Adipose-derived adult stem cells: isolation,characterization, and differentiation potential

JM Gimble1 and F Guilak2

1Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA, USA2Department 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 adipose-

derived 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.

Keywords

adipose tissue, stem cell, multipotent, tissue engineering.

IntroductionThe 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 applica-

tion 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 hemato-

poietic stem cells (HSCs) and mesenchymal stem cells

(MSCs) or stromal cells [1/4]. Each has the potential toexpress the surface proteins and phenotype of committed

cell lineages in response to appropriate combinations of

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 progres-

sive 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.

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

70808, USA.

Cytotherapy (2003) Vol. 5, No. 5, 362/369

2003 ISCT DOI: 10.1080/14653240310003026

ADAS cell isolation methodologyHistologic and electron microscopic studies identified

putative adipocyte progenitor cells in situ within embry-

onic and adult adipose tissues (reviewed in Nnodim

1987[17]). The earliest progenitors displayed fibroblastic

characteristics, with an abundant endoplasmic retic-

ulum 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 sur-

rounded 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, triio-

dothyronine, biotin, insulin, pantothenate), the cells accu-

mulated 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 tempera-

ture 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.

ADAS cell immunophenotypeThe 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 groupshave 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 mole-cule 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), intercellularadhesion 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.

j Receptor molecules. The ADAS cells express the

hyaluronate (CD44) and transferrin (CD71) receptors.

j Surface enzymes. The ADAS cells express the neutral

endopeptidase (CD10 or common acute lymphocytic

leukemia antigen CALLA), aminopeptidase (CD13),

and ecto 5? nucleotidase (CD73).j Extracellular matrix proteins and glycoproteins. The

ADAS cells produce Type I and Type III collagens,

osteopontin, ostenectin, Thy-1 (CD90), and MUC-18

(CD146).

j Skeletal proteins. The ADAS cells express intracellular

a smooth muscle actin, and vimentin.j Hematopoietic cell markers. The ADAS cells do not

express the hematopoietic markers CD14, CD31, or

CD45.

j Complement regulatory proteins. The ADAS cells were

positive for decay accelerating factor (CD55) and

complement protectin (CD59).

j 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

Adipose-derived adult stem cells 363

analysis, the use of MAbs detecting different epitopes on

the same surface protein, and sensitivity differences

between immunohistochemical and flow cytometric detec-

tion 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: whileADAS cells do not express NCAM (CD56), muscle-

derived 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 potentialMusculoskeletal tissues

Chondrocyte */ cartilage repairHuman ADAS cells can display the biochemical markers

associated with mature chondrocytes [25,30,33/35]. Withthe 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, whenADAS 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 chondro-

genic 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 osteo-

genic 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 promotechondrogenesis [33]. Further work is needed to establish

culture conditions to maximize the ADAS cells chondro-

genic 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 weight-

bearing joint.

Myocyte */ skeletal muscle repairADAS 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 cul-

tured 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 repairADAS cells differentiate into osteoblast-like cells in the

presence of ascorbate, b-glycerophosphate, dexamethasoneand 1,25 vitamin D3 [38,39]. Over a 2/4 week period invitro , both human and rat ADAS cells deposit calcium

phosphate mineral within their extracellular matrix, and

express osteogenic genes and proteins */ including alka-line phosphatase, bone morphogenic proteins and their

receptors, osteocalcin, osteonectin, and osteopontin

[25,30,38/40]. In vivo , ADAS cells embedded in porouscubes 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 pre-

cursors. 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.

364 JM Gimble and F Guilak

Soft tissues

Adipocyte */ soft tissue cosmesisHuman 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 gluco-

corticoid receptor agonist), indomethacin or thiazolidine-

dione (a peroxisome proliferator activated receptor gligand), pantothenate, biotin, and/or triiodothyronine

[20,21,24,25,30,43]. After 7/10 days, the human ADAScells 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 quanti-

fied 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 poros-

ity 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 refine-

ment, 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] trans-

planted autologous ADAS cells to repair a rectovaginal

fistula in a patient suffering from Crohns disease and

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 demon-

strated 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 injuryThere 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 neuronal-

associated 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 andin 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 hemato-

poietic 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 cellfactor [58]. Consistent with these observations, human

ADAS cells promote the differentiation of CD34

hematopoietic stem cells in in vitro co-culture systems

Adipose-derived adult stem cells 365

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 conjunc-

tion with hematopoietic stem-cell transplantation. The

addition of ADAS cell infusions may improve and accel-

erate 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 in-

born 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 challengesCurrently, physicians rely on pharmacologic agents and

surgical interventions to treat acute and chronic clinical

conditions. Cell therapy is limited primarily to hematolo-

gic 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, cen-

tral 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 pre-

clinical research and development, some of which is

outlined below.

The manufacture of human ADAS cells must demon-

strate 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, differ-

entiating 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, incorporat-

ing vascular endothelial growth factor and other angio-

genic cytokines, may address this issue [65]. Significant

clinical advances have been made concerning the trans-

plantation of human BM-derived MSCs [58/63]. Physi-cians 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

366 JM Gimble and F Guilak

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 fibro-

blasts 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.

ConclusionsOne 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 non-

invasive method, can differentiate along multiple cell-

lineage pathways, are transplantable in an autologous

manner, and could be manufactured in a controlled,

large-scale manner in accordance with regulatory guide-

lines.

Further studies are necessary before ADAS cells can be

used clinically. In particular, investigators need to demon-

strate 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.

AcknowledgementsThe authors acknowledge support from the following

granting agencies: Artecel Sciences, Inc., the North

Carolina Biotechnology Center, the Kenan Instititute for

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.

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