Long-term self-renewing human epicardial cells generated from pluripotent stem cells under defined xeno-free conditions

Abstract

The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair, underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here, we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines, including a WT1-2A-eGFP knock-in reporter line, under chemically-defined, xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells, resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo, as determined by morphological and functional assays, including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies.

Gene Editing to Generate Versatile Human Pluripotent Stem Cell Reporter Lines for Analysis of Differentiation and Lineage Tracing

“…(A): Schematic diagram of the engineering strategy to generate lineage‐specific Cre reporter hPSC lines, and representative brightfield and fluorescent images (B) of the parental Cre reporter line before and after TAT‐Cre recombinase treatment. (C): RNA sequencing analysis of representative transcription factors expressed constitutively or enriched in human pluripotent stem cells (hPSCs), hPSC‐derived mesoendoderm (MEN), endoderm (END), mesoderm (MES), and ectoderm (ECT). (D): eGFP was knocked into the endogenous Oct4 and T loci using CRISPR/Cas9, and representative brightfield and fluorescent images of GFP signal driven by endogenous Oct4 and T (brachyury) promoters are shown.…”

Directed differentiation and long-term maintenance of epicardial cells derived from human pluripotent stem cells under fully defined conditions

“…To address these issues, we used a WT1-2A-eGFP knock-in hPSC line to show that temporal modulation of canonical Wnt signaling via small molecules is sufficient for epicardial induction from hPSCs under chemically-defined, xeno-free conditions, and that TGF-β inhibitor treatment permits long-term expansion of hPSC-derived epicardial cells 22 . After 48 days of expansion, the hPSC-derived epicardial cells exhibited uniform expression of epicardial markers and retained the capacity to differentiate to fibroblasts and smooth muscle cells.…”

“…This protocol uses a completely defined, growth factor- and xeno-free system and applies temporal modulation of Wnt/β-catenin signaling via small molecules. This protocol is based on our earlier reports of cardiac progenitor and epicardial differentiation 5,22 and is composed of four major stages: (steps 1–8) induction of cardiac progenitors from hPSCs by temporal modulation of canonical Wnt signaling under defined, albumin-free conditions, (steps 9–14) directed differentiation of cardiac progenitors to pro-epicardial then epicardial cells by Gsk3 inhibitor treatment, (steps 15 A-C) long-term maintenance of hPSC-derived epicardial cells under chemically defined conditions in the presence of a TGFβ inhibitor, and (steps 15 D) in vitro differentiation of epicardial cells to fibroblasts and SMCs. This protocol will enable efficient production of human epicardial cells for development and disease research, drug screening and testing, and advancing cardiac cellular therapies.…”

“…On day 6, hPSC-derived cardiac progenitor cells are singularized and seeded onto gelatin- or Synthemax-coated plates in albumin-containing LaSR 13 basal medium (Advanced DMEM/F12 with 2.5 mM GlutaMAX and 100μg/mL ascorbic acid) or albumin-free RPMI/Vc/Ins medium (100 μg/mL ascorbic acid and1 μg/mL insulin) as needed. When cultured in these media without any treatment, the cardiac progenitor cells will spontaneously differentiate into functional contracting cardiomyocytes 22 . In order to direct these Nkx2.5+Isl1+ cardiac progenitor cells to an epicardial cell fate, activation of canonical Wnt signaling by Gsk3 inhibitors, such as CHIR99021, is performed for 48 hr from day 7 to day 9.…”

Stem cell culture: Simply derived epicardial cells

“…So far, little is known of correlations between the iPSC differentiation process in vitro and the developmental biology of various tissue and cell types in vivo . In an effort that begins to address this gap in knowledge, Sean Palecek and colleagues report in Nature Biomedical Engineering a chemically defined, xeno-free method for the generation and expansion of epicardial cells derived from human PSCs (hPSCs) 2 .…”