Proteomic analysis of hypoxia and non-hypoxia secretome mesenchymal stem-like cells from human breastmilk

Body: 

Saudi Journal of Biological Sciences 28 (2021) Aug;28(8):4399-4407

Proteomic analysis of hypoxia and non-hypoxia secretome mesenchymal stem-like cells from human breastmilk

Sri Lilidjanti Widjaja 1 2Harsono Salimo 2Indah Yulianto 3Soetrisno 4

Abstract

Introduction: Breastmilk contains proteins and cells which have stem cell properties. The human breastmilk stem cell mimick mesenchymal stem cells and expresses pluripotency genes. The protein level of breastmilk is high in colostrum and gradually subsides in the first year of lactation. The mesenchymal stem cells from breastmilk can be an alternative source of stem cells that can potentially affect cardiovascular therapy. This study aimed to identify the proteomic analysis of secretome mesenchymal stem-like cells under hypoxia compared to non-hypoxia from human breastmilk stem cells.

Material and methods: The human breastmilk was collected from six healthy breastfeeding women and transported to the laboratory under aseptic conditions. The breastmilk cells were isolated then cultured. After 72 h, the human breastmilk stem cells reached confluence then cleaned up and isolated in serum-free media (spheroid) to allow serial passaging every 48 h. The acquisition stem cell was made with flow cytometry. The cells were divided into hBSC secretomes under hypoxia (A) and non-hypoxia (B) and analyzed for LC-MS to identify the peptide structure.

Results: The human breastmilk cells contained several mesenchymal stem-like cells in density 2.4 × 106 cell/mL for hypoxia and 2 × 106 cell/mL for non-hypoxia conditions. The human breastmilk stem cell surface markers derived from the third cell passage process were 93.77% for CD44, 98.69% for CD73, 88.45% for CD90, and 96.30% for CD105. The protein level of secretome mesenchymal stem -like cells under hypoxia was measured at 5.56 μg/mL and 4.28 μg/mL for non-hypoxia. The liquid chromatography-mass spectrometry analysis identified 130 and 59 peptides from hypoxia and non-hypoxia of the human breastmilk stem cell secretome sequentially. Some important proteomics structures were found in the hypoxic human breastmilk stem cell secretome, such as transforming growth factor-β, VE-cadherin, and caspase.

Conclusion: The human breastmilk cells contain mesenchymal stem-like cells and a high concentration of CD44, CD73, CD90, and CD105 as surface markers at third passage culture. The hypoxic hBSC secretome produces a higher protein level compare to non-hypoxia. The transforming growth factor -β was found in the hypoxic hBSC secretome as a modulator of VEGF-mediated angiogenesis.

Keywords: AFP, Alpha-Fetoprotein; ATP, Adenosine Triphosphate; BD, Becton Dickinson; BMPR-II, Bone morphogenetic protein type II; BSA, Bovine Serum Albumin; EHD3, EH Domain-containing Protein 3; FACS, Fluorescence-Activated Cell Sorting; FBS, Fetal Bovine Serum; HIF-1α, Hypoxia Inducible Factor-1α; Hypoxia; IGF1, Insulin-like Growth Factor 1; LALBA, α-Lactalbumin; LC-MS; LC-MS, Liquid Chromatography-Mass Spectrometry; LF, Lactoferrin; MAPK, Mitogen-Activated Protein Kinase; MPS, Multi Proliferative Supplement; MPZL1, Myelin Protein Zero-like Protein 1; MSC, Mesenchymal Stem Cell; Mesenchymal stem-like cell; PBS, Phosphate-buffered Saline; SDS, Sodium Dodecyl Sulfate; SMA, Smooth Muscle Actin; SMAD, Signals Mothers Against the Decapentaplegic; Secretome; TGF-β, Transforming Growth Factor-Beta; VEGF, Vascular Endothelial Growth Factor; cDNA, complementary Deoxyribonucleic Acid; hBSC; hBSC, Human Breastmilk Stem Cell; mRNA, messenger Ribonucleic Acid.

DOI: 10.1016/j.sjbs.2021.04.034

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