The epigenetic promotion of osteogenic differentiation of human adipose-derived stem cells by the genetic and chemical blockade of histone demethylase LSD1

Abstract

Human adipose-derived stem cells (hASCs) are a highly attractive source in bone tissue engineering. It has become increasingly clear that chromatin regulators play an important role in cell fate determination. However, how osteogenic differentiation of hASCs is controlled by epigenetic mechanisms is not fully understood. Here we use genetic tools and chemical inhibitors to modify the epigenetic program of hASCs and identify lysine-specific demethylase 1 (LSD1), a histone demethylase that specifically catalyzes demethylation of di- and mono- methyl histone H3 lysine 4 (H3K4me2/1), as a key regulator in osteogenic differentiation of hASCs. Specifically, we demonstrated that genetic depletion of LSD1 with lentiviral strategy for gene knockdown promoted osteogenic differentiation of hASCs by cell studies and xenograft assays. At the molecular level, we found that LSD1 regulates osteogenesis-associated genes expression through its histone demethylase activity. Significantly, we demonstrated LSD1 demethylase inhibitors could efficiently block its catalytic activity and epigenetically boost osteogenic differentiation of hASCs. Altogether, our study defined the functional and biological roles of LSD1 and extensively explored the effects of its enzymatic activity in osteogenic differentiation of hASCs. A better understanding of how LSD1 influences on osteogenesis associated epigenetic events will provide new insights into the modulation of hASCs based cell therapy and improve the development of bone tissue engineering with epigenetic intervention.

Introduction

Human adipose-derived stem cells (hASCs), one type of mesenchymal stem cells, possess a high plasticity to differentiate into multiple lineages including cartilage, bone, muscle and adipose in the presence of appropriate signaling factors and culture conditions [1], [2], [3], [4], [5]. Currently, hASCs have become a highly attractive source in tissue engineering and cell-based therapy of damaged bone defects, spinal fusion and skeletal reconstruction bone [2], [6], [7]. The main reason lies in the fact that hASCs can be obtained easily from adipose tissues carrying a more abundant and accessible pool of mesenchymal stem cells with a less invasive and less expensive procedures [8], [9], [10].

The critical issue for application of stem cells in tissue engineering is the initiation and control of cellular differentiation in a precise and appropriate manner. Recent evidence suggests that epigenetic regulation including DNA methylation and histone modification plays a key role in fate maintenance and lineage commitment of embryonic stem cells as well as mesenchymal stromal/stem cells [11], [12], [13], [14], [15], [16]. Slight variations of these epigenetic components might result in the changes of local chromatin configuration or nuclear architecture, impose flexible but precise control over the expression of the important regulatory genes, and eventually influence on cell fate determination without changes of the DNA sequences. Unlike genetic alterations, epigenetic changes are reversible and accessible to be regulated, and as such, drugs that target critical epigenetic regulators in cell commitment or differentiation are being overwhelmingly investigated [17], [18].

Lysine-specific demethylase 1 (LSD1/KDM1A) is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that catalyzes mono- and di-methyl moieties removal from histone H3 lysine 4 (H3K4) [19], [20], [21]. Despite progress in understanding the dynamic histone methylation regulation and in revealing the diverse molecular interactions for LSD1, the biological function of LSD1 is just beginning to be uncovered. Recent studies have linked LSD1 to certain high-risk tumors [22], [23], [24], [25], [26], [27], [28], [29], [30]. Moreover, LSD1 has been identified as one of the chromatin regulators implicated in the control of early embryogenesis [15], [31], [32], [33]. In addition, it was reported that LSD1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation [34]. Indeed, within the framework of the so-called epigenetic therapies, there is a growing interest in LSD1 as a potential drug target [26], [35], [36], [37], [38]. However, whether histone methylation associated epigenetic events impacted by LSD1 and currently developed LSD1 inhibitors can contribute to mesenchymal stem cells differentiation as well as bone tissue engineering are largely unknown.

Here, our study focused on investigating the functional role and the molecular mechanism of histone H3K4 demethylase LSD1, especially its catalytic activity in osteogenic differentiation of hASCs.