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Background Organoids are three-dimensional in vitro-grown cell clusters that recapitulate key features of native organs. In regenerative medicine, organoid technology represents a promising approach for the replacement of severely damaged organs, such as the pancreas in patients with type 1 diabetes. Isolation human pancreas organoids (hPOs) in chemically defined serum-free culture media would be a major milestone for this approach. Methods Starting from discarded pancreatic tissues, we developed a large-scale process for obtaining clinically relevant quantities of undifferentiated organoids, obviating enzymatic digestion and operator-dependent pancreatic ducts picking steps. hPO identity was characterized by molecular and flow cytometry analysis. Results This work demonstrates that it is possible to obtain a large-scale production of organoids. We introduced some innovations in the isolation, expansion, and freezing of hPOs from five donors. First of all, the choice of the starting material (islet-depleted pancreas) that allows obtaining a high quantity of hPOs at low passages. On the other hand, we introduced mechanical dissociation and we eliminated the picking step to exclude the operator-depending steps, without affecting the success of the culture (100% success rate). Another important improvement was to replace R-spondin-1 (Rspo1) conditioned medium with Rspo1 recombinant molecule to obtain a well-defined composition of the expansion medium. Finally, we implemented a GMP-compliant freezing protocol. hPOs showed exponential growth with diameter and area that increased three- and eight-fold in 7 days, respectively. Immunophenotypic profile and gene expression analysis revealed that hPOs were composed of ductal (82.33 ± 8.37%), acinar (2.80 ± 1.25%) cells, and pancreatic progenitors (5.81 ± 2.65%). Conclusion This work represents a milestone for a GMP-compliance hPO production and, ultimately, their clinical application as a type 1 diabetes therapy.

Human multipotent mesenchymal stromal cells (MSC) are isolated from a plethora of tissue sources for cell therapy purposes. In 2006, the International Society for Cellular Therapy (ISCT) published minimal guidelines to define MSC identity. Nevertheless, many independent studies demonstrated that cells meeting the ISCT criteria possessed heterogeneous phenotypes and functionalities, heavily influenced by culture conditions. In this study, human MSC derived from many adult (bone marrow and adipose tissue) or fetal (cord blood, Wharton’s jelly, umbilical cord perivascular compartment and amniotic fluid) tissues were investigated. Their immunophenotype was analyzed to define consistent source-specific markers by extensive flow cytometry analysis and real-time qRT-PCR. CD271+ subpopulations were detected in adult MSC, whereas NG2 was significantly more expressed in fetal MSC but failed validation on independent samples coming from an external laboratory. The highest number of CD271+ adult MSC were detected soon after isolation in serum-based culture conditions. Furthermore, heterogeneous percentages of CD271 expression were found in platelet lysate-based or serum-free culture conditions. Finally, CD271+ adult MSC showed high clonogenic and osteogenic properties as compared to CD271− cells. To conclude, in this phenotype-function correlation study CD271+ subpopulation confers heterogeneity on adult MSC, confirming the need of more specific markers to address MSC properties.

Surgical repair of large muscular defects requires the use of autologous graft transfer or prosthetic material. Naturally derived matrices are biocompatible materials obtained by tissue decellularization and are commonly used in clinical practice. Despite promising applications described in the literature, the use of acellular matrices to repair large defects has been only partially successful, highlighting the need for more efficient constructs. Scaffold recellularization by means of tissue engineering may improve not only the structure of the matrix, but also its ability to functionally interact with the host. The development of such a complex construct is challenging, due to the complexity of the native organ architecture and the difficulties in recreating the cellular niche with both proliferative and differentiating potential during growth or after damage. In this study, we tested a mouse decellularized diaphragmatic extracellular matrix (ECM) previously described by our group, for the generation of a cellular skeletal muscle construct with functional features. The decellularized matrix was stored using different conditions to mimic the off-the-shelf clinical need. Pediatric human muscle precursors were seeded into the decellularized scaffold, demonstrating proliferation and differentiation capability, giving rise to a functioning three-dimensional skeletal muscle structure. Furthermore, we exposed the engineered construct to cardiotoxin injury and demonstrated its ability to activate a regenerative response in vitro promoting cell self-renewal and a positive ECM remodeling. Functional reconstruction of an engineered skeletal muscle with maintenance of a stem cell pool makes this a promising tool toward future clinical applications in diaphragmatic regeneration. Stem Cells Translational Medicine 2019;8:858–869

The therapeutic potential of mesenchymal stem cell (MSC) extracellular vesicles (EV) is currently under investigation in many pathological contexts. Both adult and perinatal MSC are being considered as sources of EV. Herein, we address antigen expression of cord blood and bone marrow MSC and released EV to define an identity and quality parameter of MSC EV as a medicinal product in the context of clinical applications. The research focuses on EV-shuttled neural/glial antigen 2 (NG2), which has previously been detected as a promising surface marker to distinguish perinatal versus adult MSC. Indeed, NG2 was significantly more abundant in cord blood than bone marrow MSC and MSC EV. Ultracentrifuge-isolated EV were then challenged for their pro-angiogenic properties on an xCELLigence system as quality control. NG2+ cord blood MSC EV, but not bone marrow MSC EV, promote bFGF and PDGF-AA proliferative effect on endothelial cells. Likewise, they successfully rescue angiostatin-induced endothelial cell growth arrest. In both cases, the effects are NG2-dependent. These results point at NG2 as an identity and quality parameter for cord blood MSC EV, paving the way for their clinical translation.

Metabolism and mitochondrial biology have gained a prominent role as determinants of stem cell fate and function. In the context of regenerative medicine, innovative parameters predictive of therapeutic efficacy could be drawn from the association of metabolic or mitochondrial parameters to different degrees of stemness and differentiation potentials. Herein, this possibility was addressed in human mesenchymal stromal/stem cells (hMSC) previously shown to differ in lifespan and telomere length. First, these hMSC were shown to possess significantly distinct proliferation rate, senescence status and differentiation capacity. More potential hMSC were associated to higher mitochondrial (mt) DNA copy number and lower mtDNA methylation. In addition, they showed higher expression levels of oxidative phosphorylation subunits. Consistently, they exhibited higher coupled oxygen consumption rate and lower transcription of glycolysis-related genes, glucose consumption and lactate production. All these data pointed at oxidative phosphorylation-based central metabolism as a feature of higher stemness-associated hMSC phenotypes. Consistently, reduction of mitochondrial activity by complex I and III inhibitors in higher stemness-associated hMSC triggered senescence. Finally, functionally higher stemness-associated hMSC showed metabolic plasticity when challenged by glucose or glutamine shortage, which mimic bioenergetics switches that hMSC must undergo after transplantation or during self-renewal and differentiation. Altogether, these results hint at metabolic and mitochondrial parameters that could be implemented to identify stem cells endowed with superior growth and differentiation potential.

Highlights • Seventeen EBMT centers participated to a questionnaire on MSC manufacturing. • 88% of centers manufacture MSC from bone marrow and only 2 centers from umbilical cord. • Human platelet lysate has replaced bovine serum as culture medium supplement. • Release criteria extensively differ among centers. • The results highlight the need to harmonize MSC manufacturing., The immunosuppressive properties of mesenchymal stromal cells (MSC) have been successfully tested to control clinical severe graft-versus host disease and improve survival. However, clinical studies have not yet provided conclusive evidence of their efficacy largely because of lack of patients’ stratification criteria. The heterogeneity of MSC preparations is also a major contributing factor, as manufacturing of therapeutic MSC is performed according to different protocols among different centers. Understanding the variability of the manufacturing protocol would allow a better comparison of the results obtained in the clinical setting among different centers. In order to acquire information on MSC manufacturing we sent a questionnaire to the European Society for Blood and Marrow Transplantation centers registered as producing MSC. Data from 17 centers were obtained and analyzed by means of a 2-phase questionnaire specifically focused on product manufacturing. Gathered information included MSC tissue sources, MSC donor matching, medium additives for ex vivo expansion, and data on MSC product specification for clinical release. The majority of centers manufactured MSC from bone marrow (88%), whilst only 2 centers produced MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as medium supplement. 59% of centers used only third-party MSC, whilst only 1 center manufactured exclusively autologous MSC. The large majority of these facilities (71%) administered MSC exclusively from frozen batches. Aside from variations in the culture method, we found large heterogeneity also regarding product specification, particularly in the markers used for phenotypical characterization and their threshold of expression, use of potency assays to test MSC functionality, and karyotyping. The initial data collected from this survey highlight the variability in MSC manufacturing as clinical products and the need for harmonization. Until more informative potency assays become available, a more homogeneous approach to cell production may at least reduce variability in clinical trials and improve interpretation of results.

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.

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.

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

In recent years, multiple studies have investigated the biology and clinical applications of mesenchymal stem cells (MSCs), trying to define their markers, and elucidate their effects in animal models. MSCs are available from different tissues, and the use of placental-derived MSCs (PMSCs) for treating a variety of disorders is on the forefront. Herein, we discuss the most recent findings regarding the standardization of their isolation procedure and phenotype, along with advantages and limitations of their use. We also discuss the safety of the placental cell products, including the issue of senescence and mutagenesis of PMSCs, and efficacy from preclinical studies.

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.

International audience; The aim of this study was to assess the immune modulatory properties of human mesenchymal stromal cells obtained from bone marrow (BM-MSCs), fat (ASCs) and cord blood (CB-MSCs) in the presence of a novel hydroxyapatite and tricalcium-phosphate (HA/TCP) biomaterial as scaffold for MSC delivery. In resting conditions, a short-term culture with HA/TCP did not modulate the anti-apoptotic and suppressive features of the various MSC types towards T, B and NK cells; in addition, when primed with inflammatory cytokines, MSC maintained or not on HA/TCP similarlyincreased their suppressive capacities. The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype with up-regulation of OSTERIX and OSTEOCALCIN, similarly to what obtained with dexamethasone and, to a higher extent, BMP-4 treatment. MSC-derived osteoblasts did not trigger immune cell activation, but were less efficient than undifferentiated MSCs in inhibiting stimulated T and NK cells. Interestingly, their suppressive machinery included not only the activation of IDO, which plays a central role in T-cell inhibition, but also COX-2 that was not significantly involved in immune modulatory effect of human undifferentiated MSCs. COX-2 is significantly involved in bone healing, suggesting that its induction by HA/TCP could also contribute to the therapeutic activity of MSC for bone tissue engineering.

Mesenchymal stem/stromal cells (MSCs) and MSC-like multipotent stem/progenitor cells have been widely investigated for regenerative medicine and deemed promising in clinical applications. In order to further improve MSC-based stem cell therapeutics, it is important to understand the cellular kinetics and functional roles of MSCs in the dynamic regenerative processes. However, due to the heterogeneous nature of typical MSC cultures, their native identity and anatomical localization in the body have remained unclear, making it difficult to decipher the existence of distinct cell subsets within the MSC entity. Recent studies have shown that several blood-vessel-derived precursor cell populations, purified by flow cytometry from multiple human organs, give rise tobona fideMSCs, suggesting that the vasculature serves as a systemic reservoir of MSC-like stem/progenitor cells. Using individually purified MSC-like precursor cell subsets, we and other researchers have been able to investigate the differential phenotypes and regenerative capacities of these contributing cellular constituents in the MSC pool. In this review, we will discuss the identification and characterization of perivascular MSC precursors, including pericytes and adventitial cells, and focus on their cellular kinetics: cell adhesion, migration, engraftment, homing, and intercellular cross-talk during tissue repair and regeneration.

Focal segmental glomerulosclerosis (FSGS) is the most frequent acquired renal condition resulting in end stage kidney disease in children. We describe a cell therapy treatment with human allogeneic bone marrow mesenchymal stem cells (MSC) in a 13-year-old patient developing recurrent FSGS after renal transplantation, which was not responding to conventional therapy. This treatment relied on the following measurements:clinical and laboratory evaluation of renal function, proteome array, biopsy, short tandem repeat assay. Before MSC treatment, the patient needed weekly plasmapheresis to achieve proteinuria-to-creatininuria ratio below 5. After three MSC infusions without adverse events, the patient has a stable renal function and the proteinuria target was reached without plasmapheresis. In addition, some circulating inflammatory factors decreased and their levels were still low after one year. This is the first report of an MSC treatment in an FSGS patient. Even though different factors may have contributed to the clinical results, after MSC infusion a stable reduction in the serum level of several inflammatory factors has been registered and the patient does not need anymore plasmapheresis to keep proteinuria under control. In addition, this encouraging single case let us identify some putative efficacy biomarkers that could be of clinical interest in chronic kidney diseases.

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.

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.

Cord blood (CB) is a valid alternative to mobilized peripheral blood (mPB) as a source of hematopoietic stem cells for clinical transplantation. The qualitative differences among CD133+ cells from these sources have been demonstrated by in vitro and in vivo studies but the molecular phenotype of these cells is only partially understood. In view of better exploiting the peculiarities and advantages of both stem cell sources, we performed a comparative analysis of gene expression of CD133+ cells from CB and mPB using Affymetrix HG-U133A arrays. The CD133+ cells were selected using the MiniMacs immunomagnetic separation system (Miltenyi Biotec) from 6 CB units and 6 mPB samples. Since microarray results are strictly dependent on the purity of the cell population and on the RNA quality, rigorous quality controls were applied. The purity of CD133+ cells was more than 93% in all samples. In addition, after extraction, the RNA was purified by the RNeasy system (Qiagen) and the RNA quality was evaluated using the RNA 6000 Nano LabChip kit (Agilent Technologies). Given the low amount of RNA due to the limited numbers of CB CD133+ cells, we used a double-cycle amplification protocol for both sources to obtain biotin-labeled target for hybridization starting from as low as 100 ng of total RNA. Probe level data have been converted to expression values using Affymetrix MAS 5.0 algorithm. Differentially expressed genes have been identified using Significance Analysis of Microarrays (SAM). The cut-off for significance has been determined tuning the Delta parameter on the false positive rate and controlling the q-value for the gene list. We found that mPB CD133+ cells expressed cell cycle driving, transcription factor and DNA synthesis genes at distinctly higher levels than CB CD133+ cells, indicating a higher cycling activity in mPB CD133+ cells. Furthermore apoptosis driving genes were up-regulated in mPB CD133+ cells, probably due to G-CSF mobilization. Conversely in CB CD133+ cells we found an overexpression of genes involved in immunoresponse and immunomodulation. This finding could justify the peculiar immunological properties of CB. Finally, many genes related to different metabolic pathways such as alcohol, sterol, nucleotide, lipid and sugar biosynthesis, were found up-regulated in CB CD133+ cells, maybe due to the neonatal origin of these cells. In conclusion, mPB CD133+ cells include a more committed and cycling population while CB CD133+ represent a cell subset with higher potentialities probably due to their immaturity. This suggest that even if CB has a lower number of stem cells, CB CD133+ cells are more susceptible to different stimuli and potential fates.

The transplantation of two cord blood (CB) units obtained from unrelated donors (double CBT) is an effective strategy for adult patients with hematologic malignancies. Sustained hematopoiesis after double CBT is usually derived from a single donor, and only a few transplantation recipients displaying a stable mixed donor-donor chimerism have been reported. We investigated the mechanisms underlying single-donor predominance in double CBT by studying in vitro the role of the graft-versus-graft cell-mediated immune effect in two-way mixed-lymphocyte culture, along with the contribution of differential hematopoietic progenitor (HP) potency in HP mixed cultures. Results for the two-way mixed-lymphocyte culture showed that despite the weak and variable alloantigen-specific cytotoxic potential displayed by CB mononuclear cells, an immune-mediated dominance for one of the two CB units was detected in the majority of experiments. Alloantigen-induced cytotoxic activity was directed toward both CB-HP and phytohemagglutinin (PHA)-activated T lymphoblastoid cells. The CB unit with the higher fold expansion of CD34(+) cells in single-expansion culture was prevalent in the HP mixed-expansion culture, as shown by DNA chimerism evaluation. Based on these data, we hypothesize that the dominant CB unit is able to develop prevalent cytotoxic activity toward activated lymphocytes of the other CB unit, thereby preventing them from exerting alloantigen-specific cytotoxic potential against both activated lymphocytes and HPs of the dominant unit. In accordance with this hypothesis, we propose the evaluation of alloantigen-induced cytotoxic activity generated in two-way mixed-lymphocyte culture and directed toward PHA-activated T lymphoblastoid cells as a tool to identify the potentially predominant CB unit before double CBT.

Advanced therapy medicinal products represent a new generation of medicinal products for regenerative medicine. Since the implementation of the EU regulation for this innovative class of drugs, the academic and hospital institutions have played a central role in their development and manufacture. For these institutions that are not familiar with the industrial context, being in compliance with the pharmaceutical standards is extremely challenging. This report describes how we dealt with some specific issues during our hospital-based GMP experience. Furthermore, we identify as a future perspective the consistent stimulating contribution that a public entity can ensure for advanced therapy medicinal product development and licensing.

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.