Yonsei Med J.  2008 Oct;49(5):819-827. 10.3349/ymj.2008.49.5.819.

Use of Long-term Cultured Embryoid Bodies May Enhance Cardiomyocyte Differentiation by BMP2

• Kim YY
• Ku SY
• Jang J
• Oh SK
• Kim HS
• Kim SH
• Choi YM
• Moon SY

• KMID: 2158201
• DOI: http://doi.org/10.3349/ymj.2008.49.5.819

Abstract

PURPOSE
Human embryonic stem cells (hESCs) can proliferate for a prolonged period and differentiate into cardiomyocytes in vitro. Recent studies used bone morphogenetic protein 2 (BMP2) to generate cardiomyocytes from hESCs, however, all those studies used early embryoid bodies (EBs) and did not retrieve cardiomyocytes with a high yield. In this study, we treated long-term cultured EBs with BMP2 in order to promote differentiation into cardiomyocytes from hESCs. MATERIALS AND METHODS: hESC lines, including SNUhES3 and SNUhES4, were used in this study. Undifferentiated hESC colonies were detached to form EBs and cultured for up to 30 days. These long-term cultured EBs were differentiated into cardiomyocytes in serum-containing media. In our protocol, BMP2 was applied for 5 days after attachment of EBs. Cardiac specific markers, beating of differentiated cells and electron microscopic (EM) ultrastructures were evaluated and analyzed. RESULTS: Compared to 10-day or 20-day EBs, 30-day EBs showed a higher expression level of cardiac specific markers, Nkx2.5 and a-myosin heavy chain (alphaMHC). Treatment of BMP2 increased expression of cardiac troponin (cTn) I and a-actinin when evaluated at 20 days after attachment of 30-day EBs. Beating of differentiated cells was observed from 7 to 20 days after attachment. Moreover, EM findings demonstrated fine structures such as Z bands in these differentiated cardiomyocytes. These long-term cultured EBs yielded cardiomyocytes with an efficiency of as high as 73.6% when assessed by FACS. CONCLUSION: We demonstrated that the use of long-term cultured EBs may enhance differentiation into cardiomyocytes from hESCs when treated with BMP2.

Keyword

Bone morphogenetic protein 2; cardiomyocytes; cell differentiation; embryoid bodies; embryonic stem cells; long-term

MeSH Terms

Bone Morphogenetic Protein 2/*pharmacology
Cell Culture Techniques
*Cell Differentiation
Cell Line
Cell Proliferation
Embryonic Stem Cells/cytology/*drug effects
Humans
Myocytes, Cardiac/*cytology
Pluripotent Stem Cells/cytology/drug effects
Signal Transduction

Figure

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  Fig. 1 A schematic presentation of in vitro differentiation strategy for induction of cardiomyocytes from human embryonic stem cells (hESCs), using long-term cultured embryoid bodies (EBs) treated with BMP2.

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  Fig. 2 Expressions of pluripotent hESC- and cardiac specific markers in hESCs. (upper panel) Alkaline phosphatase (AP), Oct4 and SSEA4 were highly expressed in undifferentiated hESCs, Bars: 500 µm. Lower panel) Tra-1-60 and Tra-1-81 were also highly expressed in undifferentiated hESCs. However, cardiac troponin (cTn) I and α-myosin heavy chain (αMHC), cardiac specific markers, were not expressed in undifferentiated hESCs. Merged picture showed only nuclei stained with DAPI. Bars: 200 µm.

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  Fig. 3 Evaluation of mesoderm- and cardiac-specific markers in long-term cultured EBs. (A) Morphology of a 30-day EB (a) in suspension and (b) after attachment. Bars: 500 µm. (B) Expressions of cardiac lineage markers in spontaneously differentiated cells from SNUhES3 (upper panel) and SNUhES4 (lower panel). At 20 days after plating, differentiated cells from both hESC lines were positively stained with cardiomyocyte-specific markers, Nkx2.5, αMHC and cTn I, however, their expression was not high and showed a scattered pattern. Bars: 200 µm. (C) Relative expression of mesoderm- and cardiac-lineage markers in long-term cultured EBs from SNUhES3 by Q-PCR. 10-, 20- and 30-day EBs were compared in terms of expression of Brachyury, Nkx2.5 and αMHC, which showed the highest level at day 30 for all three markers.

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  Fig. 4 Evaluation of mesoderm- and cardiac specific markers in cardiomyocytes differentiated from BMP2-treated cells (SNUhES3). (A) Morphologies of long-term cultured embryoid bodies (EBs) and cardiomyocytes differentiated from BMP2-treated cells. Phase contrast microscopic appearance of 30-day EBs (a and e) in suspension and (b and f) after attachment, of hESC-derived cardiomyocytes 4 days (c) and 11 days after attachment (g), and contractile clusters derived from BMP2-treated differentiated cells (d and h) Bar: a-g, 500 µm; (h) 200 µm. (B) Expression of cardiac specific markers in differentiated cardiomyocytes. Differentiated cells were evaluated 20 days after plating and nuclei were stained with DAPI. Top panel, expression of transcription factor Nkx2.5 and protein αMHC; Middle panel, expression of Nkx2.5 and cTn I; Bottom panel, expression of Nkx2.5 and α-actinin (arrow of Fig 4A-h). Bars: 100 µm. (C) Expression of Nkx2.5 and smooth muscle actin (SMA), which is abundant in peripheral cells of cardiomyocyte cluster (yellow box of Fig. 4A-g). Bars: 100 µm. (D) Relative expression of cardiac lineage markers evaluated by Q-PCR. Differentiated cells showed significant expression of Brachyury, Nkx2.5 and αMHC. At 10 ng/mL of BMP2, αMHC expression was the highest, and beating cells were observed in this group (Suppl. Fig. 1). (E) Ultrastructures of differentiated cells observed by transmission electron microscopy (TEM). Red arrows, (a) Z bodies (dense spots), which are the precursors of Z bands. (b) unorganized Z bands. Many unorganized, unconnected bands were observed in many parts. (c and d) organized Z bands. Magnification, a, × 12K; b, × 30K; c, × 60K; d, × 100K.

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  Fig. 5 FACS analysis of cardiac lineage markers, Nkx2.5 and αMHC, in BMP2-induced differentiated cells from plated EBs. As high as 73.6% of differentiated cells were positive for both cardiac markers, evaluated 20 days after plating (Suppl. Fig. 1). Moving image of hESC-derived cardiomyocytes from long-term cultured EBs treated with BMP2. Images were recorded under phase contrast microscope (× 200), and contractile region showed regular contractions with a frequency of 1 beat/second.

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