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Cardiac differentiation using either the growth factor– or the small 4 ]- p6 q' Y0 _* l. J4 o# H# |
molecule–based differentiation protocols proceeds rapidly. A rela-
! a+ Z$ p1 ~3 ]' t. M8 C: _tively pure ( >95%) population of brachyury-expressing mesen-
5 C% f# k2 s' C! t1 G$ cdoderm cells can be detected after 1 d of differentiation. Gene 3 ]; f- l# L9 m: r2 `3 c: A
expression of the cardiac transcription factors NKX2-5 (ref. 19)
; q% G! o+ [4 O- X4 oand ISL1 (refs. 20 and 21) begins at day 4, with the protein detect-
) k4 W7 ^% v0 n: T" h9 b/ X& Kable at day 5 (Fig. 2b). Cardiac troponin T (cTnT) can be readily ; A; T" |5 _ P2 L1 h
detected at day 8 of differentiation. The presence of cardiomyocytes 8 C" \# s2 h: t7 i. w
can be easily established by visual observation of spontaneously
3 v% S8 K+ F Y4 B: G0 i9 ~. ocontracting regions. The first beating cluster of cells can be observed % i' M- p' R2 @
between days 8 and day 10, depending on the individual cell line " T9 B) a, z J, E1 ?( I
used. Robust spontaneous contraction occurs by day 12. Cardiac
8 @+ D) R- t4 P; I) n- G" N# Ymarker protein expression after onset of contractions can also be
( W S, } u/ T' z) w& Xassessed by immunostaining or be quantified with flow cytometry. |
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发表于 2016-7-4 09:17
