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.
