To characterize BM-derived stem/progenitor cells in the mouse cornea, we performed HSC transplantation.
14 15 BM cells were harvested from femurs and tibias of 8- to 12-week-old eGFP mice. Single-cell suspensions of donor cells were prepared by repeated serial passage through a 23-gauge needle. To deplete mature hematopoietic cells, the BM cells were incubated with lineage-specific antibodies (B220, CD3, Gr-1, Mac-1, and TER 119) for 30 minutes at 4°C. After washing with PBS containing 2% fetal bovine serum, the cells were incubated with sheep anti-rat immunomagnetic beads (Dynabeads M-450 coupled to sheep anti-rat IgG; Dynal, Great Neck, NY). Cells not bound to the immunobeads were further purified for Sca-1
+ cells. The purity of lineage
− cells was higher than 92% in all experiments. After negative selection of mature hematopoietic and immune cells, positive selection of Sca-1
+ cells was performed as just described. After negative and positive selection, the purity of lin
− Sca-1
+ cells of all the eGFP
+ cells exceeded 95%
(Fig. 1) .
14 15 To obtain high cell purity, samples were applied twice to columns in each experiment. The resultant 10
4 lin
− Sca-1
+ cells were transplanted into C57BL/6 mice within 2 days of their birth. The HSC transplant recipients were maintained under special pathogen-free conditions for 4 weeks. Successful HSC transplantation was confirmed by the identification of GFP
+ cells in the peripheral blood at 4 weeks after transplantation. At 4 to 5 months after HSC transplantation, six mice were used for histologic and immunohistochemical studies.