Gene amplification is a genetic process where the copy number of certain genes increases, without a proportional increase of the genome. Gene amplification is not only a hallmark of genetic alterations in tumor cells but is also well described and studied since decades as physiological mechanism in amphibians and flies. For example, during the development of Drosophila melanogaster and Xenopus laevis, specific DNA regions are amplified within defined developmental time windows (1).

There is growing evidence that gene amplification plays a physiological role during differentiation of stem and progenitor cells. Previously we described gene amplification during differentiation of mesenchymal stem cells towards osteoblasts and adipocytes (2). Interestingly, gene amplifications that arise during stem cell differentiation often overlap with those found in the corresponding tumor types. For example, the genes CDK4 and MDM2 were shown to undergo co-amplification during the differentiation of mesenchymal stem cells (MSCs) into adipocytes, mirroring the high-level amplifications characteristic of liposarcoma (3). Similarly, several studies on chondrosarcoma have reported MYC gene amplification on chromosome 8, with array-CGH analyses demonstrating that MYC amplification occurs more frequently in higher-grade and dedifferentiated chondrosarcomas (4,5). In addition, genomic profiling of chondrosarcoma revealed several amplified chromosome regions including regions on chromosomes 5, 12, 19 and 22(6). Notably, amplification of CDK4 without accompanying MDM2 co-amplification has also been documented in at least one chondrosarcoma case, further underscoring the heterogeneity and potential clinical relevance of these genetic events (7).

Mesenchymal stem cells (MSCs) possess significant potential for therapeutic applications in tissue regeneration. One of the most ambitious investigations is probably regeneration of articular cartilage since many health restrictions are based on cartilage destruction (8-10). Understanding both the regenerative capacity and the potential risks caused by genetic changes that are associated with directing hMSCs toward a chondrogenic lineage is therefore essential. Here we set out to investigate whether gene amplifications are detectable during chondrogenic differentiation of human mesenchymal stem cells (hMSCs).

HMSCs were differentiated in U-bottom 96-wells and differentiation was confirmed by toluidine blue staining and RNA-seq analysis of chondrogenic differentiation specific genes SOX9, COL2A1 and ACAN (17,18). Already at 5 days post differentiation induction cells revealed three-dimensional cell growth as spheres and high intensity of proteoglycan specific toluidine blue staining (Fig. 1). Expression analysis of two independent biological replicates revealed an increase of SOX9 expression after 2 days and a further increase after 7 days of differentiation and an increase of COL2A1 and ACAN expression after 5 and 7 days of differentiation (Fig. 2). To detect gene amplifications, we analyzed bulk DNA from hMSCs differentiated toward chondrocytes for 1, 2, 5, and 7 days, as well as from undifferentiated hMSCs serving as controls. Amplification was assessed by quantitative PCR (TaqMan) in four replicates, and data were evaluated using the CopyCaller software (Applied Biosystems).

For our investigation we selected genes previously found to be amplified during osteoblast and adipocyte differentiation and genes that were known to be amplified in tumors derived from chondrocytes including genes on chromosome 12, chromosome 22 and chromosome 8 (2,6). Copy number was determined for genes AGAP2, CDK4 and MDM2 localized on chromosome 12, CPT1B and SHANK3 localized on chromosome 22 and TRIB1 and MYC localized on chromosome 8. As shown in Fig. 3 after 1 day of differentiation none of the investigated genes revealed a copy number increase. After 2 days of differentiation CDK4, CPT1B and SHANK 3 revealed a 60% copy number increase, AGAP2 and TRIB1 exhibited a 20% copy number increase; MDM2 and MYC remained unchanged (Fig. 3). After 5 days of differentiation AGAP2 and MDM2 displayed increases, whereas CDK4, CPT1B and SHANK3, which were highest at 2 days, decreased and MYC still remained unchanged. After 7 days of differentiation CDK4, CPT1B, SHANK3, and TRIB1 decreased further, AGAP2 increased, and no further copy number change was detected for MDM2. MYC showed no change throughout the 7-day differentiation period.

Previous published investigations on gene amplification during differentiation revealed a high heterogeneity of gene amplification between single cells. During osteoblast differentiation for example, cells with high level amplification were detected next to cells with normal copy number and during neural differentiation composition and copy number of co-amplified genes varied between neighboring cells (2,19). One possible reason for the differences in gene copy numbers is differences in the degree of differentiation upon differentiation induction. After five days upon differentiation induction, we performed laser microdissection on hMSCs after toluidine blue staining for detection of proteoglycan production that is indicative for chondrocyte differentiation (Fig. 4A-D). Toluidine blue stain revealed heterogenous blue intensities with areas of grey (Fig. 4A), light blue (Fig. 4B), blue (Fig. 4C) and dark blue (Fig. 4D) staining as result of different amounts of produced proteoglycans indicating different stages of differentiation.

In total we laser microdissected 20 areas for each blue intensity as shown in Fig. 4A-D. Although the laser microdissected regions were comparable in size the amount of isolated DNA varied between 1.8 ng for grey and light blue areas and 6 ng for blue and dark blue areas. Copy analysis was done by TaqMan qPCR with 250 pg laser microdissected DNA for each experiment. Analyses were performed using three biological replicates, each with two technical replicates, and undifferentiated hMSC DNA served as the control. Copy number variations for AGAP2, CDK4, CPT1B, TRIB1, and SHANK3 are shown in Fig. 4E, and corresponding calculated P-values were summarized in Tables I and II. A significantly increased CDK4 copy number was observed in cells from grey and light blue regions, corresponding to absent or low proteoglycan content. CPT1B copy number was significantly elevated in blue regions, indicative of proteoglycan deposition. In addition, an increase in SHANK3 copy number was detected in blue regions, reflecting higher proteoglycan levels. Interestingly, dark blue areas with highest proteoglycan levels revealed no significant copy number increase for the investigated genes. AGAP2 showed no copy number increase in the laser microdissected probes for all staining intensities. TaqMan analysis of MDM2 revealed no results for technical reasons. Unfortunately, the very low amount of DNA from microdissected areas is a limitation of this study since chondrocyte marker analysis was not possible.

Gene amplification has long been associated with tumorigenesis and multidrug resistance, although its physiological role was first recognized in Drosophila melanogaster during eggshell development (20,21). Here, we identified gene amplifications in hMSCs during chondrogenic differentiation. Previous fluorescence-in-situ-hybridization (FISH) analyses of hMSCs differentiating into adipocytes and osteoblasts revealed heterogenous populations, with amplified and non-amplified cells coexisting (2). However, FISH analysis proved challenging for chondrocyte differentiation due to the three-dimensional growth pattern and proteoglycan production, which interfere with FISH. Here, we investigated gene amplification during the first seven days of chondrocyte differentiation using qPCR and Taq Man assays targeting genes previously shown to be amplified during osteoblast and adipocyte differentiation, as well as genes that were known to be amplified in chondrosarcoma (2,6).

DNA sequence amplification, as defined by Bostock CJ in 1986, refers to any event that increases a gene's copy number per haploid genome above the level characteristic for that organism (1). In accordance with a criterion defined in our recent study (22), qPCR-derived copy number values exceeding 2.3 were considered indicative of gene amplification. To distinguish gene amplification from whole-chromosome duplication, we included genes located on the same chromosomes (AGAP2, CDK4, and MDM2 on chromosome 12; CPT1B and SHANK3 on chromosome 22; and TRIB1 and MYC on chromosome 8).

During chondrocyte differentiation, qPCR analysis revealed amplification of several investigated genes at days 2 and 5, followed by a decline by day 7. To assess stage-specific amplification, we performed laser microdissection of toluidine blue-stained areas after five days of differentiation, when proteoglycan deposition was evident. Cells were grown on PET membrane slides, enabling precise isolation of regions with varying staining intensities.

Amplification patterns differed across differentiation stages: CDK4 showed the highest copy number in regions lacking and low proteoglycans, indicating early activation, whereas SHANK3 peaked in areas with strong proteoglycan staining, suggesting stage-specific amplification. Regions with the most intense staining displayed no significant amplifications for all tested genes, consistent with cessation of amplification during terminal differentiation. Although AGAP2 increased in bulk DNA at days 5 and 7, it was undetectable in microdissected samples, and MDM2 yielded no results, likely due to technical limitations of microdissection and DNA isolation. A possible reason for failure of MDM2 qPCR amplification could be the very low amount of DNA per reaction (250 pg) and degradation effects during laser microdissection and DNA isolation. In this respect we would like to mention that gene amplification analysis using microdissected samples is a starting point to address gene amplifications in a heterogenous cell population but has limitations because of low DNA yield and degradation during laser exposure. Future studies need to be expanded to increase DNA yield and to have the possibility to test more genes on gene amplification including chondrocyte marker genes and additional genome regions. Furthermore, although CPT1B and SHANK3 are both located on chromosome 22, their distinct amplification patterns across microdissected regions argues against whole-chromosome duplication and instead support gene-specific amplification events.

Chondrosarcoma development is closely linked to the dysregulation of normal chondrogenic differentiation pathways. Chondrosarcoma is increasingly viewed as a disease in which molecular alterations accumulate along the differentiation trajectory of chondrogenic cells rather than arising independently of cartilage development. During early differentiation mutations alter proliferation and differentiation of chondrocytes and very likely support malignant transformation. Those early mutations were supposed to alter the IHH/PTHrP and IDH1/IDH2 pathways (23). Late-stage mutations were suggested to involve cell cycle regulating pathways including p53 and RB (23). As early as 1980 Kreicbergs et al (24) found that many highly malignant and metastasizing chondrosarcoma were hyperploid. In this respect it is interesting that we found CDK4 amplifications predominantly during early differentiation stages and no significant gene amplifications during the latest differentiation stage. So early differentiation associated mutations can lead to unscheduled CDK4 amplification with known growth advantages whereas late differentiation mtations especially TP53 mutations probably can disturb proper cease of gene amplification and lead to genome instability.

In addition, for normal chondrocyte differentiation of mesenchymal stem cells, a precise timing of induction and silencing of SOX9 expression is necessary (25). Here we found an increasing SOX9 expression during differentiation with highest SOX9 expression after 7 days. Posttranscriptional regulation of SOX9 expression through altered miR-145 expression was described as epigenetic regulation of SOX9 in human chondrosarcoma (26). We can speculate if this regulation occurs late during differentiation maybe a loss of SOX9 gene function thereby leads to additional genome instability, polyploidy and further promotes tumorigenesis.

Despite tumor development chondrocyte differentiation may also affect Osteoarthritis (OA) and extracellular matrix (ECM) dysregulation. OA is mainly a disease of extracellular matrix (ECM) imbalance (27). Therefore, any gene affecting chondrocyte proliferation and metabolism of ECM proteins, could potentially contribute to disease susceptibility or progression.

Chondrocytes were the main producer of ECM proteins. During OA progression chondrocytes secrete inflammatory cytokines that were involved in disruption of cartilage homeostasis and cartilage degradation (28). Several studies report an association between OA and fibrosis (29). During normal chondrocyte differentiation CDK4 amplification likely necessary for regulating the balance between proliferation and differentiation. Since CDK4 gene amplification is no more detectable in late differentiation stages and as mentioned above for chondrosarcoma development mutations can lead to growth advantage of cells with unscheduled CDK4 amplification resulting in proliferation of chondrocytes and imbalances of ECM proteins and inflammatory cytokines.

CPT1B is a rate-limiting enzyme for mitochondrial fatty-acid β-oxidation and was recently shown to be highly expressed in myoblasts of OA patients (30). During normal differentiation CPT1B amplification is probably necessary for an enhanced metabolism and for OA development the involvement is still unknown. SHANK3 is known as a postsynaptic scaffold protein in neurons and an involvement in normal cartilage development is unknown. We can only speculate that during normal chondrocyte differentiation SHANK3 is necessary for cytoskeletal organization or cell-matrix communication but a functional role in OA development remains unclear.

In conclusion, we observed in both bulk and microdissected analyses that gene amplification is an early, transient process during chondrogenic differentiation, diminishing as cells mature-likely to preserve genomic stability. Since this study has limitations, further orthogonal validation methods including single cell analysis were needed. Future studies should explore mechanisms that regulate amplification cessation and their implications for regenerative therapies. Nevertheless, the current work provides valuable insights into advantages and capabilities including risks of these cells.