IntroductionRecent reports suggest that bone marrow-derived cells can be detected in regenerated muscle tissue of mice. [1][2][3][4] In addition, bone marrow transplantation in the dystrophin mutant (mdx) mouse, a model for Duchenne muscular dystrophy (DMD), resulted in the detection of donor cell-derived dystrophin in affected muscle fibers. 5,6 The presence of donor cells in muscle fibers was also observed in a patient with DMD who received an allogeneic marrow transplantation at one year of age. 7 However, in both the mdx mouse and the patient with DMD, the contribution of donor cells to affected muscle was very low and did not impact muscle function. 7,8 Whether this low frequency event represents transdifferentiation of marrow stem cells into muscle or rare fusion events remains controversial. [9][10][11] Resolving this controversy is critical for understanding the real potential and limitations of marrow transplantation. In addition, understanding how marrow cells contribute, even as rare events, to myogenesis is an important step toward developing strategies to increase the frequency of these events. We hypothesized that, regardless of the mechanism, bone marrowderived cells could be therapeutically valuable if a higher frequency of donor cell contribution to myogenesis could be achieved. This concept is supported by studies in which wild-type mesoangioblast cells were shown to rescue the ␣-sarcoglycan null dystrophic mice through myogenic fusion. 12 To identify human marrow cells with myogenic fusion potential we investigated the ability of human cells to contribute to myogenesis using first a xenogeneic in vitro model combining mouse C2C12 myoblasts with various subsets of human marrow cells, and second using an in vivo regeneration model. C2C12 cells represent an early stage of muscle differentiation. Depending on the culture conditions, they can either proliferate as myoblasts or differentiate into myotubes. 13 Previous studies have shown that coculturing C2C12 cells with dermal fibroblasts resulted in cell fusion and the subsequent correction of the dysgenic membrane in regenerating muscle. [14][15][16] The myogenic potential of neural stem cells was also demonstrated by coculture with C2C12 cells. 17,18 In this report we cocultured human hematopoietic and nonhematopoietic bone marrow cells with C2C12 and found that stromal cells have a significantly greater potential to contribute to myotube formation than enriched populations of hematopoietic progenitor cells, and that this contribution is due to cell fusion. Human stromal cells also contributed to regenerating muscle in vivo in the nonobese diabetic-severe combined immunodeficient (NOD/SCID) ␤2m Ϫ/Ϫ mouse. Materials and methods Human cell preparationsHuman CD34 ϩ cells from cadaveric marrow were provided by the Cellular Therapy Laboratory, Fred Hutchinson Cancer Research Center (FHCRC). Flow sorting was used to prepare CD34 ϩ and CD34 ϩ /CD38 Ϫ populations, both more than 95% pure. A more differentiated CD33 ϩ population was expanded from the CD34...

Trophoblast cells are specialized extra-embryonic cells present only in eutherian mammals. They play a major role in the implantation and placentation processes. To understand better the molecular mechanisms that control the development and function of trophoblast cells, we sought to identify the transcription factors that regulate murine adenosine deaminase (ADA) gene expression in the placenta. Here we report a detailed characterization of a placenta-specific footprinting region (FP1) in the Ada placental regulatory element. The sequence of FP1 was mapped by DNase I footprinting and was found to match a consensus AP-2 transcription factor-binding site. Electrophoretic mobility shift assays demonstrated that FP1 interacted with AP-2-like proteins. Further analysis using AP-2 antibody confirmed that AP-2 protein was indeed present in the placenta and bound to FP1. Mutation at the AP-2 site in FP1 abolished the ability of the Ada placental regulatory element to bind AP-2 proteins and failed to target chloramphenicol acetyltransferase reporter gene expression to placentas in transgenic mice, indicating that AP-2 is required for Ada expression in the placenta. In addition, RNase protection assays demonstrated that AP-2␥ was the predominant AP-2 family member expressed in the placenta. In situ hybridization analysis revealed that AP-2␥ expression was enriched in the trophoblast lineage throughout development, suggesting that AP-2␥ may be critical for trophoblast development and differentiation.

Murine adenosine deaminase (ADA) is a ubiquitous purine catabolic enzyme whose expression is subject to developmental and tissue-specific regulation. ADA is enriched in trophoblast cells of the chorioallantoic placenta and is essential for embryonic and fetal development. To begin to understand the genetic pathway controlling Ada gene expression in the placenta, we have identified and characterized a 770-base pair fragment located 5.4 kilobase pairs upstream of the Ada transcription initiation site, which directs reporter gene expression to the placenta of transgenic mice. The expression pattern of the reporter gene reflected that of the endogenous Ada gene in the placenta. Sequence analysis revealed potential binding sites for bHLH and GATA transcription factors. DNase I footprinting defined three protein binding regions, one of which was placenta-specific. Mutations in the potential protein binding sites and footprinting regions resulted in loss of placental expression in transgenic mice. These findings indicate that multiple protein binding motifs are necessary for Ada expression in the placenta.

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