Mutual interaction between Human Multipotent Adult Progenitor Cells and NK cells

Human Multipotent Adult Progenitor Cells (hMAPCs) are isolated from bone marrowwith more extensive expansion capacity compared to human Mesenchymal Stem Cells(hMSCs), and with the ability to differentiate into endothelium. Like hMSCs, hMAPCsinhibit T cell proliferation induced by alloantigens. In this study we tested theinteraction between hMAPCs and natural killer (NK) cells. We assessed thesusceptibility of hMAPCs to NK cell-mediated lysis and the immunomodulation ofhMAPCs on NK cell function during IL-2-driven stimulation and cytolytic effectorphase. Human MAPCs express the ligands PVR and ULBP-2/5/6, which are recognizedby activating NK cell receptors. However, they also express MHC class I molecules,which induce inhibitory signals in NK cells. Freshly isolated NK cells at differenteffector:target ratios did not kill hMAPCs as assessed by an MTT and 51Cr-releaseassay, while hMAPCs impaired the cytotoxic activity of resting NK cells against theNK-sensitive K562 leukemia cell line. By contrast, IL-2 stimulated NK cells werecapable of killing hMAPCs and pre-activated NK cells were not influenced during theircytotoxic effector function against K562 cells by hMAPCs. When added during the 6-day pre-activation phase with IL-2, hMAPCs dose-dependently reduced NK cellproliferation in an IDO-dependent manner, but they did not influence the induction ofcytotoxic capacity by IL-2. This study indicates that human MAPCs mutually interactwith NK cells.status: publishe

Monitoring the bystander killing effect of human multipotent stem cells for treatment of malignant brain tumors

Tumor infiltrating stem cells have been suggested as a vehicle for the delivery of a suicide gene towards otherwise difficult to treat tumors like glioma. We have used herpes simplex virus thymidine kinase expressing human multipotent adult progenitor cells in two brain tumor models (hU87 and Hs683) in immune-compromised mice. In order to determine the best time point for the administration of the codrug ganciclovir, the stem cell distribution and viability were monitored in vivo using bioluminescence (BLI) and magnetic resonance imaging (MRI). Treatment was assessed by in vivo BLI and MRI of the tumors. We were able to show that suicide gene therapy using HSV-tk expressing stem cells can be followed in vivo by MRI and BLI. This has the advantage that (1) outliers can be detected earlier, (2) GCV treatment can be initiated based on stem cell distribution rather than on empirical time points, and (3) a more thorough follow-up can be provided prior to and after treatment of these animals. In contrast to rodent stem cell and tumor models, treatment success was limited in our model using human cell lines. This was most likely due to the lack of immune components in the immune-compromised rodents.status: publishe

Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes

: MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8(+) cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was-even after major histocompatibility complex class I upregulation-insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8(-)CD69(+) T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs.status: publishe

Human Multipotent Adult Progenitor Cells Are Nonimmunogenic and Exert Potent Immunomodulatory Effects on Alloreactive T-Cell Responses

Multipotent Adult Progenitor Cells (MAPCs) are bone marrow-derived non-hematopoietic stem cells with a broad differentiation potential and extensive expansion capacity. A comparative study between human mesenchymal stem cells (hMSCs) and human MAPCs (hMAPCs) has shown that hMAPCs have clearly distinct phenotypical and functional characteristics from hMSCs. In particular, hMAPCs express lower levels of MHC class I than hMSCs and can not only differentiate into typical mesenchymal cell types, but can also differentiate in vitro and in vivo into functional endothelial cells. The use of hMSCs as cellular immunomodulatory stem cell products gained much interest since their immunomodulatory capacities in vitro became evident over the last decade. Currently, the clinical grade stem cell product of hMAPCs is already used in clinical trials to prevent graft-versus host disease (GVHD), as well as for the treatment of acute myocardial infarct, ischemic stroke, andCrohn's disease. Therefore we studied the immune phenotype, immunogenicity andimmunosuppressive effect of hMAPCs in vitro. We demonstrated that hMAPCs are non immunogenic for T cell proliferation and cytokine production. In addition, hMAPCs exert strong immunosuppressive effects on T cell alloreactivity, and on T cell proliferation induced by mitogens and recall antigens. This immunomodulatory effect was not MHC-restricted, which makes off-the-shelf use promising. The immunosuppressive effect of hMAPCs is partially mediated via soluble factors and dependent on indoleamine 2,3-dioxygenase (IDO) activity. At last, we isolated hMAPCs, the clinical grade stem cell product of hMAPCs, named MultiStem, and hMSCs from one single donor and observed that both the immunogenicity and the immunosuppressive capacities of all three stem cell products are comparable in vitro.In conclusion, hMAPCs have potent immunomodulatory properties in vitro and can serve as a valuable cell source for the clinical use of immunomodulatory cellular stem cell product.status: publishe

Immunoregulatory effects of multipotent adult progenitor cells in a porcine ex vivo lung perfusion model

Primary graft dysfunction (PGD) is considered to be the end result of an inflammatory response targeting the new lung allograft after transplant. Previous research has indicated that MAPC cell therapy might attenuate this injury by its paracrine effects on the pro-/anti-inflammatory balance. This study aims to investigate the immunoregulatory capacities of MAPC cells in PGD when administered in the airways.status: publishe

Controlling and monitoring stem cell safety in vivo in an experimental rodent model

Adult stem cells have been investigated increasingly over the past years for multiple applications. Although they have a more favorable safety profile compared to pluripotent stem cells, they are still capable of self-renewal and differentiate into several cell types. We investigated the behavior of Oct4-positive (Oct4(+) ) and Oct4-negative (Oct4(-) ) murine or rat bone marrow (BM)-derived stem cells in the healthy brain of syngeneic mice and rats. Engraftment of mouse and rat Oct4-positive BM-derived hypoblast-like stem cells (m/rOct4(+) BM-HypoSCs) resulted in yolk-sac tumor formation in the healthy brain which was monitored longitudinally using magnetic resonance imaging (MRI) and bioluminescence imaging (BLI). Contrast enhanced MRI confirmed the disruption of the blood brain barrier. In contrast, m/r Oct4-negative BM-derived multipotent adult progenitor cells (m/rOct4(-) BM-MAPCs) did not result in mass formation after engraftment into the brain. mOct4(+) BM-HypoSCs and mOct4(-) BM-MAPCs were transduced to express enhanced green fluorescent protein, firefly luciferase (fLuc), and herpes simplex virus-thymidine kinase to follow up suicide gene expression as a potential "safety switch" for tumor-forming stem cells by multimodal imaging. Both cell lines were eradicated efficiently in vivo by ganciclovir administration indicating successful suicide gene expression in vivo, as assessed by MRI, BLI, and histology. The use of suicide genes to prevent tumor formation is in particular of interest for therapeutic approaches where stem cells are used as vehicles to deliver therapeutic genes. Stem Cells 2014;32:2833-2844.status: publishe

Reprogramming of Human Multipotent Adult Progenitors into induced endodermal progenitor like cells by defined transcription Factors

Reprogramming of Human Multipotent Adult Progenitors into induced endodermal progenitor like cells by defined transcription Factors Rangarajan Sambathkumar, Philip Roelandt1, Manoj kumar1, Ana Rita Mestre Rosa1, Fanos Tadessa Woldemariyam1, Sumitava Dastidar2, Qing Cai1, Eric Kalo1, Valerie Roobrouck3, Satish Khurana1, Marijke Faas5, Paul de Vos5, Catherine M Verfaillie1. New sources of cells are needed for both therapy of diabetes (beta cells) and creation of hepatocytes (for toxicology and metabolization studies). One possibility are induced pluripotent stem cells generated from fibroblasts and then differentiated towards the lineage of interest. One drawback, definitely for clinical therapies is the possible persistence of pluripotent cells that might form teratomas. An alternative approach is to de-differentiate somatic cells to an intermediate progenitor, such as endodermal progenitors, which can then be committed to beta-cells and / or hepatocytes. We previously reported that using 16 TFs, human multipotent adult progenitor cells (MAPCs) could be transdifferentiated in cells with epithelial morphology, that could be expanded long term and expressed (mes)endodermal genes. However, more mature endodermal genes were also expressed including Albumin (ALB). We therefore tested a combination of 14 TF, which now resulted in iENDO cells; expandable cells with (mes)endodermal endogenous gene expression but without mature endodermal gene expression. Using adapted protocols for pancreatic endocrine cell differentiation and hepatocyte differentiation, we demonstrated that 14 TF iEndo cells could be committed to endocrine pancreatic endoderm, expressing PDX1, NGN3, PAX4, NKX2.2, NEUROD1, MAFA and MAFB, but not mature beta cell characteristics (no Insulin expressed). During hepatocyte differentiation, hepatoblast genes such as AFP, ALB, AAT were induced >1200 fold. Inability to create fully committed progenitors at this point may be because the differentiation conditions are not yet fully optimized. A second possibility is that the OCT4 transgene used in the preprogramming remains expressed. Currently we are testing if doxycycline inducible overexpression of OKSM combined with the other 10 TFs may support full differentiation towards beta cells and hepatocytes.Acknowledgments: This work is funded by Dutch Diabetes foundation, Netherlands and funds from KU Leuven. Disclosures: Catherine Verfaillie is an advisor to ReGenesys.status: publishe

Generation of hepatocytes and pancreatic endocrine cells from human induced endodermal progenitor like cells

Rangarajan Sambathkumar, Philip Roelandt1, Manoj Kumar1, Ana Rita Mestre Rosa1, Fanos Tadessa Woldemariyam1, Renate Akkerman1, Sumitava Dastidar2, Qing Cai1, Eric Kalo1, Valerie Roobrouck3, Satish Khurana1, Marijke Faas5, Paul de Vos5, Catherine M Verfaillie1. New sources of cells are needed for both therapy of diabetes (beta cells) and creation of hepatocytes (for toxicology and metabolization studies). One possibility are induced pluripotent stem cells generated from fibroblasts and then differentiated towards the lineage of interest. One drawback, definitely for clinical therapies is the possible persistence of pluripotent cells that might form teratomas. An alternative approach is to de-differentiate somatic cells to an intermediate progenitor, such as endodermal progenitors, which can then be committed to beta-cells and / or hepatocytes. We previously reported that using 16 TFs, human multipotent adult progenitor cells (MAPCs) could be transdifferentiated in cells with epithelial morphology, that could be expanded long term and expressed (mes)endodermal genes. However, more mature endodermal genes were also expressed including Albumin (ALB). We therefore tested a combination of 14 TF, which now resulted in iENDO cells; expandable cells with (mes)endodermal endogenous gene expression but without mature endodermal gene expression. Using adapted protocols for pancreatic endocrine cell differentiation and hepatocyte differentiation, we demonstrated that 14 TF iEndo cells could be committed to endocrine pancreatic endoderm, expressing PDX1, NGN3, PAX4, NKX2.2, NEUROD1, MAFA and MAFB, but not mature beta cell characteristics (no Insulin expressed). During hepatocyte differentiation, hepatoblast genes such as AFP, ALB, AAT were induced. Inability to create fully committed progenitors at this point may be because the differentiation conditions are not yet fully optimized. A second possibility is that the OCT4 transgene used in the preprogramming remains expressed. Currently we are testing if doxycycline inducible overexpression of OKSM combined with the other 10 TFs may support full differentiation towards beta cells and hepatocytes. Preliminary results suggest that upon transplantation of undifferentiated 14TFs iENDO cells under the kidney capsule of SCID mice, cells are differentiating to the hepatocyte lineage In vivo. Studies wherein iENDO cells differentiated to endocrine pancreatic cells are evaluated following grafting in vivo are pending.Acknowledgments: This work is funded by Dutch Diabetes foundation, Netherlands and funds from KU Leuven. Disclosures: Catherine Verfaillie is an advisor to ReGenesys.status: publishe