Late-onset cerebellar ataxia and a new frameshift L2HGDH mutation in a Chinese adult with L-2-hydroxyglutaric aciduria: a case report.

L-2-Hydroxyglutaric aciduria (L2HGA) is a rare, autosomal recessive neurometabolic disease, which presents with elevated L-2-hydroxyglutarate acid. Generally, L2HGA appear as slowly progressing central nervous system function deterioration during infancy, and a rapid progression in adulthood is uncommon for the syndrome's classic phenotype.

Pluronic F-127 Hydrogel Loaded with Human Adipose-Derived Stem Cell-Derived Exosomes Improve Fat Graft Survival via HIF-1α-Mediated Enhancement of Angiogenesis.

Purpose: Autologous fat grafting is playing an increasingly important role in plastic surgery. However, high absorption and low survival of autologous fat grafts limit their clinical application. This study aimed to investigate whether human adipose-derived stem cell-derived exosomes (hASC-Exos) encapsulated in a PF-127 hydrogel can improve the survival of autologous fat grafts and to elucidate the underlying mechanisms. Patients and Methods: Exosomes were isolated from hASCs and identified using transmission electron microscopy, nanoparticle tracking analysis and Western blotting. We performed functional assays in vitro to assess the effect of hASC-Exos on proliferation, migration, and tube formation as well as their regulatory role in the HIF-1α/VEGF signaling pathway. hASC-Exos encapsulated in the PF-127 hydrogel were used as an in vivo autologous fat graft model. The effects of the PF-127 hydrogel/hASC-Exos and the role of the HIF-1α/VEGF signaling pathway in promoting angiogenesis in an autologous fat grafting model were assessed. Results: hASC-Exos were taken up by human umbilical vein endothelial cells and enhanced their proliferation, migration, and tubule formation in vitro. The effects of hASC-Exos on promoting angiogenesis were mediated by the HIF-1α/VEGF signaling pathway. Moreover, we fabricated a PF-127 hydrogel for the sustained release of hASC-Exos, and in vivo results showed that hASC-Exos encapsulated in PF-127 hydrogel improved the survival of autologous fat grafts. Conclusion: Our findings indicated that hASC-Exos encapsulated in PF-127 hydrogel serve as a key regulator of angiogenesis by activating the HIF-1α/VEGF signaling pathway and provide a promising strategy for autologous fat grafting treatment.

A hospital-based retrospective study: early recognition of neuronal surface antibodies by clinical manifestations and CSF inflammation indicators in patients with unexplained epilepsy.

BACKGROUND: There is a lack of actual and comprehensive data on the detection rate of neuronal surface antibodies in patients with unexplained epilepsy in China. Thus, we attempted to analyze the differences in clinical manifestations, cerebrospinal fluid (CSF) characteristics, seizure types and other aspects of antibody-positive and negative patients, to identify suspected antibody-positive epilepsy patients. METHODS: In total, 137 inpatients with unexplained epilepsy were consecutively included, and neuronal surface antibodies (NSAbs) were detected by serological and/or CSF evaluations. The clinical features and seizure characteristics were analyzed between the NSAb-positive and negative patients. In addition, patients were divided into four groups based on CSF and blood antibody titers. CSF cell count and protein content were analyzed in relation to antibody titers. RESULTS: There were 45 (32.8%) patients tested positive for antibodies. Multivariate analyses revealed that age, mental status changes or memory deterioration, CSF protein, CSF cell count, treatment, days of hospitalization, outcome, duration of symptoms before hospitalization, status epilepticus, and number of antiepileptic drugs were significantly associated with the NSAb-positive group and changes in inflammatory indicators in routine CSF analysis were associated with antibody titers. CONCLUSIONS: A relatively high proportion of patients with unexplained epilepsy have positive NSAbs. Patients with the above clinical characteristics need to be highly suspected of NSAbs positivity and should be tested for antibodies in time to assist treatment. The decrease of CSF cell count and protein content has suggestive value for the decrease of antibody titer, which should be evaluated in the follow-up.

Three-dimensional spheroid formation of adipose-derived stem cells improves the survival of fat transplantation by enhance their therapeutic effect.

Adipose-derived stem cells (ADSCs) have important applications in basic research, especially in fat transplantation. Some studies have found that three-dimensional (3D) spheroids formed by mesenchymal stem cells have enhanced therapeutic potential. However, the fundamental basics of this effect are still being discussed. ADSCs were harvested from subcutaneous adipose tissues and 3D spheroids were formed by the automatic aggregation of ADSCs in a non-adhesive 6-well plate. Oxygen glucose deprivation (OGD) was used to simulate the transplantation microenvironment. We found that 3D culture of ADSCs triggered cell autophagy. After inhibiting autophagy by Chloroquine, the rates of apoptosis were increased. When the 3D ADSC-spheroids were re-planked, the number of senescent ADSCs decreased, and the proliferation ability was promoted. In addition, there were more cytokines secreted by 3D ADSC-spheroids including VEGF, IGF-1, and TGF-ß. After adding the conditioned medium with human umbilical vein endothelial cells (HUVECs), 3D ADSC-spheroids were more likely to promote migration, and tube formation, stimulating the formation of new blood vessels. Fat grafting experiments in nude mice also showed that 3D ADSC-spheroids enhanced survival and neovascularization of fat grafts. These results suggested that 3D spheroids culturing of ADSCs can increase the therapeutic potential in fat transplantation.

MiR-24-3p regulates the differentiation of adipose-derived stem cells toward pericytes and promotes fat grafting vascularization.

Adipose-derived stem cells (ADSCs) enhance fat graft survival by promoting neovascularization. The mechanism that promotes ADSCs differentiation toward pericytes was not known. We treated ADSCs with conditional medium (CM) from endothelial cells (ECs) or human recombinant transforming growth factor ß (TGF-ß) to induce differentiation into pericytes. Pericytes markers, including platelet-derived growth factor receptor ß (PDGFRß), alpha-smooth muscle actin (α-SMA), and desmin, were examined. Pericytes differentiation markers, migration, and their association with ECs were examined in ADSCs transfected with miR-24-3p mimics and inhibitors. Bioinformatics target prediction platforms and luciferase assays were used to investigate whether PDGFRß was directly targeted by miR-24-3p. In vivo, fat mixed with ADSCs transfected with miR-24-3p mimics or inhibitors was implanted subcutaneously on the lower back region of nude mice. Fat grafts were harvested and analyzed at 2, 4, 6, and 8 weeks. Results showed that endogenous TGF-ß derived from CM from EC or human recombinant TGF-ß promoted migration, association with ECs, and induced expression of pericyte markers (PDGFRß, α-SMA, Desmin) in ADSCs. MiR-24-3p directly targeted PDGFRß in ADSCs by lucifer reporter assays. Inhibition of miR-24-3p promoted pericytes differentiation, migration, and association with ECs in ADSCs. Inhibition of miR-24-3p in ADSCs promoted survival, integrity, adipocyte viability, vascularization, pericytes association with ECs, and reduced fibrosis, whereas overexpression of miR-24-3p in ADSCs yielded the opposite results. Collectively, TGF-ß released by ECs induced ADSCs differentiation toward pericytes through miR-24-3p. Downregulation of miR-24-3p in ADSCs induced survival, integrity, adipocyte viability, vascularization, pericytes association with ECs, and reduced fibrosis after fat grafting.

Acetyl l-carnitine protects adipose-derived stem cells against serum-starvation: regulation on the network composed of reactive oxygen species, autophagy, apoptosis and senescence.

Adipose-derived stem cells (ADSCs) play an important role in cell therapy and regenerative medicine. However, local nutritional deficiency often limits therapeutical effect of the transplanted cells. Acetyl l-carnitine (ALC) is a common energy metabolism regulator and free radical scavenger. This study investigated the effect of ALC on ADSCs exposed to severe serum-deprivation and explored the relative machanisms. Treating with 1 mM ALC improved proliferation and alleviated senescence of starved cells, accompanied with reduced reactive oxygen species (ROS) and increased protein expression of SOD1 and catalase. In addition, ALC inhibited apoptosis but increased starvation-induced autophagy, which might be related to the regulation of phases of dissociation of Bcl-2-Beclin1 and Bcl-2-Bax complexes. Evidence obtained by replacing ALC with N-acetylcysteine (N-AC) suggested that ROS might be the central inducer of autophagy, apoptosis and senescence. There was a difference between ALC and N-AC in the protection mechanism, that was, compared with N-AC, ALC maintained autophagy well at the same time as anti-oxidation. Inhibition of autophagy by 3-methyladenine (3-MA) partially offset the protective effect of ALC. However, despite low-level ROS and enhanced autophagy, ALC with high concentration (10 mM) markedly aggravated cell apoptosis and senescence, thus losing cytoprotection and even causing damage.

Substrate coated with autologous decellularized extracellular matrix facilitates in vitro spreading of spheroid from adipose-derived stem cells through regulating ERK1/2-MMP2/9 pathway.

Adipose-derived stem cells (ADSCs) are easily available and play an important role in regenerative medicine. In recent years, Cell spheroid models have been in the spotlight because of their various advantages and physiological proximity. Promoting the spreading of ADSCs spheroids may improve the therapeutic effect the transplanted ADSCs. In this study, we prepared autologous decellularized extracellular matrix (d-ECM) and ADSCs spheroids, and investigated in vitro spreading of the spheroids on the d-ECM-coated substrate. In addition, the effect of d-ECM powder (ECM-P) on the aggregation of ADSCs was analyzed in a three-dimensional (3D) culture system. The results showed that d-ECM accelerated the spreading of spheroids, and promoted the migration and proliferation of the surrounding monolayer cells, accompanied by ERK1/2 activation and an increase in the expression of MMP2 and MMP9. In addition, ECM-P facilitated the aggregation of free cells in 3D culture in a concentration-dependent way. The spheroid spreading and cell aggregation were both prevented by ERK1/2 selective inhibitor PD98059. Our data suggest that the d-ECM substrate and its derivant may regulate the transformation between ADSCs spheroids and the monolayer or free cells, and ERK1/2 signalling pathway may be involved in these processes.

Exosomes Derived From Human Adipose-Derived Stem Cells Inhibit Lipogenesis Involving Hedgehog Signaling Pathway.

Since obesity impairs wound closure and adipose-derived exosomes (ADEs) regulate wound healing in clinical applications, we hypothesized that ADEs may inhibit adipogenesis of adipose-derived stem cells (ADSCs) to reduce the adverse effects of obesity on wound healing. Hedgehog (Hh) signaling has been previously shown to inhibit adipogenesis in ADSCs. The present study aimed to determine the role of ADEs in the adipogenesis of ADSCs and the Hh signaling pathway. ADSCs collected from human adipose tissues were co-cultured with ADEs and treated with an adipogenic inducer. qRT-PCR showed that ADEs could inhibit adipogenic differentiation of ADSCs and activate Hh signaling. The differences in the mRNA expression profiles of genes related to Hh signaling between the groups that were exposed to either high fat or low fat indicated that increased Hh signaling activation is necessary but not sufficient to inhibit adipogenic differentiation in the ADSC differentiation process. The Hh signaling pathway can be activated effectively by ADEs, especially during high-fat exposure after treatment with ADEs. Oil Red O staining of adipocytes suggested that ADEs inhibited not only adipogenic differentiation, but also lipogenesis in ADSCs. Overall, targeted activation of Hh signaling by ADEs reduced lipid accumulation in ADSCs and may be explored for clinical applications.

Autologous decellularized extracellular matrix promotes adipogenic differentiation of adipose derived stem cells in low serum culture system by regulating the ERK1/2-PPARγ pathway.

High viability and further adipogenic differentiation of adipose-derived stem cells (ADSCs) are fundamental for engraftment and growth of the transplanted adipose tissue. It has been demonstrated that extracellular matrix (ECM) regulates cell proliferation and differentiation by interacting with ERK1/2 signalling pathway. In this study, we prepared autologous decellularized extracellular matrix (d-ECM) and explored its effect on the proliferation and adipogenic ability of ADSCs in low serum culture. We found that 2% foetal bovine serum (FBS) in growth medium inhibited cell viability and DNA replication, and decreased mRNA and protein levels of PPARγ and C/EPBα compared with 10% FBS. Correspondingly, after 14-days adipogenic induction, cells cultured in 2% FBS possessed lower efficiency of adipogenesis and expressed less adipocyte differentiation markers ADIPOQ and aP2. On the contrary, the d-ECM-coated substrate continuously promoted the expression of PPARγ, and regulated the phosphorylation of ERK1/2 in different manners during differentiation. Pretreatment with ERK1/2 inhibitor PD98059 neutralized the effects of d-ECM, which suggested d-ECM might regulate the adipogenesis of ADSCs through ERK1/2-PPARγ pathway. In addition, d-ECM was revealed to regulate the transcription and expression of stemness-associated genes, such as OCT4, NANOG and SOX2, in the undifferentiated ADSCs, which might be related to the initiation of differentiation.

Autologous decellularized extracellular matrix protects against H2O2-induced senescence and aging in adipose-derived stem cells and stimulates proliferation in vitro.

Background: Adipose-derived stem cells have attracted significant interest, especially in stem cell therapy and regenerative medicine. However, these cells undergo gradual premature senescence in long-term cultures, which are essential for clinical applications that require cell-assisted lipotransfer or tissue repair. Methods: Since the extracellular matrix forms the microenvironment around stem cells in vitro and regulates self-renewal and multipotency in part by slowing down stem cell aging, we evaluated its potential to protect against senescence, using H2O2-induced adipose-derived stem cells as a model. Results: We found that supplementing cultures with decellularized extracellular matrix harvested from the same cells significantly promotes proliferation and reverses signs of senescence, including decreased multipotency, increased expression of senescence-associated ß-galactosidase, and accumulation of reactive oxygen species. Conclusion: These findings suggest a novel approach in which an autologous decellularized extracellular matrix is used to prevent cellular senescence to enable the use of adipose-derived stem cells in regenerative medicine.

Stromal vascular fraction cells plus sustained release VEGF/Ang-1-PLGA microspheres improve fat graft survival in mice.

Autologous fat transplantation is increasingly applied in plastic and reconstructive surgery. Stromal vascular fraction cells (SVFs) combined with angiogenic factors, such as VEGF (vascular endothelial growth factor A) and Ang-1 (angiogenin-1), can improve angiogenesis, which is a critical factor for graft survival. However, direct transplant with such a mixture is insufficient owing to the short half-life of angiogenic factors. In this study, we evaluated whether a double sustained release system of VEGF/ANG-1-PLGA (poly (lactic-co-glycolic acid)) microspheres plus SVFs can improve angiogenesis and graft survival after autologous fat transplantation. VEGF/ANG-1-PLGA-sustained release microspheres were fabricated by a modified double emulsion-solvent evaporation technique. Human aspirated fat was mixed with SVF suspension plus VEGF/ANG-1 sustained release microspheres (Group C), SVF suspension (Group B) alone, or Dulbecco's modified Eagle's medium as the control (Group A). Eighteen immunocompromised nude mice were injected with these three mixtures subcutaneously at random positions. After 8 weeks, the mean volume of grafts was greater in the SVFs plus VEGF/ANG-1-PLGA group than in the control and SVFs groups (1.08 ± 0.069 ml vs. 0.62 ± 0.036 ml, and 0.83 ± 0.059 ml, respectively). Histological assessments showed that lower fibrosis, but greater microvascular density in the SVFs plus VEGF/ANG-1-PLGA group than in the other groups, though the SVFs group also had an appropriate capillary density and reduced fibrosis. Our findings indicate that SVFs plus VEGF/ANG-1-PLGA-sustained release microspheres can improve angiogenesis and graft survival after autologous fat transplantation.

Effects of VEGF-ANG-1-PLA nano-sustained release microspheres on proliferation and differentiation of ADSCs.

The improvement of fat graft viability might depend on the presence of multipotent resident adipose derived stem cells (ADSCs) which is the important component of stromal vascular fraction (SVF). Vascular endothelial growth factor (VEGF) and angiogenin-1 (Ang-1) are responsible for neovascularization. However, their half-life is too short to produce a biological effect. We thus investigated whether VEGF-ANG-1-polylactic acid (PLA) microspheres could enhance the angiogenic properties of ADSCs. PLA microspheres containing VEGF and ANG-1 were prepared by in vitro ultrasonic emulsification and characterized according to their encapsulation efficiency (EE), drug-loading rate (DL), particle size, and drug release. The systemic toxicity of empty loaded nanospheres (NPs) and the ability of these microspheres to promote the proliferation and differentiation of ADSCs were evaluated. The EE and DL were above 86% and 0.0288%, respectively [corrected].The drug release was completed after 20 days. Systemic toxicity was verified in ADSCs that received the unloaded NPs. It was observed that ADSCs treated with VEGF-ANG-1-PLA microspheres had an increase in the proliferation and the number of CD31 positive cells. ADSCs proliferation and differentiation toward endothelial cells (ECs) could be enhanced by the addition of VEGF-ANG-1-PLA nano-sustained release microspheres.

Rapamycin Promotes the Survival and Adipogenesis of Ischemia-Challenged Adipose Derived Stem Cells by Improving Autophagy.

BACKGROUND/AIMS: Ischemia is one of the main causes of the high rate of absorption of transplanted autologous fat. Autophagy allows cells to survive by providing energy under starvation. Rapamycin has been found to play a role in promoting autophagy. In this study, we investigated whether rapamycin participates in the survival and adipogenesis of ischemia-challenged adipose-derived stem cells (ADSCs) by regulating autophagy. METHODS: Before the cells were exposed to oxygen-glucose deprivation (OGD), a simulated ischemic microenvironment, the level of autophagy was reduced or increased by lentiviral transfection with short hairpin RNA targeting microtubule-associated protein 1-light chain 3 gene (shRNA-LC3) or treatment with rapamycin, respectively. The level of autophagy was assessed by western blotting, transmission electron microscopythen the apoptosis ratio was determined through terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and flow cytometry. Adipogenesis was further evaluated by oil red O staining and the expressions level of some specific proteins for adipocytes. RESULTS: shRNA-LC3 and rapamycin treatment effectively decreased and improved the level of autophagy in cells with or without OGD challenge, respectively. In addition, autophagy inhibition increased the apoptosis rate and activated caspase-3 expression level in response to OGD, and these were markedly inhibited by rapamycin preconditioning. During adipogenesis, autophagy inhibition decreased not only oil droplet accumulation but also lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor gamma (PPARγ) expression in cells with or without OGD challenge. However, autophagy promotion by rapamycin increased oil droplet accumulation and LPL and PPARγ expression. CONCLUSIONS: Rapamycin may promote the survival and adipogenesis of ischemia-challenged ADSCs by upregulating autophagy.

[Research progress of growth factor sustained-release microspheres in fat transplantation].

Objective: To review the research progress of growth factor sustained-release microspheres in fat transplantation. Methods: The recently published 1iterature at home and abroad related the growth factor sustained-release microspheres in fat transplantation was reviewed and analyzed. Results: The sustained-release microsphere carrier materials include natural polymer materials and synthetic polymer materials.The sustained-release complexes of different microsphere materials with different growth factors can promote the vascularization of transplanted fat in a timely manner, improve the survival rate of grafts, and reduce the incidence of complications such as liquefaction, calcification, and necrosis. Conclusion: The growth factor sustained-release microspheres have the characteristics of persistence and controllability, which is a research hotspot in the field of fat transplantation and has broad application prospects.