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.
Highlights
Microtia and anotia are rare congenital defects affecting development of the outer ear (Luquetti et al, 2012; Cox et al, 2014)
This study has shown that following chondrogenic differentiation in 3D cultures, cartilage stem/progenitor cells (CSPCs) derived from microtic ear cartilage remnants display differences in their spontaneous spatial organization as compared to normal ear CSPCs, which are not readily apparent in 2D cultures
Microtic ear cartilages were compared with developing normal ear and pediatric auricular cartilage to assess whether microtic cartilages display an immature phenotype
Summary
Microtia (small ear) and anotia (absent external ear) are rare congenital defects affecting development of the outer ear (Luquetti et al, 2012; Cox et al, 2014). They can occur as part of a syndrome, such as hemifacial microsomia, Goldenhar syndrome, Treacher Collins, Nager, and CHARGE, or independently, with the mechanisms underlying the defect largely unknown (Luquetti et al, 2012). Different degrees of reduction in size and malformed shape are observed among microtic ears depending on the severity of the malformation, with anotia and the most severe cases of microtia requiring surgical reconstruction. There is a need for carrying out a more systematic analysis of microtic cartilages both in vivo and in vitro
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