hESC Banking Research Articles

High\xa0quality clinical\xa0grade human embryonic stem cell lines derived from fresh discarded embryos

BackgroundHuman embryonic stem cells (hESCs) hold tremendous promise for cell replacement therapies for a range of degenerative diseases. In order to provide cost-effective treatments affordable by public health systems, HLA-matched allogeneic tissue banks of the highest quality clinical-grade hESCs will be required. However only a small number of existing hESC lines are suitable for clinical use; they are limited by moral and ethical concerns and none of them apply Good Manufacturing Practice (GMP) standards to the earliest and critical stages of gamete and embryo procurement. We thus aimed to derive new clinical grade hESC lines of highest quality from fresh surplus GMP grade human embryos.MethodsA comprehensive screen was performed for suitable combinations of culture media with supporting feeder cells or feeder-free matrix, at different stages, to support expansion of the inner cell mass and to establish new hESC lines.ResultsWe developed a novel two-step and sequential media system of clinical-grade hESC derivation and successfully generated seven new hESC lines of widely varying HLA type, carefully screened for genetic health, from human embryos donated under the highest ethical and moral standards under an integrated GMP system which extends from hESC banking all the way back to gamete and embryo procurement.ConclusionsThe present study, for the first time, reports the successful derivation of highest-quality clinical-grade hESC lines from fresh poor-quality surplus human embryos generated in a GMP-grade IVF laboratory. The availability of hESC lines of this status represents an important step towards more widespread application of regenerative medicine therapies.

Quality Assurance in Stem Cell Banking: Emphasis on Embryonic and Induced Pluripotent Stem Cell Banking.

For quality assurance (QA) in stem cell banking, a planned system is needed to ensure that the banked products, stem cells, meet the standards required for research, clinical use, and commercial biotechnological applications. QA is process oriented, avoids, or minimizes unacceptable product defects, and particularly encompasses the management and operational systems of the bank, as well as the ethical and legal frameworks. Quality control (QC ) is product oriented and therefore ensures the stem cells of a bank are what they are expected to be. Testing is for controlling, not assuring, product quality, and is therefore a part of QC , not QA. Like QA, QC is essential for banking cells for quality research and translational application (Schwartz et al., Lancet 379:713-720, 2012). Human embryonic stem cells (hESCs), as cells derived from donated supernumerary embryos from in vitro fertilization (IVF) therapy, are different from other stem cell types in resulting from an embryo that has had two donors . This imposes important ethical and legal constraints on the utility of the cells, which, together with quite specific culture conditions, require special attention in the QA system. Importantly, although the origin and derivation of induced pluripotent stem cells (iPSCs ) differ from that of hESCs, many of the principles of QA for hESC banking are applicable to iPSC banking (Stacey et al., Cell Stem Cell 13:385-388, 2013). Furthermore, despite differences between the legal and regulatory frameworks for hESC and iPSC banking between different countries, the requirements for QA are being harmonized (Stacey et al., Cell Stem Cell 13:385-388, 2013; International Stem Cell Banking Initiative, Stem Cell Rev 5:301-314, 2009).

Cryopreservation of human embryonic stem cells by a programmed freezer with an oscillating magnetic field

Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, are an important source of cells for regenerative medicine. The immediate obstacles that need to be addressed are the poor cell survival rate of hESCs and their cell quality after cryopreservation. In this study, we used the Cell Alive System (CAS) which combines a programmed freezer with an oscillating magnetic field to reduce cryo-injury during the freezing process. The hESC clumps suspended in freezing medium were divided into three groups: (i) cells frozen by a conventional freezing container, Mr. Frosty and kept in a −80°C freezer (MF); (ii) cells frozen to −32°C by CAS, and then transferred to a −80°C freezer (CAS); (iii) cells frozen to −32°C by CAS, and then transferred to a pre-cooled Mr. Frosty and kept in a −80°C freezer (CAS-MF) for overnight. All cryovials were placed in liquid nitrogen for one week, and hESCs were then thawed and cultured on feeder for 7days. The results of alkaline phosphatase (AP) staining showed that the attachment efficiency of the cells cryopreserved by CAS and CAS-MF was significantly higher (29.0% and 44.0%) than in the MF method (7.0%). Furthermore, we confirmed the cells cryopreserved using CAS-MF could be subcultured while expressing pluripotent markers, differentiate into three germ layers, and maintain a normal karyotype. These results demonstrate that the use of CAS-MF offers an efficient method of hESC banking.

Strategy for the creation of clinical grade hESC line banks that HLA‐match a target population

Here, we describe a pre-derivation embryo haplotyping strategy that we developed in order to maximize the efficiency and minimize the costs of establishing banks of clinical grade hESC lines in which human leukocyte antigen (HLA) haplotypes match a significant proportion of the population. Using whole genome amplification followed by medium resolution HLA typing using PCR amplification with sequence-specific primers (PCR-SSP), we have typed the parents, embryos and hESC lines from three families as well as our eight clinical grade hESC lines and shown that this technical approach is rapid, reliable and accurate. By employing this pre-derivation strategy where, based on HLA match, embryos are selected for a GMP route on day 3–4 of development, we would have drastically reduced our cGMP laboratory running costs.

An Efficient, Economical Slow-Freezing Method for Large-Scale Human Embryonic Stem Cell Banking

Human embryonic stem cells (hESCs) are one of the most interesting cell types for tissue engineering, cell therapy, basic scientific research, and drug screening. Fast advancement in these areas requires the availability of large amounts of safe and well-characterized hESCs from hESC banks. Therefore, optimized freezing protocols, allowing the cryopreservation of large amounts of hESC without direct contact with liquid nitrogen, need to be established. In this study, 6 different cryoprotector combinations [dimethylsulfoxide (DMSO), ethylene glycol, and hydroxyethylstarch (HES)] combined with 2 different application methods were screened with the VUB01 cell line, to establish a new slow-freezing protocol with high recovery rates and a good expansion capacity. Our best conditions were confirmed in 4 other hESC lines: H1, H9, 181, and UGent2. To our knowledge, this is the first time that HES is evaluated as a cryoprotector for hESCs. The use of 5% DMSO+5% HES combined with a new detachment protocol leads to efficient hESC cryopreservation. This protocol involves treating the hESC colonies with cell dissociation solution, a mild dissociation solution uncommonly used for hESC culture. A recovery ratio ranging from 45.5% to 168.2% was obtained, and these were significantly different from the other tested conditions (Student's t-test, P<0.05). The cryopreserved hESCs were morphologically comparable to control cells, exhibited a good expansion profile, were positive for pluripotent expression markers, and could still differentiate into the 3 germ layers. This new protocol allows efficient and economical hESC cryopreservation, ideal for hESC banking.

Banking on [Pluri] Potential

Wrestling with issues of provenance, pluripotency, and performance, stem cell banks do more than just freeze and ship cell vials.

Governing stem cell banks and registries: Emerging Issues

The expansion of national and international research efforts in stem cell research is increasingly paired with the trend of establishing stem cell banks and registries. In jurisdictions crossing the spectrum of restrictive to liberal stem cell policies, banks and registries are emerging as an essential resource for transnational access to quality-controlled and ethically sourced stem cell lines. In this study, we report the preliminary findings of a survey of stem cell banks participating in the International Stem Cell Forum's International Stem Cell Banking Initiative (ISCBI). The questionnaire circulated to all ISCBI members addressed both general issues surrounding research policies (e.g., national policies regulating the permissibility of conducting embryonic stem cell research (hESCR)) and, more specifically, issues relating to the governance of stem cell banking projects. The results of the questionnaire were complemented by scholarly research conducted by the authors. This article provides an overview of the current international hESC banking landscape (I). For this purpose, the policy and governance approaches adopted in the surveyed stem cell banks at the national level will be analyzed and areas of convergence and variance will be identified (II). It is beyond the scope of this paper to provide a comprehensive analysis of the wide range of possible governance approaches, policy responses, and their implications. However, we want to provide a starting point for discussion surrounding key questions and challenges as concerns provenance, access, and deposit of hESC lines (III). Finally, while our analysis is focused on research grade hESCs, the lessons to be gleaned from this examination will encourage further thought, analysis, and research into the issues raised in the banking and governance of other sources of stem cell lines (e.g., SCNT, parthenogenesis, iPs) (IV).

Centralized Banks for Human Embryonic Stem Cells: A Worthwhile Challenge

Centralized banking of human embryonic stem (hES) cells is an endeavor that can benefit individual research efforts and enhance international collaboration but is complicated by the fact that the science is rapidly evolving in an environment of heterogeneous laws, guidelines, and ethical standards. Written from the vantage point of regulatory professionals, this article provides an overview of the benefits of and challenges facing hESC banking enterprises in general with a focus on a global centralized banking effort.

A highly homozygous and parthenogenetic human embryonic stem cell line derived from a one-pronuclear oocyte following in vitro fertilization procedure

Homozygous human embryonic stem cells (hESCs) are thought to be better cell sources for hESC banking because their human leukocyte antigen (HLA) haplotype would strongly increase the degree of matching for certain populations with relatively smaller cohorts of cell lines. Homozygous hESCs can be generated from parthenogenetic embryos, but only heterozygous hESCs have been established using the current strategy to artificially activate the oocyte without second polar body extrusion. Here we report the first successful derivation of a human homozygous ESC line (chHES-32) from a one-pronuclear oocyte following routine in vitro fertilization treatment. chHES-32 cells express common markers and genes with normal hESCs. They have been propagated in an undifferentiated state for more than a year (>P50) and have maintained a stable karyotype of 46, XX. When differentiated in vivo and in vitro, chHES-32 cells can form derivatives from all three embryonic germ layers. The almost undetectable expression of five paternally expressed imprinted genes and their HLA genotype identical to the oocyte donor indicated their parthenogenetic origin. Using genome-wide single-nucleotide polymorphism analysis and DNA fingerprinting, the homozygosity of chHES-32 cells was further confirmed. The results indicated that 'unwanted' one-pronuclear oocytes might be a potential source for human homozygous and parthenogenetic ESCs, and suggested an alternative strategy for obtaining homozygous hESC lines from parthenogenetic haploid oocytes.

Establishing Standards for the Characterization of Human Embryonic Stem Cell Lines

As of August 2005, 22 human embryonic stem cell (hESC) lines listed on the National Institutes of Health (NIH) hESC Registry were being distributed to investigators. At a June 2005 meeting of NIH-supported hESC researchers, we proposed that a set of shared standards should be available in order to characterize the cells unambiguously in multiple laboratories. Here, we elaborate such a plan to identify a set of standard methods and to initiate collaborative efforts to validate the standards. The standard assays we propose should be comprehensive enough to ensure that hESC banks can provide a consistent and reliable product for NIH researchers, and inexpensive enough that individual laboratories can afford to use at least some of the methods routinely in their laboratories. We expect that as data accumulate and standards evolve, a core set of tests will become the norm for routine assessment of hESC cultures and that these tests will lay the groundwork for clinical applications of these cells.

An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells.

Human embryonic stem cells (hESCs) promise to revolutionize reparative medicine through their potential in developing cell replacement therapies for diseases like diabetes and parkinsonism. Most of the existing hESC lines available for research, including all National Institutes of Health-registered lines, have been derived and maintained on mouse embryonic fibroblast feeders in the presence of xenoproteins. For future clinical application, many more hESC lines derived and grown in current good manufacturing practice, good tissue culture practice, and xeno-free conditions need to be developed. Concurrently, effective cryopreservation methods that prevent or limit the accidental contact of hESCs with nonsterile liquid nitrogen during periods of long-term storage have to be formulated. We describe a safe, xeno-free cryopreservation protocol for hESCs involving vitrification in closed sealed straws using human serum albumin as opposed to fetal calf serum as the main protein source in the cryoprotectant and long-term storage in the vapor phase of liquid nitrogen. After thaw, hESCs exhibited high thaw-survival rates and low differentiation rates, remained pluripotent, and maintained normal diploid karyotypes throughout extended passage. The cryopreservation technique we describe here should complement xeno-free culture conditions for hESCs already in refinement and will prove very useful for the setting up of hESC banks throughout the world.

Human embryonic stem cells possess immune-privileged properties.

Human embryonic stem cells (hESCs) are envisioned to be a major source for cell-based therapies. Efforts to overcome rejection of hESCs include nuclear transfer and collection of hESC banks representing the broadest diversity of major histocompatability complex (MHC) polymorphorisms. Surprisingly, immune responses to hESCs have yet to be experimentally evaluated. Here, injection of hESCs into immune-competent mice was unable to induce an immune response. Undifferentiated and differentiated hESCs failed to stimulate proliferation of alloreactive primary human T cells and inhibited third-party allogeneic dendritic cell-mediated T-cell proliferation via cellular mechanisms independent of secreted factors. Upon secondary rechallenge, T cells cocultured with hESCs were still responsive to allogeneic stimulators but failed to proliferate upon re-exposure to hESCs. Our study demonstrates that hESCs possess unique immune-privileged characteristics and provides an unprecedented opportunity to further investigate the mechanisms of immune response to transplantation of hESCs that may avoid immune-mediated rejection.