From Marrow to Brain: Expression of Neuronal Phenotypes in Adult Mice Timothy R. Brazelton, Fabio M. V. Rossi, Gilmor I. Keshet, and Helen M. Blau

Supplementary Material

Supplemental text corresponding to (14): Bone marrow transplantation
Marrow was sterilely isolated from 8- to 10-week-old male C57BL/6 transgenic mice (13) that ubiquitously expressed enhanced green fluorescent protein (GFP) and non-GFP, C57BL/6 control mice (Stanford). Donor mice were killed by cervical dislocation, were briefly immersed in 70% ethanol, and had their skin peeled back from a midline, circumferential incision. After the femurs, tibias, and humeri were removed, all muscle was scraped away with a razor blade and the bones were placed in 10 mL of calcium and magnesium-free, Hank's balanced salt solution (HBSS, Irvine Scientific) with 2% fetal bovine serum (FCS) on ice for up to 90 min. In a tissue culture hood, the tips of the bones were removed and a 25 gauge needle containing 1 mL of ice-cold HBSS with 2% FCS was inserted into the marrow cavity and used to wash the marrow out into a sterile culture dish. Marrow fragments were dissociated by titurating through the 25 gauge needle and the resulting suspension was filtered through sterile 70 mm nitex mesh. The filtrate was cooled on ice, spun for 5 min at 250g, and the pellet was resuspended in ice-cold HBSS with 2% FCS to 4.8 x 10⁷ nucleated cells per ml. Simultaneously, 8- to 10-week-old C57BL/6 mice (Stanford) were lethally irradiated with two doses of 475 cGy three hours apart. Each irradiated recipient received 125 ml of the unfractionated marrow cell suspension by tail vein injection within 2 hours of the second irradiation dose.

Supplemental text corresponding to (15): FACS analysis of CNS
Isolated brains were minced with a razor blade, washed twice in HBSS (Irvine Scientific), resuspended in 10 ml of PPD solution [2.5 U/ml Papain (Worthington Biochemicals), 250 U/ml DNAse I (Worthington Biochemicals), 1 U/ml Dispase II (Boehringer Mannheim), 12.4 mM MgSO₄ in HBSS] and incubated at 37°C for 30 min. Following the incubation, 2 ml of FBS was added and the fragments were dissociated by pipetting. The solution was filtered through a 40-mm sieve (BD Biosciences) and washed 3 times in DME containing 20% FBS. Samples were resuspended in 1 ml of PBS containing 5% FBS (FBS/PBS) and incubated on ice for 15 min with TriChrome (TC)-conjugated rat antibody to mouse CD11b (1:100, Caltag) and allophycocyanin (APC)-conjugated rat antibody to mouse CD45 (1:100, Pharmingen) or with isotype-matched TC- and APC- conjugated control antibodies. After washing once in FBS/PBS, the cells were resuspended in 1 ml of FBS/PBS and analyzed on a Moflo (Cytomation) or a Facscalibur (BD Biosciences) flow cytometer using the Flojo software package (Treestar).

Supplemental text corresponding to (20) - Laser scanning confocal microscopic analysis
Each GFP⁺ cell was analyzed for antibody staining by three-dimensional, confocal laser scanning microscopy (Molecular Dynamics Multiprobe 2010 and Zeiss 510). No GFP⁺ cells were seen in the olfactory bulbs of control mice transplanted with unlabeled bone marrow. Data was collected with sequential laser excitation to eliminate any possibility of bleedthrough. In order to conclusively demonstrate colocalization, confocal parameters (e.g., minimal pinhole sizes) were selected to minimize the thickness of the calculated optical section to 0.3 to 04 mm despite the lower resolution images produced with these parameters. Such thin optical sections were obtained every 0.3 to 1 mm and used to reconstruct a three dimensional representation of each cell for morphological characterization.

Supplemental text corresponding to (24): Immunocytochemistry
Eight or 12 weeks after bone marrow transplant, experimental mice that had received GFP⁺ bone marrow and age-matched control mice that had received unlabeled bone marrow were perfused with 25 mL of 4°C sodium phosphate buffer (pH 7.4) followed by 25 mL of 4°C 1.5% paraformaladehyde(PF)/0.1% glutaraldehyde in phosphate buffer (pH 7.4). Brains were placed in 1.5% PF/0.1% glutaraldehyde/20% sucrose at 4°C overnight and snap frozen in TISSUE-TEK O.C.T. compound (Sakura Finetek). Twenty- to 40-mm thick coronal sections were cyrosectioned at -20°C from between Bregma -4.1 to -3.6 mm and were stained as floating sections with antibodies against NeuN (1:4000, MAB377, Chemicon), 200 kD neurofilament (1:400, AB1989, Chemicon), class III b-microtubulin (1:1000, TUJ1, Covance), GFAP (1:2000, Dako), F4/80 (1:800, Caltag), and CD45 (1:1000, Pharmingen). All sections were blocked with 25% normal goat serum 0.25% Triton and antibody to CD16/32 (1:400, Pharmingen) for 1 hour. Sections were incubated with primary and secondary antibodies for 48 hours each at 4°C in blocking solution. Goat antibody to mouse and goat antibody to rabbit (1:800, Molecular Probes) were conjugated to Texas Red or Cy5 and were used as secondary antibodies. Control brains stained with isotype control primary antibodies and with Texas Red or Cy5 secondary antibodies did not display positive staining.

Supplemental text corresponding to (27): P-CREB staining
Brains were obtained from three mice (two mice at 8 weeks of age and one mouse at 6 months of age) after transplantation with GFP⁺ marrow. Mice were anesthetized with Methoxyflurane, killed by surgical decapitation, and the olfactory bulbs were rapidly isolated and incubated in Tyrode containing 2mM Ca²⁺, 1mM Mg²⁺, 10 mM glycine, and 100 mM glutamate for 15 min at room temperature. Tissue was fixed in 1.5% paraformaldehyde in 4mM EGTA for 2 hours at room temperature and was sliced to yield coronal sections. The samples were blocked and permeabilized in 0.3% Triton-X 100, 3% BSA (bovine serum albumin), monoclonal antibody to mouse CD16/CD32 (Pharmingen, 1:400), and 100 mM glycine in PBS for 30 min at 37°C, and then for an additional 30 min at room temperature. Staining was performed with polyclonal antibody to phospho-CREB (UBI, 1:800) and monoclonal antibody NeuN (Chemicon, 1:300) in the blocking and permeabilizing solution for 14 hours at room temperature followed by five washes in 0.3 % Triton-X 100 and 3% BSA in PBS. After 2 hours of incubation with the secondary antibodies (Texas Red, goat antibody to rabbit, 1:800, Molecular Probes; Cy5, goat antibody to mouse, 1:800, Amersham) at room temperature, the tissues were washed five times over 2 hours, mounted with 60% glycerin and were visualized by confocal microscopy.

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• Movie 1
  
• Movie 2
  Rotating movies of a group of bone marrow-derived, GFP⁺ cells coexpressing both TuJ1 and NF-H. (Movie 1) Staining with antibody to class III b-tubulin (TuJ1, red) reveals cells surrounded by TuJ1 staining, several of which are bone marrow-derived (green). (Movie 2) The same group of bone marrow-derived cells (green) as in movie 1 stained with antibody to the 200-kD isoform of neurofilament (NF-H, blue).