This protocol allows efficient generation of integration-free iPS cells from a small amount of peripheral blood (<1 ml). Peripheral blood mononuclear cells (PBMCs) are cultured to expand the erythroblast (EB) population. They are then used to derive iPS cells using four recombinant Sendai viral vectors (CytotuneTM, Life Technologies), expressing the four reprogramming factors Oct4, Sox2, Kfl4 and c-Myc.
The directed differentiation of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) into hepatocytes could facilitate a rational study of the molecular mechanisms underlying human liver development as well as provide a renewable source of exogenous hepatocytes for drug toxicity testing and cell-based therapeutics. Moreover, if hepatocytes were produced from hiPSCs originating from patients with inborn errors of hepatic metabolism, such cells could be used for modeling liver disease. Here we present a step-wise protocol for efficiently and reproducibly inducing the differentiation of hepatocytes from human pluripotent stem cells under highly defined conditions.
Prolonged culture of pluripotent and multipotent stem cells exposes the cells to strong selection pressures, often resulting in genomic alterations. Any genetic manipulation of the cells may further jeopardize their genomic stability. Genomic aberrations affect the differentiation capacity of stem cells, their stem cell identity and their tumorigenicity, and should thus be routinely evaluated for their proper use in basic research and in clinical trials. Here we review the common methods currently available to analyze the genomic integrity of stem cells, and present a recently developed method for the evaluation of the genomic integrity of stem cells by their gene expression profiles. We describe the principles of this method, provide guidelines for its implementation, and discuss its advantages and limitations compared to other available methods. Introduction: Genomic instability in stem cells Stem cells grown in culture are exposed to strong selection pressures that often results in genomic alterations, varying in size from point mutations, through copy number changes in small genomic elements (e.g. amplification of repetitive sequences and retroelement mobility), to large chromosomal aberrations, trisomies and monosomies (Lefort et al., 2009). While these aberrations are assumed to occur randomly, only those that confer a selective advantage would prevail and ultimately take over the culture. Different culturing conditions, such as media composition, cell passaging techniques, and freeze-thaw cycles, may affect the nature and the frequency of the acquired aberrations; however, no culturing technique is immune to genomic instability, and it is thus considered, for most practical purposes, a “necessary evil” that does not seriously compromise the utility of aberrant cells in basic science. Stem cells acquire genomic changes throughout their expansion in culture. Much attention has been drawn in recent years to the genomic aberrations acquired by human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), from the resolution of point mutations to the resolution of whole-chromosome trisomies (Baker et al., 2007; Ben-David et al., 2011; Gore et al., 2011; Hussein et al., 2011; Laurent et al., 2011; Mayshar et al., 2010). More recently, we have shown that human adult stem cells that are expanded in culture are also prone to acquire chromosomal aberrations (Ben-David et al., 2011). In both pluripotent and multipotent stem cells, the genomic aberrations are eventually acquired in a large portion of the cell lines, and the aberrant cells can take over the population within very few passages (Ben-David et al., 2011; Mayshar et al., 2010), making the genomic instability of human stem cells a prevalent phenomenon in need of proper evaluation and consideration. Copyright: c © 2012 Uri Ben-David and Nissim Benvenisty. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ∗To whom correspondence should be addressed. E-mail: nissimb@cc.huji.ac.il Last revised March 28, 2012. Published June 10, 2012. This chapter should be cited as: Ben-David, U., and Benvenisty, N., Analyzing the genomic integrity of stem cells (June 10, 2012), StemBook, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook.1.150.1, http://www.stembook.org.
Assessing pluripotency in human cells is inherently an intractable problem. In animal systems, pluripotency can be verified through direct means: pluripotent stem cells can be introduced into an developing embryo and thus the cellular developmental potential of any given in vitro preparation can be directly determined by observing the amount of chimaerism or viability of organisms partially or fully derived from in vitro stem cells (Nagy, Rossant et al., 1993: PMID8378314).
Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the potential to become the source materials for cell-based therapy, so the quality of the stem cells has great impact on how the cells could be utilized in future applications. Derivation and maintenance conditions have critical role determining the iPSC quality, mainly due to the involvement of animal products and feeder cells. We developed a simple procedure to derive and maintain hiPSCs in chemically defined media.
IntrodutionWhen embryonic stem cells are differentiated in the presence of activin A in serum-free conditions, an endoderm progenitor population defined by the coexpression of either Brachyury, Foxa2 and c-Kit, or c-Kit and Cxcr4 is generated.Specification of these progenitors with bone morphogenetic protein-4 in combination with basic fibroblast growth factor and activin A results in the development of hepatic populations highly enriched (45-70%) for cells that express the alpha-fetoprotein and albumin proteins.Protocol 1. Prepare ES cells on matrigel plate to deplete MEFs.a. Prepare 1:3 matrigel plate: cool down tissue culture plate and pipettes on ice; thaw 1:3 matrigel in fridge or on ice.Carefully apply matrigel onto plate to cover the bottom and then suck off excessive matrigel.Keep everything cool when plate matrigel.Put matrigel plates on ice for 30 min.Suck off excessive matrigel again and put matrigel-coated plates in 37 • C incubator for 30 min.b.Treat confluent ES cells with TrypLE for 2-3 min at 37 • C, use 4 ml TrypLE for 10 cm dish or 1 ml for 1 well of 6-well plate, and then suck out TrypLE.Wash with wash media (IMDM + Glu + P/S + 0.1% BSA) for 2-3 times.Add 4 ml hES media for 10 cm dish or 1ml for 1 well of 6-well plate, and scrape ES cells off the bottom of plate with cell scraper.Pipette up and down for 3-5 times and check under microscope until you get 5-10-cell clumps.c.Plate cells onto matrigel-coated plates.Cells will get ready for differentiation in 1 day @ 1:1 split; 2 days @ 1:2 split and 3 days @ 1:3 split.2. Setup T0: Differentiation is performed at 5% O 2 , 5% CO 2 .Cells are ready to go when they are 70-80% confluent.Wash cells twice with wash media and apply appropriate volume of T0 media to cell, such as 10 ml for 10 cm dish, 2 ml for 1well in 6-well dish.3. Wash cells with wash media and change media everyday until T5.It's normal to see huge cell death in the first several days.Detailed differentiation media recipe is attached in the next page.4. To proceed to liver differentiation, dissociate T5 cells with Accutase @ 37 • C for 3 min.Rinse with wash media twice.Add 1 ml wash media to each well of 6-well plate and scrape cells off the dish.Collect T5 cells,
CytoTuneTM-iPS Reprogramming System uses vectors based on replication in competent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. In contrast to many available protocols, which rely on viral vectors that integrate into the genome of the host cell, the CytoTuneTM Reprogramming System uses vectors that are non-integrating and remain in the cytoplasm (i.e., they are zero-footprint). In addition, the host cell can be cleared of the vectors and reprogramming factor genes by exploiting the cytoplasmic nature of SeV and the functional temperature sensitivity mutations introduced into the key viral proteins. The CytoTuneTM-iPS Reprogramming Kit contains four SeV-based reprogramming vectors, each capable of expressing one of the four Yamanaka factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and are optimized for generating iPSCs from human somatic cells. The reprogramming vectors in this kit have been engineered to increase biological and environmental safety.
Over the last decade, with the advent of new techniques and technologies in modern molecular biology, our understanding of the underlying mechanisms responsible for organ differentiation has developed rapidly. Despite this, our knowledge of these signaling pathways is still far from complete. Some of these advances, such as the creation of transgenic mouse models, have given us new tools to help us understand the interactions of the various transcription factors that are responsible for the creation of various cell types from a single cell type during embryogenesis. This knowledge then gives rise to the concept of creating new ways to manipulate stem cells in order to correct the deficiencies present in various disease processes. Here, we present work that focuses specifically on pancreatic development. The ultimate goal of our research in studying the mechanisms of the basic differentiation of pancreatic precursor cells is to gain the knowledge necessary to be able to engineer stem cells specifically into β-cells in the treatment of diabetes.
IntroductionThis protocol was developed in the Salk STEM Cell Core to enable researchers to consistently and reproducibly produce reprogrammed iPS cells, the initial idea came via word of mouth reports of its effectiveness to increase the efficiency of viral transduction.It has most commonly been used on retrovirus and lentivirus factors.Initial evaluation of this method showed 5-10× increase in transduction efficiency. Flow ChartPlate 50-100K fibroblast cells per well of a 6 well plate.Change media to fresh fibroblast media, *See steps 8-9 Spin at 800xg for 1 hour at RT *See steps 5-7 Incubate plate at 37°C overnight Repeat steps 5-9 Aspirate media and add 2 ml of 4F Retro virus + Polybrene Passage transduced fibroblasts from 6w plate to a 10 cm of MEFS *See step 12 Feed everyday with WiCell hES media.
Diabetes is a metabolic disease characterized by chronic hyperglycemia. Polygenic diabetes, which encompasses type-1 and type-2 diabetes, is the most prevalent kind of diabetes and is caused by a combination of different genetic and environmental factors, whereas rare phenotype monogenic diabetes is caused by a single gene mutation. Monogenic diabetes includes Neonatal diabetes mellitus and Maturity-onset diabetes of the young. The majority of our current knowledge about the pathogenesis of diabetes stems from studies done on animal models. However, the genetic difference between these creatures and humans makes it difficult to mimic human clinical pathophysiology, limiting their value in modeling key aspects of human disease. Human pluripotent stem cell technologies combined with genome editing techniques have been shown to be better alternatives for creating in vitro models that can provide crucial knowledge about disease etiology. This review paper addresses genome editing and human pluripotent stem cell technologies for in vitro monogenic diabetes modeling.
C. elegans germline stem cells are a particularly simple system for analysis of stem cell regulation. Their well-defined mesenchymal niche consists of a single cell, the Distal Tip Cell, which uses Notch signaling to maintain a pool of germline stem cells. Downstream of Notch signaling a post-transcriptional regulatory network dictates self-renewal or differentiation. The major self-renewal hub of that network is FBF, a conserved RNAbinding protein and conserved stem cell regulator. FBF represses mRNAs encoding key regulators of germline differentiation (entry into the meiotic cell cycle, sperm or oocyte specification) as well as established regulators of somatic differentiation. Transcriptional and post-transcriptional mechanisms also control totipotency in the C. elegans germline. The key C. elegans GSC regulators are conserved broadly, making this system a paradigm for stem cell regulation.
Human embryonic stem cells and human induced-pluripotent stem cells are uniquely defined by their pluripotent differentiation potential and endless self-renewing ability.This capability to become any somatic cell type within the human body has garnered significant attention and interest in the fields of cell biology and regenerative medicine.In studying these promising cells, quality-control assays that can characterize their pluripotency and determine the tumorigenicity of their therapeutic progenies become critical.The most rigorous and arguably accurate among current assays is teratoma formation in vivo.This chapter will provide a brief description of teratoma biology, discuss its clinical relevance, and cover methods of forming, monitoring, and analyzing teratomas.The protocols outlined in this chapter have been extensively utilized in peer-reviewed literature.
The cell therapy industry is rapidly growing with several new products approved for clinical use over the past few years and many more currently in clinical trials. Nonetheless, a number of challenges remain in getting cell therapies to the market and into routine use. For instance, cell therapy bioprocesses still need to be optimised, but if these therapies are to be widely adopted there must also be clear routes for their delivery from the site of manufacture to the clinic and the patients. This review explores the currently available routes, the factors that need to be taken into consideration when choosing a route and the challenges that remain with respect to cell therapy logistics.
Dual SMAD inhibition takes a confluent, feeder free culture of hPSCs and rapidly differentiates them into early neurectoderm (Chambers et al., 2009). This rapid differentiation is caused by blocking the two signaling pathways that utilize SMADs for transduction: BMP and TGFB. Oct4 is extinguished and Pax6 expression has begun by around day 7–8, depending on the line used. This neurectoderm can be passaged to become rosettes or can be patterning to become many other types of neural cells (for example, see Fasano et al., 2010).
Flow Chart Instructions 1. Accutase treat hESCs (on an MEF monolayer) for 30-45 minutes, until all colonies are single cells.2. Pipet Accutase into 15 ml conical with hESC media.at least two volumes of hESC to one volume of Accutase
While many clinician-and industry-led autologous cellular therapies are demonstrating benefits to patients in clinical trials, few products have been commercially approved.Progress towards production and commercialization still faces substantial translational challenges under existing regulatory frameworks.Manufacturing and supply of more-than-minimally manipulated (MTMM) autologous cell based therapies presents a number of unique challenges driven by complex supply logistics and the need to scale-out production to multiple manufacturing sites or potentially near to the patient within hospital settings.The existing regulatory structure in Europe and the U.S imposes a requirement to establish and maintain comparability.Under a single market authorisation this is likely to become an insurmountable burden for the roll-out of manufacturing processes to more than two or three sites unless new enabling manufacturing and regulatory science can be established to bridge the comparability challenge. IntroductionCell based therapies fall into two broad classes, those derived from a patient's own cells (autologous or 'one to one' therapies) and those derived from a donor's cells (allogeneic or 'one to many' therapies).This distinction drives the product safety and efficacy model and the approaches to manufacturing, transportation and clinical delivery of the product.In turn, this dictates the technical and regulatory challenges for developing and commercialising safe, effective and reproducible cell based therapies at the required scale and cost.Over the last ten years there has been a steady increase in the clinical development of autologous cell products.An increasing number of clinician-and industry-led autologous cellular therapies are demonstrating benefits to patients
