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Gene-repaired iPS cells as new alternative for patient with OI type II

Poster · March 2023

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Patrycja Alicja Rozwadowska Anna Trybus

Medical University of Silesia in Katowice Medical University of Silesia in Katowice
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Agnieszka Fus-Kujawa Karolina Bajdak-Rusinek

Medical University of Silesia in Katowice Medical University of Silesia in Katowice
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 Gene-repaired iPS cells as new alternative for patient with OI type II
Patrycja Rozwadowska 1,2, Anna Trybus 1,2, Agnieszka Fus-Kujawa 1, Karolina Bajdak-Rusinek 1
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

This work was supported by the Polish National Science Centre contract no. UMO-2020/37/N/NZ2/01125.

BACKGROUND
Induced Pluripotent Stem Cells (iPSCs) were obtained in 2006 by Yamanaka's research team for the first time. The procedure involved delivery
of Yamanaka factors: Oct 3/4, Sox2, c-Myc, and Klf4 into somatic cells by retroviral transduction. iPSCs do not cause ethical controversies and
represent a single source of patient-specific somatic cells of any type. Gene repaired iPSCs by homologous recombination are a novel, valuable
tool for personalised gene therapy and regenerative medicine, as well as other medical areas. One of the potential gene therapy targets might
be Osteogenesis imperfecta (OI) type II, a lethal disease caused by collagen encoding gene mutation.

THE AIM Control cells Patient-derived cells

The aim of the study was to prove that gene-repaired iPSCs obtained Ectoderm
(TUJI)
from patients’ fibroblasts with Osteogenesis imperfecta (OI) type II are
capable of differentiation into functional cell lines: adipocytes, 12 days
osteoblasts and chondrocytes. Mesoderm
(SMA)
MATERIALS AND METHODS
12 days
In this trial, fibroblasts derived from a patient with OI type II have
been successfully reprogrammed into iPSCs using Yamanaka Endoderm
(AFP)
factors. Those cells were assigned to repair mutation in COL1A1 gene
23 days
by homologous recombination using star polymer as a carrier of
genetic material. Clones with repaired mutation were selected using Fig 3. State of iPSCs differentiation into 3 germ layers confirmation with β-III
gDNA sequencing. tubulin (Ectoderm), smooth muscle actin (Mesoderm) and α-fetoprotein
del TGGTGCTCC
(Endoderm).
g:18047-18055
Fibroblasts with OI mutation Control Control

Reference sequence
Substitution g:18062 C/T
NG_007400.1
Repaired substitution g:18062 C/T
CD73 CD105

iPSCs with repaired mutations DNA samples 100

DNA with repaired mutation 79
DNA with non-repaired mutation 15
Positive control 6 CD90

iPSCs without repaired mutations

Fig 1. Selection of clones with repaired mutation.

iPSCs show 90% viability after treatment with star polymers. The Fig 4. Flow cytometry analysis confirming differentiation into MSCs.
pluripotency state has been confirmed using specific markers: Tra-1- Mesenchymal Stem Cells were succesfully differentiated into
60 and SSEA-4. State of iPSCs differentiation into 3 germ layers has adipocytes, osteoblasts and chondrocytes. The cells were stained
been confirmed with β-III tubulin (Ectoderm), smooth muscle actin with Oil Red O, Alizarin Red S and Alcian blue, respectively in order to
(Mesoderm) and α-fetoprotein (Endoderm). Gene-repaired iPSCs confirm the phenotype.
Osteoblasts Adipocytes
Adipocytes Chondrocytes
were differentiated into Mesenchymal Stem Cells. The
immunophenotype was confirmed in flow cytometric analysis using
specific Mesenchymal Markers: CD73, CD90 and CD105. Control

RESULTS

Tra1-60 SSEA-4
Control Patient-derived cells Control Patient-derived cells
Repaired
iPSCs

Fig 5. In the last step cells were stained on day 14 in order to confirm
conclusively cell differentiation. Alizarin Red S, Oil Red O and Alcian Blue stains
were used.
The results of this experiment show that there is a possibility to
induce pluripotent state in a patient's somatic cells using Yamanaka
factors. These cells show expression of pluripotency markers and,
Fig 2. The pluripotency state confirmation with markers: Tra-1-60 and SSEA-4 after mutation repair, are able to differentiate into more specific cell
on day 19. lines.

CONCLUSIONS
This study is a proof of concept that it is possible to obtain functional cell lines from gene-repaired iPSCs. These cells may have potential in
gene therapy as model of genetic diseases and in vivo therapies. Further studies are required to confirm immunocompatibility and the
efficiency of iPSCs delivery in vivo for future clinical use.
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