ReviewMitochondria in stem cells
Introduction
Stem cells have potential for numerous biomedical applications, including therapeutic cell replacement to repair damaged body organs, as tools for studying genetic defects and testing drugs, and as models for studying cell differentiation and early development. Scientists will need to have a thorough understanding of basic stem cell properties before such high stakes goals can be achieved.
There are three types of stem cells: the pluripotent embryonic stem (ES) cells that have the potential to differentiate into any type of body cell, the multipotent cells derived from adult tissue including umbilical cord blood and amniotic fluid which can differentiate into a limited number of cell types of their own lineage, e.g., mesoderm only, and the precursor cells which are adult stem cells committed to differentiation. For a review of the somewhat complicated classification scheme for adult stem cell and precursor cells, see Raff (2003). Investigators need to carefully catalog and compare the properties of these three types of cells. While it is possible that they share common properties, it is also likely that significant differences will be found.
While stem cell research has progressed rapidly since the initial report of human ES cell isolation (Thomson et al., 1998), the majority of published articles have focused primarily on three general areas. Many reports have examined the expression of various genes that are believed to indicate the pluripotency status of stem cells (so-called “stemness”), such as alkaline phosphatase, Oct 4 or SSEA 4. Researchers must verify that the expression of such markers is stable for each cell line during prolonged periods of cell culture in order to verify that the pluripotent condition has not been lost. Another major focus involves physiological cell culture and microenvironment conditions that lead to uniform directed differentiation of stem cells into specific cell types. The third research thrust focuses on reducing possible xenogenic complications arising from cell culture techniques. Human ES cells are typically grown on a layer of mitotically-inactive fibroblasts (commonly mouse fetal fibroblasts), called a “feeder layer”, in the presence of fetal calf serum supplements. Although this approach has been moderately successful in supporting proliferation of ES cells, it is fraught with problems. For example, it has been demonstrated that mouse cell surface antigens have translocated into the plasma membrane of some ES cell lines (Martin et al., 2005), and the potential for transfer of murine or bovine viruses into human ES cells also exists (Tailor et al., 1999). These xenogenic concerns will preclude use of many human ES cell lines for therapeutic purposes. To avoid these complications, several laboratories have reported some success in culturing ES cells either on human fibroblasts, without using any feeder layer, and/or without fetal serum (Richards et al., 2002, Hovatta et al., 2003, Amit et al., 2004, Heins et al., 2004, Rosler et al., 2004, Valbuena et al., 2006), but these culture conditions are technically challenging and not yet widely used. There is the possibility that growing ES cells without feeder layers will not constitute superior growth conditions (Richards et al., 2004), and there is the possibility that cells acquire different biological properties under such culture conditions.
The fundamental importance of stem cell stability in culture conditions, and the consequent maintenance of pluripotency or multipotency, has led to searches for indicators of what is commonly referred to as “stemness”. Expression of such markers might indicate not only if the stem cells are normal, but also whether they are fully capable of differentiation. There is a serious lack of research on functional cellular markers of stem cells, such as ultrastructural morphology, metabolic profiles or cell signaling pathways. General features of stem cell function need to be identified so that deviations from the normal pattern could then be used to eliminate defective cell lines from further use. Such properties will also be important in comparing cell lines among different laboratories. The phenotypic stability of ES lines cultured for prolonged periods of time must also be addressed because they may not be chromosomally stable after prolonged cell culture passage in the absence of feeder cells (Ludwig et al., 2006).
While most stem cell studies have focused on the activity of the nuclear genome, characteristics of the mitochondrial genome have been largely ignored. Cells from embryos created by in vitro fertilization (IVF) procedures, the source of ES cells, have been reported to exhibit various forms of mitochondrial DNA (mtDNA) mutations, and it is not known if metabolic functions of stem cells are affected by high copy number of mtDNA point mutations or mitochondrial deletions (Gibson et al., 2006). There is a distinct possibility that many of the ES cell lines currently available for study do not represent high quality stem cells. Considering that mutations in mitochondrial DNA have been linked to a wide range of disorders, including diabetes, cardiovascular disease and cancer (Chinney et al., 2002; Liu et al., 2002, Maitra et al., 2005, Birch-Machin, 2006), it is surprising that the mitochondrial properties of stem cells have been largely overlooked. These concerns raise the possibility that the use of aberrant stem cell lines, whether from embryonic or adult sources, for therapeutic cell replacement could lead to the development of cancer. ES cells and cancer cells share several traits, including unlimited self renewal capabilities and the ability to generate a diverse range of other cell types. The possible presence of stem cell populations in tumors (Huntley and Gilliland, 2005, Clarke and Fuller, 2006) has many implications for the diagnosis and treatment of cancers, since the most effective way to eliminate the disease would be to target cancer stem cells for destruction. Thus, mtDNA anomalies could have widespread implications for the biomedical applications of stem cells as well as for studies on their behavior in vitro.
In this review, we examine mitochondrial properties in early-stage embryos, the source of ES cells, as well as in adult stem cells and differentiating precursor cells.
Section snippets
Mitochondrial localization in early-stage embryos and stem cells
ES cells are derived from the inner cell mass (ICM) of the pre-implantation blastocyst stage (Fig. 1). Descriptions of mitochondria in a number of human and mouse ES cell lines using electron microscopy report that these cells have few mitochondria with poorly developed cristae (Sathananthan et al., 2001, Baharvand and Matthaei, 2003, Oh et al., 2005, Cho et al., 2006). The human ES cell line HSF6 was reported as having few mitochondria that tend to localize in small perinuclear groups (see
Metabolic characterizations of stem cells
Prior to embryo implantation and subsequent in vivo vascularization, embryonic cells are contained in a hypoxic environment within the uterine lumen (Fisher and Bavister, 1993). Because this environment is not conducive to ATP synthesis through OXPHOS, the embryonic cells rely on anaerobic metabolism to meet their energy demands (Brown, 1992). It is therefore not surprising that ES cells have few mitochondria that lack cristae development. It has been shown that the ATP content of blastomeres
The significance of perinuclear arrangement of mitochondria
The possible functional significance of the perinuclear arrangement of mitochondria in stem cells deserves further examination. Numerous types of differentiated cells have also been reported to exhibit perinuclear arrangements of mitochondria, including fibroblasts (Yaffe, 1999), pancreatic ascinar cells (Johnson et al., 2003, Bruce et al., 2004), and astrocytes and neurons (Collins et al., 2002). The addition of ES and adult stem cells to this list reopens the question “Is there a benefit for
Expression of mtDNA regulatory proteins in early stage embryos and stem cells
For most species, it is not known at what stage of early embryo development mtDNA replication begins. In mice, reports suggest that no mtDNA replication occurs in the early cleavage stages (Ebert et al., 1988, Piko and Taylor, 1987) although one report suggests a short burst of mtDNA replication in the mouse two-cell stage with no corresponding increase in mtDNA copy number (McConnell and Petrie, 2004). mtDNA replication has been reported in the blastocyst stage in both the mouse (Thundathil et
Mitochondrial mutations in oocytes, embryos and stem cells
A growing concern in the area of Assisted Reproductive Technology is that mitochondrial mutations and deletions have been found at high frequencies in oocytes and embryos, and these may be passed on to the derived ES cell lines and subsequent differentiated tissues (Harvey et al., 2007). The extent to which these mitochondrial mutations perturb mitochondrial functions is unclear, but it has been suggested that mitochondrial instability and lack of repair mechanisms may be associated with poor
Future directions
Little is known about the mechanisms regulating mtDNA replication, repair mechanisms or transcription during the differentiation of ES cells. Numerous questions need to be addressed in order to develop a full understanding of the role of mitochondrial activity in stem cells. Important questions include: (1) When does mtDNA replication occur during the differentiation process, and does this correlate with an increase in mtDNA copy number? (2) Does the timing of mtDNA replication correlate with
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