Mesenchymal stem cells expanded in human platelet lysate display a decreased inhibitory capacity on T‐ and NK‐cell proliferation and function

The use of fetal bovine serum (FBS) for the culture and expansion of mesenchymal stromal cells (MSCs) limits their possible clinical applications. Although some recent studies recommended substituting FBS with human platelet lysate (HPL) for the expansion of MSCs for clinical use, the functional capacity of the expanded cells has only been partially explored. 10% FBS and two other commercial FBS‐containing media (MesenCult and MesenPro) were compared with 10% HPL‐containing medium for their ability to support MSCs expansion and immunomodulation. We demonstrate that HPL sustained MSC proliferation and expansion in vitro. However, the cumulative cell numbers recovered were comparable with those obtained in MesenPro medium. Moreover, we show that HPL alters the expression of some relevant MSC surface molecules, namely the DNAM‐1 ligands PVR and Nectin‐2, the NKG2D ligand ULBP3, the adhesion molecules CD49d and αvβ3 and the fibroblast‐associated protein. In addition, MSCs cultured in HPL displayed impaired inhibitory capacity on T‐cell proliferation to alloantigen and NK‐cell proliferation and cytotoxicity. Finally, they showed decreased constitutive PGE2 production while IL‐6, IL‐8 and RANTES secretion were upregulated. These results imply some limitations in the use of HPL for the expansion of MSCs to be used as immunomodulators in clinical applications.

[1]  K. Wood,et al.  Mesenchymal stromal cells: facilitators of successful transplantation? , 2010, Cell stem cell.

[2]  R. Handgretinger,et al.  Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM. , 2006, Cytotherapy.

[3]  J. Krieger,et al.  TRANSPLANTATION AND CELLULAR ENGINEERING: Adipose tissue mesenchymal stem cell expansion in animal serum‐free medium supplemented with autologous human platelet lysate , 2009, Transfusion.

[4]  Wolfgang Wagner,et al.  Replicative senescence-associated gene expression changes in mesenchymal stromal cells are similar under different culture conditions , 2010, Haematologica.

[5]  A. Xiang,et al.  Mesenchymal stem cell as salvage treatment for refractory chronic GVHD , 2010, Bone Marrow Transplantation.

[6]  M. Introna,et al.  Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts , 2007, Bone Marrow Transplantation.

[7]  Andrej-Nikolai Spiess,et al.  Accelerated and safe expansion of human mesenchymal stromal cells in animal serum‐free medium for transplantation and regenerative medicine , 2007, Journal of cellular physiology.

[8]  F. Locatelli,et al.  Killer Ig-like receptor-mediated control of natural killer cell alloreactivity in haploidentical hematopoietic stem cell transplantation. , 2011, Blood.

[9]  F. Locatelli,et al.  Haploidentical hemopoietic stem cell transplantation for the treatment of high-risk leukemias: how NK cells make the difference. , 2009, Clinical immunology.

[10]  A. Cometa,et al.  Phenotypical/functional characterization of in vitro-expanded mesenchymal stromal cells from patients with Crohn's disease. , 2009, Cytotherapy.

[11]  A. Cometa,et al.  Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors , 2009, Haematologica.

[12]  J. Blanco,et al.  Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification. , 2008, Experimental hematology.

[13]  L. Moretta,et al.  MSCs inhibit monocyte-derived DC maturation and function by selectively interfering with the generation of immature DCs: central role of MSC-derived prostaglandin E2. , 2009, Blood.

[14]  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.

[15]  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.

[16]  D. Strunk,et al.  Preparation of pooled human platelet lysate (pHPL) as an efficient supplement for animal serum-free human stem cell cultures. , 2009, Journal of visualized experiments : JoVE.

[17]  Philippe Bierling,et al.  Osteoblastic differentiation of human mesenchymal stem cells with platelet lysate. , 2010, Biomaterials.

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

[19]  D. Nachbaur,et al.  Dendritic cells in allogeneic hematopoietic stem cell transplantation , 2005, Leukemia & lymphoma.

[20]  J. S. San Miguel,et al.  Chronic Graft-Versus-Host Disease , 2012, Drugs.

[21]  A. Villa,et al.  Characterization of platelet lysate cultured mesenchymal stromal cells and their potential use in tissue-engineered osteogenic devices for the treatment of bone defects. , 2010, Tissue engineering. Part C, Methods.

[22]  Jessica M Malenfant,et al.  CD107a as a functional marker for the identification of natural killer cell activity. , 2004, Journal of immunological methods.

[23]  Seong-Yeol Park,et al.  Mesenchymal stromal cells inhibit graft-versus-host disease of mice in a dose-dependent manner. , 2010, Cytotherapy.

[24]  A. Reinisch,et al.  Isolation and animal serum free expansion of human umbilical cord derived mesenchymal stromal cells (MSCs) and endothelial colony forming progenitor cells (ECFCs). , 2009, Journal of visualized experiments : JoVE.

[25]  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.

[26]  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 .

[27]  X. Holy,et al.  Platelet lysates promote mesenchymal stem cell expansion: A safety substitute for animal serum in cell‐based therapy applications , 2005, Journal of cellular physiology.

[28]  A. Reinisch,et al.  Platelet-derived growth factors for GMP-compliant propagation of mesenchymal stromal cells. , 2009, Bio-medical materials and engineering.

[29]  C. Huet,et al.  New approach to radiation burn treatment by dosimetry-guided surgery combined with autologous mesenchymal stem cell therapy. , 2007, Regenerative medicine.

[30]  W. Marasco,et al.  Safety and complications reporting on the re-implantation of culture-expanded mesenchymal stem cells using autologous platelet lysate technique. , 2010, Current stem cell research & therapy.

[31]  A. Reinisch,et al.  Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum. , 2008, Tissue engineering. Part C, Methods.

[32]  M. Krampera,et al.  Mesenchymal stem cells for clinical application , 2010, Vox sanguinis.

[33]  F. Locatelli,et al.  Characterisation of CTL directed towards non-inherited maternal alloantigens in human cord blood , 1999, Bone Marrow Transplantation.

[34]  E. Andreu,et al.  Comparison of ex vivo expansion culture conditions of mesenchymal stem cells for human cell therapy , 2009, Transfusion.

[35]  Peter Harrison,et al.  Platelet a-granules , 1993 .

[36]  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.

[37]  O. Ringdén,et al.  Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms. , 2005, Experimental cell research.

[38]  Paul J. Harrison,et al.  Platelet alpha-granules. , 1993, Blood reviews.

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

[40]  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.

[41]  Karl Kashofer,et al.  Human platelet lysate can replace fetal bovine serum for clinical‐scale expansion of functional mesenchymal stromal cells , 2007, Transfusion.

[42]  G. Lucchini,et al.  Platelet-lysate-expanded mesenchymal stromal cells as a salvage therapy for severe resistant graft-versus-host disease in a pediatric population. , 2010, Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation.

[43]  C. Centeno,et al.  Regeneration of meniscus cartilage in a knee treated with percutaneously implanted autologous mesenchymal stem cells. , 2008, Medical hypotheses.

[44]  Andreas Reinisch,et al.  Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application. , 2007, Regenerative medicine.

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

[46]  R. Biassoni,et al.  Identification and Molecular Characterization of Nkp30, a Novel Triggering Receptor Involved in Natural Cytotoxicity Mediated by Human Natural Killer Cells , 1999, The Journal of experimental medicine.

[47]  R. Biassoni,et al.  Identification and Molecular Characterization of NKp 30 , a Novel Triggering Receptor Involved in Natural Cytotoxicity Mediated by Human Natural Killer Cells , 1999 .

[48]  P. Bourin,et al.  Mesenchymal Stem Cells for Therapeutic Purposes , 2009, Transplantation.

[49]  A. Kiani,et al.  Treatment of refractory acute GVHD with third-party MSC expanded in platelet lysate-containing medium , 2009, Bone Marrow Transplantation.

[50]  F. Locatelli,et al.  Natural killer alloeffector responses in haploidentical hemopoietic stem cell transplantation to treat high-risk leukemias. , 2010, Tissue antigens.

[51]  F. Rodeghiero,et al.  Effect of platelet lysate on the functional and molecular characteristics of mesenchymal stem cells isolated from adipose tissue. , 2011, Current stem cell research & therapy.