Col2-GFP reporter marks chondrocyte lineage and chondrogenesis during mouse skeletal development

Grant, T. Dawn; Cho, Jay; Ariail, K.; Weksler, Nicole B.; Smith, Randall W.; Horton, William A.

doi:10.1002/(sici)1097-0177(200006)218:2<394::aid-dvdy12>3.0.co;2-i

Cited by 50 publications

(43 citation statements)

References 31 publications

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“…To efficiently derive a cell source with sufficient chondrogenic capacity, we developed a protocol to isolate subpopulations of cells at the end of micromass culture based on GFP expression under the control of a previously described cartilage-specific Col2 regulatory element (32). This approach allowed us to demonstrate the significance of starting cell population homogeneity in recreating tissues with cartilage-specific physiological properties.…”

Section: Discussionmentioning

confidence: 99%

“…To accomplish this, we induced chondrogenic differentiation by treating micromass cultures with BMP-4 and used flow cytometry to sort cells expressing green fluorescent protein (GFP) under control of the chondrocyte-specific type II collagen (Col2) promoter/enhancer (32). In addition to biochemical and mechanical characterization of the tissue-engineered constructs, we examined the potential of differentiated and purified iPSCs to be used for functional cartilage repair using an in vitro cartilage defect model system.…”

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confidence: 99%

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Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

Diekman

Christoforou

Willard

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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250

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The development of regenerative therapies for cartilage injury has been greatly aided by recent advances in stem cell biology. Induced pluripotent stem cells (iPSCs) have the potential to provide an abundant cell source for tissue engineering, as well as generating patient-matched in vitro models to study genetic and environmental factors in cartilage repair and osteoarthritis. However, both cell therapy and modeling approaches require a purified and uniformly differentiated cell population to predictably recapitulate the physiological characteristics of cartilage. Here, iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP− cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilagespecific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twicepassaged GFP+ cells. Finally, atomic force microscopy analysis showed increased microscale elastic moduli associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage. This study demonstrates the potential use of iPSCs for cartilage defect repair and for creating tissue models of cartilage that can be matched to specific genetic backgrounds.chondrocyte | bone morphogenetic proteins | transforming growth factor-beta | cartilage micromechanics | cartilage regeneration

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

Diekman

Christoforou

Willard

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

232

250

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“…A Col II reporter mouse was used, with expression of green fluorescent protein (GFP) controlled by the type II collagen promoter/enhancer (Grant et al, 2000;Cho et al, 2002;Gareau et al, 2004). GFP ϩ/Ϫ mice were identified at 14 days by observing the fluorescence of cartilage taken in an ear punch, obviating the need for formal genotyping.…”

Section: Col Ii/gfp Mice: Phenotype Identification and Preparation Fomentioning

confidence: 99%

In vivo delivery of fluoresceinated dextrans to the murine growth plate: Imaging of three vascular routes by multiphoton microscopy

et al. 2005

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Bone elongation by endochondral ossification occurs through the differentiation cascade of chondrocytes of cartilaginous growth plates. Molecules from the systemic vasculature reach the growth plate from three different directions: epiphyseal, metaphyseal, and a ring vessel and plexus associated with the perichondrium. This study is an analysis of the real-time dynamics of entrance of fluoresceinated tracers of different molecular weights into the growth plate from the systemic vasculature and tests the hypothesis that molecular weight is a key variable in the determination of both the directionality and the extent of tracer movement into the growth plate. Multiphoton microscopy was used for direct in vivo imaging of the murine proximal tibial growth plate in anesthetized 4-to 5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter. Mice were given an intracardiac injection of either fluorescein (332.3 Da) or fluoresceinated dextrans of 3, 10, 40, 70 kDa, singly or sequentially. For each tracer, directionality and rate of arrival, together with extent of movement within the growth plate, were imaged in real time. For small molecules (up to 10 kDa), vascular access from all three directions was observed and entrance was equally permissive from the metaphyseal and the epiphyseal sides. Within our detection limit (a few percent of vascular concentration), 40 kDa and larger dextrans did not enter. These results have implications both for understanding systemic and paracrine regulation of growth plate chondrocytic differentiation, as well as variables associated with effective drug delivery to growth plate chondrocytes. Key words: growth plate; vasculature; multiphoton microscopy; endochondral ossificationBone elongation in children occurs by endochondral ossification in cartilaginous growth plates at the ends of long bones. Chondrocytic differentiation in the growth plate begins with clonal expansion of stem cells, continues during cellular proliferation, and ends with cellular enlargement, followed by death and replacement by bone. The kinetics of chondrocytic activity in each stage and of the regulatory transitions between them determine the rate of bone elongation achieved. As in any cellular growth system, nutrients are required to sustain the process, and multiple endocrine and paracrine inputs regulate at each stage. For growth plate cartilage, the role of the extracellular matrix also must be considered, not only as a substantive contributor to elongation per se, but through its potential role as a communication link among chondrocytes of the growth plate, and between growth plate chondrocytes and cells of surrounding tissues such as the peri-

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“…Expression of green fluorescent protein under control of the collagen 2 promotor in transgenic mice was used previously to purify all chondrocytes from rib cages but did not allow the detection, quantification, and fractionation of growth plate chondrocytes. (14) To identify new antigens that allow cell sorting, we conducted whole-genome transcriptome analysis of dissected distinct zones of juvenile mouse growth plate and thereby discovered several differentially expressed genes coding for cell surface proteins. A subset of these proteins was selected to set up a multiparametric cell-sorting approach that allows the qualitative and quantitative assessment of growth plate composition and the preparative separation of all distinct chondrocyte subpopulations.…”

Section: Introductionmentioning

confidence: 99%

Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status

Belluoccio¹,

Etich²,

Rosenbaum³

et al. 2010

Journal of Bone and Mineral Research

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Axial growth of long bones occurs through a coordinated process of growth plate chondrocyte proliferation and differentiation. This maturation of chondrocytes is reflected in a zonal change in gene expression and cell morphology from resting to proliferative, prehypertrophic, and hypertrophic chondrocytes of the growth plate followed by ossification. A major experimental limitation in understanding growth plate biology and pathophysiology is the lack of a robust technique to isolate cells from the different zones, particularly from small animals. Here, we report on a new strategy for separating distinct chondrocyte populations from mouse growth plates. By transcriptome profiling of microdissected zones of growth plates, we identified novel, zone-specific cell surface markers and used these for flow cytometry and immunomagnetic cell separation to quantify, enrich, and characterize chondrocytes populations with respect to their differentiation status. This approach provides a novel platform to study cartilage development and characterize mouse growth plate chondrocytes to reveal unique cellular phenotypes of the distinct subpopulations within the growth plate. ß

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Col2-GFP reporter marks chondrocyte lineage and chondrogenesis during mouse skeletal development

Cited by 50 publications

References 31 publications

Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells

In vivo delivery of fluoresceinated dextrans to the murine growth plate: Imaging of three vascular routes by multiphoton microscopy

Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status

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