Clinical-grade mesenchymal stromal cells produced under various good manufacturing practice processes differ in their immunomodulatory properties: standardization of immune quality controls.

Clinical-grade mesenchymal stromal cells (MSCs) are usually expanded from bone marrow (BMMSCs) or adipose tissue (ADSCs) using processes mainly differing in the use of fetal calf serum (FCS) or human platelet lysate (PL). We aimed to compare immune modulatory properties of clinical-grade MSCs using a combination of fully standardized in vitro assays. BMMSCs expanded with FCS (BMMSC-FCS) or PL (BMMSC-PL), and ADSC-PL were analyzed in quantitative phenotypic and functional experiments, including their capacity to inhibit the proliferation of T, B, and NK cells. The molecular mechanisms supporting T-cell inhibition were investigated. These parameters were also evaluated after pre-stimulation of MSCs with inflammatory cytokines. BMMSC-FCS, BMMSC-PL, and ADSC-PL displayed significant differences in expression of immunosuppressive and adhesion molecules. Standardized functional assays revealed that resting MSCs inhibited proliferation of T and NK cells, but not B cells. ADSC-PL were the most potent in inhibiting T-cell growth, a property ascribed to interferon-γ-dependent indoleamine 2,3-dioxygenase activity. MSCs did not stimulate allogeneic T cell proliferation but were efficiently lysed by activated NK cells. The systematic use of quantitative and reproducible validation techniques highlights differences in immunological properties of MSCs produced using various clinical-grade processes. ADSC-PL emerge as a promising candidate for future clinical trials.

[1]  N. Rouas-Freiss,et al.  Human Leukocyte Antigen‐G5 Secretion by Human Mesenchymal Stem Cells Is Required to Suppress T Lymphocyte and Natural Killer Function and to Induce CD4+CD25highFOXP3+ Regulatory T Cells , 2008, Stem cells.

[2]  D. Strunk,et al.  Human Alternatives to Fetal Bovine Serum for the Expansion of Mesenchymal Stromal Cells from Bone Marrow , 2009, Stem cells.

[3]  J. Ringe,et al.  Specific plasma membrane protein phenotype of culture-amplified and native human bone marrow mesenchymal stem cells. , 2008, Blood.

[4]  G. Orive,et al.  Safety and efficient ex vivo expansion of stem cells using platelet-rich plasma technology. , 2013, Therapeutic delivery.

[5]  P. Bourin,et al.  A first approach for the production of human adipose tissue-derived stromal cells for therapeutic use. , 2011, Methods in molecular biology.

[6]  L. Moretta,et al.  Mesenchymal stem cells inhibit natural killer-cell proliferation, cytotoxicity, and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2. , 2008, Blood.

[7]  L. Moretta,et al.  Mesenchymal stem cells expanded in human platelet lysate display a decreased inhibitory capacity on T‐ and NK‐cell proliferation and function , 2011, European journal of immunology.

[8]  R. Cancedda,et al.  Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway , 2005, European journal of immunology.

[9]  S. Messinger,et al.  Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial. , 2012, JAMA.

[10]  M. Strioga,et al.  Same or not the same? Comparison of adipose tissue-derived versus bone marrow-derived mesenchymal stem and stromal cells. , 2012, Stem cells and development.

[11]  R. Romieu-Mourez,et al.  Mesenchymal stromal cells cross-present soluble exogenous antigens as part of their antigen-presenting cell properties. , 2009, Blood.

[12]  J. Rossignol,et al.  A role for heme oxygenase-1 in the immunosuppressive effect of adult rat and human mesenchymal stem cells. , 2007, Blood.

[13]  M. Krampera,et al.  Mesenchymal stromal cell ‘licensing’: a multistep process , 2011, Leukemia.

[14]  L. Moretta,et al.  Expression of the DNAM-1 ligands, Nectin-2 (CD112) and poliovirus receptor (CD155), on dendritic cells: relevance for natural killer-dendritic cell interaction. , 2006, Blood.

[15]  R. Childs,et al.  Fetal and adult multipotent mesenchymal stromal cells are killed by different pathways. , 2011, Cytotherapy.

[16]  A. Ignatius,et al.  GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSC , 2012, PloS one.

[17]  L. Moretta,et al.  Mesenchymal stem cells inhibit natural killer – cell proliferation , cytotoxicity , and cytokine production : role of indoleamine 2 , 3-dioxygenase and prostaglandin E 2 , 2008 .

[18]  Yunhui Liu,et al.  Platelet-derived growth factor BB promotes the migration of bone marrow-derived mesenchymal stem cells towards C6 glioma and up-regulates the expression of intracellular adhesion molecule-1 , 2009, Neuroscience Letters.

[19]  Asha A. Nair,et al.  High‐Resolution Molecular Validation of Self‐Renewal and Spontaneous Differentiation in Clinical‐Grade Adipose‐Tissue Derived Human Mesenchymal Stem Cells , 2014, Journal of cellular biochemistry.

[20]  A. Uccelli,et al.  Mesenchymal stem cells in health and disease , 2008, Nature Reviews Immunology.

[21]  A. Barbero,et al.  Fibroblast growth factor 2 and platelet-derived growth factor, but not platelet lysate, induce proliferation-dependent, functional class II major histocompatibility complex antigen in human mesenchymal stem cells. , 2010, Arthritis and rheumatism.

[22]  Jacques Galipeau,et al.  Human MSC suppression correlates with cytokine induction of indoleamine 2,3-dioxygenase and bystander M2 macrophage differentiation. , 2012, Molecular therapy : the journal of the American Society of Gene Therapy.

[23]  C. Choong,et al.  PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. , 2008, Blood.

[24]  R. Romieu-Mourez,et al.  Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-γ licensing , 2011, Cytotherapy.

[25]  F. Benvenuto,et al.  Human mesenchymal stem cells modulate B-cell functions. , 2006, Blood.

[26]  L. Cosmi,et al.  Toll‐Like Receptors 3 and 4 Are Expressed by Human Bone Marrow‐Derived Mesenchymal Stem Cells and Can Inhibit Their T‐Cell Modulatory Activity by Impairing Notch Signaling , 2008, Stem cells.

[27]  C. Ricordi,et al.  Mesenchymal stromal (stem) cells to improve solid organ transplant outcome: lessons from the initial clinical trials , 2013, Current opinion in organ transplantation.

[28]  S. Perez,et al.  Characterization of the Optimal Culture Conditions for Clinical Scale Production of Human Mesenchymal Stem Cells , 2006, Stem cells.

[29]  Tao Wang,et al.  The multiple functional roles of mesenchymal stem cells in participating in treating liver diseases , 2014, Journal of cellular and molecular medicine.

[30]  S. Rutella,et al.  EDITOR’S QUIZ: GI SNAPSHOT , 2007, Gut.

[31]  P. Libby,et al.  CD40L induces inflammation and adipogenesis in adipose cells – a potential link between metabolic and cardiovascular disease , 2010, Thrombosis and Haemostasis.

[32]  R. Handgretinger,et al.  Human multipotent mesenchymal stromal cells use galectin-1 to inhibit immune effector cells. , 2010, Blood.

[33]  A. Ignatius,et al.  Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components , 2012, Cytotherapy.

[34]  E. Lombardo,et al.  Human adipose-derived stem cells impair natural killer cell function and exhibit low susceptibility to natural killer-mediated lysis. , 2012, Stem cells and development.

[35]  L. Sensebé,et al.  [Adipose-derived stromal cells: history, isolation, immunomodulatory properties and clinical perspectives]. , 2015, Annales de chirurgie plastique et esthetique.

[36]  S. Kyurkchiev,et al.  Adipose tissue-derived mesenchymal stem cells are more potent suppressors of dendritic cells differentiation compared to bone marrow-derived mesenchymal stem cells. , 2009, Immunology letters.

[37]  Pierre Tattevin,et al.  Enhanced indoleamine 2,3-dioxygenase activity in patients with severe sepsis and septic shock. , 2010, The Journal of infectious diseases.

[38]  Alan J Thompson,et al.  Autologous mesenchymal stem cells for the treatment of secondary progressive multiple sclerosis: an open-label phase 2a proof-of-concept study , 2012, The Lancet Neurology.

[39]  R. Zhao,et al.  Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. , 2008, Cell Stem Cell.

[40]  K. Tarte,et al.  Functional alteration of the lymphoma stromal cell niche by the cytokine context: role of indoleamine-2,3 dioxygenase. , 2009, Cancer research.

[41]  M. Gobbi,et al.  Interaction between Human NK Cells and Bone Marrow Stromal Cells Induces NK Cell Triggering: Role of NKp30 and NKG2D Receptors1 , 2005, The Journal of Immunology.

[42]  Karin Tarte,et al.  Good manufacturing practices production of mesenchymal stem/stromal cells. , 2011, Human gene therapy.

[43]  G. Dini,et al.  Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study , 2008, The Lancet.

[44]  A. Hmadcha,et al.  Standard requirement of a microbiological quality control program for the manufacture of human mesenchymal stem cells for clinical use. , 2014, Stem cells and development.

[45]  A. Santoni,et al.  DNAM-1 ligand expression on Ag-stimulated T lymphocytes is mediated by ROS-dependent activation of DNA-damage response: relevance for NK-T cell interaction. , 2011, Blood.

[46]  W. Weimar,et al.  The effect of rabbit antithymocyte globulin on human mesenchymal stem cells , 2013, Transplant international : official journal of the European Society for Organ Transplantation.

[47]  J. Vendrell,et al.  Stromal stem cells from adipose tissue and bone marrow of age‐matched female donors display distinct immunophenotypic profiles , 2011, Journal of cellular physiology.

[48]  P. Oefner,et al.  Tryptophan catabolism is associated with acute GVHD after human allogeneic stem cell transplantation and indicates activation of indoleamine 2,3-dioxygenase. , 2011, Blood.

[49]  D. Prockop,et al.  Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury , 2010, Proceedings of the National Academy of Sciences.

[50]  M. Toungouz,et al.  Characterization and functionality of the CD200-CD200R system during mesenchymal stromal cell interactions with T-lymphocytes. , 2012, Immunology letters.

[51]  S. Shi,et al.  Mesenchymal Stem Cell-Based Tissue Regeneration is Governed by Recipient T Lymphocyte via IFN-γ and TNF-α , 2011, Nature Medicine.

[52]  Hélène Rouard,et al.  Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation. , 2010, Blood.

[53]  Daniel A. De Ugarte,et al.  Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow. , 2003, Immunology letters.

[54]  常松 日奈子.  Fibroblast growth factor-2 enhances NK sensitivity of hepatocellular carcinoma cells , 2013 .

[55]  G. Rogler,et al.  Inducible CD40 expression mediates NFκB activation and cytokine secretion in human colonic fibroblasts , 2003, Gut.

[56]  S. Setty,et al.  IFN‐γ activation of mesenchymal stem cells for treatment and prevention of graft versus host disease , 2008, European journal of immunology.

[57]  P. Delafontaine,et al.  Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. , 2009, Cell stem cell.

[58]  K. Tarte,et al.  Immunological characterization of multipotent mesenchymal stromal cells--The International Society for Cellular Therapy (ISCT) working proposal. , 2013, Cytotherapy.

[59]  L. PayneNatalie,et al.  Mesenchymal stem cells and immunomodulation , 2014 .

[60]  E. Alici,et al.  Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data , 2014, Stem Cell Research & Therapy.

[61]  Sergio Romagnani,et al.  Role for Interferon‐γ in the Immunomodulatory Activity of Human Bone Marrow Mesenchymal Stem Cells , 2006 .

[62]  Sergio Romagnani,et al.  Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. , 2006, Stem cells.

[63]  A. Sbarbati,et al.  Adipose‐Derived Mesenchymal Stem Cells Ameliorate Chronic Experimental Autoimmune Encephalomyelitis , 2009, Stem cells.

[64]  Y. Kato,et al.  Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF. , 2001, Biochemical and biophysical research communications.

[65]  O. Ringdén,et al.  Analysis of Tissues Following Mesenchymal Stromal Cell Therapy in Humans Indicates Limited Long‐Term Engraftment and No Ectopic Tissue Formation , 2012, Stem cells.

[66]  K. Tarte,et al.  Comparative study of immune regulatory properties of stem cells derived from different tissues. , 2013, Stem cells and development.

[67]  M. Picco.  Autologous bone marrow-derived mesenchymal stromal cells in the treatment of fistulising Crohn's disease , 2011 .

[68]  P. A. Lay,et al.  Post-translational Regulation of Human Indoleamine 2,3-Dioxygenase Activity by Nitric Oxide* , 2007, Journal of Biological Chemistry.

[69]  A. Martini,et al.  Bone Marrow‐Derived Mesenchymal Stem Cells Induce Both Polyclonal Expansion and Differentiation of B Cells Isolated from Healthy Donors and Systemic Lupus Erythematosus Patients , 2008, Stem cells.

[70]  A. Cometa,et al.  Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell‐therapy approaches: Further insights in the search for a fetal calf serum substitute , 2007, Journal of cellular physiology.

[71]  M. Pozzobon,et al.  Immune regulatory properties of CD117(pos) amniotic fluid stem cells vary according to gestational age. , 2015, Stem cells and development.

[72]  D. Prockop,et al.  Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. , 2006, Cytotherapy.

[73]  Mélanie Gadelorge,et al.  CD146 expression on mesenchymal stem cells is associated with their vascular smooth muscle commitment , 2013, Journal of cellular and molecular medicine.

[74]  D. Mougiakakos,et al.  Multipotent mesenchymal stromal cells and the innate immune system , 2012, Nature Reviews Immunology.

[75]  N. Rouas-Freiss,et al.  Concise review: Combining human leukocyte antigen G and mesenchymal stem cells for immunosuppressant biotherapy , 2013, Stem cells.

[76]  S. Lehmann,et al.  Cell specific differences between human adipose-derived and mesenchymal-stromal cells despite similar differentiation potentials. , 2008, Experimental cell research.

[77]  G. Shurin,et al.  Early growth response-2 signaling mediates immunomodulatory effects of human multipotential stromal cells. , 2014, Stem cells and development.

[78]  Yufang Shi,et al.  Species Variation in the Mechanisms of Mesenchymal Stem Cell‐Mediated Immunosuppression , 2009, Stem cells.

[79]  L. Moretta,et al.  Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. , 2006, Blood.

[80]  K. Tarte,et al.  Effects of a ceramic biomaterial on immune modulatory properties and differentiation potential of human mesenchymal stromal cells of different origin. , 2015, Tissue engineering. Part A.