Effects of oxygen concentration on differentiation of human IPS cells into cartilage tissue

Abstract

Purpose: Cartilage is a type of tissue characterized by poor self-repair capacity due to the absence of blood vessels and nerve tissue. As such, cartilaginous tissue does not self-repair after sustaining extensive damage. Treatments to address cartilaginous tissue damage include bone perforation, osteochondral column transplantation, and autologous cultured cartilage transplantation. Each of these treatment methods has achieved a degree of success, but problems related to the number of procedures required and the quality of regenerated tissue remain to be overcome. In recent years, regenerative medical techniques have come to be regarded as promising new therapy options. Among these, induced pluripotent stem cells (iPSCs) have attracted attention as a potential new source of cells for therapeutic uses. iPSCs exhibit high self-renewal capacity and have excellent potential as a cell source owing to their maintenance of the ability to undergo cell division while remaining undifferentiated. Although the formation of cartilage tissues from iPSCs has been established, regenerative medicines utilizing iPSCs also face obstacles, such as long culture periods, the risk of oncogenesis, and compromised tissue purity. We hypothesized that if cartilaginous tissues could be prepared from iPSCs in a hypoxic environment, it would be possible to produce tissues more quickly than under a stable oxygen environment. In this study, the effects of oxygen concentration on the differentiation of human iPSC cells (hiPSCs) into cartilage were studied. Methods: The established hiPSC line Toe was maintained in feeder-free medium in 6-well dishes coated with laminin. The hiPSCs formed high-density cell colonies consisting of 1-2 × 105 cells at 10 days after the start of maintenance. Subsequently, chondrogenic differentiation of the iPSCs was induced. The hiPSCs were initially differentiated into mesendodermal cells in mesendodermal medium for 3 days (M-phase). On day 3, the medium was changed to chondrogenic medium, hiPSCs were cultured in chondrogenic medium for 10 days (C-phase) to induce differentiation into chondrocytes. Then, four groups were formed based on oxygen concentration in the M-phase and C-phase: NN (21%, 21%), HN (5%, 21%), NH (21%, 5%), and HH (5%, 5%). On day 14, total RNA was extracted from differentiated cells. The expression of sox9, aggrecan, and col2a1, which are cartilage differentiation markers, and of T and FOXF1, which are undifferentiated mesoderm markers, was assessed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Moreover, cells were detached and digested to form a single-cell suspension using trypsin for flowcytometry, and the sox9-positive cell rate was measured. Results: In qRT-PCR analysis, sox9 expression was higher in NH than in NN and HN groups. col2a1 expression was higher in NH than in the other groups. Aggrecan expression was higher in NH group compared with HN group. T and FOXF1 expression was higher in HN group than NH group, and the groups cultured in hypoxic condition on C-phase (NH and HH groups) were lower than the groups cultured in normoxic condition on C-phase (NN and HN groups). In flowcytometry analysis, sox9-positive cell rate was higher in the NH and HH groups (5% C-phase oxygen concentration) than in the NN and HN groups (21% C-phase oxygen concentration). Conclusions: Chondrocyte differentiation was promoted by culture under hypoxic conditions during the production of cartilaginous tissues from human embryonic stem (ES) cells. Thus, it is possible that a hypoxic environment may also promote differentiation of cartilaginous tissue from iPSCs. In this study, a period of culturing in a hypoxic environment was provided during differentiation from hiPSCs into cartilage tissue. C-phase culturing with the oxygen concentration of 5% raised cartilage differentiation markers and lowered undifferentiated mesoderm markers. An oxygen concentration of 5% promotes differentiation from hiPSCs into cartilage tissue and raises purity levels. Therefore, adjusting the culture period or oxygen concentration (hypoxic environment) may allow engineering of high-purity cartilage tissues in a short time.