Original Article
TISSUE ENGINEERING: Part A Volume 00, Number 00, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2014.0015
Incorporation of Gold-Coated Microspheres into Embryoid Body of Human Embryonic Stem Cells for Cardiomyogenic Differentiation Tae-Jin Lee, PhD,1 Seokyung Kang, MS,2 Gun-Jae Jeong, BS,2 Jeong-Kee Yoon, BS,2 Suk Ho Bhang, PhD,3 Jaesur Oh, BS,2 and Byung-Soo Kim, PhD1,2,4
Human embryonic stem cells (hESCs) are a useful cell source for cardiac regeneration by stem cell therapy. In this study, we show that incorporation of gold-coated microspheres into hESC-derived embryoid bodies (EBs) enhances the cardiomyogenic differentiation process of pluripotent embryonic stem cells. A polycaprolactone (PCL) microsphere surface was coated with gold. Either gold-coated PCL microspheres (AuMS) or PCL microspheres (MS) were incorporated into hESC-derived EBs. AuMS and MS were not cytotoxic. AuMS promoted the expression of genes for mesodermal and cardiac mesodermal lineage cells, both of which are intermediates in the process of cardiac differentiation of hESCs on day 4 and the expression of cardiomyogenic differentiation markers on day 14 compared to MS. AuMS also enhanced gene expression of cardiac-specific extracellular matrices. Incorporation of gold-coated MS into hESC-derived EBs may provide a new platform for inducing cardiomyogenic differentiation of pluripotent embryonic stem cells. growth factors.14 In this protocol, EBs are formed in the presence of a low level of bone morphogenetic protein 4 (BMP4) for 24 h and then treated with optimized levels of BMP4, fibroblast growth factor 2 (FGF2), and activin A, which are mesoderm induction factors, on day 1–4. Then, cardiac specification factors, vascular endothelial growth factor (VEGF), and dickkopf homolog 1 (DKK1) are added on day 4–8 and VEGF, DKK1, and FGF2 from day 8 onward.14 Previously, it was reported that culture on electrically conductive materials enhances cardiomyogenic differentiation of multipotent mesenchymal stem cells (MSCs) and a differentiated phenotype of cardiomyocytes. MSCs cultured on gold-coated collagen nanofibers expressed enhanced levels of cardiomyogenic markers such as the atrial natriuretic peptide cardiac hormone and Nkx 2.5.15 Neonatal rat cardiomyocytes cultured on conductive gels, in which gold nanoparticles were homogenously dispersed, exhibited increased expression of connexin 43, a gap junction protein in differentiated cardiomyocytes.16 However, no study has reported on the effect of electrically conductive materials on cardiomyogenic differentiation of pluripotent hESCs. Previously reported electrically conductive gel- or nanofibertype scaffolds, on which MSCs or cardiomyocytes were cultured for enhancement of cardiomyogenic differentiation or a differentiated phenotype of cardiomyocytes,15,16 would not be appropriate for the culture of EBs of hESCs.
Introduction
S
tem cell-based therapy has emerged as a new way to treat the damaged heart.1 A variety of adult stem cells, including bone marrow-derived stem cells,2,3 adiposederived stem cells,4 resident cardiac stem cells,5 and umbilical cord blood stem cells,6 have been used to regenerate the myocardium. However, these adult stem cells generated very small numbers of new cardiomyocytes.7 Heart repair with adult stem cells is accomplished by paracrine factors secreted by the transplanted stem cells rather than cardiomyogenic differentiation of the stem cells.7 Alternatively, human embryonic stem cells (hESCs) can be used for cardiac regeneration as they can differentiate into cardiomyocytes.8,9 Cardiomyocytes derived from hESCs are known to have structural and functional properties similar to native cardiomyocytes and the ability to integrate into the recipient heart upon transplantation.8–12 Therefore, the development of efficient methods for cardiomyogenic differentiation of hESCs would be critical for hESC therapy for cardiac regeneration. Cardiomyogenic differentiation of hESCs has been achieved by both embryoid body (EB) formation and growth factor treatment. EBs create a suitable environment for hESC differentiation into cells of the three germ layers.13 Yang et al. induced cardiomyogenic differentiation of hESCs by using
1 Engineering Research Institute and 2School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea. 3 School of Chemical Engineering, Sungkyunkwan University, Suwon, Republic of Korea. 4 Institute of Bioengineering, Institute of Chemical Processes, Seoul National University, Seoul, Republic of Korea.
1
2
In this study, we hypothesized that gold substrate incorporation into EBs would enhance cardiomyogenic differentiation of hESCs. To facilitate dispersion of conductive materials in hESC-derived EBs and contact between cells in EBs and conductive materials, electrically conductive microspheres (MS) were fabricated by coating the surfaces of polycaprolactone (PCL) MS with gold and were dispersed in hESC-derived EBs. The gold-coated PCL microsphere (AuMS)-incorporated EBs were then induced to differentiate to cardiac lineage using the Yang protocol,14 and the differentiation efficacy was compared with that of the Yang protocol (the control group). We chose PCL because PCL has been approved by the Food and Drug Administration for human use17 and has a long degradation time,18 which would be suitable for maintaining the gold-coated surface for a long culture period (14 days). Noncoated MS were also dispersed in hESC-derived EBs, which served as another control. Materials and Methods hESC culture
SNUhES31 (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, Korea), a hESC line, was maintained as undifferentiated ESCs by feeder-free culturing on human recombinant vitronectin (Life Technologies, Carlsbad, CA)-coated (0.5 mg/ cm2) culture dishes in the Essential 8 medium (Life Technologies), as previously described.19 The culture medium was changed daily. hESCs were passaged every week. The hESC colonies were fragmented into uniform sizes using the STEMPRO EZPassage (Life Technologies). Fabrication of MS and AuMS
PCL MS were fabricated from PCL (molecular weights = 65,000 Da; Sigma, St. Louis, MO), using a previously described water/oil method.20 Briefly, 2 g of PCL was dissolved in 40 mL of methylene chloride (Mallinckrodt, Inc., Phillipsburg, NJ). The oil emulsion was immediately poured into a beaker containing 400 mL of a 4% (w/v) polyvinyl alcohol (molecular weight = 30,000–70,000 Da; Sigma) solution and then re-emulsified using an overhead propeller (Eurostar, Ika, Germany) for 4 h at 1200 rpm. After the solvent evaporated, the MS were filtered to a size range of a 40-mm strainer (BD Bioscience, San Jose, CA), washed five times with distilled water, and lyophilized using a freeze dryer for 3 days. AuMS were prepared by coating the MS with gold using a sputter coating apparatus (Eiko, Tokyo, Japan) for 5 min. Characterization of MS and AuMS
The morphology of MS and AuMS was examined by scanning electron microscopy (SEM, JSM-6701F; JEOL, Tokyo, Japan). Energy-dispersive spectroscopy (EDS, INCA Energy; Oxford Instruments Analytical Ltd., Bucks, United Kingdom) was used to identify and semiquantitatively characterize the gold element on MS surfaces. Formation of EBs and AuMS-incorporated EBs
EB, MS-incorporated EBs (EB-MS), and AuMSincorporated EBs (EB-AuMS) were formed by forced aggregation of hESCs using AggreWell 400 inserts (Stem Cell
LEE ET AL.
Technologies, Vancouver, Canada). Briefly, hESC colonies were dissociated into single cells with accutase (Millipore, Temecula, CA). For EB formation, dissociated 1.2 · 106 hESCs in 2 mL of EB medium, a FGF2-depleted ESC culture medium composed of Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco BRL, Gaithersburg, MD) supplemented with 20% (v/v), knockout serum replacement (Life Technologies), 1% nonessential amino acid (Life Technologies), 0.1 mM b-mercaptoethanol (Sigma), and 0.2% primocin (InvivoGen, San Diego, CA) were plated onto AggreWell, which contained approximately 1200 wells per insert. For EB-MS or EB-AuMS formation, dissociated 1.2 · 106 hESCs and 4.8 · 106 MS (hESC: MS = 1:4, EB-MS group) or AuMS (hESC: AuMS = 1:4, EB-AuMS group) in 2 mL of EB medium were plated onto AggreWell inserts, which were then centrifuged at 100 g for 5 min. After 24 h of culture, EBs, EB-MS, and EB-AuMS were retrieved and transferred to lowattachment culture dishes (BD Bioscience). Characterization of EBs, MS-incorporated EBs, and AuMS-incorporated EBs
EBs, MS-incorporated EBs, and AuMS-incorporated EBs were examined using a light microscope (Model IX71; Olympus, Tokyo, Japan) after 24 h of culture. Incorporation of MS and AuMS in EBs was analyzed by DiI (Sigma) staining of MS and AuMS. Briefly, AuMS and MS were stained by DiI for 2 h. DiI-labeled MS or AuMS and hESCs were added to AggreWell for EB formation. After 24 h of EB formation, the EBs and AuMS-incorporated EBs were embedded in the Optimal Cutting Temperature compound (Tissue-Tek; Sakura Finetk, Torrance, CA) and frozen at - 20C. Sections (10 mm in thickness) were made using a Cryostat Cryocut Microtome (Leica, CM3050S, Nussloch, Germany) and then fixed using 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature. The sections were stained with 4,6 diamidino2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) and examined using a fluorescence microscope (Model IX71; Olympus). The surface and cross section of EBs and AuMS-incorporated EBs were examined using SEM ( JSM-6701F; JEOL, Tokyo, Japan). The interface between cells and AuMS in AuMS-incorporated EBs was examined using transmission electron microscopy (TEM, JEM1010; JEOL). Viability and apoptotic activity of hESCs in EBs, MS-incorporated EBs, and AuMS-incorporated EBs
The viability of hESCs in EBs, MS-incorporated EBs, and AuMS-incorporated EBs was analyzed by the live and dead assay. Live and dead cells were detected with fluorescein diacetate (FDA; Sigma) and ethidium bromide (Sigma), respectively, after 24 h of culture. EBs, MS-incorporated EBs, and AuMS-incorporated EBs were incubated in the FDA/ethidium bromide (5 and 10 mg/mL, respectively) solution for 5 min at 37C and then washed twice in PBS. Dead cells were stained orange due to the nuclear permeability to ethidium bromide. Viable cells, capable of converting the nonfluorescent FDA into fluorescein, stained green. After staining, the samples were examined using a fluorescence microscope (Model IX71; Olympus). The cell viability was quantified by (green + red) area divided by red
GOLD MICROSPHERES ENHANCE CARDIOMYOGENIC DIFFERENTIATION
area and expressed in percentage. The apoptotic activity of hESCs in EBs, MS-incorporated EBs, and AuMS-incorporated EBs was analyzed by reverse transcription–polymerase chain reaction (RT-PCR). Total RNA was extracted from the EB, EB-MS, and EB-AuMS on day 1. RNA was reversetranscribed into cDNA. The expression of BCL-XL, BAX, and b-ACTIN mRNA was evaluated by RT-PCR using selfprepared primers and determined by subsequent agarose gel electrophoresis. PCR was carried out for 40 cycles consisting of denaturing (94C, 30 s), annealing (60C, 30 s), and extension (72C, 45 s), with final extension at 72C for 7 min. The PCR products were visualized by electrophoresis on 1.5% (w/v) agarose gels with ethidium bromide staining. Gels were read using a gel documentation system (GL 2200 PRO Imaging system; CARESTREAM, Rochester, NY). The primer sequences are shown in Table 1 Cardiomyogenic differentiation of hESCs
EB, EB-MS, and EB-AuMS were induced to undergo cardiomyogenic differentiation, as previously described.14 Briefly, EBs, EB-MS, and EB-AuMS were transferred to a low-attachment culture dish (BD Bioscience), then treated with the following cytokines: BMP4 (0.5 ng mL - 1) on day 0–1; BMP4 (10 ng mL - 1), FGF2 (5 ng mL - 1), and activin A (3 ng mL - 1) on day 1–4; DKK1 (150 ng mL - 1) and VEGF (10 ng mL - 1) on day 4–8; VEGF (10 ng mL - 1), DKK1 (150 ng mL - 1), and FGF2 (5 ng mL - 1) on day 8–14. All cytokines were purchased from R&D systems (Minneapolis, MN). Quantitative real-time RT-PCR of hESCs
On day 4 and 14, the mRNA expression of hESCs was analyzed using quantitative real-time RT-PCR (qRT-PCR). On day 4, the expression of mesodermal, endodermal, and ectodermal genes was evaluated. On day 14, cardiac and extracellular matrix (ECM) gene expression was evaluated. Total RNA was extracted from the differentiated hESCs. RNA was reverse-transcribed into cDNA. The expression of mRNA was evaluated by qRT-PCR. qRT-PCR was performed using the StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA) with FAST SYBR Green PCR master mix (Applied Biosystems). Each cycle consisted of the following times and temperatures: 94C for 3 s and 60C for 30 s. The primer sequences are shown in Table 1. Immunocytochemistry of cardiomyogenically differentiated hESCs
After the induction of cardiomyogenic differentiation for 14 days, EB, EB-MS, and EB-AuMS were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. For immunocytochemistry, hESCs in EB, EB-MS, and EB-AuMS were stained with the anti-human cardiac troponin T (cTnT) antibody (Abcam, Cambridge, United Kingdom). The staining result was visualized using the fluorescein isothiocyanateconjugated secondary antibody ( Jackson Immuno Research Laboratories, West Grove, PA). The cells were counterstained with DAPI (Vector Laboratories, Burlingame, CA) and examined using a fluorescence microscope (Model IX71; Olympus). The relative cardiomyocyte yield was determined by dividing cTnT( + ) area by DAPI( + ) area ([cTnT( + ) area/DAPI( + ) area] · 100%).
3
Table 1. Human-Specific Primers Used for RT-PCR and qRT-PCR Analyses Gene
Primer
b-actin
Sense 5¢- GCA CTC TTC CAG CCT TCC TTC C -3¢ Antisense 5¢- TCA CCT TCA CCG TTC CAG TTT TTT -3¢ BAX Sense 5¢- GTG CAC CAA GGT GCC GGA AC -3¢ Antisense 5¢- TCA GCC CAT CTT CTT CCA GA -3¢ BCL-XL Sense 5¢- CGG GCA TTC AGT GAC CTG AC -3¢ Antisense 5¢- TCA GGA ACC AGC GGT TGA AG -3¢ GAPDH Sense 5¢- GTC GGA GTC AAC GGA TTT GG -3¢ Antisense 5¢- GGG TGG AAT CAA TTG GAA CAT -3¢ BRACHYURY Sense 5¢- CAG TGA CTT TTT GTC GTG GCA -3¢ (T) Antisense 5¢- CCA ACT GCA TCA TCT CCA CA -3¢ M-cadherin Sense 5¢- CGC AAC TGG ACA TGC CAC T -3¢ Antisense 5¢- AGA GAA ACT CTT CCC ACC CC -3¢ MESP2 Sense 5¢- CGC CTG GGC ATC TTC TAC TAA -3¢ Antisense 5¢- AGG GAA GGT ACC CGC TAT CTA -3¢ AFP Sense 5¢- TTC CAT AAG GAT CTG TGC CA -3¢ Antisense 5¢- TCA GCA AAG CAG ACT TCC TGT -3¢ TUBB3 Sense 5¢- TTC CTG CAC TGG TAC ACG G -3¢ Antisense 5¢- TGC GAG CAG CTT CAC TTG -3¢ NKX2-5 Sense 5¢-GCA GAG ACC TCC CGT TTT GTT -3¢ Antisense 5¢- GCC ACC GAC ACG TCT CAC T -3¢ a-MHC Sense 5¢-GCC CCG CCC CAC AT -3¢ Antisense 5¢- CCG GAT TCT CCC GTG ATG -3¢ b-MHC Sense 5¢- CCA CCC AAG TTC GAC AAA ATC -3¢ Antisense 5¢- CGT AGC GAT CCT TGA GGT TGT A -3¢ cTnT Sense 5¢- CAG GAT CAA CGA TAA CCA GAA AGT C -3¢ Antisense 5¢- GTG AAG GAG GCC AGG CTC TA -3¢ CONNEXIN 43 Sense 5¢- ACT GGC GAC AGA AAC AAT TCT TC -3¢ Antisense 5¢- TTC TGC ACT GTA ATT AGC CCA GTT -3¢ COLLAGEN Sense 5¢- CAG CCG CTT CAC CTA CAG C -3¢ TYPE I Antisense 5¢- TTT TGT ATT CAA TCA CTG TCT T -3¢ COLLAGEN Sense 5¢- GGG AAT GGA GCA AAA CAG TCT T -3¢ TYPE III Antisense 5¢- CCA ACG TCC ACA CCA AAT TCT -3¢ COLLAGEN Sense 5¢- TGT CCA ATA TGA AAA CCG TAA TYPE IV AGT G -3¢ Antisense 5¢- CAC TAT TGA AAG CTT ATC GCT GTC TT-3¢ FIBRONECTIN Sense 5¢- TCC ACG GGA GCC TCG AA -3¢ Antisense 5¢- ACA ACC GGG CTT GCT TTG -3¢ LAMININ Sense 5¢- CAC AAC AAC ATT GAC ACG ACA GA -3¢ Antisense 5¢- GCT GGA GGG CAT CAC CAT AGT -3¢
RT-PCR, reverse transcription–polymerase chain reaction; qRT-PCR, quantitative real-time RT-PCR.
Statistical analysis
All quantitative data are expressed as the mean – standard deviation. Statistical analysis was performed by analysis of variance using a Bonferroni test. A p-value < 0.05 was considered statistically significant. Results
The fabricated MS and AuMS had a spherical morphology (Fig. 1A). The diameter was approximately 5–15 mm. EDS analysis showed that gold was coated on the PCL MS surface in AuMS (Fig. 1B). EDS spectra confirmed gold coating on AuMS, whereas gold element was not detected on MS (Fig. 1C).
4
LEE ET AL.
FIG. 1. Preparation and characterization of gold-coated polycaprolactone (PCL) microspheres (MS). (A) SEM images of noncoated PCL MS and gold-coated PCL microspheres (AuMS). (B) Energy-dispersive spectroscopy (EDS) analysis for gold element on the surfaces of MS and AuMS. Yellow indicates gold. Yellow dots in the MS were background. Scale bars indicate 10 mm in the upper panel and 2 mm in the lower panel in (A) and 8 mm in (B). (C) EDS spectra of AuMS and MS. SEM, scanning electron microscopy. Color images available online at www.liebertpub.com/tea A ratio of hESCs:AuMS of 1:4 was used to incorporate AuMS into hESC-derived EBs. A ratio of hESCs:AuMS of 1:2 did not exhibit positive effects of AuMS on cardiomyogenic differentiation (data not shown). After 24 h, EBs, MS-incorporated EBs (EB-MS), and AuMS-incorporated
EBs (EB-AuMS) had a spherical shape (Fig. 2A). To confirm dispersion of MS and AuMS in EBs, MS and AuMS were labeled with the red fluorescence dye, DiI, before incorporation into EBs. In MS-incorporated EBs, MS and AuMS were dispersed throughout the EBs (Fig. 2B). The
FIG. 2. Incorporation of PCL MS or gold-coated PCL microsphere (AuMS) into hESC-derived embryoid bodies (EBs). (A) Light microscopic images of EBs and EBs incorporated with either PCL microspheres (EB-MS) or gold-coated PCL microspheres (EB-AuMS) on day 1. (B) Merged images of EBs and EBs incorporated with either DiI(red)-labeled MS or AuMS on day 1, showing incorporation of MS into hESC-derived EBs. (C) Fluorescence images of EBs and EBs incorporated with DiI(red)-labeled AuMS. The nuclei were stained with DAPI (blue). (D) SEM images of surface and cross section of EB and EB-AuMS. (E) A TEM image of EB-AuMS. Black dots (arrow) indicate gold. Scale bars indicate 200 mm in (A) and (B), 100 mm in (C), 10 mm in (D), and 100 nm in (E). Color images available online at www.liebertpub.com/tea
GOLD MICROSPHERES ENHANCE CARDIOMYOGENIC DIFFERENTIATION
5
FIG. 3. Effects of AuMS incorporated into EBs on viability and apoptotic activity of hESCs in EB-AuMS. (A) Fluorescence images of EBs and EBs incorporated with either MS (EB-MS) or AuMS (EB-AuMS) and stained with FDA and EB on day 1. Green and orange-red colors indicate viable and dead cells, respectively. The scale bars indicate 200 mm. (B) Reverse transcription– polymerase chain reaction (RT-PCR) analysis for proapoptotic (BAX) and antiapoptotic (BCL-XL) mRNA expression of EBs and EBs incorporated with either MS (EB-MS) or AuMS (EB-AuMS) on day 1. Color images available online at www .liebertpub.com/tea
fluorescence microscopic examination confirmed the dispersion of AuMS in EBs (Fig. 2C). SEM images showed that EB and EB-AuMS had a spherical shape, and the core of the EB was filled with hESCs and/or AuMS (Fig. 2D). The TEM examination revealed that gold was coated on the surface of AuMS and was in contact with hESCs in EBAuMS (Fig. 2E). To determine whether AuMS incorporation into EBs affects the viability and apoptotic activity of hESCs in the EBs, the viability and apoptotic activity of hESCs in EB, EB-MS, and EB-AuMS were compared. Viability was analyzed based on live and dead assays using FDA/ethidium bromide staining. It showed no difference in hESC viability among EB, EB-MS, and EB-AuMS (96.5% – 0.5%, 96.1% – 1.5%, and 96.3% – 0.9% respectively, Fig. 3A). In addition, the expression of an apoptotic gene (BAX) and an antiapoptotic gene (BCL-XL) did not differ among EB, EBMS, and EB-AuMS (Fig. 3B). In the process of cardiomyogenic differentiation of hESCs in EBs, hESCs first differentiated into mesodermal cells and endodermal cells, and then the mesodermal cells differentiated into cardiogenic lineages (Fig. 4).21 Endodermal cells promote the mesodermal cell differentiation into cardiogenic lineages by interacting with the mesodermal cells.21 The mRNA expression of mesoderm, endoderm,
and ectodermal genes was evaluated on day 4. The dispersion of AuMS in hESC-derived EBs enhanced the expression of mesodermal genes (T and M-cadherin), a cardiac mesoderm gene (MESP2), and an endodermal gene (AFP) compared to the other groups (Fig. 5A–C). However, ectodermal gene (TUBB3) expression was not significantly different among all groups (Fig. 5D). We examined whether incorporation of AuMS into hESCderived EBs enhances cardiomyogenic gene expression of the hESCs by qRT-PCR assay on day 14. The EB-AuMS group exhibited the upregulated expression of cardiomyogenic transcriptional factor (NKX2-5), early cardiomyocyte markers (a-MHC, b-MHC), a late cardiomyogenic marker (cTnT), and a gap junction gene (CONNEXIN 43) compared with the other groups (Fig. 6A–D). Immunocytochemistry revealed that the cells in the EB-AuMS group expressed a higher amount of cTnT protein compared with the other groups (Fig. 6E). The relative cardiomyocyte yields, which were determined by dividing the cTnT( + ) area by DAPI( + ) area, were 3.4% in EB, 10.3% in EM-MS, and 28.6% in EB-AuMS. The incorporation of AuMS into hESC-derived EBs also enhanced gene expression of cardiac-specific ECMs of the cells. Cardiomyocytes derived from hESCs were reported to be encased in a network of ECMs such as fibronectin,
FIG. 4. A schematic diagram describing a stepwise model for in vitro cardiomyogenic differentiation of hESCs in EBs and soluble factors used in each differentiation stage, based on Moon et al.21 Color images available online at www.liebertpub.com/tea
6
LEE ET AL.
FIBRONECTIN, and LAMININ) genes compared with the other groups (Fig. 7). Discussion
FIG. 5. Enhanced mesodermal, cardiac mesodermal, and endodermal differentiation of hESCs in EBs by AuMS incorporation into EBs. (A) Mesodermal, (B) cardiac mesodermal, (C) endodermal, and (D) ectodermal gene expressions in EBs and EBs incorporated with either MS (EB-MS) or AuMS (EBAuMS) were evaluated by quantitative real-time RT-PCR (qRT-PCR) on day 4. The values were normalized to the levels of the EB group (n = 3, *p < 0.05 vs. EB, #p < 0.05 vs. EB-MS). Color images available online at www.liebertpub.com/tea laminin, and collagen, which are the major components of the cardiac ECMs.22,23 The qRT-PCR assay on day 14 showed that cells in the EB-AuMS group expressed significantly higher levels of cardiac-specific ECM (COLLAGEN TYPE I, COLLAGEN TYPE III, COLLAGEN TYPE IV,
Culture on electrically conductive scaffolds (nanofibers and gels) enhanced cardiomyogenic differentiation of multipotent MSCs and a differentiated phenotype of cardiomyocytes. This approach can be applied to cardiomyogenic differentiation of pluripotent hESCs. However, these scaffolds would not be appropriate for in vitro cardiomyogenic differentiation of hESCs because this phenomenon is achieved by culture in the form of EBs. Incorporation of electrically conductive MS within hESC-derived EBs would be an efficient way to culture hESCs in contact with electrically conductive scaffolds. In this study, we successfully fabricated electrically conductive MS by coating the MS surface with gold. The incorporation of AuMS in hESC-derived EBs significantly enhanced mesodermal and endodermal lineage differentiation (Fig. 5) and cardiac lineage differentiation (Fig. 6). Compared to the previous Yang protocol (the EB group in this study), AuMS incorporation upregulated mRNA expression of NKX2-5 by 450%, a-MHC by 230%, b-MHC by 1300%, cTnT by 30%, and CONNEXIN 43 by 49% (Fig. 6A–D). The enhancement of mesodermal lineage differentiation by AuMS promoted cardiomyogenic differentiation of hESCs. In the cardiomyogenic differentiation process of hESCs, six differentiation steps were proposed, which were epithelial to mesenchymal transition, mesoderm differentiation, mesoderm speciation, cardiac specification, cardiomyocyte differentiation, and electrical maturation.24 The mesoderm specification includes mesodermal and cardiac mesodermal differentiation.24 Therefore, mesoderm and cardiac mesodermal differentiation is one of the important steps in cardiomyogenic differentiation of hESCs. EB-AuMS enhanced the expression of mesoderm (T- and M-cadherin) and cardiac mesoderm genes (MESP2) compared to the other groups, including the previous protocol (Yang protocol, the EB group in this study, Fig. 5A, B). MESP2 was reported to be essential for cardiac mesoderm development.25 Therefore,
FIG. 6. Enhanced cardiac lineage differentiation of hESCs in EBs by AuMS incorporation into EBs. mRNA expressions of (A) cardiac-specific transcription factors, (B) early cardiomyocyte markers, (C) late cardiomyocyte marker, and (D) a cell–cell junction protein of EBs and EBs incorporated with either MS (EB-MS) or AuMS (EB-AuMS) were evaluated by qRT-PCR on day 14. The values were normalized to the levels of the EB group (n = 3, *p < 0.05 vs. EB, #p < 0.05 vs. EB-MS). (E) Immunocytochemistry for cTnT (green), a late cardiomyocyte marker, in EB, EB-MS, and EB-AuMS at day 14. The nuclei were stained with DAPI (blue). The scale bars indicate 100 mm. Color images available online at www.liebertpub.com/tea
GOLD MICROSPHERES ENHANCE CARDIOMYOGENIC DIFFERENTIATION
7
FIG. 7. Enhanced mRNA expression of cardiacspecific ECMs in hESCderived EBs by AuMS incorporation into EBs. The mRNA expressions of cardiac-specific ECMs in EBs and EBs incorporated with either MS (EB-MS) or AuMS (EBAuMS) were evaluated by qRT-PCR at day 14. The values were normalized to the levels of the EBs (n = 3, *p < 0.05 vs. EB, #p < 0.05 vs. EB-MS). Color images available online at www .liebertpub.com/tea the enhanced cardiomyogenic differentiation of hESCs by AuMS (Fig. 6) may be attributed to the enhanced mesodermal and cardiac mesodermal lineage gene expression. Enhancement of endodermal differentiation by AuMS may also contribute to the enhanced cardiomyogenic differentiation of hESCs. It has been shown that endodermal cells around mesodermal cells induce cardiomyogenic differentiation of the mesodermal cells in the process of cardiomyogenic differentiation of hESCs.21 During cardiomyogenic differentiation of hESCs, endoderm markers were reported to increase concurrently with mesoderm markers.26 In another study, coculture with an endoderm-derived cell line (END-2 cells) induced cardiac differentiation of hESCs.9 These studies suggest that enhanced endodermal differentiation promotes cardiomyogenic differentiation of hESCs in the cardiomyogenic differentiation process. AuMS can be used as an electrically conductive material to enhance the cardiomyogenic cell phenotype. Previously, cardiomyocyte culture in gold nanoparticle-dispersed hydroxyethyl methacrylate hydrogels enhanced CONNEXIN 43 expression by 60% compared to culture in nonconductive hydrogels.16 In this study, AuMS enhanced CONNEXIN 43 expression in hESCs by 50% compared to the EB group (Fig. 6D). CONNEXIN 43 regulates cell–cell communication, which influences electrical coupling, and promotes contractile behavior.27–29 CONNEXIN 43 helps cardiac cells sense and respond to their in vivo environment.16 Therefore, the use of AuMS for in vitro cardiomyogenic differentiation of hESCs may improve electrical coupling of the hESC-derived cells with recipient myocardial cells following transplantation through the enhanced CONNEXIN 43 expression. Also, AuMS promoted cardiomyogenic differentiation of hESCs. Previously, it was reported that culture of human MSCs on composite scaffolds of PCL and carbon nanotube (CNT) enhanced expression of cardiomyogenic genes and proteins.30 CNT offers electrical conductivity to PCL scaffold,30 indicating that conductive materials may enhance the cardiomyogenic differentiation of stem cells. This, gold in the AuMS, may play a role in the enhanced cardiomyogenic differentiation of hESCs. In summary, incorporation of AuMS into hESC-derived EBs was an efficient way to promote the cardiomyogenic differentiation process of hESCs. On day 4, AuMS promoted differentiation of hESCs into mesodermal, cardiac
mesodermal, and endodermal lineages, which are the intermediate cells in the cardiomyogenic differentiation process of hESCs. On day 14, AuMS enhanced the expression of cardiomyogenic differentiation markers. In addition, AuMS enhanced gene expression of cardiac-specific ECMs. However, we did not observe beating cells following induction of cardiomyogenic differentiation with AuMS, indicating that our method does not induce full differentiation into contractile cardiomyocytes. Incorporation of AuMS into hESC-derived EBs could be a new platform to promote the cardiomyogenic differentiation process of hESCs. Acknowledgments
This study was supported by a grant (2013036054) from the National Research Foundation of Korea and a grant (HI12C0199) from the Korean Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea. Disclosure Statement
No competing financial interests exist. References
1. Habib, M., Caspi, O., and Gepstein, L. Human embryonic stem cells for cardiomyogenesis. J Mol Cell Cardiol 45, 462, 2008. 2. Orlic, D., Kajstura, J., Chimenti, S., Jakoniuk, I., Anderson, S.M., Li, B., Pickel, J., McKay, R., Nadal-Ginard, B., Bodine, D.M., Leri, A., and Anversa, P. Bone marrow cells regenerate infarcted myocardium. Nature 410, 701, 2001. 3. Tang, X.L., Rokosh, D.G., Guo, Y., and Bolli, R. Cardiac progenitor cells and bone marrow-derived very small embryonic-like stem cells for cardiac repair after myocardial infarction. Circ J 74, 390, 2010. 4. Planat-Be´nard, V., Menard, C., Andre´, M., Puceat, M., Perez, A., Garcia-Verdugo, J.M., Pe´nicaud, L., and Casteilla, L. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res 94, 223, 2004. 5. Unno, K., Jain, M., and Liao, R. Cardiac side population cells: moving toward the center stage in cardiac regeneration. Circ Res 110, 1355, 2012. 6. Schlechta, B., Wiedemann, D., Kittinger, C., Jandrositz, A., Bonaros, N.E., Huber, J.C., Preisegger, K.H., and Kocher, A.A. Ex-vivo expanded umbilical cord blood stem cells
8
7. 8.
9.
10.
11.
12.
13.
14.
15.
16. 17. 18.
19.
LEE ET AL.
retain capacity for myocardial regeneration. Circ J 74, 188, 2010. Mignone, J.L., Kreutziger, K.L., Paige, S.L., and Murry, C.E. Cardiogenesis from human embryonic stem cells. Circ J 74, 2517, 2010. He, J.Q., Ma, Y., Lee, Y., Thomson, J.A., and Kamp, T.J. Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization. Circ Res 93, 32, 2003. Mummery C., Ward-van Oostwaard, D., Doevendans, P., Spijker, R., van den Brink, S., Hassink, R., van der Heyden, M., Opthof, T., Pera, M., de la Riviere, A.B., Passier, R., and Tertoolen, L. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107, 2733, 2003. Kehat, I., Khimovich, L., Caspi, O., Gepstein, A., Shofti, R., Arbel, G., Huber, I., Satin, J., Itskovitz-Eldor, J., and Gepstein, L. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol 22, 1282, 2004. Xue, T., Cho, H.C., Akar, F.G., Tsang, S.Y., Jones, S.P., Marba´n, E., Tomaselli, G.F., and Li, R.A. Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes: insights into the development of cell-based pacemakers. Circulation 111, 11, 2005. Shiba, Y., Fernandes, S., Zhu, W.Z., Filice, D., Muskheli, V., Kim, J., Palpant, N.J., Gantz, J., Moyes, K.W., Reinecke, H., Van Biber, B., Dardas, T., Mignone, J.L., Izawa, A., Hanna, R., Viswanathan, M., Gold, J.D., Kotlikoff, M.I., Sarvazyan, N., Kay, M.W., Murry, C.E., and Laflamme, M.A. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature 489, 322, 2012. Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., and Benvenisty, N. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 6, 88, 2000. Yang, L., Soonpaa, M.H., Adler, E.D., Roepke, T.K., Kattman, S.J., Kennedy, M., Henckaerts, E., Bonham, K., Abbott, G.W., Linden, R.M., Field, L.J., and Keller, G.M. Human cardiovascular progenitor cells develop from a KDR + embryonic-stem-cell-derived population. Nature 453, 524, 2008. Orza, A., Soritau, O., Olenic, L., Diudea, M., Florea, A., Rus Ciuca, D., Mihu, C., Casciano, D., and Biris, A.S. Electrically conductive gold-coated collagen nanofibers for placental-derived mesenchymal stem cells enhanced differentiation and proliferation. ACS Nano 5, 4490, 2011. You, J.O., Rafat, M., Ye, G.J., and Auguste, D.T. Nanoengineering the heart: conductive scaffolds enhance connexin 43 expression. Nano Lett 11, 3643, 2011. Tang, Z.G., Callaghan, J.T., and Hunt, J.A. The physical properties and response of osteoblasts to solution cast films of PLGA doped polycaprolactone. Biomaterials 26, 6618, 2005. Lam, C.X., Hutmacher, D.W., Schantz, J.T., Woodruff, M.A., and Teoh, S.H. Evaluation of polycaprolactone scaffold degradation for 6 months in vitro and in vivo. J Biomed Mater Res A 90, 906, 2009. Chen, G., Gulbranson, D.R., Hou, Z., Bolin, J.M., Ruotti, V., Probasco, M.D., Smuga-Otto, K., Howden, S.E., Diol, N.R., Propson, N.E., Wagner, R., Lee, G.O., AntosiewiczBourget, J., Teng, J.M., and Thomson, J.A. Chemically
20.
21.
22.
23. 24.
25. 26.
27.
28.
29.
30.
defined conditions for human iPSC derivation and culture. Nat Methods 8, 424, 2011. Kang, S.W., Yang, H.S., Seo, S.W., Han, D.K., and Kim, B.S. Apatite-coated poly(lactic-co-glycolic acid) microspheres as an injectable scaffold for bone tissue engineering. J Biomed Mater Res A 85, 747, 2008. Moon, S.H., Ban, K., Kim, C., Kim, S.S., Byun, J., Song, M.K., Park, I.H., Yu, S.P., and Yoon, Y.S. Development of a novel two-dimensional directed differentiation system for generation of cardiomyocytes from human pluripotent stem cells. Int J Cardiol 168, 41, 2013. van Laake, L.W., van Donselaar, E.G., Monshouwer-Kloots, J., Schreurs, C., Passier, R., Humbel, B.M., Doevendans, P.A., Sonnenberg, A., Verkleij, A.J., and Mummery, C.L. Extracellular matrix formation after transplantation of human embryonic stem cell-derived cardiomyocytes. Cell Mol Life Sci 67, 277, 2010. Moore, L., Fan, D., Basu, R., Kandalam, V., and Kassiri, Z. Tissue inhibitor of metalloproteinases (TIMPs) in heart failure. Heart Fail Rev 17, 693, 2012. Burridge, P.W., Keller, G., Gold, J.D., and Wu, J.C. Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell 10, 16, 2012. Kitajima, S., Takagi, A., Inoue, T., and Saga, Y. MesP1 and MesP2 are essential for the development of cardiac mesoderm. Development 127, 3215, 2000. Bettiol, E., Sartiani, L., Chicha, L., Krause, K.H., Cerbai, E., and Jaconi, M.E. Fetal bovine serum enables cardiac differentiation of human embryonic stem cells. Differentiation 75, 669, 2007. Oyamada, M., Kimura, H., Oyamada, Y., Miyamoto, A., Ohshika, H., and Mori, M. The expression, phosphorylation, and localization of connexin 43 and gap-junctional intercellular communication during the establishment of a synchronized contraction of cultured neonatal rat cardiac myocytes. Exp Cell Res 212, 351, 1994. Ando, M., Katare, R.G., Kakinuma, Y., Zhang, D., Yamasaki, F., Muramoto, K., and Sato, T. Efferent vagal nerve stimulation protects heart against ischemia-induced arrhythmias by preserving connexin43 protein. Circulation 112, 164, 2005. Bupha-Intr, T., Haizlip, K.M., and Janssen, P.M. Temporal changes in expression of connexin 43 after load-induced hypertrophy in vitro. Am J Physiol Heart Circ Physiol 296, H806, 2009. Crowder, S.W., Liang, Y., Rath, R., Park, A.M., Maltais, S., Pintauro, P.N., Hofmeister, W., Lim, C.C., Wang, X., and Sung, H.J. Poly(e-caprolactone)-carbon nanotube composite scaffolds for enhanced cardiac differentiation of human mesenchymal stem cells. Nanomedicine (Lond) 8, 1763, 2013.
Address correspondence to: Byung-Soo Kim, PhD School of Chemical and Biological Engineering Seoul National University Seoul 151-744 Republic of Korea E-mail: [email protected] Received: January 7, 2014 Accepted: July 23, 2014 Online Publication Date: September 5, 2014
TISSUE ENGINEERING: Part A Volume 00, Number 00, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2014.0015
Incorporation of Gold-Coated Microspheres into Embryoid Body of Human Embryonic Stem Cells for Cardiomyogenic Differentiation Tae-Jin Lee, PhD,1 Seokyung Kang, MS,2 Gun-Jae Jeong, BS,2 Jeong-Kee Yoon, BS,2 Suk Ho Bhang, PhD,3 Jaesur Oh, BS,2 and Byung-Soo Kim, PhD1,2,4
Human embryonic stem cells (hESCs) are a useful cell source for cardiac regeneration by stem cell therapy. In this study, we show that incorporation of gold-coated microspheres into hESC-derived embryoid bodies (EBs) enhances the cardiomyogenic differentiation process of pluripotent embryonic stem cells. A polycaprolactone (PCL) microsphere surface was coated with gold. Either gold-coated PCL microspheres (AuMS) or PCL microspheres (MS) were incorporated into hESC-derived EBs. AuMS and MS were not cytotoxic. AuMS promoted the expression of genes for mesodermal and cardiac mesodermal lineage cells, both of which are intermediates in the process of cardiac differentiation of hESCs on day 4 and the expression of cardiomyogenic differentiation markers on day 14 compared to MS. AuMS also enhanced gene expression of cardiac-specific extracellular matrices. Incorporation of gold-coated MS into hESC-derived EBs may provide a new platform for inducing cardiomyogenic differentiation of pluripotent embryonic stem cells. growth factors.14 In this protocol, EBs are formed in the presence of a low level of bone morphogenetic protein 4 (BMP4) for 24 h and then treated with optimized levels of BMP4, fibroblast growth factor 2 (FGF2), and activin A, which are mesoderm induction factors, on day 1–4. Then, cardiac specification factors, vascular endothelial growth factor (VEGF), and dickkopf homolog 1 (DKK1) are added on day 4–8 and VEGF, DKK1, and FGF2 from day 8 onward.14 Previously, it was reported that culture on electrically conductive materials enhances cardiomyogenic differentiation of multipotent mesenchymal stem cells (MSCs) and a differentiated phenotype of cardiomyocytes. MSCs cultured on gold-coated collagen nanofibers expressed enhanced levels of cardiomyogenic markers such as the atrial natriuretic peptide cardiac hormone and Nkx 2.5.15 Neonatal rat cardiomyocytes cultured on conductive gels, in which gold nanoparticles were homogenously dispersed, exhibited increased expression of connexin 43, a gap junction protein in differentiated cardiomyocytes.16 However, no study has reported on the effect of electrically conductive materials on cardiomyogenic differentiation of pluripotent hESCs. Previously reported electrically conductive gel- or nanofibertype scaffolds, on which MSCs or cardiomyocytes were cultured for enhancement of cardiomyogenic differentiation or a differentiated phenotype of cardiomyocytes,15,16 would not be appropriate for the culture of EBs of hESCs.
Introduction
S
tem cell-based therapy has emerged as a new way to treat the damaged heart.1 A variety of adult stem cells, including bone marrow-derived stem cells,2,3 adiposederived stem cells,4 resident cardiac stem cells,5 and umbilical cord blood stem cells,6 have been used to regenerate the myocardium. However, these adult stem cells generated very small numbers of new cardiomyocytes.7 Heart repair with adult stem cells is accomplished by paracrine factors secreted by the transplanted stem cells rather than cardiomyogenic differentiation of the stem cells.7 Alternatively, human embryonic stem cells (hESCs) can be used for cardiac regeneration as they can differentiate into cardiomyocytes.8,9 Cardiomyocytes derived from hESCs are known to have structural and functional properties similar to native cardiomyocytes and the ability to integrate into the recipient heart upon transplantation.8–12 Therefore, the development of efficient methods for cardiomyogenic differentiation of hESCs would be critical for hESC therapy for cardiac regeneration. Cardiomyogenic differentiation of hESCs has been achieved by both embryoid body (EB) formation and growth factor treatment. EBs create a suitable environment for hESC differentiation into cells of the three germ layers.13 Yang et al. induced cardiomyogenic differentiation of hESCs by using
1 Engineering Research Institute and 2School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea. 3 School of Chemical Engineering, Sungkyunkwan University, Suwon, Republic of Korea. 4 Institute of Bioengineering, Institute of Chemical Processes, Seoul National University, Seoul, Republic of Korea.
1
2
In this study, we hypothesized that gold substrate incorporation into EBs would enhance cardiomyogenic differentiation of hESCs. To facilitate dispersion of conductive materials in hESC-derived EBs and contact between cells in EBs and conductive materials, electrically conductive microspheres (MS) were fabricated by coating the surfaces of polycaprolactone (PCL) MS with gold and were dispersed in hESC-derived EBs. The gold-coated PCL microsphere (AuMS)-incorporated EBs were then induced to differentiate to cardiac lineage using the Yang protocol,14 and the differentiation efficacy was compared with that of the Yang protocol (the control group). We chose PCL because PCL has been approved by the Food and Drug Administration for human use17 and has a long degradation time,18 which would be suitable for maintaining the gold-coated surface for a long culture period (14 days). Noncoated MS were also dispersed in hESC-derived EBs, which served as another control. Materials and Methods hESC culture
SNUhES31 (Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, Korea), a hESC line, was maintained as undifferentiated ESCs by feeder-free culturing on human recombinant vitronectin (Life Technologies, Carlsbad, CA)-coated (0.5 mg/ cm2) culture dishes in the Essential 8 medium (Life Technologies), as previously described.19 The culture medium was changed daily. hESCs were passaged every week. The hESC colonies were fragmented into uniform sizes using the STEMPRO EZPassage (Life Technologies). Fabrication of MS and AuMS
PCL MS were fabricated from PCL (molecular weights = 65,000 Da; Sigma, St. Louis, MO), using a previously described water/oil method.20 Briefly, 2 g of PCL was dissolved in 40 mL of methylene chloride (Mallinckrodt, Inc., Phillipsburg, NJ). The oil emulsion was immediately poured into a beaker containing 400 mL of a 4% (w/v) polyvinyl alcohol (molecular weight = 30,000–70,000 Da; Sigma) solution and then re-emulsified using an overhead propeller (Eurostar, Ika, Germany) for 4 h at 1200 rpm. After the solvent evaporated, the MS were filtered to a size range of a 40-mm strainer (BD Bioscience, San Jose, CA), washed five times with distilled water, and lyophilized using a freeze dryer for 3 days. AuMS were prepared by coating the MS with gold using a sputter coating apparatus (Eiko, Tokyo, Japan) for 5 min. Characterization of MS and AuMS
The morphology of MS and AuMS was examined by scanning electron microscopy (SEM, JSM-6701F; JEOL, Tokyo, Japan). Energy-dispersive spectroscopy (EDS, INCA Energy; Oxford Instruments Analytical Ltd., Bucks, United Kingdom) was used to identify and semiquantitatively characterize the gold element on MS surfaces. Formation of EBs and AuMS-incorporated EBs
EB, MS-incorporated EBs (EB-MS), and AuMSincorporated EBs (EB-AuMS) were formed by forced aggregation of hESCs using AggreWell 400 inserts (Stem Cell
LEE ET AL.
Technologies, Vancouver, Canada). Briefly, hESC colonies were dissociated into single cells with accutase (Millipore, Temecula, CA). For EB formation, dissociated 1.2 · 106 hESCs in 2 mL of EB medium, a FGF2-depleted ESC culture medium composed of Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco BRL, Gaithersburg, MD) supplemented with 20% (v/v), knockout serum replacement (Life Technologies), 1% nonessential amino acid (Life Technologies), 0.1 mM b-mercaptoethanol (Sigma), and 0.2% primocin (InvivoGen, San Diego, CA) were plated onto AggreWell, which contained approximately 1200 wells per insert. For EB-MS or EB-AuMS formation, dissociated 1.2 · 106 hESCs and 4.8 · 106 MS (hESC: MS = 1:4, EB-MS group) or AuMS (hESC: AuMS = 1:4, EB-AuMS group) in 2 mL of EB medium were plated onto AggreWell inserts, which were then centrifuged at 100 g for 5 min. After 24 h of culture, EBs, EB-MS, and EB-AuMS were retrieved and transferred to lowattachment culture dishes (BD Bioscience). Characterization of EBs, MS-incorporated EBs, and AuMS-incorporated EBs
EBs, MS-incorporated EBs, and AuMS-incorporated EBs were examined using a light microscope (Model IX71; Olympus, Tokyo, Japan) after 24 h of culture. Incorporation of MS and AuMS in EBs was analyzed by DiI (Sigma) staining of MS and AuMS. Briefly, AuMS and MS were stained by DiI for 2 h. DiI-labeled MS or AuMS and hESCs were added to AggreWell for EB formation. After 24 h of EB formation, the EBs and AuMS-incorporated EBs were embedded in the Optimal Cutting Temperature compound (Tissue-Tek; Sakura Finetk, Torrance, CA) and frozen at - 20C. Sections (10 mm in thickness) were made using a Cryostat Cryocut Microtome (Leica, CM3050S, Nussloch, Germany) and then fixed using 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature. The sections were stained with 4,6 diamidino2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) and examined using a fluorescence microscope (Model IX71; Olympus). The surface and cross section of EBs and AuMS-incorporated EBs were examined using SEM ( JSM-6701F; JEOL, Tokyo, Japan). The interface between cells and AuMS in AuMS-incorporated EBs was examined using transmission electron microscopy (TEM, JEM1010; JEOL). Viability and apoptotic activity of hESCs in EBs, MS-incorporated EBs, and AuMS-incorporated EBs
The viability of hESCs in EBs, MS-incorporated EBs, and AuMS-incorporated EBs was analyzed by the live and dead assay. Live and dead cells were detected with fluorescein diacetate (FDA; Sigma) and ethidium bromide (Sigma), respectively, after 24 h of culture. EBs, MS-incorporated EBs, and AuMS-incorporated EBs were incubated in the FDA/ethidium bromide (5 and 10 mg/mL, respectively) solution for 5 min at 37C and then washed twice in PBS. Dead cells were stained orange due to the nuclear permeability to ethidium bromide. Viable cells, capable of converting the nonfluorescent FDA into fluorescein, stained green. After staining, the samples were examined using a fluorescence microscope (Model IX71; Olympus). The cell viability was quantified by (green + red) area divided by red
GOLD MICROSPHERES ENHANCE CARDIOMYOGENIC DIFFERENTIATION
area and expressed in percentage. The apoptotic activity of hESCs in EBs, MS-incorporated EBs, and AuMS-incorporated EBs was analyzed by reverse transcription–polymerase chain reaction (RT-PCR). Total RNA was extracted from the EB, EB-MS, and EB-AuMS on day 1. RNA was reversetranscribed into cDNA. The expression of BCL-XL, BAX, and b-ACTIN mRNA was evaluated by RT-PCR using selfprepared primers and determined by subsequent agarose gel electrophoresis. PCR was carried out for 40 cycles consisting of denaturing (94C, 30 s), annealing (60C, 30 s), and extension (72C, 45 s), with final extension at 72C for 7 min. The PCR products were visualized by electrophoresis on 1.5% (w/v) agarose gels with ethidium bromide staining. Gels were read using a gel documentation system (GL 2200 PRO Imaging system; CARESTREAM, Rochester, NY). The primer sequences are shown in Table 1 Cardiomyogenic differentiation of hESCs
EB, EB-MS, and EB-AuMS were induced to undergo cardiomyogenic differentiation, as previously described.14 Briefly, EBs, EB-MS, and EB-AuMS were transferred to a low-attachment culture dish (BD Bioscience), then treated with the following cytokines: BMP4 (0.5 ng mL - 1) on day 0–1; BMP4 (10 ng mL - 1), FGF2 (5 ng mL - 1), and activin A (3 ng mL - 1) on day 1–4; DKK1 (150 ng mL - 1) and VEGF (10 ng mL - 1) on day 4–8; VEGF (10 ng mL - 1), DKK1 (150 ng mL - 1), and FGF2 (5 ng mL - 1) on day 8–14. All cytokines were purchased from R&D systems (Minneapolis, MN). Quantitative real-time RT-PCR of hESCs
On day 4 and 14, the mRNA expression of hESCs was analyzed using quantitative real-time RT-PCR (qRT-PCR). On day 4, the expression of mesodermal, endodermal, and ectodermal genes was evaluated. On day 14, cardiac and extracellular matrix (ECM) gene expression was evaluated. Total RNA was extracted from the differentiated hESCs. RNA was reverse-transcribed into cDNA. The expression of mRNA was evaluated by qRT-PCR. qRT-PCR was performed using the StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA) with FAST SYBR Green PCR master mix (Applied Biosystems). Each cycle consisted of the following times and temperatures: 94C for 3 s and 60C for 30 s. The primer sequences are shown in Table 1. Immunocytochemistry of cardiomyogenically differentiated hESCs
After the induction of cardiomyogenic differentiation for 14 days, EB, EB-MS, and EB-AuMS were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. For immunocytochemistry, hESCs in EB, EB-MS, and EB-AuMS were stained with the anti-human cardiac troponin T (cTnT) antibody (Abcam, Cambridge, United Kingdom). The staining result was visualized using the fluorescein isothiocyanateconjugated secondary antibody ( Jackson Immuno Research Laboratories, West Grove, PA). The cells were counterstained with DAPI (Vector Laboratories, Burlingame, CA) and examined using a fluorescence microscope (Model IX71; Olympus). The relative cardiomyocyte yield was determined by dividing cTnT( + ) area by DAPI( + ) area ([cTnT( + ) area/DAPI( + ) area] · 100%).
3
Table 1. Human-Specific Primers Used for RT-PCR and qRT-PCR Analyses Gene
Primer
b-actin
Sense 5¢- GCA CTC TTC CAG CCT TCC TTC C -3¢ Antisense 5¢- TCA CCT TCA CCG TTC CAG TTT TTT -3¢ BAX Sense 5¢- GTG CAC CAA GGT GCC GGA AC -3¢ Antisense 5¢- TCA GCC CAT CTT CTT CCA GA -3¢ BCL-XL Sense 5¢- CGG GCA TTC AGT GAC CTG AC -3¢ Antisense 5¢- TCA GGA ACC AGC GGT TGA AG -3¢ GAPDH Sense 5¢- GTC GGA GTC AAC GGA TTT GG -3¢ Antisense 5¢- GGG TGG AAT CAA TTG GAA CAT -3¢ BRACHYURY Sense 5¢- CAG TGA CTT TTT GTC GTG GCA -3¢ (T) Antisense 5¢- CCA ACT GCA TCA TCT CCA CA -3¢ M-cadherin Sense 5¢- CGC AAC TGG ACA TGC CAC T -3¢ Antisense 5¢- AGA GAA ACT CTT CCC ACC CC -3¢ MESP2 Sense 5¢- CGC CTG GGC ATC TTC TAC TAA -3¢ Antisense 5¢- AGG GAA GGT ACC CGC TAT CTA -3¢ AFP Sense 5¢- TTC CAT AAG GAT CTG TGC CA -3¢ Antisense 5¢- TCA GCA AAG CAG ACT TCC TGT -3¢ TUBB3 Sense 5¢- TTC CTG CAC TGG TAC ACG G -3¢ Antisense 5¢- TGC GAG CAG CTT CAC TTG -3¢ NKX2-5 Sense 5¢-GCA GAG ACC TCC CGT TTT GTT -3¢ Antisense 5¢- GCC ACC GAC ACG TCT CAC T -3¢ a-MHC Sense 5¢-GCC CCG CCC CAC AT -3¢ Antisense 5¢- CCG GAT TCT CCC GTG ATG -3¢ b-MHC Sense 5¢- CCA CCC AAG TTC GAC AAA ATC -3¢ Antisense 5¢- CGT AGC GAT CCT TGA GGT TGT A -3¢ cTnT Sense 5¢- CAG GAT CAA CGA TAA CCA GAA AGT C -3¢ Antisense 5¢- GTG AAG GAG GCC AGG CTC TA -3¢ CONNEXIN 43 Sense 5¢- ACT GGC GAC AGA AAC AAT TCT TC -3¢ Antisense 5¢- TTC TGC ACT GTA ATT AGC CCA GTT -3¢ COLLAGEN Sense 5¢- CAG CCG CTT CAC CTA CAG C -3¢ TYPE I Antisense 5¢- TTT TGT ATT CAA TCA CTG TCT T -3¢ COLLAGEN Sense 5¢- GGG AAT GGA GCA AAA CAG TCT T -3¢ TYPE III Antisense 5¢- CCA ACG TCC ACA CCA AAT TCT -3¢ COLLAGEN Sense 5¢- TGT CCA ATA TGA AAA CCG TAA TYPE IV AGT G -3¢ Antisense 5¢- CAC TAT TGA AAG CTT ATC GCT GTC TT-3¢ FIBRONECTIN Sense 5¢- TCC ACG GGA GCC TCG AA -3¢ Antisense 5¢- ACA ACC GGG CTT GCT TTG -3¢ LAMININ Sense 5¢- CAC AAC AAC ATT GAC ACG ACA GA -3¢ Antisense 5¢- GCT GGA GGG CAT CAC CAT AGT -3¢
RT-PCR, reverse transcription–polymerase chain reaction; qRT-PCR, quantitative real-time RT-PCR.
Statistical analysis
All quantitative data are expressed as the mean – standard deviation. Statistical analysis was performed by analysis of variance using a Bonferroni test. A p-value < 0.05 was considered statistically significant. Results
The fabricated MS and AuMS had a spherical morphology (Fig. 1A). The diameter was approximately 5–15 mm. EDS analysis showed that gold was coated on the PCL MS surface in AuMS (Fig. 1B). EDS spectra confirmed gold coating on AuMS, whereas gold element was not detected on MS (Fig. 1C).
4
LEE ET AL.
FIG. 1. Preparation and characterization of gold-coated polycaprolactone (PCL) microspheres (MS). (A) SEM images of noncoated PCL MS and gold-coated PCL microspheres (AuMS). (B) Energy-dispersive spectroscopy (EDS) analysis for gold element on the surfaces of MS and AuMS. Yellow indicates gold. Yellow dots in the MS were background. Scale bars indicate 10 mm in the upper panel and 2 mm in the lower panel in (A) and 8 mm in (B). (C) EDS spectra of AuMS and MS. SEM, scanning electron microscopy. Color images available online at www.liebertpub.com/tea A ratio of hESCs:AuMS of 1:4 was used to incorporate AuMS into hESC-derived EBs. A ratio of hESCs:AuMS of 1:2 did not exhibit positive effects of AuMS on cardiomyogenic differentiation (data not shown). After 24 h, EBs, MS-incorporated EBs (EB-MS), and AuMS-incorporated
EBs (EB-AuMS) had a spherical shape (Fig. 2A). To confirm dispersion of MS and AuMS in EBs, MS and AuMS were labeled with the red fluorescence dye, DiI, before incorporation into EBs. In MS-incorporated EBs, MS and AuMS were dispersed throughout the EBs (Fig. 2B). The
FIG. 2. Incorporation of PCL MS or gold-coated PCL microsphere (AuMS) into hESC-derived embryoid bodies (EBs). (A) Light microscopic images of EBs and EBs incorporated with either PCL microspheres (EB-MS) or gold-coated PCL microspheres (EB-AuMS) on day 1. (B) Merged images of EBs and EBs incorporated with either DiI(red)-labeled MS or AuMS on day 1, showing incorporation of MS into hESC-derived EBs. (C) Fluorescence images of EBs and EBs incorporated with DiI(red)-labeled AuMS. The nuclei were stained with DAPI (blue). (D) SEM images of surface and cross section of EB and EB-AuMS. (E) A TEM image of EB-AuMS. Black dots (arrow) indicate gold. Scale bars indicate 200 mm in (A) and (B), 100 mm in (C), 10 mm in (D), and 100 nm in (E). Color images available online at www.liebertpub.com/tea
GOLD MICROSPHERES ENHANCE CARDIOMYOGENIC DIFFERENTIATION
5
FIG. 3. Effects of AuMS incorporated into EBs on viability and apoptotic activity of hESCs in EB-AuMS. (A) Fluorescence images of EBs and EBs incorporated with either MS (EB-MS) or AuMS (EB-AuMS) and stained with FDA and EB on day 1. Green and orange-red colors indicate viable and dead cells, respectively. The scale bars indicate 200 mm. (B) Reverse transcription– polymerase chain reaction (RT-PCR) analysis for proapoptotic (BAX) and antiapoptotic (BCL-XL) mRNA expression of EBs and EBs incorporated with either MS (EB-MS) or AuMS (EB-AuMS) on day 1. Color images available online at www .liebertpub.com/tea
fluorescence microscopic examination confirmed the dispersion of AuMS in EBs (Fig. 2C). SEM images showed that EB and EB-AuMS had a spherical shape, and the core of the EB was filled with hESCs and/or AuMS (Fig. 2D). The TEM examination revealed that gold was coated on the surface of AuMS and was in contact with hESCs in EBAuMS (Fig. 2E). To determine whether AuMS incorporation into EBs affects the viability and apoptotic activity of hESCs in the EBs, the viability and apoptotic activity of hESCs in EB, EB-MS, and EB-AuMS were compared. Viability was analyzed based on live and dead assays using FDA/ethidium bromide staining. It showed no difference in hESC viability among EB, EB-MS, and EB-AuMS (96.5% – 0.5%, 96.1% – 1.5%, and 96.3% – 0.9% respectively, Fig. 3A). In addition, the expression of an apoptotic gene (BAX) and an antiapoptotic gene (BCL-XL) did not differ among EB, EBMS, and EB-AuMS (Fig. 3B). In the process of cardiomyogenic differentiation of hESCs in EBs, hESCs first differentiated into mesodermal cells and endodermal cells, and then the mesodermal cells differentiated into cardiogenic lineages (Fig. 4).21 Endodermal cells promote the mesodermal cell differentiation into cardiogenic lineages by interacting with the mesodermal cells.21 The mRNA expression of mesoderm, endoderm,
and ectodermal genes was evaluated on day 4. The dispersion of AuMS in hESC-derived EBs enhanced the expression of mesodermal genes (T and M-cadherin), a cardiac mesoderm gene (MESP2), and an endodermal gene (AFP) compared to the other groups (Fig. 5A–C). However, ectodermal gene (TUBB3) expression was not significantly different among all groups (Fig. 5D). We examined whether incorporation of AuMS into hESCderived EBs enhances cardiomyogenic gene expression of the hESCs by qRT-PCR assay on day 14. The EB-AuMS group exhibited the upregulated expression of cardiomyogenic transcriptional factor (NKX2-5), early cardiomyocyte markers (a-MHC, b-MHC), a late cardiomyogenic marker (cTnT), and a gap junction gene (CONNEXIN 43) compared with the other groups (Fig. 6A–D). Immunocytochemistry revealed that the cells in the EB-AuMS group expressed a higher amount of cTnT protein compared with the other groups (Fig. 6E). The relative cardiomyocyte yields, which were determined by dividing the cTnT( + ) area by DAPI( + ) area, were 3.4% in EB, 10.3% in EM-MS, and 28.6% in EB-AuMS. The incorporation of AuMS into hESC-derived EBs also enhanced gene expression of cardiac-specific ECMs of the cells. Cardiomyocytes derived from hESCs were reported to be encased in a network of ECMs such as fibronectin,
FIG. 4. A schematic diagram describing a stepwise model for in vitro cardiomyogenic differentiation of hESCs in EBs and soluble factors used in each differentiation stage, based on Moon et al.21 Color images available online at www.liebertpub.com/tea
6
LEE ET AL.
FIBRONECTIN, and LAMININ) genes compared with the other groups (Fig. 7). Discussion
FIG. 5. Enhanced mesodermal, cardiac mesodermal, and endodermal differentiation of hESCs in EBs by AuMS incorporation into EBs. (A) Mesodermal, (B) cardiac mesodermal, (C) endodermal, and (D) ectodermal gene expressions in EBs and EBs incorporated with either MS (EB-MS) or AuMS (EBAuMS) were evaluated by quantitative real-time RT-PCR (qRT-PCR) on day 4. The values were normalized to the levels of the EB group (n = 3, *p < 0.05 vs. EB, #p < 0.05 vs. EB-MS). Color images available online at www.liebertpub.com/tea laminin, and collagen, which are the major components of the cardiac ECMs.22,23 The qRT-PCR assay on day 14 showed that cells in the EB-AuMS group expressed significantly higher levels of cardiac-specific ECM (COLLAGEN TYPE I, COLLAGEN TYPE III, COLLAGEN TYPE IV,
Culture on electrically conductive scaffolds (nanofibers and gels) enhanced cardiomyogenic differentiation of multipotent MSCs and a differentiated phenotype of cardiomyocytes. This approach can be applied to cardiomyogenic differentiation of pluripotent hESCs. However, these scaffolds would not be appropriate for in vitro cardiomyogenic differentiation of hESCs because this phenomenon is achieved by culture in the form of EBs. Incorporation of electrically conductive MS within hESC-derived EBs would be an efficient way to culture hESCs in contact with electrically conductive scaffolds. In this study, we successfully fabricated electrically conductive MS by coating the MS surface with gold. The incorporation of AuMS in hESC-derived EBs significantly enhanced mesodermal and endodermal lineage differentiation (Fig. 5) and cardiac lineage differentiation (Fig. 6). Compared to the previous Yang protocol (the EB group in this study), AuMS incorporation upregulated mRNA expression of NKX2-5 by 450%, a-MHC by 230%, b-MHC by 1300%, cTnT by 30%, and CONNEXIN 43 by 49% (Fig. 6A–D). The enhancement of mesodermal lineage differentiation by AuMS promoted cardiomyogenic differentiation of hESCs. In the cardiomyogenic differentiation process of hESCs, six differentiation steps were proposed, which were epithelial to mesenchymal transition, mesoderm differentiation, mesoderm speciation, cardiac specification, cardiomyocyte differentiation, and electrical maturation.24 The mesoderm specification includes mesodermal and cardiac mesodermal differentiation.24 Therefore, mesoderm and cardiac mesodermal differentiation is one of the important steps in cardiomyogenic differentiation of hESCs. EB-AuMS enhanced the expression of mesoderm (T- and M-cadherin) and cardiac mesoderm genes (MESP2) compared to the other groups, including the previous protocol (Yang protocol, the EB group in this study, Fig. 5A, B). MESP2 was reported to be essential for cardiac mesoderm development.25 Therefore,
FIG. 6. Enhanced cardiac lineage differentiation of hESCs in EBs by AuMS incorporation into EBs. mRNA expressions of (A) cardiac-specific transcription factors, (B) early cardiomyocyte markers, (C) late cardiomyocyte marker, and (D) a cell–cell junction protein of EBs and EBs incorporated with either MS (EB-MS) or AuMS (EB-AuMS) were evaluated by qRT-PCR on day 14. The values were normalized to the levels of the EB group (n = 3, *p < 0.05 vs. EB, #p < 0.05 vs. EB-MS). (E) Immunocytochemistry for cTnT (green), a late cardiomyocyte marker, in EB, EB-MS, and EB-AuMS at day 14. The nuclei were stained with DAPI (blue). The scale bars indicate 100 mm. Color images available online at www.liebertpub.com/tea
GOLD MICROSPHERES ENHANCE CARDIOMYOGENIC DIFFERENTIATION
7
FIG. 7. Enhanced mRNA expression of cardiacspecific ECMs in hESCderived EBs by AuMS incorporation into EBs. The mRNA expressions of cardiac-specific ECMs in EBs and EBs incorporated with either MS (EB-MS) or AuMS (EBAuMS) were evaluated by qRT-PCR at day 14. The values were normalized to the levels of the EBs (n = 3, *p < 0.05 vs. EB, #p < 0.05 vs. EB-MS). Color images available online at www .liebertpub.com/tea the enhanced cardiomyogenic differentiation of hESCs by AuMS (Fig. 6) may be attributed to the enhanced mesodermal and cardiac mesodermal lineage gene expression. Enhancement of endodermal differentiation by AuMS may also contribute to the enhanced cardiomyogenic differentiation of hESCs. It has been shown that endodermal cells around mesodermal cells induce cardiomyogenic differentiation of the mesodermal cells in the process of cardiomyogenic differentiation of hESCs.21 During cardiomyogenic differentiation of hESCs, endoderm markers were reported to increase concurrently with mesoderm markers.26 In another study, coculture with an endoderm-derived cell line (END-2 cells) induced cardiac differentiation of hESCs.9 These studies suggest that enhanced endodermal differentiation promotes cardiomyogenic differentiation of hESCs in the cardiomyogenic differentiation process. AuMS can be used as an electrically conductive material to enhance the cardiomyogenic cell phenotype. Previously, cardiomyocyte culture in gold nanoparticle-dispersed hydroxyethyl methacrylate hydrogels enhanced CONNEXIN 43 expression by 60% compared to culture in nonconductive hydrogels.16 In this study, AuMS enhanced CONNEXIN 43 expression in hESCs by 50% compared to the EB group (Fig. 6D). CONNEXIN 43 regulates cell–cell communication, which influences electrical coupling, and promotes contractile behavior.27–29 CONNEXIN 43 helps cardiac cells sense and respond to their in vivo environment.16 Therefore, the use of AuMS for in vitro cardiomyogenic differentiation of hESCs may improve electrical coupling of the hESC-derived cells with recipient myocardial cells following transplantation through the enhanced CONNEXIN 43 expression. Also, AuMS promoted cardiomyogenic differentiation of hESCs. Previously, it was reported that culture of human MSCs on composite scaffolds of PCL and carbon nanotube (CNT) enhanced expression of cardiomyogenic genes and proteins.30 CNT offers electrical conductivity to PCL scaffold,30 indicating that conductive materials may enhance the cardiomyogenic differentiation of stem cells. This, gold in the AuMS, may play a role in the enhanced cardiomyogenic differentiation of hESCs. In summary, incorporation of AuMS into hESC-derived EBs was an efficient way to promote the cardiomyogenic differentiation process of hESCs. On day 4, AuMS promoted differentiation of hESCs into mesodermal, cardiac
mesodermal, and endodermal lineages, which are the intermediate cells in the cardiomyogenic differentiation process of hESCs. On day 14, AuMS enhanced the expression of cardiomyogenic differentiation markers. In addition, AuMS enhanced gene expression of cardiac-specific ECMs. However, we did not observe beating cells following induction of cardiomyogenic differentiation with AuMS, indicating that our method does not induce full differentiation into contractile cardiomyocytes. Incorporation of AuMS into hESC-derived EBs could be a new platform to promote the cardiomyogenic differentiation process of hESCs. Acknowledgments
This study was supported by a grant (2013036054) from the National Research Foundation of Korea and a grant (HI12C0199) from the Korean Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea. Disclosure Statement
No competing financial interests exist. References
1. Habib, M., Caspi, O., and Gepstein, L. Human embryonic stem cells for cardiomyogenesis. J Mol Cell Cardiol 45, 462, 2008. 2. Orlic, D., Kajstura, J., Chimenti, S., Jakoniuk, I., Anderson, S.M., Li, B., Pickel, J., McKay, R., Nadal-Ginard, B., Bodine, D.M., Leri, A., and Anversa, P. Bone marrow cells regenerate infarcted myocardium. Nature 410, 701, 2001. 3. Tang, X.L., Rokosh, D.G., Guo, Y., and Bolli, R. Cardiac progenitor cells and bone marrow-derived very small embryonic-like stem cells for cardiac repair after myocardial infarction. Circ J 74, 390, 2010. 4. Planat-Be´nard, V., Menard, C., Andre´, M., Puceat, M., Perez, A., Garcia-Verdugo, J.M., Pe´nicaud, L., and Casteilla, L. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res 94, 223, 2004. 5. Unno, K., Jain, M., and Liao, R. Cardiac side population cells: moving toward the center stage in cardiac regeneration. Circ Res 110, 1355, 2012. 6. Schlechta, B., Wiedemann, D., Kittinger, C., Jandrositz, A., Bonaros, N.E., Huber, J.C., Preisegger, K.H., and Kocher, A.A. Ex-vivo expanded umbilical cord blood stem cells
8
7. 8.
9.
10.
11.
12.
13.
14.
15.
16. 17. 18.
19.
LEE ET AL.
retain capacity for myocardial regeneration. Circ J 74, 188, 2010. Mignone, J.L., Kreutziger, K.L., Paige, S.L., and Murry, C.E. Cardiogenesis from human embryonic stem cells. Circ J 74, 2517, 2010. He, J.Q., Ma, Y., Lee, Y., Thomson, J.A., and Kamp, T.J. Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization. Circ Res 93, 32, 2003. Mummery C., Ward-van Oostwaard, D., Doevendans, P., Spijker, R., van den Brink, S., Hassink, R., van der Heyden, M., Opthof, T., Pera, M., de la Riviere, A.B., Passier, R., and Tertoolen, L. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107, 2733, 2003. Kehat, I., Khimovich, L., Caspi, O., Gepstein, A., Shofti, R., Arbel, G., Huber, I., Satin, J., Itskovitz-Eldor, J., and Gepstein, L. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol 22, 1282, 2004. Xue, T., Cho, H.C., Akar, F.G., Tsang, S.Y., Jones, S.P., Marba´n, E., Tomaselli, G.F., and Li, R.A. Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular cardiomyocytes: insights into the development of cell-based pacemakers. Circulation 111, 11, 2005. Shiba, Y., Fernandes, S., Zhu, W.Z., Filice, D., Muskheli, V., Kim, J., Palpant, N.J., Gantz, J., Moyes, K.W., Reinecke, H., Van Biber, B., Dardas, T., Mignone, J.L., Izawa, A., Hanna, R., Viswanathan, M., Gold, J.D., Kotlikoff, M.I., Sarvazyan, N., Kay, M.W., Murry, C.E., and Laflamme, M.A. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature 489, 322, 2012. Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., and Benvenisty, N. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 6, 88, 2000. Yang, L., Soonpaa, M.H., Adler, E.D., Roepke, T.K., Kattman, S.J., Kennedy, M., Henckaerts, E., Bonham, K., Abbott, G.W., Linden, R.M., Field, L.J., and Keller, G.M. Human cardiovascular progenitor cells develop from a KDR + embryonic-stem-cell-derived population. Nature 453, 524, 2008. Orza, A., Soritau, O., Olenic, L., Diudea, M., Florea, A., Rus Ciuca, D., Mihu, C., Casciano, D., and Biris, A.S. Electrically conductive gold-coated collagen nanofibers for placental-derived mesenchymal stem cells enhanced differentiation and proliferation. ACS Nano 5, 4490, 2011. You, J.O., Rafat, M., Ye, G.J., and Auguste, D.T. Nanoengineering the heart: conductive scaffolds enhance connexin 43 expression. Nano Lett 11, 3643, 2011. Tang, Z.G., Callaghan, J.T., and Hunt, J.A. The physical properties and response of osteoblasts to solution cast films of PLGA doped polycaprolactone. Biomaterials 26, 6618, 2005. Lam, C.X., Hutmacher, D.W., Schantz, J.T., Woodruff, M.A., and Teoh, S.H. Evaluation of polycaprolactone scaffold degradation for 6 months in vitro and in vivo. J Biomed Mater Res A 90, 906, 2009. Chen, G., Gulbranson, D.R., Hou, Z., Bolin, J.M., Ruotti, V., Probasco, M.D., Smuga-Otto, K., Howden, S.E., Diol, N.R., Propson, N.E., Wagner, R., Lee, G.O., AntosiewiczBourget, J., Teng, J.M., and Thomson, J.A. Chemically
20.
21.
22.
23. 24.
25. 26.
27.
28.
29.
30.
defined conditions for human iPSC derivation and culture. Nat Methods 8, 424, 2011. Kang, S.W., Yang, H.S., Seo, S.W., Han, D.K., and Kim, B.S. Apatite-coated poly(lactic-co-glycolic acid) microspheres as an injectable scaffold for bone tissue engineering. J Biomed Mater Res A 85, 747, 2008. Moon, S.H., Ban, K., Kim, C., Kim, S.S., Byun, J., Song, M.K., Park, I.H., Yu, S.P., and Yoon, Y.S. Development of a novel two-dimensional directed differentiation system for generation of cardiomyocytes from human pluripotent stem cells. Int J Cardiol 168, 41, 2013. van Laake, L.W., van Donselaar, E.G., Monshouwer-Kloots, J., Schreurs, C., Passier, R., Humbel, B.M., Doevendans, P.A., Sonnenberg, A., Verkleij, A.J., and Mummery, C.L. Extracellular matrix formation after transplantation of human embryonic stem cell-derived cardiomyocytes. Cell Mol Life Sci 67, 277, 2010. Moore, L., Fan, D., Basu, R., Kandalam, V., and Kassiri, Z. Tissue inhibitor of metalloproteinases (TIMPs) in heart failure. Heart Fail Rev 17, 693, 2012. Burridge, P.W., Keller, G., Gold, J.D., and Wu, J.C. Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell 10, 16, 2012. Kitajima, S., Takagi, A., Inoue, T., and Saga, Y. MesP1 and MesP2 are essential for the development of cardiac mesoderm. Development 127, 3215, 2000. Bettiol, E., Sartiani, L., Chicha, L., Krause, K.H., Cerbai, E., and Jaconi, M.E. Fetal bovine serum enables cardiac differentiation of human embryonic stem cells. Differentiation 75, 669, 2007. Oyamada, M., Kimura, H., Oyamada, Y., Miyamoto, A., Ohshika, H., and Mori, M. The expression, phosphorylation, and localization of connexin 43 and gap-junctional intercellular communication during the establishment of a synchronized contraction of cultured neonatal rat cardiac myocytes. Exp Cell Res 212, 351, 1994. Ando, M., Katare, R.G., Kakinuma, Y., Zhang, D., Yamasaki, F., Muramoto, K., and Sato, T. Efferent vagal nerve stimulation protects heart against ischemia-induced arrhythmias by preserving connexin43 protein. Circulation 112, 164, 2005. Bupha-Intr, T., Haizlip, K.M., and Janssen, P.M. Temporal changes in expression of connexin 43 after load-induced hypertrophy in vitro. Am J Physiol Heart Circ Physiol 296, H806, 2009. Crowder, S.W., Liang, Y., Rath, R., Park, A.M., Maltais, S., Pintauro, P.N., Hofmeister, W., Lim, C.C., Wang, X., and Sung, H.J. Poly(e-caprolactone)-carbon nanotube composite scaffolds for enhanced cardiac differentiation of human mesenchymal stem cells. Nanomedicine (Lond) 8, 1763, 2013.
Address correspondence to: Byung-Soo Kim, PhD School of Chemical and Biological Engineering Seoul National University Seoul 151-744 Republic of Korea E-mail: [email protected] Received: January 7, 2014 Accepted: July 23, 2014 Online Publication Date: September 5, 2014
