Comparison of a xeno-free and serum-free culture system for human embryonic stem cells with conventional culture systems

As a renewable cell source for future regenerative medicine applications, the derivation, passaging, and culture of hESCs have attracted widespread attention [23]. To generate therapeutically safe and usable hESC derivatives for clinical cell treatment, all animal-derived materials which are a potential risk for infection transmitted by animal pathogens must be eliminated during the process [24].

The major sources of xeno-contamination are the presence of animal feeders and the use of either FBS or serum replacement (SR) in the culture medium [4]. Many groups have previously claimed successful culture of hESCs on hFFs and other feeders [7, 12, 14, 25–28]. Hovatta et al. [27] used commercially available human foreskin fibroblasts for hESC propagation. Crook et al. [29] proposed the proof of concept for the generation of six clinical-grade hESC lines using hFFs as feeder cells. hFFs isolated from infant foreskin have a distinct advantage over MEFs. In addition to the xenobiotic issue, hFFs can be cultured for up to 62 passages before senescence, while MFFs can only be propagated for five passages [7, 30]. Moreover, little ethical concern is considered over the acquirement of foreskin tissue for the generation of feeder cells.

Another obstacle to overcome was the presence of FBS or SR in the culture medium. Several studies described the use of HS as serum supplement to culture hESCs. However, it has been proved previously that commercial xeno-free SRs and human sera are inappropriate for long-term culture of hESCs [21]. Some attempts to use HS as a medium supplement failed due to spontaneous differentiation [12, 26, 31]. It is important to point out that HS may be involved in the interactions between secreted factors from the feeder cells. Therefore, it is highly desirable to employ defined components rather than complex mixtures [32–35]. The establishment of a chemically defined xeno-free and feeder-free culture system is advantageous for exact identification of factors secreted by feeder layers or signal pathways existing in them. Thus, xeno-free commercially available CDM was employed in the culture system.

Our data demonstrate that hESCs can be successfully derived and cultured long term in the XF-HFF/CDM system while maintaining their undifferentiated state. Furthermore, teratoma analysis demonstrated that hESCs maintain their pluripotency and differentiation potential, which were comparable with hESCs cultured in the conventional hESC culture medium. The use of CDM with hFF cells has been described previously [17]. On the basis of that work, we further presented the differences of culture systems for hESCs. We tested four culture systems and two hESC lines, and little variation was observed between hESCs cultured in CDM and KSR medium in maintaining undifferentiated states of hESCs. On the other hand, data obtained by flow cytometry and qRT-PCR demonstrated that feeder cells supported the growth of undifferentiated hESCs more efficiently than HS-matrix when cells were presented in CDM. The HS-matrix, which contains hyaluronic acid, fibronectin, and vitronectin, as well as other unknown human factors, supports the attachment and growth of undifferentiated cells [36]. Stojkovic et al. [37] suggested that HS used as a matrix maintained pluripotency and genomic stability of hESCs. Meng et al. [38] pointed that extracellular matrix isolated from foreskin fibroblasts has an advantage in the process of long-term xeno-free hESC culture. However, feeder-free cultures usually presented a higher degree of spontaneous differentiation than conventional culture along with a higher concentration of exogenous bFGF, which indicated that feeder-free derivation and culture were not suitable for developing transplantable hESC derivatives [11]. The results in this manuscript also proved that the HS-matrix/CDM system could not maintain the undifferentiated state and differentiated potential of hESCs. Although the molecular and developmental mechanisms controlling the pluripotency and differentiation of hESCs are largely unknown, feeder cells may provide a better, more stable environment, and thus they can secrete the unique proteins that participate in cell growth, extracellular matrix formation, and remodeling [1, 39].

The results demonstrated that CDM provided an effective replacement for HS medium, comparable with KSR medium, to support hESC self-renewal and pluripotency. This observation suggests that CDM and KSR medium culture conditions supply similar growth factors or signal pathways for hESC growth. CDM is more suitable than serum-containing medium for studies that aimed at identifying the exogenous signals required for undifferentiated hESC growth. XF-HFF/HS were unable to maintain undifferentiated of hESCs in long-term growth.

Exogenous bFGF has been identified as a key factor involved in self-renewal of hESCs [40–43], and some studies have indicated that FGF proteins are sensitive to thermal denaturation, while fibroblasts likely secrete either protease inhibitors or binding proteins that modulate bFGF stability [42, 44]. In our work, we found that bFGF was unstable in 37 °C culture conditions. Although the concentrations of bFGF declined in all culture conditions, they were higher in cofeeder conditions than in feeder-free conditions. The data indicated that fibroblast is likely to secrete some proteins which modulate the stability of bFGF [42], and also indicated that the stability of bFGF was not the only factor responsible for the successful culture of hESCs.

Lysophosphatidic acid (LPA) as the key lipid is a simple phospholipid mediator which plays an important role in the regulation of cell proliferation, migration, and survival of multiple cell types [45, 46]. Todorova et al. [47] proposed that LPA was involved in the expression of the Ca2⁺-dependent early response gene c-myc, which was a key gene implicated in ESC self-renewal and pluripotency. Therefore, LPA increased the proliferation rate of mESCs. Liu et al. demonstrated that LPA induced the expression of the erythroid biomarkers in cultured human hematopoietic stem cells (hHSCs) under plasma-free conditions. In addition, LPA was also reported to enhance osteogenic differentiation of human mesenchymal stem cells [48]. These results suggested that LPA may play a critical role in cell fate determination. Hence, we suspected that HFF-1 cells could secrete LPA to support the long-term culture.