Effects of Extreme High Temperatures on Proliferation, Cell Cycle, Cell Differentiation and ROS of Adipose-Derived Mesenchymal Stromal Cells


 Backgrounds: Global warming has led to extreme temperatures in different latitudinal regions, resulting in the extinction of a large number of species. This study focuses on the effects of extreme high temperatures on cell proliferation, cell cycle, cell differentiation and mitochondria activity in human adipose-derived mesenchymal stromal cells (hADSCs). Methods: hADSCs were divided into three groups and incubated in 37°C, 39°C and 40°C environment for 5 hours of exposure each day, and then to 37°C circumstances for further incubation. Cell surface markers, cell cycle, cell proliferation activity, mitochondrial activity and cell polarization were detected and analyzed by flow cytometry, CCK-8 assay, ROS and JC-1 staining respectively on the 1 st and 3 rd day of cell culture; osteogenic and adipogenic differentiation ability of hASCs was analyzed by staining after 21 days of osteogenic and adipogenic differentiation induction culture. Results The results of this study showed that hASCs grown under high temperature conditions had restricted growth activity, blocked S and G2 phases of the cell cycle, reduced cytokinesis and impaired mitochondrial activity, while their osteogenic differentiation ability and membrane potential depolarization were enhanced. Conclusions: hADSCs were subjected to high temperature stimulation with restricted growth activity, reduced cell division, impaired mitochondrial activity, significant cell depolarization and enhanced osteogenic differentiation, and these results were closely related to the pathogenic mechanisms of skin aging and heat stroke due to outdoor sun exposure.


Introduction 42
Recently, global warming has led to extreme temperatures in different latitudinal 43 regions. Some studies have reported that high temperatures from temperate and 44 tropical regions may result in wildlife experiencing a dramatic increase in disease risk 45 [1] . As anthropogenic climate change continues to worsen, the risks to biodiversity will 46 increase over time, and future projections suggest that a potentially catastrophic 47 decline in global biodiversity is imminent [2] , and these studies suggest that high 48 temperature environments pose a serious threat to both animals and people. 49 Previous reports have shown that high temperature radiation can cause skin aging 50 and slow down lipid metabolism. Ken Kobayashi et al [3] showed that lactating mice, 51 under moderately high temperature conditions at 39℃, induced higher lactation 52 capacity of mammary epithelial cells (MECs) through control of STAT5 and STAT3 53 signaling. In contrast, prolonged exposure to 41℃ gave rise to a decrease in milk 54 production capacity through inactivation of STAT5 and a decrease in the total number 55 of MECs. It has also been shown that when rats are exposed to high temperature 56 (50℃), lipolysis in adipose tissue is inhibited due to their high body temperature, 57 while intravascular lipolysis is activated [4] . Therefore, understanding the response of 58 human cells to high temperature environment can help us to make positive responses 59 to future environmental changes. 60 With climate change, the increasing incidence of heat-related deaths has been a 61 frequent concern. The 2019 Global Burden of Disease, Injury, and Risk Factors Study 62 demonstrates the non-optimal temperature as one of the top 10 causes of death 63 globally. Globally, 5 million deaths from 2000 to 2019 are associated with abnormal 64 temperatures [6] . For example, a common illness, heat stroke (HS) is a life-threatening 65 disease defined as exposure to excessive hyperthermia at core temperatures above 66 40°C and resulting in a systemic inflammatory response syndrome [7] . HS occurs when multiple tissues and organs are damaged, allowing the secretion of 68 pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) and 69 interleukin-6 (IL-6), and ultimately systemic inflammation [8] . However, the specific 70 pathogenic mechanisms need to be further investigated.

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Adipose tissue is a highly metabolically active endocrine organ, which plays an 72 important role in energy storage, energy balance and metabolic regulation [9] . 73 However, many statistics in recent years have shown that body temperature in healthy 74 adults is gradually decreasing [10][11] , suggesting that some changes in adipose tissue in 75 the body are occurring with environmental changes or induced. Therefore, studying 76 the changes in cellular properties in adipose tissue in abnormal environments will help 77 us better understand the effects of environmental changes on the human body.      The P4 cells were adjusted to 10 6 cells per tube and centrifuged at 250g for 5 min.

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The supernatant was discarded, and the cells were fixed in 70% ethanol at 4°C for 127 more than 2 h and centrifuged at 250g for 5 min. 100 μL of RNaseA was added to the   133 The hADSCs of P4 were inoculated into gelatinized 6-well plates. When the cells were confluent to 60-70%, the osteogenic differentiation induction medium was 135 changed every 3 days, and after 2-4 weeks of induction, alizarin red staining was 136 performed; When the cells were confluent to 100%, they were replaced with lipogenic

Reactive oxygen species (ROS) and JC-1 staining assay 150
The hADSCs of P4 were stained with JC-1 dye and ROS probe to determine the 151 mitochondrial membrane potential and intracellular ROS content, respectively.   169 The cell morphology of hADSCs was observed under different temperature 170 gradient treatments, and no morphological differences were observed in hADSCs on 171 the 1 st day (Fig 1). When the cells grew to the 3 rd day, the cells in the 37℃ group of hADSCs treated at 37°C, 39°C and 40°C groups, respectively. The results showed 183 that CD90, CD73 and CD105 expression rates were above 90%, while CD34 and 184 CD45 expression rates were <3% (Fig 2). Statistical analysis showed that CD90 and 185 CD73 were not statistically different between groups on the 1 st day and the 3 rd day,   Values bar were expressed as mean±SEM, *P < 0.05. 203 204 The cell cycle of hADSCs in each group was measured by flow cytometry, and 205 the results showed (Fig 4)   respectively. Values bar were expressed as mean±SEM, *P < 0.05, **P < 0.01, ***P < 0.001.   The results showed that the depolarization was more pronounced at higher 262 temperatures. Global warming will affect ecosystems as well as human health in multiple ways, 273 and these impacts are expected to rise dramatically with increasing warming, with 274 estimates that the number of people at climate-related risk will increase by hundreds 275 of millions by 2050. However, it remains difficult to predict the human impacts of the 276 complex interplay of mechanisms driven by warming [12] . At present, the impact of 277 climate warming on human survival and in animals is still poorly understood.  [16] . In contrast, the 294 expression of CD34 and CD45 decreased and was <5%, suggesting that the cells still 295 have the characteristics of mesenchymal stromal cells [17] . Analysis of cell 296 proliferation viability showed that at 24 h after passaging, the proliferation capacity of 297 hADSCs in the 39°C environment was significantly higher than the other two groups, 298 and by 72 h, the proliferation capacity of cells in the 37°C group was slightly higher 299 than the others, showing that the change in environmental high temperature caused a 300 short-term rapid proliferation of hADSCs, while the opposite proliferation trend 301 occurred by 72 h. Further analysis in terms of cell cycle showed that at the 3 rd day of 302 cell growth, the G1 phase (pre-DNA synthesis phase) was reduced and the S phase 303 (DNA replication phase) and G2 phase (late DNA synthesis phase) were significantly 304 higher in the 39°C and 40°C environments compared to the 37°C group, illustrating 305 that the S and G2 phases of the cells were blocked and the cell division ability was 306 reduced, which were consistent with the formation of a gradual decrease in cell 307 proliferation activity at the 3 rd day.

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The multidirectional differentiation potential of stromal cells is one of the showed that with increasing temperature, mitochondrial oxidative stress ROS content 376 increases, demonstrating increased production and release of mitochondrial oxides 377 and inhibited activity.

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The results of ROS and JC-1 assays were in line with the mechanistic studies in 379 humans when heat stroke occurs, such as in heat stroke, cells in the body are in a hypoxic state, ATP is reduced, Ca 2+ concentration is increased, followed by elevated 381 ROS and oxidative stress, leading to apoptosis, tissue necrosis and autophagy, and 382 eventually multi-organ dysfunction and individual death [23][24] . Interestingly, the 383 results in this experiment showed that ROS was elevated and mitochondrial activity 384 was reduced when hADSCs cells were in a high temperature environment; the cell 385 membrane depolarization was severe during JC-1 staining, which may also lead to