RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem... Show moreRAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development, from early CD7(-)CD5(-) to CD4(+)CD8(+). The impaired differentiation was accompanied by an increase in CD7(-)CD56(+)CD33(+) natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells, whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements, normalized T cell development, and corrected the NK cell-like phenotype. In conclusion, we succeeded in generating an iPSC-based RAG2-SCID model, which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks. Show less
Garza-Rodea, A.S. de la; Velde-van Dijke, I. van der; Boersma, H.; Goncalves, M.A.F.V.; Bekkum, D.W. van; Vries, A.A.F. de; Knaan-Shanzer, S. 2012
BACKGROUND Mesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are... Show moreBACKGROUND Mesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are capable of extensive proliferation without showing chromosomal aberrations. Large numbers of hMSCs can thus be acquired from small samples of easily obtainable tissues like fat and bone marrow. MSCs can contribute to regeneration indirectly by secretion of cytokines or directly by differentiation into specialized cell types. The latter mechanism requires their long-term acceptance by the recipient. Although MSCs do not elicit immune responses in vitro, animal studies have revealed that allogeneic and xenogeneic MSCs are rejected. METHODOLOGY/PRINCIPAL FINDINGS We aim to overcome MSC immune rejection through permanent down-regulation of major histocompatibility complex (MHC) class I proteins on the surface of these MHC class II-negative cells through the use of viral immune evasion proteins. Transduction of hMSCs with a retroviral vector encoding the human cytomegalovirus US11 protein resulted in strong inhibition of MHC class I surface expression. When transplanted into immunocompetent mice, persistence of the US11-expressing and HLA-ABC-negative hMSCs at levels resembling those found in immunodeficient (i.e., NOD/SCID) mice could be attained provided that recipients' natural killer (NK) cells were depleted prior to cell transplantation. CONCLUSIONS/SIGNIFICANCE Our findings demonstrate the potential utility of herpesviral immunoevasins to prevent rejection of xenogeneic MSCs. The observation that down-regulation of MHC class I surface expression renders hMSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make hMSCs non-immunogenic and thereby universally transplantable. Show less
Garza-Rodea, A.S. de la; Velde, I. van der; Boersma, H.; Goncalves, M.A.F.V.; Bekkum, D.W. van; Vries, A.A.F. de; Knaan-Shanzer, S. 2011
Mesenchymal stromal cells (MSCs) are attractive for cellular therapy of muscular dystrophies as they are easy to procure, can be greatly expanded ex vivo, and contribute to skeletal muscle repair... Show moreMesenchymal stromal cells (MSCs) are attractive for cellular therapy of muscular dystrophies as they are easy to procure, can be greatly expanded ex vivo, and contribute to skeletal muscle repair in vivo. However, detailed information about the contribution of bone marrow (BM)-derived human MSCs (BM-hMSCs) to skeletal muscle regeneration in vivo is very limited. Here, we present the results of a comprehensive study of the fate of LacZ-tagged BM-hMSCs following implantation in cardiotoxin (CTX)-injured tibialis anterior muscles (TAMs) of immunodeficient mice. beta-Galactosidase-positive (beta-gal(+)) human mouse hybrid myofibers (HIVIs) were counted in serial cross sections over the full length of the treated TAMs of groups of mice at monthly intervals. The number of human cells was estimated using chemiluminescence assays. While the number of human cells declined gradually to about 10% of the injected cells at 60 days after transplantation, the number of HMs increased from day 10 onwards, reaching 104 +/- 39.1 per TAM at 4 months postinjection. beta-gal(+) cells and HMs were distributed over the entire muscle, indicating migration of the former from the central injection site to the ends of the TAMs. The identification of HMs that stained positive for human spectrin suggests myogenic reprogramming of hMSC nuclei. In summary, our findings reveal that BM-hMSCs continue to participate in the regeneration/remodeling of CTX-injured TAMs, resulting in +/- 5% HMs at 1 months after damage induction. Moreover, donor-derived cells were shown to express genetic information, both endogenous and transgenic, in recipient myofibers. Show less
Garza-Rodea, A.S. de la; Verweij, M.C.; Boersma, H.; Velde-van Dijke, I. van der; Vries, A.A.F. de; Hoeben, R.C.; ... ; Knaan-Shanzer, S. 2011
