Wound healing in athymic nude mice with tissue engineered skin substitute prepared by human ADSC on
human acellular amniotic membrane.
Background: Tissue engineered (TE) skin offers promising alternative treatment in wound healing of acute and
chronic skin injuries. In acute situations limited autografts from donor skin and risks of rejection with allograft
have instigated the development of TE skin. Use of autologous keratinocytes as epidermal substitute attached to
either synthetic biomaterial or fibroblast derived dermal substitute has already started in clinics. ADSC is an
attractive source of abundant stem cells in the field of tissue engineering and regenerative medicine for repair
and regeneration tissues and organs. Human amniotic membrane (AM) has been used in treating burns since last
century. AM has anti-fibroblastic, ati-angiogenic property and also reported to reduce scarring. We aim to
produce TE skin substitute with the use of decellularised human AM matrix that can emulate dermis with human
adipose derived epithelial like cells for reepithelialization.
Methods: Human AM (n=6) was decellularized by treating with hypotonic buffer and then with 0.03% sodium
dodecyl sulphate (SDS). Nuclear debris of cells was washed away with 0.0004% deoxyribonuclease (DNase).
The decellularised (DC) AM were characterized by hematoxylin and eosine (H&E), DNA quantification and
electron microscopy. Human adipose derived stem cells (ADSC) were isolated from fat tissue (n=5) and
EPCAM positive cells were selected using magnetic nanoparticles. Adipose derived EPCAM positive cells
(passage1-2) further seeded (1x103 cells/cm2) on decellularized AM for 2 and 4 weeks. For in vivo
characterization, TE skin and DC AM was further orthotopically transplanted to full thickness skin wounds
(1.5cm x 1.5cm) in athymic nude mice and observed for four weeks.
Results: Complete decellularization of the AM was achieved in three DC cycles. We found that the isolated
EpCAM+ cells from ADSCs stained positive for different epithelial markers such as CK-7, CK-8, CK-18, CK19, Claudin-1 and ZO-2 with immunofluoroscence staining and FACS analysis. Extensive epithelial like cellular
in-growth forming well integrated TE skin was observed in HE stained in vitro TE skin. Immunohistochemical
examination of basal layer of in vitro TE skin showed positive staining for CK-18 and CK-19 (epithelial layer)
and tight junction claudin-1. This TE skin was transplanted into athymic mice for in vivo characterization. All
animals were healthy and there was no sign of red swelling, necrosis and exudation in both, TE skin group and
DC AM group after four weeks. After four weeks, surfaces of both grafts were taken over by the skin and
appeared pink-white, soft and normal in its texture, whereas the control group wound was with a scar. HE and
MT stainings shows that in all three groups, keratinocytes were migrated around the wound boundaries, and
acted like the barrier function of the epidermis by closing the wound. Regeneration of dermal matrix under the
healed epidermis formed skin appendages like hair follicles, sweat and sebaceous glands in TE skin and DC AM
group after four weeks of Tx. However in control group wound, dermal matrix in healed epidermis had fibrosis
like scar observed in MT staining. Further in TE skin group, transition of human like skin with thicker
epithelium and prominent rete ridges in a healed epidermis and normal mouse epidermis (thin and lacks rete
ridges) was observed.
Conclusion: Our findings suggest that, adipose derived stem cells can be differentiated into epithelial like cells.
Results from the animal studies further confirm the regeneration of TE skin in both epithelial layer and also in
dermal layer. Use of autologous adipose derived stem cells for tailor-made TE skin would be promising
treatment for acute and chronic skin injuries.
Background
Stem cell is characterized by its ability to undergo unlimited or prolonged self-renewal
and also have the capacity to differentiate along multiple lineage pathways [1]. The two
broad types of stem cells are embryonic stem cells and adult stem cells which are found in
adult tissues. Despite the fact that embryonic stem cells exhibit nearly unlimited potential
to differentiate in vitro and in vivo than adult stem cells, its use in regenerative medicine
and cell therapy is limited due to increased ethical issues associated with the application
of embryonic stem cells [2]. On the other hand, adult stem cells are not totipotent,
however they are pluripotent. It was believed that adult stem cells exhibit their
differentiation capacity limited to their tissue of origin, but recent findings have shown
that, they can differentiate into cells of mesodermal, endodermal and ectodermal origin[39] . Adult stem cells are found in the tissue in rare populations localized in small niches
[10]. Since the identification of pluripotent stem cells in the bone marrow 40 years ago,
bone marrow stem cells (BMSCs) have been used widely due to their high differentiation
potential in vitro. However bone marrow aspiration is a painful procedure and it may
leave donor site morbidity as a result [11]. Also BMSCs are present at low frequency in
the bone marrow and numbers of harvested cells are even low [2]. There is a need to find
an alternative which is reliable and abundant cell source, which can be harvested by
minimally invasive procedure, ability to differentiate into multiple cell lineage pathways
in a reproducible manner and importantly not ethical, legal, political, scientific and
clinical safety concern.
In light of this, ADSC is an attractive source of abundant Mesenchymal stem cells (MSCs)
in the field of tissue engineering and regenerative medicine for repair and regeneration of
acute and chronically damaged tissues and organs. ADSCs can be isolated in higher
number from fat tissue wastes resulting from reconstructive plastic surgery i.e. liposuction
aspirates, large tissue resection or from subcutaneous fat and can be easily expanded in
vitro [12]. Adipose tissue is made up of primarily with adipocytes and other cell types that
surrounds and supports them and upon stepwise isolation they called as stromal vascular
fraction (SVF) [2]. ADSCs share similar properties like BMSCs, upon isolation they have
the ability to differentiate into adipocytes, osteoblasts, chondrocytes, myocytes,
endothelial cells, hematopoetic cells, hepatocytes and neural cells [1, 11, 13-20]. In
another study ADSCs appeared to be immunoprivileged [21, 22], which helps to prevent
severe graft versus host disease in vitro and in vivo and unlike BMSCs they appear to be
more genetically stable in long term culture [23, 24]. Furthermore ADSCs have advanced
into ongoing clinical trials in many countries for the treatment of many diseases [2]. Due
to the safety and efficacy of ADSCs over other adult stem cells attracts the interest of
using them for different tissue engineering applications.
Materials and methods
All protocols used in the present study were approved by the local Ethics Committee.
Discarded adipose tissue weighing app. 70-100 g was collected during surgery with the
informed consent of the patients (n=5), and approval by the hospital.
1. Isolation of adipose derived stem cells
In Brief, immediately after collecting samples of fat or liposuction aspirates (70-100g)
were transported from hospital to the laboratory in ice cold Phosphate-buffered saline
(PBS) containing 1% penicillin/streptomycin (P/S, Invitrogen, USA). After receiving the
adipose tissue (AT) sample, all further procedures were performed under laminar air flow
hood. AT was then washed extensively with PBS containing 1% P/S. Connective tissue
was removed with the help of sterile scalpels and forceps and AT sample was kept in a
sterile container containing 70 ml DMEM (Lonza, Belgium) medium supplemented with
0.5% collagenase type II (Sigma life science, USA) and 1% P/S. AT sample was minced
using sterile scissor and mixed with pipette up and down several times to further
disintegrate aggregates of AT. Then it was transferred into another sterile container and
kept for digestion at 37oC for 30 min under agitation. After digestion for 30 min,
collagenase type II activity was neutralized by sterile 50 ml PBS containing 10% human
AB serum (Invitrogen, USA) and 1% P/S and again suspension was mixed well with the
pipette up and down several times. The suspension was then filtered through sterile gauze
pieces and filtrate was centrifuged at 1500 RPM for 15 min to separate floating mature
adipocytes and stromal- vascular fraction (SVF). Supernatant was discarded and pellet
was resuspended in PBS+ 10% human AB serum and centrifuged again at 1500 RPM for
10 min. The centrifugation step was repeated again to separate ADSCs from primary
adipocytes and to wash away collagenase type II. ADSCs were counted and cultured in
vitro in DMEM medium supplemented with 10% human AB serum, 1% L-glutamine
(Invitrogen, USA), 1% P/S overnight.
2. Primary ADSCs culture
ADSCs were seeded at 4000/cm2 in a flask treated previously with human placental
collagen which was washed in sterile PBS. Seeded cells were cultured in DMEM medium
which consist of 10% human AB serum, 1% L-glutamine and 1% P/S. ADSCs cells were
incubated at 37oC under 5% CO2 and medium was changed after 24 hrs. At confluency, of
60–70%, the cells were trypsinized with 1x Trypsin-EDTA (Invitrogen, USA) and further
used for isolation of EpCAM positive selection with magnetic nanoparticles.
3. Isolation of EpCAM positive cells from ADSCs using magnetic beads
The cells were positively selected using a Human EpCAM positive selection kit with
magnetic nanoparticles (Stem Cell Technologies, Vancouver, Canada) according to the
manufacturer’s instructions. Briefly, cell suspension in a concentration of 1x108 cells/ml,
was prepared in a cold buffer containing 2% FBS (Invitrogen, USA) and 2mM EDTA
(Sigma-Aldrich, USA) in PBS. Cells were passed through a 70 µm Nylon cell strainer
(BD Bioscience, Sweden) to ensure a single cell suspension. Cells were then placed in 12
x 75 mm polystyrene tube in a volume of not more than 1 ml. Then the cell selection
cocktail and EasySep magnet nanoparticles were added according to the manufacturer’s
instructions. Cell suspension was brought to a total volume of 2.5 ml using the buffer.
Three magnetic separations of 5 minutes each were carried out using EasySep magnet
(Stem Cell Technologies, Vancouver, Canada) according to manufacturer’s instructions.
Both cell fractions EpCAM positive and EpCAM negative were resuspended in PBS+10%
human AB serum and centrifuged at 1500 RPM for 8 min. EpCAM negative fraction was
frozen down and EpCAM positive fraction was plated at a density of 5 x 104 cells/well in
T75 culture flasks treated previously with human placental collagen and washed with
sterile PBS. Cells were cultured in EpiLife medium-Cat. No. MEPI500CA (Life
technologies corporation, NY, USA) supplemented with Human Keratinocyte Growth
Supplement- HKGS- Cat. No.S-001-5(Life technologies corporation, NY, USA), 10%
human AB serum, 1% L-glutamine, 1% P/S. The culturing medium was changed every
third day until the cells reached their confluency. At confluency, of 60–70%, the cells
were trypsinized with 1% Trypsin-EDTA (Invitrogen, USA) and further cultured at a
density of 2x103/cm2 for four to five passages. At every passage, cells were taken for
characterization by Flow cytometer (FACS) and Immunocytochemistry (ICC).
4. Characterization of EpCAM+ Cells from ADSCs
Immunocytochemistry (ICC)
EpCAM+ cells were cultured at a density of 1x103 cells/well in an 8-chamber culture slide
(Becton Dickinson, USA). The medium was changed on a regular basis until the cells
became 90% confluent. ICC staining was carried out for characterization of EpCAM+
cells. Cells were fixed in 30% acetone in methanol. Wells were washed with PBS/Tween
20 (Medicago, Sweden) for 3 x 5min. Then cells were incubated in blocking serum (5%
goat serum in 1% BSA) for 30 min. The following non-conjugated monoclonal antibodies
were used and incubated overnight at 4oC : anti-CK 18, anti-CK19, anti-CK7, anti-CK8
(Abcam Inc. USA), anti-claudin1and anti- ZO -2 (Invitrogen corporation, USA). All the
primary antibodies were diluted as per manufacturer’s recommendation. The next day, all
wells were washed three times with PBS/Tween 20 for 3x 5 min. FITC conjugated Alexa
flour 488 goat anti mouse secondary antibody (Invitrogen corporation, USA) was
incubated for 30 min at room temperature. Again all wells were washed three times with
PBS/Tween 20 for 5 min. Finally cells were counterstained with 4',6-Diamidino-2Phenylindole, Dihydrochloride (DAPI) with mounting media- VECTASHIELD H-1500
(Vector laboratories, USA). Experiments were accompanied by negative and positive
control staining to detect possible nonspecific signals. Negative controls were processed
by replacing the primary antibody with diluents only.
Flow cytometric analysis (FACS)
Characterization of EpCAM+ cells were also done by using flow cytometry. The cells
were stained and incubated for 30 min at 4oC with monoclonal antibodies to EpCAM
(Santacruz, USA), anti-CK 18,anti-CK7, anti-CK8 (Abcam Inc. USA), α-smooth muscle
actin (eBiosciences), CD 31(Sinobilogical Inc. China), E-cadherin (eBiosciences).
Fluorochrome-conjugated mouse immunoglobulins were used as isotype controls. The
stained cells were then analyzed on a FACS (Millipore) equipped with Guava software 1.1
(Millipore) for data analysis.
5. Preparation of acellular scaffold
Human placenta and amniotic membrane (n=5) were obtained after normal delivery and
written consent from close relatives and hospital as approved by the local ethical
committee. The placenta and associated membranes were washed several times with
PBS+1%P/S. The human amniotic membrane (AM) was separated from the underlying
chorion placental surface with blunt dissection. Decellularization of AM was achieved by
using three different protocols. In the first protocol, separated AM was immersed in the
hypotonic tris buffer solution (Merck, Germany), pH 8.0 for 4hrs at room temperature
with gentle agitation. Tissue was then transferred to 0.03% sodium dodecyl sulphate
(SDS, Invitrogen, USA) in distilled water (D/W) for 24 hrs at room temperature with
gentle agitation. Nuclear debris were washed away by incubating tissue in 0.0004%
deoxyribonuclease (DNase, Sigma life science, USA) in PBS containing magnesium and
calcium at 37oC for 3hrs with constant agitation. After each chemical treatment tissue was
washed with PBS for several times to wash away chemical remnants. Terminally the
tissue was sterilized using 0.1% Peracetic acid (sigma) in PBS (pH 7.2) for 3hrs at RT
with agitation at 140 rpm. Finally tissue was washed several times with PBS and kept in
sterile container containing PBS + 1%P/S. All decellularizing chemicals were enriched
with 5mM ethylenediaminetetraacetic acid (EDTA), 0.4 mM Phenylmethylsulfonyl
fluoride (PMSF, MP Biochemicals, France) and 0.2% sodium azide (Sigma-Aldrich,
Japan). This protocol was repeated three times to get complete acellular AM scaffold. A
comparison was made in the decellularization (DC) method by replacing 1% Tri-n-butyl
phosphate (TNBP, VWR, Sweden) and 1% sodium deoxycholate (SDC, Sigma life
science, Japan) with SDS however all other conditions were same. At the end of each DC
cycle, biopsies were taken for the histological analysis to see presence of nuclei and DNA
quantification.
6. Characterization of decellularized human amniotic membrane (AM)
The decellularizsed AM was characterized by histological staining with hematoxylin and
eosine (H&E) and DNA quantification.
H&E staining
Tissue samples were fixed in 10% phosphate buffer formalin (Histolab, Sweden) for 24
hours at room temperature. Then they were washed in distilled water, dehydrated in
graded alcohol, embedded in paraffin and sectioned using microtome at 5µm thickness.
For frozen sections, tissue samples were snap-frozen in OCT compound (Tissue Tek,
USA) at -800C, sectioned at 5µm thickness with cryotome. Adjacent sections were cut and
stained with H&E (Histolab, Sweden) as per manufacturer’s instructions to check
presence of nuclei and morphological changes.
DNA Quantification
DNA quantification (Qiagen, Germany) was also performed to detect the amount of DNA
left after decellularization. Briefly, tissue samples (approximately 10– 15 mg, n=15) were
incubated overnight in proteinase K at 560C. Buffers provided with kit were added to the
samples and centrifuged in the DNeasy mini spin column to selectively bound the DNA to
DNeasy membrane (silica-membrane-based). Contaminants and enzyme inhibitors were
removed by two efficient washing steps. The purified DNA was further processed for
spectrophotometric quantification at 260 nm to determine the concentrations of residual
DNA in the decellularized tissue as compared to normal tissue. DNA quantification
procedure was followed by manufacturer’s (QIAamp DNA Mini Kit, Qiagen, Basel,
Switzerland) instructions.
7. Preparation of TE skin with EpCAM+ cells
Samples of DC AM (2x2 cm, n=13) were placed in sterile 6 well plates (Becton
Dickinson, USA). EpCAM+ cells from early passage (1o-2o) were mixed in 500 µl
complete EpiLife medium and seeded on each piece of DCamniotic membrane
respectively at density of 1x106 and incubated for 30 min at 37oC under 5% CO2. After 30
min, 3ml of complete EpiLife medium was added in each well and incubated for 4 weeks
at 37oC under 5% CO2. DC AM incubated in complete EpiLife medium without cells was
used as negative control. The culture medium was changed after every 3rd day and a piece
of a tissue was taken after each week for histological analysis.
In vitro TE skin characterization
The recellularized TE skin (after 2, 3 and 4 weeks) was characterized by histological
staining (HE) and immunohistochemical staining.
Briefly, tissue samples were fixed in 10% phosphate buffer formalin (Histolab, Sweden)
for 24 hours at room temperature. They were washed in distilled water, dehydrated in
graded alcohol, embedded in paraffin and sectioned using microtome at 5µm thickness.
Slides were then deparaffinised in xylene and rehydrated in graded alcohol, washed in
D/W. Immunohistochemistry was performed by the avidin-biotin-peroxidase complex
method. Antigen retrieval was achieved by incubating the slides in 10 mM citrate buffer
(Merck, Germany), pH 6.0 in a thermostatic bath at 95 oC for 30 min. The endogenous
biotin was bolcked by using avidin-biotin blocking kit (Vector lab Inc. USA) and
nonspecific binding was blocked by normal horse serum. The slides were incubated over
night at 4 oC with anti-human CK-18, CK-19, Claudin-1 and EpCAM (Abcam Inc,
cambridge, MA). All antibodies were used at a dilution as per manufactures instructions.
The slides were then washed three times with PBS/Tween 20 for 3x5 min. Endogenous
peroxidase activity was quenched by 3% H2O2 in PBS. Biotinylated horse anti-mouse
secondary antibody (Vector lab Inc. USA) was incubated for 40 min at room temperature.
After washing (4 x PBS/Tween 20), color was developed using 3,3'-diaminobenzidine
(DAB; Vector lab Inc. USA). Finally, nuclei were stained with Mayers’s
hematoxylin(Histolab, Sweden). Stained slides were dehydrated cleared in xylene and
mounted in di-n-butyl phthalate xyline (DPX; VWR, Sweden). Experiments were
accompanied by negative and positive control staining to detect possible nonspecific
signals. Negative controls were processed by replacing the primary antibody with diluents
only. Normal AM tissues were used as a positive controls to detect possible nonspecific
signals in the staining.
8. In-vivo transplantation of TE skin
Two full thickness skin wounds measuring 1.5cm x 1.5cm were created on the
dorsolateral aspect of each athymic nude mice (BABL/C nude, n=10). The defected
wounds were divided into TE skin group, a DC AM group and a control group. TE skin
piece and DC AM piece was orthotopically placed on respective wounds on each side and
wound margins were secured with 7-0 sutures (Ethicon, USA). A control wound was
made on any one side of the back of the mice. All wounds were dressed with a piece of
sterile gauze with petroleum jelly and the grafted site was covered with dressing. All mice
were left undisturbed until 5 days after the surgery after which the dressings were
removed. All mice were observed for two weeks after the operation and photographs were
taken every week. The wound perimeters were traced at the time of surgery and at weekly
intervals until 2 weeks by visual observation. Two weeks after operation, animals were
euthanized with prolonged inhalation of isoflurane and the grafts were collected from
further analysis.
In-vivo TE skin characterization
Grafts from the mouse skin (after 2 weeks) were characterized by histological
stainings(HE) and Masson’s trichrome staining.
Tissue samples were snap-frozen in OCT compound (Tissue Tek, USA) at -800C,
sectioned at 5µm thickness with cryotome and stained for HE as mentioned earlier.
Masson-trichrome staining
Masson-trichrome staining was used to detect collagen, morphology of tissue and fibrosis
in tissue. The sections from cryotome was fixed in 30% acetone in methanol for 10 mins
in -20oC, washed in D/W for 5 min and re-fixed overnight at room temperature in Bouin’s
fixative. Further staining was followed by using the Masson’s Trichrome staining kit (cat
No. 25088-1, Polysciences Inc., USA). The dyes employed during the staining procedure
stained the collagen fibers blue, nuclei black and the connective tissue and muscle fibers
red.
Results
1. In-vitro culture
Viability of EpCAM+ cells (n=5), isolated from ADSCs were more than 95% as per trypan
blue staining. We found that the isolated EpCAM+ cells from ADSCs stained positive for
different epithelial markers suc as CK-7, CK-8, CK-18, CK-19 and Claudin-1with
immunofluoroscence staining. In this, small population was positive for CK-7, CK-8 but larger
numbers of cells were positive for CK-18 and CK-19. These results were further confirmed by
FACS. In FACS analysis we found that these cells were positive for CK-7, CK-8, CK-18 and
small pulation was also positive for pan epithelial cell marker, i.e. anti-EpCAM antibody. Cells
were negative for endotheial cell marker CD31.
2. Characterization of DC AM
Complete decellularization of the AM was achieved in three DC cycles in all three
protocols. After DC, AM looked white, semi-transparent but retained its size and tensile
strength similar to the native/normal AM. Histologically, AM was decellularizsed
completely in all three DC protocols, without residual cells and cell fragments. DNA
quantification results showed that AM decellularized with SDC and SDS protocols
achieved good nucleic acid depletion (30ng/mg and 40ng/mg of tissue respectively) than
TnBP protocol (95ng/mg of tissue). For this reason, AM decellularized with SDC protocol
was used for recellularization and in-vivo transplantation in mice.
3. In-vitro TE skin
After culturing DC AM with EpCAM+ cells for two weeks in 6 well plates, the tissue
looked thick however shrinkage was observed in the specimens. Extensive epithelial like
cellular in-growth forming well integrated TE skin was observed in HE stained tissues. In
H-E staining we also observed that these stratified epithelial like cells were cued on the
edge of the tissue. Further, Engraftment of EpCAM+ cells in DC AM were verified by the use
of antibodies specific for human epithelial cell. Immunohistochemical examination of basal layer
of TE skin showed positive staining for CK-18 and CK-19 (epithelial layer) resembling to native
AM. Tight junction antibody claudin-1 was also positive in TE skin. However EpCAM antibody
staining was negative in TE skin.
4. In-vivo TE skin Tx
All animals were healthy and there was no sign of red swelling, necrosis and exudation in
both, TE skin group and DC AM group for two weeks. At day 5 postoperatively i.e. after
removal of dressing from the Tx site, both TE skin and DC AM were attached to the
wound and to the surrounding margins of native mouse skin. The surfaces of both grafts
were dry and well keratinized. After two weeks, surfaces of both grafts were taken over by
the skin and appeared pink-white, soft and normal in its texture, whereas the control group
wound was with a scar. Also while collecting the grafts from the animals after two weeks,
we could see that TE skin wound (inside part) was seen completely absorbed by native
skin with new infiltrating blood vessels and was not adhered to the muscle of the mice.
However control wound was adhered to the muscle of mice. HE and Masson’s trichrome
staining showed that all wounds re-epithelialized 2 weeks after Tx without complications.
2 weeks after Tx in all three groups, keratinocytes were migrated around the wound
boundaries, and acted like the barrier function of the epidermis by closing the wound.
Also there was no significant sign of acute inflammation or rejection reaction. Dermal
matrix under the healed epidermis had sweat and sebaceous glands developed in TE skin
and DC AM group after 2 weeks of Tx. However in control group wound, dermal matrix
in healed epidermis had fibrosis like scar observed in MT staining. Healed epidermis in
TE skin group had 6-7 layers of epithelial cell but in control and DC AM group, only had
3-4 epithelial cell layers. Similar to the normal nude mice skin, formation of papille-like
structure of epithelial lining above basement membrane was observed in TE skin group
but it was absent in control and DC AM group.
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