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Mesenchymal stem cells (MSCs) are multipotent cells, originally derived from the embryonic mesenchyme, and able to differentiate into connective tissues such as bone, fat, cartilage, tendon, and muscle. Furthermore, MSCs derived from adipose tissue ADSC (Adipose derived Stem Cells) show a great potential for degenerative diseases treatment. In this study, we designed a series of experiments based on real-time rt-QPCR to validated a commercially available kit able to explore changes in gene expression under osteogenic, adipogenic and chondrogenic differentiation of human ADSC. Initially, we selected a better indicators of trilineage differentiation by using third passages of cultured ADSC from stromal vascular fraction (SVF) isolated from fresh adipose tissue by enzymatic digestion. On the basis of statistically significant results ACAN, FABP4A and Col11a1 were chosen as indicators of chondrogenic, adipogenic and osteogenic differentiation respectively. An in-vitro aging analysis was then performed to evaluate the ADSC passage with the highest differentiation potential. Total RNA extraction from induced differentiation and controls ADSC from passage 2-6 and relative quantifications of mRNA expression of selected genes were performed according to rt-PCR kits tested. The chondrogenic differentiation test showed equivalent ∆∆Ct values for ACAN detection for cell passages ranging from P3 to P6, proving that they can be considered as equivalent samples for differentiation assays evaluation. For what concerns adipogenic differentiation and FABP4 detection, similar results were observed in all the cell passages tested; on the contrary only passage P6 showed suitable ∆∆Ct values for Col11a1 detection for osteogenic differentiation evaluation. In conclusion, we have validated a suitable real-time rt-QPCR protocol for osteogenic, chondrogenic and adipogenic ADSC differentiation ability evaluation in-vitro.

Some bacterial pathogens can manipulate the angiogenic response, suppressing or inducing it for their own ends. In humans, Bartonella henselae is associated with cat-scratch disease and vasculoproliferative disorders such as bacillary angiomatosis and bacillary peliosis. Although endothelial cells (ECs) support the pathogenesis of B. henselae, the mechanisms by which B. henselae induces EC activation are not completely clear, as well as the possible contributions of other cells recruited at the site of infection. Mesenchymal stromal cells (MSCs) are endowed with angiogenic potential and play a dual role in infections, exerting antimicrobial properties but also acting as a shelter for pathogens. Here, we delved into the role of MSCs as a reservoir of B. henselae and modulator of EC functions. B. henselae readily infected MSCs and survived in perinuclearly bound vacuoles for up to 8 days. Infection enhanced MSC proliferation and the expression of epidermal growth factor receptor (EGFR), Toll-like receptor 2 (TLR2), and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), proteins that are involved in bacterial internalization and cytokine production. Secretome analysis revealed that infected MSCs secreted higher levels of the proangiogenic factors vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF-7), matrix metallopeptidase 9 (MMP-9), placental growth factor (PIGF), serpin E1, thrombospondin 1 (TSP-1), urokinase-type plasminogen activator (uPA), interleukin 6 (IL-6), platelet-derived growth factor D (PDGF-D), chemokine ligand 5 (CCL5), and C-X-C motif chemokine ligand 8 (CXCL8). Supernatants from B. henselae-infected MSCs increased the susceptibility of ECs to B. henselae infection and enhanced EC proliferation, invasion, and reorganization in tube-like structures. Altogether, these results indicate MSCs as a still underestimated niche for persistent B. henselae infection and reveal MSC-EC cross talk that may contribute to exacerbate bacterium-induced angiogenesis and granuloma formation.

Background B henselae is in humans the aetiologic agent of cat-scratch disease and of the vasculoproliferative disorders bacillary angiomatosis and bacillary peliosis. Although endothelial cells are crucial in the pathogenesis other cell types function as reservoir and contribute to pathological angiogenesis. Among them, mesenchymal stromal cells (MSCs) can sense pathogens and mount an appropriate cytokine/chemokine response through different Pattern Recognition Receptors (PRRs). MSCs exert direct antimicrobial effector function but may also shelter bacteria such as M. tuberculosis. Methods Adipose-derived MSCs were infected with B. henselae and analyzed for bacterial persistence by gentamicin protection assay, immunohistochemistry and immunofluorescence. Involvement of PRRs in bacterial infection was evaluated through gene and protein expression analysis. The effect of infection on MSC proliferation, apoptosis and release of soluble factors was assessed. The role of infected-MSC conditioned medium in promoting Bartonella infection of endothelial cells and angiogenesis was demonstrated using respectively gentamicin protection assay and different pro-angiogenic assays including spheroid, wound healing and morphogenesis. Results B. henselae can readily infect MSCs and survive in perinuclear bound vacuoles for up to 8 days. Bartonella infection stimulates MSC proliferation and upregulation of EGFR and of the two pattern recognition receptors (PRRs) TLR2 and NOD1. Specific inhibition of EGFR reduces bacterial internalization and treatment with anti-TLR2 neutralizing antibody or EGFR/NOD1 inhibitors significantly downmodulates CXCL8 production. Secretome analysis shows that, in addition to CXCL8, infected MSCs secrete higher levels of the proangiogenic factors VEGF, FGF-7, MMP-9, PIGF, serpin E1, TSP-1, uPA, IL-6, CCL5 and PDGF-D. Importantly, supernatants from B. henselae-infected MSCs increase the susceptibility of ECs to B. henselae infection while enhancing EC proangiogenic potential. Conclusions Altogether, these findings indicate that MSCs constitute a novel niche for B. henselae, which favors the persistence of vascular proliferative disorders.

Mesenchymal stromal cells (MSCs) exert immunosuppressive effects on immune cells including dendritic cells (DCs). However, many details of the bidirectional interaction of MSCs with DCs are still unsolved and information on key molecules by which DCs can modulate MSC functions is limited. Here, we report that osteopontin (OPN), a cytokine involved in homeostatic and pathophysiologic responses, is constitutively expressed by DCs and regulated in the DC/MSC cocultures depending on the activation state of MSCs. Resting MSCs promoted OPN production, whereas the production of OPN was suppressed when MSCs were activated by proinflammatory cytokines (i.e., TNF-α, IL-6, and IL-1β). OPN induction required cell-to-cell contact, mediated at least in part, by β1 integrin (CD29). Conversely, activated MSCs inhibited the release of OPN via the production of soluble factors with a major role played by Prostaglandin E2 (PGE2). Accordingly, pretreatment with indomethacin significantly abrogated the MSC-mediated suppression of OPN while the direct addition of exogenous PGE2 inhibited OPN production by DCs. Furthermore, DC-conditioned medium promoted osteogenic differentiation of MSCs with a concomitant inhibition of adipogenesis. These effects were paralleled by the repression of the adipogenic markers PPARγ, adiponectin, and FABP4, and induction of the osteogenic markers alkaline phosphatase, RUNX2, and of the bone-anabolic chemokine CCL5. Notably, blocking OPN activity with RGD peptides or with an antibody against CD29, one of the OPN receptors, prevented the effects of DC-conditioned medium on MSC differentiation and CCL5 induction. Because MSCs have a key role in maintenance of bone marrow (BM) hematopoietic stem cell niche through reciprocal regulation with immune cells, we investigated the possible MSC/DC interaction in human BM by immunohistochemistry. Although DCs (CD1c+) are a small percentage of BM cells, we demonstrated colocalization of CD271+ MSCs with CD1c+ DCs in normal and myelodysplastic BM. OPN reactivity was observed in occasional CD1c+ cells in the proximity of CD271+ MSCs. Altogether, these results candidate OPN as a signal modulated by MSCs according to their activation status and involved in DC regulation of MSC differentiation.

Background The development of dermal scaffolds is of major interest in reconstructive surgery. Human Acellular Dermal Matrices (HADMs) provides biomechanical support and elicits new tissue formation. The use of allograft dermis is limited by its immunogenic characteristics. Our research group has focused on the use of human alloplastic glycerolized reticular dermis. Objective The dermal grafts were subjected to two different decellularization protocols in parallel, in order to compare the efficacy in the elimination of residual DNA. Methods It was compared the incubation of the dermis in NaOH (0.06 N) and in the standard culture medium "Dulbecco Modified Eagle Medium" (DMEM). The samples were incubated in the specific medium for 8 weeks. The newly developed real-time TaqMan® MGB-PCR assay was applied for both the detection and absolute quantification of residual DNA. Results It was observed that the level of residual DNA decreased until time T3 and remained constant until time T8. Moreover, there was no statistical difference between treatment with DMEM or NaOH 0.06 N as to the amount of residual DNA. Conclusions Decellularization methods, DMEM or NaOH 0.06 N do not affect DNA recovery. The proposed approach offers an alternative method to quantify residual DNA in HADM samples.

Osteoarthritis is characterized by loss of articular cartilage also due to reduced chondrogenic activity of mesenchymal stem cells (MSCs) from patients. Adipose tissue is an attractive source of MSCs (ATD-MSCs), representing an effective tool for reparative medicine, particularly for treatment of osteoarthritis, due to their chondrogenic and osteogenic differentiation capability. The treatment of symptomatic knee arthritis with ATD-MSCs proved effective with a single infusion, but multiple infusions could be also more efficacious. Here we studied some crucial aspects of adipose tissue banking procedures, evaluating ATD-MSCs viability, and differentiation capability after cryopreservation, to guarantee the quality of the tissue for multiple infusions. We reported that the presence of local anesthetic during lipoaspiration negatively affects cell viability of cryopreserved adipose tissue and cell growth of ATD-MSCs in culture. We observed that DMSO guarantees a faster growth of ATD-MSCs in culture than trehalose. At last, ATD-MSCs derived from fresh and cryopreserved samples at −80°C and −196°C showed viability and differentiation ability comparable to fresh samples. These data indicate that cryopreservation of adipose tissue at −80°C and −196°C is equivalent and preserves the content of ATD-MSCs in Stromal Vascular Fraction (SVF), guaranteeing the differentiation ability of ATD-MSCs.

Vitamin D receptor (VDR) mediates many genomic and non-genomic effects of vitamin D. Recently, the mitochondrial effects of vitamin D have been characterized in many cell types. In this article, we investigated the importance of VDR not only in mitochondrial activity and integrity but also in cell health. The silencing of the receptor in different healthy, non-transformed, and cancer cells initially decreased cell growth and modulated the cell cycle. We demonstrated that, in silenced cells, the increased respiratory activity was associated with elevated reactive oxygen species (ROS) production. In the long run, the absence of the receptor caused impairment of mitochondrial integrity and, finally, cell death. Our data reveal that VDR plays a central role in protecting cells from excessive respiration and production of ROS that leads to cell damage. Because we confirmed our observations in different models of both normal and cancer cells, we conclude that VDR is essential for the health of human tissues.

Autologous epidermal cell cultures (CEA) represent a possibility to treat extensive burn lesions, since they allow a significative surface expansion which cannot be achieved with other surgical techniques based on autologous grafting. Moreover currently available CEA preparations are difficult to handle and their take rate is unpredictable. This study aimed at producing and evaluating a new cutaneous biosubstitute made up of alloplastic acellular glycerolized dermis (AAGD) and CEA to overcome these difficulties. A procedure that maintained an intact basement membrane was developed, so as to promote adhesion and growth of CEA on AAGD. Keratinocytes were seeded onto AAGD and cultured up to 21 days. Viability tests and immunohistochemical analysis with specific markers were carried out at 7, 14, and 21 days, to evaluate keratinocyte adhesion, growth, and maturation. Our results support the hypothesis that this newly formed skin substitute could allow its permanent engraftment in clinical application.

Cell viability assessment in allograft skin is an essential step to ensure a supply of good quality allograft skin for clinical repair of wounds. It is widely recognised that ‘take’ of allografts is strongly influenced grafted by tissue viability. The aim of this study was to set-up storage protocols that maintain high viability of the allograft after harvest, treatment and storage. In this study, the viability of post-mortem allografts (n=350) harvested from 35 different donors, was investigated using the MTT salt assay. The conditions of preparation and storage of the allograft included: 1. Fresh skin samples (about 12, 30, and 60 h after harvesting). 2. The same specimens (stored at 4 and 37 °C) tested for at least 1 month. 3. Samples after cryopreservation and thawing. 4. Thawed specimens tested daily for at least 6 days. Parallel histomorphological analysis performed, under each of these conditions, showed a correlation between changes in structure and changes in viability as measured by the MTT quantitative assay. The viability index (VI) of skin is expressed as the ratio between the optical density (O.D.) produced in the MTT assay by the skin sample and its weight in grams. The percentage viability index is the ratio of the VI of the fresh sample (considered as 100% viability) and the value of specimens from the same harvest batch after storage or cryopreservation. The results indicated that samples tested within 12–30 h from harvesting have an average viability index of about 75 with little variation. Samples tested within 60 h have an average viability index of 40, showing a viability decrease of about 50%. A protocol to treat skin within a maximum of 30 h was, therefore, set-up. The data suggested that skin stored at 37 °C, undergoes a viability increase during the first 2 days after harvesting. However, the viability under these conditions then decreased very quickly. After 6 days of preservation at this temperature the samples were no longer viable (PVI = 0). The tissue structure started to become damaged after 3 days. On the other hand, skin stored at 4 °C, showed a very slow viability decrease. After 15 days, viability was still almost 25% of the fresh sample. The tissue architecture showed no signs of damage under these conditions until day 7 from harvesting. MTT analysis was performed on the specimens cryopreserved with DMSO at 10%. These measurements were compared to viability assessment of the same fresh skin samples (considered as 100%) that were analysed within 30 h from harvesting. The average PVI of thawed skin was 54% of the fresh sample. This result demonstrates that the viability of cryopreserved skin is comparable to the viability of fresh skin stored at 4 °C for 4 days. The PVI of thawed skin samples decreased dramatically within 24 h, and had reached 0% within 6 days.

Genetic experiments established that p63 is crucial for the development and maintenance of pluri-stratified epithelia and KLF4 for the barrier function of the skin. KLF4 is one of the factors that reprogram differentiated cells to iPS. We investigated the relationship between p63 and KLF4 using RNA interference, overexpression, chromatin immunoprecipitation and transient transfections with reporter constructs. We find that p63 directly represses KLF4 in normal keratinocytes (KCs) by binding to upstream promoter sites. Unlike p63, KLF4 levels are high in the upper layers of human skin and increase upon differentiation of KCs in vitro. In HaCaT KCs, which harbor two mutant alleles of p53, inactivation of p63 and of mutant p53 leads to KLF4 repression. p63 and p53 mutants are bound to sites in the KLF4 core promoter. Importantly, expression of the H179Y and R282Q p53 mutants in primary KCs is sufficient to activate endogenous KLF4. Finally, immunohistochemical analysis of tissue arrays confirms increased coexpression of KLF4 and mutant p53 in squamous cell carcinomas. Our data indicate that suppression of KLF4 is part of the growth-promoting strategy of p63 in the lower layers of normal epidermis, and that tumor-predisposing p53 mutations hijack p63 to a different location on the promoter, turning it into an activator of this reprogramming factor.

C/EBPs are a family of B-Zip transcription factors -TFs- involved in the regulation of differentiation in several tissues. The two most studied members -C/EBPα and C/EBPβ- play important roles in skin homeostasis and their ablation reveals cells with stem cells signatures. Much less is known about C/EBPδ which is highly expressed in the granular layer of interfollicular epidermis and is a direct target of p63, the master regular of multilayered epithelia. We identified C/EBPδ target genes in human primary keratinocytes by ChIP on chip and profiling of cells functionally inactivated with siRNA. Categorization suggests a role in differentiation and control of cell-cycle, particularly of G2/M genes. Among positively controlled targets are numerous genes involved in barrier function. Functional inactivation of C/EBPδ as well as overexpressions of two TF targets -MafB and SOX2- affect expression of markers of keratinocyte differentiation. We performed IHC on skin tumor tissue arrays: expression of C/EBPδ is lost in Basal Cell Carcinomas, but a majority of Squamous Cell Carcinomas showed elevated levels of the protein. Our data indicate that C/EBPδ plays a role in late stages of keratinocyte differentiation. © 2010 Borrelli et al.

The transcription factor p63, member of the p53 family, is crucial for epithelial development. An RNAi screening identified the apoptotic gene Procaspase-8 as a target activated by p63. The caspase-8 inhibitor FLIP is also under p63 control. We analysed and detailed the direct transactivation through the use of RNAi, reporter assays, ChIPs, western blots, confocal studies in HaCat, as well as in primary human keratinocytes. The direct DeltaNp63 regulation of these targets was confirmed in vivo using transgenic DeltaNp63 mice under the K5 promoter, as compared with p63 knockout mice, and in vitro in normal human primary keratinocytes following UV irradiation. Lowering the steady state of p63 protein levels changes the relative ratio of FLIP isoforms, causing the activation of the expressed, inactive Procaspase-8, into the active isoform thus triggering the proapoptotic cascade. Therefore, p63 fine-tunes the Procaspase-8-FLIP pro- and antiapoptotic pathway in keratinocytes.

Summary Background Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of lymphomas where the tumour population emerges within a multiple subclone pattern. Mycosis fungoides (MF) and Sezary syndrome (SS) are characterized by the expansion of clonal CD4+/CD45RO+ memory T cells. Lymphomatoid papulosis (LyP) is a chronic, lymphoproliferative disorder included in the CD30+ primary CTCL spectrum. Several studies have suggested a role of viral infection for super-antigenic activation of T lymphocytes; however, evidence of their association with CTCLs is still lacking. Human herpesvirus (HHV) 7 is a CD4+ T-lymphotropic herpesvirus; its restricted cellular tropism and the ability to induce cytokine production in infected cells could make it an important pathogenic cofactor in lymphoproliferative disorders. Objectives To investigate the presence of HHV7 DNA on CTCL and healthy skin donors (HD). Methods We used quantitative real time polymerase chain reaction to evaluate the potential pathogenic role of HHV7. Results Twenty-seven of 84 (32·1%) HD were positive for HHV7 DNA. Twenty-one of 148 (14·2%) patients with CTCLs were positive for HHV7 DNA: nine of 39 (23·1%) SS, six of 14 (42·9%) CD30+ CTCLs and six of 24 (25·0%) LyP, and HHV7 DNA was negative in all 71 patients with MF. Conclusions These results seem to exclude a pathogenic role of HHV7 in CTCLs, suggesting the possibility of skin as a latency site.

Hypertrophic scarring is a skin disorder characterized by persistent inflammation and fibrosis that may occur after wounding or thermal injury. Altered production of cytokines and growth factors, such as TGF-beta, play an important role in this process. Activin A, a member of the TGF-beta family, shares the same intra-cellular Smad signalling pathway with TGF-beta, but binds to its own specific transmembrane receptors and to follistatin, a secreted protein that inhibits activin by sequestration. Recent studies provide evidences of a novel role of activin A in inflammatory and repair processes. The aim of this study was to evaluate the importance of activin A and follistatin expression in the different phases of scar evolution. Immunostaining of sections obtained from active phase hypertrophic scars (AHS) revealed the presence of a high number of alpha-SMA(+) myofibroblasts and DC-SIGN(+) dendritic cells coexpressing activin A. Ex-vivo AHS fibroblasts produced more activin and less follistatin than normal skin or remission phase hypertrophic scar (HS) fibroblasts, both in basal conditions and upon TGF-betas stimulation. We demonstrate that fibroblasts do express activin receptors, and that this expression is not affected by TGF-betas. Treatment of HS fibroblasts with activin A induced Akt phosphorylation, promoted cell proliferation, and enhanced alpha-SMA and type I collagen expression. Follistatin reduced proliferation and suppressed activin-induced collagen expression. These results indicate that the activin/follistatin interplay has a role in HS formation and evolution. The impact of these observations on the understanding of wound healing and on the identification of new therapeutic targets is discussed.

International audience; p63 is a developmentally regulated transcription factor related to p53. It is involved in the development of ectodermal tissues, including limb, skin and in general, multilayered epithelia. The DeltaNp63alpha isoform is thought to play a 'master' role in the asymmetric division of epithelial cells. It is also involved in the pathogenesis of several human diseases, phenotypically characterized by ectodermal dysplasia. Our understanding of transcriptional networks controlled by p63 is limited, owing to the low number of bona fide targets. To screen for new targets, we employed chromatin immunoprecipitation from keratinocytes (KCs) coupled to the microarray technology, using both CpG islands and promoter arrays. The former revealed 96 loci, the latter yielded 85 additional genes. We tested 40 of these targets in several functional assays, including: (i) in vivo binding by p63 in primary KCs; (ii) expression analysis in differentiating HaCaT cells and in cells overexpressing DeltaNp63alpha; (iii) promoter transactivation and (iv) immunostaining in normal tissues, confirming their regulation by p63. We discovered several new specific targets whose functional categorization links p63 to cell growth and differentiation.

p63 is a transcription factor involved in the development of ectodermal tissues, including limb, skin and, in general, multilayered epithelia. We identified both activated and repressed genes in human keratinocytes via gene expression profiling of p63-depleted cells and validated 21 new primary targets by RT-PCR and ChIP location analysis. The p63 isoforms differentially activate or repress selected promoters. ChIPs in primary keratinocytes indicate that p63 targets are generally shared with p53, but some are p63-specific. Several growth suppressors are among repressed genes. The newly identified genes belong to pathways of growth and differentiation and are regulated in HaCaT differentiation and in stratification of human skin.

Background Genetic experiments have clarified that p63 is a key transcription factor governing the establishment and maintenance of multilayered epithelia. Key to our understanding of p63 strategy is the identification of target genes. We perfomed an RNAi screening in keratinocytes for p63, followed by profiling analysis. Results C/EBPδ, member of a family with known roles in differentiation pathways, emerged as a gene repressed by p63. We validated C/EBPδ as a primary target of ΔNp63α by RT-PCR and ChIP location analysis in HaCaT and primary cells. C/EBPδ is differentially expressed in stratification of human skin and it is up-regulated upon differentiation of HaCaT and primary keratinocytes. It is bound to and activates the ΔNp63 promoter. Overexpression of C/EBPδ leads to alteration in the normal profile of p63 isoforms, with the emergence of ΔNp63β and γ, and of the TA isoforms, with different kinetics. In addition, there are changes in the expression of most p63 targets. Inactivation of C/EBPδ leads to gene expression modifications, in part due to the concomitant repression of ΔNp63α. Finally, C/EBPδ is found on the p63 targets in vivo by ChIP analysis, indicating that coregulation is direct. Conclusion Our data highlight a coherent cross-talk between these two transcription factors in keratinocytes and a large sharing of common transcriptional targets.