Protein O-mannosylation is crucial for human mesencyhmal stem cells fate.

Human mesenchymal stem cells (MSC) are promising cell types in the field of regenerative medicine. Although many pathways have been dissected in the effort to better understand and characterize MSC potential, the impact of protein N- or O-glycosylation has been neglected. Deficient protein O-mannosylation is a pathomechanism underlying severe congenital muscular dystrophies (CMD) that start to develop at the embryonic developmental stage and progress in the adult, often in tissues where MSC exert their function. Here we show that O-mannosylation genes, many of which are putative or verified glycosyltransferases (GTs), are expressed in a similar pattern in MSC from adipose tissue, bone marrow, and umbilical cord blood and that their expression levels are retained constant during mesengenic differentiation. Inhibition of the first players of the enzymatic cascade, POMT1/2, resulted in complete abolishment of chondrogenesis and alterations of adipogenic and osteogenic potential together with a lethal effect during myogenic induction. Since to date, no therapy for CMD is available, we explored the possibility of using MSC extracellular vesicles (EVs) as molecular source of functional GTs mRNA. All MSC secrete POMT1 mRNA-containing EVs that are able to efficiently fuse with myoblasts which are among the most affected cells by CMD. Intriguingly, in a pomt1 patient myoblast line EVs were able to partially revert O-mannosylation deficiency and contribute to a morphology recovery. Altogether, these results emphasize the crucial role of protein O-mannosylation in stem cell fate and properties and open the possibility of using MSC vesicles as a novel therapeutic approach to CMD.

Mesenchymal stem cells encapsulated into biomimetic hydrogel scaffold gradually release CCL2 chemokine in situ preserving cytoarchitecture and promoting functional recovery in spinal cord injury.

Spinal cord injury (SCI) is an acute neurodegenerative disorder caused by traumatic damage of the spinal cord. The neuropathological evolution of the primary trauma involves multifactorial processes that exacerbate the pathology, worsening the neurodegeneration and limiting neuroregeneration. This complexity suggests that multi-therapeutic approaches, rather than any single treatment, might be more effective. Encouraging preclinical results indicate that stem cell-based treatments may improve the disease outcome due to their multi-therapeutic ability. Mesenchymal Stem Cells (MSCs) are currently considered one of the most promising approaches. Significant improvement in the behavioral outcome after MSC treatment sustained by hydrogel has been demonstrated. However, it is still not known how hydrogel contribute to the delivery of factors secreted from MSCs and what factors are released in situ. Among different mediators secreted by MSCs after seeding into hydrogel, we have found CCL2 chemokine, which could account for the neuroprotective mechanisms of these cells. CCL2 secreted from human MSCs is delivered efficaciously in the lesioned spinal cord acting not only on recruitment of macrophages, but driving also their conversion to an M2 neuroprotective phenotype. Surprisingly, human CCL2 delivered also plays a key role in preventing motor neuron degeneration in vitro and after spinal cord trauma in vivo, with a significant improvement of the motor performance of the rodent SCI models.

Engineered stromal layers and continuous flow culture enhance multidrug resistance gene transfer in hematopoietic progenitors.

We recently reported that in stroma-free cultures 11-33% of clonogenic cells derived from a bulk long-term culture [long-term culture-clonogenic cells (LTC-CC)] could be transduced by supernatant exposure or coculture of human CD34+ progenitors with MDR retroviral producer line A12M1. We reasoned that a stromal cell layer may generate niches in which LTC-CC could enter in the S-phase, thus becoming a more accessible target for gene delivery. In static culture studies in flasks, human engineered stromal cell line L87/4 or stromal murine M2-10B4 cells were used as feeder after irradiation, and CD34+ cells from either cord blood or peripheral blood of mobilized cancer patients were exposed to MDR supernatant for 7 consecutive days before 5-week culture for LTC-CC evaluation. In continuous flow perfusion culture studies, CD34+ cells were seeded over irradiated stromal murine M2-1OB4 cells and exposed to MDR supernatant for 7 days before LTC-CC evaluation. In mock-transduced controls, <5% of LTC-CC were found to he viable after exposure to 10 ng/ml Taxol. In cells exposed to MDR supernatant in static stroma cultures, 68 +/- 4% of seeded LTC-CC were found to be drug resistant and express MDR mRNA as evaluated by reverse transcription-PCR analysis of single colonies. The addition of cytokines did not further enhance transfer efficiency. After MDR retroviral exposure in continuous flow cultures, 88 +/- 5% of LTC-CC were found to be drug resistant (P < 0.01 versus static stroma culture). P-glycoprotein expression in CD34+ cells was evaluated using flow cytometry and found to he higher after continuous flow versus static cultures. Finally, very high levels of P-glycoprotein expression after MDR supernatant exposure in the presence of stroma were confirmed by APAAP staining of cultured cells. We conclude that engineered stromal cell layers and continuous flow culture conditions can significantly enhance retroviral-mediated gene transfer into human hematopoietic progenitor cells.

"Stem cell candidates" purified by liquid culture in the presence of Steel factor, IL-3, and 5FU are strictly stroma-dependent and have myeloid, lymphoid, and megakaryocytic potential.

Berardi et al. (Science 1995; 267:105) reported recently that combined cytokine stimulation and antimetabolite treatment were able to isolate cells with characteristics of hemopoietic stem cells. Bone marrow (BM) low-density cells were cultured for 1 week in the presence of Steel factor (SF), IL-3, and the antimetabolite 5-FU. Following this approach one in 10(5) BM cells were purified. These cells showed no clonogenic potential in soft gel assays but presented a striking myeloid-lymphoid potential as long-term culture-initiating cells (LTC-ICs). We investigated this new "stem cell candidate." following a similar approach we purified one in 55,000/130,000 cells from cord blood and peripheral blood in mobilized cancer patients. These cells displayed no clonogenic potential in methylcellulose assays in the presence of different cytokine combinations and generated very few or no clonogenic progenitors in liquid cultures in the presence of cytokines. When seeded on layers derived from the murine BM stromal cell line M2-10B4, 38-86% of the cells purified using this approach generated multilineage progenitors acting as LTC-lCs. In a different series of studies, after 5-week culture on human BM stromal cell line L87/4 layers, cells were forced to selected lineage differentiation by culture in the presence of low concentrations of SF and high concentrations of lineage-specific cytokines such as Flt3-ligand (myeloid and pre-B cell differentiation), Tpo (megakaryocytic differentiation). IL-7, and IL-2 (pre-B and NK differentiation). After 12-day culture under these conditions, generation of myeloid, pre-B, megakaryocytic, and NK progenitors was assessed by immunohistochemistry, flow cytometry, and mRNA expression of CD7, 14, 19, 41b, S6, and 61. We conclude that this procedure for multilineage progenitor cell purification is simple and effective and could have major implications for gene transfer and stem cell transplantation.

Cord blood plasma-mediated ex vivo expansion of hematopoietic progenitor cells.

Cord blood (CB) plasma has previously been found to augment the replating capacity of CB-derived hematopoietic progenitors. In the present study, we observed a 2.8 +/- 0.3 and a 1.8 +/- 0.2-fold expansion of CFU-GM in cultures of CB cells without growth factors but supplemented by CB plasma or maternal blood (MB) plasma, respectively. In the absence of growth factors, CFU-GM expansion did not occur in CB cell cultures supplemented with fetal calf serum (FCS), peripheral blood (PB) plasma, PB plasma plus concentrations of IL-6 similar to those previously reported in CB plasma and in bone marrow (BM) cell cultures supplemented with FCS, CB, MB or PB plasma. In the presence of stem cell factor (SCF), IL-3 and IL-11, an expansion of CFU-GM was observed in CB and BM cell cultures supplemented by either CB, MB or PB plasma or FCS. Nevertheless, progenitor expansion was significantly superior in CB cell cultures supplemented by CB plasma (8.7 +/- 0.7, 2.4 +/- 0.3 and 2.6 +/- 0.3-fold expansions for CFU-GM, BFU-E and CFU-Mix, respectively). In conclusion, an unknown factor(s) present in CB plasma, probably capable of crossing the placenta, has an effect alone and in the presence of growth factors in supporting ex vivo expansion of CB progenitors.

A quality system for placental blood banking.

A Quality System for Placental Blood Banking aimed at the transplantation of haematopoietic stem cells to related and unrelated allogeneic recipients is described. It includes the organizational structure, procedures, processes and resources needed to implement quality management. The Quality System described in this article is based on ISO 9002, a model for quality assurance in production, installation and servicing developed in 1987 and revised in 1994 by the International Organization for Standardization. ISO 9002 includes 20 clauses that provide guidance for the implementation of the Quality System. The development of the Quality System is started by the Placental Blood Bank Medical Director with the definition of a General Quality Plan including: (1) the written description of the Mission, Objectives, Technical and Organizational Policies, and Staff Organization Chart; (2) the definition and acquisition of adequate financial, human and structural resources; (3) the appointment of a Quality System Head, who must identify the Placental Blood Banking process together with the Placental Blood Bank personnel; implement a documentation plan; identify quality indicators; start regular internal audit; report audit results to the Medical Director for review. Following staff training and qualification, the Quality System is launched. The Placental Blood Bank can then undergo audit by an external inspector and be finally certified for compliance to ISO 9002. The Quality System must be maintained and subjected to external audit at regular intervals so that certification is confirmed.

Evaluation of the effect of cryopreservation on ex vivo expansion of hematopoietic progenitors from cord blood.

In previous studies, we identified a cytokine cocktail including thrombopoietin, Flt-3 ligand, interleukin (IL)-6 and IL-11 in serum-free medium, suitable to induce significant and sustained ex vivo expansion of primitive hematopoietic stem cells (HSCs) from cord blood (CB) for up to 10 weeks. The aim of the present study was to evaluate the effects of cryopreservation on ex vivo expansion of HSCs and their committed progenitors. CD34+ cells were purified from CB units, each of which was processed in part as such and in part as cryopreserved and thawed, then expanded for 5 weeks in serum-free medium with the cytokine cocktail described above. We determined the number of nucleated cells (NC), CD34+, CD34+/38(-)/33(-), CD34+/61+, CD61+ cells and the clonogenic potential. After 2 weeks the median fold expansion of NC, CD34+ and CD34+/38(-)/33(-) cells was around two log both with fresh and cryopreserved CB and the expansion continued similarly until week 5. Our data suggest that this serum free protocol induces similar ex vivo expansion of HSCs and their committed progenitors from both fresh and cryopreserved CB. Our findings can be useful in view of clinical applications, since CB used for transplantation is stored in the cryopreserved state.

Gene transfer-mediated generation of drug-resistant hemopoiesis.

Autologous- or allogeneic-bone marrow transplantation are increasingly used to overcome the myelosuppressive effects of high dose chemotherapy administered to cancer patients. Transfer of the multidrug resistance (MDR) gene in hemopoietic progenitors has been proposed as a tool to administer higher and possibly more curative doses of chemotherapy. Murine models have demonstrated that retrovirus-mediated MDR transfer in bone marrow cells can render animals resistant to myeloablative doses of Taxol, and in vitro studies have shown that MDR-transduced human CD34+ cells can generate drug-resistant multipotential hemopoietic progenitors such as long term culture-initiating cells. Given these results, phase I clinical trials are currently under way to evaluate feasibility and treatment-related toxicity of MDR gene transfer in cancer patients by means of safe retroviral vectors. Finally, Taxol treatment of MDR transduced mice and human CD34+ cells have indicated that MDR is a dominant selectable marker in vitro and in vivo, and vectors carrying both MDR and non selectable genes such as beta-globin or glucocerebrosidase could be used in the next future for gene therapy of inherited disorders like thalassemia or Gaucher disease.

Psychological and symptom distress in terminal cancer patients with met and unmet needs.

This study identified the needs of terminal cancer patients, investigated the factors associated with unmet needs, and assessed psychological and symptom distress associated with unsolved needs. Ninety-four patients were randomly selected from 324 patients admitted for palliative care in 13 Italian centers. Two self-administered questionnaires (the Symptom Distress Scale and the Psychological Distress Inventory) were administered to all the patients. Patients needs were identified using a semi-structured interview, aimed at exploring five areas: physiological needs, safety needs, love and belonging needs, self-esteem needs, self-fulfillment needs. A content analysis of the answers defined 11 needs, and identified patients with unmet needs. The most frequent unmet needs were symptom control (62.8%), occupational functioning (62.1%), and emotional support (51.7%). The less frequently reported needs were those related to personal care (14.6%), financial support (14.1%), and emotional closeness (13.8%). Low functional state was significantly associated with a high proportion of patients with unmet needs of personal care, information, communication, occupational functioning, and emotional closeness. Patients with unmet needs showed significantly higher psychological and symptom distress for most needs. This study provides some suggestions about the concerns that should be carefully considered during the late stage of cancer.

Correlation between inorganic (heavy metals) and organic (PCBs and PAHs) micropollutant concentrations during sewage sludge composting processes.

The nature and congener composition of PCBs and PAHs present in sewage sludge composting processes was investigated. These studies included analysis of the most significant process parameters (such as pH, temperature, weight percentage variation) and in addition heavy metals whose typical composting speciation and behaviour were also considered in order to better understand organic compound time profiles. The significant correlation found between Pb, Cd, Cu and PCBs and between PAHs and Hg implies that quite a strong adsorption of PCBs onto organic matter takes place and also provides evidence for the volatilisation of PAHs. Chemical characteristics of inorganic species and organic compounds are summarised to account for the observed correlation and time trend profiles. Moreover, single congener concentrations demonstrate that the number of Cl substituents for PCBs and condensed benzene rings for PAHs determine to what extent they can be broken down for biodegradation and removed through volatilisation respectively.

Sentinel node biopsy in breast cancer: the experience of Brescia Civic Hospital.

The accuracy of the sentinel node technique in the evaluation of axillary node involvement in breast cancer was evaluated in 83 consecutive patients with monofocal T1-2 carcinoma, who were clinically N0 and who underwent lymphoscintigraphy with 99mTc-colloid integrated with intraoperative sentinel node detection by a portable probe. Lymphoscintigraphy revealed at least one sentinel node in 75 patients (90.4%), always identified by the probe. In eight patients (9.6%) the sentinel node was detected neither by lymphoscintigraphy nor by the probe. All removed lymph nodes were analyzed by hematoxylin-eosin histology and the sentinel node by immunostaining. In 28/75 patients (37.3%) at least one metastatic axillary lymph node was detected; in 16 of the 28 N+ subjects (57%) only the sentinel node was positive. The false negative rate (sentinel node negative/other axillary lymph nodes positive) was 17.85% (5/28 patients). In 9/23 patients (39%) micrometastases were found in the sentinel node only. In conclusion, specific sentinel node positivity in 57% of cases supports the validity of the sentinel node concept. Moreover, nine patients would have been considered N0 by standard hematoxylin-eosin histology without sentinel node-aided immunostaining. A 17.8% false negative rate calls for caution in patients with negative sentinel nodes.

Cord blood banking for stem cell transplant.

Umbilical cord blood has been recently used as a source of hematopoietic progenitor cells for transplantation of pediatric patients. This study was performed to evaluate the feasibility of a cord blood bank for unrelated transplant. When the umbilical cord was clamped within 20 seconds after delivery, it was possible to collect 86 +/- 25 ml of cord recipients with more than 2000 CFU-GM/kg; 53% of cord blood samples were found to contain enough CFU-GM for engraftment in 50-70 kg adult patients.

[The role of intraoperative parathyroid hormone assay in the surgical management of hyperparathyroidism]. / II ruolo della valutazione del paratormone intraoperatorio nel trattamento chirurgico degli iperparatiroidismi.

Surgical management of primary hyperparathyroidism has undergone several chances in recent years and historically has required bilateral neck exploration with identification of the parathyroid adenoma together with three normal glands. The intraoperative hormone assay allows a more limited procedure by confirming complete removal of hypersecreting tissue. The Authors report surgical treatment of 24 consecutive hyperparathyroidism and conclude that evaluation of intraoperative hormone assay accurately predicts the determination of adequacy of resection and the correct outcome of surgery in patients with parathyroid adenomas.

Pre-culturing human adipose tissue mesenchymal stem cells under hypoxia increases their adipogenic and osteogenic differentiation potentials.

OBJECTIVES: Hypoxia is an important factor in many aspects of stem-cell biology including their viability, proliferation, differentiation and migration. We evaluated whether low oxygen level (2%) affected human adipose tissue mesenchymal stem-cell (hAT-MSC) phenotype, population growth, viability, apoptosis, necrosis and their adipogenic and osteogenic differentiation potential. MATERIALS AND METHODS: hAT-MSCs from four human donors were cultured in growth medium under either normoxic or hypoxic conditions for 7 days and were then transferred to normoxic conditions to study their differentiation potential. RESULTS: Hypoxia enhanced hAT-MSC expansion and viability, whereas expression of mesenchymal markers such as CD90, CD73 and endothelial progenitor cell marker CD34, remained unchanged. We also found that pre-culturing hAT-MSCs under hypoxia resulted in their enhanced ability to differentiate into adipocytes and osteocytes. CONCLUSIONS: This protocol could be useful for maximizing hAT-MSC potential to differentiate in vitro into the adipogenic and osteogenic lineages, for use in plastic and reconstructive surgery, and in tissue engineering strategies.

Autologous fat injection to face and neck: from soft tissue augmentation to regenerative medicine.

Minimally-invasive autologous fat injection of the head and neck region can be considered a valid alternative to major invasive surgical procedures both for aesthetic and functional purposes. The favourable outcomes of autologous fat injection in otolaryngological practice are due to the filling of soft tissue and, mainly, to the potential regenerative effect of adipose-derived mesenchymal stem cells. Herewith, some important biological preliminary remarks are described underlying the potential of autologous fat injection in regenerative medicine, and personal experience in using it for both consolidated clinical applications, such as fat grafting to the face and vocal fold augmentation in the treatment of glottic incompetence, and more recent applications including the treatment of post-parotidectomy Frey syndrome and velopharyngeal insufficiency.

Intracerebroventricular administration of human umbilical cord blood cells delays disease progression in two murine models of motor neuron degeneration.

The lack of effective drug therapies for motor neuron diseases (MND), and in general for all the neurodegenerative disorders, has increased the interest toward the potential use of stem cells. Among the cell therapy approaches so far tested in MND animal models, systemic injection of human cord blood mononuclear cells (HuCB-MNCs) has proven to reproducibly increase, although modestly, the life span of SOD1G93A mice, a model of familial amyotrophic lateral sclerosis (ALS), even if only few transplanted cells were found in the damaged areas. In attempt to improve the potential efficacy of these cells in the central nervous system, we examined the effect and distribution of Hoechst 33258-labeled HuCB-MNCs after a single bilateral intracerberoventricular injection in two models of motor neuron degeneration, the transgenic SOD1G93A and wobbler mice. HuCB-MNCs significantly ameliorated symptoms progression in both mouse models and prolonged survival in SOD1G93A mice. They were localized in the lateral ventricles, even 4 months after administration. However, HuCB-MNCs were not found in the spinal cord ventral horns. This evidence strengthens the hypothesis that the beneficial role of transplanted cells is not due to cell replacement but is rather associated with the production and release of circulating protective factors that may act both at the central and/or peripheral levels. In particular, we show that HuCB-MNCs release a series of cytokines and chemokines with antiinflammatory properties that could be responsible of the functional improvement of mouse models of motor neuron degenerative disorders.

Met-driven invasive growth involves transcriptional regulation of Arhgap12.

Invasive growth is a complex biological program triggered by hepatocyte growth factor (HGF) through its tyrosine kinase receptor encoded by the Met proto-oncogene. The program involves-besides proliferation-cell dissociation, motility and invasiveness, controlled by intracellular signals impinging on PI3K and on the small G-proteins of the Rac/Rho family. The mechanism(s) unbalancing Rac/Rho activation are still not completely clarified. Here, we describe a functional link between HGF and Arhgap12, a gene encoding a previously uncharacterized protein of the RhoGAP family. We identified Arhgap12 as a transcriptional target of HGF, through a novel gene trapping strategy. We found that Arhgap12 mRNA and protein are robustly suppressed by HGF treatment, but not by serum. Arhgap12 displayed GTPase activating protein (GAP) activity towards Rac1 and, upon overexpression, impaired cell scattering, invasion and adhesion to fibronectin in response to HGF. Consistently, Arhgap12 silencing by RNA interference selectively increased the scatter and adhesion responses. These data show that HGF-driven invasive growth involves transcriptional regulation of a Rac1-specific GAP.

Assessment of selective homing and contribution to vessel formation of cryopreserved peripherally injected bone marrow mononuclear cells following experimental myocardial damage.

In view of a potential clinical use we aimed this study to assess the selective homing to the injured myocardium and the definitive fate of peripherally injected labeled and previously cryopreserved Bone Marrow Mononuclear cells (BMMNCs). The myocardial damage (cryoinjury) was produced in 59 rats (45 treated, 14 controls). From 51 donor rats 4.4 x 10(9) BMMNCs were isolated and cryopreserved (slow-cooling protocols); the number of CD34+ and the viability of pooled cells was assessed by flow-cytometry analysis before and after cryopreservation and simulated delivery through a 23G needle. Seven days after injury, BMMNCs were thawed, labeled with PKH26 dye and peripherally injected (20 x 10(6) cells in 500 microl) in recipient rats. Two weeks after experimental injury, the heart, lungs, liver, kidneys, spleen and thymus were harvested to track transplanted cells. Except a small amount in the spleen, PKH26+ cells were found only in the infarcted myocardium of the treated animals. Typical vascular structures CD34+ were found in the infarcted areas of all animals; treated rats showed a significantly higher number of these structures if compared with untreated. Morphological ultra-structural examination of infarcted areas confirmed in treated rats the presence of early-stage PKH26+ vascular structures derived from injected BMMNCs. The estimated mean CD34+ cells loss due to the cryopreservation procedure and to the system of delivery was 0.24% and 0.1%, respectively, confirming the feasibility of the procedure. This study supports the possible therapeutic use of cryopreserved peripherally injecetd BMMNCs as a source of CD34+ independent vascular structures following myocardial damage.