[Ubiquitin-specific protease 42 regulates osteogenic differentiation of human adipose-derived stem cells].

OBJECTIVE: To explore the effect of ubiquitin-specific protease 42 (USP42) on osteogenic differentiation of human adipose-derived stem cells (hASCs) in vivo and in vitro. METHODS: A combination of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy. Alkaline phosphatase (ALP) staining and quantification, alizarin red S (ARS) staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group (knockdown group and overexpression group) and the control group. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of osteogenesis related genes in the experimental group and control group, and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group. Nude mice ectopic implantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo. RESULTS: The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group, and those in overexpression group were significantly higher than those in control group. After 7 days of osteogenic induction, the ALP activity in the knockdown group was significantly higher than that in the control group, and ALP activity in overexpression group was significantly lower than that in control group. After 14 days of osteogenic induction, ARS staining was significantly deeper in the knockdown group than in the control group, and significantly lighter in overexpression group than in the control group. The results of qRT-PCR showed that the mRNA expression levels of ALP, osterix (OSX) and collagen type Ⅰ (COLⅠ) in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction, and those in overexpression group were significantly lower than those in control group. The results of Western blotting showed that the expression levels of runt-related transcription factor 2 (RUNX2), OSX and COLⅠ in the knockout group were significantly higher than those in the control group at 14 days after osteogenic induction, while the expression levels of RUNX2, OSX and COLⅠ in the overexpression group were significantly lower than those in the control group. Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group. CONCLUSION: Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vivo, and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs, and USP42 can provide a potential therapeutic target for bone tissue engineering.

The HIFIA/LINC02913/IGF1R axis promotes the cell function of adipose-derived mesenchymal stem cells under hypoxia via activating the PI3K/AKT pathway.

OBJECTIVE: Promoting angiogenesis is crucial for tissue repair. Adipose-derived mesenchymal stem cells (ADSCs) are endowed with the ability of paracrine secretion of various angiogenic cytokines and the differentiation potential into endothelium-like cells to directly participate in angiogenesis. ADSCs are key seed cells for promoting angiogenesis in regenerative medicine and tissue engineering. This study aimed to explore the role and mechanism of C9orf106 (LINC02913) in the angiogenesis of ADSCs. METHODS: The microarray dataset GSE12884 was analyzed to identify the differentially expressed lncRNAs in ADSCs under normoxia and hypoxia. The expression of the key genes was detected using qRT-PCR, western blot assay (western blot), and immunofluorescence (IF) staining. The adipogenic ability and tube formation ability of ADSCs was detected using oil red O staining and tube formation assay, respectively. The regulatory relationship between hypoxia-inducible factor-1alpha (HIF1A) and LINC02913 was verified using chromatin immunoprecipitation (ChIP) assay and dual-luciferase reporter gene assay. A skin wound healing nude mice model was established. Hematoxylin and eosin (H&E) staining was applied to detect pathological skin damage. Immunohistochemistry (IHC) staining was used to determine the level of CD31 in skin tissues. RESULTS: LINC02913 expression was decreased in ADSCs under hypoxia; LINC02913 overexpression inhibited the proliferation, adipogenic ability, endothelial differentiation ability, and tube formation ability of ADSCs. ChIP assay and dual-luciferase reporter gene assay results showed that HIF1A could directly bind to the LINC02913 promoter region to inhibit its transcription. Through RNAact prediction and analysis of the correlation with LINC02913 expression, it was found that IGF1R may directly interact with LINCO02913. The HIF1A/LINC02913/IGF1R axis could activate the PI3K/AKT pathway to promote the biological function of ADSCs. Hypoxia-ADSCs significantly promoted vascularization in the wounded skin. The regulatory effect of LINC02913/IGF1R axis on hypoxia-ADSCs treated skin wound healing were verified. CONCLUSION: The HIF1A/LINC02913/IGF1R axis promoted the proliferation, adipogenic ability, and tube formation ability of ADSCs under hypoxia via activating the PI3K/AKT pathway.

Autologous adipose-derived mesenchymal stem cell therapy reverses detrusor underactivity: open clinical trial.

BACKGROUND: Detrusor underactivity is a disease that can cause chronic urinary tract infection, urinary tract infection, urinary retention and kidney failure and has no effective treatment in traditional medicine. The present research evaluated the effects of cell therapy with adipose tissue-derived stem cells on the treatment of detrusor underactivity in men. METHODS: Nine male patients diagnosed with a clinical and urodynamic diagnosis of detrusor underactivity were evaluated and underwent two transplants via cystourethroscopy, with 2 × 106 cells/transplant, performed by intravesical injection at five points on the bladder body above the vesical trigone. RESULTS: Cell therapy increased the maximum flow from 7.22 ± 1.58 to 13.56 ± 1.17, increased the mean flow from 3.44 ± 0.74 to 5.89 ± 0.45, increased the urinated volume from 183.67 ± 49.28 to 304.78 ± 40.42 and reduced the residual volume in the uroflowmetry exam from 420.00 ± 191.41 to 118.33 ± 85.51; all of these changes were significant (p < 0.05). There were also significant increases (p < 0.05) in maximum flow (from 7.78 ± 0.76 to 11.56 ± 1.67), maximum detrusor pressure (from 20.22 ± 8.29 to 41.56 ± 5.75), urinary volume (from 244 ± 27.6 to 418.89 ± 32.73) and bladder contractility index (from 44.33 ± 4.85 to 100.56 ± 8.89) in the pressure flow study. Scores on the International Consultation on Incontinence Questionnaire decreased from 11.44 ± 1.43 to 3.78 ± 0.78 after cell therapy, which indicates an improvement in quality of life and a return to daily activities. No complications were observed in the 6-month follow-up after cell therapy. Before treatment, all patients performed approximately five intermittent clean catheterizations daily. After cell therapy, 7/9 patients (77.78%) did not need catheterizations, and the number of catheterizations for 2/9 patients (22.28%) was reduced to two catheterizations/day. CONCLUSIONS: The results indicate that stem cell therapy led to improvements in voiding function. Cell therapy with adipose tissue-derived stem cells is safe and should be considered a new therapeutic option for the treatment of detrusor underactivity. Trial registration ISRCTN, ISRCTN23909398; Registered 15 March 2021-Retrospectively registered, https://doi.org/10.1186/ISRCTN23909398.

ADSCs-exo attenuates hepatic ischemia-reperfusion injury after hepatectomy by inhibiting endoplasmic reticulum stress and inflammation.

Hepatic ischemia-reperfusion (I/R) injury commonly occurs during liver surgery. Exosomes from adipose-derived stem cells (ADSCs-exo) induce a hepatoprotective effect during hepatic I/R injury. This study aimed to investigate the possible mechanism by which ADSCs-exo attenuates hepatic I/R injury in rats. Rats were randomly divided into four groups: Sham, I30R + PH, ADSCs, and ADSCs-exo groups. Liver tissues were collected immediately after 24 h of reperfusion for further analyses. The content of inflammatory factors in liver tissue was detected using enzyme-linked immunosorbent assay. The pathological changes in liver tissue were analyzed using HE staining. Transmission electron microscopy was used to visualize the ultrastructural changes of hepatocytes. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to detect the expression of endoplasmic reticulum stress (ERS)-related genes and proteins. Liver histomorphology and hepatocyte ultrastructure changes improved after ADSCs-exo treatment. Moreover, ADSCs-exo treatment significantly downregulated tumor necrosis factor-α, interleukin-1ß (IL-1ß), and IL-6 levels while upregulating IL-10 levels. Western blot analysis suggested that the protein expressions of GRP78, p-PERK, p-eIF2α, p-IRE1α, XBP1s, ATF-6, ATF-4, CHOP, p-JNK, cleaved-Caspase-3, cleaved Caspase-9, and cleaved Caspase-12 significantly decreased after ADSCs-exo treatment. RT-qPCR results demonstrated that mRNA expression of GRP78, IRE1α, XBP1, ATF-6, ATF-4, CHOP, JNK, Caspase-3, Caspase-9, and Caspase-12 markedly reduced after ADSCs-exo treatment. In conclusion, ADSCs-exo protects against hepatic I/R injury after hepatectomy by inhibiting ERS and inflammation. Therefore, ADSCs-exo can be considered as a viable option for the treatment of hepatic I/R injury.

Adipose tissue-derived Muse cells promote autophagy and oxidative stress tolerance in human epidermal melanocytes.

To investigate the effect of human adipose tissue-derived multilineage-differentiating stress-enduring (Muse) cells on the oxidative stress injury of human epidermal melanocytes (HEMs) in vitro. HEMs were treated with H2O2 to establish an oxidative stress injury model and then were co-cultured with adipose tissue-derived Muse cells. Immunohistochemistry, flow cytometry and Western blotting were used to assess changes in autophagy flux, apoptosis, expression of melanin synthesis related proteins and proliferation of melanocytes. Our findings demonstrate that co-culture with Muse cells significantly increased the tolerance of HEMs to oxidative stress, enhanced autophagy flux and reduced apoptosis. The expression of proteins related to the formation of melanin increased as did cell proliferation. Treatment with the autophagy inhibitor, 3-methyladenine (3MA), partially counteracted the improvement of oxidative stress tolerance in melanocytes elicited by co-culture with Muse cells. Muse cells promote autophagy and oxidative stress tolerance of melanocytes.

The preliminary study of exosomes derived from thymosin beta 4-treated adipose-derived stem cells in fat grafting.

BACKGROUND: The retention rate in autologous fat grafting is an increasing concern for surgeons and patients. Our previous research verified that thymosin beta 4 (Tß4) positively affected fat survival, while the mechanism was unknown. The endothelial cells (ECs) and exosomes derived from adipose-derived stem cells (ADSCs) were regarded to play a critical role in fat transplantation. OBJECTIVE: This study aimed to evaluate the effect of exosomes derived from Tß4-treated ADSCs on EC proliferation and to identify the exosomal microRNA (miRNA) profile compared with the Tß4-untreated group. Additionally, this research intended to recognize the related molecules and signaling pathways in the Tß4-treated group with potential roles in fat transplants. METHODS: ADSCs were collected from patients who underwent liposuction surgery. Depending on whether the medium was supplemented with exogenous Tß4 or not, exosomes derived from cultured ADSCs were divided into the Tß4-Exos group and Con-Exos group. Exosome uptake and cell counting kit-8 (CCK-8) assays assessed the influence of Tß4-Exos on EC proliferation. The exosomal miRNAs of the two groups were analyzed by next-generation sequencing. With the criteria at the |log2 (fold change)| ≥ 1 and p-value < 0.05, up-regulated and down-regulated differentially expressed miRNAs (DEMs) were obtained. Prediction databases were used to predict the downstream mRNAs for DEMs. And then, overlapping genes for the up-regulated DEMs and the down-regulated were screened out, followed by enrichment analysis, protein-protein interaction network construction, and the gene cluster and hub gene identification. RESULTS: ADSCs were obtained from four female patients. The exosome uptake and CCK-8 assays showed that the Tß4-Exos could increase cell growth rate compared with the control group (DMEM-H + PBS). In Tß4-Exos and Con-Exos groups, 2651 exosomal miRNAs were recognized, with 80 up-regulated and 99 down-regulated DEMs according to the criteria. After the prediction, 621 overlapping genes for the up-regulated and 572 for the down-regulated DEMs were screened. The subsequent bioinformatics analysis found specific molecules and pathways related to the positive effect on fat survival. CONCLUSIONS: The exosomes derived from Tß4-treated ADSCs probably positively affect EC proliferation. Compared with the Con-Exos group, several exosomal DEMs, genes, and pathways were distinguished. These findings of this exploratory study provide the potential direction for future in-depth research on fat grafting.

Systematic review on spheroids from adipose-derived stem cells: Spontaneous or artefact state?

Three-dimensional (3D) cell cultures represent the spontaneous state of stem cells with specific gene and protein molecular expression that are more alike the in vivo condition. In vitro two-dimensional (2D) cell adhesion cultures are still commonly employed for various cellular studies such as movement, proliferation and differentiation phenomena; this procedure is standardized and amply used in laboratories, however their representing the original tissue has recently been subject to questioning. Cell cultures in 2D require a support/substrate (flasks, multiwells, etc.) and use of fetal bovine serum as an adjuvant that stimulates adhesion that most likely leads to cellular aging. A 3D environment stimulates cells to grow in suspended aggregates that are defined as "spheroids." In particular, adipose stem cells (ASCs) are traditionally observed in adhesion conditions, but a recent and vast literature offers many strategies that obtain 3D cell spheroids. These cells seem to possess a greater ability in maintaining their stemness and differentiate towards all mesenchymal lineages, as demonstrated in in vitro and in vivo studies compared to adhesion cultures. To date, standardized procedures that form ASC spheroids have not yet been established. This systematic review carries out an in-depth analysis of the 76 articles produced over the past 10 years and discusses the similarities and differences in materials, techniques, and purposes to standardize the methods aimed at obtaining ASC spheroids as already described for 2D cultures.

Live-seq enables temporal transcriptomic recording of single cells.

Single-cell transcriptomics (scRNA-seq) has greatly advanced our ability to characterize cellular heterogeneity1. However, scRNA-seq requires lysing cells, which impedes further molecular or functional analyses on the same cells. Here, we established Live-seq, a single-cell transcriptome profiling approach that preserves cell viability during RNA extraction using fluidic force microscopy2,3, thus allowing to couple a cell's ground-state transcriptome to its downstream molecular or phenotypic behaviour. To benchmark Live-seq, we used cell growth, functional responses and whole-cell transcriptome read-outs to demonstrate that Live-seq can accurately stratify diverse cell types and states without inducing major cellular perturbations. As a proof of concept, we show that Live-seq can be used to directly map a cell's trajectory by sequentially profiling the transcriptomes of individual macrophages before and after lipopolysaccharide (LPS) stimulation, and of adipose stromal cells pre- and post-differentiation. In addition, we demonstrate that Live-seq can function as a transcriptomic recorder by preregistering the transcriptomes of individual macrophages that were subsequently monitored by time-lapse imaging after LPS exposure. This enabled the unsupervised, genome-wide ranking of genes on the basis of their ability to affect macrophage LPS response heterogeneity, revealing basal Nfkbia expression level and cell cycle state as important phenotypic determinants, which we experimentally validated. Thus, Live-seq can address a broad range of biological questions by transforming scRNA-seq from an end-point to a temporal analysis approach.

Adipose­derived mesenchymal stem cell­derived HCAR1 regulates immune response in the attenuation of sepsis.

Sepsis serves as a leading cause of admission to and death of patients in the intensive care unit (ICU) and is described as a systemic inflammatory response syndrome caused by abnormal host response to infection. Adipose­derived mesenchymal stem cells (ADSCs) have exhibited reliable and promising clinical application potential in multiple disorders. However, the function and the mechanism of ADSCs in sepsis remain elusive. In the present study, the crucial inhibitory effect of ADSC­derived hydroxy­carboxylic acid receptor 1 (HCAR1) on sepsis was identified. Reverse transcription quantitative­PCR determined that the mRNA expression of HCAR1 was reduced while the mRNA expression of Toll­like receptor 4 (TLR4), major histocompatibility complex class II (MHC II), NOD­like receptor family pyrin domain containing 3 (NLRP3), and the levels of interleukin­1ß (IL­1ß), tumor necrosis factor­α (TNF­α), interleukin­10 (IL­10), and interleukin­18 (IL­18) were enhanced in the peripheral blood of patients with sepsis. The expression of HCAR1 was negatively correlated with TLR4 (r=­0.666), MHC II (r=­0.587), and NLRP3 (r=­0.621) expression and the expression of TLR4 was positively correlated with NLRP3 (r=0.641), IL­1ß (r=0.666), TNF­α (r=0.606), and IL­18 (r=0.624) levels in the samples. Receiver operating characteristic (ROC) curve analysis revealed that the area under the ROC curve (AUC) of HCAR1, TLR4, MHC II and NLRP3 mRNA expression was 0.830, 0.853, 0.735 and 0.945, respectively, in which NLRP3 exhibited the highest diagnostic value, and the AUC values of IL­1ß, IL­18, TNF­α, and IL­10 were 0.751, 0.841, 0.924 and 0.729, respectively, in which TNF­α exhibited the highest diagnostic value. A sepsis rat model was established by injecting lipopolysaccharide (LPS) and the rats were randomly divided into 5 groups, including a normal control group (NC group; n=6), a sepsis model group (LPS group; n=6), an ADSC transplantation group (L + M group; n=6), a combined HCAR1 receptor agonist group [L + HCAR1 inducer (Gi) + M group; n=6], and a combined HCAR1 receptor inhibitor group [L + HCAR1 blocker (Gk) + M group; n=6]. Hematoxylin and eosin staining determined that ADSCs attenuated the lung injury of septic rats and ADSC­derived HCAR1 enhanced the effect of ADSCs. The expression of HCAR1, TLR4, MHC II, NLRP3, IL­1ß, IL­18 and TNF­α levels were suppressed by ADSCs and the effect was further induced by ADSC­derived HCAR1. However, ADSC­derived HCAR1 induced the levels of anti­inflammatory factor IL­10. The negative correlation of HCAR1 expression with TLR4, MHC II, and NLRP3 expression in the peripheral blood and lung tissues of the rats was then identified. It is thus concluded that ADSC­derived HCAR1 regulates immune response in the attenuation of sepsis. ADSC­derived HCAR1 may be a promising therapeutic strategy for sepsis.

LncRNA MEG3 induces endothelial differentiation of mouse derived adipose-derived stem cells by targeting MiR-145-5p/KLF4.

BACKGROUND: The present study aimed to investigate the mechanisms through which long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) affected the endothelial differentiation of mouse derived adipose-derived stem cells (ADSCs). MATERIALS AND METHODS: ADSCs were isolated and identified by specific surface marker detection. The effects of lncRNA MEG3 on endothelial differentiation of ADSCs were also detected via quantitative PCR, western blotting, immunofluorescence and Matrigel angiogenesis assays. In addition, using target gene prediction tools and luciferase reporter assays, the downstream target gene was demonstrated. RESULTS: LncRNA MEG3 targeted and reduced the expression levels of microRNA-145-5p (miR-145-5p), which upregulated the expression levels of Krüppel like factor 4 (KLF4), promoting endothelial differentiation of ADSCs. CONCLUSION: LncRNA MEG3 induced endothelial differentiation of ADSCs by targeting miR-145-5p/KLF4, which may provide novel insights to illustrate the mechanism of endothelial differentiation of ADSCs.

Dose-dependent dominance: How cell densities design stromal cell functions during soft tissue healing.

Regular soft tissue healing relies on the well-organized interaction of different stromal cell types with endothelial cells. However, spatiotemporal conditions might provoke high densities of one special stromal cell type, potentially leading to impaired healing. Detailed knowledge of the functions of rivaling stromal cell types aiming for tissue contraction and stabilization as well as vascular support is mandatory. By the application of an in vitro approach comprising the evaluation of cell proliferation, cell morphology, myofibroblastoid differentiation, and cytokine release, we verified a density-dependent modulation of these functions among juvenile and adult fibroblasts, pericytes, and adipose-derived stem cells during their interaction with microvascular endothelial cells in cocultures. Results indicate that juvenile fibroblasts rather support angiogenesis via paracrine regulation at the early stage of healing, a role potentially compromised in adult fibroblasts. In contrast, pericytes showed a more versatile character aiming at angiogenesis, vessel stabilization, and tissue contraction. Such a universal character was even more pronounced among adipose-derived stem cells. The explicit knowledge of the characteristic functions of stromal cell types is a prerequisite for the development of new analytical and therapeutic approaches for impaired soft tissue healing. The present study delivers new considerations concerning the roles of rivaling stromal cell types within a granulation tissue, pointing to extraordinary properties of pericytes and adipose-derived stem cells.

TCF7L2 plays a complex role in human adipose progenitor biology, which might contribute to genetic susceptibility to type 2 diabetes.

INTRODUCTION: Non-coding genetic variation at TCF7L2 is the strongest genetic determinant of type 2 diabetes (T2D) risk in humans. TCF7L2 encodes a transcription factor mediating the nuclear effects of WNT signaling in adipose tissue (AT). In vivo studies in transgenic mice have highlighted important roles for TCF7L2 in adipose tissue biology and systemic metabolism. OBJECTIVE: To map the expression of TCF7L2 in human AT, examine its role in human adipose cell biology in vitro, and investigate the effects of the fine-mapped T2D-risk allele at rs7903146 on AT morphology and TCF7L2 expression. METHODS: Ex vivo gene expression studies of TCF7L2 in whole and fractionated human AT. In vitro TCF7L2 gain- and/or loss-of-function studies in primary and immortalized human adipose progenitor cells (APCs) and mature adipocytes (mADs). AT phenotyping of rs7903146 T2D-risk variant carriers and matched controls. RESULTS: Adipose progenitors (APs) exhibited the highest TCF7L2 mRNA abundance compared to mature adipocytes and adipose-derived endothelial cells. Obesity was associated with reduced TCF7L2 transcript levels in whole subcutaneous abdominal AT but paradoxically increased expression in APs. In functional studies, TCF7L2 knockdown (KD) in abdominal APs led to dose-dependent activation of WNT/ß-catenin signaling, impaired proliferation and dose-dependent effects on adipogenesis. Whilst partial KD enhanced adipocyte differentiation, near-total KD impaired lipid accumulation and adipogenic gene expression. Over-expression of TCF7L2 accelerated adipogenesis. In contrast, TCF7L2-KD in gluteal APs dose-dependently enhanced lipid accumulation. Transcriptome-wide profiling revealed that TCF7L2 might modulate multiple aspects of AP biology including extracellular matrix secretion, immune signaling and apoptosis. The T2D-risk allele at rs7903146 was associated with reduced AP TCF7L2 expression and enhanced AT insulin sensitivity. CONCLUSIONS: TCF7L2 plays a complex role in AP biology and has both dose- and depot-dependent effects on adipogenesis. In addition to regulating pancreatic insulin secretion, genetic variation at TCF7L2 might also influence T2D risk by modulating AP function.

circ_0003204 regulates the osteogenic differentiation of human adipose-derived stem cells via miR-370-3p/HDAC4 axis.

Human adipose-derived stem cells (hASCs) are a promising cell type for bone tissue regeneration. Circular RNAs (circRNAs) have been shown to play a critical role in regulating various cell differentiation and involve in mesenchymal stem cell osteogenesis. However, how circRNAs regulate hASCs in osteogenesis is still unclear. Herein, we found circ_0003204 was significantly downregulated during osteogenic differentiation of hASCs. Knockdown of circ_0003204 by siRNA or overexpression by lentivirus confirmed circ_0003204 could negatively regulate the osteogenic differentiation of hASCs. We performed dual-luciferase reporting assay and rescue experiments to verify circ_0003204 regulated osteogenic differentiation via sponging miR-370-3p. We predicted and confirmed that miR-370-3p had targets in the 3'-UTR of HDAC4 mRNA. The following rescue experiments indicated that circ_0003204 regulated the osteogenic differentiation of hASCs via miR-370-3p/HDAC4 axis. Subsequent in vivo experiments showed the silencing of circ_0003204 increased the bone formation and promoted the expression of osteogenic-related proteins in a mouse bone defect model, while overexpression of circ_0003204 inhibited bone defect repair. Our findings indicated that circ_0003204 might be a promising target to promote the efficacy of hASCs in repairing bone defects.

Adipose Mesenchymal Stromal Cell-Derived Exosomes Prevent Testicular Torsion Injury via Activating PI3K/AKT and MAPK/ERK1/2 Pathways.

Adipose mesenchymal stromal cell-derived exosomes (ADSC-Exos) have shown great potential in the treatment of oxidative stress induced by ischemia-reperfusion injury. However, alleviation of testicular torsion injury by ADSC-Exos has not been reported. Therefore, we investigated the protective effect of ADSC-Exos against testicular torsion-detorsion injury. ADSC-Exos were isolated by ultracentrifugation and injected into torsion-detorsion-affected testes of rats. H&E staining and sperm quality were used to evaluate the therapeutic effects of ADSC-Exos, and tissue oxidative stress was measured by determining MDA and SOD levels. In addition, TUNEL staining and immunohistological analysis (Ki67, Cleaved Caspase-3, IL-6, IL-10, CCR7, and CD163) were used to clarify the effects of ADSC-Exos on spermatogenic cell proliferation, apoptosis, and the inflammatory microenvironment in vivo. Possible signaling pathways were predicted using sequencing technology and bioinformatics analysis. The predicted signaling pathways were validated in vitro by assessing the proliferation (EdU assay), migration (transwell assay and scratch test), and apoptosis (flow cytometry, TUNEL staining, and western blotting) of spermatogenic cells. The results showed that ADSC-Exos alleviated testicular torsion-detorsion injury by attenuating oxidative stress and the inflammatory response. In addition, ADSC-Exos promoted the proliferation and migration of spermatogenic cells and inhibited their apoptosis by activating the PI3K/AKT and MAPK/ERK1/2 signaling pathways.

Mineralized collagen as a bioactive ink to support encapsulation of human adipose stem cells: A step towards the future of bone regeneration.

Bioprinting - printing with incorporated living cells - has earned special attention on tissue engineering approaches, aiming to closer reproduce the 3D microenvironment of the target tissue. However, it raises extra complexity related to the need to use cell-friendly printing conditions that still comply with material printing fidelity. Inspired by the composite nano structural organization of mineralized tissues, this work reports the efficiency of the chemical approach followed to in situ mineralize blue shark skin collagen, at a nano scale level, to ultimately produce stable inks. The influence of initial cellular density was evaluated by assessing three different concentrations (2.5, 5 and 7.5 × 106 cells·ml-1) of human adipose stem cells (hASC), with the higher density of encapsulated cells presenting improved viability in a long culture term. Immunodetection of osteogenic-related markers, like RUNX2 and osteopontin, 21 days after cell culture in basal conditions confirmed the potential of the ink to be applied for osteogenic purposes, which may be associated with the success of the cell-to-ink interaction and the Ca2+ ions released from the co-precipitated hydroxyapatite. A combination of mineralized shark collagen, alginate and hASC is thus proposed as a bioactive bioink with potential properties for regeneration of bone tissue.

Inhibition of RIP3 increased ADSC viability under OGD and modified the competency of adipogenesis, angiogenesis, and inflammation regulation.

Adipose-derived stem cells (ADSCs) showed decreased cell viability and increased cell death under oxygen-glucose deprivation (OGD). Meanwhile, vital necroptotic proteins, including receptor-interacting protein kinase (RIP) 3 (RIP3) and mixed lineage kinase domain-like pseudokinase (MLKL), were expressed in the early stage. The present study aims to explore the effect of necroptosis inhibition on ADSCs. ADSCs were obtained from normal human subcutaneous fat and verified by multidirectional differentiation and flow cytometry. By applying cell counting kit-8 (CCK-8), calcein/propidium iodide (PI) staining and immunostaining, we determined the OGD treatment time of 4 h, a timepoint when the cells showed a significant decrease in viability and increased protein expression of RIP3, phosphorylated RIP3 (pRIP3) and phosphorylated MLKL (pMLKL). After pretreatment with the inhibitor of RIP3, necroptotic protein expression decreased under OGD conditions, and cell necrosis decreased. Transwell assays proved that cell migration ability was retained. Furthermore, the expression of the adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) and quantitative analysis of Oil Red O staining increased in the inhibitor group. The expression of vascular endothelial growth factor-A (VEGFA) and fibroblast growth factor 2 (FGF2) and the migration test suggest that OGD increases the secretion of vascular factors, promotes the migration of human umbilical vein endothelial cells (HUVECs), and forms unstable neovascularization. ELISA revealed that inhibition of RIP3 increased the secretion of the anti-inflammatory factor, interleukin (IL)-10 (IL-10) and reduced the expression of the proinflammatory factor IL-1ß. Inhibition of RIP3 can reduce the death of ADSCs, retain their migration ability and adipogenic differentiation potential, reduce unstable neovascularization and inhibit the inflammatory response.

Transposon-mediated glial cell line-derived neurotrophic factor overexpression in human adipose tissue-derived mesenchymal stromal cells: A potential approach for neuroregenerative medicine?

Glial cell line-derived neurotrophic factor (GDNF) has neuroprotective effects and may be a promising candidate for regenerative strategies focusing on neurodegenerative diseases. As GDNF cannot cross the blood-brain barrier to potentially regenerate damaged brain areas, continuous in situ delivery with host cells is desired. Here, a non-viral Sleeping Beauty transposon was used to achieve continuous in vitro overexpression of GDNF in immune-privileged human adipose tissue-derived mesenchymal stromal cells (GDNF-tASCs). In addition, in vivo survival, tolerance, and effectiveness of transfected cells were tested in a very mild 6-hydroxydopamine (6-OHDA)-induced dopamine depletion rat model by means of intrastriatal injection on a sample basis up to 6 months after treatment. GDNF-tASCs showed vast in vitro gene overexpression up to 13 weeks post-transfection. In vivo, GDNF was detectable 4 days following transplantation, but no longer after 1 month, although adipose tissue-derived mesenchymal stromal cells (ASCs) could be visualized histologically even after 6 months. Despite successful long-term in vitro GDNF overexpression and its in vivo detection shortly after cell transplantation, the 6-OHDA model was too mild to enable sufficient evaluation of in vivo disease improvement. Still, in vivo immunocompatibility could be further examined. ASCs initially induced a pronounced microglial accumulation at transplantation site, particularly prominent in GDNF-tASCs. However, 6-OHDA-induced pro-inflammatory immune response was attenuated by ASCs, although delayed in the GDNF-tASCs group. To further test the therapeutic potential of the generated GDNF-overexpressing cells in a disease-related context, a follow-up study using a more appropriate 6-OHDA model is needed.

Adipose-Derived Stem Cells from Type 2 Diabetic Rats Retain Positive Effects in a Rat Model of Erectile Dysfunction.

Erectile dysfunction is a common complication associated with type 2 diabetes mellitus (T2DM) and after prostatectomy in relation to cancer. The regenerative effect of cultured adipose-derived stem cells (ASCs) for ED therapy has been documented in multiple preclinical trials as well as in recent Pase 1 trials in humans. However, some studies indicate that diabetes negatively affects the mesenchymal stem cell pool, implying that ASCs from T2DM patients could have impaired regenerative capacity. Here, we directly compared ASCs from age-matched diabetic Goto-Kakizaki (ASCGK) and non-diabetic wild type rats (ASCWT) with regard to their phenotypes, proteomes and ability to rescue ED in normal rats. Despite ASCGK exhibiting a slightly lower proliferation rate, ASCGK and ASCWT proteomes were more or less identical, and after injections to corpus cavernosum they were equally efficient in restoring erectile function in a rat ED model entailing bilateral nerve crush injury. Moreover, molecular analysis of the corpus cavernosum tissue revealed that both ASCGK and ASCWT treated rats had increased induction of genes involved in recovering endothelial function. Thus, our finding argues that T2DM does not appear to be a limiting factor for autologous adipose stem cell therapy when correcting for ED.

Characterization and functional analysis of the adipose tissue-derived stromal vascular fraction of pediatric patients with osteogenesis imperfecta.

Pediatric patients with Osteogenesis Imperfecta (OI), a heritable connective tissue disorder, frequently suffer from long bone deformations. Surgical correction often results in bone non-unions, necessitating revision surgery with autogenous bone grafting using bone-marrow-derived stem cells (BM-SC) to regenerate bone. BM-SC harvest is generally invasive and limited in supply; thus, adipose tissue's stromal vascular fraction (SVF) has been introduced as an alternative stem cell reservoir. To elucidate if OI patients' surgical site dissected adipose tissue could be used as autologous bone graft in future, we investigated whether the underlying genetic condition alters SVF's cell populations and in vitro differentiation capacity. After optimizing SVF isolation, we demonstrate successful isolation of SVF of pediatric OI patients and non-OI controls. The number of viable cells was comparable between OI and controls, with about 450,000 per gram tissue. Age, sex, type of OI, disease-causing collagen mutation, or anatomical site of harvest did not affect cell outcome. Further, SVF-containing cell populations were similar between OI and controls, and all isolated SVF's demonstrated chondrogenic, adipogenic, and osteogenic differentiation capacity in vitro. These results indicate that SVF from pediatric OI patients could be used as a source of stem cells for autologous stem cell therapy in OI.