Mechanical stretch preconditioned adipose-derived stem cells elicit polarization of anti-inflammatory M2-like macrophages and improve chronic wound healing.

Transplantation of adipose-derived stem cells (ASCs) is a promising option in the field of chronic wounds treatment. However, the effectiveness of ASCs therapies has been hampered by highly inflammatory environment in chronic wound areas. These problems could be partially circumvented using efficient approaches that boost the survival and anti-inflammatory capacity of transplanted ASCs. Here, by application of mechanical stretch (MS), we show that ASCs exhibits increased survival and immunoregulatory properties in vitro. MS triggers the secretion of macrophage colony stimulating factor (M-CSF) from ASCs, a chemokine that is linked to anti-inflammatory M2-like macrophages polarization. When the MS-ASCs were transplanted to chronic wounds, the wound area yields significantly faster closure rate and lower inflammatory mediators, largely due to macrophages polarization driven by transplanted MS-ASCs. Thus, our work shows that mechanical stretch can be harnessed to enhance ASCs transplantation efficiency in chronic wounds treatment.

Insights into the role of adipose-derived stem cells and secretome: potential biology and clinical applications in hypertrophic scarring.

Scar tissue is the inevitable result of repairing human skin after it has been subjected to external destructive stimuli. It leads to localized damage to the appearance of the skin, accompanied by symptoms such as itching and pain, which reduces the quality of life of the patient and causes serious medical burdens. With the continuous development of economy and society, there is an increasing demand for beauty. People are looking forward to a safer and more effective method to eliminate pathological scarring. In recent years, adipose-derived stem cells (ADSCs) have received increasing attention from researchers. It can effectively improve pathological scarring by mediating inflammation, regulating fibroblast proliferation and activation, and vascular reconstruction. This review focuses on the pathophysiological mechanisms of hypertrophic scarring, summarizing the therapeutic effects of in vitro, in vivo, and clinical studies on the therapeutic effects of ADSCs in the field of hypertrophic scarring prevention and treatment, the latest application techniques, such as cell-free therapies utilizing ADSCs, and discussing the advantages and limitations of ADSCs. Through this review, we hope to further understand the characterization of ADSC and clarify the effectiveness of its application in hypertrophic scarring treatment, so as to provide clinical guidance.

Isolation of Nuclei from Human Intermuscular Adipose Tissue and Downstream Single-Nuclei RNA Sequencing.

Intermuscular adipose tissue (IMAT) is a relatively understudied adipose depot located between muscle fibers. IMAT content increases with age and BMI and is associated with metabolic and muscle degenerative diseases; however, an understanding of the biological properties of IMAT and its interplay with the surrounding muscle fibers is severely lacking. In recent years, single-cell and nuclei RNA sequencing have provided us with cell type-specific atlases of several human tissues. However, the cellular composition of human IMAT remains largely unexplored due to the inherent challenges of its accessibility from biopsy collection in humans. In addition to the limited amount of tissue collected, the processing of human IMAT is complicated due to its proximity to skeletal muscle tissue and fascia. The lipid-laden nature of the adipocytes makes it incompatible with single-cell isolation. Hence, single nuclei RNA sequencing is optimal for obtaining high-dimensional transcriptomics at single-cell resolution and provides the potential to uncover the biology of this depot, including the exact cellular composition of IMAT. Here, we present a detailed protocol for nuclei isolation and library preparation of frozen human IMAT for single nuclei RNA sequencing. This protocol allows for the profiling of thousands of nuclei using a droplet-based approach, thus providing the capacity to detect rare and low-abundant cell types.

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells.

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

Transdifferentiation of fibroblasts into muscle cells to constitute cultured meat with tunable intramuscular fat deposition.

Current studies on cultured meat mainly focus on the muscle tissue reconstruction in vitro, but lack the formation of intramuscular fat, which is a crucial factor in determining taste, texture, and nutritional contents. Therefore, incorporating fat into cultured meat is of superior value. In this study, we employed the myogenic/lipogenic transdifferentiation of chicken fibroblasts in 3D to produce muscle mass and deposit fat into the same cells without the co-culture or mixture of different cells or fat substances. The immortalized chicken embryonic fibroblasts were implanted into the hydrogel scaffold, and the cell proliferation and myogenic transdifferentiation were conducted in 3D to produce the whole-cut meat mimics. Compared to 2D, cells grown in 3D matrix showed elevated myogenesis and collagen production. We further induced fat deposition in the transdifferentiated muscle cells and the triglyceride content could be manipulated to match and exceed the levels of chicken meat. The gene expression analysis indicated that both lineage-specific and multifunctional signalings could contribute to the generation of muscle/fat matrix. Overall, we were able to precisely modulate muscle, fat, and extracellular matrix contents according to balanced or specialized meat preferences. These findings provide new avenues for customized cultured meat production with desired intramuscular fat contents that can be tailored to meet the diverse demands of consumers.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

A hybrid construct of decellularized matrix and fibrin for differentiating adipose stem cells into insulin-producing cells, an optimized in vitro assessment.

The generation of insulin-producing cells (IPCs) is an attractive approach for replacing damaged ß cells in diabetic patients. In the present work, we introduced a hybrid platform of decellularized amniotic membrane (dAM) and fibrin encapsulation for differentiating adipose tissue-derived stem cells (ASCs) into IPCs. ASCs were isolated from healthy donors and characterized. Human AM was decellularized, and its morphology, DNA, collagen, glycosaminoglycan (GAG) contents, and biocompatibility were evaluated. ASCs were subjected to four IPC differentiation methods, and the most efficient method was selected for the experiment. ASCs were seeded onto dAM, alone or encapsulated in fibrin gel with various thrombin concentrations, and differentiated into IPCs according to a method applying serum-free media containing 2-mercaptoethanol, nicotinamide, and exendin-4. PDX-1, GLUT-2 and insulin expression were evaluated in differentiated cells using real-time PCR. Structural integrity and collagen and GAG contents of AM were preserved after decellularization, while DNA content was minimized. Cultivating ASCs on dAM augmented their attachment, proliferation, and viability and enhanced the expression of PDX-1, GLUT-2, and insulin in differentiated cells. Encapsulating ASCs in fibrin gel containing 2 mg/ml fibrinogen and 10 units/ml thrombin increased their differentiation into IPCs. dAM and fibrin gel synergistically enhanced the differentiation of ASCs into IPCs, which could be considered an appropriate strategy for replacing damaged ß cells.

Hypoxic adipose stem cell-derived exosomes carrying high-abundant USP22 facilitate cutaneous wound healing through stabilizing HIF-1α and upregulating lncRNA H19.

Hypoxic preconditioning has been recognized as a promotive factor for accelerating cutaneous wound healing. Our previous study uncovered that exosomal lncRNA H19, derived from adipose-derived stem cells (ADSCs), plays a crucial role in orchestrating cutaneous wound healing. Herein, we aimed to explore whether there is a connection between hypoxia and ADSC-derived exosomes (ADSCs-exos) in cutaneous wound healing. Exosomes extracted from ADSCs under normoxic and hypoxic conditions were identified using transmission electron microscope (TEM) and particle size analysis. The effects of ADSCs-exos on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8, EdU, wound healing, and tube formation assays. Expression patterns of H19, HIF-1α, and USP22 were measured. Co-immunoprecipitation, chromatin immunoprecipitation, ubiquitination, and luciferase reporter assays were conducted to confirm the USP22/HIF-1α/H19 axis, which was further validated in a mice model of skin wound. Exosomes extracted from hypoxia-treated ADSCs (termed as H-ADSCs-exos) significantly increased cell proliferation, migration, and angiogenesis in H2O2-exposed HUVECs, and promoted cutaneous wound healing in vivo. Moreover, H-ADSCs and H-ADSCs-exos, which exhibited higher levels of H19, were found to be transcriptionally activated by HIF-1α. Mechanically, H-ADSCs carrying USP22 accounted for deubiquitinating and stabilizing HIF-1α. Additionally, H-ADSCs-exos improved cell proliferation, migration, and angiogenesis in H2O2-triggered HUVECs by activating USP22/HIF-1α axis and promoting H19 expression, which may provide a new clue for the clinical treatment of cutaneous wound healing.

3D Porous Edible Scaffolds from Rye Secalin for Cell-Based Pork Fat Tissue Culturing.

Cellular agriculture holds hope for a sustainable alternative to conventional meat, yet multiple technical challenges remain. These include the large-scale production of edible scaffolds and culturing methods for fat tissues, which are key to meat texture, flavor, and nutritional values. Herein. we disclose our method in the facile fabrication of sponge-like plant protein scaffolds by applying commercial sugar cubes as highly permeable templates. The prepared secalin scaffolds feature a high porosity of 85-90%, fully interconnected pores, and high water stability. The mechanical properties of scaffolds could be tuned by varying sugar-to-protein weight ratio and post-water annealing treatment. Moreover, murine preadipocytes (3T3-L1) and porcine adipose-derived stem cells (ADSCs) readily infiltrate, adhere, proliferate, and differentiate on the secalin scaffolds to develop a fat tissue morphology. A cultured fat tissue was produced by culturing porcine ADSCs for 12 days, which remarkably resembles conventional porcine subcutaneous adipose tissue in appearance, texture, flavor, and fatty acid profiles. The research demonstrates the great potential of sponge-like secalin scaffolds for cultured fat tissue production.

Modeling alcohol-associated liver disease in humans using adipose stromal or stem cell-derived organoids.

Alcohol-associated liver disease (ALD) is a prevalent liver disease, yet research is hampered by the lack of suitable and reliable human ALD models. Herein, we generated human adipose stromal/stem cell (hASC)-derived hepatocellular organoids (hAHOs) and hASC-derived liver organoids (hALOs) in a three-dimensional system using hASC-derived hepatocyte-like cells and endodermal progenitor cells, respectively. The hAHOs were composed of major hepatocytes and cholangiocytes. The hALOs contained hepatocytes and nonparenchymal cells and possessed a more mature liver function than hAHOs. Upon ethanol treatment, both steatosis and inflammation were present in hAHOs and hALOs. The incubation of hALOs with ethanol resulted in increases in the levels of oxidative stress, the endoplasmic reticulum protein thioredoxin domain-containing protein 5 (TXNDC5), the alcohol-metabolizing enzymes ADH1B and ALDH1B1, and extracellular matrix accumulation, similar to those of liver tissues from patients with ALD. These results present a useful approach for understanding the pathogenesis of ALD in humans, thus facilitating the discovery of effective treatments.

[Research progress in regulation of hair growth by dermal adipose tissue].

Objective: To summarize the dynamic and synchronized changes between the hair cycle and dermal adipose tissue as well as the impact of dermal adipose tissue on hair growth, and to provide a new research idea for the clinical treatment of hair loss. Methods: An extensive review of relevant literature both domestic and international was conducted, analyzing and summarizing the impact of dermal adipose precursor cells, mature dermal adipocytes, and the processes of adipogenesis in dermal adipose tissue on the transition of hair cycle phases. Results: Dermal adipose tissue is anatomically adjacent to hair follicles and closely related to the changes in the hair cycle. The proliferation and differentiation of dermal adipose precursor cells promote the transition of hair cycle from telogen to anagen, while mature adipocytes can accelerate the transition from anagen to catagen of the hair cycle by expressing signaling molecules, with adipogenesis in dermal adipose tissue and hair cycle transition signaling coexistence. Conclusion: Dermal adipose tissue affects the transition of the hair cycle and regulates hair growth by secreting various signaling molecules. However, the quantity and depth of existing literature are far from sufficient to fully elucidate its prominent role in regulating the hair cycle, and the specific regulatory mechanisms needs to be further studied.

Deletion of CD44 promotes adipogenesis by regulating PPARγ and cell cycle-related pathways.

CD44, a cell surface adhesion receptor and stem cell biomarker, is recently implicated in chronic metabolic diseases. Ablation of CD44 ameliorates adipose tissue inflammation and insulin resistance in obesity. Here, we investigated cell type-specific CD44 expression in human and mouse adipose tissue and further studied how CD44 in preadipocytes regulates adipocyte function. Using Crispr Cas9-mdediated gene deletion and lentivirus-mediated gene re-expression, we discovered that deletion of CD44 promotes adipocyte differentiation and adipogenesis, whereas re-expression of CD44 abolishes this effect and decreases insulin responsiveness and adiponectin secretion in 3T3-L1 cells. Mechanistically, CD44 does so via suppressing Pparg expression. Using quantitative proteomics analysis, we further discovered that cell cycle-regulated pathways were mostly decreased by deletion of CD44. Indeed, re-expression of CD44 moderately restored expression of proteins involved in all phases of the cell cycle. These data were further supported by increased preadipocyte proliferation rates in CD44-deficient cells and re-expression of CD44 diminished this effect. Our data suggest that CD44 plays a crucial role in regulating adipogenesis and adipocyte function possibly through regulating PPARγ and cell cycle-related pathways. This study provides evidence for the first time that CD44 expressed in preadipocytes plays key roles in regulating adipocyte function outside immune cells where CD44 is primarily expressed. Therefore, targeting CD44 in (pre)adipocytes may provide therapeutic potential to treat obesity-associated metabolic complications.

A Gelatin Hydrogel Nonwoven Fabric Combined With Adipose Tissue-Derived Stem Cells Enhances Subcutaneous Islet Engraftment.

Subcutaneous islet transplantation is a promising treatment for severe diabetes; however, poor engraftment hinders its prevalence. We previously revealed that a gelatin hydrogel nonwoven fabric (GHNF) markedly improved subcutaneous islet engraftment. We herein investigated whether the addition of adipose tissue-derived stem cells (ADSCs) to GHNF affected the outcome. A silicone spacer sandwiched between two GHNFs with (AG group) or without (GHNF group) ADSCs, or a silicone spacer alone (Silicone group) was implanted into the subcutaneous space of healthy mice at 6 weeks before transplantation, then diabetes was induced 7 days before transplantation. Syngeneic islets were transplanted into the pretreated space. Intraportal transplantation (IPO group) was also performed to compare the transplant efficiency. Blood glucose, intraperitoneal glucose tolerance, immunohistochemistry, and inflammatory mediators were evaluated. The results in the subcutaneous transplantation were compared using the Silicone group as a control. The results of the IPO group were also compared with those of the AG group. The AG group showed significantly better blood glucose changes than the Silicone and the IPO groups. The cure rate of AG group (72.7%) was the highest among the groups (GHNF; 40.0%, IPO; 40.0%, Silicone; 0%). The number of vWF-positive vessels in the subcutaneous space of the AG group was significantly higher than that in other groups before transplantation (P < 0.01). Lectin angiography also showed that the same results (P < 0.05). According to the results of the ADSCs tracing, ADSCs did not exist at the transplant site (6 weeks after implantation). The positive rates for laminin and collagen III constructed around the transplanted islets did not differ among groups. Inflammatory mediators were higher in the Silicone group, followed by the AG and GHNF groups. Pretreatment using bioabsorbable scaffolds combined with ADSCs enhanced neovascularization in subcutaneous space, and subcutaneous islet transplantation using GHNF with ADSCs was superior to intraportal islet transplantation.

Migrasomes from adipose derived stem cells enrich CXCL12 to recruit stem cells via CXCR4/RhoA for a positive feedback loop mediating soft tissue regeneration.

BACKGROUND: Adipose-derived stem cells (ASCs) represent the most advantageous choice for soft tissue regeneration. Studies proved the recruitment of ASCs post tissue injury was mediated by chemokine CXCL12, but the mechanism by which CXCL12 is generated after tissue injury remains unclear. Migrasomes are newly discovered membrane-bound organelles that could deliver CXCL12 spatially and temporally in vivo. In this study, we sought to investigate whether migrasomes participate ASC-mediated tissue regeneration. METHODS: Discrepant and asymmetrical soft tissue regeneration mice model were established, in which HE staining, immunofluorescent staining, western blot and qPCR were conducted to confirm the role of CXCL12 and migrasomes in ASC-mediated tissue regeneration. Characterization of ASC-derived migrasomes were carried out by confocal microscopy, scanning electron microscopy, transmission electron microscopy as well as western blot analysis. The function and mechanism of migrasomes were further testified by assisting tissue regeneration with isolated migrasomes in vivo and by in vitro transwell combined with co-culture system. RESULTS: Here, we show for the first time that migrasomes participate in soft tissue regeneration. ASCs generate migrasomes enriched with CXCL12 to mediate tissue regeneration. Migrasomes from ASCs could promote stem cells migration by activating CXCR4/RhoA signaling in vivo and in vitro. Chemoattracted ASCs facilitate regeneration, as demonstrated by the upregulation of an adipogenesis-associated protein. This positive feed-back-loop creates a favorable microenvironment for soft tissue regeneration. Thus, migrasomes represent a new therapeutic target for ASC-mediated tissue regeneration. CONCLUSIONS: Our findings reveal a previously unknown function of ASCs in mediating tissue regeneration by generating migrasomes. The ASC-derived migrasomes can restore tissue regeneration by recruiting stem cells, which highlighting the potential application of ASC-derived migrasomes in regenerative medicine.

The use of stem cells in the treatment of mastitis in dairy cows.

Mastitis is a multifactorial inflammatory disease. The increase in antibiotic resistance of bacteria that cause mastitis means that cattle breeders would prefer to reduce the use of antibiotics. Recently, therapies using mesenchymal stem cells (MSCs) from various sources have gained significant interest in the development of regenerative medicine in humans and animals, due to their extraordinary range of properties and functions. The aim of this study was to analyze the effectiveness of an allogeneic stem cells derived from bone marrow (BMSC) and adipose tissue (ADSC) in treating mastitis in dairy cattle. The research material consisted of milk and blood samples collected from 39 Polish Holstein-Friesian cows, 36 of which were classified as having mastitis, based on cytological evaluation of their milk. The experimental group was divided into subgroups according to the method of MSC administration: intravenous, intramammary, and intravenous + intramammary, and according to the allogeneic stem cells administered: BMSC and ADSC. The research material was collected at several time intervals: before the administration of stem cells, after 24 and 72 h, and after 7 days. Blood samples were collected to assess hematological parameters and the level of pro-inflammatory cytokines, while the milk samples were used for microbiological assessment and to determine the somatic cells count (SCC). The administration of allogeneic MSCs resulted in a reduction in the total number of bacterial cells, Staphylococcus aureus, bacteria from the Enterobacteriaceae group, and a systematic decrease in SCC in milk. The therapeutic effect was achieved via intravenous + intramammary or intramammary administration.

Extremely low frequency-electromagnetic fields promote chondrogenic differentiation of adipose-derived mesenchymal stem cells through a conventional genetic program.

Progressive cartilage deterioration leads to chronic inflammation and loss of joint function, causing osteoarthritis (OA) and joint disease. Although symptoms vary among individuals, the disease can cause severe pain and permanent disability, and effective therapies are urgently needed. Human Adipose-Derived Stem Cells (ADSCs) may differentiate into chondrocytes and are promising for treating OA. Moreover, recent studies indicate that electromagnetic fields (EMFs) could positively affect the chondrogenic differentiation potential of ADSCs. In this work, we investigated the impact of EMFs with frequencies of 35 Hertz and 58 Hertz, referred to as extremely low frequency-EMFs (ELF-EMFs), on the chondrogenesis of ADSCs, cultured in both monolayer and 3D cell micromasses. ADSC cultures were daily stimulated for 36 min with ELF-EMFs or left unstimulated, and the progression of the differentiation process was evaluated by morphological analysis, extracellular matrix deposition, and gene expression profiling of chondrogenic markers. In both culturing conditions, stimulation with ELF-EMFs did not compromise cell viability but accelerated chondrogenesis by enhancing the secretion and deposition of extracellular matrix components at earlier time points in comparison to unstimulated cells. This study showed that, in an appropriate chondrogenic microenvironment, ELF-EMFs enhance chondrogenic differentiation and may be an important tool for supporting and accelerating the treatment of OA through autologous adipose stem cell therapy.

Exosomes Secreted by Wharton's Jelly-Derived Mesenchymal Stem Cells Promote the Ability of Cell Proliferation and Migration for Keratinocyte.

Mesenchymal stem cells (MSCs) isolated from Wharton's jelly (WJ-MSCs) and adipose tissue (AD-MSCs) are alternative sources for bone marrow-derived MSCs. Owing to their multiple functions in angiogenesis, immune modulation, proliferation, migration, and nerve regeneration, MSC-derived exosomes can be applied in "cell-free cell therapy". Here, we investigated the functional protein components between the exosomes from WJ-MSCs and AD-MSCs to explain their distinct functions. Proteins of WJ-MSC and AD-MSC exosomes were collected and compared based on iTRAQ gel-free proteomics data. Results: In total, 1695 proteins were detected in exosomes. Of these, 315 were more abundant (>1.25-fold) in AD-MSC exosomes and 362 kept higher levels in WJ-MSC exosomes, including fibrinogen proteins. Pathway enrichment analysis suggested that WJ-MSC exosomes had higher potential for wound healing than AD-MSC exosomes. Therefore, we treated keratinocyte cells with exosomes and the recombinant protein of fibrinogen beta chain (FGB). It turned out that WJ-MSC exosomes better promoted keratinocyte growth and migration than AD-MSC exosomes. In addition, FGB treatment had similar results to WJ-MSC exosomes. The fact that WJ-MSC exosomes promoted keratinocyte growth and migration better than AD-MSC exosomes can be explained by their higher FGB abundance. Exploring the various components of AD-MSC and WJ-MSC exosomes can aid in their different clinical applications.

Knockdown of LAP2α inhibits adipogenesis of human adipose-derived stem cells and ameliorates high-fat diet-induced obesity.

Adipogenesis, a pivotal cellular process involving the differentiation of mesenchymal stem cells (MSCs) to mature adipocytes, plays a significant role in various physiological functions. Dysregulation of adipogenesis is implicated in conditions such as obesity. However, the complete molecular understanding of adipogenesis remains elusive. This study aimed to uncover the novel role of lamina-associated polypeptide 2 alpha (LAP2α) in human adipose-derived stem cells (hASCs) adipogenesis and its impact on high-fat diet (HFD)-induced obesity and associated metabolic disturbances. LAP2α expression was assessed during the adipogenic differentiation of hASCs using RT-qPCR and western blotting. The functional role of LAP2α in adipogenesis was explored both in vitro and in vivo through loss- and gain-of-function studies. Moreover, mice with HFD-induced obesity received lentivirus injection to assess the effect of LAP2α knockdown on fat accumulation. Molecular mechanisms underlying LAP2α in adipogenic differentiation were investigated using RT-qPCR, Western blotting, immunofluorescence staining, and Oil Red O staining. LAP2α expression was upregulated during hASCs adipogenic differentiation. LAP2α knockdown hindered adipogenesis, while LAP2α overexpression promoted adipogenic differentiation. Notably, LAP2α deficiency resisted HFD-induced obesity, improved glucose intolerance, mitigated insulin resistance, and prevented fatty liver development. Mechanistically, LAP2α knockdown attenuated signal transducer and activator of transcription 3 (STAT3) activation by reducing the protein level of phosphorylated STAT3. A STAT3 activator (Colivelin) counteracted the negative impact of LAP2α deficiency on hASCs adipogenic differentiation. Taken together, our current study established LAP2α as a crucial regulator of hASCs adipogenic differentiation, unveiling a new therapeutic target for obesity prevention.

The immunoregulatory property of mesenchymal stem cells in Crocin treatment by expression modulation of microRNA-155, microRNA-21, microRNA-23b, microRNA-126a, and their target inflammatory genes.

Mesenchymal stem cells (MSCs) have high priority in clinical applications for treatment of immune disorders because of their immunomodulatory function. A lot of researches have currently been undertaken to enhance the stemness capacities of the cells and pick an excellent type of MSCs for clinical approaches. This study aims to assess the immunomodulatory related MicroRNAs (miRNAs) expression as well as their target genes in both adipose derived stem cells (Ad-SCs) and dental pulp derived stem cell (DP-SCs) in the presence or lack of Crocin (saffron plant's bioactive compound). For this purpose, first MSCs were extracted from adipose and dental pulp tissues, and then their mesenchymal nature was confirmed using flow cytometry and differentiation tests. Following the cell treatment with an optimal-non-toxic dose of Crocin (Obtained by MTT test), the expression of 4 selected immunomodulatory-related micro-RNAs (Mir-126, -21, -23, and-155) and their target genes (PI3K/ Akt 1 and 2/ NFKB and RELA) were assessed by RT-PCR. Our findings revealed that miRNA-23 and miRNA-126 were up-regulated in both types of cells treated with Crocin, while in the other side, miRNA-21 and miRNA-155 were down-regulated in DP-SCs and were up-regulated in Ad-SCs under treatment. Moreover, the real-time PCR results indicated that Crocin could significantly down regulate the expression of PI3K/ Akt1/ Akt2/ NFKB/ RELA genes in DP-SCs and PI3K/Akt2 genes in Ad-SCs and up regulate the expression of Akt1/ NFKB/ RELA genes in recent cells. Based on the analysis of the obtained data, the immunoregulatory effects of Crocin were higher in DP-SCs than in Ad-SCs. In conclusion, Crocin could control essential signaling pathways related to the inflammation by regulating the expression of related- miRNAs genes that play a key function in the immune regulation pathways in MSCs. Our findings can give an understanding of the mechanisms by which Crocin enhances the immunomodulatory feature of MSCs. According to the research findings, DP-SCs are probably a better immunomodulator in Crocin treatment than Ad-SCs and it may be helpful for MSCs selection in clinical applications for modulation or treatment of autoimmune disorders.

Viscoelastic hydrogels regulate adipose-derived mesenchymal stem cells for nucleus pulposus regeneration.

Low back pain is a leading cause of disability worldwide, often attributed to intervertebral disc (IVD) degeneration with loss of the functional nucleus pulposus (NP). Regenerative strategies utilizing biomaterials and stem cells are promising for NP repair. Human NP tissue is highly viscoelastic, relaxing stress rapidly under deformation. However, the impact of tissue-specific viscoelasticity on the activities of adipose-derived stem cells (ASC) remains largely unexplored. Here, we investigated the role of matrix viscoelasticity in regulating ASC differentiation for IVD regeneration. Viscoelastic alginate hydrogels with stress relaxation time scales ranging from 100 s to 1000s were developed and used to culture human ASCs for 21 days. Our results demonstrated that the fast-relaxing hydrogel significantly enhanced ASCs long-term cell survival and NP-like extracellular matrix secretion of aggrecan and type-II collagen. Moreover, gene expression analysis revealed a substantial upregulation of the mechanosensitive ion channel marker TRPV4 and NP-specific markers such as SOX9, HIF-1α, KRT18, CDH2 and CD24 in ASCs cultured within the fast-relaxing hydrogel, compared to slower-relaxing hydrogels. These findings highlight the critical role of matrix viscoelasticity in regulating ASC behavior and suggest that viscoelasticity is a key parameter for novel biomaterials design to improve the efficacy of stem cell therapy for IVD regeneration. STATEMENT OF SIGNIFICANCE: Systematically characterized the influence of tissue-mimetic viscoelasticity on ASC. NP-mimetic hydrogels with tunable viscoelasticity and tissue-matched stiffness. Long-term survival and metabolic activity of ASCs are substantially improved in the fast-relaxing hydrogel. The fast-relaxing hydrogel allows higher rate of cell protrusions formation and matrix remodeling. ASC differentiation towards an NP-like cell phenotype is promoted in the fast-relaxing hydrogel, with more CD24 positive expression indicating NP committed cell fate. The expression of TRPV4, a molecular sensor of matrix viscoelasticity, is significantly enhanced in the fast-relaxing hydrogel, indicating ASC sensing matrix viscoelasticity during cell development. The NP-specific ECM secretion of ASC is considerably influenced by matrix viscoelasticity, where the deposition of aggrecan and type-II collagen are significantly enhanced in the fast-relaxing hydrogel.