Catia Orrico

Common Tasks in Microscopic and Ultrastructural Image Analysis Using ImageJ

Cooperation between research communities and software-development teams has led to the creation of novel software. The purpose of this paper is to show an alternative work method based on the usage of ImageJ (http://rsb.info.nih.gov/ij/), which can be effectively employed in solving common microscopic and ultrastructural image analysis tasks. As an open-source software, ImageJ provides the possibility to work in a free-development/sharing world. Its very “friendly” graphical user interface helps users to manage and edit biomedical images. The on-line material such as handbooks, wikis, and plugins leads users through various functions, giving clues about potential new applications. ImageJ is not only a morphometric analysis software, it is sufficiently flexible to be adapted to the numerous requirements tasked in the laboratories as routine as well as research demands. Examples include area measurements on selectively stained tissue components, cell count and area measurements at single cell level, immunohistochemical antigen quantification, and immunoelectron microscopy gold particle count.

Thoracic aortas from multiorgan donors are suitable for obtaining resident angiogenic mesenchymal stromal cells.

The clinical use of endothelial progenitor cells is hampered by difficulties in obtaining an adequate number of functional progenitors. This study aimed to establish whether human thoracic aortas harvested from healthy multiorgan donors can be a valuable source of angiogenic progenitors. Immunohistochemical tissue studies showed that two distinct cell populations with putative stem cell capabilities, one composed of CD34+ cells and the other of c‐kit+ cells, are present in between the media and adventitia of human thoracic aortas. Ki‐67+ cells with high growth potential were located in an area corresponding to the site of CD34+ and c‐kit+ cell residence. We thus isolated cells (0.5 ∼ 2.0 × 104 aortic progenitors per 25 cm2) which, upon culturing, coexpressed molecules of mesenchymal stromal cells (i.e., CD44+, CD90+, CD105+) and showed a transcript expression of stem cell markers (e.g., OCT4, c‐kit, BCRP‐1, Interleukin‐6) and BMI‐1. Cell expansion was adequate for use in a clinical setting. A subset of cultured cells acquired the phenotype of endothelial cells in the presence of vascular endothelial growth factor (e.g., increased expression of KDR and von Willebrand factor positivity), as documented by flow cytometry, immunofluorescence, electron microscopy, and reverse transcription‐polymerase chain reaction assays. An in vitro angiogenesis test kit revealed that cells were able to form capillary‐like structures within 6 hours of seeding. This study demonstrates that thoracic aortas from multiorgan donors yield mesenchymal stromal cells with the ability to differentiate in vitro into endothelial cells. These cells can be used for the creation of an allogenic bank of angiogenic progenitors, thus providing new options for restoring vascularization at ischemic sites.

Ultrastructural Characteristics of Human Mesenchymal Stromal (Stem) Cells Derived from Bone Marrow and Term Placenta

Human mesenchymal stromal (stem) cells (hMSCs) isolated from adult bone marrow (BM-hMSCs) as well as amnion (AM-hMSCs) and chorion (CM-hMSCs) term placenta leaves were studied by transmission electron microscopy (TEM) to investigate their ultrastructural basic phenotype. At flow cytometry, the isolated cells showed a homogeneous expression of markers commonly used to identify hMSCs, i.e., CD105, CD44, CD90, CD166, HLA-ABC positivities, and CD45, AC133, and HLA-DR negativities. However, TEM revealed subtle yet significant differences. BM-hMSCs had mesenchymal features with dilated cisternae of rough endoplasmic reticulum (rER) and peripheral collections of multiloculated clear blisters; this latter finding mostly representing complex foldings of the plasma membrane could be revelatory of the in situ cell arrangement in the niche microenvironment. Unlike BM-hMSCs, CM-hMSCs were more primitive and metabolically quiescent, their major features being the presence of rER stacks and large peripheral collections of unbound glycogen. AM-hMSCs showed a hybrid epithelial–mesenchymal ultrastructural phenotype; epithelial characters included non-intestinal-type surface microvilli, intracytoplasmic lumina lined with microvilli, and intercellular junctions; mesenchymal features included rER profiles, lipid droplets, and well-developed foci of contractile filaments with dense bodies. These features are consistent with the view that AM-hMSCs have a pluripotent potential. In conclusion, this study documents that ultrastructural differences exist among phenotypically similar hMSCs derived from human bone marrow and term placenta leaves; such differences could be revelatory of the hMSCs in vitro differentiation potential and may provide useful clues to attempt their in situ identification.

DnaK from Bifidobacterium animalis subsp. lactis is a surface-exposed human plasminogen receptor upregulated in response to bile salts.

Bifidobacterium animalis subsp. lactis lives in the gastrointestinal tract of most mammals, including humans. Recently, for the probiotic strain B. animalis subsp. lactis BI07, a dose-dependent plasminogen-binding activity was demonstrated and five putative plasminogen-binding proteins were identified. Here we investigated the role of surface DnaK as a B. animalis subsp. lactis BI07 plasminogen receptor. DnaK was visualized on the bacterial cell surface by transmission electron microscopy. The His-tagged recombinant DnaK protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. The capability to tolerate physiological concentrations of bile salts is a crucial feature for an intestinal symbiont micro-organism. By proteome analysis we demonstrated that the long-term exposure of B. animalis subsp. lactis BI07 to bile salts results in the upregulation of important surface plasminogen receptors such as DnaK and enolase. Moreover, adaptation of B. animalis subsp. lactis BI07 to physiological concentrations of bile salts significantly increased its capacity to interact with the host plasminogen system. By enhancing the bacterial capacity to interact with the host plasminogen, the gut bile environment may facilitate the colonization of the human host by B. animalis subsp. lactis BI07.

Angiogenic Potential of Human Dental Pulp Stromal (STEM) Cells

Dental pulp is a heterogeneous microenviroment where unipotent progenitor and pluripotent mesenchymal stem cells cohabit. In this study we investigated whether human Dental Pulp Stromal (Stem) Cells (DP-SCs) committed to the angiogenic fate. DP-SCs showed the specific mesenchymal immunophenotypical profile positive for CD29, CD44, CD73, CD105, CD166 and negative for CD14, CD34, CD45, in accordance with that reported for bone marrow-derived SCs. The Oct-4 expression in DP-SCs, evaluated through RT-PCR analysis, increased in relation with the number of the passages in cell culture and decreased after angiogenic induction. In agreement with their multipotency, DP-SCs differentiated toward osteogenic and adipogenic commitments. In angiogenic experiments, differentiation of DP-SCs, through Vascular Endothelial Growth Factor (VEGF) induction, was evaluated by in vitro matrigel assay and by cytometric analysis. Accordingly, endothelial-specific markers like Flt-1 and KDR were basally expressed and they increased after exposure to VEGF together with the occurrence of ICAM-1 and von Willebrand Factor positive cells. In addition, VEGF-induced DP-SCs maintained endothelial cell-like features when cultured in a 3-D fibrin mesh, displaying focal organization into capillary-like structures. The DP-SC angiogenic potential may prove a remarkable tool for novel approaches to developing tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.

Mesenchymal stem cell interaction with a non-woven hyaluronan-based scaffold suitable for tissue repair

The fabrication of biodegradable 3‐D scaffolds enriched with multipotent stem cells seems to be a promising strategy for the repair of irreversibly injured tissues. The fine mechanisms of the interaction of rat mesenchymal stem cells (rMSCs) with a hyaluronan‐based scaffold, i.e. HYAFF®11, were investigated to evaluate the potential clinical application of this kind of engineered construct. rMSCs were seeded (2 × 106 cells cm−2) on the scaffold, cultured up to 21 days and analysed using appropriate techniques. Light (LM), scanning (SEM) and transmission (TEM) electron microscopy of untreated scaffold samples showed that scaffolds have a highly porous structure and are composed of 15‐µm‐thick microfibres having a rough surface. As detected by trypan blue stain, cell adhesion was high at day 1. rMSCs were viable up to 14 days as shown by CFDA assay and proliferated steadily on the scaffold as revealed by MTT assay. LM showed rMSCs in the innermost portions of the scaffold at day 3. SEM revealed a subconfluent cell monolayer covering 40 ± 10% of the scaffold surface at day 21. TEM of early culture showed rMSCs wrapping individual fibres with regularly spaced focal contacts, whereas confocal microscopy showed polarized expression of CD44 hyaluronan receptor; TEM of 14‐day cultures evidenced fibronexus formation. Immunohistochemistry of 21‐day cultures showed that fibronectin was the main matrix protein secreted in the extracellular space; decorin and versican were seen in the cell cytoplasm only and type IV collagen was minimally expressed. The expression of CD90, a marker of mesenchymal stemness, was found unaffected at the end of cell culture. Our results show that HYAFF®11 scaffolds support the adhesion, migration and proliferation of rMSCs, as well as the synthesis and delivery of extracellular matrix components under static culture conditions without any chemical induction. The high retention rate and viability of the seeded cells as well as their fine modality of interaction with the substrate suggest that such scaffolds could be potentially useful when wide tissue defects are to be repaired as in the case of cartilage repair, wound healing and large vessel replacement.

Multidistrict human mesenchymal vascular cells: pluripotency and stemness characteristics.

BACKGROUND AIMS The presence of ectopic tissues in the pathologic artery wall raises the issue of whether multipotent stem cells may reside in the vasculature itself. Recently mesenchymal stromal cells (MSC) have been isolated from different human vascular segments (VW MSC), belying the previous view that the vessel wall is a relatively quiescent tissue. METHODS Resident multipotent cells were recovered from fresh arterial segments (aortic arches, thoracic and femoral arteries) collected in a tissue-banking facility and used to establish an in situ and in vitro study of the stemness features and multipotency of these multidistrict MSC populations. RESULTS Notch-1+, Stro-1+, Sca-1+ and Oct-4+ cells were distributed along an arterial wall vasculogenic niche. Multidistrict VW MSC homogeneously expressed markers of stemness (Stro-1, Notch-1 and Oct-4) and MSC lineages (CD44, CD90, CD105, CD73, CD29 and CD166) whilst they were negative for hematopoietic and endothelial markers (CD34, CD45, CD31 and vWF). Each VW MSC population had characteristics of stem cells, i.e. a high efflux capability for Hoechst 33342 dye and the ability to form spheroids when grown in suspension and generate colonies when seeded at low density. Again, VW MSC cultured in induction media exhibited adipogenic, chondrogenic and leiomyogenic potential but less propensity to osteogenic differentiation, as documented by histochemical, immunohistochemical, molecular and electron microscopy analysis. CONCLUSIONS Overall, these findings may enlighten the physiopathologic mechanisms of vascular wall diseases as well as having potential implications for cellular, genetic and tissue engineering approaches to treating vascular pathologies when these are unresponsive to medical and surgical therapies.

Isolation of stem/progenitor cells from normal lung tissue of adult humans.

Objectives:  This study aimed to isolate and characterize stem/progenitor cells, starting from normal airway epithelia, obtained from human adults.

Architectural Organization and Functional Features of Early Endothelial Progenitor Cells Cultured in a Hyaluronan-Based Polymer Scaffold

Neovascularization can be improved using polymer scaffolds supporting endothelial progenitor cells (EPCs). The aim of the present study was to investigate whether human early EPCs (eEPCs) could be efficiently cultured in a hyaluronan-based non-woven mesh (HYAFF-11). eEPCs were seeded on HYAFF-11 at the density of 1 x 10(6)/cm(2) and cultured with endothelial differentiating factors for 3 weeks. After 24 h, nearly 90% of EPCs were adherent. Cell viability, evaluated by methyltetrazolium test, was greater in HYAFF-11 than on the most commonly used fibronectin-coated dishes, even if a progressive decline in viability was observed starting from approximately the second week of culture. eEPCs easily migrated to and aggregated on the scaffold. Evidence of active protein synthesis and features of endothelial differentiation, including cellular transcytotic channels and micropinocytotic vesicles, was revealed using electron microscopy, immunofluorescence, and reverse transcriptase polymerase chain reaction analysis. eEPCs cultured in the scaffold also showed a certain angiogenic activity, as demonstrated by hepatocyte growth factor transcription and vascular endothelial growth factor secretion. In conclusion, eEPCs can migrate and adhere inside HYAFF-11, maintain their pre-endothelial phenotype, and express angiogenic factors, especially within the first week of growth. These results indicate that non-woven HYAFF-11 could be a promising candidate as a vehicle for eEPCs for regenerative medicine applications.

Dysfunctional Vasa Vasorum in Diabetic Peripheral Artery Obstructive Disease with Critical Lower Limb Ischaemia

OBJECTIVES AND DESIGN To establish whether in diabetic patients with peripheral artery obstructive disease (PAOD) vasa vasorum (vv) neoangiogenesis is altered with increased arterial damage. MATERIALS Thirty-three patients with PAOD and critical lower limb ischaemia, 22 with type II diabetes. METHODS Immunohistochemistry for endothelial cell markers (CD34 and von Willebrand Factor); real-time reverse transcription polymerase chain reaction (RT-PCR) to quantify arterial wall expression of vascular endothelial growth factor (VEGF); enzyme-linked immunosorbent assay (ELISA) to assess blood VEGF; flow cytometry to detect circulating endothelial cells (CECs). RESULTS Patients with PAOD and diabetes have a higher frequency (60% vs. 45%) of advanced atherosclerotic lesions and a significant reduction (p = 0.0003) in CD34(+) capillaries in the arterial media. Adventitial neoangiogenesis was increased equally (CD34(+) and vWF(+)) in all patients. Likewise, all patients have increased CEC and VEGF concentration in the blood as well as in-situ VEGF transcript expression. CONCLUSIONS Patients with PAOD have remarkable arterial damage despite increased in-situ and circulating expression of the pro-angiogenic VEGF; a dysfunctional vv angiogenesis was seen in diabetics which also showed a higher frequency of parietal damage; it is suggested that in diabetic arterial wall, injury is worsened by vv inability to finalise an effective VEGF-driven arterial wall neoangiogenesis.