Final report

GENETIC AND EPIGENETIC MECHANISMS INVOLVED IN STEM CELL SENESCENCE

SENSTEM

FINAL REPORT

Reporting period: 13/05/2022 - 12/04/2024

PN-III-P1-1.1-TE-2021-0864, TE no. 19 ⁄ 2022

Senolytics are a class of drugs designed to specifically target senescent cells and to interfere with the anti-apoptotic pathways of senescent cells, allowing the cells to undergo apoptosis, having great potential for understanding and treating diseases associated with aging. In this project we treated stem cells with 2 senolytic drugs spermidine and metformin to identify the genetic and epigenetic changes that occur in senescent stem cells.

Our results showed that cellular senescence is controlled by epigenetic mechanisms involving the methyltransferases DNMT1, ATF2, KAT1, CREB2, HAT1 and KAT2A; histone methyltransferases EHMT2 and KMT2A; proteins with a role in the phosphorylation of histones AURKB and RPS6KA3, those with a role in the ubiquitination of histones UBE2A and USP16; histone demethylases KDM1A, and MBD2 and histone deacetylases HDAC1, HDAC4, HDAC7.

Signaling pathway analysis showed that miR-22-3p, miR-92a-3p, miR-196a-5p, miR-24-3p, miR-100-5p, miR-103a-3p, miR-23b-3p, miR-15b-5p identified in senescent stem cells showed their involvement in processes such as senescence, cell death, cell cycle regulation, apoptosis regulation, G1-S transition regulation.

Genes involved in cell cycle regulation, such as p16INK4A, eNOS, and cytoskeletal elements, such as β-actin, showed increased expression in senescent stem cells, while HMGB1, cMyc, Bcl2 showed a slight increase, and p21, and MDM2 showed a low expression, suggesting that the main signaling pathway in senescent stem cells is the p16INK4A pathway.

Metformin and spermidine can be used as senolytic agents, our results demonstrated that are capable to reduce the SASP phenotype (Senescence Associate Secretory Phenotype) by decreasing the inflammatory proteins such as IL-1α, IL-6, IL-8, CXCR2, IGF and IFN.

Dissemination of results

• National and international conferences and congresses

1. The Biochemistry Global Summit, 7-14 July 2022, Lisboan, Portugal. Molecular and electrophysiological profile of induced stem cell senescence. F. Iordache, A. Ionescu, L. Stanca, C. Curutiu, C.M. Istrate, A.I. Serban.
2. 47th Congress of the European Federations of Biochemistry "47th FEBS Congress", Tours, France, 08 -12 July 2023. PCR array profiling of senescent amniotic mesenchymal stem cells. F. Iordache, A. Petcu, L. Stanca, A.M. Pisoschi, O. GeicuI L. Bilteanu, A.I. Serbian.
3. Conference of the Romanian Electron Microscopy Society - C.R.E.M.S., October 18-21, 2023, Cluj-Napoca, Romania. Morphological changes induced by senescence in amniotic fluid stem cells. Florin Iordache, Adriana (Ionescu) Petcu.
4. Simpozion “Academicina Nicolae Cajal” 28-29 March 2024, The Romanian Academy Library, Bucharest, Romania. Epigenetic interactions involved in differentiation and senescence of stem cells. Florin Iordache, Adriana Petcu, Loredana Stanca, Aurelia Magdalena Pisoschi, Ovidiu Geicu, Liviu Bilteanu, Andreea Iren Serban.

• Scientific papers

1. Iordache, F.; Petcu, A.; Pisoschi, A.M.; Stanca, L.; Geicu, O.I.; Bilteanu, L.; Curuțiu, C.; Amuzescu, B.; Serban, A.I. PCR Array Profiling of miRNA Expression Involved in the Differentiation of Amniotic Fluid Stem Cells toward Endothelial and Smooth Muscle Progenitor Cells. Int. J. Mol. Sci. 2024, 25, 302. https://doi.org/10.3390/ijms25010302. (Impact factor = 5.6).
2. F. Iordache, A. (Ionescu) Petcu, A. Magdalena Pisoschi, L. Stanca, O.I. Geicu, L. Bilteanu, C. Curuțiu, A. I. Serban. Epigenetic interactions involved in senescence of stem cells. International Journal of Molecular Sciences, in press. (Impact factor = 5.6).

Scientific report II / 2023

SCIENTIFIC REPORT

STAGE No. 2

Reporting period: 11/12/2022 - 10/12/2023

PN-III-P1-1.1-TE-2021-0864, TE no. 19 ⁄ 2022

Genetic and epigenetic mechanisms involved in stem cell senescence

SENSTEM

Scientific report II /2023

Epigenetic changes play a significant role in stem cell fate determination of AFSC in terms of senescence and differentiation into multiple lineages. There is a close correlation between DNA methylation, histone modifications and non-coding miRNAs during the epigenetic control of stem cell differentiation; however, until now, the precise mechanisms are not yet clear. The objective of stage no. 2 "Discovery and validation of new epigenetic markers in senescent AFSC" had the following activities:

• Activity 2.1 Isolation and quantification of DNA from senescent and non-senescent AFSC.
• Activity 2.2 Microarray data analysis, interpretation, and dissemination of results.
• Activity 2.3 Validation of the new senescence markers and dissemination of the results.
• Activity 2.4 Project management and dissemination of results.

Following these activities, the array results showed a significant increase in the DNA methyltransferases DNMT1, ATF2, KAT1, CREB2, HAT1 and KAT2A in senescent cells. Histone methyltransferases EHMT2 and KMT2A also showed a significant increase in senescent cells. The proteins involved in the phosphorylation of histones AURKB and RPS6KA3, those involved in the ubiquitination of histones UBE2A and USP16, histone demethylases KDM1A and MBD2, and histone deacetylases HDAC1, HDAC4, HDAC7 increased in senescent AFSCs.

The analysis of signaling pathways showed that, after applying the differentiation and senescence protocols, the expression of miRNAs was different, EPC expressing 9 specific miRNAs (hsa-miR-125a-5p, hsa-miR-125b-5p, hsa-miR-181a -5p, hsa-miR-103a-3p, hsa-miR-129-5p, hsa-miR-210-3p, hsa-let-7c-5p, hsa-let-7a-5p, hsa-let-7d-5p), SPC only 2 specific miRNAs (has-miR-20a-5p, has-miR-155-5p) and 7 miRNAs (miR-92-3p, miR-196a-5p, miR-100-5p, miR-103a- 3p, miR-23b-3p, miR-375-3p, miR-15b-5p) were expressed in senescent cells.

Dissemination of results PHASE II /2023

National and international conferences and congresses:

1. 47th Congress of the European Federations of Biochemistry "47th FEBS Congress", Tours, France, 08 -12 July 2023. PCR array profiling of senescent amniotic mesenchymal stem cells. F. Iordache, A. Petcu, L. Stanca, A.M. Pisoschi, O. GeicuI L. Bilteanu, A.I. Serbian.
2. Conference of the Romanian Electron Microscopy Society - C.R.E.M.S., October 18-21, 2023, Cluj-Napoca, Romania. Morphological changes induced by senescence in amniotic fluid stem cells. Florin Iordache, Adriana (Ionescu) Petcu.

Scientific papers:

1. F. Iordache, A. (Ionescu) Petcu, A. Magdalena Pisoschi, L. Stanca, O.I. Geicu, L. Bilteanu, C. Curuțiu, B. Amuzescu, A. I. Serban. PCR array profiles the expression of miRNAs involved in differentiation of amniotic fluid stem cells towards endothelial and smooth muscle progenitor cells. International Journal of Molecular Sciences, in press.

Scientific report I / 2022

Executive Unit for the Financing of Higher Education, Research, Development and Innovation (UEFISCDI)

PN-III-P1-1.1-TE-2021-0864, nr. TE 19 ⁄ 2022

May 2022-April 2024 

PROJECT TITLE

Genetic and epigenetic mechanisms involved in stem cell senescence (SENSTEM)

Scientific report  I / 2022

Senescent cells are characterized by a secretory phenotype that exerts pleiotropic effects in the surrounding tissue, disrupting tissue homeostasis. In addition to DNA damage, excessive mitogenic signaling is the other major stressor associated with senescence.

The objective of phase I of the project was "Obtaining and characterizing senescent cells from stem cells isolated from amniotic fluid (AFSC)" and had the following activities:

Activity 1. Cultivation of AFSCs in specific cell media to obtain a homogeneous population of mesenchymal stem cells. Obtaining mesenchymal stem cells from amniotic fluid (AFSC) was achieved by centrifuging the amniotic fluid for 10 min at 400xg and cultivating the sediment in BIOAMF-2/AmnioMax culture media, supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. After 5-7 days the medium was changed to remove non-adherent cells, and then the medium was changed 2 times a week. When the cells reached confluence, the were passaged and used for research at passage 1-3 (young AFSCs). Senescent AFSCs were obtained by maintain the cells for six weeks in culture without passages.

Activity 2. Morphological and phenotypic characterization of primary and senescent AFSCs. The morphological characterization of AFSC was performed by optical and fluorescence microscopy and the SA-β-Gal test. The immunophenotypic characterization was performed by flow cytometry. The morphological aspect observed by fluorescence microscopy showed that AFSCs have a fibroblast-like phenotype specific to adherent mesenchymal cells, with spindle cells, with multiple extensions, suggesting an active metabolism. Senescent AFSCs presented a similar morphology, but with the presence of vacuoles in the cytoplasm, which suggests a decrease in cellular metabolism. The results showed that the cells isolated from the amniotic fluid are stem cells, having an increased expression of the pluripotency markers SSEA-1 (100%), SSEA-4 (98%), TRA1-60 (100%) and TRA1-81 (96 %) in young AFSC and senescent AFSCs (SSEA-1 (78%), SSEA-4 (82%), TRA1-60 (84%) and TRA1-81 (76%)). The obtained results showed that after 24 hours of incubation at 37°C with the X-Gal substrate, senescent AFSCs were stained blue, specific staining for senescence.

Activity 3. Analysis of the molecular pathways involved in the determination of AFSC senescence. This activity was carried out using the qRT-PCR technique. Genes involved in cell cycle regulation, such as p16INK4A, eNOS, and cytoskeletal elements, such as β-actin, showed increased expression in senescent AFSCs (71.7-fold for p16INK4A, 22-fold for eNOS and 45-fold β-actin), while HMGB1 (2.6 -fold), cMyc (1.7-fold), Bcl2 (1.4-fold) showed a slight increase, and p21, and MDM2 showed a low expression.

Activity 4. Investigation of the senescence-associated secretory phenotype (SASP). The investigation of the senescence-associated secretory phenotype (SASP) was carried out by quantifying the expression level of molecules with an inflammatory role (IL-1α, IL-6, IL-8, TGF-β, NF-κB p65, eNOS) in senescent cells compared to young AFSC. After six consecutive weeks in culture without passage, senescent AFSCs showed increased expression of IL-1α (43.5-fold), IL-6 (21-fold), IL-8 (84-fold), TGF-β (24-fold), NF-kB (p65) (15.3-fold), and eNOS (22-fold).

RESULTS DISEMINATION FASE I/2022

Conferences

1. F. Iordache, A. Ionescu, L. Stanca, C. Curutiu, C.M. Istrate, A.I. Serban. Molecular and electrophysiological profile of induced stem cell senescence. The Biochemistry Global Summit, 7-14 July 2022, Lisboan, Portugal.