ISSN Print 2713-2757
ISSN Online 2713-2765
Extreme Medicine
SCIENTIFIC AND PRACTICAL REVIEWED JOURNAL OF FMBA OF RUSSIA
ORIGINAL RESEARCH
In vitro assessment of immunogenicity in chondrocytes obtained from the B2M knockout induced pluripotent stem cells
About authors
1 Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of the Federal Medical Biological Agency, Moscow, Russia
2 Koltzov Institute of Developmental Biology of Russian Academy of Sciences, Moscow, Russia
Correspondence should be addressed: Anna A. Barinova
Malaya Pirogovskaya, 1а, 119435, Moscow, Russia; ur.liam@la.anna.avonirab
About paper
Funding: chondrocyte-like cells were obtained from iPSCs within the framework of the State Assignment #122032300191-2 “Organoid-22”. Immunocytochemical and PCR-based assessment of chondrogenic markers in the iPSC-derived chondrocytes, as well as assessment of immunogenicity of these chondrocyte-like cells were conducted within the framework of the RSF project #22-15-00250 “Comparison of chondrogenic potential of the cartilage tissue obtained using primary chondrocyte culture and using differentiated derivatives of induced pluripotent stem cells”.
Acknowledgements: the authors express their gratitude to M. Bogomyakova, L. Belikova, Laboratory of Cell Biology, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of FMBA of Russia, for assistance in planning and conducting the experiment.
Author contribution: Barinova AA — immunological tests, manuscript preparation and writing, search for literature; Pikina AS — collecting chondrocytes from the patient and iPSC differentiation into chondrocyte-like cells, immunohistochemistry; Golubinskaya PA — search for literature; Ruchko ES — conducting PCR; Eremeev AV — manuscript editing.
Compliance with ethical standards: the study was approved by the Ethics Committee of the Lopukhin Federal Research and Clinical Center of PhysicalChemical Medicine of FMBA of Russia (protocol № 2019/02 dated 09 April 2019).
Received: 2023-11-15
Accepted: 2024-01-15
Published online: 2024-02-05
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Fig. 1. Immunohistochemical assessment of monolayer cultures. The cell nuclei are stained blue, the studied markers are stained red and green. The scale bar size (shown in yellow) is 100 µm for iCh and iCh_dB2M, 1000 µm for iPS
Fig. 2. Relative expression of chondrocytic markers in the human chondrocyte lines (Chondro), B2M knockout (iCh_dB2M) (А) and wild-type chondrocyte-like cells (iCh) (B). * — significant differences in gene expression relative to human chondrocytes, р < 0.05, unpaired Student’s t-test
Fig. 3. Flow cytometry analysis of the CD8+ Т cell activation. А. Gating scheme. B. Percentage of activated CD8+ Т cells during co-cultivation with iPS (on the left) and iCh-dB2M (on the right). C. Comparative analysis of CD8+ Т cell activation after the 5-day co-cultivation with the target cell lines. D. Comparative analysis of CD8+ Т cell activation after the 24 h co-cultivation with the target cell lines. * — p < 0.05; * * — p < 0.01, unpaired Student’s t-test
Fig. 4. Comparative analysis of CD8+ Т cell degranulation after the 5 h co-cultivation with the target cell lines. Unpaired Student’s t-test, * — p < 0.05; * * — p < 0.01
Table 1. Использованные в работе праймеры
