Simplified three-dimensional culture system for long-term expansion of embryonic stem cells

Table 1 Mouse primer sets used in quantitative real time polymerase chain reaction

Target-gene	Forward (5’- 3’)	Reverse (5’-3’)	Product size (bp)
Oct4	TCTGTTCCCGTCACTGCTCT	TGTCTACCTCCCTTGCCTTG	96
Nanog	GCAAGCGGTGGCAGAAAAAC	GCAATGGATGCTGGGATACTCA	92
Klf4	AAGCCAAAGAGGGGAAGAAG	CAGTGGTAAGGTTTCTCGCC	146
Sox2	CGAACTGGAGAAGGGGAGAG	AAGCGTTAATTTGGATGGGA	165
Col1	GCAGGTTCACCTACTCTGTC	CTTGCCCCATTCATTTGTCT	62
Col2	ACCCCCAGGTGCTAATGG	AACACCTTTGGGACCATCTTT	76
Myog	CCTAAAGTGGAGATCCTGCG	GTGGGAGTTGCATTCACTGG	147
Nestin	AGACAGTGAGGCAGATGAG	CTCTCAGCTGTGGTGGTGAA	224
Brachyury (T)	CACACCACTGACGCACAC	GAGGCTATGAGGAGGCTTTG	132
FGF5	GCTCAATGATCAGAAGGAGGA	TCAGCTGGTCTTGAATGAGG	175
GATA4	GATGGGACGGGACACTACCTG	ACCTGCTGGCGTCTTAGATTT	309
Gapdh	GCACAGTCAAGGCCGAGAAT	GCCTTCTCCATGGTGGTGAA	151
β-Actin	AGCCATGTACGTAGCCATCC	CTCTCAGCTGTGGTGGTGAA	228

Table 2 Effects of thiol substitutions of Dex-SH on the rate of scaffold degradation

Degree of thiol substitution of Dex-SH	Time in degradation (d)
4%	10 ± 2
6%	13 ± 4
7.5%	22 ± 5
12%	28 ± 6
30%	35 ± 5
33%	37 ± 5
34%	37 ± 5

Table 3 Cell concentration for optimal growth of embryonic stem cells under 2-D and 3-D culture conditions

Culture conditions	Cell concentration
	(cells/mL)	(cells/cm2)
2-D Culture	---	3 × 104 to 5 × 104[33]
3-D Culture	1 × 104	1 × 103

3-D: Three-dimensional; 2-D: Two-dimensional.