Although there are several reports on differentiation of human embryonic stem cells to dopaminergic neurons, notable heterogeneity exists in the reported yields of tyrosine hydroxylase (TH)-positive cells. For benchmarking performance and efficiency standards in future applications of hESC-derived dopaminergic neurons, there is thus a dire need of well-defined directed differentiation protocols. Pal et al. [Pal et al. 2009 Exp Biol Med (Maywood) 234:1230-3] demonstrated predisposition of HUES9 towards ectodermal lineage, but the directed differentiation of HUES9 to dopaminergic neurons has not yet been reported. Therefore, we report here a simple two-step protocol using suitable ECM and serum-free induction medium for generating dopaminergic cells from HUES9-derived embryoid bodies. Flow cytometry analysis of the neural progenitors obtained after the first step gave an enriched yield of cells immune-positive for nestin (99.6 ± 0.1%), musashi12 (98.1 ± 1.5%) and Sox2 (95.4 ± 2.6%). Most of these cells also expressed the proliferation marker Ki67 (83.8 ± 1.5%), whereas the presence of the undifferentiated stem cell marker Oct4 was negligible. In the second step, when these neural progenitors were exposed to midbrain cues sonic hedgehog and fibroblast growth factor 8 along with bFGF, the differentiated cells showed an upregulation of dopaminergic-related transcription factors Nurr1 and Engrailed1. Immunocytochemistry and flow cytometry analysis showed that these differentiated cells were positive for the mature neuronal marker Map2ab (96.2 ± 1.5%) and dopaminergic neuronal marker TH (71.9 ± 4.4%). Thus, the data demonstrate novel findings of the directed differentiation of HUES9 to dopaminergic neurons using well-defined serum-free nutrient supplements.

Background:Articular cartilage (AC) injuries and malformations are commonly noticed because of trauma or age-related degeneration. Many methods have been adopted for replacing or repairing the damaged tissue. Currently available AC repair methods, in several cases, fail to yield good-quality long-lasting results, perhaps because the reconstructed tissue lacks the cellular and matrix properties seen in hyaline cartilage (HC).Purpose:To reconstruct HC tissue from 2-dimensional (2D) and 3-dimensional (3D) cultures of AC-derived human chondrocytes that would specifically exhibit the cellular and biochemical properties of the deep layer of HC.Study Design:Descriptive laboratory study.Methods:Two-dimensional cultures of human AC–derived chondrocytes were established in classical medium (CM) and newly defined medium (NDM) and maintained for a period of 6 weeks. These cells were suspended in 2 mm–thick collagen I gels, placed in 24-well culture inserts, and further cultured up to 30 days. Properties of chondrocytes, grown in 2D cultures and the reconstructed 3D cartilage tissue, were studied by optical and scanning electron microscopic techniques, immunohistochemistry, and cartilage-specific gene expression profiling by reverse transcription polymerase chain reaction and were compared with those of the deep layer of native human AC.Results:Two-dimensional chondrocyte cultures grown in NDM, in comparison with those grown in CM, showed more chondrocyte-specific gene activity and matrix properties. The NDM-grown chondrocytes in 3D cultures also showed better reproduction of deep layer properties of HC, as confirmed by microscopic and gene expression analysis. The method used in this study can yield cartilage tissue up to approximately 1.6 cm in diameter and 2 mm in thickness that satisfies the very low cell density and matrix composition properties present in the deep layer of normal HC.Conclusion:This study presents a novel and reproducible method for long-term culture of AC-derived chondrocytes and reconstruction of cartilage tissue with properties similar to the deep layer of HC in vitro.Clinical Relevance:The HC tissue obtained by the method described can be used to develop an implantable product for the replacement of damaged or malformed AC, especially in younger patients where the lesions are caused by trauma or mechanical stress.