Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture

Table 1 Propagation and differentiation of HES3 to cardiomyocytes using different culture methods

Culture conditions	MNL hESC cultures	hESC/MC aggregate spinner cultures
Propagation (7 days)
Expansion fold (propagation)	10.3 ± 0.4	15.4 ± 0.4
% Pluripotent cells
Tra-1-60-positive	93.1 ± 1.4	96.2 ± 1.7
mAb84-positive	99.4 ± 0.5	98.6 ± 1.7
Aggregate sizes (μm)	NA	316 ± 11
Differentiation to cardiomyocytes (20 days)
	MNL	MC-Rp	MC-SS	MC-AS	MC-Sp
Expansion fold (differentiation)	2.0 ± 0.2	2.2 ± 0.1	2.1 ± 0.2	2.0 ± 0.2	2.3 ± 0.1
% beating aggregates	Beating sheet	65.7 ± 1.8 (beating foci on plate)	66.5 ± 2.6	53.4 ± 1.5	73.6 ± 2.8
Beating aggregate size (μm)	NA	NA	1063 ± 53	581 ± 54	655 ± 13
% Cardiomyocytes
MHC-positive	45.7 ± 4.6	43.1 ± 1.2	42.6 ± 1.9	42.8 ± 1.6	47.7 ± 1.9
cTnT-positive	51.1 ± 0.5	53.1 ± 0.9	45.7 ± 2.9	42.4 ± 0.2	56.1 ± 1.4
CM/hESC	3.8 ± 0.2	1.2 ± 0.2	1.0 ± 0.2	0.8 ± 0.1	9.6 ± 0.3^a
CM/ml (×x10⁶)	0.5 ± 0.02	0.2 ± 0.02	0.1 ± 0.02	0.1 ± 0.01	1.9 ± 0.05^b
Total expansion fold (propagation + differentiation)	20.0 ± 0.8	32.8 ± 1.1	31.8 ± 2.1	29.3 ± 2.5	34.3 ± 0.9^c

HES3 cells were propagated as either monolayer cultures (MNL) or human embryonic stem cell (hESC)/microcarrier (MC) aggregates for 7 days. Five differentiation culture regimes were tested. Data presented are mean ± standard deviation (n = 3 to 4). In HES3 cultures: ^asignificant difference between MC-Sp and MNL (P < 0.05), and between MC-Sp and the other three regimes (P < 0.01), in terms of cardiomyocytes produced per initial hESC seeded (CM/hESC); ^bsignificant differences (P < 0.01) between MC-Sp and the other four regimes in terms of cardiomyocyte density (CM/ml); ^csignificant difference between MC-Sp and MNL (P < 0.05) in terms of total expansion fold achieved. CM, number of cardiomyocytes (cTnT-positive cells) in the final cell population; Total expansion fold, cell expansion from seeding the hESC culture in the propagation phase, to final CM harvesting (day 20) in the differentiation phase. cTnT, Cardiac troponin T; MC-AS, hESC/MC aggregates in agitated suspension cultures; MC-Rp, monolayer replated cultures; MC-Sp, hESC/MC aggregates in spinner cultures; MC-SS, hESC/MC aggregates in static suspension cultures; MHC, myosin heavy chain; NA, not available.

ISSN: 1757-6512