Adipose tissue-derived stromal cells (ADSC) express oligodendrocyte and myelin markers, but they do not function as oligodendrocytes.

Mesenchymal cells cultured from the vasculo-stromal fraction of adipose tissue (ADSC) show adult stem cell characteristics and several groups have claimed generating neural cells from them. However, we have observed that many markers commonly used for the identification of neural cells are spontaneously expressed by ADSC in culture. In the present study, we have examined the expression of characteristic oligodendrocyte molecules in cultured ADSC, aiming to test if myelinating cells could be generated from accessible non-neural adult tissues. In basal growth conditions, rat ADSC spontaneously expressed CNPase, MBP, MOG, protein zero, GAP43, Sox10, and Olig2, as shown by immunocytrochemistry and western blot. A small population of cultured ADSC expressed membrane galactocerebroside (O1 antibody), but no cell stained with O4 antibody. RT-PCR analyses showed the expression of CNPase, MBP, DM20, and low levels of Olig2, Sox10, and Sox2 mRNA by rat ADSC. When rat ADSC were treated with combinations of factors commonly used in neural-inducing media (retinoic acid, dbcAMP, EGF, basic FGF, NT3, and/or PDGF), the number of O1-positive cells changed, but in no case, mRNA expression of Sox10 and Olig2 transcription factors approached CNS oligodendrocyte levels. In co-culture with rat dorsal root ganglion neurons, no sign of axonal myelination by rat ADSC was observed. These studies show that the expression of oligodendrocyte traits by cultured ADSC is not a proof of functional competence as oligodendroglia and suggest that in culture conditions, ADSC acquire intermediate, uncommitted phenotypes.

Electric stimulation at 448 kHz promotes proliferation of human mesenchymal stem cells.

BACKGROUND/AIMS: Capacitive-resistive electric transfer (CRET) is a non invasive electrothermal therapy that applies electric currents within the 400 kHz - 450 kHz frequency range to the treatment of musculoskeletal lesions. Evidence exists that electric currents and electric or magnetic fields can influence proliferative and/or differentiating processes involved in tissue regeneration. This work investigates proliferative responses potentially underlying CRET effects on tissue repair. METHODS: XTT assay, flow cytometry, immunofluorescence and Western Blot analyses were conducted to asses viability, proliferation and differentiation of adipose-derived stem cells (ADSC) from healthy donors, after short, repeated (5 m On/4 h Off) in vitro stimulation with a 448-kHz electric signal currently used in CRET therapy, applied at a subthermal dose of 50 µA/mm(2) RESULTS: The treatment induced PCNA and ERK1/2 upregulation, together with significant increases in the fractions of ADSC undergoing cycle phases S, G2 and M, and enhanced cell proliferation rate. This proliferative effect did not compromise the multipotential ability of ADSC for subsequent adipogenic, chondrogenic or osteogenic differentiation. CONCLUSIONS: These data identify cellular and molecular phenomena potentially underlying the response to CRET and indicate that CRET-induced lesion repair could be mediated by stimulation of the proliferation of stem cells present in the injured tissues.

Oligodendroglia Generated From Adult Rat Adipose Tissue by Direct Cell Conversion.

Obtaining oligodendroglial cells from dispensable tissues would be of great interest for autologous or immunocompatible cell replacement therapy in demyelinating diseases, as well as for studying myelin-related pathologies or testing therapeutic approaches in culture. We evaluated the feasibility of generating oligodendrocyte precursor cells (OPCs) from adult rat adipose tissue by expressing genes encoding transcription factors involved in oligodendroglial development. Adipose-derived mesenchymal cells were lentivirally transduced with tetracycline-inducible Sox10, Olig2, Zfp536, and/or Nkx6.1 transgenes. Immunostaining with the OPC-specific O4 monoclonal antibody was used to mark oligodendroglial induction. O4- and myelin-associated glycoprotein (MAG)-positive cells emerged after 3 weeks when using the Sox10 + Olig2 + Zfp536 combination, followed in the ensuing weeks by GFAP-, O1 antigen-, p75NTR (low-affinity NGF receptor)-, and myelin proteins-positive cells. The O4+ cell population progressively expanded, eventually constituting more than 70% of cells in culture by 5 months. Sox10 transgene expression was essential for generating O4+ cells but was insufficient for inducing a full oligodendroglial phenotype. Converted cells required continuous transgene expression to maintain their glial phenotype. Some vestigial characteristics of mesenchymal cells were maintained after conversion. Growth factor withdrawal and triiodothyronine (T3) supplementation generated mature oligodendroglial phenotypes, while FBS supplementation produced GFAP+- and p75NTR+-rich cultures. Converted cells also showed functional characteristics of neural-derived OPCs, such as the expression of AMPA, NMDA, kainate, and dopaminergic receptors, as well as similar metabolic responses to differentiation-inducing drugs. When co-cultured with rat dorsal root ganglion neurons, the converted cells differentiated and ensheathed multiple axons. We propose that functional oligodendroglia can be efficiently generated from adult rat mesenchymal cells by direct phenotypic conversion.

Senolytic activity of small molecular polyphenols from olive restores chondrocyte redifferentiation and promotes a pro-regenerative environment in osteoarthritis.

Articular cartilage and synovial tissue from patients with osteoarthritis (OA) show an overactivity of connexin43 (Cx43) and accumulation of senescent cells associated with disrupted tissue regeneration and disease progression. The aim of this study was to determine the effect of oleuropein on Cx43 and cellular senescence for tissue engineering and regenerative medicine strategies for OA treatment. Oleuropein regulates Cx43 promoter activity and enhances the propensity of hMSCs to differentiate into chondrocytes and bone cells, reducing adipogenesis. This small molecule reduce Cx43 levels and decrease Twist-1 activity in osteoarthritic chondrocytes (OACs), leading to redifferentiation, restoring the synthesis of cartilage ECM components (Col2A1 and proteoglycans), and reducing the inflammatory and catabolic factors mediated by NF-kB (IL-1ß, IL-6, COX-2 and MMP-3), in addition to lowering cellular senescence in OACs, synovial and bone cells. Our in vitro results demonstrate the use of olive-derived polyphenols, such as oleuropein, as potentially effective therapeutic agents to improve chondrogenesis of hMSCs, to induce chondrocyte re-differentiation in OACs and clearing out senescent cells in joint tissues in order to prevent or stop the progression of the disease.

Targeting of chondrocyte plasticity via connexin43 modulation attenuates cellular senescence and fosters a pro-regenerative environment in osteoarthritis.

Osteoarthritis (OA), a chronic disease characterized by articular cartilage degeneration, is a leading cause of disability and pain worldwide. In OA, chondrocytes in cartilage undergo phenotypic changes and senescence, restricting cartilage regeneration and favouring disease progression. Similar to other wound-healing disorders, chondrocytes from OA patients show a chronic increase in the gap junction channel protein connexin43 (Cx43), which regulates signal transduction through the exchange of elements or recruitment/release of signalling factors. Although immature or stem-like cells are present in cartilage from OA patients, their origin and role in disease progression are unknown. In this study, we found that Cx43 acts as a positive regulator of chondrocyte-mesenchymal transition. Overactive Cx43 largely maintains the immature phenotype by increasing nuclear translocation of Twist-1 and tissue remodelling and proinflammatory agents, such as MMPs and IL-1ß, which in turn cause cellular senescence through upregulation of p53, p16INK4a and NF-κB, contributing to the senescence-associated secretory phenotype (SASP). Downregulation of either Cx43 by CRISPR/Cas9 or Cx43-mediated gap junctional intercellular communication (GJIC) by carbenoxolone treatment triggered rediferentiation of osteoarthritic chondrocytes into a more differentiated state, associated with decreased synthesis of MMPs and proinflammatory factors, and reduced senescence. We have identified causal Cx43-sensitive circuit in chondrocytes that regulates dedifferentiation, redifferentiation and senescence. We propose that chondrocytes undergo chondrocyte-mesenchymal transition where increased Cx43-mediated GJIC during OA facilitates Twist-1 nuclear translocation as a novel mechanism involved in OA progression. These findings support the use of Cx43 as an appropriate therapeutic target to halt OA progression and to promote cartilage regeneration.

Dynamics of plasma membrane surface related to the release of extracellular vesicles by mesenchymal stem cells in culture.

Extracellular vesicles (exosomes and shedding vesicles) released by mesenchymal stem cells (MSCs) are regarded as a storable, cell-free alternative with comparable therapeutic potential to their parent cells. Shedding vesicles originate as bulges on the cell surface but little is known about their turnover or how their formation can be stimulated. We have used atomic force microscopy (AFM) to follow the formation dynamics of bulges in living adipose tissue-derived MSCs. AFM images showed that, in general, MSCs present hundreds of nanosized protrusions on their surface with life spans of 10-20 min. Scanning electron microscopy confirmed those images and showed that bulges are also formed on filamentous processes. Extracellular vesicles deposited on the culture surface have comparable sizes to those of bulges showing up on the cell surface. The amount of protrusions on cells treated with progesterone or PDGF-BB, two treatments that stimulate the secretion of extracellular vesicles in MSCs, was evaluated by AFM. Measurements of the cross-area at 50 nm over the cell surface provided estimates of the amount of protrusions and showed that these values increased with the stimulating treatments. Our study suggests that shedding vesicles constitute a large population of the extracellular vesicle pool.

Cellular characterization of epidermal growth factor-expanded free-floating neurospheres.

Neural stem cells proliferate in liquid culture as cell clusters (neurospheres). This study was undertaken to characterize the epidermal growth factor (EGF)-expanded free-floating neurospheres derived from rat fetal striatum. We examined the ultrastructural and antigenic characteristics of these spheres. They consisted of two cell types, electron-dense and electron-lucent cells. Lucent cells were immunopositive to actin, vimentin, and nestin, whereas dense cells were immunopositive to actin, weakly positive to vimentin, and nestin-negative. Neurospheres contained healthy, apoptotic, and necrotic cells. Healthy cells were attached to each other by adherens junctions. They showed many pseudopodia and occasionally a single cilium. Sphere cells showed phagocytic capability because healthy cells phagocytosed the cell debris derived from dead cells in a particular process that involves the engulfment of dying cells by cell processes from healthy cells. Sphere cells showed a cytoplasmic and a nuclear pool of fibroblast growth factor (FGF) receptors. They expressed E- and N-cadherin, alpha- and beta-catenin, EGF receptor, and a specific subset of FGF receptors. Because sphere cells expressed this factor in the absence of exogenous FGF-2, we propose that they are able to synthesize FGF-2.

Dopaminergic modulation of neuropathic pain: analgesia in rats by a D2-type receptor agonist.

Experimental studies have shown that dopaminergic mechanisms can modulate both nociception and chronic pain perception, but such property is not exploited pharmacologically at the clinical level. We have previously shown that levodopa produces D2-receptor-mediated antiallodynic effects in rats with peripheral mononeuropathy. Here, we test the effects of a D2-type receptor (D2R) agonist, quinpirole, on neuropathic pain in rats. Allodynic responses to cooling and light touch were measured in the hind limbs of rats with chronic constriction injury of one sciatic nerve. Single intraperitoneal injection of quinpirole (1 mg/kg) totally inhibited cold and tactile allodynic responses for over 3 and 48 h, respectively. At that dose, quinpirole had no effect on nocifensive responses to heat. Lumbar intrathecal injection of quinpirole produced short-term inhibition of the responses to cold and tactile stimuli, suggesting that spinal mechanisms may contribute to the antiallodynic activity of quinpirole. Chronic subcutaneous infusion of quinpirole by implanted Alzet pumps (0.025 mg/kg·day) provided a slowly progressing inhibition of cold and tactile allodynic responses, which re-emerged after the pumps were removed. These experiments show the involvement of dopaminergic systems in the modulation of chronic allodynias and provide experimental support for proposing the use of D2R agonists for neuropathic pain relief.

Use of transduced adipose tissue stromal cells as biologic minipumps to deliver levodopa for the treatment of neuropathic pain: possibilities and limitations.

Subarachnoidal grafting of monoamine-producing cells has been used with success to treat chronic pain in animal models. In the search for a source of autologous transplantable cells, capable of delivering neuroactive substances to the cerebrospinal fluid (CSF) to treat pain, we have tested adipose tissue-derived stromal cells (ADSCs) transduced to produce levodopa. Intrathecally grafted ADSCs survive for long term adhered to spinal cord and nerve root meninges. Cultured ADSCs were retrovirally transduced with tyrosine hydroxylase (TH) and/or GTP cyclohydroxylase 1 (GCH1) genes and stably expressed them for at least 6 weeks in culture. Singly transduced cultures did not produce measurable levodopa but doubly transduced or a mixture of singly transduced ADSCs were able to efficiently synthesize and release levodopa. When 0.5-1 x 10(6) TH- and GCH1-expressing ADSCs were intrathecally grafted in rats, elevated levels of levodopa and dopamine metabolites were found in CSF at 3 days, although at lower concentrations than expected. Unexpectedly, no levodopa was measurable in CSF at 6 days. In a rat model of neuropathic pain, intrathecal grafting of doubly transduced cells did not produce antiallodynic effects at 2 or 6 days, even when histological analysis revealed the presence of weak TH-immunoreactive subarachnoidal cell clusters. These results suggested that doubly transduced cells could indeed function as biological minipumps to enhance the dopaminergic neurotransmission at the spinal cord level but transgenes were rapidly silenced after intrathecal grafting. Transgene silencing was mimicked in culture by serum deprivation for 3 days. Serum addition at this point recovered transgene expression in just 6 h, as did, to a smaller degree, dbcAMP or histone deacetylase inhibitors. Transgene expression silencing in serum deprivation conditions was prevented by 5'-terminal IRES sequences. The present study does not discard the use of transduced cells as a strategy to treat chronic pain but shows that controlling transgene silencing in implanted cells needs to be achieved first.