Mariateresa Giuliano

Human CD34 + /CD90 + ASCs Are Capable of Growing as Sphere Clusters, Producing High Levels of VEGF and Forming Capillaries

Background Human adult adipose tissue is an abundant source of mesenchymal stem cells (MSCs). Moreover, it is an easily accessible site producing a considerable amount of stem cells. Methodology/Principal Findings In this study, we have selected and characterized stem cells within the stromal vascular fraction (SVF) of human adult adipose tissue with the aim of understanding their differentiation capabilities and performance. We have found, within the SVF, different cell populations expressing MSC markers – including CD34, CD90, CD29, CD44, CD105, and CD117 – and endothelial-progenitor-cell markers – including CD34, CD90, CD44, and CD54. Interestingly, CD34+/CD90+ cells formed sphere clusters, when placed in non-adherent growth conditions. Moreover, they showed a high proliferative capability, a telomerase activity that was significantly higher than that found in differentiated cells, and contained a fraction of cells displaying the phenotype of a side population. When cultured in adipogenic medium, CD34+/CD90+ quickly differentiated into adipocytes. In addition, they differentiated into endothelial cells (CD31+/VEGF+/Flk-1+) and, when placed in methylcellulose, were capable of forming capillary-like structures producing a high level of VEGF, as substantiated with ELISA tests. Conclusions/Significance Our results demonstrate, for the first time, that CD34+/CD90+ cells of human adipose tissue are capable of forming sphere clusters, when grown in free-floating conditions, and differentiate in endothelial cells that form capillary-like structures in methylcellulose. These cells might be suitable for tissue reconstruction in regenerative medicine, especially when patients need treatments for vascular disease.

Human CD34+ stem cells produce bone nodules in vivo

Abstract.  Objectives: The aim of this study was to select and provide enough stem cells for quick transplantation in bone engineering procedures, avoiding any in vitro expansion step. Materials and Methods: Dental germ pulp, collected from 25 healthy subjects aged 13–20 years, were subjected to magnetic‐activated cell sorting to select a CD34+ stem cell population capable of differentiating into pre‐osteoblasts. These cells were allowed to adhere to an absorbable polylactic–coglycolic acid scaffold for 30 min, without any prior expansion, and the CD34+ cell‐colonized scaffolds were then transplanted into immunocompromised rats, subcutaneously. Results: After 60 days, analysis of recovered transplants revealed that they were formed of nodules of bone, of the same dimensions as the original scaffold. Bone‐specific proteins were detected by immunofluorescence, within the nodules, and X‐ray diffraction patterns revealed characteristic features of bone. In addition, presence of platelet endothelial cell adhesion molecule and von Willebrand factor immunoreactivity were suggestive of neo‐angiogenesis and neovasculogenesis taking place within nodules. Importantly, these vessels were HLA‐1+ and, thus, clearly human in origin. Conclusions: This study indicates that CD34+ cells obtained from dental pulp can be used for engineering bone, without the need for prior culture expanding procedures. Using autologous stem cells, this schedule could be used to provide the basis for bone regenerative surgery, with limited sacrifice of tissue, low morbidity at the collection site, and significant reduction in time needed for clinical recovery.

Innovative fermentation strategies for the production of extremophilic enzymes

A new type of microfiltration (MF) bioreactor, developed in our laboratory, was investigated for use in improving efficiency of the production of extremophilic enzymes. In spite of the difficulties in cultivating hyperthermophiles, we achieved, in 300 h fermentation, more than 38 g/l dry weight of Sulfolobus solfataricus using a MF technique, and we demonstrated that the activity of alcohol dehydrogenase (ADH), as the reporter enzyme, was not affected by cell density. However, hyperthermophile cultivation is difficult to scale up because of evaporation and the very low growth rate. Thus, to achieve high productivity we cultivated, in the MF bioreactor, recombinant mesophilic hosts engineered for the production of two thermophilic enzymes, namely, trehalosyldextrin-forming enzyme (SsTDFE) and trehalose-forming enzyme (SsTFE) from Sulfolobus solfataricus. The traditional Luria-Bertani broth used for recombinant Escherichia coli growth was replaced with a semidefined medium. The latter was used in both the batch and the MF experiments, and the ratio of complex components (e.g., yeast extract and tryptone) to a simple carbon source (glycerol) was decreased during the fed-batch phase to further decrease the medium cost in view of industrial applications. The bioprocess developed was able to improve productivity 500 fold for rSsTFE and 60 fold for rSsTDFE with respect to the wild type cultivated in MF mode. Comparisons with another recombinant enzyme, alpha-glucosidase (rSsalphagly), from Sulfolobus solfataricus produced in our MF bioreactor are reported.

Gene Expression Profile of Patients with Mayer-Rokitansky-Küster-Hauser Syndrome: New Insights into the Potential Role of Developmental Pathways

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is a rare disease characterized by congenital aplasia of uterus and vagina. Although many studies have investigated several candidate genes, up to now none of them seem to be responsible for the aetiology of the syndrome. In our study, we identified differences in gene expression profile of in vitro cultured vaginal tissue of MRHKS patients using whole-genome microarray analysis. A group of eight out of sixteen MRKHS patients that underwent reconstruction of neovagina with an autologous in vitro cultured vaginal tissue were subjected to microarray analysis and compared with five healthy controls. Results obtained by array were confirmed by qRT-PCR and further extended to other eight MRKHS patients. Gene profiling of MRKHS patients delineated 275 differentially expressed genes, of which 133 downregulated and 142 upregulated. We selected six deregulated genes (MUC1, HOXC8, HOXB2, HOXB5, JAG1 and DLL1) on the basis of their fold change, their differential expression in most patients and their relevant role in embryological development. All patients showed upregulation of MUC1, while HOXB2 and HOXB5 were downregulated, as well as Notch ligands JAG1 and DLL1 in the majority of them. Interestingly, HOXC8 was significantly upregulated in 47% of patients, with a differential expression only in MRKHS type I patients. Taken together, our results highlighted the dysregulation of developmental genes, thus suggesting a potential alteration of networks involved in the formation of the female reproductive tract and providing a useful clue for understanding the pathophysiology of MRKHS.

A combined fermentative-chemical approach for the scalable production of pure E. coli monophosphoryl lipid A

Lipid A is the lipophilic region of lipopolysaccharides and lipooligosaccharides, the major components of the outer leaflet of most part of Gram-negative bacteria. Some lipid As are very promising immunoadjuvants. They are obtained by extraction from bacterial cells or through total chemical synthesis. A novel, semisynthetic approach to lipid As is ongoing in our laboratories, relying upon the chemical modification of a natural lipid A scaffold for the fast obtainment of several other lipid As and derivatives thereof. The first requisite for this strategy is to have this scaffold available in large quantities through a scalable process. Here, we present an optimized fed-batch fermentation procedure for the gram-scale production of lipid A from Escherichia coli K4 and a suitable phenol-free protocol for its purification. A study for regioselective de-O-phosphorylation reaction was then performed to afford pure monophosphoryl lipid A with an attenuated endotoxic activity, as evaluated by cytokine production in human monocytic cell line THP-1 in vitro. The reported method for the large-scale obtainment of monophoshoryl lipid A from the fed-batch fermentation broth of a recombinant strain of E. coli may permit the access to novel semisynthetic lipid A immunoadjuvant candidates.

A Semisynthetic Approach to New Immunoadjuvant Candidates: Site‐Selective Chemical Manipulation of Escherichia coli Monophosphoryl Lipid A

A semisynthetic approach to novel lipid A derivatives from Escherichia coli (E. coli) lipid A is reported. This methodology stands as an alternative to common approaches based exclusively on either total synthesis or extraction from bacterial sources. It relies upon the purification of the lipid A fraction from fed-batch fermentation of E. coli, followed by its structural modification through tailored, site-selective chemical reactions. In particular, modification of the lipid pattern and functionalization of the phosphate group as well as of the sole primary hydroxyl group were accomplished, highlighting the unusual reactivity of the molecule. Preliminary investigations of the immunostimulating activity of the new semisynthetic lipid A derivatives show that some of them stand out as promising, new immunoadjuvant candidates.

A time-lapse approach to examine chromium and nickel effects on wound healing in vitro.

Chromium and nickel cause allergic contact dermatitis, a common biological skin response to sensitizing agents. This study used a conventional in vitro wounding model to study the impact of sensitizing agents on the innate immune response of human keratinocytes. Experiments were designed to evaluate the involvement of specific Toll-like receptors and metalloproteinases as effectors molecules downstream, at a molecular level. Further, keratinocytes were co-cultured with monocytes (THP-1 cells) to reproduce an inductive stimulus on monocytes made by metals. Human keratinocytes (HaCat) were grown on plates covered with collagen type I, chemically treated, and then mechanically injured with a sterile pipette tip. Restoration of the monolayer integrity was monitored by time-lapse video microscopy. Effector gene expression was evaluated by real-time PCR. The presence of chromium significantly dropped the rate of wound closure, while nickel-induced hyper-proliferation ended in an acceleration of the healing process, an event that does not occur in vivo. This latter outcome led to considering nickel as an unsuitable example for use in the experimental model. Focusing solely on the chromium aspect of this study, RNA profiles of selected molecular markers were generated to ascertain if the detrimental stimulus from chromium was eliminated or persisted both in keratinocytes alone and/or during co-cultures of keratinocytes and monocytes. Monocytes accelerated the process of wound repair. This in vitro experimental model highlighted the involvement of innate immunity in response to chromium and might be useful for test molecules of therapeutic interest for the treatment of skin lesions. However, the experience with nickel reveals that there are limitations to the utility of this wound model system after all.