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Introduction:

Embryonic stem cells are by definition the master cells capable of differentiating into every type of cells either in vitro or in vivo. Several lines of evidence suggest, however, that adult stem cells and even terminally differentiated somatic cells under appropriate microenvironmental cues are able to be reprogrammed and contribute to a much wider spectrum of differentiated progeny than previously anticipated. This has been demonstrated by using tissue- specific stem cells, which like embryonic stem cells do not express CD45 as an exclusive hematopoietic marker (skin, adipose, cord blood and bone marrow- derived stem cells). On the other side, there is a great number of reports which demonstrate that hematopoietic cells (CD45) from different sources (peripheral blood, cord blood, bone marrow) are also able to cross the tissue boundaries and give rise to the cells of the other germ layers. Herein we discuss the differentiation and reprogramming potential of both hematopoietic and non- hematopoietic stem cells along endodermal, mesodermal and neuroectodermal lineage and their importance for regenerative medicine.

6-Hydroxydopamine (6-OHDA) was used for ex vivo purging of bone marrow from neuroblastoma cells before autologous transplantation. However, this concept failed because of the rapid autoxidation of 6-OHDA, which leads to the generation of cytotoxic reactive oxygen species (ROS), mainly in the incubation medium before 6-OHDA can be incorporated by neuroblastoma cells. We based our experiments on the theory that, in contrast, 6-fluorodopamine (6-FDA), which is slowly converted to 6-OHDA at neutral pH, is able to enter neuroblastoma cells via the noradrenaline transporter (NA-T). Therefore, most ROS are generated inside the target cells. Small amounts of ascorbate prevent the extracellular conversion of 6-FDA to 6-OHDA without affecting its cytotoxicity, leading to an even more selective effect of 6-FDA. We conclude that 6-FDA is a promising substance for selective destruction of NA-T-positive neuroblastoma cells. Med. Pediatr. Oncol. 35:612–615, 2000. © 2000 Wiley-Liss, Inc.

Bader: Medac: Patents & Royalties, Research Funding; Cellgene: Consultancy; Neovii: Research Funding; Riemser: Research Funding; Novartis: Consultancy, Speakers Bureau. Kuci:Medac: Patents & Royalties. Kuci:Medac: Patents & Royalties. Bug:Amgen: Honoraria; Jazz Pharmaceuticals: Other: Travel Grant; Neovii: Other: Travel Grant; Astellas Pharma: Other: Travel Grant; Janssen: Other: Travel Grant; Celgene: Honoraria; Novartis Pharma: Honoraria, Research Funding. Lang:Miltenyi Biotec: Patents & Royalties, Research Funding. Sykora:Aventis-Behring: Research Funding; medac: Research Funding. Seifried:Medac: Other: BSD owns IP and is contract manufacturer; Uniqure BV: Research Funding. Bonig:Kiadis Pharma: Consultancy.

Introduction:Since the seminal paper of LeBlanc in 2008, despite several negative studies, the scientific community has retained optimism with respect to the usefulness of mesenchymal stroma cells (MSCs) in refractory acute graft-versus-host disease (GvHD). A prevailing theme of past studies was that, while pediatric GvHD responded to MSCs, adult GvHD did not. As reported, we developed proprietary protocols GMP-quality MSC production from bone marrow (BM) mononuclear cells expanded in platelet lysate-enriched media.

Abstract

Mesenchymal stromal cells (MSCs) are non- hematopoietic multipotent cells which can be derived from bone marrow mononuclear cells either by plastic adherence (PA-MSCs) or by a positive selection with antibodies against cell surface antigens expressed by MSC-progenitor cells (CD271, CD73, CD146, CD105, CD166, SSEA-4 and recently GD2). In this study, we compare the phenotype, proliferation potential, differentiation potential, the cytokine expression pattern and inhibitory potential of MSC- derived by plastic adherence and MSCs derived from positively selected CD271+bone marrow mononuclear cells (BM-MNCs). According to CFU-F assay, the enriched CD271+BM cells possess a significantly higher frequency (2952 per 1×106BM cells) compared to PA-MSCs (21 per 1×106BM cells). Phenotypically, both populations expressed high levels of common MSC antigens such as CD73, CD105, CD44, CD166, CD90, HLA-Class I and were negative for CD34, CD133, and CD14. Compared to PA-MSCs, CD271+BM cells after the isolation express high levels of the hematopoietic antigen CD45 which is down- regulated within the first passage and HLA-DR, which remains constant through many passages. These cells have a 10- to 1000-fold higher proliferation capacity compared to PA- MSCs. However, both populations differentiated in vitroalong adipogenic, chondrogenic and osteogenic lineage. In MLR, both populations significantly suppressed the proliferation of PHA- stimulated allogeneic T- lymphocytes at the ratio 10: 1 (MSCs:T-cells). However, CD271+BM- derived MSCs were more efficient in secreting IFN-γ, IL-1β, IL-2, IL-4, GM-CSF and TNF-α, whereas PA- MSCs secreted significantly more IL-6 and IL-8 than CD271+BM- derived MSCs. Ongoing in vivo studies with immunodeficient NOD/SCID mice will show the whole potential of both population in the improvement of engraftment of mobilized peripheral blood hemaatopoietic CD133+cells. Based on their higher frequency and proliferation capacity we suggest that CD271+BM- cells may represent a better source than PA- MSCs in order to generate bulk quantities of MSCs for clinical applications.

Mesenchymal stromal cells (MSCs) are non- hematopoietic multipotent cells which can be derived from bone marrow mononuclear cells either by plastic adherence (PA-MSCs) or by a positive selection with antibodies against cell surface antigens expressed by MSC-progenitor cells (CD271, CD73, CD146, CD105, CD166, SSEA-4 and recently GD2). In this study, we compare the phenotype, proliferation potential, differentiation potential, the cytokine expression pattern and inhibitory potential of MSC- derived by plastic adherence and MSCs derived from positively selected CD271+ bone marrow mononuclear cells (BM-MNCs). According to CFU-F assay, the enriched CD271+ BM cells possess a significantly higher frequency (2952 per 1×106 BM cells) compared to PA-MSCs (21 per 1×106 BM cells). Phenotypically, both populations expressed high levels of common MSC antigens such as CD73, CD105, CD44, CD166, CD90, HLA-Class I and were negative for CD34, CD133, and CD14. Compared to PA-MSCs, CD271+ BM cells after the isolation express high levels of the hematopoietic antigen CD45 which is down- regulated within the first passage and HLA-DR, which remains constant through many passages. These cells have a 10- to 1000-fold higher proliferation capacity compared to PA- MSCs. However, both populations differentiated in vitro along adipogenic, chondrogenic and osteogenic lineage. In MLR, both populations significantly suppressed the proliferation of PHA- stimulated allogeneic T- lymphocytes at the ratio 10: 1 (MSCs:T-cells). However, CD271+ BM- derived MSCs were more efficient in secreting IFN-γ, IL-1β, IL-2, IL-4, GM-CSF and TNF-α, whereas PA- MSCs secreted significantly more IL-6 and IL-8 than CD271+ BM- derived MSCs. Ongoing in vivo studies with immunodeficient NOD/SCID mice will show the whole potential of both population in the improvement of engraftment of mobilized peripheral blood hemaatopoietic CD133+ cells. Based on their higher frequency and proliferation capacity we suggest that CD271+ BM- cells may represent a better source than PA- MSCs in order to generate bulk quantities of MSCs for clinical applications.