Lysophosphatidic acid protects human mesenchymal stromal cells from differentiation-dependent vulnerability to apoptosis

Abstract

The survival of transplanted cells and their resulting efficacy in cell-based therapies is markedly impaired due to serum deprivation and hypoxia (SD/H) resulting from poor vascularization within tissue defects. Lysophosphatidic acid (LPA) is a platelet-derived growth factor with pleiotropic effects on many cell types. Mesenchymal stromal cells (MSC) exhibit unique secretory and stimulatory characteristics depending on their differentiation state. In light of the potential of MSC in cell-based therapies, we examined the ability of LPA to abrogate SD/H-induced apoptosis in human MSC at increasing stages of osteogenic differentiation in vitro and assessed MSC survival in vivo. Undifferentiated MSC were rescued from SD/H-induced apoptosis by treatment with both 25 and 100 μM LPA. However, MSC conditioned with osteogenic supplements responded to 25 μM LPA, and cells conditioned with dexamethasone-containing osteogenic media required 100 μM LPA. This rescue was mediated through LPA1 in all cases. The addition of 25 μM LPA enhanced vascular endothelial growth factor (VEGF) secretion by MSC in all conditions, but VEGF availability was not responsible for protection against apoptosis. We also showed that codelivery of 25 μM LPA with MSC in alginate hydrogels significantly improved the persistence of undifferentiated MSC in vivo over 4 weeks as measured by bioluminescence imaging. Osteogenic differentiation alone was protective of SD/H-induced apoptosis in vitro, and the synergistic delivery of LPA did not enhance persistence of osteogenically induced MSC in vivo. These data demonstrate that the capacity of LPA to inhibit SD/H-induced apoptosis in MSC is dependent on both the differentiation state and dosage. This information will be valuable for optimizing osteogenic conditioning regimens for MSC before in vivo implementation.

FIG. 1.

Osteogenic differentiation of mesenchymal stromal cells (MSC) grown in growth medium (GM), osteogenic media (OM), and OM+ for 7 days as determined by (A) intracellular alkaline phosphatase (ALP) activity and (B) RUNX2 expression. **p<0.001 versus GM and OM, (n=4).

FIG. 2.

Lysophosphatidic acid (LPA) receptor expression is significantly affected by the MSC differentiation state. (A) LPAR1 expression is significantly increased by serum deprivation and hypoxia (SD/H) in OM and OM+, while (B) LPAR2 expression is higher in SD/H for all media conditions. SD/H also increases expression of (C) LPAR4 and (D) LPAR5, but not to statistically significant levels. LPAR1 and LPAR2 expression was significantly higher in GM and OM+ than OM. LPAR3 was not detected MSC in any media type. *p<0.05 versus control, **p<0.01 versus control.

FIG. 3.

SD/H induces apoptosis in MSC in GM after 24 h. (A) Representative images of MSC in 21% O₂ with 10% fetal bovine serum (FBS). (B) MSC exposure to SD/H results in significant loss of cells from the culture dish that is partially rescued by the addition of (C) 25 μM LPA. Images taken at 100×; scale bar represents 100 μm. (D) Quantification of cell number by DNA content confirms visual assessment. *p<0.001 versus control, **p<0.001 versus SD/H (n=3).

FIG. 4.

LPA rescues MSC from SD/H-induced apoptosis in a dose-dependent manner that varies by differentiation state as induced by the following: (A) GM, (B) OM, and (C) OM+. Data are normalized to RLU from cells in 21% O₂ and media containing 10% FBS. *p<0.01 (n=6).

FIG. 5.

(A) LPA significantly increases production of vascular endothelial growth factor (VEGF) by MSC in GM and OM+, which is mitigated by the addition of Ki16425 (n=4). However, (B) the addition of VEGF does not affect MSC survival under SD/H (n=6). *p<0.0001 versus control, **p<0.01 versus SD/H, ^#p<0.001 versus SD/H+25 μM LPA, and ***p<0.01 versus LPA(−).

FIG. 6.

LPA promotes cell survival of MSC over 4 weeks in vivo. (A) BLI reveals that more GM-preconditioned MSC-Luc survive over 28 days when delivered with 25 μM LPA (top right) compared with untreated cells (top left). OM+ preconditioned MSC-Luc (bottom left) have higher persistence over time, but also exhibit improved survival in LPA-containing gels (bottom right). (B) Quantification of luminescence over time shows that LPA significantly improves survival of GM MSC-Luc. OM+ conditioning alone had a protective effect over 28 days, regardless of LPA treatment. Data are normalized to luminescence from gels containing undifferentiated cells within each animal at each time point. (C) Representative CD90 staining from gels containing GM-conditioned MSC confirms poor cell survival at 28 days that is (D) improved with the addition of LPA. (E, F) MSC conditioned in OM+ persisted at 28 days both in the absence (E) or presence (F) of LPA. Magnification is 100×; scale bar represents 100 μm. **p<0.01 versus GM, ***p<0.001 versus GM (n=6). Color images available online at www.liebertpub.com/tea