Turning Blood into Brain: Cells Bearing Neuronal Antigens Generated in Vivo from Bone Marrow
Tóm tắt
Từ khóa
Tài liệu tham khảo
Williams K. C., Hickey W. F., Curr. Top. Microbiol. Immunol. 202, 221 (1995).
Azizi S. A., Stokes D., Augelli B. J., DiGirolamo C., Prockop D. J., Proc. Natl. Acad. Sci. U.S.A. 95, 3908 (1998).
PU.1 null mice were reconstituted as follows. Adult male mice (8 to 24 weeks old) were killed and both femurs were removed under sterile conditions. The muscle was removed and the ends of the bones were cut off with a scalpel. The remaining central portion of the femur was placed into Dulbecco's modified Eagle's medium (DMEM) (Gibco Gaithersburg MD) containing 10% fetal bovine serum (Gibco). Marrow cells from each femur were flushed out with medium. A suspension of the bone marrow cells was prepared by pushing the marrow and medium through 18-gauge 21-gauge and 25-gauge needles consecutively. The cell suspension was centrifuged at 300 g for 8 min and the supernatant was discarded. The cells were washed in DMEM without serum and an aliquot was removed for NeuN immunostaining. For the transplantation experiments the remainder of live cells was centrifuged and resuspended in DMEM without serum. For immunostaining of acutely isolated bone marrow cells see supplemental methods (25). Bone marrow transplants were performed as follows. At birth each female neonate was given an intraperitoneal injection of a 0.05-ml suspension that contained 1 × 10 7 male bone marrow cells (equivalent to one adult mouse). Approximately 0.05 to 0.5% of the total number of the marrow cellularity are hematopoietic stem cells and ∼0.125% are stromal cells (42–44). All pups were given subcutaneous injections of enrofloxacin for 2 weeks as previously reported (15) to help reduce the incidence of infection.
Supplemental Web material is available at www.sciencemag.org/cgi/content/full/290/5497/1779/DC1.
Tissues were collected as follows. Reconstituted and normal mice were killed at the appropriate age with carbon dioxide gas. Tissues were collected and immediately stored at −80°C until used. ISH histochemistry and immunohistochemistry were performed as follows. Fresh frozen brain sections (12 μm thick) or acutely isolated bone marrow cells were fixed with 2 to 4% paraformaldehyde and immunostained with the neuronal nuclear marker NeuN [monoclonal immunoglobulin G1 1:1000 dilution (Chemicon Temecula CA)]. The antibody was detected by using the Mouse on Mouse kit (Innogenex San Ramon CA) and subsequent deposition of biotinylated tyramide preceding the ISH. After the ISH streptavidin-546 Alexa dye conjugate (Molecular Probes Eugene OR) was added to bind the biotin. Immediately following the deposition of the tyramide nonradioactive ISH was performed on the same sections to detect the Y chromosome by using a 1.5-kb RNA probe pY3531B that was generated against a repeat sequence of the mouse Y chromosome (17) and labeled with digoxigenin–uridine 5′-triphosphate (for technical details see ). After several washes the digoxigenin was developed using an antibody to digoxigenin conjugated to either alkaline phosphatase (1:1500 dilution) or peroxidase (1:400 dilution) (Roche Pharmaceuticals Indianapolis IN). The antibody to digoxigenin was then visualized with either 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) as substrate (purple precipitate with light microscopy) or tyramide–fluorescein isothiocyanate (FITC) (NEN Boston MA) (green fluorescence). Subsequently cell nuclei were stained with 4′ 6-diamidino-2-phenylindole (DAPI) (blue fluorescence). Representative sections from transplanted mice were double-labeled with NeuN and NSE [polyclonal 1:10 000 dilution (Polysciences Warrington PA)] antibodies. Primary antibodies were visualized with an Alexa 594 antibody to mouse (NeuN 1:1000 dilution Molecular Probes) or Alexa 488 secondary antibodies to rabbit (NSE 1:500 dilution Molecular Probes).
B. Alberts et al. Molecular Biology of the Cell (Garland New York ed. 2 1989).
J. D. Watson Molecular Biology of the Gene (Benjamin Menlo Park CA ed. 3 1976).
Vinores S. A., Herman M. M., Rubinstein L. J., Marangos P. J., J. Histochem. Cytochem. 32, 1295 (1984).
Morrison S. J., Weissman I. L., Proc. Assoc. Am. Physicians 107, 187 (1995).
Heumann D., Leuba G., Rabinowicz T., J. Hirnforsch. 18, 483 (1977).
E.M. dedicates this report to the memory of János Szentágothai (1912–94) anatomist statesman romantic artist and mentor who helped me understand the difference between looking at tissue sections and seeing the secrets they hold. The authors would like to express their sincere thanks to R. Dreyfus for his help with the conventional microscopy and C. L. Smith and R. Cohen for their help with the confocal microscopy. We are also grateful to M. Brownstein R. Cohen H. Gainer L. Hudson and M. Palkovits for their helpful suggestions and support throughout the work. These studies were supported by NIH grant AI30656 to R.A.M.