U.S. patent application number 11/742963 was filed with the patent office on 2008-11-06 for composite for thermo-sensitive cell-tissue transplanted scaffold and use thereof.
This patent application is currently assigned to TAIPEI VETERANS GENERAL HOSPITAL. Invention is credited to Shih-Hwa Chiou, Han-Tzo Lin, Cherng-Kang Perng, Yi-Ping Yang.
Application Number | 20080274054 11/742963 |
Document ID | / |
Family ID | 39939666 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080274054 |
Kind Code |
A1 |
Chiou; Shih-Hwa ; et
al. |
November 6, 2008 |
Composite for Thermo-Sensitive Cell-Tissue Transplanted Scaffold
and Use thereof
Abstract
A composite comprising a stem cell; a biodegradable layer, which
can provide an environment for the stem cell to grow and to
differentiate, and; a N-isopropylacrylamide (NIPAAm), which can
polymerize with the biodegradable layer and possess the
temperature-responsive character for easy stripping. This invention
also provides a method of treating a subject with a skin defect by
covering the composite of the present invention on the skin defect
of the subject in need of such treatment. Furthermore, using this
composite with different growth factors, stem cells can be induced
to differentiate into skin-related, neuronal cells, neuron, and
insulin-positive cells in biodegradable scaffolds as well as
transplanted graft. Finally, this invention also provides a quick
and convenient method of monitoring cell growth or tissue
engineering in an animal.
Inventors: |
Chiou; Shih-Hwa; (Taipei
City, TW) ; Perng; Cherng-Kang; (Taipei City, TW)
; Lin; Han-Tzo; (Taipei City, TW) ; Yang;
Yi-Ping; (Taipei City, TW) |
Correspondence
Address: |
WPAT, PC;INTELLECTUAL PROPERTY ATTORNEYS
2030 MAIN STREET, SUITE 1300
IRVINE
CA
92614
US
|
Assignee: |
TAIPEI VETERANS GENERAL
HOSPITAL
Taipei City
TW
|
Family ID: |
39939666 |
Appl. No.: |
11/742963 |
Filed: |
May 1, 2007 |
Current U.S.
Class: |
424/9.1 ;
424/93.7; 435/29 |
Current CPC
Class: |
A61P 17/02 20180101;
C12N 2539/10 20130101; C12N 2533/52 20130101; A61P 17/00 20180101;
Y10T 442/2525 20150401; A61L 27/60 20130101; Y10T 442/2926
20150401; A61L 27/3834 20130101; C12N 5/0068 20130101; A61K 35/12
20130101; A61F 2/105 20130101; A61L 27/50 20130101 |
Class at
Publication: |
424/9.1 ;
424/93.7; 435/29 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61K 49/00 20060101 A61K049/00; A61P 17/02 20060101
A61P017/02; C12Q 1/02 20060101 C12Q001/02 |
Claims
1. A composite comprising: (a) a stem cell which differentiates
into mature normal cell, osteocyte, chondrocyte, adipocyte,
epithelium cell, epidermis-related cell, keratocyte, neuron,
neuronal cell, insulin-positive cell, glucagon-positive cell, or
tissues thereof; (b) a biodegradable layer for the stem cell to
grow and differentiation, wherein the layer contains material
selected from the group consisting of gelatin, fibronectin,
collagen, laminin, bFGF, EGF, insulin, progesterone, glucose, SDF
and thymosin beta-4; and (c) a N-isopropylacrylamide (NIPAAm),
which polymerizes with the biodegradable layer to provide the
feature of thermo-sensitive response for easy stripping.
2. The composition of claim 1, wherein the biodegradable layer is
selected from the group consisting of gelatin, fibronectin,
collagen, laminin, bFGF and EGF.
3. The composition of claim 1, wherein the stem cell is selected
from the group consisting of (a) adult stem cell derived from bone
marrow, umbilical tissues, or placenta; (b) neural stem cell; and
(c) embryomic stem cell.
4. The composition of claim 1, wherein the N-isopropylacrylamide
(NIPAAm) further polymerizes with a cover.
5. The composition of claim 4, wherein the cover is PP
non-woven.
6. A method for preparing a composite of claim 1 comprising: (a)
irradiating a NIPAAm solution with a UV light; (b) crosslinking the
NIPAAm-grafted a cover in gelatin solution by the glutaraldehyde
crosslinking agent; (c) freezing and drying the NIPAAm-grafted
cover; (d) immersing the dried NIPAAm-grafted cover into the
glutaraldehyde solution to produce a crosslinked gelatin hydrogel;
(e) treating the crosslinked gelatin hydrogel with a glycine
aqueous solution to block non-reacted aldehyde groups, and; (f)
cultivating a stem cell on the crosslinked gelatin hydrogel.
7. The method of claim 6, wherein the biodegradable layer is
selected from the group consisting of gelatin, fibronectin,
collagen, laminin, bFGF and EGF.
8. The method of claim 6, wherein the stem cell is selected from
the group consisting of (a) adult stem cell derived from bone
marrow, umbilical tissues, or placenta; (b) neural stem cell; and
(c) embryomic stem cell.
9. The method of claim 6, wherein the UV light is 50-2000 W UV
light.
10. The method of claim 6, wherein NIPAAm-grafted PP non-woven in
step (c) is dried out.
11. The method of claim 6, which further washes with the
double-distilled water after step (e).
12. The method of claim 6, wherein the cover is a PP non-woven.
13. A method of treating a subject with a skin defect comprises
covering the composite of claim 1 on the skin defect of the subject
in need of such treatment.
14. The method of claim 13 wherein the skin defect is burn, trauma
or wound.
15. The method of claim 14 wherein the wound is resulted from a
surgery or a burn.
16. The method of claim 13 wherein the N-isopropylacrylamide
(NIPAAm) further polymerizes with a cover.
17. The method of claim 16, wherein the cover is easily peeled by
treating under room temperature on the cover.
18. A method of monitoring cell growth or tissue engineer in an
animal comprising applying the composite to the animal in need of
such monitoring, wherein the composite comprises a stem cell
labeled by a marker; a biodegradable layer, which can provide an
environment for the stem cell to grow, differentiate, and; a
N-isopropylacrylamide (NIPAAm), which can polymerize with the
biodegradable layer.
19. The method of claim 18, wherein the monitoring is directed to
development induction of skin-related, pancreas islet-related
cells, neural cell, or neurons.
20. The method of claim 19 wherein the pancreas islet-related cells
is like insulin- and glucagon-positive cells.
21. The method of claim 18, wherein the marker is green, red or
blue fluorescent protein gene.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a composite for thermo-sensitive
cell-tissue transplanted scaffold and use thereof.
DESCRIPTION OF PRIOR ART
[0002] Skin is not just a passive barrier for fluid loss and
mechanical injury but a complex organ in the human body. Skin
injuries and defects may be caused by burn, trauma, cancer, or
other diseases. For a small skin defect, primary or secondary
healing is typically the simplest and most effective management.
Autologous skin graft, however, is the standard treatment for large
skin defects such as a major burn or trauma. The disadvantages of
an autologous skin graft are possible donor site morbidity and the
limitation of available skin amount especially in the case of
subjects with extensive burns.
[0003] Skin tissue engineering is a possible solution for treating
extensive skin defects. The ultimate goal of skin tissue
engineering is to restore the complete functions of native skin,
but until now the structures and functions of skins are only
partially restored in known methods. Full-thickness skin
regeneration by means of tissue engineering requires a material to
restore the epidermal barrier function and dermal properties of
mechanical stability and elasticity. The combination of cultivated
keratinocytes and dermal substitutes in vitro is the current
approach with variable success.
[0004] Scaffold-guided tissue regeneration involves seeding highly
porous biodegradable scaffolds with donor cells and/or growth
factors, then culturing and implanting the scaffolds to induce and
direct the growth of new tissue. The goal is for the cells to
attach to the scaffold, then replicate, differentiate (i.e.,
transform from a non-specific state into a cell exhibiting the
functions of the target tissue), and organize into normal healthy
tissue as the scaffold degrades. This method has been used to
create various tissue analogs including skin, cartilage, bone,
liver, nerve, vessels, etc. For example, donor bone cells have been
cultured on hydoxyapatite (HA) ceramic scaffolds to create
small-scale bone. This scaffold is prepared using sea coral. The
microstructure of coralline HA is very similar to the mineralized
component of natural bone ECM. The diameter of the pores in this
structure is about 250 micrometers, or approximately three times
the diameter of a human hair. While HA is osteogenic, there is
still a need for better scaffolding materials which satisfy a range
of goals related to strength, toughness, osetoinductivity,
osteoconductivity, controlled degradation, and inflammatory
response.
[0005] To date, only tissue engineered skin has been
commercialized. There are still numerous issues to be addressed and
challenges to overcome, for scaffold guided tissue generation to be
extensible to the creation of more complex, large-scale structures.
One key barrier is how to manufacture scaffolds that exhibit
spatially controlled distributions of cells, growth factors, and
scaffold materials and microstructures. Scaffold-based processes
have limitations in addressing these needs due, in part, to
restrictions of the manufacturing methods used to synthesize and
seed scaffolds. Some of these limitations are summarized in the
table below.
[0006] In recent years, temperature-responsive material such as
thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) has been
applied in tissue engineering. Harimoto M et a/., disclosed a novel
approach for achieving double-layered cell sheets co-culture in an
article entitled: overlaying endothelial cell sheets onto monolayer
hepatocytes utilizing temperature-responsive culture dishes (J
Biomed Mater Res. Dec. 5, 2002; 62(3):464-70). Yamato M., et al.,
disclosed thermo-responsive culture dishes allow the intact harvest
of multilayered keratinocyte sheets without dispase by reducing
temperature (Tissue Eng. Aug. 7, 2001:(4):473-80). Hsiue G. H.
et.al., disclosed corneal endothelial reconstruction with a
bioengineered cell sheet (Transplantation, Feb. 15,
2006;81(3):473-6). (All of these references are herein incorporated
by reference in their entirety.) However, the development of the
above is still unsatisfied in commercialization due to their
undesirable properties. All referenced patents, applications and
literatures are incorporated herein by reference in their entirety.
Furthermore, where a definition or use of a term in a reference,
which is incorporated by reference herein is inconsistent or
contrary to the definition of that term provided herein, the
definition of that term provided herein applies and the definition
of that term in the reference does not apply. The invention may
seek to satisfy one or more of the above-mentioned desire. Although
the present invention may obviate one or more of the
above-mentioned desires, it should be understood that some aspects
of the invention might not necessarily obviate them.
SUMMARY OF THE INVENTION
[0007] The present invention provides a composite comprising (a) a
stem cell which differentiates into mature normal cell, osteocyte,
chondrocyte, adipocyte, epithelium cell, epidermis-related cell,
keratocyte, neuron, neural cell, insulin-positive cell,
glucagons-positive cell, or tissues thereof; (b) a biodegradable
layer for the stem cell to grow and differentiation, wherein the
layer contains material selected from the group consisting of
gelatin, fibronectin, collagen, laminin, bFGF, EGF, insulin,
progesterone, glucose, SDF and thymosin beta-4; and (c) a
N-isopropylacrylamide (NIPAAm), which polymerizes with the
biodegradable layer to provide the feature of thermo-sensitive
response for easy stripping.
[0008] This invention also provides a method for preparing a
composite of the present invention comprising: (a) irradiating a
NIPAAm solution with a UV light; (b) crosslinking the
NIPAAm-grafted a cover in gelatin solution by the glutaraldehyde
crosslinking agent; (c) freezing and drying the NIPAAm-grafted
cover; (d) immersing the dried NIPAAm-grafted cover into the
glutaraldehyde solution to produce a crosslinked gelatin hydrogel;
(e) treating the crosslinked gelatin hydrogel with a glycine
aqueous solution to block non-reacted aldehyde groups, and (f)
cultivating a stem cell on the crosslinked gelatin hydrogel.
[0009] The present invention further provides a method of treating
a subject with a skin defect comprises covering the composite of
the present invention on the skin defect of the subject in need of
such treatment.
[0010] Various objects, features, aspects and advantages of the
present invention will become more apparent from the following
detailed description of preferred embodiments of the invention,
along with the accompanying drawings in which like numerals
represent like components.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows (a) schematic diagram of the chemical reaction
of surface immobilized gelatin, plasma activation, PNIPAAm
graft-polymerization, and gelatin immobilize on PNIPAAm by GA
crosslink agent. (b) Scanning electrical microscope (SEM) study
showed the composite of the trilayer (gelatin/PNIPAAm/PP non-woven)
cell-transferred scaffold after freeze-dried. (c) the flow chart of
preparing the composite of the present invention. VOL:Ventilation
outer layer, ESI: Easy striped (NiPPAm) interface. (d) the
illustration of crosslinking gelatin with collagen fiber.
[0012] FIG. 2 shows the effects of temperature variation on
swelling ratio of the tested hydrogel. ( solid circle: Pure PNIPAAm
hydrogel, .box-solid. solid square: The trilayer cell-transferred
scaffold).
[0013] FIG. 3 shows isolation and Characteristic of adult human
bone marrow stem cells (hBMSCs). (a) hBMSCs were isolated and
usually appeared as spindle-shaped cells (bar: 30 .mu.m). (b) BMSCs
were cultured for 10th passage and labeled with monoclonal
antibodies of CD45, CD34, AC133, CD13, CD49b, CD105, CD81, SH3, and
SH4 for by FACS analysis.
[0014] FIG. 4 shows the cell growth and proliferation rate of
hBMSCs in the different passage. The hBMSCs isolated from 5
different donors were expanded in vitro and passaged every 7 days
for more than 30 passages.
[0015] FIG. 5 shows (a) the GFP-positive hBMSC cells were detected
by in vitro GFP imaging system. (b) BMSCs-GFP was cultured on
gelatin scaffold with pNIPAAm. (c) hBMSCs were found in the gelatin
scaffold by the H&E stain. Arrows: the tight conjunctions of
cell-to-cell (bar: 50 .mu.m). (d) The longitudinal section of the
multi-layer adult human bone marrow stem cell-transferred scaffold
by SEM. (e) The cell viability of hBMSCs-GFP cultured on gelatin
scaffold with pNIPAAm. (f) The elevation of growth rate of
hBMSCs-GFP on gelatin scaffold with pNIPAAm by in vitro GFP imaging
system. (*: p<0.05; **: p<0.01 compared with 10.sup.4
BMSC-GFP).
[0016] FIG. 6 shows determination of (a-f) wound size and (c, d)
the growth rate of hBMSCs-GFP in skin-defect nude mice by using in
vivo GFP imaging system.
[0017] FIG. 7 shows (a) the measurement of GFP-positive areas by
using in vivo GFP-positive. (*: p<0.05, compared with Day 3) (b)
The measurement of skin-defect size. (*: p<0.05, **: p<0.01,
compared with control; control: without any treatment).
[0018] FIG. 8 shows the examination of histological survey and for
the detection of the epithelial differentiation of hBMSCs by
western blotting assay. (a) and (b) The histology examination of
the healed wound with scaffold at day 21. Arrow: small vessel (bar:
150 .mu.m). (b) The detection of human markers for CD13, CD105,
pan-cytokeratin, and E-cadherin by western blotting. (c) The
expression ratio of human CD13, CD105, pan-cytokeratin, and
E-cadherin. (*: p<0.05; **: p<0.01 compared with day 0).
[0019] FIG. 9 shows evaluation of human stem cell and cytokeratin
markers by western blotting. (a) The protein expressions of human
CD13, CD105, pan-cytokeratin, and E-cadherin were measured in the
transplanted site of hBMSCs-GFP/gelatin/pNIPAAm-treated mice. (b)
The expression ratio of human CD13, CD105, pan-cytokeratin, and
E-cadherin in the different treatment groups at Day 0, Day 7, Day
14, and Day 21. (*: p<0.05; **: p<0.01 compared with day
0).
[0020] FIG. 10 shows (a) the distribution of stem cells in scaffold
by using H&E staining. (b) the observation of insulin
expression in stem cells by using immunofluorescent staining
method. (c) the nuclei position of alive cell as a control group is
labeled by the use of DAPI method. (d) PCR result of the gene
expression related to the differentiation of pancreatic stem cells
in scaffold (Left is control groups and right is induction
groups).
[0021] FIG. 11 illustrates induction of placenta-derived
multipotent stem cells (PDMSCs) differentiated into insulin- and
glucagons-positive cells. In the Figure (a) PDMSCs appeared as
spindle-shaped cells with scant cytoplasm and granules around the
nuclei; (b) the result showed that PDMSCs positively expressed the
mRNA of Nestin, Oct. 4, and Nanog; and (C) the flow cytometry
analysis for PDMSCs.
[0022] FIG. 12 shows (A) the protein expression of insulin in these
spheroid bodies derived from PDMSCs, (B) the numbers of the
insulin-positive cells gradually increased in SB-PDMSCs for 4 weeks
in pancreatic selection medium culture; (C) both of signals of
insulin (red fluorescence) and glucagon (green fluorescence)
co-expressed in SB-PDMSCs; and (D) the percentages of of
insulin-positive cells in PDMSCs.
[0023] FIG. 13 shows (A) the evidence of the renal subcapsular
space in SCID mice providing a microenvironment suitable for
endocrine cells differentiation; (B) the result of ex vivo biopsy
and histological examination of the transplanted SB-PDMSCs; (C) the
immunfluorescent result of insulin (red fluorescence)- and glucagon
(green fluorescence)-positive cells in the SB-PDMSC-derived tissues
of the subrenal site in SCID mice; and (D) blood glucose examined
every two days before and after the transplantation.
[0024] FIG. 14 shows induction of embryonic stem cell
differentiated into insulin-positive cells. In this figure, (A) and
(B) saturated undifferentiated ES cells as hanging drops by using
the medium for undifferentiated ES cells without LIF supplement;
(C) the result of the positive signals for glucagon (green color);
(D) insulin (red color); and (E) immunofluorescent assay (merged
imaging) after induction.
[0025] FIG. 15 shows (A) the hNSCs and (B) neurospheres (solid
arrow) seeded thereafter into the medium with gelatin scaffold; (C)
after five passages; (D) the growth and development of these hNSCs
in gelatin scaffold reviewed by H&E; and (E) hNSCs
proliferation ability of three cases in different conditions, (F)
Dot blot hybridization to examine the MAP2 gene expression in
scaffold. Equal total RNA (20 ug) was used for each examination,
(G) Dot blot hybridization to examine the NeuN gene expression in
scaffold. Equal total RNA (20 ug) was used for each
examination.
[0026] FIG. 16 shows (A) original wound; (B) original wound
confined by plastic ring; (C) the recovery of wound under natural
light, (D) the recovery of wound under in vivo fluorescent
microscopy; (E) the condition of outer layer of thermo-sensitive
scaffold being peeled after treatment of cold water; and (F) the
recovery of wound in nude mice.
[0027] FIG. 17 shows the proliferation and differentiation of
dermis derived stem cells matrixgel, scaffold alone or scaffold
with formulated medium (FM). In this figure, (A) The MTT assays
were used to evaluate the cells proliferation ability of three
cases in different conditions, (B) Dot blot hybridization to
examine the E-cadherin gene expression in scaffold. Equal total RNA
(20 ug) was used for each examination, (C) Dot blot hybridization
to examine the keratin gene expression in scaffold. Equal total RNA
(20 ug) was used for each examination.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Recently, mesenchymal stem cells (MSC) or stromal cells,
derived from the human bone marrow, can be purified and cultivated
based on their self-renewal ability to adhere to plastic and
differentiate into connective tissue lineages of
mesodermal--osteocyte, chondrocyte, and adipocyte. Adult human bone
marrow stem cells (hBMSCs) that are of multi-lineaged potential and
showed the ability to differentiate can be cultivated into cell
types of all three germ layers. Moreover, the present invention
also established a biomaterial system to transfer skin and
keratocyte with temperature-sensitive materials. In this invention,
the differentiation potential of epidermis and the capability of
skin wound healing were investigated, and the green fluorescence
protein (GFP) gene was transduced into the hBMSCs by using the
murine stem cell viral vector. The GFP-labeled hBMSCs were then
cultured on the gelatin scaffold with the polypropylene non-woven
N-isopropylacrylamide (pNIPAAm), and the GFP signals in the healing
processes of skin-defect animal model were further monitored. This
novel method provided a transferred system for cell therapy and
while maintaining its temperature-sensitive property and the ease
of peeling by lower-temperature treatment.
[0029] Accordingly, the present invention provides a composite
comprising: [0030] (a) a stem cell which differentiates into mature
normal cell, osteocyte, chondrocyte, adipocyte, epithelium cell,
epidermis-related cell, keratocyte, neuron, neuronal cell,
insulin-positive cell, or tissues thereof; [0031] (b) a
biodegradable layer for the stem cell to grow and differentiation,
wherein the layer contains material selected from the group
consisting of gelatin, fibronectin, collagen, laminin, bFGF, EGF,
insulin, progesterone, glucose, SDF and thymosin beta-4; and [0032]
(c) a N-isopropylacrylamide (NIPAAm), which polymerizes with the
biodegradable layer to provide the feature of thermo-sensitive
response for easy stripping.
[0033] In the present invention, the stem cell can differentiate
into mature normal cell, osteocyte, chondrocyte, adipocyte,
epithelium cell, epidermis-related cell, keratocyte, neuron,
neuronal cell, insulin-positive cell, or tissues thereof. In the
preferred embodiment, the stem cell is selected from the group
consisting of (a) adult stem cell derived from bone marrow,
umbilical tissues, or placenta; (b) neural stem cell; and (c)
embryomic stem cell.
[0034] In the present invention, the biodegradable layer contains
material selected from the group consisting of gelatin,
fibronectin, collagen, laminin, bFGF, EGF, insulin, progesterone,
glucose, SDF and thymosin beta-4. The preferred embodiment of the
layer contains gelatin, fibronectin or collagen type 1. In the
preferred embodiment of the layer contains gelatin or collagen type
1.
[0035] The formulated medium (FM) used for different stem cells is
illustrated in Table I.
TABLE-US-00001 TABLE I the formulated medium (FM) used in the
present invention Type of stem cells Formulated medium (FM) in
scaffold Bone marrow DMEM medium, gelatin (5%), fibronectin (1%),
collagen mesenchymal stem cells (1%), laminin (1%), bFGF (10
ng/ml), thymosin (10 ng/ml), SDF (10 ng/ml), 10% fetal calf serum
or human serum Dermis derived DMEM medium, gelatin (5%),
fibronectin (1%), collagen mesenchymal stem cells (1%), laminin
(1%), bFGF (10 ng/ml), EGF (10 ng/ml), SDF (10 ng/ml), 10% fetal
calf serum or human serum neuron stem cells DMEM/F12, bFGF (10 ng),
serum free pancreatic stem cells DMEM/F12, insulin (100 ug/ml),
transferrin (20 nmol/L), progesteron (60 umol/L), glucose 25 ug/ml,
10% Fetal calf serum
[0036] To facilitate performing the peeling action and to protect
the composite of the present invention, the N-isopropylacrylamide
(NIPAAm) can further polymerize with a cover to form a protective
layer. In a preferred embodiment, the cover is PP non-woven.
[0037] The present invention further provides a method for
preparing a composite of the present invention comprising: [0038]
(a) irradiating a NIPAAm solution with a UV light; [0039] (b)
crosslinking the NIPAAm-grafted a cover in gelatin solution by
glutaraldehyde crosslinking agent; [0040] (c) freezing and drying
the NIPAAm-grafted cover; [0041] (d) immersing the dried
NIPAAm-grafted cover into the glutaraldehyde solution to produce a
crosslinked gelatin hydrogel; [0042] (e) treating the crosslinked
gelatin hydrogel with a glycine aqueous solution to block
non-reacted aldehyde groups, and; [0043] (f) cultivating a stem
cell on the crosslinked gelatin hydrogel.
[0044] In a preferred embodiment of the present invention, the UV
light is 50-2000 W UV light. In a more preferred embodiment of the
present invention, the UV light is 100-500 UV light. In a further
more preferred embodiment of the present invention, the UV light is
150-250 UV light.
[0045] NIPAAm-grafted PP non-woven in step (c) is dried out. To
further remove non-reacted aldehyde groups, the method of present
invention further washes with the double-distilled water after step
(e).
[0046] The present invention further provides a method of treating
a subject with a skin defect, the method comprises covering the
composite of the present invention on the skin defect of the
subject in need of such treatment.
[0047] The term "skin defect" used herein includes, but not limited
to, burn, trauma or wound. In a preferred embodiment, the wound is
resulted from surgery or burn.
[0048] To facilitate performing the peeling action and to protect
the composite of the present invention, the N-isopropylacrylamide
(NIPAAm) can further polymerize with a cover to form a protective
layer. In a preferred embodiment, the cover is PP non-woven. The
cover is easily peeled by treating under room temperature (such as
25.degree. C.) on the cover.
[0049] The term "subject" used herein includes, but not limited to,
an animal. In a preferred embodiment, the subject is a mammal. In a
more preferred embodiment, the subject is a human.
[0050] The present invention further provides a method of
monitoring cell growth or tissue engineering in an animal, the
method comprises applying the composite to the animal in need of
such monitoring, wherein the composite comprises a stem cell
labeled by a marker; a biodegradable layer, which can provide an
environment for the stem cell to grow, differentiate, and; a
N-isopropylacrylamide (NIPAAm), which can polymerize with the
biodegradable layer.
[0051] The monitoring method of the present invention can be
applied to monitor cell development or tissue engineering in an
animal under in vitro or in vivo condition. In the preferred
embodiment, the monitoring method can monitor development induction
of skin-related, pancreas islet-related cells (such as like
insulin- and glucagon-positive cells), neural cell, or neurons.
[0052] The marker used herein includes, but not limited to, a
fluorescent protein gene such as green, red or blue fluorescent
protein.
EXAMPLE
[0053] The examples below are non-limiting and are merely
representative of various aspects and features of the present
invention.
Example 1
Materials and Methods
[0054] Preparation of Easy-Peeling Scaffold
[0055] Polypropylene (PP) non-woven was cut into strips (2.times.3
cm.sup.2and washed in distilled water and the 95% ethanol solution.
The N-isopropylacrylamide (NIPAAm) was supplied by Eastman Kodack
Co. Ltd USA; the ammonium persulfate (APS) was purchased from Wako
Pure Chemical Industry Co., Ltd. The
N,N,N',N'-tetra-methylethylene-diamine (TEMED) was purchased from
Fluka and N,N'-methylene-bis-acrylamide (NMBA) from Sigma. Other
agents or drugs were all of chemical grade. The PP nonwoven was
placed into a plasma treatment system with a rotating substrate
holder. Then the graft polymerization of the 10 wt % NIPAAm
solution was carried out with a 1000 W UV light irradiated for 30
minutes. The NIPAAm-grafted PP non-woven was chemically crosslinked
in gelatin solution by the GA crosslinking agent. The
NIPAAm-grafted PP non-woven was placed into a -80.degree. C.
refrigerator for three hours and further placed into a freeze-dryer
for 1-2 days to completely dry the sample. Finally, the dry sample
was immersed into the glutaraldehyde solution to process the
crosslinking reaction overnight. After that, the crosslinked
gelatin hydrogel was further treated with the 0.1 M glycine aqueous
solution to block the non-reacted aldehyde groups, and then washed
three times with the double-distilled water. The preparation was
illustrated in FIGS. 1(a) and (c).
[0056] Easy-Peeling Scaffold Property Evaluation
[0057] Swelling behavior of the hydrogel in the distilled water was
observed and measured by the soaking weight variation in a
temperature-controlled water bath. The temperature of the water
bath was adjusted within 1050.degree. C. The swelling ratio was
calculated according to the following equation:
Swelling ratio=(Ws-W0)/W0
where W0 and Ws indicated weights of dried and swollen samples
respectively. The strip-off strength of pNIPAAm hydrogel was tested
by using the elongation machine (NEXYGEN). Samples were tightly
clipped by the clamping apparatus which was placed on both sides of
the samples.
[0058] Bone Marrow Stem Cells Culture
[0059] The Declaration of Helsinki was used and has been reviewed
by the Institutional Review Committee at Taipei Veterans General
Hospital. The bone marrow (BM) was obtained from 5 healthy adult
donors following informed consents (ages 17 to 63). The BM
mononuclear cells (MNC) were obtained by Ficoll-Paque density
gradient centrifugation (Lymphoprep, 1.073 g/ml; Sigma). Cells
(5.times.10.sup.5) obtained from the BM MNCs were negative for CD45
and glycophorin A after depletion of CD45.sup.+ and glycophorin
A.sup.+ cells by micromagnetic beads (MACS, Stem Cell
Technologies). These cells were then plated in the human
fibronectin (FN)-coated (5 ng/ml, Sigma) 96-well plates. The
expansion medium consisted of Dulbecco's modified Eagle's medium
with 1 g/l of glucose (DMEM-LG, Gibco) and 10% fetal bovine serum
(FBS; Gibco) supplemented with 10 ng/ml bFGF, 10 ng/ml EGF, 10
ng/ml PDGF-BB (R&D), 100 units/ml penicillin, and 1,000
.mu.g/ml streptomycin, and 2 mM L-glutamine (Gibco). Once the
adherent cells were more than 50% confluent, they were removed with
the 0.25% trypsin-EDTA (Sigma) and replated at a 1:3 dilution under
the same culture condition. The cell densities were maintained
between 1.times.10.sup.3 and 3.times.10.sup.3 cells/cm.sup.2.
[0060] Immunophenotypic Analysis.
[0061] For the hBMSC cell surface antigen phenotyping, sixth- to
eighth-passage cells were detached and stained with anti-CD13
(Chemicon), CD34 (Chemicon), CD44 (Chemicon), CD45 (Chemicon),
CD49b (Chemicon), CD81 (Chemicon), AC133 (Chemicon), SH2, SH3
(DAKO), and secondary fluorescein (FITC)- or phycoerythrin
(PE)-coupled antibodies (Chemicon). The hBMSCs were fixed with 2%
paraformaldehyde until analysis using the FACSCalibur apparatus
(Becton Dickinson).
[0062] GFP Gene Delivery by MSCV Retrovirus
[0063] The cDNA plasmids of MIGFP, PMD, and VZV-G of murine stem
cell retroviral vectors (MSCV; a gift from Shih C C and Yee J K;
City of Hope, Duarte, Calif., USA) have been described previously.
To generate the GFP retroviral supernatants, 293 cells were
transiently transfected by calcium phosphate-mediated
coprecipitation with 5 .mu.g of the plasmids. The cells were fed at
the 24.sup.th hour with postinfection, and the retroviral
supernatant was used at the 48.sup.th hour. The cell continued to
produce high-titer retrovirus for another 2 days. Supernatant was
used if needed for additional experiments. The supernatant was
collected, brought to 8 .mu.g of polybrene per ml-10 mM HEPES, and
filtered with a 0.45 .mu.m pore-sized filter. The hBMSC cells for
infection were washed and trypsinized. They then were plated at
10.sup.6 cells per well of a six-well dish and centrifuged. The
medium was removed, and the retroviral supernatant was added at 1
ml/10.sup.6 cells.
[0064] Animal Study and In Vivo GFP Imaging
[0065] The animal experiment followed and obeyed the "Principles of
laboratory animal care" of Taipei Veterans General Hospital and
National Yang-Ming University. After anaesthetizing with the 40
mg/kg pentobarbital intraperitoneal injection, a circular skin
defect with a 2-cm diameter including panniculous carnosus was
created on the back skin of the nude mice (8 w/o).
[0066] The defect was covered by the scaffold with the hBMSC
cultured in vitro for 2 to 3 weeks and surrounded by a plastic ring
to prevent epithelialization to adjacent skin. The hBMSC group mice
also received the in vivo GFP imaging evaluation. The excitation
filter of 470 nm with a lamp supply of optical lighting of 150
watts (Southern California Services, USA) was used as an excited
light source (470 nm) to project on the foci of the GFP-positive
cells of the living mice and ex vivo transplanted tissues. The GFP
imaging capture and photography were based on the record of the
digital camera (Olympus) through optical configuration of dissected
microscope (SZ60; Olympus) with a 515 nm viewing (emission) filter.
The growth size of the xenografts was plotted and analyzed with the
software of Image-Pro Plus (Media Cybernetics, USA).
[0067] Western Blot Assay
[0068] The scaffolds with hBMSCs at the the skin defect were
harvested at days 0, 7, 14, and 21 day of post-transplantation. The
cell lysates were prepared as described. Fifteen PL of the sample
was boiled at 95.degree. C. for 5 minutes and separated on a 10%
SDS-PAGE. The proteins were transferred to a Hybond-ECL
Nitrocellulose paper (Amersham) by the wet-transfer system. The
monoclonal antibodies (mAB) used to identify protein products were
anti-human cytokeratin mAB (Chemicon), anti-human E-cadherin mAB
(Chemicon), anti-human CD13 mAB (Chemicon), anti-human CD105 mAB
(Chemicon), and anti-.beta.-actin mAB (Chemicon). The reactive
protein bands were detected by the ECL detection system
(Amersham).
[0069] Statistical Analysis
[0070] Statistical analysis was performed by using the ANOVA test.
The results were reported as mean .+-.SD. A p<0.05 was
considered to be statistically significant.
Example 2
Easy-Peeling Scaffold Property Evaluation
[0071] As showed in FIG. 1(b), the composite of the trilayer
(gelatin/pNIPAAm/PP non-woven) cell-transferred scaffold was
demonstrated by scanning electrical microscope. As showed in FIG.
2, the thermo-sensitive response behavior and swelling ratio
variation between the pure pNIPAAm hydrogel and the trilayer
scaffold showed a similar trend. Furthermore, they all revealed the
equivalent lower critical solution temperature (LCST) point at
around 32.degree. C. It demonstrated that the pNIPAAm hydrogel
exhibited its temperature-sensitive property even if it was grafted
onto the surface of different substrates. The trilayer scaffold
after the freeze-dried treatment exhibited the higher force (57
Newton) for PP mesh being separated from the scaffold. When
immersed this composite in room-temperature water for 2-3 hours to
the swelling state, the strip-off force decreased to 1 Newton.
However, when it was put in hot water (>34.degree. C.), the
force increased to 7 Newton (Table II). The result showed that the
PP membrane could attach to the gelatin scaffold firmly when
applied to skin defects when the body temperature is around
37.degree. C. After the scaffold was healed to the wound bed, the
PP membrane could be easily stripped off by the lower-temperature
treatment.
TABLE-US-00002 TABLE II The stripped strength of pNIPAAm composites
at different elongation state Average Status stripped force (N)
Freeze-Dried 57.36 .+-. 3.07 Swelled at low-temperature
(<25.degree. C.) 1.27 .+-. 0.21 Swelled at high-temperature
(>34.degree. C.) 7.28 .+-. 0.36
Example 3
Bone Marrow Stem Cells (BMSCs) Culture
[0072] As showed in FIG. 3(a), the protocol for isolating the
hBMSCs from fresh human BM of five donors was used. hBMSCs usually
appeared as spindle-shaped cells with scant cytoplasm and had
granules around the nuclei. Within 7 days, the cultures of hBMSCs
were nearly confluent and were passaged at 1:3. As showed in FIG.
3(b), the flow cytometry analysis revealed that the hBMSCs were
strongly positive for CD13, CD49b, CD105, CD81, SH3, and SH4, but
negative for CD45, CD34, and AC133. As showed in FIG. 4, the hBMSC
morphology and phenotype remained unchanged for more than 30 cell
passages [n=5]. To further monitor the tumor cell proliferation in
vitro and in vivo, GFP gene was transduced into hBMSCs using MSCV.
As showed in FIG. 5(a), the GFP-positive hBMSCs (hBMSCs-GFP) were
then sorted by flow cytometry. The hBMSCs-GFP stably passaged and
expressed the markers of mesenchymal stem cells to 30.sup.th
passage (Table III).
TABLE-US-00003 TABLE III The percentage of immunophenotypically
positive MSCs at different passages P5 P10 P15 P20 P25 P30 CD13 90%
88% 92% 91% 87% 85% CD49b 87% 86% 88% 87% 85% 86% CD105 89% 85% 84%
90% 87% 88% CD34 0.5% 0% 0.3% 0% 0.5% 0.2% CD45 0% 1% 0% 0.5% 1%
0.5% P: cell passage
Example 4
In Vitro Culture of GFP-Labeled hBMSC on the Easy-Peeling
Scaffold
[0073] To further develop an easy-peeling scaffold with the adult
human bone marrow stem cells, the hBMSCs-GFP was cultivated by
using temperature-responsive culture surface-gelatin/pNIPAAm. By
using in vitro GFP imaging system, the results indicated in FIGS.
5(b) and 5(f) showed that 5.times.10.sup.5 hBMSCs-GFP can grow and
become confluent on the gelatin scaffold (3.14 cm.sup.2) with
pNIPAAm after 7 day culture. The histological study indicated in
FIG. 5(c) showed that hBMSC-GFP can attach at gelatin scaffold with
pNIPAAm. As showed in FIG. 5(d), the longitudinal section of SEM
study further revealed that the multi-layer structure of
hBMSC-transferred membrane (hBMSC/gelatin scaffold with pNIPAAm)
could be formed and exhibit the whole integrity for transplanted
manipulation. Furthermore, the cell viability and proliferation of
hBMSCs-GFP were analyzed by using MTT reduction assay. As showed in
FIG. 5(e), there were no significant difference between hBMSC group
and hBMSC-GFP group [p>0.05].
Example 5
Animal Study and In Vivo GFP Imaging
[0074] To examine the utility of the hBMSC-GFP/gelatin scaffold
with the pNIPAAm in the wound healing, the skin-defect model of
nude mice as showed in FIGS. 6 was used and evaluated by the GFP
imaging. The original wound as showed in FIGS. 6(a) and 6(b) was
3.14 cm.sup.2 confined by the plastic ring. The in vivo GFP imaging
technique was used to analyze the sizes of the skin defects of nude
mice in FIGS. 6(b), 6(c) and 6(d) after the transplantation of
hBMSC-GFP/gelatin scaffold with pNIPAAm. The green signals of GFP
imaging in FIGS. 6(c) and 6(d) were clearly detected in the skin
defect of the hBMSC-GFP mice. As showed in FIG. 7(a), the size of
the GFP-positive area at days 3, 7, 14, and 21 were 0.90.+-.0.18,
2.10.+-.0.22, 3.00.+-.0.23, and 3.14.+-.0.25 cm in the hBMSC-GFP
group.
[0075] As showed in FIG. 7(b), the wound sizes of all 3 groups were
no significantly different among the groups after 3 days of the
transplantation [FIG. 7(b)]. At day 7, the wound size of the
transplanted hBMSC/gelatin scaffold group decreased to 0.90.+-.0.22
cm and was significant smaller than the control group (n=12;
2.00.+-.0.19 cm.sup.2, p<0.001) and the scaffold-only group
(n=12; 1.50.+-.0.20 cm.sup.2, p<0.05). At Day 14, the wound of
the hBMSC group had healed completely. However, the wound size of
the control and scaffold-only groups were still having 0.40.+-.0.08
and 0.20.+-.0.09 cm.sup.2 of skin defects, respectively. At day 21,
the wounds in the 3 groups were all healed.
Example 6
Histology and Western blot Assay of hBMSC/Gelatin Scaffold With
pNIPAAm and Healing Mice
[0076] The histology examination of the healed wound was performed
with the hBMSC-GFP/gelatin scaffold at day 21. The result indicated
in FIG. 8(a) showed the transplanted cells under the scaffold had
formed the epithelium, connect tissues, and small vessels [FIG.
8(a)]. To further characterize the differentiation ability of the
transplanted hBMSC, the stem cell markers-human CD13 and human
CD105, keratinocyte markers-human pan-cytokeratin and human
E-cadherin were evaluated. Compared to the pre-transplantation
level, the expression of the human CD13 in the transplanted
hBMSC/gelatin scaffold of nude mice significantly decreased and
declined at days 7, 14, and 21 in a time-dependent manner.
Similarly, the expression of human CD105 in the transplanted site
was significantly decreased at days 14 and 21. More importantly,
the expressions of human pan-cytokeratin and human E-cadherin as
showed in FIGS. 8(b), 8(c) and FIG. 9(b) were detected in the site
of hBMSC/gelatin scaffold and significantly increased at days 7, 14
and 21.
Example 7
Evaluation of Preferred Biodegradable Layer
[0077] After testing various mixture of gelatin and glutaraldehyde
corsslinking reaction, 2.5% glutaraldehyde, the layer produced by
5% gelatin and 5% type I collagen provided best crosslinking
efficacy under lower temperature crosslinking reaction. All
reaction was controlled at 4.degree. C. over 36 hours. To further
construct the different micro-environments for stem cell growth and
differentiation, the gelatin polymer scaffold was made of 5% gel
form in warmed DMEM/F12 medium with fibronectin, collagen, laminin,
bFGF, or EGF growth factors at 37.degree. C. Due to the special
functional group (--NH) in the gelatin gel, it can be further
chemically cross-linked with gelatin via cross-linking fibronectin,
collagen, laminin, bFGF, or EGF growth factors.
Example 8
Differentiation of Pancreatic Islet-Like Insulin-Positive Cells
Induced by hBMSCs in the Biodegradable Layer of Scaffold Contained
with Fibronectin, Laminin, and Type I Collagen
[0078] hBMSCs cells were cultivated in cell layer in scaffold
(contained fibronectin, laminin, and type I collagen), and then
added with 10 mL of CMRL 1066 (5.6 mmoL/L glucose, Gibco.TM., USA)
media plus 2% Fetal bovine serum (FBS, Biological Industries,
Israel), ITSFn medium (composed: 1:1 of DMEM/F12, 0.6% Glucose, 25
.mu.g/mL Insulin, 100 .mu.g/mL Transferrin, 20 nmoL/L Progesterone,
60 .mu.moL/L Putrescine, 30 mmoL/L Selenium chloride, 2 mmoL/L
Glutamine, 3 mmoL/L Sodium bicarbonate, 5 mmoL/L HEPES buffer, 2
.mu.g/mL Heparin, 20 ng/mL human epidermal growth factor (EGF), 20
ng/mL human basic fibroblastic growth factor (b-FGF) and 20 ng/mL
human hepatocyte growth factors, all growth factors were purchased
from PerproTech, Israel). After 30 days, the ability of secreting
insulin and cell proportion made by hBMSCs in scaffold were
examined by immunological staining. As depicted in FIG. 10, PCR
results of the gene expression indicated the differentiation of
pancreatic stem cells in scaffold. The RNA expressions of insulin,
glucagon and somatostatin were all detectable in the
differentiating hBMSCs, but not observed in undifferentiated hBMSCs
(FIG. 10a). Furthermore, the pancreatic islet markers of insulin
(FIG. 10c), glucagon and somatostatin were showed the positive
immune reactivity in the spheroid body (FIGS. 10b, 10c and 10d) of
aggregated differentiated hBMSCs by using immunofluorescent
staining.
Example 9
Induction of Placenta-Derived Multipotent Stem Cells (PDMSCs)
Differentiated into Insulin-Positive Cells
[0079] This example followed the tenets of the Declaration of
Helsinki and informed consent was obtained from the donor subjects.
The tissues of human term placenta were dissected and digested by
collagenase P (Roche) with HEPES-buffered saline for 7 h at
37.degree. C. The dissociated cells obtained from human placenta
were negative for CD45 and glycophorin-A after depletion of
CD45.sup.+ and glycophorin-A.sup.+ cells by micromagnetic beads
(MACS). These cells were then plated in human fibronectin
(FN)-coated (5 ng/ml, Sigma) 96-well plates. Expansion medium
consisted of Dulbecco's modified Eagle's medium with 1 g/l of
glucose (DMEM-LG, Gibco) and 10% fetal bovine serum (FBS; Gibco)
supplemented with 10 ng/ml bFGF, 10 ng/ml EGF, 10 ng/ml PDGF-BB
(R&D), 100 units/ml penicillin, and 1,000 .mu.g/ml
streptomycin, and 2 mM L-glutamine (Gibco). In the example,
placenta-derived multipotent stem cells (PDMSCs) were isolated as a
follow-up to our previous protocol of negative immunoselection
(CD45 and glycophorin-A). PDMSCs usually appeared as spindle-shaped
cells with scant cytoplasm and granules around the nuclei (FIG.
11A). Within 7 days, PDMSC cultures were nearly confluent and were
passaged 1:3. By using RT-PCR, the result showed that PDMSCs
positively expressed the mRNA of Nestin, Oct.-4, and Nanog (FIG.
11B). Flow cytometry analysis revealed that PDMSCs were strongly
positive (signal; the red line shift right of the control; the
solid curve) for CD13, CD29, CD49b, CD51, CD105, and CD166, but
were negative for CD45, CD34, MHC I, MHC II, and cKit (FIG.
11C).
[0080] By using serum-free pancreatic selection medium
(DMEM+EGF+bFGF+HGF+ITS) for 4 weeks of culture, a monolayer
appeared, and spindle-like PDMSCs gradually formed 3D
spheroid-bodies (SB-PDMSCs; FIG. 12A). To further evaluate the
protein expression of insulin in these spheroid bodies derived from
PDMSCs (SB-PDMSCs; FIG. 12A), immunofluorescent assay was used. The
results showed that the numbers of the insulin-positive cells were
gradually increased in SB-PDMSCs for 4 weeks in pancreatic
selection medium culture (FIGS. 12B and 12D). The pancreatic islet
markers of insulin exhibited a high positive rate and percentage of
immune reactivity in the spheroid body of aggregated SB-PDMSCs by
immunofluorescent staining (FIGS. 12B and 12D). Moreover, both of
signals of insulin (red fluorescence) and glucagon (green
fluorescence) co-expressed in SB-PDMSCs (FIG. 12C). In contrast,
the percentages of insulin-positive cells in PDMSCs
(undifferentiated type) were lower and stably expressed in 4-week
culture (FIG. 12D).
[0081] SCID mice with STZ pretreatment were employed to examine the
restoration of normoglycemia in the differentiated SB-PDMSCs
derived from PDMSCs in xenotransplantation. The renal subcapsular
space (FIG. 13A) in SCID mice has been demonstrated to provide a
microenvironment suitable for endocrine cells differentiation. A
total of 2.times.10.sup.5 SB-PDMSCs were implanted into the
subcapsular space of the left kidney (n=6 each). After 4 weeks, ex
vivo biopsy and histological study revealed that transplanted
SB-PDMSCs can proliferate and grow solid tissues in the subrenal
site (FIGS. 13A and 13B). The immunfluorescent study further
confirmed that insulin (red fluorescence)- and glucagon (green
fluorescence)-positive cells were detected in the SB-PDMSC-derived
tissues of the subrenal site in SCID mice (FIG. 13C). Blood glucose
was examined every two days before and after the transplantation
(FIG. 13D). Although the blood glucose was reduced in both groups
of transplanted animals in comparison to the untreated control
group, a significantly lower blood glucose was observed in the
group of the SB-PDMSC implanted animals (p<0.05; FIG. 13D). The
results show that SB-PDMSCs restored the blood glucose to a nearly
normal level in the STZ pretreated SCID mice.
Example 10
Induction of Embryonic Stem Cell Differentiated into Insulin- and
Glucagons-Positive Cells
[0082] The murine embryonic stem cell strain of BALB/c mice
(ESC26GJ constructed by Chiou et al during passages 25 and 50 was
used in the present study). This strain that has been transferred
by pCX-EGFP can express green fluorescence constitutively.
Undifferentiated murine stem cells were cultured on mitomycin
C-treated STO (mouse embryonic fibroblast cell line, ATCC CRL-1503
passages 15-35 were utilized) and supplied to Dulbecco's modified
Eagle's medium (DMEM, 4500 mg glucose/L) containing 15% fetal
bovine serum (FBS, HyClone defined and tested batches or ES cell
grade), 1% nonessential amino acid, 0.1 mmol/L
.beta.-mercaptoethanol, 100 U/mL penicillin, 100 .mu.g/mL
streptomycin (all from GIBCO-BRL) and leukemia inhibitory factor
(LIF, R&D). Cells were cultured in a humidified chamber
containing a 5% CO.sub.2/air mixture at 37.degree. C., subcultured
every 3 d and changed medium twice a day. Alkaline phosphatase
detection kit (Chemicon) was utilized to examine the quality of
undifferentiated ES every 5 passages. Approximately 70% saturated
undifferentiated ES cells were harvested and aggregated as hanging
drops with the density of 3000 cells/per drop for 4 d (using the
medium for undifferentiated ES cells without LIF supplement; FIGS.
14A and 14B). To further induce ES cells differentiated into
insulin-positive cells, the new serum-free ITSFn medium (composed:
1:1 of DMEM/F12, 0.6% Glucose, 25 .mu.g/mL Insulin, 100 .mu.g/mL
Transferrin, 20 nmoL/L Progesterone, 60 .mu.moL/L Putrescine, 30
nmoL/L Selenium chloride, 2 mmoL/L Glutamine, 3 mmoL/L Sodium
bicarbonate, 5 mmoL/L HEPES buffer, 2 .mu.g/mL Heparin, 20 ng/mL
human epidermal growth factor (EGF), 20 ng/mL human basic
fibroblastic growth factor (b-FGF) and 20 ng/mL human hepatocyte
growth factors were used and place on new dish. After 28 induction,
the positive signals for glucagon (green color; FIG. 14C) and
insulin (red color; FIG. 14D) were detected by immunofluorescent
assay (FIG. 14E; merged imaging).
Example 11
Differentiation of Neural Cells Induced by Using Cultivated Neural
Stem Cells in Scaffold Contained Basic Fibroblast Growth Factor
(bFGF)
[0083] The human neural stem cell (hNSC) could be a useful donor
tissue for neural regeneration by transplantation after central
nervous system damage and spinal injury. However, the difficulty in
ex vivo cell culture with this biomaterial graft is still unsolved:
hNSC cannot develop and differentiate well in conventional
two-dimensional culture media. More recent studies have focused on
biodegradable material, such as gelatin scaffolds, to provide
better adhesion and growth for hNSC before transplantation. Herein,
gelatin polymer scaffolds coated with basic fibroblast growth
factor (bFGF) were used to form a three-dimensional structure of
hNSCs. Tissues from the lateral ventricle wall were dissociated and
incubated in HBSS containing collagenase for 10 minutes at
37.degree. C. Dissociated cells were then centrifuged at 150 g for
5 minutes. The enzyme solution was removed and replaced with
serum-free culture media composed of DMEM (GIBCO) and F-12 nutrient
(1:1), including bFGF (10 ng/mL, R&D Systems), Hepes buffer (5
mmol), glucose (0.6%), sodium bicarbonate (3 mmol), and glutamine
(2 mmol). After 2 week culture, some stem cells (FIG. 15A)
aggregated and formed spheroid-like bodies, called neurospheres.
These neurospheres (FIG. 15B) were obtained in suspension and
cultivated them by seeding into gelatin scaffolds (FIG. 15C). The
gelatin polymer scaffold (Sigma, Type A, powder) was made of 5% gel
form in warmed DMEM/F12 medium with bFGF at 37.degree. C. Due to
the special functional group (--NH) in the gelatin gel, it can be
further chemically cross-linked with gelatin via cross-linking bFGF
growth factor. We observed that gelatin scaffolds provided a good
microenvironment for the adhesion and growth of hNSCs to form
neural network-like structures. Upon morphologic analysis, the
scaffold may also help the proliferation and differentiation of
hNSCs to become neurons. By MTT assay, better cell proliferation
was found in the group when bFGF (EGF) was coated in gelatin
scaffold as well (FIG. 15E).Gelatin polymer scaffolds were coated
with basic fibroblast growth factor (bFGF) to form a
three-dimensional structure of hNSCs. This example provides a
potential new cell transfer technology. Nontoxic and biodegradable
gelatin scaffolds coated with bFGF formed a three-dimensional
microenvironment facilitating proliferation and differentiation of
hNSCs before transplantation.
Example 12
Examination on Temperature-Sensitive Biological Transplanted
Scaffold in In Vivo Animal Fluorescent Cells
[0084] As showed in FIG. 16, eight-week-old nude mice were
anesthetized by 40 mg/kg pentobarbital intraperitoneal injection.
Around 2 cm diameter of trauma was made on the back of the mice.
After inserting into the trauma with hBMSCs-GFP (stem cell
constitutively expressed green fluorescent protein gene) cultivated
in temperature-sensitive biological transplanted scaffold over 3
weeks, trauma periphery was blocked by plastic circle to avoid
causing epithelialization (FIGS. 16a and 16b). Skin regeneration in
body was examined by fluorescent in vivo monitoring system. The
results of GFP imaging were consistent with the clinical
observation and histology survey (FIGS. 16e and 16f). The
excitation filter of 470 nm with a lamp supply of optical lighting
of 150 watts (Southern California Services, USA) was used as an
excited light source (470 nm) to project on the foci of the
GFP-positive cells of the living mice and ex vivo transplanted
tissues. The GFP imaging capture and photography is based on the
record of the digital camera (Olympus) through optical
configuration of dissected microscope (SZ60; Olympus) with a 515 nm
viewing (emission) filter. Furthermore, the hBMSC group mice
received the in vivo GFP imaging evaluation, and results showed
that the GFP-positive signals (FIG. 16c) were significantly
detected in the regenerated skin tissues with transplanted the
scaffold with hBMSCs-GFP (FIGS. 16c and 16d).
[0085] Herein it demonstrated the incorporation of GFP reporter
gene into the genome of hBMSC as markers of proliferation both in
cells and in living animals. Without adding any exogenous
substrates or antibodies, the constitutive GFP signals at the
living cellular level as well as in vivo transplanted graft could
be visualized directly and quickly under specific excitation
wavelength for noninvasive, real time, and in situ observation. In
addition, in vitro and in vivo GFP imaging systems provide a new
imaging modality for understanding the differentiation process and
the effective expression of stem cell in wound healing.
[0086] While the invention has been described and exemplified in
sufficient detail for those skilled in this art to make and use it,
various alternatives, modifications, and improvements should be
apparent without departing from the spirit and scope of the
invention.
[0087] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned, as well as those inherent therein.
The embryos, animals, and processes and methods for producing them
are representative of preferred embodiments, are exemplary, and are
not intended as limitations on the scope of the invention.
Modifications therein and other uses will occur to those skilled in
the art. These modifications are encompassed within the spirit of
the invention and are defined by the scope of the claims.
* * * * *