In addition recent findings indicate that the

In addition, recent findings indicate that the majority of in vitro expanded cells undergo apoptosis upon implantation (Stegen et al., 2016). A cause may be the need to instantly adjust to a compromised environment, since current culture conditions represent a rather non-physiological and abundant nutritional source. Optimized culture conditions for clinically relevant progenitors are therefore essential for the in vitro preparation of cell-based constructs and should maintain cell survival, allow efficient growth factor treatments without batch-to-batch variation, and meet the regulatory guidelines (Dimarakis and Levicar, 2006). We developed a serum-free pre-conditioning regime in a CDM in order to adapt/prepare progenitor cells for differentiation and the subsequent in vivo environment. Of note, pre-conditioning in CDM led to an increased DNA content per cell, but no cell proliferation or change in karyotype profile. This suggests the onset of endoreplication, a phenomenon where cells replicate the nuclear genome in the absence of cell division, leading to elevated DNA content and enhanced cell potential in order to save energy (Edgar and Orr-Weaver, 2001). This was further supported by increased mRNA transcript levels of the cell cycle markers CCNE1, CDK1, and BIRC5 (Banyai et al., 2016; Britton and Edgar, 1998; Yamamoto et al., 2008). Although the mechanism of endoreplication is not yet fully understood, studies in mice have shown improved membrane transporter to stress and/or limited energy sources, typically seen during tissue regeneration (Pandit et al., 2013; Sher et al., 2013). Although more studies are needed in order to elucidate whether cells from the periosteum adapt this mechanism after the initial proliferative burst in fracture healing, this may be a potential cause of their remarkable regenerative capacity (Roberts et al., 2015), a phenomenon partially replicated by the pre-conditioning.
Experimental Procedures Pre-conditioning in CDM or GM as a control was carried out for 6 days. Directly following pre-conditioning, BMP-2 stimulation and/or aggregation was carried out for an additional 6 days. In vivo implantation was carried out ectopically and orthotopically in NMRInu/nu mice. Cell material was encapsulated in collagen type 1 gels for implantation. In vivo development of the implanted constructs was studied for up to 8 weeks. Detailed experimental procedures are provided in the Supplemental Information. The ethical committee for human medical research (KU Leuven) approved all procedures, and informed consent was obtained from the patients. All procedures on animal experiments were approved by the local ethical committee for animal research (KU Leuven). The animals were housed according to the guidelines of the Animalium Leuven (KU Leuven).
Author Contributions
Acknowledgments This work is part of Prometheus, the KU Leuven R&D division for skeletal tissue engineering. http://www.kuleuven.be/prometheus. The authors would like to thank M. V.d. Broeck, K. Bosmans, L. Ceysens, S. Bellinkx, and W.L. Tam for excellent technical assistance. The research leading to these results has been funded by: IWT-SBO-111545, FP/2007–2013/ERC grant agreement nos. 249191, FWO (12S6817N, 12O8715N, 12C8214N, 12I6216N, and 12G2715N), KU Leuven (PDMK/14/203), and NWO (14328). Nano-computed tomography images were generated in the X-ray computed tomography facilities of the Department of Materials Engineering of the KU Leuven, financed by the Hercules Foundation (project AKUL 09/001: Micro- and nano-CT for the hierarchical analysis of materials).
Introduction Tissue engineering technology has become one of the most prospective therapeutic approaches for bone regeneration in bone defects (Guan et al., 2012; Petite et al., 2000). As a type of adult mesenchymal stem cells (MSCs), human adipose-derived mesenchymal stem cells (hASCs) are capable of self-renewal and differentiation into cells such as osteoblasts, chondrocytes, and adipocytes (Zuk et al., 2002). Because they can be obtained from adipose tissue in abundance by means of a minimally invasive procedure, hASCs are a valuable source of adult MSCs for bone tissue engineering and bone regeneration (Bosnakovski et al., 2005). Therefore, how to effectively promote the osteogenic differentiation of hASCs has become dramatically important in bone tissue engineering.