c-MYC-Independent Nuclear Reprogramming Favors Cardiogenic Potential of Induced Pluripotent Stem Cells

Abstract

Induced pluripotent stem cell (iPS) technology has launched a new platform in regenerative medicine aimed at deriving unlimited replacement tissue from autologous sources through somatic cell reprogramming using stemness factor sets. In this way, authentic cardiomyocytes have been obtained from iPS and recently demonstrated in proof-of-principle studies to repair infarcted heart. Optimizing the cardiogenic potential of iPS progeny would ensure a maximized yield of bioengineered cardiac tissue. Here, we reprogrammed fibroblasts in the presence or absence of c-MYC to determine if the acquired cardiogenicity is sensitive to the method of nuclear reprogramming. Using lentiviral constructs that expressed stemness factors SOX2, OCT4, and KLF4 with or without c-MYC, iPS clones generated through fibroblast reprogramming demonstrated indistinguishable characteristics for 5 days of differentiation with similar cell morphology, growth rates, and chimeric embryo integration. However, 4-factor c-MYC dependent nuclear reprogramming produced iPS progeny that consistently prolonged the expression of pluripotent Oct-4 and Fgf4 genes and repressed cardiac differentiation. In contrast, 3-factor c-MYC-less iPS clones efficiently up-regulated pre-cardiac (CXCR4, Flk-1, and Mesp1/2) and cardiac (Nkx2.5, Mef2c, and Myocardin) gene expression patterns. In fact, 3-factor iPS progeny demonstrated early and robust cardiogenesis during in vitro differentiation with consistent beating activity, sarcomere maturation, and rhythmical intracellular calcium dynamics. Thus, nuclear reprogramming independent of c-MYC enhances production of pluripotent stem cells with innate cardiogenic potential.

ΔNp63 regulates select routes of reprogramming via multiple mechanisms

“…1 Mouse and human iPSCs are typically produced via forced expression of various combinations of transcription factors, most often Oct4, Sox2, Klf4 and c-Myc (OSKM) or without c-Myc (OSK). [2][3][4][5] Moreover, reprogramming by only two factors can be achieved, especially on less differentiated cells. [6][7][8][9][10] For mechanistic studies of reprogramming, mouse embryo fibroblasts (MEFs) are the most commonly used source of somatic cells.…”

Mesenchymal Stem Cells: Possibilities of New Treatment Options

“…Moreover, cell fate specifi cation and maturation can be selectively altered via manipulation of endogenous developmental signaling pathways (Rathjen and Rathjen 2003 ;Meyer et al 2009 ) . The ability to differentiate iPS cells in vitro to specifi c lineages effi ciently and reproducibly has been achieved using those described protocols with certain modifi cations (Dimos et al 2008 ;Narazaki et al 2008 ;Tateishi et al 2008 ;Wernig et al 2008 ;Ebert et al 2009 ;Hu and Zhang 2009 ;Jin et al 2009 ;Karumbayaram et al 2009 ;Pfannkuche et al 2009 ;Senju et al 2009 ;Tanaka et al 2009 ;Taura et al 2009b ;Zhang et al 2009b ;Carvajal-Vergara et al 2010 ;Comyn et al 2010 ;Dick et al 2010 ;Gamm and Meyer 2010 ;Huang et al 2010 ;Kaichi et al 2010 ;Lamba et al 2010 ;Lee et al 2010 ;Martinez-Fernandez et al 2010 ;Parameswaran et al 2010 ;Swistowski et al 2010 ;Teramura et al 2010 ;Zhou et al 2010 ) .…”

“…Examples are auditory retinal cells (Jin et al 2009 ;Comyn et al 2010 ;Lamba et al 2010 ;Parameswaran et al 2010 ) , cardiomyocytes (Narazaki et al 2008 ;Pfannkuche et al 2009 ;Tanaka et al 2009 ;Zhang et al 2009b ;CarvajalVergara et al 2010 ;Kaichi et al 2010 ;Martinez-Fernandez et al 2010 ) , insulinsecreting islet-like clusters (Tateishi et al 2008 ) , motor neurons (Dimos et al 2008 ;Ebert et al 2009 ;Hu and Zhang 2009 ;Karumbayaram et al 2009 ) , dopaminergic neurons (Wernig et al 2008 ;Cai et al 2010 ;Swistowski et al 2010 ) , auditory spinal ganglion neurons (Nishimura et al 2009 ) , smooth muscle cells (Taura et al 2009b ;Xie et al 2009 ) , vascular endothelial cells (Taura et al 2009b ) , dendritic cells and macrophages (Senju et al 2009(Senju et al , 2010 , adipocytes (Tashiro et al 2009 ;Taura et al 2009a ) , osteoblasts (Tashiro et al 2009 ) , hematopoietic cells (Hanna et al 2007 ;Eminli et al 2009 ;Kaufman 2009 ;Lu et al 2009 ;Okabe et al 2009 ;Raya et al 2009 ;Kaneko et al 2010 ) , and endothelial progenitor cells (Xu et al 2009 ;Abaci et al 2010 ;…”

Generation of Patient Specific Stem Cells: A Human Model System

“…A comparison between the cardiac differentiation potential of hESCs and iPSCs concluded that the difference between the two cell sources were no greater than the known differences between different hESC lines and that iPSCs thus should be a viable alternative as an autologous cell source (Zhang et al, 2009). Furthermore, a recent study demonstrated that reprogramming excluding c-MYC yielded iPSCs which efficiently up-regulated a cardiac gene expression pattern and showed spontaneous beating in contrast to iPSCs reprogrammed with four factors including c-MYC (Martinez-Fernandez et al, 2010). On the transcriptional level, beating clusters from both iPSCs and hESCs were found to be similarly enriched for cardiac genes, although a small difference in their global gene expression profile was noted .…”