**4.** *In vitro* **long-term culture associated alterations of MSCs**

#### **4.1 Morphological and immunophenotypic alterations of MSCs**

It is well known that MSCs demonstrate biological alterations in the course of *in vitro* long-term culture. Different tissue derived MSCs may present different morphological and immunophenotypic characteristics in the expansion culture. At present, there is lack of a unifying definition for the "passage" of MSCs. Morphological changes are continuous during the long-term culture and expansion of BM-MSCs [38–40], which display a fibroblast-like appearance at early passages while the flattened and larger morphology as well as a visible increase of cellular granularity in late passages. For example, one previous study showed that human BM-MSCs were consistent with a morphological appearance from passage 1 to passage 6–8 and beyond that period such cells became large and flat [40].

During further cultivation, MSCs demonstrate the altered common immunological surface markers. Comparison of the early and late passages of BM-MSCs reveals that no differences are observed between passage 2 and 6 MSCs in expression of CD44, CD90, CD105, HLA-ABC, and HLA-DR, while CD106 is downregulated in MSCs of passage 6 [41]. Research has also reported that the expression pattern of the common surface markers maintains consistently with consecutive passaging up to passage 8 of BM-MSCs [40]. In contrast, the positive expression of the common surface markers such as CD73, CD90 and CD105 presents at the passage 30 of human adipose-derived MSCs (AD-MSCs) [42] and human umbilical cord MSCs (UC-MSCs) [43].

#### **4.2 Alterations of proliferation and differentiation of MSCs**

MSCs exhibit a high proliferation rare at lower passage and, however, the rapid growth kinetics decrease gradually with consecutive cell passaging. A linear correlation is observed between cumulative population doubling and days in culture up to passage 6–8 of human BM-MSCs and the passage-dependent decrease in the proliferation rate is also observed beyond that period [40]. A reduction in the proliferation in the course of long-term cultivation has been reported in human dental pulp tissue-derived MSCs [44] and human tonsil-derived MSCs [45].

The differentiation ability of human BM-MSCs vary in long-term culture manifested by the significant reduction in expression levels of the osteogenic markers, such as ALP and osteocalcin, and adipogenic markers, such as fatty acid binding protein-4 and lipoprotein lipase at the late passages [45]. It has been reported that 25% samples of BM-MSCs from different donors in the 8th passage and the 20% in the 10th passage lost their osteogenic differentiation potential [46]. Similarly, 10% BM-MSC samples in the 6th passage, the 50% in the 8th passage, and the 60% in the 10th passage also lost their adipogenic differentiation [46]. In contrast, an *in vitro* differentiation study has also reported that the potential of adipogenesis decreases in higher passages (from the 5th passage) whereas the propensity for osteogenesis increases in the long-term culture [39].

#### **4.3 Replicative senescence during long-term culture expansion of MSCs**

Replicative senescence is known as the irreversible growth arrest of the mitotic cells and is induced by telomere shortening. The expression of senescence markers such as senescence-associated β-galactosidase, heterochromatin protein-1, and p16INK4a increase during aging [47, 48]. Molecular damage and epigenetic alterations occur in aging stem cells [49], which can result in the impairment of stem cell function.

There are various signaling pathways involved in the senescence of MSCs, including oxidative damage [50, 51], age-related defects [52], and senescence associated up-regulation of microRNAs [53]. MSC senescence can be observed with long-term *in vitro* cultivation [54, 55], thus suggesting that a certain proportion of MSCs may undergo senescence during culture expansion. *In vitro* long-term culture of MSCs can induce continuous changes in gene expression [39, 56]. The expression levels of the senescence related genes, such as p16, p21 and p53, increase gradually in MSCs in the course of *in vitro* culture expansion [57]. DNA-methylation changes in MSCs during long-term culture have been investigated as an important epigenomic feature of replicative senescence of MSCs [58–60]. DNA-methylation changes may affect the proliferation and differentiation of MSCs. Differential methylation patterns of gene and miRNAs show between early-passage (passage 5) and late-passage (passage 15) MSCs [60]. Some genes that are hypermethylated at passage 5 present the lower mRNA expression than does these hypermethylated at passage 15 and vise versa [60].

Senescent cells secrete a complex combination of interleukins, chemokines, growth factors, proinflammatory/inflammatory cytokines, which compose the senescence-associated secretory phenotype (SASP) [61, 62]. One previous report has shown that conditioned medium (CM) collected from senescent BM-MSC culture at passage 10 is able to trigger senescence in young cells [63]. The key factors of senescent MSC CM needed for triggering senescence in the young MSCs have been characterized as insulin-like growth factor binding proteins 4 and 7, which are linked to cellular senescence and apoptosis [63]. Similarly, monocyte chemoattractant proten-1 (MCP-1), as a dominant component of the SASP, is markedly increased in the conditioned medium of the late-phase MSCs and MCP-1 treatment significantly increase the senescence phenotypes of umbilical cord blood-derived MSCs via its cognate receptor chemokine receptor 2 signaling cascade [64]. Senescence-associated changes

*Isolation and Expansion of Mesenchymal Stem/Stromal Cells, Functional Assays and Long-Term… DOI: http://dx.doi.org/10.5772/intechopen.100286*

are observed in the metabolome of MSCs during replicative senescence, including down-regulation of nicotinamide ribonucleotide and up-regulation of orotic acid, which may be used to monitor the cellular senescent state during culture expansion of MSCs [65].

#### **4.4** *In vitro* **long-term culture associated spontaneous transformation of MSCs**

Sarcoma represents a very heterogeneous group of relatively rare tumors and a variety of different studies have investigated to support the MSC origin of sarcoma. There are a number of cellular and molecular mechanisms of MSC transformation for better understanding of MSCs' contribution to sarcomagenesis [66]. The majority of published research articles indicate that various sarcoma types have been shown MSCs' origin. Several group have reported spontaneous transformation in human and murine MSCs after long term culture [67–70]. For example, one study has reported that murine BM-MSCs are spontaneously transformed at passage 29 under standard conditions and that these transformed MSCs are able to generate fibrosarcoma in immunocompromised mice [70]. Accumulated chromosomal abnormalities, such as chromosome instability, chromosomal imbalances and aneuploidy, are suggested to be associated with the transformation of BM MSCs [70]. Indeed, chromosomal aberrations (chromosomal level) in *in vitro* cultures of human MSCs have been reported in previous studies, including human BM-MSCs after passage 4 [71], human AD-MSCs from passage 5 [72], and UC-MSCs from passage 5 [73].

There are also studies that have not detected the transformation of MSCs in long-term culture [74–76]. One previous study reports that human BM-MSCs do not undergo malignant transformation after long-term *in vitro* culture for up to 44 weeks and these cells maintain a normal karyotype [75]. In agree with the previous report [75], another study has described the occurrence of aneuploidy in cultivated human MSCs without evidence of transformation either *in vitro* or *in vivo* [76]. In addition, Røsland G.V. *et al* have reported that human BM-MSC spontaneous transformation phenomenon occurred in consequence of the cross-contamination between the transformed human MSCs and human cancer cells [77]. To date, there is no solid evidence for the transformation of different sarcoma subtypes from MSCs and it leaves an uncertainty for MSCs with the ability to spontaneously transform.
