fig3: Limb SP cells express ALCAM. (A) LMCs were stained with Hoechst 33342 and analyzed for expression of ALCAM, PECAM-1, and CD45 in the SP cell fraction. (a) Hoechst 33342 staining and emission pattern of LMCs. SP cells were indicated in the enclosed region. (b) Hoechst 33342 staining and emission pattern of LMCs in the presence of verapamil. (B) A fluorescence histogram shows the ALCAM staining profile of the SP gated with the PECAM-1−CD45− fraction. The percentages of ALCAM+ cells are representative of triplicate experiments.

Mentions: To analyze ALCAM expression on stem cell fraction in limb bud, we examined limb SP cells. SP cells are known to be pluripotent stem cells. To identify SP cells in the limb, initial studies were conducted to establish optimal Hoechst dye staining conditions (data not shown). Incubation of LMCs with 2.5 μg/ml of Hoechst 33342 for 90 min consistently identified a population similar to that of murine BM SP cells (Fig. 3 A a, and data not shown). SP cells represented 0.58 ± 0.37% in freshly prepared LMCs. Goodell et al. had previously shown that the Hoechst SP profile in murine BM was blocked by staining in the presence of verapamil, indicating that the faint staining of SP cells was at least partially due to the efflux of Hoechst dye mediated by ABC transporters (31). Hoechst staining of LMCs was also sensitive to verapamil (Fig. 3 A b). The percentages of ALCAM+ cells in PECAM-1−CD45− gated SP cells were 32.9 ± 9.0% (Fig. 3 B). Thus, ALCAM+ cells were enriched about fourfold in SP cell population.

Arai F, Ohneda O, Miyamoto T, Zhang XQ, Suda T - J. Exp. Med. (2002)

Bottom Line: Furthermore, the addition of ALCAM-Fc or CD6-Fc to the metatarsal culture, the invasion of the blood vessels to a cartilage was inhibited.Our findings indicate that ALCAM+ perichondrial cells participate in vascular invasion by recruiting osteoclasts and vessels.These findings suggest that perichondrium might serve as a stem cell reservoir and play an important role in the early development of a bone and bone marrow.

Affiliation: Department of Cell Differentiation, Institute of Molecular Embryology and Genetics (IMEG), Kumamoto University School of Medicine, Japan.

Abstract: Perichondrium in fetal limb is composed of undifferentiated mesenchymal cells. However, the multipotency of cells in this region and the role of perichondrium in bone marrow formation are not well understood. In this report, we purified and characterized perichondrial cells using a monoclonal antibody against activated leukocyte cell adhesion molecule (ALCAM) and investigated the role of perichondrial cells in hematopoietic bone marrow formation. ALCAM is expressed on hematopoietic cells, endothelial cells, bone marrow stromal cells, and mesenchymal stem cells and mediates homophilic (ALCAM-ALCAM)/heterophilic (ALCAM-CD6) cell adhesion. Here we show by immunohistochemical staining that ALCAM is expressed in perichondrium. ALCAM+ perichondrial cells isolated by FACS exhibit the characteristics of mesenchymal stem cells. ALCAM+ cells can differentiate into osteoblasts, adipocytes, chondrocytes, and stromal cells, which can support osteoclastogenesis, hematopoiesis, and angiogenesis. Furthermore, the addition of ALCAM-Fc or CD6-Fc to the metatarsal culture, the invasion of the blood vessels to a cartilage was inhibited. Our findings indicate that ALCAM+ perichondrial cells participate in vascular invasion by recruiting osteoclasts and vessels. These findings suggest that perichondrium might serve as a stem cell reservoir and play an important role in the early development of a bone and bone marrow.