Mesenchymal stem cells (MSCs) show promise for treating immune disorders and degenerative diseases. However, targeting MSCs to damaged tissue sites is challenging. The researchers hypothesized that engineering MSCs to express leukocyte adhesion molecules could enhance their homing ability. They coupled a recombinant form of P-selectin glycoprotein ligand-1 (PSGL-1), which mediates leukocyte rolling, to MSCs. This non-covalently coupled the PSGL-1 to the MSC surface. MSCs modified with PSGL-1 were then able to tether and roll on endothelial cells under flow, mimicking leukocyte behavior, suggesting this approach may help target MSCs to sites of injury and inflammation.

1. Surface coupling of P-selectin glycoprotein ligand-1 on mesenchymal
stem cells enables leukocyte-like cell tethering & rolling
o Mesenchymal stem cell (MSC) therapy has shown
great promise in the treatment of many immune
disorders and degenerative diseases such as graft-
versus-host-disease, multiple sclerosis, myocardial
infarction and liver cirrhosis.
o In target organs, MSCs repair tissue by acting as a
source of paracrine factors that promote cell survival
and growth, regulate vascular permeability and
promote immune-tolerance.
o These multipotent cells may also integrate into the
host and differentiate into various tissue types
including bone, adipose, cardiac, cartilage, muscle
and the myelosupportive stroma.
o Allogeneic MSCs can serve as off-the-shelf treatment
since they do not display signs of rejection, even in the
absence of immunosuppressive therapy.
Sriram Neelamegham1,2, Chi Lo1, Techung Lee3, Aristotelis Antonopoulos4, Anne Dell4, and Stuart Haslam4
1 Department of Chemical and Biological Engineering, 2 The NY State Center for Excellence in Bioinformatics and Life Sciences, and 3 Department of Biochemistry
The State University of New York, Buffalo, NY, 14260, USA
4 Department of Life Sciences, Imperial College, South Kensington Campus, London, SW7 2AZ, UK
Hypothesis
Background
Methods
Results and Discussion
Conclusions
o MSC therapy requires targeting of cells to the tissue repair site
with high efficiency. Two potential delivery routes:
 Local infusion to damaged tissue
 Systemic infusion proximal to the therapeutic site
o Systemic infusion has the advantage of being minimally invasive,
however:
 A large number of MSCs must be perfused at sites of injury
and inflammation
 Engraftment rate is low and downstream occlusion may
occur
 Non-specific distribution to other organs may occur
o Hypothesis: Engineering natural leukocyte adhesion
molecules onto MSC surface can enhance cell homing to sites
of injury which are often also inflamed.
o Specifically, non-covalent coupling of functional recombinant
PSGL-1 onto MSCs can enable leukocyte-like
capture/tethering and rolling on stimulated endothelial cells.
Gnecchi, M. et al., Vascul
Pharmacol, 2012. 57(1): p. 48-55.
Acknowledgements: T32 NIH Ruth L. Kirschstein Postdoctoral Research Training Grant, NYSTEM (NY State Stem Cell initiative) and NIH (National Heart, Lung and Blood Institute)
Reference: Lo et al. ”The use of surface immobilization of P-selectin glycoprotein ligand-1 on mesenchymal stem cells to facilitate selectin mediated cell tethering and rolling”, 34:8213–8222, Biomaterials,
November 2013.
1. Human embryonic kidney 293T (‘HEK’) cells were
transduced with α1,3 fucosyltranferase FUT7-DsRed
fusion protein virus to generate red HEK[FUT7+] (see
micrograph).
19Fc and 19Fc[FUT7+] were then expressed in wild-type
‘HEK’ and HEK[FUT7+] cells respectively.
2. HEK293T cells and MSCs coupled to 19Fc/19Fc[FUT7+]
using palmitated protein G chemistry
o A non-covalent, non-viral and simple strategy is presented for the coupling of functional selectin-ligands on stem cells. It does not alter MSC viability and
multipotency.
o The construct 19Fc[FUT7+] contains the physiological selectin-ligand that is expressed at the N-terminus of PSGL-1. Coupling 19Fc[FUT7+] to MSCs
enables both cell capture/tethering from continuous flow and retention at wall shear stress > 10 dyn/cm2.
o Unlike previous work where MSCs could only be captured onto selectin-bearing substrates at low or no-flow conditions, the current work presents a ‘glycan
engineering’ strategy to enable leukocyte-like capture and rolling. This method may be applied to target heterologous cells to sites of inflammation.
E-selectin
2 dyn/cm2
*
*
P-selectin
1 dyn/cm2
(0) (0) (0)
(0) (0)
0
20
40
60
80
100
Interactingcells(cells/mm2)
0
20
40
60
80
100
Interactingcells(cells/mm2)
Lo et al.
Figure 5
anti-E-selectin(CL2/6)
anti-PSGL-1(KPL1)anti-PSGL-1(KPL1)
anti-P-selectin(G1)
E-selectin
2 dyn/cm2
*
*
P-selectin
1 dyn/cm2
(0) (0) (0)
(0) (0)
0
20
40
60
80
100
Interactingcells(cells/mm2)
0
20
40
60
80
100
Interactingcells(cells/mm2)
Lo et al.
Figure 5
anti-E-selectin(CL2/6)
anti-PSGL-1(KPL1)anti-PSGL-1(KPL1)
anti-P-selectin(G1)
FIGURE 4
A, B. MSCs coupled with 19Fc[FUT7+] interacted with P-selectin bearing CHO-P
(panel A) and E-selectin bearing HUVEC cells (panel B) in a specific manner (* P
< 0.001). Coupling cells with anti-P-selectin mAb G1 or anti-E-selectin mAb
CL2/6 did not result in cell capture from flow.
C. MSC-PPG-19Fc[FUT7+] were captured onto stimulated HUVECs up to 2-4
dyn/cm2.
D. Once captured, cells were robustly retained on stimulated HUVECs at all shear
stresses tested.
(* P < 0.01 with respect to MSC or MSC-PPG cells. )
FIGURE 3
A. Concentration of 19Fc[FUT7+] on MSCs titrated by varying the concentration of PPG used for
immobilization.
B. 19Fc[FUT7+] bound to MSCs decreased gradually over a period of days.
C. Coupling strategy resulted in immobilization of glycoprotein on cell-surface as seen in confocal
image. The MSC coupling procedure did not affect viability or multipotency (data not shown)
Selectin mediated leukocyte tethering
and rolling
Therapeutic potential of Mesenchymal Stem Cells
HEK [FUT7+]
Viscometer Microfluidics flow chamber
3. P- and E-selectin mediated cell adhesion function
measured using cone-plate viscometer and microfluidic
parallel plate flow chamber.
PSGL-1 is a mucinous glycoprotein with a selectin-
ligand located at its N-terminus (red box). 19Fc
consists of the first 19 a. acids of mature PSGL-1
followed by a human IgG tail.
PSGL-1:
19Fc:
FIGURE 1
A. 19Fc/19Fc[FUT7+] purified from cell culture supernatant using Ni-chelate chromatography
was a 68 kDa dimeric protein as shown under non-reducing (’NR’) and reducing (‘R’)
conditions.
B. Glycomics analysis of O-glycans (left panels) and N-glycans (right panels) from 19Fc (top
panels) and 19Fc[FUT7+] (bottom panels). SLeX structure at m/z=1879.8 is prominent in O-
glycans of 19Fc[FUT7+] only (bottom left panel).
19Fc/19Fc[FUT7+] Expression and Characterization
B
His tag19aa PSGL-1 Human Fc
HEK
HEK[FUT7+]
19Fc[FUT7+]
FUT7 FUT7
FUT7
FUT7
FUT7
FUT7
FUT7
FUT7
FUT7
FUT7
FUT7FUT7
FUT7
FUT7
FUT7
FUT7-DsRED
Lentivirus
19Fc Lentivirus
A
CMV R U5
5’ LTR
U3 R U5
3’LTR
CMV PSGLpp19Fc
19Fc
37kDa
75kDa
NR: Non-reduced
R: Reduced
NR NRR R NR NRR R NR NRR R
Silver stain PSGL-1 Human IgG
CMV U5
5’ LTR 3’ LTR
U3DsREDCMV FUT7 U5
19Fc
19Fc[FUT7+]
O-glycan
O-glycan
19Fc
19Fc[FUT7+]
N-glycan
N-glycan
Mass (m/z)
Mass (m/z) Mass (m/z)
Mass (m/z)
%Intensity%Intensity
%Intensity%Intensity
Lo et al.
Figure 1
A B
NHS-Palmitate
NHS
Palmitate-Protein G (PPG)
A
HEK
HEK-PPG
19Fc
HEK-PPG-
19Fc[FUT7+]
Alexa488 on
HEK293T
HEK-PPG
HEK-PPG-
19Fc[FUT7+]
DICFITC
HEK
D
0
300
600
900
1200
0 100 200 300
Goatanti-mouse
Alexa488(MFI)
PPG conc (µg/ml)
50 µg/ml
100 µg/ml
Protein G
Lo et al.
Figure 2
FITCSignal
(MFI)
0
20
40
60
80
100
0
200
400
600
800
GαH IgG (H+L) [F(ab')₂]
GαH IgG (F(ab')₂) [whole]
FITCSignal
(MFI)
NHS-Palmitate
NHS
Palmitate-Protein G (PPG)
A
B
C
HEK
HEK-PPG
19Fc
HEK-PPG-
19Fc[FUT7+]
Alexa488 on
HEK293T
HEK-PPG
HEK-PPG-
19Fc[FUT7+]
DICFITC
HEK
D
0
300
600
900
1200
0 100 200 300
Goatanti-mouse
Alexa488(MFI)
PPG conc (µg/ml)
50 µg/ml
100 µg/ml
Protein G
Lo et al.
Figure 2
FITCSignal
(MFI)
0
20
40
60
80
100
0
200
400
600
800
GαH IgG (H+L) [F(ab')₂]
GαH IgG (F(ab')₂) [whole]
FITCSignal
(MFI)
0
20
40
60
80
100
0 1 2 3
HEK-plateletadhesion(%)
Time (min)
HEK
HEK-PPG
HEK-PPG-19Fc
HEK-PPG-19Fc[FUT7⁺]
HEK-PPG-19Fc[FUT7⁺] + PSGL-1 blocking
E-selectin
*
anti-E-selectin(CL2/6)
1 dyne/cm2
0
20
40
60
80
Interactingcells
(cells/mm2)
P-selectin
*
anti-PSGL-1(KPL1)
(0)(0)
1 dyne/cm2
0
20
40
60
80
Interactingcells
(cells/mm2)
Lo et al.
Figure 3
anti-PSGL-1(KPL1)
0
20
40
60
80
100
0 1 2 3
HEK-plateletadhesion(%)
Time (min)
HEK
HEK-PPG
HEK-PPG-19Fc
HEK-PPG-19Fc[FUT7⁺]
HEK-PPG-19Fc[FUT7⁺] + PSGL-1 blocking
E-selectin
*
anti-E-selectin(CL2/6)
1 dyne/cm2
0
20
40
60
80
Interactingcells
(cells/mm2)
P-selectin
*
anti-PSGL-1(KPL1)
(0)(0)
1 dyne/cm2
0
20
40
60
80
Interactingcells
(cells/mm2)
Lo et al.
Figure 3
anti-PSGL-1(KPL1)
0
20
40
60
80
100
0 1 2 3
HEK-plateletadhesion(%)
Time (min)
HEK
HEK-PPG
HEK-PPG-19Fc
HEK-PPG-19Fc[FUT7⁺]
HEK-PPG-19Fc[FUT7⁺] + PSGL-1 blocking
E-selectin
*
anti-E-selectin(CL2/6)
1 dyne/cm2
0
20
40
60
80
Interactingcells
(cells/mm2)
P-selectin
*
anti-PSGL-1(KPL1)
(0)(0)
1 dyne/cm2
0
20
40
60
80
Interactingcells
(cells/mm2)
Lo et al.
Figure 3
anti-PSGL-1(KPL1)
FIGURE 2
A. Dose dependent immobilization on 19Fc[FUT7+] on HEK cells detected using flow cytometry.
B. Surface immobilization confirmed by confocal microscopy.
C. Majority of protein G sites on cells are occupied by 19Fc[FUT7+].
D. 19Fc[FUT7+] - HEK cells bound P-selectin on activated platelets in the viscometer assay.
E., F. 19Fc[FUT7+] but not 19Fc bearing cells bound P- (panel E) and E- (panel F) selectin bearing
substrates. Cell adhesion was blocked by anti-PSGL-1 mAb KPL-1 and anti-E-selectin mAb CL2/6. *
indicates P < 0.01.
A B C
D E F
19Fc[FUT7+] coupling to Mesenchymal Stem Cells
MSC-PPG
MSC-
PPG-19Fc[F
UT7+]
DICFITC
MSC
0
200
400
600
800
0 100 200 300
Goatanti-mouse
Alexa488(MFI)
PPG conc (µg/ml)
50 µg/ml
100 µg/ml
0
50
100
150
200
0 2 4 6 8
Alkaline
phosphatase
activity(%)
Days after treatment
MSC
MSC-PPG
MSC-PPG-19Fc[FUT7⁺]
0
50
100
0 2 4 6 8
Cellviability(%)
Days after treatment
MSC
MSC-PPG
MSC-PPG-19Fc[FUT7⁺]
Alexa488 on
MSC
0
200
400
600
0 20 40 60 80
Time (h)
MSC
MSC-PPG
MSC-PPG-19Fc[FUT7⁺]
Lo et al.
Figure 4
GαHIgG(H+L)
[F(ab’)₂]#(MFI)
MSC-PPG
MSC-
PPG-19Fc[F
UT7+]
DICFITC
MSC
0
200
400
600
800
0 100 200 300
Goatanti-mouse
Alexa488(MFI)
PPG conc (µg/ml)
50 µg/ml
100 µg/ml
0
50
100
150
200
0 2 4 6 8
Alkaline
phosphatase
activity(%)
Days after treatment
MSC
MSC-PPG
MSC-PPG-19Fc[FUT7⁺]
0
50
100
0 2 4 6 8
Cellviability(%)
Days after treatment
MSC
MSC-PPG
MSC-PPG-19Fc[FUT7⁺]
Alexa488 on
MSC
0
200
400
600
0 20 40 60 80
Time (h)
MSC
MSC-PPG
MSC-PPG-19Fc[FUT7⁺]
Lo et al.
Figure 4
GαHIgG(H+L)
[F(ab’)₂]#(MFI)
A B C
A B
C D
19Fc[FUT7+] enhances MSC cell adhesion to P-
and E-selectin19Fc[FUT7+] enabled HEK293T adhesion to P- and E-selectin