Int J Stem Cells.  2020 Jul;13(2):257-267. 10.15283/ijsc20027.

Effect of Conditioned Medium from Human Umbilical Cord-Derived Mesenchymal Stromal Cells on Rejuvenation of Nucleus Pulposus Derived Stem/Progenitor Cells from Degenerated Intervertebral Disc

• Zeng X
• Lin J
• Wu H
• Yu J
• Tu M
• Cheang LH
• Zhang J

• KMID: 2504338
• DOI: http://doi.org/10.15283/ijsc20027

Abstract

Background and Objectives
Mesenchymal stromal cells (MSCs)-based treatment for degeneration of intervertebral disc (IVD) has been proposed recently. We here addressed whether MSC secreted factors can rejuvenate nucleus pulposus- derived stem/progenitor cells from degenerated disc (D-NPSCs) in vitro.
Methods and Results
We analyzed the expression of MSCs and NP cell specific surface markers, pluripotency related genes, multilineage potential and cell proliferative capacity of D-NPSCs upon incubation with the conditioned medium which was collected from the umbilical cord derived MSCs (UCMSCs). Our results indicated that the conditioned medium restore the stemness of D-NPSCs by up-regulating the expression level of CD29 and CD105, pluripotency related genes OCT4 and Nanog, and NP progenitor marker Tie2. The increased stemness was accompanied by promoted cell proliferative capacity and improved osteogenic and chondrogenic differentiation potential.
Conclusions
Our findings suggested that the UCMSCs derived conditioned medium might be used to rejuvenate the degenerated NP stem/progenitor cells.

Keyword

Conditioned medium; Mesenchymal stromal cells; Nucleus pulposus derived stem/progenitor cells; Intervertebral disc degeneration

Figure

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  Fig. 1 The MSC and NP cell surface markers expression were analysis by flow cytometry after 72 h treatment with UCMSCs-CM. (A) MSC positive surface markers, (B) MSC negative surface markers, (C) NP cell surface marker CD24. The data are presented as the means±SD, n=3. *p<0.05, compare with D-NPSCs group. UCMSCs were positive for CD29, CD44, CD73, CD90 and CD105 at rates greater than 95%. For D-NPSCs, the expression of CD29, and CD105 showed interindividual variability but all at rates lower than 95%, however, CD29 and CD105 in CM group were at rates greater than 95%, and the CM group revealed a very similar expression pattern of surface markers with UCMSCs group; Cells in all three groups were negative for the hematopoietic stem cell surface markers CD11b, CD14, CD34, CD45 and HLA-DR. Cells in all three groups expressed CD24 at low rates, while the rate in D-NPSCs significantly higher than that in both CM group and UCMSCs group.

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  Fig. 2 Proliferation capability analysis of cells in the three groups. (A) Growth curves, (B) Cell viability was analyzed by cell counting kit-8 on the 1st, 3rd, 5th, and 7th day. The data are expressed as mean±SD, n=3, *p<0.01 vs. D-NPSCs. (A) Both UCMSCs and CM group were accelerated rapidly during days 3∼5 (logarithmic phase), and slowed down thereafter (stationary phase), while D-NPSCs in the days 3, cells proliferated slowly and then entered the logarithmic growth phase, which continued for 5∼6 days, and reached cell growth plateau in 9∼13 days. (B) Cells from CM group exhibited a greatly increased OD value compared with D-NPSCs group at day 3, 5 and 7.

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  Fig. 3 EdU proliferation assay after 72 h treatment with UCMSCs-CM. (A) EdU incorporated cells in the three groups. (B) Comparative analysis of the percentage of EdU incorporated cells in the three groups. Scale bar=1000 μm (magnification 40×). The data are presented as the means±SD, n=3. *p<0.01, compare with D-NPSCs group. (A) Cells in D-NPSCs group had markedly lower proportion of EdU incorporated cell than CM group and UCMSCs group. (B) Further quantitative analysis also revealed that the percentage of EdU incorporated cell was significantly lower in D-NPSCs than that in CM group and UCMSCs group.

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  Fig. 4 The cell cycles (A) and apoptosis (B) were assessed after 72 h treatment with UCMSCs-CM by flow cytometry. The data are expressed as means±SD, n=3, *p<0.05 vs. D-NPSCs group. CM group had significantly higher percentages of cells in the S phases and lower percentages of cells in the G1/G0 phase than D-NPSCs group, and showed a similarity with UCMSCs group (A). The cell apoptosis rate in CM group was significantly decreased compared with D-NPSCs group, and tended to be higher compared with UCMSCs group (B). Data are presented as the means±SD, n=3. *p<0.05, compare with D-NPSCs group.

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  Fig. 5 Multipotent differentiation potential analysis after 72 h treatment with UCMSCs-CM. (A) Osteogenic differentiation for 21 days, Scale bar=100 μm (magnification 100×), (B) adipogenic differentiation for 21 days, Scale bar=100 μm (magnification 100×), (C) chondrogenic differentiation for 21 days, Scale bar=50 μm (magnification 200×). The data are presented as the means±SD, n=3. *p<0.01, vs. D-NPSCs group. D-NPSCs exhibited few calcium deposition stained by Alizarin red S, whereas the cells from CM group displayed larger and more intensely stained mineralized nodules though it presented less intensely staining than UCMSCs. (A) There were no significant difference in Oil red O positive staining area between the CM group and D-NPSCs group, both appeared to form less fat drops than UCMSCs as shown in (B); Cells from CM group produced more intensely stained extracellular matrix than D-NPSCs group, showed similar intensity levels with UCMSCs group. (C) For osteogenic and chondrogenic differentiation, further quantitative analysis also revealed that the percentage of area stained positively was significantly lower in D-NPSCs group than that in both CM group and UCMSCs group.

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  Fig. 6 qRT-PCR for ACAN, COL2A1, SOX9, CD24, Tie2, OCT4 and NANOG was performed after 72 h treatment with UCMSCs-CM and data were presented as relative ratio to GAPDH. The data are expressed as mean±SD. n=3, *p<0.01 vs. D-NPSCs. The expression levels of NP cell phenotypic markers, SOX9, COL2A1, ACAN and CD24 in CM group were significantly lower than those in D-NPSCs, while the level of Tie2, OCT4 and NANOG were significantly upregulated in CM group compared to D-NPSCs group.

Reference

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