culturing-human-pluripotent-neural-stem-cells-an-enclosed-cell

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Human pluripotent stem cells have the unique properties of being able to proliferate indefinitely in their undifferentiated state and to differentiate into any somatic cell type. These cells are thus posited to be extremely useful for furthering our understanding of both normal and abnormal human development, providing a human cell preparation that can be used to screen for new reagents or therapeutic agents, and generating large numbers of differentiated cells that can be used for transplantation purposes. Critical among the applications for the latter are diseases and injuries of the nervous system, medical approaches to which have been, to date, primarily palliative in nature. Differentiation of human pluripotent stem cells into cells of the neural lineage, therefore, has become a central focus of a number of laboratories. This has resulted in the description in the literature of several dozen methods for neural cell differentiation from human pluripotent stem cells. Among these are methods for the generation of such divergent neural cells as dopaminergic neurons, retinal neurons, ventral motoneurons, and oligodendroglial progenitors. In this review, we attempt to fully describe most of these methods, breaking them down into five basic subdivisions: (1) starting material, (2) induction of loss of pluripotency, (3) neural induction, (4) neural maintenance and expansion, and (5) neuronal/glial differentiation. We also show data supporting the concept that undifferentiated human pluripotent stem cells appear to have an innate neural differentiation potential. In addition, we evaluate data comparing and contrasting neural stem cells differentiated from human pluripotent stem cells with those derived directly from the human brain.

Differentiation of neural lineage cells from human pluripotent stem cells.

Menschliche pluripotenten Stammzellen haben die einzigartigen Eigenschaften des Seins auf unbestimmte Zeit in ihrem undifferenzierte vermehren und in jede Art von somatischen Zellen zu unterscheiden. Diese Zellen sind daher &auml;u&szlig;erst n&uuml;tzlich zur F&ouml;rderung unseres Verst&auml;ndnisses sowohl normalen und abnormalen menschlichen Entwicklung, Bereitstellung einer menschlichen Zelle-Zubereitung, die benutzt werden kann, postuliert Bildschirm f&uuml;r neue Reagenzien oder Therapeutika und Generierung zahlreicher differenzierte Zellen, die Transplantation Zwecken verwendet werden k&ouml;nnen. Kritisch zu den Anwendungen f&uuml;r Letztere sind Erkrankungen und Verletzungen des Nervensystems, medizinische, die Ans&auml;tze, denen bis dato vor allem palliative in der Natur gewesen. Differenzierung des menschlichen pluripotenten Stammzellen in Zellen der neuronalen Abstammung, ist daher ein zentraler Schwerpunkt Forschungsinstitute geworden. Dies f&uuml;hrte in der Beschreibung in der Literatur von mehreren Dutzend Methoden f&uuml;r neuronale Zelldifferenzierung von menschlichen pluripotenten Stammzellen. Dazu geh&ouml;ren Methoden f&uuml;r die Generierung von solchen abweichenden neuralen Zellen als dopaminergen Neuronen, Netzhaut Neuronen, ventral Motoneurons und oligodendroglial Vorl&auml;uferzellen. In diesem Bericht, versuchen wir, die meisten dieser Methoden, brechen sie in f&uuml;nf grundlegende Teilbereiche vollst&auml;ndig zu beschreiben: (1) ab Material und Induktion (2) der Verlust der Pluripotenz, (3) Neuronale Induktion, (4) Neuronale Wartung und Erweiterung (5) Neuronale/Gliazellen Differenzierung. Wir zeigen auch Daten zur Unterst&uuml;tzung des Konzepts, das undifferenzierte menschlichen pluripotenten Stammzellen scheinen eine angeborene neuronale Differenzierung Potenzial zu haben. Dar&uuml;ber hinaus Werten wir Daten vergleichen und kontrastierenden neuralen Stammzellen von menschlichen pluripotenten Stammzellen unterschieden, mit denen das menschliche Gehirn direkt abgeleitet.

Differenzierung der neuronalen Linie Zellen aus menschlichen pluripotenten Stammzellen.

Culturing human embryonic stem cells (hESCs) requires a significant commitment of time and resources. It takes weeks to establish a culture, and the cultures require daily attention. Once hESC cultures are established, they can, with skill and the methods described, be kept in continuous culture for many years. hESC lines were originally derived using very similar culture medium and conditions as those developed for the derivation and culture of mouse ESC lines. However, these methods were suboptimal for hESCs and have evolved considerably in the years since the first hESC lines were derived. Compared with mouse ESCs, hESCs are very difficult to culture - they grow slowly, and most importantly, since we have no equivalent assays for germline competence, we cannot assume that the cells that we have in our culture dishes are either stable or pluripotent. This makes it far more critical to assay the cells frequently using the characterization methods, such as karyotyping, immunocytochemistry, gene expression analysis, and flow cytometry, provided in this manual.

Traditional human embryonic stem cell culture.

This protocol describes the culture of human pluripotent stem cells (PSCs) under feeder-free conditions in a commercially available, chemically defined, growth medium, using Matrigel as a substrate and the enzyme solution Accutase for single-cell passaging. This system is strikingly different from traditional PSC culture, where the cells are co-cultured with feeder cells and in medium containing serum replacement. PSCs cultured in this new system have a different morphology than those cultured on feeder cells but retain their characteristic pluripotency. This feeder-free PSC culture system is conceptually similar to feeder-free systems that use mouse embryonic fibroblast (MEF)-conditioned medium (MEF-CM) and Matrigel substratum. Instead of MEF-CM, a very complex and undefined medium, this new system uses StemPro SFM, a chemically defined medium that permits enzymatic passaging with Accutase to disaggregate the colonies into single cells. Accutase passaging has been used in conjunction with Stempro in our hands for 20+ passages without detectable karyotypic abnormalities. We will also review techniques for adapting cultures previously grown on MEFs, routine passaging of the cells, and cryopreservation.

Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging.

Embryoid body (EB) formation is a traditional method of inducing differentiation of pluripotent stem cells (PSCs). It is a routine in vitro test of pluripotency as well as the first stage in many differentiation protocols targeted toward the production of a specific lineage or cellular population, as in neural differentiation (see Chapters 29 and 30). The induction of differentiation via EB formation is fairly straightforward. However, depending on the specific PSC culture conditions - substrate, feeders, medium, and eventual cell type of interest - various methods are applied in order to most routinely obtain healthy EB cultures.

The generation of embryoid bodies from feeder-based or feeder-free human pluripotent stem cell cultures.

Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials.

Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling.

The autism spectrum disorders (ASDs) comprise a set of neurodevelopmental disorders that are, at best, poorly understood but are the fastest growing developmental disorders in the United States. Because animal models of polygenic disorders such as the ASDs are difficult to validate, the derivation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming offers an alternative strategy for identifying the cellular mechanisms contributing to ASDs and the development of new treatment options. Access to statistically relevant numbers of ASD patient cell lines, however, is still a limiting factor for the field. We describe a new resource with more than 200 cell lines (fibroblasts, iPSC clones, neural stem cells, glia) from unaffected volunteers and patients with a wide range of clinical ASD diagnoses, including fragile X syndrome. We have shown that both normal and ASD-specific iPSCs can be differentiated toward a neural stem cell phenotype and terminally differentiated into action-potential firing neurons and glia. The ability to evaluate and compare data from a number of different cell lines will facilitate greater insight into the cause or causes and biology of the ASDs and will be extremely useful for uncovering new therapeutic and diagnostic targets. Some drug treatments have already shown promise in reversing the neurobiological abnormalities in iPSC-based models of ASD-associated diseases. The ASD Stem Cell Resource at the Children's Hospital of Orange County will continue expanding its collection and make all lines available on request with the goal of advancing the use of ASD patient cells as disease models by the scientific community.

The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery.

Mucopolysaccharidosis type I (MPS I) is an inherited α-L-iduronidase (IDUA, I) deficiency in which glycosaminoglycan (GAG) accumulation causes progressive multisystem organ dysfunction, neurological impairment, and death. Current MPS I mouse models, based on a NOD/SCID (NS) background, are short-lived, providing a very narrow window to assess the long-term efficacy of therapeutic interventions. They also develop thymic lymphomas, making the assessment of potential tumorigenicity of human stem cell transplantation problematic. We therefore developed a new MPS I model based on a NOD/SCID/Il2rγ (NSG) background. This model lives longer than 1 year and is tumor-free during that time. NSG MPS I (NSGI) mice exhibit the typical phenotypic features of MPS I including coarsened fur and facial features, reduced/abnormal gait, kyphosis, and corneal clouding. IDUA is undetectable in all tissues examined while GAG levels are dramatically higher in most tissues. NSGI brain shows a significant inflammatory response and prominent gliosis. Neurological MPS I manifestations are evidenced by impaired performance in behavioral tests. Human neural and hematopoietic stem cells were found to readily engraft, with human cells detectable for at least 1 year posttransplantation. This new MPS I model is thus suitable for preclinical testing of novel pluripotent stem cell-based therapy approaches.

Article	Year
Differentiation of neural lineage cells from human pluripotent stem cells. Methods (San Diego, Calif.)\| PubMed ID: 18593611	2008
Traditional human embryonic stem cell culture. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 21822870	2011
Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 21822872	2011
The generation of embryoid bodies from feeder-based or feeder-free human pluripotent stem cell cultures. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 21822890	2011
Process-based expansion and neural differentiation of human pluripotent stem cells for transplantation and disease modeling. Journal of neuroscience research\| PubMed ID: 23893392	2013
The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery. Stem cells translational medicine\| PubMed ID: 25273538	2014
A novel, long-lived, and highly engraftable immunodeficient mouse model of mucopolysaccharidosis type I. Molecular therapy. Methods & clinical development\| PubMed ID: 26052536	2015
The Autism Spectrum Disorders Stem Cell Resource at Children's Hospital of Orange County: Implications for Disease Modeling and Drug Discovery. Stem cells translational medicine\| PubMed ID: 26508786	2015

Alexander E. Stover

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