Cardiac differentiation

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In vitro differentiation of hPSCs to CMs mimics the stages of embryonic cardiac develop- ment. A simplified model of the CM lineage differentiation process and of the transcription factors, cell-surface markers and signaling pathways affecting this process is presented in Table 1.

Table 1. Simplified model shows the differentiation of hPSCs toward the CM lineage and the transcription factors and cell-surface markers identified in this process. Some of the best- characterized signaling pathways that are responsible for the sequential transitions in cell fate are shown. BMP4, bone morphogenetic protein 4; Dkk1, dickkopf Wingless and Integrase-1 (WNT) signaling pathway inhibitor; NKX2.5, NK2 transcription factor-related gene, locus 5;

Gata4, GATA binding protein 4; MIXL1, homeodomain protein; Mesp1, mesoderm posterior 1;

SSEA, stage-specific embryonic antigen; EpCAM, epithelial cell adhesion molecule; Isl1, Insu- lin gene enhancer 1; Tbx, T-box transcription factor; NCAM, neuronal cell adhesion molecule;

KDR, kinase insert domain receptor; PDGFR, platelet-derived growth factor receptor; SIRPA, signal regulatory protein-α; VCAM, vascular cell adhesion molecule. Table modified from (Mummery et al., 2012).

Signaling pathways Cell stage Transcription

factors Cell-surface markers

Epithelial-mesenchymal transition

BMP4, Activin A, FGF2, Wnt3a

Insulin inhibition

BMP4, Activin A, FGF2, Wnt3a

Dkk1

hPSC Oct4, Nanog, Sox2 Tra-1-60, SSEA4, EpCAM

Mesoderm

Progenitor Oct4 NCAM, SSEA1

Precardiac Mesoderm

Brachyury T, MIXL1

Cardiac

Mesoderm Mesp1 KDR, PDGFRα

Heart Speci-

fic Progenitor Nkx2.5, Gata4

(Tvx5/Isl1/Tbx20) SIRPA

Embryonic

CM Nkx2.5, Gata4 SIRPA, VCAM-1

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hPSCs can be differentiated into CMs using several techniques. CMs can be derived from hESCs and iPSCs by spontaneous differentiation in EBs, in which cells are cultured in a suspen- sion. Differentiation efficiency has usually been under 10%. (Kehat et al., 2001) The EB proto- col has been further tested with different medias such as APEL and STEMPro-34 that include growth factors such as BMP4, basic fibroblast growth factor (bFGF), activin A, vascular endo- thelial growth factor (VEGF), dickkopf homolog 1 (DKK1), stem cell factor (SCF) and WNT3a (Elliott et al., 2011; Yang et al., 2008). The differentiation process is dependent on the time of addition and on the removal of growth factors. EB formation efficiency has also been enhanced by methods that control EB size. In the spin EB method, hESCs are cultured in a suspension and aggregated by centrifugation to control the aggregate size precisely (Ng et al., 2005). Microfab- rication of microwells with defined sizes also provides a way to control the size of the EBs (Khademhosseini et al., 2006), plating cells onto micropatterned ECM (Matrigel) matrix is an- other option for controlling their size (Bauwens et al., 2008).

hPSCs can be differentiated into CMs by co-culturing stem cells with mouse endodermal- like cells (END-2) in the absence of serum and with ascorbic acid (Mummery et al., 2003;

Passier et al., 2005). The role of END-2 cells in this cardiac differentiation method remains unclear; however, the removal of insulin and secretion of prostaglandin I2 (PGI2) by END-2 cells are thought to influence the differentiation process (Xu et al., 2008). Differentiation has also been successful with END-2-conditioned medium instead of co-culturing (Graichen et al., 2008).

Another cardiac differentiation method for hPSCs is monolayer cultures. hESCs are cultured on Matrigel as a monolayer, and activin A and BMP4 induce a cardiogenic fate with a differen- tiation efficiency of over 30% when used at the beginning of the differentiation protocol (Laflamme et al., 2007). Single-cell dissociated hESCs on Matrigel have been differentiated into CMs with APEL medium containing cytokines such as BMP4, activing A and WNT3a that in- duce cardiac mesoderm (Ng et al., 2008). In the matrix sandwich differentiation method, hPSCs are differentiated into CMs by layering stem cells that were cultured as monolayers on Matrigel and Matrigel is subsequently overlaid; the matrix sandwich can also be combined with applica- tion of growth factors such as Activin A, BMP4 and bFGF (Zhang et al., 2012). Monolayer differentiation methods based on small-molecule activation and canonical Wnt signaling inhibi- tion by treatment with CHIR99021 or IWP2 have been shown to produce high-yield CMs (Lian et al., 2012). CHIR99021 is an aminopyrimidine, which activates Wnt signaling and initiates

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mesodermal differentiation by inhibiting glycogen synthase kinase 3β (GSK-3β). After initiation, Wnt signaling is inhibited by some specific Wnt inhibitors (IWP-4, KY02111, XAV939, Wnt- C59), which direct the mesodermal cells toward CMs. (Burridge et al., 2014; Lian et al., 2012;

Minami et al., 2012)

In direct reprogramming, transcription factors are used to differentiate cells directly into other differentiated cell types. Direct reprogramming of mouse post-natal cardiac or dermal fibroblasts into functional induced CMs (iCMs) in vitro has been achieved by expressing three transcription factors: GATA binding protein 4 (Gata4), myocyte-specific enhancer factor 2C (Mef2c), and T-box transcription factor 5 (Tbx5) (GMT factors) (Ieda et al., 2010). In addition, GMT factors combined with a basic helix-loop-helix transcription factor (Hand2) (GMHT fac- tors) have been able to convert fibroblasts into functional beating iCMs (Song et al., 2012) as well as a combination of miRNAs (Jayawardena et al., 2012). Upon cardiac injury, resident non-myocytes in the murine heart can be reprogrammed into CM-like cells in vivo by retroviral delivery of GMT factors (Qian et al., 2012). With human cells, neither GMT nor GHMT have been able to reprogram human fibroblasts into iCMs alone; however, combinations of these factors with other factors have seemed more promising (Fu et al., 2013; Nam et al., 2013; Wada et al., 2013). For example, GHT factors without Mef2C but with myocardin and two muscle- specific miRNAs could reprogram human fibroblasts into iCMs (Nam et al., 2013).