Human Platelet Lysate Maintains Stemness of Umbilical Cord-Derived Mesenchymal Stromal Cells and Promote Lung Repair in Rat Bronchopulmonary Dysplasia

Abstract

Mesenchymal stromal cells (MSCs) show potential for treating preclinical models of newborn bronchopulmonary dysplasia (BPD), but studies of their therapeutic effectiveness have had mixed results, in part due to the use of different media supplements for MSCs expansion in vitro. The current study sought to identify an optimal culture supplement of umbilical cord-derived MSCs (UC-MSCs) for BPD therapy. In this study, we found that UC-MSCs cultured with human platelet lysate (hPL-UCMSCs) were maintained a small size from Passage 1 (P1) to P10, while UC-MSCs cultured with fetal bovine serum (FBS-UCMSCs) became wide and flat. Furthermore, hPL was associated with lower levels of senescence in UC-MSCs during in vitro expansion compared with FBS, as indicated by the results of β-galactosidase staining and measures of senescence-related genes (CDKN2A, CDKN1A, and mTOR). In addition, hPL enhanced the proliferation and cell viability of the UC-MSCs and reduced their doubling time in vitro. Compared with FBS-UCMSCs, hPL-UCMSCs have a greater potential to differentiate into osteocytes and chondrocytes. Moreover, using hPL resulted in greater expression of Nestin and specific paracrine factors (VEGF, TGF-β1, FGF2, IL-8, and IL-6) in UC-MSCs compared to using FBS. Critically, we also found that hPL-UCMSCs are more effective than FBS-UCMSCs for the treatment of BPD in a rat model, with hPL leading to improvements in survival rate, lung architecture and fibrosis, and lung capillary density. Finally, qPCR of rat lung mRNA demonstrated that hPL-UCMSCs had lower expression levels of inflammatory factors (TNF-α and IL-1β) and a key chemokine (MCP-1) at postnatal day 10, and there was significant reduction of CD68⁺ macrophages in lung tissue after hPL-UCMSCs transplantation. Altogether, our findings suggest that hPL is an optimal culture supplement for UC-MSCs expansion in vitro, and that hPL-UCMSCs promote lung repair in rat BPD disease.

Keywords: bronchopulmonary dysplasia; human platelet lysate; lung repair; mesenchymal stromal cells; senescence.

FIGURE 1

Growth supplement hPL could largely reduce the senescence of UC-MSCs in vitro expansion compared with FBS. (A) Schematic of protocols used for UC-MSCs in vitro expansion with either FBS-supplemented (10% FBS) or hPL-supplemented (5% hPL) media for up to 10 passages. Representative images showing the morphology of UC-MSCs changes notably as a result of serial passaging (from P1 to P10) between the FBS- and hPL-supplemented groups (B); and the cell diameters in both groups were measured (C). Scale bars, 100 μm (40×) or 200 μm (200×). At Passage 10, the cell apoptosis of FBS-UCMSCs and hPL-UCMSCs were analyzed using flow cytometry by staining with Annexin V and 7AAD reagent (D); and the percentage of Annexin V⁺ cells was compared between these two groups (E). At Passage 10, the cell senescence of FBS-UCMSCs and hPL-UCMSCs were analyzed by staining with β-galactosidase reagent (F). Scale bars, 200 μm; and the count of SA-β-Gal positive cells in high field (100×) was compared (G). (H) The mRNA expressions of senescence-related gene (CDKN2A, CDKN1A, P53, and mTOR) in P10 FBS-UCMSCs and hPL-UCMSCs were analyzed by qPCR. Data are shown as mean ± SEM. n = 3–5. ^∗∗p < 0.01, ^∗∗∗p < 0.001. ns, not significant.

FIGURE 2

Growth supplement hPL maintain the stemness of UC-MSCs in vitro expansion compared with FBS. (A) Growth curves of P4 FBS-UCMSCs and hPL-UCMSCs were assessed by direct counting for 5 days. Three replicates were performed at each time point. (B) The cell viability of FBS-UCMSCs and hPL-UCMSCs in each point was investigated. (C) P4 UC-MSCs cultured with hPL represented lower doubling time (DT) than that in FBS, as analyzed by a formula: DT = t × [log 2/(log N_t–logN₀)], where N_t is the number of harvested cells, N₀ is the number of seeded cells and t is the culture time. (D) The expressions of cell surface makers on P4 FBS-UCMSCs and hPL-UCMSCs were detected by flow cytometry. (E) Representative stained images showed the osteogenic, adipogenic, and chondrogenic potentials of P4 FBS-UCMSCs and hPL-UCMSCs, as confirmed by Alizarin red, Oil red O, and toluidine blue, respectively. Scale bars, 200 or 500 μm. The Nestin expression of FBS-UCMSCs and hPL-UCMSCs at P4 was analyzed by flow cytometry (F); and the percentage of Nestin⁺ cells in the two groups was showed (G). (H) The paracrine factors (growth factors and chemokines) that expressed by P4 FBS-UCMSCs and hPL-UCMSCs were compared using qPCR. Data are shown as mean ± SEM. n = 3–4. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 3

Schematic of protocols used for rat BPD establishment, and survival rate, body weight, and lung histology analysis. (A) Newborn rat pups were exposed to hyperoxia (HYRX; 95% O₂) for 10 days during PN1 to PN10; rat pups that remained in normoxic (NRMX) conditions (21% O₂) were compared as control group. During PN1 to PN10, the survival rate (B) and body weight (C) in each point were recorded. Rat lung tissue were harvested at PN10 and lung sections were stained with H&E (D). Scale bars, 200 μm. Quantification of mean linear intercept (MLI) in each group represents a surrogate of average air space diameter (E). Data are shown as mean ± SEM. n = 5–10. ^∗∗p < 0.01.

FIGURE 4

Superior therapeutic effects of hPL-UCMSCs versus FBS-UCMSCs on lung restoration in BPD model. (A) Schematic of protocols used for model establishment, administration of saline and UC-MSCs, and function analysis. During PN1 to PN10, the survival rate (B) and body weight (C) in each point were recorded among saline and UC-MSCs (FBS-UCMSCs or hPL-UCMSCs) treatment group. Rat lung tissue were harvested at PN10 and lung sections were stained with H&E (D). Scale bars, 200 μm. Quantification of mean linear intercept (MLI) in each group represents a surrogate of average air space diameter (E). Collagen deposition of lung sections were assessed by staining for Masson’s trichrome (F). Scale bars, 200 μm. Collagen deposition was used as surrogate of fibrosis and was reported as percent of septal area (G). Data are shown as mean ± SEM. n = 5–10. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 5

More hPL-UCMSCs were maintained in lung tissue than FBS-UCMSCs. (A) The biodistribution of hPL-UCMSCs and FBS-UCMSCs in rat lung during PN4 to PN10 was detected by In Vivo Imaging System. Rat lung tissue were harvested at PN10 (B), and the count of administrated cm-DiL⁺ UC-MSCs in lung sections were evaluated from five random high field (C). Scale bars, 100 μm. Data are shown as mean ± SEM. n = 5. ^∗∗p < 0.01.

FIGURE 6

Human platelet lysate-human umbilical cord-derived mesenchymal stromal cells treatment rescued hyperoxia-induced loss of microvessels and suppressed macrophages-induced inflammatory response. Microvessels in lung sections were analyzed by staining with von Willebrand factor (vWF) at PN10 (A); vWF-positive vessels that smaller than 50 μm were counted in five random views (B). Scale bars, 100 μm. (C) The expression of vascular endothelial growth factor (VEGF) in lung tissue was analyzed by qPCR. (D) Rat TGF-β is related to lung fibrosis, so the TGF-β expression in treatment group was analyzed. TNF-α (E) and IL-1β (F) are the major factors in lung inflammation post-BPD, and their expressions were analyzed. (G) Monocyte chemoattractant protein-1 (MCP-1) can recruit monocytes from blood vessel to lung tissues, the MCP-1 expression was also evaluated. CD68⁺ inflammatory macrophages in the lung tissue at PN10 post-BPD were determined under fluorescence microscopy (H); and calculated from five random fields of view for each experiment using a double-blind method (I). Scale bars, 100 μm. Data are shown as mean ± SEM. n = 5–10. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 7

The overview of the work (graphical abstract).