Engineered Microenvironments for Maturation of Stem Cell Derived Cardiac Myocytes

Abstract

Through the use of stem cell-derived cardiac myocytes, tissue-engineered human myocardial constructs are poised for modeling normal and diseased physiology of the heart, as well as discovery of novel drugs and therapeutic targets in a human relevant manner. This review highlights the recent bioengineering efforts to recapitulate microenvironmental cues to further the maturation state of newly differentiated cardiac myocytes. These techniques include long-term culture, co-culture, exposure to mechanical stimuli, 3D culture, cell-matrix interactions, and electrical stimulation. Each of these methods has produced various degrees of maturation; however, a standardized measure for cardiomyocyte maturation is not yet widely accepted by the scientific community.

Keywords: Cardiac Myocytes; Differentiation; Maturation; Stem cells; Tissue Engineering..

Figure 1

Schematic of engineered microenvironments used to mature human pluripotent stem cell-derived cardiomyocytes.

Figure 2

Fluorescence image of (A) early stage PSC-CM (day ~30) and (B) late stage PSC-CM (day ~100). Stained for α-actinin and phalloidin, indicating an increase in sarcomeric organization and myofibril density with increased time in culture. Scale bar 25 μm . Reproduced with permission from John Wiley and Sons. (C) Median, maximum, and minimum sarcomere distance of human fetal cardiomyocytes (hFetal-CM), and hiPSC-CM and hESC-CM in standard BPEL media, cardiomyocyte media (CA), and maturation media (MM). ns = non-significant; *P < 0.05; **P < 0.01; ***P < 0.001 . Reproduced with permission from Elsevier. (D) Late stage PSC-CM (day ~100) were fixed, stained, and imaged using fluorescence microscopy then compared to early stage PSC-CM (day ~30) for (E) %multinucleation, (F) sarcomere length, (G) cell perimeter, (H) cell area, and (I) circularity . Reproduced with permission from John Wiley and Sons.

Figure 3

(A) Fluorescence image of neonatal rat cardiomyocytes in static conditions and after 24 h of cyclic stretch (1 Hz). Stained for Cx43 (green) and N-Cadherin (red) and counterstained with DAPI (blue). Junction proteins display an unorganized distribution in static conditions compared to localized distribution after cyclic stretch . (B) Average contraction stress in mN/mm² of hESC-CM, hiPSC-CM, and human fetal cardiomyocytes at 14 weeks, 17 weeks, and 19 weeks. ns = non-significant; *P < 0.05; **P < 0.01; ***P < 0.001 . Reproduced with permission from Elsevier. (C) Contraction stress map for a contracting day 30 hPSC-CM showing the range and localizations of contraction stresses . This is an open access article distributed under the Creative Commons Attribution License. (D) Volume percentages occupied by the nucleus, sarcomere, and mitochondria in non-stimulated and stimulated neonatal rat ventricles, and native heart. (E) Frequency of intercalated discs (IC) and gap junctions in non-stimulated and stimulated neonatal rat ventricles and native heart. * denotes statistically significant differences between the groups (P <0.05; Tukey's post hoc test with one-way ANOVA) . Copyright 2004 National Academy of Sciences.

Figure 4

(A) Electrically induced Ca²⁺ of fetal left ventricular cardiomyocytes (FLV-CMs), H1-CMs, and HES2-CMs. Tracings of basal Ca²⁺ and Ca²⁺ transients. Bar graphs of (B) basal Ca²⁺, (C) amplitude, (D) maximum upstroke velocity, and (E) maximum decay velocity of Ca²⁺ transients. *P < 0.05 versus FLV-CM . Reproduced with permission from John Wiley and Sons. (F) AP of PSC-CM in standard BPEL media, cardiomyocyte media (CA), and maturation media (MM) . Reproduced with permission from Elsevier.

Figure 5

(A) Genetic profile of early stage (~30 days) hESC-CM and hiPSC-CM, late stage (~100 days) hESC-CM and hiPSC-CM, and the adult human heart. Late stage cardiomyocytes show upregulation of MYH7 (β-myosin heavy chain), MYH6 (α-myosin heavy chain), GJA1 (connexin-43), HCN4 (hyperpolarization-activated K+ channel), and SERCA (sarcoendoplasmic reticulum ATPase). *P < 0.05 versus corresponding early-stage PSC-CMs. ∔P <0.05 versus adult human heart . Reproduced with permission from John Wiley and Sons. (B-G) Engineered human tissue (EHT) developed from neonatal rat cardiomyocytes compared to fetal, neonatal, and adult cardiomyocytes. Expression of (B) α-cardiac actin, (C) α-skeletal actin, (D) α-sarcomeric protein, (E) α-myosin heavy chain (MHC), (F) β-MHC, (G) α-/β-MHC. Note in the murine system α-MHC is predominantly present in the adult heart while β-MHC has a greater presence in the fetal heart. In the human system both α-MHC and β-MHC have increased expression in the adult heart. *P < 0.05 versus EHT day 0 (B, C, F, and G) and EHT day 3 (D) or between indicated columns (C, E, and G); ANOVA followed by Bonferroni multiple comparison test . Reproduced with permission from Wolters Kluwer Health, Inc.

Figure 6

Human pluripotent stem cell-derived cardiomyocytes (hPS-CM) at early stage and late stage display marked differences from each other and adult human CM . Reproduced with permission from John Wiley and Sons.