TGF-ß signaling in stromal cells acts upstream of FGF-10 to regulate epithelial stem cell growth in the adult lung.

Tissue resident mesenchymal stromal cells (MSCs) contribute to tissue regeneration through various mechanisms, including the secretion of trophic factors that act directly on epithelial stem cells to promote epithelialization. However, MSCs in tissues constitute a heterogeneous population of stromal cells and different subtypes may have different functions. In this study we show that CD166(neg) and CD166(pos) lung stromal cells have different proliferative and differentiative potential. CD166(neg) lung stromal cells exhibit high proliferative potential with the capacity to differentiate along the lipofibroblastic and myofibroblastic lineages, whereas CD166(pos) lung stromal cells have limited proliferative potential and are committed to the myofibroblastic lineage. Moreover, we show that CD166(pos) lung stromal cells do not share the same epithelial-supportive capacity as their CD166(neg) counterparts, which support the growth of lung epithelial stem cell (EpiSPC) colonies in vitro. In addition, ex vivo expansion of lung stromal cells also resulted in the loss of epithelial-supportive capacity, which could be reinstated by inhibition of the TGF-ß signaling pathway. We show that epithelial-supportive capacity correlated with the level of FGF-10 expression and the reactivation of several lung development-associated genes. In summary, these studies suggest that TGF-ß signaling in stromal cells acts upstream of FGF-10 to regulate epithelial stem cell growth in the adult lung.

Application of autologous bone marrow derived mesenchymal stem cells to an ovine model of growth plate cartilage injury.

Injury to growth plate cartilage in children can lead to bone bridge formation and result in bone growth deformities, a significant clinical problem currently lacking biological treatment. Mesenchymal stem/stromal cells (MSC) offer a promising therapeutic option for regeneration of damaged cartilage, due to their self renewing and multi-lineage differentiation attributes. Although some small animal model studies highlight the therapeutic potential of MSC for growth plate repair, translational research in large animal models, which more closely resemble the human condition, are lacking. Our laboratory has recently characterised MSCs derived from ovine bone marrow, and demonstrated these cells form cartilage-like tissue when transplanted within the gelatin sponge, Gelfoam, in vivo. In the current study, autologous bone marrow MSC were seeded into Gelfoam scaffold containing TGF-beta1, and transplanted into a surgically created defect of the proximal ovine tibial growth plate. Examination of implants at 5 week post-operatively revealed transplanted autologous MSC failed to form new cartilage structure at the defect site, but contributed to an increase in formation of a dense fibrous tissue. Importantly, the extent of osteogenesis was diminished, and bone bridge formation was not accelerated due to transplantation of MSCs or the gelatin scaffold. The current study represents the first work that has utilised this ovine large animal model to investigate whether autologous bone marrow derived MSC can be used to initiate regeneration at the injured growth plate.

Heat shock protein-90 beta is expressed at the surface of multipotential mesenchymal precursor cells: generation of a novel monoclonal antibody, STRO-4, with specificity for mesenchymal precursor cells from human and ovine tissues.

Mesenchymal stromal cells (MSCs) and their precursor cells (MPCs) can proliferate and differentiate into multiple mesodermal and some ectodermal and endodermal tissues. Culture-expanded MSCs are currently being evaluated as a possible cell therapy to replace/repair injured or diseased tissues. While a number of mAb reagents with specificity to human MSCs, including STRO-1, STRO-3 (BLK ALP), CD71 (SH2, SH3), CD106 (VCAM-1), CD166, and CD271, have facilitated the isolation of purified populations of human MSCs from primary tissues, few if any mAb reagents have been described that can be used to isolate equivalent cells from other species. This is of particular relevance when assessing the tissue regenerative efficacy of MSCs in large immunocompetent, preclinical animal models of disease. In light of this, we sought to generate novel monoclonal antibodies (mAb) with specific reactivity against a cell surface molecule that is expressed at high levels by MSCs from different species. Using CD106 (VCAM-1)-selected ovine MSCs as an immunogen, mAb-producing hybridomas were selected for their reactivity to both human and ovine MSCs. One such hybridoma, termed STRO-4, produced an IgG mAb that reacted with <5% of human and ovine bone marrow (BM) mononuclear cells. As a single selection reagent, STRO-4 mAb was able to enrich colony-forming fibroblasts (CFU-F) in both human and ovine BM by 16- and 8-folds, respectively. Cells isolated with STRO-4 exhibited reactivity with markers commonly associated with MSCs isolated by plastic adherence including CD29, CD44, and CD166. Moreover, when placed in inductive culture conditions in vitro, STRO-4(+) MSCs exhibited multilineage differentiation potential and were capable of forming a mineralized matrix, lipid-filled adipocytes, and chondrocytes capable of forming a glycosaminoglycan-rich matrix. Biochemical analysis revealed that STRO-4 identified the beta isoform of heat shock protein-90 (Hsp90beta). In addition to identifying an antibody reagent that identifies a highly conserved epitope expressed by MSCs from different species, our study also points to a potential role for Hsp90beta in MSC biology.

Characterisation and developmental potential of ovine bone marrow derived mesenchymal stem cells.

Since discovery, significant interest has been generated in the potential application of mesenchymal stem cells or multipotential stromal cells (MSC) for tissue regeneration and repair, due to their proliferative and multipotential capabilities. Although the sheep is often used as a large animal model for translating potential therapies for musculoskeletal injury and repair, the characteristics of MSC from ovine bone marrow have been inadequately described. Histological and gene expression studies have previously shown that ovine MSC share similar properties with human and rodents MSC, including their capacity for clonogenic growth and multiple stromal lineage differentiation. In the present study, ovine bone marrow derived MSCs positively express cell surface markers associated with MSC such as CD29, CD44 and CD166, and lacked expression of CD14, CD31 and CD45. Under serum-deprived conditions, proliferation of MSC occurred in response to EGF, PDGF, FGF-2, IGF-1 and most significantly TGF-alpha. While subcutaneous transplantation of ovine MSC in association with a ceramic HA/TCP carrier into immunocomprimised mice resulted in ectopic osteogenesis, adipogenesis and haematopoietic-support activity, transplantation of these cells within a gelatin sponge displayed partial chondrogenesis. The comprehensive characterisation of ovine MSC described herein provides important information for future translational studies involving ovine MSC.

Intramembranous ossification mechanism for bone bridge formation at the growth plate cartilage injury site.

Salter's type III and type IV growth plate injuries often induce bone bridge formation at the injury site. To understand the cellular mechanisms, this study characterized proximal tibial transphyseal injury in rats. Histologically, bony bridge trabeculae appeared on day 7, increased on day 10, and became well-constructed on day 14 with marrow. Prior to and during bone bridging, there was no cartilage proteoglycan metachromatic staining and no collagen-X immunostaining at the injury site, nor was there any up-regulation of BrdU-labelled chondrocyte proliferation at the adjacent physeal cartilage, suggesting no new cartilage formation at the injury site. However, infiltration of vimentin-immunopositive mesenchymal cells from metaphysis and epiphysis was apparent on day 3, with the mesenchymal population being prominent on days 7 and 10 and subsided on day 14. Among these infiltrates were osteoprogenitor precursors expressing osteoblast differentiation factor (cbf-alpha1) on day 3, along with some cbf-alpha1+ osteoblast-like cells lining bone trabeculae on days 7 and 10. Some mesenchymal cells and trabecula-lining cells were also alkaline phosphatase-immunopositive, further suggesting their osteoblast differentiation. From day 7 onwards, some trabecula-lining cells became osteocalcin-producing mature osteoblasts. These results suggest that bone bridge formation after growth plate injury occurs directly via intramembranous ossification through recruitment of marrow-derived osteoprogenitor cells.

Identification of a second presenilin gene in zebrafish with similarity to the human Alzheimer's disease gene presenilin2.

Presenilins play prominent roles in the molecular pathogenesis of Alzheimer's disease and during embryo development. We have isolated a zebrafish presenilin orthologue (pre2), which shows a high degree of sequence identity to the human PS2 protein. Zebrafish pre2 is maternally and ubiquitously expressed during early embryo development, whereas Pre2 protein expression is initiated between 6 and 12 hours post fertilisation (hpf), suggesting strict regulation of pre2 translation. pre2 expression is especially high in neural-crest-derived melanocytes.