Thermoresponsive Worms for Expansion and Release of Human Embryonic Stem Cells

Abstract

The development of robust suspension cultures of human embryonic stem cells (hESCs) without the use of cell membrane disrupting enzymes or inhibitors is critical for future clinical applications in regenerative medicine. We have achieved this by using long, flexible, and thermoresponsive polymer worms decorated with a recombinant vitronectin subdomain that bridge hESCs, aiding in hESC's natural ability to form embryoid bodies (EBs) and satisfying their inherent requirement for cell-cell and cell-extracellular matrix contact. When the EBs reached an optimal upper size where cytokine and nutrient penetration becomes limiting, these long and flexible polymer worms facilitated EB breakdown via a temperature shift from 37 to 25 °C. The thermoresponsive nature of the worms enabled a cyclical dissociation and propagation of the cells. Repeating the process for three cycles (over eighteen days) provided a >30-fold expansion in cell number while maintaining pluripotency, thereby providing a simple, nondestructive process for the 3D expansion of hESC.

A Critical Appraisal of RAFT-Mediated Polymerization-Induced Self-Assembly

“…These thermoresponsive worms were used in combination with PNIPAM functionalized with cell-binding vitronectin protein to bridge and aggregate human embryonic stem cells (hESCs), allowing 3D cell growth and exploiting the thermoresponsive properties to allow breakdown and subsequent reformation of the hESC aggregates. 133 Similarly, Davis et al examined the RAFT aqueous emulsion polymerization of styrene but, in this case, used a poly(di(ethylene glycol) ethyl ether methacrylate- co - N -(2-hydroxypropyl)methacrylamide) (P(DEGMA- co -HPMAc)) stabilizer. 134 Cooling from 70 to 23 °C, below the cloud point temperature of the thermoresponsive stabilizing block, led to restructuring of the copolymer assemblies from spheres to worm-like nanoparticles or vesicles.…”

“…While such post-polymerization modification undoubtedly adds both cost and complexity to PISA syntheses, this approach may yet be cost-effective for certain high-value biomedical applications suggested for block copolymer nanoparticles. 123,133,150−152 Nevertheless, the relatively high cost, intrinsic color, and malodorous nature of the sulfur-based RAFT CTAs may well prove to be detrimental to the development of next-generation paints and coatings or even relatively “high-value” cosmetics additives. On the other hand, it is perhaps worth emphasizing that a series of RAFT-synthesized star copolymers have already been commercialized by the Lubrizol Corporation as high-performance thickeners for automotive engine oils.…”

Smart hybrid materials by conjugation of responsive polymers to biomacromolecules

“…2d). The latter approach has been widely exploited for bioconjugation of responsive polymers [51][52][53][54][55] . Thiols also participate in several particularly efficient addition reactions with alkenes, alkynes, or α-β unsaturated carbonyl compounds (that is, Michael addition) to yield stable thioether linkages 56,57 (Fig.…”

Molecular characterisation of stromal populations derived from human embryonic stem cells: Similarities to immortalised bone marrow derived stromal stem cells

“…Significant progress has been made in designing and validating efficient differentiation protocols of hESC into a number of cell lineages including osteoblastic cells (Abdallah Basem et al, 2011, Irion et al, 2008). Additionally, new technologies allowing the expansion of pluripotent stem cells in bioreactors, without enzymatic interventions, will allow a large number of PSC to be generated prior to differentiation and transplantation (Chen et al, 2014). While the most interesting application of hESC-derived differentiated cells is as a universal off-the-shelf product for allogenic use in cell therapy, further research about the in vivo functions of hESC-derived differentiated cells need to be conducted both in animal models and in humans.…”