Dynamic Click Hydrogels for Xeno‐Free Culture of Induced Pluripotent Stem Cells

Abstract

3D cell-laden hydrogels are increasingly used for in vitro and ex vivo cell expansion, controlled stem cell differentiation, and regenerative medicine. [1] Hydrogels can be fabricated by derivatives of purely synthetic polymers (e.g., poly(ethylene glycol) or PEG) and/or naturally derived biomacromolecules (e.g., gelatin, hyaluronic acid, or heparin). [2] Tumor basement membrane derived Matrigel is routinely used as a convenient matrix for 3D cell culture owing to its inherent cell affinity provided by laminin, collagen IV, and residual growth factors. However, Matrigel contains ill-defined and batch-to batch variability of extracellular matrix (ECM) components that may present challenges for studying the contributions of individual matrix cues on cell fate processes. To this end, synthetic PEG-based hydrogels afford a blank slate in which a bottom-up approach can be used to impart precise biochemical and biophysical properties in the cell-laden network. Advances in material chemistry have also enabled the conjugation of a variety of ECM motifs to the otherwise inert PEG-based network, as well as for spatiotemporal manipulation of matrix properties to facilitate the study of cell fate processes within a highly defined and controllable microenvironment. Chemically defined PEG-based hydrogels are particularly advantageous for studying iPSC behaviors in 3D. [3] For example, PEG hydrogels formed by Michael-type additions have proven effective in cell encapsulation owing to the reaction specificity between vinyl sulfone/maleimide (VS/MAL) and thiol (SH) moieties. Hubbell and colleagues utilized VS-SH reactions to determine the role of integrin activation on embryonic stem cell (ESC) self-renewal. [4] Lim et al. later use PEG-based VS-SH hydrogels for maintaining stemness of three human ESC lines. [5] Nonetheless, the reaction rates of Michael-type additions may be difficult to control and vary greatly from seconds to hours, depending on the type of Michael donor-acceptor pairs (e.g., VS-SH ≈ 30-120 min and MAL-SH ≈tens of seconds). [4a] Alternatively, enzymatic crosslinking provides excellent cytocompatibility and tunable gelation kinetics for in situ cell encapsulation. For instance, Xeno-free, chemically defined poly(ethylene glycol) (PEG)-based hydrogels are being increasingly used for in vitro culture and differentiation of human induced pluripotent stem cells (hiPSCs). These synthetic matrices provide tunable gelation and adaptable material properties crucial for guiding stem cell fate. Here, sequential norbornene-click chemistries are integrated to form synthetic, dynamically tunable PEG-peptide hydrogels for hiPSCs culture and differentiation. Specifically, hiPSCs are photoencapsulated in thiol-norbornene hydrogels crosslinked by multiarm PEG-norbornene (PEG-NB) and proteaselabile crosslinkers. These matrices are used to evaluate hiPSC growth under the influence of extracellular matrix properties. Tetrazine-norbornene (Tz-NB) click reaction is then employed to dynamically stiffen the cell-laden hydrogels. Fast ...

Structurally Dynamic Hydrogels for Biomedical Applications: Pursuing a Fine Balance between Macroscopic Stability and Microscopic Dynamics

“…By encapsulating iPSCs in thiol-norbornene cross-linked PEG hydrogels, the authors demonstrated that dynamic hydrogels resulted in remarkably higher cell survival than static hydrogels did. 367 You et al developed a photodegradable "heparin photogel" to differentiate mouse ESCs into definitive endoderm. To fabricate such hydrogels, thiolated heparin molecules were cross-linked by PEGs with bifunctional acryl-terminated photolabile o-nitrobenzyl moieties.…”

“…Pluripotent stem cells include both embryonic stem cells (ESCs) derived from the inner cell mass of blastocyst-stage embryos and induced pluripotent stem cells (iPSCs) reprogrammed from mature, terminally differentiated cells. , These cells are traditionally cultured on feeder layers of mouse embryonic fibroblasts or reconstituted basement membrane derived from mouse sarcoma (e.g., Matrigel), − although a growing number of xeno-free, chemically defined dynamic hydrogels have been developed for the in vitro culture of these cells in recent years. − …”

Hydrogel Models with Stiffness Gradients for Interrogating Pancreatic Cancer Cell Fate

“…Taking advantage of the dual reactivity of the norbornene group towards thiol and tetrazine moieties, our group has recently reported orthogonal PEG-peptide thiol-norbornene hydrogels for culture and differentiation of human-induced pluripotent stem cells (hiPSCs). In particular, hiPSC-laden hydrogels were dynamically stiffened using tetrazine-modified macromers [ 81 ].…”

Facile Synthesis of Rapidly Degrading PEG-Based Thiol-Norbornene Hydrogels

“…PEG-based thiol-norbornene hydrogels are known for their high cytocompatibility for in situ cell encapsulation. ,,, The PEGNB CA hydrogels also showed excellent cytocompatibility, as exemplified by the encapsulation of human induced pluripotent stem cells (hiPSC), dental pulp stem cells (DPSC), and PANC-1 human pancreatic cancer cells (Figure ). All hydrogels were cross-linked by matrix metalloproteinase (MMP)-sensitive peptide linker (i.e., KCGPQG*IWGQCK, *cleavage site) to permit cell-mediated matrix remodeling and immobilized with 1 mM RGDS peptide to support cell survival.…”