Influence of donor age on expansion and differentiation

MSC can be isolated from almost every tissue, from adult organisms (Friedenstein, 1974; Pittenger, 1999) but also from fetal tissues (In 't Anker, 2004), suggesting that they occupy a ubiquitous niche. Most studies used human MSC from younger donors, i.e. younger than 30 or 50 years of age, hypothesizing that increasing age affects expansion and differentiation potential (Jiang, 2008; Stolzing, 2006; Zhou, 2008c). Similar to expression of stem cell markers within MSC populations, analysis of the relevance of donor age on MSC numbers in the bone marrow, and expansion and differentiation potentials showed controversial results in the literature. Some studies demonstrated clear differences in telomere lengths, and in vitro expansion and differentiation potentials between MSC from younger and elder donors (Mendes, 2002; Parsch, 2004; Stolzing, 2008; Zhou, 2008c), whereas some other data showed no age-related differences (D'Ippolito, 1999; Oreffo, 1998; Scharstuhl, 2007;

Stenderup, 2001; Suva, 2004; Tokalov, 2007a).

Telomere shortening and reduction of telomerase activity have been correlated to cellular ageing (Aubert, 2008; Lansdorp, 2008). Telomeres are repetitive DNA

sequences located at both ends of linear chromosomes. They provide a protective mechanism to preserve chromosomal integrity and conceal the chromosomal ends from the DNA repair machinery that would otherwise identify them as double strand breaks (Verdun, 2007). Telomere length is maintained in germline and most embryonic stem cells / tissue by the enzyme called telomerase. Some fetal tissue-derived MSC express telomerase activity (Guillot, 2007). Telomerase activity was also described in certain adult stem cells (Beltrami, 2007; D'Ippolito, 2004; Hermann, 2006; Ingram, 2004; Kucia, 2007b; Tateishi, 2007; Zeng, 2006), and within MSC populations (Gronthos, 2003; Parsch, 2004; Yanada, 2006). Other studies, however, reported absence of telomerase activity in cultured MSC (Banfi, 2002; Graakjaer, 2007; Isenmann, 2007; Zimmermann, 2003). It has been shown that in cells expressing telomerase, its activity decreases with time in culture and population doubling numbers, resulting in telomere shortening (Baxter, 2004; Bonab, 2006;

Isaikina, 2006; Izadpanah, 2006; Mareschi, 2006; Shibata, 2007). Shibata demonstrated that telomere shortening, expansion potential (population doubling numbers) and expression of p16^INK4A (a cyclin kinase inhibitor) are closely related.

With increasing population doublings, expression of p16^INK4A increased and induced concomitantly beta-galactosidase, a senescence marker. Knock-down of p16^INK4A expression reversed senescence and induced massive proliferation (Shibata, 2007).

In most studies using MSC isolated from donors differing in age, telomerase activity and telomere length decreased with increased donor age (Baxter, 2004; Kastrinaki, 2008), although this was not always statistically significant (Shibata, 2007). This pattern was also correlated with reduction in expansion potential (Baxter, 2004;

Kastrinaki, 2008) and appearance of senescence (Shibata, 2007). However, in MSC from older donors (50-79 years old patients), no age-dependent reduction of telomere length was documented (Parsch, 2004). Such discrepancies in telomerase activity might not be solely due to differences in donor age but also to culture conditions. Insemann et al suggested that culture medium containing high levels of serum inhibits telomerase activity (Isenmann, 2007).

The importance of telomerase activity in MSC for proliferation and differentiation has been shown in MSC isolated from telomerase knock-out mice. Their expansion potential was dramatically reduced to half of passage numbers and time in culture before senescence,when compared to wild type (Liu, 2004). Telomerase knock-out MSC did not differentiate into adipocytes and chondrocytes, even at early passages, demonstrating an association between telomerase activity and differentiation

potential. These observations were confirmed in mice deficient for telomerase and Werner helicase, a mouse model for premature aging. These mice developed low bone mass and early osteoporosis, a consequence of defective differentiation of MSC into osteoblasts (Pignolo, 2008).

Age-related reduction in differentiation potential was documented in several studies.

Zhou et al demonstrated a reduction in proliferation rate, increased doubling time, reduced osteogenic differentiation, increased apoptosis and beta-galactosidase expressing senescent cells in human MSC isolated from older compared to younger donors (Zhou, 2008c). Similar results were shown for MSC isolated from human vertebral bodies (D'Ippolito, 1999) and rat bone marrow (Stolzing, 2006; Zheng, 2007). Stolzing et al showed that MSC from older animals had reduced expansion and differentiation potential and increased apoptosis, probably secondary to accumulation of oxidative damage to lipids and proteins and up-regulation of p53.

Chondrogenic differentiation was also impaired in MSC from older rats. Microarray data showed that most genes related to cartilaginous extra-cellular matrix production and secretion were down-regulated in MSC from old animals (Zheng, 2007). In contrast to these results, several studies showed no impact of age or inflammatory diseases on differentiation potential of MSC. In studies using rat MSC from bone marrow of 4 to 48 week-old animals, older animals exhibit lower frequencies of MSC within the bone marrow, but these cells showed similar amplification and differentiation characteristics (Tokalov, 2007a; Tokalov, 2007b). In human MSC isolated from femoral heads of patients aged 24 to 92, undergoing total hip replacement due osteoarthritis, hip dysplasia or trauma, no age- or etiology-related differences were observed in cell yields, expansion potential and chondrogenesis (Scharstuhl, 2007; Suva, 2004). Expression profiles comparing MSC from fetal, infant and adult rhesus monkeys showed however down-regulation of genes related to proliferation and up-regulation of genes associated to mesodermal tissue differentiation with increasing age (Hacia, 2008). Similarly, in human MSC, Jiang et al showed that with increasing age, osteogenic genes were down-regulated whereas genes related to adipogenesis were up-regulated (Jiang, 2008).

In vitro culture and expansion of MSC harbor the risk of accumulation of translocations and aneuploidy (Izadpanah, 2008; Wang, 2005c). Furthermore, MSC derived from older donors, or after prolonged times in culture transform spontaneously into potential sarcomatoid cells with p53 point mutations and other changes in tumor suppressors and oncogenes expression (Li, 2007b; Rubio, 2005;

Rubio, 2008). Likewise, they seem to get “older” artificially when expanded in Petri dishes, which may raise some concern about potential detrimental effects of expansion for clinical applications (Banfi, 2002).

Thus, aging, both “in vivo and in vitro” generally affects mesenchymal stem cells, decreases their expansion and differentiation potentials, and increases the risk of spontaneous degeneration into cancer inducing, sarcomatoid “stem” cells. For clinical applications, the use of MSC derived from younger donors (<30 years old), or from UCB and umbilical cord matrix, with limited in vitro expansion and culture would increase safety for recipient patients and maintain broader differentiation potential.