ReviewAging of mesenchymal stem cells
Introduction
In recent years, multipotent stem cells in adult tissue have received considerable attention. The term mesenchymal stem cells (MSC) was popularized by Caplan (Gao et al., 2001), in reference to work by Friedenstein and Owen (Friedenstein et al., 1970) describing a plastic-adherent fibroblastic cell isolated by Percoll density centrifugation, reactive with monoclonal antibodies SH2 and SH3.
The adjective ‘mesenchymal’ is fraught with some ambiguity since ‘mesenchyme’ describes tissue of mesodermal origin, the middle embryological germ layer, giving rise to the musculoskeletal, blood, vascular and urinogenital systems, and to connective tissue (including dermis). Thus, developmentally speaking, the term ‘mesenchymal’ should include both blood and connective tissue cells.
In practice however, only the latter cells are usually described as being derived from MSC and considered distinct from haematopoietic stem cells (HSC), which are responsible for the development, maintenance, and regeneration of blood forming tissues [reviewed by Chen (2002)]. It is quite possible that MSC and HSC have a common precursor in the elusive “haemangioblasts” (Sabin, 1920), in the cells identified by the group of Verfaillie originally termed “mesodermal progenitor” (Reyes et al., 2001), later “multipotent adult progenitor” (MAPC) (Young and Black, 2004) cells, or in “pluripotent stem cells” (Howell et al., 2003), or ‘tissue committed stem cells’ (TCSC) (Ratajczak et al., 2004). However, this is contentious and the physiological relevance of these cells remains to be demonstrated.
Cells with non-haematopoietic multipotency can ultimately differentiate into multiple cell lineages including osteoblasts (Jaiswal et al., 1997), adipocytes (Purpura et al., 2004), chondrocytes (Johnstone et al., 1998), myoblasts (Wakitani et al., 1995), and early progenitors of neural cells (Deng et al., 2001). Such cells can be isolated from umbilical cord blood (Lee et al., 2004, Hou et al., 2003), connective tissue (Young et al., 1995), skin (Shih et al., 2005), synovium fluid (Jones et al., 2004), fat (De Ugarte et al., 2003), the placenta (Waller et al., 1995) and even teeth (Nakashima and de Crombrugghe, 2003), but most commonly they are taken from marrow of various bones.
Accordingly, the nomenclature is not consistent. Designations for cells with non-haematopoietic multipotency have included “colony-forming-unit-fibroblasts”, “stromal (stem) cells”, “bone marrow (stromal) cells”, “skeletal stem cells”, “mesodermal progenitor cells”, “non-haematopoietic stem cells”, “(bone marrow) stem cells”, “mesenchymal progenitor cells” and others (see Baksh et al., 2004, Young and Black, 2004 for enumeration). There is also an understandable tendency to designate such cells as “pre-(lineage-under-investigation)” cells (e.g. pre-osteoblast etc.). It has also been suggested that MSC are simply pericytes (Nakashima and de Crombrugghe, 2003).
Some of the inconsistencies surrounding the identification of MSC arise from the fact that specific markers have not yet been agreed on. In the absence of a universal antigenic indication (analogous to CD34+ for HSC) and an universal assay (analogous to the repopulation assays for HSC) MSC are often identified simply by testing a cultures’ differentiation potential into colony forming units (CFU) indicative of proliferative capacity (see below) and into several lineages of mesenchymal tissue as defined above (Pittenger et al., 1999). Also, the ability to adhere to tissue culture plastic and a fibroblast-like morphology are taken as characteristic markers for MSC (Prockop, 1997).
Recently, different surface markers have been associated with MSC including D7fib (Jones et al., 2002), Stro1 (Stenderup et al., 2001), CD45 and glycophorin A (Jones et al., 2004, Pittenger et al., 1999, Reyes et al., 2001), BMPR1a (Zvaifler et al., 2000) (for comparative analysis, see Young and Black, 2004).
Further complications for comparative review arise when different sources, extraction and cultivation methods are used. Even when narrowing sources to bone marrow (as in this review), the site of extraction is reported to influence cell behaviour: e.g. MSC from alveolar bone show less chondrogenic and adipogenic potential compared to iliac bone (Matsubara et al., 2004). Isolation is usually conducted by density centrifugation (sometimes enhanced by gradient solutions) to obtain the mononuclear fraction of marrow cells and by using the widely reported ability of MSC to adhere to tissue culture plastic (Sekiya et al., 2002). Newer methods employ magnetic beads (Stenderup et al., 2001) or FACS sorting (Fickert et al., 2003) in conjunction with antibodies to the proposed MSC markers above. Additionally, widely differing standards regarding serum composition, culture conditions, and growth factor application in MSC cultivation exist. Differing conditions can lead to enrichment of different subsets of MSC with differing clonogenic potential. All these potential deviation points in current methods are summarised in Table 1.
For the purposes of this review, MSC will be defined as post-embryonic, bone-marrow derived cells, naturally capable of multipotent differentiation into connective tissue of non-haematopoietic lineage; in particular bone, ligaments, tendons, fibres, cartilage, and adipose tissue. (Compare the cell type called PPIMSC by Young and Black (2004).)
If the definition of MSC is elusive, a definition of aging is even more daunting. The patchwork nature of different research foci, coupled with fundamental uncertainties about the nature and the evolutionary role of the aging process preclude a common definition. In cytological study, further challenges arise in distinguishing between aging in vivo and prolonged cultivation in vitro that might or might not simulate ‘true’ aging (‘in vitro aging’).
We adopt a definition of aging as “the sum of primary restrictions in regenerative mechanisms of multicellular organisms” (Sames and Stolzing, 2005). This definition highlights the involvement of MSC in cell replenishment and thus in influencing lifespan.
Aging can be conceptually distinguished from senescence, with the latter emphasising the cellular level. Here, we adopt the definition of Campisi (2000) that equates senescence with replicative senescence (Hayflick and Moorhead, 1961) by defining it as “an essentially irreversible arrest of cell division”. Unlike apoptotic cells, senescent cells remain alive, despite a derangement of function (Itahana et al., 2001). Cellular senescence is a complex phenotype that entails changes in both function and replicative capacity. Different experimental protocols, culture conditions, and cell types yield different kinds of senescence. Generally, senescent cells display a characteristic enlarged, flattened morphology and are characterized by an irreversible G1 growth arrest involving the repression of genes that drive cell cycle progression and the upregulation of cell cycle inhibitors like p53/p21 and p16/RB. It was suggested that there are notable distinctions between senescent states induced by the p53 and p16/RB pathways; there is an emerging consensus that senescence occurs via one pathway or the other, with p53 mediating senescence due primarily to telomere dysfunction and DNA damage and the p16/RB pathway mediating senescence due primarily to oncogenes, chromatin disruption, and various stresses (Campisi, 2005).
It has been speculated that senescence may lead to arrested regeneration in tissues and thus to organ failure and death (Knapowski et al., 2002). One degenerative factor in senescence is the accumulation of damage in the cell (Gao et al., 2001, von Zglinicki et al., 2001). Furthermore, senescent cells secrete factors including degradative enzymes, inflammatory cytokines, and growth factors that stimulate tissue aging and tumorigenesis (Krtolica and Campisi, 2003).
Nonetheless, there are persistent doubts about the phenomenon's relevance for in vivo aging (Hornsby, 2002). Critical tissues such as cardiac or neural tissue divides little if at all. Furthermore, senescence in metazoans composed entirely of postmitotic cells is just as predictable and robust as that in metazoans containing mitotic cells (Effros et al., 2005). The immediate relevance of senescence is likely closer to cancer than to other aging-related developments.
The capacity of MSC to effect tissue and organ regeneration is still not well understood. MSC will populate a wide variety of tissues after systemic infusion (Gao et al., 2001).
Recent evidence suggests that the process of tissue repair is driven by tissue-specific progenitor cells which are replenished by MSC from bone marrow by migration–differentiation (Shake et al., 2002), fusion (Kotton et al., 2001, Prockop et al., 2003b) or the provision of a stromal support network (Alexanian and Kurpad, 2005, Sheng et al., 1998). Involvement of bone marrow-derived stem cells has been demonstrated in the regeneration of a number of organs/tissues including bone (Shirley et al., 2005), skin (Mori et al., 2005), liver (Grompe, 2005), kidney (Okada, 2005) and muscle (Natsu et al., 2004). Consequently, any loss in numbers or functionality with age would have profound consequences for the maintenance of tissue viability (Pelicci, 2004)
Section snippets
Morphology
In vitro aged MSC are reportedly bigger (Baxter et al., 2004, Mauney et al., 2004, Stenderup et al., 2003) than their young counterparts; they exhibit more podia and spread further (Mauney et al., 2004) and contain more actin stress fibers (Stenderup et al., 2003). Increase in cell size is often associated with senescence (Dimri et al., 1995, Hayflick and Moorhead, 1961).
MSC from older patients show no spindle-formed (young) MSC morphology in culture, whereas MSC from young donors exhibit the
Extrinsic and intrinsic MSC aging
When considering the link between aging and MSC, we are faced with two interrelated components: The effect of aging on MSC themselves, and the contribution of MSC to the aging of the organism. In this, a primary concern is to determine whether MSC are aging internally (intrinsic theory) or if the cells are driven into proliferative silence by changes within the surrounding tissue (extrinsic theory).
For practical reasons, aging of MSC tends to be studied in monolayer cultures of purified or
Collating findings
In collating current findings on age markers in MSC, the emerging picture is curiously consistent in its inhomogeneity. A few methodological difficulties in MSC analysis have been discussed, and in the context of aging-related research further difficulties are presented by the inclusion of differently defined age groups, variations in sex and disease status of donors.
However, evidence suggests that many of the apparent inconsistencies are likely attributable to observations made in Section 1.
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