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Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes

  • Authors:
    • Paula E. Schwarz
    • Magali Cucchiarini
    • Shusruto Rishik
    • Andreas Keller
    • Eckart Meese
    • Ulrike Fischer
  • View Affiliations / Copyright

    Affiliations: Department of Human Genetics, Saarland University, D‑66421 Homburg, Saarland, Germany, Center of Experimental Orthopedics, Saarland University Medical Center, D‑66421 Homburg, Saarland, Germany, Chair of Clinical Bioinformatics, Saarland University, D‑66123 Saarbrücken, Saarland, Germany
    Copyright: © Schwarz et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 346
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    Published online on: October 23, 2026
       https://doi.org/10.3892/etm.2026.13341
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Abstract

For decades, gene amplification was described as an attribute of tumor cells and as a physiological mechanism to increase gene copy numbers for the higher protein demand during the development of amphibians and flies. Notably, gene amplification has since been detected in normal mammalian cells during differentiation. Numerous amplified genes detected in tumor cells overlap with amplified genes detected during stem cell differentiation. Since stem cells have a valuable potency in regenerative therapies and since cartilage regeneration is a highly demanded therapeutic strategy, the current study investigated gene amplification dynamics during the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). Using quantitative PCR, copy number changes were analyzed for genes previously implicated in differentiation, as well as genes amplified in chondrosarcoma, including CDK4, MDM2, AGAP2, CPT1B, SHANK3, TRIB1 and MYC. Amplifications were transient and stage‑specific: CDK4, CPT1B and SHANK3 exhibited the highest copy number increases at day 2, followed by a gradual decline by day 7, whereas AGAP2 and MDM2 increased later in differentiation. Laser microdissection of toluidine blue‑stained areas revealed heterogeneity in amplification patterns: CDK4 amplification was prominent in areas lacking or showing moderate proteoglycan deposition; CPT1B amplification occurred in regions with absent, moderate or intense proteoglycan deposition; and SHANK3 amplification was restricted to areas with intense proteoglycan deposition. Notably, regions with the strongest proteoglycan staining exhibited no gene amplification, suggesting that gene amplification is an early, transient event that diminishes as differentiation progresses. These findings highlight gene amplification as a mechanism during chondrogenesis, potentially critical for early differentiation stages and genome stability in mature cells.
View Figures

Figure 1

Proteoglycan and spheroid formation
during chondrocyte differentiation. Human mesenchymal stem cells
were differentiated in U-bottom wells, and spheroid formation
together with proteoglycan deposition using toluidine blue staining
was monitored over 14 days. (A) Proteoglycan staining by toluidine
blue was detectable as early as day 2 in the absence of spheroid
formation. (B) Spheroid formation became evident by day 5. Strong
toluidine blue staining was observed in all spheroids at days (C) 7
and (D) 14. (E) An unstained spheroid at day 14 is shown as a
reference. Images were taken at 20x magnification.

Figure 2

Expression of SOX9,
COL2A, and ACAN during chondrocyte differentiation.
RNA-seq data from two biological replicates (shown as 1. and 2. on
the left) are visualized using Integrative Genomics Viewer. (A)
Undifferentiated hMSCs exhibited minimal SOX9 expression;
levels increased at days 2 and 5 and reached a maximum at day 7,
consistent with chondrocyte differentiation. (B) COL2A1 and
(C) ACAN expression were undetectable at day 2 but became
apparent at day 5 and peaked at day 7, further confirming
successful chondrogenic differentiation. X-axis indicates
chromosome localization in Mb and y-axis RNA-seq results
represented as read-height.

Figure 3

Descriptive amplification analysis of
AGAP2, CDK4, MDM2, CPT1B,
SHANK3, TRIB1 and MYC using qPCR during
chondrogenic differentiation. Amplification of AGAP2,
CDK4, MDM2, CPT1B, SHANK3, TRIB1
and MYC was analyzed by qPCR using TaqMan copy number assays
and RNAseP was used as reference TaqMan assay. DNA from
undifferentiated hMSCs (CTRL) served as standard for normal diploid
copy number with copy number values indicated on the y-axis and
normal diploid copy number is referred as 2. Copy numbers are shown
as mean from four technical replicates from one experiment (3
pooled spheroid samples) with vertical lines indicating the range.
Chondrogenic-induced hMSC cells were analyzed at four time points
after differentiation induction (1, 2, 5 and 7 days). CPT1B
and SHANK3 revealed the highest copy numbers of all
investigations after 2 days of differentiation and a decrease until
7 days. CDK4 revealed also a high copy number after 2 days
of differentiation induction (green) and a decrease until 7 days.
TRIB1 revealed a copy number increase only 2 days after
differentiation induction. AGAP2 and MDM2 revealed a
copy number increase only after 5 and 7 days. MYC revealed
no copy number increase at any time point after differentiation
induction. qPCR, quantitative PCR.

Figure 4

Amplification analysis of
AGAP2, CDK4, CPT1B, SHANK3 and
TRIB1 after laser microdissection. hMSCs were differentiated
towards chondrocytes for 5 days on PET frame slides for laser
microdissection. Cells were seeded at high density, and slide was
stained with toluidine blue for proteoglycan detection during
differentiation. Different areas of staining intensity could be
detected and were defined as areas with (A) grey cells, (B) light
blue cells, (C) blue cells and (D) dark blue cells, all indicating
stages during the differentiation towards chondrocytes. In total
for each area type twice 20 areas were laser microdissected and
used for DNA isolation. Experiments were done in three biological
replicates. Examples of laser microdissected areas are circled in
white and scale bars represent 150 µm. (E) Results of amplification
analysis of our quantitative PCR using TaqMan copy number assays
and RNAseP as reference are shown as diagram with
AGAP2, CDK4 and CPT1B SHANK3 and TRIB1.
DNA from undifferentiated hMSCs (CTRL) served as standard for
normal diploid copy number with copy number values indicated on the
y-axis and normal diploid copy number is referred as 2. Copy
numbers are presented as means of two technical replicates across
three biological replicates (N=6), with error bars representing
standard deviation. *P<0.05. CDK4 revealed the
highest copy number in areas with grey and light blue cells
indicating gene amplification at the beginning of differentiation.
CPT1B revealed high copy numbers in grey, light blue and
blue cells indicating gene amplification during differentiation and
SHANK3 revealed the highest copy numbers in areas with blues
cells indicating gene amplifications during later differentiation
progression. AGAP2 and TRIB1 revealed no copy number
increase. In areas with dark blues cells no significant
amplification was detectable indicating a reduction of gene
amplification towards final differentiation. hMSC, human
mesenchymal stem cell.
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Copy and paste a formatted citation
Spandidos Publications style
Schwarz PE, Cucchiarini M, Rishik S, Keller A, Meese E and Fischer U: Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes. Exp Ther Med 32: 346, 2026.
APA
Schwarz, P.E., Cucchiarini, M., Rishik, S., Keller, A., Meese, E., & Fischer, U. (2026). Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes. Experimental and Therapeutic Medicine, 32, 346. https://doi.org/10.3892/etm.2026.13341
MLA
Schwarz, P. E., Cucchiarini, M., Rishik, S., Keller, A., Meese, E., Fischer, U."Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes". Experimental and Therapeutic Medicine 32.6 (2026): 346.
Chicago
Schwarz, P. E., Cucchiarini, M., Rishik, S., Keller, A., Meese, E., Fischer, U."Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes". Experimental and Therapeutic Medicine 32, no. 6 (2026): 346. https://doi.org/10.3892/etm.2026.13341
Copy and paste a formatted citation
x
Spandidos Publications style
Schwarz PE, Cucchiarini M, Rishik S, Keller A, Meese E and Fischer U: Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes. Exp Ther Med 32: 346, 2026.
APA
Schwarz, P.E., Cucchiarini, M., Rishik, S., Keller, A., Meese, E., & Fischer, U. (2026). Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes. Experimental and Therapeutic Medicine, 32, 346. https://doi.org/10.3892/etm.2026.13341
MLA
Schwarz, P. E., Cucchiarini, M., Rishik, S., Keller, A., Meese, E., Fischer, U."Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes". Experimental and Therapeutic Medicine 32.6 (2026): 346.
Chicago
Schwarz, P. E., Cucchiarini, M., Rishik, S., Keller, A., Meese, E., Fischer, U."Gene amplification during differentiation of mesenchymal stem cells towards chondrocytes". Experimental and Therapeutic Medicine 32, no. 6 (2026): 346. https://doi.org/10.3892/etm.2026.13341
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