Characterization of primary placental mesenchymal stem cells and cytotrophoblasts
It has been more than 10 years since primary human placenta-derived mesenchymal stem cells were isolated, identified, and used by our research team [20-25]. After isolation for 3-7 days, some short spindle-shaped cells, more long spindle-shaped cells, and few cobblestone-like cells gradually crawled out of the minced placental tissues and expanded to form a single clone. After being cultured for 15-21 days, the isolated hPDMSCs were confluent and passaged. Beginning at the 3rd passage (P3), the cells exhibited fibroblast-like shapes and whirlpool-clonal growth (Fig. 1a). P3 hPDMSCs were used for phenotype identification by flow cytometry and multilineage differentiation potentials. The flow cytometry analysis showed that hPDMSCs were positive for the mesenchymal stem cell markers CD73, CD90, and CD105, but negative for the hematopoietic stem cell markers CD34 and CD45 (Fig. 1b). hPDMSCs differentiated toward endotheliocytes, osteoblasts, and adipocytes in the corresponding differentiation medium. The differentiated endothelial cells were stained positive with von Willebrand factor (vWF), osteoblasts with alkaline phosphatase, and adipocytes with oil red O (Fig. 1c). Compared with middle-pregnancy and full-term placental cells, early-pregnancy MSCs exhibited shorter spindle-like shapes and had stronger proliferative abilities. However, their surface marker expression and differentiation abilities were not significantly different (data not shown).
At the same time, primary early-pregnancy cytotrophoblasts were successfully isolated by enzymatic digestion and purified by Percoll centrifugation and differential adhesion. These cells were positive for cytokeratin 7 (CK7, trophoblast marker) and negative for Vimentin (mesenchymal cell marker), similar to that of the HTR-8 cell line (Fig. 1d). After being cultured for 3-5 days, the cells expanded into multiple epithelial-like cell clones or multiple nuclear-fused syncytiotrophoblasts (Fig. 1e). However, with multiple passages, CTBs were gradually replaced with MSCs. Therefore, primary CTBs that were cultured for 1-2 days after isolation were used to collect conditioned medium for follow-up experiments.
In our study, full-term placental tissue, early-pregnancy primary CTBs, and hPDMSCs from different gestational periods (early, middle, and full-term) were cultured to collect conditioned medium at different time points (placental cells-derived conditioned medium: 6, 12, 24, 48, and 72 hours; placental tissue-derived conditioned medium: 1, 3, 5, 7, 10, and 14 days). The effect of the different conditioned media on HUVECs angiogenesis was analyzed in vitro.
CM from placental cells or placental tissue promoted HUVECs proliferation
To investigate the effect of CM derived from CTBs, hPDMSCs, or placental tissue on HUVECs proliferation, HUVECs were cultured in placental cell-derived CM for 6 days or placental tissue-derived CM for 48 hours, and then the number of cells was calculated by Countstar. The results are shown in Fig. 2.
In the placental-cells-CM groups (early-CTBs, early-hPDMSCs, middle-hPDMSCs, and term-hPDMSCs), the logarithmic growth phase appeared earlier (placental-cells-CM groups: from approximately the 2nd day; control group: from the 3rd day) and lasted longer (placental cells groups: 2 or 3 days, control group: probably 2 days) than those of the control group. From the 2nd day to the 5th day, the number of HUVECs cultured in CM from different placental cells was greater than that of cells cultured only in DMEM (control group). The effect of CM collected at different time points was different: compared with that of the control group, the effect of CM collected at 24 or 48 hours on proliferation was the best, but the effect of CM collected at 6 hours was not obvious. However, HUVECs exhibited slow growth, and many cell fragments were produced at the later stage of proliferation in the 72-hour group (Fig. 2a-d). The number of expanded HUVECs on the 6th day of culture in 24-hour CM was plotted, and the results are shown in Fig. 2e. Compared with the control group, there were significant differences in all placental cell-derived CM groups (control group: 4.207±0.117; early-CTBs-CM group: 7.273±0.255, early-hPDMSCs-CM group: 7.197±0.134, middle-hPDMSCs-CM group: 7.17±0.0601, term-hPDMSCs-CM group: 6.78±0.15). In comparing the different placental cell types, the number of HUVECs cultured in the early-CTBs-CM group was higher than that in the term-hPDMSCs-CM group, but there was no significant difference among the remaining groups.
In the long-term cultured placental tissue-CM groups, especially in the 10- and 14-day CM groups, HUVECs often showed cell cycle arrest or fragmentation in the second-half of the proliferation assay (the 3rd to the 6th day). Therefore, the number of cells was only measured in the first two days (Fig. 2f). The results showed that in the short-term cultured placental tissue-CM groups (1-, 3-, and 5-day group), the number of HUVECs grew slowly at first and then rapidly after 24 hours. Compared with that of the control group, the number of HUVECs at 36 and 48 hours was significantly increased in the 1-, 3-, 5-, and 7-day placental tissue-CM groups. In the long-term cultured placental tissue-CM groups (10 and 14 days), HUVECs grew faster in the early stage of the proliferation assay (at 12-36 hours), and the cell number decreased in the later stage (at 36-48 hours). The possible reason was that there were large amounts of cellular impurities or excessive metabolites in the conditioned medium from the long-term culture of placental tissue.
In all conditioned medium derived from placental cells or placental tissue, there was no obvious effect on proliferation within 24 hours, and so the experimental index was measured within 24 hours in the subsequent experiments.
CM from placental cells or placental tissue promoted HUVECs adhesion
Adhesion is an important cellular process. In our study, a cell adhesion assay was performed. HUVECs were suspended in different CM and seeded into 96-well plates coated with the corresponding CM, and the number of adherent HUVECs was measured after culture for 2 hours.
The results suggested that, compared with that of the control group, the number of adherent cells increased in all groups treated with placenta cells-derived CM collected at 24 and 48 hours (control group: 43.87±7.75; early-CTBs-CM 24-hour group: 96.33±17.11, 48-hour group: 90.4±20.36; early-hPDMSCs-CM 24-hour group: 86.6±12.09, 48-hour group: 81.33±16.42; middle-hPDMSCs-CM 24-hour group: 85.53±14.81, 48-hour group: 87.2±18.82; term-hPDMSCs-CM 24-hour group: 75.67±15.85, 48-hour group: 83.13±12.87). Among the 72-hour groups, only early-hPDMSCs-CM and early-CTBs-CM enhanced adhesion (control group: 43.87±7.75; early-CTBs-CM 72-hour group: 68±14.58; early-hPDMSCs-CM 72-hour group: 72.73±17.14; middle-hPDMSCs-CM 72-hour group: 62.67±8.78; term-hPDMSCs-CM 72-hour group: 62.33±10.1), as shown in Fig. 3b.
The graphical analysis of the different placental cell types as the abscissa is shown in Fig. 3c. Among the different placental cell type groups, the adhesion-promoting effect of the term-hPDMSCs-CM group was weaker than that of the remaining three cell type groups, but there was no significant difference among these three groups. Within each cell type, CM collected at 24 and 48 hours had the best adhesion-promoting effect, which was almost higher than that of the other time points (except the 24- and 72- hour groups of early-hPDMSCs-CM and the 48- and 72- hour term-hPDMSCs-CM groups).
The CM that was collected at different time points was used as the abscissa for plot analysis (Fig. 3c). The results showed that CM collected at 24 and 48 hours was better than that collected at other time points, but there was no significant difference between them. Within each time point, there was no significant difference among the four placental cell types at the 48 hours, but the effect of early-CTBs-CM was better than that of term-hPDMSCs-CM at the 24 hours.
In the placental tissue groups, compared with the control group, short-term tissue culture groups and long-term tissue culture groups had stronger adhesion-promoting effects. The effect of the 7-day group was the most significant, which was better than that of the 1-, 3-, or 14-day group, but was not obvious compared with that of the 5- or 10-day group (control group: 41.8±9.886; placental tissue-derived CM 1-day group: 69.53±14.17, 3-day group: 99.8±26.12, 5-day group: 111.3±32.19, 7-day group: 131.3±30.67, 10-day group: 118.26±25.82, 14-day group: 85.87±27.32) (Fig. 3e).
CM from placental cells or placental tissue promoted HUVECs migration
In addition to proliferation and adhesion, HUVECs migration and invasion are two key processes associated with angiogenesis. To assess the effect of CM from different placental cells or placental tissue on HUVECs migration, the horizontal and vertical migration capacities of HUVECs were measured by wound healing assays and Transwell assays, respectively.
In the scratch wound healing assay, confluent HUVECs were scratched and incubated in placental CM for 8 hours, and then the closure distance of the scratch, also known as the cell horizontal migration distance, was measured. The results are shown in Fig. 4.
All placenta cell-derived CM collected at 24, 48, and 72 hours increased the closure distance of HUVECs compared with that of control medium (control group: 113.3±7.243; early-CTBs-CM 24-hour group: 301.3±31.21, 48-hour group: 290.7±35.99, 72-hour group: 207.4±14.99; early-hPDMSCs-CM 24-hour group: 308.3±41.51, 48-hour group: 289.73±36.21, 72-hour group: 194.3±29.59; middle-hPDMSCs-CM 24-hour group: 276.9±31.91, 48-hour group: 277.1±35.41, 72-hour group: 194±30.1; term-hPDMSCs-CM 24-hour group: 236.3±24.3, 48-hour group: 254.9±23.19, 72-hour group: 175.6±21.29) (Fig. 4a&b).
Fig. 4 shows the plot analysis with the different placental cell types as the abscissa. Comparative analysis between the different placental cell type groups revealed that the migration-promoting effect of term-hPDMSCs-CM was weaker than that of the other three groups, but there was no significant difference among them. Within each placental cell type group, 24 and 48 hours had the best promigratory effect, but there was no difference among them (Fig. 4c).
CM collected at different time points was used as the abscissa to analyze the variables. The results showed that the CM of all placental cell types collected at 24 and 48 hours was better than that collected at other time points, but there were no differences between them. Within each time point group, in the 24-hour group, the term-hPDMSCs-CM group had a weaker effect than the other three groups, and the early-hPDMSCs-CM group has a better effect than the middle-hPDMSCs-CM group, but there was no significant difference among the other groups. In the 48-hour groups, early-CTBs and early-hPDMSCs were better than term-hPDMSCs (Fig. 4d).
The placental tissue-derived CM that was collected at all the time points (1, 3, 5, 7, 10, and 14 days) had better promigratory effects than that of the control group. The 7-day group had the most robust effect, which was significantly better than that of the 1-, 3-, and 14-day groups, but there was no significant difference compared with that of the 5- and 10-day groups (control group: 113.3±7.243; placental tissue-derived CM 1-day group: 182.5±21.794, 3-day group: 270.8±68.13, 5-day group: 338.3±58.32, 7-day group: 427.1±70.26, 10-day group: 362.5±28.68, 14-day group: 209.8±31.16) (Fig. 4e).
A transwell migration assay was used to evaluate the vertical migration ability of HUVECs. HUVECs migrated from the upper chamber to the lower surface of the membrane through 8 μm pores in response to different conditioned medium from placental cells or placental tissue in the lower chamber. After being incubated for 18 hours, the cells that migrated to the lower side of the membrane were stained, counted, and analyzed. The results are shown in Fig. 5.
Compared with that of control CM, CM collected from all placental-cells at 24, 48, and 72 hours increased the number of migrated HUVECs (control group: 106.3±14.4; early-CTBs-CM 24-hour group: 216.1±18.56, 48-hour group: 248.6±39.54, 72-hour group: 193.2±37.37; early-hPDMSCs-CM 24-hour group: 202.8±22.31, 48-hour group: 252.1±26.73, 72-hour group: 163.6±19.69; middle-hPDMSCs-CM 24-hour group: 191.2±28.25, 48-hour group: 191.1±25.75, 72 hours:162.2±22.4; term-hPDMSCs-CM 24hours: 163.7±28.56, 48-hour group: 190.3±18.17, 72-hour group: 155.3±19.7) (Fig. 5b).
Using the different placental cell types as the abscissa, the graphical analysis is shown in Fig. 5c. Comparative analysis of the different placental cell types revealed that the promigratory effects of the early-CTBs-CM and early-hPDMSCs-CM groups were stronger than those of the middle-hPDMSCs-CM and term-hPDMSCs-CM groups. However, there was no significant difference among the early placental cell-derived CM groups, and there was no difference between middle- and term-hPDMSCs CM groups. Comparative analysis within the groups and among all four placental cell types showed that the promotion of vertical migration by CM obtained at 24 and 48 hours was robust (only the 48-hour group had the best effect among the term-hPDMSCs-CM groups), and there was no significant difference between them (except 24 hours, which was better than 48 hours in the early-hPDMSCs-CM groups). Compared with CM at other time points, both 24 hours and 48 hours were stronger than most of the other groups (except that in the early-CTBs-CM, middle-hPSMSCs-CM and term-hPDMSCs-CM groups, there was no difference between 24 hours and 72 hours, and in the middle-hPDMSCs-CM and term-hPDMSCs-CM groups, there was no difference between 48 hours and 72 hours) (Fig. 5c).
The CM collected at different time points was used as the abscissa to analyze the variables. The results are shown in Fig. 5d. Conditioned medium collected at 48 hours had the best effect among the different time points. Comparative analysis within each time point group showed that in the 24-hour CM group, the promigratory effects of early-CTBs-CM and early-hPDMSCs-CM were better than that of term-hPDMSCs-CM; in the 48-hour CM group, the effects of early-CTBs-CM and early-hPDMSCs–CM were stronger than those of middle-hPDMSCs-CM and term-hPDMSCs-CM; and in the 72-hour CM group, early-CTBs-CM was better than term-hPDMSCs-CM (Fig. 5d).
In the placental tissue-derived CM groups, compared with the control group, all groups had stronger effects on promoting migration, and the effect of the 7-day group was the strongest (control group: 106.3±11.06; placental tissue-derived CM 1-day group: 317.8±37.07, 3-day group: 413.8±22.43, 5-day group: 492.9±43.27, 7-day group: 554.5±39.47, 10-day group: 496±51.75, 14-day group: 224.4±44.3) (Fig. 5e).
CM from placental cells or placental tissue promoted HUVECs invasion
Invasion is a subsequent step of migration and is a complex multistep process that involves the ability of cells to move through a 3D matrix. The transwell invasion assay was used to analyze the ability of HUVECs to degrade the extracellular matrix and move through the basement membrane of blood vessels towards a cytokine gradient. HUVECs were seeded into the upper chamber, which was coated with Matrigel. After being incubated for 18 hours, the invasive HUVECs were fixed, stained, photographed, and analyzed. The results are shown in Fig. 6c.
Compared with the control group, placental cell-derived CM collected at 24 and 48 hours increased the number of cells that degraded the Matrigel and moved through the membrane. CM derived from both early-CTBs and early-hPDMSCs that was collected at 72 hours also enhanced the invasive effect (control group: 87±20.49; early-CTBs-CM 24-hour group: 258.8±31.84, 48-hour group: 213.6±25.39, 72-hour group: 161.7±28.96; early-hPDMSCs-CM 24-hour group: 242.8±46.12, 48-hour group: 332.2±64.05, 72-hour group: 234.7±60.03; middle-hPDMSCs-CM 24-hour group: 222±24.06, 48-hour group: 204.2±35.94; term-hPDMSCs-CM 24-hour group: 201.9±45.02, 48-hour group: 194.1±45.04) (Fig. 6b).
Using the different placental cell types as the abscissa, the graphical analysis is shown in Fig. 6c. Comparative analysis between the different placental cell types revealed that the number of invasive HUVECs in the early-hPDMSCs-CM group was the highest among all the CM groups, and there was no significant difference among the other three groups. Comparative analysis within each group showed that the invasion-promoting effect of CM collected at 24 hours was the strongest in the early-CTBs-CM group. In the three hPDMSCs-CM groups, the effect of CM collected at 24 and 48 hours was better than that of CM collected at other time points; moreover, there was no significant difference between 24 and 48 hours (Fig. 6c).
The different time points were used as the abscissa for plot analysis. The results are shown in Fig. 6d. The effect of CM collected at 24 and 48 hours was the best among the different time points, and there was no significant difference between 24 and 48 hours. The intragroup comparison showed that in the 24-hour group, the effects of early-CTBs-CM and early-hPDMSCs-CM were better than that of term-hPDMSCs-CM. However, in the 48- and 72-hour groups, early-hPDMSCs-CM was the best among the four placental cell type groups (Fig. 6d).
In the placental tissue-derived CM groups, compared with the control group, short-term culture groups and long-term culture groups had stronger effects on promoting invasion. The 7-day CM had the strongest effect, which was significantly better than that of 1-, 3-, or 14-day CM, but there was no significant difference compared with that of 5- or 10-day CM (control group: 87±20.49; placental tissue-derived CM 1-day group: 174.3±22.48, 3-day group: 268.7±94.5, 5-day group: 413.3±88.97, 7-day group: 467.4±83.92, 10-day group: 317.2±63.66, 14-day group: 135.5±44.32) (Fig. 6e).
The angiogenic effect from CM of placental cells or placental tissue
The in vitro Matrigel tube formation assay is generally used to assess the angiogenic ability of HUVECs. HUVECs were suspended in different CM and directly seeded onto Matrigel. DMEM supplemented with VEGF-A was used as the positive control, and DMEM without any additive was used as the negative control. After being incubated for 15 hours, the capillary-like tube was photographed under a microscope, and the total length of the tube was quantified using ImageJ software (Fig. 7a). As shown in Fig. 7b, compared with the negative control CM derived from the four placental cell types at 24, 48, and 72 hours had obvious proangiogenic effects, and at 12 hours, only CM from the early-CTBs and early-hPDMSCs promoted angiogenesis (control group: 4038±318.3; early-CTBs-CM 12-hour group: 8343±1232, 24-hour group: 9656±1232, 48-hour group: 10417±2443, 72-hour group: 7105±1293; early-hPDMSCs-CM 12-hour group: 8586±1448, 24-hour group: 10033±1784, 48-hour group: 9022±1563, 72-hour group: 7663±1809; middle-hPDMSCs-CM 24-hour group: 7962±2216, 48-hour group: 8815±1304, 72-hour group:7012±1804; term-hPDMSCs-CM 24-hour group: 76027±1372, 48-hour group: 8107±1928, 72-hour group: 6964±2141) (Fig. 7b).
The results of the graphical analysis with the different placental cell types as the abscissa are shown in Fig. 7c. Comparative analysis between the different placental cell types revealed that the total tube lengths of HUVECs incubated in early-CTBs-CM and early-hPDMSCs-CM were longer than those incubated in middle-hPDMSCs-CM and term-hPDMSCs-CM, and there was no significant difference between the early-CTBs-CM and early-hPDMSCs-CM groups or between the middle-hPDMSCs-CM and term-hPDMSCs-CM groups. Comparative analysis within the groups showed that the angiogenic effects of 24- and 48-hour CM were stronger than those of CM collected at the other time points among the placental cell types, although the differences among some groups were not statistically significant (Fig. 7c).
The results of the plot analysis, using different time points as the abscissa, are shown in Fig. 7d. Comparative analysis between the different time points showed that the promoting effect of 24- and 48-hour CM was strongest among the time points, but there was no significant difference between them. Comparative analysis within each time point showed that in the 12- and 24-hour groups, the effects of early-CTBs-CM and early-hPDMSCs-CM were stronger than that of term-hPDSMCs-CM, and early-hPDMSCs-CM was stronger than middle-hPDMSCs-CM. In the 48-hour group, the effect of early-CTBs-CM was better than that of term-hPDMSCs-CM (Fig. 7d).
In the in vitro Matrigel tube formation assay with placental tissue-derived CM, compared with that of the control group, there was an obvious proangiogenic effect in all of the placental tissue-derived CM groups. The 7-day CM group had the most significant proangiogenic effect of all the CM groups (control group: 4038±318.3; placental tissue-derived CM 1-day group: 5502±1389, 3-day group: 7672±1368, 5-day group: 11556±1745, 7-day group: 13864±2312, 10-day group: 7249±1409, 14-day group: 4476±1068) (Fig. 7e). The results were the same as those for adhesion, migration, and invasion.
Angiogenic factor expression in placental cells or placental tissue
It is well known that the biological process of blood vessel formation includes cell proliferation, adhesion, migration, invasion, and angiogenesis. According to our results, placental cell-derived or placental tissue-derived CM can promote HUVECs proliferation, adhesion, migration, invasion, and tube formation to varying degrees, thus enhancing angiogenesis. The effects of 24- and 48-hour placental cells-derived CM or 7-day placental tissue-derived CM were the strongest. To further investigate the mechanism, the levels of 43 conventional angiogenic factors in 24-hour placental cells-derived CM and in 7-day placental tissue-derived CM were measured with a human angiogenesis antibody array (RayBiotech, USA). The results are shown in Fig. 8.
Among these 43 factors, in placental tissue-derived CM, the 5 factors with the highest levels were GRO, IL-6, IL-8, CXCL-5, and MCP-1 (ratio to the positive control: >1); the levels of the following 3 factors were moderate: TIMP-1, TIMP-2, and angiostatin (0.5~1); and the ratios of the following 6 factors ranged from 0.1 to 0.5: angiogenin, bFGF, TPO, VEGF-A, MMP-1, and uPAR. In addition, the ratios of 4 factors ranged from 0.1 to 0.05 (PLGF, CCL5, ANGPT2, and GM-CSF). Among these 18 factors, the levels of CXCL-5 and angiostatin were increased in placental tissue-derived CM but were low or undetectable in hPDMSCs-CM or early-CTBs-CM. The levels of other factors in placental tissue-derived CM were significantly higher than those of placental cells-derived CM (except MMP-1 and PLGF). Among the different placental cell types, the levels of PLGF, TPO, and VEGF-A were higher in early-CTBs-CM, while the levels of GRO and IL-6 in early-hPDMSCs-CM or middle-hPDMSCs-CM were significantly higher than those in early-CTBs-CM and term-hPDMSCs-CM. IL-8, MCP-1, TIMP-1, TIMP-2, angiogenin, bFGF, MMP-1, uPAR, CCL5, ANGPT2, and GM-CSF were all present in both early-CTBs-CM and hPDMSCs-CM, but the concentrations of these factors in placental cells need to be further confirmed (Fig. 8a-d). The protein-protein interactions of these 17 secreted factors in the placenta (except for angiostatin) were analyzed by STRING11.0. In the graph, some factor names were different ('bFGF': FGF2; 'GRO': CXCL1; 'IL-8': CXCL8; 'MCP-1': CCL2; 'PLGF': PGF; TPO-THPO: thrombopoietin; 'GMCSF': CSF2; 'uPAR': PLAUR). The interaction network is shown in Fig. 8e. The thickness of the connecting lines represented the strength of the interaction between the factors. All 17 of these factors were related to each other, and multiple proteins had multiple action points associated with cell activity. These results can provide ideas for subsequent experiments.