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; Knaan-Shanzer, S.; Bekkum, D.W. van 2011
Stem cell-based therapies represent promising approaches for the treatment of incurable diseases and tissue injury due to the capacity of these cells to self-renew and differentiate into... Show moreStem cell-based therapies represent promising approaches for the treatment of incurable diseases and tissue injury due to the capacity of these cells to self-renew and differentiate into specialized mature cells. Understanding the interaction of stem cells with the disease/injured environment and their contribution to the repair process by release of regeneration-promoting signals or by differentiation into the lost cell types is crucial for the advancement of their clinical application. This thesis focuses on one stem cell population, the mesenchymal stem cells (MSCs) from human and in particular on their role and utility in skeletal muscle regeneration. For this purpose, several in vivo tissue damage models were employed (i.e. cardiotoxin-induced injury, pressure ulcers, and subcutaneous implants of minced skeletal muscle). Most of the experiments were performed in immunocompromised mice (i.e. NOD/SCID) to avoid immunological rejection of the human cells. Furthermore, we described the downregulation of MHC class I protein expression on the surface of human MSCs by retroviral vectors encoding a herpesviral immunoevasin (i.e. the US11). This renders human MSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make human MSCs non-immunogenic and thereby universally transplantable. 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
Streptozotocin is widely used to induce diabetes in laboratory animals through multiple low-dose or single high-dose intraperitoneal injections. HPLC analysis has shown that the composition of the... Show moreStreptozotocin is widely used to induce diabetes in laboratory animals through multiple low-dose or single high-dose intraperitoneal injections. HPLC analysis has shown that the composition of the solution may change considerably during the first 2 h after dissolution due to equilibration of the 2 anomers (alpha and beta) of streptozotocin. Because of the drug's alleged instability in solution, the typical recommendation is to administer streptozotocin within 10 min after dissolution. We compared the induction of diabetes in NOD/SCID mice by injection of a single high dose of freshly made or anomer-equilibrated streptozotocin solution. Solutions were prepared from dry compound containing 85% of the alpha anomer, which is the more toxic of the 2. Body weight and nonfasting blood glucose levels were measured weekly for 8 wk. Both solutions induced long-term hyperglycemia, but blood glucose levels and mortality were higher and damage to pancreatic islands more pronounced in the mice receiving freshly prepared solution. A small proportion of mice did not respond in both treatment groups. If stored at 4 degrees C in the dark, the anomer-equilibrated solution retains its biologic activity for at least 40 d; under those conditions the streptozotocin content decreases by 0.1% daily, as determined by HPLC. Anomer-equilibrated streptozotocin solution has several practical advantages, and we recommend its use as standard for the induction of experimental diabetes because this practice may improve reproducibility and comparison of results between different laboratories. Show less
Garza-Rodea, A.S. de la; Knaaen-Shanzer, S.; Hartigh, J.D. den; Verhaegen, A.P.L.; Bekkum, D.W. van 2010
Streptozotocin is widely used to induce diabetes in laboratory animals through multiple low-dose or single high-dose intraperitoneal injections. HPLC analysis has shown that the composition of the... Show moreStreptozotocin is widely used to induce diabetes in laboratory animals through multiple low-dose or single high-dose intraperitoneal injections. HPLC analysis has shown that the composition of the solution may change considerably during the first 2 h after dissolution due to equilibration of the 2 anomers (a and P) of streptozotocin. Because of the drug's alleged instability in solution, the typical recommendation is to administer streptozotocin within 10 min after dissolution. We compared the induction of diabetes in NOD/SCID mice by injection of a single high dose of freshly made or anomer-equilibrated streptozotocin solution. Solutions were prepared from dry compound containing 85% of the (x anomer, which is the more toxic of the 2. Body weight and nonfasting blood glucose levels were measured weekly for 8 wk. Both solutions induced long-term hyperglycemia, but blood glucose levels and mortality were higher and damage to pancreatic islands more pronounced in the mice receiving freshly prepared solution. A small proportion of mice did not respond in both treatment groups. If stored at 4 degrees C in the dark, the anomer-equilibrated solution retains its biologic activity for at least 40 d; under those conditions the streptozotocin content decreases by 0.1% daily, as determined by HPLC. Anomer-equilibrated streptozotocin solution has several practical advantages, and we recommend its use as standard for the induction of experimental diabetes because this practice may improve reproducibility and comparison of results between different laboratories. Show less
