U.S. patent application number 11/425949 was filed with the patent office on 2007-08-02 for composition comprising separated or proliferated cells from umbilical cord blood for treating developmental and/or chronic lung disease.
This patent application is currently assigned to Samsung Life Public Welfare Foundation. Invention is credited to Yun Sil Chang, Won Soon Park, Yoon-Sun Yang.
Application Number | 20070178073 11/425949 |
Document ID | / |
Family ID | 38322300 |
Filed Date | 2007-08-02 |
United States Patent
Application |
20070178073 |
Kind Code |
A1 |
Chang; Yun Sil ; et
al. |
August 2, 2007 |
Composition Comprising Separated or Proliferated Cells from
Umbilical Cord Blood for Treating Developmental and/or Chronic Lung
Disease
Abstract
The present invention relates to a composition comprising
separated or proliferated cells from umbilical cord blood for
treating developmental and/or chronic lung disease, more precisely
a composition containing separated or proliferated cells from
umbilical cord blood for intratracheal administration for treating
developmental and/or chronic lung disease. The cells of the
composition of the present invention have excellent activities of
proliferation and differentiation, so that the extraction,
separation and selection of a tissue donor are all very easy.
Besides, the composition of the invention can be very effectively
used for the treatment of developmental and/or chronic diseases by
intratracheal administration.
Inventors: |
Chang; Yun Sil; (Seoul,
KR) ; Park; Won Soon; (Seongnam-si, KR) ;
Yang; Yoon-Sun; (Seoul, KR) |
Correspondence
Address: |
GREENLEE WINNER AND SULLIVAN P C
4875 PEARL EAST CIRCLE, SUITE 200
BOULDER
CO
80301
US
|
Assignee: |
Samsung Life Public Welfare
Foundation
Seoul
KR
Medipost Co., Ltd.
Seoul
KR
|
Family ID: |
38322300 |
Appl. No.: |
11/425949 |
Filed: |
June 22, 2006 |
Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61K 35/51 20130101;
A61K 35/16 20130101; A61K 35/28 20130101; A61K 45/06 20130101; A61K
35/28 20130101; A61K 35/16 20130101; A61P 43/00 20180101; A61P
11/00 20180101; A61K 35/51 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/14 20060101
A61K035/14 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 2006 |
KR |
10-2006-0009625 |
Claims
1. A composition for treating developmental and/or chronic lung
disease comprising cells separated or proliferated from umbilical
cord blood.
2. The composition for treating developmental and/or chronic lung
disease according to claim 1, wherein the cells are one or more
cells selected from a group consisting of monocytes, such as
autologous hematopoietic stem cells, and mesenchymal stem cells
separated from umbilical cord blood and mesenchymal stem cells
sub-cultured and amplified from umbilical cord blood derived
mesenchymal stem cells.
3. The composition for treating developmental and/or chronic lung
disease according to claim 1, which additionally includes a medium
for those separated and proliferated cells.
4. The composition for treating developmental and/or chronic lung
disease according to claim 3, wherein the medium is selected from a
group consisting of DMEM, MEM, alpha-MEM, McCOys 5A medium (Gibco),
Eagle's basal medium, CMRL medium, Glasgow minimum essential
medium, Ham's F-12 medium, Iscove's modified Dulbecco's medium,
Liebovitz' L-15 medium and RPMI 1640 medium.
5. The composition for treating developmental and/or chronic lung
disease according to claim 1, wherein the composition is
intratracheally administered into a patient.
6. The composition for treating developmental and/or chronic lung
disease according to claim 1, wherein the chronic lung disease is
selected from a group consisting of cystic fibrosis, chronic
obstructive pulmonary disease or pulmonary dysplasia.
7. The composition for treating development and/or chronic lung
disease according to claim 1, wherein the composition includes one
or more subsidiary components selected from a group consisting of
serum, antibiotics and antifungal agents.
8. The composition for treating developmental and/or chronic lung
disease according to claim 1, wherein the composition includes one
or more subsidiary components selected from a group consisting of
fetal bovine serum, horse serum, human serum, Penicillin G,
streptomycin sulfate, amphotericin B, gentamycin and nystatin.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition comprising
separated or proliferated cells from umbilical cord blood for
treating developmental and/or chronic lung disease, more precisely
a composition containing separated or proliferated cells from
umbilical cord blood for intratracheal administration for treating
developmental and/or chronic lung disease.
BACKGROUND ART
[0002] The developmental and/or chronic lung disease includes adult
chronic obstructive lung disease (COPD) such as cystic fibrosis,
emphysema, and bronchopulmonary dysplasia of infant or premature
baby, etc. The seriousness of these diseases is that there is no
specific treatment modality even though the severeness and
chronicity of these diseases cannot be disregarded. The
bronchopulmonary dysplasia is a chronic lung disease developed
during the treatment of respiratory failure in a newborn infant or
a premature baby. Recent progress of treating a premature baby
increases the incidence of this untreatable disease (Avery M E et
al., Pediatrics 79: 26-30, 1987). Not only this disease is regarded
as a major cause of death of a newborn infant or a premature baby,
but also among survivors, long-term hospitalization is required and
serious side effects including pulmonary hypertension must be a
worry. Even after discharge from hospital, re-hospitalization rate
of bronchopulmonary dysplasia infants is usaually more than 50%
because they are more susceptible for acute viral bronchiolitis and
pneumonia, and various infectious problems. It is also known that
bronchopulmonary dysplasia might be progressed to bronchial asthma
because of continuous bronchial hyper-sesntivity (Coalson J J.
Semin Neonatol, 8:73-81, 2003) and associated with a serious
long-term neurodevelopmental sequalae such as cerebral palsy
(Bregman J and Farrell E E, Clin Perinatol, 19: 673-94, 1992).
[0003] An effective treatment method for bronchopulmonary dysplasia
has not been developed, yet. It has been a major approach for the
treatment of bronchopulmonary dysplasia to reduce barotrauma or
volutrauma caused by positive pressure ventilation or reduce oxygen
concentration during the artificial ventilation for a new born- and
a premature baby. In addition, steroid has been used to prevent
and/or treat inflammation in the damaged lung. However, steroid
treatment is now limited in relation to the recent reports that the
use of steroid might be responsible for later abnormal
neurodevelopmental prognosis, especially increase in cerebral
palsy, etc (Committee on Fetus and Newborn, Pediatrics, 109: 330-8,
2002).
[0004] Recently an anticipation for the treatment using stem cells
having a potential to be differentiated into every organs has been
rising. However, the transplantation of embryonic stem cells which
have excellent differentiation potential has serious technical and
ethical problems, that is generation of uncontrollable teratoma,
developmental problems caused by genomic imprinting, and questions
of moral and ethics. Therefore, the application of embryonic stem
cells seems to be limited and instead adult stem cells have been
moved into the center of our interest.
[0005] Among adult stem cells, stem cells of hematopoietic system
are a major target, stem cells of Hematopoietic system in bone
marrow, which is their source, are generally grouped into two;
hematopoietic stem cells and mesenchymal stem cells.
[0006] It has been already known that the hematopoietic stem cells
in bone marrow have plasticity, suggesting that they are
differentiated into not only hematopoietic system cells but also
other organ cells (Gussoni E, Soneoka Y et al. Nature.,
401:390-394, 1999; Petersen B E et al., Science. 284:1168-1170,
1999; Mezey E et al., Science. 290:1779-1782, 2000; Krause D S et
al., Cell., 105:369-377, 2001), which is though not very common so
that there is a doubt of biological usefulness, and some reports
that was resulted from cell fusion, not from direct differentiation
(Wagers A J et al., Science., 297:2256-2259, 2002).
[0007] Whereas, mesenchymal stem cells separated from bone marrow
of an adult mouse, which were then named `multipotent adult
progenitor cell (MAPC)` are very capable of being differentiated
into all three germ layers, ectoderm, mesoderm and endoderm, and in
fact they were proved when the stem cells were injected into
blastocyst of a mouse to be differentiated into almost every organ
cells. These cells were reported to bear embryonic stem cell
markers such as OCT-4, Rex 1 and SSEA-1 (Jiang Y et al., Nature.
418:41-49, 2002).
[0008] It is expected that the similar stem cells can be separated
from human bone marrow to be applied to cell therapy for diseases
and damages (Reyes M et al., Blood. 98:2615-2625, 2001; Woodbury D
et al., J Neurosci Res., 61:364-370, 2000). However, hematopoietic
stem cell and mesenchymal stem cell regions are reduced in bone
marrow according to aging (Geiger H and Van Zant G. Nat Immunol.,
3:329-333, 2002), and bone marrow extraction itself is distressing,
which is disadvantage for actual clinical application. Thus,
alternatives have been searched.
[0009] Umbilical cord is the line connecting a mother and a fetus
through which nutrition is provided and wastes is excreted, and the
blood inside is called umbilical cord blood. The umbilical cord
blood seems to be the most appropriate alternative for bone marrow
for hematopoietic stem cell extraction because it contains more
primitive stem cells, compared with those in bone marrow, and cell
extraction therefrom is much easier.
[0010] The transplantation of hematopoietic stem cells extracted
from umbilical cord blood has been clinically applied since 1980s,
based on such advantages, compared with bone marrow, as higher
hematopoietic proliferation activity which means more hematopoietic
stem cells per unit volume (Szilvassy S J et al., Blood.
98:2108-2115, 2001), less HLA incompatibility which means less
graft versus host reactions (Rocha V et al., N Engl J Med.,
342:1846-1854, 2000), simple and easy, and less invasive during
extraction (Rubinstein P et al., N Engl J Med., 339:1565-1577,
1998) and remarkably lower risks of autologous marrow
transplantation in case of various types of cancer or other
diseases. In particular, umbilical cord blood bank has been opened
recently, providing services of preservation and amplification of
umbilical cord blood and their cells, which triggers the clinical
practice for transplantation of hematopoietic stem cells of
umbilical cord blood.
[0011] It is controversial whether mesenchymal stem cells
particularly MAPC-like cells that have excellent differentiation
potential into various organ cells reside in umbilical cord blood.
If mass-production of such mesenchymal stem cells or MAPC-like
cells is possible from umbilical cord blood, it would be an
epoch-making discovery in cell therapy and cell & tissue
regenerative medicine. It has been predicted based on the
primitiveness of stem cells of umbilical cord blood that MAPC-like
cells exist more in umbilical cord blood than in bone marrow.
Recently mesenchymal stem cells were successfully separated from
umbilical cord blood (Erices A et al., Br J Haematol., 109:235-242,
2000) and further proved to have MAPC cell-level multipotency
enabling the differentiation into osteoblasts, lipocytes and nerve
cells ex vivo (Lee O K et al., Blood., 103:1669-1675, 2004). At
first, it was a common belief that the number of mesenchymal cells
to be taken from umbilical cord blood was small and the
proliferation of them was very difficult. But, according to recent
reports, it has been proved that umbilical cord blood can provide
huge number of mesenchymal stem cells and thereby ex vivo
amplification of these cells is also possible.
[0012] It has been reported that these cells still possess
multipotency after being amplified, and can be differentiated into
osteoblasts, chondroblasts, lipocytes and nerve cells ex vivo, and
nerve cells, cartilage and bone cells, hematopoietic cells and stem
cells in vivo (Kogler G et al., J Exp Med., 200:123-135, 2004).
[0013] Methodologically, umbilical cord blood extracted from the
real placental tissue can be most ideal source for autologous and
homologous stem cells and these stem cells obtained thereby can be
used directly or after amplifying stage whenever and as many as
required.
[0014] However, there has been no attempt to apply umbilical cord
blood derived stem cell transplantation to developmental and/or
chronic lung disease. There are a few experimental reports that the
adult bone marrow stem cells transplanted into a mouse with
pneumonia induced by irradiation were differentiated into bronchial
cells and type II pneumonocyte (Theise N D et al. Exp Hematol.,
30:1333-1338, 2002) and reduced bleomycin induced pulmonary
fibrosis in adult animal models (Ortiz L et al. Proc Natl Acad Sci
USA, 100:8407-8411, 2003).
[0015] There are some patents about disease treatment using the
umbilical cord blood originated cells. For example, Korean Patent
Publication No. 2003-0015160 describes a composition for the
treatment of articular cartilage damage comprising cell components
separated, proliferated and differentiated from umbilical cord
blood and a medium containing thereof. And, Korean Patent
Publication No. 2005-0105467 describes a method for treating
myelodysplastic syndrome and myelosclerosis by the administration
of umbilical cord blood-originated stem cells. However, there have
been no descriptions on the therapeutic effect of umbilical cord
blood-derived stem cell transplantation in developmental and/or
chronic lung disease.
[0016] Thus, the present inventors established bronchopulmonary
dysplasia model by administering high concentrated oxygen
continuously, and then administered a composition of the invention
into the airway. As a result, alveoli were increased in their
numbers and developed normally. In conclusion, the present
inventors completed this invention by confirming that the
composition of the present invention can be effectively used for
the treatment of developmental and/or chronic lung disease.
DISCLOSURE
[Technical Problem]
[0017] It is an object of the present invention to provide a
composition comprising stem cells of umbilical cord blood for the
treatment of developmental and/or chronic lung disease, for which
no specific treatment method has been reported, yet.
[Technical Solution]
[0018] The present invention provides a composition for treating
developmental and/or chronic lung disease comprising separated and
proliferated cells from umbilical cord blood.
[0019] Umbilical cord blood, the origin of therapeutic cells of the
invention, is defined as blood taken from umbilical vein connecting
placenta and a fetus. Umbilical cord blood is a natural by-product
of childbirth. The umbilical cord blood is much easier to obtain
than general mesenchymal tissue like bone marrow requiring several
steps of operation and it is also very easy to find a donor because
umbilical cord blood deposit industry is developing steadily and
infra has already been established. In addition, umbilical cord
blood-originated cells are the one that does not express
histocompatibility antigen HLA-DR (class II) which is the major
cause of rejection after tissue- or organ transplantation. Thus,
these cells can minimize the immune response according to
transplantation, for example rejection against transplanted tissue
or organ, suggesting that autologous or homologous umbilical cord
blood transplantation is very useful and effective.
[0020] Extraction and separation of umbilical cord blood is as
follows. In the case of normal vaginal delivery, right after
childbirth, placenta is still in the womb and umbilical vein is
expelled out. Thus, umbilical cord blood is extracted from the
exposed umbilical vein. In the case of cesarian section, after
placental separation from uterus
[0021] Umbilical cord blood is taken ex vivo from umbilical vein.
According to the present invention, umbilical cord blood is
extracted from the expelled umbilical vein right after childbirth
by aseptic manipulation. At this time, umbilical cord blood can be
taken either before or after placental separation from uterus.
Particularly, from the secured umbilical vein, umbilical cord blood
is taken into umbilical cord blood sampling bag containing
anticoagulant by using a sampling needle.
[0022] A composition of the present invention contains one or more
cells selected from a group consisting of monocytes such as
autologous hematopoietic stem cells and mesenchymal stem cells
derived from the umbilical cord blood, mesenchymal stem cells
derived from the umbilical cord blood, and mesenchymal stem cells
sub-cultured and amplified from the mesenchymal stem cells. The
mesenchymal stem cells derived from the umbilical cord blood are
multipotent, unlike the typical stromal cells of bone marrow,
suggesting that they are able to be differentiated into mesenchymal
tissues such as bone, cartilage, adipose tissue, muscle, tendon,
etc., under a proper condition. Umbilical cord blood derived
mesenchymal stem cells have ability of self-renewal, suggesting
that they are capable of proliferating under a proper condition,
and might exhibit anti-inflammation activity when transplanted.
Compared with those cells originated from mesenchymal stem cells
derived from general mesenchymal tissues such as bone marrow,
muscle and skin, these more primitive cells have far much excellent
cell proliferation, and differentiation, and regulation substance
secreting capacity.
[0023] To separate and culture mesenchymal stem cells derived from
umbilical cord blood, any method described in previous literatures
including Korean Patent Publication No. 2003-0069115 (Pittinger M F
et al. Science, 284: 143-7, 1999; Lazarus H M et al. Bone Marrow
Transplant, 16: 557-64, 1995) can be used and here is one example.
The extracted umbilical cord blood is centrifuged to separate
monocytes, which are then washed several times to eliminate
impurities. The washed monocytes are cultured in a culture vessel
with proper density, then the cells are proliferated with forming a
single layer. Among these proliferating cells, those who are
homogeneous and forming a colony with spindle shape, which can be
observed by phase contrast microscopy, are mesenchymal stem cells.
When these cells are proliferated, sub-cultures are performed until
the cells are amplified enough.
[0024] For the cryopreservation of cells of the present invention,
the conventional method well-known to those in the art can be used
(Doyle et al., 1995), that is, a medium for the cryopreservation is
composed of 10-20% FBS (fetal bovine serum), 10% DMSO
(dimethylsulfoxide) and 5-10% glycerol and cells are suspended at
the concentration of 1.times.10.sup.6.about.5.times.10.sup.6 cells
per 1 Ml of medium.
[0025] The cell suspension is distributed into glass- or plastic
ampoules for deep freezing, and then the ampoules are sealed and
put in a deep freezer programmed to proper temperature. At this
time, it is preferred to use a freeze-program allowing -1.degree.
C./min of temperature change in order to reduce cell damage during
thawing.
[0026] When the temperature of the ampoule reached -180.degree. C.,
it is transferred into a liquid nitrogen tank. Cells can be stored
therein for several years and their viability has to be checked at
least every 5 years.
[0027] To thaw these cells, the ampoule has to be transferred from
the liquid nitrogen tank into a 37.degree. C. water bath quickly.
The thawed cells in the ampoule are placed in a culture vessel
containing a medium supplemented with 10% FBA and 5% ES quickly
under the aseptic condition.
[0028] According to the present invention, cell concentration is
1.0.times.10.sup.5-1.0.times.10.sup.9 cells/Ml, and
1.0.times.10.sup.6-1.0.times.10.sup.8 cells/Ml is more preferred
and 1.0.times.10.sup.7 cells/Ml is most preferred. A composition of
the present invention is preferably intratracheally-administered as
close to lung tissues as possible, to increase therapeutic effect
by improved accessibility, compared with the conventional cell
transplantation using intravenous injection.
[0029] The present inventors centrifuged the extracted umbilical
cord blood to separate monocytes, then the separated cells were
cultured with a proper density in a culture vessel. When the cells
were grown to proper density, sub-cultures were performed. The
present inventors established a bronchopulmonary dysplasia model in
neonatal rats by administering high-concentrated oxygen
continuously from the birth. Bronchoalveolar lavage were obtained
and the lungs were extracted from the animal model and stained. As
a result, rats with bronchopulmonary dysplasia exhibited increased
respiratory rate, poor weight gain, and chronic inflammatory
reactions with monocytic infiltration and fibrosis with
over-proliferated interstitial fibroblasts in the lung (see FIG.
1-FIG. 3). Besides, radial alveolar count (RAC) representing the
number of alveoli was significantly decreased mean linear intercept
(MLI) representing the size of alveoli was remarkably increased,
and resultantly the ratio of RAC to MLI, an alveolar development
index, was significantly reduced, compared with that in the wild
type normal rat.
[0030] The composition of the invention comprising cells labeled
with red fluorescent PKH26 was intratracheally administered and
then lung tissue of the rat was observed with a fluorescent
microscope. As a result, the cells included in the composition of
the invention safely located in the lung (see FIG. 5). Damage in
the lung of the rat intratracheally administered with the
composition of the invention (HT) was alleviated (see FIG. 1-FIG.
3) and the ratio of RAC to MLI was increased (see FIG. 4).
[0031] The composition of the present invention can additionally
include a medium of cells separated or proliferated from umbilical
cord blood. The medium was to suspend cells and any medium of
general cell culture mediums, such as DMEM, MEM, alpha-MEM, McCOys
5A medium (Gibco), Eagle's basal medium, CMRL medium, Glasgow
minimum essential medium, Ham's F-12 medium, Iscove's modified
Dulbecco's medium, Liebovitz' L-15 medium, RPMI 1640 medium, etc,
can be used. Among these media, DMEM supplemented with 10% FBS is
preferred.
[0032] The cell culture medium of the present invention can
additionally include one or more subsidiary components, for
example, fetal bovine serum, horse serum or human serum,
antibiotics such as Penicillin G or streptomycin sulfate and
antifungal agent such as amphotericin B, gentamycin or nystatin to
prevent microorganism contamination.
[0033] The composition of the present invention includes the
effective dosage enough to induce alveolar development by one time
or several times administration. The composition can also be mixed
with other stem cells for successful transplantation and other
purposes. The composition can be used as not-frozen or can be
frozen for the next use. To freeze the composition, a standard
cryopresservative agent (ex: DMSO, glycerol, Epilife.TM. or cell
freezing medium (Cascade Biologics)) is added to the cells.
[0034] The cells separated and proliferated from unfrozen umbilical
cord blood are carried by infusion bag (ex: a product Baxter,
Becton-Dickinson, Medcep, National Hospital Products or
Terumo).
[0035] The present invention further provides a method for treating
developmental and/or chronic lung disease using a composition
comprising separated or proliferated cells from umbilical cord
blood.
[0036] The method of the present includes a step of administration
to a patient. The administration route is direct engraftment or
transplantation to the lesion of the lung or transplantation or
injection into airway, but not always limited thereto. Both
non-surgical administration using catheter and surgical
administration such as injection or transplantation after
thoracotomy are possible, but non-surgical administration using
catheter is more preferred. Intravenou injection, one of the
conventional methods for transplantation of hematopoietic stem
cells, can be also used.
DESCRIPTION OF DRAWINGS
[0037] The application of the preferred embodiments of the present
invention is best understood with reference to the accompanying
drawings, wherein:
[0038] FIG. 1-FIG. 3 are photographs illustrating that the
pathological findings about lung tissues of neonatal rats induced
with bronchopulmonary dysplasia, one of the developmental and/or
chronic lung diseases, after treating with the method of the
invention. FIG. 1 represents the normal control (NC), FIG. 2
represents the hyperoxia-exposed group (HC), and FIG. 3 represents
the hyperoxia-exposed and umbilical cord blood mesenchymal stem
cell transplanting group (HT).
[0039] FIG. 4 is a graph illustrating the alveolar development in
the lung of a neonatal rat induced with bronchopulmonary dysplasia,
one of the developmental and/or chronic lung disease, after
treating with the method of the invention,
[0040] NC: normal control
[0041] HC: hyperoxia-exposed group (bronchopulmonary dysplasia)
[0042] HT: hyperoxia-exposed and umbilical cord blood mesenchymal
stem cell transplanting group
[0043] *significant difference with NC group (p<0.05)
[0044] #significant difference with HC group (p<0.05)
[0045] FIG. 5 is a photograph illustrating that the therapeutic
cell component was safely located by the method of the invention in
the lung of a neonatal rat induced with bronchopulmonary
dysplasia,
[0046] Blue: nuclei stained with DAPI,
[0047] Red: labeled with PKH26
MODE FOR INVENTION
[0048] Practical and presently preferred embodiments of the present
invention are illustrative as shown in the following Examples.
[0049] However, it will be appreciated that those skilled in the
art, on consideration of this disclosure, may make modifications
and improvements within the spirit and scope of the present
invention.
EXAMPLE 1
Intratracheal Transplantation in Bronchopulmonary Dysplasia
Model
<1-1> Separation and Culture of Cells
[0050] The therapeutic cells of the invention were separated and
cultured as follows. The extracted umbilical cord blood was
centrifuged to separate monocytes. The separated cells were washed
several times to eliminate impurities. The washed cells were
cultured with a proper density in a culture vessel, observing the
proliferation of mesenchymal stem cells with forming a monolayer.
Then, when the cells were proliferated enough, sub-culture was
performed until enough cell numbers were obtained (Yang S E et al.,
Cytotherapy, 6(5): 476-86, 2004).
<1-2> Intratracheal Transplantation in Bronchopulmonary
Dysplasia Model
[0051] The therapeutic cells of the invention were intratracheally
transplanted in bronchopulmonary dysplasia model. To prepare the
bronchopulmonary dysplasia model, high-concentrated oxygen was
administered from right after birth and for 14 days into neonatal
rats which were given birth by timed-pregnant Sprague-Dawley rats.
To expose to hyperoxia, the rat was put in a 50 l acryl box, in
which humidity and temperature were controlled as 40-60% and
23-26.degree. C. respectively under 1 a.p, then the box was
saturated with 100% oxygen by 10 l/min for the first 10 minutes.
When the oxygen saturation reached 95%, which was measured by
oxygen analyzer, 100% oxygen was refluxed by 2.5 l/min, during
which the oxygen saturation was measured continuously to keep the
oxygen saturation more than 95%. To avoid pulmonary edema caused by
oxygen toxicity nursing rat dams were switched between room air and
95% oxygen every 24 hour.
[0052] Intratracheal administration of umbilical cord blood
mesenchymal stem cells was performed using 26-gauge needle on the
5.sup.th day from birth by the concentration of 5.83.times.10.sup.5
cells/0.05 ml after confirming air flow of trachea in the midline
area of the neck.
[0053] On the 14.sup.th day, the rats were anesthetized by
intraperitoneal ketamine injection. After fixing the limbs,
thoracotomy was performed to expose the heart and the lung.
Transcardiac perfusion with saline was done by introducing a
23-gage needle into the left ventricle, and followed by the right
atrial paracentesis. A 24-gauge catheter was inserted
intratracheally, followed by tight clamp of the right main bronchus
and excised cardiopulmonary tissues. 0.5 ml of saline was
administered by using 1 cc syringe through the inserted catheter
and then bronchoalveolar lavage fluid was obtained by suction. 10%
formalin was administered in the left lung under the pressure of 12
cmH.sub.2O through the inserted catheter, followed by
extraction.
[0054] The lung tissue sections fixed with 10% formalin for 24
hours were embedded in paraffin, which were then cut by 5 um thick
and stained with hematoxylin eosin, followed by observation under
optical microscope. From the observation, the number of
neutrophils, the level of fibrosis, the cell numbers and thickness
of alveolar septa and pulmonary interstitium, and the presence or
absence of pulmonary edema were investigated, and radial alveolar
count (RAC) representing newly-formed saccules and alveoli and mean
linear intercept (MLI) measuring the size of alveoli were measured
and their ratio was determined.
[0055] To measure the RAC, a vertical line was drawn from terminal
bronchiole to the nearest fibrovascular septum, and the number of
saccules between the terminal bronchiole and the fibrovascular
septum was counted under the optical microscope (.times.40). MLI
was calculated using 1 mm ruler with counting the number of septum.
Randomly selected different areas were observed under the
microscope 10 times for each area and the resulting values were
averaged for further statistic analysis.
[0056] Rats with bronchopulmonary dysplasia induced by
hyperoxia-exposure showed increased respiratory rate and poor
weight gain. The pathology of their lung showed chronic
inflammatory reaction accompanied by the increase of monocytes such
as alveolar macrophages and lymphocytes, and fibrosis accompanied
by over-proliferation of interstitial fibroblasts (FIG. 1-FIG.
3).
[0057] The lung tissue of a bronchopulmonary dysplasia induced rats
(HC), compared with that of normoxia control rats (NC) RAC,
indicating the number of alveoli (Husain A N and Hassel R G Pediatr
Pathol., 13:475-484, 1993), was significantly decreased, and MLI,
indicating the size of alveoli (Dunnill M S., Thorax
1962;17;320-328), was remarkably increased. As a result, the ratio
of RAC to MLI, which is an index for alveolar development (Husain A
N et al., Hum Pathol., 29:710-717, 1998), was significantly
decreased. The damage in pathology were significantly alleviated in
the lung of the rats intratracheally administered with the
therapeutic cell composition (HT) for the prevention and treatment
of bronchopulmonary dysplasia (FIG. 1-FIG. 3), and their ratio of
RAC to MLI was markedly increased (FIG. 4).
[0058] The composition of the invention comprising cells labeled
with red fluorescent PKH26 was intratracheally administered and
then lung tissue of the rat with bronchopulmonary dysplasia was
observed with a fluorescent microscope. As a result, the cells
included in the composition of the invention safely located in the
lung (FIG. 5). Therefore, it has been confirmed that intra-tracheal
transplantation of the therapeutic cells of the invention is very
effective way to treat developmental and/or chronic lung
disease.
INDUSTRIAL APPLICABILITY
[0059] The present invention provides a therapeutic composition
comprising separated or proliferated cells from umbilical cord
blood for treating developmental and/or chronic lung disease.
According to the present invention, when the composition of the
invention is administered directly into the airway, suggesting that
the composition is administered near the lung tissues, the
therapeutic effect is increased owing to its accessibility. In
addition, umbilical cord blood, a by-product of childbirth, can be
obtained much easier than general mesenchymal tissue such as bone
marrow, which requires several times of surgical operations. It is
another advantage of using umbilical cord blood that deposit
industry of umbilical cord blood has already been active, meaning
infra is established and thereby donor is easily searched. Further
the cells separated from umbilical cord blood do not express
histocompatibility antigen HLA-DR (Class II), which is the major
cause of rejection after organ transplantation. Therefore,
umbilical cord blood originated cells do not induce or minimize
rejection, which is a huge barrier for transplantation. Not only
autologous umbilical cord blood but also homologous umbilical cord
blood can be used. The composition of the present invention, thus,
can be effectively used for the treatment of developmental and/or
chronic lung disease.
[0060] Those skilled in the art will appreciate that the
conceptions and specific embodiments disclosed in the foregoing
description may be readily utilized as a basis for modifying or
designing other embodiments for carrying out the same purposes of
the present invention. Those skilled in the art will also
appreciate that such equivalent embodiments do not depart from the
spirit and scope of the invention as set forth in the appended
claims.
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