Gene Targeting and Embryonic Stem Cells

Jim Mcwhir, Alison Thomson

• Publisher: Taylor & Francis
• Year: 2004
• ISBN:
  9781135319427
• Edition: 1
• Topic: Ciencias biológicas
• Subtopic: Biotecnología

Gene Targeting and Embryonic Stem Cells is a practical guide designed for the rapidly growing number of researchers who are moving into this field. Provides details on how to culture, transfect and differentiate established cell lines, and how to isolate new cell lines. Gene targeting experiments are described for a number of cell types, including ungulate fetal fibroblasts, murine ES cells, human embryonal carinoma cells and human ES cells, and include protocols for gene-targeting vectors, DNA transfection and RNA interference. The recent isolation of human embryonic stem cells and the potential of these cells for therapeutic applications has generated an entirely new methodology. Similarly, gene targeting methodology has recently been extended to nuclear donor cells in ungulate species. This volume will be invaluable for both new and established researchers in the field of human embryonic stem cells, and to biotech companies engaged in the production of transgenic proteins in livestock, xenotransplantation and the development of animal models.

1

An overview of gene targeting and stem cells

Jim McWhir, Alison J.Thomson and David Russell

DOI: 10.4324/9780203489246-1

At first glance this might appear an odd grouping of techniques. Yet, at least in the special case of embryonic stem cells, gene targeting and stem cell culture are commonly undertaken in the same lab. To some of us, though we really know better, it seems as though murine embryonic stem (mES) cells were expressly designed for genetic manipulation. They do everything we ask of them: grow indefinitely, accept incoming DNA, maintain a relatively stable karyotype and then go on to make chimeric animals and to pass modified genes through the germ line. With the help of heterosis, and a tetraploid host embryo to provide the extraembryonic membranes, mouse ES cells can even regenerate an entire animal (1). These extraordinary properties have enabled the generation of precise genetic modifications in mice by gene targeting followed by germ line chimerism, allowing us to ask important questions about gene function and to model human genetic disease.

There is another side to ES cells that is not necessarily related to their genetic manipulation. They have the inherent potential to give rise to virtually all cell types, thereby providing tools for the possible treatment of metabolic or degenerative disease. Since the isolation of the first human ES (hES) cells in 1998 (2) there has been a dramatic increase in the number of new workers who are beginning to culture these cells for the first time. Sadly, we cannot yet say of human ES cells, even tongue in cheek that they were ‘designed for genetic manipulation’–at the present time they remain difficult to work with. It is also unclear to what extent the various cell lines available differ, to what extent they adapt to changes in their culture regime and if such adaptations have functional or tumorigenic consequences. We hope that the protocols herein will provide a useful basis for the further refinement to culture systems that will be so necessary to resolve these issues and to achieve regulatory acceptance.

But why have we included a chapter on nuclear transfer? The historical context of this book is the post Dolly biology of challenged conventions. If the nucleus of an adult differentiated cell can support development to term and beyond (3), what else may be possible? So nuclear transfer is perhaps only one manifestation of this broader phenomenon of nuclear reprograming that may eventually offer access to all of the varied cell fates encoded within a single nucleus, effectively making all cells potential ‘stem cells’. Adding gene targeting to this equation renders all genetic lesions repairable, in principle, in all tissues.

1.1 The murine embryonic stem cell system

Chapters 2 and 3 describe the use of murine ES cells in gene targeting and nuclear transfer respectively. ES cell lines were first isolated by Evans and Kaufman (4), and independently, by Gail Martin (5) in 1981. Their experiments were inspired by the properties of the stem cells of germ cell tumors known as embryonal carcinoma or EC cells (see Chapter 6). EC cells had been shown to contribute to somatic tissues when injected into the blastocoel cavity of murine preimplantation embryos, but rarely if ever, contributed to the germ line. It was evident that if that deficiency could be overcome by deriving similar cells directly from the embryo, it might provide a route to germ line modification.

The process of ES isolation in mice can be characterized as the serial disaggregation of the undifferentiated component of an embryonic explant (most commonly a 3.5-day blastocyst). In permissive strains this process eventually gives rise to ES lines at a frequency of up to 30% depending upon level of expertise. The unsuccessful explants succumb to differentiation, generally to trophectoderm, followed by senescence. The process of ES isolation may be enhanced in mice by ovariectomy followed by supplementation with progesterone to induce delay of implantation (4). Certain mouse strains are more difficult or impossible to obtain lines from using conventional techniques, but do yield lines under conditions in which the differentiating component of the explant is removed, either by microdissection (6), by selective ablation (7, 8), or is forestalled by inhibition of the ERK/MEK pathway (9).

Control of the undifferentiated growth of murine ES cells is now relatively well understood, in large part due to the work of Hans Scholer and colleagues (10) and of Austin Smith and his colleagues (11, 12), in unravelling the control of mES self-renewal and differentiation. Three transcription factors are involved in controlling mES fate in vitro. The same factors in vivo control the fate of the epiblast from which ES cells arise. Oct4 is required in vivo for regulation of cell fate during the first differentiation event leading to trophectoderm and in vitro for the self-renewal of ES cells (11). Stat3 activation by leukemia inhibitory factor (LIF) is also required to sustain mES/epiblast self-renewal (12, 13), and Nanog (14) is subsequently required in vivo to maintain the epiblast, its down-regulation triggering the second differentiation event that leads to parietal/visceral endoderm.

1.2 Human ES cells

There are many reasons why the isolation of additional human ES lines remains a high priority. Foremost among these is the near certainty that existing lines could not obtain regulatory approval for clinical application due to the nature of their derivation. The use of undefined medium including serum and the requirement for coculture of embryonic explants on a feeder layer of murine embryonic fibroblasts means that existing lines would be treated for regulatory purposes as xenografts. Additional reasons include the desirability of banking hES lines of diverse major histocompatibility complex (MHC) haplotype in order to minimize the problem of graft rejection and the utility for research purposes of the derivation of lines carrying specific genetic lesions. Chapter 7 describes the methods currently and most commonly in use for the successful isolation of hES lines.

The excitement attached to hES cells arises from their potential application in medicine. How much of this potential can be substantiated? Undirected differentiation of hES cells arises simply by their removal from feeders or conditioned medium. The real challenge is to direct that differentiation and to purify large numbers of functional cell types or their progenitors, ultimately by specific induction of a single differentiation pathway in defined media. The protocols provided in Chapter 8 provide a basis for the further development of directed differentiation of hES cells.

Zhang et al. (15) reported a transition of embryoid bodies to neural rosettes, which are subsequently harvested by selective dissociation as free-floating aggregates that generate both neurons and glia. Reubinoff et al. (16) observed expression of neural markers when hES cells were maintained in culture without passage or replenishment of feeders and in the absence of basic fibroblast growth factor (bFGF). These cells were then replated in serum-free medium to form free-floating spherical structures similar to neurospheres isolated from adult brain tissue. Both groups performed engraftment studies into the lateral ventricle of neonate rats and showed that transplanted cells migrated into many brain regions. However, it is unclear if these cells are functional. In a third study (17), a wide variety of ES-derived neural cell types were demonstrated in vitro and shown to have action potential. A limitation of these techniques, is the absence of effective protocols for the derivation of enriched populations of the most clinically relevant cell types in the central nervous system (CNS)—dopaminergic neurons (Parkinson’s disease), cholinergic neurons (Alzheimer’s) and oligodendrocytes for repair of myelination defects.

Several groups have reported the enhanced generation of functional cardiomyocytes from hES cells after treatment with 5-aza-2’-deoxycyridine (18, 19) (a demethylation reagent), enrichment by Percoll gradient separation (18), and treatment with retinoic acid (RA) (20). Human ES cells also respond to osteogenic factors, giving rise to mineralizing osteoblasts with a similar time course to that observed with marrow stromal cells (21). Spontaneous differentiation of hES cells includes cells with characteristics of insulin-producing ß-cells (22). Although this is an encouraging first step, these putative ES-derived islet cells remain small numbers of cells interspersed among a mixed population. Hepatocyte differentiation from hES cells is important both in regenerative medicine and for drug and toxicity testing, and an expandable population of ES-derived cells with the features of hepatocytes has recently been observed following treatment with sodium butyrate (23). Endothelial cells have been purified from differentiating hES cells using antibodies to platelet endothelial cell-adhesion molecule 1 (PECAM1) (24) and were seen to form vessel-like structures in matrigel and hematopoietic precursor cells arise when hES cells are cocultured with the murine bone marrow cell line S17 or the yolk sac endothelial cell line C1620 (25).

The general features of human ES cells in the undifferentiated state are thoroughly described in Chapters 6–8 and are not fully reprised here. Suffice it to say that there are important dissimilarities with murine ES cells. For example hES cells are not responsive to exogenous LIF and do not express the SSEA1 epitope characteristic of murine ES cells. The control of hES self-renewal and differentiation is not as well understood as it is for mES cells. However, recent results may have provided a way to short circuit the very elaborate signal transduction pathways controlling ES fate in both species. Pharmacological inhibition of glycogen synthase kinase 3 (GSK3) was shown to be sufficient to sustain ES self-renewal for 7 days in the absence of additional cytokines (26). It remains to be seen if that effect is maintained with prolonged culture. If so, and if hES cells can be shown to be unaffected in other respects following this treatment (e.g. tumorigenesis) then it may soon be possi...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Contents
• Preface
• Abbreviations
• 1 An Overview of Gene Targeting and Stem Cells
• 2 Advances in Gene Targeting in Murine Embryonic Stem Cells
• 3 Nuclear Transfer with Murine Embryonic Stem Cells
• 4 Gene Targeting in Sheep
• 5 RNA Interference
• 6 Human Embryonal Carcinoma Cells: Surrogate Tools in the Study of Human Embryonic Stem Cells
• 7 Isolation of Human Embryonic Stem Cells
• 8 In Vitro Culture and Differentiation of Human Embryonic Stem Cells
• 9 Transfection of Human Embryonic Stem Cells
• Index