Human ASC Research Articles

Subcutaneous injection of adipose stromal cell-secretome improves renal function and reduces inflammation in established acute kidney injury

BackgroundAdipose stromal cells (ASC) are a form of mesenchymal stromal cells that elicit effects primarily via secreted factors, which may have advantages for the treatment of injury or disease. Several previous studies have demonstrated a protective role for MSC/ASC on mitigating acute kidney injury but whether ASC derived factors could hasten recovery from established injury has not been evaluated.MethodsWe generated a concentrated secretome (CS) of human ASC under well-defined conditions and evaluated its ability to improve the recovery of renal function in a preclinical model of acute kidney injury (AKI) in rats. 24 h following bilateral ischemia/reperfusion (I/R), rats were randomized following determination of plasma creatinine into groups receiving vehicle -control or ASC-CS treatment by subcutaneous injection (2 mg protein/kg) and monitored for evaluation of renal function, structure and inflammation.ResultsRenal function, assessed by plasma creatinine levels, recovered faster in ASC-CS treated rats vs vehicle. The most prominent difference between the ASC-CS treated vs vehicle was observed in rats with the most severe degree of initial injury (Pcr > 3.0 mg/dl 24 h post I/R), whereas rats with less severe injury (Pcr < 2.9 mg/dl) recovered quickly regardless of treatment. The quicker recovery of ASC-treated rats with severe injury was associated with less tissue damage, inflammation, and lower plasma angiopoietin 2. In vitro, ASC-CS attenuated the activation of the Th17 phenotype in lymphocytes isolated from injured kidneys.ConclusionsTaken together, these data suggest that ASC-CS represents a potent therapeutic option to improve established AKI.

KLF4 interacts with TXNIP to modulate the pyroptosis in ulcerative colitis via regulating NLRP3 signaling.

Ulcerative colitis (UC) is one of the most common diseases in the gastrointestinal tract related to abnormal inflammation. Pyroptosis, which is characterized by the formation of inflammasome, activation of caspase-1, and separation of N- and C-terminus of gasdermin D (GSDMD), and may be involved in the pathogenesis of IBD. Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor expressed in differentiated epithelial cells. KLF4 mediates proinflammatory signaling in macrophages. Here, we tested whether KLF4 is functional in pyroptosis of UC. In human UC tissues and/or lipopolysaccharide (LPS)/adenosine 5-triphosphate (ATP) stimulation human colon epithelial cells, KLF4, TXNIP, Cleave-Caspase-1, and GSDMD expression were detected through quantitative reverse transcription polymerase chain reaction (PCR), immunohistochemical and western blot assay. Interleukin (IL)-1β and IL-18 levels were quantified by enzyme-linked immunosorbent assay. We successfully constructed a KLF4-silenced colon epithelial cell line using an adenovirus vector. We apply the UCSC and JASPAR to predict the KLF4 binding sites in the promoter region of TXNIP. In human UC tissues and/or LPS/ATP stimulation human colon epithelial cells, KLF4, TXNIP, Caspase-1, and GSDMD expression level were significantly elevated via quantitative reverse transcription PCR, immunohistochemical and western blot assay. Moreover, We identified that there is an interaction between KLF4 and TXNIP through Yeast double hybrid assay and CO-IP assay. We successfully constructed a KLF4-silenced human intestinal epithelial cell line. In LPS/ATP stimulation KLF4-silenced human intestinal epithelial cells, KLF4, TXNIP, Cleave Caspase-1, ASC, and GSDMD expression level were significantly decreased via quantitative reverse transcription PCR. Our results confirm that KLF4 can positively regulate the expression of TXNIP and regulate the pyroptosis process of UC through the TXNIP/NLRP3 pathway.

Serum apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) is a novel stroke biomarker

BackgroundApoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) is a promising stroke biomarker. However, a large study of human serum ASC has not yet to be reported; additionally, the diagnostic value of prehospital concentration and practicality of ASC remains unknown. MethodsWe recruited 774 Chinese stroke patients, including 523 with ischemic stroke (IS) and 251 with hemorrhagic stroke (HS) within 14 days following symptom onset in the emergency department, alongside 481 healthy individuals and 64 cognitive impairment patients as controls. Serum ASC concentrations were determined using automated chemiluminescence immunoassay, exploring the relationship between serum ASC concentration and subtypes, severity, and sampling timepoints of stroke. ResultsASC concentrations were significantly higher in stroke patients compared with all controls (P < 0.001). HS patients had greater ASC concentrations than IS patients (P < 0.05). With increasing ASC concentration, the proportion of severe cases increased. The area under the receiver operating characteristic curve (AUC) for differentiating between healthy individuals and stroke patients in the hyperacute phase was 0.78; this markedly improved (0.90) when considering samples from healthy individuals and patients with subarachnoid hemorrhage (SAH) ≤ 3 h from last-known-well (LKW). ConclusionsSerum ASC is a valuable biomarker for stroke differentiation and aids in the clinical diagnosis of stroke severity and subtypes.

Limited Efficacy of Adipose Stromal Cell Secretome-Loaded Skin-Derived Hydrogels to Augment Skin Flap Regeneration in Rats.

Insufficient vascularization is a recurring cause of impaired pedicled skin flap healing. The administration of adipose tissue-derived stromal cells' (ASCs') secretome is a novel approach to augment vascularization. Yet, the secretome comprised of soluble factors that require a sustained-release vehicle to increase residence time. We hypothesized that administration of a hydrogel derived from decellularized extracellular matrix (ECM) of porcine skin with bound trophic factors from ASCs enhances skin flap viability and wound repair in a rat model. Porcine skin was decellularized and pepsin-digested to form a hydrogel at 37°C. Conditioned medium (CMe) of human ASC was collected, concentrated 20-fold, and mixed with the hydrogel. Sixty Wistar rats were included. A dorsal skin flap (caudal based) of 3 × 10 cm was elevated for topical application of DMEM (group I), a prehydrogel with or without ASC CMe (groups II and III), or ASC CMe (group IV). After 7, 14, and 28 days, perfusion was measured, and skin flaps were harvested for wound healing assessment and immunohistochemical analysis. Decellularized skin ECM hydrogel contained negligible amounts of DNA (11.6 ± 0.6 ng/mg), was noncytotoxic and well tolerated by rats. Irrespective of ASC secretome, ECM hydrogel application resulted macroscopically and microscopically in similar dermal wound healing in terms of proliferation, immune response, and matrix remodeling as the control group. However, ASC CMe alone increased vessel density after 7 days. Porcine skin-derived ECM hydrogels loaded with ASC secretome are noncytotoxic but demand optimization to significantly augment wound healing of skin flaps.

Synergy of human platelet lysate and laminin to enhance the neurotrophic effect of human adipose-derived stem cells

Synergy of human platelet lysate and laminin to enhance the neurotrophic effect of human adipose-derived stem cells

Angiogenic factors secreted from human ASC spheroids entrapped in an alginate-based hierarchical structure via combined 3D printing/electrospinning system

Human adipose-derived stem cell spheroids have been widely used in the treatment or regeneration of damaged skin tissues, and their success is believed to be due in part to angiogenic factors released from the spheroids. To achieve the sustained release of bioactive components from implanted spheroids within a defective area, the use of a biocompatible scaffolding biomaterial is required. In this study, we developed an alginate-based scaffolding structure, which was processed using three-dimensional printing and electrospinning for use as a spheroid-entrapping structure. A micro-sized alginate strut and electrospun alginate nanofibers functioned not only to firmly entrap the spheroids, but also to enable the stable release of various angiogenic and wound healing-related factors. We also demonstrated the function of these factors using a tube-forming assay and found that conditioned media from the spheroid-scaffold group improved capillary-like structure formation in human umbilical vein endothelial cells compared to the single cell-scaffold group. Our results suggest that this spheroid-entrapping alginate hybrid structure could represent a new platform for stem cell therapy using spheroid transplantation.

HMGA2 Modulates the TGFβ/Smad, TGFβ/ERK and Notch Signaling Pathways in Human Lens Epithelial-Mesenchymal Transition.

Multiple signaling pathways coordinately promote epithelial-mesenchymal transition (EMT) in lens epithelial cells (LECs), where transforming growth factor beta (TGFβ)-mediated signaling plays a central role. But the mechanism of crosstalk among these pathways remains obscure. The objective of this study is to investigate the regulatory effect of the high mobility group protein A2 (HMGA2) on the signaling pathways in lens fibrosis. The human anterior capsulorhexis specimens were collected. The human SRA01/04 LEC line was cultured and treated with recombinant human TGFβ2 (5ng/ml). For inhibition of signaling pathways, a selective inhibitor SB431542, U0126 or DAPT was added to LECs respectively. The specific small interfering RNA (siRNA) were transfected to LECs for gene silence. The mRNAs expressions were measured by realtime PCR and the proteins expressions were determined by western blot and immunofluorescent staining. HMGA2 and EMT markers α-smooth muscle actin (SMA), fibronectin (FN) and collagen type I (Col I) were overexpressed in human ASC specimens and TGFβ2 stimulated EMT in LECs. While blockage of EMT by a selective inhibitor of TGFβ/Smad, TGFβ/extracellular signal-regulated kinase (ERK) or Notch signaling pathway could significantly inhibited HMGA2 protein expression. And silence of HMGA2 by siRNA could significantly inhibit TGFβ2 induced expression of EMT markers including FN, Col I, collagen type IV (Col IV), key transcription factors Snail and Slug, and remarkably upregulate the epithelial markers E-cadherin and tight junction protein (ZO-1). In addition, silence of HMGA2 gene could abrogate TGFβ2 induced phosphorylation of Smad2, Smad3 as well as ERK1/2. Blockage of HMGA2 could also inhibit the upregulation of Jagged1, Notch2, and Notch3 induced by TGFβ2. This study indicated that HMGA2 functions as a shared effector in TGFβ2- induced lens fibrosis, modulating the signaling network necessary for EMT in a positive feedback loop.

Assay for high-throughput screening of inhibitors of the ASC-PYD inflammasome core filament.

The protein ASC is a central component of most inflammasome complexes, forming functional oligomeric filaments that activate large amounts of pro-caspase-1 for further IL-1β processing and the induction of Gasdermin D-dependent cell death. The central role of inflammasomes in the innate immune response pose them as new molecular targets for therapy of diverse acute, chronic and inherited autoinflammatory pathologies. In recent years, an increasing number of molecules were proposed to modulate inflammasome signalling by interacting with different components of inflammasome complexes. However, the difficult in vitro reconstitution of the inflammasome has limited the development of specific on-target biochemical assays for compound activity confirmation and for drug discovery in high throughput screening setups. Here we describe a homogeneous, pH-based ASC oligomerization assay that employs fluorescence anisotropy (FA) to monitor the in vitro filament formation of the PYD domain of human ASC. The absence of additional solubility tags as well as of proteolytic enzymes to initiate the filament reaction makes this assay suitable for testing the direct effect of small molecules on filament formation in high throughput format. The ability of the assay to detect modulators of filament formation was confirmed by using a non-filament forming PYD mutant. The high and reproducible Z’-factor of 0.7 allowed to screen 10,100 compounds by high-throughput screening (HTS) aiming to identify inhibitors of ASC filament. While none of these molecules was able to inhibit ASC filament formation in vitro, the assay is directly amenable to screen other compound classes or validate candidate molecules from other screens.

Abstract P6-07-05: Interactions between adipocyte stem cells and normal or tumoral mammary epithelial cells. Potantial role of BRCA status and estrogen pathway

Abstract Prophylactic mastectomies are more and more frequent. As a result, breast reconstruction is also more frequent and mammary lipofilling often used to improve the aesthetic results. Contradictory cliniacal data have been published concerning the risk of breast cancer after using this technique. It is possible that Adipose-Derived Stem / Stromal Cells (ADSC or ASC), present in the lipofilling may interact with normal or tumoral epithelial mammary cells. In this context, we studied the effect of conditioned media of human ASC on different tumor breast lines (MCF7, T47D, ZR75-1, SKBR3, SUM159). The ASCs were extracted from non-cancer patients with identical body mass index (BRCA1 and BRCA2 mutated or wild-type). We observed that the conditioned media of ASC induced the proliferation of luminal, Her2 and basal-like tumor breast lines. This proliferative effect is more important when ASCs are differentiated and the BRCA mutation status has no impact on it. Finally, we have demonstrated an estrogen signaling within the ASCs which is set up during their differentiation. Differentiated ASCs express ERα and produce 17β-estradiol (E2). This estrogenic pathway seems to be established independently of the BRCA mutational status. The proliferative effect on luminal mammary lines induced by conditioned media of ASC could pass through this estrogenic signaling, but remains to be confirmed. These data highlight the fact that adipose-derived stem cells in mammary lipofilling should only be used after a cancer has been eliminated, as there are still some uncertainties concerning their implication on carcinogenesis. Citation Format: Benderra M-A, Zaoui M, Atlan M, Ferrand N, Denis JA, Leban M, Lamari F, Larsen AK, Sabbah M, Gligorov J. Interactions between adipocyte stem cells and normal or tumoral mammary epithelial cells. Potantial role of BRCA status and estrogen pathway [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-07-05.

Human and Autologous Adipose-derived Stromal Cells Increase Flap Survival in Rats Independently of Host Immune Response.

There is a rising interest in adipose-derived stromal cells for clinical use; however, it is unknown whether freshly isolated stromal cells (SVF) or culture-expanded cells (ASCs) are more efficacious. We therefore aimed to compare the 2 cellular therapies in an in vivo model of angiogenesis, the ischemic flap in rats, which induces acute ischemia. We also aimed to determine the importance of cell presence and the host immune response. A total of 96 rats (n = 12 in each group) were used, and in each rat, a caudally based random flap measuring 2 × 7 cm was made. The study was conducted in 3 phases. First, each rat was treated with human SVF cells, human ASCs, or vehicle. Second, each rat was treated with human SVF, human SVF lysate, or vehicle. Finally, each rat was treated with rat (autologous) SVF cells or vehicle. Flap survival, vessel density, and stromal cell retention were evaluated after 7 days. The mean survival rates for SVF treatment regardless of human or autologous origin were significantly increased as compared with the control group. Adipose stem/stromal cell and SVF lysate injection did not increase flap survival. Vessel density was increased for human and rat SVF and human ASC but not for SVF lysate. Human cells were not detected in the flaps after 7 days. Flap survival increased with SVF treatment regardless of human or autologous origin, suggesting that increased flap survival is independent of the host immune response. All cell injections lead to increased vessel density, but it did not necessarily lead to increased flap survival. Further research should elaborate which molecular events make SVF treatment more efficacious than ASC.

Adipose-derived mesenchymal stem/stromal cells: from the lab bench to the basic concepts for clinical translation

In the last years, much work has shown that the most effective repair system of the body is represented by stem cells, which are defined as undifferentiated precursors that own unlimited or prolonged self-renewal ability, which also have the potential to transform themselves into various cell types through differentiation.All tissues that form the body contain many different types of somatic cells, along with stem cells that are called ‘mesenchymal stem (or stromal) cells’ (MSC). In certain circumstances, some of these MSC migrate to injured tissues to replace dead cells or to undergo differentiation to repair it.The discovery of MSC has been an important step in regenerative medicine because of their high versatility. Moreover, the finding of a method to isolate MSC from adipose tissue, so called ‘adipose-derived mesenchymal stem cells’ (ASC), which share similar differentiation capabilities and isolation yield that is greater than other MSC, and less bioethical concerns compared to embryonic stem cells, have created self-praised publicity to procure almost any treatment with them. Here, we review the current techniques for isolation, culture and differentiation of human ASC (hASC), and describe them in detail. We also compile some advantages of the hASC over other stem cells, and provide some concepts that could help finding strategies to promote their therapeutic efficiency.

Recombinant human collagen-based microspheres mitigate cardiac conduction slowing induced by adipose tissue-derived stromal cells.

BackgroundStem cell therapy to improve cardiac function after myocardial infarction is hampered by poor cell retention, while it may also increase the risk of arrhythmias by providing an arrhythmogenic substrate. We previously showed that porcine adipose tissue-derived-stromal cells (pASC) induce conduction slowing through paracrine actions, whereas rat ASC (rASC) and human ASC (hASC) induce conduction slowing by direct coupling. We postulate that biomaterial microspheres mitigate the conduction slowing influence of pASC by interacting with paracrine signaling.AimTo investigate the modulation of ASC-loaded recombinant human collagen-based microspheres, on the electrophysiological behavior of neonatal rat ventricular myocytes (NRVM).MethodUnipolar extracellular electrograms, derived from microelectrode arrays (8x8 electrodes) containing NRVM, co-cultured with ASC or ASC loaded microspheres, were used to determine conduction velocity (CV) and conduction heterogeneity. Conditioned medium (Cme) of (co)cultures was used to assess paracrine mechanisms.ResultsMicrospheres did not affect CV in control (NRVM) monolayers. In co-cultures of NRVM and rASC, hASC or pASC, CV was lower than in controls (14.4±1.0, 13.0±0.6 and 9.0± 1.0 vs. 19.5±0.5 cm/s respectively, p<0.001). Microspheres loaded with either rASC or hASC still induced conduction slowing compared to controls (13.5±0.4 and 12.6±0.5 cm/s respectively, p<0.001). However, pASC loaded microspheres increased CV of NRVM compared to pASC and NRMV co-cultures (16.3±1.3 cm/s, p< 0.001) and did not differ from controls (p = NS). Cme of pASC reduced CV in control monolayers of NRVM (10.3±1.1 cm/s, p<0.001), similar to Cme derived from pASC-loaded microspheres (11.1±1.7 cm/s, p = 1.0). The presence of microspheres in monolayers of NRVM abolished the CV slowing influence of Cme pASC (15.9±1.0 cm/s, p = NS vs. control).ConclusionThe application of recombinant human collagen-based microspheres mitigates indirect paracrine conduction slowing through interference with a secondary autocrine myocardial factor.

Targeting endogenous proteins for degradation through the affinity-directed protein missile system.

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel–Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins.

Human and feline adipose-derived mesenchymal stem cells have comparable phenotype, immunomodulatory functions, and transcriptome

BackgroundAdipose-derived mesenchymal stem cells (ASCs) are a promising cell therapy to treat inflammatory and immune-mediated diseases. Development of appropriate pre-clinical animal models is critical to determine safety and attain early efficacy data for the most promising therapeutic candidates. Naturally occurring diseases in cats already serve as valuable models to inform human clinical trials in oncologic, cardiovascular, and genetic diseases. The objective of this study was to complete a comprehensive side-by-side comparison of human and feline ASCs, with an emphasis on their immunomodulatory capacity and transcriptome.MethodsHuman and feline ASCs were evaluated for phenotype, immunomodulatory profile, and transcriptome. Additionally, transwells were used to determine the role of cell-cell contact in ASC-mediated inhibition of lymphocyte proliferation in both humans and cats.ResultsSimilar to human ASCs, feline ASCs were highly proliferative at low passages and fit the minimal criteria of multipotent stem cells including a compatible surface protein phenotype, osteogenic capacity, and normal karyotype. Like ASCs from all species, feline ASCs inhibited mitogen-activated lymphocyte proliferation in vitro, with or without direct ASC-lymphocyte contact. Feline ASCs mimic human ASCs in their mediator secretion pattern, including prostaglandin E2, indoleamine 2,3 dioxygenase, transforming growth factor beta, and interleukin-6, all augmented by interferon gamma secretion by lymphocytes. The transcriptome of three unactivated feline ASC lines were highly similar. Functional analysis of the most highly expressed genes highlighted processes including: 1) the regulation of apoptosis; 2) cell adhesion; 3) response to oxidative stress; and 4) regulation of cell differentiation. Finally, feline ASCs had a similar gene expression profile to noninduced human ASCs.ConclusionsFindings suggest that feline ASCs modulate lymphocyte proliferation using soluble mediators that mirror the human ASC secretion pattern. Uninduced feline ASCs have similar gene expression profiles to uninduced human ASCs, as revealed by transcriptome analysis. These data will help inform clinical trials using cats with naturally occurring diseases as surrogate models for human clinical trials in the regenerative medicine arena.

182 - Noninvasively Measured Human Brain Glutathione and Ascorbate Concentrations in Healthy Aging and in Alzheimer's Disease

Numerous studies have found lower levels of antioxidants in the blood and cerebrospinal fluid of elderly adults. Oxidative stress is involved in Alzheimer’s disease (AD) pathogenesis and progression. Increased sensitivity of magnetic resonance technology has made it possible to noninvasively quantify concentrations of the antioxidants glutathione (GSH) and ascorbate (Asc) in intact human brain. Ultra-high field magnetic resonance spectroscopy (MRS) was used to measure concentrations of GSH and ascorbate in the occipital cortex (OCC) and posterior cingulate cortex (PCC) of seventeen young adults, sixteen cognitively normal older adults, and eight patients with AD. The short-echo-time MRS approach inherently retained numerous resonances, from which a profile of 15 neurochemicals was measured. For the first time, the concentration of GSH was found to be lower (p = 0.02) in the OCC of the elderly in a way that is not contingent upon the transverse relaxation rate. Healthy aging tended to impact neurochemicals associated with excitotoxicity (i.e. N-acetylaspartate) in the OCC, whereas those more closely linked to reactive astrogliosis (i.e.,myo-inositol, creatine and choline) were impacted in the PCC. The concentration of Asc was lower (p = 0.005) in the PCC of the patients with AD than in the healthy older adults. This is the first time human brain Asc has been studied noninvasively in patients with AD. In patients, one of the neurochemicals associated with reactive astrogliosis (myo-inositol) was higher in the OCC and two were higher in the PCC (i.e., myo-inositol and choline). Taken together, these outcomes show that both aging and AD impact brain regions differentially. These findings fill part of the gap in understanding how the brain deteriorates throughout aging and AD. Ultra-high field ultra-short-echo-time MRS supplies hitherto missing steps along the pathway to preventing and treating cognitive decline, i.e. means to identify which antioxidant is compromised, in which brain region, in whom, and whether intervention can achieve normalization.

A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.

Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASC(CARD) interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASC(PYD) filaments for the first time. These data revealed that cross-linking of ASC(PYD) filaments via ASC(CARD) mediates the assembly of ASC foci.

Development &amp; performance assessment of a new ATMP for cartilage tissue engineering

Event Abstract Back to Event Development & performance assessment of a new ATMP for cartilage tissue engineering Alain Morais1, 2, Cristina Correia3, Carlos A. Vilela1, 2, Ana C. Gertrudes3, David Learmonth3, Joaquim M. Oliveira1, 2, Rui A. Sousa3 and Rui L. Reis1, 2, 3 1 University of Minho, 3B’s Research Group—Biomaterials, Biodegradables and Biomimetics, Department of Polymer Engineering, Portugal 2 University of Minho, ICVS/3B’s—PT Government Associate Laboratory, Portugal 3 Stemmatters SA, Portugal Introduction: Joint disorders are responsible of reduced quality of life and high socio-economic burden in our progressively ageing and physically active population. Even though huge efforts on the development of cartilage repair strategies, presently no approach has demonstrate full efficiency. The present study aimed to create an innovative advanced-therapy medicinal product (ATMP) where human adipose-derived stem/stromal cells (hASCs)[1] encapsulated within a novel hydrogel based on methacrylated gellan gum (mimsys G)[2] will develop cartilage-like tissue. These complete xeno-free 3D approach was developed and in vitro experiment were conducted under static and dynamic conditions, the latest using a rotational dual chamber bioreactor[3]. Preliminary in vivo assay using rabbit model of cartilage defect was based on autologous and allogenic approaches. Both in vitro and in vivo experiments were established to evaluate the performance of this system on focal cartilage tissue engineering. Materials and Methods: Human ASC (hASC xeno-free, irisbiosciences), isolated in GMP conditions, were expanded in xeno-free media until sub-confluency. mimsys G hydrogel (mimsys®G, irisbiosciences) was prepared to yield a 2% w/V solution. Cells were encapsulated at 10x106 cells/mL, ionically crosslinked and cultured with or without chondrogenic induction for 21 days. For dynamic growth, a rotational dual chamber bioreactor was used to control the perfusion and flow gradient within the cellular scaffolds. Cell viability was determined by live/dead assay, and cell metabolic activity by MTS reduction. A in vivo study evaluated regeneration of focal cartilage lesions on a rabbit knee model for 8 weeks, using autologous and allogenic approaches. Rabbit ASCs (rASCs), isolated from fat pad, and hASCs were encapsulated into mimsys G prior administration into the cartilage defect. Chondrogenesis was assessed on both in vitro and in vivo samples by histological staining of cartilage matrix using safranin O and immunolocalization of collagen type II. Expression of chondrogenic-related genes (aggrecan, collagen type II and sox-9) were also determined. Results: In vitro analysis of the new ATMP demonstrated high viability of the cells and progressive metabolic activity along static and dynamic culture. Additionally, intense cartilage matrix deposition was detected through up-regulation of chondrogenic maker Collagen II gene and protein (Immunohistochemistry) by end of culture (Fig.1). For the in vivo study, both autologous and allogenic approaches demonstrated the establishment of lateral and subchondral integration (Fig.2). Fig. 1 Fig. 2 Fig.1. In vitro Chondrogenesis – Collagen II gene (A) and protein (B) expression at day 0 and day 21, after chondrogenic induction in static culture conditions. Fig 2. In vivo study – Safranin-O staining after regeneration of cartilage lesion. Discussion and Conclusions: The proposed innovative ATMP demonstrated intense cartilage development in vitro through evaluation of specific markers. Preliminary in vivo data demonstrates promising repair performance in focal cartilage lesions, suggesting a valuable approach for further therapeutic exploitation. The authors thank the financial support under NORTE-07-0202-FEDER-023189 / ARTICULATE - Desenvolvimento de novos produtos e terapias regenerativas para o tratamento de patologias articulares / ADI

Adipose-derived Stem Cell Conditioned Media Extends Survival time of a mouse model of Amyotrophic Lateral Sclerosis.

Adipose stromal cells (ASC) secrete various trophic factors that assist in the protection of neurons in a variety of neuronal death models. In this study, we tested the effects of human ASC conditional medium (ASC-CM) in human amyotrophic lateral sclerosis (ALS) transgenic mouse model expressing mutant superoxide dismutase (SOD1G93A). Treating symptomatic SOD1G93A mice with ASC-CM significantly increased post-onset survival time and lifespan. Moreover, SOD1G93A mice given ASC-CM treatment showed high motor neuron counts, less activation of microglia and astrocytes at an early symptomatic stage in the spinal cords under immunohistochemical analysis. SOD1G93A mice treated with ASC-CM for 7 days showed reduced levels of phosphorylated p38 (pp38) in the spinal cord, a mitogen-activated protein kinase that is involved in both inflammation and neuronal death. Additionally, the levels of α-II spectrin in spinal cords were also inhibited in SOD1G93A mice treated with ASC-CM for 3 days. Interestingly, nerve growth factor (NGF), a neurotrophic factor found in ASC-CM, played a significant role in the protection of neurodegeneration inSOD1G93A mouse. These results indicate that ASC-CM has the potential to develop into a novel and effective therapeutic treatment for ALS.

Extracellular Calcium Modulates Chondrogenic and Osteogenic Differentiation of Human Adipose-Derived Stem Cells: A Novel Approach for Osteochondral Tissue Engineering Using a Single Stem Cell Source.

We have previously shown that elevating extracellular calcium from a concentration of 1.8 to 8 mM accelerates and increases human adipose-derived stem cell (hASC) osteogenic differentiation and cell-mediated calcium accretion, even in the absence of any other soluble osteogenic factors in the culture medium. However, the effects of elevated calcium on hASC chondrogenic differentiation have not been reported. The goal of this study was to determine the effects of varied calcium concentrations on chondrogenic differentiation of hASC. We hypothesized that exposure to elevated extracellular calcium (8 mM concentration) in a chondrogenic differentiation medium (CDM) would inhibit chondrogenesis of hASC when compared to basal calcium (1.8 mM concentration) controls. We further hypothesized that a full osteochondral construct could be engineered by controlling local release of calcium to induce site-specific chondrogenesis and osteogenesis using only hASC as the cell source. Human ASC was cultured as micromass pellets in CDM containing transforming growth factor-β1 and bone morphogenetic protein 6 for 28 days at extracellular calcium concentrations of either 1.8 mM (basal) or 8 mM (elevated). Our findings indicated that elevated calcium induced osteogenesis and inhibited chondrogenesis in hASC. Based on these findings, stacked polylactic acid nanofibrous scaffolds containing either 0% or 20% tricalcium phosphate (TCP) nanoparticles were electrospun and tested for site-specific chondrogenesis and osteogenesis. Histological assays confirmed that human ASC differentiated locally to generate calcified tissue in layers containing 20% TCP, and cartilage in the layers with no TCP when cultured in CDM. This is the first study to report the effects of elevated calcium on chondrogenic differentiation of hASC, and to develop osteochondral nanofibrous scaffolds using a single cell source and controlled calcium release to induce site-specific differentiation. This approach holds great promise for osteochondral tissue engineering using a single cell source (hASC) and single scaffold.

Preventive effects of CTLA4Ig-overexpressing adipose tissue–derived mesenchymal stromal cells in rheumatoid arthritis

Background aimsRheumatoid arthritis is a systemic autoimmune disorder. In this study, we first compared the therapeutic effects of syngeneic and xenogeneic adipose tissue–derived stem cells on a collagen-induced arthritis mouse model. Second, we investigated the synergistic preventive effects of CTLA4Ig and adipose tissue–derived mesenchymal stromal cells (ASCs) as a therapeutic substance. MethodsArthritis was induced in all groups except for the normal, saline (N) group, using chicken type II collagen (CII). Animals were divided into C (control, saline), H (hASCs), M (mASCs) and N groups (experiment I) and C, H, CT (CTLA4Ig-overexpressing human ASC [CTLA4Ig-hASCs]) and N groups (experiment II), according to transplanted material. Approximately 2 × 106 ASCs or 150 μL of saline was intravenously administered on days 24, 27, 30 and 34, and all animals were killed on days 42 to 44 after CII immunization. ResultsAnti-mouse CII autoantibodies were significantly lower in the H, M and CT groups than in the C group. Cartilage damage severity score and C-telopeptide of type II collagen were significantly lower in the CT group than in the C group. The serum levels of IL-6 were significantly lower in the H, M and CT groups than in the C group. The serum levels of keratinocyte chemoattractant were significantly lower in the CT group than the C group. ConclusionsThere were similar effects of ASCs on the decrease of anti-mouse CII autoantibody levels between syngeneic and xenogeneic transplantations, and CTLA4Ig-hASCs showed synergistic preventive effects compared with non-transduced hASCs.