Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA

Abstract

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.

Figure 1

Expression of VEGF receptors in the human Isl1⁺ progenitors. (A) Frozen sections from a human fetal heart at gestation week 9 were stained for DAPI (scale bar = 500 μm), Isl1, endothelial cell-specific markers: CD144, vWF, VEGF-A receptor 1 (Flt1) or 2 (KDR), or neurofilament (scale bars = 50 μm and 10 μm). Isl1⁺ cells are indicated by white asterisks (scale bar = 100 μm) and colocalization of Isl1 and EC markers are indicated by white arrows (scale bar = 10 μM). (B) Schematic diagram showing the Isl1 lineage-tracing construct in human ESCs. (C) Differentiation protocol used to derive the Isl1⁺ progenitors from human ESCs and to examine, which angiocrine factor (X) is responsible for endothelial differentiation of the progenitors. (D) FACS analyses showing expression of VEGFR1 or VEGFR2 in day-4 or day-7 human Isl1⁺ progenitors.

Figure 2

VEGF-A is the most abundantly expressed angiocrine factor in human fetal hearts and endothelial differentiation of the human Isl1⁺ progenitors is KDR dependent. (A) qPCR profiling showing expression levels of angiocrine factors derived from CD144⁺CD31⁺ ECs purified from outflow tract of the 10-week human fetal hearts, expression levels were compared with those of the noncardiac, human cord blood-derived outgrowth endothelial cells (OECs, value on y axis = 1). (B) FACS analyses to determine the endothelial differentiation efficiency by factor X following treatment from day 7-12. (C) FACS analyses on the proliferation rate of the human Isl1-cre eGFP⁺ cells following VEGF-A treatment from day 4-7, and the endothelial differentiation efficiency without VEGF-A, with VEGF-A or with VEGF-A+KDR inhibitor (SU5614) from day 7-14. (D) Clonal assay was performed by culturing the day-7 human Isl1-cre eGFP⁺ cells on MEFs with VEGF-A (n = 33) for 3 days. Result was normalized to cells without VEGF-A treatment. (E) qPCR data showing endothelial differentiation of the day-7 human Isl1-cre eGFP⁺ cells in the presence of VEGF-A or VEGF-A+SU5614 for 7 days. Result was normalized to cells with VEGF-A alone (value on y axis =1).

Figure 3

VEGF-A-treated human Isl1⁺ progenitors express EC markers and secrete angiocrine factors in a similar pattern to the human outflow tract-derived ECs. (A) FACS analyses on the purified CD144⁺CD31⁺ ECs from the human Isl1-cre eGFP⁺ cells (Isl1-ECs) in the presence of VEGF-A from day 7-21. (B, C) Correlation of angiocrine gene expression between qPCR data obtained from the day-7 purified human Isl1-cre eGFP⁺ cells (Isl1 progenitors) or Isl1-ECs in the presence of VEGF-A from day 7-21 and (B) the purified CD144⁺CD31⁺ ECs from the outflow tract of human fetal hearts at 10 weeks of gestation (OFT-ECs) or (C) the noncardiac, human cord blood-derived outgrowth endothelial cells (OECs) (n = 5).

Figure 4

Endothelial differentiation of the human Isl1⁺ progenitors can be achieved by repeated transfections of the VEGF-A modRNA. (A) FACS result showing expression of mCherry in the day-7 purified human Isl1-cre eGFP⁺ cells 24 h post transfection of the mCherry modRNA (1 μg/10⁶ cells). (B) Quantification of mCherry expression by FACS showing percentage of mCherry⁺ Isl1-cre eGFP⁺ cells following 0-150 h post transfection. (C) ELISA analyses using VEGF-A-containing supernatant (refreshed 6 h before collection) cultured with the day-7 purified human Isl1-cre eGFP⁺ cells following 0-72 h post transfection of different concentrations of the VEGF-A modRNA. (D) Trypan blue staining showing the survival percentage of the day-7 purified human Isl1-cre eGFP⁺ cells following 24 h post transfection of different concentrations of the VEGF-A modRNA. (E) ELISA analyses showing the rate of VEGF-A secreted by the day-7 purified human Isl1-cre eGFP⁺ cells (supernatant refreshed 6 h before collection) following two rounds of daily transfections of 1 μg/10⁶ cells with VEGF-A modRNA. (F) FACS analyses showing the efficiency of CD144⁺CD31⁺ EC differentiation by one transfection or repeated transfections (following medium change) of the VEGF-A modRNA compared to the use of VEGF-A protein 3 or 7 days post treatment.

Figure 5

VEGF-A drives differentiation of the human Isl1⁺ progenitors toward an EC lineage, but away from the SMC lineage in vivo. (A) Schematic diagram of the experimental design. (B-U) Frozen sections from matrigel plugs incubated with the day-7 purified human Isl1-cre eGFP⁺ cells in the presence of (B-K) vehicle or (L-U) VEGF-A modRNA were stained for eGFP with smooth muscle- or endothelial cell-specific markers (scale bar in C = 100 μm, scale bar in M = 50 μm, scale bars in all the remaining panels = 25 μm). (V) Quantification analyzed by ImageJ showing number of double positive cells (eGFP⁺SMMHC⁺, eGFP⁺Vimentin⁺ or eGFP⁺CD31⁺) per unit area of the matrigel plug following treatment with vehicle or VEGF-A modRNA.

Figure 6

VEGF-A drives endothelial differentiation and promotes survival via increased proliferation and reduced apoptosis of the human Isl1⁺ progenitors in vivo. (A) FACS analyses to determine the percentage of CD144⁺CD31⁺ ECs differentiated from the human Isl1-cre eGFP⁺ cells isolated from matrigel plugs following incubation in the presence of vehicle or VEGF-A modRNA 2 weeks post s.c. implantation. (B, C) Cell counting to determine the percentage of proliferation (eGFP⁺Ki67⁺ by immunostaining), apoptosis (eGFP⁺Tunel⁺ by immunostaining) or survival (total eGFP⁺ cells by FACS) of the human Isl1-cre eGFP⁺ cells in the vehicle- or VEGF-A modRNA-treated matrigel plugs two weeks post s.c. implantation. (D) Model for the proposed cell fate switch of the human Isl1⁺ progenitors following VEGF-A treatment.