Embryonic stem cell rescue of tremor and ataxia in myelin-deficient shiverer mice

Abstract

Transplantation of neural precursor cells has been proposed as a possible approach for replacing missing or damaged central nervous system myelin. Neonatal and adult myelin-deficient shiverer (shi) mice, bearing a mutation of the myelin basic protein (MBP) gene, have been used extensively as hosts for testing cell engraftment, migration, and myelination, but relatively little progress has been made in reversing shi motor deficits. Here we describe a prenatal cell replacement strategy, showing that embryonic stem cells injected into shi blastocyst embryos can generate chimeric mice with strong and widespread immunoreactive MBP expression throughout the brain and a behavioral (motor) phenotype that appears essentially rescued.

Fig. 1

Coronal brain sections at two rostrocaudal levels labeled for immunoreactive MBP from homozygous shi mice (shi/shi) (A,D), a homozygous shi mouse genetically rescued with the wild-type MBP transgene (shi/shi; MBP/MBP) (B,E), and chimeras derived from ES cell-injected shi blastocysts (ES cell-rescued shi Chimera) (C,F). Scale bar represents 2 mm.

Fig. 2

Agarose gel electrophoretic analysis of PCR amplification for the shi and WT alleles, displayed as 380- and 169-bp amplification products, respectively, from brain genomic DNA of shi/shi (Lane 2), B6C3F1 (Lane 3), Ini1+/− (Lane 4), shi/shi; MBP/MBP (Lane 5), and the 5 “rescued” shi chimeric (1, 2, 3, 4 and 5; Lanes 6 – 10) mice. The size of the amplification products was determined by reference to the 100-bp DNA ladder (Lanes 1 and 12). For negative control, water was used in place of genomic DNA as template (Lane 11).

Fig. 3

Coronal brain sections labeled for immunoreactive MBP from a homozygous shi mouse genetically rescued with the wild-type MBP transgene (shi/shi; MBP/MBP) (A), a “rescued” chimeric mouse (“Rescued” shi Chimera) (B) and a chimeric mouse with severe motor deficits (“Intermediate” shi Chimera) (C) derived from ES cell-injected shi blastocysts. cc, corpus callosum; LV, lateral ventricle. Scale bar represents 0.2 mm.

Fig. 4

Representative rostrocaudal maps of MBP immunoreactivity from two “intermediate” shi chimeras with behavioral phenotypes that were relatively more (top) or less (bottom) severe. Shown are coronal brain sections at atlas [16] levels 0.2 mm, −0.2 mm, −2.4 mm, −3.3 mm, −5.2 mm, −6.4 mm, and −7.5 mm from bregma.

Fig. 5

Dual immuno- and histochemical labeling of single sections, showing (A) an immunoreactive MBP patch along with punctate cellular X-gal reaction products from an “intermediate” chimeric mouse (MBP, red; X-gal, blue), and (B–D) co-localization of immunoreactive Olig1, GFAP, or NeuN (brown) with X-gal (blue, arrows) in presumptive oligodendrocytes (B), astroglia (C), and neurons (D) from a “rescued” chimeric mouse. Scale bars represent 50 μm.