The fate of mesenchymal stem cells transplanted into immunocompetent neonatal mice: implications for skeletal gene therapy via stem cells  Christopher Niyibizi, Sujing Wang, Zhibao Mi, Paul D Robbins  Molecular Therapy  Volume 9, Issue 6, Pages 955-963 (June 2004) DOI: 10.1016/j.ymthe.2004.02.022 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Whole-body image of a neonatal mouse that was infused with GFP+ cells at 5 and 7 days after cell infusion. GFP+ cells were infused into neonatal mice at 2 days after birth. The mice were euthanized and examined under a fluorescence microscope. (A) MSCs transduced with DFG-eGFP-Zeor and selected for GFP+ cells; 100% of the cells are GFP positive. (B) Distribution of the GFP+ cells infused in a neonatal mouse at 5 days postinfusion and (C) at 7 days postinfusion. At 5 days most of the cells are located in the upper extremities and at 7 days, the GFP+ cells appear to be distributed throughout the entire mouse. (A) Original magnification 100×, (B and C) original magnification 10×. Molecular Therapy 2004 9, 955-963DOI: (10.1016/j.ymthe.2004.02.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Morphological appearance of GFP+ cells recovered from lungs at 7, 60, and 90 days and liver at 7 days post-cell infusion. The cells recovered from liver exhibit a morphological appearance distinct from those recovered from lung. Cells recovered from lung appear to possess a distinct morphological appearance at each different time point. It was not determined in the present study if the cells underwent differentiation. Original magnification 100×. Molecular Therapy 2004 9, 955-963DOI: (10.1016/j.ymthe.2004.02.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Morphological appearance of the GFP+ cells recovered from bone. Femurs were harvested from the recipient mice at 25 days postinfusion, cut into small pieces, and placed in culture. (A) A large number of GFP+ cells are growing out of the bone chips recovered from the recipient mice. (B) GFP+ cells recovered from bone and selected in a medium supplemented with zeocin. The recovered cells exhibit a morphological appearance distinct from those recovered from the lung and liver. Original magnification 100×. Molecular Therapy 2004 9, 955-963DOI: (10.1016/j.ymthe.2004.02.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Morphological appearance of the GFP+ cells recovered from bone at 35 days postinfusion and after two rounds of recycling in bone. (A) The cells exhibit a spindle-shaped morphology and are smaller than the cells recovered from the lung. (B) All the cells were CD105 positive. These cells were found in bone and cartilage only when they were reinjected into neonatal mice. Original magnification 100×. Molecular Therapy 2004 9, 955-963DOI: (10.1016/j.ymthe.2004.02.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Morphological appearance of the GFP+ cells recovered from cartilage. Rib and knee joint cartilage was harvested from the recipient mince, minced into small pieces, and placed in tissue culture in a medium supplemented with zeocin. (A) Morphological appearance of the cells observed under phase contrast and (B) identical cells observed under fluorescence. The cells exhibit a chondrocytic morphological appearance and express high levels of GFP. Original magnification 100×. Molecular Therapy 2004 9, 955-963DOI: (10.1016/j.ymthe.2004.02.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Collagen synthesis by the GFP+ cells recovered from cartilages of recipient mice. (A) GFP+ cells. (B) Immunofluorescence localization for type II collagen in GFP+ cells using a secondary antibody conjugated with Cy3. (C) Immunoblot for type II collagen synthesized by the GFP+ cells from cartilage. The antibody immunoreacts with type II collagen standard, a protein band with migration identical to that of the standard type II collagen synthesized by the GFP+ cells recovered from the cartilage. The antibody does not cross-react with type I collagen chains (I). The data demonstrate that the GFP+ cells recovered from cartilages of recipient mice are chondrocytes. Molecular Therapy 2004 9, 955-963DOI: (10.1016/j.ymthe.2004.02.022) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions