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Modeling Normal and Pathological Ear Cartilage in vitro Using Somatic Stem Cells in Three-Dimensional Culture.

ABSTRACT: Microtia (underdeveloped ear) is a rare congenital dysmorphology affecting the development of the outer ear. Although human microtic cartilage has not been fully characterized, chondrogenic cells derived from this tissue have been proposed as a suitable source for autologous auricular reconstruction. The aim of this study was to further characterize native microtic cartilage and investigate the properties of cartilage stem/progenitor cells (CSPCs) derived from it. Two-dimensional (2D) systems are most commonly used to assess the chondrogenic potential of somatic stem cells in vitro, but limit cell interactions and differentiation. Hence here we investigated the behavior of microtic CSPCs in three-dimensional spheroid cultures. Remarkable similarities between human microtic cartilages from five patients, as compared to normal cartilage, were observed notwithstanding possibly different etiologies of the disease. Native microtic cartilage displayed poorly defined perichondrium and hyper-cellularity, an immature phenotype that resembled that of the normal developing human auricular cartilage we studied in parallel. Crucially, our analysis of microtic ears revealed for the first time that, unlike normal cartilage, microtic cartilages are vascularized. Importantly, CSPCs isolated from human microtic and normal ear cartilages were found to recapitulate many characteristics of pathological and healthy tissues, respectively, when allowed to differentiate as spheroids, but not in monolayer cultures. Noteworthily, starting from initially homogeneous cell pellets, CSPC spheroids spontaneously underwent a maturation process in culture, and formed two regions (inner and outer region) separated by a boundary, with distinct cell types that differed in chondrogenic commitment as indicated by expression of chondrogenic markers. Compared to normal ear-derived spheroids, microtic spheroids were asymmetric, hyper-cellularized and the inner and outer regions did not develop properly. Hence, their organization resembled that of native microtic cartilage. Together, our results identify novel features of microtic ears and highlight the importance of 3D self-organizing in vitro systems for better understanding somatic stem cell behavior and disease modeling. Our observations of ear-derived chondrogenic stem cell behavior have implications for choice of cells for tissue engineered reconstructive purposes and for modeling the etiopathogenesis of microtia.

SUBMITTER: Zucchelli E

PROVIDER: S-EPMC7402373 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Modeling Normal and Pathological Ear Cartilage <i>in vitro</i> Using Somatic Stem Cells in Three-Dimensional Culture.

Zucchelli Eleonora E Birchall Martin M Bulstrode Neil W NW Ferretti Patrizia P

Frontiers in cell and developmental biology 20200728

Microtia (underdeveloped ear) is a rare congenital dysmorphology affecting the development of the outer ear. Although human microtic cartilage has not been fully characterized, chondrogenic cells derived from this tissue have been proposed as a suitable source for autologous auricular reconstruction. The aim of this study was to further characterize native microtic cartilage and investigate the properties of cartilage stem/progenitor cells (CSPCs) derived from it. Two-dimensional (2D) systems ar ...[more]

PMID: 32850801

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Project description:Defining the final size and geometry of engineered tissues through precise control of the scalar and vector components of tissue growth is a necessary benchmark for regenerative medicine, but it has proved to be a significant challenge for tissue engineers. The growth plate cartilage that promotes elongation of the long bones is a good model system for studying morphogenetic mechanisms because cartilage is composed of a single cell type, the chondrocyte; chondrocytes are readily maintained in culture; and growth trajectory is predominately in a single vector. In this cartilage, growth is generated via a differentiation program that is spatially and temporally regulated by an interconnected network composed of long- and short-range signaling mechanisms that together result in the formation of functionally distinct cellular zones. To facilitate investigation of the mechanisms underlying anisotropic growth, we developed an in vitro model of the growth plate cartilage by using neonatal mouse growth plate chondrocytes encapsulated in alginate hydrogel beads. In bead cultures, encapsulated chondrocytes showed high viability, cartilage matrix deposition, low levels of chondrocyte hypertrophy, and a progressive increase in cell proliferation over 7 days in culture. Exogenous factors were used to test functionality of the parathyroid-related protein-Indian hedgehog (PTHrP-IHH) signaling interaction, which is a crucial feedback loop for regulation of growth. Consistent with in vivo observations, exogenous PTHrP stimulated cell proliferation and inhibited hypertrophy, whereas IHH signaling stimulated chondrocyte hypertrophy. Importantly, the treatment of alginate bead cultures with IHH or thyroxine resulted in formation of a discrete domain of hypertrophic cells that mimics tissue architecture of native growth plate cartilage. Together, these studies are the first demonstration of a tunable in vitro system to model the signaling network interactions that are required to induce zonal architecture in growth plate chondrocytes, which could also potentially be used to grow cartilage cultures of specific geometries to meet personalized patient needs.

Dataset Information

Modeling Normal and Pathological Ear Cartilage in vitro Using Somatic Stem Cells in Three-Dimensional Culture.

Publications

Modeling Normal and Pathological Ear Cartilage <i>in vitro</i> Using Somatic Stem Cells in Three-Dimensional Culture.

Similar Datasets

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