Creation of an open-access, mutation-defined fibroblast resource for neurological disease research

Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community.     

SNP panel identification assay (SPIA): a genetic-based assay for the identification of cell lines

Translational research hinges on the ability to make observations in model systems and to implement those findings into clinical applications, such as the development of diagnostic tools or targeted therapeutics. Tumor cell lines are commonly used to model carcinogenesis. The same tumor cell line can be simultaneously studied in multiple research laboratories throughout the world, theoretically generating results that are directly comparable. One important assumption in this paradigm is that researchers are working with the same cells. However, recent work using high throughput genomic analyses questions the accuracy of this assumption. Observations by our group and others suggest that experiments reported in the scientific literature may contain pre-analytic errors due to inaccurate identities of the cell lines employed. To address this problem, we developed a simple approach that enables an accurate determination of cell line identity by genotyping 34 single nucleotide polymorphisms (SNPs). Here, we describe the empirical development of a SNP panel identification assay (SPIA) compatible with routine use in the laboratory setting to ensure the identity of tumor cell lines and human tumor samples throughout the course of long term research use.     

Derivation, characterization, differentiation, and registration of seven human embryonic stem cell lines (VAL-3, -4, -5, -6M, -7, -8, and -9) on human feeder

Derivation of human embryonic stem cell lines has been a remarkable scientific achievement during the last decade. Human embryonic stem cells are regarded as an unlimited cell source for replacement therapy in regenerative medicine. Clearly, the scientific community requires proper derivation, characterization, and registration with the purpose of making them available for research and future medical applications worldwide. In this paper, we report our derivation work as the Valencian Node of the Spanish Stem Cell Bank in the generation, characterization, and registration of VAL-3, -4, -5, -6M, -7, -8, and 9 (www.isciii/htdocs/terapia/terapia_bancocelular.jsp). The derivation process was performed on microbiologically tested and irradiated human foreskin fibroblasts and designed to minimize contact with xeno-components in knockout Dulbecco’s modified Eagle’s medium supplemented with knockout serum replacement and basic fibroblast growth factor. Fingerprinting of the cell lines was performed to allow their identification and traceability. All lines were expressed at the mRNA and specific protein markers for undifferentiation and were found to be negative for classical differentiation markers such as neurofilament heavy chain (ectoderm), renin (mesoderm), and amylase (endoderm). All lines displayed high levels of telomerase activity and were shown to successfully overcome cryopreservation and thawing. Finally, we demonstrated the potential to differentiate in vitro (embryoid body formation) and in vivo (teratoma formation) into cell types from all three germ layers. Teratoma derived from all human embryonic stem cell lines present similar morphological features except VAL-8 that display more aggressive tumor behavior with a larger proportion of solid tissues, as opposed to cyst formation in the other cell lines.     

Cell lines authentication and <Emphasis Type="Italic">mycoplasma</Emphasis> detection as minimun quality control of cell lines in biobanking

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.     

Role of the embryology laboratory in the human embryonic stem cell line derivation process

With the introduction of regenerative medicine and cell therapy programmes by means of human embryonic stem cells (hESC), several research centres have begun projects of derivation of hESC lines. In some stem cell banks, such as the Andalusian Stem Cell Bank, the law also permits the creation of these cell lines. Therefore, the recovery of cryopreserved embryos, their culture and the subsequent derivation to hESC lines requires a suitable embryology laboratory and specialized and highly qualified staff. Moreover, new techniques, from therapeutic nuclear transfer, need this type of laboratory and staff, too. Several International Associations have drawn up some guidelines for laboratories where embryos are manipulated and they reflect the physical space, the staff and the equipment needed in these kinds of laboratories. Nevertheless, we can see that these guidelines do not distinguish between IVF laboratories and other laboratories that obtain hESC lines, so it would be convenient to make a distinction. Following these guidelines, we have tried to draw up concurrent aspects applicable to areas of embryology within stem cell banks. So, the design and the specific implementation programmes for these areas and other research centres with this area but which do not use IVF techniques is vital to develop embryonic cell lines in optimum conditions for future therapeutic applications, although maybe it is rather premature to standardize this type of research.     

Systematic analysis of the ability of stromal cell lines derived from different murine adult tissues to support maintenance of hematopoietic stem cells in vitro.

Hematopoietic stem cells interact with a complex microenvironment both in vivo and in vitro. In association with this microenvironment, murine stem cells are maintained in vitro for several months. Fibroblast-like stromal cells appear to be important components of the microenvironment, since several laboratories have demonstrated that cloned stromal cell lines support hematopoiesis in vitro. The importance of the tissue of origin of such cell lines remains unknown, since systematic generation of stromal cell lines from adult tissues has never been accomplished. In addition, the capacity of stromal cell lines to support reconstituting stem cell has not been examined. We have previously described an efficient and rapid method for the immortalization of primary bone marrow stromal cell lines (Williams et al., Mol. Cell. Biol. 8:3864-3871, 1988) which can be used to systematically derive cell lines from multiple tissues of the adult mouse. Here we report the immortalization of primary murine lung, kidney, skin, and bone marrow stromal cells using a recombinant retrovirus vector (U19-5) containing the simian virus large T antigen (SV40 LT) and the neophosphotransferase gene. The interaction of these stromal cells with factor-dependent cells Patterson-Mix (FDCP-Mix), colony forming units-spleen (CFU-S), and reconstituting hematopoietic stem cells was studied in order to analyze the ability of such lines to support multipotent stem cells in vitro. These studies revealed that stromal cell lines from these diverse tissues were morphologically and phenotypically similar and that they quantitatively bound CFU-S and FDCP-Mix cells equally well. However, only those cell lines derived from bone marrow-supported maintenance of day 12 CFU-S in vitro. One lung-derived stromal cell line, ULU-3, supported the survival of day 8 CFU-S, but not the more primitive CFU-S12. A bone marrow-derived stromal cell line, U2, supported the survival of long-term reconstituting stem cells for up to 3 weeks in vitro as assayed by reconstitution 1 year post-transplant. These studies suggest that adherence of HSC to stromal cells is necessary but not sufficient for maintenance of these stem cell populations and that bone marrow provides specific signals relating to hematopoietic stem cell survival and proliferation.     

Establishment and characterization of human embryonic stem cell lines, Turkey perspectives

Human embryonic stem cells (hESC), which are derived from the inner cell mass (ICM) of blastocyst stage embryos, are of great importance because of their unpredictable two unique features: their differentiation ability into all types of cells derived from three germ layers and their potentially unlimited capacity of self renewing with stable karyotype. These distinguished properties make hESC very promising cell source for regenerative medicine, tissue replacement therapies, and drug screening studies as well as genomics. However, due to the several technical problems, such as risk of teratoma formation, immune response, and unknown genetic pathways for lineage specific differentiation, and ethical drawbacks of their using in clinical treatments, hESC researches are still waiting to advance beyond to animal trials and drug studies. During the last decade, more than 300 new hESC lines have been derived and published by researchers worldwide. However, despite their similar well-known unique properties, recent studies reported that hESC lines have very individual properties and are differed from each other with regards to their differentiation ability and gene expression profiles. Therefore, all hESC lines should be characterized in detail and then registered in a stem cell bank for generating global database. In this report, the characteristic of hESC lines, which were established in Istanbul Memorial Hospital between 2003 and 2005, and derivation methods were described in detail to inform researchers and to facilitate new prospective cooperative studies.     

Stem cell plasticity, cell fusion, and transdifferentiation

One of the most contentious issues in biology today concerns the existence of stem cell plasticity. The term "plasticity" refers to the capacity of tissue-derived stem cells to exhibit a phenotypic potential that extends beyond the differentiated cell phenotypes of their resident tissue. Although evidence of stem cell plasticity has been reported by multiple laboratories, other scientists have not found the data persuasive and have remained skeptical about these new findings. This review will provide an overview of the stem cell plasticity controversy. We will examine many of the major objections that have been made to challenge the stem cell plasticity data. This controversy will be placed in the context of the traditional view of stem cell potential and cell phenotypic diversification. What the implications of cell plasticity are, and how its existence may modulate our present understanding of stem cell biology, will be explored. In addition, we will examine a topic that is usually not included within a discussion of stem cell biology--the direct conversion of one differentiated cell type into another. We believe that these observations on the transdifferentiation of differentiated cells have direct bearing on the issue of stem cell plasticity, and may provide insights into how cell phenotypic diversification is realized in the adult and into the origin of cell phenotypes during evolution.     

小鼠胚胎干细胞建系技术研究进展

目前,对小鼠胚胎干细胞的研究较为深入,并已成为研究细胞分化及信号转导、新基因发现及功能鉴定、器官发生、人类疾病和药物开发等的有效手段。胚胎干细胞建系是一项基础性工作。虽然技术日趋成熟,有些品系小鼠的胚胎干细胞建系已是常规技术,但不同品系小鼠胚胎干细胞的建系效率仍有很大差异,建系途径和方法各有特点,一个品系胚胎干细胞的建系方法不一定都适用于其他品系。本文从小鼠胚胎干细胞建系的途径、分离操作技术、培养体系等方面进行综述,并就与之相关的有些问题提出思考和对策。     

Cell line cross-contamination: How aware are mammalian cell culturists of the problem and how to monitor it?

HeLa was the first human cell line established (1952) and became one of the most frequently used lines because of its hardiness and rapid growth rate. During the next two decades, the development of other human cell lines mushroomed. One reason for this became apparent during the 1970s, when it was demonstrated that many of these cell lines had been overgrown and replaced by fast-growing HeLa cells inadvertently introduced into the original cultures. Although the discovery of these "HeLa contaminants" prompted immediate alarm, how aware are cell culturists today of the threat of cell line cross-contamination? To answer this question, we performed a literature search and conducted a survey of 483 mammalian cell culturists to determine how many were using HeLa contaminants without being aware of their true identity and how many were not using available means to ensure correct identity. Survey respondents included scientists, staff, and graduate students in 48 countries. HeLa cells were used by 32% and HeLa contaminants by 9% of survey respondents. Most were also using other cell lines; yet, only about a third of respondents were testing their lines for cell identity. Of all the cell lines used, 35% had been obtained from another laboratory instead of from a repository, thus increasing the risk of false identity. Over 220 publications were found in the PubMed database (1969-2004) in which HeLa contaminants were used as a model for the tissue type of the original cell line. Overall, the results of this study indicate a lack of vigilance in cell acquisition and identity testing. Some researchers are still using HeLa contaminants without apparent awareness of their true identity. The consequences of cell line cross-contamination can be spurious scientific conclusions; its prevention can save time, resources, and scientific reputations.     

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.     

Generation of reference cell lines,media, and a process platform for CHO cell biomanufacturing

Due to the favorable attributes of Chinese hamster ovary (CHO) cells for therapeutic proteins and antibodies biomanufacturing, companies generate proprietary cells with desirable phenotypes. One key attribute is the ability to stably express multi-gram per liter titers in chemically defined media. Cell, media, and feed diversity has limited community efforts to translate knowledge. Moreover, academic, and nonprofit researchers generally cannot study “industrially relevant” CHO cells due to limited public availability, and the time and knowledge required to generate such cells. To address these issues, a university-industrial consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) has acquired two CHO “reference cell lines” from different lineages that express monoclonal antibodies. These reference cell lines have relevant production titers, key performance outcomes confirmed by multiple laboratories, and a detailed technology transfer protocol. In commercial media, titers over 2 g/L are reached. Fed-batch cultivation data from shake flask and scaled-down bioreactors is presented. Using productivity as the primary attribute, two academic sites aligned with tight reproducibility at each site. Further, a chemically defined media formulation was developed and evaluated in parallel to the commercial media. The goal of this work is to provide a universal, industrially relevant CHO culture platform to accelerate biomanufacturing innovation.     

Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells

The past few years have seen remarkable progress in our understanding of embryonic stem cell (ES cell) biology. The necessity of examining human ES cells in culture, coupled with the wealth of genomic data and the multiplicity of cell lines available, has enabled researchers to identify critical conserved pathways regulating self-renewal and identify markers that tightly correlate with the ES cell state. Comparison across species has suggested additional pathways likely to be important in long-term self-renewal of ES cells including heterochronic genes, microRNAs, genes involved in telomeric regulation, and polycomb repressors. In this review, we have discussed information on molecules known to be important in ES cell self-renewal or blastocyst development and highlighted known differences between mouse and human ES cells. We suggest that several additional pathways required for self-renewal remain to be discovered and these likely include genes involved in antisense regulation, microRNAs, as well as additional global repressive pathways and novel genes. We suggest that cross species comparisons using large-scale genomic analysis tools are likely to reveal conserved and divergent paths required for ES cell self-renewal and will allow us to derive ES lines from species and strains where this has been difficult.     

Invitromatics,invitrome, and invitroomics: introduction of three new terms for in vitro biology and illustration of their use with the cell lines from rainbow trout

The literature on cell lines that have been developed from rainbow trout (RT) (Oncorhynchus mykiss) is reviewed to illustrate three new terms: invitromatics, invitrome, and invitroomics. Invitromatics is defined as the history, development, characterization, engineering, storage, and sharing of cell lines. RT invitromatics differs from invitromatics for humans and other mammals in several ways. Nearly all the RT cell lines have developed through spontaneous immortalization. No RT cell line undergoes senescence and can be described as being finite, whereas many human cell lines undergo senescence and are finite. RT cell lines are routinely grown at 18–22°C in free gas exchange with air in basal media developed for mammalian cells together with a supplement of fetal bovine serum. An invitrome is defined as the grouping of cell lines around a theme or category. The broad theme in this article is all the cell lines that have ever been created from O. mykiss, or in other words, the RT invitrome. The RT invitrome consists of approximately 55 cell lines. These cell lines can also be categorized on the basis of their storage and availability. A curated invitrome constitutes all the cell lines in a repository and for RT consists of 11 cell lines. These consist of epithelial cell lines, such as RTgill-W1, and fibroblast cell lines, such as RTG-2. RTG-2 can be purchased from a scientific company and constitutes the commercial RT invitrome. Cell lines that are exchanged between researchers are termed the informally shared invitrome and for RT consists of over 35 cell lines. Among these is the monocyte/macrophage cell line, RTS11. Cell lines whose existence is in doubt are termed the zombie invitrome, and for RT, approximately 12 cell lines are zombies. Invitroomics is the application of cell lines to a scientific problem or discipline. This is illustrated with the use of the RT invitrome in virology. Of the RT invitrome, RTG-2 was the most commonly used cell line to isolate viruses. Fifteen families of viruses were studied with RT invitrome. RT cell lines were best able to support replication of viruses from the Herpesviridae, Iridoviridae, Birnaviridae, Togaviridae, and Rhabdoviridae families.     

Embryonic liver cells and permanent lines as models for hepatocyte and bile duct cell differentiation

Analysis of liver cells during development is facilitated by the possibility of complementing in vivo analysis with experiments on cultured cells. In this review, we discuss results from several laboratories concerning bipotential hepatic stem cells from mouse (HBC-3, H-CFU-C, MMH and BMEL), rat (rhe14321) and primate (IPFLS) embryos. Several groups have used fluorescence-activated cell sorting to identify clonogenic bipotential cells; others have derived bipotential cell lines by plating liver cell suspensions and cloning. The bipotential cells, which probably originate from hepatoblasts, can differentiate as hepatocytes or bile duct cells, and undergo morphogenesis in culture. Disparities in differentiation can be explained by distinct medium compositions, extracellular matrix coated culture surfaces, and gene expression detection methods. Potential applications of these cell lines are discussed.     

Derivation of 30 human embryonic stem cell lines—improving the quality

We have derived 30 human embryonic stem cell lines from supernumerary blastocysts in our laboratory. During the derivation process, we have studied new and safe method to establish good quality lines. All our human embryonic stem cell lines have been derived using human foreskin fibroblasts as feeder cells. The 26 more recent lines were derived in a medium containing serum replacement instead of fetal calf serum. Mechanical isolation of the inner cell mass using flexible metal needles was used in deriving the 10 latest lines. The lines are karyotypically normal, but culture adaptation in two lines has been observed. Our human embryonic stem cell lines are banked, and they are available for researchers.     

Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product-free environment

The protocols described here are comprehensive instructions for deriving human embryonic stem (hES) cell lines in xeno-free conditions from cryopreserved embryos. Details are included for propagation, cryopreservation and characterization. Initial derivation is on feeder cells and is followed by adaptation to a feeder-free environment; competent technicians can perform these simplified methods easily. From derivation to cryopreservation of fully characterized initial stocks takes 3-4 months. These protocols served as the basis for standard operating procedures (SOPs), with both operational and technical components, that we set to meet good manufacturing practice (GMP) and UK regulatory body requirements for derivation of clinical-grade cells. As such, these SOPs are currently used in our current GMP compliant facility to derive hES cell lines ab initio, in an animal product-free environment; these lines are suitable for research and potentially for clinical use in cell therapy. So far, we have derived eight clinical-grade lines, which will be freely available to the scientific community after submission/accession to the UK Stem Cell Bank.     

The untapped potential of the GENSAT mice—A valuable resource for developmental biology

Gene Expression Nervous System Atlas (GENSAT) transgenic mice express EGFP, tdTomato, or Cre recombinase in a wide range of cell types. The mice and the bacterial artificial chromosome transgenes are available from repositories (MMRRC or CHORI), thereby making these resources readily available to the research community. This resource of 1,386 transgenic lines was developed and validated for neuroscience research. However, GENSAT mice have many potential applications in other contexts including studies of development outside of the CNS. The cell type‐specific expression of fluorescent proteins in these mice has been used to identify cells in living embryos, in living embryo explants, and in stem or progenitor cell populations in postnatal tissues. The large number of fluorescent protein driver lines generated by GENSAT greatly expands the range of cell type markers that can be used for live cell sorting. In addition, the GENSAT project has generated 278 new Cre driver lines. This review provides an overview of the GENSAT lines and information for identifying lines that may be useful for a particular application. I also provide a review of the few published cases in which GENSAT mice have been used for studies of embryonic development or analysis of stem/progenitor cells in nonneural tissues. genesis 54:245–256, 2016. © 2016 Wiley Periodicals, Inc.     

Stem cell colloquy: conclusion

The stem cell data presented and discussed during the symposium raise the hope that important medical progress can be made in several fields: neuro-degenerative diseases, those linked to cellular deficit, some aspects of aging linked to cellular degeneration, and the treatment of cancers that may harm normal tissues at risk of being infiltrated by malignant cells. Three main types of stem cells are available. (i) Those present in normal adult tissue: contrary to what was believed, some data suggest that certain adult stem cells have a great plasticity (they can differentiate into cells different from those in tissues from which they were taken) and can proliferate in vitro without losing their properties. Nevertheless, their use faces several obstacles: in ill or elderly subjects, then these cells can be limited in number or not multiply well in vitro. In this case, auto-grafting of the cells cannot be used. They must be sought in another subject, and allo-grafting causes difficult and sometimes insoluble problems of immunological tolerance. (ii) Embryonic stem cells from surplus human embryos, obtained by in vitro fertilisation, which the parents decide not to use: these cells have a great potential for proliferation and differentiation, but can also encounter problems of immunological intolerance. (iii) Cells obtained from cell nuclear transfer in oocytes: these cells are well tolerated, since they are genetically and immunologically identical to those of the host. All types of stem cells can be obtained with them. However, they do present problems. For obtaining them, female oocytes are needed, which could lead to their commercialization. Moreover, the first steps for obtaining these cells are identical to those used in reproductive cloning. It therefore appears that each type of cell raises difficult scientific and practical problems. More research is needed to overcome these obstacles and to determine which type of stem cell constitutes the best solution for each type of disease and each patient. There are three main ethical problems: (a) to avoid the commercialization of stem cells and oocytes (this can be managed through strict regulations and the supervision of authorized laboratories); (b) to avoid that human embryos be considered as a mere means to an end (they should only be used after obtaining the informed consent of the parents; the conditions of their use must be well defined and research programs must be authorized); (c) to avoid that research on stem cell therapy using cell nuclear replacement opens the way to reproductive cloning (not only should reproductive cloning be firmly forbidden but authorization for cell nuclear transfer should be limited to a small number of laboratories). Overall, it appears that solutions can be found for administrative and ethical problems. Harmonisation of international regulations would be desirable in this respect, in allowing at the same time each country to be responsible for its regulations. A last ethical rule should be implemented, not to give patients and their families false hopes. The scientific and medical problems are many, and the solutions will be long and difficult to find. Regenerative medicine opens important avenues for research, but medical progress will be slow.