Stefania Niada

Systemic Administration of Human Adipose-Derived Stem Cells Reverts Nociceptive Hypersensitivity in an Experimental Model of Neuropathy

Over the last decade, it has been proved that mesenchymal stem cells (MSCs) elicit anti-inflammatory effects. MSCs from adipose tissue (hASCs) differentiate into cells of the mesodermal lineage and transdifferentiate into ectodermal-origin cells. Although there are various etiologies to chronic pain, one common feature is that painful states are associated with increased inflammation. We believe in hASCs as a therapeutic tool also in pathologies involving neuroinflammation and neuronal tissue damage. We have investigated the effect of hASCs injected in a model of neuropathic pain [(mouse sciatic nerve chronic constriction injury (CCI)]. hASCs from 5 donors were characterized, and no major differences were depicted. hASCs were cryopreserved and grown on demand. About 1×10(6), 3×10(6), and 6×10(6) hASCs were intravenously injected into normal immunocompetent mice. No mouse died, and no macroscopic toxicity or behavioral changes were observed, confirming the safety of hASCs. hASCs, intravenously (i.v.) injected into C57BL/6 mice when the neuropathic pain was already established, induced a significant reduction in mechanical allodynia and a complete reversion of thermal hyperalgesia in a dose-response fashion, already 1 day after administration. Moreover, the hASCs effect can be boosted by repeated administrations, allowing a prolonged therapeutic effect. Treatment decreased the level of the CCI-induced proinflammatory cytokine interleukin (IL)-1β and activated the anti-inflammatory cytokine IL-10 in the lesioned nerve. hASCs treatment also restored normal inducible nitric oxide synthase expression in the spinal cord of CCI animals. Our data suggest that hASCs are worthy of further studies as an anti-inflammatory therapy in the treatment of neuropathic pain or chronic inflammatory diseases.

Stemness and osteogenic and adipogenic potential are differently impaired in subcutaneous and visceral adipose derived stem cells (ASCs) isolated from obese donors.

Today adipose tissue is not just considered as the primary energy storage organ, but it is also recognized as an important endocrine tissue and an abundant source of mesenchymal stem cells (adipose-derived stem cells, ASCs). During the last decade, several studies have provided preclinical data on the safety and efficacy of ASCs, supporting their use in cell-based therapy for regenerative medicine purposes. Little is known about the effect of obesity on ASCs properties. Since ASCs differentiation and proliferation are determined by their niche, the differences in body fat distribution and the obesity-related co-morbidities may have several consequences. In this study we compared ASCs of subcutaneous adipose tissue from obese (obS-ASCs) and non-obese (nS-ASCs) donors in order to compare their immunophenotype and osteogenic and adipogenic potential. Moreover, in order to evaluate the possible difference between subcutaneous and visceral fat, obS-ASCs were also compared to ASCs derived from visceral adipose tissue of the same obese donors (obV-ASCs). Our results show that subcutaneous and visceral ASCs derived from obese donors have an impaired cell proliferation, clonogenic ability and immunophenotype. Nevertheless, obS-ASCs are able to differentiate toward osteogenic and adipogenic lineages, although to a small extent with respect to non-obese donors, whereas obV-ASCs lose most of their stem cell characteristics, including multi-differentiation potential. Taken together our findings confirm that not all ASCs present the same behavior, most likely due to their biological microenvironment in vivo. The specific stimuli which can play a key role in ASCs impairment, including the effects of the obesity-related inflammation, should be further investigated to have a complete picture of the phenomenon.

Chemical and genetic blockade of HDACs enhances osteogenic differentiation of human adipose tissue-derived stem cells by oppositely affecting osteogenic and adipogenic transcription factors

The human adipose-tissue derived stem/stromal cells (hASCs) are an interesting source for bone-tissue engineering applications. Our aim was to clarify in hASCs the role of acetylation in the control of Runt-related transcription factor 2 (Runx2) and Peroxisome proliferator activated receptor (PPAR) γ. These key osteogenic and adipogenic transcription factors are oppositely involved in osteo-differentiation. The hASCs, committed or not towards bone lineage with osteoinductive medium, were exposed to HDACs chemical blockade with Trichostatin A (TSA) or were genetically silenced for HDACs. Alkaline phosphatase (ALP) and collagen/calcium deposition, considered as early and late osteogenic markers, were evaluated concomitantly as index of osteo-differentiation. TSA pretreatment, useful experimental protocol to analyse pan-HDAC-chemical inhibition, and switch to osteogenic medium induced early-osteoblast maturation gene Runx2, while transiently decreased PPARγ and scarcely affected late-differentiation markers. Time-dependent effects were observed after knocking-down of HDAC1 and 3: Runx2 and ALP underwent early activation, followed by late-osteogenic markers increase and by PPARγ/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPARγ target genes, respectively. Noteworthy, HDACs knocking-down favoured the commitment effect of osteogenic medium. Our results reveal a role for HDACs in orchestrating osteo-differentiation of hASCs at transcriptional level, and might provide new insights into the modulation of hASCs-based regenerative therapy.

Porcine adipose-derived stem cells from buccal fat pad and subcutaneous adipose tissue for future preclinical studies in oral surgery

IntroductionAdipose-derived stem cells (ASCs) are progenitor cells used in bone tissue engineering and regenerative medicine. Despite subcutaneous adipose tissue being more abundant, the buccal fat pad (BFP) is easily accessible for dentists and maxillofacial surgeons. For this reason, considering the need for preclinical study and the swine as an optimal animal model in tissue engineering applications, we compared the features of porcine ASCs (pASCs) from both tissue-harvesting sites.MethodsASCs were isolated from interscapular subcutaneous adipose tissue (ScI) and buccal fat pads of six swine. Cells were characterized for their stemness and multipotent features. Moreover, their osteogenic ability when cultured on titanium disks and silicon carbide-plasma-enhanced chemical vapor-deposition fragments, and their growth in the presence of autologous and heterologous serum were also assessed.ResultsIndependent of the harvesting site, no differences in proliferation, viability, and clonogenicity were observed among all the pASC populations. Furthermore, when induced toward osteogenic differentiation, both ScI- and BFP-pASCs showed an increase of collagen and calcified extracellular matrix (ECM) production, alkaline phosphatase activity, and osteonectin expression, indicating their ability to differentiate toward osteoblast-like cells. In addition, they differentiated toward adipocyte-like cells, and chondrogenic induced pASCs were able to increase glycosaminoglycans (GAGs) production over time. When cells were osteoinduced on synthetic biomaterials, they significantly increased the amount of calcified ECM compared with control cells; moreover, titanium showed the osteoinductive effect on pASCs, also without chemical stimuli. Finally, these cells grew nicely in 10% FBS, and no benefits were produced by substitution with swine serum.ConclusionsSwine buccal fat pad contains progenitor cells with mesenchymal features, and they also osteo-differentiate nicely in association with synthetic supports. We suggest that porcine BFP-ASCs may be applied in preclinical studies of periodontal and bone-defect regeneration.

Adult Stem Cell as New Advanced Therapy for Experimental Neuropathic Pain Treatment

Neuropathic pain (NP) is a highly invalidating disease resulting as consequence of a lesion or disease affecting the somatosensory system. All the pharmacological treatments today in use give a long lasting pain relief only in a limited percentage of patients before pain reappears making NP an incurable disease. New approaches are therefore needed and research is testing stem cell usage. Several papers have been written on experimental neuropathic pain treatment using stem cells of different origin and species to treat experimental NP. The original idea was based on the capacity of stem cell to offer a totipotent cellular source for replacing injured neural cells and for delivering trophic factors to lesion site; soon the researchers agreed that the capacity of stem cells to contrast NP was not dependent upon their regenerative effect but was mostly linked to a bidirectional interaction between the stem cell and damaged microenvironment resident cells. In this paper we review the preclinical studies produced in the last years assessing the effects induced by several stem cells in different models of neuropathic pain. The overall positive results obtained on pain remission by using stem cells that are safe, of easy isolation, and which may allow an autologous transplant in patients may be encouraging for moving from bench to bedside, although there are several issues that still need to be solved.

Mesenchymal Stem Cells from Bichat's Fat Pad: In Vitro Comparison with Adipose-Derived Stem Cells from Subcutaneous Tissue

Abstract Adipose-derived stem/stromal cells (ASCs) are progenitor cells used in bone tissue engineering and regenerative medicine. Since Bichats fat pad is easily accessible for dentists and maxillo-facial surgeons, we compared the features of ASCs from Bichats fat pad (BFP-ASCs) with human ASCs from subcutaneous adipose tissue (SC-ASCs). BFP-ASCs isolated from a small amount of tissue were characterized for their stemness and multidifferentiative ability. They showed an important clonogenic ability and the typical mesenchymal stem cell immunophenotype. Moreover, when properly induced, osteogenic and adipogenic differentiation markers, such as alkaline phosphatase activity, collagen deposition and lipid vacuoles formation, were promptly observed. Growth of both BFP-ASCs and SC-ASCs in the presence of human serum and their adhesion to natural and synthetic scaffolds were also assessed. Both types of ASCs adapted rapidly to human autologous or heterologous sera, increasing their proliferation rate compared to standard culture condition, and all the cells adhered finely to bone, periodontal ligament, collagen membrane, and polyglycol acid filaments that are present in the oral cavity or are commonly used in oral surgery. At last, we showed that amelogenin seems to be an early osteoinductive factor for BFP-ASCs, but not SC-ASCs, in vitro. We conclude that Bichats fat pad contains BFP-ASCs with stemness features that are able to differentiate and adhere to biological supports and synthetic materials. They are also able to proliferate in the presence of human serum. For all these reasons we propose BFP-ASCs for future therapies of periodontal defects and bone regeneration.

Hypoxia Promotes the Inflammatory Response and Stemness Features in Visceral Fat Stem Cells From Obese Subjects

Low‐grade chronic inflammation is a salient feature of obesity and many associated disorders. This condition frequently occurs in central obesity and is connected to alterations of the visceral adipose tissue (AT) microenvironment. Understanding how obesity is related to inflammation may allow the development of therapeutics aimed at improving metabolic parameters in obese patients. To achieve this aim, we compared the features of two subpopulations of adipose‐derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of two subpopulations of ASC from the same isolation sites of non‐obese individuals. In particular, the behavior of ASC of obese versus non‐obese subjects during hypoxia, which occurs in obese AT and is an inducer of the inflammatory response, was evaluated. Obesity deeply influenced ASC from visceral AT (obV‐ASC); these cells appeared to exhibit clearly distinguishable morphology and ultrastructure as well as reduced proliferation, clonogenicity and expression of stemness, differentiation and inflammation‐related genes. These cells also exhibited a deregulated response to hypoxia, which induced strong tissue‐specific NF‐kB activation and an NF‐kB‐mediated increase in inflammatory and fibrogenic responses. Moreover, obV‐ASC, which showed a less stem‐like phenotype, recovered stemness features after hypoxia. Our findings demonstrated the peculiar behavior of obV‐ASC, their influence on the obese visceral AT microenvironment and the therapeutic potential of NF‐kB inhibitors. These novel findings suggest that the deregulated hyper‐responsiveness to hypoxic stimulus of ASC from visceral AT of obese subjects may contribute via paracrine mechanisms to low‐grade chronic inflammation, which has been implicated in obesity‐related morbidity. J. Cell. Physiol. 231: 668–679, 2016.

Genome-wide DNA methylation profiling of recurrent and non-recurrent chordomas

Chordomas are an aggressive rare type of malignant bone tumors arising from the remnant of the notochord. Chordomas occur mainly in vertebral bones and account for 1–4% of malignant bone tumors. Management and treatment of chordomas are difficult as they are resistant to conventional chemotherapy; therefore, they are mainly treated with surgery and radiation therapy. In this study, we performed DNA methylation profiling of 26 chordomas and normal nucleus pulposus samples plus UCH-1 chordoma cell line using the Illumina Infinium HumanMethylation450 BeadChips. Combined bisulfite restriction analysis and bisulfite sequencing was used to confirm the methylation data. Gene expression was analyzed using RT-PCR before and after 5-aza-2’-deoxycytidine (5-azaDC) treatment of chordoma cell lines. Analysis of the HumanMethylation450 BeadChip data led to the identification of 8,819 loci (2.9%) that were significantly differentially methylated (>0.2 average β-value difference) between chordomas and nucleus pulposus samples (adjusted P < 0.05). Among these, 5,868 probes (66.5%) were hypomethylated, compared to 2,951 (33.5%) loci that were hypermethylated in chordomas compared to controls. From the 2,951 differentially hypermethylated probes, 33.3% were localized in the promoter region (982 probes) and, among these, 104 probes showed cancer-specific hypermethylation. Ingenuity Pathway Analysis indicates that the cancer-specific differentially methylated loci are involved in various networks including cancer disease, nervous system development and function, cell death and survival, cellular growth, cellular development, and proliferation. Furthermore, we identified a subset of probes that were differentially methylated between recurrent and non-recurrent chordomas. BeadChip methylation data was confirmed for these genes and gene expression was shown to be upregulated in methylated chordoma cell lines after treatment with 5-azaDC. Understanding epigenetic changes in chordomas may provide insights into chordoma tumorigenesis and development of epigenetic biomarkers.

Repair of osteochondral defects in the minipig model by OPF hydrogel loaded with adipose-derived mesenchymal stem cells.

AIM Critical knee osteochondral defects in seven adult minipigs were treated with oligo(polyethylene glycol)fumarate (OPF) hydrogel combined with autologous or human adipose-derived stem cells (ASCs), and evaluated after 6 months. METHODS Four defects were made on the peripheral part of right trochleas (n = 28), and treated with OPF scaffold alone or pre-seeded with ASCs. RESULTS A better quality cartilage tissue characterized by improved biomechanical properties and higher collagen type II expression was observed in the defects treated by autologous or human ASC-loaded OPF; similarly this approach induced the regeneration of more mature bone with upregulation of collagen type I expression. CONCLUSION This study provides the evidence that both porcine and human adipose-derived stem cells associated to OPF hydrogel allow improving osteochondral defect regeneration in a minipig model.

Therapeutic effect of human adipose-derived stem cells and their secretome in experimental diabetic pain

Painful neuropathy is one of the complications of diabetes mellitus that adversely affects patients’quality of life. Pharmacological treatments are not fully satisfactory, and novel approaches needed. In a preclinical mouse model of diabetes the effect of both human mesenchymal stromal cells from adipose tissue (hASC) and their conditioned medium (hASC-CM) was evaluated. Diabetes was induced by streptozotocin. After neuropathic hypersensitivity was established, mice were intravenously injected with either 1 × 106 hASC or with CM derived from 2 × 106 hASC. Both hASC and CM (secretome) reversed mechanical, thermal allodynia and thermal hyperalgesia, with a rapid and long lasting effect, maintained up to 12 weeks after treatments. In nerves, dorsal root ganglia and spinal cord of neuropathic mice we determined high IL-1β, IL-6 and TNF-α and low IL-10 levels. Both treatments restored a correct pro/antinflammatory cytokine balance and prevented skin innervation loss. In spleens of streptozotocin-mice, both hASC and hASC-CM re-established Th1/Th2 balance that was shifted to Th1 during diabetes. Blood glucose levels were unaffected although diabetic animals regained weight, and kidney morphology was recovered by treatments. Our data show that hASC and hASC-CM treatments may be promising approaches for diabetic neuropathic pain, and suggest that cell effect is likely mediated by their secretome.