ROLE OF PIEZO-1 IN MESENCHYMAL STEM CELLS Sanjucta Adak1,2,3| Bithiah Grace Jaganathan1,2,3 | Stem Cell and Cancer Biology (SCBG) Lab1, BioSciences and BioEngineering (BSBE)2, Indian Institute of Technology (IIT) Guwahati3
Aim and Focus: Mesenchymal Stem Cells (MSCs) which are characterized as multipotent and categorized as adult stem cells, have been extensively investigated since their discovery for their potential to treat a plethora of diseases. Obtaining a better understanding of the biology of MSCs is critical to the development of MSC-based regenerative medicine and the improvement of efficacy of this bio-drug.
Yoda1 + Dooku1 Dooku1 Yoda1 Untreated As all tissues in the human body experience various mechanical cues, the cells respond to these mechanical signals with biological responses targeted towards Fig.2: Phase contrast images of Oli-Red O stained MSCs after 14 days of sustenance of the individual cells, their respective tissues and ultimately the differentiation in mixed (osteogenic + adipogenic) induction media as well as entire human body. The cells detect mechanics via mechanically activated ion channels, one of which is the Piezo1 cation channel, which brings about continuously kept in the four treatment conditions intracellular influx of Ca2+ ions. This study focuses on elucidation of how MSCs sense and respond to their mechanical environment via this ion channel. Specifically, the behavioral changes due the mechano-transduction in MSCs as a result of pharmacological activation and competitive inhibition of Piezo-1 channel by the usage of Yoda-1 (5µM) and Dooku-1 (5µM), respectively is being looked into. The proliferation, migration speed, integrin expression, osteogenic and adipogenic lineage commitment tendency of MSCs after treatment with either or both these drugs, is being monitored in this study.
Scope of the Project: Mechanical force constitutes an essential factor for osteogenesis and bone homeostasis. Thus, a greater understanding of the role of Piezo-1 in MSCs can aid the usage of these cells as a therapy for skeletal diseases.
Concept:
Fig 3: Quantification of expression of integrin molecules in MSCs by FACS after being kept under the four treatment conditions for 48 hours Untreated
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Architecture of Piezo 1 channel (4.8 Å) cryo-electron microscopy
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Materials and Methods:
Bone marrow derived MSCs were cultured in-vitro and used for all the experiments Quantification of integrin molecules and phosphorylated extracellularsignal-regulated kinase 1/2 (pERK1/2) expression was carried out by usage of fluorescence associated cell sorting (FACS) after processing the cells via standardized protocols FACS data was analysed using FCS Express 7 software Migration assay was carried out by seeding MSCs in 12 well culture dishes, creation of a scratch between adhered cells by usage of sterile micro-tip, refilling drug treated media in the wells and then monitoring the closure of the scratch for the next 28 hours. Migration speed of MSCs were determined by analysis of phase contrast images in T-Scratch software 14-day differentiation was carried out by keeping MSCs in mixed (osteogenic + adipogenic) induction media, along with continuous treatment with the drugs
Results:
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Fig. 5: Migration speed of MSCs in the four treatment conditions as calculated by T-Scratch software
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Fig.4: Phase contrast images for the determination of migration speed of MSCs under the four treatment conditions
Fig. 6: Quantification of expression levels of pERK1/2 which belongs to the mechanoresponsive, mitogen activated protein kinase (MAPK) pathway
Conclusion: A marked reduction in proliferation, adipogenic differentiation, migration speed and integrin expression, upon treatment of MSCs with Yoda-1 which activates the Piezo-1 ion channel pERK1/2 expression levels were enhanced in MSCs upon treatment with Yoda-1
Cell Counts after 48 hours of drug treatment 525000
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Future perspective:
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To ascertain the expression levels of intermediate proteins in several other mechanoresponsive signaling cascades which might get affected upon Piezo-1 activation or inhibition in MSCs. To analyze morphological and functional alterations in the cytoskeleton of MSCs upon Piezo-1 activation or inhibition
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Contact Details sanjucta.adak93@gmail.com
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Fig.1: Proliferation analysis (Cell Counts): MSCs were treated with the aforementioned drugs and their combination for 48 hours, after which their counts were determined with the help of a hemocytometer
Acknowledgments The author expresses sincere gratitude towards all lab members of the SCBG lab, BSBE, IIT Guwahati.