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.

Full chimaeric CAR.CIK from patients engrafted after allogeneic haematopoietic cell transplant: Feasibility, anti-leukaemic potential and alloreactivity across major human leukocyte antigen barriers.

Cytokine-induced killer lymphocytes (CIK) are a promising alternative to conventional donor lymphocyte infusion (DLI), following allogeneic haematopoietic cell transplantation (HCT), due to their intrinsic anti-tumour activity and reduced risk of graft-versus-host disease (GVHD). We explored the feasibility, anti-leukaemic activity and alloreactive risk of CIK generated from full-donor chimaeric (fc) patients and genetically redirected by a chimeric antigen receptor (CAR) (fcCAR.CIK) against the leukaemic target CD44v6. fcCAR.CIK were successfully ex-vivo expanded from leukaemic patients in complete remission after HCT confirming their intense preclinical anti-leukaemic activity without enhancing the alloreactivity across human leukocyte antigen (HLA) barriers. Our study provides translational bases to support clinical studies with fcCAR.CIK, a sort of biological bridge between the autologous and allogeneic sources, as alternative DLI following HCT.

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.

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.

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.

Congenital Hypopigmentary Disorders with Multiorgan Impairment: A Case Report and an Overview on Gray Hair Syndromes.

The term congenital hypopigmentary disorders refers to a wide group of heterogeneous hereditary diseases, clinically characterized by inborn pigmentary defects of the iris, hair, and/or skin. They include Gray Hair Syndromes (GHSs), a rare group of autosomal recessive genodermatosis hallmarked by inborn silvery gray hair. GHSs encompass Griscelli, Chediak⁻Higashi, Elejalde, and Cross syndromes, which are all characterized by a broad spectrum of severe multisystem disorders, including neurological, ocular, skeletal, and immune system impairment. In this manuscript, we describe in detail the clinical, trichoscopic, and genetic features of a rare case of Griscelli syndrome; moreover, we provide an overview of all the GHSs known to date. Our report highlights how an accurate clinical examination with noninvasive methods, like trichoscopy, may play a crucial rule in diagnosis of rare and potentially lethal genetic syndromes such as Griscelli syndrome, in which timely diagnosis and therapy may modify the clinical course, quality of life, and likelihood of survival.

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.

Correction to: 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.

Following publication of the original article [1], the authors reported that all of the authors' names were processed incorrectly so that their given and family names were interchanged. In this Correction the correct author names are shown. The original publication of this article has been corrected.

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.

Human mesenchymal stromal cell transplantation modulates neuroinflammatory milieu in a mouse model of amyotrophic lateral sclerosis.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs), after intraparenchymal, intrathecal and endovenous administration, have been previously tested for cell therapy in amyotrophic lateral sclerosis in the SOD1 (superoxide dismutase 1) mouse. However, every administration route has specific pros and cons. METHODS: We administrated human MSCs (hMSCs) in the cisterna lumbaris, which is easily accessible and could be used in outpatient surgery, in the SOD1 G93A mouse, at the earliest onset of symptoms. Control animals received saline injections. Motor behavior was checked starting from 2 months of age until the mice were killed. Animals were killed 2 weeks after transplantation; lumbar motoneurons were stereologically counted, astrocytes and microglia were analyzed and quantified after immunohistochemistry and cytokine expression was assayed by means of real-time polymerase chain reaction. RESULTS: We provide evidence that this route of administration can exert strongly positive effects. Motoneuron death and motor decay were delayed, astrogliosis was reduced and microglial activation was modulated. In addition, hMSC transplantation prevented the downregulation of the anti-inflammatory interleukin-10, as well as that of vascular endothelial growth factor observed in saline-treated transgenic mice compared with wild type, and resulted in a dramatic increase in the expression of the anti-inflammatory interleukin-13. CONCLUSIONS: Our results suggest that hMSCs, when intracisternally administered, can exert their paracrine potential, influencing the inflammatory response of the host.

Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein-free Good Manufacturing Practice-compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS). Because the use of human-derived blood materials alleviates immunologic risks but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL). METHODS: Bone marrow samples were plated and expanded in α-minimum essential medium with 10% of three culture supplements: HPL, iHPL and FBS, at the same time. MSC morphology, growth and immunophenotype were analyzed at each passage. Karyotype, tumorigenicity and sterility were analyzed at the third passage. Statistical analyses were performed. RESULTS: The MSCs cultivated in the three different culture conditions showed no significant differences in terms of fibroblast colony-forming unit number, immunophenotype or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than did FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics that may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or karyotype modifications were observed. CONCLUSIONS: We demonstrated that iHPL is safer than HPL and represents a good, Good Manufacturing Practice-compliant alternative to FBS for MSC clinical production that is even more advantageous in terms of cellular growth and stemness.

Validation of analytical methods in compliance with good manufacturing practice: a practical approach.

BACKGROUND: The quality and safety of cell therapy products must be maintained throughout their production and quality control cycle, ensuring their final use in the patient. We validated the Lymulus Amebocyte Lysate (LAL) test and immunophenotype according to International Conference on Harmonization Q2 Guidelines and the EU Pharmacopoeia, considering accuracy, precision, repeatability, linearity and range. METHODS: For the endotoxin test we used a kinetic chromogenic LAL test. As this is a limit test for the control of impurities, in compliance with International Conference on Harmonization Q2 Guidelines and the EU Pharmacopoeia, we evaluated the specificity and detection limit.For the immunophenotype test, an identity test, we evaluated specificity through the Fluorescence Minus One method and we repeated all experiments thrice to verify precision. The immunophenotype validation required a performance qualification of the flow cytometer using two types of standard beads which have to be used daily to check cytometer reproducibly set up. The results were compared together.Collected data were statistically analyzed calculating mean, standard deviation and coefficient of variation percentage (CV%). RESULTS: The LAL test is repeatable and specific. The spike recovery value of each sample was between 0.25 EU/ml and 1 EU/ml with a CV% < 10%. The correlation coefficient (≥ 0.980) and CV% (< 10%) of the standard curve tested in duplicate showed the test's linearity and a minimum detectable concentration value of 0.005 EU/ml.The immunophenotype method performed thrice on our cell therapy products is specific and repeatable as showed by CV% inter -experiment < 10%. CONCLUSIONS: Our data demonstrated that validated analytical procedures are suitable as quality controls for the batch release of cell therapy products.Our paper could offer an important contribution for the scientific community in the field of CTPs, above all to small Cell Factories such as ours, where it is not always possible to have CFR21 compliant software.

Evaluating Ecological Impact and Sustainability in the Manufacturing of Advanced Therapies: Comparative Analysis of Greenhouse Gas Emissions in the Production of ATMPs in Open and Closed Systems.

The primary aim of this systematic analysis is to highlight opportunities to improve the environmental impact of advanced therapy medicinal products (ATMP) manufacturing. We have compared the Greenhouse Gas (GHG) emissions expressed in CO2eq of a classic clean room open system (AinB) Cell Factory versus a comparable closed system equipped with isolators (AinD). We have therefore outlined a theoretical situation to simulate the use of a closed system with an equivalent production output to that obtained in the Cell Factory (CF) of the Regina Margherita Children's Hospital. Open and closed systems for ATMPs have been compared as regards energy requirements, ecological footprints, and costs by analyzing a hypothetic cell production cycle of 21 days. The results demonstrate energy saving and a reduction of 52% in GHG emissions using closed systems per process cycle. Moreover, a reduction in production costs in an isolator setting is also evident. This study shows that the closed system solution has evident advantages compared with the open one.

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.

Managing leukapheresis in adult and pediatric patients eligible for chimeric antigen receptor T-cell therapy: suggestions from an Italian Expert Panel.

Chimeric antigen receptor (CAR) T-cell therapy relies on T cells engineered to target specific tumor antigens such as CD-19 in B-cell malignancies. In this setting, the commercially available products have offered a potential long-term cure for both pediatric and adult patients. Yet manufacturing CAR T cells is a cumbersome, multistep process, the success of which strictly depends on the characteristics of the starting material, i.e., lymphocyte collection yield and composition. These, in turn, might be affected by patient factors such as age, performance status, comorbidities, and previous therapies. Ideally, CAR T-cell therapies are a one-off treatment; therefore, optimization and the possible standardization of the leukapheresis procedure is critical, also in view of the novel CAR T cells currently under investigation for hematological malignancies and solid tumors. The most recent Best Practice recommendations for the management of children and adults undergoing CAR T-cell therapy provide a comprehensive guide to their use. However, their application in local practice is not straightforward and some grey areas remain. An Italian Expert Panel of apheresis specialists and hematologists from the centers authorized to administer CAR T-cell therapy took part in a detailed discussion on the following: 1) pre-apheresis patient evaluation; 2) management of the leukapheresis procedure, also in special situations represented by low lymphocyte count, peripheral blastosis, pediatric population <25 kg, and the COVID-19 outbreak; and 3) release and cryopreservation of the apheresis unit. This article presents some of the important challenges that must be faced to optimize the leukapheresis procedure and offers suggestions as to how to improve it, some of which are specific to the Italian setting.

Validation of analytical methods in GMP: the disposable Fast Read 102® device, an alternative practical approach for cell counting.

BACKGROUND: The quality and safety of advanced therapy products must be maintained throughout their production and quality control cycle to ensure their final use in patients. We validated the cell count method according to the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use and European Pharmacopoeia, considering the tests' accuracy, precision, repeatability, linearity and range. METHODS: As the cell count is a potency test, we checked accuracy, precision, and linearity, according to ICH Q2. Briefly our experimental approach was first to evaluate the accuracy of Fast Read 102® compared to the Bürker chamber. Once the accuracy of the alternative method was demonstrated, we checked the precision and linearity test only using Fast Read 102®. The data were statistically analyzed by average, standard deviation and coefficient of variation percentages inter and intra operator. RESULTS: All the tests performed met the established acceptance criteria of a coefficient of variation of less than ten percent. For the cell count, the precision reached by each operator had a coefficient of variation of less than ten percent (total cells) and under five percent (viable cells). The best range of dilution, to obtain a slope line value very similar to 1, was between 1:8 and 1:128. CONCLUSIONS: Our data demonstrated that the Fast Read 102® count method is accurate, precise and ensures the linearity of the results obtained in a range of cell dilution. Under our standard method procedures, this assay may thus be considered a good quality control method for the cell count as a batch release quality control test. Moreover, the Fast Read 102® chamber is a plastic, disposable device that allows a number of samples to be counted in the same chamber. Last but not least, it overcomes the problem of chamber washing after use and so allows a cell count in a clean environment such as that in a Cell Factory. In a good manufacturing practice setting the disposable cell counting devices will allow a single use of the count chamber they can then be thrown away, thus avoiding the waste disposal of vital dye (e.g. Trypan Blue) or lysing solution (e.g. Tuerk solution).

Dissection of the biphasic nature of hypoxia-induced motogenic action in bone marrow-derived human mesenchymal stem cells.

Hypoxic conditions have been reported to facilitate preservation of undifferentiated mesenchymal stem cell (MSC) phenotype and positively affect their colony-forming potential, proliferation, and migration/mobilization. In this study, designed to dissect mechanisms underlying hypoxia-dependent migration of bone marrow-derived human MSC (hMSC), signal transduction, and molecular mechanisms were evaluated by integrating morphological, molecular, and cell biology techniques, including the wound healing assay (WHA) and modified Boyden's chamber assay (BCA) to monitor migration. Exposure of hMSCs to moderate hypoxia resulted in a significant increase of migration of hMSCs in both WHA (from 6 to 20 hours) and BCA (within 6 hours). Mechanistic experiments outlined the following sequence of hypoxia-dependent events: (a) very early (15 minutes) increased generation of intracellular reactive oxygen species (ROS), which (b) was sufficient to switch on activation of extracellular regulated kinase 1/2 and c-Jun N-terminal protein kinase 1/2, found to be relevant for the early phase of hMSC migration; (c) hypoxia inducible factor-1 (HIF-1)-dependent increased expression of vascular endothelial growth factor (VEGF) (facilitated by ROS) and its progressive release that was responsible for (d) a delayed and sustained migration of hMSCs. These results suggest that hypoxia-dependent migration relies on a previously unrecognized biphasic scenario involving an early phase, requiring generation of ROS, and a delayed phase sustained by HIF-1-dependent expression and release of VEGF.

Mesenchymal stromal cell transplantation in amyotrophic lateral sclerosis: a long-term safety study.

BACKGROUND AIMS: Mesenchymal stem cells/marrow stromal cells (MSC) represent a promising tool for stem cell-based clinical trials in amyotrophic lateral sclerosis (ALS). We present the results of long-term monitoring of 19 ALS patients enrolled in two phase I clinical trials of autologous MSC transplantation. METHODS: Nineteen patients (11 male and eightfemale) with ALS were enrolled in two consecutive phase I clinical trials. The patients were followed-up for 6-9 months and then treated with autologous MSC isolated from bone marrow and implanted into the dorsal spinal cord with a surgical procedure. The patients were monitored regularly before and after transplantation with clinical, psychological and neuroradiologic assessments every 3 months, at the tertiary referral ALS center in Novara (Italy), until death. RESULTS: Follow-up brain magnetic resonance imaging (MRI) revealed no structural changes (including tumor formation) relative to the baseline throughout the follow-up. There was no deterioration in the psychosocial status and all patients coped well. No clear clinical benefits were detected in these patients but the recruitment and selection of appropriate patients into larger trials will be needed to test the efficacy of the treatment. CONCLUSIONS: This study is the first to show the safety of MSC transplantation in the central nervous system during a follow-up of nearly 9 years, and is in support of applying MSC-based cellular clinical trials to neurodegenerative disorders.