Generating high-purity cardiac and endothelial derivatives from patterned mesoderm using human pluripotent stem cells

Abstract

Human pluripotent stem cells (hpscs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and requires experience in hPSC culture and differentiation techniques. Building on lessons taken from early development, this monolayer-directed differentiation protocol uses different concentrations of activin A and bone morphogenetic protein 4 (BMP4) to polarize cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm. this differentiation platform provides a basis for generating distinct cardiovascular progenitor populations that enable the derivation of cardiomyocytes and functionally distinct endothelial cell (EC) subtypes from cardiogenic versus hemogenic mesoderm with high efficiency without cell sorting. ecs derived from cardiogenic and hemogenic mesoderm can be matured into >90% CD31+/VE-cadherin+ definitive ECs. To test the functionality of ECs at different stages of differentiation, we provide methods for assaying the blood-forming potential and de novo lumen-forming activity of ECs. To our knowledge, this is the first protocol that provides a common platform for directed differentiation of cardiomyocytes and endothelial subtypes from hPSCs. This protocol yields endothelial differentiation efficiencies exceeding those of previously published protocols. Derivation of these cell types is a critical step toward understanding the basis of disease and generating cells with therapeutic potential.

Chromatin and Transcriptional Analysis of Mesoderm Progenitor Cells Identifies HOPX as a Regulator of Primitive Hematopoiesis

“…We recently generated a differentiation method involving modulation of activin A and BMP4 stimulation to direct anterior vs posterior-like mesoderm that gives rise to cell populations reflecting cardiogenic fates vs. blood forming lineages, respectively, from hPSCs (Palpant et al, 2017; Palpant et al, 2015b). Using well established cues from developmental biology we can generate cardiogenic mesoderm, an anterior primitive streak lineage that is directed primarily through stimulation of activin A and gives rise to cardiomyocytes with high efficiency.…”

Single-cell RNA-seq of human induced pluripotent stem cells reveals cellular heterogeneity and cell state transitions between subpopulations

“…In recent decades, the isolation of pluripotent stem cells, first in mouse followed by human 50 (Evans and Kaufman, 1981;Thomson et al, 1998), and the more recent discovery of deriving pluripotent stem cells from somatic cell types (iPSCs) (Takahashi and Yamanaka, 2006), is a means to study lineage-specific mechanisms underlying development and disease to broaden our capacity for biological therapeutics (Palpant et al, 2017). Pluripotent stem cells are capable of unlimited selfrenewal and can give rise to specialized cell types based on stepwise changes in the transcriptional 55 networks that orchestrate complex fate choices from pluripotency into differentiated states.…”

Regulatory T-cells are required for neonatal heart regeneration

“…Our results showed that hCD2 + Treg, supernatant of Treg or AREG significantly increased %Ki67 + cTnT + cells among total cTnT + cells (Fig 6B). To recapitulate the same effect on human cardiomyocytes, we differentiated beating cardiomyocytes from human embryonic stem cells as previously described 31 (hESC-CM, Fig 6C). We cocultured relatively mature, less proliferative hESC-CM (day 76) with supernatant of Treg cultures or recombinant human AREG protein for 3 days.…”

“…For human cardiomyocytes, cardiomyocytes were derived from hESCs as previously described 31 . Cardiomyocytes were generated with a sequential administration of growth factors (Fig 6C).…”