DIRECT DIFFERENTIATION OF HUMAN UMBILICAL CORD BLOOD DERIVED

MESENCHYMAL STEM CELL INTO A SCHWANN- CELL PHENOTYPE

(STUDY IN VITRO)
Indah Yulianto *., Yuyun Rindiastuti H **
Dermato Venereology Department Medical Faculty Sebelas Maret University *
Doctoral Programme National South Korea University **

Abstract :
Cell based therapy have promising functional recovery for peripheral nerve repair,Schwann
cells are important components of the peripheral glia that form myelin, serving as the
microenvironment of nerve fibers in peripheral nervous system (PNS). Although Schwann
cells(SCs) and bone marrow derived mesenchymal stromal cells (BM-MSCs) are the main
cell source for nerve tissue engineering, the clinical application is limited because of donor
site morbidity, the invasive procedures and the decreased number SCs and BM-MSCs.
Human umbilical cord blood derived mesenchymal stem cells (HUCB-MSCs) could be a
promising cell source for nerve tissue engineering because they are easily eccessibly and their
use has no ethical issues. Schwanns cells (SCs) are essential facilitators for peripheral nerve
regeneration following injury, they release supporting neurotrophic factors that provide both
physical support and guidance. In vitro, these cells are slowing growing, hence not well
suited to a tissue engineering approach to nerve repair. Further more the expansion of native
Schwann cells to obtain a sufficient number of cells for clinical application within reasonable
period is technically difficult. Therefore, a method to induce easily accessible and highly
proliferative cells to differentiate into Schwann cell properties would be very practical and is
highly desirable. The purpose of our study was to direct differentiate mesenchymal sem cells
(MSCs) into Schwann cells with neural growth factor (NGF) induction.
Keywords : Schwann cells, differentiation, peripheral nerve regeneration, mesenchymal stem
cells, umbilical cord, growth factor.
Objective :
Analyze the optimal concentration and time of Schwann cells differentiation from human
umbilical cord blood (HUCBs) stem cells with neural growth factor (NGF) induction.
Introduction
Although the peripheral nervous system (PNS) and central nervous system (CNS) are
categorized into nervous tissue, nerve axons of the PNS are capable of regenerating after
damage while such regeneration does not normally occur in CNS
[1]
. The difference in the
regenerative capacity of the PNS and CNS is explained mainly by differences in the
properties of their glial cells (Figure.1)
[2]
. Schwann Cells one of the most important
components of the peripheral glia that forms myelin, serve as a favorable micro-environment
for the repair of damaged nerve fibers in PNS (Figure 1)
[2,3,4]




Fig.1. Overview of CNS and PNS responses after injury : (A). After CNS injury, microglia and macrophages
are recruited to the lesion site, secreting various cytokines and growth factors, while repoving necrotic tissue.
The formation of a glial scar by reactive astrocytes and fibrous scar by invading meningeal fibroblasts in the
lesion center limits further damage by suppressing inflammation and preserve the function of the salvageable
tissue. The presence of inhibitory molecules accumulating in the scar tissue inhibits regeneration of damage
axons.
(B). Following PNS injury, monocytes and macrophages are recruited to the lesion and immediately
phagocytose axon and myelin debris. Schwann cells differentiate, proliferate and align longitudinally to form a
scaffold (bands of Bngner) guiding regenerating PNS axons. (C) CNS injury leads to secondary degeneration
which is often accompanied by a cerebral spinal fluid-filled cyst. The presence of collagenous tissue and
inhibitory molecules which attach to the ECM of the fibrous scar are the main components for the failure
regeneration. (D) Axon successfully innervate their targets while Schwann cells produce new myalin to insulate
the regenerated axons after PNS injury. (According : Jessica Schira : Transplantation from human umbilical
cord blood to improve regeneration after spinal cord injury, Inaugural Dissertation,December 2010)
[4]
.
Normally, peripheral myelin, as well as myelinating Schwann cells, inhibit axonal
regeneration, but the cellular cascades that occur after damage initiate the removal of
damaged myelin and the dedifferentiated Schwanns cells actively produce factors that
strongly promote the growth of regeneratig axons.
[5,6]
Schwann cells that have been
collected in vitro and transplanted are known to support axonal regeneration in the damaged
nervous system. These cells overcome the inhibitory environment to elicit axonal
regeneration and construct myelin in the CNS
[7,8)
. For these reasons, Schwann cells are
concidered one of the most suitable cell types for inducing axonal regeneration in both the
PNS and CNS.
Although Schwann cell collection and transplantation are effective for the treatment of neural
diseases, isolation of Schwann cells causes new damage to other peripheral nerve sgments,
causing undesirable iatrogenic injury to the donor. Furthermore, expansion of Schwann cells
to obtain an adequate cell number for clinical application within a reasonable period of time
is difficult. Therefore, a method to induce easily accessible and highly proliferative cells to
differentiate into cells with Schwann cell properties wwould be very practical and is highly
desirable. Here in this review, we focus on the potential of HUCBs to trans-differentiate into
functional Schwann cells, by neural growth factor induction.
Materials and Methods
Separation and culture of HUCBs
After our protocol had been determined and institutional review board approval obtained,
HUCBs were obtained, using sterile syringes, from the umbilical veins immediate after full-
term deliveries. All the sample collected after obtaining written informed consent. The blood
sample volume was 50 to 100 ml.
Aspirated blood was diluted 1:1 with Hanks balanced salt solution (HBSS) and isolation
without Ficoll-Hypaque density gradient. Cells were initially plated into 25 cm
2
cultures
flasks at density 1 x 10
6
cell/cm
2
and sub culture every 2 weeks up to 4 passage.
Mesenchymal stem cells (MSCs) population were sorted using CD 105 magnetic cell sorting.
The fourth passage of CD 105 positive cells divided into 3 groups: control, neural growth
factor (NGF) 5 ng/ml and NGF 10 ng/ml. Induction which observe every 3 hours until 18
hours. Immunocytochemistry using nestin, glial fibrillary acidic protein (GFAP) and neuron
specific - III tubulin antibody were used to analyze Schwann cells expression in each group
and each time of evaluation. The level of expression was analyzed by Friedman and anova
statistical test to compare means between groups.
Result
It was obtained 2,35 X 10
7
cells/cm
2
nucleated cells after cell isolation without ficoll. In all
cultures adherent cells were observed 24 hours after being cultured. Cells presented
mesenchymal morphology. In most of the cultures, cell proliferation occurs in the first week.
HUCB stem cell differentiation into NPC performed at the fourth passage when cells are in a
phase of transient amplifying cells that have the capacity of a state of optimal differentiation.
In this research, the neural growth factor induction of 5ng/ml dan10ng/ml, this study
compared the expression of NPC in three groups: control group, group 5ng/ml, and 10ng/ml.
each group comes from one source of umbilical cord blood culture on the fourth passage,
each group consisting of 18 subgroups of cells were observed every 3 hours and assessed the
expression of nestin, GFP, and Beta III tubulin.
At the fourth passage, cell were turn into transient amplifying stage which potential to
differentiate into Schwann cell with NGF induction. There were significant result of nestin,
GFAP, and early neuronal differentiation marker TUJ1 expression betwen control, NGF
5ng/ml, and NGF 10ng/ml groups which were indicated with P=0.012; P=0.014; P=0.010
using friedman test (Tabel 1).
Glial nerve outgrowth and Schwann cells indicated with high expression level of GFAP and
neuron specific - III tubulin were observed at 10ng/ml and 18 hours.(Figure 5,6)






Antibody
expression
N Mean SD Friedman P
Nestin
18 5361.44
2390.94 14.62 .012
GFAP
18 4321.89
1838.51 14.24 .014
III tubulin
18 5074.06
2016.07 15.00 .010





Tabel 1. Statistical analysis between groups




Staining By Hematoxillin
Eosin
Staining By Sirus red Staining by neural Specific
Enulase















Figure 2 : Series of light micrographs showing the result of HUCBs cultures, has
transdifferentiation to mesenchymal stem cells
(Cultures were then treated with 5 ng/ml and 10ng/ml NGF)





Figure 3: Mesenchymal stem cells-derived Human Umbilical Blood Cord, before induce by
NGF (arrow)


Figure 4 : Confocal photographs showing that MSC-derived HUCB in neurobasal media
supplemented with NGF 5ng/ml, the highest result Expression Schwann cell marker Nestin,
in 3 hours to passages-4 over untreated control cells.


Figure 5: Confocal photographs showing that MSCs-derived HUCB in neurobasal media
supplementaed with NGF 5ng/ml. Increased the expression of Schwann cell marker glial
fibrillary acidic protein (GFAP) highest for 18 hours to passage-4 over untreated control
cells




Figure 6: Confocal photographs showing that MSCs-derived HUCB in neurobasal media
supplemented with NGF 5 ng/ml. Increased the expression of Schwann cell marker III tubulin for
18 hours to passage-4 over untreated control cells.

Observations antibody expression levels in each treatment group performed by using a photo
camera types Olympus 3.2 under the microscope at a magnification of 40 times the field of
view. Quantification of the expression levels of antibodies made by software IMGE J8.
Based on these calculations obtained the highest nestin expression (5 ng/ml) in the three-hour
increment, GFAP in the 18-hours and III-tubulin in the 18 hours (by 10 ng / ml-NGF)



Graph 1. Nestin expression in the control group, NGF 5ng/ml, and 10ng/ml NGF. Indicated on the
graph 4 highest nestin expression was found in the first three hours of treatment and in group III
treatment with NGF levels 10ng/ml.sedangkan lowest nestin expression obtained at the 18th hour in
the control group.
NGF 5ng/ml Control NGF 10 ng/ml

Graph 2 : GFAP expression in the control group, NGF 5ng/ml, and 10ng/ml NGF. Shown the
highest GFAP expression obtained at the 18 hrs treatment and in treatment group III with 10ng/ml
NGF levels. GFAP expression while the lowest was found in the first three hours of treatment in the
control group.


Graph 3 : Beta III tubulin expression in the control group, NGF 5ng/ml, and 10ng/ml NGF. Indicated
on the graph 3 BETA III tubulin expression highest obtained at the 18 hrs treatment and in treatment
group III with 10ng/ml NGF levels. Beta III tubulin expression while the lowest was found in the first
three hours of treatment
Conclusion :
Human umbilical cord blood (HUCB) is the potential source of stem cells which could differentiate
into Schwann cells using neural growth factor (NGF) induction. NGF 10 ng/ ml concentration at 18
hpurs were the best condition to generate Schwann cells in peripheral glial nerve component.

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