Impaired neutrophil-mediated cell death drives Ewing's Sarcoma in the background of Down syndrome.

Introduction: Ewing Sarcoma (EWS) has been reported in seven children with Down syndrome (DS). To date, a detailed assessment of this solid tumour in DS patients is yet to be made. Methods: Here, we characterise a chemo-resistant mediastinal EWS in a 2-year-old DS child, the youngest ever reported case, by exploiting sequencing approaches. Results: The tumour showed a neuroectodermal development driven by the EWSR1-FLI1 fusion. The inherited myeloperoxidase deficiency of the patient caused failure of neutrophil-mediated cell death and promoted genomic instability. Discussion: In this context, the tumour underwent genome-wide near haploidisation resulting in a massive overexpression of pro-inflammatory cytokines. Recruitment of defective neutrophils fostered rapid evolution of this EWS.

The exposure to extremely low frequency electromagnetic-fields inhibits the growth and potentiates the sensitivity to chemotherapy of bidimensional and tridimensional human osteosarcoma models.

We previously established a thermodynamical model to calculate the specific frequencies of extremely low frequency-electromagnetic field (ELF-EMF) able to arrest the growth of cancer cells. In the present study, for the first time, we investigated the efficacy of this technology on osteosarcoma, and we applied a precise frequency of the electromagnetic field on three human osteosarcoma cell lines, grown as adherent cells and spheroids. We evaluated the antitumour efficacy of irradiation in terms of response to chemotherapeutic treatments, which is usually poor in this type of cancer. Importantly, the results of this novel combinatorial approach revealed that the specific exposure can potentiate the efficacy of several chemotherapeutic drugs, both on bidimensional and tridimensional cancer models. The effectiveness of cisplatinum, methotrexate, ifosfamide and doxorubicin was greatly increased by the concomitant application of the specific ELF-EMF. Moreover, our experiments confirmed that ELF-EMF inhibited the proliferation and modulated the mitochondrial metabolism of all cancer models tested, whereas mesenchymal cells were not affected. The latter finding is extremely valuable, given the importance of preserving the cell reservoir necessary for tissue regeneration after chemotherapy. Altogether, this novel evidence opens new avenues to the clinical applications of ELF-EMF in oncology.

Low-density cultured cartilage cells expanded in platelet lysate present distinct features to develop an innovative clinical treatment for diffuse cartilage lesions.

PURPOSE: Chondrocyte-based cell therapies are effective for the treatment of chondral lesions, but remain poorly indicated for diffuse lesions in the context of early osteoarthritis (OA). The aim of this study was to develop a protocol to obtain chondroprogenitor cells suitable for the treatment of diffuse chondral lesions within early OA. METHODS: Cartilage cells were expanded at low density in human platelet lysate (hPL). A test was performed to exclude senescence. The expression of surface cluster of differentiation 146, cluster of differentiation 166, major histocompatibility complex (MHC)-I and MHC-II and of genes of interest were evaluated, as well as the trophic potential of these cells, by the assessment of lubricin and matrix production. The immunomodulatory potential was assessed through their co-culture with macrophages. RESULTS: Cartilage cells expanded at low density in hPL showed higher proliferation rate than standard-density cells, no replicative senescence, low immunogenicity and expression of lubricin. Moreover, they presented an increased expression of chondrogenic and antihypertrophic markers, as well as a superior matrix deposition if compared to cells cultured at standard density. Cartilage cells induced on macrophages an upregulation of CD206, although a higher increase of CD163 expression was observed in the presence of low-density cells. CONCLUSIONS: These findings lay the grounds to explore the clinical usefulness of low-density cultured cartilage cells to treat diffuse lesions in early OA joints for both autologous and allogenic use. LEVEL OF EVIDENCE: Not applicable.

State of the Art and New Trends from the Second International StemNet Meeting.

The Second International StemNet (Federation of Stem Cell Research Associations) meeting took place on 18-20 October 2023 in Brescia (Italy), with the support of the University of Brescia and the Zooprophylactic Institute of Lombardy and Emilia Romagna. The program of the meeting was articulated in nine sections: (1) Biomedical Communication in Italy: Critical Aspects; (2) StemNet Next Generation Session; (3) Cell-Free Therapies; (4) Tips and Tricks of Research Valorisation; (5) Stem Cells and Cancer; (6) Stem Cells in Veterinary Applications; (7) Stem Cells in Clinical Applications; (8) Organoids and 3D Systems; (9) induced pluripotent stem cells (iPCS) and Gene Therapy. National and International speakers presented their scientific works, inspiring debates and discussions among the attendees. The participation in the meeting was high, especially because of the young researchers who animated all the sessions and the rich poster session.

A New Paclitaxel Formulation Based on Secretome Isolated from Mesenchymal Stem Cells Shows a Significant Cytotoxic Effect on Osteosarcoma Cell Lines.

BACKGROUND: Osteosarcoma (OS) represents a rare cancer with an unfavorable prognosis that needs innovative treatment. The aim was to isolate a secretome from mesenchymal stem cells (MSCs) that are treated with paclitaxel (PTX)-containing microvesicles as a drug delivery system and analyze its cytotoxic effects on OS cell lines (SJSA, MG63, and HOS). METHODS: Three batches of secretome (SECR-1, SECR-2, and SECR-3) were produced from three bone marrow (BM) MSCs samples treated for 24 h with 15 µg/mL of PTX or with a standard medium. The viability of the OS cell lines after 5 days of exposure to SECR-1-2-3 (pure and diluted to 1:2 and 1:4) was analyzed with an MTT assay. The same SECR batches were analyzed with high-performance liquid chromatography (HPLC) and with a nanoparticle tracking assay (NTA). RESULTS: A statistically significant decrease in the viability of all OS cell lines was observed after treatment with SECR-PTX 1-2-3 in a dose-response manner. The NTA analyses showed the presence of nanoparticles (NPs) with a mean size comparable to that of extracellular vesicles (EVs). The HPLC analyses detected the presence of PTX in minimal doses in all SECR batches. CONCLUSIONS: This proof-of-concept study showed that the conditioned medium isolated from MSCs loaded with PTX had a strong cytotoxic effect on OS cell lines, due to the presence of EV and PTX.

State of the Art and New Trends from the 2022 Gism Annual Meeting.

The 2022 Italian Mesenchymal Stem Cell Group (Gruppo Italiano Staminali Mesenchimali, GISM) Annual Meeting took place on 20-21 October 2022 in Turin (Italy), with the support of the University of Turin and the City of Health and Science of Turin. The novelty of this year's meeting was its articulation, reflecting the new structure of GISM based on six sections: (1) Bringing advanced therapies to the clinic: trends and strategies, (2) GISM Next Generation, (3) New technologies for 3D culture systems, (4) Therapeutic applications of MSC-EVs in veterinary and human medicine, (5) Advancing MSC therapies in veterinary medicine: present challenges and future perspectives, (6) MSCs: a double-edged sword: friend or foe in oncology. National and international speakers presented their scientific works with the aim of promoting an interactive discussion and training for all attendees. The atmosphere was interactive, where ideas and questions between younger researchers and senior mentors were shared in all moments of the congress.

Effect of mesenchymal stromal cell transplantation on long-term survival in amyotrophic lateral sclerosis.

BACKGROUND AIMS: Thanks to their immunomodulatory, tissue-protective and regenerative properties, mesenchymal stromal cells (MSCs) are a promising approach for amyotrophic lateral sclerosis (ALS); however, trials are limited and few follow-up studies have been published. This post-hoc analysis aims to describe the potential long-term effects of MSCs in ALS, analyzing data from two phase 1 clinical trials in ALS patients conducted by our group in 2002 and 2006. METHODS: We conducted two consecutive phase 1 prospective, open, pilot clinical trials, enrolling a total of 19 ALS patients. We followed patients for the duration of the disease. For each patient, we used the European Network to Cure ALS (ENCALS) survival prediction model to retrospectively calculate the expected survival at diagnosis. We then compared the predicted disease duration with the observed survival, analyzing patients at a single-patient level. RESULTS: Using the ENCALS model, we predicted short survival in one patient, intermediate survival in three patients, long survival in three patients and very long survival in 12 patients. The difference between predicted and observed survival for the whole group was significant and demonstrated a mean predicted survival of 70.79 months (standard deviation [SD], 27.53) and a mean observed survival of 118.8 months (SD, 89.26) (P = 0.016). Based on the monthly ALS Functional Rating Scale-Revised progression rate (median, 0.64/month), we considered 10 of 19 patients slow progressors and nine of 19 patients fast progressors. Of the slow progressors, eight of 10 (80%) had significantly increased disease duration compared with predicted, and only two (20%) had decreased estimated disease duration. By contrast, five of nine (55%) fast progressors had increased disease duration, whereas four (45%) had decreased disease duration. To date, four patients are still alive. CONCLUSIONS: The current study represents the first very long-term analysis of survival as an effect of MSC focal transplantation in the central nervous system of ALS patients, demonstrating that MSC transplantation could potentially slow down ALS progression and improve survival. Due to the interindividual variability in clinical course, at the current state of our knowledge, we cannot generalize the results, but these data provide new insights for planning the next generation of efficacy MSC clinical trials in ALS.

Stroma-derived miR-214 coordinates tumor dissemination.

BACKGROUND: Tumor progression is based on a close interaction between cancer cells and Tumor MicroEnvironment (TME). Here, we focus on the role that Cancer Associated Fibroblasts (CAFs), Mesenchymal Stem Cells (MSCs) and microRNAs (miRs) play in breast cancer and melanoma malignancy. METHODS: We used public databases to investigate miR-214 expression in the stroma compartment of primary human samples and evaluated tumor formation and dissemination following tumor cell injections in miR-214 overexpressing (miR-214over) and knock out (miR-214ko) mice. In addition, we dissected the impact of Conditioned Medium (CM) or Extracellular Vesicles (EVs) derived from miR-214-rich or depleted stroma cells on cell metastatic traits. RESULTS: We evidence that the expression of miR-214 in human cancer or metastasis samples mostly correlates with stroma components and, in particular, with CAFs and MSCs. We present data revealing that the injection of tumor cells in miR-214over mice leads to increased extravasation and metastasis formation. In line, treatment of cancer cells with CM or EVs derived from miR-214-enriched stroma cells potentiate cancer cell migration/invasion in vitro. Conversely, dissemination from tumors grown in miR-214ko mice is impaired and metastatic traits significantly decreased when CM or EVs from miR-214-depleted stroma cells are used to treat cells in culture. Instead, extravasation and metastasis formation are fully re-established when miR-214ko mice are pretreated with miR-214-rich EVs of stroma origin. Mechanistically, we also show that tumor cells are able to induce miR-214 production in stroma cells, following the activation of IL-6/STAT3 signaling, which is then released via EVs subsequently up-taken by cancer cells. Here, a miR-214-dependent pro-metastatic program becomes activated. CONCLUSIONS: Our findings highlight the relevance of stroma-derived miR-214 and its release in EVs for tumor dissemination, which paves the way for miR-214-based therapeutic interventions targeting not only tumor cells but also the TME.

Quantification of fecal adenovirus viral load and correlation with Vesikari Score in children with acute gastroenteritis.

BACKGROUND: Human adenoviruses (HAdVs) are an important cause of acute respiratory tract infections, conjunctivitis, hemorrhagic cystitis, and gastroenteritis. In addition to enteric serotypes 40 and 41, some serotypes belonging to subgroups A, B, and C have also been implicated to be etiological agents of gastroenteritis among infants and young children. The Vesikari Scoring System (VSS) is the severity scale that was originally developed to evaluate the effectiveness and efficacy of rotavirus vaccines on 20 points. The aim of this study was to evaluate and compare the diagnostic value of the VSS with HAdVs genome quantification in fecal samples collected from hospitalized children with acute gastroenteritis. METHODS: A total of 137 fecal specimens (69 male and 68 female) were tested for HAdVs. The samples were collected from under-five-year-old children with acute gastroenteritis in pediatric Hospital Regina Margherita of Turin in Italy. RESULTS: A total of 69 out of 137 (50.3%) samples were associated with HAdV genomic detection with a mean viral load of 1.08×1011±9.02×1011 genomes/mg fecal specimens. The samples were grouped on the basis of Mild VSS and Moderate VSS and the HAdV viral load was calculated in the two groups. No statistical differences were observed between two groups (P=0.6123 calculated by Mann-Whitney Test). CONCLUSIONS: Our results did not show a difference in mean viral load between the group with mild VVS and moderate VVS.

Improving Osteosarcoma Treatment: Comparative Oncology in Action.

Osteosarcoma (OSA) is the most common pediatric malignant bone tumor. Although surgery together with neoadjuvant/adjuvant chemotherapy has improved survival for localized OSA, most patients develop recurrent/metastatic disease with a dismally poor outcome. Therapeutic options have not improved for these OSA patients in recent decades. As OSA is a rare and "orphan" tumor, with no distinct targetable driver antigens, the development of new efficient therapies is still an unmet and challenging clinical need. Appropriate animal models are therefore critical for advancement in the field. Despite the undoubted relevance of pre-clinical mouse models in cancer research, they present some intrinsic limitations that may be responsible for the low translational success of novel therapies from the pre-clinical setting to the clinic. From this context emerges the concept of comparative oncology, which has spurred the study of pet dogs as a uniquely valuable model of spontaneous OSA that develops in an immune-competent system with high biological and clinical similarities to corresponding human tumors, including in its metastatic behavior and resistance to conventional therapies. For these reasons, the translational power of studies conducted on OSA-bearing dogs has seen increasing recognition. The most recent and relevant veterinary investigations of novel combinatorial approaches, with a focus on immune-based strategies, that can most likely benefit both canine and human OSA patients have been summarized in this commentary.

A New Human Platelet Lysate for Mesenchymal Stem Cell Production Compliant with Good Manufacturing Practice Conditions Preserves the Chemical Characteristics and Biological Activity of Lyo-Secretome Isolated by Ultrafiltration.

Recently, we proposed a Good Manufacturing Practice (GMP)-compliant production process for freeze-dried mesenchymal stem cell (MSC)-secretome (lyo-secretome): after serum starvation, the cell supernatant was collected, and the secretome was concentrated by ultrafiltration and freeze-dried, obtaining a standardized ready-to-use and stable powder. In this work, we modified the type of human platelet lysate (HPL) used as an MSC culture supplement during the lyo-secretome production process: the aim was to verify whether this change had an impact on product quality and also whether this new procedure could be validated according to GMP, proving the process robustness. MSCs were cultured with two HPLs: the standard previously validated one (HPL-E) and the new one (HPL-S). From the same pool of platelets, two batches of HPL were obtained: HPL-E (by repeated freezing and thawing cycles) and HPL-S (by adding Ca-gluconate to form a clot and its subsequent mechanical wringing). Bone marrow MSCs from three donors were separately cultured with the two HPLs until the third passage and then employed to produce lyo-secretome. The following indicators were selected to evaluate the process performance: (i) the lyo-secretome quantitative composition (in lipids and proteins), (ii) the EVs size distribution, and (iii) anti-elastase and (iv) immunomodulant activity as potency tests. The new HPL supplementation for MSCs culture induced only a few minimal changes in protein/lipid content and EVs size distribution; despite this, it did not significantly influence biological activity. The donor intrinsic MSCs variability in secretome secretion instead strongly affected the quality of the finished product and could be mitigated by concentrating the final product to reach a determined protein (and lipid) concentration. In conclusion, the modification of the type of HPL in the MSCs culture during lyo-secretome production induces only minimal changes in the composition but not in the potency, and therefore, the new procedure can be validated according to GMP.

A New Human Platelet Lysate for Mesenchymal Stem Cell Production Compliant with Good Manufacturing Practice Conditions.

Mesenchymal stem cells (MSCs) are classified as advanced therapy medicinal products, a new category of GMP (good manufacturing practice)-compliant medicines for clinical use. We isolated MSCs from 5 bone marrow (BM) samples using human platelet lysate (HPL) instead of foetal bovine serum (FBS). We used a new method of HPL production consisting of treating platelet (PLTs) pools with Ca-Gluconate to form a gel clot, then mechanically squeezing to release growth factors. We compared the new HPL (HPL-S) with the standard (HPL-E) obtained by freezing/thawing cycles and by adding heparin. HPL-S had not PLTs and fibrinogen but the quantity of proteins and growth factors was comparable to HPL-E. Therefore, HPL-S needed fewer production steps to be in compliance with GMP conditions. The number of colonies forming unit-fibroblasts (CFU-F) and the maintenance of stem markers showed no significant differences between MSCs with HPL-E and HPL-S. The cumulative population doubling was higher in MSCs with HPL-E in the earlier passages, but we observed an inverted trend of cell growth at the fourth passage. Immunophenotypic analysis showed a significant lower expression of HLA-DR in the MSCs with HPL-S (1.30%) than HPL-E (14.10%). In conclusion, we demonstrated that HPL-S is an effective alternative for MSC production under GMP conditions.

A Novel Xeno-Free Method to Isolate Human Endometrial Mesenchymal Stromal Cells (E-MSCs) in Good Manufacturing Practice (GMP) Conditions.

The cyclic regeneration of human endometrium is guaranteed by the proliferative capacity of endometrial mesenchymal stromal cells (E-MSCs). Due to this, the autologous infusion of E-MSCs has been proposed to support endometrial growth in a wide range of gynecological diseases. We aimed to compare two different endometrial sampling methods, surgical curettage and vacuum aspiration biopsy random assay (VABRA), and to validate a novel xeno-free method to culture human E-MSCs. Six E-MSCs cell samples were isolated after mechanical tissue homogenization and cultured using human platelet lysate. E-MSCs were characterized for the colony formation capacity, proliferative potential, and multilineage differentiation. The expression of mesenchymal and stemness markers were tested by FACS analysis and real-time PCR, respectively. Chromosomal alterations were evaluated by karyotype analysis, whereas tumorigenic capacity and invasiveness were tested by soft agar assay. Both endometrial sampling techniques allowed efficient isolation and expansion of E-MSCs using a xeno-free method, preserving their mesenchymal and stemness phenotype, proliferative potential, and limited multi-lineage differentiation ability during the culture. No chromosomal alterations and invasive/tumorigenic capacity were observed. Herein, we report the first evidence of efficient E-MSCs isolation and culture in Good Manufacturing Practice compliance conditions, suggesting VABRA endometrial sampling as alternative to surgical curettage.

Organotypic spinal cord cultures: An in vitro 3D model to preliminary screen treatments for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a severe neuromuscular disease affecting children, due to mutation/deletion of survival motor neuron 1 (SMN1) gene. The lack of functional protein SMN determines motor neuron (MN) degeneration and skeletal muscle atrophy, leading to premature death due to respiratory failure. Nowadays, the Food and Drug Administration approved the administration of three drugs, aiming at increasing the SMN production: although assuring noteworthy results, all these therapies show some non-negligible limitations, making essential the identification of alternative/synergistic therapeutic strategies. To offer a valuable in vitro experimental model for easily performing preliminary screenings of alternative promising treatments, we optimized an organotypic spinal cord culture (derived from murine spinal cord slices), which well recapitulates the pathogenetic features of SMA. Then, to validate the model, we tested the effects of human Mesenchymal Stem Cells (hMSCs) or murine C2C12 cells (a mouse skeletal myoblast cell line) conditioned media: 1/3 of conditioned medium (obtained from either hMSCs or C2C12 cells) was added to the conventional medium of the organotypic culture and maintained for 7 days. Then the slices were fixed and immunoreacted to evaluate the MN survival. In particular we observed that the C2C12 and hMSCs conditioned media positively influenced the MN soma size and the axonal length respectively, without modulating the glial activation. These data suggest that trophic factors released by MSCs or muscular cells can exert beneficial effects, by acting on different targets, and confirm the reliability of the model. Overall, we propose the organotypic spinal cord culture as an excellent tool to preliminarily screen molecules and drugs before moving to in vivo models, in this way partly reducing the use of animals and the costs.

Genetic and Epigenetic Characterization of a Discordant KMT2A/AFF1-Rearranged Infant Monozygotic Twin Pair.

The KMT2A/AFF1 rearrangement is associated with an unfavorable prognosis in infant acute lymphocytic leukemia (ALL). Discordant ALL in monozygotic twins is uncommon and represents an attractive resource to evaluate intrauterine environment-genetic interplay in ALL. Mutational and epigenetic profiles were characterized for a discordant KMT2A/AFF1-rearranged infant monozygotic twin pair and their parents, and they were compared to three independent KMT2A/AFF1-positive ALL infants, in which the DNA methylation and gene expression profiles were investigated. A de novo Q61H NRAS mutation was detected in the affected twin at diagnosis and backtracked in both twins at birth. The KMT2A/AFF1 rearrangement was absent at birth in both twins. Genetic analyses conducted at birth gave more insights into the timing of the mutation hit. We identified correlations between DNA methylation and gene expression changes for 32 genes in the three independent affected versus remitted patients. The strongest correlations were observed for the RAB32, PDK4, CXCL3, RANBP17, and MACROD2 genes. This epigenetic signature could be a putative target for the development of novel epigenetic-based therapies and could help in explaining the molecular mechanisms characterizing ALL infants with KMT2A/AFF1 fusions.

Precision Medicine in Osteosarcoma: MATCH Trial and Beyond.

Osteosarcoma (OS) is a rare bone malignant tumour with a poor prognosis in the case of recurrence. So far, there is no agreement on the best systemic therapy for relapsed OS. The availability of next generation sequencing techniques has recently revolutionized clinical research. The sequencing of the tumour and its matched normal counterpart has the potential to reveal a wide landscape of genetic alterations with significant implications for clinical practice. The knowledge that the genomic profile of a patient's tumour can be precisely mapped and matched to a targeted therapy in real time has improved the development of precision medicine trials (PMTs). PMTs aiming at determining the effectiveness of targeted therapies could be advantageous for patients with a tumour refractory to standard therapies. Development of PMTs for relapsed OS is largely encouraging and is in its initial phase. Assessing OS features, such as its rarity, its age distribution, the technical issues related to the bone tissue origin, and its complex genomic landscape, represents a real challenge for PMTs development. In this light, a multidisciplinary approach is required to fully exploit the potential of precision medicine for OS patients.

Human Endogenous Retrovirus-H and K Expression in Human Mesenchymal Stem Cells as Potential Markers of Stemness.

OBJECTIVE: The human endogenous retroviruses (HERVs) are endogenous retroviruses that were inserted into the germ cell DNA of humans over 30 million years ago. Insertion of HERVs into the chromosomal DNA can influence a number of host genes in various modes during human evolution and their proviral long terminal repeats can participate in the transcriptional regulation of various cellular genes. Our aim was to evaluate the pol gene expression of HERV-K and HERV-H in mesenchymal stem cells (MSCs) in relation with the expression of stemness genes such as NANOG, OCT-4, and SOX-2. METHODS: MSCs were isolated from bone marrow of healthy donors and expanded until the 5th passage in α-MEM with 10% fetal bovine serum. HERV-K, HERV-H pol gene, NANOG, OCT-4, SOX-2, and GAPDH expression was quantified by real-time PCR in MSCs during the expansion. RESULTS: HERV-K and HERV-H expression was always higher at p1 compared to other passages and this difference reached a high statistical significance when passage p1 was compared with passage 3. In addition, NANOG, OCT-4, and SOX-2 expression at p1 was significantly higher than their expression at p3. Pearson's test demonstrated a strong correlation between the expression of HERV-K and HERV-H and the expression of NANOG, OCT-4, and SOX-2. CONCLUSIONS: Our findings showed that HERV-K and H were concurrently expressed with pluripotency biomarkers NANOG, OCT-4, and SOX-2.

In Vitro Mesenchymal Progenitor Cell Expansion is a Predictor of Transplant-related Mortality and acute GvHD III-IV After Bone Marrow Transplantation in Univariate Analysis: A Large Single-Center Experience.

Mesenchymal stromal cells (MSCs) are multipotent stem cells able to differentiate into mesenchymal origin tissue and support the growth of hematopoietic stem cells. In order to understand the role of MSCs infused in bone marrow grafts, 53 consecutive patients were analyzed for engraftment, acute and chronic graft-versus-host disease (GvHD), transplant-related mortality (TRM), relapse incidence, and overall survival. The MSC content was measured as MSC expansion at the second passage. When in vitro-expanded MSC (cumulative population doubling at second passage, cPDp2) values were stratified according to the median value (2.2-fold increase), the univariate analysis showed a significant difference in TRM (23% vs. 3.8%, P=0.05.) and in acute GvHD III-IV incidence (12% vs. 4%, P=0.04), while the multivariate analysis did not confirm its independent role. No clinical parameters in donors and recipients were identified as predictors of cPDp2 expansion. Our study suggests a role for short-term ex vivo-expanded MSCs in reduced aGVHD III-IV incidence and TRM in univariate analysis. A multicenter, larger study is warranted to confirm these data.

Analysis of Mesenchymal Stromal Cell Engraftment After Allogeneic HSCT in Pediatric Patients: A Large Multicenter Study.

The mesenchymal stem cell (MSC) role after allogeneic hematopoietic stem cell transplantation (HSCT) is still a matter of debate; in particular, MSC engraftment in recipient bone marrow (BM) is unclear. A total of 46 patients were analyzed for MSC and hemopoietic stem cell engraftment after HSCT. The majority of patients had the BM as the stem cell source, and acute leukemia was the main indication for HSCT. Mesenchymal and hematopoietic stem cell chimerism analysis was carried out through specific polymorphic tandemly repeated regions. All patients reached complete donor engraftment; no evidence of donor-derived MSC engraftment was noted. Our data indicate that MSCs after HSCT remain of recipient origin despite the following: (i) myeloablative conditioning; (ii) the stem cell source; (iii) the interval from HSCT to BM analysis; (iv) the underlying disease before HSCT; and (v) the patients' or the donors' age at HSCT.

Cytokines induced killer cells produced in good manufacturing practices conditions: identification of the most advantageous and safest expansion method in terms of viability, cellular growth and identity.

BACKGROUND: Cytokine-induced killer (CIK) cells are a very promising cell population raising growing interest in the field of cellular antitumor therapy. The aim of our study was to validate the most advantageous expansion method for this advanced therapy medicinal product (ATMP) and to translate it from preclinical field to good manufacturing practices (GMP). GMP ensures that ATMP are consistently produced and controlled to the quality standards required to their intended use. For this reason, the use of the xenogenic sera tended to be minimized by GMP for their high variability and the associated risk of transmitting infectious agents. RESULTS: We decided to replace Fetal Bovine Serum (FBS), largely used as medium supplement for CIKs expansion, with other culture media. Firstly, Human Serum (HS) and Human Pool Plasma (HPP) were tested as medium supplements giving not compliant results to acceptance criteria, established for CIKs, probably for the great batch to batch variability. Consequently, we decided to test three different serum free expansion media: X-VIVO 15, (largely used by other groups) and Tex Macs and Cell Genix GMP SCGM: two GMP manufactured media. We performed a validation consisting in three run-sand even if the small number of experiments didn't permit us to obtained statistical results we demonstrated that both X-VIVO 15 and Tex Macs fulfilled the quality standards in terms of cellular growth, viability and identity while Cell Genix GMP SCGM resulted not compliant as it caused some technical problems such as high mortality. CONCLUSION: In conclusion, these preclinical validation data lay the bases for a GMP-compliant process to improve the CIKs expansion method.