Acoustic vibration promotes in vitro expansion of human embryonic stem cells.

Am J Stem Cells 2024;13(3):143-151 www.AJSC.us /ISSN:2160-4150/AJSC0156130 Original Article Acoustic vibration promotes in vitro expansion of human embryonic stem cells Xiangyue Hu1,2, Haoyun Duan2, Dulei Zou2,3, Chunxiao Dong2,3, Yani Wang2, Yao Wang2, Zongren Li4, Zongyi Li1,2 Shandong First Medical University (Shandong Academy of Medical Sciences), Jinan 250000, Shandong, China; Eye Institute of Shandong First Medical University, State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Qingdao 266071, Shandong, China; 3Eye Institute of Shandong First Medical University, Eye Hospital of Shandong First Medical University (Shandong Eye Hospital), Jinan 250021, Shandong, China; 4970 Hospital of Chinese PLA Joint Logistic Support Force, Weihai 264200, Shandong, China 1 2 Received February 26, 2024; Accepted May 6, 2024; Epub June 15, 2024; Published June 30, 2024 Abstract: Objectives: This study aimed to investigate the effect of acoustic vibration on the pluripotency of human embryonic stem cells (hESCs) and evaluate cell proliferation and self-renewal ability post-treatment. Methods: The human ES cell line H1 was used for the experiments. hESCs were treated with an acoustic vibration device. Their proliferative ability was subsequently detected using a colony formation assay, while the expression of pluripotencyrelated markers was detected via immunofluorescence staining. Finally, changes in gene expression levels were examined using quantitative polymerase chain reaction (qPCR) in the presence of appropriate primers. Results: Compared with normal cells in the control group, the morphology of experimental cells subjected to acoustic vibration did not significantly change. Contrastingly, the colony-forming efficiency of the experimental cells significantly increased. Immunofluorescence staining results showed the cells in experimental group were positive for the pluripotency markers NANOG, octamer-binding transcription factor 4 gene (OCT4), and SRY (sex determining region Y)-box 2 (SOX2). In addition, the expression levels of pluripotency genes NANOG, OCT4, SOX2, and Yes-associated protein (YAP)-related genes were up-regulated following acoustic vibration. Conclusions: Our results revealed that acoustic vibration enhanced the proliferative ability of hESCs and increased the expression levels of NANOG, OCT4, SOX2, and YAP-related genes, indicating that acoustic vibration can optimize the self-renewal ability of hESCs and that the YAP signaling pathway may play a critical role in the functional process of acoustic vibration. Keywords: Human embryonic stem cells, acoustic vibration, pluripotency, proliferation, YAP Introduction Human embryonic stem cells (hESCs) exhibit remarkable self-renewal capacity and potential to differentiate into various cell types, rendering them highly promising for the research of tissue development, pathological mechanisms, and human therapeutics [1, 2]. As is well documented, the pluripotency of hESCs is intricately regulated by epigenetic modifications, core stemness transcription factors, signaling pathways, and histone modifications [3-5]. Its maintenance relies on key transcription factors, such as SRY (sex determining region Y)-box 2 (SOX2), octamer-binding transcription factor 4 (OCT4), and NANOG [6]. Despite extensive research, the mechanisms governing pluripo- tency and self-renewal of embryonic stem cells remain elusive. Various approaches, including modification of culture protocols [7], incorporating different factors [8-10], alteration of metabolic patterns [11], manipulation of RNA processing, and implementation of reprogramming techniques [12, 13], have been explored to sustain the pluripotency of embryonic stem cells in vitro. The application of acoustic vibrations as a mechanical stimulus has emerged as a promising approach for mediating complex cell behaviors and directing stem cell differentiation [14]. Indeed, it has been extensively applied in cellular research and disease treatment [15, 16], including the generation of pluripotent stem https://doi.org/10.62347/PJFC2708 Acoustic vibration promotes hESCs proliferation cells using ultrasound [17], induction wound healing in vitro [18], promotion of mesenchymal stem cell proliferation, differentiation, and migration [19], as well as exosome secretion through low-intensity pulsed ultrasound (LIPUS) [20, 21], the acceleration of spinal healing [22], promotion of nerve regeneration and recovery [23, 24], the induction of neurogenesis, and the treatment of traumatic brain injury, mental disorders, and Alzheimer’s disease [25, 26]. Notably, sound waves exert their influence on tissue homeostasis and regeneration by modulating intercellular communication, thereby impacting the onset and progression of diseases [27]. Acoustic vibration offers numerous advantages, such as streamlined instrumentation and experimental setup, enhanced operational resolution, and flexibility. This versatile and non-invasive technique represents an ideal method for conducting cellular research [18, 28-30]. The Yes-associated protein (YAP) pathway functions as a sensor and mediator of mechanical signals in the cell microenvironment. It is highly sensitive to a wide range of mechanical stimuli, enabling the transduction of these signals into cell-specific biological signals and consequent modulation of cellular responses that promote the expression of target genes by activating the transcription factor TEAD PDZ binding motif (TAZ) [31-33]. Specifically, YAP1 is vital for the self-differentiation and renewal of stem cells, as well as the long-term proliferation and survival of hESCs in vitro [34, 35]. This study aimed to investigate the impact of acoustic vibration treatment on hESCs culture and elucidate the underlying mechanism. We hypothesized that acoustic vibration could enhance hESCs pluripotency and promote proliferation and thus provide novel promising strategies for stem cell culture. Material and methods Cell culture Human embryonic stem cell line H1 was donated by Professor Zhengqin Yin of Army Medical University (Chongqing, China). H1 cells were seeded onto plates coated with Matrigel (Corning 354277) and incubated in mTeSR1 medium (STEMCELL Technology) until the next 144 passage, which was performed every 5-7 days using ReLeSR™ (STEMCELL Technology, 05872). The cells were incubated at 37°C in a humidified atmosphere with 5% CO2. The medium was renewed daily. Cells were treated with acoustic vibration This experiment was carried out using an acoustic vibration device (Beijing Yishengkang, China) comprising a regulating acoustic display, an acoustic transmission box, and an infinite input sound head. The acoustic output frequency of the device ranged from 20 Hz to 4000 Hz, whilst the acoustic intensity ranged from 0 to 100 dB. In the present study, the mode was employed at 3 intensity levels, with an acoustic output frequency of 450-500 Hz and an acousti (...truncated)