Abstract
The maintenance and expansion of human embryonic stem cells (ESCs) in two-dimensional (2-D) culture is technically challenging, requiring routine manipulation and passaging. We developed three-dimensional (3-D) scaffolds to mimic the in vivo microenvironment for stem cell proliferation. The scaffolds were made of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups, which self-assembled via a Michael addition reaction. When primed ESCs (H9 cells) were mixed with PEG polymers, they were encapsulated and grew for an extended period, while maintaining their viability, self-renewal, and differentiation potential both in vitro and in vivo. Three-dimensional (3-D) self-assembling scaffold-grown cells displayed an upregulation of core pluripotency genes, OCT4, NANOG, and SOX2. In addition, the expression of primed markers decreased, while the expression of naïve markers substantially increased. Interestingly, the expression of mechanosensitive genes, YAP and TAZ, was also upregulated. YAP inhibition by Verteporfin abrogated the increased expression of YAP/TAZ as well as core and naïve pluripotent markers. Evidently, the 3-D culture conditions induced the upregulation of makers associated with a naïve state of pluripotency in the primed cells. Overall, our 3-D culture system supported the expansion of a homogenous population of ESCs and should be helpful in advancing their use for cell therapy and regenerative medicine.
Highlights
Pluripotency is defined by the potential of a cell to differentiate into germline cells as well as the cells of all three germ layers [1,2]
H9 cells encapsulated in self-assembling scaffolds composed of polyethylene glycol (PEG)-8-SH and PEG-8-Acr polymers grew for extended periods without requiring routine passaging or manipulation
H9 induce the naïve state of pluripotency in primed Embryonic stem cells (ESCs) [40], we investigated the effect of yes-associated protein (YAP) incells using VP,H9 which a YAP
Summary
Pluripotency is defined by the potential of a cell to differentiate into germline cells as well as the cells of all three germ layers [1,2]. Pluripotency ranges from the formation of the epiblast until gastrulation and lineage commitment, resulting in a population of cells representing a range of pluripotent states [3,4]. Pluripotency in vitro is determined by the developmental stage from which the cell line is derived and by culture conditions [5,6,7,8] with distinct naïve and primed states of pluripotency corresponding to in vivo pre-implantation and post-implantation epiblast cells, respectively [4]. Embryonic stem cells (ESCs) display unlimited self-renewal and differentiation potential in vitro [9], making them an ideal source for the development of cell therapies and regenerative medicine applications. ESCs are typically grown as a monolayer in two-dimensional (2-D) plastic culture plates coated with extracellular matrix (ECM) components
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