Understanding Osteogenic Nodule Formation From Single Embryonic Stem Cell-Derived Progenitors
Open Access
- Author:
- Woll, Nicole Leanor
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 22, 2007
- Committee Members:
- Sarah Bronson, Committee Chair/Co-Chair
Mala Chinoy, Committee Member
Henry Joseph Donahue, Committee Member
Ralph Lauren Keil, Committee Member - Keywords:
- embryonic stem cells
osteoblasts
bone
differentiation - Abstract:
- The process of bone formation can be observed in vitro in the form of a mineralized nodule. Mesenchymal stem cells (MSCs), the immediate precursors to osteoprogenitors, are found in the marrow, and likely in numerous other sites throughout the organism. Differentiation of these progenitors, when placed into culture under appropriate conditions, proceeds through characteristic stages of commitment, proliferation, matrix secretion, and mineral deposition during a period of 3-4 weeks. We have developed an embryonic stem cell (ESC)-derived osteogenic culture system. ESCs are allowed to form embryoid bodies (EBs) that are then disrupted after two days and plated as single cells at densities as low as 25 cells/cm2. By eight days post plating, a significant percentage of the colonies have morphology characteristic of other types of osteogenic cultures. By three weeks in culture, these colonies go on to form layered nodules. Generally 60% of the colonies are layered, mineralized nodules. This thesis provides three lines of evidence for osteogenesis in these ESC-derived cultures: (i) cell and colony morphology as revealed by phase contrast microscopy, (ii) mineralization of extracellular matrix as revealed by von Kossa staining, and (iii) QRT-PCR analysis of cDNA from entire plates and individual colonies revealing expression of genes characteristic of, and specific for, osteoblasts. For QRT-PCR analysis, we have isolated RNA from entire plates and individual colonies at different stages of the differentiation process to investigate the expression of genes characteristic of the osteoblast lineage, as well as genes characteristic of other lineages and stem/progenitor cells. Analysis of gene expression in the first week revealed that brachyury, Twist-2, Runx2, and Sox-9 are up-regulated by day 4 of the osteogenic culture, with osterix expression increasing by day 7. Runx2, osterix, type I collagen, and Bglap1, all important for osteogenesis, are transcriptionally up-regulated by day 14 and continue to increase in expression out to 21 days. QRT-PCR analysis of amplified RNA from individual colonies indicates that it is the morphologically osteogenic colonies that are expressing mRNA characteristic of the osteoblast lineage and allows us to further categorize colonies into hematopoietic, osteogenic, adipogenic, myogenic or a mixture of mesenchymal lineages. The goal of this project has been to define the timing and molecular basis of commitment in ESC-derived osteogenic cultures. We hypothesize that there is a critical decision point involving mesenchymal lineages that occurs somewhere before day 3 of the osteogenic culture. In order to investigate the commitment of progenitor cells to the osteogenic lineage, we have developed methods permitting overexpression and knockdown of Runx2 mRNA. Runx2 is a transcription factor necessary for bone formation and is postulated to be the transcriptional switch of osteoblast differentiation. Three methods of inducing Runx2 expression have been utilized: (i) up-regulation of Runx2 by bone morphogenetic protein 2 (BMP-2), (ii) constitutive overexpression of Runx2 constructs, and (iii) tetracycline-inducible overexpression of Runx2 constructs. For the knockdown of Runx2 mRNA expression, we have utilized both a constitutive and tetracycline-inducible short hairpin RNA (shRNA) system, which employs components of the pSUPERIOR RNAi System„§. The overexpression studies suggest that before cells commit to the osteogenic lineage, overexpression of Runx2 is detrimental to the osteogenic lineage. The loss-of-functions studies suggest that there is a critical time point between the ESC and day 2 embryoid body (D2EB), in which induction of shRNA for Runx2 is also detrimental to osteogenesis. This method of initiating osteogenesis from murine ESC cultures, where individual EB-derived progenitors are plated at a low density such that the potential and differentiation of a single cell can be determined, is the only described method that allows for the observation and manipulation of the commitment stage of mesengenesis from single murine embryonic progenitors. It also shows great promise for unraveling the mechanisms of commitment to the osteogenic lineage, as well as exploring the therapeutic potential of cells of this lineage.