The influence of osteogenic differentiation on the stem-like properties of adipose derived stem cells – an RT-qPCR study

Adipose Derived Stem Cells are an effective source of mesenchymal progenitors and were first described in 2001 [2]. This class of cells presents mesenchymal stem cells-like characteristics and present proliferation and differentiation potential, and self-renewal ability. These attributes grant them multiple and diverse clinical application, serving as a promising tool for gene-based therapies, such as tissue engineering and regenerative medicine [3]. Not only ADSCs are multipotent and can differentiate into the tri-germ cell lineages, but also show paracrine activity impacting immune responses [4]. They can differentiate into various cell types, specifically osteocytes, but also adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic cells, muscle cells and hepatocytes [5, 6, 7, 8]

ADSCs are readily accessible and widely available. Isolated through a minimally invasive procedure from adipose depots, they can be found at diverse body location, where they served various functions, including energy homeostasis. They can be obtained upon surgeries from otherwise waste tissues, like after excision of fat tissue or liposuction. [9]. In addition, due to the possibility to isolate many ADSCs, in vitro proliferation can be performed in a short time period, resulting in cells showing more predictable results [1]. For this study ADSCs were obtained from waste material following routing sterilization procedures of dogs.

Due to its qualities, adipose tissue has been studied in many different medical fields and is thought to be a powerful source of stem cells. The capability of ADSCs to differentiate towards various cell lineages and the opportunity of directing this differentiation, increases their possible clinical applications and their employability in different therapies and treatments of diseases [10].

ADSCs show great potential in tissue regeneration, specifically bone reconstruction upon trauma, poor bone formation or the removal of cancer tissue, and more generally in situations when bone cells lack regenerative capacity. Although many techniques already exist for bone tissue augmentation and repair, they are usually costly and present high risk of morbidity at donor site and risk of rejection [11]. The osteogenic potential of ADSCs, together with their ability to regulate the release of growth factor and cytokines with anti-inflammatory and angiogenic properties, has opened new prospects for bone tissue engineering and regeneration [12]. Different cases of implementation of bone regeneration therapies with ADSCs delivered positive results, showing ossification with little or no side effects [13]. As the potential of ADCS in orthopedic application is investigated, a deeper understanding of the mechanism underlying the osteoblastic differentiation of ADSC and its influence on the expression of mesenchymal stem cell markers could result in novel techniques and applications in tackling a diverse range of bone-related diseases.

The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. As the study was based on a remnant waste material, the Bioethical Committee approval was not necessary.

Regarding the osteogenic differentiation ability of ADSCs, multiple studies have reported the pathways, genes, proteins, and hormones involved in the process. Some factors influencing the commitment of ADSCs have been identified, such as the parathyroid hormone, PTH1-34, and the Notch proteins, a family of key regulator ligands usually involved in osteogenesis [15,16]. Moreover, the evaluation of osteogenic marker expression during ADSCs cultures was performed, resulting in the identification of genes such as CBFA1. However, since stem cells grow in vivo in a multifactorial environment presenting diverse biochemical and mechanical signals affecting their functioning and properties, which are subject to constant changes, it is very complicated to elucidate the mechanisms underlying niche cues and the connection between them. Moreover, their osteogenic potential has been investigated for other possible clinical utilizations, such as in dental placement strictly upon tooth extraction in healthy dogs [17]. In fact, the proliferation and osteogenic potential of ADSCs are expected to show great capacity to implement bone formation upon diseases or trauma, providing a resourceful alternative in reconstructive surgeries. As the results obtained so far appear ambiguous, the need for the understanding of their basic functioning is clear [18, 19, 20, 21]. However, promising results were obtained from the treatment of dogs with a combination of ASC-derived cell sheets and platelet-rich fibrin membranes transplantation, presenting elevated re-osseointegration percentage and increased new bone formation [22]. Although, in a different study, the application of ADSCs for dental implants did not present any substantial improvements [23]. This study aimed to analyse the expression of MSC specific markers before and after in vitro differentiation of ASCs. Three positive and three negative markers were analysed, CD105, CD73, CD90, CD34, CD14 and CD45 [24]. There were significant differences detected in the expression of all of the genes, with most of them exhibiting notable downregulation. CD45, was not detected in any of the analysed sample groups.

CD14 (Cluster of differentiation 14) is gene encoding a protein mostly produced by macrophages, playing a role in the proper functioning of the immune system. It was proven to aid the organism in detection of bacterial pathogens, through the binding of lipopolysaccharides (LPS), especially the pathogen-associated molecular protein (PAMP) [25]. In the human body, it can be found in two forms, soluble and attached to the cellular membrane. During LPS detection, this protein acts as a co-receptor with Toll-like receptor 4 and MD-2 [26]. It was also found to be able to bind other molecules associated with pathogens, aiding the recognition process [27]. In the context of stem cells, this protein was found to be a regulator of differentiation in porcine spermatogonia [28]. Furthermore, CD14⁺ macrophages were found to participate in chondrogenesis of synovium-associated stem cells in osteoarthritis [29]. Finally, single-nucleotide polymorphisms of CD14 were associated with outcomes of bone marrow stem cell transplantations [30].

Summarising, in this study, the subset of genes defined in literature as MSC markers underwent significant alterations in expression after osteogenic differentiation of ASCs. Downregulation of most of the genes was most probably associated with loss of stem-like properties over the time of in vitro culture and due to lineage commitment. In turn, a significant increase in the expression of CD14 could be an indicator of culture and differentiation stress on the occurrence of immune-response-like symptoms in the cultured cells.

Overall, the above described changes confirm the success of differentiation, as well as suggest that this process significantly lowers the stem-like ability of ASCs. This knowledge should serve as a reference for further molecular and clinical studies, possibly aiding the understanding of the internal mechanisms governing the differentiation and stemness of ASCs, to enable their widespread and safe application in regenerative medicine.