(GCL) NSG mouse muscle groups from 6-month-old animals never injected with human cells and injured with cardiotoxin show expression of human spectrin in regenerating myofibers. antibodies that recognize human, but not mouse, proteins. Here we show that one Rabbit polyclonal to KLF4 antibody specific to human spectrin, which is commonly used to evaluate the efficacy of transplanted human cells in mouse muscle, OSI-930 detects myofibers in muscles of NOD/mice, or nude mice, irrespective of whether they were injected with human cells. These reactive clusters are regenerating myofibers, which are normally present in dystrophic tissue and the spectrin OSI-930 antibody is likely recognizing utrophin, which contains spectrin-like repeats. Therefore, caution should be used in interpreting data based on detection of single human-specific proteins, and evaluation of human stem cell engraftment should be performed using multiple human-specific labeling strategies. Introduction Stem cells of human origin are considered of potential therapeutic value for a number of diseases. Stem cells directly isolated from either discarded or consented human tissue specimens, or generated via reprogramming of adult cells, require rigorous testing in preclinical models for both safety and efficacy. Preclinical testing is facilitated by the availability of immune-deficient murine models, such as (Flanagan, 1966; Pantelouris, 1968), NOD/(Shultz or NOD scid gamma) mice (Shultz and models bred into the NSG or NOD/background (Darabi and mice were generated by breeding the mutation into mice with a NOD/background for more than 10 generations (Lapan nude mice were generated as previously described (Partridge mice, as previously described (Lapan nude mice, aged 4 weeks. Tibialis anterior muscles were cryoinjured, or irradiated with 18?Gy 3 days before cryoinjury and grafting as described previously (Brimah and NSG mice, consecutive skeletal muscle tissue sections (10?m) were collected on Tissue Tack microscope slides (Polysciences, Warrington, PA). Various methods of fixation were tested: slides were fixed for 3?min in 100% methanol at room temperature; or fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15?min at room temperature followed by 3?min of permeabilization with 0.5% Triton X-100; or left OSI-930 to air dry for 30?min. After fixation, sides were washed once in PBS and blocked in 10% fetal bovine serum (FBS)CPBSC0.1% Triton X-100 for 30?min at room temperature. Slides were stained with polyclonal rabbit anti-dystrophin (CAP6-10, diluted 1:2000) (Lidov nude mice, muscles were frozen in isopentane chilled in liquid nitrogen and 7-m cryosections were cut throughout the muscles. Sections were rehydrated in PBS for 5?min, and then incubated in M.O.M. blocking reagent (Vector Laboratories, Peterborough, UK) diluted in 10% normal goat serum (Vector Laboratories, Peterborough, UK) in PBS for 1?hr at room temperature, according to the manufacturer’s instructions. Sections were then stained with primary antibodies to human spectrin (mouse monoclonal, diluted 1:20; Vector Laboratories, Peterborough, UK), human lamin A/C (mouse monoclonal, diluted 1:100; Vector Laboratories, Peterborough, UK), neonatal myosin (mouse monoclonal, diluted 1:50, BF34; Borrione recipient mice and the muscles were examined 1C2 months after injection. Immunostaining with mouse anti-human spectrin and rabbit anti-dystrophin (this antibody recognizes both human and mouse dystrophin) revealed the presence of myofibers positive for both proteins (Fig. 1ACC), which demonstrated that human donor cells had fused to these myofibers. In different sections, spectrin-reactive myofibers appeared to contain human-derived nuclei based on the expression of human-specific lamin A/C (Fig. 1DCF), confirming the presence of injected human cells. In addition, clusters or individual human spectrin-positive myofibers that were negative for dystrophin expression were also seen (Fig. 1GCI and Supplementary Fig. S1; supplementary data are available online at www.liebertpub.com/hum). Given the low abundance of the dystrophin transcript and an estimated time of 16?hr to transcribe the entire molecule (Tennyson muscles 45 days after injection of 1105 MCAM+ human fetal cells. (ACC) Coexpression of human spectrin and dystrophin (antibody recognizes both mouse and human proteins) documents human cell engraftment in myofibers. (DCF) Human spectrin-positive myofibers contain nuclei expressing human-specific lamin A/C. (GCI) Clusters of small myofibers reactive to human spectrin but negative for dystrophin expression. H-spectrin, human spectrin; h-lamin A/C, human lamin A/C. Scale bar: 50?m To further study the nature of these human spectrin-reactive myofibers and determine whether this expression unequivocally marks the engraftment of donor human cells, tissue sections of 2- to 4-month-old NOD/mice not injected with.