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Secreted matrix metalloproteinase 1 as a marker for assessment of
bone marrow cell migration enhanced by tonsil-derived
mesenchymal stem cells

Hee-Yeon Kim, Hee-Soo yoon, and Joo-Won Park*
Department of Biochemistry, College of Medicine, Ewha Womans University, Seoul 07084, Republic of Korea
INTRODUCTION RESULTS
Bone marrow transplantation is a procedure to substitute diseased (A) (B)
bone marrow with healthy bone marrow stem cells. Bone marrow
transplantation is performed to treat several types of cancers
including hematologic malignancies, and other immune-related
diseases in which abnormal bone marrow is the pathologic
mechanism. To achieve optimal bone marrow engraftment, migration (C) (D)
of donor bone marrow cells towards the recipient’s bone marrow is
critical. Although the enhanced engraftment of bone marrow cells by
co-administration of tonsil-derived mesenchymal stem cells (TMSCs)
has been previously reported, the efficiency can be various
depending on TMSC donors. Conditioned medium from TMSCs
enhanced bone marrow cell migration in different degrees depending
on donors. Analyzing transcriptome and proteome profiles of TMSCs
from 25 donors revealed higher levels of matrix metalloproteinase 1 Figure1. RNA-seq data analysis of TMSC. (A) Sort TMSC based on migration.
(MMP1) in TMSCs with high efficacy for bone marrow cell migration. Compared to the control group, the group with improved hematopoietic stem
cell mobility by 200% or more was classified as High, and the group with
MMP1 knockdown in TMSCs abrogated the supportive efficacy of improved mobility by 150% or less was classified as Low. (B) Show migration
TMSCs in bone marrow cell migration. This study suggests secreted of High and Low group. (C) No difference in between each group’s doubling
MMP1 can be used as a marker to evaluate the efficacy of TMSCs in time. (D) Heatmap of hierarchical clustering indicate differentially expressed
enhancing bone marrow cell migration. gene between TMSC High and TMSC Low. Red indicates up-regulation and
Blue indicates down-regulation. (E) Dendrogram of hierarchical clustering
Keyword : Tonsil, Bone marrow, Matrix metalloproteinase 1 indicates the interclass correlation between two TMSC groups.
(A) (B)
Methods and Materials
Cell culture
Human TMSCs were isolated from palatine tonsils, as described previously,
and were culture in Dul-becco’s modified Eagle’s medium(DMEM)
supplemented with 10% foetal bovine serum(FBS)(Corning, NY, USA)and 1%
penicillin/streptomycin(P/S). The experimental procedures were approved by (C)
the Institutional Review Board of Ewha Womans University Mokdong Hospital
(Seoul, South Korea, ECT-11–53-02). Human adipose tissue-derived
mesenchymal stem cells (AMSCs) were purchased from Invitrogen (Carlsbad,
CA, USA) and cultured as described previously. Human BMSCs were
purchased from PromoCell (Heidelberg, Germany). Cells of passages 5–8 were
utilized in the present study. Figure2. Genetic analysis. (A) Low groups expression gene. (B) High groups
expression gene. (C) Venn diagrams of common expression gene.
Collection of conditioned media(CM)
To collect the Cm from human tonsil-derived mesenchymal stem cell(TMSCs) (A) * * * (B) 1 .5 * * * * *
were cultured to 80% confluency in serum-free DMEM in a 100mm dish and
incubation at 37℃ with 5% 2 . The medium was harvested after 48h, 2 0 0 L o w 1 .0
centrifuge at 1000rpm 5min to remove cellular debris, and filtered through a 1 5 0 H ig h C e ll c o u n t(*1 0 ^ 4 )
0.2um syringe filter(Sartorius Stedim, Germany). CM was concentrated using a M M P 1 (n g /m l) 1 0 0 0 .5
3-kDa Amicon Ultra centrifugal filter unit(EMD Milliore, Darmastadt, Germany) 5 0
by centrifugation at 5000G at 4℃ for 1h. 0 .0
0 N g a tiv e c o n tro l T M S C 3 T M S C 1 0 T M S C 2 4
Bone marrow cell isolation T M S C 3 T M S C 1 0 T M S C 1 1 T M S C 2 1 T M S C 2 4 T M S C 2 5 T M S C 4 T M S C 9 T M S C 1 8 T M S C 2 2 P o s itiv e c o n tro l T M S C 3 -s iM M P 1 T M S C 1 0 -s iM M P 1 T M S C 2 4 -s iM M P 1
After euthanizing 8-week-old male C57BL/6 mice, the bone marrows of both Figure3. MSC migration correlates with MMP1. (A) MMP1 expression amount
legs of the mouse is harvested. The harvested bone marrow cells were washed of each TMSC CM. (B) Migration of TMSC CM and MMP1-RNAi-transfected
with 70% ethanol and PBS. Both ends of the bone marrow were cut in serum- TMSC CM analyze with or without 20ng/ml of SDF-1α.
free RPMI1640 media. The serum-free RPMI1640 penetrates through the cut A C E G
bone marrow to separate the cell. Filter the media with the separated cells on a
70um strainer (SPL, Pocheon, Korea). After centrifugation at 300G for 5
minutes, red blood cells are disrupted with ACK lysing buffer to obtain
hematopoietic stem cells.
B D F H
In vitro migration assay
To evaluate the effect of TMSC on MSC migration, bone marrow cells were
isolated from 8week old male C57BL/6 mice. Bone marrow cells (1*10^6) were
seeded along with 100 ul of TMSC CM in the upper chamber of 24well tissue
culture insert with a 5um pore size membrane.(Costar, Merak SA, Germany), Figure4. Cell microscope images of migration. (A) Negative control without
and the lower chambers were filled with 600ul of the mixture of serum free SDF-1α. (B) Positive control with SDF-1α(20ng/ml). (C, E, G) Migration
medium and SDF1-alph(20ng/ml). MSC migration was compared by counting progressed to conditioned medium for TMSC3, TMSC10 and TMSC24. (D, F,
only viable cells by staining cells with Trypan blue after incubation at 37℃ for H) MMP1-RNAi-transfected TMSC3,TMC10, TMSC24 migration progressed to
4hours. conditioned medium.
CONCLUSION
Bone marrow cell migration is important to achieve bone marrow engraftment. In this study, TMSC assists in bone marrow migration and through analysis of TMSC RNA-
seq data and proteomics, MMP1 showing high efficacy in cell migration is revealed and presented as a maker for evaluating TMSC efficacy. Pearson correlation coefficient
and unsupervised hierarchical clustering was used to analyze RNA-seq data to show the difference of two TMSC groups. As a result, it shows that the distance in between
High and Low group is far. Each sample from both groups show the shortest distance and separation of High and Low. Since each group’s sample creates paired set, the
difference of donor deviation is suggested. By comparing MMP1 expression in each TMSC-Conditioned medium, the High group It was confirmed that the MMP1
expression level was higher than that of the Low group. A few samples were selected in the high group and migration experiments were conducted using MMP1
knockdown Conditioned medium and non-MMP1-knockdown Conditioned medium. As a result, significant results were demonstrated in the MMP1-knockdown sample and
non-MMP1-knockdown sample. We revealed the correlation between MMP1 and TMSC; and propose MMP1 as a TMSC maker.

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