Method for inducing dedifferentiation of adipose cells

Abstract

The invention investigates the influence of hyperosmotic pressure on fat cells, and discloses the effect of hyperosmotic pressure in inducing the release of mitochondrial extracellular vesicles (MEV). This release then enhances the secretion of the proinflammatory cytokine TNF-alpha, which is crucial during the stress response, thereby activating the Wnt / beta-catenin signaling pathway for fat cell dedifferentiation. By means of the method, MEV release and TNF-alpha secretion are induced through hyperosmosis, Wnt / beta-catenin signals are finally adjusted, fat cell dedifferentiation is promoted, and the method has the hopeful treatment significance.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 598,153 filed on November 13, 2023 and U.S. Formal Patent Application No. 18 / 619,199 filed on March 28, 2024, and the disclosures of which are incorporated herein by reference in their entirety. Reference sequence list

[0002] The submitted sequence listing file named "P2989CN00_sequence list.xml" was created on July 18, 2024, using the ST.26XML file format, with a file size of 23.3KB, and is incorporated into this article by full reference. Technical Field

[0003] The present invention relates to the fields of cell biology, biochemistry, protein analysis, biomedical research and potentially drug development. Background Art

[0004] Adipocytes, of mesenchymal origin, are considered important energy storage units and key endocrine sources for regulating systemic metabolism. Despite the important physiological functions of adipose tissue, hypertrophy and excessive proliferation of adipose tissue lead to obesity, which is a major risk factor for a range of diseases, including abnormal lipid metabolism, cardiovascular disease, type 2 diabetes, and cancer. At the cellular level, the development of obesity is associated with the disturbance of adipogenesis, that is, the differentiation of directed preadipocytes into mature adipocytes. Interestingly, adipocytes do not remain in a static stage of terminal differentiation after adipogenesis. In fact, mature adipocytes exhibit a high degree of plasticity and can revert to a state similar to multipotent progenitor cells. This is often referred to as adipocyte dedifferentiation and has been widely observed in vitro and in vivo. For example, during late pregnancy and lactation, mammary adipocytes lose lipid droplets and dedifferentiate into fibroblast-like preadipocytes, and then redifferentiate into adipocytes after lactation. 1 In addition, recent studies have observed adipocyte dedifferentiation in the development of different malignancies, which is associated with the Wnt signaling pathway. 2-3 and Notch signaling pathway 4 A deeper understanding of the signal transduction pathways that regulate the balance of adipocyte differentiation / dedifferentiation will be the key to effectively prevent and treat obesity and related diseases.

[0005] Adipogenesis regulation mechanisms have been extensively studied. However, relatively little is known about the molecular mechanisms of adipocyte dedifferentiation. In general, pro-adipogenic and anti-adipogenic genes / pathways play opposite roles in adipocyte dedifferentiation. For example, C / EBPβ and PPARγ are key pro-adipogenic genes that are usually inhibited to promote adipocyte dedifferentiation. As a PPARγ agonist, rosiglitazone can block adipocyte dedifferentiation and prevent tumor development in a Notch-driven liposarcoma mouse model. 4 In contrast, canonical Wnt / β-catenin signaling ligands are thought to inhibit adipogenesis and have been identified as enhancers of dedifferentiation. Overexpression of Wnt1 inhibits adipogenic differentiation of progenitor cells, whereas exogenous application of Wnt3a can induce dedifferentiation of mouse 3T3-L1 and human adipocytes. 2,5-6 In addition to the Wnt signaling pathway, the Notch, TGF-β, and TNF-α signaling pathways have been reported to inhibit adipogenesis and promote adipocyte dedifferentiation in different contexts. 4,6-8 The detailed mechanism of how different signaling pathways synergistically regulate adipocyte differentiation deserves further study.

[0006] A variety of methods have been developed to induce dedifferentiation of adipocytes, which has helped to explore the molecular and cellular mechanisms in more depth. Initial studies have shown that the "ceiling culture" technique can cause mature adipocytes to lose lipid droplets and give rise to progenitor-like cells, namely dedifferentiated adipocytes (DFAT cells). 9 . Activation of the TGF-β1 signaling pathway as well as different types of collagen can regulate DFAT cells in ceiling culture systems. Recent studies have shown that physical changes in the microenvironment, including matrix stiffening, increased compressive forces, and elevated osmotic pressure, can affect cell fate and adipocyte dedifferentiation. 10-11 . Thus, hypertonic treatment has been shown to induce osmotic stress and promote adipocyte dedifferentiation. Considering the cell abundance and easy operation, hypertonicity-induced DFAT shows great potential and is expected to revolutionize the landscape of regenerative medicine. During the permetic reprogramming of adipocytes, activation of the anti-adipogenic Wnt / β-catenin signaling pathway was observed.

[0007] However, the mechanism of how the hyperosmotic state activates the Wnt / β-catenin signaling pathway and drives cell dedifferentiation remains unclear. The present invention addresses this need. Summary of the invention

[0008] The present invention aims to elucidate the molecular mechanism of osmotic-induced adipocyte reprogramming.

[0009] In the first aspect, the present invention provides a method for inducing adipocyte dedifferentiation, comprising exposing one or more adipocytes to a hypertonic solution; and inducing the release of mitochondrial extracellular vesicles (MEVs) from one or more adipocytes to the extracellular environment. The released mitochondrial extracellular vesicles enhance the secretion of a series of inflammatory genes in one or more adipocytes, which activate the Wnt / β-catenin signaling pathway, thereby driving the dedifferentiation of adipocytes.

[0010] In one aspect, the panel of inflammatory genes includes TNF-α, IL-6, RIP1, CEBPA, and MCP-1.

[0011] Preferably, the inflammatory genes include TNF-α and IL-6.

[0012] In one aspect, the one or more adipocytes primarily include isolated adipocytes, adipocytes derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), and adipocytes from different anatomical locations.

[0013] In another aspect, the one or more adipocytes include 3T3-L1 or adipose stromal vascular fraction (SVF)-derived adipocytes.

[0014] In one aspect, the adipocyte count of the one or more adipocytes is reduced by at least 3.5-fold after the hypertonic treatment.

[0015] In another aspect, hyperosmotic treatment resulted in a 40-60% decrease in the expression of the adipogenic markers C / EBPβ, PPAR-γ, and adiponectin.

[0016] In another aspect, hypertonic treatment results in a 10-20% increase in the expression of surface markers associated with dedifferentiated adipocytes.

[0017] In one aspect, the hypertonic solution is formulated to have a higher hypertonic pressure than the surrounding environment, and the hypertonic solution includes a culture medium and 2% PEG 300. The culture medium can be DMEM. The hypertonic solution also includes an additive, including a preservative, a stabilizer, or a drug.

[0018] In one aspect, the one or more adipocytes treated with the hypertonic solution exhibit osteogenic and chondrogenic redifferentiation capabilities.

[0019] In another aspect, the method further comprises adding a small molecule compound to reduce apoptosis of one or more adipocytes induced by hyperosmotic treatment. The small molecule compound comprises 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine.

[0020] In another aspect, the expression of lipogenic genes in one or more adipocytes is reduced, accompanied by an increase in the expression of marker genes. Lipogenic genes include C / EBPβ, PPAR-γ, and adiponectin. Marker genes include Smad 9, Esrrb, and Sox2.

[0021] In another aspect, the one or more adipocytes exhibit osteogenic and chondrogenic redifferentiation capabilities.

[0022] The present invention established a powerful cell model in which high osmotic pressure effectively induced dedifferentiation of 3T3-L1 and adipose vascular stromal fraction (SVF)-derived adipocytes. Subsequently, proteomic analysis was performed on extracellular vesicles (EVs) released from dedifferentiated adipocytes subjected to hyperosmotic treatment. The results showed that high osmotic pressure prompted adipocytes to release mitochondria through extracellular vesicles, thereby enhancing the secretion of TNF-α cytokines during stress response. Importantly, hyperosmotic pressure-induced extracellular vesicles and TNF-α played a key role in activating the Wnt / β-catenin signaling pathway that drives adipocyte dedifferentiation. In general, the present invention defines a novel mitochondria-TNF-α axis, an axis mechanism that potentially regulates the Wnt / β-catenin signaling pathway and adipocyte dedifferentiation.

[0023] Compared with the prior art, the present method provides a more effective and controllable method for generating mitochondrial extracellular vesicles (MEVs). By applying a hypertonic shock to adipocytes, the controlled release of mitochondrial extracellular vesicles containing mitochondrial components is enhanced. The present invention provides a platform for drug development and screening, using mitochondrial extracellular vesicles to study intercellular communication, signaling pathways and drug responses. In short, the present invention provides a new method for enhancing the production of mitochondrial extracellular vesicles through hyperosmotic pressure, thereby making it potential for application in disease diagnosis, treatment, regenerative medicine and tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Embodiments of the present invention are described in more detail below with reference to the accompanying drawings, in which:

[0025] Figure 1A Describe the experimental workflow diagram. Figure 1B Representative images of adipocytes cultured under hypertonic conditions for the indicated times are depicted. Scale bar: 100 μm. Figure 1C Depicted are quantifications of the loss of distinct lipid droplets from adipocytes over time under the indicated culture conditions. Error bars represent standard deviation (n=3 independent experiments). Figure 1D Depicted are Oil Red O staining images and quantitative results of adipocytes seven days after the indicated treatments. Figure 1E Depicted are box plots showing the aspect ratio of adipocytes after 4 days of control or hypertonic treatment (n=200 cells). Figure 1FDepicted are the results of RT-qPCR analysis of C / EBPβ, PPARγ, and adiponectin expression in isotonic and hyperosmotic treated adipocytes.

[0026] Figure 2A The results of CD13 immunostaining in control and hypertonic treated 3T3-L1 adipocytes are depicted, and the percentage of cells with positive CD13 labeling is quantified. Scale bar: 20 μm. Figure 2B Results of immunostaining for Endoglin in control and hyperosmotic treated 3T3-L1 adipocytes are depicted, and the percentage of cells with positive Endoglin labeling is quantified. Scale bar: 20 μm.

[0027] Figure 3A Representative images depicting alkaline phosphatase (ALP) staining showing the osteogenic potential of dedifferentiated 3T3-L1 adipocytes. Figure 3B Depicts the results of RT-qPCR analysis of Runx2 and ALP expression after osteogenic induction in isotonic and hyperosmotic treated 3T3-L1 adipocytes. Figure 3C Depicted are the results of RT-qPCR analysis of Smad3, Col2a1, and Sox9 expression after chondrogenic induction in isotonic and hyperosmotic treated 3T3-L1 adipocytes.

[0028] Figure 4A Depicted are the results of quantitative proteomic analysis of extracellular vesicles (EVs) from isotonic control and hypertonic cultured 3T3-L1 adipocytes. Figure 4B Figure 2. Venn diagram comparing proteins in control and hypertonic EVs of adipocytes. Figure 3. Right depicts Gene Ontology (GO) term analysis of 25 hypertonic unique proteins. Figure 4C Scatter plot showing the results of differential analysis of the EV proteome in the control and hyperosmotic conditions. The diagonal dashed lines indicate areas with a ten-fold difference in abundance. Figure 4D GO term analysis results depicting upregulated proteins in hypertonic EVs, showing the top five cellular components. Figures 4E-4F Depicted are MitoTracker Green and DAPI staining of 3T3-L1 adipocytes treated with the indicated treatments over 8 hours. The relative intensity of the green channel was quantified. Scale bar is 10 μm. Figure 4G Representative transmission electron microscopy (TEM) images depicting hyperosmotic EVs from 3T3-L1 adipocytes. Arrows indicate inclusion of mitochondrial fragments. Figure 4H Depicted are the results of immunoblot analysis of the indicated proteins in cell lysates and EVs from 3T3-L1 adipocytes subjected to the indicated treatments.

[0029] Figure 5Depicted are the levels of NDUFA9 and CD81 in 3T3-L1 adipocytes assessed by immunoblot analysis after the indicated treatments, including the isotonic+DMSO group, the isotonic+GW4869 group, the hyperosmotic+DMSO group, or the hyperosmotic+GW4869 group.

[0030] Fig. 6A Figure 3 EVs from control or hypertonic cultured 3T3-L1 adipocytes were used to treat primary 3T3-L1 adipocytes cultured isotonically. Figure 6B Depicted are the expression of C / EBPβ, PPARγ, and adiponectin in 3T3-L1 adipocytes treated with EVs from the indicated culture conditions as analyzed by RT-qPCR. Figure 6C Depicted are the expression of TNF-α and IL-6 in 3T3-L1 adipocytes treated with EVs isolated from adipocytes in the indicated culture conditions, as analyzed by RT-qPCR.

[0031] Fig. 7A Depicted are the expression of RIP1, CEBPA, and MCP-1 in 3T3-L1 adipocytes treated with EVs isolated from adipocytes in the indicated culture conditions, as analyzed by RT-qPCR. Figure 7B Depicted are the expression of RIP1, CEBPA, and MCP-1 in 3T3-L1 adipocytes subjected to the indicated treatments as analyzed by RT-qPCR.

[0032] Fig. 8A Depicted is the expression of TNF-α in 3T3-L1 adipocytes receiving EVs from the indicated treatments analyzed by RT-qPCR. Figure 8B Depicted are the expression of TNF-α and IL-6 in 3T3-L1 adipocytes subjected to the indicated treatments as analyzed by RT-qPCR.

[0033] Fig.9A Depicted are the expressions of inflammatory factors associated with TNF-α in 3T3-L1 adipocytes subjected to the indicated treatments for 2 hours, as analyzed by RT-qPCR. Fig. 9B Depicted are the expression of TNF-α protein levels analyzed by ELISA in the culture medium of 3T3-L1 adipocytes subjected to the indicated treatments for 8 hours.

[0034] Fig. 10A Depicted are the expressions of Smad9, Esrrb, and Sox2 in adipocytes subjected to the indicated treatments as analyzed by RT-qPCR. Fig. 10B Total cell lysates of EV-treated 3T3-L1 adipocytes cultured with isotonic or hypertonic adipocytes were analyzed by immunoblotting for the indicated proteins. Fig. 10C Total cell lysates of 3T3-L1 adipocytes cultured under the indicated conditions were subjected to immunoblot analysis of the indicated proteins. Fig. 10D Representative images depicting immunostaining of active β-catenin in 3T3-L1 adipocytes in the control group or after hyperosmotic treatment. Fig. 10E Depicted are the quantitative results of active β-catenin staining intensity in adipocyte nuclei after control or hyperosmotic treatment for 24 h. Fig.10F Depicted 3T3-L1 adipocytes were treated under the indicated conditions for 24 h, and cell lysates were subjected to immunoblot analysis of the indicated proteins. Figure 10G Depicted are representative results of phosphatidylserine (annexin V) / PI flow cytometric analysis of adipocytes receiving the indicated treatments. Fig. 10H Bar graphs depicting the proportion of early apoptotic cells in adipocytes subjected to the indicated treatments.

[0035] Fig.11A Depicted are representative images of adipocytes treated with BML-284 for the indicated times. Scale bar, 100 μm. Fig. 11B Depicted are the quantification of adipocytes that lost all distinct lipid droplets over seven days in control or BML-284 culture conditions. Error bars represent standard deviation (n = three independent replicates). Fig. 11C Images and quantification of Oil Red O staining depict adipocytes that have lost all clear lipid droplets seven days after the indicated treatments. Fig.11D Box plots depicting the aspect ratios of the indicated adipocytes in control or after BML-284 treatment (n=200 cells). Fig.11E RT-qPCR analysis results depicting the expression of C / EBPβ, PPARγ, and adiponectin in adipocytes after control or BML-284 treatment. Fig.11F RT-qPCR analysis results depicting the expression of pluripotency markers Smad9, Esrrb, and Sox2 in adipocytes after control or BML-284 treatment.

[0036] Fig. 12A Depicted are the results of immunostaining for CD13 in control and BML 284-treated 3T3-L1 adipocytes and the percentage of cells with positive CD13 labeling is shown. Scale bar: 20 μm. Fig. 12B The results of immunostaining for endoglin in control and BML 284-treated 3T3-L1 adipocytes are depicted, and the percentage of cells with positive endoglin labeling is shown. Scale bar: 20 μm.

[0037] Fig.13ART-qPCR analysis results depicting the expression of Smad3, Col2a1, and Sox9 after chondrogenic induction in isotonic and BML-284-treated adipocytes. Fig. 13B Representative images depicting alkaline phosphatase (ALP) staining show the osteogenic potential of dedifferentiated 3T3-L1 adipocytes after control or BML-284 treatment, and the expression of alkaline phosphatase was analyzed by RT-qPCR after osteoinduction of control or BML-284-treated 3T3-L1 adipocytes. Fig. 13C Depicts representative results of phosphatidylserine (annexin V) / PI flow cytometry analysis of adipocytes that received the indicated treatments. The bar graph shows the proportion of early apoptotic cells in adipocytes that received the indicated treatments

[0038] Fig.14 Working model depicting adipocyte dedifferentiation induced by hyperosmotic pressure. DETAILED DESCRIPTION

[0039] definition

[0040] Throughout this specification, unless otherwise specified herein, the word "comprise" or variations such as "comprises" or "comprising" should be understood to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers. It should also be noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as "comprises / comprised / comprising" may have the meanings ascribed to them in U.S. patent law; for example, they allow for non-explicit recitation of elements but exclude elements that exist in the prior art or that affect the basic or novel characteristics of the invention.

[0041] Throughout the present specification and claims, unless otherwise indicated herein, the word "include" or variations such as "includes" or "including", will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or groups of integers.

[0042] As used herein, the terms "substantially," "essentially," "approximately," and "about" are used to describe and explain a small change. When used in conjunction with an event or circumstance, the terms may refer to the situation where the event or circumstance occurs exactly, as well as the situation where the event or circumstance occurs approximately. For example, when used in conjunction with a numerical value, these terms may include a range of variation less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%.

[0043] References in this specification to "one embodiment", "an exemplary embodiment", etc. indicate that the described embodiment may include certain features, structures, or characteristics, but each embodiment may not necessarily include the certain features, structures, or characteristics. Moreover, such phrases do not necessarily refer to the same embodiment. In addition, when a certain feature, structure, or characteristic is described in conjunction with an embodiment, it should be understood that it is within the knowledge of a technician in the field to affect such features, structures, or characteristics in conjunction with other embodiments, whether or not explicitly described.

[0044] In the preparation methods described herein, the steps may be performed in any order without departing from the principles of the invention unless a time sequence or an operational order is explicitly described. In a claim, it should be stated that one step is performed first and then several other steps are performed, which means that the first step is performed before any other steps, but the other steps may be performed in any appropriate order unless the sequence is further listed in the other steps. For example, a claim element listing "step A, step B, step C, step D, and step E" should be understood as step A being performed first and step E being performed last, and steps B, C, and D may be performed in any order between steps A and E, and the order is still within the literal scope of the claim process. A given step or subset of steps may also be repeated.

[0045] Other definitions of selected terms used herein can be found in the detailed description of the entire invention and application. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs.

[0046] MEV enhances proinflammatory TNF-α signaling in an autocrine manner, which in turn activates β-catenin and drives adipocyte dedifferentiation. Adipocytes have the potential to dedifferentiate into a multipotent progenitor state.

[0047] Recent studies have shown that hyperosmotic stress can induce adipocyte dedifferentiation, which provides an attractive approach for producing regenerative tools. Alleviating mitochondrial stress can inhibit MEV release and hyperosmotic-induced adipocyte dedifferentiation. However, the molecular mechanisms underlying hyperosmotic-induced adipocyte reprogramming remain unclear.

[0048] Therefore, the present invention provides a method for inducing adipocyte dedifferentiation, comprising exposing one or more adipocytes to a hypertonic solution; and inducing the release of mitochondrial extracellular vesicles (MEVs) from one or more adipocytes to the extracellular environment. The released mitochondrial extracellular vesicles enhance the secretion of a series of inflammatory genes in one or more adipocytes, which activate the Wnt / β-catenin signaling pathway, thereby driving the dedifferentiation of adipocytes. The present invention defines a new signaling pathway that promotes the multipotent dedifferentiation of adipocytes.

[0049] In one embodiment, the adipocytes may primarily include isolated adipocytes, adipocytes derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), adipocytes from different anatomical locations (e.g., subcutaneous adipocytes, visceral adipocytes, or brown adipocytes).

[0050] Preferably, the adipocytes may be 3T3-L1 cells or adipose vascular stromal fraction (SVF)-derived adipocytes.

[0051] The present invention further studies the mechanism of hyperosmotic-induced adipocyte reprogramming. Hyperosmotic pressure causes adipocytes to release mitochondrial extracellular vesicles, which in turn enhance the secretion of TNF-α, a proinflammatory cytokine, during stress response in an autocrine manner.

[0052] A hypertonic solution has a higher concentration of solutes than the surrounding environment or another solution. Such a solution may contain culture medium and 2% PEG 300 to help maintain a stable pH level. The solute is an additive.

[0053] In one embodiment, the additive may include a preservative, a stabilizer, or a drug.

[0054] MEVs have shown potential in modulating cellular metabolism, enhancing mitochondrial function, and influencing diverse cellular pathways. Specifically: (1) Regulation of cell metabolism: MEVs encapsulate enzymes and metabolic regulators that are essential for cell metabolism. When MEVs are internalized by recipient cells, they affect metabolic pathways such as glycolysis, oxidative phosphorylation, and fatty acid oxidation. (2) Enhanced mitochondrial function: MEVs typically carry components necessary for optimizing mitochondrial function. These components may include mitochondrial DNA (mtDNA), mitochondrial RNA (mtRNA), and proteins involved in mitochondrial biogenesis, fusion-fission dynamics, and electron transport chain activity. Delivery of these components to recipient cells can enhance mitochondrial integrity, increase ATP production, and generally improve cellular energy metabolism. (3) Effects on cell signaling pathways: MEVs act as effective regulators of cell signaling pathways by transporting signaling molecules such as growth factors, cytokines, and microRNAs. (4) Therapeutic potential: Engineered MEVs or cargo molecules derived from MEVs can be customized to deliver therapeutic agents, genetic material, or metabolic regulators to specific cell types or tissues. In addition, MEVs isolated from stem cells or other sources may have regenerative properties, providing opportunities for innovative tissue repair or regeneration strategies.

[0055] MEV may activate the Wnt / β-catenin signaling pathway, which plays a key role in regulating adipogenesis, adipocyte differentiation, and metabolic homeostasis. TNF-α has been shown to be essential for the activation of the Wnt / β-catenin signaling pathway. Activation of this pathway may lead to changes in gene expression patterns that promote adipocyte dedifferentiation and alter cellular metabolism. The Wnt / β-catenin signaling pathway is initiated when Wnt proteins bind to their receptors on the cell membrane, which leads to the stabilization of β-catenin, allowing it to translocate to the nucleus to interact with transcription factors, thereby initiating the transcription of specific genes. In adipocytes, activation of the Wnt / β-catenin signaling pathway may lead to changes in the expression patterns of various genes, including genes involved in adipocyte differentiation and metabolism. TNF-α is essential for the activation of the Wnt / β-catenin signaling pathway because TNF-α promotes the stability of β-catenin, thereby enhancing its accumulation and transcriptional activity in the nucleus. Therefore, TNF-α can regulate adipocyte dedifferentiation and cellular metabolism by regulating the activity of this signaling pathway.

[0056] In addition to activating the Wnt / β-catenin signaling pathway, MEVs may also participate in several other signaling pathways that together contribute to the hyperosmotic-induced adipocyte reprogramming process. For example, MEVs may affect the AMP-activated protein kinase (AMPK) pathway, which is a major regulator of cellular energy balance. Activation of AMPK can lead to increased fatty acid oxidation, glucose uptake, and mitochondrial biogenesis, thereby affecting adipocyte metabolism and function. MEVs may affect the activation of the NF-κB signaling pathway, which is a key regulator of inflammation and immune responses. In addition, MEVs may activate the MAPK / ERK signaling pathway, which regulates multiple cellular processes such as cell proliferation, differentiation, and inflammation. Activation of this pathway can regulate the phenotype and function of adipocytes and may contribute to hyperosmotic-induced adipocyte reprogramming. In addition, MEVs may also regulate the PI3K / Akt signaling pathway, which regulates cell survival, proliferation, and metabolism. Activation of the Akt signaling pathway can promote adipocyte dedifferentiation and alter cell metabolism in response to hyperosmotic stress-induced stress.

[0057] One potential limitation of hypertonic treatment is its tendency to induce apoptosis. However, the present invention provides an effective solution to this problem by directly activating the Wnt / β-catenin signaling pathway using BML-284, while promoting adipocyte dedifferentiation and alleviating apoptosis. BML-284 has been shown to directly activate the Wnt / β-catenin signaling pathway.

[0058] BML-284 is a small molecule compound that acts as an inhibitor of glycogen synthase kinase 3β (GSK-3β). GSK-3β is a serine / threonine kinase that plays a key role in multiple cellular processes, including glycogen metabolism, cell proliferation, apoptosis, and regulation of gene transcription. In the Wnt / β-catenin signaling pathway, GSK-3β phosphorylates β-catenin, targeting it for degradation in the proteasome, thereby keeping the pathway in an inactive state. It does so by inhibiting the activity of GSK-3β, an enzyme that normally phosphorylates β-catenin, making it a target for degradation. By inhibiting GSK-3β, BML-284 prevents the degradation of β-catenin, which stabilizes and accumulates in the cytoplasm. Stabilized β-catenin is then transported to the nucleus, where it interacts with transcription factors of the T-cell factor / lymphocyte enhancer factor (TCF / LEF) family to initiate transcription of Wnt target genes. These target genes are involved in multiple cellular processes, including cell survival, proliferation, and differentiation. Thus, by directly activating the Wnt / β-catenin signaling pathway, BML-284 is able to counteract the apoptotic effects induced by hyperosmotic treatment. This activation contributes to cell survival and may enhance adipocyte dedifferentiation, resulting in a dual benefit in the hyperosmotic-induced cellular response.

[0059] In summary, the present invention reveals the mechanism of multipotent dedifferentiation of adipocytes, providing great prospects and hope for the application of regenerative medicine. The present invention identifies a new signal transduction pathway, including hyperosmotic-induced mitochondrial stress, exosome-mediated mitochondrial emission, TNF-α secretion, and Wnt / β-catenin activation, which promotes adipocyte dedifferentiation. In addition, the present invention demonstrates that the use of BML-284 to directly activate the Wnt / β-catenin signaling pathway can effectively induce adipocyte differentiation while overcoming the apoptotic effect of hyperosmotic treatment.

[0060] Examples

[0061] Example 1

[0062] Hyperosmotic pressure induces adipocyte dedifferentiation in 3T3-L1 cells

[0063] Recent studies have shown that physical compression caused by hypertonic treatment can lead to dedifferentiation of adipocytes from primary sources. 11 However, this phenomenon has not yet been validated in cell line models.

[0064] To address this question, an examination was performed to evaluate whether hyperosmotic treatment could induce dedifferentiation in the established 3T3-L1 adipocyte model. Figure 1A , a diagram of the experimental workflow is shown. Adipose vascular stromal fraction (SVF)-derived adipocytes served as a positive control. After adipogenic culture of 3T3-L1 and SVF cells, adipocytes were treated with hyperosmotic medium for an additional 7 days, resulting in the appearance of visible lipid droplet accumulation and adipocytes with round morphology. Dedifferentiated cells were isolated for osteogenic differentiation.

[0065] Adipocyte dedifferentiation was initiated by hyperosmotic treatment of adipocytes in medium containing 2% PEG-300 as described previously. 11-12 After four to seven days of hyperosmotic treatment, approximately 40–70% of 3T3-L1 and SVF adipocytes lost lipid droplets, a significantly higher proportion than that of the control isotonic treatment ( Figure 1B and 1C After 7 days of treatment, Oil Red O staining was performed to confirm a decrease in fat content and adipocyte number ( Figure 1D At the same time, it was observed that the aspect ratio of cells in the hypertonic treatment group increased significantly ( Figure 1E ), which is consistent with the elongated morphology observed in previous studies on dedifferentiated adipocytes. In other words, dedifferentiated adipocytes will exhibit an elongated morphology.

[0066] To verify the dedifferentiation of 3T3-L1 adipocytes, the expression of adipocyte marker genes was further evaluated. Compared with the isotonic control group, the hyperosmotic-treated 3T3-L1 adipocytes showed a 40-60% decrease in the expression of adipogenic markers, including C / EBPβ, PPAR-γ, and adiponectin ( Figure 1F ). At the same time, immunostaining techniques were used to study CD13 and Endoglin, which are surface markers associated with dedifferentiated adipocytes. Quantitative analysis showed that the expression of CD13 and Endoglin in hyperosmotic-treated adipocytes was significantly higher than that in the isotonic control group ( Figure 2A-2B ). Collectively, these results indicate that hyperosmotic treatment can effectively induce dedifferentiation of 3T3-L1 adipocytes.

[0067] References: The disclosures of the following references are incorporated by reference

[0068] Example 2

[0069] The ability of hyperosmotically treated 3T3-L1 adipocytes to redifferentiate into osteogenic and chondrogenic cell lineages

[0070] An important functional feature of dedifferentiated adipocytes is their ability to redifferentiate into other mesenchymal cell lineages. Next, hyperosmotically treated 3T3-L1 adipocytes were evaluated for their ability to redifferentiate into osteogenic and chondrogenic pathways.

[0071] Bone formation induction promoted the expression of alkaline phosphatase (ALP) in 3T3-L1 adipocytes treated with hyperosmotic pressure ( Figures 3A-3C ), which is a marker of bone-forming cell lineage. At the same time, the expression of Runx2, another bone-forming marker ( Figure 3B In addition, after chondrogenesis induction in 3T3-L1 adipocytes treated with hyperosmotic pressure, the expression of chondrogenic marker genes increased, including Smad3, Col2a1, and Sox9 ( Figure 3C ). It is noteworthy that the control cells subjected to isotonic treatment did not show differentiation ability of bone formation or chondrogenesis, which is consistent with the adipogenesis of 3T3-L1 cells. Overall, it can be concluded that hyperosmotic treatment effectively induced the dedifferentiation of 3T3-L1 adipocytes from multipotent cells.

[0072] Example 3

[0073] Microvesicles released from adipocytes under hypertonic conditions

[0074] Cellular transformation is often accompanied by autocrine signaling events. Extracellular vesicles (EVs) are important mediators of intercellular communication in many biological contexts, including cancer. To investigate the potential role of EVs in regulating hyperosmotic dedifferentiation of adipocytes, EVs were isolated from isotonic and hyperosmotically treated 3T3-L1 adipocytes.

[0075] according to Figures 4A-4H , hypertonic treatment induces adipocytes to expel mitochondria via EVs. Their protein compositions were compared using LC-MS analysis ( Figure 4A ). Overall, 359 and 486 proteins (identified by ≥2 unique peptides) were detected in EVs obtained from hyperosmotic-treated and isotonic-treated 3T3-L1 adipocytes, respectively. 334 common proteins were found in both EV populations, while 25 proteins were uniquely identified in hyperosmotic EVs ( Figure 4B Interestingly, analysis of the gene ontology (GO) terms for these 25 hyperosmotic-unique proteins revealed an enrichment for mitochondrial components ( Figure 4B and Table 1), components host enzyme complexes for oxidative phosphorylation and play key roles in cellular ATP production and energy homeostasis.

[0076] Furthermore, quantitative analysis identified 47 proteins that were upregulated more than tenfold in hyperosmotic EVs ( Figure 4C ). Notably, GO term analysis attributed more than 50% of hyperosmotic upregulated EV proteins to mitochondrial components ( Figure 4D ), including NDUFA9 and UQCRC2, which are components of NADH-ubiquinone oxidoreductase (complex I) and ubiquinone-cytochrome c reductase (complex III) on the inner mitochondrial membrane, respectively. These results suggest that adipocytes excrete mitochondrial components through exosomes under hyperosmotic treatment.

[0077] Table 1 - Representative GO terms for cellular components of unique proteins in hypertonic EVs

[0078] Hyperosmotic conditions are known to disrupt cellular ion homeostasis and reduce the electron potential of mitochondria, thereby inhibiting ATP production. Therefore, adipocyte mitochondrial status was assessed using MitoTracker Green (MTG), a fluorescent probe that accumulates in mitochondria of living cells in an electron potential-dependent manner. Hyperosmotic treatment significantly attenuated MTG staining of mitochondria in adipocytes compared to isotonic controls. Pyruvate is known to relieve mitochondrial stress by increasing the electron potential, and application of 1 mM pyruvate to hyperosmotic treatment effectively restored MTG staining ( Figures 4E-4F ). These results suggest that hyperosmotic treatment may lead to disturbance of electron potential and mitochondrial stress in adipocytes.

[0079] Recent studies have shown that a range of cells can expel damaged mitochondria from their cytoplasm 14-15 Therefore, a hypothesis was proposed that adipocytes release mitochondria via EVs when mitochondria are subjected to hyperosmotic stress.

[0080] To investigate this phenomenon, EVs released from 3T3-L1 adipocytes under hyperosmotic treatment were examined using electron microscopy (EM). Figure 4G The folded membrane structure inside the EVs was shown to resemble mitochondrial cristae. At the same time, hyperosmotic treatment also increased the levels of mitochondrial proteins VDAC and NDUFA9 in EVs of 3T3-L1 adipocytes, while these levels were reduced by pyruvate, alleviating mitochondrial stress ( Figure 4H Meanwhile, no changes in the levels of the pan-EV marker CD81 were observed, suggesting that the effects were specific to mitochondrial fractions.

[0081] Furthermore, GW4869, a compound that blocks EV secretion, significantly suppressed the EV level of NDUFA9 induced by hyperosmotic treatment ( Figure 5 ). This result supports the idea that mitochondrial components are released via the EV pathway under hyperosmotic stress. Taken together, these results suggest that hyperosmotic treatment causes mitochondrial stress in adipocytes, which results in the excretion of mitochondrial components in EVs. These vesicles are called mitochondrial extracellular vesicles (MEVs).

[0082] Example 4

[0083] Role of inflammatory pathways in promoting hyperosmotic-induced adipocyte dedifferentiation

[0084] Transfer of mitochondrial contents via extracellular vesicles (EVs) has been shown to stimulate metabolic and inflammatory responses in recipient cells. Thus, mitochondrial EVs may function as an autocrine factor regulating adipocyte dedifferentiation.

[0085] To test this, MEVs were isolated from hyperosmotically treated 3T3-L1 adipocytes and then applied to primary 3T3-L1 adipocytes cultured under isotonic conditions ( Fig. 6A ). The results showed that hyperosmotic and mitochondrial extracellular vesicles activated the TNF-α signaling pathway in 3T3-L1 adipocytes. After 72 hours, RT-qPCR analysis showed that the expression of adipogenic markers including C / EBPβ, PPAR-γ and adiponectin was significantly reduced ( Figure 6B ). These results suggest that MEVs may be mediators of hyperosmotic-induced adipocyte dedifferentiation.

[0086] It has been demonstrated that extracellular mitochondria can promote inflammatory processes. Subsequent studies aimed to determine whether hypertonic MEV could affect inflammatory pathways in adipocytes. Indeed, application of hypertonic MEV to 3T3-L1 adipocytes significantly induced the expression of a range of inflammatory genes, including TNF-α, IL-6, RIP1, CEBPA, and MCP-1 ( Figure 6C and Fig. 7A In particular, a significant 4-fold and 8-fold induction of key inflammatory genes TNF-α and IL-6 was observed after 2 h of MEV treatment, respectively. The induction levels of these two genes remained high up to 8 h after treatment, indicating that hyperosmotic MEVs have a lasting effect. Importantly, alleviating mitochondrial stress with pyruvate prevented hyperosmotic EV-induced TNF-α expression ( Fig. 8A ). Given that pyruvate reduced the mitochondrial content in EVs, these results suggest that hypertonic MEVs may stimulate inflammatory signaling in adipocytes. Subsequently, the inflammatory response of adipocytes under hypertonic culture conditions was investigated. Similar to hypertonic EV treatment, 2 h of hypertonic culture induced the expression of inflammatory genes TNF-α, IL-6, RIP1, CEBPA, and MCP-1 in 3T3-L1 adipocytes ( Figure 8B and Figure 7B ).

[0087] Interestingly, after 8 h of hypertonic culture, the expression levels of TNF-α, IL-6, CEBPA, and MCP-1 decreased significantly. This is in contrast to the persistent effects of hypertonic EVs and may be due to the adaptation of adipocytes to the relatively mild hypertonic stress. Notably, the addition of pyruvate to the hypertonic culture abolished the induction of TNF-α and IL-6 expression ( Fig.9A ), further supporting the role of mitochondrial stress in the hyperosmotic stress effect. It is worth noting that ELISA experiments showed that the level of TNF-α protein increased in the culture medium of hyperosmotic-treated adipocytes ( Fig. 9B ). Collectively, these results suggest that hyperosmotic stress and mitochondrial EVs are able to activate inflammatory signaling in adipocytes.

[0088] Example 5

[0089] Role of TNF-α and Wnt / β-catenin signaling pathways in hyperosmotic-induced adipocyte dedifferentiation

[0090] Previous studies have highlighted the anti-adipogenic activity of the TNF-α inflammatory signaling pathway 8,16 To investigate the role of TNF-α in hyperosmotic-induced adipocyte dedifferentiation, 20 ng / ml of TNF-α neutralizing antibody was added to 3T3-L1 adipocytes cultured under hyperosmotic conditions.

[0091] Figures 10A-10HThe results showed that TNF-α activated β-catenin and apoptosis in adipocytes under hyperosmotic treatment. Compared with the control antibody, the TNF-α neutralizing antibody significantly inhibited the expression of stem cell genes induced by hyperosmotic treatment, including Esrrb and Sox2. Although not significant, the expression of Smad 9 was also slightly reduced after neutralization of TNF-α in dedifferentiated adipocytes ( Fig. 10A ). These results suggest a key role for TNF-α in hyperosmotic-induced adipocyte dedifferentiation.

[0092] The Wnt / β-catenin pathway is also known to inhibit adipogenesis and promote dedifferentiation of mature adipocytes. In fact, activation of β-catenin has been reported to be one of the causes of hyperosmotic dedifferentiation of adipocytes. 11 Evaluations were subsequently performed to determine the effects of hyperosmotic EV treatment on β-catenin in adipocytes.

[0093] Immunoblotting experiments confirmed that after 24 hours of hypertonic MEV treatment, β-catenin was stabilized in 3T3-L1 adipocytes, and non-phosphorylated (active) β-catenin accumulated ( Fig. 10B This was also observed in 3T3-L1 adipocytes after 24 hours of hypertonic culture ( Fig. 10C ). At the same time, immunostaining showed the accumulation of active β-catenin in the nucleus under hypertonic culture conditions ( Figures 10D-10E ). Therefore, in addition to inducing TNF-α, hyperosmotic stress and mitochondrial EVs also activated the anti-adipogenic Wnt / β-catenin signaling pathway.

[0094] TNF-α has been reported to induce stabilization of β-catenin and inhibit adipogenesis 8 Therefore, we evaluated the importance of hyperosmotic activation of β-catenin by TNF-α during adipocyte dedifferentiation. Fig.10F The application of TNF-α neutralizing antibody significantly blocked the stabilization of total and non-phosphorylated (active) β-catenin in hyperosmotic culture. These findings suggest that TNF-α plays an important role in hyperosmotic activation of the Wnt / β-catenin signaling pathway, which regulates adipocyte dedifferentiation.

[0095] Example 6

[0096] Hyperosmotic-induced adipocyte dedifferentiation and apoptosis: evaluation and therapeutic intervention with BML-284

[0097] Hyperosmotic dedifferentiation of adipocytes usually requires culturing in 2% PEG-300 for 7 days. Given that TNF-α is considered a proinflammatory cytokine that can induce apoptosis in adipocytes, the apoptotic status of adipocytes was assessed using Annexin V-FITC / PI staining. Figure 10G-10H , compared with the isotonic control, the number of early apoptotic cells was significantly increased by Annexin V staining. At the same time, no difference in the number of PI-positive cells was observed in the early apoptotic stage, which may be attributed to the loss of these cells during the washing process before harvesting. In conclusion, prolonged hyperosmotic treatment may lead to dedifferentiation of adipocytes accompanied by apoptosis.

[0098] To circumvent the apoptotic effects of hyperosmotic and TNF-α-induced adipocyte dedifferentiation, BML-284, a small molecule compound that stabilizes β-catenin and directly activates the Wnt / β-catenin signaling pathway, was used. 3T3-L1 and SVF adipocytes were treated with BML-284 (10 mM) under isotonic culture conditions, and their lipid droplets and morphological changes were subsequently evaluated. Strikingly, after 4 days of BML-284 treatment, there was a significant reduction in visible lipid droplets ( Figures 11A-11B In 3T3-L1 adipocytes, the number of visible lipid droplets decreased by 40%, while in SVF adipocytes, the reduction reached 80%. In 3T3-L1 and SVF adipocytes treated with BML-284, the number of cells with spindle morphology increased significantly ( Fig. 11C These results suggest the anti-adipogenic activity of BML-284, which is further supported by the reduction of Oil Red staining ( Figures 11C-11D ). At the same time, in cells treated with BML-284, the expression of lipogenic genes (C / EBPβ, PPAR-γ, and adiponectin) was significantly reduced, while the expression of marker genes (Smad 9, Esrrb, and Sox2) and pluripotency proteins (CD13 and endothelin) was significantly increased ( Figures 11E-11F and Figures 12A-12B Importantly, redifferentiation experiments showed that the expression of osteogenic and chondrogenic markers was increased in adipocytes treated with BML-284 after the corresponding induction treatment ( Figures 13A-13B ). Collectively, these results suggest that BML-284 may lead to adipocyte dedifferentiation by directly activating the β-catenin signaling pathway.

[0099] Subsequent steps involved comparing the effects of BML-284 and hyperosmotic treatment on apoptosis. Notably, while hyperosmotic treatment induced significant levels of apoptosis in 3T3-L1 adipocytes, BML-284 treatment did not result in significant apoptosis ( Fig. 13C ). Collectively, these results suggest that BML-284 treatment can effectively induce adipocyte dedifferentiation while circumventing the apoptotic effects of hyperosmotic treatment, and revealing the mechanisms that determine apoptotic / dedifferentiation fate will pave the way for the effective use of adipocytes for regenerative medicine ( Fig.14 ).

[0100] Osmotic stress can occur under various physiological or pathological conditions and have an impact on adipocytes. For example, hyperosmotic stress in adipocytes has been reported to inhibit insulin signaling and induce insulin resistance. Zhu et al. pointed out that adipocytes in breast cancer tissue may undergo adipose-mesenchymal transition (AMT), generating multiple cell types that constitute the inflammatory and cancer-promoting stroma. 17 Although the mechanisms of adipocyte-mesenchymal transition (AMT) remain unclear, it is noteworthy that osmotic stress and compression in breast cancer have been reported to activate β-catenin signaling and induce adipocyte dedifferentiation with the potential for multilineage redifferentiation. 11 . These findings suggest that hyperosmotic MEVs mediate β-catenin activation and induce adipocyte dedifferentiation in response to osmotic stress. Our results suggest that hyperosmotic MEVs mediate β-catenin activation and adipocyte dedifferentiation in response to osmotic stress. Our results suggest that hyperosmotic MEVs mediate β-catenin activation and adipocyte dedifferentiation in response to osmotic stress. It will be of interest to further investigate whether, in vivo, mitochondrial hyperosmotic stress and adipocyte MEVs are involved in the malignant adipocyte-mesenchymal transition in breast adipose tissue. This may provide potential therapeutic targets for the breast cancer microenvironment.

[0101] Furthermore, recent studies have shown that various cells can expel mitochondria to maintain homeostasis. For example, et al. found that cardiomyocytes release damaged mitochondria into extracellular vesicles to maintain normal cardiac function 18 . Here we report that hyperosmotic treatment of adipocytes may lead to mitochondrial excretion mediated by EVs. Mechanistically, fluctuations in cellular osmotic pressure may disturb ion homeostasis and electron potential in mitochondria, leading to damaged components. This view is supported by data showing that pyruvate is able to reduce hyperosmotic MEVs. Similarly, Rosina et al. reported that heat-stimulated brown adipocytes excrete a portion of dysfunctional mitochondria into EVs to prevent failure of the thermogenic program. 14 . The cellular processes and mechanisms underlying the release of mitochondrial components by EVs are still unclear, and it will be interesting to compare the composition and activity of MEVs from different conditions in inducing adipocyte dedifferentiation. Accumulating evidence also suggests that adipocyte dedifferentiation plays an important role in tissue homeostasis. Zhang et al. reported that subcutaneous adipose tissue undergoes dedifferentiation and redifferentiation during the hair cycle and wound healing. 19 Consistent with this, lineage tracing and single-cell RNA sequencing revealed adipocyte-derived myofibroblasts during wound healing 20 It is worth noting that wound healing is associated with the activation of inflammatory cytokines, including TNF-α.

[0102] Studies by et al. and Rosina et al. have shown that immune cells such as macrophages play a key role in clearing expelled mitochondria and maintaining homeostasis of the source tissue. Therefore, how MEVs interact with the inflammatory / immune system to induce adipocyte dedifferentiation in vivo requires further investigation.

[0103] Example 7

[0104] Materials and methods

[0105] Cell culture

[0106] 3T3-L1 (ATCC CL-173) cells were cultured in DMEM medium (Thermo Fisher, product number: 11965126) containing 10% FBS, 2 mM L-glutamine and 100 mg / ml penicillin-streptomycin.

[0107] The present invention uses C57BL / 6J mice and follows a 12:12 hour light cycle. All mouse experiments were conducted in accordance with the protocol approved by the Institutional Animal Research Ethics Committee of City University of Hong Kong and the Department of Health of the Government of the Hong Kong Special Administrative Region. Female mice aged 6 to 8 weeks were anesthetized by inhalation of 4% isoflurane, and then the inguinal and retroperitoneal adipose tissue were removed. The tissue was washed in sterile PBS and minced. Subsequently, the tissue fragments were incubated in type II collagenase (Sigma, product number C6885) at 37°C for 1 hour and then passed through a sterile 100 micron filter. SVF was obtained after centrifugation at 1000rpm for 5 minutes. Culture medium (DMEM, containing 10% FBS and 1% penicillin-streptomycin, both from Thermo Fisher) was added to suspend the cells and then cultured in a 100mm culture dish. The next day, the culture medium was replaced to remove dead and non-adherent cells.

[0108] 3T3-L1 and SVF differentiation

[0109] 1.25 × 10 53T3-L1 cells were cultured at 37°C and 5% CO2. 10 μg / ml insulin (MedChemExpress, product number HY-P0035), 1 μM dexamethasone (MedChemExpress, product number HY-14648), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (MedChemExpress, product number HY-12318) and 2 μM rosiglitazone (Sigma, product number R2408-10MG) (differentiation medium) were added to the culture medium to form a differentiation medium for 2 consecutive days of culture. The differentiation of SVF was induced by the differentiation medium. SVF cells were incubated in the differentiation medium for 5 days. The culture medium was then replaced with DMEM medium containing 10% FBS, 1×P / S and 10 μg / ml insulin, incubated for 48 hours, and then replaced with culture medium.

[0110] Oil Red O staining

[0111] Adipocytes were stained with Oil Red O staining kit (Solarbio, product number: G1262) solution according to the manufacturer's instructions to detect lipid droplets. Specifically, cells were fixed with 10% formaldehyde for 20 minutes at room temperature and then washed with 60% isopropanol for 5 minutes. Subsequently, ORO solution was used for staining at room temperature for 20 minutes to allow the dye to bind to lipid droplets. Finally, the cells were washed with distilled water to eliminate any residual dye.

[0112] Hyperosmotic-induced dedifferentiation of adipocytes

[0113] To induce hyperosmotic pressure, a culture medium solution containing 2% polyethylene glycol 300 (PEG 300) was added to adipocytes (3T3-L1 or SVF). Specifically, adipocytes were exposed to a hyperosmotic dedifferentiation medium, which was supplemented with 10% FBS and 1% penicillin-streptomycin from DMEM (provided by Thermo Fisher) and 2% polyethylene glycol-300 (MedChemExpress, product number: HY-Y0873) and maintained at 37°C and 5% CO2. The culture medium was replaced every 3 days, and care was taken to avoid shaking the culture dish to allow the hyperosmotic medium to diffuse slowly in the culture dish.

[0114] Osteogenic differentiation

[0115] The dedifferentiated adipocytes were then examined for their osteogenic and chondrogenic redifferentiation abilities. To induce osteogenic differentiation, 1×106 cells were seeded in a 6-well plate and incubated overnight at 37°C and 5% CO2. The medium was then replaced with StemPro Osteogenic Differentiation Medium (Thermo Fisher, Product Number: A1007201), and the cells were maintained in this medium for up to 14 days, with the medium replaced every 3 days. This medium contains specific factors that promote differentiation into the osteoblast lineage, enabling the cells to deposit mineralized extracellular matrix and express osteogenic markers.

[0116] Alkaline phosphatase assay

[0117] On day 9 of the reprogramming process, alkaline phosphatase (ALP) staining was performed using an alkaline phosphatase detection kit (Sigma, product number: SCR004) according to the manufacturer's instructions. Staining was performed to detect the activity of alkaline phosphatase (ALP), a pluripotency marker, and to assess the success of the reprogramming process.

[0118] MitoTracker staining

[0119] To stain cells with MitoTracker (CST, Catalog No. FM 9074), the 1 mM stock solution should be diluted directly into normal growth medium to obtain staining concentrations in the 400 nM range. Live cells are incubated at 37°C for 15 minutes. Following incubation, live cell imaging is required to visualize staining patterns within the cells.

[0120] qPCR gene expression analysis

[0121] Total RNA was isolated using an RNA extraction kit (Takara, product number 9767) following the manufacturer's recommended protocol. PrimeScript RT reagent kit (Takara, product number RR047A) was used to synthesize first-strand cDNA for RT-PCR. Briefly, a 10 μl reaction system containing 2 μl of 5× PrimeScript RT Master Mix (Perfect Real Time), RNase-free distilled water, and RNA solution with a total RNA amount of 500 ng was transferred to the ABI ProFlex PCR System (2×96 wells) for reverse transcription. RT-PCR was performed using the Ex-Taq PCR Kit (Takara, product number RR820A) according to the manufacturer's instructions. All RT-qPCR primer sequences are included in Table 2.

[0122] Table 2 - Primer sequences used for qPCR validation

[0123] Immuno / fluorescence labeling and microscopy

[0124] 12 hours before the start of the experiment, cells were plated onto glass coverslips in 12-well culture dishes and then cultured overnight at 37°C and 5% CO2. Cells were fixed with 2% paraformaldehyde in PBS at 4°C for 10 minutes. Next, cells were incubated in 1% BSA / PBS for 1 hour or permeabilized in 0.1% Triton X-100 / PBS for 10 minutes and then blocked with 1% BSA / PBS. Subsequently, cells were incubated with CD13 primary antibody (Santa Cruz, product number: sc-13536, 1:100) or Endoglin primary antibody (Santa Cruz, product number: sc-18838, 1:100) at 4°C overnight. After washing three times in 0.5% Tween / PBS, cells were incubated with secondary antibody (CST, product number: 8890S) in 1% BSA / PBS (1:400) for 1.5 hours at room temperature in the dark. After further washing, cells were mounted with DAPI (Thermo Fisher, Product No.: 62247) and observed using a Nikon A1HD25 confocal microscope.

[0125] Immunoblotting

[0126] To obtain the lysate, cells were washed twice with cold PBS and incubated at 4°C with gentle rotation for 1 hour, and lysed using 1 mL of RIPA buffer (Beyotime, product number: P0013B). Next, the lysate was centrifuged at 12,000 g for 20 minutes at 4°C, and the resulting supernatant was used for protein concentration determination. The protein extracts were then separated by SDS-PAGE and transferred to NC membranes (Bio-Rad, product number 1620112). The membrane was incubated with antibodies against VDAC (CST, product number 4866s, 1:1000), NDUFA9 (Abcam, product number ab14713, 1:1000), CD81 (Santa Cruz, product number sc-166029, 1:1000), non-phosphorylated (active) β-Catenin (CST, product number 8814, 1:2000), β-Catenin (CST, product number 9562, 1:1000), and β-actin (Santa Cruz, product number sc-47778, 1:1000) at 4°C overnight. After incubation with appropriate secondary antibodies (Abcam, product number ab205719; Thermo Fisher, product number 31460, 1:4000), protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Bio-Rad, product number 170-5061) and captured using Bio-red ChemiDoc.

[0127] Cell drug treatment

[0128] To inhibit EV secretion, adipocytes were treated with 10 μM GW4869 (Sigma-Aldrich, Product No. D1692) under isotonic and hypertonic culture conditions, respectively. An equal volume of DMSO was added to the control sample. After two days, the supernatant was collected for EV isolation and analysis.

[0129] To alleviate mitochondrial stress, adipocytes were treated with 1 mM pyruvate (Sigma-Aldrich, Product No. 107360) in hypertonic medium. An equal volume of DMSO was added to control samples. After treatment, cells were stained with MTG or the supernatant was collected for EV isolation and analysis.

[0130] EV isolation and protein digestion

[0131] 3T3-L1 adipocytes were treated separately with isotonic and hypertonic medium. After 48 hours of culture, the corresponding conditioned medium was collected. EVs were isolated by sequential ultracentrifugation, including centrifugation at 300g for 10 minutes to remove the supernatant, centrifugation at 2000g for 15 minutes, centrifugation at 10000g for 70 minutes, and finally collecting the precipitate by centrifugation at 120000g for 2 hours. The precipitate was washed once with PBS and then precipitated by centrifugation at 120000g for 70 minutes. The purified EV precipitate was vacuum dried and then resuspended to 100μL in a buffer containing 8M urea (Sigma, product number U1250) and 50mM NH4HCO3 (Alfa Aesar, product number 14249). The resuspended suspensions each contained approximately 80μg of protein, as determined by the DC protein assay (Bio-Rad, product number 5000114). The following solution digestion for LC-MS / MS analysis was performed. Briefly, EV protein suspension was reduced with 20 mM DTT at 95 °C for 10 min and then alkylated with 50 mM IAA for 30 min at room temperature in the dark. The solution was then diluted with 50 mM NH4HCO3 buffer (pH 8.0) to reduce the urea concentration to less than 1 M. MS-grade trypsin (Thermo Fisher, product number 90058) coupled to Lyc-C protease was added to the solution (pH 8.0) at an enzyme / protein ratio of 1:25 to digest the proteins overnight at 37 °C. The resulting peptide samples were desalted using a C18 patch (Thermo Fisher, product number 87784) and resuspended with 0.1% formic acid buffer (Thermo Fisher, product number 85178) for LC-MS / MS analysis.

[0132] LC-MS / MS analysis

[0133] Peptide samples were analyzed using an EASY-nLC 1200 system coupled to a Q Exactive HF mass spectrometer (Thermo Scientific). Each injection of 6 μL of sample containing approximately 0.5 μg of peptide was separated via a C18 nanocolumn (250 nm, 75 μm, 3 μm, PepSep, Denmark) (Thermo Fisher, product number 87784) at a flow rate of 250 nl / min. A 75-min reverse phase gradient was achieved using mobile phase A (0.1% formic acid in ultrapure water) and mobile phase B (0.1% formic acid / 80% acetonitrile in ultrapure water). MS recording was performed in the range of 350 to 1800 m / z with a mass resolution of 120,000. Positive ion mode was used with a spray voltage of 2000 V and a spray temperature of 320 °C. The resolution of dd-MS2 was 30,000, and the AGC target was set to 1 × 105 The maximum integration time was set to 60 ms and the number of cycles was 12. The isolation window was 1.6 m / z and the fixed first mass was 120.0 m / z.

[0134] Database Search

[0135] Raw files created by XCalibur 4.0.27 (Thermo Fisher) software were analyzed in the Sequest HT node using Proteome Discoverer software (version 2.2, Thermo Fisher) against the UniProt mouse protein database. The mass tolerances for precursors and fragments were set to 10 ppm and 0.02 Da, respectively. A maximum of two undigested tryptic cleavage sites were allowed. Carboxymethylation (C) was set as a static modification, and oxidation (M) and acetylation (protein N-terminus) were set as variable modifications. The false discovery rate (FDR) for peptide spectrum matching (PSM) and peptide identification was determined at the 1% level based on the q value using the Percolator algorithm. For label-free quantification, the Minora Feature Detector node was used in the processing workflow, and the Precursor Ions Quantifier node and Feature Mapper node were used in the consensus workflow. In Proteome Discoverer, the quantitative values ​​were normalized based on the total peptide intensity of the sample.

[0136] Transmission electron microscopy analysis of extracellular vesicles

[0137] In 0.1M cocodiamine buffer (pH 7.2), extracellular vesicles (EVs) were fixed with a solution of 5% formaldehyde and 2% glutaraldehyde and then left overnight at 4°C. After washing with 0.1M cocodiamine buffer, the samples were fixed with a 2% osmium tetroxide solution in 0.1M cocodiamine buffer for 2 hours at room temperature in the dark. After washing again with 0.1M cocodiamine buffer, the samples were dehydrated with a series of ethanol solutions of increasing concentrations (30%, 50%, 70%, 80%, 90%, 95% and 100%), followed by further dehydration with 70% and 100% acetone. The samples were then immersed in Spurr resin, gradually increasing concentrations to 25%, 50%, 75% and 100%. After 2 days of polymerization at 70°C, ultrathin sections were prepared using a diamond knife and collected on 100-mesh grids coated with butvar (EMS, product number FF100-CU), followed by 10 minutes of contrast staining with UranyLess (EMS, product number 22409) and 1 minute of lead citrate staining. Finally, the samples were imaged using a transmission electron microscope (Philips Technai12) to observe the internal structure and morphology of extracellular vesicles (EVs).

[0138] Industrial Applicability:

[0139] The dedifferentiation of adipocytes from pluripotency has the potential to revolutionize regenerative medicine. However, its potential application is limited by unclear mechanisms and low efficiency. Mitochondrial extracellular vesicles act as an early warning signal and play a protective role in various physiological and pathological processes. The generated MEVs can be used as biomarkers for disease diagnosis and monitoring, providing valuable insights into the status of cells and tissues. Therefore, the present invention has therapeutic applications in regenerative medicine and tissue engineering, promoting processes such as cell proliferation, angiogenesis, and extracellular matrix remodeling.

[0140] abbreviation SVF: stromal vascular fraction EVs: Extracellular vesicles MEVs: mitochondrial extracellular vesicles DFAT: Dedifferentiated adipocytes ACK: Ammoniacal Potassium Chloride PEG 300: Polyethylene glycol 300 ALP: Alkaline phosphatase C / EBPβ:CCAAT / enhancer binding protein β PPARγ: peroxisome proliferator-activated receptor γ CD13: Aminopeptidase N Runx2: RUNX family transcription factor 2 Smad3: maternal 12-cell adenoma-associated protein 3 Col2a1: type II collagen α1 chain Sox9: SRY-box transcription factor 9 ATP: Adenosine triphosphate NDUFA9:NADH:ubiquinone redoxin reductase subunit A9UQCRC2:ubiquinone-cytochrome C reductase core protein 2 VDAC: Voltage Dependent Aperture Channel CD81: 81 cluster-differentiated protein TNF-α: Tumor necrosis factor-α IL-6: interleukin 6 RIP1: receptor interacting serine / threonine protein 1 CEBPA:CCAAT / enhancer binding protein alpha MCP-1: Monocyte chemoattractant protein-1 Esrrb: estrogen-related receptor beta Sox2: sex determining region Y-box 2 Smad9: maternal 12-cell adenoma-associated protein 9

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Claims

1. A method for inducing adipocyte dedifferentiation, characterized in that: include: exposing one or more adipocytes to a hypertonic solution; as well as Inducing the release of mitochondrial extracellular vesicles from the one or more adipocytes to The extracellular environment The released mitochondrial extracellular vesicles enhance the secretion of a series of inflammatory genes in the one or more adipocytes, and the series of inflammatory genes activate the Wnt / β-catenin signaling pathway, thereby driving the Dedifferentiation of adipocytes.

2. The method of claim 1, wherein the panel of inflammatory genes comprises TNF-α, IL-6, RIP1, CEBPA and MCP-1.

3. The method of claim 1, wherein the one or more adipocytes include isolated adipocytes, adipocytes derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), and adipocytes from different anatomical locations. 4 . The method of claim 1 , wherein the one or more adipocytes comprise 3T3-L1 or adipose stromal vascular fraction (SVF)-derived adipocytes.

5. The method of claim 1, wherein the adipocyte count of the one or more adipocytes is reduced by at least 3.5-fold after the hypertonic treatment.

6. The method of claim 5, wherein the hyperosmotic treatment results in a 40-60% decrease in the expression of the adipogenic markers C / EBPβ, PPAR-γ, and adiponectin.

7. The method of claim 5, wherein the hypertonic treatment results in a 10-20% increase in the expression of surface markers associated with dedifferentiated adipocytes. 8 . The method according to claim 1 , wherein the hypertonic solution is formulated to have a higher hypertonic pressure than the surrounding environment, and the hypertonic solution comprises a culture medium and 2% PEG 300.

9. The method of claim 8, wherein the hypertonic solution further comprises an additive, the additive comprising a preservative, a stabilizer or a drug.

10. The method of claim 1, wherein the one or more adipocytes treated with the hypertonic solution exhibit osteogenic and chondrogenic redifferentiation capabilities.

11. The method of claim 1, further comprising adding a small molecule compound to mitigate apoptosis of the one or more adipocytes induced by the hyperosmotic treatment.

12. The method of claim 11, wherein the small molecule compound comprises 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine.

13. The method of claim 11, wherein the one or more adipocytes have reduced expression of adipogenic genes accompanied by increased expression of marker genes.

14. The method of claim 13, wherein the lipogenic genes include C / EBPβ, PPAR-γ, and adiponectin.

15. The method of claim 13, wherein the marker genes include Smad 9, Esrrb and Sox2.

16. The method of claim 11, wherein the one or more adipocytes exhibit osteogenic and chondrogenic redifferentiation capabilities.

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