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Cytotherapy (2003) Vol. 5, No.

5, 362 /369

Adipose-derived adult stem cells: isolation,


characterization, and differentiation potential
JM Gimble1 and F Guilak2
1

Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA, USA
Department of Orthopaedic Surgery, Division of Orthopaedic Surgery, Duke University Medical Center, Durham, NC, USA

Summary
Adipose tissue is an abundant, accessible, and replenishable source of
adult stem cells that can be isolated from liposuction waste tissue by
collagenase digestion and differential centrifugation. These adiposederived adult stem (ADAS) cells are multipotent, differentiating along
the adipocyte, chondrocyte, myocyte, neuronal, and osteoblast lineages,
and can serve in other capacities, such as providing hematopoietic

support and gene transfer. ADAS cells have potential applications for
the repair and regeneration of acute and chronically damaged tissues.
Additional pre-clinical safety and efficacy studies will be needed before
the promise of these cells can be fully realized.

Introduction

chemical agents, cytokines, and hormones. Recent studies


indicate that nascent stem cells exist within other adult
tissues, including the brain, dermis, periosteum, skeletal
muscle, synovium, trabecular bone, and vasculature [1/
15]; however, the most abundant and accessible source of
adult stem cells is the adipose tissue.
Several physiologic and pathologic observations have
pointed to the presence of a population we have identified
as adipose-derived adult stem (ADAS) cells in human
adipose tissue. For example, well-nourished humans store
their excess calories in their adipose tissue not only
through an increase in the adipocyte cell volume, but
also through an expansion of the number of differentiated
adipocytes. This suggests that a pool of adipocyte
progenitors exists within the adult fat tissue. However,
their differentiation is not restricted to the adipocyte
lineage; patients suffering from the rare disorder progressive osseous heteroplasia form ectopic bone within their
subcutaneous adipose tissue, indicating the presence of
multipotent progenitor or stem cells at that site [16].
This review will focus on recent literature describing
the isolation, characterization, and differentiation potential
of human ADAS cells. We conclude with a brief discussion
of their potential clinical applications.

The field of tissue engineering proposes to repair and


regenerate damaged organs using a combination of cells,
biomaterials, and cytokines. The availability of human cells
capable of differentiation along multiple lineage pathways
has limited the progress and development of these
modalities. Stem cells and progenitor cells offer a potential
answer to this dilemma. By definition, stem cells are able to
self-renew, exist in an undifferentiated or unspecialized
state, and are capable of differentiation or specialization
along multiple lineages. Progenitor cells, in contrast,
display less capacity for self-renewal, and are committed
to differentiation along a particular pathway or pathways.
While embryonic stem cells exhibit apparently unlimited
differentiation potential in vitro and in vivo, their application is limited by ethical, legal, and political concerns, as
well as by scientific issues of safety and efficacy.
Stem cells derived from adult tissues offer an alternative
approach that circumvents many of these concerns. It is
well established that human BM contains both hematopoietic stem cells (HSCs) and mesenchymal stem cells
(MSCs) or stromal cells [1 /4]. Each has the potential to
express the surface proteins and phenotype of committed
cell lineages in response to appropriate combinations of

Keywords
adipose tissue, stem cell, multipotent, tissue engineering.

Correspondence to: Jeffrey M Gimble, Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Rd, Baton Rouge, LA
70808, USA.
2003 ISCT

DOI: 10.1080/14653240310003026

Adipose-derived adult stem cells

363

ADAS cell isolation methodology

ADAS cell immunophenotype

Histologic and electron microscopic studies identified


putative adipocyte progenitor cells in situ within embryonic and adult adipose tissues (reviewed in Nnodim
1987[17]). The earliest progenitors displayed fibroblastic
characteristics, with an abundant endoplasmic reticulum and large nucleus relative to the cytoplasmic volume.
Upon further development, the cells displayed small
lipid vacuoles within the cytoplasm, and a paranuclear
localization of their mitochondria. This progressed to
the appearance of a signet ring cell, characterized by
the presence of a single large lipid vacuole surrounded by a thin rim of cytoplasm and an eccentric
nucleus.
In a classic study, Rodbell [18] presented the first in vitro
isolation method for mature adipocytes and progenitors
from rat epididymal fat pad. He minced the tissue into
small fragments, digested it at 378C with collagenase Type
I, and separated the cellular components by differential
centrifugation. The supernatant contained the mature
adipocytes, which floated due to their high lipid content.
The pellet contained the stromal vascular components,
which included the putative adipocyte progenitor cells in
addition to hematopoietic lineage cells.
Van, Roncari, Deslex, Hauner and others modified this
approach to isolate human adipocyte progenitors [19 /21].
When they cultured the stromal vascular components in
the presence of inductive factors (dexamethasone, triiodothyronine, biotin, insulin, pantothenate), the cells accumulated lipid vacuoles and expressed the adipogenic
enzymes lipoprotein lipase and glycerol-3-phosphate
dehydrogenase.
In recent years, an increasing number of patients have
elected to undergo liposuction procedures for cosmetic
reasons. The lipoaspirate waste tissue is finely minced, and
forms an excellent starting material for ADAS cell isolation
as the shear forces exerted during the suction process do
not significantly alter cell viability [22,23]. Several groups
have developed and refined procedures starting with
liposuction material to isolate human ADAS cells [22 /
26]. Katz and colleagues [26] have developed a temperature controlled, self-contained system for performing the
collagenase digestion and tissue washing steps. Mechanical
devices such as this offer advantages for large-scale
isolation and manufacturing quality control issues in the
future.

The immunophenotype of undifferentiated human ADAS


cells cultured in vitro has been examined using flow
cytometric and immunohistochemical methods [25,27 /
30]. Independent groups have identified highly consistent,
although not identical, expression profiles of cell-surface
proteins on ADAS cells [25,27 /30]. One or more groups
have examined the following categories of proteins.
j

Adhesion molecules. The ADAS cells consistently


express the tetraspan protein (CD9), integrins b1
(CD29) and a4 (CD49d), intercellular adhesion molecule 1 (ICAM-1; CD54), endoglin (CD105), vascular
cell adhesion molecule (VCAM; CD106), and activated
lymphocyte cell adhesion molecule (ALCAM; CD166).
The integrins ab (CD11b) and b2 (CD18), intercellular
adhesion molecule 3 (ICAM-3; CD50), neural cell
adhesion molecule (NCAM; CD56), and endothelial
selectin (E-selectin; CD62) are not present on the
ADAS cell surface.
Receptor molecules. The ADAS cells express the
hyaluronate (CD44) and transferrin (CD71) receptors.
Surface enzymes. The ADAS cells express the neutral
endopeptidase (CD10 or common acute lymphocytic
leukemia antigen CALLA), aminopeptidase (CD13),
and ecto 5? nucleotidase (CD73).
Extracellular matrix proteins and glycoproteins. The
ADAS cells produce Type I and Type III collagens,
osteopontin, ostenectin, Thy-1 (CD90), and MUC-18
(CD146).
Skeletal proteins. The ADAS cells express intracellular
a smooth muscle actin, and vimentin.
Hematopoietic cell markers. The ADAS cells do not
express the hematopoietic markers CD14, CD31, or
CD45.
Complement regulatory proteins. The ADAS cells were
positive for decay accelerating factor (CD55) and
complement protectin (CD59).
Histocompatibility Ags. The ADAS cells were positive
for the Class I histocompatibility protein HLA-ABC
and negative for the Class II protein, HLA-DR.

Some discrepancies exist. For example, while Gronthos


et al. [27] detected CD34 and VCAM (CD106) on ADAS
cells, Zuk et al. [30] did not. Likewise, while Zuk et al. [30]
detected Stro-1, Gronthos et al. [27] did not. These
discrepancies could reflect differences in cell isolation
methods, how long the cells were cultured prior to

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JM Gimble and F Guilak

analysis, the use of MAbs detecting different epitopes on


the same surface protein, and sensitivity differences
between immunohistochemical and flow cytometric detection methods [25,27 /30].
The immunophenotype of ADAS cells resembles that
reported for other adult stem cells prepared from human
BM (mesenchymal stem cells), and skeletal muscle
[25,27,29 /31]. Nevertheless, differences do exist: while
ADAS cells do not express NCAM (CD56), musclederived adult stem cells do [27,29]. Indeed, a direct
comparison of ADAS cells and BM-derived adult stem
cells did not reveal identical protein patterns [30]. A single
gene microarray study has compared undifferentiated
ADAS and BM adult stem cells using a panel of 28 genes
[32]. Although no significant differences were detected, it
is clear that this area needs additional investigation.

ADAS cell differentiation potential


Musculoskeletal tissues
Chondrocyte */ cartilage repair
Human ADAS cells can display the biochemical markers
associated with mature chondrocytes [25,30,33 /35]. With
the addition of transforming growth factor-b (TGF-b),
ascorbate, and dexamethasone, ADAS cell will secrete the
extracellular matrix proteins of cartilage, collagen Type II,
collagen Type VI, and aggrecan when maintained in an
appropriate 3D matrix for 1 /2 weeks in vitro .
This is achieved by suspending the ADAS cells in a
calcium alginate gel at a concentration of 4 to 10 million
cells/mL, or by maintaining approximately 0.25 million
ADAS cells as a micromass pellet [33 /35]. However, when
ADAS cells were maintained in a monolayer under the
same chondrogenic culture conditions, the expression of
chondrocytic markers was present but reduced [34].
Importantly, human ADAS cells can retain their chondrogenic phenotype in vivo when implanted s.c. in non-obese
diabetic (NOD)/ SCID mice for up to 12 weeks, based on
analyses of alginate matrices [34].
A recent in vitro study compared the expression profile
of 28 genes between human ADAS cells and human BM
stromal cells under chondrogenic conditions [32]. In
monolayer, the two cell types displayed a similar gene
expression profile of adipogenic, chondrogenic, and osteogenic markers; however, when cultured as micromass
pellets, the BM stromal cells exhibited a greater expression
of chondrogenic gene markers relative to ADAS cells [32].
Further in vitro studies have optimized culture concentra-

tions of dexamethasone, ascorbate, and TGF-b to promote


chondrogenesis [33]. Further work is needed to establish
culture conditions to maximize the ADAS cells chondrogenic potential in vitro . With that accomplished, it will be
necessary next to use the modified cells to repair a full
thickness articular cartilage defect in vivo at a weightbearing joint.

Myocyte */ skeletal muscle repair


ADAS cells demonstrate in vitro evidence of differentiation
along each of the myocyte lineage pathways. In the
presence of horse serum, human ADAS cells express
myoD and myogenin, transcription factors regulating
skeletal muscle differentiation [25,30,36,37]. When cultured under these conditions, the ADAS cells fuse, form
multi-nucleated myotubes, and express protein markers of
the skeletal myocyte lineage, such as myosin light chain
kinase. This suggests that ADAS cells can be used to repair
damaged skeletal muscle in combination with appropriate
biomaterials.

Osteoblast */ bone defect repair


ADAS cells differentiate into osteoblast-like cells in the
presence of ascorbate, b-glycerophosphate, dexamethasone
and 1,25 vitamin D3 [38,39]. Over a 2 /4 week period in
vitro, both human and rat ADAS cells deposit calcium
phosphate mineral within their extracellular matrix, and
express osteogenic genes and proteins */ including alkaline phosphatase, bone morphogenic proteins and their
receptors, osteocalcin, osteonectin, and osteopontin
[25,30,38 /40]. In vivo, ADAS cells embedded in porous
cubes of hydroxyapatite/tricalcium phosphate form bone
as s.c. implants in immunodeficient mice [41,42]. New
osteoid, derived from the human ADAS cells, is present
within a 6-week incubation period [41].
This finding suggests that ADAS cells will have
therapeutic applications in bone repair. In particular,
human ADAS cells may benefit osteoporotic and other
patients with reduced numbers of native osteoblast precursors. A composite of ADAS cells and a biomaterial
scaffold or matrix may accelerate repair at a fracture site in
elderly patients undergoing an orthopedic procedure. One
of the next steps in the application of ADAS cells for bone
repair will involve testing this concept in vivo using a large,
weight-bearing animal model.

Adipose-derived adult stem cells

Soft tissues
Adipocyte */ soft tissue cosmesis
Human ADAS cells have the ability to return to what is
believed to be their original differentiation pathway,
adipogenesis. This is accomplished in vitro using induction
cocktails containing insulin, methylisobutylxanthine (a
phosphodiesterase inhibitor resulting in elevated cyclic
AMP levels), hydrocortisone or dexamethasone (a glucocorticoid receptor agonist), indomethacin or thiazolidinedione (a peroxisome proliferator activated receptor g
ligand), pantothenate, biotin, and/or triiodothyronine
[20,21,24,25,30,43]. After 7 /10 days, the human ADAS
cells contain vacuoles filled with neutral lipid, as detected
by Oil Red O or Nile Red staining, secrete increased
amounts of the adipocyte protein leptin, and transcribe
adipogenic mRNAs such as the fatty acid binding protein,
aP2, and lipoprotein lipase [21,24,25,30,43]. Some of these
parameters (leptin, aP2 mRNA levels) have been quantified and found to increase by several hundred-fold during
the differentiation process [24,43].
When grown on appropriate biomaterial scaffolds,
ADAS cells form new fat depots in vivo. In a variety of
animal models (murine, porcine), undifferentiated and/or
adipocyte differentiated ADAS cells have been implanted
s.c. with alginate coupled to RGD peptides, collagen, fibrin
glue, hyaluronic acid, poly glycolic acid/ poly lactic acid
(PGLA), and polytetrafluoroethylene to form fat [44 /52].
It is likely that the ability of nutrients to reach the
embedded ADAS cells influences their differentiation.
Studies demonstrate that biomaterials with greater porosity display improved adipogenic function in vivo [46]. In
nude mice, human preadipocytes performed better in
terms of adipocyte differentiation when loaded into a
porous hyaluronate sponge compared to denser unwoven
hyaluronate or collagen sponges [46]. With further refinement, it may be possible to use these approaches for
clinical cosmetic and reconstructive procedures.

Smooth muscle and cardiac myocyte


Since human ADAS cells express a-smooth muscle actin,
they may prove to be of value in the repair of smooth
muscle defects in the gastrointestinal and urinary tracts.
Indeed, surgeons have begun to explore this possibility. In
a case report, Garcia-Olmo and colleagues [53] transplanted autologous ADAS cells to repair a rectovaginal
fistula in a patient suffering from Crohns disease and

365

observed good closure of the fistula. These authors


conclude that autologous adult stem cells may prove to
be a valuable surgical therapeutic tool.
Studies using BM-derived adult stem cells have demonstrated their ability to differentiate into cardiac myocytes
[54]. A recent report demonstrates that ADAS cells
exposed to 5 azacytadine in vitro also differentiate along
the cardiac myocyte pathway [55]. After a 3-week period,
spontaneously beating cells expressing the cardiomyocyte
specific protein, troponin I, appear in culture [55]. These
preliminary reports lend promise to the concept that
ADAS cells can be used to regenerate cardiac tissues
damaged through infarctions or ischemic injury.

Other lineages and functions


Neuronal */ spinal cord and peripheral nervous system injury
There is preliminary evidence to suggest that human
ADAS cells can display neuronal and/or oligodendrocytic
markers. When exposed in vitro to antioxidants in the
absence of serum, human and murine ADAS cells take on a
bipolar morphology, similar to that of neuronal cells [56].
This is accompanied by expression of the neuronalassociated proteins nestin, intermediate filament M, and
Neu N, as well as glial fibrillary acidic protein (GFAP) */
a protein associated with oligodendrocyte differentiation
[56]. Exposure of ADAS cells to indomethacin, insulin, and
isobutylmethylxanthine results in a similar phenotype
[30,57].
Further in vitro studies are needed to characterize
ADAS cell neurogenesis with respect to neurophysiologic
and/or neurochemical signal transduction properties. This
work should lead to in vivo analyses assessing the ability of
ADAS cells to accelerate the regeneration of the central or
peripheral nervous system following a traumatic injury.
Hematopoietic support */ hematopoietic stem cell transplant and
in vitro expansion
Human ADAS cells express some of the same adhesive
proteins on their surface that BM stromal cells use to
support the proliferation and differentiation of hematopoietic stem cells [27]. In addition, ADAS cells in culture
secrete many of the cytokines produced by BM stromal
cells; these including M-CSF, GM-CSF, tumor necrosis
factor-a (TNFa), IL-6, IL-7, IL-8, IL-11, and stem cell
factor [58]. Consistent with these observations, human
ADAS cells promote the differentiation of CD34 
hematopoietic stem cells in in vitro co-culture systems

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JM Gimble and F Guilak

along the B-cell, T-cell, and myeloid lineages (Storms RW,


Green P, Potiny S, et al . in preparation). This suggests that
human ADAS cells have potential applications in conjunction with hematopoietic stem-cell transplantation. The
addition of ADAS cell infusions may improve and accelerate hematopoietic stem-cell engraftment in recipients
who have undergone BM ablation. Similar approaches
employing human BM stromal cells or MSCs are currently
undergoing clinical trials in the treatment of patients
receiving high-dose chemotherapy or suffering from inborn errors of metabolism [59 /63].
Gene therapy
It is possible to transduce ADAS cells with viral vectors to
introduce exogenous DNA. Adenoviral, herpes simplex
virus, lentiviral, and retroviral vectors all infect ADAS cells
in vitro [26,64, Halvorsen, Bond and Gimble, unpublished
observations]. Although initial proof of principle studies
was limited to marker genes (green fluorescent protein,
Lac Z) [[26], Halvorsen, Bond and Gimble, unpublished
observations], the field is advancing. Katz and colleagues
have demonstrated the introduction of the basic fibroblast
growth factor (bFGF) gene into ADAS cells and observed
peak secretion of the bFGF protein 3 days following viral
transduction [26]. Lentiviral vectors demonstrate the
greatest transduction efficiency [64].
It may prove possible to use ADAS cells as cell carriers
for gene delivery. Theoretically, prior to transplantation,
the ADAS cells can be exposed to high titers of viral vector
in vitro . This avoids the need to deliver high titers of the
viral vector directly to the patient. Before considering the
clinical application of ADAS cells as a gene carrier,
additional in vitro and pre-clinical animal testing is
necessary.

Future applications and challenges


Currently, physicians rely on pharmacologic agents and
surgical interventions to treat acute and chronic clinical
conditions. Cell therapy is limited primarily to hematologic disorders, however, there is a growing body of evidence
supporting a broader application of this modality. Human
adipose tissue is an abundant and accessible source of adult
stem cells. It has the potential to supply the large number
of cells required for therapeutic intervention. ADAS cells
have potential applications in the treatment of acute and
chronic musculoskeletal disorders, cosmetic surgery, central nervous system injury, and other conditions. Before

ADAS cells can be used to treat patients, they will need to


be scrutinized by the Food and Drug Administration for
both safety and efficacy. This will require significant preclinical research and development, some of which is
outlined below.
The manufacture of human ADAS cells must demonstrate in vitro quality assurances to assure safety of the
finished product. This will be facilitated through the
development of a self-contained and closed culture system
for ADAS cells, to reduce the likelihood of contamination
by bacterial, fungal or viral pathogens. In addition, a closed
system permits continuous monitoring of culture levels of
oxygen, lactate, and glucose; this will enhance cell
proliferation and viability, and reduce costs. As concerns
regarding bovine spongiform encephelopathy (BSE) grow,
there are further reasons to culture ADAS cells for all
clinical therapeutic applications in serum-free media.
To extend the shelf-life of the ADSA cells, it will be
necessary to optimize cryopreservation methods and to
fully evaluate their ability to maintain functional, differentiating ADAS cells over time. In vivo pre-clinical animal
trials will be required to document that undifferentiated or
differentiated ADAS cells do not cause adverse events,
either locally at the site of implantation or at distant sites.
Further attention must also be given to improving the
vascularity of ADAS cell implants. In the absence of an
adequate blood supply, the size of a viable tissue implant
will be only a few 100 m, due to limited diffusion of
oxygen and nutrients. Improved biomaterials, incorporating vascular endothelial growth factor and other angiogenic cytokines, may address this issue [65]. Significant
clinical advances have been made concerning the transplantation of human BM-derived MSCs [58 /63]. Physicians have transplanted autologous MSCs in breast-cancer
patients receiving high-dose chemotherapy and improved
their engraftment by MSCs [58,61]. In similar trials,
clinical investigators have transplanted allogeneic MSCs
to treat patients suffering from leukemia [63] and such
genetic disorders as osteogenesis imperfecta [59,60] and
glycogen storage diseases [62].
In contrast, there are limited published clinical studies
to date that have used autologous ADAS cells for
transplantation [53]. Autologous cells reduce the risk of
immune rejection by the host and of transfer of infectious
agents. Nevertheless, the possible use of allogeneic ADAS
cells would have a significant economic and practical
impact on the field. If a single adipose tissue donor could

Adipose-derived adult stem cells

provide ADAS cells for multiple recipients, the cost of


production would be decreased markedly and physicians
would have access to an off the shelf product, eliminating
the need to plan ahead and perform an elective liposuction
to obtain autologous ADAS cells. Animal studies will be
required to determine the feasibility of allogeneic ADAS
cell transplantation, however, some supporting evidence
already exists from other human cell systems.
Bartholomew and colleagues [66] reported that human
BM-derived MSCs expressed low levels of surface Class II
histocompatibility proteins, and did not elicit MLR. Kern
and colleagues [67] reported that human foreskin fibroblasts in a 3D collagen matrix did not increase their surface
Class II histocompatibility protein expression in response
to IFN; in contrast, the same cells in monolayer culture
increased this protein significantly in response to IFN.
These studies suggest that human adult stem cells may not
initiate an immune response under appropriate conditions
in vivo . Further study of the hosts immune response
to undifferentiated and differentiated ADAS cells is
warranted.

Conclusions
One of the major challenges facing the emerging field of
regenerative medicine is a reliable source of cells for tissue
repair. Human ADAS cells meet many of the requirements
required of the ideal cell for tissue engineering. They are
available in quantities of hundreds of million cells
per individual, accessible through a relatively noninvasive method, can differentiate along multiple celllineage pathways, are transplantable in an autologous
manner, and could be manufactured in a controlled,
large-scale manner in accordance with regulatory guidelines.
Further studies are necessary before ADAS cells can be
used clinically. In particular, investigators need to demonstrate the safety and efficacy of ADAS cells in animal
models, either alone or in combination with biomaterial
scaffolds. In addition, manufacturing and quality assurance
issues need to be addressed. Although challenges remain,
the ADAS cell holds significant promise for future
applications.

Acknowledgements
The authors acknowledge support from the following
granting agencies: Artecel Sciences, Inc., the North
Carolina Biotechnology Center, the Kenan Instititute for

367

Engineering, Technology and Science, and NIH grants


AR43876, AG15768, AR48182 & AR49294.
Thanks also to Hani Awad, Lyndon Cooper, Geoffery
Erickson, Beverly Fermor, Yuan-Di Halvorsen, Kevin
Hicok, Henry Rice, Kristine Safford, Robert Storms,
Quinn Wickham, and all the former members of the
R&D staff at Artecel Sciences for their contributions.

References
1 Bianco P, Robey PG. Stem cells in tissue engineering. Nature
2001;414:118 /21.
2 Bianco P, Riminucci M, Gronthos S et al . Bone marrow stromal
stem cells: nature, biology and potential applications. Stem Cells
2001;19:1133 /40.
3 Caplan AI. The mesengenic process. Clin Plast Surg
1994;21:429 /35.
4 Weissman IL, Anderson DJ, Gage F. Stem and progenitor cells:
origins, phenotypes, lineage commitments, and transdifferentiations. Ann Rev Cell Dev Biol 2001;17:387 /403.
5 Adachi N, Sato K, Usas A et al . Muscle derived cell based ex
vivo gene therapy for the treatment of full thickness articular
cartilage defects. J Rheumatol 2002;29:1920 /30.
6 Doherty DJ, Ashton BA, Walsh S et al . Vascular pericytes express
osteogenic potential in vitro and in vivo . J Bone Miner Res
1998;13:828 /38.
7 De Bari C, Dell Accio F, Tylzenowski P et al . Multipotent
mesenchymal stem cells from adult synovial membrane. Arthritis
Rheum 2001;44:1928 /42.
8 Gao J, Dennis JE, Solchaga LA et al . Tissue engineered
fabrication of osteochondral composite graft using rat bone
marrow derived mesenchymal stem cells. Tissue Eng
2001;7:363 /71.
9 Hanada K, Solchaga LA, Caplan AI et al . BMP2 induction and
TGFb1 modulation of rat periosteal cell chondrogenesis. J Cell
Biochem 2001;81:284 /91.
10 Nishimura K, Solchaga LA, Caplan AI et al . Chondroprogenitor
cells of synovial tissue. Arthritis Rheum 1999;42:2631 /7.
11 Nicholl SB, Wedrychowska A, Smith NR et al . Modulation of
proteoglycan and collagen profiles in human dermal fibroblasts
by high density micromass culture and treatment with lactic
acid suggests change to a chondrogenic phenotype. Connect
Tissue Res 2001;42:59 /69.
12 Noth U, Oszycska AM, Tuli R et al . Multilineage mesenchymal
differentiation potential of human trabecular bone derived cells.
J Orthop Res 2002;20:1060.
13 Sottile V, Halleux C, Bassilana F et al . Stem cell characteristics
of human trabecular bone derived cells. Bone 2002;30:699 /704.
14 Young HE, Steele TA, Bray RA et al . Human pluripotent and
progenitor cells display cell surface cluster differentiation
markers CD10, CD13, CD56 and MHC Class I. Proc Soc Exp
Biol Med 1999;221:63 /71.
15 Young HE, Steele TA, Bray RA et al . Human reserve pluripotent
mesenchymal stem cells are present in the connective tissues of

368

16

17
18

19

20

21

22

23

24

25

26
27

28

29

30
31

32

JM Gimble and F Guilak

skeletal muscle and dermis derived from fetal, adult, and


geriatric donors. Anat Rec 2001;264:51 /62.
Shore EM, Ahn J, Jan de Beur S et al . Paternally inherited
inactivating mutations of the GNAS1 gene in progressive
osseous heteroplasia. New Engl J Med 2002;396:99 /106.
Nnodim JO. Development of adipose tissues. Anat Rec
1987;219:331 /7.
Rodbell M. Metabolism of isolated fat cells. Effects of hormone
on fat metabolism and lipolysis. J Biol Chem 1964;239:
375 /80.
Van RLR, Bayliss CE, Roncari DAK. Cytological and enzymological characterization of adult human adipocyte precursors in
culture. J Clin Invest 1976;58:699 /704.
Deslex S, Negrel F, Vannier C et al . Differentiation of human
adipocyte precursors in a chemically defined serum-free
medium. Int J Obes 1986;10:19 /27.
Hauner H, Entenmann G, Wabitsch M et al . Promoting effect of
glucocorticoids on the differentiation of human adipocyte
precursor cells cultured in a chemically defined medium. J
Clin Invest 1989;84:1663 /70.
Moore JH Jr, Kolaczynski JW, Morales LM et al . Viability of fat
obtained by syringe suction lipectomy: effects of local anesthesia
with lidocaine. Aesthetic Plast Surg 1995;19:335 /9.
Lalikos JF, Li YQ , Roth TP et al . Biochemical assessment of
cellular damage after adipocyte harvest. J Surg Res 1997;70:
95 /100.
Halvorsen YDC, Bond A, Sen A et al . Thiazolidinediones and
glucocorticoids synergistically induce differentiation of human
adipose tissue stromal cells: biochemical, cellular and molecular
analysis. Metabolism 2001;50:407 /13.
Zuk PA, Zhu M, Mizuno H et al . Multilineage cells from human
adipose tissue: implications for cell-based therapies. Tissue Eng
2001;7:211 /28.
Katz AJ, Llull R, Hedrick MH et al . Emerging approaches to the
tissue engineering of fat. Clin Plas Surg 1999;26:587 /603.
Gronthos S, Franklin DM, Leddy HA et al . Characterization of
surface protein expression on human adipose tissue-derived
stromal cells. J Cell Physiol 2001;189:54 /63.
Williams SK, Rose DG, Jarrell BE. Liposuction derived human
fat used for vascular sodding contains endothelial cells and not
mesothelial cells as the major cell type. J Vasc Surg 1994;19:
916 /23.
Young C, Jarrell BE, Hoying JB et al . A porcine model for
adipose tissue-derived endothelial cell transplantation. Cell
Transplant 1992;1:293 /8.
Zuk PA, Zhu M, Ashjian P et al . Human adipose tissue is a
source of multipotent stem cells. Mol Biol Cell 2002;13:4279 /95.
Pittenger MF, Mackay AM, Beck SC et al . Multilineage
potential of adult human mesenchymal stem cells. Science
1999;284:143 /7.
Winter A, Breit S, Parch D et al . Cartilage-like gene expression
in differentiated human stem cell spheroids. A comparison of
bone marrow-derived and adipose tissue-derived stromal cells.
Arth Rheum 2003;48:418 /29.

33 Awad HA, Halvorsen, Gimble JM, et al . The effects of TGF-b1


and dexamethasone on the growth and chondrogenic differentiation of adipose derived stromal cells. Tissue Eng In press.
34 Erickson GR, Gimble JM, Franklin D et al . Chondrogenic
potential of adipose tissue-derived stromal cells in vitro and in
vivo . Biochem Biophys Res Commun 2002;290:763 /9.
35 Wickham QM, Erickson GR, Gimble JM, et al . Multipotent
human adult stem cells derived from infrapatellar fat pad of the
knee. Clin Orth Rel Res In press.
36 Mizuno H. The myogenic potential of human processed
lipoaspirates */ Part I: Morphological, immunohistochemical
analysis and gene expression. J Japan Soc Plastic Recontr Surg
2001;21:427 /36.
37 Mizuno H, Zuk PA, Zhu M et al . Myogenic differentiation by
human processed lipoaspirate cells. Plast Reconstr Surg
2002;109:199 /209.
38 Halvorsen Y-DC, Wilkison WO, Gimble JM. Adipose-derived
stromal cells */ their utility and potential in bone formation. Int
J Obes Rel Metab Dis 2000;24(Suppl.):S41 /4.
39 Halvorsen YDC, Franklin DM, Bond AL et al . Extracellular
matrix mineralization and osteoblast gene expression by human
adipose tissue-derived stromal cells. Tissue Eng 2001;7:729 /41.
40 Huang JI, Beanes SR, Zhu M et al . Rat extramedullary adipose
tissue as a source of osteochondrogenic progenitor cells. Plast
Reconstr Surg 2002;109:1033 /41.
41 Hicok KC, du Laney TV, Zhou YS, et al . Human adipose derived
adult stem cells produce osteoid in vivo . Tissue Eng . In press.
42 Justesen J, Pedersen SB, Stenderup K, et al . Subcutaneous
adipocytes can differentiate into bone forming cells in vitro and
in vivo . Under review.
43 Sen A, Lea-Currie YR, Sujkowski D et al . The adipogenic
potential of human adipose derived stromal cells from multiple
donors is heterogeneous. J Cell Biochem 2001;81:312 /9.
44 Halberstadt C, Austin C, Rowley J et al . A hydrogel material for
plastic and reconstructive applications injected into the subcutaneous space of a sheep. Tissue Eng 2002;8:309 /19.
45 von Heimburg D, Zachariah S, Heschel I et al . Human
preadipocytes seeded on freeze-dried collagen scaffolds investigated in vitro and in vivo . Biomaterials 2001a;22:429 /38.
46 von Heimburg D, Zachariah S, Low A et al . Influence of different
biodegradable carriers on the in vivo behavior of human adipose
precursor cells. Plast Reconstr Surg 2001b;108:411 /20.
47 von Heimburg D, Ulrich D, Hemmrich K et al . Soft tissue
engineering by the implantation of autologous adipose precursor
cells into the rabbit ear */ pathophysiology in adipose tissue
transplantation. Clin Exper Plast Surg (Riv Ital Chir Plastica)
2002;33:127 /32.
48 Kral JG, Crandall DL. Development of a human adipocyte
synthetic polymer scaffold. Plast Reconstr Surg 1999;104:1732 /8.
49 Patrick CW Jr, Chauvin PB, Reece GP. Preadipocyte seeded
PLGA scaffolds for adipose tissue engineering. Tissue Eng
1999;5:139 /51.
50 Patrick CW Jr. Adipose tissue engineering: the future of breast
and soft tissue reconstruction following tumor resection. Semin
Surg Oncol 2000;19:302 /11.

Adipose-derived adult stem cells

51 Patrick CW Jr, Zheng B, Johnston C et al . Long-term implantation of preadipocyte-seeded PLGA scaffolds. Tissue Eng
2002;8:283 /93.
52 Schoeller T, Lille S, Wechselberger G et al . Histomorphologic
and volumetric analysis of implanted autologous preadipocyte
cultures suspended in fibrin glue: a potential new source for
tissue augmentation. Aesth Plast Surg 2001;25:57 /63.
53 Garcia-Olmol D, Garcia-Arranz M, Garcia LG, et al . Autologous
stem cell transplantation for treatment of rectovaginal fistula in
perianal Crohns disease: a new cell-based therapy. Int J Colorectal
Dis . In press.
54 Toma C, Pittenger MF, Cahill KS et al . Human mesenchymal
stem cells differentiate to a cardiomyocyte phenotype in the
adult murine heart. Circulation 2002;105:93 /8.
55 Rangappa S, Fen C, Lee EH et al . Transformation of adult
mesenchymal stem cells isolated from the fatty tissue into
cardiomyocytes. Ann Thorac Surg 2003;75:775 /9.
56 Safford KM, Hicok KC, Safford SD et al . Neurogenic differentiation of murine and human adipose-derived stromal cells.
Biochem Biophys Res Commun 2002;294:371 /9.
57 Ashjian PH, Elbarbary AS, Edmonds B et al . In vitro differentiation of human processed lipoaspirate cells into early neural
progenitors. Plast Recontstr Surg 2003;111:1922 /31.
58 Koc ON, Lazarus HM. Mesenchymal stem cells: heading into
the clinic. Bone Marrow Transplant 2001;27:235 /9.
59 Horwitz EM. Marrow mesenchymal cell transplantation for
genetic disorders of bone. Cytotherapy 2001;3:399 /401.
60 Horwitz EM, Gordon PL, Koo WK et al . Isolated allogeneic
bone marrow-derived mesenchymal cells engraft and stimulate

61

62

63

64

65
66

67

369

growth in children with osteogenesis imperfecta: implications for


cell therapy of bone. Proc Natl Acad Sci USA 2002;99:8932 /7.
Koc ON, Gerson SL, Cooper BW et al . Rapid hematopoietic
recovery after coinfusion of autologous-blood stem cells and
culture expanded marrow mesenchymal stem cells in advanced
breast cancer patients receiving high-dose chemotherapy. J Clin
Oncol 2000;18:307 /16.
Koc ON, Day J, Nieder M et al . Allogeneic mesenchymal stem
cell infusion for treatment of metachromatic leukodystrophy
(MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplant 2002;30:215 /22.
Lee ST, Jang JH, Cheong JW et al . Treatment of high-risk acute
myelogenous leukaemia by myleoablative chemoradiotherapy
followed by co-infusion of T cell-depleted haematopoietic stem
cells and culture-expanded marrow mesenchymal stem cells
from a related donor with one fully mismatched human
leucocyte antigen haplotype. Br J Haematol 2002;118:1128 /31.
Morizono K, De Ugarte DA, Zhu M et al . Multilineage cells
from adipose tissue as gene delivery vehicles. Hum Gene Ther
2003;14:59 /66.
Ennett AB, Mooney DJ. Tissue engineering strategies for in vivo
neovascularisation. Expert Opin Biol Ther 2002;2:805 /18.
Bartholomew A, Sturgeon C, Siatskas M et al . Mesenchymal
stem cells suppress lymphocyte proliferation in vitro and
prolong skin graft survival in vivo . Exp Hematol 2002;30:42 /8.
Kern A, Liu K, Mansbridge J. Modification of fibroblast ginterferon responses by extracellular matrix. Invest Dermatol
2001;117:112 /8.