Adipose-derived Mesenchymal Stem Cells Research Articles

Adipose-derived Mesenchymal Stem Cell Transplantation Improves Ovarian Function and Oocyte Quality in Aged Mice.

Age-related decline in the number of ovulations and ovum quality are major causes of female infertility, and stem cells have been reported to be effective in tissue regeneration. However, current therapeutic modalities are inadequate. This study investigated the effects of adipose-derived mesenchymal stem cells (ASCs) on ovarian functions in aged mice. Following the characterization of ASCs using flow cytometry, the effects of ASCs on the number of ovulations, fertilization rate, and blastocyst-formation rate were investigated. In addition, the number of ovarian follicles and serum anti-Müllerian hormone (AMH) levels were examined. ASCs marked with Kusabira Orange were used to examine the location after cell administration. The quality of ovulated oocytes was analyzed using next-generation RNA sequencing. ASCs showed characteristics of mesenchymal stem cells and were distributed to various organs, including the ovarian stroma. The transplantation resulted in increased number of oocytes and ovulation in the ovaries and increased AMH values. Genetic analysis revealed improved oocyte quality and increased fertilization and blastocyst-formation rates. ASC therapy may be effective in improving fertility in older women.

Mesenchymal stromal/stem cell tissue source and in vitro expansion impact extracellular vesicle protein and miRNA compositions as well as angiogenic and immunomodulatory capacities.

Recently, therapies utilizing extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) have begun to show promise in clinical trials. However, EV therapeutic potential varies with MSC tissue source and in vitro expansion through passaging. To find the optimal MSC source for clinically translatable EV-derived therapies, this study aims to compare the angiogenic and immunomodulatory potentials and the protein and miRNA cargo compositions of EVs isolated from the two most common clinical sources of adult MSCs, bone marrow and adipose tissue, across different passage numbers. Primary bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (ASCs) were isolated from adult female Lewis rats and expanded in vitro to the indicated passage numbers (P2, P4, and P8). EVs were isolated from the culture medium of P2, P4, and P8 BMSCs and ASCs and characterized for EV size, number, surface markers, protein content, and morphology. EVs isolated from different tissue sources showed different EV yields per cell, EV sizes, and protein yield per EV. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of proteomics data and miRNA seq data identified key proteins and pathways associated with differences between BMSC-EVs and ASC-EVs, as well as differences due to passage number. In vitro tube formation assays employing human umbilical vein endothelial cells suggested that both tissue source and passage number had significant effects on the angiogenic capacity of EVs. With or without lipopolysaccharide (LPS) stimulation, EVs more significantly impacted expression of M2-macrophage genes (IL-10, Arg1, TGFβ) than M1-macrophage genes (IL-6, NOS2, TNFα). By correlating the proteomics analyses with the miRNA seq analysis and differences observed in our in vitro immunomodulatory, angiogenic, and proliferation assays, this study highlights the trade-offs that may be necessary in selecting the optimal MSC source for development of clinical EV therapies.

Osteogenic Differential Expression of Rabbit Adipose-Derived Mesenchymal Stem Cells by PPARγ Transfection Coated with Nanoparticles

Adipose-derived stem cells (ADSCs) have advantages in utilization. Nanoparticles (NP) are degradable, nontoxic and have good biocompatibility, besides, they can encapsulate and transport water-soluble, fat-soluble and deoxyribonucleic acid. This study explored the influence of NP-coated PPARγ transfection on osteogenic differentiation of rabbit ADSCs. After preparing PPARγ transfection system coated with nanoparticles, Petri dishes containing the same amount of cells were divided at the beginning of cell culture into blank group (not transfected), control group and observation group (transfected with PPARγ coated NP). Alkaline phosphatase (ALP) staining and Western Blot were done to detect the contents of lipoprteinlipase (LPL), Runx2 and serum osteocalcin (BGP) protein. The levels of Runx2, BGP and PPARγ mRNA were measured by RT-PCR. After ADSCs cells were stained, a small number of cells adhered to each other, and the cells were mainly fibroblast-like and spindle-shaped, and proliferated in the form of colonies. The levels of Runx2 and BGP in the observation group were significantly lower than those in the control group (p < 0.05) after the ADSCs cells were cultured for 7 and 14 days. The expression of LPL and PPARγ mRNA in the observation group was higher control group (p < 0.05). Transfection of PPARγ coated with nanoparticles inhibited the osteogenic differentiation of rabbit ADSCs by inhibiting the expression of Runx2 and BGP and promoting the expression of LPL and PPARγ.

Research on the role of exosomes secreted by immortalized adipose-derived mesenchymal stem cells differentiated into pericytes in the repair of high glucose-induced retinal vascular endothelial cell damage

Diabetic retinopathy, a leading cause of vision impairment, is marked by microvascular complications in the retina, including pericyte loss, a key indicator of early-stage disease. This study explores the therapeutic potential of exosomes derived from immortalized adipose-mesenchymal stem cells differentiated into pericyte-like cells in restoring the function of mouse retinal microvascular endothelial cells damaged by high glucose conditions, thereby contributing to the understanding of early diabetic retinopathy intervention strategies. To induce immortalized adipose-mesenchymal stem cells differentiation into pericyte-like cells, the study employed pericyte growth supplement. And confirmed the success of cell differentiation through the detection of α-smooth muscle actin and neural/glial antigen 2 expression by Western blot and immunofluorescence. Exosomes were isolated from the culture supernatant of immortalized adipose-mesenchymal stem cells using ultracentrifugation and characterized through Western blot for exosomal markers (CD9, CD81, and TSG101), transmission electron microscopy, and nanoparticle tracking analysis. Their influence on mouse retinal microvascular endothelial cells under high glucose stress was assessed through various functional assays. Findings revealed that exosomes, especially those from pericyte-like immortalized adipose-mesenchymal stem cells, were efficiently internalized by retinal microvascular endothelial cells and effectively counteracted high glucose-induced apoptosis. These exosomes also mitigated the rise in reactive oxygen species levels and suppressed the migratory and angiogenic properties of retinal microvascular endothelial cells, as demonstrated by Transwell and tube formation assays, respectively. Furthermore, they preserved endothelial barrier function, reducing hyperglycemia-induced permeability. At the molecular level, qRT-PCR analysis showed that exosome treatment modulated the expression of critical genes involved in angiogenesis (VEGF-A, ANG2, MMP9), inflammation (IL-1β, TNF-α), gap junction communication (CX43), and cytoskeletal regulation (ROCK1), with the most prominent effects seen with exosomes from pericyte-like immortalized adipose-mesenchymal stem cells. High glucose increased the expression of pro-angiogenic and pro-inflammatory markers, which were effectively normalized post-exosome treatment. In conclusion, this research highlights the reparative capacity of exosomes secreted by pericyte-like differentiated immortalized adipose-mesenchymal stem cells in reversing the detrimental effects of high glucose on retinal microvascular endothelial cells. By reducing apoptosis, oxidative stress, inflammation, and abnormal angiogenic behavior, these exosomes present a promising avenue for therapeutic intervention in early diabetic retinopathy. Future studies can focus on elucidating the precise molecular mechanisms and exploring their translational potential in vivo.

Differentiation of Human Adipose-derived Stem Cells to Exosome-affected Neural-like Cells Extracted from Human Cerebrospinal Fluid Using Bioprinting Process.

Advancement in tissue engineering has provided novel solutions for creating scaffolds as well as applying induction factors in the differentiation of stem cells. The present research aimed to investigate the differentiation of human adipose-derived mesenchymal stem cells to neural-like cells using the novel bioprinting method, as well as the effect of cerebrospinal fluid exosomes. In the present study, the extent of neuronal proliferation and differentiation of adipose- derived stem cells were explored using the MTT method, immunocytochemistry, and real-- time PCR in the scaffolds created by the bioprinting process. Furthermore, in order to investigate the veracity of the identity of the CSF (Cerebrospinal fluid) derived exosomes, after the isolation of exosomes, dynamic light scattering (DLS), scanning electron microscopy (SEM), and atomic force microscopy (AFM) techniques were used. MTT findings indicated survivability and proliferation of cells in the scaffolds created by the bioprinting process during a 14-day period. The results obtained from real-time PCR showed that the level of MAP2 gene (Microtubule Associated Protein 2) expression increased on days 7 and 14, while the expression of the Nestin gene (intermediate filament protein) significantly decreased compared to the control. The investigation to confirm the identity of exosomes indicated that the CSF-derived exosomes had a spherical shape with a 40-100 nm size. CSF-derived exosomes can contribute to the neuronal differentiation of adipose- derived stem cells in alginate hydrogel scaffolds created by the bioprinting process.

The Optimal Effective Dose of Adipose-Derived Stem Cell Exosomes in Wound Healing.

Although the effect of adipose-derived mesenchymal stem cell exosomes (ADSC-exos) on wound healing with different doses are shown in various studies, efficient and sufficient doses of ADSC-exos are still unknown. The study aimed to determine the optimal dose of ADSC-exos in wound healing. The 45 Sprague-Dawley rats were randomly divided into five groups, with seven animals in each. After dorsal circular defects were created, each wound was injected as follows: group 1: saline, group 2: 10 μg/mL of ADSC-exos, group 3: 100 μg/mL of ADSC-exos, group 4: 200 μg/mL of ADSC-exos, and group 5: 400 μg/mL of ADSC-exos. The effects of ADSC-exos on epithelization, angiogenesis, and collagen formation were analyzed macroscopically, histopathologically, and immunohistochemically on day 14. A total of 200 μg/mL and 400 μg/mL ADSC-exos groups had higher epithelial tongue length, epithelial tongue area, and angiogenesis scores than the other groups. Although there was no statistical difference in fibrosis scores among groups, collagen fibers were becoming well-organized as the ADSC-exos doses increased. While the wound area was clinically smaller in the 200 μg/mL ADSC-exos group, there was no statistically significant difference among groups on day 14. A total of 200 μg/mL of ADSC-exos was found to be the adequate and effective dose for re-epithelialization and angiogenesis in cutaneous wound healing. Moreover, the collagen density increased with a more regular pattern in the 200 μg/mL group, which can be important in scar regulation.

Dampened inflammatory response in oral ulcer after topical therapy of adipose mesenchymal stem cell secretome

ObjectivesResearch has demonstrated that modulating inflammation can significantly accelerate the healing of oral ulcers. Our study focused on the adipose mesenchymal stem cell secretome (AdMSCS), which is rich in immunoregulatory molecules capable of dampening the immune response and interfering with inflammatory pathways. We assessed both inflammatory pathway expression and macrophage phenotypes at the sites of oral ulcers. MethodsWe induced oral ulcers in the inferior fornix mucosa of 20 healthy male Wistar rats (Rattus norvegicus). These subjects were treated topically with adipose MSC metabolite (AdMSCM) oral gel three times daily, for durations of 3 and 7 days. We performed immunohistochemical analyses to evaluate the expression of Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-κB) p65 at the ulcer sites. Additionally, we assessed macrophage polarization by examining the ratio of M2/M1 macrophages, identified through CD68+Φ (M1) and CD163+Φ (M2) cells. Data were analyzed using one-way analysis of variance, followed by post-hoc Tukey's Honestly Significantly Difference test. ResultsApplication of AdMSCM oral gel significantly reduced the expression of TLR4 and NF-κB p65. This treatment also enhanced macrophage polarization towards the anti-inflammatory M2 phenotype at the ulcer sites (p < 0.05). ConclusionThe topical application of AdMSCM oral gel effectively modulates the inflammatory response, enhancing healing processes in the oral ulcer rat model. This suggests its potential utility as a therapeutic agent in managing oral ulcers.

Porous gelatin-based phosphorylated scaffold: Microstructure, cell response and osteogenic differentiation of human adipose-derived mesenchymal stem cells

To repair the highly organized composite bone tissue, the design and development of biomimetic three-dimensional scaffolds is an active strategy. Here, we describe the fabrication route of a bioengineered gelatin-based scaffold for potential application in bone tissue engineering. First, phosphorus-containing polyurethane (PU–P) polymer was synthesized by polymerization starting from phosphorous polyol and methylene diisocyanate (DMI). Second, a gelatin/phosphorus-containing polyurethane (Gelatin/PU-P) scaffold was prepared by solution mixing, ultrasonication, and freeze-drying of gelatin and PU-P polymer. Next, prepared scaffold samples were characterized by FT-IR, SEM microscopy, porosity measurement, swelling, degradability, and mechanical tests. The scaffold with a porosity of about 78 % was achieved, and at the same time, it had good dimensional stability so that it only lost 43.61 % of its initial weight after 35 days immersing in PBS. Moreover, Gelatin/PU-P showed flexibility, large elongations at break (420 %) and high tensile strength (14 MPa). Finally, biological assessments revealed that the scaffold significantly supported the viability of Adipose-derived mesenchymal stem cells (ADMS). High ALP activity, enhancement in mineralization, and greater expression of osteogenic gene markers in cells grown on the Gelatin/PU-P confirmed the osteoinductivity of the scaffold. These findings suggested that Gelatin/PU-P can be used as a bioactive scaffold for bone tissue engineering.

Advances in Adipose-derived Mesenchymal Stem Cells for Burn Wound Healing

Advances in Adipose-derived Mesenchymal Stem Cells for Burn Wound Healing

Optimization of differentiation conditions for porcine adipose-derived mesenchymal stem cells and analysis of fatty acids in cultured fat

Cultured fat is an important part of cultured meat, and the ability of adipose-derived mesenchymal stem cells (ADSCs) to differentiate into mature adipose tissue affects the quality of cultured fat. Thus, the primary aim of this study was to screen for combinations of differentiation-inducing factors (DIF) using single-factor experiment and orthogonal experimental design under two-dimensional culture conditions for ADSCs. The results showed that a combination of DIF consisting of 1 μmol/L dexamethasone, 0.1 mmol/L 3-isobutyl-1-methylxanthine, 10 μg/mL insulin, 0.1 mmol/L indomethacin, and 2 μmol/L rosiglitazone was a good choice for the differentiation of ADSCs. An combination of DIF was applied to the preparation of cultured fat with collagen as scaffolds. Forty-eight fatty acids were detected in cultured fat by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Among them, the content of twenty-one fatty acids in cultured fat was significantly higher than that of conventional porcine subcutaneous adipose tissue (P < 0.05), and the content of 14 fatty acids was not significantly different (P > 0.05). The ratio of ω-6 polyunsaturated fatty acids content to ω-3 polyunsaturated fatty acids content was 1.23:1, which meant cultured fat was beneficial for human health. This study provides a method to improve the differentiation ability of ADSCs while also providing a reference for indicating the nutritional value of cultured fat.

Rapid and Controllable Multilayer Cell Sheet Assembly via Biodegradable Nanochannel Membranes

AbstractThe ability to precisely arrange and control the assembly of diverse cell types into intricate 3D structures remains a critical challenge in tissue engineering. Herein, a versatile and programmable 3D cell sheet assembly is described technology by developing a biodegradable nanochannel (BNC) membrane to fulfill this unmet need. This membrane, hierarchically assembled from 2D nanomaterial aggregates, exhibits both exceptional fluid permeability and rapid biodegradation under physiological conditions. The unique properties of the BNC membrane enable precise spatial and temporal control over cell assembly, facilitating the creation of complex 3D cellular architectures. The BNC membrane is integrated with a programmable negative‐pressure‐based cell assembly strategy to form single and multicellular 3D sheets in a highly controllable manner. To demonstrate the feasibility and translatability of this technology in the field of tissue engineering approaches to screen stem cell‐derived therapeutics with “core–shell” macrophage‐fibroblast multicellular patterns and treat murine diabetic skin wounds via scaffold‐free 3D adipose‐derived mesenchymal stem cell (ADMSC) sheets are devised. In summary, the results demonstrate that the BNC membrane‐based 3D cell sheet assembly approach significantly advances current tissue engineering capabilities, offering substantial potential for both regenerative medicine applications and the development of physiologically relevant disease models.

Transcriptional profiling of fracture-associated cytokines and growth factors identifies transcriptional regulation of osteogenic genes by recombinant-human IL1β

Signaling molecules secreted during bone fracture healing stimulate molecular and cellular changes to promote healing and tissue regeneration. Molecular characterization of cytokines and growth factors can provide insight into their function and potential application as biologics or drug targets for bone tissue regeneration. Previous studies have identified the expression of IL1B, IL6, IL34, IGF1, ANGPT1, and TGFB3 during osteogenic differentiation and facture healing. In this study, we evaluated the transcriptome of adipose-derived mesenchymal stromal cells (AMSCs) following stimulation with these biological factors and examined their role in osteogenic differentiation. Stimulation of AMSCs with recombinant human (rh)-IL1B led to the largest gene expression changes compared to the other signaling factors including 1411 induced and 541 repressed genes (>2-fold). Rh-IL1B regulated genes included inflammatory (IL1B, IL6, CCL2) and osteogenic (BMP2, RUNX2, TWIST2) genes. Rh-IL1B also induced the expression of BMP2 in bone marrow-derived mesenchymal stromal cells (BMSCs) and bone digest cells. Although no changes in osteogenic differentiation of AMSCs was observed, low concentrations of rh-IL1B (0.1 ng/mL) promoted alkaline phosphatase activity of differentiated bone digest cells. Further, rh-IL1B significantly induced the secretion of BMP2 from these cells. Together this data provides insight into the molecular function of fracture-associated biological factors.

Hmga2 knockdown enhances osteogenic differentiation of adipose-derived mesenchymal stem cells and accelerates bone defect healing in mice

To investigate the role of high-mobility group AT-hook 2 (HMGA2) in osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) and the effect of Hmga2 knockdown for promoting bone defect repair. Bioinformatics studies using the GEO database and Rstudio software identified HMGA2 as a key factor in adipogenic-osteogenic differentiation balance of ADSCs. The protein-protein interaction network of HMGA2 in osteogenic differentiation was mapped using String and visualized with Cytoscape to predict the downstream targets of HMGA2. Primary mouse ADSCs (mADSCs) were transfected with Hmga2 siRNA, and the changes in osteogenic differentiation of the cells were evaluated using alkaline phosphatase staining and Alizarin red S staining. The expressions of osteogenic markers Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and osteocalcein (OCN) in the transfected cells were detected using RT-qPCR and Western blotting. In a mouse model of critical-sized calvarial defects, mADSCs with Hmga2-knockdown were transplanted into the defect, and bone repair was evaluated 6 weeks later using micro-CT scanning and histological staining. GEO database analysis showed that HMGA2 expression was upregulated during adipogenic differentiation of ADSCs. Protein-protein interaction network analysis suggested that the potential HMGA2 targets in osteogenic differentiation of ADSCs included SMAD7, CDH1, CDH2, SNAI1, SMAD9, IGF2BP3, and ALDH1A1. In mADSCs, Hmga2 knockdown significantly upregulated the expressions of RUNX2, OPN, and OCN and increased cellular alkaline phosphatase activity and calcium deposition. In a critical-sized calvarial defect model, transplantation of mADSCs with Hmga2 knockdown significantly promoted new bone formation. HMGA2 is a crucial regulator of osteogenic differentiation in ADSCs, and Hmga2 knockdown significantly promotes osteogenic differentiation of ADSCs and accelerates ADSCs-mediated bone defect repair in mice.

PCL reinforced nano strontium hydroxyapatite-Butea monosperma-scaffold for bone regeneration

Biocomposite materials, composed of biodegradable polymers and supplements, are promising for bone regenerative matrices due to their mechanical strength and excellent osteoconductivity. This study focuses on fabricating electrospun composite scaffolds using the bark extract of Butea monosperma (BM), nano strontium hydroxyapatite (nSrHA), and polycaprolactone (PCL). Four types of scaffolds were prepared: PCL (control), BM-PCL, nSrHA-PCL, and BM-nSrHA-PCL. The scaffolds were evaluated through various techniques: bright field microscopy for standardization, SEM for structural evaluation, and SEM-EDX for elemental analysis. Additionally, mechanical strength, release study, degradation, and protein adsorption were assessed. Furthermore, cytotoxicity, proliferation, and cytocompatibility of the scaffolds were evaluated using rat adipose-derived mesenchymal stem cells (rADMSCs) through SEM, MTT assay, LDH assay, DAPI staining, and Direct contact assay. SEM analysis confirmed that all four fabricated scaffolds were nano-fibrous in nature. The incorporation of phytochemicals and nSrHA was confirmed via SEM-EDX. Mechanical testing revealed that the BM-nSrHA-PCL scaffolds had superior mechanical properties compared to the control PCL scaffolds. Protein adsorption studies indicated enhanced protein binding in BM-nSrHA-PCL scaffolds. Degradation studies showed that BM-nSrHA-PCL scaffolds had a controlled degradation rate suitable for tissue engineering applications. Cytotoxicity tests confirmed that all scaffolds were noncytotoxic. Proliferation and cytocompatibility assays demonstrated that BM-nSrHA-PCL scaffolds significantly promoted cell proliferation compared to other scaffold types. These results suggest that the BM-nSrHA-PCL scaffolds exhibit improved mechanical properties, protein adsorption, controlled degradation, and enhanced cell proliferation, making them promising candidates for bone tissue engineering applications.

Biological characteristics of tissue engineered-nerve grafts enhancing peripheral nerve regeneration

BackgroundA favorable regenerative microenvironment is essential for peripheral nerve regeneration. Neural tissue-specific extracellular matrix (ECM) is a natural material that helps direct cell behavior and promote axon regeneration. Both bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ADSCs) transplantation are effective in repairing peripheral nerve injury (PNI). However, there is no study that characterizes the in vivo microenvironmental characteristics of these two MSCs for the early repair of PNI when combined with neural tissue-derived ECM materials, i.e., acellular nerve allograft (ANA).MethodsIn order to investigate biological characteristics, molecular mechanisms of early stage, and effectiveness of ADSCs- or BMSCs-injected into ANA for repairing PNI in vivo, a rat 10 mm long sciatic nerve defect model was used. We isolated primary BMSCs and ADSCs from bone marrow and adipose tissue, respectively. First, to investigate the in vivo response characteristics and underlying molecular mechanisms of ANA combined with BMSCs or ADSCs, eighty-four rats were randomly divided into three groups: ANA group, ANA+BMSC group, and ANA+ADSC group. We performed flow cytometry, RT-PCR, and immunofluorescence staining up to 4 weeks postoperatively. To further elucidate the underlying molecular mechanisms, changes in long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) were systematically investigated using whole transcriptome sequencing. We then constructed protein–protein interaction networks to find 10 top ranked hub genes among differentially expressed mRNAs. Second, in order to explore the effectiveness of BMSCs and ADSCs on neural tissue-derived ECM materials for repairing PNI, sixty-eight rats were randomized into four groups: ANA group, ANA+BMSC group, ANA+ADSC group, and AUTO group. In the ANA+BMSC and ANA+ADSC groups, ADSCs/BMSCs were equally injected along the long axis of the 10-mm ANA. Then, we performed histological and functional assessments up to 12 weeks postoperatively.ResultsThe results of flow cytometry and RT-PCR showed that ANA combined with BMSCs exhibited more significant immunomodulatory effects, as evidenced by the up-regulation of interleukin (IL)-10, down-regulation of IL-1β and tumor necrosis factor-alpha (TNF-α) expression, promotion of M1-type macrophage polarization to M2-type, and a significant increase in the number of regulatory T cells (Tregs). ANA combined with ADSCs exhibited more pronounced features of pro-myelination and angiogenesis, as evidenced by the up-regulation of myelin-associated protein gene (MBP and MPZ) and angiogenesis-related factors (TGF-β, VEGF). Moreover, differentially expressed genes from whole transcriptome sequencing results further indicated that ANA loaded with BMSCs exhibited notable immunomodulatory effects and ANA loaded with ADSCs was more associated with angiogenesis, axonal growth, and myelin formation. Notably, ANA infused with BMSCs or ADSCs enhanced peripheral nerve regeneration and motor function recovery with no statistically significant differences.ConclusionsThis study revealed that both ANA combined with BMSCs and ADSCs enhance peripheral nerve regeneration and motor function recovery, but their biological characteristics (mainly including immunomodulatory effects, pro-vascular regenerative effects, and pro-myelin regenerative effects) and underlying molecular mechanisms in the process of repairing PNI in vivo are different, providing new insights into MSC therapy for peripheral nerve injury and its clinical translation.

Synthesis and Systematic Investigation of Lepidiline A and Its Gold(I), Silver(I), and Copper(I) Complexes Using In Vitro Cancer Models and Multipotent Stem Cells.

The imidazole alkaloid lepidiline A from the root of Lepidium meyenii has a moderate to low in vitro anticancer effect. Our aim was to extend cytotoxicity investigations against a panel of cancer cells, including multidrug-resistant cancer cells, and multipotent stem cells. Lepidiline A is a N-heterocyclic carbene precursor, therefore a suitable ligand source for metal complexes. Thus, we synthesized lepidiline A and its copper(I), gold(I), and silver(I) complexes and tested them against ovarian, gastrointestinal, breast, and uterine cancer cells and bone marrow-derived and adipose-derived mesenchymal stem cells. Lepidiline A and its copper complex demonstrated moderate cytotoxicity, while silver and gold complexes exhibited significantly enhanced and consistent cytotoxicity against both cancer and stem cell lines. ABCB1 in the multidrug-resistant uterine sarcoma line conferred significant resistance against lepidiline A and the copper-lepidiline A complex, but not against the silver and gold complexes. Our results indicate that only the copper complex induced a significant and universal increase in the production of reactive oxygen species within cells. In summary, binding of metal ions to lepidiline A results in enhanced cytotoxicity with the nature of the metal ion playing a critical role in determining its properties.

Cellular In Vitro Responses Induced by Human Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles Obtained from Suspension Culture.

Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have been described to have important roles in tissue regeneration, including tissue repair, control of inflammation, enhancing angiogenesis, and regulating extracellular matrix remodeling. MSC-EVs have many advantages for use in regeneration therapies such as facility for dosage, histocompatibility, and low immunogenicity, thus possessing a lower possibility of rejection. In this work, we address the potential activity of MSC-EVs isolated from adipose-derived MSCs (ADMSC-EVs) cultured on cross-linked dextran microcarriers, applied to test the scalability and reproducibility of EV production. Isolated ADMSC-EVs were added into cultured human dermal fibroblasts (NHDF-1), keratinocytes (HaCat), endothelial cells (HUVEC), and THP-1 cell-derived macrophages to evaluate cellular responses (i.e., cell proliferation, cell migration, angiogenesis induction, and macrophage phenotype-switching). ADMSC viability and phenotype were assessed during cell culture and isolated ADMSC-EVs were monitored by nanotracking particle analysis, electron microscopy, and immunophenotyping. We observed an enhancement of HaCat proliferation; NHDF-1 and HaCat migration; endothelial tube formation on HUVEC; and the expression of inflammatory cytokines in THP-1-derived macrophages. The increased expression of TGF-β and IL-1β was observed in M1 macrophages treated with higher doses of ADMSC-EVs. Hence, EVs from microcarrier-cultivated ADMSCs are shown to modulate cell behavior, being able to induce skin tissue related cells to migrate and proliferate as well as stimulate angiogenesis and cause balance between pro- and anti-inflammatory responses in macrophages. Based on these findings, we suggest that the isolation of EVs from ADMSC suspension cultures makes it possible to induce in vitro cellular responses of interest and obtain sufficient particle numbers for the development of in vivo concept tests for tissue regeneration studies.

Isolation of the Stromal Vascular Fraction Using a New Protocol with All Clinical-Grade Drugs: From Basic Study to Clinical Application.

The purpose of this study was to evaluate the yield, viability, clinical safety, and efficacy of the stromal vascular fraction (SVF) separated with a new protocol with all clinical-grade drugs. SVF cells were isolated from lipoaspirate obtained from 13 participants aged from 30 to 56 years by using a new clinical protocol and the laboratory protocol. The cell yield, viability, morphology, mesenchymal stem cell (MSC) surface marker expression, and differentiation abilities of the SVF cells harvested from the two protocols were compared. Furthermore, three related clinical trials were conducted to verify the safety and efficiency of SVF cells isolated by the new clinical protocol. There were no significant differences in the yield, viability, morphology, and differentiation potential of the SVFs isolated with the clinical protocol and laboratory protocol. Adipose-derived mesenchymal stem cell (ASC) surface marker expression, including that of CD14, CD31, CD44, CD90, CD105, and CD133, was consistent between the two protocols. Clinical trials have demonstrated the effectiveness of the SVF isolated with the new clinical protocol in improving skin grafting, promoting mechanical stretch-induced skin regeneration and improving facial skin texture. No complications occurred. SVF isolated by the new clinical protocol had a noninferior yield and viability to that of the SVF separated by the laboratory protocol. SVFs obtained by the new protocol can be safely and effectively applied to improve skin grafting, promote mechanical stretch-induced skin regeneration, and improve facial skin texture. The trials were registered with the ClinicalTrials.gov (NCT03189628), the Chinese Clinical Trial Registry (ChiCTR2000039317), and the ClinicalTrials.gov (NCT02546882). All the three trials were not patient-funded trials. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Effect of microcapsules’ mechanical stability on the encapsulated cells’ behavior

Stem cell microencapsulation offers immunoprotection, utilizing natural polymers such as sodium alginate (SA), chitosan, and derivatives such as trimethyl chitosan (TMC). Additive minerals improve microcapsule properties and cell differentiation. Layering increases mechanical strength and shields cells from the host’s immune system. This study explores the impact of incorporating nano-hydroxyapatite (nHA) and graphene oxide (GO) into the three-layer alginate–TMC–alginate microcapsules housing human adipose mesenchymal stem cells (haMSCs). The experimental design focused on optimizing the mechanical stability, with the ideal GO and nHA contents identified as 0.81% and 34% w/w, respectively. Rheological analysis revealed enhanced mechanical consistency, evidenced by higher storage modulus and lower loss factor in the optimized sample. Viability of the microencapsulated haMSCs, assessed through acridine orange/propidium iodide staining and MTT assays, affirmed the nontoxic nature of the microcapsules. Osteogenic potential was evaluated via alkaline phosphatase (ALP) activity measurement and immunocytochemistry staining. Microcapsules containing nHA and GO exhibited significantly elevated ALP activity, underscoring the pivotal role of these additives in enhancing mesenchymal stem cell differentiation. The highest osteocalcin deposition occurred in the optimized and SA/nHA 40% w/w groups in the differentiation medium. This comprehensive investigation highlights the potential of three-layer microcapsules with nHA and GO for bone tissue engineering applications.

Synovial mesenchymal stem cells secrete more lubricin than adipose mesenchymal stem cells after injection into rat osteoarthritis knees

Intra-articular injection of mesenchymal stem cells (MSCs) is envisioned as a solution for knee osteoarthritis (OA). Although synovial MSCs (SyMSCs) are promising for cartilage regeneration, the clinical choice is usually adipose MSCs (AdMSCs). However, the similarities/differences in the mode of action between SyMSCs and AdMSCs remain unclear. Here, we compared factors secreted by human SyMSCs and AdMSCs after injection into OA knees. Human SyMSCs or AdMSCs were injected into the knees of rat partial meniscectomy models. The next day, the knee joints were collected to analyze the distribution of injected MSCs and transcriptome changes in the human MSCs and rat synovium. Non-injected MSCs were mixed with rat synovium as a control. After injection, no difference was apparent in intra-articular distribution of the SyMSCs or AdMSCs. RNA sequencing demonstrated an enrichment of cytokine-cytokine receptor interaction-related genes in both human SyMSCs and AdMSCs after injection. Differentially expressed genes (DEGs) specific to SyMSCs were associated with cartilage matrix synthesis and homeostasis. PCR analysis of the matrisome-related DEGs showed significantly higher expression of PRG4 in SyMSCs than in AdMSCs after injection. Immunostaining also confirmed a significantly greater expression of lubricin by SyMSCs than by AdMSCs. These findings indicate that SyMSCs will be a more promising treatment for OA.