Camilla Siciliano

The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs.

Optimization of the isolation and expansion method of human mediastinal–adipose tissue derived mesenchymal stem cells with virally inactivated GMP-grade platelet lysate

Mesenchymal stem cells (MSCs) are adult multipotent cells currently employed in several clinical trials due to their immunomodulating, angiogenic and repairing features. The adipose tissue is certainly considered an eligible source of MSCs. Recently, putative adipose tissue derived MSCs (ADMSCs) have been isolated from the mediastinal depots. However, very little is known about the properties, the function and the potential of human mediastinal ADMSCs (hmADMSCs). However, the lack of standardized methodologies to culture ADMSCs prevents comparison across. Herein for the first time, we report a detailed step by step description to optimize the isolation and the expansion methodology of hmADMSCs using a virally inactivated good manufacturing practice (GMP)-grade platelet lysate, highlighting the critical aspects of the procedure and providing useful troubleshooting suggestions. Our approach offers a reproducible system which could provide standardization across laboratories. Moreover, our system is time and cost effective, and it can provide a reproducible source of adipose stem cells to enable future studies to unravel new insights regard this promising stem cell population.

Role of NOX2 in mediating doxorubicin-induced senescence in human endothelial progenitor cells

Senescence exerts a great impact on both biological and functional properties of circulating endothelial progenitor cells (EPCs), especially in cardiovascular diseases where the physiological process of aging is accelerated upon clinical administration of certain drugs such as doxorubicin. EPC impairment contributes to doxorubicin-induced cardiotoxicity. Doxorubicin accelerates EPC aging, although mechanisms underlying this phenomenon remain to be fully clarified. Here we investigated if Nox2 activity is able to modulate the premature senescence induced in vitro by doxorubicin in human EPCs. Results showed that in conditioned media obtained from late EPC cultures, the levels of interleukin-6, isoprostanes and nitric oxide bioavailability were increased and reduced respectively after 3h of doxorubicin treatment. These derangements returned to physiological levels when cells were co-treated with apocynin or gp91ds-tat (antioxidant and specific Nox2 inhibitors, respectively). Accordingly, Nox2 activity resulted to be activated by doxorubicin. Importantly, we found that Nox2 inhibition reduced doxorubicin-induced EPC senescence, as indicated by a lower percentage of β-gal positive EPCs. In conclusion, Nox2 activity efficiently contributes to the mechanism of oxidative stress-induced increase in premature aging conferred by doxorubicin. The importance of modulation of Nox2 in human EPCs could reveal a useful tool to restore EPC physiological function and properties.

Cardiosphere Conditioned Media Influence the Plasticity of Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

Nowadays, cardiac regenerative medicine is facing many limitations because of the complexity to find the most suitable stem cell source and to understand the regenerative mechanisms involved. Mesenchymal stem cells (MSCs) have shown great regenerative potential due to their intrinsic properties and ability to restore cardiac functionality, directly by transdifferentiation and indirectly by paracrine effects. Yet, how MSCs could respond to definite cardiac-committing microenvironments, such as that created by resident cardiac progenitor cells in the form of cardiospheres (CSs), has never been addressed. Recently, a putative MSC pool has been described in the mediastinal fat (hmADMSCs), but both its biology and function remain hitherto unexplored. Accordingly, we investigated the potential of hmADMSCs to be committed toward a cardiovascular lineage after preconditioning with CS-conditioned media (CCM). Results indicated that CCM affects cell proliferation. Gene expression levels of multiple cardiovascular and stemness markers (MHC, KDR, Nkx2.5, Thy-1, c-kit, SMA) are significantly modulated, and the percentage of hmADMSCs preconditioned with CCM and positive for Nkx2.5, MHC, and KDR is significantly higher relative to FBS and explant-derived cell conditioned media (EDCM, the unselected stage before CS formation). Growth factor-specific and survival signaling pathways (i.e., Erk1/2, Akt, p38, mTOR, p53) present in CCM are all equally regulated. Nonetheless, earlier BAD phosphorylation (Ser112) occurs associated with the CS microenvironment (and to a lesser extent to EDCM), whereas faster phosphorylation of PRAS40 in FBS, and of Akt (Ser473) in EDCM and 5-azacytidine occurs compared to CCM. For the first time, we demonstrated that the MSC pool held in the mediastinal fat is adequately plastic to partially differentiate in vitro toward a cardiac-like lineage. Besides, we have provided novel evidence of the potent inductive niche-like microenvironment that the CS structure can reproduce in vitro. hmADMSCs can represent an interesting tool in order to exploit their possible role in cardiovascular diseases and treatment.

Suitability of Human Tenon’s Fibroblasts as Feeder Cells for Culturing Human Limbal Epithelial Stem Cells

Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon’s fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63α), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63α+LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ΔNp63α than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-β3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture.

Influence of Egr-1 in Cardiac Tissue-Derived Mesenchymal Stem Cells in Response to Glucose Variations

Mesenchymal stem cells (MSCs) represent a promising cell population for cell therapy and regenerative medicine applications. However, how variations in glucose are perceived by MSC pool is still unclear. Since, glucose metabolism is cell type and tissue dependent, this must be considered when MSCs are derived from alternative sources such as the heart. The zinc finger transcription factor Egr-1 is an important early response gene, likely to play a key role in the glucose-induced response. Our aim was to investigate how short-term changes in in vitro glucose concentrations affect multipotent cardiac tissue-derived MSCs (cMSCs) in a mouse model of Egr-1 KO (Egr-1−/−). Results showed that loss of Egr-1 does not significantly influence cMSC proliferation. In contrast, responses to glucose variations were observed in wt but not in Egr-1−/− cMSCs by clonogenic assay. Phenotype analysis by RT-PCR showed that cMSCs Egr-1−/− lost the ability to regulate the glucose transporters GLUT-1 and GLUT-4 and, as expected, the Egr-1 target genes VEGF, TGFβ-1, and p300. Acetylated protein levels of H3 histone were impaired in Egr-1−/− compared to wt cMSCs. We propose that Egr-1 acts as immediate glucose biological sensor in cMSCs after a short period of stimuli, likely inducing epigenetic modifications.

Beta2-adrenergic signaling affects the phenotype of human cardiac progenitor cells through EMT modulation

Graphical abstract Figure. No Caption available. ABSTRACT Human cardiac progenitor cells (CPCs) offer great promises to cardiac cell therapy for heart failure. Many in vivo studies have shown their therapeutic benefits, paving the way for clinical translation. The 3D model of cardiospheres (CSs) represents a unique niche‐like in vitro microenvironment, which includes CPCs and supporting cells. CSs have been shown to form through a process mediated by epithelial‐to‐mesenchymal transition (EMT). &bgr;2‐Adrenergic signaling significantly affects stem/progenitor cells activation and mobilization in multiple tissues, and crosstalk between &bgr;2‐adrenergic signaling and EMT processes has been reported. In the present study, we aimed at investigating the biological response of CSs to &bgr;2‐adrenergic stimuli, focusing on EMT modulation in the 3D culture system of CSs. We treated human CSs and CS‐derived cells (CDCs) with the &bgr;2‐blocker butoxamine (BUT), using either untreated or &bgr;2 agonist (clenbuterol) treated CDCs as control. BUT‐treated CS‐forming cells displayed increased migration capacity and a significant increase in their CS‐forming ability, consistently associated with increased expression of EMT‐related genes, such as Snai1. Moreover, long‐term BUT‐treated CDCs contained a lower percentage of CD90+ cells, and this feature has been previously correlated with higher cardiogenic and therapeutic potential of the CDCs population. In addition, long‐term BUT‐treated CDCs had an increased ratio of collagen‐III/collagen‐I gene expression levels, and showed decreased release of inflammatory cytokines, overall supporting a less fibrosis‐prone phenotype. In conclusion, &bgr;2 adrenergic receptor block positively affected the stemness vs commitment balance within CSs through the modulation of type1‐EMT (so called “developmental”). These results further highlight type‐1 EMT to be a key process affecting the features of resident cardiac progenitor cells, and mediating their response to the microenvironment.

Human Lung Spheroids as In Vitro Niches of Lung Progenitor Cells With Distinctive Paracrine and Plasticity Properties

Basic and translational research on lung biology has discovered multiple progenitor cell types, specialized or facultative, responsible for turnover, renewal, and repair. Isolation of populations of resident lung progenitor cells (LPCs) has been described by multiple protocols, and some have been successfully applied to healthy human lung tissue. We aimed at understanding how different cell culture conditions may affect, in vitro, the phenotype of LPCs to create an ideal niche‐like microenvironment. The influence of different substrates (i.e., fibronectin, gelatin, laminin) and the impact of a three‐dimensional/two‐dimensional (3D/2D) culture switch on the biology of LPCs isolated as lung spheroids (LSs) from normal adult human lung biopsy specimens were investigated. We applied a spheroid culture system as the selective/inductive step for progenitor cell culture, as described in many biological systems. The data showed a niche‐like proepithelial microenvironment inside the LS, highly sensitive to the 3D culture system and significantly affecting the phenotype of adult LPCs more than culture substrate. LSs favor epithelial phenotypes and LPC maintenance and contain cells more responsive to specific commitment stimuli than 2D monolayer cultures, while secreting a distinctive set of paracrine factors. We have shown for the first time, to our knowledge, how culture as 3D LSs can affect LPC epithelial phenotype and produce strong paracrine signals with a distinctive secretomic profile compared with 2D monolayer conditions. These findings suggest novel approaches to maintain ex vivo LPCs for basic and translational studies. Stem Cells Translational Medicine 2017;6:767–777

The adipose tissue of origin influences the biological potential of human adipose stromal cells isolated from mediastinal and subcutaneous fat depots

Indirect evidence suggests that adipose tissue-derived stromal cells (ASCs) possess different physiological and biological variations related to the anatomical localization of the adipose depots. Accordingly, to investigate the influence of the tissue origin on the intrinsic properties of ASCs and to assess their response to specific stimuli, we compared the biological, functional and ultrastructural properties of two ASC pools derived from mediastinal and subcutaneous depots (thoracic compartment) by means of supplements such as platelet lysate (PL) and FBS. Subcutaneous ASCs exhibited higher proliferative and clonogenic abilities than mediastinal counterpart, as well as increased secreted levels of IL-6 combined with lower amount of VEGF-C. In contrast, mediastinal ASCs displayed enhanced pro-angiogenic and adipogenic differentiation properties, increased cell diameter and early autophagic processes, highlighted by electron microscopy. Our results further support the hypothesis that the origin of adipose tissue significantly defines the biological properties of ASCs, and that a homogeneric function for all ASCs cannot be assumed.

M2muscarinic receptors inhibit cell proliferation and migration in urothelial bladder cancer cells

The role of muscarinic receptors in several diseases including cancer has recently emerged. To evaluate the hypothesis that muscarinic acetylcholine receptors may play a role in bladder cancer as well as in other tumor types, we investigated their expression in bladder tumor specimens. All examined samples expressed the M1, M2 and M3 receptor subtypes. We also found that the level of M2 transcripts, but not those of M1 or M3, significantly increased with the tumor histologic grade. In view of these results, we proceeded to investigate whether the M2 agonist Arecaidine had any effect on in vitro cell growth and migration of T24 cells, a bladder tumor cell line expressing the muscarinic receptors, including the M2 subtype. We observed that Arecaidine significantly reduced T24 and 5637 cell proliferation and migration in a concentration dependent manner. The silencing of M2 receptor by siRNA in T24 and 5637 cell lines showed the inability of Arecaidine (100 μM) to inhibit cell proliferation after 48 hours, whereas the use of M1 and M3 antagonists in T24 appeared not to counteract the Arecaidine effect, suggesting that the inhibition of cell proliferation was directly dependent on M2 receptor activation. These data suggest that M2 muscarinic receptors may play a relevant role in bladder cancer and represent a new attractive therapeutic target.