Selective inhibition of inositol hexakisphosphate kinases (IP6Ks) enhances mesenchymal stem cell engraftment and improves therapeutic efficacy for myocardial infarction

Zheng Zhang1,2*, Dong Liang1*, Xue Gao3*, Chuanxu Zhao1, Xing Qin1, Yong Xu4, Tao Su1, Dongdong Sun1, WeijieLi1, Haichang Wang1, Bing Liu1#, Feng Cao1,4#

Supplemental Data

Supplemental methods

Isolation, culture and identification of MSCs

BM-MSCs were isolated from Tg(Fluc-egfp) mice and expanded with a modified procedure as described previously. In brief, bone marrow was flushed from the femoral and tibia of adult reportertransgenic mice with phosphate-buffered saline(PBS). After passing through a 70 μm strainer and centrifugation at 1200 rpm for 5 min, the cell pellet was resuspended in DMEM supplemented with20% fetal bovine serum (FBS) and 1% penicillin/streptomycin medium. All plated cells were allowed to grow for 4 passages beforetransplantation to avoid contamination with other cell types.

Characteristics of MSCs

MSCs were characterized asCD29+, CD44+, CD90+, CD31-,CD34-,SMA-,C-kit-, and CD45-using flow cytometry. Briefly, after being incubated with 1 μL monoclonal PE-conjugated antibodies against CD markers (BD, San Jose, CA, USA) for 1h, MSCs were processed through a FACS Calibur system (BD, San Jose, CA, USA) according to the manufacturer’sprotocol.Cells were gated according to their high fluorescence.

For adipogenic differentiation, adipogenic media (αMEM with 10% FCS, 1% antibiotics, 50 μM indomethacin (Sigma), 0.5 mM IBMX (Sigma) and 1 μM dexamethasone) was added to the confluent layer of MSCs for 21 days.The adipogenic cells were stained with a working solution of oil red O for 15 minutes at roomtemperature.

Osteogenic differentiation of MSCswas induced by culturing cells inosteogenic medium (OM, 10% FBS, 0.1 μMdexamethasone, 10mMβ-glycerophosphate, and 0.2 mM ascorbic acid in α-MEM) for 21 days, and thedegree of extracellular matrix calcification was estimated using analizarin red S stain.

Cell viability assay

The cell viability of MSCs was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay as described27. Briefly, MSCs were plated in 96-well plates at 1x104/well. After H/SD with different doses of TNP (0.5μM, 1μM, 5μM and 10μM), MSCs were incubated with MTT solution (5 g/L,Sigma) at 37°C for 4h. Then the medium was removedand 200μl dimethyl sulphoxide (DMSO) was added toeach well. The absorbance was determined ata wavelength of 490 nm.

To validate the viability results of MSCswith MTT assay, we assessed cell viability with Bioluminescence imaging(BLI) using the IVIS Kinetic system29(Caliper, Hopkinton, MA, USA)29. Briefly, MSCs were plated in 24-well plates (5×104 per well) andadministeredwith PBS or different doses of TNP (0.5μM, 1μM, 5μM and 10μM) for 24h, followed by either hypoxia or normal conditions for 6h. After administration of D-luciferin(4.5ug/mL), peak signal expressed as photons per second per centimeter square per steridian(photons/s/cm2/sr) was measured using theIVIS Xenogen Kineticsystem (Caliper Life Sciences, USA).

Measurement of MSCs apoptosis induced by H/SD injury

MSCs apoptosis was determined using an AnnexinV-FITC/PI Kit (Merck)according to the manufacturer’s instructions.Briefly, cells werecollected and resuspended in 200 μl of binding buffer. 10 μl ofAnnexin V solution was added to the cell suspension and incubatedfor 30 minutes at room temperature. The cells were then incubatedwith 5μl propidine iodide (PI) and were immediately analyzedon a FACSC-LSR (Becton, Dickinson and Company, San Jose,CA). Flowcytometry data were analyzed with FlowJo software.Caspase-3 activity was measured using a Caspase-3 Assay kit (Clontech, Mountain View, Calif) according to the manufacturer’s instructions.

RNA interference

RNA interference was performed as the manufacture's protocols. Briefly, 2×105cells were seeded in 6-well plates and allowed to grow to 50% confluence. Then cells were transfected with siRNA or control siRNAs (sc-37007) and the medium was changed to DMEM supplemented with 10% FBS. The cells were allowed to grow for another 18h before collected for the following Western blot analysis.

Western blot assay

Western blotting was performed following standard protocol(16). Equal amounts of protein (50 μg/lane) were separated by electrophoresis on 12% SDS-PAGE gels in a Tris/HCl buffer system at 120 V for 90 min, and sequentially electrophoretically transferred to nitrocellulose (NC) membranes.After blocking with Tris-Buffered Saline Tween-20 (TBST) containing 5% milk (TBST: milk), NC membranes were subjected to immunoblotting with primary antibodiesagainst Akt(1:500), p-Akt (Thr 308, Ser 4731:500), p-Bad (1:500), Bad (1:500), Bcl-2 (1:500), Bax (1:500) and β-actin (1:2000)over night at 4°C. All of the antibodies werepurchased from cell signal technology. Blots bands were visualized with an enhanced chemiluminescenesystem (Amersham Bioscience, Buchinghamshire,UK) after incubated with appropriate secondary antibody conjugated with horseradish peroxidase at 37°C for 60 min.Densitometric analysis of Western blots was carried out using VisionWorks LS, version 6.7.1.

Determination of VEGF, FGF2, IGF-1 and HGF

The concentrations ofVEGF, FGF2, IGF-1 and HGF secretedby MSCs were determined byenzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions as previously described(27). All samples and standards were measured induplicate.

Evaluation of MSC engraftment

To evaluate the survival of MSCsin ischemic myocardium, mice were sacrificed at2 daysand 14 days after MSC transplantationrespectively.The hearts were harvested and rapidly (within a minute) fixed in 4%paraformaldehyde. Serial sections were stained with an FITC labeled anti-eGFP antibody and 4,6-diamidino-2-phenylindole (DAPI). Cardiomyocytes were stained with an anti-cTn I antibody. Engrafted MSCs were confirmed by identification of eGFP expression under fluorescent microscopy. The data were expressed as thepercentage of GFP+/DAPI in 5 slides obtained from 5 frozen sections. All of these assays were performed in a blinded manner.

Determination of apoptosis in the heart

Apoptosis in the heart was determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay 48h after MI as previously described(16). In brief, the hearts were harvested and rapidly (within a minute) fixed in 4% paraformaldehyde. Serial sections were prepared and stained with fluorescein-dUTP using an assay kit (In Situ Cell Death Detection Kit, Roche Diagnostics). Additional staining was performed using a monoclonal antibody against troponin I (cTnI, Santa Cruz) for the identification of myocardium. The percentage of apoptotic cells in border zone was termed as “apoptotic index.” All of these assays were performed in a blinded manner.

Supplemental figure legend

SupplementalFigure. 1. Characterization of MSCs expressing firefly luciferase-enhanced green fluorescence protein (GFP) reporter gene

(A) Flow cytometry results shows that MSCswere uniformly negative for CD31,CD45,SMA, C-kit, CD34,and positive for CD29, CD44, CD90. (B) Fibroblast-like shapedMSCs uniformly express GFP within the cytosol (i，ii).MSCs can be induced to differentiate intoadipocytes and osteoblasts in vitro which was assessed by oil red O staining (iii) and alizarin S staining (iv) respectively (scale bar, 100μm). (C) MSCs constitutively express firefly luciferase (fLuc). Ex vivo BLI shows a linear relationship between cell number and bioluminescence imaging signal.

Supplemental Figure.2. Akt regulated Bax expressions in MSCs.

(A)Representative blots of Akt and Bax expressions in MSCs in all groups. (B) The semi-quantitative analysis of Akt and Bax in all groups. Data expressed as mean±SEM. n=5, *p<0.05.

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