The promotion of stemness and pluripotency following feeder-free culture of embryonic stem cells on collagen-grafted 3-dimensional nanofibrous scaffold
Introduction
Embryonic stem cells (ESCs) were first isolated in 1981 from mouse blastocysts [1], [2] and subsequently from human blastocysts [3], [4]. These pluripotent cells were found to be capable of both unlimited self-renewal and differentiation into ectodermal, endodermal, and mesodermal derivatives. Traditionally, mouse ESCs (mESCs), human ESCs and induced pluripotent stem (iPS) cells were cultured on mouse embryonic fibroblast (MEF) feeder cells in serum containing media, which can supply the requisite matrix and synthesize differentiation inhibitory factors such as LIF [5]. The concern over xenogeneic contaminants from the mouse feeder cells may restrict stem cell based therapy [6]. Regardless of the source, feeder cells are derived from primary tissues and therefore have a limited life span. Moreover, MEF feeder cells at passages 7 or more do not support the pluripotency of ESCs. Therefore, fresh batches of feeders have to be prepared which consequently leads to potential batch-to-batch variations [7]. In recent years, in order to reduce xeno-components several feeder-free culture systems using Matrigel™ matrix, MEF-conditioned media, and growth factors have been reported for maintaining human ESCs [6], [8]. In spite of this progress, several significant disadvantages still exist. Exposure to animal pathogens through MEF-conditioned medium or Matrigel matrix is still a possibility [9]. On the other hand, the derivation, proliferation, and pluripotency of ESCs depend on their microenvironment, including the chemical and physical properties of the extracellular matrix (ECM) and the presence of growth factors [10]. Since ECM plays an important role in the regulation of cell growth and differentiation in addition to its structural function a lot of interests have been paid to mimic the characteristics of natural ECM to facilitate cell seeding, adhesion, proliferation, and differentiation [11].
Over the past decade, the electrospinning method has been applied to fabricate nanofibrous scaffolds with the structural features of the ECM [12], [13], [14]. Moreover, naturally occurring polymers extracted from the native ECM such as, gelatin and collagen have been widely used to modify the synthetic material to improve the surface properties and cell–scaffold interactions [15], [16], [17], [18].
Polyethersulfone (PES) is a non-biodegradable polymer which is widely used as a membrane material in hemodialysis, filtration and bioreactor technology [19], [20]. Although there are some reports on the ability of different cell types to adhere and proliferate on PES membranes [21], [22], [23], there are few reports on stem cell culture on the PES membranes [19]. Recently, our research group has successfully fabricated the plasma treated and collagen-grafted PES nanofibrous scaffolds [24], [25]. Shabani et al. found enhanced proliferation, differentiation, and infiltration of unrestricted somatic stem cells (USSC) cultured on the collagen-grafted PES nanofibrous scaffolds [25]. However, there are no studies available to demonstrate the behavior of embryonic stem cells on PES nanofibers. The goal of this study was to analyze the maintenance of stemness and pluripotency of mESCs following feeder-free culture on collagen-grafted PES electrospun nanofibrous scaffolds.
Section snippets
Derivation and culture of mESCs Line
The derivation and culture of mESCs were performed as described by Bryja et al. [26]. Briefly, The mESCs were derived from 3.5-day-old blastocyst-stage mouse embryos, which were collected and plated individually on mitomycin C mitotically inactivated mouse embryonic fibroblast feeder (MEF) layers in gelatin-coated 96-well culture plates. The mESC medium was composed of Knockout Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% Knockout serum, penicillin (100 U/ml)/streptomycin
Characterization of the mESCs cultured on MEF
In this study we established a mouse embryonic stem cell line derived from hybrid F1 blastocyst of C57BL/6 and BALB/c mice. Then we characterized the mESCs cultured on MEF in the presence of LIF after 10 passages. Moreover, the mESCs were analyzed for in vitro and in vivo pluripotency (Fig. 2). The mESCs cultured on MEF indicated positive staining for alkaline phosphatase, Oct-4, and SSEA-1. RT-PCR analysis indicated the expression of embryonic stem cell specific genes such as Oct-4, Nanog,
Discussion
Mouse embryonic fibroblasts secrete numerous substances such as ECM and cytokines that are beneficial for maintenance of ESCs self-renewal and pluripotency. However, the use of feeder cells involves several time-consuming steps, such as generation of MEFs from mouse embryos, inactivation of cells, and removal of feeder cells from ESCs. In addition, batch variability and xenobiotic contamination are the problems encountered by many labs [28]. The colonization and maintenance of ES cells in an
Conclusion
In this report, we evaluated the maintenance of stemness and pluripotency following Feeder-free culture of mESCs on collagen-grafted PES nanofibrous scaffold. Here we demonstrate a successful feeder-free culture system in which mESCs can be maintained in an undifferentiated state and thereby eliminating the need of feeder layers for in vitro culture of embryonic stem cells. Furthermore, PES-COL nanofibrous scaffold could be a good candidate for feeder-free culture of human embryonic stem cells
Acknowledgments
We acknowledge funding for this study provided by the Iranian Council of Stem Cell Technology (Tehran, Iran) and Stem Cell Technology Research Center (Tehran, Iran). The authors would like to thank Athena Hajarizadeh and Maryam Abbasi for technical assistance.
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