Tunable Surface Repellency Maintains Stemness and Redox Capacity of Human Mesenchymal Stem Cells

Abstract

Human bone marrow derived mesenchymal stem cells (hMSCs) hold great promise for regenerative medicine due to their multipotent differentiation capacity and immunomodulatory capabilities. Substantial research has elucidated mechanisms by which extracellular cues regulate hMSC fate decisions, but considerably less work has addressed how material properties can be leveraged to maintain undifferentiated stem cells. Here, we show that synthetic culture substrates designed to exhibit moderate cell-repellency promote high stemness and low oxidative stress—two indicators of naïve, healthy stem cells—in commercial and patient-derived hMSCs. Furthermore, the material-mediated effect on cell behavior can be tuned by altering the molar percentage (mol %) and/or chain length of poly(ethylene glycol) (PEG), the repellant block linked to hydrophobic poly(ε-caprolactone) (PCL) in the copolymer backbone. Nano- and angstrom-scale characterization of the cell-material interface reveals that PEG interrupts the adhesive PCL domains in a chain-length-dependent manner; this prevents hMSCs from forming mature focal adhesions and subsequently promotes cell–cell adhesions that require connexin-43. This study is the first to demonstrate that intrinsic properties of synthetic materials can be tuned to regulate the stemness and redox capacity of hMSCs and provides new insight for designing highly scalable, programmable culture platforms for clinical translation.

Aging Donor-Derived Human Mesenchymal Stem Cells Exhibit Reduced Reactive Oxygen Species Loads and Increased Differentiation Potential Following Serial Expansion on a PEG-PCL Copolymer Substrate

“…Flow cytometry was performed with this kit to evaluate expression of CD14, CD20, CD34, CD45, CD73, CD90, and CD105. All donors were CD14/20/34/45 negative and CD73/90/105 positive at greater than 99% of the sample population as described in our previous report by Balikov et al [ 31 ]. All patients provided informed consent for use of their bone marrow aspirates for research purposes.…”

“…X and Y refer to the mole percent fraction of PEG and PCL, respectively. Based on prior work in our group [ 31 ], we utilized a 5% PEG–95% PCL copolymer in which the PEG block was 2000 Da in size [ 32 ]. This polymer served as a favorable hMSC culture substrate in that in vivo-like cell morphologies were adopted by the cells in addition to reinstatement of an in vivo surface marker, STRO-1, and lowered ROS load compared to hMSCs cultured on TCPS.…”

“…Non-adhesive cells obtained from the filtered bone marrow aspirate (e.g., hematopoietic stem cells) were removed by media aspiration and gentle media washes. The adhesive cells were grown to confluence before being evaluated for appropriate positive and negative MSC markers at passage 1 (refer to Balikov et al [ 31 ]). Cells were frozen following a standard stem cell culture method utilizing 70% complete media, 20% FBS (fetal bovine serum), and 10% DMSO (dimethyl sulfoxide) prior to serial passaging.…”

“…Prior work in our lab discovered that carefully tuning of PEG-PCL copolymer composition manipulated the homeostasis of hMSCs towards a more potent and potentially therapeutic phenotype [ 31 ]. Of note, this previous work also demonstrated that cells on PEG-PCL materials exhibited a significantly reduced proliferation rate compared to those grown on TCPS, although cells on both materials were proliferative (approximately 40% on TCPS versus 10–20% on PEG-PCL).…”

“…Using these studies as inspiration, we set out to demonstrate a cheap, easily-reproducible, and effective culture platform that could maintain stem cell phenotype and functional capacity over serial passaging. In previous work, we found that by using a poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) copolymer film as a culture substrate, human bone marrow-derived MSCs maintained high stemness, retained key surface protein markers lost during in vitro culture (STRO-1), contained low reactive oxygen species load, and adopted decreased proliferation rates compared to conventional TCPS plates [ 31 ]. Morphologically, the moderate repellency of specific PEG-PCL (poly(ethylene glycol)-poly(ε-caprolactone)) film compositions created an optimal interface that allowed hMSCs to bind the amorphous PCL domains while the hydrophilic PEG domains forced the cells to aggregate into spheroids that were morphologically similar to marginating hMSCs in the bone marrow.…”