Cozzarelli Prize Winner: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

Department of Chemical and Biological Engineering, University of Wisconsin, Madison, WI 53706, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/2012; 109(27):E1848-57. DOI: 10.1073/pnas.1200250109
Source: PubMed


Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. Here we show that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs under defined, growth factor-free conditions. shRNA knockdown of β-catenin during the initial stage of hPSC differentiation fully blocked cardiomyocyte specification, whereas glycogen synthase kinase 3 inhibition at this point enhanced cardiomyocyte generation. Furthermore, sequential treatment of hPSCs with glycogen synthase kinase 3 inhibitors followed by inducible expression of β-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually (up to 98%) pure functional human cardiomyocytes from multiple hPSC lines. The robust ability to generate functional cardiomyocytes under defined, growth factor-free conditions solely by genetic or chemically mediated manipulation of a single developmental pathway should facilitate scalable production of cardiac cells suitable for research and regenerative applications.

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Available from: Kexian Zhu, Oct 30, 2014
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    • "In the case of mesoderm, several protocols have been developed to derive mature tissues stemming from this lineage, including muscle, blood, and urogenital cells (Kee and Reijo Pera, 2008; Ng et al., 2008; Lian et al., 2012; Taguchi et al., 2014). While these studies have demonstrated the potential of hPSC-derived mesodermal tissues for cell replacement therapies, these protocols result in the generation of heterogeneous cell populations, some with tumor forming potential, which limits their clinical utility. "
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    ABSTRACT: The field of tissue engineering entered a new era with the development of human pluripotent stem cells (hPSCs), which are capable of unlimited expansion whilst retaining the potential to differentiate into all mature cell populations. However, these cells harbor significant risks, including tumor formation upon transplantation. One way to mitigate this risk is to develop expandable progenitor cell populations with restricted differentiation potential. Here, we used a cellular microarray technology to identify a defined and optimized culture condition that supports the derivation and propagation of a cell population with mesodermal properties. This cell population, referred to as intermediate mesodermal progenitor (IMP) cells, is capable of unlimited expansion, lacks tumor formation potential, and, upon appropriate stimulation, readily acquires properties of a sub-population of kidney cells. Interestingly, IMP cells fail to differentiate into other mesodermally-derived tissues, including blood and heart, suggesting that these cells are restricted to an intermediate mesodermal fate.
    Full-text · Article · Nov 2015 · eLife Sciences
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    • "Bioactive small molecules have been applied to the field of hESC research with success. Many such studies have applied small molecules as modulators of lineage-specific differentiations (Borowiak et al., 2009; Chen et al., 2009, 2012; Gonzalez et al., 2011a; Lian et al., 2012; Mahmood et al., 2010). Other studies have exploited small molecules as chemical probes to uncover novel molecular mechanisms underlying hESC pluripotency or differentiation (Chen et al., 2006; Xu et al., 2010; Zhu et al., 2009). "
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    ABSTRACT: Chemical biology methods such as high-throughput screening (HTS) and affinity-based target identification can be used to probe biological systems on a biomacromolecule level, providing valuable insights into the molecular mechanisms of those systems. Here, by establishing a human embryonal carcinoma cell-based HTS platform, we screened 171,077 small molecules for regulators of pluripotency and identified a small molecule, Displurigen, that potently disrupts hESC pluripotency by targeting heat shock 70-kDa protein 8 (HSPA8), the constitutively expressed member of the 70-kDa heat shock protein family, as elucidated using affinity-based target identification techniques and confirmed by loss-of-function and gain-of-function assays. We demonstrated that HSPA8 maintains pluripotency by binding to the master pluripotency regulator OCT4 and facilitating its DNA-binding activity.
    Full-text · Article · Nov 2015 · Stem Cell Reports
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    • "By including the Wnt inhibitor dickkopf-related protein 1 (DKK-1)da Wnt inhibitor that binds LRP6[23,24]dduring the stage of cardiac mesoderm induction and specification, the CM yield was increased to >50%[25]. Furthermore, Lian and colleagues robustly generated directed differentiation cultures with yields of up to 98% CMs by sequential treatment with a small molecule Wnt agonist, CHIR99021, followed by a small molecule Wnt antagonist, Wnt inhibitor of Wnt production 4 (IWP4)[26,27]. More recently, differentiation strategies further manipulating cellular signaling haveshown control over individual cardiac cell types such as atrial, ventricular and nodal cells[28e30]. "
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    ABSTRACT: Wingless-related integration site (Wnt) signaling has proven to be a fundamental mechanism in cardiovascular development as well as disease. Understanding its particular role in heart formation has helped to develop pluripotent stem cell differentiation protocols that produce relatively pure cardiomyocyte populations. The resultant cardiomyocytes have been used to generate heart tissue for pharmaceutical testing, and to study physiological and disease states. Such protocols in combination with induced pluripotent stem cell technology have yielded patient-derived cardiomyocytes that exhibit some of the hallmarks of cardiovascular disease and are therefore being used to model disease states. While FDA approval of new treatments typically requires animal experiments, the burgeoning field of tissue engineering could act as a replacement. This would necessitate the generation of reproducible three-dimensional cardiac tissues in a well-controlled environment, which exhibit native heart properties, such as cellular density, composition, extracellular matrix composition, and structure-function. Such tissues could also enable the further study of Wnt signaling. Furthermore, as Wnt signaling has been found to have a mechanistic role in cardiac pathophysiology, e.g. heart attack, hypertrophy, atherosclerosis, and aortic stenosis, its strategic manipulation could provide a means of generating reproducible and specific, physiological and pathological cardiac models.
    Full-text · Article · Nov 2015 · Biochemical and Biophysical Research Communications
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