated in a 0.1% gelatin-coated dish in knockout Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% knockout serum replacement, 1% GlutaMAX ® , 0.55 m M β-mercaptoethanol, 1% nonessential amino acids (Invi-trogen, 11140-050), 50 U penicillin ml -1 (Invitrogen), 50 μg streptomycin ml -1 (Invitrogen), and 8 ng hbFGF ml -1 (Invitrogen) or 6xHis-hbFGF and Trx-6xHis-hbFGF fusion proteins. Subsequently, hESC and hiPSC colonies with ES cell morphology were selected for subculture and maintained by standard methods. ...
To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.
