A phase I study of autologous mesenchymal stromal cells for severe steroid-dependent nephrotic syndrome.

BACKGROUNDSevere forms of idiopathic nephrotic syndrome (INS) require prolonged immunosuppressive therapies and repeated courses of high-dose glucocorticoids. Mesenchymal stromal cells (MSCs) have promising immunomodulatory properties that may be employed therapeutically to reduce patient exposure to medications and their side effects.METHODSWe performed a phase I open-label trial assessing safety and feasibility of autologous bone marrow-derived MSCs (BM-MSCs) in children and young adults with severe forms of steroid-dependent nephrotic syndrome. Following autologous BM-MSC preparation and infusion, oral immunosuppression was tapered. Safety, efficacy, and immunomodulatory effects in vivo were monitored for 12 months.RESULTSSixteen patients (10 children, 6 adults) were treated. Adverse events were limited and not related to BM-MSC infusions. All patients relapsed during follow-up, but in the 10 treated children, time to first relapse was delayed (P = 0.02) and number of relapses was reduced (P = 0.002) after BM-MSC infusion, compared with the previous 12 months. Cumulative prednisone dose was also reduced at 12 months compared with baseline (P < 0.05). No treatment benefit was observed in adults.In children, despite tapering of immunosuppression, clinical benefit was mirrored by a significant reduction in total CD19+, mature, and memory B cells and an increase in regulatory T cells in vivo up to 3-6 months following BM-MSC infusionCONCLUSIONTreatment with autologous BM-MSCs is feasible and safely reduces relapses and immunosuppression at 12 months in children with severe steroid-dependent INS. Immunomodulatory studies suggest that repeating MSC infusions at 3-6 months may sustain benefit.TRIAL REGISTRATIONEudraCT 2016-004804-77.FUNDINGAIFA Ricerca Indipendente 2016-02364623.

Development and Validation of a New LC-MS/MS Bioanalytical Method for the Simultaneous Determination of Levodopa, Levodopa Methyl Ester, and Carbidopa in Human Plasma Samples.

Levodopa (L-DOPA) treatment, combined with the administration of dopa-decarboxylase inhibitors (DDCIs), is still the most effective symptomatic treatment of Parkinson's disease (PD). Although its efficacy in the early stage of the disease has been confirmed, its complex pharmacokinetics (PK) increases the variability of the intra-individual motor response, thus amplifying the risk of motor/non-motor fluctuations and dyskinesia. Moreover, it has been demonstrated that L-DOPA PK is strongly influenced by several clinical, therapeutic, and lifestyle variables (e.g., dietary proteins). L-DOPA therapeutic monitoring is therefore crucial to provide personalized therapy, hence improving drug efficacy and safety. To this aim, we have developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify L-DOPA, levodopa methyl ester (LDME), and the DDCI carbidopa in human plasma. The compounds were extracted by protein precipitation and samples were analyzed with a triple quadrupole mass spectrometer. The method showed good selectivity and specificity for all compounds. No carryover was observed, and dilution integrity was demonstrated. No matrix effect could be retrieved; intra-day and inter-day precision and accuracy values met the acceptance criteria. Reinjection reproducibility was assessed. The described method was successfully applied to a 45-year-old male patient to compare the pharmacokinetic behavior of an L-DOPA-based medical treatment involving commercially available Mucuna pruriens extracts and an LDME/carbidopa (100/25 mg) formulation.

Potency assays and biomarkers for cell-based advanced therapy medicinal products.

Advanced Therapy Medicinal Products (ATMPs) based on somatic cells expanded in vitro, with or without genetic modification, is a rapidly growing area of drug development, even more so following the marketing approval of several such products. ATMPs are produced according to Good Manufacturing Practice (GMP) in authorized laboratories. Potency assays are a fundamental aspect of the quality control of the end cell products and ideally could become useful biomarkers of efficacy in vivo. Here we summarize the state of the art with regard to potency assays used for the assessment of the quality of the major ATMPs used clinic settings. We also review the data available on biomarkers that may substitute more complex functional potency tests and predict the efficacy in vivo of these cell-based drugs.

A comprehensive report of long-term stability data for a range ATMPs: A need to develop guidelines for safe and harmonized stability studies.

BACKGROUND AIMS: Advanced therapy medicinal products (ATMPs) are novel drugs based on genes, cells or tissues developed to treat many different diseases. Stability studies of each new ATMP need to be performed to define its shelf life and guarantee efficacy and safety upon infusion, and these are presently based on guidelines originally drafted for standard pharmaceutical drugs, which have properties and are stored in conditions quite different from cell products. The aim of this report is to provide evidence-based information for stability studies on ATMPs that will facilitate the interlaboratory harmonization of practices in this area. METHODS: We have collected and analyzed the results of stability studies on 19 different cell-based experimental ATMPs, produced by five authorized cell factories forming the Lombardy "Plagencell network" for use in 36 approved phase I/II clinical trials; most were cryopreserved and stored in liquid nitrogen vapors for 1 to 13 years. RESULTS: The cell attributes collected in stability studies included cell viability, immunophenotype and potency assays, in particular immunosuppression, cytotoxicity, cytokine release and proliferation/differentiation capacity. Microbiological attributes including sterility, endotoxin levels and mycoplasma contamination were also analyzed. All drug products (DPs), cryopreserved in various excipients containing 10% DMSO and in different primary containers, were very stable long term at <-150°C and did not show any tendency for diminished viability or efficacy for up to 13.5 years. CONCLUSIONS: Our data indicate that new guidelines for stability studies, specific for ATMPs and based on risk analyses, should be drafted to harmonize practices, significantly reduce the costs of stability studies without diminishing safety. Some specific suggestions are presented in the discussion.

Third-party bone marrow-derived mesenchymal stromal cell infusion before liver transplantation: A randomized controlled trial.

Mesenchymal stromal cells (MSC) have emerged as a promising therapy to minimize the immunosuppressive regimen or induce tolerance in solid organ transplantation. In this randomized open-label phase Ib/IIa clinical trial, 20 liver transplant patients were randomly allocated (1:1) to receive a single pretransplant intravenous infusion of third-party bone marrow-derived MSC or standard of care alone. The primary endpoint was the safety profile of MSC administration during the 1-year follow-up. In all, 19 patients completed the study, and none of those who received MSC experienced infusion-related complications. The incidence of serious and non-serious adverse events was similar in the two groups. Circulating Treg/memory Treg and tolerant NK subset of CD56bright NK cells increased slightly over baseline, albeit not to a statistically significant extent, in MSC-treated patients but not in the control group. Graft function and survival, as well as histologic parameters and intragraft expression of tolerance-associated transcripts in 1-year protocol biopsies were similar in the two groups. In conclusion, pretransplant MSC infusion in liver transplant recipients was safe and induced mild positive changes in immunoregulatory T and NK cells in the peripheral blood. This study opens the way for a trial on possible tolerogenic efficacy of MSC in liver transplantation. ClinicalTrials.gov identifier: NCT02260375.

Protective Effects of Human Nonrenal and Renal Stromal Cells and Their Conditioned Media in a Rat Model of Chronic Kidney Disease.

Mesenchymal stromal cells (MSCs) are emerging as a novel therapeutic option for limiting chronic kidney disease progression. Conditioned medium (CM) containing bioactive compounds could convey similar benefits, avoiding the potential risks of cell therapy. This study compared the efficacy of nonrenal and renal cell-based therapy with the corresponding CM in rats with renal mass reduction (RMR). Infusions of human kidney stromal cells (kPSCs) and CM-kPSCs, but not umbilical cord (uc) MSCs or CM-ucMSCs, reduced proteinuria and preserved podocyte number and nephrin expression in RMR rats. Glomerular fibrosis, microvascular rarefaction, and apoptosis were reduced by all treatments, while the peritubular microvascular loss was reduced by kPSCs and CM-kPSCs treatment only. Importantly, kPSCs and CM-kPSCs reduced NG2-positive pericytes, and all therapies reduced α-smooth muscle actin expression, indicating reduced myofibroblast expansion. Treatment with kPSCs also significantly inhibited the accumulation of ED1-positive macrophages in the renal interstitium of RMR rats. These findings demonstrate that the CM of ucMSCs and kPSCs confers similar renoprotection as the cells. kPSCs and CM-kPSCs may be superior in attenuating chronic renal injury as a cell source.

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Therapeutic potential of stromal cells of non-renal or renal origin in experimental chronic kidney disease.

BACKGROUND: Mesenchymal stromal cell (MSC)-based therapy is a promising strategy for preventing the progression of chronic kidney disease (CKD), with the potential to induce tissue regeneration. In search of the best cellular source we compared, in the rat model of adriamycin (ADR) nephropathy, the regenerative potential of human stromal cells of non-renal origin, such as bone marrow (bm) MSCs and umbilical cord (uc) MSCs, with that of newly discovered stromal cells of renal origin, the kidney perivascular cells (kPSCs) known to exhibit tissue-specific properties. METHODS: The therapeutic effect of repeated infusions of human bmMSCs, ucMSCs, kPSCs (1.5 × 106 cells/rats) or conditioned medium from ucMSCs was studied in athymic rats with ADR-induced nephropathy (7.9 mg/kg). The ability of the three stromal cell populations to engraft the damaged kidney was evaluated by detecting the presence of human nuclear antigenpos cells. Glomerular podocyte loss and endothelial damage, sclerotic lesions and inflammation were assessed at 14 and 28 days. In-vitro experiments with a transwell system were performed to investigate the effects of different stromal cell populations on parietal epithelial cells (PECs) activated or not with albumin or angiotensin II for 24 h. RESULTS: Infusions of non-renal and renal stromal cells resulted in a comparable engraftment into the kidney, in the peritubular areas and around the glomerular structures. All three cell populations limited podocyte loss and glomerular endothelial cell injury, and attenuated the formation of podocyte and PEC bridges. This translated into a reduction of glomerulosclerosis and fibrosis. Human ucMSCs had an anti-inflammatory effect superior to that of the other stromal cells, reducing macrophage infiltration and inducing polarisation towards the M2 macrophage phenotype. Conditioned medium from ucMSCs shared the same renoprotective effects of the cells. Consistent with in-vivo data, bmMSCs and kPSCs, but even more so ucMSCs, limited proliferation, migratory potential and extracellular matrix production of activated PECs, when cultured in a transwell system. CONCLUSIONS: Our data indicate that either non-renal or renal stromal cells induce renal tissue repair, highlighting ucMSCs and their conditioned medium as the most reliable clinical therapeutic tool for CKD patients.

Mesenchymal stromal cells from human umbilical cord prevent the development of lung fibrosis in immunocompetent mice.

Lung fibrosis is a severe condition resulting from several interstial lung diseases (ILD) with different etiologies. Current therapy of ILD, especially those associated with connective tissue diseases, is rather limited and new anti-fibrotic strategies are needed. In this study, we investigated the anti-fibrotic activity in vivo of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC). Adult immunocompetent C57BL/6 mice (n. = 8 for each experimental condition) were injected intravenously with hUC-MSC (n. = 2.5 × 105) twice, 24 hours and 7 days after endotracheal injection of bleomycin. Upon sacrifice at days 8, 14, 21, collagen content, inflammatory cytokine profile, and hUC-MSC presence in explanted lung tissue were analyzed. Systemic administration of a double dose of hUC-MSC significantly reduced bleomycin-induced lung injury (inflammation and fibrosis) in mice through a selective inhibition of the IL6-IL10-TGFß axis involving lung M2 macrophages. Only few hUC-MSC were detected from explanted lungs, suggesting a "hit and run" mechanism of action of this cellular therapy. Our data indicate that hUC-MSC possess strong in vivo anti-fibrotic activity in a mouse model resembling an immunocompetent human subject affected by inflammatory ILD, providing proof of concept for ad-hoc clinical trials.

Utility of routine evaluation of sterility of cellular therapy products with or without extensive manipulation: Best practices and clinical significance.

BACKGROUND: We analyzed the results of routine sterility testing performed in our center over the last 10 years, in the context both hematopoietic stem cell transplantation (HSCT) and Advanced Therapeutic Medicinal Products (ATMPs). METHODS: For sterility tests 14-day cultures were performed in culture media detecting aerobic and anaerobic microorganisms. RESULTS: In this study, 22/1643 (1.3%) of apheretic products for autologous or allogeneic HSCT were contaminated, whereas 14/73 bone marrow (BM) harvests (17.8%) were positive. In 22 cases, the contaminated HSCs were infused to patients, but there was no evidence of any adverse impact of contamination on the hematologic engraftment or on infections. Indeed none of the five positive hemocultures detected in patients following infusion could be linked to the contaminated stem cell product. Our Cell Factory also generated 286 ATMPs in good manufacturing practice (GMP) conditions since 2007 and all final products were sterile. In three cases of mesenchymal stromal cell expansions, the starting BM harvests were contaminated, but the cell products at the end of expansion were sterile, presumably thanks to the presence of an antibiotic in the culture medium. DISCUSSION: The decreased rate of contamination of cell harvests observed with time suggests that routine sterility testing and communication of the results to the collecting centers may improve clinical practices. Furthermore, we recommend the use of antibiotics in the medium for ATMP expansion, to decrease the likelihood of expanding microorganisms within clean rooms. Finally we discuss the costs of sterility testing of ATMPs by GMP-approved external laboratories.

A dual-phase xenon TPC for scintillation and ionisation yield measurements in liquid xenon.

A small-scale, two-phase (liquid/gas) xenon time projection chamber (Xurich II) was designed, constructed and is under operation at the University of Zürich. Its main purpose is to investigate the microphysics of particle interactions in liquid xenon at energies below 50 keV, which are relevant for rare event searches using xenon as target material. Here we describe in detail the detector, its associated infrastructure, and the signal identification algorithm developed for processing and analysing the data. We present the first characterisation of the new instrument with calibration data from an internal 83 m Kr source. The zero-field light yield is 15.0 and 14.0 photoelectrons/keV at 9.4 and 32.1 keV, respectively, and the corresponding values at an electron drift field of 1 kV/cm are 10.8 and 7.9 photoelectrons/keV. The charge yields at these energies are 28 and 31 electrons/keV, with the proportional scintillation yield of 24 photoelectrons per one electron extracted into the gas phase, and an electron lifetime of 200  µ s. The relative energy resolution, σ / E , is 11.9 and 5.8% at 9.4 and 32.1 keV, respectively using a linear combination of the scintillation and ionisation signals. We conclude with measurements of the electron drift velocity at various electric fields, and compare these to literature values.

Multiple intracerebroventricular injections of human umbilical cord mesenchymal stem cells delay motor neurons loss but not disease progression of SOD1G93A mice.

Stem cell therapy is considered a promising approach in the treatment of amyotrophic lateral sclerosis (ALS) and mesenchymal stem cells (MSCs) seem to be the most effective in ALS animal models. The umbilical cord (UC) is a source of highly proliferating fetal MSCs, more easily collectable than other MSCs. Recently we demonstrated that human (h) UC-MSCs, double labeled with fluorescent nanoparticles and Hoechst-33258 and transplanted intracerebroventricularly (ICV) into SOD1G93A transgenic mice, partially migrated into the spinal cord after a single injection. This prompted us to assess the effect of repeated ICV injections of hUC-MSCs on disease progression in SOD1G93A mice. Although no transplanted cells migrated to the spinal cord, a partial but significant protection of motor neurons (MNs) was found in the lumbar spinal cord of hUC-MSCs-treated SOD1G93A mice, accompanied by a shift from a pro-inflammatory (IL-6, IL-1ß) to anti-inflammatory (IL-4, IL-10) and neuroprotective (IGF-1) environment in the lumbar spinal cord, probably linked to the activation of p-Akt survival pathway in both motor neurons and reactive astrocytes. However, this treatment neither prevented the muscle denervation nor delayed the disease progression of mice, emphasizing the growing evidence that protecting the motor neuron perikarya is not sufficient to delay the ALS progression.

Human mesenchymal stromal cells transplanted into mice stimulate renal tubular cells and enhance mitochondrial function.

Mesenchymal stromal cells (MSCs) are renoprotective and drive regeneration following injury, although cellular targets of such an effect are still ill-defined. Here, we show that human umbilical cord (UC)-MSCs transplanted into mice stimulate tubular cells to regain mitochondrial mass and function, associated with enhanced microtubule-rich projections that appear to mediate mitochondrial trafficking to create a reparative dialogue among adjacent tubular cells. Treatment with UC-MSCs in mice with cisplatin-induced acute kidney injury (AKI) regulates mitochondrial biogenesis in proximal tubuli by enhancing PGC1α expression, NAD+ biosynthesis and Sirtuin 3 (SIRT3) activity, thus fostering antioxidant defenses and ATP production. The functional role of SIRT3 in tubular recovery is highlighted by data that in SIRT3-deficient mice with AKI, UC-MSC treatment fails to induce renoprotection. These data document a previously unrecognized mechanism through which UC-MSCs facilitate renal repair, so as to induce global metabolic reprogramming of damaged tubular cells to sustain energy supply.Mesenchymal stromal cells drive renal regeneration following injury. Here, the authors show that human mesenchymal stromal cells, when transplanted into mice with acute kidney injury, stimulate renal tubular cell growth and enhance mitochondrial function via SIRT3.

Phenotypical and Functional Characteristics of In Vitro-Expanded Adipose-Derived Mesenchymal Stromal Cells From Patients With Systematic Sclerosis.

Mesenchymal stromal cells (MSCs) have received attention as an ideal source of regenerative cells because of their multipotent differentiation potential. Adipose tissue is an attractive source of MSCs. Recent studies have shown that autologous fat grafting may be effective in the treatment of systemic sclerosis (SSc), but no specific study exists that aimed at investigating whether adipose tissue-derived stromal cells (ADSCs) from SSc patients maintain normal phenotypic and functional characteristics. The purpose of the current study was to investigate whether ADSCs from patients with SSc (SSc-ADSCs) are phenotypically and functionally identical to those from healthy controls (HC-ADSCs). Adipose tissue samples were obtained from 10 patients with SSc and from 8 HCs. Both MSC populations were evaluated for their capacity to (a) express specific MSC surface antigens by flow cytometry analysis, (b) proliferate, (c) differentiate along the adipogenic and osteogenic lineages, (d) suppress in vitro lymphocyte proliferation induced by a mitogenic stimulus, and (e) support endothelial cell (EC) tube formation. ADSCs from SSc patients and HCs showed similar surface phenotype and multilineage differentiation capabilities. In PBMC proliferation inhibition assays, no significant differences were observed between SSc- and HC-ADSCs. Using ADSC/EC cocultures, both SSc- and HC-ADSCs improved tube formation by both HC- and SSc-ECs. This effect was enhanced under hypoxic conditions in all of the cocultures. SSc-ADSCs exhibited the same phenotypic pattern, proliferation and differentiation potentials, and immunosuppressive properties as those from HCs. The proangiogenic activity shown by SSc-ADSCs, namely, under hypoxic conditions, suggests that autologous ADSC grafting may represent a possible therapeutic option for SSc.

Thrombospondin-1 promotes mesenchymal stromal cell functions via TGFß and in cooperation with PDGF.

Mesenchymal stromal cells (MSC) are characterized by unique tropism for wounded tissues, high differentiating capacity, ability to induce tissue repair, and anti-inflammatory and immunoregulatory activities. This has generated interest in their therapeutic use in severe human conditions as well as in regenerative medicine and tissue engineering. Identification of factors involved in the regulation of MSC proliferation, migration and differentiation could provide insights into the pathophysiological regulation of MSC and be exploited to optimize clinical grade expansion protocols for therapeutic use. Here we identify thrombospondin-1 (TSP-1) as a major regulator of MSC. TSP-1 induced MSC proliferation. This effect was mediated by TSP-1-induced activation of endogenous TGFß, as shown by the inhibitory effects of anti-TGFß antibodies and by the lack of activity of TSP-2 - that does not activate TGFß. Moreover, TSP-1 strongly potentiated the proliferative and migratory activity of PDGF on MSC. TSP-1 directly bound to PDGF, through a site located within the TSP-1 type III repeats, and protected the growth factor from degradation by MSC-derived proteases, hence increasing its stability and bioavailability. The studies presented here identify a more comprehensive picture of the pleiotropic effect of TSP-1 on MSC behavior, setting the basis for further studies aimed at investigating the possible use of PDGF and TSP-1 in the in vitro expansion of MSC for therapeutic applications.

Longitudinal tracking of triple labeled umbilical cord derived mesenchymal stromal cells in a mouse model of Amyotrophic Lateral Sclerosis.

The translational potential of cell therapy to humans requires a deep knowledge of the interaction between transplanted cells and host tissues. In this study, we evaluate the behavior of umbilical cord mesenchymal stromal cells (UC-MSCs), labeled with fluorescent nanoparticles, transplanted in healthy or early symptomatic transgenic SOD1G93A mice (a murine model of Amyotrophic Lateral Sclerosis). The double labeling of cells with nanoparticles and Hoechst-33258 enabled their tracking for a long time in both cells and tissues. Whole-body distribution of UC-MSCs was performed by in-vivo and ex-vivo analyses 1, 7, 21 days after single intravenous or intracerebroventricular administration. By intravenous administration cells were sequestered by the lungs and rapidly cleared by the liver. No difference in biodistribution was found among the two groups. On the other hand, UC-MSCs transplanted in lateral ventricles remained on the choroid plexus for the whole duration of the study even if decreasing in number. Few cells were found in the spinal cord of SOD1G93A mice exclusively. No migration in brain parenchyma was observed. These results suggest that the direct implantation in brain ventricles allows a prolonged permanence of cells close to the damaged areas and makes this method of tracking reliable for future studies of efficacy.

Transplanted Umbilical Cord Mesenchymal Stem Cells Modify the In Vivo Microenvironment Enhancing Angiogenesis and Leading to Bone Regeneration.

Umbilical cord mesenchymal stem cells (UC-MSCs) show properties similar to bone marrow mesenchymal stem cells (BM-MSCs), although controversial data exist regarding their osteogenic potential. We prepared clinical-grade UC-MSCs from Wharton's Jelly and we investigated if UC-MSCs could be used as substitutes for BM-MSCs in muscoloskeletal regeneration as a more readily available and functional source of MSCs. UC-MSCs were loaded onto scaffolds and implanted subcutaneously (ectopically) and in critical-sized calvarial defects (orthotopically) in mice. For live cell-tracking experiments, UC-MSCs were first transduced with the luciferase gene. Angiogenic properties of UC-MSCs were tested using the mouse metatarsal angiogenesis assay. Cell secretomes were screened for the presence of various cytokines using an array assay. Analysis of implanted scaffolds showed that UC-MSCs, contrary to BM-MSCs, remained detectable in the implants for 3 weeks at most and did not induce bone formation in an ectopic location. Instead, they induced a significant increase of blood vessel ingrowth. In agreement with these observations, UC-MSC-conditioned medium presented a distinct and stronger proinflammatory/chemotactic cytokine profile than BM-MSCs and a significantly enhanced angiogenic activity. When UC-MSCs were orthotopically transplanted in a calvarial defect, they promoted increased bone formation as well as BM-MSCs. However, at variance with BM-MSCs, the new bone was deposited through the activity of stimulated host cells, highlighting the importance of the microenvironment on determining cell commitment and response. Therefore, we propose, as therapy for bone lesions, the use of allogeneic UC-MSCs by not depositing bone matrix directly, but acting through the activation of endogenous repair mechanisms.

Molecular events underlying interleukin-6 independence in a subclone of the CMA-03 multiple myeloma cell line.

We explored the molecular mechanisms involved in the establishement of CMA-03/06, an IL-6-independent variant of the multiple myeloma cell line CMA-03 previously generated in our Institution. CMA-03/06 cells grow in the absence of IL-6 with a doubling time comparable with that of CMA-03 cells; neither the addition of IL6 (IL-6) to the culture medium nor co-culture with multipotent mesenchymal stromal cells increases the proliferation rate, although they maintain the responsiveness to IL-6 stimulation as demonstrated by STAT1, STAT3, and STAT5 induction. IL-6 independence of CMA-03/06 cells is not apparently due to the development of an autocrine IL-6 loop, nor to the observed moderate constitutive activation of STAT5 and STAT3, since STAT3 silencing does not affect cell viability or proliferation. When compared to the parental cell line, CMA-03/06 cells showed an activated pattern of the NF-κB pathway. This finding is supported by gene expression profiling (GEP) analysis identifying an appreciable fraction of modulated genes (28/308) in the CMA-03/06 subclone reported to be involved in this pathway. Furthermore, although more resistant to apoptotic stimuli compared to the parental cell line, CMA-03/06 cells display a higher sensibility to NF-κB inhibition induced by bortezomib. Finally, GEP analysis suggests an involvement of a number of cytokines, which might contribute to IL-6 independence of CMA-03/06 by stimulating growth and antiapoptotic processes. In conclusion, the parental cell-line CMA-03 and its variant CMA-03/06 represent a suitable model to further investigate molecular mechanisms involved in the IL-6-independent growth of myeloma cells.