PRELIMINARY DATA & METHODS
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Comparing the young ECM and aged ECM
(A) Steps to preparing y- and aECM. (B) Von Kossa (top) and Oil Red O (bottom) staining of y- and aMSC cultures kept on y- and aECM in osteogenic or adipogenic induction media. (C) ECM height maps by AFM (Scale = 35μm) show changes in fibrillar organization, thus highlighting that aging alters ECM organization (D) Graph showing mechanical properties of y- and aECM. (E) Indicates that >85% of unique components in yECM are associated with HSPC niche and table showing yECM-specific proteins (F) Western Blots showing CCN1 is significantly reduced in aging stromal cells and their produced matrices compared to young cells. GAPDH was used on Western Blots to confirm matrix decellularization, and CCN1/GAPDH band intensity was expressed as an OD ratio.
Understanding if differences between the yECM and aECM alter MSC Biology
(A/B) MSC expansion and immunophenotype significantly increase when cultured on yECM. (C) yECM promotes RUNX2 mRNA expression (D) aECM promotes PPARγ mRNA expression in yMSCs.
METHODS
Utilizing HSPCs isolated from cord blood units, I evaluated HSPC function using colony-forming unit assays and characterized them immunophenotypically through flow cytometry at input and following 7 days of culture on either tissue culture-treated (TCP), young ECM (yECM), or aged ECM (aECM) wells. Nucleated cord blood cells were used in a colony-forming unit (CFU) assay on 2% methylcellulose stimulated with recombinant human EPO, IL-3, GM-CSF and SCF and cultured at 5% oxygen. The numbers of CFU-GM, BFU-E, and CFU-GEMM were collected. Flow cytometry was performed with phenotyping markers APC mouse anti-human CD34, PE mouse anti-human CD38, PE-CF594 mouse anti-human CD45RA, PerCP-Cy5.5 rat anti-human CD49f, PE-Cy7 mouse anti-human CD10, and BV421 mouse anti-human CD90.
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