IJHOSCR
Original Article
International Journal of Hematology- Oncology and Stem Cell Research
Expansion of human cord blood hematopoietic
stem/progenitor cells in three-dimensional
Nanoscaffold coated with Fibronectin
1
2
2
Seyed Hadi Mousavi , Saeid Abroun , Masoud Soleimani , Seyed Javad Mowla
3
1
PhD student of Hematology, Department of Hematology and Blood Banking, Faculty of Medical Sciences, Tarbiat Modares University,
Tehran, Iran
2
Associated Professor of Hematology, Department of Hematology and Blood Banking, Faculty of Medical Sciences, Tarbiat Modares
University, Tehran, Iran
3
Associated Professor of Genetic, Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Corresponding Author: Saeid Abroun, PhD. Associated professor of hematology, Department of Hematology and Blood Banking, Faculty of
Medical Sciences, Tarbiat Modares University, Tehran, Iran
Tel: +98-21-82884563
Fax: +98-21-82884507
Email: abroun@modares.ac.ir
Received: 25, Aug, 2014
Accepted: 15, Dec, 2014
ABSTRACT
Background: Allogeneic hematopoietic stem cell transplantation is used in the treatment of patients suffering
from hematologic and non-hematologic disorders, but the application is limited by the identification of a
suitable donor. Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cell (HSC)
transplantation. Despite all advantages, the limited cell dose is one of the major obstacles. Ex-vivo expansion
of HSC is an alternative way to overcome this problem.
Materials and Methods: In this study, polycaprolactone (PCL) scaffold coated with fibronectin (3D) is
compared to routine cell culture system (two dimensional, 2D) used for cell culture.1×104 cord blood CD34+
cells isolated by MACS were seeded on PCL scaffold and allowed to expand for 10 days. Before and after this
period, total cells, CD34+ cells, CFC assay and CXCR4 expression were evaluated.
Results: Our findings demonstrated that 3D scaffold produced a 58-fold expansion of total cells compared to
2D cultures (38-fold expansion). Also CD34+ cells in 3D compare to 2D cell culture was 40-fold and 2.66 fold
increased, respectively; this difference was statistically significant (p<0.05). Moreover, total number of
colonies in the 3D scaffold was higher than those of 2D cell culture system, but no statistically significant
difference was observed. Higher expression of CXCR4 in 3D compared to 2D showed better homing of cells
that were cultured in 3D scaffold (p<0.05).
Conclusion: PCL scaffold coated with fibronectin had higher number of total cells and CD34+cells than 2D
routine culture system. Findings revealed that 3D is a proper cell culture system for hematopoietic stem cell
expansion, compared to 2D.
Keywords: Cord Blood Stem Cell Transplantation, Hematopoietic Stem Cells, Tissue Engineering, 3D culture
INTRODUCTION
Allogeneic hematopoietic stem cell (HSC)
transplantation is used for treatment of several
hematologic disorders and immune deficiencies, but
the major obstacle to the use of this source is
finding suitable donors for most patients.1 Umbilical
cord blood is rich in HSCs and is an alternative
source for hematopoietic stem cell transplantation.
Despite the advantages of umbilical cord blood such
as lower incidence of GVHD after transplantation,
little or no risk for donors, low risk of
contamination, less HLA restriction and easy access,
the major disadvantage remains delayed
engraftment which results in delayed immune
IJHOSCR 9(2) - ijhoscr.tums.ac.ir – April, 1, 2015
IJHOSCR, 1 April 2015. Volume 9, Number 2
reconstitution and high rate of mortality, compared
to BM source. 2-5 Cell dose of cord blood is the most
important factor for transplantation,6 and to
overcome this limitation several efficient strategies
such as ex-vivo expansion by 3D scaffold,7 coculture by mesenchymal stem cell,8 bioreactor9 and
etc., have been developed. Hematopoietic stem
cells in bone marrow are in a specific place named
i he ells.10 Bone marrow established a balance
between stem cell self-renewal and differentiation.
It seems that the design of scaffold that mimics all
the essential characteristics of BM niche can
maintain stem cells in a self-renewable state and
influence the differentiation of stem cells.
Moreover, interaction between stem cells and their
niches can modulate HSC functions in vitro.11 The
fate of stem cells are affected by several factors
such as hormones, cytokines, extracellular matrix
(ESM)and cell interaction with other cells and
tissues.12 ECM component has a crucial role in HSC
niche. Adhesiveness or interaction between HSCs
and cell adhesion molecule providing homing or
retaining HSCs in bone marrow niche are provided
by these elements.12,13 Most of the in vitro cell
culture systems currently used for expansion of
hematopoietic stem cells are 2D systems.14 It is
important that 3D scaffold, producing sufficient cell
number, retains the capacity for self-renewal and
maintains proliferation of HSCs in cell culture
medium.Fabrication of 3D scaffolds is one of the
effective methods to promote cell growth and
provide structural support.15
Today, different 3D scaffolds have been used for
ex-vivo expansion of HSC including nanofiber mesh,
porous matrices, woven and non-woven fabrics and
microspheres. These fibers have different materials
such as Polydimethylsiloxane (PDMS), poly-L-lactide
acid (PLLA), Poly lactic-co-glycolic acid (PLGA),
Polycaprolactone
(PCL)
and
polyethylene
16
terephthalate (PET). The purpose of this study is
to establish the new 3D culture system by using
specific nanofiber and polycaprolactone (PCL)
coated with fibronectin for ex-vivo expansion of
cord blood hematopoietic stem cells.
MATERIALS AND METHODS
Isolation of CB-CD34+ progenitors
3D culture of cord blood hematopoietic stem cells
After obtaining informed consent, human cord
blood was collected from donors. For isolation
CD34+ cells, mononuclear cells (MNC) were
separated by Ficoll-Hypaque gradient centrifugation
(density 1.077 g/mL, Sigma) and then MACS CD34+
cell isolation kit was used (miltenyibiotec, USA).
Briefly, after centrifugation (Eppendorf) and cell
counting, 300µl buffer was added up to 108 total
cells. Then, 100 µl blocker and 100 µl CD34 micro
beads were added, mixed and incubated in 40C for
30 minutes. Cells were washed with buffer and
centrifuged at 300×g for 10 minutes. Next, the
supernatants were aspirated completely and
resuspended in 500 µl buffer. MS column was
placed in a magnetic field and rinsed with buffer.
Cell suspension was then applied onto the column.
The column was washed, removed from the
separator and placed into 15ml falcon tube.
Afterwards, the buffer was added to the column
and then the magnetically labeled cells were
immediately flushed out by firmly pushing the
plunger into the column. culture medium was
prepared by Stem line II serum-free media(Sigma,
Germany) supplemented with recombinant human
stem cell factor (SCF, 50 ng/mL, Peprotech, UK),
recombinant human thrombopoetin (TPO, 50
ng/mL, Gibco, USA) and recombinant human FMSlike tyrosine kinas 3 (FLT-3, 50 ng/mL, Gibco, USA).
Cell seeding into 3D scaffolds and control
PCL scaffolds were sterilized in 70% ethanol,
washed by PBS and then dried overnight under
sterile condition. Scaffolds were placed in 24-well
polystyrene plates (Grainger) and coated with
fibronectin in 50µg/ml concentration for 24h at 40C.
Then, we removed fibronectin solution and seeded
cells. In each well, 1×104CD34+ cells suspended in
250 µl culture medium were added. Plate was
incubated at 370C in 5% Co2 for 10 days. Half of the
medium was exchanged every 48h with fresh
medium and cells were counted.
Immunophenotype analysis
To evaluate the ability of HSC expansion and
differentiation, cells were removed from scaffold
and then the morphology of cells was checked and
73
International Journal of Hematology Oncology and Stem Cell Research
ijhoscr.tums.ac.ir
Seyed Hadi Mousavi, et al.
IJHOSCR, 1 April. Volume 9, Number 2
stained with monoclonal antibody CD34-PE and
CD45-FITC (Stem cell technology, Canada) against
human epitope. The tubes were incubated in 40C for
30 minutes. Isotype control was used to set
compensation and confirm the specificity. 10000
events were acquired on a Partec PAS flow
cytometer. Flow Jo software was used for data
analysis.
Colony assay
After 10 days of expansion, 10000 of expanded
cells were harvested and transferred to 2 ml of
methylcellulose medium (H4435, stem cell
technology, Canada) plated in 6-well dishes
(Grainger). H4435 contains 1% methylcellulose in
Is o e’s MDM (IMDM, sigma, Germany). After 14
days incubation in 370C and 5% CO2, CFU-GM, BFUE/CFU-E and CFU-GEMM (CFU-Mix) colonies were
counted.
Gene expression
To evaluate the homing ability of expanded cells
before and after expansion, CXCR4 expression was
assessed by qPCR. RNA isolated by Trizol (Sigma,
Germany) protocol. Briefly, 1ml Trizol was added
and mixed well with every 106 cells; then 200µl
chloroform was added and centrifuged at 40C for 15
minutes in 12000 rpm. The aqueous phase was
transferred to a new tube; 500µl isopropanol was
added and centrifuged at 40C for 10 minutes in
12000 rpm. The supernatant was then removed and
only RNA plate was washed by 75% ethanol for 5
minutes in 40C for 7500 rpm. The supernatant was
then removed and 20µl DEPC water was added to
RNA. To evaluate RNA quality, the value of OD260/280
and the concentration of RNA were measured using
Nano Drop (Nano Drop 2000c) system. cDNA
synthesis was performed by Thermo Scientific kit.
For this purpose, 200 ng total RNA was mixed with
1µl oligo dt primer, 0.5 µl AMV enzyme, 4 µl buffer
4X, 0.5 µl RNAse inhibitor and 1 µl dNTP in tube and
then nuclease free water was added up to a final
volume of 20 µl. After mixing well, the tube was
placed in a thermocycler using the following
program: 10 min in 25 0C, 1h in 420C and 10 min 750C.
After synthesis of cDNA, 1 µl cDNA was added and
mixed by real-time master mix (Ampilicon,
Denmark), gene-specific primers for CXCR4 and
GAPDH control gene (Table 1). It was then watered
up to 10 µl and placed in real-time PCR (ABI, USA) in
the following program: 15 in 95 0C (1 cycle), 20 sec
in 95 0C and 60 sec in 60 0C (40 cycles).
Statistical analysis
Statistical analysis performed by T-test using Graph
Pad prism6 software. Also, Real time PCR data was
analyzed by REST.2009 software.
RESULTS
Flowcytometry analysis
The purity of CD34+ cell cells isolated from cord
blood before and after expansion in 3D scaffold was
93% (Figure1) and 65.66±5.35%, respectively. It was
reported 7±0.86% in the control group, showing
statically significant difference (Figure2) (p<0.05).
Figure 1: Purity of CD34+ cells before expansion. After isolation of cord
blood cells, the surface marker CD34 was assessed immediately. A)
Isotypecontrol, B) Percentage of CD34+ cells from MNC isolated cord
blood
74
International Journal of Hematology Oncology and Stem Cell Research
ijhoscr.tums.ac.ir
IJHOSCR, 1 April 2015. Volume 9, Number 2
Expansion of cultured cells in 3D PCL coated with
fibronectin microenvironment
After a 10-day expansion culture in serum-free
medium, CD34+CB cells that cultured in 2D culture
wells showed 38±3.3 fold expansion of total cells
and 2.6±0.23 fold expansion of CD34+ cells (Figure
3). Fibronectin-coated PCL scaffold resulted in
significant improvement in expansion of CD34+ cells
with 58±1.63 fold expansion of total cells and
38.8±1.79 fold expansion of CD34+ cells (p <0.05).
Colony assay
To quantitate the clonogenic potential of
hematopoietic stem cells and progenitors in the
expanded cells, CFU assays were performed on cells
expanded under PLC coated with fibronectin
scaffold and plastic dish culture conditions (2D)
before and after 10 days of culture. The BFU-E/CFUE count in 3D scaffold, 2D culture system and
primary cells was 90.67±7.76, 59±3.27 and 53±7.78,
respectively. These differences were statistically
significant (<0.05). The CFU-GM count in 3D scaffold
(58±2.16) was higher than primary (47±2.94) and 2D
culture system (54.33±8.73), but these differences
were not statistically significant (p>0.05). CFUGEMM colony counts in all cell culture systems
were not statically significant (p>0.05). Also, total
colonies in 3D and 2D culture systems were higher
than primary cell culture (157.67±8.18, 121±4.55
and 107.67±6.13, respectively) and 3D was higher
than 2D cell culture system, showing statically
significant difference (p<0.05) (Figure 4).
Evaluation of HSC homing
To evaluate homing and migration of expanded
cells, assessment of homing factors such as CXCR4
can show the status of homing. CXCR-4 relative
expression in 3D scaffold and in 2D cell culture
system compared to before cell culture system had
increased 7.2 fold and decreased -0.8 fold,
respectively. CXCR-4 relative expression in 3D
scaffold increased 8.6 fold compared to 2D culture
(Figure 5).
DISCUSSION
By the first successful UCBT and thereinafter
establishment of cord blood banks, UCB was
increasingly used in the treatment of hematologic
3D culture of cord blood hematopoietic stem cells
and non-hematologic disorders. UCB is a valuable
source of HSCT which can be used as an alternative
for hematopoietic stem cell transplantation, as they
are easily accessible and more readily available
when a suitable donor is unavailable. Despite all
advantages, the limited cell dose is one of the major
obstacles. One of the best ways to overcome this
problem is expansion of HSCs focusing on
maintaining self-renewability and stemness.
In this research, 3D scaffold coated with
fibronectin was used as it had a higher capacity for
HSC expansion and lower differentiation compared
to 2D cell culture system. Moreover, flow
cytometric
analysis
demonstrated
lower
differentiation in 3D scaffold compared to the
control group. The cause of this difference improves
the interaction of cells and mimicking of
physiochemical,
cellular
and
natural
microenvironment (niche) in 3D scaffold. 3D
compartment is used to elevate the surface- tovolume ratio to increase cell interaction.
In 2D cell culture system, this interaction capacity
is low and limited between neighbor cells.18 In
another study, different scaffolds with variable
coating material were used for cord blood
expansion. Ehring et al. showed that cytomatrix
(tantalum-coated porous biomaterial) system
produced a great number of CD34+ cells compared
to 2D cell culture and culture in presence of bonemarrow stromal cells. Also, CD34+ and CD45+
increased 3-fold in cytomatrix and 1.3 and 1.6
increase in 2D cell culture in analysis by
flowcytometry, respectively. 19 Ferreira et al.(2012)
demonstrated that 3D scaffolds (PCL, fibrin and
collagen) with and without co-culture of cord blood
mesenchymal stem cells had large number of CD34+
cells
compared
to
2D
cell
culture
microenvironment.15 In another study, Feng et al.
showed that Fibronectin-conjugated PET film
resulted in a significant increase of CD34+ and total
cell expansion compared to 2D cell culture.20 Also,
our results support the findings of previous studies
and showed that 3D scaffold system had a better
environment for expansion of CD34+ and total cells
compared to 2D cell culture system. Our results
showed that total cells and CD34+ cells in 3D
scaffold were increased by 58 and 38-fold,
respectively.
75
International Journal of Hematology Oncology and Stem Cell Research
ijhoscr.tums.ac.ir
Seyed Hadi Mousavi, et al.
IJHOSCR, 1 April. Volume 9, Number 2
Figure 2: Percentage of CD34 + cells after a10-days extension on scaffold PCL nanofibers coated with fibronectin and the two-dimensional environment. A)
Isotype control in 2Denvironment, B) The percentage of CD34 + cells in the two-dimensional environment, C) Isotype control in3-D environment, D) The
percentage of CD34 + cells in three-dimensional environment.
Colony-forming potential depended on number of
hematopoietic stem cells and progenitor cells.
According to higher number of HSCs in 3D scaffold,
it seems that total colonies in 3D culture system
were higher than those of 2D culture system. Ehring
et al. (2003) showed the CFU-GM count in 3D
culture system was higher than routinely cell
culture system. Another study demonstrated that
scaffolds conjugated with fibronectin and collagen
had more total colonies than 2D cell culture system
but this difference was not statistically significant.19
Our results in CFC assay were similar to the abovementioned studies. In 3D culture system, the total
number of colonies was higher than that of 2D cell
culture system, but no statistically significant
difference was found. Also, BFU-E/CFU-E colony
numbers were higher in 3D when compared with
those of 2D cell culture, but CFU-GM colony
numbers were lower in 3D than 2D.
Expression of genes involving in homing of HSCs
allows them to be placed in proper site. But, cell
culture system may change their properties. The
culture systems that can maintain the potential and
homing properties of HSCs such as BM are better
than others. Therefore, in this study, we evaluated
CXCR4 expression in 3D culture system.
76
International Journal of Hematology Oncology and Stem Cell Research
ijhoscr.tums.ac.ir
IJHOSCR, 1 April 2015. Volume 9, Number 2
3D culture of cord blood hematopoietic stem cells
Figure 3: Effects of culture system on conventional and PCL nanofibers scaffold coated with fibronectin (2D versus 3D) on expansion of hematopoietic
stem cells. 10000 cells of CD34 + cord blood (93%) culture on the conventional and scaffolds for 10 days in serum-free culture medium and analysis of the
environment were taken. A) Total cell fold expansion, B) Percentage of CD34+, C): CD34+ cells fold expansion.
Ehring et al. reported that this epitope was
expressed on a large percentage of CD34 cells in
cytomatrix cell culture model.19 Our results
demonstrated a 5.5-fold increase in CXCR4
expression in PCL scaffold coated with fibronectin
compared to 2D cell culture model.
CONCLUSION
In this study, we found that 3D PCL scaffolds
coated by fibronectin were ideally suited for the
expansion of HSC. The use of this scaffold resulted
in higher cell proliferation rates, increased adhesion
and better homing cell.
77
International Journal of Hematology Oncology and Stem Cell Research
ijhoscr.tums.ac.ir
Seyed Hadi Mousavi, et al.
IJHOSCR, 1 April. Volume 9, Number 2
Figure 4: CFU numbers and progenitor percentage (CFU-GM, BFU-E/CFU-E, CFU-GEMM) based on total CFU analyzed for cells expanded in different
culture system for 10 days
Table 1: Primer sequence of genes
Figure 5: The changes in the expression of CXCR-4 after 10 days culture
in 3D compare to 2D
Genes
Primer sequence
CXCR-4 F
TGAACCCCATCCTCTATGCTT
CXCR-4 R
GATGAATGTCCACCTCGCTTT
GAPDH-F
TGCACCACCAACTGCTTAGC
GAPDH-R
GGCATGGACTGTGGTCATGAG
ACKNOWLEDGEMENT
This paper is based on research done for the PhD
thesis and was supported by Deputy of Research
Affairs and Faculty of Medical Sciences at Tarbiat
Modares University.
We would like to thank Dr. Zarrabi, Chief Director of
Royan Stem Cell Technology Company (Royan Cord
Blood Bank), and Mr. Janzamin for his assistance
with flow cytometry.
78
International Journal of Hematology Oncology and Stem Cell Research
ijhoscr.tums.ac.ir
IJHOSCR, 1 April 2015. Volume 9, Number 2
REFERENCES
1. Rocha V, Wagner Jr JE, Sobocinski KA, et al. Graftversus-host disease in children who have received a cordblood or bone marrow transplant from an HLA-identical
sibling. New England Journal of Medicine. 2000;
342(25):1846-54.
2. Barker JN, Krepski TP, DeFor TE, et al. Searching for
unrelated donor hematopoietic stem cells: availability
and speed of umbilical cord blood versus bone marrow.
Biology of Blood and Marrow Transplantation.
2002;8(5):257-60.
3. Dalle J, Duval M, Moghrabi A, et al. Results of an
unrelated transplant search strategy using partially HLAmismatched cord blood as an immediate alternative to
HLA-matched
bone
marrow.
Bone
marrow
transplantation. 2004; 33(6):605-11.
4. Davey S, Armitage S, Rocha V, et al. The London Cord
Blood Bank: analysis of banking and transplantation
outcome. British journal of haematology. 2004;
125(3):358-65.
5. Zarrabi M, Mousavi SH, Abroun S, et al. Potential uses
for cord blood mesenchymal stem cells. Cell Journal
(Yakhteh). 2014; 15(4):274.
6. Stanevsky A, Goldstein G, Nagler A. Umbilical cord
blood transplantation: pros, cons and beyond. Blood
reviews. 2009; 23(5):199-204.
7. Sachlos E, Czernuszka J. Making tissue engineering
scaffolds work. Review: the application of solid freeform
fabrication technology to the production of tissue
engineering scaffolds. Eur Cell Mater. 2003;5(29):39-40.
8. Robinson S, Ng J, Niu T, et al. Superior ex vivo cord
blood expansion following co-culture with bone marrowderived mesenchymal stem cells. Bone marrow
transplantation. 2006; 37(4):359-66.
9. Cabral J. Ex vivo expansion of hematopoietic stem
cells in bioreactors. Biotechnology Letters. 2001;
23(10):741-51.
10. Wilson A, Trumpp A. Bone-marrow haematopoieticstem-cell niches. Nature Reviews Immunology. 2006;
6(2):93-106.
11. Wilson A, Oser GM, Jaworski M, et al. Dormant and
“elf‐Re e i g He atopoieti “te
Cells a d Their
Niches. Annals of the New York Academy of Sciences.
2007; 1106(1):64-75.
12. Liu H, Lin J, Roy K. Effect of 3D scaffold and dynamic
culture condition on the global gene expression profile of
mouse embryonic stem cells. Biomaterials. 2006;
27(36):5978-89.
13. Vazin T, Schaffer DV. Engineering strategies to
emulate the stem cell niche. Trends in biotechnology.
2010; 28(3):117-24.
3D culture of cord blood hematopoietic stem cells
14. Even-Ram S, Yamada KM. Cell migration in 3D
matrix. Current opinion in cell biology. 2005; 17(5):52432.
15. Ventura Ferreira MS, Jahnen-Dechent W, Labude N,
et al. Cord blood-hematopoietic stem cell expansion in
3D fibrin scaffolds with stromal support. Biomaterials.
2012; 33(29):6987-97.
16. Hu J, Ma PX. Nano-fibrous tissue engineering
scaffolds capable of growth factor delivery.
Pharmaceutical research. 2011; 28(6):1273-81.
17. Gluckman E, Rocha V. History of the clinical use of
umbilical cord blood hematopoietic cells. Cytotherapy.
2005; 7(3):219-27.
18. Dhandayuthapani B, Yoshida Y, Maekawa T, et al.
Polymeric scaffolds in tissue engineering application: a
review. International Journal of Polymer Science.
2011;2011.
19. Ehring B, Biber K, Upton T, et al. Expansion of HPCs
from cord blood in a novel 3D matrix. Cytotherapy.
2003;5(6):490-9.
20. Feng Q, Chai C, Jiang XS, et al. Expansion of engrafting
human hematopoietic stem/progenitor cells in
three‐di e sio al s affolds ith surfa e‐i
o ilized
fibronectin. Journal of Biomedical Materials Research
Part A. 2006;78(4):781-91.
79
International Journal of Hematology Oncology and Stem Cell Research
ijhoscr.tums.ac.ir