Engineered Microenvironment for Manufacturing Human Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells

Abstract

SummaryHuman pluripotent stem cell-derived vascular smooth muscle cells (hPSC-VSMCs) are of great value for disease modeling, drug screening, cell therapies, and tissue engineering. However, producing a high quantity of hPSC-VSMCs with current cell culture technologies remains very challenging. Here, we report a scalable method for manufacturing hPSC-VSMCs in alginate hydrogel microtubes (i.e., AlgTubes), which protect cells from hydrodynamic stresses and limit cell mass to <400 μm to ensure efficient mass transport. The tubes provide cells a friendly microenvironment, leading to extremely high culture efficiency. We have shown that hPSC-VSMCs can be generated in 10 days with high viability, high purity, and high yield (∼5.0 × 108 cells/mL). Phenotype and gene expression showed that VSMCs made in AlgTubes and VSMCs made in 2D cultures were similar overall. However, AlgTube-VSMCs had higher expression of genes related to vasculature development and angiogenesis, and 2D-VSMCs had higher expression of genes related to cell death and biosynthetic processes.

Generation of a Purified iPSC-Derived Smooth Muscle-like Population for Cell Sheet Engineering

“…Modulation of these cues can readily be explored to further enhance mature/contractile marker expression in our iPSC-derived cells. For example, a recent publication described a three-dimensional culture system for culturing and expanding iPSC-derived smooth muscle-like cells, which resulted in increased expression of contractile markers relative to the two-dimensional culture system (Lin et al., 2018b). Other studies utilized pulsatile flow/stretch bioreactors on engineered vascular grafts to modulate SMC phenotype (Niklason et al., 1999, Syedain et al., 2011), which could be adopted to enhance the contractile phenotype of our iPSC-SMC cell sheets.…”

A Human Pluripotent Stem Cell-Based Screen for Smooth Muscle Cell Differentiation and Maturation Identifies Inhibitors of Intimal Hyperplasia

“…Previous SMC differentiation protocols used serum or serum replacement products that contain animal components (Bajpai et al., 2012, Cao et al., 2013, Cheung et al., 2012, Dash et al., 2015, Karamariti et al., 2013, Lin et al., 2019, Patsch et al., 2015, Wang et al., 2014, Wanjare et al., 2013, Yang et al., 2016, Zhang et al., 2011). Here, we performed a stepwise optimization to further improve SMC differentiation efficiency in completely defined, xeno-free medium (Figures S3 and 3A).…”

“…Transforming growth factor β1 (TGF-β1) and platelet-derived growth factor BB (PDGF-BB) are widely used for the differentiation of SMCs from human pluripotent stem cells (PSCs) (Bajpai et al., 2012, Cao et al., 2013, Cheung et al., 2012, Dash et al., 2015, Karamariti et al., 2013, Lin et al., 2019, Patsch et al., 2015, Wang et al., 2014, Wanjare et al., 2013, Yang et al., 2016, Zhang et al., 2011). However, upregulation of PDGF and TGF-β signaling has been shown to promote the switching of SMC phenotypes from contractile to synthetic, contributing to intimal hyperplasia (Muto et al., 2007, Nabel et al., 1993, Newby and Zaltsman, 2000, Raines, 2004, Suwanabol et al., 2011, Wolf et al., 1994).…”

Aortic “Disease-in-a-Dish”: Mechanistic Insights and Drug Development Using iPSC-Based Disease Modeling

“…The collagen gel contraction assay is a typical one to assess functionality of VSMCs by measuring the reduction in gel area and has been applied to both primary and stem cell-derived VSMCs (Oishi et al, 2000;Sinha et al, 2006;van den Akker et al, 2012;Lee et al, 2019). Newer models employ the use of bioreactors for scale-up, and 3D hydrogel disks are prepared by mixing multiple cell types like VSMCs and ECs with collagen, and contraction assayed over 30 min to 1 h (Lin et al, 2019). Vascular rings, a 3D structure comprising VSMCs, can be created relatively quickly and changes in circumference or force generation can be assayed in response to contractile agonists (Bi et al, 2005;Dash et al, 2016).…”

“…This automated system was utilized by another group for cardiomyocyte differentiation, and found success with producing a maximum of 3 × 10 9 cardiomyocytes per batch (Denning et al, 2016). A recent method describing high-yield derivation of VSMCs based on an existing protocol (Patsch et al, 2015) was described, where VSMCs were derived in alginate hydrogel tubes (Lin et al, 2019). This method yielded 5 × 10 8 cells/ml in 10 days; as a result, bioengineering methods could rely on such advances for producing high numbers of cells.…”