FLI1 and PKC co-activation promote highly efficient differentiation of human embryonic stem cells into endothelial-like cells

Abstract

Rationale-endothelial cells (ECs) play important roles in various regeneration processes and can be used in a variety of therapeutic applications, such as cardiac regeneration, gene therapy, tissue-engineered vascular grafts and prevascularized tissue transplants. ECs can be acquired from pluripotent and adult stem cells. To acquire ECs from human embryonic stem cells (hESCs) in a fast, efficient and economic manner. We established a conditional overexpression system in hESCs based on 15 transcription factors reported to be responsible for hematopoiesis lineage. Among them, only overexpression of FLI1 could induce hESCs to a hematopoietic lineage. Moreover, simultaneous overexpression of FLI1 and activation of PKC rapidly and efficiently induced differentiation of hESCs into induced endothelial cells (iECs) within 3 days, while neither FLI1 overexpression nor PKC activation alone could derive iECs from hESCs. During induction, hESCs differentiated into spindle-like cells that were consistent in appearance with ECs. Flow cytometric analysis revealed that 92.2–98.9% and 87.2–92.6% of these cells were CD31+ and CD144+, respectively. Expression of vascular-specific genes dramatically increased, while the expression of pluripotency genes gradually decreased during induction. iECs incorporated acetylated low-density lipoproteins, strongly expressed vWF and bound UEA-1. iECs also formed capillary-like structures both in vitro and in vivo. RNA-seq analysis verified that these cells closely resembled their in vivo counterparts. Our results showed that co-activation of FLI1 and PKC could induce differentiation of hESCs into iECs in a fast, efficient and economic manner.

The LINC00961 transcript and its encoded micropeptide, small regulatory polypeptide of amino acid response, regulate endothelial cell function

“…Several groups have demonstrated the ability to differentiate ECs from human embryonic stem cells (hESC). 5–7 This protocol yields ECs that are relatively immature and express genes that are somewhat distinct from those of mature ECs from various vascular beds, 8 highlighting the importance of understanding the molecular mechanisms controlling both general and specialized EC differentiation, specification, and function. These derived ECs have been extensively proven to be functional both in vitro , by the ability to form capillary-like networks on Matrigel 7 and in vivo , by their ability to improve vascular density and perfusion in a murine model of hind limb ischaemia (HLI).…”

Hematopoietic aging: Cellular, molecular, and related mechanisms

“…Embryonic stem cell (ESC) transplantation has greatly reduced the number of senile cells in bone marrow and improved the cell composition of the hematopoietic niche. [ 84 ] Therefore, reprogrammed ESCs are used to treat ischemic diseases because of their unique proliferation, survival characteristics, and hematopoietic derivation. Leung et al [ 85 ] developed a cell culture system and found that hematopoietic stem/progenitor cells (HSPCs) modified with ESC or iPSC genes produced myeloid and lymphoid hematopoietic cells with an enhanced colony-forming ability and hemoglobin expression to promote hematopoiesis in elderly individuals.…”

Endothelial Progenitor Cells Produced From Human Pluripotent Stem Cells by a Synergistic Combination of Cytokines, Small Compounds, and Serum-Free Medium

“…To overcome these hurdles, human pluripotent stem cells (hPSCs) can be differentiated into endothelium. Attempts at efficient in vitro endothelial differentiation of hPSCs have been conducted for at least 10 years (Choi et al, 2009;Park et al, 2010;Vodyanik et al, 2010;Joo et al, 2011;Li et al, 2011;Tatsumi et al, 2011;Adams et al, 2013;Prasain et al, 2014;Sahara et al, 2014;Zhang et al, 2014;Bao et al, 2015;Patsch et al, 2015;Sriram et al, 2015;Kitajima et al, 2016;Ye et al, 2016;Harding et al, 2017;Olmer et al, 2018;Suknuntha et al, 2018;Zhao et al, 2018). This strategy has the potential to ensure a consistent and unlimited source of ECFCs for in vitro studies and regenerative medicine.…”

“…Second, monolayer differentiation is a more feasible approach, with higher efficiency in a shorter time. There are multiple monolayer differentiation protocols that vary in both medium and cytokine supplement (Park et al, 2010;Joo et al, 2011;Tatsumi et al, 2011;Orlova et al, 2014;Prasain et al, 2014;Sahara et al, 2014;Bao et al, 2015;Patsch et al, 2015;Sriram et al, 2015;Kitajima et al, 2016;Harding et al, 2017;Zhao et al, 2018). Most monolayer protocols use single-cell seeding, small-clump seeding using ethylenediaminetetraacetic acid (EDTA), or larger-clump seeding using a needle.…”