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Background A growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). Results The GMP-compliant protocol defined in this work allows at least 4.11 × 109 Treg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function. Conclusions These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases.

During the last decade it has been demonstrated that mesenchymal progenitors are present and can be isolated also from cord blood (CB). Recently, we managed to set up a standard protocol allowing the isolation of mesenchymal stromal cells (MSCs) with high proliferative potential and multiple differentiation capabilities, whereas the generation rate of MSC-initiating colonies could still be further improved. Herein, we strikingly succeeded in defining some simple and basic culture conditions based on the use of a chemically defined medium that increased the colony isolation efficiency up to almost 80% of processed CB units. Importantly, this result was achieved irrespective of CB unit white blood cell content and time elapsed from delivery, two limiting parameters involved with processing CB units. Thus, this high efficiency is guaranteed without strict selection of the starting material. In addition, since we are profoundly concerned about how different culture conditions can influence cell behavior, we devoted part of this study to in-depth characterization of the established CB-MSC populations to confirm their stemness features in this novel isolation and culture system. Therefore, an extended study of their immunophenotype, including classical pericytic markers, and a detailed molecular analysis addressing telomere length and also stemness-related microRNA contribution were performed. In summary, we propose a straightforward, extremely efficient, and reliable approach to isolate and expand thoroughly characterized CB-MSCs, even when poor-quality CB units are the only available source, or there is no space for an isolation to fail.

In a recent work, our group showed the existence of two distinct mesenchymal stem cell (MSC) subsets within human umbilical cord blood. One less proliferative and short-living (SL-CBMSC), the other with higher growth rate and long-living (LL-CBMSC), and therefore better suited for regenerative medicine applications. We examined whether LL-CBMSC possess peculiar paracrine properties able to affect angiogenesis or inflammatory processes. It was shown for the first time that pro-angiogenic, proliferation-stimulating and tissue repairing factors were released at high level not only as soluble cytokines, but also as mRNA precursors embedded in membrane vesicles. The combination of this primary (proteic factors interacting with surface receptors) and delayed (mRNA transferred and translated via vesicle fusion and cargo release) interaction in endothelial target cells resulted in strong blood vessel induction with the development of capillary-like structures. In addition, LL-CBMSC dynamically modulated their release of pro-angiogenic and anti-inflammatory factors in an in vitro model of damage. In conclusion, LL-CBMSC synthesize and secrete multiple factors that may be attuned in response to the status of the target cell, a crucial requisite when paracrine mechanisms are needed at onset of tissue regeneration.

Mesenchymal stromal cells (MSC) for cellular therapy in European Union are classified as advanced therapy medicinal products (ATMPs), and their production must fulfill the requirements of Good Manufacturing Practice (GMP) rules. Despite their classification as medicinal products is already well recognized, there is still a lack of information and indications to validate methods and to adapt the noncompendial and compendial methods to these peculiar biological products with intrinsic characteristics that differentiate them from classic synthetic or biologic drugs. In the present paper, we present the results of the validation studies performed in the context of MSC development as ATMPs for clinical experimental use. Specifically, we describe the validation policies followed for sterility testing, endotoxins, adventitious viruses, cell count, and immunophenotyping. Our work demonstrates that it is possible to fully validate analytical methods also for ATMPs and that a risk-based approach can fill the gap between the prescription of the available guidelines shaped on traditional medicinal products and the peculiar characteristics of these novel and extremely promising new drugs.

Background The simultaneous measurement of multiple cytokines in parallel by using multiplex proteome arrays (MPA) is of great interest to understanding the inflammatory response following myocardial infarction; however, since cytokines are pleiotropic and redundant, increase of information throughput (IT) attained by measuring multiple cytokines remain to be determined. We aimed this study to assess the IT of an MPA system designed to assess 8 cytokines – commercially available at the time of the study – serum levels, before (control state) and after experimental myocardial cryoinjury (activated state) in rats. Methods By assuming that redundant information do not generally increase the IT, we derived Entropy ( H ) and Redundancy ( R ) of information by using formulas of Shannon modified accordingly, where a high IT (high H and low R ) corresponds to a low level of correlation between cytokines and vice versa for a low IT. The maximum theoretical level of IT and the contribution of each cytokine were also estimated. Results In control state, no significant correlations were found between cytokines showing high IT; on the contrary, in activated state, several significant correlations were found supporting a complex cross-talk pattern between cytokines with low IT. Using as reference the maximum theoretical level of IT, in activated state, H was reduced of 67.0% and R was increased of 77.4% supporting a reduction of IT. Furthermore, the contribution of individual cytokines to H value of MPA was variable: in control state, IL-2 gave the most contribution to H value, conversely during activated state IL-10 gave most contribution. Finally during activated state, IL-1β was the only cytokine strongly correlated with values of all other cytokines, suggesting a crucial role in the inflammatory cascade. Conclusions Paradoxically, by analyzing an MPA system designed for redundant analytes such as cytokines, translating the Shannon's information theory from the field of communication to biology, the IT system in our model deteriorates during the activation state by increasing its redundancy, showing maximum value of entropy in the control conditions. Finally, the study of the mutual interdependence between cytokines by the contribution to the IT may allow formulating alternative models to describe the inflammatory cascade after myocardial infarction.

Background aims Growing evidence supports the therapeutic potential of bone marrow (BM)-derived stem/progenitor cells for end-stage liver disease (ESLD). We recently demonstrated that CD133+ stem/progenitor cell (SPC) reinfusion in patients with ESLD is feasible and safe and improve, albeit transiently, liver function. However, the mechanism(s) through which BM-derived SPCs may improve liver function are not fully elucidated. Methods Here, we characterized the circulating SPCs compartment of patients with ESLD undergoing CD133+ cell therapy. Next, we set up an in vitro model mimicking SPCs/liver microenvironment interaction by culturing granulocyte colony-stimulating factor (G-CSF)-mobilized CD133+and LX-2 hepatic stellate cells. Results We found that patients with ESLD show normal basal levels of circulating hematopoietic and endothelial progenitors with impaired clonogenic ability. After G-CSF treatment, patients with ESLD were capable to mobilize significant numbers of functional multipotent SPCs, and interestingly, this was associated with increased levels of selected cytokines potentially facilitating SPC function. Co-culture experiments showed, at the molecular and functional levels, the bi-directional cross-talk between CD133+ SPCs and human hepatic stellate cells LX-2. Human hepatic stellate cells LX-2 showed reduced activation and fibrotic potential. In turn, hepatic stellate cells enhanced the proliferation and survival of CD133+ SPCs as well as their endothelial and hematopoietic function while promoting an anti-inflammatory profile. Discussion We demonstrated that the interaction between CD133+ SPCs from patients with ESLD and hepatic stellate cells induces significant functional changes in both cellular types that may be instrumental for the improvement of liver function in cirrhotic patients undergoing cell therapy.

Human mesenchymal stem cells (MSCs) are multipotent cells offering valuable hopes for the treatment of degenerative diseases. MSCs can be found among differentiated cells in many tissues and organs but, unfortunately, their phenotypic similarity hinders a robust cell characterization and discrimination from diverse tissue harvests. MicroRNAs (miRNAs) are crucial managers of gene expression with intriguing and still poorly known roles in stem cell maintenance and differentiation. To identify miRNAs that can discriminate among MSCs, we performed a whole-genome comparative miRNA expression profiling analysis on adipose (AD), bone marrow (BM) and cord blood (CB) derived MSCs, all three considered among the most promising in the field of regenerative medicine. miRNA expression patterns were very similar, meeting their extensive phenotypic and functional overlaps. An in-depth comparison of the few most differentially expressed miRNAs allowed the identification of a highly restricted molecular signature consisting of 5 BMMSC, 11 ADMSC and 11 CBMSC specific miRNAs. Functional analysis of their validated targets allowed the identification of an "environmental-niche memory" for BMMSC and an "epithelial" commitment for ADMSC, providing new insights into the molecular mechanisms discriminating between these MSCs, a crucial element to identify the most appropriate stem cell source for clinical application.

Cell-based therapies promise important developments for regenerative medicine purposes. Adipose tissue and the adipogenic process has become central to an increasing number of translational efforts in addition to plastic and reconstructive surgical applications. In recent experimental clinical trials, human mesenchymal stem cells (MSC) have been proven to be well tolerated because of their low immunoreactivity. MSC are multipotent cells found among mature cells in different tissues and organs with the potentiality to differentiate in many cell types, including osteocytes, chondrocytes and adipocytes, thus being a suitable cell source for tissue engineering strategies. We compared the adipogenic potential of MSC originated from two adult sources as fat pads and bone marrow, and from four foetal sources as umbilical cord blood, Wharton's jelly, amniotic fluid and preterm umbilical cord perivascular cells. Surprisingly, adult MSC displayed higher differentiation capacities confirmed by gene expression analysis on a selected panel of adipogenesis-related genes. Further, an in-depth molecular analysis highlighted the early and vigorous activation of the PPARγ transcription factor-cascade in adipose-derived MSC that resulted to be both delayed and reduced in foetal MSC accounting for their lack of adipogenic potential. Thus, MSC show a different degree of phenotypic plasticity depending on the source tissue, that should be taken into consideration for the selection of the most appropriate MSC type for specific tissue regeneration purposes.

Hematopoietic stem and progenitor cells (HSPCs) emerge and develop adjacent to blood vessel walls in the yolk sac, aorta-gonad-mesonephros region, embryonic liver, and fetal bone marrow. In adult mouse bone marrow, perivascular cells shape a "niche" for HSPCs. Mesenchymal stem/stromal cells (MSCs), which support hematopoiesis in culture, are themselves derived in part from perivascular cells. In order to define their direct role in hematopoiesis, we tested the ability of purified human CD146(+) perivascular cells, as compared with unfractionated MSCs and CD146(-) cells, to sustain human HSPCs in coculture. CD146(+) perivascular cells support the long-term persistence, through cell-to-cell contact and at least partly via Notch activation, of human myelolymphoid HSPCs able to engraft primary and secondary immunodeficient mice. Conversely, unfractionated MSCs and CD146(-) cells induce differentiation and compromise ex vivo maintenance of HSPCs. Moreover, CD146(+) perivascular cells express, natively and in culture, molecular markers of the vascular hematopoietic niche. Unexpectedly, this dramatic, previously undocumented ability to support hematopoietic stem cells is present in CD146(+) perivascular cells extracted from the nonhematopoietic adipose tissue.

Growing evidence supports the therapeutic potential of bone marrow (BM)-derived stem/progenitor cells for end-stage liver disease (ESLD). We recently demonstrated that CD133Here, we characterized the circulating SPCs compartment of patients with ESLD undergoing CD133We found that patients with ESLD show normal basal levels of circulating hematopoietic and endothelial progenitors with impaired clonogenic ability. After G-CSF treatment, patients with ESLD were capable to mobilize significant numbers of functional multipotent SPCs, and interestingly, this was associated with increased levels of selected cytokines potentially facilitating SPC function. Co-culture experiments showed, at the molecular and functional levels, the bi-directional cross-talk between CD133We demonstrated that the interaction between CD133

Multi-lineage progenitors, e.g. mesenchymal stem cells, persist in adult developed organs, making a windfall for the cell therapist but an enigma for stem cell biologists. Recent results from our own and other laboratories show that the ancestor of these elusive adult stem cells is likely to be found in the perivascular area, explaining the ubiquitous distribution of these cells in the body. We have prospectively identified and purified vascular pericytes in multiple human organs and shown that these cells are potent mesodermal progenitors that give rise to genuine mesenchymal stem cells in culture. Pericytes can differentiate into diverse cell lineages, but also secrete multiple paracrine growth factors/cytokines, which likely explains in part their robust regenerative potential.

Objectives: Adipose tissue in vocal fold lipoinjection is currently used to treat patients affected by laryngeal hemiplegia or anatomical defects. The aim of this study has been to evaluate the efficacy of this clinical strategy, by long‐term follow‐up of the patients and to investigate whether the fat samples used to treat them contain a stem cell population with a wide differentiation potential. Materials and methods: Fat samples harvested from 12 patients affected by severe breathy dysphonia who had undergone vocal fold lipoinjection were analysed by immunocytochemistry, by flow cytometry and reverse transcription‐polymerase chain reaction, and the isolated adipose derived mesenchymal stem cells (ADMSCs) were evaluated in order to define their ability to produce soluble factors possibly involved in tissue regeneration, and to differentiate towards different lineages. Results: ADMSCs were efficiently and successfully isolated from all of the samples. They were positive for SSEA‐4, an embryonic marker recently identified on bone marrow MSCs and which could explain their high differentiation plasticity. Molecular analysis showed that these cells also expressed Oct‐4, Runx‐1 and ABCG‐2, which characterize the stem cell state, and a number of other specific lineage markers. Flow cytometry revealed mesenchymal markers expressed on ADMSCs and identified a subpopulation characterized by CD146(+)/34(−)/45(−) cells consistent with perivascular/pericyte‐like cells. Osteogenic, adipogenic and endothelial tissue differentiation were obtained. Conclusions: Our results confirmed the therapeutic efficacy of this clinical approach and showed that adipose tissue, administered to patients in order to restore glottic competence, contains mesenchymal stem cells.

Introduction.Crohn’s disease (CD) is a chronic inflammatory auto-immune bowel disease (IBD) characterized by segmental transmural inflammation. In 1/3 of patients, CD is complicated by perianal fistulas which rarely heal spontaneously or after medical treatment. Disappointing results have been obtained in anti-inflammatory drug-based (TNF-inhibitors) clinical trials so that 2/3 of patients either do not respond or loose their response. Pre-clinical studies and preliminary clinical trials have suggested that, due to their immunomodulatory properties, mesenchymal stromal cell (MSC)-based therapy hold promising potential in the IBD treatment. Ongoing studies are aimed at evaluating the proper dose and mode of MSC administration needed for more effective results. Human platelet lysate (HPL) has been proposed as a valid FBS substitute to safely expand MSCs for therapeutic purpose. A recent paper demonstrates that in MSCs HPL triggers in the secretion of factors enhancing the initial inflammatory response to the injury, a key element in the wound healing process. We recently set up a novel murine model of IBD. This model, conversely to that adopted so far, does not lead to animal death, but it presents a mild intestinal damage which makes it the most accurate to study therapeutic agent effects. The aim of our study was to characterize the effects of MSCs isolated from adipose tissue of CD patients and resuspended in HPL (adCD-MSCs/HPL) in our IBD model. Methods.Colitis was induced in C57BL/6J mice by administration of 1,5% dextran sulphate sodium (DSS) in tap water. adCD-MSCs, w/wo HPL, were administrated via enema for 3 times (1x10^6 cells/mouse/time) every other day starting on day+7 from DSS induction. Results.We found that adCD-MSCs can be easily isolated and expanded from CD patients, showing typical MSC properties. We demonstrated that HPL potentiated the adCD-MSC efficacy in ameliorating DSS-induced colitis in our model. Indeed, DSS+adCD-MSC/HPL-treated mice group showed a reduced weight loss compared to DSS+PBS mice group. Disease Activity Index, including inflammation clinical parameters (weight loss, diarrohea, rectal bleeding), was lower in DSS+adCD-MSC/HPL-treated mice at each time point starting from the end of MSC treatment. Histopathological examination of the colon distal and proximal tracts showed that adCD-MSCs/HPL significantly reduced the extension and the severity of the inflamed area and the inflammatory cells infiltration due to DSS treatment. We also found that adCDMSCs/HLP decreased systemic inflammatory mediators levels (IL-1β, IL-6, IL-17, IFN-γ, TNF-α, IL-10) in the colitic mice plasma. Finally, we set up a novel method to label adCD-MSCs. We found that Nile red staining was very efficient and stable and preserved MSC biological properties. Thus, Nile red staining can be efficiently used to track injected MSCs. Conclusions.Overall, this study validate a novel promising adCD-MSC/HPL-based therapy for refractory CD treatment.

Background The trophic, anti-apoptotic and regenerative effects of bone marrow mesenchymal stromal cells (MSC) may reduce neuronal cell loss in neurodegenerative disorders. Methods We used MSC as a novel candidate therapeutic tool in a pilot phase-I study for patients affected by progressive supranuclear palsy (PSP), a rare, severe and no-option form of Parkinsonism. Five patients received the cells by infusion into the cerebral arteries. Effects were assessed using the best available motor function rating scales (UPDRS, Hoehn and Yahr, PSP rating scale), as well as neuropsychological assessments, gait analysis and brain imaging before and after cell administration. Results One year after cell infusion, all treated patients were alive, except one, who died 9 months after the infusion for reasons not related to cell administration or to disease progression (accidental fall). In all treated patients motor function rating scales remained stable for at least six-months during the one-year follow-up. Conclusions We have demonstrated for the first time that MSC administration is feasible in subjects with PSP. In these patients, in whom deterioration of motor function is invariably rapid, we recorded clinical stabilization for at least 6 months. These encouraging results pave the way to the next randomized, placebo-controlled phase-II study that will definitively provide information on the efficacy of this innovative approach. Trial registration ClinicalTrials.gov NCT01824121

Platelet gel derived from peripheral blood is widely applied in many clinical fields of surgery as biomaterial containing growth factors with high proliferative properties. In 2010, we studied and patented a platelet gel derived from cord blood. In this study, due to the crucial role of the factors released by the platelet gel, we first extended the characterization of its releasate. Using a wide proteomic array and splitting the two components of the releasate, that is, platelets and plasma, we have been able to study their growth factor content. Interestingly, we discovered high levels of hormones and molecules able to support tissue growth in the cord blood platelet gel releasate and, in addition, higher concentrations of several angiogenic factors if compared with the peripheral blood counterpart. On the contrary, the latter was much richer in inflammatory factors. The second aim of our work was to study the effects on cell culture, immunophenotype, and function of mesenchymal stem cells exposed to these two platelet gel releasates as substitute for the animal serum. Since our findings nicely show that the use of the peripheral versus the cord blood platelet gel releasate can differently influence the mesenchymal stem cell commitment, we can suggest that in addition to its peculiar angiogenic properties cord blood platelet gel releasate shows excellent proliferative properties as cell culture supplement.

Tiziana Montemurro, Mariele Viganò, Silvia Budelli, Elisa Montelatici, Cristiana Lavazza, Luigi Marino, Valentina Parazzi, Lorenza Lazzari, Rosaria GiordanoCell Factory, Unit of Cell Therapy and Cryobiology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano, ItalyAbstract: In the 21st century scenario, new therapeutic tools are needed to take up the social and medical challenge posed by the more and more frequent degenerative disorders and by the aging of population. The recent category of advanced therapy medicinal products has been created to comprise cellular, gene therapy, and tissue engineered products, as a new class of drugs. Their manufacture requires the same pharmaceutical framework as for conventional drugs and this means that industrial, large-scale manufacturing process has to be adapted to the peculiar characteristics of cell-containing products. Our hospital took up the challenge of this new path in the early 2000s; and herein we describe the approach we followed to set up a pharmaceutical-grade facility in a public hospital context, with the aim to share the solutions we found to make cell therapy compliant with the requirements for the production and the quality control of a high-standard medicinal product.Keywords: advanced therapy medicinal product, good manufacturing practices, stem cells

Adipose-derived mesenchymal stem cells (ADMSCs) are an ideal population for regenerative medical application. Both the isolation procedure and the culturing conditions are crucial steps, since low yield can limit further cell therapies, especially when minimal adipose tissue harvests are available for cell expansion. To date, a standardized procedure encompassing both isolation sites and expansion methods is missing, thus making the choice of the most appropriate conditions for the preparation of ADMSCs controversial, especially in view of the different applications needed. In this study, we compared the effects of three different commercial media (DMEM, aMEM, and EGM2), routinely used for ADMSCs expansion, and two supplements, FBS and human platelet lysate, recently proven to be an effective alternative to prevent xenogeneic antibody transfer and immune alloresponse in the host. Notably, all the conditions resulted in being safe for ADMSCs isolation and expansion with platelet lysate supplementation giving the highest isolation and proliferation rates, together with a commitment for osteogenic lineage. Then, we proved that the high ADMSC hematopoietic supportive potential is performed through a constant and abundant secretion of both GCSF and SCF. In conclusion, this study further expands the knowledge on ADMSCs, defining their identity definition and offers potential options for in vitro protocols for clinical production, especially related to HSC expansion without use of exogenous cytokines or genetic modifications.

In regenerative medicine, human cord blood-derived multipotent mesenchymal stromal cells (CBMSCs) stand out for their biological peculiarities demonstrated in in vitro and in vivo preclinical studies. Here, we present our 9-year experience for the consistent isolation of CBMSCs. Although nearly one CB unit out of two retains the potential to give rise to MSC colonies, only 46% of them can be cultured till low passages (P≥4), but one-fourth of those reaches even higher passages (P≥8). Subsequent characterization for morphological, clonal, differentiation, and proliferation properties revealed two divergent CBMSC behaviors. In particular, a cumulative population doublings cut-off (CPD=15) was identified that undoubtedly distinguishes two growth curves, and different degrees of commitment toward osteogenesis were observed. These data clearly show the existence of at least two distinct CBMSC subsets: one mainly short-living and less proliferative (SL-CBMSCs), the other long-living, with higher growth rate, and, very importantly, with significantly (P≤0.01) longer telomere (LL-CBMSCs). Moreover, significant differences in the immunoprofile before seeding were found among CB units giving rise to LL-CBMSCs or SL-CBMSCs or showing no colony formation. Finally, all the aforementioned results provided a peculiar and useful set of parameters potentially predictive for CBMSC culture outcome.

evaluation. The area at risk (AR) was assessed either by Evans blue or by fluorescent microspheres (FMic). IS was expressed as percentage of AR not uptaking TTC. Stem cell engraftment was evaluated at 30 days by immunohistochemistry. Results: Calculation of infarcted area resulted easier, faster and more reliable when using the FMic compared with Evans blue. In many samples, the dye permeated into the tissue colouring the heart in blue and resulting in a difficult identification of non-ischemic area. At the contrary, delimitation of the AR was easier and reproducible when using FMic. Histology showed high quality Hematoxylin&Eosin and α-sarcomeric actin only in hearts injected with FMic. At two days, both tracking methods were reliable. At day 30, cells labelled with the V-CM-Dil were promptly identified, while the GFP signal was dim even when the fluorescence was amplified using anti-GFP antibodies. Conclusions: Our study provides evidences suggesting that the use of FMic represents a better alternative compared with Evans blue for AR/IS quantification. We also demonstrated that lipophilic dyes seem to work better than GFP for cell tracking.

Dental pulp stem cells (DPSC), a cell type of mesenchymal origin showing high proliferation and plasticity, are an emerging source of adult stem cells offering interesting features in view of potential applications in regenerative medicine. These features prompted us to develop a new method to cryopreserve DPSC inside a whole tooth, thus avoiding the need to purify the cells before cryopreservation and reducing the initial costs and workload of tooth banking. In this study we cryopreserved 4 human deciduous whole teeth after digging micro-channels into the tooth with an Nd:YAG laser beam (laser piercing) to allow the cryopreservative to reach the dental pulp and preserve the cells at -80°C. Then, we isolated, expanded and characterized in vitro the stem cells after tooth thawing and mechanical fracture. In parallel, we characterized cells extracted from 2 teeth cryopreserved without laser piercing and from 4 non cryopreserved, non laser pierced, freshly fractured teeth. Our data demonstrate that DPSC isolated from laser pierced cryopreserved teeth show mesenchymal stem cells morphology, immunophenotype, viability and proliferation rate similar to those of cells isolated from fresh, non cryopreserved teeth, whereas significant loss of cell viability and proliferation rate was shown by cells isolated from teeth cryopreserved without laser piercing. These data support the use of this method for prospective whole tooth banking.

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.

Mesenchymal stem cells (MSC) have been derived from different cultured human tissues, including bone marrow, adipose tissue, amniotic fluid and umbilical cord blood. Only recently it was suggested that MSC descended from perivascular cells, the latter being defined as CD146 � neuro-glial proteoglycan (NG)2 � platelet-derived growth factor-R� � ALPCD34 - CD45 - von Willebrand factor (vWF) - CD144 - . Herein we studied the properties of perivascular cells from a novel source, the foetal human umbilical cord (HUC) collected from pre-term newborns. By immunohistochemistry and flow cytometry we show that pre-term/foetal HUCs contain more perivascular cells than their full- term counterparts (2.5% versus 0.15%). Moreover, foetal HUC perivascular cells (HUCPC) express the embryonic cell markers specific embryonic antigen-4, Runx1 and Oct-4 and can be cultured over the long term. To further confirm the MSC identity of these cultured perivascular cells, we also showed their expression at different passages of antigens that typify MSC. The multilineage differentiative capac- ity of HUCPC into osteogenic, adipogenic and myogenic cell lineages was demonstrated in culture. In the perspective of a therapeutic appli- cation in chronic lung disease of pre-term newborns, we demonstrated the in vitro ability of HUCPC to migrate towards an alveolar type II cell line damaged with bleomycin, an anti-cancer agent with known pulmonary toxicity. The secretory profile exhibited by foetal HUCPC in the migration assay suggested a paracrine effect that could be exploited in various clinical conditions including lung disorders.

Hematopoietic stem cells (HSC) isolated from umbilical cord blood (UCB) were treated with ionizing radiation (IR) and sensitivity and IR induced checkpoints activation were investigated. No difference in the sensitivity and in the activation of DNA damage pathways was observed between CD133+ HSC and cells derived from them after ex vivo expansion. Chk1 protein was very low in freshly isolated CD133+ cells, and undetectable in ex vivo expanded UCB CD133+ cells. Chk1 was expressed only on day 3 of the ex vivo expansion. This pattern of Chk1 expression was corroborated in CD133+ cells isolated from peripheral blood apheresis collected from an healthy donor. Treatment with a specific Chk1 inhibitor resulted in a strong reduction in the percentage of myeloid precursors (CD33+) and an increase in the percentage of lymphoid precursors (CD38+) compared to untreated cells, suggesting a possible role for Chk1 in the differentiation program of UCB CD133+ HSC.

Background Bone Marrow (BM) progenitor cells can target the site of myocardial injury, contributing to tissue repair by neovascolarization and/or by a possible direct paracrine effect on the inflammatory cascade. Angiotensin Converting Enzyme inhibitors (ACE-I) are effective in reducing mortality and preventing left ventricular (LV) function deterioration after myocardial infarction. Methods We investigated the short term effects of BM mononuclear cells (BMMNCs) therapy on the pro-inflammatory cytokines (pro-CKs) and on LV remodelling and compared these effects over a standard ACE-I therapy in a rat model of myocardial cryodamage. Forty two adult inbread Fisher-F344 rats were randomized into three groups: untreated (UT; n = 12), pharmacological therapy (ACE-I; n = 14, receiving quinapril), and cellular therapy (BMMNCs; n = 16, receiving BMMNCs infusion). Rats underwent to a standard echocardiogram in the acute setting and 14 days after the damage, before the sacrifice. Pro-CKs analysis (interleukin (IL)1β, IL-6, tumor necrosis factor (TNF)α was performed (multiplex proteome arrays) on blood samples obtained by direct aorta puncture before the sacrifice; a control group of 6 rats was considered as reference. Results Concerning the extension of the infarcted area as well as the LV dimensions, no differences were observed among the animal groups; treated rats had lower left atrial diameters and higher indexes of LV function. Pro-Cks were increased in infarcted-UT rats if compared with controls, and significantly reduced by BMMNCs and ACE-I ; TNFα inversely correlated with LV fractional shortening. Conclusion After myocardial infarction, both BMMNCs and ACE-I reduce the pattern of pro-Ck response, probably contributing to prevent the deterioration of LV function observed in UT rats.

The Oct-4 transcription factor, a member of the POU family that is also known as Oct-3 and Oct3/4, is expressed in totipotent embryonic stem cells (ES) and germ cells, and it has a unique role in development and in the determination of pluripotency. ES may have their postnatal counterpart in the adult stem cells, recently described in various mammalian tissues, and Oct-4 expression in putative stem cells purified from adult tissues has been considered a real marker of stemness. In this context, normal mature adult cells would not be expected to show Oct-4 expression. On the contrary, we demonstrated, using reverse transcription-polymerase chain reaction (PCR) (total RNA, Poly A+), real-time PCR, immunoprecipitation, Western blotting, band shift, and immunofluorescence, that human peripheral blood mononuclear cells, genetically stable and mainly terminally differentiated cells with well defined functions and a limited lifespan, express Oct-4. These observations raise the question as to whether the role of Oct-4 as a marker of pluripotency should be challenged. Our findings suggest that the presence of Oct-4 is not sufficient to define a cell as pluripotent, and that additional measures should be used to avoid misleading results in the case of an embryonic-specific gene with a large number of pseudogenes that may contribute to false identification of Oct-4 in adult stem cells. These unexpected findings may provide new insights into the role of Oct-4 in fully differentiated cells. Disclosure of potential conflicts of interest is found at the end of this article.

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.

The majority of clinical studies on endothelial progenitor cells (EPCs) focuses on the role of these cells in cardiovascular diseases and no systematic studies exist regarding their variations in healthy subjects. In order to define the burden of angiogenesis in physiological conditions we assessed the frequency of peripheral blood endothelial colonies (PB-ECs) and their relation with other factors possibly involved in their function such as high-sensitivity C-reactive protein (hs-CRP), endothelial cell-specific mitogen factor (VEGF) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in a highly selected healthy population. A PB sample was obtained from 37/47 healthy subjects (age 40.2+/-15.0yrs; M/F 15/22) without known cardiovascular risk factors. The serum level of hs-CRP, VEGF, TIMP-1, the frequency of PB-ECs by clonogenic assay, and the number of early EPCs and late EPCs by flow cytometry analysis were evaluated. PB-ECs were formed by 40.5% of studied subjects with a mean of 0.40+/-0.82 colonies/10(6) cells. The differences in the frequency of colony formation between genders were not statistically significant. The subjects with PB-ECs were characterized by higher values of hs-CRP, when compared with those not forming colonies, 0.276+/-0.230 vs 0.095+/-0.077 mg/l (p=0.003) respectively, and of VEGF, 328.3+/-162.9 vs 202.68+/-118.53 pg/ml (p=0.02). No significant differences were found in TIMP-1 values. The EPC clonogenic potential seems to be related to hs-CRP and VEGF levels even in healthy population supporting the concept that these mediators are involved in physiological ECs function.

Circulating endothelial progenitor cells (EPCs) are believed to contribute to vascular homeostasis; unfortunately, the response of EPCs in physiological conditions remains largely unknown. Herein we report our observations of a 44-year-old healthy subject after a trek in the Himalayas that support high-altitude hypoxia and exercise oxygen demands are strong stimuli for clonogenic endothelial cell activation and activity, as shown by the increase in the number of mature EPCs and in the endothelial colony-forming unit capacity. Both of these effects were completely reverted at sea level, 45 days after the subject's trek. Am. J. Hematol. 79:76-78, 2005. 2005 Wiley-Liss, In

Background: Progressive Supranuclear Palsy (PSP) is a sporadic and progressive neurodegenerative disease which belongs to the family of tauopathies and involves both cortical and subcortical structures. No effective therapy is to date available. Methods/design: Autologous bone marrow (BM) mesenchymal stem cells (MSC) from patients affected by different type of parkinsonisms have shown their ability to improve the dopaminergic function in preclinical and clinical models. It is also possible to isolate and expand MSC from the BM of PSP patients with the same proliferation rate and immuphenotypic profile as MSC from healthy donors. BM MSC can be efficiently delivered to the affected brain regions of PSP patients where they can exert their beneficial effects through different mechanisms including the secretion of neurotrophic factors. Here we propose a randomized, placebo-controlled, double-blind phase I clinical trial in patients affected by PSP with MSC delivered via intra-arterial injection. Discussion: To our knowledge, this is the first clinical trial to be applied in a no-option parkinsonism that aims to test the safety and to exploit the properties of autologous mesenchymal stem cells in reducing disease progression. The study has been designed to test the safety of this " first-in-man" approach and to preliminarily explore its efficacy by excluding the placebo effect. Trial registration: NCT01824121

Abstract 4446 Hematopoietic stem and progenitor cells are routinely used in onco-hematological applications as consolidated approaches. Recently, these cells have been exploited also for their non-hematopoietic potential in the novel field of regenerative medicine. In fact, several clinical trials are already ongoing in the cardiovascular field, and more and more innovative applications, such as in liver disorders, are emerging to treat patients for whom no other efficient therapies are available. In Europe, the preparation of hematopoietic cells for non-hematopoietic applications must comply to medicinal products current good manufacturing practices (cGMP) and thus, the production process and the analytical methods applied in quality controls must be validated in order to demonstrate standardization and compliance with pre-defined specifications. In our hospital-based GMP facility, named Cell Factory, the first one authorized by the National Drug Agency, we have several processes ongoing for different clinical experimental trials. In particular, we produce CD133+ cells from leukapheresis (AF) products for liver chronic disease and bone marrow (BM) CD133+ cells for cardiovascular repair, strictly following the GMP rules governing medicinal product and we validated both processes as herein reported. In our hands, AF and BM need different approaches to improve the purity of the final cell product that is crucially important to have consistent clinical results. In fact, BM requires an efficient erythrocyte depletion that we obtained by optimizing a centrifugation based separation system, before the CliniMacs (Miltenyi, Gladbach, Germany) immunomagnetic purification. To validate the aseptic technique, three runs of specific media-fill protocols were set up. Finally, to validate the defined protocol, three runs of validation were performed in our class B facility for both procedures with all the quality controls on “in process” as well as on final products. BM samples from 3 patients who underwent sternotomy for by-pass surgery were collected after informed consent and processed as previously described. The results of the three validation runs were consistent in: cell dose >2×106 (2.54×106, 2.81×106 and 2.62×106), purity >70% (87.5%, 97.4% and 93.6%), viability >70% (88.5%, 90.6% and 96.4%), no growth for sterility and endotoxin AF samples from 3 patients who underwent mobilized standard-volume leukapheresis were collected and processed. The results of validation processes were consistent in: cell dose >100×106 (181×106, 138.5×106 and 178.3×106), purity >80% (91%, 90% and 92%), viability >80% (89%, 93% and 95%), for sterility no growth and for endotoxin In conclusion, all the end-points were reached and currently the cell products are provided to different clinical sites as investigational new drug, approved by the national competent authority. These products, as well as other cellular therapy products, are available upon request at our facility for experimental clinical applications (www.cellfactorymilano.com). Disclosures: No relevant conflicts of interest to declare.

Abstract 2588 Mesenchymal stem cells (MC) represent a promising population for regenerative medicine. Recently, their presence has been documented not only in bone marrow, but also in cord blood (CB), an attractive stem cell source with no ethical controversy. In this study, we investigated if CB could represent a new therapeutic tool for the treatment of severe vascular diseases. We first validated a negative immunoselection approach using a cocktail of antibodies that targets and reduces erythroid components in order to efficiently isolate low-potential (lp)-CBMSC (46% of isolation efficiency) and high-potential (hp)-CBMSC (8.1% of isolation efficiency). Specific aim of this study was to investigate the proangiogenic effect of hp-CBMSC for endothelial regenerative purposes. Previous experiments had shown that lp-CBMSC are able to grow until passage 4–5 only, with a lower differentiation rate as compared to the hp-CBMSC, therefore we used hp-CBMS. First, they were induced towards the endothelial lineage by growing them in endothelial medium supplemented with 5% FBS, 100 ng/ml Vascular Endothelial Growth Factor (VEGF) and 50 ng/ml Endothelial Growth Factor (EGF) in 24-well cell culture plates coated with fibronectin. After 3 weeks of induction, the ability of induced-cells to self-organize into capillary-like structures in the presence of basement membrane matrix was analyzed. Human umbilical vein endothelial cells (labeled with green fluorescent dye PKH67) were co-cultured with induced (A) or non-induced (B) hp-CBMSC (labeled with red fluorescent dye PKH26) in the presence of hp-CBMSC conditioned media (supernatant collected from hp-CBMSC cell culture). Interestingly, the tube formation was observed only in the presence of induced-hp-CBMSC (A) where the contribution of the endothelial (green) and mesenchymal (red) compartment in the vascular network was extremely clear and evident. In addition, to evaluate whether there was a possible endothelial contribution of the conditioned medium released by the non-induced-hp-CBMSC, an angiokit assay (Angiokit, TCS CellWorks Ltd) was performed. A strong tube formation was observed and the anastomosis-like structure was confirmed by the expression of CD31 and Von Willebrand Factor (DAB staining) in immunocytochemistry. The next step was to identify the factors released in the conditioned medium and in fact several angiogenic factors were found: HB-EGF 13.5 (8.7 – 16.7); HGF 22.4 (16.3 – 39.1); FGF 10.6 (7.0 – 13.5); VEGF 471.3 (369.3 – 590.0) (the results are expressed as median (and range) pg/mL and normalized per 1×106 seeded cells) using a cytokine array (Searchlight Multiplex Immunoassay Kits). The work is still ongoing for the quantification of the endothelial gene expression by real-time PCR for some angiogenic genes including: angiopoietin-2, the adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular endothelium-selectin (E-selectin), VCAM1 and VEGF-A in the induced versus the non-induced hp-CBMSC. In conclusion, our promising results suggest that hp-CBMSC can be considered as endothelial “drug stores” and are extremely promising paracrine effectors that could be exploited in future clinical applications for endothelial tissue regeneration. Disclosures: No relevant conflicts of interest to declare.

Mesenchymal stem cells (MSC) can be derived selectively in culture from multiple organs, an omnipresence we have recently suggested to be explained by the perivascular location of native MSC ancestors within intact tissues (Crisan et al. 2008, in press). We have now analyzed the ability of MSC extracted pro- or retrospectively from different human tissues to support hematopoiesis. MSC were either classically derived in primary cultures of umbilical cord blood (UCB) lineage-depleted mononuclear cells (n=3) or enzymatically dissociated adult adipose tissue (n=3), or grown as CD146+ NG2+ CD34-CD56- CD45- pericytes (n=2) purified by flow cytometry from fetal skeletal muscle and cultured over the long term. In both settings, identical MSC were obtained that maintained a stable CD146+ CD90+ CD73+ CD105+ CD34- CD45- surface phenotype and could differentiate into skeletal muscle, fat, bone and cartilage. CD34+ hematopoietic progenitors (n=3) immunoselected from term UCB were seeded (5×10e3cells/cm2 in triplicate) onto confluent irradiated layers of MSC derived from UCB, adipose tissue or fetal muscle pericytes (MSCu, MSCa and MSCmp, respectively) or, as a control, MS5 bone marrow stromal cells that allow the proliferation of very primitive human progenitor cells. All studies were approved by the relevant institutional regulatory board. The cells were cocultured for 5 weeks in a classical long-term culture-initiating cell assay in a complete medium (MyeloCult H5100, Stem Cell Technologies) containing hydrocortisone but no added cytokine. Wells were scored daily for the presence of cobblestone areas (CA) and half of the medium was replaced every week. Eventually, trypsinized cells from each well were characterized by flow cytometry for the expression of hematopoietic cell markers and assayed for CFC potential. After 14 days of incubation, colonies grown in semi-solid medium were scored as derived from colony forming units (CFU)-granulocyte, erythroid, macrophage, megakaryocyte (GEMM) and as high-proliferative-potential colony precursors (HPPC), the most primitive hematopoietic cell so far identified in a clonogenic assay in vitro. Within the CD45+ gate, all trypsinized cultures contained comparable percentages of CD34+lin- cells (MSCu: 51±9%; MSCa: 58±14%; MSCmp: 61±19%; MS5: 59±18%), the most immature hematopoietic cell compartment maintained during the long-term coculture. MSCu and MSCmp supported a similar cell proliferation during the whole culture while on MSCa, CA formed very rapidly and consistently but eventually decreased over the long-term culture. Interestingly, MSCu and MSCmp supported the development of the highest numbers of HPPC and of CFU giving rise to the largest GEMM colonies, as compared to MSCa that gave the same results as the control MS5 cell line. In summary, all MSCs tested were able to support hematopoiesis and CA formation, albeit with differences in growth kinetics and morphology of the colonies. Herein we show for the first time that purified human perivascular cells exhibit robust hematopoiesis support in vitro, in addition to multilineage mesodermal developmental potential. In conclusion, we demonstrate that MSC from novel sources distinct from the bone marrow are able to support hematopoiesis. These results further sustain the identity, beyond acronyms, between marrow stromal cells, long known for their support of hematopoiesis, and mesenchymal stem cells that gained more recent credit in the field of regenerative medicine because of their multilineage differentiation potential.

There are experimental evidences that Endothelial Progenitor Cells (EPCs) is a pool of cells that could proliferate and differentiate into mature endothelial cells and that may contribute to endothelial repair, but no systematic studies exist regarding physiologic variations in EP circulating pool and in CFU-Endo capacity in healthy subjects. In this regard, since the level of EPCs may be a biologic marker for vascular function, we decided to characterize primitive and circulating EPCs and their relation with gender, aging and the capacity to form CFU-Endo in a selected healthy population. Each subject underwent a screening evaluation to exclude any underlying cardiac diseases. A 20mL sample of peripheral blood was obtained (h 11:00–12:00 AM) from 15 male and 17 female age matched subjects (mean age 41.6±15.9yrs). EPCs were assessed by flow cytometric analysis on 100,000 events per sample (FACScan, Becton Dickinson). Primitive EPCs, defined as CD45−/34+/133+ and CD45−/KDR+/144−, were 8±1.8SE/uL. Circulating EPCs, defined as CD45−/146+/31+/FCSlow and CD45−/146+/144+, were 18.72±2.5SE/uL. No correlation was found between these populations and aging, gender and BMI. Instead a significant decrease in CD45−/34+/133+ (2.9±0.6SE/uL) subpopulation was observed with aging (r = 0.45; p = 0.009). CFU-Endo were performed as described by Hill JM et al. (NEJM 2003) and scored by microscopy in blind by two operators. The confirmation of endothelial-cell lineage was performed by immunostaining for endothelial-specific antibodies (CD31+, CD105+, CD144+, CD146low, CD133−, vWf-). CFU-Endo were expressed as the mean number of colonies per well in 8–25 separate determinations for each subjects and only 15 subjects gave colonies (mean 2.1±0.05SE). CFU-Endo do not correlate with aging and gender in healthy subjects confirming that endothelial injuries and cardiovascular risk factors are a crucial endothelium stimulus. In fact, recently it has been reported that higher colony counts were found in subjects with various degrees of cardiovascular risks. In conclusion CD45-/34+/133+ cell population correlates with aging but since this population represents Hemato-Endo Precursors that include a majority of endothelial progenitors and a small number of hematopoietic stem cells, further studies will be needed to identify a subpopulation strongly predictive of CFU-Endo capacity and to assess its endothelial lineage switch.

In search for new sources of mesenchymal stem cells (MSCs) for renal repair in acute kidney injury (AKI), we investigated the potential of human cord blood (CB)-MSCs to cure mice with AKI. Infusion of CB-MSCs in immunodeficient mice with cisplatin-induced AKI ameliorated both renal function and tubular cell injury, and prolonged survival. Transplanted CB-MSCs localized in peritubular areas, limited capillary alterations and neutrophil infiltration. Apoptosis reduced and tubular cell proliferation increased by virtue of stem cell capacity to produce growth factors. The reno-protective effect of CB-MSCs was further confirmed by their ability to inhibit oxidative damage and to induce the prosurvival factor Akt in tubular cells. The evidence that CB-MSCs in vitro increased the production of growth factors and inhibited IL-1β and TNFα synthesis when cocultured with damaged proximal tubular cells indicates a regenerative and anti-inflammatory action of stem cell treatment. Altogether these results highlight the potential of human CB-MSCs as future cell therapy for testing in human AKI.