Cardiomyocyte differentiation of mouse and human embryonic stem cells

Abstract

Ischaemic heart disease is the leading cause of morbidity and mortality in the western world. Cardiac ischaemia caused by oxygen deprivation and subsequent oxygen reperfusion initiates irreversible cell damage, eventually leading to widespread cell death and loss of function. Strategies to regenerate damaged cardiac tissue by cardiomyocyte transplantation may prevent or limit post-infarction cardiac failure. We are searching for methods for inducing pluripotent stem cells to differentiate into transplantable cardiomyocytes. We have already shown that an endoderm-like cell line induced the differentiation of embryonal carcinoma cells into immature cardiomyocytes. Preliminary results show that human and mouse embryonic stem cells respond in a similar manner. This study presents initial characterization of these cardiomyocytes and the mouse myocardial infarction model in which we will test their ability to restore cardiac function.

Fig. 1

Co-cultures of stem cells with the mouse visceral endoderm-like cell line END-2. (a) P19 EC in normal monolayer culture, 3 days after initiation of co-culture with END-2 cells and after 10 days, when beating muscle (B.M.) is evident. (b) mES cell line R1 in monolayer on its normal ‘feeder’ cells (SNL), 3 days after initiation of co-culture and 2 days later, when beating muscle is evident. (c) as (b), with the exception that B.M. is evident on day 7 after aggregation. (d) GCT27X human EC cell line on mouse embryonic fibroblast (MEF) feeder cells, 3 days after initiation of co-culture and after 16 days. No beating muscle is present. (e) hES cells on MEF feeders, 3 days after initiation of END-2 co-culture and beating muscle formed after 11 days.

Fig. 2

Electrophysiological characteristics of cardiomyocytes from stem cells. Repetitive action potentials recorded from spontaneously beating areas. (a) In mouse P19 EC cell-derived cardiomyocytes. (b) In an aggregate of hES-derived cardiomyocytes. (c) Phase contrast image of the beating area in the hES culture from which the recording showed in (b) was derived. (Note the height of the protruding structure where the beating region is located, 20× objective.)

Fig. 3

Isolated cardiomyocytes: (a) exhibiting sharp edges and well-defined sarcomeres in contrast with cells cultured for 2 days (b) which had disorganized sarcomeric patterning. (a) is a phase contrast image of multiple cells after isolation and fixation. (b) represents a single cell, digitally magnified 2× compared with (a).

Fig. 4

Immunocytochemistry on adult human primary atrial cardiomyocytes and stem cell-derived cardiomyocytes. Primary atrial cardiomyocytes stained positive for sarcomeric proteins including (green) α-actinin, (red) mlc-2a (a) and tropomyosin (b). Cell DNA was stained with (blue) Hoechst to distinguish normal and apoptotic cells. Cells cultured for 2 days had a disorganized tropomyosin sarcomeric patterning and diffuse antibody staining (c). mES-derived cardiomyocytes also show sharp banding when stained with α-actinin (d) but in hES-derived cardiomyocytes α-actinin is diffuse and poorly banded (not shown). (e) shows overall extensive α-actinin staining in hES-derived cardiomyocytes at low magnification.

Fig. 5

Haemodynamic assessment of left ventricular function in mice. (a) Normal loop representing the relationship between volume and pressure changes in the mouse heart: indicated are the valvular events and stages during one cycle of contraction and relaxation. (b) Pressure volume relationship 4 weeks post-myocardial infarction: note the difference in the shape of the loop and the alterations in both contraction and relaxation.