in vitro differentiation of human processed lipoaspirate cells into early neural progenitors

Experimental

In Vitro Differentiation of Human ProcessedLipoaspirate Cells into Early NeuralProgenitorsPeter H. Ashjian, M.D., Amir S. Elbarbary, M.D., Brian Edmonds, Ph.D., Daniel DeUgarte, M.D.,Min Zhu, M.D., Patricia A. Zuk, Ph.D., H. Peter Lorenz, M.D., Prosper Benhaim, M.D., andMarc H. Hedrick, M.D.Los Angeles, Calif.

Human processed lipoaspirate (PLA) cells are multi-potent stem cells, capable of differentiating into multiplemesenchymal lineages (bone, cartilage, fat, and muscle).To date, differentiation to nonmesodermal fates has notbeen reported. This study demonstrates that PLA cells canbe induced to differentiate into early neural progenitors,which are of an ectodermal origin. Undifferentiated cul-tures of human PLA cells expressed markers characteristicof neural cells such as neuron-specific enolase (NSE),vimentin, and neuron-specific nuclear protein (NeuN).After 2 weeks of treatment of PLA cells with isobutylmeth-ylxanthine, indomethacin, and insulin, about 20 to 25percent of the cells differentiated into cells with typicalneural morphologic characteristics, accompanied by in-creased expression of NSE, vimentin, and the nerve-growth factor receptor trk-A. However, induced PLA cellsdid not express the mature neuronal marker, MAP, or themature astrocyte marker, GFAP. It was also found thatneurally induced PLA cells displayed a delayed-rectifiertype K� current (an early developmental ion channel)concomitantly with morphologic changes and increasedexpression of neural-specific markers. The authors con-cluded that human PLA cells might have the potential todifferentiate in vitro into cells that represent early pro-genitors of neurons and/or glia. (Plast. Reconstr. Surg.111: 1922, 2003.)

The generation of neurons in the mamma-lian central nervous system occurs during earlydevelopment. Neuronal production is largelycomplete within a few days of birth, and theadult mammalian brain and spinal cord lackthe ability to replace neurons lost to injury or

disease. Thus, the consensus among neurosci-entists is that damage to the central nervoussystem is usually irreversible. This can be exem-plified by certain neurodegenerative states,such as amyotrophic lateral sclerosis, spinalcord trauma, Parkinson disease, and Alzheimerdisease, in which loss of a particular populationof neural cells results in circumscribed deficits.Currently, the only way to replace neural tissuelost to injury or disease is through transplanta-tion. The most important and indispensablecomponent of any strategy to replace neuraltissue is the neural progenitor cell. Long-termneural integration, remodeling, and regenera-tion will require a continuous supply of neuralprogenitor cells capable of differentiating intomature neurons and/or glial cells. Despite thediscovery of stem cell populations in the ma-ture central nervous system, an abundantsource of easily accessible neural cells islacking.

Adult stem cells have been isolated fromseveral tissue sources, including the centralnervous system, bone marrow, retina, skin, andskeletal muscle.1–6 Recent work has focused onthe utility of cells for tissue engineering anddisease management. The human bone mar-row has historically been the primary source ofadult stem cells. A subclass of bone marrow

From the Laboratory for Regenerative Bioengineering and Repair, Departments of Surgery and Orthopedics, University of California, LosAngeles, School of Medicine. Received for publication March 12, 2002; revised July 25, 2002.

Presented at the 70th Annual Scientific Meeting of the American Society of Plastic Surgeons in Orlando, Florida, November 3 through 7,2001; the 46th Annual Meeting of the Plastic Surgery Research Council in Milwaukee, Wisconsin, June 9 through 12, 2001; and the 2001 SurgicalForum of the American College of Surgeons in New Orleans, Louisiana, October 7 through 12, 2001.

Drs. Hedrick, Benhaim, and Lorenz have a financial interest in technology related to this scientific study.

DOI: 10.1097/01.PRS.0000055043.62589.05

1922

stromal cells, termed mesenchymal stem cells,has been shown to differentiate into cells ofvarious mesodermal lineages, including bone,cartilage, and fat.3 However, recent reportshave demonstrated that mesenchymal stemcells may not be restricted to the mesodermalpathway. They have been induced to differen-tiate into neural cells (ectodermal origin) invitro and in vivo.7–11 Woodbury et al.7 usedbeta-mercaptoethanol in serum free media toinduce rat and human mesenchymal stem cellsto form cells with neuron-like morphologiccharacteristics expressing the neuron markersneuron-specific enolase (NSE), neuron specificnuclear protein (NeuN), neurofilament-M, andtau. Deng et al.10 differentiated human mesen-chymal stem cells into early neural progenitorsusing isobutylmethylxanthine and dibutyryl cy-clic adenosine monophosphate.

Our laboratory has identified an alternativesource of pluripotent cells from human adi-pose tissue, termed processed lipoaspirate(PLA) cells. These cells have been shown todifferentiate in vitro into adipogenic, chondro-genic, myogenic, and osteogenic cells.12,13 Inthis study, we report that human PLA cells canbe induced to differentiate into cells with char-acteristics of early neurons and/or glia.

MATERIALS AND METHODS

Isolation and Culture of Human PLA Cells

Human adipose tissue was harvested fromhealthy donors (aged 30 to 60 years) by suctionassisted lipectomy. The samples were obtainedfrom patients with informed consent and ac-cording to a protocol approved by the Institu-tional Review Board (HSPC#98–08 011–02).Isolation and culture of human PLA were per-formed as previously described by Zuk et al.12

Briefly, lipoaspirates were washed extensivelywith equal volumes of phosphate-buffered sa-line to remove contaminating red blood cellsand debris. The extracellular matrix was subse-quently digested with 0.075% collagenase for30 minutes at 37°C. Enzyme activity was neu-tralized with 10% fetal bovine serum (Hyclone,Logan, Utah), and the cells were centrifuged at1200 � g for 5 minutes. The resulting pellet,containing PLA cells, was resuspended in com-plete culture medium consisting of Dulbecco’sModified Eagle Medium (Gibco BRL, Rock-ville, Md.), 10% fetal bovine serum, and 1%antibiotic/antimycotic. All cells were distrib-uted in 100 � 20-mm tissue culture dishes and

incubated at 37°C with 5% humidified carbondioxide. Cells were washed thoroughly withphosphate-buffered saline after 24 hours of in-cubation, and nonadherent cells were dis-carded. Fresh complete culture medium wasadded every 3 days. The cells were grown to 50to 60 percent confluency, at which point neu-ral differentiation was initiated.

Cell Surface Marker Characterization

PLA cells were isolated as described above.Cell aliquots were incubated with phyco-erythrin-conjugated CD11c and fluorescein iso-thiocyanate conjugated CD44, CD45, andCD90 antibodies (BD/Pharmingen, San Di-ego, Calif.) and were subsequently washed inlysis buffer. The unconjugated primary anti-body for CD29 (BD/Pharmingen) requiredthe addition of a phycoerythrin-conjugated sec-ondary antibody (�-immunoglobulin G, Sigma,St. Louis, Mo.). Flow cytometry was performedusing a FACSCalibur cytometer (Becton Dick-son, San Jose, Calif.). Cell surface marker ex-pression was determined by comparing fluores-cence intensity of CD antibody staining withthat of isotype control on a histogram plot.

Neural Differentiation Protocol

Subconfluent cultures of human PLA cells(passage 2) were maintained in nondifferenti-ating culture medium. To initiate neural in-duction the cells were washed with 1� phos-phate-buffered saline, and neural inductionmedia [composed of Dulbecco’s Modified Ea-gle Medium � 10% fetal bovine serum � 1%antibiotic/antimycotic � 5 �g/ml insulin (Sig-ma) � 200 �M indomethacin (Sigma) � 0.5mM isobutylmethylxanthine (Sigma)] wasadded. The induction media was replaced ev-ery 3 days, for a total differentiation time of 2weeks.

Quantification of Neural Differentiation

To determine the degree of neural differen-tiation, cells exhibiting a typical neural struc-ture (a refractile cell body with multipolar pro-cesses) were quantified. Six independentexperiments were performed in triplicate ateach time point. A Zeiss microscope with amounted digital camera was used to capturethree random nonoverlapping low-power(100�) images of each quadrant of a 100 �20-mm tissue culture dish. Cells with neuralmorphologic characteristics were expressed asa percentage of total PLA cells counted � the

Vol. 111, No. 6 / PROCESSED LIPOASPIRATE CELLS 1923

standard deviation (percentage of total PLA �SD).

Western Blot Analysis

Induced and noninduced control PLA cellswere rinsed with cold phosphate-buffered sa-line and drained. Whole cell lysates were pre-pared by adding 0.5 ml detergent based celllysis buffer, plus protease/phosphatase inhibi-tor cocktail (1:100 dilution, Sigma), and phe-nylmethylsulfonyl fluoride (final concentra-tion � 500 �M, Sigma), after which the cellswere scraped into a centrifuge tube. The cellswere further lysed by sonication. The samplewas centrifuged at 14,000 � g for 30 minutes at4°C and the supernatant was collected. Proteincontent was assayed colorimetrically (BioAssayKit; Bio-Rad, Hercules, Calif.). Fifty micro-grams of protein extract from induced andnoninduced control cells were separated on10% gradient acrylamide gel (Bio-Rad Labora-tories, Hercules, Calif.) and were electro-phoretically transferred to a polyvinyl difluo-ride membrane. Immunodetection wasperformed with antibodies to NSE (rabbit poly-clonal, 1:3000 dilution; Polysciences Inc., War-rington, Pa.), trk-A (rabbit polyclonal, 1:500;Santa Cruz Biotechnology, Santa Cruz, Calif.),vimentin (goat polyclonal, 1:1000; Santa CruzBiotechnology), MAP2 (mouse monoclonal,1:500; Leinco Technologies, Ballwin, Mo.), tau(mouse monoclonal, 1:500; Leinco), GFAP(mouse monoclonal, 1:500; Dako, Carpinte-ria, Calif.), and NeuN (mouse monoclonal,1:100; Chemicon, Temecula, Calif.). Appro-priate secondary antibodies were conjugatedto alkaline phosphatase. The membraneswere processed using enhanced chemilumi-nescence (CSPD, Tropix, Bedford, Mass.).Ten micrograms of human brain extract(University of California, Los Angeles BrainBank) was used as a positive control. Equalloading of samples was determined by stain-ing for �-actin (Santa Cruz Biotechnology,1:5000 dilution) (Table I).

Immunocytochemistry

After neural induction, the cells were fixedwith 4% buffered paraformaldehyde, incu-bated overnight with primary antibody at 4°C,incubated for 1 hour with biotinylated second-ary antibody, and then exposed to an avidin-biotin conjugate of horseradish peroxidase(Vectorstain, Vector Laboratory, Burlingame,

Calif.). Diamino benzidine served as achromagen.

Electrophysiology: Patch Clamp Recordings

Human PLA cells were cultured in neuralinduction media for 14 days on 12-mm glasscoverslips. The coverslips were placed in anacrylic recording chamber, and the chamberwas mounted on the stage of a Zeiss invertedmicroscope. The chamber was continuouslyperfused with solutions at a rate of 1 to 2ml/min. The patch pipettes were made fromthick-walled borosilicate glass on a Flaming/Brown P-87 pipette puller, to resistances of 1.5to 3 mohms. Whole-cell voltage clamp record-ings were made with a patch clamp amplifier(Axopatch 200B, Axon Instruments, FosterCity, Calif.). Voltage-clamp protocols were gen-erated and linear leak currents were subtractedwith J clamp software (www.med.yale.edu/surgery/otolar/santos/jclamp.html). Out-wardly rectifying K� currents were recorded byvoltage-clamping the cell membrane to �70mV, and stepping the test voltages between�70 mV and �10 mV in 10 mV increments for30 msec and returning to the initial holdingpotential.

RESULTS

PLA Cell Characterization

Fluorescent-activated cell sorter analysisdemonstrated that cells were negative forCD11c and CD45, cell surface markers associ-ated with lymphohemopoietic cells. In con-trast, PLA cells, like bone-marrow derived mes-enchymal stem cells, expressed CD29, CD44,and CD90, as described previously.14

Morphologic Changes of Induced PLA Cells

Human PLA were induced to differentiate inculture by incubation with media supple-

TABLE IProtein Markers and Their Neural Cell Specificity

Cell Marker* Specificity

NSE Early neuronalNeuN Early neuronalVimentin Early glialGFAP Mature glial (astrocyte)trk-A Neuronaltau Mature neuronalMAP2 Mature neuronal

* NSE, neuron specific enolase; NeuN, neuron specific nuclear protein;GFAP, glial acidic fibrillary protein; trk-A, nerve growth factor receptor; MAP2,microtubule associated protein-2.

1924 PLASTIC AND RECONSTRUCTIVE SURGERY, May 2003

mented with isobutylmethylxanthine, indo-methacin, and insulin. As early as 3 days ofneural induction, morphologic changes werenoted. Specifically, the PLA cells changed fromflat, elongated, spindle-shaped cells torounded cells with several branching exten-sions and refractile characteristics (Fig. 1).Neuronal induction was conducted for 14 days,during which time the number of cells exhib-iting the neuronal phenotype increased to amaximum of 20 to 25 percent of the total PLApopulation (Fig. 2). This maximal differentia-tion plateau was reached after 9 to 10 days inculture. The branching pattern of individualneuronal-appearing cells also became more ex-tensive during the induction process. Thebranching cells often seemed to contact nearbyundifferentiated spindle-shaped cells. Nonin-duced PLA cells maintained in culture me-dium were used as negative controls. Theseexperiments were repeated in six patients.

Western Blot Analysis

After 14 days of differentiation, protein washarvested from cells for Western blot assays.Human PLA cells maintained in culture me-dium were used as controls. Noninduced PLAcells (controls) expressed several markers char-acteristic of neural cells such as NSE, vimentin,trk-A, and NeuN. Using �-actin as a controlmarker, we observed that cells cultured in neu-ral induction media had an increased expres-sion of NSE, vimentin, and trk-A relative to thenoninduced controls, whereas the expressionof NeuN remained unchanged (Fig. 3). Be-cause NSE and NeuN are early markers forneurons and vimentin is an early marker forglia, our data suggest that human PLA cellsmay have the potential to differentiate in vitrointo early progenitors of neurons and/or glia.However, no expression of the mature astro-cyte marker, GFAP, or the mature neuronalmarkers, MAP2 and tau, was noted in eitherthe induced or noninduced PLA cells (data notshown).

Immunocytochemical Analysis of Cell MarkerExpression

To further characterize neuronal differenti-ation, we fixed neurally induced cultures after2 weeks and stained them for the neuronalmarker NSE. Nondifferentiated control PLAcells expressed low levels of NSE. PLA cells thatexhibited a flat, fibroblast structure stainedlightly for NSE, whereas PLA that exhibited a

FIG. 1. Morphologic changes exhibited by PLA cells follow-ing treatment with isobutylmethylxanthine, indomethacin, andinsulin. (Above) Noninduced PLA cells have an elongated, flat,spindle-shaped structure similar to that of fibroblasts. (Centerand below) As early as 2 days postinduction, cells exhibiting aneural-like structure were observed (arrows). These cells devel-oped characteristic rounded cell bodies with several branchingextensions. Images were taken at 200� magnification.


neural structure stained positively (Fig. 4, aboverow). To investigate neuronal characteristics

further, we stained differentiated cultures forNeuN, a neuron-specific marker expressed inearly postmitotic neurons. A subset of cells withcharacteristic neuronal structure stained posi-tive for NeuN (Fig. 4, second row from above).Immunostaining was also conducted for thebiologically active nerve-growth factor recep-tor, trk-A. After 2 weeks of induction, PLA cellsstained for trk-A, whereas noninduced PLAcells had no trk-A expression (Fig. 4, third rowfrom above). However, induced PLA cells re-vealed no expression of the neuronal markerMAP-2 or the mature glial marker GFAP (Fig.4, second row from below, and below row).

Electrophysiology

After 14 days of differentiation, cells express-ing neuron-like morphologic characteristicsdisplayed voltage-dependent outward currentsthat activated after a brief delay and did notseem to become inactivated during the depo-larization (Fig. 5, above). Moreover, the steady-state current-voltage relation exhibited out-ward rectification (Fig. 5, below) similar to thatobserved for classic delayed-rectifier K� chan-nels of the mammalian node of Ranvier. Noinward currents were observed at this stage ofdifferentiation.

DISCUSSION

It is well established that various adult tissuescontain stem cells, which are capable of regen-erating damage in the tissues in which they

FIG. 2. Plot of neural differentiation as a function of time. PLA cells were treated withisobutylmethylxanthine, indomethacin, and insulin for up to 2 weeks. For quantitative anal-ysis, cells with typical neural morphologic characteristics were counted and calculated as apercentage of total PLA. A plateau level of differentiation, approximately 22 percent, wasreached at about 9 days of treatment. Data represent means � SD.

FIG. 3. Western blot analysis for the expression of NSE,trk-A, NeuN, GFAP, and vimentin in undifferentiated andneurally induced PLA cells. Comparable levels of �-actin weredetected, indicating equal loading of samples. Human brainextract was used as a positive control (lane HB). Lane U rep-resents PLA cells maintained in noninductive control media.Lane I represents PLA cells induced in isobutylmethylxan-thine, indomethacin, and insulin for 2 weeks.


FIG. 4. Immunohistochemical analysis of neurally induced PLA cells. Control PLA and induced PLA were stained withanti-NSE (above), anti-trk-A (second row from above), anti-NeuN (third row from above), anti-MAP2 (second row from below), andanti-GFAP (below). After treatment with isobutylmethylxanthine, indomethacin, and insulin for 2 weeks, increased expressionof NSE, trk-A, and NeuN was noted. However, there was a lack of expression of the neuronal marker, MAP2, and the astrocytesmarker, GFAP. Only cells that exhibited a neural-like structure stained positive; PLA cells with a flattened, spindle-like structuredid not.


reside. However, several recent studies haveshown that adult stem cells may not be as lim-ited by lineage as was once thought and, infact, display profound plasticity.5,11 We havepreviously demonstrated that human PLA cellsisolated from adult adipose tissue contain cellswith the potential to differentiate into variousmesodermal lineages.12,13 In this study, we dem-onstrate that human PLA cells are capable ofdifferentiating into cells that express severalspecific neural proteins and that morphologi-cally resemble immature neurons or glial cells.

There are significant potential benefits to theclinical use of neuronal cells derived fromPLA. PLA cells are readily accessible in largequantities with minimal morbidity, overcomingthe risks of obtaining neural stem cells fromthe subependymal layer of the brain. They alsoprovide a renewable population of cells thatcan be easily expanded in culture medium.

Our induction protocol used isobutylmethyl-xanthine, indomethacin, and insulin. Isobutyl-methylxanthine, a phosphodiesterase inhibi-tor, results in the elevation of intracellular

FIG. 5. Electrophysiologic evaluation of induced PLA cells by whole-cell voltage clamprecordings. (Above) Current responses to a series of voltage commands evoked from aholding potential �70 mV in a patch-clamped induced PLA cell. In response to depolar-izing voltage commands, time-dependent outward currents were observed after a briefdelay. No inward currents were detected. (Below) The current-voltage relationship is plottedfor current values near the end of the depolarization (dotted line) for the cell shown in theabove panel. The relationship indicates that the currents are likely to be mediated byoutwardly rectifying K� channels.


cyclic adenosine monophosphate (a neuralstimulus for various cell types, including mes-enchymal stem cells, prostate carcinoma cells,and glioma cells).10,15,16 Indomethacin, an in-hibitor of cyclooxygenase, has been shown topromote neural cell survival after ischemic in-jury to the central nervous system.17,18 Insulinhas been shown to promote the maturation ofdifferentiating neocortical cells in ratbrains.19,20

Before neural induction, human PLA cellshave a flat, spindle-like structure, similar tothat of fibroblasts. Characterization of humanPLA cells using fluorescent-activated cell sorteranalysis demonstrates that they are negative forthe cell surface markers, CD11c and CD45,indicating that our population does not con-tain hemopoietic precursors. Under neural in-duction conditions, PLA cells take on the mor-phologic characteristics of neural cells, andthese structural changes are accompanied bythe increased expression of neural markersNSE, trk-A, and vimentin. These changes areconsistent with previous reports by Woodburyet al.7 and Deng et al.,10 who used bone-marrowmesenchymal stem cells despite different in-duction conditions. Immunostaining demon-strated increased expression of the neuralmarkers NSE, trk-A, and NeuN relative tocontrols.

Western blot analysis showed that undiffer-entiated PLA cells expressed NeuN, NSE, vi-mentin, and trk-A. The finding that PLA cellsexpressed low levels of NSE is consistent withprevious studies showing low levels of NSE ex-pression in noninduced cells of bone marroworigin.7,10 The level of expression of the neuro-nal markers NSE and trk-A and the early astro-glial marker, vimentin, increased significantlyafter 2 weeks of induction in isobutylmethyl-xanthine/indomethacin/insulin.21 The level ofexpression of NeuN remained unchanged overthis same time period. In a study conducted bySanchez-Ramos et al.,8 nondifferentiated mes-enchymal stem cells expressed NeuN, and thelevel of expression did not increase after neu-ronal induction using retinoic acid and brainderived nerve factor. PLA cell cultures, bothpreneural and postneural induction, failed toexpress the mature astrocyte marker GFAP orthe mature neuronal markers tau or MAP2.The finding that induced PLA cells expressedNeuN and failed to express MAP2 and tau isconsistent with the temporal genesis of variousproteins in a maturing neuron. NeuN is a rel-

atively early marker, being expressed at a pointwhen the cell becomes postmitotic and ini-tiates terminal differentiation.22 On the otherhand, microtubule associated proteins, such asMAP2 and tau, are expressed at a distinctlylater time in neuronal development.23 Ourdata also correlates with the findings of Denget al.10 with regard to mesenchymal stem cells,which showed increased expression of NSEand vimentin after neural induction using iso-butylmethylxanthine/dibutyryl cyclic adeno-sine monophosphate (Table II).

The genesis of specific ion channels is afundamental property of neurons that allowsthem to respond to incoming signals throughreceptor potentials and to transmit this infor-mation to the axon terminals by propagatingaction potentials. The action potential is pri-marily the result of the sequential activation ofvoltage-gated Na� and K� channels, respec-tively. Typically, the expression of K� channelsprecedes that of Na� (and Ca2�) channels dur-ing neuronal development.24–26 Recent studieshave demonstrated voltage-dependent rectifi-cation of K� currents in neuronally differenti-ated bone marrow stromal cells using 5-azacy-tidine.27 We report here that induced humanPLA cells clearly exhibited a similar delayedrectifier K� current, indicating the presence ofvoltage-dependent K� channels and correlat-ing to the temporal development of specificion channels in maturing neurons.

TABLE IINeural Differentiation

(PLA versus Mesenchymal Stem Cells)*

Cell TypeInduction

Media

Human MSC Human PLA Cells(IBMX/Indo-

methacin/Insulin)�-Mercaptoethanol† IBMX/dcAMP‡

NSE � � �NeuN � �NF-M � 0trk-A � �tau � 0 0GFAP 0 0 0MAP2 0 0Vimentin � �

IBMX, isobutylmethylxanthine; dcAMP, dibutyrylcyclic adenosine mono-phosphate; NF-M, neurofilament-M; 0, no staining detected in either the non-induced or neurally induced groups; �, expression detected in noninducedPLA and/or increased expression after induction.

* For definition of other abbreviations, see Table I.† Data from Woodbury, D., Schwarz, E. J., Prockop, D. J., and Black,

I. B. Adult rat and human bone marrow stromal cells differentiate into neu-rons. J. Neurosci. Res. 61: 364, 2000. Immunohistochemistry and/or Westernblots were used for the analysis.

‡ Data from Deng, W., Obrocka, M., Fischer, I., and Prockop, D. J. In vitrodifferentiation of human marrow stromal cells into early progenitors of neuralcells by conditions that increase intracellular cyclic AMP. Biochem. Biophys. Res.Commun. 282: 148, 2001. Immunohistochemistry and/or Western blots wereused for the analysis.


In summary, after induction with isobutyl-methylxanthine, indomethacin, and insulin,human PLA cells differentiated into early neu-ronal and/or glial progenitors. There was noexpression of mature neuronal or glial mark-ers. However, the fact that human PLA cellsexpress NeuN, in addition to the increasedexpression of several early neuronal and glialmarkers, is not proof that these cells will ulti-mately differentiate into mature neurons thatare capable of undertaking complex electro-physiologic and synaptic functions. Furtherstudies are needed to evaluate various in vitroculture conditions (i.e., co-culture and growthfactors) and in vivo models (i.e., central ner-vous system injury model) to examine if PLAcells will eventually form mature neuronsand/or glial cells and participate in centralnervous system integration and repair. Still,these findings suggest that the adipose com-partment is a source of cells that are potentiallyuseful for the treatment of neurologic diseases.

Marc H. Hedrick, M.D.Division of Plastic and Reconstructive SurgeryUCLA School of Medicine64-140 Center for Health Sciences10833 Le Conte AvenueLos Angeles, Calif. [email protected]

ACKNOWLEDGMENTS

The study was funded by the American Society for Aes-thetic Plastic Surgery, Plastic Surgery Educational Founda-tion, Orthopedic Hospital Foundation, Severin WundermanFamily Foundation, and the American College of Surgeons.We are grateful to Dr. Harry Vinters for providing humanbrain tissue (University of California, Los Angeles, Alzhei-mer’s Disease Research Center Brain Bank, funded by PHS50AG16570). We also appreciate Drs. John Fraser, Zeni Alfonso,and Felix Schweizer for their assistance in flow cytometry andelectrophysiology. Special thanks to Dr. Wuyi Kong for hercontinued guidance in the planning and execution of theseexperiments.

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in vitro differentiation of human processed lipoaspirate cells into early neural progenitors

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