IRE1a-Induced FilaminA Phosphorylation Enhances Migration of Mesenchymal Stem Cells Derived from Multiple Myeloma Patients.

Multiple myeloma (MM) is an aggressive malignancy that shapes, during its progression, a pro-tumor microenvironment characterized by altered protein secretion and the gene expression of mesenchymal stem cells (MSCs). In turn, MSCs from MM patients can exert an high pro-tumor activity and play a strong immunosuppressive role. Here, we show, for the first time, greater cell mobility paralleled by the activation of FilaminA (FLNA) in MM-derived MSCs, when compared to healthy donor (HD)-derived MSCs. Moreover, we suggest the possible involvement of the IRE1a-FLNA axis in the control of the MSC migration process. In this way, IRE1a can be considered as a good target candidate for MM therapy, considering its pro-survival, pro-osteoclast and chemoresistance role in the MM microenvironment. Our results suggest that IRE1a downregulation could also interfere with the response of MSCs to MM stimuli, possibly preventing cell-cell adhesion-mediated drug resistance. In addition, further investigations harnessing IRE1a-FLNA interaction could improve the homing efficiency of MSC as cell product for advanced therapy applications.

Supporting mentalizing in primary school children: the effects of thoughts in mind project for children (TiM-C) on metacognition, emotion regulation and theory of mind.

Mentalization is a useful ability for social functioning and a crucial aspect of mentalizing is emotion regulation. Literature suggests programmes for children and adults to increase mentalizing abilities useful both for emotional and social competences. For this reason, the issue of how to prompt children's mentalization has started to attract researchers' attention, supporting the importance of the interpersonal dimension for the individual differences in the developmental of mentalization. The TiM (Thoughts in Mind) Project, a training programme based on the explanation of mentalization mechanisms and designed for adults, deals with emotion regulation. Starting from the TiM Project, this study tests the effects of the TiM Child (TiM-C) training programme, over a control training programme, a conversational training designed for the school context, in children attending Year 2 of primary school. We designed a training based on narratives, followed by multiple-choice questions and conversations about mental states. Our results revealed significant improvements over the training period only in the TiM-C Project group for Metacognition, Emotion Regulation Strategies and a Theory of Mind task. As far as the educational implications, our findings suggest that it is possible to enhance mentalization through activities at school by promoting not only the understanding of the relations between mind and emotion, but also metacognitive skills.

Modified mesenchymal stem cells in cancer therapy: A smart weapon requiring upgrades for wider clinical applications.

Mesenchymal stem stromal cells (MSC) are characterized by the intriguing capacity to home toward cancer cells after systemic administration. Thus, MSC can be harnessed as targeted delivery vehicles of cytotoxic agents against tumors. In cancer patients, MSC based advanced cellular therapies were shown to be safe but their clinical efficacy was limited. Indeed, the amount of systemically infused MSC actually homing to human cancer masses is insufficient to reduce tumor growth. Moreover, induction of an unequivocal anticancer cytotoxic phenotype in expanded MSC is necessary to achieve significant therapeutic efficacy. Ex vivo cell modifications are, thus, required to improve anti-cancer properties of MSC. MSC based cellular therapy products must be handled in compliance with good manufacturing practice (GMP) guidelines. In the present review we include MSC-improving manipulation approaches that, even though actually tested at preclinical level, could be compatible with GMP guidelines. In particular, we describe possible approaches to improve MSC homing on cancer, including genetic engineering, membrane modification and cytokine priming. Similarly, we discuss appropriate modalities aimed at inducing a marked cytotoxic phenotype in expanded MSC by direct chemotherapeutic drug loading or by genetic methods. In conclusion, we suggest that, to configure MSC as a powerful weapon against cancer, combinations of clinical grade compatible modification protocols that are currently selected, should be introduced in the final product. Highly standardized cancer clinical trials are required to test the efficacy of ameliorated MSC based cell therapies.

Nucleofection of Adipose Mesenchymal Stem/Stromal Cells: Improved Transfection Efficiency for GMP Grade Applications.

Nucleofection (NF) is a safe, non-viral transfection method, compatible with Good Manufacturing Practice guidelines. Such a technique is useful to improve therapeutic effectiveness of adipose tissue mesenchymal stem cells (ASC) in clinical settings, but improvement of NF efficiency is mandatory. Supernatant rich in growth factors (SRGF) is a clinical-grade medium additive for ASC expansion. We showed a dramatically increased NF efficiency and post-transfection viability in ASC expanded in presence of SRGF (vs. fetal bovine serum). SRGF expanded ASC were characterized by increased vesicle endocytosis but lower phagocytosis properties. SRGF increased n-6/n-3 ratio, reduced membrane lipid raft occurrence, and lowered intracellular actin content in ASC. A statistical correlation between NF efficiency and lipid raft availability on cell membranes was shown, even though a direct relationship could not be demonstrated: attempts to selectively modulate lipid rafts levels were, in fact, limited by technical constraints. In conclusion, we reported for the first time that tuning clinical-grade compatible cell culture conditions can significantly improve ASC transfection efficiency by a non-viral and safe approach. A deep mechanistic characterization is extremely complex, but we can hypothesize that integrated changes in membrane structure and intracellular actin content could contribute to explain SRGF impact on ASC NF efficiency.

miR-335-laden B Cell-Derived Extracellular Vesicles Promote SOX4-Dependent Apoptosis in Human Multiple Myeloma Cells.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow. Despite novel therapies, MM still remains an incurable cancer and new strategies are needed. Increased expression of the transcription factor Sex-determining region Y-related high-mobility-group box transcription factor 4 (SOX4) has been correlated with tumor development and progression through a variety of distinct processes, including inhibition of apoptosis, increased cell invasion and metastasis, and induction and maintenance of cancer-initiating cells. The role of SOX4 in MM is largely unknown. Since SOX4 is a known target of miR-335, we used miR-335 to assess whether SOX4 modulation could promote apoptosis in MM cells. Using an MM cell model we show that miR-335 acts both on SOX4-related genes (AKT, PI3K) and hypoxia-inducible factor 1-alpha (Hif1-α). In addition, we show miR-335-laden extracellular vesicles induced in B cells (iEVs) are also effective in targeting SOX4, causing apoptosis. Collectively, we propose that miR-335-laden iEVs could be developed as a novel form of gene therapy in MM.

Telerehabilitation and Wellbeing Experience in Children with Special Needs during the COVID-19 Pandemic.

Social distancing due to the COVID-19 pandemic represented a golden opportunity to implement telerehabilitation for clinical groups of children. The present study aims to show the impact that telerehabilitation had on the experience of well-being of children with special needs being treated at the Foundation IRCCS Neurological Institute 'C. Besta' in Milan (Specific Learning Disorders and Cerebral Palsy diagnosis); it aims to do so by comparing it with experiences of those who did not undertake telerehabilitation despite the diagnosis during the pandemic, and with typically developing children. Results show that the three groups differed in the Support, Respect and Learning dimensions of well-being experience. Post hoc comparisons revealed that children with Specific Learning Disorders and Cerebral Palsy scored higher than normotypical children in Support and in Respect scales. Furthermore, children who experienced telerehabilitation showed the highest scores on the Learning scale in comparison with the other two groups. These results support the importance of reorganizing care and assistance by integrating telemedicine, which seems to have fostered a positive experience of well-being in people with special needs, particularly in the perception of a supportive environment that respects psychological needs.

Ablation of collagen VI leads to the release of platelets with altered function.

Hemostatic abnormalities and impaired platelet function have been described in patients affected by connective tissue disorders. We observed a moderate bleeding tendency in patients affected by collagen VI-related disorders and investigated the defects in platelet functionality, whose mechanisms are unknown. We demonstrated that megakaryocytes express collagen VI that is involved in the regulation of functional platelet production. By exploiting a collagen VI-null mouse model (Col6a1-/-), we found that collagen VI-null platelets display significantly increased susceptibility to activation and intracellular calcium signaling. Col6a1-/- megakaryocytes and platelets showed increased expression of stromal interaction molecule 1 (STIM1) and ORAI1, the components of store-operated calcium entry (SOCE), and activation of the mammalian target of rapamycin (mTOR) signaling pathway. In vivo mTOR inhibition by rapamycin reduced STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell autonomous, because transplantation of lineage-negative bone marrow cells from Col6a1-/- mice into lethally irradiated wild-type animals showed the same alteration in SOCE and platelet activation seen in Col6a1-/- mice. Peripheral blood platelets of patients affected by collagen VI-related diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, displayed increased expression of STIM1 and ORAI1 and were more prone to activation. Altogether, these data demonstrate the importance of collagen VI in the production of functional platelets by megakaryocytes in mouse models and in collagen VI-related diseases.

Comparison on Well-Being, Engagement and Perceived School Climate in Secondary School Students with Learning Difficulties and Specific Learning Disorders: An Exploratory Study.

Reading and writing skills influence the social status of students, exerting effects not only on learning, but also on wellbeing. This study aimed to assess the impact of diagnosis of specific learning disorder on well-being in secondary-school students, comparing students with a diagnosis of specific learning disorder (SLD-group), students showing learning difficulties without diagnosis (LD-group) and students without learning difficulties (control-group). Students were tested with neuropsychological screening tests in order to identify learning difficulties and were further assessed by means of psychological and school well-being questionnaires. The results show that LD group perceive themselves as having a low sense of mastery and autonomy, less interest and engagement in daily activities and low peer social support than their schoolmates. This result highlights, for the LD group, a low well-being experience, which is not observed in the SLD and control groups. On the contrary, SLD group students do not differ from control group students in any dimensions except for the perceived parents' support and involvement in school life, in which the SLD group show the highest scores. This work underlines the importance of having a diagnosis as it seems to work as a protective factor for both the psychological and school well-being of the student.

Learning to Wait and Be Altruistic: Testing A Conversational Training in Economic Education for Primary School Children.

Individual economic competence is important but increasingly challenging to manage due to the growing complexity of the nature of economic decisions people must make and the substantial impacts of some of these decisions on their lives. Decision-making ability develops from childhood and is closely related to specific economic components and prosocial behaviour such as fairness, altruism, and delay of gratification. However, while there are financial-education programs for children and young people focusing on financial products, few studies have examined training for the psychological abilities underlying economic decision-making. To promote those psychological skills that contribute to a more socially effective decision-making, we designed and tested a conversational-based training program for primary school children using reflective thinking. A total of 110 (male = 47, female = 63) children aged 8 to 10 years (Mean age = 9.71 years) from two schools in Northern Italy participated in the study with 55 children in a training group and 55 in a control group. All participated in pre-tests measuring their socio-economic background and economics-related skills and abilities. The training group were told stories relaying values of fairness, altruism, and delayed gratification. Both groups participated in task-based post-tests relating to fairness, altruism, and delayed gratification. Results revealed that children in the training group showed significant improvement at the post-test in altruistic and investment behaviour, showing the training efficacy, suggesting that similar programs could be implemented in primary schools as foundational teaching of economics and fiscal responsibility.

Exploring the relation between maternal mind-mindedness and children's symbolic play: A longitudinal study from 6 to 18 months.

Relations between mothers' mind-mindedness (appropriate attunement to their infants' internal states) at 6 and 12 months and infants' early symbolic play during infant-mother pretense at 12 and 18 months were investigated in a sample of 43 mothers and infants. Mothers' appropriate mind-related comments were associated with average level, length, complexity, and maturity level of symbolic play. Specific sub-categories of appropriate mind-related comments were identified as independent predictors of children's symbolic play. Appropriate comments about desires and cognitions at 6 months were associated with average level and length of episodes, as well as with maturity level of symbolic play at 12 months. Longitudinal stability in the appropriateness and content of mothers' mind-related comments was also investigated. The results are discussed in terms of the proposal that attunement to specific types of internal state should vary as a function of infant age in order to index mind-mindedness.

Adipose mesenchymal stromal/stem cells expanded by a GMP compatible protocol displayed improved adhesion on cancer cells in flow conditions.

BACKGROUND: Adipose tissue derived mesenchymal stromal/stem cells (ASC) can be expanded using supernatant rich in growth factors (SRGF) as Good Manufacturing Practice compatible additive, instead of fetal bovine serum (FBS). After transendothelial migration, ASC can migrate to cancer masses where they can release active substances. Due to their homing and secretion properties ASC can be used as targeted drug delivery vehicles. Nevertheless, the fraction of ASC actually reaching the tumor target is limited. The impact of culture conditions on ASC homing potential on cancer cells is unknown. METHODS: In dynamic in vitro conditions, we perfused FBS or SRGF ASC in flow chambers coated with collagen type I and fibronectin or seeded with endothelial cells or with HT1080, T98G and Huh7 cancer cells. Expression of selected adhesion molecules was evaluated by standard cytofluorimetry. Dynamic intracellular calcium concentration changes were evaluated in microfluidic and static conditions. RESULTS: When compared to FBS ASC, not specific adhesion of SRGF ASC on collagen type I and fibronectin was lower (-33.9%±12.2% and -45.3%±16.9%), while on-target binding on HT1080 and T98G was enhanced (+147%±8% and 120.5%±5.2%). Adhesion of both FBS and SRGF ASC on Huh7 cells was negligible. As confirmed by citofluorimetry and by function-blocking antibody, SRGF mediated decrease of CD49a expression accounted for lower SRGF-ASC avidity for matrix proteins. Upon stimulation with calcium ionophore in static conditions, mobilization of intracellular calcium in SRGF ASC was greater than in FBS ASC. In dynamic conditions, upon adhesion on matrix proteins and HT1080 cells, SRGF ASC showed marked oscillatory calcium concentration changes. CONCLUSIONS: SRGF can enhance specific ASC binding capacity on selected cancer cells as HT1080 (fibrosarcoma) and T98G (glioblastoma) cells. Upon cell-cell adhesion, SRGF ASC activate intracellular responses potentially improving cell secretion functions. SRGF ASC could be considered as suitable drug delivery vehicle for cancer therapy.

An Online Tool to Assess Sentence Comprehension in Teenagers at Risk for School Exclusion: Evidence From L2 Italian Students.

This study presents a web-based sentence comprehension test aimed at identifying high school students who are at risk for a language delay. By assessing linguistic skills on a sample of high school students with Italian as an L2 and their monolingual peers, attending a vocational school, we were able to identify a subgroup of L2 students with consistent difficulties in sentence comprehension, though their reading skills were within the average range. The same subgroup revealed to experience a lack of support within the school context, suggesting that poor L2 skills might be a critical variable to consider in order to identify students at risk for school exclusion. Regarding the test, accuracy to the on-line sentence comprehension task was significantly predicted by reading abilities and vocabulary skills, thus indicating that this test might represent a rapid but efficient way to assess linguistic abilities at school. We recommend that establishing a valid and practical procedure for the evaluation of linguistic skills in bilingual students who struggle with their L2 is the first step toward promoting social inclusion in the multilingual classroom, in order to increase their ability to actively participate in school and social activities.

Tell Me a Story: Socio-Emotional Functioning, Well-Being and Problematic Smartphone Use in Adolescents With Specific Learning Disabilities.

Although Specific Learning Disabilities (SLD) are described as specific difficulties in one or more academic areas, often socio-emotional problems are also reported to be related to well-being and school engagement. Moreover, recent evidence shows that emotional problems and reduced social support predict problematic use of new technologies, such as a smartphone, that can, in turn, increase these problems. In this study, we aimed to investigate socio-emotional functioning and its relation to well-being, school engagement, and problematic smartphone use. Social and emotional skills of 19 adolescents with a diagnosis of SLD and 19 control adolescents were assessed through a narrative test; adolescents were requested to narrate complete stories elicited by pictures representing social situations. Information on well-being and problematic smartphone use were collected through questionnaires. The comparison between groups showed differences in cognitive-social skills, although no significant differences in emotional functioning were found. Additionally, the perception of the social environment as supportive and trustworthy was related to general well-being for both groups, whereas the perception of limits and rules set by the adult world appeared to be related to a decreased investment in learning processes only for the SLD students. Finally, correlation analysis showed that smartphone use was associated with reduced perception of social support and to a decreased ability to understand and solve social situations. These results assert the critical role played by social information processing and social support in terms of well-being in adolescence, and contribute to enhancing knowledge of the mechanisms underlying problematic smartphone use in a clinical sample.

The Impact of School Climate on Well-Being Experience and School Engagement: A Study With High-School Students.

The aim of this work is to investigate the factors promoting students' engagement at school and supporting their well-being experience. According to the Positive Education there is a strong relationship between school environment and student's well-being. Moreover, the quality of the school climate perceived by the students was found to influence engagement in school activities, as well. In this study, 153 students (M = 67) attending 10th grade were presented with tests and questionnaires to assess individual assets (personality traits, literacy skills), emerging appraisals (school-climate, well-being experience) and emerging actions (school engagement), according to the Student Well-Being Model. Path analysis showed that the best model does include neither individual assets nor direct effect of school climate on engagement, as the effect of school climate on engagement is mediated by well-being experience. The main result is that school climate has been confirmed as an important factor to be considered to improve engagement in school activities, but it is effective only when its influence can modify the well-being experience of the students. Moreover, the lack of significant effects of individual assets in the model suggests that improving school climate means to support well-being experience and, indirectly school engagement, irrespective to learning abilities and personality traits. This work encourages working in/with schools to implement positive education programs that support and sustain a positive school climate and culture for school-community wellbeing.

Factor H interferes with the adhesion of sickle red cells to vascular endothelium: a novel disease-modulating molecule.

Sickle cell disease is an autosomal recessive genetic red cell disorder with a worldwide distribution. Growing evidence suggests a possible involvement of complement activation in the severity of clinical complications of sickle cell disease. In this study we found activation of the alternative complement pathway with microvascular deposition of C5b-9 on skin biopsies from patients with sickle cell disease. There was also deposition of C3b on sickle red cell membranes, which is promoted locally by the exposure of phosphatidylserine. In addition, we showed for the first time a peculiar "stop-and-go" motion of sickle cell red blood cells on tumor factor-α-activated vascular endothelial surfaces. Using the C3b/iC3b binding plasma protein factor Has an inhibitor of C3b cell-cell interactions, we found that factor H and its domains 19-20 prevent the adhesion of sickle red cells to the endothelium, normalizing speed transition times of red cells. We documented that factor H acts by preventing the adhesion of sickle red cells to P-selectin and/or the Mac-1 receptor (CD11b/CD18), supporting the activation of the alternative pathway of complement as an additional mechanism in the pathogenesis of acute sickle cell related vaso-occlusive crises. Our data provide a rationale for further investigation of the potential contribution of factor H and other modulators of the alternative complement pathway with potential implications for the treatment of sickle cell disease.

Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media.

BACKGROUND: The stromal vascular fraction (SVF) derived from adipose tissue contains adipose-derived stromal/stem cells (ASC) and can be used for regenerative applications. Thus, a validated protocol for SVF isolation, freezing, and thawing is required to manage product administration. To comply with Good Manufacturing Practice (GMP), fetal bovine serum (FBS), used to expand ASC in vitro, could be replaced by growth factors from platelet concentrates. METHODS: Throughout each protocol, GMP-compliant reagents and devices were used. SVF cells were isolated from lipoaspirates by a standardized enzymatic protocol. Cells were cryopreserved in solutions containing different albumin or serum and dimethylsulfoxide (DMSO) concentrations. Before and after cryopreservation, we analyzed: cell viability (by Trypan blue); immunophenotype (by flow cytometry); colony-forming unit-fibroblast (CFU-F) formation; and differentiation potential. ASC, seeded at different densities, were expanded in presence of 10% FBS or 5% supernatant rich in growth factors (SRGF) from platelets. The differentiation potential and cell transformation grade were tested in expanded ASC. RESULTS: We demonstrated that SVF can be obtained with a consistent yield (about 185 × 103 cells/ml lipoaspirate) and viability (about 82%). Lipoaspirate manipulation after overnight storage at +4 °C reduced cell viability (-11.6%). The relative abundance of ASC (CD34+CD45-CD31-) and endothelial precursors (CD34+CD45-CD31+) in the SVF product was about 59% and 42%, respectively. A period of 2 months cryostorage in autologous serum with added DMSO minimally affected post-thaw SVF cell viability as well as clonogenic and differentiation potentials. Viability was negatively affected when SVF was frozen at a cell concentration below 1.3 × 106 cells/ml. Cell viability was not significantly affected after a freezing period of 1 year. Independent of seeding density, ASC cultured in 5% SRGF exhibited higher growth rates when compared with 10% FBS. ASC expanded in both media showed unaltered identity (by flow cytometry) and were exempt from genetic lesions. Both 5% SRGF- and 10% FBS-expanded ASC efficiently differentiated to adipocytes, osteocytes, and chondrocytes. CONCLUSIONS: This paper reports a GMP-compliant approach for freezing SVF cells isolated from adipose tissue by a standardized protocol. Moreover, an ASC expansion method in controlled culture conditions and without involvement of animal-derived additives was reported.

Body Aesthetic Preference in Preschoolers and Attraction to Canons Violation: An Exploratory Study.

Sensitivity to canons of beauty as represented in the human body-and as typically defined in the Western Culture-has been poorly studied in children. Current literature shows that infants as young as about three months are sensitive to the human body structure and its parts. Using a sample of 54 three- to five-year-old children, the present study investigated preference for drawings representing the "canonical" body structure, contrasting these with drawings showing the same bodies, but where the relation between trunk and legs was modified. It was hypothesized that preference for the canonical body structures would emerge as early as three years, increasing with age. Results only partially supported the hypothesis: while three-year-olds showed a significant preference for the canonical body structures as predicted, a significant preference reversal was found for the four-year-olds, with a tendency to return to preferring the canonical body at five years. The results are discussed in light of research findings associated with developmental theories hallmarking visual art perception in children.

Impedance biosensor for real-time monitoring and prediction of thrombotic individual profile in flowing blood.

A new biosensor for the real-time analysis of thrombus formation is reported. The fast and accurate monitoring of the individual thrombotic risk represents a challenge in cardiovascular diagnostics and in treatment of hemostatic diseases. Thrombus volume, as representative index of the related thrombotic status, is usually estimated with confocal microscope at the end of each in vitro experiment, without providing a useful behavioral information of the biological sample such as platelets adhesion and aggregation in flowing blood. Our device has been developed to work either independently or integrated with the microscopy system; thus, images of the fluorescently labeled platelets are acquired in real-time during the whole blood perfusion, while the global electrical impedance of the blood sample is simultaneously monitored between a pair of specifically designed gold microelectrodes. Fusing optical and electrical data with a novel technique, the dynamic of thrombus formation events in flowing blood can be reconstructed in real-time, allowing an accurate extrapolation of the three-dimensional shape and the spatial distribution of platelet thrombi forming and growing within artificial capillaries. This biosensor is accurate and it has been used to discriminate different hemostatic conditions and to identify weakening and detaching platelet aggregates. The results obtained appear compatible with those quantified with the traditional optical method. With advantages in terms of small size, user-friendliness and promptness of response, it is a promising device for the fast and automatic individual health monitoring at the Point of Care (POC).

Prospective thinking and decision making in primary school age children.

In this study, we seek to widen our understanding of the developmental processes underlying bargaining behaviour in children addressing the concept of prospective thinking. We argue that the emergence of the capacity to think prospectively about future outcomes or behaviours in response to current actions is a required precedent to strategic decision making. To test this idea, we compared 6, 8 and 10 years old children's performance on three tasks: the ultimatum game assessing fairness/inequality aversion, the marshmallow task, an intertemporal choice task evaluating the ability to delay gratification, and the dictator game assessing altruism. The children's socio-demographic and cognitive variables were also evaluated. We hypothesized that development of strategic thinking in the ultimatum game is related to an increased ability to delay gratification - given that both tasks require looking at prospective benefits - and, crucially, not to altruism, which benefits from immediate selfless reward. Our results confirmed our hypothesis suggesting that increased strategic planning with age would also stem from the development of competencies like prospective thinking.

Standardization of platelet releasate products for clinical applications in cell therapy: a mathematical approach.

BACKGROUND: Standardized animal-free components are required for manufacturing cell-based medicinal products. Human platelet concentrates are sources of growth factors for cell expansion but such products are characterized by undesired variability. Pooling together single-donor products improves consistency, but the minimal pool sample size was never determined. METHODS: Supernatant rich in growth factors (SRGF) derived from n = 44 single-donor platelet-apheresis was obtained by CaCl2 addition. n = 10 growth factor concentrations were measured. The data matrix was analyzed by a novel statistical algorithm programmed to create 500 groups of random data from single-donor SRGF and to repeat this task increasing group statistical sample size from n = 2 to n = 20. Thereafter, in created groups (n = 9500), the software calculated means for each growth factor and, matching groups with the same sample size, the software retrieved the percent coefficient of variation (CV) between calculated means. A 20% CV was defined as threshold. For validation, we assessed the CV of concentrations measured in n = 10 pools manufactured according to algorithm results. Finally, we compared growth rate and differentiation potential of adipose-derived stromal/stem cells (ASC) expanded by separate SRGF pools. RESULTS: Growth factor concentrations in single-donor SRGF were characterized by high variability (mean (pg/ml)-CV); VEGF: 950-81.4; FGF-b: 27-74.6; PDGF-AA: 7883-28.8; PDGF-AB: 107834-32.5; PDGF-BB: 11142-48.4; Endostatin: 305034-16.2; Angiostatin: 197284-32.9; TGF-ß1: 68382-53.7; IGF-I: 70876-38.3; EGF: 2411-30.2). In silico performed analysis suggested that pooling n = 16 single-donor SRGF reduced CV below 20%. Concentrations measured in 10 pools of n = 16 single SRGF were not different from mean values measured in single SRGF, but the CV was reduced to or below the threshold. Separate SRGF pools failed to differently affect ASC growth rate (slope pool A = 0.6; R2 = 0.99; slope pool B = 0.7; R2 0.99) or differentiation potential. DISCUSSION: Results deriving from our algorithm and from validation utilizing real SRGF pools demonstrated that pooling n = 16 single-donor SRGF products can ameliorate variability of final growth factor concentrations. Different pools of n = 16 single donor SRGF displayed consitent capability to modulate growth and differentiation potential of expanded ASC. Increasing the pool size should not further improve product composition.