Paracrine Effect of Human Adipose-Derived Stem Cells on Lymphatic Endothelial Cells

Figures & data

Table 1. Experimental conditions used for LEC growth.

	Culture media
DMEM0.1%	DMEM + 0.1% FBS
DMEM10%	DMEM + 10% FBS
hASC-CM	Conditioned hASC medium with added DMEM + 0.1% FBS (1:1)

DMEM: Dulbecco’s modified Eagle medium; FBS: Fetal bovine serum; hASC-CM: Human adipose-derived stem cell-conditioned medium; LEC: Lymphatic endothelial cell.

Table 2. Primers used in this work.

Gene name	Accession number	Primer sequence 5′–3′	Melting temperature (°C)
Rn_ GAPDH	AF106860.2	Fw CATCACCATCTTCCAGGA	59.8
		Rev GACTCCACGACATACTCA	59.5
Rn_ β2M	NM_012512.2	Fw GGTGACCGTGATCTTTCT	60.4
		Rev GGCGAGAGTACACTTGAA	60.4
Rn_ RPL13	NM_031101.1	Fw GAGGTGCCCTACAGTTAG	60.2
		Rev TTCTTGTGGATACCAGCC	60.5
Rn_ PROX1	NM_00107201.1	Fw CCAAGGTTCAGAGCAGGATG	60.8
		Rev CATGGCATCTTCATACGAGTTC	59.6
Rn_ VEGFR3	AF402786.1	Fw GTCTTCTTCTGGGTCCTCCTCC	62.7
		Rev GGGTCCATGATGATGGACAG	61.2
Hs_ VEGFA ^†	NM_001025366.2	Fw CAAGTGGTCCCAGGCTGC	62.9
		Rev CTGGAAGATGTCCACCAGGG	62.8
Hs_ VEGFC ^†	NM_005429.4	Fw TGTGTCCGTCTACAGATGT	61.9
		Rev GGCAGGAAGTGTGATTGG	62.2
Hs_ IL6 ^†	M14584.1	Fw ACTCACCTCTTCAGAACG	60.4
		Rev CCTCTTTGCTGCTTTCAC	60.0

† For these genes, primers were designed in a conserved region between human and rat.

Figure 1. ELISA quantification of VEGFA, VEGFC and IL6.

(A) VEGFA, VEGFC protein evaluation in human adipose-derived stem cell-conditioned medium. (B) IL6 protein evaluation in human adipose-derived stem cell-conditioned medium. Results given in pg/ml, are expressed as mean ± SE. n = 3 subjects.

Figure 2. Effect of human adipose-derived stem cell-conditioned medium treatment on lymphatic endothelial cell culture.

(A, C & E) Representative images of cells after 24h of serum starvation (t0). (B) Cells maintained in Dulbecco’s modified Eagle medium (DMEM)_0.1% for 48h of culture (t48). (D) Cells maintained for 48h of culture in DMEM_10%. (F & G) Cells maintained for 48h of culture in human adipose-derived stem cell-conditioned medium. (G) Lymphatic endothelial cells maintained in human adipose-derived stem cell-conditioned medium were able to organize vessel-like structure (*). Scalebar 200 µm.

Figure 3. Evaluation of lymphatic endothelial cell density.

Cells, expressed as number of cells/mm², are counted at t0, baseline condition, and t48. For DMEM_0.1% and DMEM_10%, 20 fields/each were evaluated; for hASC-CM, 48 fields/each were analyzed.

**Data statistically significant, p < 0.01, paired t-test t48 vs proper t0 n = 5 for DMEM_0.1% and DMEM_10%, n = 12 for hASC-CM.

^§§Data statistically significant, p < 0.01, unpaired t-test vs DMEM_0.1% t48 h, n = 17.

^##Data statistically significant, p < 0.01, unpaired t-test vs DMEM_10% t48 h n = 17.

DMEM: Dulbecco’s modified Eagle medium; hASC-CM: Human adipose-derived stem cell conditioned medium.

Figure 4. Evaluation of lymphatic endothelial cell density.

hASC-CM: Human adipose-derived stem cell conditioned medium.

*p < 0.05.

Figure 5. Immunostaining assay of lymphatic endothelial cells and vessel like structures.

Representative image of positive LYVE1 expressing cells (red staining) in t48 human adipose-derived stem cell conditioned medium treated lymphatic endothelial cells. Blue DAPI staining indicates nuclei. (a’) shows higher magnification of (a), which highlights the red staining delimiting the borders of vessel-like structure.

* = newly formed vessel-like structure.

Figure 6. Gene expression evaluation.

Prox1 and VegfR3 expression were significantly upregulated by hASC-CM treatment.

**p < 0.01 and *p < 0.05, respectively vs DMEM_10%, unpaired t-test, n = 12.

DMEM: Dulbecco’s modified Eagle medium; hASC-CM: Human adipose-derived stem cell conditioned medium.