In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Abstract

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) - are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

Figure 1:. Schematic representation of in vitro specification of heart field-specific cardiac progenitor cells.

mESCs form spheroids within 48 h. Then exposure to Activin A and BMP4 for 40 h will lead to mesodermal induction. Cardiac progenitor cells develop approximately 36 h later. Progenitors of the second or first heart field can be sorted using fluorescent activated cell sorting. Second heart field cells are marked by Tbx1-RFP expression vs first heart field that are marked by Hcn4-GFP. Alternatively, Isl1-RFP marks CPCs and using live immunostaining against Cxcr4 one can sort Isl1+, Cxcr4+ vs Isl1+, Cxcr4- CPCs that represent second vs first heart field cells respectively.

Figure 2:. Representative image of cardiac spheroids after CPC specification.

RFP marks Tbx1+ and GFP marks Hcn4+ CPCs. The two cell populations are formed in close proximity in a complimentary pattern. Scale bars = 50 μm.

Figure 3:. Flow cytometric analysis of cardiac spheroids after exposure to different concentrations of Activin A and BMP4.

Adjusting the concentrations of the two morphogens leads to different percentages of Tbx1+ and Hcn4+ CPCs. The two populations were mainly affected by adjusting BMP4 concentration.

Figure 4:. Flow cytometric analysis of cardiac progenitor cells expressing Isl1 and are immunostained for Cxcr4.

Cardiac progenitors were first gated based on their Isl1 expression and then Isl1+, Cxcr4+ vs Isl1+, Cxcr4- cells were sorted.

Figure 5:. Flow cytometric analysis of cells derived from heart field-specific CPCs stained for cardiac Troponin T.

(A) Consistent with the higher myogenic potential of FHF cells, a high percentage of Hcn4-GFP+ cells differentiate to myocytes. (B) Analysis of all mESC-derived cardiomyocytes, where the vast majority are Hcn4-GFP+. (C) Cxcr4- CPCs differentiate to a higher percentage of cardiomyocytes.

Figure 6:. Representative cytometric analyses of failed/low efficiency in vitro differentiations.

(A) Flow cytometry analysis after 132 h of differentiation showing no formation of Hcn4-GFP cells and a very low percentage of Tbx1-RFP+ cells. (B) Low differentiation efficiency of mESCs expressing very low levels of Isl1.