Molecular Mechanisms and Therapeutic Implications of Human Pericyte-like Adipose-Derived Mesenchymal Stem Cells in an In Vitro Model of Diabetic Retinopathy.

The blood-retinal barrier (BRB) is strongly compromised in diabetic retinopathy (DR) due to the detachment of pericytes (PCs) from retinal microvessels, resulting in increased permeability and impairment of the BRB. Western blots, immunofluorescence and ELISA were performed on adipose mesenchymal stem cells (ASCs) and pericyte-like (P)-ASCs by co-cultured human retinal endothelial cells (HRECs) under hyperglycemic conditions (HG), as a model of DR. Our results demonstrated that: (a) platelet-derived growth factor receptor (PDGFR) and its activated form were more highly expressed in monocultured P-ASCs than in ASCs, and this expression increased when co-cultured with HRECs under high glucose conditions (HG); (b) the transcription factor Nrf2 was more expressed in the cytoplasmic fraction of ASCs and in the P-ASC nuclear fraction, under normal glucose and, even more, under HG conditions; (c) cytosolic phospholipase A2 activity and prostaglandin E2 release, stimulated by HG, were significantly reduced in P-ASCs co-cultured with HRECs; (d) HO-1 protein content was significantly higher in HG-P-ASCs/HRECs than P-ASCs/HRECs; and (e) VEGF-A levels in media from HG-co-cultures were reduced in P-ASCs/HRECs with respect to ASCs/HRECs. The data obtained highlighted the potential of autologous differentiated ASCs in future clinical applications based on cell therapy to counteract the damage induced by DR.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Functional Roles of Connexins and Gap Junctions in Osteo-Chondral Cellular Components.

Gap junctions (GJs) formed by connexins (Cxs) play an important role in the intercellular communication within most body tissues. In this paper, we focus on GJs and Cxs present in skeletal tissues. Cx43 is the most expressed connexin, participating in the formation of both GJs for intercellular communication and hemichannels (HCs) for communication with the external environment. Through GJs in long dendritic-like cytoplasmic processes, osteocytes embedded in deep lacunae are able to form a functional syncytium not only with neighboring osteocytes but also with bone cells located at the bone surface, despite the surrounding mineralized matrix. The functional syncytium allows a coordinated cell activity through the wide propagation of calcium waves, nutrients and anabolic and/or catabolic factors. Acting as mechanosensors, osteocytes are able to transduce mechanical stimuli into biological signals that spread through the syncytium to orchestrate bone remodeling. The fundamental role of Cxs and GJs is confirmed by a plethora of investigations that have highlighted how up- and downregulation of Cxs and GJs critically influence skeletal development and cartilage functions. A better knowledge of GJ and Cx mechanisms in physiological and pathological conditions might help in developing therapeutic approaches aimed at the treatment of human skeletal system disorders.

Protective Effects of Human Pericyte-like Adipose-Derived Mesenchymal Stem Cells on Human Retinal Endothelial Cells in an In Vitro Model of Diabetic Retinopathy: Evidence for Autologous Cell Therapy.

Diabetic retinopathy (DR) is characterized by morphologic and metabolic alterations in endothelial cells (ECs) and pericytes (PCs) of the blood-retinal barrier (BRB). The loss of interendothelial junctions, increased vascular permeability, microaneurysms, and finally, EC detachment are the main features of DR. In this scenario, a pivotal role is played by the extensive loss of PCs. Based on previous results, the aim of this study was to assess possible beneficial effects exerted by adipose mesenchymal stem cells (ASCs) and their pericyte-like differentiated phenotype (P-ASCs) on human retinal endothelial cells (HRECs) in high glucose conditions (25 mM glucose, HG). P-ASCs were more able to preserve BRB integrity than ASCs in terms of (a) increased transendothelial electrical resistance (TEER); (b) increased expression of adherens junction and tight junction proteins (VE-cadherin and ZO-1); (c) reduction in mRNA levels of inflammatory cytokines TNF-α, IL-1ß, and MMP-9; (d) reduction in the angiogenic factor VEGF and in fibrotic TGF-ß1. Moreover, P-ASCs counteracted the HG-induced activation of the pro-inflammatory phospho-ERK1/2/phospho-cPLA2/COX-2 pathway. Finally, crosstalk between HRECs and ASCs or P-ASCs based on the PDGF-B/PDGFR-ß axis at the mRNA level is described herein. Thus, P-ASCs might be considered valuable candidates for therapeutic approaches aimed at countering BRB disruption in DR.

Adult stem cell niches for tissue homeostasis.

Adult stem cells are fundamental to maintain tissue homeostasis, growth, and regeneration. They reside in specialized environments called niches. Following activating signals, they proliferate and differentiate into functional cells that are able to preserve tissue physiology, either to guarantee normal turnover or to counteract tissue damage caused by injury or disease. Multiple interactions occur within the niche between stem cell-intrinsic factors, supporting cells, the extracellular matrix, and signaling pathways. Altogether, these interactions govern cell fate, preserving the stem cell pool, and regulating stem cell proliferation and differentiation. Based on their response to body needs, tissues can be largely classified into three main categories: tissues that even in normal conditions are characterized by an impressive turnover to replace rapidly exhausting cells (blood, epidermis, or intestinal epithelium); tissues that normally require only a basal cell replacement, though able to efficiently respond to increased tissue needs, injury, or disease (skeletal muscle); tissues that are equipped with less powerful stem cell niches, whose repairing ability is not able to overcome severe damage (heart or nervous tissue). The purpose of this review is to describe the main characteristics of stem cell niches in these different tissues, highlighting the various components influencing stem cell activity. Although much has been done, more work is needed to further increase our knowledge of niche interactions. This would be important not only to shed light on this fundamental chapter of human physiology but also to help the development of cell-based strategies for clinical therapeutic applications, especially when other approaches fail.

Lactate Rewrites the Metabolic Reprogramming of Uveal Melanoma Cells and Induces Quiescence Phenotype.

Uveal melanoma (UM), the most common primary intraocular cancer in adults, is among the tumors with poorer prognosis. Recently, the role of the oncometabolite lactate has become attractive due to its role as hydroxycarboxylic acid receptor 1 (HCAR1) activator, as an epigenetic modulator inducing lysine residues lactylation and, of course, as a glycolysis end-product, bridging the gap between glycolysis and oxidative phosphorylation. The aim of the present study was to dissect in UM cell line (92.1) the role of lactate as either a metabolite or a signaling molecule, using the known modulators of HCAR1 and of lactate transporters. Our results show that lactate (20 mM) resulted in a significant decrease in cell proliferation and migration, acting and switching cell metabolism toward oxidative phosphorylation. These results were coupled with increased euchromatin content and quiescence in UM cells. We further showed, in a clinical setting, that an increase in lactate transporters MCT4 and HCAR1 is associated with a spindle-shape histological type in UM. In conclusion, our results suggest that lactate metabolism may serve as a prognostic marker of UM progression and may be exploited as a potential therapeutic target.

The Role of Epigenetics in Neuroinflammatory-Driven Diseases.

Neurodegenerative disorders are characterized by the progressive loss of central and/or peripheral nervous system neurons. Within this context, neuroinflammation comes up as one of the main factors linked to neurodegeneration progression. In fact, neuroinflammation has been recognized as an outstanding factor for Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and multiple sclerosis (MS). Interestingly, neuroinflammatory diseases are characterized by dramatic changes in the epigenetic profile, which might provide novel prognostic and therapeutic factors towards neuroinflammatory treatment. Deep changes in DNA and histone methylation, along with histone acetylation and altered non-coding RNA expression, have been reported at the onset of inflammatory diseases. The aim of this work is to review the current knowledge on this field.

Ghrelin peptide improves glial conditioned medium effects on neuronal differentiation of human adipose mesenchymal stem cells.

The influences of ghrelin on neural differentiation of adipose-derived mesenchymal stem cells (ASCs) were investigated in this study. The expression of typical neuronal markers, such as protein gene product 9.5 (PGP9.5) and Microtubule Associated Protein 2 (MAP2), as well as glial Fibrillary Acid Protein (GFAP) as a glial marker was evaluated in ASCs in different conditions. In particular, 2 µM ghrelin was added to control ASCs and to ASCs undergoing neural differentiation. For this purpose, ASCs were cultured in Conditioned Media obtained from Olfactory Ensheathing cells (OEC-CM) or from Schwann cells (SC-CM). Data on marker expression were gathered after 1 and 7 days of culture by fluorescence immunocytochemistry and flow cytometry. Results show that only weak effects were induced by the addition of only ghrelin. Instead, dynamic ghrelin-induced modifications were detected on the increased marker expression elicited by glial conditioned media. In fact, the combination of ghrelin and conditioned media consistently induced a further increase of PGP9.5 and MAP2 expression, especially after 7 days of treatment. The combination of ghrelin with SC-CM produced the most evident effects. Weak or no modifications were found on conditioned medium-induced GFAP increases. Observations on the ghrelin receptor indicate that its expression in control ASCs, virtually unchanged by the addition of only ghrelin, was considerably increased by CM treatment. These increases were enhanced by combining ghrelin and CM treatment, especially at 7 days. Overall, it can be assumed that ghrelin favors a neuronal rather than a glial ASC differentiation.

Effects of High Glucose Concentration on Pericyte-Like Differentiated Human Adipose-Derived Mesenchymal Stem Cells.

A pericyte-like differentiation of human adipose-derived mesenchymal stem cells (ASCs) was tested in in vitro experiments for possible therapeutic applications in cases of diabetic retinopathy (DR) to replace irreversibly lost pericytes. For this purpose, pericyte-like ASCs were obtained after their growth in a specific pericyte medium. They were then cultured in high glucose conditions to mimic the altered microenvironment of a diabetic eye. Several parameters were monitored, especially those particularly affected by disease progression: cell proliferation, viability and migration ability; reactive oxygen species (ROS) production; inflammation-related cytokines and angiogenic factors. Overall, encouraging results were obtained. In fact, even after glucose addition, ASCs pre-cultured in the pericyte medium (pmASCs) showed high proliferation rate, viability and migration ability. A considerable increase in mRNA expression levels of the anti-inflammatory cytokines transforming growth factor-ß1 (TGF-ß1) and interleukin-10 (IL-10) was observed, associated with reduction in ROS production, and mRNA expression of pro-inflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), and angiogenic factors. Finally, a pmASC-induced better organization of tube-like formation by retinal endothelial cells was observed in three-dimensional co-culture. The pericyte-like ASCs obtained in these experiments represent a valuable tool for the treatment of retinal damages occurring in diabetic patients.

Connexin expression decreases during adipogenic differentiation of human adipose-derived mesenchymal stem cells.

Adipose-derived stem cells (ASCs) represent a valuable tool for regenerative medicine being able to differentiate toward several cell lines, such as adipocytes, chondrocytes and osteocytes. During ASC adipogenic differentiation, changes in connexin (Cx) expression were evaluated in the present study. Three different Cxs were investigated: Cx43, Cx32 and Cx31.9. Cx43 is the most abundant in human tissues, Cx32 is prevalently found in nervous tissue and Cx31.9 is found at the myocardial level. Human ASCs undergoing adipogenic differentiation were isolated from raw lipoaspirate and characterized as mesenchymal stem cells. After multiple days of culture (1, 7, 14, 21 and 28 days), adipogenic differentiation was assessed by Oil Red O staining and Acetyl-CoA carboxylase (ACC) levels by western blotting. Cx expression was evaluated by western blotting at the same time points. In treated ASCs, lipidic vacuoles were detected from day 7 of treatment. Their number and size progressively increased over the entire period of observation. A parallel increase of ACC expression was also found. Lower levels of Cx expression were detected during adipogenic differentiation. Such decreases were particularly evident for Cx32, already after the first day of treatment. Cx31.9 and Cx43 also decreased, but starting from day 7. Our results suggest that ASCs may initially be equipped with a variety of Cxs, which is not surprising assuming their multipotential differentiation ability. Although some Cxs may be selectively enhanced depending on specific induction strategies toward different tissues, they seem markedly downregulated during adipogenic differentiation.

Functional role of mesenchymal stem cells in the treatment of chronic neurodegenerative diseases.

Mesenchymal stem cells (MSCs) can differentiate into not only cells of mesodermal lineages, but also into endodermal and ectodermal derived elements, including neurons and glial cells. For this reason, MSCs have been extensively investigated to develop cell-based therapeutic strategies, especially in pathologies whose pharmacological treatments give poor results, if any. As in the case of irreversible neurological disorders characterized by progressive neuronal death, in which behavioral and cognitive functions of patients inexorably decline as the disease progresses. In this review, we focus on the possible functional role exerted by MSCs in the treatment of some disabling neurodegenerative disorders such as Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease, and Parkinson's Disease. Investigations have been mainly performed in vitro and in animal models by using MSCs generally originated from umbilical cord, bone marrow, or adipose tissue. Positive results obtained have prompted several clinical trials, the number of which is progressively increasing worldwide. To date, many of them have been primarily addressed to verify the safety of the procedures but some improvements have already been reported, fortunately. Although the exact mechanisms of MSC-induced beneficial activities are not entirely defined, they include neurogenesis and angiogenesis stimulation, antiapoptotic, immunomodulatory, and anti-inflammatory actions. Most effects would be exerted through their paracrine expression of neurotrophic factors and cytokines, mainly delivered at damaged regions, given the innate propensity of MSCs to home to injured sites. Hopefully, in the near future more efficacious cell-replacement therapies will be developed to substantially restore disease-disrupted brain circuitry.

Neural differentiation of human adipose-derived mesenchymal stem cells induced by glial cell conditioned media.

Adipose-derived mesenchymal stem cells (ASCs) may transdifferentiate into cells belonging to mesodermal, endodermal, and ectodermal lineages. The aim of this study was to verify whether a neural differentiation of ASCs could be induced by a conditioned medium (CM) obtained from cultures of olfactory ensheathing cells (OECs) or Schwann cells (SCs). ASCs were isolated from the stromal vascular fraction of adipose tissue and expanded for 2-3 passages. They were then cultured in OEC-CM or SC-CM for 24 hr or 7 days. At each stage, the cells were tested by immunocytochemistry and flow cytometer analysis to evaluate the expression of typical neural markers such as Nestin, PGP 9.5, MAP2, Synapsin I, and GFAP. Results show that both conditioned media induced similar positive effects, as all tested markers were overexpressed, especially at day 7. Overall, an evident trend toward neuronal or glial differentiation was not clearly detectable in many cases. Nevertheless, a higher tendency toward a neuronal phenotype was recognized for OEC-CM (considering MAP2 increases). On the other hand, SC-CM would be responsible for a more marked glial induction (considering GFAP increases). These findings confirm that environmental features can induce ASCs toward a neural differentiation, either as neuronal or glial elements. Rather than supplementing the culture medium by adding chemical agents, a "more physiological" condition was obtained here by means of soluble factors (cytokines/growth factors) likely released by glial cells. This culture strategy may provide valuable information in the development of cell-based therapeutic approaches for pathologies affecting the central/peripheral nervous system.

Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation.

The Low-Affinity Nerve Growth Factor Receptor (LNGFR), also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271+ and CD271- mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271+ and CD271- mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271+, CD271- mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271+ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271+ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate.

A Novel Pumping Principle for a Total Artificial Heart.

OBJECTIVE: Total artificial hearts (TAH) serve as a temporary treatment for severe biventricular heart failure. The limited durability and complication rates of current devices hamper long-term cardiac replacement. The aim of this study was to assess the feasibility of a novel valveless pumping principle for a durable pulsatile TAH (ShuttlePump). METHODS: The pump features a rotating and linearly shuttling piston within a cylindrical housing with two in- and outlets. With a single moving piston, the ShuttlePump delivers pulsatile flow to both systemic and pulmonary circulation. The pump and actuation system were designed iteratively based on analytical and in silico methods, utilizing finite element methods (FEM) and computational fluid dynamics (CFD). Pump characteristics were evaluated experimentally in a mock circulation loop mimicking the cardiovascular system, while hemocompatibility-related parameters were calculated numerically. RESULTS: Pump characteristics cover the entire required operating range for a TAH, providing 2.5-9 L/min of flow rate against 50-160 mmHg arterial pressures at stroke frequencies of 1.5-5 Hz while balancing left and right atrial pressures. FEM analysis showed mean overall copper losses of 8.84 W, resulting in a local maximum blood temperature rise of <2 K. The CFD results of the normalized index of hemolysis were 3.57 mg/100 L, and 95% of the pump's blood volume was exchanged after 1.42 s. CONCLUSION AND SIGNIFICANCE: This study indicates the feasibility of a novel pumping system for a TAH with numerical and experimental results substantiating further development of the ShuttlePump.

Multiobjective Optimization of Rotodynamic Blood Pumps: The Use Case of a Cavopulmonary Assist Device.

Comprehensive optimization of rotodynamic blood pumps (RBPs) requires the consideration of three partially conflicting objectives: size, hemocompatibility, and motor efficiency. Optimizing these individual objectives independently, the potential of multiobjective optimizations often remains untapped. This study aimed at the multiobjective optimization of an RBP for cavopulmonary support accounting for all three objectives simultaneously. Hydraulic and electromagnetic design spaces were characterized using computational fluid dynamics and computational electromagnetics, respectively. Design variables included secondary flow gap widths, impeller diameters, and stator heights. The size objective encompassed the RBP widths and heights, the hemocompatibility objective was a weighted composite measure of well-established metrics, and the motor objective was determined by motor losses. Multiobjective optimization was performed through Pareto analysis. 81 designs were considered, and 21 Pareto-optimal designs were identified. The Pareto analysis indicated that hemocompatibility performance could be improved by 72.4% with a concomitant 1.5% reduction in the baseline pump volume. This, however, entailed an increase in motor losses by 0.2 W, while still meeting design requirements, with maximum local temperature rises remaining below 0.4 K. The multiobjective optimization led to a Pareto front, demonstrating the feasibility to improve hemocompatibility at reduced pump volume, however, at the cost of a diminished yet still acceptable motor performance.

Microglia and glioblastoma heterocellular interplay sustains tumour growth and proliferation as an off-target effect of radiotherapy.

Glioblastoma (GBM), a WHO grade IV glioma, is a malignant primary brain tumour for which combination of surgery, chemotherapy and radiotherapy is the first-line approach despite adverse effects. Tumour microenvironment (TME) is characterized by an interplay of cells and soluble factors holding a critical role in neoplastic development. Significant pathophysiological changes have been found in GBM TME, such as glia activation and oxidative stress. Microglia play a crucial role in favouring GBM growth, representing target cells of immune escape mechanisms. Our study aims at analysing radiation-induced effects in modulating intercellular communication and identifying the basis of protective mechanisms in radiation-naïve GBM cells. Tumour cells were treated with conditioned media (CM) derived from 0, 2 or 15 Gy irradiated GBM cells or 0, 2 or 15 Gy irradiated human microglia. We demonstrated that irradiated microglia promote an increase of GBM cell lines proliferation through paracrine signalling. On the contrary, irradiated GBM-derived CM affect viability, triggering cell death mechanisms. In addition, we investigated whether these processes involve mitochondrial mass, fitness and oxidative phosphorylation and how GBM cells respond at these induced alterations. Our study suggests that off-target radiotherapy modulates microglia to support GBM proliferation and induce metabolic modifications.

Differentiation of human adipose stem cells into neural phenotype by neuroblastoma- or olfactory ensheathing cells-conditioned medium.

Olfactory ensheathing cells (OECs) are known to be capable of continuous neurogenesis throughout lifetime and are a source of multiple trophic factors important in central nervous system regeneration. B104 neuroblastoma cells are recognized to induce differentiation of neural stem cells into oligodendrocyte precursor cells. Therefore, the aim of this study was to verify if conditioned medium (CM) obtained from OECs or B104 cells was capable of inducing differentiation of adipose tissue-derived mesenchymal stem cells (AT-MSCs) to a neuronal phenotype. In order to this goal, immunocytochemical procedures and flow cytometry analysis were used and some neural markers, as nestin, protein gene product 9.5 (PGP 9.5), microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), and neuron cell surface antigen (A2B5) were examined 24 h and 7 days after the treatment. The results showed that both OECs- or B104-CM treated AT-MSCs express markers of progenitor and mature neurons (nestin, PGP 9.5 and MAP2) in time-dependent manner, display morphological features resembling neuronal cells, and result negative for GFAP and A2B5, astrocyte and oligodendrocyte markers, respectively. This study demonstrated that AT-MSCs can be influenced by the environment, indicating that these cells can respond to environmental cues also versus a neuronal phenotype.

PI3K p110γ overexpression in idiopathic pulmonary fibrosis lung tissue and fibroblast cells: in vitro effects of its inhibition.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease whose molecular pathogenesis remains unclear. In a recent paper, we demonstrated a key role for the PI3K pathway in both proliferation and differentiation into myofibroblasts of normal human lung fibroblasts treated with TGF-ß. In this research, we assessed the expression of class I PI3K p110 isoforms in IPF lung tissue as well as in tissue-derived fibroblast cell lines. Moreover, we investigated the in vitro effects of the selective inhibition of p110 isoforms on IPF fibroblast proliferation and fibrogenic activity. IHC was performed on normal and IPF lung tissue. Expression levels of PI3K p110 isoforms were evaluated by western blot and flow cytometry analysis. Fibroblast cell lines were established from both normal and IPF tissue and the effects of selective pharmacological inhibition as well as specific gene silencing by small interfering RNAs were studied in vitro. No significant differences between normal and IPF tissue/tissue-derived fibroblasts were observed for the expression of PI3K p110 α, ß and δ isoforms whereas p110γ was more greatly expressed in both IPF lung homogenates and ex vivo fibroblast cell lines. Myofibroblasts and bronchiolar basal cells in IPF lungs exhibited strong immunoreactivity for p110γ. Positive staining for the markers of proliferation proliferating cell nuclear antigen and cyclin D1 was also shown in cells of fibrolastic foci. Furthermore, both p110γ pharmacological inhibition and gene silencing were able to significantly inhibit proliferation rate as well as α-SMA expression in IPF fibroblasts. Our data suggest that PI3K p110γ isoform may have an important role in the etio-pathology of IPF and can be a specific pharmacological target.

Decrease of apoptosis markers during adipogenic differentiation of mesenchymal stem cells from human adipose tissue.

Although the proliferation and differentiation of mesenchymal stem cells (MSCs) from adipose tissue (AT) have been widely studied, relatively little information is available on the underlying mechanism of apoptosis during the adipogenic differentiation. Thus, the aim of this study was to analyze how the expression of some apoptotic markers is affected by in vitro expansion during adipogenic differentiation of AT-MSCs. The cultures incubated or not with adipogenic medium were investigated by Western blot at 7, 14, 21, and 28 days for the production of p53, AKT, pAKT, Bax, PDCD4 and PTEN. MSCs were recognized for their immunoreactivity to MSC-specific cell types markers by immunocytochemical procedure. The effectiveness of adipogenic differentiation was assessed by staining with Sudan III and examination of adipogenic markers expression, such as PPAR-γ and FABP, at different time points by Western blot. The adipogenic differentiation medium led to the appearance, after 7 days, of larger rounded cells presenting numerous vacuoles containing lipids in which it was evident a red-orange staining, that increased in size in a time-dependent manner, parallel to an increase of the levels of expression of PPAR-γ and FABP. More than 50 % of human MSCs were fully differentiated into adipocytes within the four-week induction period. The results showed that during adipogenic differentiation of AT-MSCs the PI3K/AKT signaling pathway is activated and that p53, PTEN, PDCD4, and Bax proteins are down-regulated in time-dependent manner. Our data provide new information on the behavior of some apoptotic markers during adipogenic differentiation of AT-MSCs to apply for tissues repair and regeneration.

Adipose-Derived Mesenchymal Stromal Cells: A Tool for Bone and Cartilage Repair.

The osteogenic and chondrogenic differentiation ability of adipose-derived mesenchymal stromal cells (ASCs) and their potential therapeutic applications in bone and cartilage defects are reported in this review. This becomes particularly important when these disorders can only be poorly treated by conventional therapeutic approaches, and tissue engineering may represent a valuable alternative. Being of mesodermal origin, ASCs can be easily induced to differentiate into chondrocyte-like and osteocyte-like elements and used to repair damaged tissues. Moreover, they can be easily harvested and used for autologous implantation. A plethora of ASC-based strategies are being developed worldwide: they include the transplantation of freshly harvested cells, in vitro expanded cells or predifferentiated cells. Moreover, improving their positive effects, ASCs can be implanted in combination with several types of scaffolds that ensure the correct cell positioning; support cell viability, proliferation and migration; and may contribute to their osteogenic or chondrogenic differentiation. Examples of these strategies are described here, showing the enormous therapeutic potential of ASCs in this field. For safety and regulatory issues, most investigations are still at the experimental stage and carried out in vitro and in animal models. Clinical applications have, however, been reported with promising results and no serious adverse effects.