Differential Effects of the Extracellular Microenvironment on Human Embryonic Stem Cell Differentiation into Keratinocytes and Their Subsequent Replicative Life Span

Abstract

Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-Kert(ACC)) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-Kert(AFF) were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-Kert(AFF) showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-β1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during differentiation of hESCs on subsequent replicative life span and cellular senescence of the differentiated keratinocytes, with implications for use of these cells for applications in tissue engineering and regenerative medicine.

Epithelial-mesenchymal transition: An emerging target in tissue fibrosis

“…The microenvironment can determine the cell phenotype. 55,56 In chronic disease, the microenvironment of the damage site changes, exhibiting characteristics of hypoxia,…”

Pluripotent stem cells: An in vitro model for nanotoxicity assessments

“…A stem cell-based approach provides a platform to study, diagnose and treat various diseases, including Alzheimer's disease, Parkinson's disease, cardiovascular disease, diabetes and amyotrophic lateral sclerosis (also known as Lou Gehrig's disease) (Merkle and Eggan, 2013). Promising features of PSCs have been highlighted in various biomedical applications, such as: (1) genome regulation and cell behavior; (2) therapeutic applications in the regeneration of organs/tissues; (3) drug screening, cytotoxicity and genotoxicity testing, and gene therapy; and (4) as an in vitro model to investigate aging (Movahednia et al, 2015). In theory, reprogramming technology allows researchers to convert easily accessible cells (such as skin cells) from a donor biopsy to reflect disease conditions in culture dishes.…”

Potential applications of keratinocytes derived from human embryonic stem cells

“…Further analysis indicated that the decellularized matrix of hESC-derived fibroblasts is highly-enriched in dermoepidermal junction (DEJ) proteins such as laminin, type I, type IV and type VII collagens and fibronectin and was able to further enhance differentiation of hESCs into almost pure population of K14 + cells (>90%) [32,35]. However, using this autogenic feeder-free system, hESC-derived keratinocytes were not able to expand for multiple passages and suffered cell senescence after 8 PD, thus implying the differential effects of the culture microenvironment during epidermal commitment of hESCs on the subsequent replicative potential of hESC-derived keratinocytes [35]. All studies to date related to differentiation of keratinocyte from hESCs are summarized in Table 1.…”

“…Moreover, the conclusion of formation of cornified epithelium in conventional "organotypic" culture system as the hallmark of epidermal keratinocytes for hESCderived p63 + /K14 + has been challenged. The formation of stratum corneum-like structure was observed in urothelial and tracheobronchial epithelial cells upon exposure to air-liquid interface, albeit at various degree of K10 matrix extracted from hESC-derived junction-like substrates to establish hESC-derived cells in feeder- [35] fibroblasts or on hESC-derived efficient epidermal generation. free condition as compared fibroblast feeder.…”