In skeletal development, cells of the chondrocyte and osteoblast lineage undergo serial steps of proliferation and differentiation, and give rise to matrix-producing cells that drive bone growth. The goal of this research project is to reveal stem/progenitor cells in the chondrocyte and osteoblast lineage in terms of their origin, distribution, regulated kinetics and genetic profiles in vivo.
Specific Aim 1. Stem-like chondrocytes at the top of the postnatal epiphyseal growth plate cartilage: In endochondral bone formation, chondrocytes in the specific regions termed growth plates continue to proliferate postnatally, providing engines for bone lengthening. Slowly dividing cells at the top o the growth plate probably share some characteristics of postnatal stem cells. First, existence of self-renewing chondrocytes that are the sources of all other chondrocytes in the growth plate will be demonstrated by a lineage-tracking experiment using a chondrocyte-specific inducible CreERt and a fluorescent reporter system with a long chase period. Second, the genetic make-up of label-retaining cells at the top of the growth plate will be characterized based on cDNA microarrays. A chondrocyte-specific pulse-chase experiment will be performed to identify slowly replicating cells based on a doxycycline-regulatable Tet-off system and a histone 2B-bound EGFP (H2B-EGFP) label. Label-retaining and non-label-retaining chondrocytes will be isolated by fluorescent activated cell sorting (FACS). Genes specifically expressed in label-retaining chondrocytes will be tested for their gene expression during development by in situ hybridization, using probes identified in microarray experiments comparing the label-retaining and rapidly proliferating chondrocytes.
Specific Aim 2. Early cells early in the osteoblast lineage Osteoblast differentiation of "mesenchymal stem cells" is regulated by transcription factors Runx2 and Osterix (Osx) expressed early after commitment to the osteoblast lineage. Msx2 is putatively upstream of these two transcription factors. Nestin has been recently shown to be a marker of mesenchymal stem cells. Heterogeneity, origin and self-renewal of the "mesenchymal stem cell" population in vivo will be investigated by a combined lineage-tracking experiment based on a double fluorescent system using Nestin-EGFP;Nestin-/Osx-/Runx2-/Msx2-CreERt;Rosa26-CAG-tdTomato reporter mice. Double positive self-renewing and single positive descendant populations of interest will be isolated by FACS to analyze genes specifically upregulated in each population.
Specific Aim 3. Common stem/progenitor cells of the chondrocyte and the osteoblast lineage and their function: Inducible CreERt BAC transgenic mouse in which CreERt expression is regulated by the promoter of one of the commonly upregulated genes of Aim 1 and 2 will be created. To understand the role of these cells during skeletal development, the CreERt mice will be crossed with inducible diphtheria toxin receptor (iDTR) mice. Diphtheria toxin will be administered at various times of development, and disruption on normal skeletogenesis will be monitored to elucidate the role of these progenitors in vivo.

Public Health Relevance

The prospective findings of this research project will give important insights into the property and the role of stem/progenitor cells in skeletal development. This information will become a valuable tool for understanding the mechanisms of various dental and craniofacial deformities and developing novel diagnosis categories and treatment modalities.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Career Transition Award (K99)
Project #
1K99DE022564-01
Application #
8279758
Study Section
NIDCR Special Grants Review Committee (DSR)
Program Officer
Frieden, Leslie A
Project Start
2012-04-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
1
Fiscal Year
2012
Total Cost
$137,970
Indirect Cost
$10,220
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Ono, Noriaki; Ono, Wanida; Mizoguchi, Toshihide et al. (2014) Vasculature-associated cells expressing nestin in developing bones encompass early cells in the osteoblast and endothelial lineage. Dev Cell 29:330-9
Ono, Noriaki; Ono, Wanida; Nagasawa, Takashi et al. (2014) A subset of chondrogenic cells provides early mesenchymal progenitors in growing bones. Nat Cell Biol 16:1157-67
Mizoguchi, Toshihide; Pinho, Sandra; Ahmed, Jalal et al. (2014) Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development. Dev Cell 29:340-9
Usumi-Fujita, Risa; Hosomichi, Jun; Ono, Noriaki et al. (2013) Occlusal hypofunction causes periodontal atrophy and VEGF/VEGFR inhibition in tooth movement. Angle Orthod 83:48-56