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CD44 alternative splicing profile - a novel molecular basics discovery of somatic cells reprogramming

Poster · March 2023

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Anna Trybus Patrycja Alicja Rozwadowska

Medical University of Silesia in Katowice Medical University of Silesia in Katowice
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 CD44 alternative splicing profile - a novel molecular basics discovery of somatic cells
reprogramming
Anna Trybus 1,2, Patrycja Rozwadowska 1,2, Gabriela Bronkowska 1,2
1 Department of Medical Genetics, Faculty of Medical Sciences in Katowice, Medical University of Silesia, Katowice, 18 Medyków Street, 40-752 Katowice, Poland
2 Students Scientific Society, Faculty of Medical Sciences in Katowice, Medical University of Silesia, Katowice, Poland

INTRODUCTION RESULTS
Induced pluripotent stem cells (iPSCs) are the products of A) PDF B) iPSC colony
reprogramming somatic cells to an embryonic-like state. The process
of transduction into iPSCs is not yet sufficiently understood
molecularly to be fully controlled. The differentiation marker for
partially and fully reprogrammed cells is an adhesion protein, CD44,
which is subject to alternative splicing. The functions performed by the
different CD44 isoforms in pluripotency are not fully understood. It has 200μm 200μm 200μm

already been recognized that changes in the pattern of alternative Fig. 3. Morphology of A) Primary Dermal Fibroblasts and B) iPS cells on day
splicing of various factors have a significant impact on the process of 35, passage 12. Scale bars = 200 µm.
obtaining iPSCs. Learning the expression profile of the different CD44
Undifferentiated A) Osteogenesis B) Chondrogenesis C) Adipogenesis
variants will allow a better understanding of the mechanisms occurring
during cellular reprogramming and may contribute to a better
efficiency of obtaining fully reprogrammed iPSCs. This is particularly
important for diseases for which no effective treatments have been
developed and are considered incurable. 200μm 200μm 200μm 200μm

Fig. 4. Confirmation of iPSCs ability to osteogenesis, adipogenesis and

A) chondrogenesis with staining: A) Alizain Red S, B) Alcian blue C) Oil Red O on day 14.

RT-PCR results
Relative mRNA values showed that fibroblasts express high levels of
B) CD44 total.

Fig 1. Human CD44 structure. This protein is encoded by a single gene, which in
humans consists of 20 exons A) pre-mRNA, B) CD44s mRNA.

OBJECTIVES
The aim of this study was to analyse CD44 variants expression profile
in the process of somatic cells reprogramming into iPSCs, in order to
understand molecular aspects of this process. Fig. 5. PDF express high levels of CD44 mRNA relatively to iPSCs.

MATERIALS AND METHODS Interestingly, predominantly standard isoform (CD44s) was expressed
Cells in fibroblasts, whereas mainly other variants (CD44v2-v10) were
Cells were cultured in Dulbecco’s Modified Eagle’s Medium. present in iPSCs.
Furthermore, fibroblasts were incubated in Mouse Embryonic
Fibroblast Conditioned Medium, serum free medium for the feeder
independent culture of human pluripotent stem cells, and were
assigned to be reprogrammed into iPSCs by introduction of Thomson
factors in form of proteins, with STAR polymer as carrier.

RNA isolation and quantitative RT-PCR

Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen, Hilden,
Germany, 74136) according to the manufacturer's protocol. cDNA was
synthesized from 1 µg RNA with the RevertAid First Strand cDNA
Synthesis Kit (Thermo Scientific, Karlsruhe, Germany, K1621) according
to the manufacturer's instructions. Relative expression levels were
measured in triplicates in a Roche Light Cycler 480 using Power SYBR
Green PCR Master Mix (Applied Biosystems, Darmstadt, Germany,
4368702), 300 µM primers (Fig 2.) and 1/15 cDNA stock. Values were Fig. 6. CD44s variant dominates in fibroblasts (PDF), while showing low expression in
calculated using the Pfaffl method and normalized to those of ACTB. iPSCs. Other variants dominate in iPSCs despite low CD44 total expression level,
Target mRNA Forward primer Reverse primer
suggesting their possible role in pluripotency.
ACTB 5’-GCCCTGAGGCACTCTTCCA-3’ 5’-TTGCGGATGTCCACGTCA-3’

CD44 total 5’-ATAATTGCCGCTTTGCAGGTGTATT-3’ 5’-ATAATGGCAAGGTGCTATTGAAAGCCT-3’

CONCLUSIONS, DISCUSSION
CD44s 5’-ATAATAAAGGAGCAGCACTTCAGGA-3’ 5’-ATAATTGTGTCTTGGTCTCTGGTAGC-3’
CD44v2
These results indicate a significant difference in the profile of CD44
5’-ATAATCAGCAACTGAGACAGCAACCAA-3 5’ ATAATAACCAATCCCAGGTTTCTTGCC-3’

CD44v3 5’-ATAATGGCTGGGAGCCAAATGAAGAAA-3’ 5’-ATAATCATCATCATCAATGCCTGATCCAGA-3’

variants in fibroblasts and iPSCs. These findings represent a novel
CD44v4
5’-ATAATCAGTGGAACCCAAGCCATTCAA-3’ 5’-ATAATCCTTGTGGTTGTCTGAAGTAGCAC-3’
discovery of the molecular basis of reprogramming that has not been
CD44v5 5’-ATAATGAAACTGGAACCCAGAAGCACA-3’ 5’-ATAATTGATGCTCATGGTGAATGAGGG-3’
previously described. The research work presented here suggests that
CD44v6
5’-ATAATCAGAAGGAACAGTGGTTTGGCA-3’ 5’-ATAATGTCTTCTTTGGGTGTTTGGCGA-3’
the CD44 protein and its variants should be considered in the
CD44v7 5’-ATAATTGCAAGGAAGGACAACACCAAG-3’ 5’-ATAATGGGTGTGAGATTGGGTTGAAGA-3’
development of more effective control of cell differentiation.
CD44v8
5’-ATAATACGCTTCAGCCTACTGCAAA-3’ 5’-ATAATAAGAGGTCCTGTCCTGTCCAAA-3’;
However, further studies are needed to confirm the role of alternative
CD44v9
CD44 splicing in somatic cell reprogramming, especially in the context
5’-ATAATGAGCTTCTCTACATCACATGAAGGC-3’ 5’-ATAATGTCAGAGTAGAAGTTGTTGGATGGTC-3’

CD44v10
of differences between somatic and pluripotent cells.
5’-ATAATACCTCTCATTACCCACACACGA-3’ 5’-ATAATTAGCTGAGGTCACTGGGATGAA-3’.

Funded by Polish Ministry of Education and Science

Fig. 2. Primers used in RT – PCR for CD44 variants' mRNA. (Contract Number SKN/SP/533512/2022)
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