Tissue Engineering And Nanotheranostics

“9.61x6.69” b2815 Tissue Engineering and Nanotheranostics

Directed Differentiation of Human Pluripotent Stem Cells 91

in early stages of differentiation prevented the formation of cardio-

myocytes while late stage inhibition did not prevent cardiomyocyte

formation, illustrating a biphasic influence and the critical role of

Wnt/β-catenin signaling for mesoderm induction.^57 Willems et al.

tested the efficacy of several small molecule inhibitors of the Wnt/β-

catenin pathway to promote cardiomyocyte differentiation post meso-

derm induction.^58 Using Activin A, BMP4, and bFGF to promote

mesoderm specification, they performed extensive small molecule

screening and identified several small molecules (IWR-1, 53AH,

IWP-3, and XAV939) whose addition was sufficient to generate

cardiomyocytes from hESC derived mesodermal cells.^58

In 2012, Lian et al. developed a fully defined protocol for highly

efficient differentiation of cardiomyocytes using chemical manipula-

tion of the Wnt/β-catenin pathway (Fig. 2).59,60 This protocol used

dense monolayer culture with mesodermal induction by activation of

the Wnt/β-catenin pathway via small molecule Gsk3 inhibitor,

CHIR99021.59,60 Inhibition of the Wnt/β-catenin pathway was

accomplished by addition of a small molecule porcupine inhibitor:

IWP-2, IWP-4 or C59.59,60 This differentiation strategy yielded 98%

cardiac troponin T positive cells, illustrating high efficiency without

an extra purification step, and its efficacy was demonstrated with both

hESCs and iPSCs.^60 This protocol provided a defined and simple

strategy to generate cardiomyocytes; however scale-up challenges and

further delineation of the molecular underpinnings of this process

would benefit future applications of this technology.

Fig. 2. Schematic of the chemically defined directed differentiation protocol devel-
oped by Lian et al. using chemical modulators of Wnt/β-catenin pathway
activity.59,60