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Progenitors Systemically Transplanted into Neonatal Mice Localize to Areas of Active Bone Formation In Vivo: Implications of Cell Therapy for Skeletal Disease.

Wang, et al. Stem Cells 2006; 24:1896-1878
Summary by Feryal Ahmad

LAY SUMMARY

Mesenchymal stem cells (MSC) have the potential to differentiate into a variety of cell types, including osteoblasts, bone cells that produce Type I collagen. Type I collagen is defective in patients with osteogenesis imperfecta (OI), a genetic disease that is characterized by fragile bones. To determine if MSCs engraft to skeletal tissue of developing neonatal mice affected by OI, Wang, et al., systemically injected the cells into homozygous (two defective genes) and heterozygous (one defective gene) mice. The transplanted cells were found to be present in limb bones of the recipient mice. The cells localized to areas of active bone growth, in addition to other areas of the bone (see figure 1). The authors speculate that the donor cells may participate in bone formation. The donor cells isolated from the bone appeared to be osteoblasts, while those from the bone marrow appeared to be osteoprogenitors or the precursors of osteoblasts. Although MSCs have been shown to differentiate into osteoblasts in vivo, not a sufficient number of cells engraft and differentiate to produce detectable amount of Type I collagen.

Wang, et al, conclude a sufficient number of systemically introduced MSC progenitors will engraft to skeletal tissue in vivo, differentiate into osteoblasts, and may participate in bone formation in vivo of developing animals with skeletal disease. However, cells with a tendency to migrate to and engraft in skeletal tissue with high efficiency will have to be isolated and utilized

SCIENTIFIC SUMMARY

Mesenchymal stem cells (MSC) have the potential to differentiate into a variety of cell types, including, but not limited to, osteoblasts, chondrocytes, and adipocytes. Previous studies have shown injection of MSC directly into tissue may give rise to cells with phenotypes similar to or the same as the target tissue or organ. However, varied results have been documented for systemically delivered cells, which have been shown to engraft in organs or tissues of the recipients and which may or may not engraft in skeletal tissue. In this study, Wang, et al., purport MSC progenitor cells engraft in skeletal tissue after systemic introduction to homozygous and heterozygous neonatal osteogenesis imperfecta (OI) mice.

Osteogenesis imperfecta, also known as brittle bone disease, is a genetic skeletal disease characterized by fragile bones that break easily even under normal load. Type I collagen is defective in OI patients and the normal collagen heterotrimer formed by a1(I) and a2(I) are either deficient or formed improperly. Although previous studies in mice and children with OI have demonstrated systemically delivered MSC may engraft in bone and contribute to bone growth, their location post-transplantation and contribution to bone growth is unknown. Wang, et al, have previously reported MSC enhanced with green fluorescence protein (GFP) and Zeocin-resistant genes injected into normal developing mice migrate to and engraft in bones. Serial passage of recovered donor cells resulted in isolation of progenitor cells that had a tendency to engraft in bones of developing mice. These retrieved MSC progenitor cells were used in the current study.

The MSCs were verified to be pluripotent based on their ability to differentiate into cells of adipogenic and osteogenic lineages. Neonatal mice were examined at 14 and 28 days after injection with GFP+ MSCs via superficial temporal vein. The neonates were 2-day old and were γ-irradiated. The GFP+ MSCs were observed in the femur, tibia, and forelimb of mice. Histological analyses of femur and tibia showed the GFP+ cells localized to the surface of spicules in spongiosa below the growth plate of the epiphysis (see Figure 1). The authors speculate these results indicate the donor cells may participate in bone formation. Donor GFP+ cells were also found on surface of endosteum, in bone marrow, and in cortical bone (see Figure 1). Furthermore, engraftment in homozygous OI mice was found to be more prevalent than in heterozygous mice, though the authors speculate this may be due to the increased space available in the bones of homozygous OI mice. GFP+ cells were also found to engraft to lung.

Morphological appearance and genetic analysis showed the GFP+, Zeocin-resistant recovered cells from bone, bone marrow, and lung differed. Those from bone have osteoblast morphological appearance and express osteoblast-specific genes, including those of mature osteoblasts, while cells from bone marrow are osteoprogenitors, potentially serving as a reservoir for osteoblasts during growth and repair. Cells retrieved from lungs have fibroblast morphological appearance and express genes for lung surfactants A and D. Fluorescence-activated cell sorting (FACS) analysis confirmed none of the isolated cells were of endogenous source. From a separate experiment, FACS also showed donor cells constituted only ~1% of non-selected harvested cells from bone.

Although the MSCs have been shown to differentiate into osteoblasts in vivo, the total cells that engraft and differentiate to produce detectable amount of Type I collagen were insufficient. This observation was further evident after detectable amount of collagen heterotrimers was observed in femurs by increased cells.

Wang, et al, conclude a sufficient number of systemically introduced MSCs will engraft to skeletal tissue in vivo, differentiate into osteoblasts, and may participate in bone formation of animals with skeletal disease. However, cells with a tendency to migrate and engraft in skeletal tissue with high efficiency will have to be isolated and utilized. They also suggest this may explain the failure of systemically delivered MSC to engraft to skeletal tissue as reported in literature.

Comments:

In this study, Wang, et al., serially passaged MSC in normal mice for in vivo selection of donor cells that engraft to bone. The authors termed these cells as “osteoprogenitors.” However, data presented in the paper indicates these cells, although were progenitors, were not osteoprogenitors, which is particularly evidenced by the fact that these cells were able to differentiate towards adipogenic lineage with appropriate medium. Despite this mislabeling, the authors failed to show systemically injected MSC progenitors will engraft to skeletal tissue and contribute to growth. To begin, Wang, et al., did not stain for or identify blood vessels within the vicinity of engrafted GFP+ cells, where endogenous MSC may reside and potentially contribute to bone growth. Furthermore, the mice in this experiment were sub-lethally irradiated and many died either immediately or a few days after cell injection. It is not known whether the mice died because of inability to recover from irradiation or from an immune response elicited by donor cells, possibly due to presence of GFP, a compound known to cause rapid immune response. Lastly, the authors did not report engraftment of donor cells to areas other than skeletal or lung tissue. Consequently, the engraftment tendency of MSC progenitor cells has not been thoroughly investigated. Taking all of these shortcomings into consideration, these results do not provide enough information to be of relevance for human treatment of OI or other skeletal diseases, but forms the basis for future studies in this clinically relevant disorder of the bone.