Three dimensional collagen scaffolds promote iPSC induction with higher pluripotency
KeywordsiPSC Nuclear Transfer Tirone Mouse iPSCs Extracellular Matrix Secretion
Extracellular environment plays a role in regulating stem cell fates and three dimensional (3D) scaffolds can be utilized to mimic the internal environment in vitro. Currently, many types of cells have been cultured in 3D conditions but only few studies have focused on reprogramming in a 3D environment. 3D culture systems provide circumstances that can better simulate native conditions which are comprised of distinctive cell morphology, oxygen levels, extracellular matrix secretion and concentration gradients of signaling factors (Keung et al., 2010; Gu et al., 2016).
Herein, we used collagen, the major composition of the extracellular matrix (Di Lullo et al., 2002), that serves as scaffolds to offer porous 3D surrounding to mimic in vivo environments (Song et al., 2015) and to explore the role of 3D conditions in reprogramming. In this study, we investigated the effect of 3D collagen scaffolds on the reprogramming of mouse embryonic fibroblasts (MEFs) and pig embryonic fibroblasts (PEFs). MEFs could be successfully converted into mouse induced pluripotent stem cells (iPSCs) in 3D collagen scaffolds. After long time incubation, the results demonstrated that 3D conditions increased reprogramming efficiency with high levels of pluripotency in comparison with the conventional 2D method. Another reprogramming method, nuclear transfer (NT), was also detected with high improved efficiency when using the MEFs from 3D as nuclear donor. In addition, reprogramming inhibitors namely p21 and B-cell translocation gene 2 (Btg2), were suppressed during cultivation in 3D collagen scaffolds.
To further confirm the role of 3D collagen scaffolds in reprogramming, pig iPSCs, commonly difficult to silent their exogenous activation, were derived in 3D conditions from PEFs. PEFs were directly reprogrammed in 3D collagen scaffolds and typical colonies can be observed by SEM (Fig. S1A). Pig iPSCs were cultured in 3D conditions and longer lasting cell viability was observed in 3D compared to 2D conditions (Fig. S1B). The core problem of pig iPSCs is the persistent expression of transgenic genes (Petkov et al., 2015). Our result suggested that the expressions of exogenous, Oct4 and c-Myc, were down-regulated compared to 2D condition (Figure S1C). The endogenous genes of stemness, Oct4, Sox2, Rex1 and Nanog, were up-regulated when cells were reprogrammed in 3D collagen scaffolds (Figure S1D). The down-regulation of the exogenous genes and up-regulation of the endogenous genes (Fig. S1C and S1D) may bring forward a new approach for reprogramming without Doxycycline (Fujishiro et al., 2013).
Additionally, NT is part of the classic reprogramming methods. NT efficiency is affected by the conditions of the donor cells (Blelloch et al., 2006). In order to understand the influence of 3D culture on NT, MEFs cultured in 3D collagen scaffolds were digested into single cells and served as donors for NT (Fig. 2D). The results were compared and they performed significantly different from 2D group of embryo developments, which included 2-cell, 4-cell, morulae and blastocyst (Table S1). The single cells digested from 3D appeared smooth with no filopodia visible around the edges (Fig. 2E). This suggests that filopodia may not play a role in the proliferation in 3D system, which is different from the present 2D studies (Twarock et al., 2010; Arjonen et al., 2011). During a revision of this work, an independent study (Caiazzo et al., 2016) also reported an iPSCs generation in 3D microenvironments, which supports part of our work. However, we used 3D porous scaffolds whereas they have developed a 3D encapsulation culture.
In summary, the relationship between 3D conditions and stem cell has attracted considerable attentions (Gu et al., 2015). Our study may provide a novel and useful avenue for stem cell research.
This work was supported by grants from National Basic Research Program of China (Grant NO .2011CB965001 to L.L.) and Strategic Priority Research Program of Chinese Academy of Sciences (Grant No. XDA01020101 to Q.Z.).
Jie Hao, Qi Zhou, Qi Gu, He Zhu, Lei Chen, Ling Shuai, Jinhui Fang, Jun Wu, Lei Liu, Wei Li, and Jianwu Dai declare that they have no conflict of interest.
All studies were carried out in accordance with the Guidelines of the Care and Use of Laboratory Animals established by Beijing Association for Laboratory Animal Science. All experiments were approved by the Institute of Zoology, Chinese Academy of Sciences.
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