PLOS ONE

Surface Fucosylation of Human Adipose-Derived Stem Cells (ASCs) Augments

Homing to Bone
--Manuscript Draft--

Manuscript Number: PONE-D-13-31438

Article Type: Research Article

Full Title: Surface Fucosylation of Human Adipose-Derived Stem Cells (ASCs) Augments
Homing to Bone

Short Title: In vitro glycan engineering of CD44

Corresponding Author: Hongtao Tian

Union Hospital
Wuhan, Hubei CHINA

Keywords: Glycan Engineering; ASC; CD44; cell migration; Osteotropism

Abstract: There has been growing interest in the clinical use of adipose-derived stem cells
(ASCs), which are connective tissue progenitor cells. One of the greatest challenges in
stem cell-based tissue engineering and other cellular therapies is delivering large
number of viable stem cells with high efficiency. Homing of ASCs at a low efficiency is
due to lack of pertinent stem cell homing factors on their surface. Cellular recruitment
to bone marrow happens within specialized marrow vessels that permanently express
E-selectin, which recognizes sialofucosylated determinants on its various ligands. In
our study, we demonstrated that human ASCs do not express E-selectin ligands, but
express a CD44 glycoform bearing α-2,3-sialyl modifications. Using an α-1,3-
fucosyltransferase under appropriate enzymatic conditions, we converted the inborn
CD44 glycoform on ASCs into hematopoietic cell E-selectin/L-selectin ligand (HCELL),
which rendered powerful E-selectin binding under static or flow conditions without
negative effects on cell viability, proliferation or multipotency. The enhancement of the
homing abilities of HCELL+ ASCs was confirmed by transplantation into NOD/SCID
mice. Twelve weeks after transplantation, only few human osteoblasts localized to the
endosteum were detected in the mice received HCELL+ ASCs. These data reveal that
the enforced expression of HCELL confers tropism to bone and ex vivo fucosylation
might be a simple and effective method to enhance stem cell homing to bone marrows
of patients receiving stem cell-based therapies.

Order of Authors: Xin Jin

Xianzhe Liu

Wei Tong

Rabi M. Dhakal

Jian Wang

Shuhua Yang

Hongtao Tian

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Ethics Statement Human abdomen subcutaneous adipose tissue was obtained from five healthy women
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institutional review board or equivalent informed consent was obtained from each participant. We obtained umbilical cords
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Click here to download Manuscript: Surface Fucosylation of Human Adipose-Derived stem Cells (ASCs) Augments Homing to Bone.do

Surface Fucosylation of Human Adipose-Derived Stem Cells (ASCs) Augments

Homing to Bone

Xin Jin1#, Xianzhe Liu1#, Wei Tong1, Rabi M. Dhakal1, Jian Wang1, Shuhua Yang1*, Hongtao

Tian1*

1
Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of

Science and Technology, Wuhan, Hubei, China

* E-mail: jimmyking119@163.com or yangshunion@163.com

#
These authors contributed equally to this work.

1
ABSTRACT

There has been growing interest in the clinical use of adipose-derived stem cells (ASCs),

which are connective tissue progenitor cells. One of the greatest challenges in stem

cell-based tissue engineering and other cellular therapies is delivering large number of viable

stem cells with high efficiency. Homing of ASCs at a low efficiency is due to lack of pertinent

stem cell homing factors on their surface. Cellular recruitment to bone marrow happens within

specialized marrow vessels that permanently express E-selectin, which recognizes

sialofucosylated determinants on its various ligands. In our study, we demonstrated that

human ASCs do not express E-selectin ligands, but express a CD44 glycoform bearing

α-2,3-sialyl modifications. Using an α-1,3-fucosyltransferase under appropriate enzymatic

conditions, we converted the inborn CD44 glycoform on ASCs into hematopoietic cell

E-selectin/L-selectin ligand (HCELL), which rendered powerful E-selectin binding under static

or flow conditions without negative effects on cell viability, proliferation or multipotency. The

enhancement of the homing abilities of HCELL+ ASCs was confirmed by transplantation into

NOD/SCID mice. Twelve weeks after transplantation, only few human osteoblasts localized

to the endosteum were detected in the mice received HCELL+ ASCs. These data reveal that

the enforced expression of HCELL confers tropism to bone and ex vivo fucosylation might be

a simple and effective method to enhance stem cell homing to bone marrows of patients

receiving stem cell-based therapies.

2
INTRODUCTION

The potential of stem cell-based therapies for the repair and regeneration of tissues and

organs provides a promising therapeutic solution for tissue regeneration and potential

treatment of numerous diseases. The utilization of embryonic stem (ES) cells and induced

pluripotent stem cells (iPSCs) in clinic is limited due to the potential problems of ethical and

technical considerations [1,2]. As mesenchymal stem cells (MSCs) are not subject to the

same limitations, they seem to be an ideal population of stem cells for cell-based tissue

engineering. Moreover, large numbers of adipose-derived stem cells (ASCs) can be easily

harvested from adipose tissue. Recent studies have shown that the use of ASCs in

regenerative medicine is not limited to mesodermal tissue but extends to both ectodermal

and endodermal tissues and organs [3,4]. These characteristics make ASCs excellent

candidates for restorative cell therapies.

Although a growing number of studies have revealed the therapeutic potential of intravenous

administration of ASCs in skeletal diseases, myocardial injuries, and immunologic disorders

[5-7], a significant limitation in the development of cellular therapies using ASCs is their poor

capability of migrating and homing to the targeted tissue following systemic administration

[8,9]. The inefficient ASCs homing is due to various factors but is typically attributed to an

absence of pertinent cell surface homing ligands, particularly E-selectin ligands and CXCR4

[10-12]. Specifically, culture expanded ASCs develop heterogeneous receptor expression

and lose some key homing ligands during cell culture, which contributes to the inefficiency of

homing. This characteristic represents a major challenge for minimally invasive ASCs-based

3
therapies that require efficient localization and retention of cells within appropriate tissues

[13]. Therefore, it prompts us to think whether engineering the inborn surface molecule of

ASCs could generate adhesion ligands and, thus, enhance the homing ability of target cells to

specific tissues following systemic infusion.

Cell migration is a complex physiological process that involves an orchestrated series of

cellular events initiated by shear-resistant adhesive interactions between circulating cells and

the vascular endothelium. This process is largely mediated by selectins and their ligands,

resulting in cell-tethering and rolling (step 1), chemokine receptor engagement and resultant

activation of integrins (step 2), firm adhesion by integrins (step 3), extravasation (step 4) [14].

The portal for cell migrate into bone is the bone marrow (BM), and in vivo studies have

revealed that selectins and their ligands are critical in governing the initial interactions of

infused cells with BM endothelial cells [15-17].

Selectins are Ca2+-dependent membrane-bound C-type lectins that bind to carbohydrate

determinants expressed on their respective ligands, which consist of sialofucosylations

containing an a-2,3-linked sialic acid substitution on galactose and an a-1,3-linked fucose

modification on N-acetylglucosamine, which are together displayed as the terminal

tetrasaccharide sialyl Lewis X (sLex) [18,19]. L-selectin is expressed on leukocytes, whereas

E-selectin and P-selectin are cytokine-inducible endothelial cell adhesion molecules.

Furthermore, in humans or mice, the expression of E-selectin on the microvasculature of the

skin and the bone marrow is permanent [20].

Among human hematopoietic progenitor cells, a particular sialofucosylated glycoform of

CD44 known as hematopoietic cell E-/L-selectin ligand (HCELL) functions as the most

4
powerful E- and L-selectin ligand which renders greater E-selectin ligand activity [20,21]. The

fact that HCELL is a glycoform of CD44 created by expression of α-1,3-fucosyltransferase

which are able to transfer fucose in α-1,3-linkage to N-acetylglucosamine raises the premise

that transient α-1,3- fucosylation of CD44 on ASCs may generate HCELL and contribute to

selectin binding capability. The proof for this hypothesis is provided by approaches that have

involved genetic and chemical modifications of cells to over-express adhesion or chemokine

receptors leading to improved homing and functional outcome in animal models [22-24].

In our study, an effective amount of an α-1,3-fucosyltransferase (e.g., fucosyltransferase V

(FTV)) and its substrate (GDP-fucose) were used to investigate whether the enforced

fucosylation of CD44 on human ASCs may improve the affinity with which the cells bind to

E-selectins, enhance homing ability and subsequently confer osteotropism following systemic

delivery.

MATERIALS AND METHODS

Isolation, expansion and multi-differentiation ability test of ASC

Human abdomen subcutaneous adipose tissue was obtained from five healthy women with

lipo-aspirated surgery and all the procedures were approved by the Human Experimentation

and Ethics Committees of Wuhan Union Hospital. The written informed consent was obtained

from each participant. Human adipose-derived stem cells (hASCs) were isolated from

adipose tissue according to our previous studies [25]. Briefly, adipose tissue was minced and

then digested with 0.075 wt% collagenase type I (Sigma-Aldrich, St. Louis, MO) at 37 °C for

45 minutes with vigorous shakeing. The top lipid layer was removed and the remaining liquid

5
portion was centrifuged at 220 g for 10 minutes at room temperature. The pellet was

suspended in DMEM/F-12 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine

serum (FBS) (Invitrogen), filtered through a cell strainer with pore size of 100 mm diameter

and plated in 100 mm diameter culture dishes at 37 °C in a 5% CO2 humidified incubator.

Upon reaching complete confluence, the isolated cells were trypsinized, and subcultured at

1:2 dilutions under the same culture condition. For all experiments, we used hASCs within the

first three passages. For osteogenic differentiation, hASCs were cultured in the presence of

DMEM/F-12 (Invitrogen) supplemented with 10% FBS, 0.1 mM dexamethasone (Sigma), 10

mM β-glycerolphosphate (Sigma) and 50 mM ascorbic acid (Sigma) for about 2 weeks. At the

end of incubation, osteogenic differentiation was detected by Alizarin red staining. For

adipogenic differentiation, hASCs were cultured in the presence of DMEM/F-12 (Invitrogen)

supplemented with 10% FBS, 1 mM dexamethasone, 0.5 mM methyl-isobutyl-xanthine

(Sigma), 10 mg/ml insulin (Invitrogen) and 100 mM indomethacin (Sigma) for 3 weeks. At the

end of the incubation, adipogenic differentiation was detected by Oil-Red-O staining.

Isolation and expansion of HUVEC

We obtained umbilical cords from three healthy delivery women and all the procedures were

approved by the Human Experimentation and Ethics Committees of Wuhan Union Hospital.

The written informed consent was obtained from each participant. Human umbilical vein

endothelial cells (HUVECs) were extracted from human umbilical veins as described by

Bruno et al [26]. The culture media was replaced every other day and cells passaged 0-5

times were used in all experiments. For adhesion and flow chamber assays, we activated

6
confluent monolayers of HUVECs to express E-selectin by pretreating them with 2 ng/ml

IL-1β and 25 ng/ml TNF-α (both from Peprotech, Rocky Hill, NJ) for 6 hours.

Fucosyltransferase V and sialidase treatment.

hASCs were incubated at 106 cells/mL for 45 minutes at room temperature with 90 mU/ml

FTV (R&D Systems Inc., Minneapolis, MN) in 1 ml Hank’s Buffered Saline Solution (HBSS)

(Invitrogen) containing 20 mM HEPES (Sigma), 0.1% human serum albumin (HAS) (Sigma)

and 1 mM GDP-fucose (Santa Cruz Biotechnology, Santa Cruz, CA). After incubation, we

washed the hASCs with HBSS containing 0.2% BSA and 20 mM HEPES. Cell viabilities were

assessed by trypan blue exclusion. Then we used untreated and FTV-treated hASCs for

experiments. In some experiments, we first treated hASCs with FTV and then subjected them

to treatment with 100 mU/ml of S. pneumoniae sialidase (Sigma) for 1 hour at 37 °C

(FTV-sialidase-hASCs).

Flow cytometry

Cells were washed with PBS/2% FBS and incubated with respective primary monoclonal

antibodies (mAbs) or with isotype control mAbs. The cells were washed three times with

PBS/2% FBS and, for indirect immunofluorescence, incubated with appropriate secondary

fluorochrome-conjugated antibody. The cells were re-washed and analyzed on a BD LSR II

instrument (BD Biosciences, San Diego, http://www.bdbiosciences.com) with CELL Quest

software (BD Biosciences). The following antibodies were used for flow cytometry:

Recombinant mouse E-selectin-Ig chimera (E-Ig) was purchased from R&D Systems. Mouse

7
Anti-Human sLex mAb (CSELX-1) was purchased from BD Pharmingen. PE-Anti-Human

CXCR4 mAb (clone 12G5), PE-Anti-Human PSGL-1 mAb (clone KPL-1), Alexa fluor

488-Anti-Human CD29 mAb (clone TS2/16), PE-Anti-Human CD44 mAb (clone BJ18),

APC-Anti-Human CD49d mAb (clone 9F10), PE-Anti-Human CD49e mAb (clone NKI-SAM-1),

FITC-Anti-Human Integrin β7 mAb (clone FIB27) and all isotype control antibodies were

purchased from Biolegend (San Diego, CA). FITC-anti-human secondary antibody and

FITC-anti-mouse secondary antibody were purchased from Abcam (Cambridge, MA).

Cell proliferation assay

A cell proliferation assay was performed with the Cell Counting Kit-8 (CCK-8, Dojindo

Laboratories, Kumamoto, Japan) according to the manufacturer's instruction. The 450 nm

absorbance was measured with a microplate reader (Bio-Rad, Richmond, CA) at 1, 2, 3, 4

days after culture initiation..

Western blot analysis

Untreated and HCELL+ hASCs were washed twice with cold PBS, added with cell lysis buffer

(Beyotime, Shanghai, China), placed on ice for 15 minutes, then centrifuged at 12,000 g for

10 minutes. The protein concentrations were determined by using an enhanced BCA Protein

assay kit (Beyotime). The supernatant was cultured with 1× SDS-PAGE loading buffer at

100°C for 5 minutes for protein denaturation. Then, Equal amounts of protein from each

sample were used for SDS-PAGE gel electrophoresis. The protein was transferred onto

PVDF membrane, blocked by 5% fat-free milk powder at room temperature for 2 hours,

8
added with primary mouse mAb CSLEX1 antibody (BD PharMingen, 1:500) and primary

rabbit mAb CD44 antibody (Abcam, clone EPR1013Y, 1:2000) and cultured at 4 °C overnight,

then added with appropriate secondary HRP-labeled antibody to isotype antibodies and

cultured at room temperature for 2 hours, and finally visualized by ECL reagent.

Cell adhesion assay

HUVEC were seeded into the wells of 96-well culture plates (Costar, Cambridge, MA), and

grown to confluence. Prior to the adhesion assay, the HUVEC monolayers were stimulated

with cytokines IL-1β (2 ng/mL) and TNF-α (25 ng/mL) for 6 hours to induce E-selectin

expression. Static adhesion assays were performed using an equal number of hASCs (1000

cells/well) per condition. hASCs after respective treatments were labeled with 5mM CFDA-SE

(Invitrogen) and overlaid on HUVEC. After 1 hour of incubation at 37 °C, remaining adherent

cells were washed with PBS, and the number of attached cells was counted using a

fluorescence microscope.

Parallel plate flow chamber adhesion assay

The E-selectin ligand activity of hASCs after each treatment was evaluated using a parallel

plate flow chamber (Glycotech, Gaithersburg, MD). Under defined shear stress,

E-selectin-mediated adhesive interactions were examined between HUVEC monolayers and

suspensions of hASCs in flow. hASCs tethering and rolling on HUVEC monolayers were

visualized by inverted video microscopy (IX51, Olympus, Tokyo, Japan) in real time using the

parallel plate flow chamber as described [27,28]. Briefly, hASCs (5 × 105 cells/ml, suspended

9
in HBSS/10 mM HEPES/2 mM CaCl2+ solution (H/H/Ca2+)) were drawn over confluent

stimulated HUVEC monolayers. Initially, the hASCs were allowed to contact the HUVEC

monolayer at a shear stress of 0.5 dyne/cm2, subsequently the flow rate was adjusted to exert

shear stress ranging from 0.5–30 dynes/cm2. The number of cells rolling to the HUVEC

monolayer was quantified in the final 15 seconds interval at shear stress of 0.5, 1, 2, 5, 10, 20

and 30 dyne/cm2. Negative control groups were prepared by adding 5 mM EDTA to the H/H

assay buffer (to chelate Ca2+ required for binding) prior to use in adhesion assays.

Short-term homing assay

To measure the efficiency of bone marrow homing, cells were labeled with DiI (Invitrogen)

according to the manufacturer’s instructions and injected intravenously into NOD/SCID mice

(Beijing HFK Bioscience Co. Ltd, Beijing, China). Male and female pathogen-free NOD/SCID

mice, 6 weeks of age, were used as recipients. An equal number of untreated hASCs,

HCELL+ hASCs or FTV-sialidase-hASCs (5 × 106 cells/mouse in 200 µl HBSS) was injected

into each mouse. Mice that received HBSS buffer alone were used to determine background

signals. Mice were euthanized 16 hours after injection, femora were removed, and marrow

suspensions were assessed for frequencies of DiI-positive cells by flow cytometry. Flow

cytometric data were analyzed and expressed as percentage of dye-positive events detected

in 200,000 cells scanned within a narrow gate that is set to include hASCs. All procedures

were reviewed and approved by the Animal Research Committee at Tongji Medical School,

Huazhong University of Science and Technology. And all efforts were made to minimize

suffering.

10
In vivo bone formation studies

Male and female pathogen-free NOD/SCID mice were used as recipients. The mice received

intravenous injections of untreated hASCs, HCELL+ hASCs (5 × 106 cells/mouse in 200 µl

HBSS) or HBSS buffer alone. Twelve weeks after transplantation, the mice were sacrificed.

The skull from each animal was harvested, fixed in 4% formalin solution, decalcified in 4%

EDTA, and snap frozen in OCT with a bath of dry ice and 2-Methylbutane. Cryosectioning

was performed on a cryostat (Cryotome E, Thermo Shandon, Pittsburgh, PA), and 5-µm

longitudinal central sections were obtained. The slides were air dried in the dark and stored in

a slide box at –20 °C until staining. All procedures were reviewed and approved by the Animal

Research Committee at Tongji Medical School, Huazhong University of Science and

Technology. And all efforts were made to minimize suffering.

Immunofluorescence

Immunofluorescent staining of bone frozen sections was performed to determine the

presence of human osteoid tissue, a rabbit polyclonal antibody to human osteocalcin (Santa

Cruz Biotechnology, clone FL-100, 1:100) was used to stain tissue sections followed by an

FITC-conjugated goat antibody to rabbit IgG secondary antibody (Sigma, 1:200). Normal goat

IgG (Santa Cruz Biotechnology) was used as isotype controls. 4’,6-diamidino-2-phenylindole

(DAPI) (Invitrogen) staining for detection of cells in sections was performed by incubating

sections with DAPI for 3 minutes followed by extensive washing. The mounted tissue

sections were observed under a confocal laser scanning microscope (Nikon A1, Nikon, Tokyo,

11
Japan).

Statistical analysis

Statistical analysis was performed using Student’s paired t-test (Graphpad Prism, Graphpad

Software, San Diego, CA), and P < 0.05 was considered to be significant. The results are

expressed as mean ± standard deviation (SD).

RESULTS

Expression of homing receptors on hASCs

We analyzed the expression of characterized hematopoietic stem/progenitor cells (HSPCs)

homing molecules on the hASCs surface. By flow cytometric analysis, hASCs positively

expressed CD44, CD49d/CD29 (VLA-4), and CD49e/CD29 (VLA-5), but did not express

detectable levels of CSLEX-1, which recognizes an sLex-like epitope (Figure 1); they also did

not express E-selectin-immunoglobulin chimera (E-Ig), PSGL-1, and CXCR4 (Figure 1).

These findings show that hASCs lack some cell surface adhesion receptors shown to be key

effectors of homing to bone marrow in hematopoietic cells, especially E-selectin ligands and

CXCR4, the chemokine receptor, which is essential for recruitment of HSPCs to bone

marrow.

Enforced α-1,3-fucosylation of hASCs generated HCELL

After enforced α-1,3-fucosylation, all hASCs stained positively with mAbs CSLEX1,

consistent with strong expression of sLex epitopes and display prominent binding of E-Ig

12
(Figure 2A and B), correlated specifically strong expression of HCELL without changes in

overall CD44 levels (Figure 2C and D). The strong reactivity to CSLEX-1 and binding of E-Ig

were abolished by treating FTV-treated hASCs with sialidase (Figure 2A and B). Western blot

analysis of cell lysates from FTV-treated hASCs using mAbs CSLEX1 confirmed the

expression of HCELL in form of about 100 kDa (Figure 2B). The prominent expression of

HCELL was eliminated by treating FTV-treated hASCs with sialidase (Figure 2B). Moreover,

no obvious differences were found between untreated and FTV-treated hASCs in cell

viabilities assessed by trypan blue exclusion (data now shown). These results demonstrate

that in vitro α-1,3-fucosylation of hASCs generates HCELL without compromising cell viability.

Enforced HCELL markly upregulated binding of hASCs with HUVECs under static

conditions.

To determine the E-selectin-ligand activity of HCELL+ hASCs under static conditions, an

adhesion assay was performed. After 1 hour of incubation, HCELL+ hASCs showed 5-fold

greater adherence to E-selectin than untreated hASCs under static conditions (Figure 3A and

B). And the unregulated binding was abolished by treating HCELL+ hASCs with S.

pneumoniae sialidase (Figure 3A and B). These data suggest that prominent HCELL

expression induces a strong binding on endothelial E-selectin under static conditions.

Created HCELL markly upregulated rolling of hASCs with HUVEC under flow

conditions.

To determine the E-selectin-ligand activity of HCELL+ hASCs under physiologic flow

13
conditions, parallel-plate flow chamber studies were performed. Exofucosylation profoundly

increased the number of rolling cells on stim-HUVEC at a shear stress of up to 30 dyn/cm2

(Figure 4). The most obvious rolling interactions were observed at the shear stresses from

0.5 to 5 dyn/cm2. The prominent E-selectin ligand activity was completely eliminated by

treatment of HCELL+ hASCs with S. pneumoniae sialidase or in the presence of EDTA

(Figure 4). These findings imply that enforced HCELL expression yields a robust rolling

response on endothelial E-selectin under hydrodynamic shear conditions.

Ex vivo fucosylation of hASCs enhanced the efficiencies of BM homing in NOD/SCID

mice

To study the effect of enforced HCELL activity on short-term homing of hASCs in vivo, hASCs

were stained using a fluorochrome tracking dye, DiI and infused through the tail vein into

NOD/SCID mice. At 16 hours after injection, the recipients were sacrificed and DiI-positive

cells in the BM were quantitated by flow cytometry. In this study, HCELL+ hASCs accumulated

in the marrow ~ 6.5-fold more efficiently than untreat hASCs (Figure 5). The prominent

infiltration was completely abolished by treating HCELL+ hASCs with sialidase (Figure 5).

These data indicate that the created HCELL on hASCs after fucosylation licenses

immigration of these cells into the bone marrow.

Infiltrated HCELL+ hASCs localized to the endosteum and turned into osteoid.

To analyze whether marrow-infiltrating HCELL+ hASCs repopulate and turn into osteoblasts,

frozen sections of mouse calvarium were observed using a confocal laser scanning

14
microscope. Twelve weeks after injection, mouse calvarial bone was harvested, decalcified,

snap frozen, sectioned and immunofluorescence staining for bone-specific protein

osteocalcin and DAPI staining to identify human osteoid tissue. Sections of bone from mice

that received untreated hASCs, FTV-sialidase-hASCs or HBSS alone completely lacked

staining by mAb to human osteocalcin, whereas bone sections of all mice that received

HCELL+ hASCs showed sparse human osteocalcin postive cells localized to the endosteal

surfaces (Figure 6). Collectively, these results show that the extravasated cells can lodge

within marrow endosteal surfaces and generate human osteoid in mouse bone marrow.

DISCUSSION

Adipose-derived stem cells (ASCs) can be easily harvested, are available in large numbers,

demonstrate a strong multi-differentiation ability and have no ethical issues related to their

use. All of these qualities make ASCs a promising option for practical regenerative medicine.

For most therapies, systemic infusion remains the practical approach of administration,

especially for treatment of systemic diseases. However, the application of ASCs in stem

cell-based therapies is limited owing to poor homing and tissue tropisms. Therefore, finding a

way to improve the trafficking and repopulation of ASCs to intended sites is a high priority for

cell therapies. Although our data showed that culture-expanded human ASCs (hASCs)

express certain cell-surface receptors that mediate homing including VLA-4/5, they do not

express some homing receptors which are reported to be functioned as key effectors of

homing to bone which are typically utilized by hematopoietic stem cells, especially E-selectin

ligands and CXCR4, which governs rolling interactions of circulating cells on activated

15
vascular endothelium. These data are in agreement with previous studies which demonstrate

that poor tissue tropisms of mesenchymal stem cell result from the lack of pertinent cell

surface homing ligands [11,12].

In the present study, we use an α-1,3-fucosyltransferase (FTV) and appropriate enzymatic

conditions to glycan engineer cultured hASCs. After ex vivo α-1,3-fucosylation, all hASCs

stained positive with mAbs CSLEX-1, which was consistent with high expression of sLex

epitopes with concomitant strong binding of E-selectin-immunoglubulin chimera. The

absence of staining following sialidase digestion showed the specificity of CSLEX1 staining

for sialofucosylated carbohydrate modifications. By using adhesion and flow chamber assays,

we showed that the enforced expression of HCELL on hASCs markedly increases E-selectin

ligand activity under static and flow conditions. Thus, the created HCELL is a functional and

potent effector in rolling and adhesion interactions, which is similar to those of native HCELL

expressed on human leukocytes and human hematopoietic stem and progenitor cells [29,30].

Furthermore, the prominent HCELL expression was transient. It declined to ~ 80% by 24h，

and undetectable by 72h (data not shown). We presume that is owing to the cell division and

the turnover of surface protein. We proved that no major changes were observed in the

expression of membrane CD44 after the fucosylation. Accordingly, after infiltration within

target tissues, it is less likely to influence the long-term functions of hASCs.

In this study, we also proved that procedures involved in the modification of hASC with FTV

do not influence multipotential differentiation capability as engineered cells could be induced

into osteoblasts or adipocytes with appropriate stimuli (Figure S1). After fucosylation, growth

characteristics of hASCs were not found to be affected as determined by proliferation

16
analyses (Figure S2). Moreover, there are no significant changes in other member markers

analyzed after the fucosylation (as in Figure 1).

The in vivo data suggested that the high expression of HCELL on hASCs after

exofucosylation enhances immigration of these cells into the bone marrow and the

extravasated cells could repopulate, seed endosteum and undergo osteogenesis without

compromising their self-renew and multipotency ability. However, the presence of human

osteocalcin positive cells was sparse. And we presume the scarcity of human osteoid tissue

in normal adult mouse calvarial bone may be caused by an inapposite local

micro-environment, the slow turnover of native bone elements under steady-state conditions,

and the necessity for seeding of suitable osteogenic niches.

Even though the prominent expression of HCELL augments homing of hASCs, we do not

expect that this will reduce the accumulation of hASCs within filtering organs such as the lung,

liver, or spleen which is frequently observed following intravenous delivery [31]. Because the

engineered hASCs are still relatively large, activated and express certain adhesion

molecules.

The proof of principle for this hypothesis is provided by other methods including chemical and

genetic modification of mesenchymal stem cells to alter the repertoire of cell surface markers

to overcome the inefficient homing ability [22,23]. Although these strategies can improve the

delivery of cells to sites of inflammation or ischemia, the feasibility and applicability of these

methods is restricted. Chemical modification is complex and may have an impact on the

expression of other surface markers, while genetic manipulation of cells may not be practical

to change the expression of multiple receptors, and potential safety in long term is also

17
concerned.

Employing the same strategy with proper modifications, other cell-specific ligands may be

presented on hASCs for specific tissue targeting by following three steps: first, identify a

target glycoconjugate acceptor on the surface of cells, then use appropriate enzymatic

reagents and conditions to generate expected epitope without significantly adverse effects,

finally use pertinent biochemical studies and functional assays to verify target modification.

In summary, the data presented in this study showed that despite the absence of CXCR4 and

other molecules of homing to bone on hASCs, efficient homing and tropism to bone can be

conferred by α-1,3-fucosylation of CD44 on cell surface to express HCELL, which is the most

potent E-selectin ligand expressed on human cells. And ex vivo fucosylation might be a

simple and effective method to enhance stem cell homing to bone marrows of patients

receiving stem cell-based therapies. Moreover, given the general expression of E-selectin at

injury or inflamed tissues [32], enforced HCELL expression may have important implications

for systemic cell delivery to pathological skeletal or nonskeletal tissues including heart,

muscle, lung, liver for therapeutic function or tissue regeneration.

18
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21
Figure Legends

Figure 1. Expression of homing receptors on hASCs. Expression of E-selectin ligand

activity (E-Ig binding), CSLEX1, CD44, PSGL-1, CXCR4, CD49d/CD29 (VLA-4),

CD49e/CD29 (VLA-5), CD49d/β7(LPAM-1) on hASCs analyzed by flow cytometric. Dotted

line is isotype control staining, black line is E-Ig chimera or specific antibody. n = 3 per group.

Figure 2. α-1,3-fucosylation of CD44 on hASCs generate HCELL without changes in

overall CD44 expression. (A) Flow cytometric analysis of CSLEX-1 and E-Ig reactivity in

untreated hASCs (Dotted line), FTV-treated hASCs (solid line) or hASCs digested with S.

pneumoniae sialidase after FTV treatment (FTV-sialidase-hASCs) (shaded histogram). (B)

Western blot analysis of CSLEX-1 reactivity of cell lysates from untreated hASCs,

FTV-treated hASCs or FTV-sialidase-hASCs. FTV treatment induced CSLEX1-reactive

sialofucosylations on a about 100-kDa glycoprotein. Proteins were normalized to β-actin. (C)

Flow cytometric analysis did not reveal major differences in the expression of CD44 between

untreated and FTV-treated hASCs. (D) Western blot confirmed no significant differences in

the expression of CD44 between untreated and FTV-treated hASCs. n = 3 per group.

Figure 3. Ex vivo fucosylation enhances firm adherence between hASCs and HUVEC

under static situations. hASCs after respective treatments were labeled with 5 mM

CFDA-SE (Invitrogen) and allowed to bind to HUVECs for 1 hour at 37 ℃ as described. (A)

Binding of hASCs to unstimulated HUVECs (control), and binding of untreated hASCs,

HCELL+ hASCs, sialidase-digested FTV-treated hASCs to cytokines stimulated HUVECs,

22
magnification: ×10. (B) Number of adherent cells per field in each group. Data are expressed

as mean ± SD from three independent experiments, **P＜0.01 for comparisons of HCELL+

hASCs to other groups.

Figure 4. α-1,3-fucosylated hASCs obviously improve rolling with endothelial

E-selectin under flow conditions. Untreated hASCs, HCELL+ hASCs or sialidase-digested

FTV-treated hASCs (FTV-sialidase-hASCs) were perfused over cytokines stimulated (stim) or

unstimulated (unstim) HUVECs at 0.5, 1, 2, 5, 10, 20 and 30 dyn/cm2. Then the number of

rolling cells was determined. To control for the specificity for E-selectin binding, EDTA was

added to the assay buffer (EDTA group) before use in adhesion assays. Data are expressed

as mean ± SD from three independent experiments, #P＜0.001 for comparisons of HCELL+

hASCs to other groups at different shear stress level.

Figure 5. In vitro fucosylation of hASCs increases homing to mouse bone marrow.

Untreated hASCs, HCELL+ hASCs or FTV-sialidase-hASCs were labeled with DiI and

injected intravenously into NOD/SCID mice. Bone marrow was analyzed 16 hours after the

injection to determine the percentage of DiI positive cells present. Mice that received HBSS

without cells were used to determine the background signal. Data are expressed as mean ±

SD from three independent experiments, **P＜0.01 for comparisons of HCELL+ hASCs to

other groups.

23
Figure 6. Marrow-infiltrating HCELL+ hASCs repopulate and turn into osteoblasts.

Confocal images of frozen calvarium sections from NOD/SCID mice that received

intravenous injections of untreated hASCs, HCELL+ hASCs, sialidase-digested FTV-treated

(FTV-sialidase-hASCs) hASCs or HBSS alone. Twelve weeks after transplantation, the mice

were killed and their skulls harvested, snap frozen and sectioned. Sections were stained for

nucleus (blue) and human osteocalcin (green); in the merged image, arrows indicate cells

staining for osteocalcin localized to endosteal surfaces. Magnification: ×40; scale bars

represent 20µm.

24
Supplemental Figure Legends

Figure S1. FTV treated hASCs retain multipotential differentiation capability.

Osteogenic differentiatiated HCELL+ hASCs were positively stained by Alzarin red.

Adipogenic differentiatiated HCELL+ hASCs were positively stained by Oil Red. Magnification:

×20.

Figure S2. Surface fucosylation does not affect proliferation of hASCs. An equal

number (1000/well) of untreated or HCELL+ hASCs was plated in 96-well plates and cultured

for 4 days. To quantify cell proliferation, Cell Counting Kit-8 was used according to

manufacturer's instructions. There was no significant difference in proliferation between

untreated and HCELL+ hASCs at indicated time point. All experiments were carried out in

triplicate and 3 independent experiments were performed.

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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Figure S1
Click here to download Supporting Information: Figure S1.tif
Figure S2
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