Heritability of in vitro phenotypes exhibited by murine adipose-derived stromal cells

Adipose-derived stromal cells (ADSCs) exhibit significant potential as therapeutic agents to promote tissue regeneration. Success of ADSC-based therapies is dependent upon efficient cell expansion in vitro as well as postinjection survival in the caustic milieu of damaged tissue. Genetic background regulates ADSC proliferative capacity and stress resistance, but the extent of the genetic effect size is not completely defined. The present study aimed to quantify phenotypic ranges and heritability of in vitro ADSC characteristics. ADSCs were isolated from mice representing 16 genetically diverse inbred mouse strains, including 12 classical inbred strains and four wild-derived strains. Cells were grown in vitro, and proliferative capacity and oxidative stress resistance were assessed. The fold change for ADSC growth ranged from 0.87 (BALB/cByJ) to 23.60 (POHN/DehJ), relative to original seeding density. The heritability of proliferative capacity was estimated to be 0.6462 (p = 9.967 × 10^?15), and this phenotype was not associated with other ADSC traits. Cell viability following H₂O₂ treatment ranged from 39.81 % (CAST/EiJ) to 91.60 % (DBA/2 J), and the heritability of this phenotype was calculated as 0.6146 (p = 1.22 × 10^?12). Relationships between cell viability and weight of the donor fat pad were also discovered. Donor genetic background is a major determinant of in vitro ADSC phenotypes. This study supports the development of forward genetics strategies to identify genes that underlie ADSC phenotypic diversity, which will inform efforts to improve cell-based therapies.     

MR Angiography,MR Imaging and Proton MR Spectroscopy In-Vivo Assessment of Skeletal Muscle Ischemia in Diabetic Rats

To prospectively evaluate the feasibility of using magnetic resonance (MR) techniques for in-vivo assessing a rat diabetic model of limb ischemia. Unilateral hind limb ischemia was induced by ligation of the iliac-femoral artery in male streptozotocin-treated and non-diabetic control rats. Four weeks after ligation, rats underwent MR Angiography (MRA), T₁-weighted and Short Time Inversion Recovery (STIR) sequences and muscle Proton MR Spectroscopy (¹H-MRS) on both hind limbs. After MR examinations, immunoblotting and immunofluorescence analysis were performed. MRA showed a signal void due to flow discontinuation distal to the artery ligation. T₁-weighted and STIR images showed, respectively, the presence of tissue swelling (p = 0.018 for non-diabetic; p = 0.027 for diabetic rats) and signal hyperintensity in tissue affected by occlusion. Mean total creatine/water for the occluded limb was significantly lower than for the non-occluded limbs in both non-diabetic (5.46×10⁻⁴ vs 1.14×10⁻³, p = 0.028) and diabetic rats (1.37×10⁻⁴ vs 1.10×10⁻³; p = 0.018). MR Imaging and ¹H-MRS changes were more pronounced in diabetic than in non-diabetic occluded limbs (p = 0.032). MR findings were confirmed by using histological findings. Combined MR techniques can be used to demonstrate the presence of structural and metabolic changes produced by iliac-femoral artery occlusion in rat diabetic model of limb ischemia.     

The evaluation of cartilage differentiations using transforming growth factor beta3 alone and with combination of bone morphogenetic protein-6 on adult stem cells

In our quest to standardize our formula for a clinical trial, transforming growth factor-beta3 (TGF-β3) alone and in combination with bone morphogenetic protein-6 (BMP-6) were evaluated for their effectiveness in cartilage differentiation. Bone Marrow Stem Cells (BMSCs) and Adipose Derived Stem Cells (ADSCs) were induced to chondrogenic lineage using two different media. Native chondrocytes served as positive control. ADSCs and BMSCs proved multipotency by tri-lineage differentiations. ADSC has significantly higher growth kinetics compare to Chondrocyte only p ≤ 0.05. Using TGF-β3 alone, BMSC revealed higher expressions for hyaline cartilage genes compare to ADSCs. Chondrocyte has significantly higher early chondrogenic markers expression to ADSCs and BMSCs, while BMSCs was only higher to ADSC at chondroadherin, p ≤ 0.0001. On mature chondrogenic markers, chondrocytes were significantly higher to ADSCs and BMSCs for aggrecan, collagen IX, sry (sex determining region y)-box9, collagen II and fibromodullin; and only to ADSC for collagen XI. BMSC was higher to ADSC for aggrecan and collagen IX, p ≤ 0.0001. The combination of TGF-β3 + BMP-6 revealed increased gene expressions on both BMSCs and ADSCs for early and mature chondrogenic markers, but no significance difference. For dedifferentiation markers, ADSC was significantly higher to chondrocyte for collagen I. Glycosaminoglycan evaluations with both formulas revealed that chondrocytes were significantly higher to ADSCs and BMSCs, but none was significant to each other, p ≤ 0.0001. Combination of 10 ng TGF-β3 with 10 ng of BMP-6 enhanced chondrogenic potentials of BMSCs and ADSCs compare to TGF-β3 alone. This could be the ideal cocktail for either cell’s chondrogenic induction.     

Effect of Leukemia Inhibitory Factor on the Myelinogenic Ability of Schwann-Like Cells Induced from Human Adipose-Derived Stem Cells

The Schwann cells (SCs) may be obtain from nerve biopsies for autologous transplantation. However, it is difficult to obtain sufficient amount of SCs for clinical applications. Human adipose-derived stem cells (ADSCs) can be induced to differentiate into Schwann-like cells (S-like cells) and used for autologous transplantation. However, effect of leukemia inhibitory factor (LIF) on the myelinogenic ability of SC-like cells induced from human ADSC is not investigated yet. The aim of this study was to evaluate of the effect of exogenous LIF on myelinogenic potential of differentiated cells in vitro. ADSCs were harvested from human fat tissue and characterized using flow cytometry. Human ADSCs were treated for sphere formation and LIF was added to terminal differentiation medium. GFAP/S100β and MBP markers were used to confirm differentiation of human ADSCs, and myelinogenic ability of SC-like cells, respectively, using both immunostaining and real-time RT-PCR analysis. The analysis for GFAP⁺/S100β⁺ revealed that LIF can increase both differentiated cells rates and the percentage of myelinating SC-like cells (p < 0.05). Our data showed that SC-like cells induced from human ADSCs were able to generate myelin when exposed to LIF and these cells could be a potential source for the treatment of peripheral and central axonal injuries.     

Autologous bone marrow cell implantation as therapeutic angiogenesis for ischemic hindlimb in diabetic rat model

The angiogenic effect induced by autologous bone marrow cell implantation (BMCI) was examined in the ischemic hindlimbs of diabetic and nondiabetic rats. Diabetes mellitus was induced by the systemic administration of streptozotocin. We investigated the production of angiogenic factors and endothelial differentiation from bone marrow cells and the native recovery of blood flow in the ischemic hindlimbs. To observe the angiogenic effect induced by BMCI treatment, 6 x 10(7) bone marrow cells were injected intramuscularly at six points into the ischemic limbs, and regional perfusion recovery was evaluated with colored microspheres 2 wk later. No difference was found between diabetic and nondiabetic rats in the release of angiogenic factors or endothelial differentiation from bone marrow cells in vitro. The levels of nitric oxide in plasma were significantly lower, and native perfusion recovery in the ischemic hindlimbs was significantly slower in the diabetic rats than in the nondiabetic rats. However, although perfusion recovery was achieved in the ischemic hindlimbs, there was no significant increase in systemic VEGF after BMCI treatment in either the diabetic or nondiabetic rats. Therefore, therapeutic angiogenesis induced by BMCI could be a safe and effective treatment for ischemic limb disease in diabetic patients.     

Sensible heat transfer and thermal windows in Dasyprocta leporina (Mammalia,Rodentia)

This study aimed to evaluate the diurnal variation of the sensible heat transfer in red-rumped agoutis (Dasyprocta leporina) bred in captivity in a semi-arid environment. In addition, we seek to identify thermal windows by infrared thermography during the daytime period (07:00, 09:00, 11:00, 14:00, and 16:00). The body surface temperature was higher in the pinna (36.84 ± 0.11 °C), followed by the hind limbs (36.55 ± 0.11 °C). These body regions were primarily responsible for heat loss by radiation (which was 10.13 ± 1.17 W m^?2 and 11.19 ± 1.17 W m^?2, respectively), and acted like biological thermal windows. Heat transfer by convection was more intense in the body trunk and hind limbs at all times of the day. Thus, sensible heat transfer is important for maintaining homeothermy in red-rumped agouti in hot environments. In conclusion, these rodents use specialized body regions (pinna and hind limbs) for heat transfer.     

Abscopal effect of boron neutron capture therapy (BNCT): proof of principle in an experimental model of colon cancer

The aim of the present study was to evaluate, for the first time, the abscopal effect of boron neutron capture therapy (BNCT). Twenty-six BDIX rats were inoculated subcutaneously with 1 × 10⁶ DHD/K12/TRb syngeneic colon cancer cells in the right hind flank. Three weeks post-inoculation, the right leg of 12 rats bearing the tumor nodule was treated with BPA-BNCT (BPA-Boronophenylalanine) at the RA-3 nuclear reactor located in Buenos Aires, Argentina, at an absorbed dose of 7.5 Gy to skin as the dose-limiting tissue. The remaining group of 14 tumor-bearing rats were left untreated and used as control. Two weeks post-BNCT, 1 × 10⁶ DHD/K12/TRb cells were injected subcutaneously in the contralateral left hind flank of each of the 26 BDIX rats. Tumor volume in both legs was measured weekly for 7 weeks to determine response to BNCT in the right leg and to assess a potential influence of BNCT in the right leg on tumor development in the left leg. Within the BNCT group, a statistically significant reduction was observed in contralateral left tumor volume in animals whose right leg tumor responded to BNCT (post-treatment/pre-treatment tumor volume <1) versus animals who failed to respond (post/pre ≥1), i.e., 13 ± 15 vs 271 ± 128 mm³. In addition, a statistically significant reduction in contralateral left leg tumor volume was observed in BNCT-responsive animals (post/pre <1) vs untreated animals, i.e., 13 ± 15 vs 254 ± 251 mm³. The present study performed in a simple animal model provides proof of principle that the positive response of a tumor to BNCT is capable of inducing an abscopal effect.     

In vitro toxicity and antidiabetic activity of a newly developed polyherbal formulation (MAC-ST/001) in streptozotocin-induced diabetic Wistar rats

The present study was designed to investigate the hypoglycemic effect of an aqueous extract of MAC-ST/001 (a new polyherbal formulation) which was given once daily to rats at different doses. The animals were divided into diabetic and nondiabetic control groups. The duration of each experiment lasted from 1 week to 1 month, and the results were compared with that of the standard hypoglycemic drug glibenclamide (10 mg/kg), which was given once daily. In this study, biochemical and histopathological parameters were studied in streptozotacin (STZ) (single intraperitoneal injection of 55 mg/kg)-induced diabetic rats. The diabetic rats showed a significant (p?<?0.05 and p?<?0.01) decrease in their body weight and serum amylase with marked elevation in blood glucose, serum cholesterol, blood urea nitrogen, creatinine, alkaline phosphatase, and serum transaminases (AST and ALT) after 1 week till the 28th day of diabetes. Cytotoxicity of MAC-ST/001 formulation was also studied on C2C12, 3T3-L1, and HepG2 cells through MTT assay. Histological examination of the liver and pancreas of normal control, diabetic control, and drug-treated rats revealed significant results. Finally, it was concluded that administration of this MAC-ST/001 extract reversed most blood and tissue changes caused by STZ-induced diabetes in rats.     

Vasoreparative Dysfunction of CD34+ Cells in Diabetic Individuals Involves Hypoxic Desensitization and Impaired Autocrine/Paracrine Mechanisms

We hypothesized that endothelial progenitor cells derived from individuals with diabetes would exhibit functional defects including inability to respond to hypoxia and altered paracrine/autocrine function that would impair the angiogenic potential of these cells. Circulating mononuclear cells isolated from diabetic (n = 69) and nondiabetic (n = 46) individuals were used to grow endothelial colony forming cells (ECFC), early endothelial progenitor cells (eEPCs) and isolate CD34⁺ cells. ECFCs and eEPCs were established from only 15% of the diabetic individuals tested thus directing our main effort toward examination of CD34⁺ cells. CD34⁺ cells were plated in basal medium to obtain cell-free conditioned medium (CM). In CM derived from CD34⁺ cells of diabetic individuals (diabetic-CM), the levels of stem cell factor, hepatocyte growth factor, and thrombopoietin were lower, and IL-1β and tumor necrosis factor (TNFα) levels were higher than CM derived from nondiabetic individuals (nondiabetic-CM). Hypoxia did not upregulate HIF1α in CD34⁺ cells of diabetic origin. Migration and proliferation of nondiabetic CD34⁺ cells toward diabetic-CM were lower compared to nondiabetic-CM. Attenuation of pressure-induced constriction, potentiation of bradykinin relaxation, and generation of cGMP and cAMP in arterioles were observed with nondiabetic-CM, but not with diabetic-CM. Diabetic-CM failed to induce endothelial tube formation from vascular tissue. These results suggest that diabetic subjects with microvascular complications exhibit severely limited capacity to generate ex-vivo expanded endothelial progenitor populations and that the vasoreparative dysfunction observed in diabetic CD34⁺ cells is due to impaired autocrine/paracrine function and reduced sensitivity to hypoxia.     

Bone tissue engineering by using a combination of polymer/Bioglass composites with human adipose-derived stem cells

Translational research in bone tissue engineering is essential for “bench to bedside” patient benefit. However, the ideal combination of stem cells and biomaterial scaffolds for bone repair/regeneration is still unclear. The aim of this study is to investigate the osteogenic capacity of a combination of poly(DL-lactic acid) (PDLLA) porous foams containing 5 wt% and 40 wt% of Bioglass particles with human adipose-derived stem cells (ADSCs) in vitro and in vivo. Live/dead fluorescent markers, confocal microscopy and scanning electron microscopy showed that PDLLA/Bioglass porous scaffolds supported ADSC attachment, growth and osteogenic differentiation, as confirmed by enhanced alkaline phosphatase (ALP) activity. Higher Bioglass content of the PDLLA foams increased ALP activity compared with the PDLLA only group. Extracellular matrix deposition after 8 weeks in the in vitro cultures was evident by Alcian blue/Sirius red staining. In vivo bone formation was assessed by using scaffold/ADSC constructs in diffusion chambers transplanted intraperitoneally into nude mice and recovered after 8 weeks. Histological and immunohistochemical assays indicated significant new bone formation in the 40 wt% and 5 wt% Bioglass constructs compared with the PDLLA only group. Thus, the combination of a well-developed biodegradable bioactive porous PDLLA/Bioglass composite scaffold with a high-potential stem cell source (human ADSCs) could be a promising approach for bone regeneration in a clinical setting.     

CircRNA-vgll3 promotes osteogenic differentiation of adipose-derived mesenchymal stem cells via modulating miRNA-dependent integrin α5 expression

Adipose-derived mesenchymal stem cells (ADSCs) are promising candidate for regenerative medicine to repair non-healing bone defects due to their high and easy availability. However, the limited osteogenic differentiation potential greatly hinders the clinical application of ADSCs in bone repair. Accumulating evidences demonstrate that circular RNAs (circRNAs) are involved in stem/progenitor cell fate determination, but their specific role in stem/progenitor cell osteogenesis, remains mostly undescribed. Here, we show that circRNA-vgll3 originating from the vgll3 locus markedly enhances osteogenic differentiation of ADSCs; nevertheless, silencing of circRNA-vgll3 dramatically attenuates ADSC osteogenesis. Furthermore, we validate that circRNA-vgll3 functions in ADSC osteogenesis through a circRNA-vgll3/miR-326-5p/integrin α5 (Itga5) pathway. Itga5 promotes ADSC osteogenic differentiation and miR-326-5p suppresses Itga5 translation. CircRNA-vgll3 directly sequesters miR-326-5p in the cytoplasm and inhibits its activity to promote osteogenic differentiation. Moreover, the therapeutic potential of circRNA-vgll3-modified ADSCs with calcium phosphate cement (CPC) scaffolds was systematically evaluated in a critical-sized defect model in rats. Our results demonstrate that circRNA-vgll3 markedly enhances new bone formation with upregulated bone mineral density, bone volume/tissue volume, trabeculae number, and increased new bone generation. This study reveals the important role of circRNA-vgll3 during new bone biogenesis. Thus, circRNA-vgll3 engineered ADSCs may be effective potential therapeutic targets for bone regenerative medicine.Subject terms: Epigenetics, Stem-cell research     

Potential use of human adipose mesenchymal stromal cells for intervertebral disc regeneration: a preliminary study on biglycan-deficient murine model of chronic disc degeneration

Introduction

Biglycan is an important proteoglycan of the extracellular matrix of intervertebral disc (IVD), and its decrease with aging has been correlated with IVD degeneration. Biglycan deficient (Bgn^−/0) mice lack this protein and undergo spontaneous IVD degeneration with aging, thus representing a valuable in vivo model for preliminary studies on therapies for human progressive IVD degeneration. The purpose of the present study was to assess the possible beneficial effects of adipose-derived stromal cells (ADSCs) implants in the Bgn^−/0 mouse model.

Methods

To evaluate ADSC implant efficacy, Bgn^−/0 mice were intradiscally (L1-L2) injected with 8x10⁴ ADSCs at 16 months old, when mice exhibit severe and complete IVD degeneration, evident on both 7Tesla Magnetic Resonance Imaging (7TMRI) and histology. Placebo and ADSCs treated Bgn^−/0 mice were assessed by 7TMRI analysis up to 12 weeks post-transplantation. Mice were then sacrificed and implanted discs were analyzed by histology and immunohistochemistry for the presence of human cells and for the expression of biglycan and aggrecan in the IVD area.

Results

After in vivo treatment, 7TMRI revealed evident increase in signal intensity within the discs of mice that received ADSCs, while placebo treatment did not show any variation. Ultrastructural analyses demonstrated that human ADSC survival occurred in the injected discs up to 12 weeks after implant. These cells acquired a positive expression for biglycan, and this proteoglycan was specifically localized in human cells. Moreover, ADSC treatment resulted in a significant increase of aggrecan tissue levels.

Conclusion

Overall, this work demonstrates that ADSC implant into degenerated disc of Bgn^−/0 mice ameliorates disc damage, promotes new expression of biglycan and increased levels of aggrecan. This suggests a potential benefit of ADSC implant in the treatment of chronic degenerative disc disease and prompts further studies in this field.     

Adverse cardiac responses to alpha-lipoic acid in a rat-diabetic model: possible mechanisms?

Alpha-lipoic acid (ALA) is widely used as an antioxidant for the treatment of diabetes and its complications; however, the pro-oxidant potential of ALA has recently been reported. This study was designed to investigate whether ALA supplementation could have pro-oxidant effects on cardiac tissues in normal and diabetic rats. Diabetes was induced by a single dose of streptozotocin (STZ; 55 mg/kg (intraperitoneal). Diabetic and normal rats were treated with ALA (100 mg kg^?1 day^?1) for 45 days. ALA supplementation resulted in oxidative protein damage as evident by significant reduction in the cardiac levels of protein thiol in ALA-treated normal rats (P?<?0.01) together with a significant elevation (P?<?0.001) in the plasma levels of advanced oxidation protein products in ALA-treated normal rats and in ALA?+?STZ-diabetic rats compared with the normal control rats. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has emerged as the major source of superoxide anion and enhanced oxidative damage in heart failure. ALA supplementation increased the myocardial immunoreactivity of p47phox subunit of NADPH oxidase in both normal nondiabetic and diabetic rats reflecting its pro-oxidant effect. Data showed that ALA supplementation failed to prevent cardiac complications in diabetic rats and led to cardiac toxicity in normal rats as indicated by pathological changes (cellular infiltration, fibrosis, and degeneration) and by the elevation of serum cardiac biomarkers compared with normal controls. The pro-oxidant effects of ALA suggest that careful selection of appropriate doses of ALA in reactive oxygen species-related diseases are critical.     

Hepatocyte Growth Factor Gene-Modified Adipose-Derived Mesenchymal Stem Cells Ameliorate Radiation Induced Liver Damage in a Rat Model

Liver damage caused by radiotherapy is associated with a high mortality rate, but no established treatment exists. Adipose-derived mesenchymal stem cells (ADSCs) are capable of migration to injured tissue sites, where they aid in the repair of the damage. Hepatocyte growth factor (HGF) is critical for damage repair due to its anti-apoptotic, anti-fibrotic and cell regeneration-promoting effects. This study was performed to investigate the therapeutic effects of HGF-overexpressing ADSCs on radiation-induced liver damage (RILD). ADSCs were infected with a lentivirus encoding HGF and HGF-shRNA. Sprague-Dawley (SD) rats received 60Gy of irradiation to induce liver injury and were immediately given either saline, ADSCs, ADSCs + HGF or ADSCs + shHGF. Two days after irradiation, a significant reduction in apoptosis was observed in the HGF-overexpressing ADSC group compared with the RILD group, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Scanning electron microscopy showed chromatin condensation after irradiation, which was ameliorated in the group that received ADSCs and was reversed in the group that received HGF-overexpressing ADSCs. HGF-overexpressing ADSCs ameliorated radiation- induced liver fibrosis through down regulation of α-SMA and fibronectin. Hepatocyte regeneration was significantly improved in rats treated with ADSCs compared with rats from the RILD group), as assessed by Ki-67 immunohistochemistry. Rats that received HGF-overexpressing ADSCs showed an even greater level of hepatocyte regeneration. HGF-overexpressing ADSCs completely blocked the radiation-induced increase in the enzymes ALT and AST. The effect of mitigating RILD was compromised in the ADSC + shHGF group compared with the ADSC group. Altogether, these results suggest that HGF-overexpressing ADSCs can significantly improve RILD in a rat model, which may serve as a valuable therapeutic alternative.     

Muscle Microdialysis to Investigate Inflammatory Biomarkers in Facioscapulohumeral Muscular Dystrophy

Recent progresses in the understanding of facioscapulohumeral muscular dystrophy (FSHD) genetics opened the way to the development of targeted therapies. However, knowledge about pathophysiology of muscle damage is still limited and there is increasing need to identify biomarkers of disease activity in the perspective of clinical trial readiness.We analyzed inflammatory mediators in the interstitial fluid of muscles with different MRI signal in FSHD patients, comparing muscles displaying early lesions on short-tau inversion recovery (STIR) sequences with normal ones. Patients with one T1-weighted normal and STIR hyperintense (STIR+) and contralateral T1-weighted and STIR normal (STIR-) lower limb muscle were asked to enter the study. Twelve consecutive patients, five controls, and one non-penetrant gene carrier underwent prolonged muscle microdialysis with high cut-off membranes. Microdialysates were analyzed using xMAP technology with a wide panel for cytokines, chemokines, and growth factors. A small number of inflammatory mediators were dysregulated in STIR+ versus STIR- and control muscles: CXCL13, upregulated in STIR+ muscles compared with controls (p < 0.01); CXCL5, downregulated in STIR+ compared with STIR- muscles (p < 0.05); and G-CSF, downregulated in STIR+ muscles compared with controls (p < 0.05). CXCL13 was also upregulated in the STIR+ muscles compared with the contralateral STIR- muscles of the same patient (p < 0.01).These results support the evidence of a selective inflammatory process taking place in STIR+ FSHD muscles. The application of microdialysis could provide insights on novel mechanisms involved in muscle damage in FSHD and in other myopathies. Further studies are needed to validate these investigated molecules as tissue and circulating biomarkers.     

Murine Adipose Tissue-Derived Stromal Cell Apoptosis and Susceptibility to Oxidative Stress In Vitro Are Regulated by Genetic Background

Adipose tissue-derived stromal cells (ADSCs) are of interest for regenerative medicine as they are isolated easily and can differentiate into multiple cell lineages. Studies of their in vitro proliferation, survival, and differentiation are common; however, genetic effects on these phenotypes remain unknown. To test if these phenotypes are genetically regulated, ADSCs were isolated from three genetically diverse inbred mouse strains- C57BL/6J (B6), BALB/cByJ (BALB), and DBA/2J (D2)- in which genetic regulation of hematopoietic stem function is well known. ADSCs from all three strains differentiated into osteogenic and chondrogenic lineages in vitro. ADSCs from BALB grew least well in vitro, probably due to apoptotic cell death after several days in culture. BALB ADSCs were also the most susceptible to the free radical inducers menadione and H₂O₂. ADSCs from the three possible F1 hybrids were employed to further define genetic regulation of ADSC phenotypes. D2, but not B6, alleles stimulated ADSC expansion in BALB cells. In contrast, B6, but not D2, alleles rescued BALB H₂O₂ resistance. We conclude that low oxidative stress resistance does not limit BALB ADSC growth in vitro, as these phenotypes are genetically regulated independently. In addition, ADSCs from these strains are an appropriate model system to investigate genetic regulation of ADSC apoptosis and stress resistance in future studies. Such investigations are essential to optimize cell expansion and differentiation and thus, potential for regenerative medicine.     

Clinical and anti-aging effect of mud-bathing therapy for patients with fibromyalgia

This study focuses on two inflammatory diseases, viz., “diabetes mellitus (DM)” that causes serious complications such as retinopathy, nephropathy, and neuropathy, and “ischemic colitis” which is evoked by DM. Ischemic colitis originates from the reduction in mesenteric blood flow to the colon with existence of the occlusive or non-occlusive reasons. Our study objective was to provide early diagnostic approach for ischemic colitis in streptozotocin (STZ)-induced diabetic rats. Sprague-Dawley rats were divided into four groups: (i) control use of 0.1 M citrate buffer, the solvent of streptozotocin (C), (ii). induced ischemia (I), (iii) rats subjected to 60 mg/kg STZ intraperitoneally to induce type 1 diabetes (D) (48 h after STZ injection, blood glucose levels >200 mg/dl were considered as diabetic), and (iv) diabetic rats subjected to intestinal ischemia (D+I). The third diabetic group (D) was not operated. At the end of the experimental period, rats were sacrificed, C-reactive protein (CRP) and calprotectin levels were measured in the serum and colon tissue specimens. Tissue specimens were also analyzed histologically. We found that serum and colon calprotectin levels were elevated in the D+I group compared to the D and/or I group alone, but relatively calprotectin levels increased in I as compared to C group in colon tissues. CRP levels were significantly increased with ischemic colitis in diabetes, while colon CRP levels were decreased. These results provide evidence for the existence of inflammation in the STZ-induced diabetic rats with ischemic colitis. In conclusion, our measurements of serum calprotectin levels of STZ-induced diabetic rats with ischemic colitis provide a practical approach for an early diagnosis of ischemic colitis. Furthermore, these biochemical analyses correlate well with the histopathologic findings of STZ-induced diabetic rats with ischemic colitis. Future studies would be desirable to further strengthen the role of calprotectin in the early diagnosis of ischemic colitis in diabetics clinical settings.     

Adipose Stromal Cells Amplify Angiogenic Signaling via the VEGF/mTOR/Akt Pathway in a Murine Hindlimb Ischemia Model: A 3D Multimodality Imaging Study

Although adipose-derived stromal cell (ADSC) transplantation has been demonstrated as a promising therapeutic strategy for peripheral arterial disease (PAD), the mechanism of action behind the observed therapeutic efficacy of ADSCs remains unclear. This study was designed to investigate the long-term outcome and therapeutic behavior of engrafted ADSCs in a murine hindlimb ischemia model using multimodality molecular imaging approaches. ADSCs (1.0×10⁷) were isolated from Tg(Fluc-egfp) mice which constitutively express dual-reporter firefly luciferase and enhanced green fluorescent protein (Fluc⁺-eGFP⁺, mADSCs^Fluc+GFP+), then intramuscularly injected into the hindlimb of BALB/c-nu mice after unilateral femoral artery ligation and excision. Abbreviated survival (∼5 weeks) of post-transplant mADSCs within the ischemic hindlimb was longitudinally monitored using noninvasive bioluminescence imaging (BLI), fluorescence imaging (FRI), and bioluminescence tomography with micro-computed tomography (BLT/micro-CT). Use of the BLT/micro-CT system enabled quantitative 3-dimensional (3D) imaging of the cells’ distribution and kinetics in vivo. Engrafted mADSCs improved blood perfusion recovery, ambulatory performance and prognosis of the ischemic hindlimb, probably by inducing angiogenesis and formation of collateral vessels, which could be visualized using laser Doppler perfusion imaging (LDPI), micro-CT angiography, vascular-cast imaging, and immunofluorescence. mADSCs augmented activation of the pro-angiogenic VEGF/mTOR/Akt pathway in vivo, even though the cells failed to incorporate into the host microvasculature as functional components. Downregulation of VEGF/mTOR/Akt signaling using small molecule inhibitors counteracted mADSC-induced angiogenesis and perfusion restoration. This study demonstrates for the first time the spatiotemporal kinetics and functional survival of transplanted mADSCs in a PAD model using in vivo 3D multimodality imaging. Our study indicates that mADSCs potentiate pro-angiogenic signal amplification via a VEGF/mTOR/Akt-dependent pathway, and thereby promote recovery from hindlimb ischemia.     

Intramyocardial injection of hypoxia-preconditioned adipose-derived stromal cells treats acute myocardial infarction: an in vivo study in swine

Hypoxic preconditioning is a promising method for improving the anti-apoptotic and paracrine signaling capabilities of adipose-derived stromal cells (ADSCs). The purpose of this study was to analyze the influence of different hypoxic conditions on ADSCs and the therapeutic effects of hypoxia-preconditioned ADSCs (HPADSCs) on an animal model of myocardial infarction (MI). For the in vitro studies, ADSCs were divided into five groups and cultured in different oxygen concentrations (1, 3, 5, 10, and 21 %). After 24 h, RT-PCR and western blots showed that 3 % oxygen preconditioning could improve the viability and cytokine secretion of the ADSCs. A Matrigel assay indicated that the HPADSC-conditioned medium could stimulate endothelial cells to form capillary-like tubes. For the in vivo studies, MI was induced by coronary occlusion in 24 mature Chinese minipigs. The animals were divided into three groups and treated by intramyocardial injection with vehicle alone (saline group), with 1?×?10⁸ ADSCs cultured in normoxic conditions (ADSCs group) or with 1?×?10⁸ ADSCs precultured in 3 % oxygen (HPADSCs group). SPECT and echocardiography demonstrated that cardiac function was improved significantly in the HPADSC transplant group compared with the vehicle control group (P?P?相似文献