Inhibition of the RhoGTPase Cdc42 by ML141 enhances hepatocyte differentiation from human adipose-derived mesenchymal stem cells via the Wnt5a/PI3K/miR-122 pathway: impact of the age of the donor

Table 1 Immunophenotyping of hADSCs derived from young and aged subjects and differentiated into hepatoblast- and hepatocyte-like cells

	(−) ML141				(+) ML141 (d–2/14)	(+) ML141 (d14/28)
	D0	D7	D14	D28	D28	D28
Differentiation of hADSCs derived from young subjects
HLA-DR	2.03 ± 0.53	0.02 ± 0.11	2.55 ± 0.11	0.51 ± 0.92
CD14⁺	0.12 ± 0.40	0.14 ± 0.24	0.15 ± 0.38	0.08 ± 0.32
CD34⁺	1.17 ± 1.88	6.03 ± 0.52	3.20 ± 0.68	1.12 ± 0.44
CD45⁺	0.06 ± 0.43	0.07 ± 0.98	0.05 ± 0.12	0.26 ± 0.75
CD73⁺	88.61 ± 3.92	72.30 ± 10.39*	37.33 ± 2.05**	19.14 ± 1.62***	16.44 ± 5.23**	21.36 ± 3.98**
CD90⁺	90.96 ± 6.80	74.43 ± 3.76*	36.41 ± 2.16***	17.09 ± 0.84***	10.38 ± 3.41***	7.33 ± 4.26***
CD105⁺	91.09 ± 7.68	53.07 ± 1.24*	40.25 ± 6.43***	20.72 ± 3.51***	14.25 ± 4.12***	15.13 ± 5.22***
Differentiation of hADSCs derived from aged subjects
HLA-DR	1.44 ± 0.37	0.33 ± 0.10	1.50 ± 0.42	1.61 ± 0.86
CD14⁺	0.11 ± 0.10	0.91 ± 0.82	0.11 ± 0.41	0.42 ± 0.13
CD34⁺	1.43 ± 0.24	1.80 ± 0.39	1.51 ± 0.80	0.98 ± 1.10
CD45⁺	0.02 ± 0.23	0.08 ± 0.34	0.03 ± 0.21	0.12 ± 0.33
CD73⁺	94.68 ± 3.58	85.07 ± 1.04	80.96 ± 4.96*^§§	70.46 ± 2.29*^§§	35.57 ± 0.71*^#	29.43 ± 3.16*^#
CD90⁺	91.41 ± 6.80	80.23 ± 3.76	75.95 ± 2.11*^§§	70.01 ± 4.08*^§§	31.43 ± 1.60*^#	35.95 ± 1.42*^#
CD105⁺	92.88 ± 3.09	71.40 ± 2.36*^§	73.03 ± 2.30*^§§	64.92 ± 3.15*^§§	34.63 ± 3.09*^#	45.57 ± 2.81*^#

Human adipose-derived mesenchymal stem cells (hADSCs) isolated from young and aged donors were cultured and differentiated as described in the Methods for 28 days. Cells were incubated with or without 10 μM ML141 following two protocols of treatment: from day −2 to day 14 of the differentiation (d-2/14) and from day 14 to day 28 of the differentiation (d14/28). Cells were collected at day 0 (MSCs) at the moment of induction of the differentiation, and days 7, 14, and 28 of the differentiation (hepatoblast-like cells and hepatocyte-like cells, respectively). Cells were labeled with fluorescence-coupled antibodies against HLA-DR, CD14, CD34, CD45, CD73, CD90, and CD105, and analyzed using a MACSQuant flow analyzer as indicated in the Methods. The results are expressed as the percentage of cell surface marker per total number of cells, and are the mean ± SEM of 19 and 20 subjects each performed in duplicate in young and aged groups, respectively. Effect of ML141 was evaluated at day 28 of the differentiation. *P < 0.05, **P < 0.01, ***P < 0.005, D14/D28 versus D0; ^§P < 0.05, ^§§P < 0.01, aged versus young; ^#P < 0.01, treated with ML141 versus untreated in the same group

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