The march of pluripotent stem cells in cardiovascular regenerative medicine

Abstract

Cardiovascular disease (CVD) continues to be the leading cause of global morbidity and mortality. Heart failure remains a major contributor to this mortality. Despite major therapeutic advances over the past decades, a better understanding of molecular and cellular mechanisms of CVD as well as improved therapeutic strategies for the management or treatment of heart failure are increasingly needed. Loss of myocardium is a major driver of heart failure. An attractive approach that appears to provide promising results in reducing cardiac degeneration is stem cell therapy (SCT). In this review, we describe different types of stem cells, including embryonic and adult stem cells, and we provide a detailed discussion of the properties of induced pluripotent stem cells (iPSCs). We also present and critically discuss the key methods used for converting somatic cells to pluripotent cells and iPSCs to cardiomyocytes (CMs), along with their advantages and limitations. Integrating and non-integrating reprogramming methods as well as characterization of iPSCs and iPSC-derived CMs are discussed. Furthermore, we critically present various methods of differentiating iPSCs to CMs. The value of iPSC-CMs in regenerative medicine as well as myocardial disease modeling and cardiac regeneration are emphasized.

Keywords: Cardiomyocytes; Cardiovascular disease; Heart failure; Regenerative medicine; Stem cell therapy; iPSCs.

Fig. 1

Generation of embryonic stem cells. A fertilized egg is allowed to develop to the blastocyst stage. The inner cell mass dissociates from the trophoblast by laser dissection or enzymatic digestion. Isolated cells are cultured in this pluripotent state for a long period of time in the presence of growth factors. The pluripotent stem cells can be differentiated into various cell lineages, such as cardiomyocytes, neurons, or liver cells

Fig. 2

Stem cell research: key dates. Genetic reprogramming started as early as 1958 with the first somatic nuclear cell transfer, demonstrating that the nucleus was responsible for the function of a cell. The derivation of the first embryonic stem cell from mice was only achieved in the early 1980s. The major breakthrough that turned world attention toward cloning and genetic manipulation happened in 1997 with the first animal cloning of the famous sheep Dolly. Soon after, in 1998, the first human embryonic stem cell was derived. Those cells remained the only pluripotent stem cells at the disposal of researchers until 2006, when Shinya Yamanaka identified the reprogramming factors capable of inducing pluripotency in adult cells. Somatic nuclear cell transfer image is courtesy of Howard Hughes Medical Institute (HHMI). Mouse ESC image is courtesy of emouseatlas.org. Dolly the sheep, human ESC, and mouse iPSC images are courtesy of wikipedia.org. ESC embryonic stem cell, iPSC induced pluripotent stem cell

Fig. 3

The integrating reprogramming method using viral transduction. The first method developed to deliver OSKM factors involved the use of retro- and lenti-viruses. These delivery modes were chosen based on their high efficiency. However, these methods require the reverse transcription of the delivered factors and their subsequent integration into the host genome, running the risk of induced genomic instability

Fig. 4

Lox site. The 8-bp core sequence is flanked by two 13-bp inverted repeats

Fig. 5

The Cre-Lox excision system. The DNA sequences for the OSKM factors are flanked by LoxP sites and delivered virally to the target cells of interest. The Cre-recombinase is delivered in parallel in a similar manner. When expressed, the Cre-recombinase excises the sequences by recombination of the two flanking LoxP sites. This excision will nevertheless leave a residual LoxP site at the site of the original insertion

Fig. 6

The PiggyBac transposition system. The PiggyBac transposase has the ability to integrate into the genomic DNA of the host cell a DNA sequence provided that it is flanked by ITR sequences. The same PiggyBac transposase can in turn excise this inserted material, leaving the genomic DNA virally unchanged. ITR inverted terminal repeat

Fig. 7

Non-integrative methods using plasmids, sendaiviruses, or RNA delivery. Non-integrative methods (DNA- or RNA-based) have been developed to overcome the increased risk of genomic instability and gene expression modifications encountered with integrative methods. When RNA-based, the mRNA is delivered without reverse transcriptase and is directly translated into proteins. The RNA can be delivered directly or using viruses. The DNA can also be directly delivered to the target cells in a form of self-replicating plasmid that will not integrate the host cell genome. The plasmid is then transcribed to mRNA for translation to proteins. O Oct3/4, S Sox2, K Klf4, M c-Myc

Fig. 8

Direct reprogramming using transcription factors or small molecules. To avoid the use of genetic material, fibroblasts can also be reprogrammed by the excessive delivery of OSKM factors in their protein form. The method consists of the incubation of fibroblasts with a large amount of OSKM factors and their internalization by forced endocytosis. The factors then bind to DNA and directly induce the reprogramming of the target cells. The use of small molecules and chemical compounds during the reprogramming process could significantly improve the efficiency of the reprogramming process

Fig. 9

In vitro differentiation of CMs from hiPSCs. Three main methods are documented for differentiation of hiPSCs into CMs. The most documented, directed cardiac differentiation, is achieved with sequential cytokine stimulation following the culture of hiPSCs in low adherent culture plates, forcing the cells to aggregate into so-called embryoid bodies. Alternatively, the same type of sequential cytokine stimulation was also proven successful when cells are kept in 2D conditions. Finally, a “natural” differentiation into CMs was documented following co-culture of hiPSCs with END-2 endothelial cells. CM cardiomyocyte, END-2 endodermal cell line-2, hiPSC human induced pluripotent stem cell

Fig. 10

Myosin heavy chain (MHC, green) and nuclear (DAPI, blue) staining of hESC-CMs without (a) and with (b) characteristic sarcomeric striation patterns, compared with c adult rat ventricular myocyte. Scale bar is 20 μm. Figure reproduced with permission of Rao and colleagues. Phenotype and developmental potential of cardiomyocytes from induced pluripotent stem cells and human embryonic stem cells. In: Ainscough J. et al. eds. Nuclear reprogramming and stem cells. Humana Press, 2011 (159). CM cardiomyocyte, hESC human embryonic stem cell

Fig. 11

a Different action potential phenotypes recorded from hESC-CMs. Figure reproduced with permission of Rao and colleagues. Phenotype and developmental potential of cardiomyocytes from induced pluripotent stem cells and human embryonic stem cells. In: Ainscough J. et al. eds. Nuclear reprogramming and stem cells. Humana Press, 2011 (159). b Diagram of an idealized adult human ventricular action potential. The phases of the action potential are labeled (phases 0–4). The predominant cardiac ion currents at each point in the action potential are labeled (I_Na = sodium current, I_to = transient outward potassium current, I_Ca = calcium current, I_Kr = rapidly activating delayed rectifier potassium current, I_Ks = slowly activating delayed rectifier potassium current, I_K1 = inward rectifier potassium current). Figure reproduced with permission of Rao and colleagues. Phenotype and developmental potential of cardiomyocytes from induced pluripotent stem cells and human embryonic stem cells. In: Ainscough J. et al. eds. Nuclear reprogramming and stem cells. Humana Press, 2011 (159). CM cardiomyocyte, hESC human embryonic stem cell, iPSC induced pluripotent stem cells

Fig. 12

Application of hiPSC technology in cardiovascular medicine. Fibroblasts can be obtained from skin biopsies and derived into iPSCs in vitro as previously discussed. iPSC differentiation into CMs allows the study of the cellular and mechanical aspects of a variety of genetic diseases. In vitro drug screening for reversion of the particular affliction can be tested on such “diseased” CMs. When derived from healthy donors, iPSCs and CMs can be used to test the cardiac toxicity of drugs. The use of “healthy” iPSC-CMs for cellular therapy is also considered as a potential application of iPSC technology in regenerative medicine. CM cardiomyocyte, iPSC induced pluripotent stem cell