COSMETIC

Adipose-Derived Stem and Progenitor Cells asFillers in Plastic and Reconstructive Surgery

Timothy A. Moseley, Ph.D.Min Zhu, M.D.

Marc H. Hedrick, M.D.

San Diego and Los Angeles, Calif.

Summary: Plastic surgeons are keenly aware of the principle “replace like withlike.” This principle underlies much of the rationale behind the clinical use ofautologous fat transplantation, despite the procedure’s drawbacks. Autologousfat transplantation is frequently used for a variety of cosmetic and reconstructiveindications not limited to posttraumatic defects of the face and body, involu-tional disorders such as hemifacial atrophy, sequelae of radiation therapy, andmany aesthetic uses such as lip and facial augmentation and wrinkle therapy.However, the limitations of fat transplantation are well known, particularly thelong-term unpredictability of volume maintenance. Regenerative cell-basedstrategies such as those encompassing the use of stem cells hold tremendouspromise for augmentation of the soft-tissue space. Preclinical studies and earlyclinical series show that adipose-derived stem cells offer the possibility of finallyfulfilling the key principle of replacing like with like as an aesthetic filler, withoutthe drawbacks of current technology. (Plast. Reconstr. Surg. 118 (Suppl.): 121S,2006.)

REGENERATIVE MEDICINE ANDSTEM CELLS

Regenerative medicine, broadly speaking,refers to the use of the body’s raw materialsof repair—namely, cells, matrix, and/or

chemical compounds—to naturally restore ap-pearance or function. This strategy appliesequally to both life-threatening diseases such asheart attack and cosmetic and reconstructive in-dications. In many respects, cells are the mostcritical and elusive members of the “regenerativetriad.” Cells, unlike many matrix macromole-cules, proteins, and small molecules, cannot bemanufactured per se. They must be harvested,purified, and in most cases replicated beforeclinical use. Furthermore, this process must beperformed in a manner such that the therapeu-tic potential of the cells is not dissipated orremoved altogether.

Stem cells have received significant attention as anideal source of regenerative-capable cells because oftheir multipotentiality and ability to replicate. Fur-thermore, stem cells have been used in a limited

fashion for decades with great clinical success. Forexample, stem cells from the bone marrow, periph-eral blood, and even umbilical cord blood have beenused to treat a variety of diseases.1,2

Although stem cell therapies can be used allo-geneically, the issue of transplant-related im-mune responses, however, limits the universalapplicability of allogenic cell treatments andstem cell therapies.3 The innate problems asso-ciated with allogenic stem cell transplantationsuch as host immune responses and implant re-jection can be avoided by the use of autologousstem cells. Furthermore, autologous stem cells,such as those derived from bone marrow or ad-ipose tissue, can be used clinically for regenera-tive cell therapy only if they can be obtained insufficient quantities. Adipose-derived stem cellscan be easily processed from lipoaspirated fatand can provide the physician with a significantquantity of multipotent stem cells for a variety oftherapeutic regenerative medicine therapies.

A BRIEF BACKGROUND OFFILLER MATERIALS

The need for fillers to “replace like with like”is found in over a century’s experience with both

From the Department of Research and Technology, Divisionof Biologics, Cytori Therapeutics, Inc., and Department ofSurgery, Division of Plastic Surgery, University of Califor-nia, Los Angeles.Received for publication January 20, 2006; accepted May12, 2006.Copyright ©2006 by the American Society of Plastic SurgeonsDOI: 10.1097/01.prs.0000234609.74811.2e

Adipose-derived stem and progenitor cellshave not been approved for use by the FDA.

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fat transplantation and synthetic materials. Com-mon materials such as paraffin, rubber, latex, andsilicone were all used starting in the early 1900s assoft-tissue fillers.4 Today, some of these or theirman-made progeny are still in use, such as poly-methylmethacrylate microbeads marketed asArteFill.5 Recently, more sophisticated biologi-cally compatible fillers have been introduced thatare made of hyaluronic acid, such as Hylaform andRestylane.6–8 All of these fillers, both synthetic andnaturally occurring, have significant limitationsthat are well known to most surgeons. Althoughthese macromolecules are used currently in theclinic as simple soft-tissue fillers, they can also beused as constructs for tissue regeneration. Bioma-terials such as these can act as a scaffold supportor matrix for surrounding, infiltrating cells or canbe mixed ex vivo with a variety of cell types, suchas adipocyte precursors7 or dermal fibroblasts9 toproduce engineered constructs for volume aug-mentation. A critical constraint of this paradigm isneovascularization of the construct. New growthof blood vessels from the surrounding tissue maytake 5 days to infiltrate and provide adequate nu-trition, which could result in graft cell death andtissue necrosis.10

Neuber was the first to publish findings re-garding the use of autologous fat transplantationin 1893. He filled scars with autologous fat andfound a reduction of transplant resorption by de-creasing graft particle size.11 Unfortunately, de-spite over 100 years of clinical use since, little hasbeen developed to improve free fat graft perfor-mance, and clinical experience has beenlackluster.12–14 Specifically, the clinical longevity ofthe graft is highly variable and the volume of largegrafts in particular decreases significantly overtime. Histologically, progressive loss of trans-planted adipocytes is noted along with a conver-sion of the graft to fibrous tissue, oftentimes withcyst formation. The presumed mechanism of tis-sue loss appears to be primarily insufficient vas-cularity and cell death. However, scientific con-firmation of this as the only mechanism involvedis limited. Other mechanisms such as mechanicaldisruption of cells, lipid-induced membrane dam-age, apoptosis, or perhaps other potential mech-anisms are possible but have not been well studied.

Clearly, however, both preclinical and clinicalstudies show that some of the transplanted graftremains, adding to soft-tissue volume. It is pre-sumed that the portion of the graft adjacent to thenative recipient blood supply nourishes the graftand sustains it until vascularization occurs. Pre-sumably, more central areas of the graft, that are

not so fortunate, die. This concept has led to de-creasing the size of fat grafts with the hope that asmaller graft will mean more fat is adjacent toviable recipient tissue, resulting in improved avail-ability and diffusion of cellular nutrition until neo-vascularization occurs.15,16

This concept has been expanded and formal-ized in the “lipostructure technique” as describedby Coleman in 1997. The lipostructure techniquedescribes the microinjection of autologous li-poaspirated fat.17,18 This technique works to pre-serve the fragile nature of the donor adipose tissueand gently transfers these small fat particles tolocations near vascularized structures and associ-ated nutrition. Implementation of this techniquehas led to improved fat grafting outcomes formany surgeons. By combining stem cell therapywith the lipostructure technique, tissue survivabil-ity could potentially be enhanced, because stemcells have been shown to enhance angiogenesis19,20

and to minimize inflammatory responses.21–23

THE BIOLOGICAL BASIS FOR SOFT-TISSUE THERAPY USING ADIPOSE-

DERIVED STEM CELLSCurrently, scientific and clinical interest is high

regarding the potential of stem cells to treat a broadrange of conditions.24 Many sources of cells havebeen identified, including bone marrow, fat, muscle,liver, skin, heart, and brain in the adult,25 in additionto fetal and embryonic sources.26 Stem cells have twobasic properties. First, stem cells have the potentialto develop or differentiate into many different celltypes in the body, such as osteoblasts, myocytes, andadipocytes.24–26 Second, stem cells, as they divide,have the potential to self-renew, making yet morestem cells. Age appears to play an important role instem cell potential. The number of stem cells able todifferentiate and their plasticity to differentiate intoany tissue appears to diminish with age.27–29 On thesurface, this may seem to support the use of“younger” stem cell types such as embryonic andfetal stem cells. Unfortunately, despite the ethicalquandaries entangling embryonic stem cell usage,there are also key biological limitations with their useas well.24 Major hurdles such as cell stability, onco-genicity, and spontaneous teratoma formation, inaddition to transplantation issues based on the re-quirement for allogenic cell use or conversely theneed for therapeutic cloning to produce autogenic-ity, represent profound impediments to clinical use,particularly for non–life-threatening indications. Asa result, the future clinical use of these cells is, at best,likely many years away.

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For the foreseeable future, adult stem cellsrepresent the most promising source of cells forplastic surgery. Bone marrow, for example, hasbeen shown to contain a population of stem cellsthat have regenerative potential. Bone marrowmesenchymal stem cells are able to support therepair and regeneration of bone, cartilage, adi-pose, heart, and even neuronal tissues.25,30 How-ever, the use of bone marrow–derived stem cellsis limited by the quantity of cells that can be col-lected from the patient and associated donor-sitepain and morbidity. Also, the cells obtained fromthe bone marrow need to be cultured and ex-panded in Good Manufacturing Practice–regu-lated tissue processing facilities for several weeksto generate enough cells for therapeutic use. Thisprocedure is expensive, heavily regulated, andfundamentally changes the biology of the cells.31

These limitations and others have stimulatedinterest in a possible therapeutic role of adipose-derived stem cells from processed lipoaspirate formany applications, including their use as soft-tis-sue fillers for plastic and reconstructivesurgery.32–34 Historically, most biology textbookslist the primary purpose of adipose tissue as areservoir of energy in the form of triglycerides andas a storehouse for fat-soluble vitamins. Only re-cently has this source of calories and vitamins beenthought of as a source of several types of regen-erative cells as well.35,36 Armed with this under-standing, many new doors are now opened to avariety of potential therapeutic strategies usingadipose tissue.

Developmentally, adipose tissue, like bonemarrow, is derived from the embryonic mesen-chyme and rapidly expands after birth by prolif-eration of the adipocyte precursor cells and anincrease in cellular size. Also like bone marrow,fibroblast-like cells in adipose tissue contain a pop-ulation of cells that can differentiate toward anumber of lineages not limited to the osteogenic,myogenic, chondrogenic, adipogenic, and neuro-genic lineages37–40 (Table 1).36,37,39,41–46 Adipose-derived stem cells can be obtained from the pro-cessing of either liposuctioned or excised fat.Despite its ease of harvest and the volume of tissueobtainable from liposuction, adipose contains 100to 1000 times more pluripotent cells on a per–cubic centimeter basis than bone marrow.39,47 The-oretically, the abundance of isolated stem andtherapeutically active cells from adipose elimi-nates the requirement of cell expansion in tissueculture facilities as is required with bone marrow–derived cells and makes these cells readily avail-able to the clinician, at the bedside. In general,

fillers currently on the market have not been suc-cessful in supporting an engineered tissue replace-ment therapy for defect repair or tissue restora-tion. Identifying the appropriate filler incombination with stem cells could provide an op-timized microenvironment in which to create anengineered tissue that can be used as a semiper-manent filler material.9

There are a myriad of reasons to suggest thatadipose-derived stem cells are perhaps the idealsource of cells for regenerative-based soft-tissuetherapies. First, their multipotentiality, especiallytheir proclivity for adipose differentiation, makesthem an alluring cell source. It is relatively simpleto achieve a high level of adipose differentiationof adipose-derived stem cells in vitro, and manystudies have developed methods to use them invivo.33,48 Second, adipose-derived stem cells seemto have a significant potential for angiogenesis andvasculogenesis, one of the fundamental limita-tions in the current technique of autologous fattransfer. Third, plastic surgeons have a significantcomfort level with the harvest and manipulation ofadipose tissue.49–53

However, early work suggests that adipose-de-rived stem cells injected into the soft tissue or intoa defect will not alone produce soft-tissue fill (M.Zhu et al., unpublished observations, 2005). Atherapeutic strategy must be used to leverage theunique aspects of the cells. One strategy is to usea tissue-engineering approach, combining cellsstimulated with certain chemical compounds andplaced into a matrix or scaffold to “manufacture”an implantable neoadipose construct. Several po-tential scaffolds combined with cells have beeninvestigated for their utility for soft-tissue treat-ment. In one example, preadipocyte cells, whichare similar if not identical to adipose-derived stemcells, were combined with the resorbable material

Table 1. The Multipotentiality of Adipose-DerivedStem Cells and the Associated References thatDescribe the Lineage Differentiation

Adipose-Derived Stem CellDifferentiation Lineage

References

Adipogenic (fat) 36,39Cardiomyogenic (cardiac muscle) 39,41Leiomyogenic (smooth muscle) 42Myogenic (skeletal muscle) 43,44Chondrogenic (cartilage) 39,43Osteogenic (bone) 39,43Neurogenic (nerves) 39,44Angiogenic (blood vessel) 45,46Hepatocytic (liver) 37Hematopoietic (blood) 45

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poly(lactide-co-glycolide acid). In this study, al-though initial results were promising, ultimateanalysis at 5 to 12 months showed a completeabsence of fat and poly(lactide-co-glycolideacid).48,54 Another similar study compared the hy-aluronic acid sponge, HYAFF 11, with collagensponges augmented with adipose precursor cells.They found that HYAFF 11 was superior to colla-gen in supporting the differentiation and expan-sion of adipose precursor cells.53 From these stud-ies and others like them, it is difficult to drawclinically meaningful conclusions. However, it sug-gests that the tissue-engineering approach may bequite difficult to accomplish successfully untilmore is known regarding the basic signaling in-volved in stem cell differentiation, cell/matrix in-teractions, and understanding of adipose-relatedmorphogen biology.

An ingenious approach was used clinically byLlull, who used autologous fat as a matrix andsupplemented the transplant with adipose-derivedstem cells. He found that this procedure couldprovide long-term (�1 year) soft-tissue volumerestoration in a patient with soft-tissue involutionon the ulnar aspect of one hand.55 Notably, thispatient had previously failed autologous fat graft-ing alone, which was performed before this adi-pose-derived stem cell procedure. This case pro-vided evidence that fat itself can provide thematrix for stem cells. The mechanism driving theclinical result is not known, but this single clinicalcase suggests new potential mechanisms beyonddifferentiation and invites new therapeutic strat-egies by which adipose-derived stem cell therapymay one day be broadly useful.

STEM CELL–SUPPLEMENTED FATGRAFTS

To study the ability of adipose-derived stemcells to provide clinical soft-tissue support, predic-tive preclinical models are required. Based on thework of Llull and other unpublished work, wedeveloped an in vivo model for characterizing andoptimizing stem cell–based methods for soft-tissueaugmentation.

In the rodent, the subdermal scalp space rep-resents a highly vascular and accessible yet de-manding subcutaneous space for testing the im-plantation of soft-tissue constructs. Using thisapproach, we have found that freshly isolated ad-ipose-derived stem cells, when mixed with minceddonor fat, can improve the longevity and volumeof the graft.56 In this preliminary study, we useddonor stem cells from the adipose tissue of Rosamice, which are transgenic for �-galactosidase

(LacZ) and that can be stained blue during his-tologic evaluation with the chromogenic substrateX-gal. The stem cells were isolated as a heterog-enous cell population from lipectomized fat thatwas enzymatically digested and pelleted by cen-trifugation to remove the buoyant fraction accord-ing to previously published prodedures.43 Freshlyisolated, not cultured, Rosa adipose-derived stemcells or cultured Rosa adipose-derived stem cellswere mixed with 100 �l of minced adipose tissuefrom B6;129 sF1/J mice and injected over theskulls of experimental athymic nude mice. The fatwas harvested at 6 months and the quantity andquality of the fat observed, as was the presence ofthe Rosa-derived cells. We found that, 6 monthsafter transplantation, the fat with freshly isolatedstem cells had a weight 2.5 times greater than thefat graft–only group (p � 0.021) (Fig. 1). Also, atrend was noted (p � 0.06) that cultured adipose-derived stem cells were greater in maintaininggraft volume than fat only. Qualitatively, hema-toxylin and eosin evaluation of the transplantedtissues at 6 months demonstrated that the graftedfat supplemented with fresh stem cells maintainedits adipocyte-rich appearance, whereas the graftedtissue without cell supplementation group had amore fibrous tissue appearance (Fig. 2). �-Galac-tosidase staining of fat with fresh adipose-derivedRosa stem cells shows that the stem cells are re-tained in the graft for at least 6 months. Further-more, immunohistochemistry of fat with the freshadipose-derived Rosa stem cells with von Wille-brand factor (factor VIII), an endothelial marker,showed an identifiable vascular support for theentire graft at 6 months. This work, although in-complete, suggests that one potential mechanismby which adipose-derived stem cells may work isthe improvement of graft blood supply. This issupported by recent work presented by Pinkernellet al., Strem et al., and others, who show convinc-ingly that adipose-derived stem cells improve car-diac function after heart attack in pigs, in part byimproving myocardial perfusion.57,58 Further stud-ies in vivo, including greater mechanistic detailand longer follow-up of the transplanted cellularconstructs, is ongoing.

Building on the clinical feasibility of using ad-ipose-derived stem cell–supplemented fat as a de-fect filler, Yoshimura and colleagues treated 23patients with either demonstrable soft-tissue de-fects or breast augmentation using stem cell–sup-plemented fat transplantation for soft-tissue fill.These data, which have yet to be published buthave been presented in both the United States andJapan, suggest that this approach may be feasible

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Fig. 1. Transplant weight of fat recovered after 6 months over the skull of nude mice normalizedto the fat-only group. The cultured cells with 100 �l of fat did not maintain a significant graft volumeover the fat-only group (p � 0.06). The freshly isolated adipose derived cells did retain a significantlylarger volume of graft tissue (p � 0.021).

Fig. 2. Histologic images of transplanted fat removed after 6 months in athymic nude mice. (Above, left) Represen-tative sample from hematoxylin and eosin–stained fat with fresh adipose-derived stem cell group. (Above, right)Representative sample from hematoxylin and eosin stain of fat-only group. (Below, left) �-Galactosidase staining offat with adipose-derived Rosa stem cells shows the cells are retained in the graft. (Below, right) Immunohistochem-istry of fat with adipose-derived Rosa stem cells with von Willebrand factor, an endothelial marker. This shows thatthere is vascular support for the entire graft.

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and effective.59 In this feasibility series, 23 variouscases were treated and followed up to 26 months,with evidence of long-term maintenance of vol-ume in patients treated for either subcutaneoussoft-tissue fill or breast enhancement (Fig. 3). Re-ported complications were limited to one patientwho has exhibited some fibrosis at the central areaof the chest over the sternum. However, despitethese results, a final determination of the successand potential complications of this early seriesmust be predicated on the complete study and itspeer-reviewed evaluation.

CONCLUSIONSAdipose tissue is plentiful and can be easily

obtained using current liposuction techniques. Al-though fat is inconsistent on its own as a defectfiller, adipose tissue can provide a source of stemcells that may be able to either enhance fat graft-ing or provide the basis for other more uniquestrategies for soft-tissue regeneration or repair.Although the property of differentiation may bean important aspect of stem cell therapeutic po-tential, other mechanisms such as vasculogenesis,arteriogenesis, angiogenesis, cell preservation, an-tiapoptosis, anti-inflammation, and others may un-derlie the potential effectiveness of adipose-de-

rived stem cells as well. Furthermore, as newtherapies are developed using adipose-derivedstem cells, the procedure of liposuction will as-sume a broader role as a preferred method ofautologous tissue and cell harvest and not just acosmetic procedure.

Marc H. Hedrick, M.D.Cytori Therapeutics, Inc.

3020 Callan RoadSan Diego, Calif. 92121mhedrick@cytoritx.com

DISCLOSURESAll authors are employees of Cytori Therapeutics,

Inc. Cytori Therapeutics is discovering and developingproprietary, cell-based therapeutics using adult stem cellsderived from adipose tissue. The company’s investiga-tional therapies target cardiovascular disease, spine andorthopedic conditions, gastrointestinal disorders, andnew approaches for aesthetic and reconstructive surgery.

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Fig. 3. Lateral and frontal views before (above) and 1 year after (below) treatment. The patient was a 28-year-old woman who hada very minor deformity of the thorax. Three hundred cubic centimeters of suctioned fat was injected together with freshly isolatedadipose-derived stem cells in each side. Adipose-derived stem cells were harvested from another aliquot of fat tissue and aspiratedfluid. The patient gained 8.5 cm in circumference at 1 year. Furthermore, at 1 year, she had soft breasts and a normal mammogram.(Photographs courtesy of Dr. Kotaro Yoshimura.)

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