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Effect of Hypoxia and Heat Inactivated Serum on Mesenchymal Stem Cell Migration
Author(s) -
Boomsma Robert A.,
Jochems Valerie K.,
Spotts Anna R.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.877.6
Subject(s) - mesenchymal stem cell , fibronectin , chemistry , stem cell , laminin , paracrine signalling , lysis , gelatin , cell migration , hypoxia (environmental) , cell , microbiology and biotechnology , andrology , biochemistry , biology , medicine , receptor , organic chemistry , oxygen
Previous studies have shown that intravenous injection of mouse bone marrow mesenchymal stem cells (MSC) attenuate the loss of function in the heart after coronary artery occlusion (Boomsma et al, Int J Cardiol 122:17 2007) and secrete paracrine factors that may be responsible for their beneficial effect (Boomsma and Geenen, PLoSONE 7:e3685, 2012). The purpose of the current study was to determine the effect of hypoxia and heat inactivated (HI) serum on MSC migration across surfaces coated with laminin or fibronectin, conditions that better mimic the situation MSC encounter as they migrate in an ischemic heart. MSC were cultured in Mesencult + 20% serum supplement (Stem Cell Technologies) under normoxic (21% O 2 + 5% CO 2 ) conditions until confluent. The cells were lifted, placed in Mesencult containing either 20% supplement or 20% HI serum, and transferred to 12 mm Millicell cell culture inserts (PCF membranes, 3 μm pores; 600,000 cells/insert) that were either uncoated, coated with gelatin‐fibronectin, or coated with laminin. The inserts were placed in 24 well plates containing the same media and incubated to allow for cell migration at 37°C in normoxic or hypoxic (1% O 2 , 5% CO 2 , 95% N 2 ) conditions. After 24 hours the cells on top of the membranes were removed and the cells on the underside of the membranes were stained with 0.9% crystal violet. The cells were lysed with 10% acetic acid and the absorbance at 560 nm was determined using a plate reader. The amount of migration was calculated as the % change in absorbance from the mean control value. Mean ± SE was calculated and significance was determined by Student's t‐test (p<0.05; n=9). When MSC were cultured in 20% supplement and normoxia, there was no significant change in migration on laminin or fibronectin compared to uncoated membranes. When cultured in HI serum, migration across uncoated membranes was 87% lower compared to supplement. However, migration in HI serum significantly increased 5‐fold on laminin and 9‐fold on fibronectin compared to uncoated membranes returning migration levels back to that found in supplement. Cells in HI serum were also cultured in hypoxic conditions. Hypoxia significantly reduced migration on uncoated (75%) and fibronectin coated (25%) membranes but was unaffected on laminin. These data show that in the absence of serum stimulatory factors, MSC migration is reduced on uncoated membranes, but laminin and fibronectin are able to increase migration in those conditions. Hypoxia reduces migration on fibronectin but not on laminin. Together, these data suggest that MSC migration in the ischemic conditions of the heart is best on fibronectin but is also promoted by laminin. Support or Funding Information VanderVelde Research Scholarship from Trinity Christian College