Concurrent Generation of Functional Smooth Muscle and Endothelial Cells via a Vascular Progenitor

Abstract

Smooth muscle cells (SMCs) and endothelial cells (ECs) are typically derived separately, with low efficiencies, from human pluripotent stem cells (hPSCs). The concurrent generation of these cell types might lead to potential applications in regenerative medicine to model, elucidate, and eventually treat vascular diseases. Here we report a robust two-step protocol that can be used to simultaneously generate large numbers of functional SMCs and ECs from a common proliferative vascular progenitor population via a two-dimensional culture system. We show here that coculturing hPSCs with OP9 cells in media supplemented with vascular endothelial growth factor, basic fibroblast growth factor, and bone morphogenetic protein 4 yields a higher percentage of CD31 + CD34+ cells on day 8 of differentiation. Upon exposure to endothelial differentiation media and SM differentiation media, these vascular progenitors were able to differentiate and mature into functional endothelial cells and smooth muscle cells, respectively. Furthermore, we were able to expand the intermediate population more than a billionfold to generate sufficient numbers of ECs and SMCs in parallel for potential therapeutic transplantations. STEM CELLS TRANSLATIONAL MEDICINE 2014;3:91-97

Smooth Muscle Precursor Cells Derived from Human Pluripotent Stem Cells for Treatment of Stress Urinary Incontinence

“…Human pSMCs are differentiated from iPSCs and ESCs (H9) using a modified, feeder cell-free, vascular progenitor protocol as previously described [22,23]. Briefly, the SCs are cultured in chemically defined media [RPMI 1640 with 1 mM Glutamax, 1% Nonessential Amino Acids, 0.1 mM bmercaptoethanol, 1% penicillin and streptomycin (Life Science Technology, Inc.)], 1% ITS (Sigma-Aldrich) supplemented with Activin A (50 ng/mL), BMP4 (50 ng/mL, Sigma), then basic fibroblast growth factor (50 ng/mL), and vascular endothelial growth factor (40 ng/mL; Invitrogen).…”

“…Contractions are induced by treating the cells with 100 mM Carbachol (Sigma-Aldrich). Images are recorded every minute for 10 min on a Nikon BioStation IM (Nikon; www.nikon.com) [22].…”

A practical guide to induced pluripotent stem cell research using patient samples

“…Although there remain many issues to be improved, some iPSC differentiation protocols are relatively straightforward, and have been successfully used by multiple groups to obtain mesoderm, [42][43][44] endoderm 45 and ectodermal [46][47][48] lineages. These protocols utilize available materials, the procedures are uncomplicated, the methods include simple cell purification steps such as sorting, and their reproducibility and usefulness have been demonstrated by other investigators.…”

“…This can become taxing if a differentiation protocol takes months from start to finish as in the case of vascular cell differentiation with a 2-month long protocol. 42 Also, unless the differentiation protocol is well established in an investigator's own hands, a portion of the obtained cells will need to be used to assess the proper phenotype. Despite a successful differentiation protocol, investigators may run into issues if these cells are to be used in functional assays.…”

Stem Cells in Functional Bladder Engineering

“…Human smooth muscle iPSCs have been generated in vitro with different efficiencies using several differentiation protocols directing the cells towards the contractile [14] or synthetic [90] SMC phenotype [60,91]. Depending on the research question asked, an appropriate protocol should be selected, and the in vitro differentiation efficiency and in vivo safety should be tested.…”