Clonogenic Analysis Reveals ReserveStem Cells in Postnatal Mammals.

II. Pluripotent Epiblastic-LikeStem Cells

HENRY E. YOUNG,1,2* CECILE DUPLAA,6 MICHAEL J. YOST,7

NICHOLAS L. HENSON,1 JULIE A. FLOYD,1 KRISTINA DETMER,1

ANGELA J. THOMPSON,1 STEVEN W. POWELL,1 T. CLARK GAMBLIN,5

KIRK KIZZIAH,1 BENJAMIN J. HOLLAND,1 ANGEL BOEV,1

J.M. VAN DE WATER,5 DAN C. GODBEE,8 STEPHANIE JACKSON,9

MARYLEN RIMANDO,10 CHAD R. EDWARDS,1 EVELINE WU,1

CHRIS CAWLEY,1 PAMELA D. EDWARDS,1 ANNA MACGREGOR,1

RYAN BOZOF,1 T. MICHELE THOMPSON,11 GEORGE J. PETRO JR.,1

HEATHER M. SHELTON,1 BETH L. MCCAMPBELL,5 JARED C. MILLS,1

FREDERICK L. FLYNT,1 TIMOTHY A. STEELE,12 MARIANNE KEARNEY,13

AMY KIRINCICH-GREATHEAD,7 WADE HARDY,1 PAUL R. YOUNG,1

AMAN V. AMIN,1 R. STEVE WILLIAMS,2 MIRANDA M. HORTON,1

SHAUN MCGUINN,1 KRISTINA C. HAWKINS,1 KURT ERICSON,7

LOUIS TERRACIO,14 CATHERINE MOREAU,6 DOUGLAS HIXSON,15

BRIAN W. TOBIN,1,2 JOHN HUDSON,4 FRANK P. BOWYER III,2

AND ASA C. BLACK JR.1,3

1Division of Basic Medical Sciences, Mercer University School of Medicine,Macon, Georgia

2Department of Pediatrics, Mercer University School of Medicine, Macon, Georgia3Department of Obstetrics and Gynecology, Mercer University School of Medicine,

Macon, Georgia4Department of Internal Medicine, Mercer University School of Medicine,

Macon, Georgia5Department of Surgery, Mercer University School of Medicine, Macon, Georgia

6INSERM U441, Pessac, France7Department of Surgery, University of South Carolina School of Medicine,

Columbia, South Carolina8Department of Emergency Medicine, LSU-Medical Center, Earl K. Long,

Baton Rouge, Louisiana9Department of Biomedical Engineering, Mercer University, Macon, Georgia

10Department of Biology, Mercer University, Macon, Georgia11Department of Family Medicine, Mountain Area Health Education Center,

Asheville, North Carolina12Des Moines University—Osteopathic Medical Center, Des Moines, Iowa

13Division of Vascular Medicine, St. Elizabeth’s Medical Center,Boston, Massachusetts

14New York University College of Dentistry, New York, New York15Department of Medicine, Brown University, Providence, Rhode Island

Grant sponsor: NIH; Grant numbers: K25-HL67097,HL072096; Grant sponsor: NASA; Grant number: CooperativeAgreement NCC5-575; Grant sponsor: Rubye Ryle Smith Chari-table Trust; Grant sponsor: MedCen Community Health Founda-tion; Grant sponsor: MorphoGen Pharmaceuticals, Inc.; Grantsponsor: Lucille M. and Henry O. Young Estate Trust; Grantsponsor: University of South Carolina Research and ProductiveScholarship Program

*Correspondence to: Henry E. Young, Ph.D., Division of BasicMedical Sciences, Mercer University School of Medicine, 1550

College St., Macon, GA 31207. Fax: 478-301-5489.E-mail: young_he@mercer.edu

Received 16 July 2003; Accepted 14 November 2003DOI 10.1002/ar.a.20000

THE ANATOMICAL RECORD PART A 277A:178–203 (2004)

© 2004 WILEY-LISS, INC.

ABSTRACTUndifferentiated cells have been identified in the prenatal blastocyst, inner cell mass,

and gonadal ridges of rodents and primates, including humans. After isolation these cellsexpress molecular and immunological markers for embryonic cells, capabilities for extendedself-renewal, and telomerase activity. When allowed to differentiate, embryonic stem cellsexpress phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin.When implanted in vivo, undifferentiated noninduced embryonic stem cells formed terato-mas. In this report we describe a cell clone isolated from postnatal rat skeletal muscle andderived by repetitive single-cell clonogenic analysis. In the undifferentiated state it consistsof very small cells having a high ratio of nucleus to cytoplasm. The clone expresses molecularand immunological markers for embryonic stem cells. It exhibits telomerase activity, whichis consistent with its extended capability for self-renewal. When induced to differentiate, itexpressed phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin.The clone was designated as a postnatal pluripotent epiblastic-like stem cell (PPELSC). Theundifferentiated clone was transfected with a genomic marker and assayed for alterations instem cell characteristics. No alterations were noted. The labeled clone, when implanted intoheart after injury, incorporated into myocardial tissues undergoing repair. The labeled clonewas subjected to directed lineage induction in vitro, resulting in the formation of islet-likestructures (ILSs) that secreted insulin in response to a glucose challenge. This study suggeststhat embryonic-like stem cells are retained within postnatal mammals and have the potentialfor use in gene therapy and tissue engineering. Anat Rec Part A 277A:178–203, 2004.© 2004 Wiley-Liss, Inc.

Key words: pluripotent stem cells; gene therapy; myocardial infarction;diabetes

Embryonic stem cells are undifferentiated precursorcells. They have been isolated from the blastocyst, innercell mass, and gonadal ridges of rodents and primates,including humans (Evans and Kaufman, 1981; Martin,1981; Thomson et al., 1995, 1998; Shamblott et al., 1998;Pera et al., 2000). After isolation and growth in vitro withinhibitory agents (i.e., leukemia inhibitory factor, ESGRO,and/or fibroblast feeder layers), these cells exhibit immu-nological and molecular markers for undifferentiated em-bryonic cells (Niwa et al., 2000; Pera et al., 2000; Pesceand Scholer, 2001; Henderson et al., 2002; Cheng et al.,2003). They exhibit telomerase activity, which is consis-tent with their extended capability for self-renewal (Liu,2000; Pera et al., 2000; Lin et al., 2003). When releasedfrom inhibitory control in vitro, these cells will spontane-ously differentiate into and exhibit phenotypic expressionmarkers for cells of ectodermal, mesodermal, and endoder-mal origin (Thomson et al., 1995, 1998; Shamblott et al.,1998; Pera et al., 2000). Thus, embryonic stem cells ex-hibit pluripotentiality, i.e., the ability of a single cell toform multiple types of tissue from all three primary germlayer lineages. Based on the unique qualities of extendedcapability for self-renewal and pluripotentiality, embry-onic stem cells have been proposed as a source of donorcells for tissue transplantation (Thomson et al., 1995,1998; Shamblott et al., 1998; Assady et al., 2001; Lumel-sky et al., 2001). Unfortunately, transplantation of undif-ferentiated embryonic stem cells in vivo has resulted thusfar in the formation of teratomas (Thomson et al., 1995,1998; Shamblott et al., 1998; Pera et al., 2000).

Recently, undifferentiated stem cells with characteris-tics similar to embryonic stem cells have been isolatedfrom postnatal mammals, including newborn to geriatric

humans. These postnatal adult stem cells have been iso-lated from brain (Jiang et al., 2002a), bone marrow (Jianget al., 2002a, 2002b; Reyes et al., 2002; Schwartz et al.,2002), blood (Zhao et al., 2003), skeletal muscle (Jiang etal., 2002a; Young, 2004; Young and Black, 2004; Young etal., 2004), and dermis (Young, 2004; Young and Black,2004; Young et al., 2004). The particular characteristicsreported for the undifferentiated adult stem cells isolatedby Young et al. included small size with a high ratio ofnucleus to cytoplasm, quiescence in serum-free definedmedium lacking inhibitory factors, extended capabilitiesfor self-renewal, expression of telomerase activity, expres-sion of embryonic markers in the undifferentiated state,and ability to form cells from all three primary germ layerlineages in vitro when treated with general and specificlineage-induction agents. Based on these characteristicsYoung et al. designated their adult undifferentiated stemcell as a pluripotent epiblastic-like stem cell (PPELSC).Young et al. (1999, 2004) proposed potential advantagesfor using adult-derived pluripotent stem cells instead ofembryonic stem cells for gene therapy and tissue engi-neering.

The current study aimed to determine if adult-derivedPPELSCs could be used for gene therapy and tissue engi-neering as proposed by Young et al. First, a pure popula-tion of adult-derived undifferentiated cells was generatedby repetitive single-cell clonogenic analysis. Second, theclone was examined for stem cell characteristics, includingpluripotentiality. Third, the clone was transfected with agenomic sequence and then reexamined in vitro for alter-ations in stem cell characteristics. Fourth, the labeledclone was implanted into the heart following injury andassessed for incorporation into tissues undergoing repair.

179ADULT PLURIPOTENT EPIBLASTIC-LIKE STEM CELLS

And fifth, the labeled clone was induced to form a three-dimensional entity composed of multiple differentiatedcells. This entity was then assayed for biological function.

MATERIALS AND METHODSThe use of animals in this study complied with the

guidelines of Mercer University, the University of SouthCarolina, and the National Research Council’s criteria forhumane animal care as outlined in “Guide for the Careand Use of Laboratory Animals,” prepared by the Instituteof Laboratory Animal Resources and published by theNational Institutes of Health (National Academy Press,1996).

Cell Harvest and CulturePostnatal Sprague-Dawley rats were euthanized using

CO2 inhalation, and the fleshy muscle bellies of the thighand leg musculature were processed for stem cell isola-tion, cultivation, and cryopreservation (Young et al., 1991,1995, 1998a, 2001a, 2004; Young, 2000, 2004).

Repetitive Single-Cell Clonogenic Analysis

Preconditioned medium. Previous cloning studieswith prenatal chicks (Young et al., 1993), prenatal mice(Rogers et al., 1995; Young et al., 1998a), and postnatalrats (Young et al., 2001a) revealed that repetitive single-cell clonogenic analysis could be achieved if individualcells were grown in medium preconditioned by highly pro-liferating cells of the same parental line. Therefore, theculture medium from stem cells undergoing postconflu-ence log-phase expansion was pooled, processed, andstored at –70 to 80°C. A 1:1 ratio of preconditioned me-dium and complete medium (CM) was used as the cloningmedium.

Cultivation past 50 population doublings. Previ-ous cloning studies in prenatal mice (Rogers et al., 1995;Young et al., 1998b) and postnatal rats (Young et al.,2001a) revealed that a higher efficacy of cloning could beachieved if cells were cultivated past 50 population dou-blings prior to cloning. When such cells were incubatedwith insulin, less than 1% of the cells displayed pheno-typic markers for differentiated cells of the various meso-dermal tissue lineages (Young, 2000, 2004). Cells werepropagated a minimum of 50 population doublings, ali-quoted, and cryopreserved.

Cloning. Frozen cells were thawed, grown past conflu-ence, harvested, and cell viability was determined. Theywere then diluted to clonal density (one cell per 5 �l) withcloning medium and plated as described (Young et al.,1998b, 2001a, 2004). The process of seeding at single-cellclonal density, propagation past confluence, culture selec-tion (i.e., retention of a stellate morphology, loss of contactinhibition, and growth past confluence), harvest, propaga-tion past confluence in six-well plates, culture selection,harvest, and cryopreservation was repeated two addi-tional times after initial cloning to ensure that each clonewas derived from a single cell. The resultant clones werepropagated, harvested, and cryopreserved (Young et al.,1993, 1998b, 2001a; Rogers et al., 1995). Each round ofcloning resulted in approximately 20 population dou-blings. Thus, three rounds of single-cell clonogenic analy-sis resulted in approximately 60 population doublings in

the resultant clones. One of the clones reported herein wasdesignated as Rat-A2B2 and had accrued a minimum of130 population doublings after initial harvest.

Capability for Extended Self-Renewal

Starting at 130 population doublings, clone Rat-A2B2was thawed and plated at 5 � 104 cells per gelatinizedT-25 flask. Cells were propagated past confluence (5–7days) and harvested. Cell numbers ranged from 5 to 6.5 �106 cells per flask, or 6–7 cell doublings per passage.Overall doubling time averaged 16–24 hr. However, thenormal growth curve for these cells consisted of twophases, a protracted 1- to 3-day lag phase and an expo-nential 3- to 5-day growth phase (Young et al., 1991).Actual doubling time during the exponential postconfluentgrowth phase approximated 12–14 hr. This was in con-trast to an 18- to 24-hr exponential preconfluent growthphase for either pluripotent mesenchymal stem cells orgerm layer lineage mesodermal stem cells that becomecontact inhibited at confluence (Young et al., 2001a,2001b, 2004). Cells were aliquoted at 106–107 cells/ml andcryopreserved. The procedure of propagation past conflu-ence, harvest, and cryopreservation was repeated througha minimum of 124 population doublings after cloning. Thelatter number combined with the starting number of 130population doublings resulted in a clone of cells that hadundergone a minimum of 254 population doublings. Atevery other passage interval from 130–254 populationdoublings, cell aliquots were removed, incubated in ourstandard insulin-dexamethasone bioassay for up to 56days, and examined morphologically, histochemically, andimmunochemically to denote any changes in phenotypicexpression within the clone.

Insulin-Dexamethasone Bioassay

The identity of specific types of progenitor and pluripo-tent cells within an unknown population of cells can beascertained by comparing the effects of treatment with aprogression factor and a general nonspecific lineage-in-duction agent (Young et al., 1992a, 1992b, 1993, 1995,1998a, 1998b, 1999, 2001a, 2001b, 2004; Lucas et al.,1993, 1995; Pate et al., 1993; Rogers et al., 1995; Warejckaet al., 1996; Young, 2000, 2004; Young and Black, 2004).Progression factors, such as insulin (at 2–5 �g/ml), accel-erate phenotypic expression in progenitor cells but haveno effect on the induction of phenotypic expression inpluripotent stem cells. By contrast, lineage-inductionagents, such as dexamethasone (at 10–10 to 10–6 M), in-duce lineage commitment and expression in pluripotentcells, but do not alter phenotypic expression in progenitorcells. Therefore, if progenitor cells alone are present in theculture, there will be no difference in either the quality orquantity of expressed phenotypes for cultures incubatedin insulin compared with those incubated with dexameth-asone. If the culture is mixed, containing both progenitorand pluripotent cells, then there will be a greater qualityand/or quantity of expressed phenotypes in culturestreated with dexamethasone than in those treated withinsulin. If the culture contains pluripotent cells alone,there will be no expressed phenotypes in cultures treatedwith insulin. Similar cultures treated with dexametha-sone will exhibit multiple expressed phenotypes.

180 YOUNG ET AL.

Phenotypic ExpressionCell types belonging to embryonic, ectodermal, mesoder-

mal, and endodermal lineages were assayed using previ-ously established morphological, histochemical, and im-munochemical procedures to denote changes inphenotypic expression markers (Young et al., 1991, 1992a,1992b, 1993, 1995, 1998a, 1998b, 1999, 2001a, 2001b;Young, 2000, 2004; Young and Black, 2004) (Table 1).

Nuclear Expressing LacZ TransfectionRat-A2B2 clone at 254 population doublings was grown on

gelatinized dishes in Eagle’s minimal essential medium(MEM) (GIBCO-BRL, Life Technologies, Cergy Pontoise,France), with 10% horse serum (Gibco-BRL), 5 mMN-2-Hydroxyethylpiperazine-N�-2-Ethane Sulfonic Acid(HEPES) (GIBCO-BRL), 50 U/ml penicillin–50 mg/ml strep-tomycin (GIBCO-BRL), and 500 U/ml recombinant humanleukemia inhibitory factor (TEBU, le Perray-en-Yvelines,France). Stable rat clonal cell lines expressing nuclear tar-geted LacZ gene (nls-LacZ) were constructed using the plas-mid pUT651 (selectable reported gene Sh ble::lacZ). Cellswere plated at 5 � 103 cells/cm2 on six-well plastic dishes(Falcon) (Becton Dickinson, Le pont-de claix, France) in se-rum-containing medium and allowed to attach overnight.The cells were then incubated overnight with 2 mg ofpUT651 using lipofectin reagent (Gibco-BRL) during 16 hr inserum-free medium (Opti-MEM, GIBCO-BRL). Transfectedcells were split 1:10 into the selection medium supplementedwith 250 mg of zeocin (Invitrogen, Netherlands). One cloneamong 12 resistant clones expressing the highest level of�-galactosidase, Rat-A2B2-Scl-40-�-galactosidase (Scl-40�),was subcloned and used for this study. �-Galactosidase ex-pression was evaluated by two techniques. After fixation in2% paraformaldehyde for 10 min at room temperature, andrinsing in phosphate-buffered saline (PBS), LacZ expressionwas evaluated by histochemical staining with the chromo-genic substrate X-Gal and by immunostaining with the poly-clonal (Chemicon, Temecula, CA) anti-�-Gal antibody(Couffinhal et al., 1997).

Telomerase AssayScl-40� clone at a minimum of 254 cell doublings was

assayed for telomerase activity. Cells were thawed, platedat 5 � 105 cells per gelatinized T-25 flask, and grown pastconfluence. Cells were harvested (Young et al., 2001a) andprocessed for telomerase activity as described by the man-ufacturer (TRAPeze Assay, Intergen).

Oct-4 Gene ExpressionOct-4 gene expression was detected by the electro-

phoretic mobility shift assay using the oligonucleotide 5�-TGTCGAATGCAAATCACTAGA-3� containing the Oct-1consensus binding site. Scl-40� clone at a minimum of 287population doublings was utilized. Cells were thawed,plated at 5 � 105 cells per gelatinized T-25 flask in stemcell propagation medium (SCPM), and grown past conflu-ence. SCPM consisted of 89% (v/v) Opti-MEM, 0.01 mM�mercaptoethanol (�ME), 1% (v/v) antibiotic-antimycoticsolution (10,000 units/ml penicillin, 10,000 �g/ml strepto-mycin, 25 �g/ml Amphotericin-B, GIBCO) (1% ab-am),and 10% SS3, at pH 7.4 (Young et al., 2004). Ten percentSS3 contained proliferative activity resembling that ofplatelet-derived growth factor (PDGF) and inductive/dif-

ferentiation-inhibitory activity resembling that of antidif-ferentiation factor (ADF) (Young, 2000, 2004; Young et al.,1998a, 2004). Cells were harvested and processed forwhole-cell extracts as previously described (Detn andLatchman, 1993). Cell aliquots (5,000 cell equivalents)were incubated for 30 min at room temperature. 32P-labeled Oct-1 oligonucleotide (1 ng) was added and themixture incubated for 30 min at room temperature beforeelectrophoresis through a 5% polyacrylamide gel. Afterdrying, bands were visualized with a phosphorimager andquantified using the accompanying software.

Phenotypic Bioassay of Scl-40�

Scl-40� clone was plated into gelatinized 96-well platesat 103 cells per well in CM and allowed to attach for 24 hr(Young et al., 2001a, 2001b). CM consisted of 89% (v/v)Opti-MEM-based medium (catalog no. 22600-050, GIBCO)containing 0.01 mM �ME (Sigma, St. Louis, MO), 1%ab-am, and 15% (v/v) SS12 (MPI, MorphoGen Pharmaceu-ticals, Inc., San Diego, CA), at pH 7.4 (Young et al.,2001b). The CM was then removed and replaced withtesting medium (TM) for 24 hr to wash out any potentialsynergistic components in the CM. TM consisted of CMwithout SS12. Then the TM was changed to one of thefollowing to determine the identity of the clone. For con-trols, TM alone was used. To identify potential lineage-committed progenitor cells, 2 �g/ml insulin (Sigma) wasadded to the TM. To identify potential pluripotent stemcells, 10–10 to 10–6 M dexamethasone (Sigma) was addedto the TM. To further identify pluripotent stem cells,1–15% selected sera (SS) shown to contain one or morebioactive factor activities (Young et al., 1998a, 1998b,2001a, 2004; Young, 2000, 2004) were added to TM con-taining 2 �g/ml insulin and 10–6 M dexamethasone. Thesera used were SS7 (17F-0218, Sigma), SS9 (90H-0701,Sigma), SS10 (MPI), and SS12 (MPI). SS7 and SS10 at10% contain PDGF-like (proliferative) ADF-like (induc-tive/differentiation-inhibitory) activities; SS12 at 15% (pH7.4) contains PDGF-like (proliferative) and leukemia-in-hibitory factor-like (inductive-inhibitory) activities; 10, 5,3, and 1% SS9 contains skeletal muscle morphogeneticprotein (Sk-MMP)-like, adipocyte morphogenetic protein(AMP)-like, bone morphogenetic protein-2 (BMP-2)-like,and endothelial inductive activities; 5, 3, and 1% SS12 (pH7.4) contains ectodermal inductive activities; 10 and 15%SS12 (pH 7.6) contains endodermal lineage-inductive ac-tivity; and 15% SS12 (pH 7.2) contains pancreatic progen-itor cell (PanPC)-inductive activity (Young et al., 2004).Control and experimental cultures were propagated for anadditional 7–56 days with medium changes every otherday. Three to 96 culture wells were used per concentrationper experiment. During the 7- to 56-day time period, thecultures were examined daily by subjective analysis andcorrelated with days of treatment and concentrations ofexogenous agents utilized.

The above experiments were then repeated utilizingthese parameters to confirm objectively the presence ofvarious established markers for phenotypic expression.Cultures were stained with an antibody to �-galactosidaseto identify nuclear-expressing LacZ-transfected cells(Couffinhal et al., 1997). Cultures were then processed perthe manufacturer’s directions or as described (Young etal., 1992b, 2001a, 2004; Young and Black, 2004) to identifycytoplasmic, cell surface, pericellular, or extracellular cell-specific phenotypic expression markers. The cells were

181ADULT PLURIPOTENT EPIBLASTIC-LIKE STEM CELLS

TABLE 1. Induction of phenotypic expression in postnatal precursor cell lines

Phenotypic markers ELSCs1 EctoSCs2 MesoSCs3 EndoSCs4 PanPCs5 ILS6

EmbryonicSSEA-17 � – – – – –SSEA-38 � – – – – –SSEA-49 � – – – – –CEA10 � – – – – –HCEA11 � – – – – –CD66e12 � – – – – –CEA-CAM-113 � – – – – –Oct-414 � nd15 nd nd nd ndTelomerase � nd � nd nd nd

EctodermNeuronal progenitor cells16 � � – – – –Neurons17 � � – – – –Ganglia18 � � – – – –Oligodendrocytes19 � � – – – –Astrocytes20 � � – – – –Synaptic vesicles21 � � – – – –Radial glial cells22 � � – – – –Keratinocytes23 � � – – – –

MesodermSkeletal muscle24 � – � – – –Smooth muscle25 � – � – – –Cardiac muscle26 � – � – – –White fat27 � – � – – –Brown fat28 � – � – – –Hyaline cartilage29 � – � – – –Articular cartilage30 � – � – – –Elastic cartilage31 � – � – – –Growth plate Cartilage32 � – � – – –Fibrocartilage33 � – � – – –Endochondral bone34 � – � – – –Intramembranous bone35 � – � – – –Tendon/Ligament36 � – � – – –Dermis37 � – � – – –Scar tissue38 � – � – – –Endothelial cells39 � – � – – –Hematopoietic cells40 � – � – – –

EndodermEndodermal progenitor cells41 � – – � – –GI epithelium42 � – – � – –Liver oval cells43 � – – � – –Liver hepatocytes44 � – – � – –Liver biliary cells45 � – – � – –Liver canalicular cells46 � – – � – –Pancreatic progenitor cells47 � – – � � –Pancreas ductal cells48 � – – � � �Pancreatic �-cells49 � – – � � �Pancreatic �-cells50 � – – � � �Pancreatic �-cells51 � – – � � �

1ELSCs, pluripotent epiblastic-like stem cells (isolated and cloned) (Young, 2004; Young and Black, 2004; Young et al., 2004;this study).2EctoSCs, germ layer lineage ectodermal stem cells (induced) (Romero-Ramos et al., 2002; Young, 2004; Young and Black,2004; Young et al., 2004).3MesoSCs, germ layer lineage mesodermal (mesenchymal) stem cells (isolated and cloned) (Young et al., 1999, 2001a,b; Young,2000, 2004).4EndoSCs, germ layer lineage endodermal stem cells (induced) (Young, 2004; Young and Black, 2004; Young et al., 2004; thisstudy).5PanPCs, pancreatic progenitor cells induced from germ layer lineage endodermal stem cells (this study).6ILS, islet-like structures induced from pancreatic progenitor stem cells (this study).Embryonic cells were identified as follows:7SSEA-1, stage-specific embryonic antigen-1, MC480, Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA (Solterand Knowles, 1978).8SSEA-3, stage-specific embryonic antigen-3, antibody MC631 (DSHB) (Damjanov et al., 1982).9SSEA-4, stage-specific embryonic antigen-4, antibody MC-813-70 (DHSB) (Lannagi et al., 1983).10CEA, carcinoembryonic antigen, (Hixson, Providence, RI) (Estrera et al., 1999).11HCEA, human carcinoembryonic antigen (Sigma) (Young et al., 2004).12CD66e, carcinoembryonic antigen (Vector, Burlingame, CA) (Kishimoto et al., 1997).13CEA-CAM1, carcinoembryonic antigen-cell adhesion molecule (Hixson) (Estrera et al., 1999).

182 YOUNG ET AL.

14Oct-4, a gene directly involved in the capacity for self-renewal and pluripotency of mammalian embryonic stem cells (Pesceand Scholer, 2001).15nd, not as yet determined.Ectodermal lineage cells were identified as follows:16Neuronal progenitor cells were identified using FORSE-1 (DSHB) for neural precursor cells (Tole et al., 1995; Tole andPatterson, 1995), RAT-401 (DSHB) for nestin (Hockfield and McKay, 1985), HNES (Chemicon, Temecula, CA) for nestin(Young et al., 2004), and MAB353 (Chemicon) for nestin (Gritti et al., 1996).17Neurons were identified using 8A2 (DSHB) for neurons (Drazba et al., 1991), S-100 (Sigma) for neurons (Baudier et al., 1986;Barwick, 1990), T8660 (Sigma) for beta-tubulin III (Banerjee et al., 1988, 1990; Joshi and Cleveland, 1990), RT-97 (DSHB) forneurofilaments (Wood and Anderton, 1981), N-200 (Sigma) for neurofilament-200 (Debus et al., 1983; Franke, et al., 1991), andSV2 (DSHB) for synaptic vesicles (Feany et al., 1992).18Ganglia were identified using TuAg1 (Hixson) for ganglion cells (Faris et al., 1990; Hixson et al., 1990).19Oligodendrocytes were identified using Rip (DSHB) for oligodendrocytes (Friedman et al., 1989) and CNPase (Sigma) foroligodendrocytes and astroglia (Sprinkle et al., 1987; Sprinkle, 1989; Reynolds et al., 1989).20Astrocytes were identified using CNPase (Sigma) for astroglia and oligodendrocytes (Sprinkle et al., 1987; Sprinkle, 1989;Reynolds et al., 1989).21Synaptic Vesicles were identified using SV2 (DSHB) for synaptic vesicles (Feany et al., 1992).22Radial Glial Cells, were identified using 40E-C (DSHB) for radial glial cells (Alvarez-Buylla et al., 1987).23Keratinocytes were identified using VM-1 (DSHB) to keratinocyte cell surface protein (Oseroff et al., 1985; Morhenn, 2002).Mesodermal lineage cells were identified as follows:24Skeletal muscle was identified as mononucleated myoblasts staining with OP137 (Calbiochem, San Diego, CA) for MyoD(Thulasi et al., 1996), F5D (DSHB) for myogenin (Wright et al., 1991), and DEU-10 (Sigma) for desmin (Debus et al., 1983),and as multinucleated spontaneously contracting structures staining with MF-20 (DSHB) for sarcomeric myosin (Bader et al.,1982), MY-32 (Sigma) for skeletal muscle fast myosin (Naumann and Pette, 1994), ALD-58 (DSHB) for myosin heavy chain(Shafiq et al., 1984), and A4.74 (DSHB) for myosin fast chain (Webster et al., 1988).25Smooth muscle was identified as mononucleated cells staining with antibodies IA4 (Sigma) for smooth muscle alpha-actin(Skalli et al., 1986) and Calp (Sigma) for calponin (Frid et al., 1992; Lazard et al., 1993).26Cardiac muscle was identified as binucleated cells co-staining with MF-20 (DSHB) � IA4 (Sigma) for sarcomeric myosin andsmooth muscle alpha actin (Eisenberg and Markwald, 1997; Eisenberg et al., 1997), MAB3252 (Chemicon) for cardiotin(Schaart et al., 1997) and MAB1548 for cardiac muscle (Chemicon).27White fat, also denoted as unilocular adipose tissue, was identified as a mononucleated cell with a peripherally-locatednucleus and containing a large central intracellular vacuole filled with refractile lipid and stained histochemically forsaturated neutral lipid using Oil Red-O (Sigma) and Sudan Black-B (Chroma-Gesellschaft, Roboz Surgical Co., Washington,DC) (Young et al., 2001a).28Brown fat, also denoted as multi-locular adipose tissue, was identified as a mononucleated cell with a centrally-locatednucleus containing multiple small intracellular vacuoles filled with refractile lipid and stained histochemically for saturatedneutral lipid using Oil Red-O (Sigma) and Sudan Black-B (Chroma-Gesellschaft) (Young, 2000; Young et al., 2001b).29–33Cartilage: structures thought to be cartilage nodules were tentatively identified as aggregates of rounded cells containingpericellular matrix halos. Cartilage nodules were confirmed by both histochemical and immunochemical staining. Histochem-ically, cartilage nodules were visualized by staining the pericellular matrix halos for proteoglycans containing glycosamino-glycan side chains with chondroitin sulfate and keratan sulfate moieties. This was accomplished using Alcian Blue (AlcianBlau 8GS, Chroma-Gesellschaft), Safranin-O (Chroma-Gesellschaft) at pH 1.0, and Perfix/Alcec Blue. Verification of glycos-aminoglycans specific for cartilage was confirmed by loss of extracellular matrix staining following digestion of the materialwith chondroitinase-AC (ICN Biomedicals, Cleveland, OH) and keratanase (ICN Biomedicals) (Young et al., 1989a,b, 2001a,b)prior to staining (negative staining control). Immunochemically, the chondrogenic phenotype was confirmed by initialintracellular staining followed by subsequent staining of the pericellular and extracellular matrices with CIIC1 (DSHB) fortype-II collagen (Holmdahl et al., 1986), HC-II (ICN Biomedicals, Aurora, OH) for type-II collagen (Burgeson and Hollister,1979; Kumagai et al., 1994), D1-9 (DSHB) for type-IX collagen (Ye et al., 1991), 9/30/8A4 (DSHB) for link protein (Catersonet al., 1985), 12/21/1C6 (DSHB) for proteoglycan-hyaluronate binding region (Caterson, 2001), and 12C5 (DSHB) for versican(Asher et al., 1995). Types of cartilage were segregated based on additional attributes.29Hyaline cartilage was identified by a perichondrial-like connective tissue surrounding the above stained cartilage nodule andhistochemical co-staining for type-I collagen (Young et al., 1989c).30Articular cartilage was identified as the above stained cartilage nodule without a perichondrial-like connective tissuecovering (Young et al., 1993).31Elastic cartilage was identified by nodular staining for elastin fibers and a perichondrial-like connective tissue surroundingthe above stained cartilage nodule and histochemical co-staining for type-I collagen (Young et al., 1989c).32Growth plate cartilage was identified by nodular staining for cartilage phenotypic markers (see above) and co-staining forcalcium phosphate using the von Kossa procedure (Young et al., 1999, 2001a,b).33Fibrocartilage was identified as three-dimensional nodules demonstrating extracellular histochemical staining for type-Icollagen (Young et al., 1989c) and co-staining for pericellular matrices rich in chondroitin sulfates A and C. The latter wereassessed by Alcian Blue pH 1.0 staining. Negative staining controls were digested prior to staining with chondroitinase-ABCor chondroitinase-AC (Young et al., 1989a,b, 2001a,b).34Endochondral bone was identified as the formation of a three-dimensional structure with progressional staining from onedisplaying chondrogenic phenotypic markers, i.e., pericellular type-II collagen, type-IX collage, chondroitin sulfate/keratansulfate glycosaminoglycans (see above) to three-dimensional nodules displaying osteogenic phenotypic markers, i.e.,WV1D1(9C5) (DSHB) for bone sialoprotein II (Kasugai et al., 1992), MPIII (DSHB) for osteopontine (Gorski et al., 1990), andthe von Kossa procedure (Silber Protein, Chroma-Gesellschaft) for calcium phosphate. In the von Kossa procedure, negativestaining controls were pre-incubated in EGTA, a specific chelator for calcium (Sigma) (Young et al., 1993, 1999, 2001a,b).

183ADULT PLURIPOTENT EPIBLASTIC-LIKE STEM CELLS

photographed using a Nikon TMS inverted-phase-con-trast/brightfield microscope coupled with a Nikon 995Coolpix digital camera for brightfield microscopy, exceptwhere noted. Photographs (�200 magnification) weretaken per antibody/stain, printed, coded for a double-blindstudy, and scored for number of cytoplasm-stained cellsvs. total number of nuclear-stained cells, to quantify in-duced expression. Each sample set of 43 photographs (n �29) was scored independently by a separate individual.Scores were tabulated and means standard error of themean (SEM) were determined. Means were analyzed byone-way analysis of variance (P 0.05) using the AB-STAT computer program (Anderson-Bell Corp., Arvada,CO).

Myocardial Infarction and Repair

Cell culture. Scl-40� was grown as described above.Once confluent, the cells were harvested and suspended in

Dulbecco’s PBS (DPBS) for injection at a concentration of2 � 106 cells/ml.

Normal rat heart model. Two hundred- to 300-gramSprague-Dawley male rats were anesthetized using xyla-zine (7 mg/kg IP), ketamine (50 mg/kg IP), and aceproma-zine (1 mg/kg IP). Intubation and ventilation were per-formed using the methods of Weksler et al. (1994). A 3-ccbolus of normal saline was given subcutaneously. Therat’s chest and abdomen were shaved, prepared, anddraped. The subxiphoid midline portion of the abdomenwas anesthetized with 0.25% lidocaine with epinephrineand opened to expose the rat’s heart. Injections were madein the apex or left ventricle of the heart. Two hundredmicroliters of stem cell suspension was injected through a25-gauge needle. Once complete, the fascia was closedwith 3.0 Vicryl suture and the skin was closed with 4.0

35Intramembranous bone was identified as a direct transition from stellate-shaped stem cells to three-dimensional nodulesdisplaying only osteogenic phenotypic markers, i.e., WV1D1(9C5) (DSHB) for bone sialoprotein II (Kasugai et al., 1992), MPIII(DSHB) for osteopontine (Gorski et al., 1990), and the von Kossa procedure (Silber Protein, Chroma-Gesellschaft) for calciumphosphate. In the von Kossa procedure, negative staining controls were pre-incubated in EGTA, a specific chelator for calcium(Sigma) (Young et al., 1993, 1999, 2001a,b).36Tendon/ligament was identified as linear structures with cellular staining for fibroblast specific protein IB10 (Sigma)(Ronnov-Jessen et al., 1992) and displaying extracellular histochemical staining for type-I collagen (Young et al., 1989c).37Dermis was identified by the presence of interwoven type-I collagen fibers (Young et al., 1989c) interspersed with spindle-shaped cells staining for fibroblast specific protein IB10 (Sigma) (Ronnov-Jessen et al., 1992) with an extracellular matrix richin chondroitin sulfate and dermatan sulfate glycosaminoglycans as assessed by Alcian Blue pH 1.0 staining. In the latterprocedure negative staining controls were digested with chondroitinase-ABC or chondroitinase-AC prior to staining (Young etal., 1989a,b, 2001a,b).38Scar Tissue was identified as interwoven type-I collagen fibers (Young et al., 1989c) interspersed with spindle-shaped cellsstaining for fibroblast specific protein IB10 (Sigma) (Ronnov-Jessen et al., 1992) with an extracellular matrix rich inchondroitin sulfate glycosaminoglycans as assessed by Alcian Blue pH 1.0 staining. In the latter procedure negative stainingcontrols were digested with chondroitinase-ABC or chondroitinase-AC prior to staining (Young et al., 1989a,b, 2001a,b).39Endothelial cells were identified by staining with antibodies P2B1 (DSHB) for CD31-PECAM (Young et al., 2001b), H-Endo(Chemicon) for CD146 (Solovey et al., 1997; St. Croix et al., 2000), P8B1 (DSHB) for VCAM (Dittel et al., 1993; Young et al.,2001b), and P2H3 (DSHB) for CD62e selectin-E (Young et al., 2001b).40Hematopoietic cells were identified using H-CD34 (Vector) for sialomucin-containing hematopoietic cells (Kishimoto et al.,1997; Young et al., 2001b); Hermes-1 (DSHB) for CD44 - hyaluronate receptor (Picker et al., 1989; Lewinsohn et al., 1990;Butcher, 2002); and H5A4 (DSHB) for CD11b-granulocytes, monocytes; and Natural Killer cells, H5H5 (DSHB) for CD43 -leukocytes, H4C4 (DSHB) for CD44 - hyaluronate receptor, H5A5 (DSHB) for CD45 - all leukocytes, and H5C6 (DSHB) forCD63 - macrophages, monocytes, and platelets (Hildreth and August, 1985; August and Hildreth, 2002).Endodermal lineage cells were identified as follows:41Endodermal progenitor cells were identified with H-AFP (Vector) and R-AFP (NORDIC, Tiburg, The Netherlands) foralpha-fetoprotein (Mujoo et al., 1983).42GI epithelium was identified with HESA (Sigma) for GI-epithelium (Young, 2004; Young and Black, 2004; Young et al.,2004).43Liver oval cells were identified with OC2 and OV6 (Hixson) for oval cells, liver progenitor cells, and biliary epithelial cells(Faris et al., 1991; Gordon et al., 2000).44Liver hepatocytes were identified with H-1 and H-4 (Hixson) for hepatocyte cell surface marker and hepatocyte cytoplasm,respectively (Walborg et al., 1985; Faris et al., 1991) and 151-IgG for liver epithelial growth factor receptor (Hubbard et al.,1985).45Liver biliary cells were identified with OC2, OC3, OC4, OC5, OC10, DPP-IV, and OV6 (Hixson) for biliary epithelial cells,liver progenitor cells, oval cells, and canalicular cells (Hixson et al., 1984, 1990, 2000; Walborg et al., 1985; Faris et al., 1991;Gordon et al., 2000).46Liver canalicular cells were identified with antibodies H4Ac19 (DSHB), DPP-IV, OV6, and LAP (Hixson) for bile canalicularcells, liver progenitor cells, biliary epithelial cells, and canalicular cell surface protein (Hixson et al., 1984, 1990, 2000;Hubbard et al., 1985; Walborg et al., 1985; Faris et al., 1991; Gordon et al., 2000).47Pancreatic progenitor cells were tentatively identified as three-dimensional structures void of chondrogenic or osteogenicphenotypic markers. This identity was confirmed by the presence phenotypic markers for pancreatic ductal cells, �-Cells,a-Cells, and d-Cells (Young, 2004; Young and Black, 2004; Young et al., 2004).48Pancreatic ductal cells were identified with cytokeratin-19 (Chemicon) to pancreatic ductal cells (Young, 2004; Young andBlack, 2004; Young et al., 2004).49Pancreatic �-cells were identified with YM-PS5088 (Accurate, Westbury, NY) an antibody to insulin (Young, 2004; Youngand Black, 2004; Young et al., 2004).50Pancreatic �-cells were identified with YM-PS087 (Accurate) an antibody to glucagon (Young, 2004; Young and Black, 2004;Young et al., 2004).51Pancreatic �-cells, were identified with 11180 (ICN) an antibody to somatostatin (Young, 2004; Young and Black, 2004;Young et al., 2004).

184 YOUNG ET AL.

nylon sutures. The rat was extubated and allowed to re-cover.

Myocardial infarction model. Animals were anes-thetized and intubated as described above. The left chestwas prepared, draped, and anesthetized with 0.25% lido-caine with epinephrine. A left anterior thoracotomy wasperformed. Once the chest was opened, the heart wasexposed. A myocardial infarction was created using gauze-tipped applicators soaked with liquid nitrogen along thedistribution of the left coronary artery. This was reappliedseveral times for 2–3 min until a uniform area of cryo-injury was created. A chest tube was created by placing a20-gauge catheter into the chest. The ribs and musclewere closed with 3.0 Vicryl suture and the skin closed with4.0 nylon. Any remaining air was aspirated through thechest tube. Triple antibiotic ointment was applied. Theanimal was then extubated and allowed to recover. Ratsthat were designated for immediate stem cell transplan-tation were injected with 200 �l of stem cell suspensionprior to closure of the chest. Those designated for delayedinjection underwent transplantation via the subxiphoidtechnique or tail vein injection at the designated time.

Experimental groups. There were five differentgroups of rats: 1) sham-operated heart control (n � 3), 2)ischemic heart control (n � 3), 3) pluripotent stem cellimplanted normal heart (n � 12), 4) pluripotent stem cellimplanted ischemic heart (n � 7), and 5) pluripotent cellsinjected in the tail vein of a rat that underwent myocardialinfarction (n � 7).

The sham-operated heart control group underwent asubxiphoid window and was injected with DPBS. Theischemic heart control underwent left thoracotomy, cryo-injury, and injection with DPBS. The tail vein injectiongroup underwent cryo-injury followed by direct injectionof cells into the tail vein. Normal hearts implanted withstem cells were harvested from one day to four weekslater. Ischemic hearts implanted with stem cells wereinjected from immediately to one-week postinjury. Thesehearts were harvested from one day to four weeks later.

Microscopy. For confocal microscopy, hearts were sec-tioned through the left ventricle, fixed, and stained forf-actin, cell nuclei, and �-galactosidase (Rockland 200-4136; 1:1,000 dilution; Gilbertsville, PA) as previouslydescribed (Price et al., 1996). For imaging, z-series werecollected at 2-�m intervals to a maximum depth of 80 �m.Images were selected showing cell location from the z-series. Sections were screened for recruitment and reten-tion of labeled nuclei/cells within noninjured tissues andthose tissues undergoing repair.

Induced Pancreatic Islets

Induction of islet-like structures. Three-dimensionalpancreatic islet-like structures (ILSs) were induced from theScl-40� clone by sequential directed lineage induction, i.e.,Scl-40� to endodermal stem cells (EndoSCs) to PanPCs toILSs. Directed lineage induction was accomplished by alter-ing the microenvironment of the cells in culture and bygrowing them in serum-free defined medium containing seraand/or growth factors specifically selected for their respec-tive endodermal-inductive (Young, 2004), pancreatic-induc-tive (Young et al., 2004), and islet-like-inductive (Bonner-Weir et al., 2000; Young et al., 2004) activities.

Glucose-mediated insulin release. The efficacy ofinsulin production in vitro by induced ILSs and nativeislets (see Fig. 8L and M) was compared at basal (5 mM)and elevated (25 mM) glucose concentrations. The result-ant induced ILSs from a starting population of 5 � 103

Scl-40� for each trial (n � 12) were used. For positivecontrols, 200 � 150 �m native pancreatic islet equivalentunits from Wistar-Furth rat pancreases were isolated foreach trial (n � 8). Induced ILSs and native islets wereincubated sequentially with TM only, followed by TM � 5mM glucose for 24 hr, followed by TM � 5 mM glucose for1 hr, followed by TM � 25 mM glucose for 1 hr. The mediawere removed and the amount of insulin secreted wasdetermined at 5 and 25 mM glucose loading by double-antibody competitive binding radioimmunoassay (RIA),using rat insulin standards and antibodies raised againstrat-specific insulin (Linco, St. Louis, MO), following themanufacturer’s directions.

Negative controls. A series of negative controls wasutilized to insure reliability of the rat-specific insulin-RIA.TM; TM with 5 and 25 �M glucose at 0, 1, and 24 hrincubation in a cell-free system; serum-free stem cell culturemedium (S-FSCCM), which contains a small amount of bo-vine insulin; S-FSCCM with lot-specific serum of bovineorigin (SS12); and S-FSCCM containing 0.1, 0.2, 0.5, 1.0, 2.0,5.0, and 10 ng/ml bovine insulin (same concentration rangeas rat insulin standards in the RIA kit) were examined.

RESULTSMultiple clones of cells isolated from adult skeletal mus-

cle and displaying stellate morphology, loss of contactinhibition, and growth past confluence were generatedusing repetitive single-cell clonogenic analysis. One suchclone, designated Rat-A2B2, was further evaluated forretention of traits characteristic of stem cells. At 130 pop-ulation doublings the cells were small with a high ratio ofnucleus to cytoplasm. They demonstrated quiescence inserum-free defined medium lacking inhibitory factors, ex-pressed embryonic markers in the undifferentiated state,and demonstrated the ability to form cells from all threeprimary germ layer lineages in vitro when treated withgeneral and specific lineage-induction agents (see Figs.1–4 and Table 1 for equivalent morphologies). Rat-A2B2was then examined for extensive capabilities for self-re-newal while maintaining pluripotency. Every other pas-sage interval from 130–254 population doublings wasevaluated for potential differences in induced phenotypicexpression of the cells. No differences were noted.

We further evaluated the Rat-A2B2 clone by transfec-tion with a nuclear-expressing LacZ to provide a perma-nent genomic label, and so designated one of its progenyclones as Rat-A2B2-Scl-40� (Scl-40�). Scl-40� was thenrescreened for retention of stem cell characteristics. Char-acteristics examined were size, ratio of nucleus to cyto-plasm, telomerase activity, extended capabilities for self-renewal, phenotypic expression in serum-free mediumlacking inhibitory agents, response to a proliferationagent, retention of embryonic stem cell markers, negativephenotypic response to incubation with a progression fac-tor, and positive phenotypic response to incubation with ageneral nonspecific lineage-induction agent. No differ-ences in stem cell characteristics were noted between theoriginal clone, Rat-A2B2, and its transfected progeny, Scl-

185ADULT PLURIPOTENT EPIBLASTIC-LIKE STEM CELLS

40�. After transfection, Scl-40� retained its small sizewith a high ratio of nucleus to cytoplasm.

Scl-40� was next examined for the presence of telomer-ase activity, an enzyme essential for increased populationdoublings. The clone was telomerase positive (Fig. 5A,lane 2). Scl-40� was then expanded to 287 populationdoublings and reexamined for pluripotency. In serum-freedefined medium lacking inhibitory factors (i.e., leukemiainhibitory factor or ADF), inductive factors, progressionfactors, and/or proliferation agents, Scl-40� remained instasis (Fig. 6A) and did not demonstrate cell proliferation,cell differentiation, and/or cell degeneration. Under thesame serum-free media conditions but in the presence of aproliferation agent, Scl-40� proliferated well past conflu-ence, demonstrating multiple overlapping confluent layersof cells (Fig. 6B), yet still expressed the Oct-4 gene (Fig. 5Band C) indicative of pluripotent embryonic stem cells.

Incubation with progression factor (i.e., 2 �g/ml insulin)in serum-free defined medium did not alter the phenotypicexpression of the Scl-40� at 287 population doublingscompared to the untreated nontransfected Rat-A2B2 con-trol clone at 130 population doublings. This suggested thatthe clone had not converted to a progenitor cell lineage dueto either extended self-renewal, the transfection proce-dure itself, or incorporation of the LacZ sequence into itsgenome. When Scl-40� was incubated in either serum-freedefined medium lacking inhibitory agents (leukemia inhibi-tory factor or ADF) or serum-free defined media containinginsulin, it expressed cytoplasmic phenotypic expressionmarkers for embryonic stem cells, i.e., stage-specific embry-onic antigen-4 (Fig. 6C) and carcinoembryonic antigen-celladhesion molecule-1 (CEA-CAM-1) (Fig. 6D) (Table 2).

In contrast, cells incubated with a general nonspecificinductive agent (i.e., 10–10 to 10–6 M dexamethasone) (Ta-ble 2) demonstrated alterations in phenotypic expression.These alterations consisted of changes in cell surface orstaining of the cytoplasm. Representative examples ofthese changes are shown in Figure 6E–M. These agentsinduced the expression of ectodermal lineage cells, i.e.,neuronal precursor cells (nestin, MAB353, Fig. 6E), gan-glion cells (synaptic vesicles, SV2, Fig. 6F), and neuroglia(oligodendrocytes, Rip, Fig. 6G); mesodermal lineage cells,i.e., skeletal muscle (sarcomeric myosin, MF-20, Fig. 6H),cartilage (type II collagen, HCII, Fig. 6I), and bone (bonesialoprotein II, WV1D1, Fig. 6J); and endodermal lineagecells, i.e., endodermal precursor cells (alpha-fetoprotein(AFP), Fig. 6K), pancreatic �-cells (insulin (INS), Fig. 6L),and liver progenitor cells, biliary cells, oval cells, andcanalicular cells (OC4, Fig. 6M). These changes werenoted whether or not SS were added that contained lin-eage-specific ectodermal, mesodermal, or endodermal in-duction agents or tissue-specific induction agents such as

Fig. 1. Rat-A2B2 incubated in TM only (A), TM with 10% SS3 (B), orTM with 2 �g/ml insulin (C and D) for either 24 hr (A) or 7 days (B–D).Morphologies and immunochemical staining as noted. Photographedwith phase-contrast (A and B) brightfield microscopy (C and D). Originalmagnifications, �200. A: Very small cells with high nuclear to cytoplas-mic ratios. B: Multiple confluent layers of cells maintaining stellate mor-phology. C: Mononucleated cells demonstrating moderate to heavystaining for stage-specific embryonic antigen-4 (SSEA-4). D: Mono-nucleated cells demonstrating moderate to heavy staining for CEA-CAM1.

186 YOUNG ET AL.

Sk-MMP, smooth muscle morphogenetic protein (Sm-MMP), AMP, fibroblast morphogenetic protein (FMP), orBMP-2.

Myocardial RepairScl-40� readily attached and grew on gelatinized plastic

tissue culture flasks. The antibody to �-galactosidase re-acted with the protein both within the nucleus and, to alesser extent, within the cell cytoplasm (Fig. 7A). Thisdemonstrated that all of the Scl-40� injected were positivefor �-galactosidase and could be readily detected followinginjection into the animals. Under gross inspection, cryo-genic infarction caused the cardiac tissue to become whit-ish gray in color in contrast to the normal deep red of theheart tissue. Tissue obtained from animals into whichScl-40� had been injected into ischemic myocardium dem-onstrated groups of living cells positive for �-galactosi-dase. One week after injection of Scl-40� into the cryo-injured heart, cells positive for �-galactosidase could belocated in the damaged myocardial tissues (Fig. 7B). Sub-sequent weeks postinjection demonstrated retention ofScl-40� in all myocardial tissues undergoing repair, i.e.,myocardium (Fig. 7C), vasculature (Fig. 7D), and connec-

tive tissue (Fig. 7E). Many of the �-galactosidase-positivecells may be seen in cross section in Figure 7C. These cellsare smaller in diameter than the endogenous myocytes,but they are similar in appearance. Inspection of normalmyocardium immediately adjacent to infarcted tissuedemonstrated few if any �-galactosidase-positive cells inthe surrounding uninjured tissues (data not shown). Tis-sue was also obtained from animals into which Scl-40�was delivered systemically via tail vein injection aftercryo-injury to the heart. A thorough inspection of theinfarcted area revealed recruitment and retention of la-beled nuclei within the myocardium (Fig. 7F) and connec-tive tissues (Fig. 7G) undergoing repair.

Pancreatic ILSsInduced PPELSCs (Scl-40�), induced EndoSCs, and in-

duced PanPCs were incubated with islet-inductive mediato ascertain their ability to form ILSs. For each cell line,103 cells were plated per well (n � 96) and treated withislet-inductive medium. The treated cultures were as-sayed for average numbers of three-dimensional ILSs gen-erated per well (SEM) and were as follows: 0.364 0.066 for the induced Scl-40�, 1.177 0.117 for the in-

Fig. 2. Rat-A2B2 incubated for seven days in TM with 10–6 M dexand 1% SS12. Morphologies and immunochemical staining as noted.Photographed with brightfield microscopy. Original magnifications,�100 (A, B, D, E, and G–I), �200 (C and F). A: Mononucleated cellsstaining for neural precursor cell expression marker (FORSE-1). B:Mononucleated cells showing intracellular staining for neurofilaments(RT-97). C: Mononucleated cells showing intracellular staining for neu-rons (8A2). D: Mononucleated cells showing intracellular staining for

neuronal nestin (Rat-401). E: Mononucleated cells showing intracellularstaining for b-tubulin-III (T8660). F: Mononucleated cells showing intra-cellular staining for oligodendrocytes (Rip). G: Mononucleated cellsshowing intracellular staining for neuronal expression marker (S-100). H:Mononucleated cells showing intracellular staining for neuronal vimentinfor radial cells and radial glial cells (40E-C). I: Mononucleated cellsshowing intracellular staining for ganglion cells (TuAg1).

187ADULT PLURIPOTENT EPIBLASTIC-LIKE STEM CELLS

Figure 3.

188 YOUNG ET AL.

duced EndoSCs, and 10.104 0.480 for the induced Pan-PCs. The increase in the number of ILSs formed by thePanPCs was statistically significant compared to that pro-duced by the Scl-40�s or the EndoSCs (P 0.05, analysisof variance).

After treatment with the islet-induction medium thecultures were stained with antibodies to insulin, glucagon,and somatostatin (Fig. 8). Scl-40� incubated with islet-inductive medium showed minimal intracellular stainingfor any of three antibodies assayed (Fig. 8A–C). InducedEndoSCs incubated with islet-inductive medium demon-strated a diffuse distribution of individual cells stainedintracellularly for insulin, glucagon, and somatostatin(Fig. 8D–F). Induced PanPCs incubated with islet-induc-tive medium demonstrated three-dimensional pancreaticILSs containing cells that exhibited intracellular stainingfor insulin, glucagon, and somatostatin (Fig. 8G–I).

Scl-40�s, induced EndoSCs, induced PanPCs, and in-duced ILSs were examined for retention of pluripotencyusing general and specific induction agents. SCl-40�s ex-pressed embryonic stem cell markers and formed 30� celltypes across all three primary germ layer lineages (Table1). Induced EndoSCs lost expression of embryonic stemcell markers, lost the ability to form cells of the ectodermallineage, and lost the ability to form cells of the mesoder-mal lineage, but retained the ability to form cells of theendodermal lineage (Table 1). Induced PanPCs lost ex-pression for endodermal progenitor cells, lost the ability toform GI epithelium, and lost the ability to form liver cells,but retained the ability to form pancreatic cells (Table 1).Induced ILSs lost the expression of PanPCs, but retainedthe expression of pancreatic ductal cells, �-cells, �-cells,and �-cells. Therefore, as the postnatal PPELSCs becomemore and more differentiated with each successive induc-tive step, they progressively lose their ability to formmultiple cell types.

The biological activity, i.e., the ability to secrete insulinin response to a glucose challenge, was then examined inthe induced ILSs (Fig. 8J and K) vs. native islets (Fig. 8Land M). ILSs secreted 22% of the amount of insulin se-creted by native islets during incubation with 5 mM glu-cose for 24 hr. When this was followed in each well by

incubation in 5 mM glucose for one hour, the ILSs secreted49% of the amount secreted by the native islets. A subse-quent incubation with 25 mM glucose for one hour re-sulted in secretion by the ILSs of 42% of the amount ofinsulin secreted by the native islets (Table 3). RIA mea-surements of negative controls confirmed rat-specific in-sulin release rather than release of medium-sequesteredbovine insulin.

DISCUSSIONThe current study examined the proposal of Young et al.

(2004) that undifferentiated PPELSCs derived fromadults have the potential for use in gene therapy andtissue engineering. Their proposal was based on the dis-covery of a population of undifferentiated precursor cells,having characteristics similar to embryonic stem cells,residing within the skeletal muscle and dermis of postna-tal humans. One of the characteristics noted for this pop-ulation was pluripotentiality for all three primary germlayer lineages. The undifferentiated human precursorcells reported by Young et al. (2004) were segregatedusing cluster of differentiation (CD) markers for the cellsurface epitopes CD10 and CD66e. There are at least twopossibilities to explain their findings. Young et al. mayhave discovered a pure population of embryonic-like stemcells residing within adult tissues. Alternatively, theymay have discovered a mixed population of germ layerlineage stem cells sharing cell surface epitopes and havingthe potential to form ectoderm, mesoderm, and endoderm.This second explanation is a distinct possibility sincethese investigators reported the discovery of both germlayer lineage mesodermal stem cells (Young et al., 2001a,2001b) and germ layer lineage ectodermal stem cells (Ro-mero-Ramos et al., 2002) residing in adult skeletal mus-cle.

Using CD markers for cell segregation is a valid proce-dure. However, the procedure itself cannot distinguishbetween a pure cell population with unique cell surfaceepitopes and a mixed cell population sharing the sameunique cell surface epitopes. To make the distinction be-tween pure and mixed cell populations, it is necessary togenerate a pure population derived from a single cell

Fig. 3. Rat-A2B2 incubated for one week (A, G, H, and J), two weeks(B–F, M, O, P, T, and U), four weeks (N and Q), six weeks (I, K, R, and S),or eight weeks (L) in TM and 10–8 M Dex (A–L, T, and U) or TM and 10–7

M Dex (M–S). Photographed with brightfield microscopy; original mag-nifications, �200 (A, C, F, H, J, M, P, and T), �100 (B, D, E, and G), or�40 (I, K, L, N, O, Q–S, and U). A: Mononucleated cells showing heavyintracellular staining for myogenin (F5D). B: Mononucleated and binucle-ated cells showing moderate to heavy intracellular staining for sarco-meric myosin (MF-20). C: Mononucleated and binucleated cells showingmoderate to heavy intracellular staining for antiskeletal muscle fast my-osin (MY-32). D: Mononucleated cells showing moderate to heavy intra-cellular staining for skeletal myosin heavy chain (ALD58). E: Mononucle-ated and binucleated cells showing heavy intracellular staining forskeletal myosin fast chain (A4.74). F: Mononucleated cells showingheavy intracellular staining for smooth muscle �-actin (IA4). G: Mono-nucleated cells showing moderate intracellular staining for cardiotin(cardiac myocytes, MAB 3252). H: Mononucleated cells demonstratingheavy intracellular staining for bone sialoprotein II (WV1D1). I: Nodule ofcells demonstrating extracellular staining for bone sialoprotein II(WV1D1). J: Mononucleated cells demonstrating moderate to heavyintracellular staining for osteopontine (MP111). K: Nodule of cells dem-

onstrating extracellular staining for osteopontine (MP111). L: Nodule ofcells demonstrating extracellular staining for calcium phosphate usingthe von Kossa procedure (vK). M: Mononucleated cells with intracellularstaining for cartilage-specific collagen pro type-II (CIIC1). N: Three nod-ules demonstrating intense extracellular staining for cartilage-specificcollagen pro type-II (CIIC1). O: Single nodule of cells demonstratingmoderate extracellular staining for cartilage-specific collagen type-II(HC-II). P: Mononucleated cells demonstrating moderate intracellularstaining for cartilage-specific collagen type-IX (D1-9). Q: Three nodulesdemonstrating extracellular staining for sulfated glycosaminoglycanchains of proteoglycans (Perfix/Alcec Blue). R: Nodule demonstratingextracellular staining for sulfated glycosaminoglycan chains of proteo-glycans (Safranin-O, pH 1.0). Individual nuclei stained with antibody to�-galactosidase (Gal-19) and visualized with 3-3�-diaminobenzidine(DAB). S: Two nodules demonstrating extracellular staining for sulfatedglycosaminoglycan chains of proteoglycans (Alcian Blue, pH 1.0). T:Mononucleated cells with moderate to heavily stained intracellular ves-icles demonstrating saturated neutral lipids (Oil Red-O), indicative ofadipocytes. U: Mononucleated cells with moderate to intensely stainedintracellular vesicles demonstrating saturated neutral lipids (SudanBlack-B), indicative of adipocytes.

189ADULT PLURIPOTENT EPIBLASTIC-LIKE STEM CELLS

Fig. 4. Rat-A2B2 incubated for one week (A, B, and E), two weeks (D,K, and L), three weeks (C, F, I, M, N, and R), four weeks (G, H, O, and Q),or five weeks (J and P) in TM with 15% SS12 and 10–6 M Dex. Morphol-ogies and immunochemical staining as noted. Photographed withbrightfield microscopy, original magnifications, �200 (A), �100 (B, D, H,K–N, and P), or �40 (C, E, I, J, Q, and R). A: Mononucleated andbinucleated cells showing intense intracellular staining for rat-specificAFP. B: Mononucleated cells showing moderate to intense intracellularstaining for rat-specific liver epithelial growth factor receptor (151-Ig).C: Nodular aggregations showing moderate intracellular staining forpro-insulin of endocrine pancreas (�-cells). D: Cellular aggregationshowing moderate to heavy intracellular staining for glucagon ofendocrine pancreas (�-cells). E: Cellular aggregation and individualdiffuse mononucleated cells showing moderate to intense intracellu-lar staining for somatostatin of endocrine pancreas (�-cells). F: Cel-lular aggregation and individual diffuse mononucleated cells showingmoderate to intense intracellular staining for ductal cells of exocrinepancreas (CK-19), �100. G: Cellular aggregation and individual dif-fuse mononucleated cells showing moderate to intense intracellularstaining for bile canalicular cells of liver (HA4c19). H: Nodule showingheavy intracellular staining for progenitor cells, biliary epithelial cells,and oval cells of liver (OC2). I: Diffuse mononucleated cells showing

moderate to heavy intracellular staining for progenitor cells and biliaryepithelial cells of liver (OC3). J: Cellular aggregation and individualdiffuse mononucleated cells showing moderate to intense intracellu-lar staining for progenitor cells and biliary epithelial cells of liver(OC4). K: Diffuse mononucleated cells showing moderate to heavyintracellular staining for progenitor cells and biliary epithelial cells ofliver (OC5). L: Diffuse mononucleated cells showing moderate tointense intracellular staining for progenitor cells and biliary epithelial cellsof liver (OC10). M: Diffuse and aggregated cells showing moderate tointense intracellular staining for cytoplasm of liver hepatocytes (H.4). N:Diffuse mononucleated cells showing moderate to intense intracellularstaining for liver hepatocyte cell surface marker (H.1). O: Diffuse andaggregated cells showing moderate to heavy intracellular staining forprogenitor cells, canalicular cells, and biliary epithelial cells of liver(DPP-IV). P: Nodular aggregate shows heavy to intense intracellularstaining for endodermal epithelial marker of liver (DESMO). Q: Nodularaggregate and diffuse cells showing moderate to heavy intracellularstaining for biliary epithelial cells, oval cells, and hepatocyte canalicularcells (HCC) of liver (OV6). R: Nodular aggregate and diffuse cells show-ing moderate to intense intracellular staining for canalicular cell surfaceprotein of liver (LAP), �100.

(Young et al., 2001a). This is in contrast to the derivationof potentially mixed populations generated from 4 cells(Pittenger et al., 1999) or generated from 10 cells (Reyesand Verfaillie, 2001). We addressed that issue by gen-erating a pure population of undifferentiated precursor

cells by repetitive single-cell clonogenic analysis follow-ing procedures previously established by Young et al.(2001a). The cells used for this clonogenic analysis wereisolated from adult skeletal muscle as described byYoung et al. (2004). Previous empirical studies by

Fig. 5. Molecular analysis of telomerase activity and Oct-4 geneexpression in LacZ-transfected adult rat PPELSC clone Scl-40�.A: Telomerase expression. Telomerase activity was detected by poly-acrylamide gel electrophoresis of cell lysates from a clone Scl-40� at254 population doublings. Cells were thawed, plated, and expanded inmedium containing PDGF-like (proliferative) and ADF-like (anti-differen-tiative/inhibitory) activities (Young, 2000, 2003). Cells were harvested(Young et al., 1999) and processed for telomerase activity as described(TRAPeze Assay, Intergen). lane 1 �, extract of telomerase-positivecells; 1 –, extraction buffer (controls); lane 2 �, test extract of Scl-40�;2 –, heat inactivated extract of Scl-40�. Note the presence of a ladderingof bands denoting the presence of telomerase activity; compare lanes 1

and 2. B: Oct-4 gene expression. Oct-4 was detected by the electro-phoretic mobility shift assay using the oligonucleotide 5�-TGTCGAATG-CAAATCACTAGA-3� containing the Oct-1 consensus binding site. Scl-40� at 287 population doublings was processed as described. Twobands that represent binding by members of the Oct family of transcrip-tion factors were obtained, as shown by the competition for binding byunlabeled Oct oligonucleotide. C: Densitometric analysis of the areacontained in the sidebar of the electrophoretic mobility shift assay inFigure 5B. Lane 1, solid line; lane 2, long dashes; lane 3, short dashes.Incubation with Oct-4-specific antibody substantially decreased the for-mation of the upper band and slightly decreased the formation of thelower band, indicating the presence of Oct-4 gene expression.

191ADULT PLURIPOTENT EPIBLASTIC-LIKE STEM CELLS

Figure 6.

192 YOUNG ET AL.

Young et al. (unpublished) revealed the existence of anautocrine-paracrine factor (APF) secreted by log-phaseexpanding cells that could maintain cells in a highlyproliferative undifferentiated state. We noted in thisstudy that single-cell clonogenic analysis of undifferen-tiated adult cells was only possible using preconditionedmedium containing APF as a component of the cloningmedium.

Examination of the resultant pure clonal population forstem cell characteristics noted small cells having a highratio of nucleus to cytoplasm, expressing embryonic cellmarkers in the undifferentiated state, having extendedcapabilities for self-renewal, and having the ability toform cells from all three primary germ layer lineages invitro when treated with general and specific lineage-in-duction agents (see Figs. 1-4 and Table 1 for equivalentmorphologies).

Next, the clone was transfected with the LacZgenomic marker. This was performed to determine iftransfection with a genomic sequence, e.g., gene ther-apy, would alter stem cell characteristics. Results fromthe nonlabeled parental clone (Rat-A2B2) were com-pared to its labeled clonal progeny (Scl-40�). There wasno discernible difference between either clone with re-spect to size, ratio of nucleus to cytoplasm, extendedcapabilities for self-renewal, telomerase activity, molec-ular and immunological embryonic markers, incubationin serum-free medium without inhibitory agents, incu-bation with a progression agent, and induced pluripo-tency across all three primary germ layer lineages usinggeneral and specific inductive agents (Figs. 5 and 6,Table 2). These results show that transfection withLacZ did not alter the clonal population’s capacity tofunction as stem cells. These data support the potentialuse of adult-derived PPELSCs as delivery vehicles forgene therapy.

Scl-40� was then implanted into rat hearts followingcryo-injury and assessed for incorporation into tissuesundergoing repair. This was accomplished using twomethods. The first method utilized direct injection into theinfarcted region of the heart. The results show recruit-ment and retention of Scl-40� in myocardial tissues un-dergoing repair (Fig. 7B). We had anticipated incorpora-tion of Scl-40� solely into myocardium; however, this wasnot the case. Scl-40� incorporated into all myocardial tis-sues undergoing repair, i.e., myocardium (Fig. 7C), vascu-

lature (Fig. 7D), and connective tissue (Fig. 7E). The sec-ond method of implantation involved systemic delivery ofthe labeled stem cells after cryo-injury via tail vein injec-tion. Interestingly, the labeled cells were able to home tothe damaged heart after ischemic injury and incorporateinto myocardium (Fig. 7F) and connective tissues (Fig.7G). This was also an unexpected finding and suggests apotentially less invasive method for stem cell delivery.These data support the recruitment and retention ofadult-derived PPELSCs for the repair of myocardial tis-sues after injury.

Lastly, Scl-40� was examined to determine if a three-dimensional biologically functional tissue could be gener-ated from adult-derived undifferentiated stem cells. Weused a directed lineage induction since, unlike embryonicstem cells, these adult-derived stem cells will not sponta-neously differentiate in the absence of inhibitory agents.Scl-40� (Fig. 8A–C) was sequentially induced to form En-doSCs (Fig. 8D–F), then pancreatic stem cells, and finallyILSs (Fig. 8G–I). This was accomplished using alterationsin the culture microenvironment and specific inductiveagents. The structures formed were then assayed for in-sulin secretion in response to a glucose challenge, compar-ing induced ILSs (Fig. 8J and K) to native pancreatic islets(Fig. 8L and M). A series of negative controls was includedto ensure that our method of measuring secreted insulinwas working properly and would only measure rat insulinsecreted into the media rather than also measuring up-take and release of bovine insulin from the medium (Ra-jagopal et al., 2003). No (bovine) insulin was detected bythe rat-specific insulin-RIA in any of the control solutionsanalyzed. Thus, as shown in Table 3, the induced ILSssecreted approximately 25–50% of the insulin secreted bynative islets under the conditions examined. Due to theextensive capabilities for self-renewal of undifferentiatedadult-derived pluripotent stem cells, these data suggestthe potential for mass production of pancreatic islets fortransplantation therapy.

Based on current and previous results, we would pro-pose that there are distinct similarities and differenceswith respect to the undifferentiated stem cells derivedfrom embryonic tissues and those derived from adult tis-sues as reported by Young et al. (this study; Young, 2004;Young and Black, 2004).

Fig. 6. Scl-40� clone incubated with antibody to �-galactosidase todemonstrate nuclear LacZ-transfected gene expression and stainedwith DAB (dark purple/black), then incubated with antibody to specificphenotypic expression markers, as noted, and counterstained with3-amino-9-ethylcarbazole (AEC) (red/orange). Embryonic-like: Scl-40�grown in serum-free medium containing 2 �g/ml insulin (C and D).Ectodermal: Scl-40� grown for one week in serum-free medium con-taining 2 �g/ml insulin, 10–6 M dexamethasone, 1% SS12 at pH 7.4 toinduce ectodermal lineage cells (E–G). Mesodermal: Scl-40� grown forone week in serum-free medium containing 2 �g/ml insulin, 10–6 Mdexamethasone, 1% SS9 at pH 7.4 to induce mesodermal lineage cells(H–J). Endodermal: Scl-40� grown for one week in serum-free mediumcontaining 2 �g/ml insulin, 10–6 M dexamethasone, 15% SS12 at pH 7.6to induce endodermal lineage cells (K–M). Original magnifications, �300(A, C–J, and M), �200 (K and L), �100 (B). A: Scl-40� grown for oneweek in serum-free medium containing 2 �g/ml insulin. Note widelydispersed mononucleated cells with no apparent cellular proliferation or

cell degeneration during culture period. B: Scl-40� grown for one weekin serum-free medium with serum containing PDGF-like (proliferative)and ADF-like (antidifferentiative/inhibitory) activities. Note multiple con-fluent layers of cells expressing nuclear �-galactosidase expression. C:MC-813-70, antibody to stage-specific embryonic antigen-4 (Lannagi etal., 1983). D: CEA-CAM-1, antibody to CEA-CAM-1 (Hixson) (Estrera etal., 1999). E: MAB353, antibody to nestin for the identification of neuro-genic progenitor cells (Gritti et al., 1996). F: SV2, antibody to synapticvesicles (Feany et al., 1992). G: Rip, antibody to oligodendrocytes (Fried-man et al., 1989). H: MF-20, antibody to sarcomeric myosin (Bader et al.,1982). I: HC-II, antibody to type-II collagen (Burgeson and Hollister,1979; Kumagai et al., 1994). J: WV1D1, antibody to bone sialoprotein II(Kasugai et al., 1992). K: R-AFP, antibody to rat-specific AFP (Mujoo etal., 1983). L: YM-PS5088, antibody to insulin-secreting �-cells (Young,2003; Young et al., 2003). M: OC4, antibody to liver progenitor cells,biliary epithelial cells, oval cells, and canalicular cells (Hixson et al., 1984,1990, 2000; Walborg et al., 1985; Faris et al., 1991; Gordon et al., 2000).

193ADULT PLURIPOTENT EPIBLASTIC-LIKE STEM CELLS

TABLE 2. Induction of phenotypic expression in rat-A2B2-scl-40

Phenotypic markersNuclearmean1 S.E.M.2

Cytoplasmicmean3 S.E.M.

Percentagephenotype4

Embryonic5

MC-813-706 5.966 0.323 4.241 0.313 71.1CEA-CAM-17 9.862 1.106 9.897 1.443 100.0

Ectodermal8Neurogenic progenitor cells9

MAB35310 34.517 1.595 12.143 1.055 35.2FORSE-111 29.414 2.167 9.724 1.474 33.1Rat-40112 33.724 1.559 7.172 0.793 21.1

Neurons8A213 24.966 1.994 7.966 1.120 31.9S-10014 35.071 1.686 9.483 1.046 27.0RT-9715 41.107 1.763 17.552 2.035 42.7N-20016 31.414 1.378 9.759 0.713 31.1SV217 34.483 1.956 14.793 1.878 42.9TuAG118 38.448 2.020 15.621 1.332 40.6

Glial cellsRip19 30.966 1.034 18.000 0.897 58.1CNPase20 34.148 2.261 10.857 1.206 31.8

Mesodermal21

Skeletal muscleOP-13722 88.179 1.495 51.138 2.916 58.0F5D23 100.138 2.749 46.517 3.020 46.5MF-2024 92.517 2.968 45.276 2.246 48.9MY-3225 101.448 3.123 57.690 3.769 56.9ALD-5826 88.586 3.412 49.966 2.880 56.4A4.7427 100.172 2.876 52.276 3.045 52.2

Smooth muscleIA428 96.897 2.382 46.448 3.294 47.9

CartilageCIIC129 96.862 2.906 51.429 2.584 53.1HC-II30 98.552 2.062 55.690 3.429 56.5D1-931 90.655 2.607 48.069 2.948 53.09/30/8A432 98.586 2.519 55.862 2.936 56.712/21/1C633 95.655 3.457 51.310 2.767 53.6

BoneWV1D134 96.000 4.399 50.069 2.884 52.2MP11135 82.931 2.640 43.207 3.389 52.1

Endodermal36

Endodermal progenitor cellsR-AFP37 83.310 3.974 51.241 2.849 61.5

Liver151-IgG38 70.724 2.707 39.966 2.617 56.5OC239 75.793 3.371 33.793 3.307 44.6OC340 67.862 2.815 29.793 2.446 43.9OC441 77.897 2.781 31.276 2.995 40.2OC542 82.793 3.139 37.759 3.122 45.6OC1043 74.133 3.636 32.931 2.617 44.4H-144 81.931 2.912 43.690 3.044 53.3H-445 75.966 3.745 31.034 3.247 40.8DPP-IV46 71.897 2.287 41.828 2.506 58.2HA4c1947 72.931 4.147 44.586 2.633 61.1OV648 78.786 1.971 44.741 1.590 56.8

PancreasYM-PS08749 66.929 2.192 41.679 2.587 62.3YM-PS508850 80.310 2.797 44.483 2.448 55.41118051 63.828 2.466 42.786 1.817 67.0

1Nuclear mean, mean number of cells within photograph with nuclear staining for �-galactosidase.2S.E.M., standard error of the mean.3Cytoplasmic mean, mean number of cells within respective photograph with cytoplasmic staining for phenotypic expressionmarker.4Percentage phenotype, percentage of �-Gal nuclear stained cells demonstrating stained cytoplasm, indicative of percentageof population displaying indicated phenotypic expression marker.5Embryonic, rat-A2B2-scl-40 grown in testing medium containing 2 �g/ml insulin.6MC-813-70, antibody to stage-specific embryonic antigen-4 (Lannagi et al., 1983).7CEA-CAM-1, antibody to carcinoembryonic antigen-cell adhesion molecule-1 (Hixson) (Estrera et al., 1999).8Ectodermal, rat-A2B2-scl-40 clone grown in testing medium containing 2 �g/ml insulin, 10�6 M dexamethasone, 1%SS12 atpH 7.4 to induce ectodermal lineage cells.

194 YOUNG ET AL.

Similarities Between Embryonic Stem Cells andAdult PPELSCs

Embryonic stem cells are of small size and demonstratehigh ratios of nucleus to cytoplasm (Martin, 1981; Sham-blott et al., 1998; Thompson et al., 1998). Both clonesexamined, Rat-A2B2 and Scl-40�, approximate a quarterof the size of the germ layer lineage mesodermal stem cellclone Rat-A2A2 (Young et al., 2001a). We have isolatedadult PPELSCs from skeletal muscle and dermal connec-tive tissue biopsy specimens taken from newborn to geri-atric humans (Young, 2004; Young and Black, 2004;Young et al., 2004). When unfixed human cells were sortedin a flow cytometer, they approximated the size of humanerythrocytes, in the range of 6–8 �m. This is in contrast tounfixed human germ layer lineage mesodermal stem cellsthat are 10–20 �m in size by flow cytometry (Young et al.,2001b).

Embryonic stem cells can be maintained in an undiffer-entiated state in serum-containing medium if an agentthat inhibits induction (leukemia inhibitory factor, ES-GRO, fibroblast feeder layers, and/or marrow stromalcells) is present within the medium (Martin, 1981; Sham-blott et al., 1998; Thompson et al., 1998; Cheng et al.,2003). We have shown a similar retention of the undiffer-entiated state for rodent and human adult PPELSCs us-ing either leukemia inhibitory factor or ADF (Young et al.,1998a, 2004; Young, 2004).

Normal differentiated diploid cells in vitro undergo afinite number of divisions before they reach a prepro-grammed state of replicative cell senescence and celldeath (Hayflick and Moorehead, 1961; Hayflick, 1963,1965). The maximum population doubling numbers fordifferentiated embryonic fibroblasts to reach this limit isreported to be proportional to the maximal life span of the

9Neurogenic progenitor cells, cells destined to become neurons or neuroglia.10MAB353, antibody to nestin for the identification of neurogenic progenitor cells (Gritti et al., 1996).11FORSE-1, antibody to neural precursor cells (Tole et al., 1995; Tole and Patterson, 1995).12Rat-401, antibody to nestin for the identification of neurogenic progenitor cells (Hockfield and McKay, 1985).138A2, antibody to neurons (Drazba et al., 1991).14S-100, antibody to neurons (Baudier et al., 1986; Barwick, 1990).15RT-97, antibody to neurofilaments (Wood and Anderton, 1981).16N-200, antibody to neurofilament-200 (Debus et al., 1983; Franke, et al., 1991).17SV2, antibody to synaptic vesicles (Feany et al., 1992).18TuAg1, antibody to ganglion cells (Faris et al., 1990; Hixson et al., 1990).19Rip, antibody to oligodendrocytes (Friedman et al., 1989).20CNPase, antibody to astroglia and oligodendrocytes (Sprinkle et al., 1987; Sprinkle, 1989; Reynolds et al., 1989).21Mesodermal, rat-A2B2-scl-40 clone grown in testing medium containing 2 �g/ml insulin, 10�6 M dexamethasone, 1%SS9 atpH 7.4 to induce mesodermal lineage cells.22OP-137, antibody to MyoD (Thulasi et al., 1996).23F5D, antibody to myogenin (Wright et al., 1991).24MF-20, antibody to sarcomeric myosin (Bader et al., 1982).25MY-32, antibody to skeletal muscle fast myosin (Naumann and Pette, 1994).26ALD-58, antibody to myosin heavy chain (Shafiq et al., 1984).27A4.74, antibody to myosin fast chain (Webster et al., 1988).28IA4, antibody to smooth muscle alpha-actin (Skalli et al., 1986).29CIIC1, antibody to type-II collagen (Holmdahl et al., 1986).30HC-II, antibody to type-II collagen (Burgeson and Hollister, 1979; Kumagai et al., 1994).31D1-9, antibody to type-IX collagen (Ye et al., 1991).329/30/8A4, antibody to cartilage link protein (Caterson et al., 1985).3312/21/1C6, antibody to cartilage proteoglycan-hyaluronate binding region (Caterson, 2001).34WV1D1, antibody to bone sialoprotein II (Kasugai et al., 1992).35MP111, antibody to osteopontine (Gorski et al., 1990).36Endodermal, rat-A2B2-scl-40 clone grown in testing medium containing 2 �g/ml insulin, 10�6 M dexamethasone, 15%SS12at pH 7.6 to induce endodermal lineage cells.37R-AFP, antibody to alpha-fetoprotein (Mujoo et al., 1983).38151-IgG, antibody to liver epithelial growth factor receptor (Hubbard et al., 1985).39OC2, antibody to oval cells, liver progenitor cells, and biliary epithelial cells (Faris et al., 1991; Gordon et al., 2000).40OC3, antibody to biliary epithelial cells, liver progenitor cells, oval cells, and canalicular cells (Hixson et al., 1984, 1990, 2000;Walborg et al., 1985; Faris et al., 1991; Gordon et al., 2000).41OC4, antibody to biliary epithelial cells, liver progenitor cells, oval cells, and canalicular cells (Hixson et al., 1984, 1990, 2000;Walborg et al., 1985; Faris et al., 1991; Gordon et al., 2000).42OC5, antibody to biliary epithelial cells, liver progenitor cells, oval cells, and canalicular cells (Hixson et al., 1984, 1990, 2000;Walborg et al., 1985; Faris et al., 1991; Gordon et al., 2000).43OC10, antibody to biliary epithelial cells, liver progenitor cells, oval cells, and canalicular cells (Hixson et al., 1984, 1990,2000; Walborg et al., 1985; Faris et al., 1991; Gordon et al., 2000).44H-1, antibody to hepatocyte cell surface marker (Walborg et al., 1985; Faris et al., 1991).45H-4, antibody to hepatocyte cytoplasm (Walborg et al., 1985; Faris et al., 1991).46DPP-IV, antibody to biliary epithelial cells, liver progenitor cells, oval cells, and canalicular cells (Hixson et al., 1984, 1990,2000; Walborg et al., 1985; Faris et al., 1991; Gordon et al., 2000).47HA4c19, antibody to bile canalicular cells of liver (Hubbard et al., 1985).48OV6, antibody to oval cells, liver progenitor cells, and biliary epithelial cells (Faris et al., 1991; Gordon et al., 2000).49YM-PS087, antibody to glucagon-secreting �-cells (Young, 2004; Young et al., 2004).50YM-PS5088, antibody to insulin-secreting �-cells (Young, 2004; Young et al., 2004).5111180, antibody to somatostatin-secreting �-cells (Young, 2004; Young et al., 2004).

195ADULT PLURIPOTENT EPIBLASTIC-LIKE STEM CELLS

Fig. 7. Laser scanning confocal microscopy of �-galactosidase-pos-itive Scl-40� in vitro and after injection in vivo. A: Scl-40� in culture ongelatin-coated tissue culture plastic. The f-actin in the cytoskeleton hasbeen stained using rhodamine phalloidin (red). The �-galactosidase hasbeen immunohistochemically labeled green using a fluoresceine isothio-cyanate (FITC) fluorophore. B: Scl-40� cells localized in normal hearttissue one week after direct injection of cells into the left ventricle (green).End views of myofibril bundles stained with rhodamine phalloidin can beseen (red). Cell nuclei (blue) are stained with topro-3 (a DNA intercalatingdye). C: Scl-40� cells localized in ischemic heart tissue one week afterdirect injection of cells into the left ventricle (green). The cells wereinjected through a subxiphoid window three days after cryo-injury. Notecluster of small �-galactosidase-positive cells among cardiac myocytesundergoing regeneration. D: Scl-40� cells localized in ischemic hearttissue two weeks after direct injection of cells into the left ventricle

(green). This �-galactosidase-positive cell has localized adjacent to acardiac blood vessel. Cell nuclei (blue) have been stained with topro-3.E: Scl-40� localized in ischemic heart tissue two weeks after directinjection of cells into the left ventricle (green). The cells were located inthe connective tissues peripheral to the injury site. The f-actin in thecytoskeleton has been stained using rhodamine phalloidin. The �-galac-tosidase has been immunochemically labeled green using an FITC flu-orophore. F: Scl-40� localized in heart tissue peripheral to the site ofcryo-injury. The cells (green) were injected one week after injury and thenleft an additional two weeks before tissue harvest. Bundles of rhodaminephalloidin-stained myofibrils can be seen (red). G: Scl-40� localized inconnective tissues immediately adjacent to the site of cryo-injury. Cellnuclei are stained with topro-3 (blue). These cells (green) were injectedsystemically into the tail vein of the rat following injury.

donor animal (Martin et al., 1970; Schneider and Mitsui,1976; Rhome, 1981). The maximal life span in terms ofpopulation doublings for differentiated embryonic fibro-blasts is 50–70 in humans (Hayflick and Moorehead,1961) and 8–10 in mice (Rhome, 1981). In contrast, undif-ferentiated embryonic stem cells demonstrate extendedcapabilities for self-renewal (Pera et al., 2000). AdultPPELSCs also exhibit extensive capabilities for self-re-newal. The current study demonstrated retention of plu-ripotency for all three primary germ layer lineages inScl-40� through a minimum of 287 population doublings.Previous studies of adult human PPELSCs noted prolifer-ation potentials through 400 population doublings with-out loss of pluripotency (Young and Black, 2004).

Telomere shortening as a mitotic clock is an acceptedtheory to explain replicative cell senescence and cell death(Harley et al., 1990; Campisi, 1997). Telomere shorteningeventually causes chromosomal instability, leading to theactivation of DNA damage response pathway followed byp53-dependent cell cycle arrest, senescence, and cell death(Vaziri and Benchimol, 1996). Telomerase was found to beactivated in embryonic germ cells and embryonic stemcells, repressed in normal somatic cells, and reactivated ina large majority of tumor cells (Liu, 2000; Pera et al., 2000;Lin et al., 2003). Both the parental clone (Rat-A2B2)(Young and Black, 2004; Young et al., 2004) and its trans-fected progeny Scl-40� (Fig. 5A) demonstrate telomeraseactivity.

The POU family transcription factor Oct-4 has beenregarded as a master regulator for initiation, mainte-nance, and differentiation of pluripotent cells (Nichols etal., 1998; Niwa et al., 2000, 2002). It is expressed intotipotent and pluripotent cells, including oocytes, earlycleavage stage embryos, the inner cell mass of the blasto-cyst, the epiblast layer, and germ cells (Scholer et al.,1990; Palmieri et al., 1994; Pesce and Scholer, 2000). It isalso present in cultured embryonic stem cells, embryonicgerm cells, and embryonal carcinoma cells (Lenardo et al.,1989; Scholer et al., 1989; Yeom et al., 1996; Brehm et al.,1998). Oct-4 downregulation is essential for mammalianembryonic stem (ES) cells to differentiate into definedlineages (Niwa et al., 2000; Pesce and Scholer, 2001).Thus, Oct-4 is absent from all differentiated somatic cellsin vitro or in vivo (Niwa et al., 2002). Scl-40� demon-strated Oct-4 expression in serum-free medium (Fig. 5Band C). Similarly, its parental line, A2B2, also demon-strated Oct-4 expression in serum-free medium (Youngand Black, 2004; Young et al., 2004). These data show thatthe adult-derived PPELSCs express the POU family tran-scription factor Oct-4 essential for the maintenance ofpluripotency in lineage-uncommitted pluripotent embry-onic stem cells. Whether the function of Oct-4 in theseadult-derived PPELSCs parallels the function of Oct-4 inembryonic stem cells remains to be elucidated.

Stage-specific embryonic antigens are cell surface mol-ecules that are developmentally regulated during earlyembryogenesis and have been used to monitor the differ-entiation status of both human- and mouse-derived em-bryonic stem cells. Undifferentiated human ES cells ex-press stage-specific embryonic antigen-3 (SSEA-3) andSSEA-4, while differentiating human ES cells expressSSEA-1. In contrast, undifferentiated mouse ES cells ex-press SSEA-1, while differentiating mouse ES cells ex-press SSEA-3 and SSEA-4 (Pera et al., 2000; Henderson etal., 2002; Cheng et al., 2003). In serum-free medium with-

out inhibitory agents, Scl-40� demonstrated SSEA-4 andCEA-CAM-1 expression (Figs. 1C and D, 6C and D; Tables1 and 2). Newborn to geriatric human PPELSCs in serum-free medium without inhibitory agents express SSEA-1,SSEA-3, SSEA-4, and CEA-CAM-1, as well as HCEA (hu-man carcinoembryonic antigen), CD66e (human carcino-embryonic antigen), and CEA (carcinoembryonic antigen)(Young, 2004; Young and Black, 2004; Young et al., 2004).These data demonstrate that adult PPELSCs in serum-free medium maintain cell surface antigens similar tothose of human embryonic stem cells.

Embryonic stem cells are able to differentiate into awide range of cell types in vitro (Thomson et al., 1998;Reubinoff et al., 2000). Scl-40�, derived by repetitive sin-gle-cell clonogenic analysis, demonstrates differentiationinto over 30 distinct cell types in vitro (Figs. 2–4, 6, 8;Table 2). Similarly, PPELSCs from newborn to geriatrichumans demonstrate differentiation thus far into over 40distinct cell types in vitro (Young, 2004; Young and Black,2004; Young et al., 2004) (Table 1). In both instances, thein vitro differentiation capabilities of these adult-derivedstem cells crossed all three primary germ layer lineages,forming cells of ectodermal, mesodermal, and endodermalorigin.

Differences Between Embryonic Stem Cells andAdult PPELSCs

However, there are also distinct differences with respectto embryonic stem cells and adult PPELSCs that suggestthat these two categories of stem cells are not equivalent.Embryonic stem cells cultured in serum-free defined me-dium in the absence of inhibitory factors (i.e., leukemiainhibitory factor, ESGRO, fibroblast feeder layer, and/ormarrow stromal layer) will spontaneously differentiateinto all somatic cells of the body (Thomson et al., 1995,1998; Shamblott et al., 1998; Pera et al., 2000). In con-trast, adult PPELSCs grown under similar conditions ofserum-free defined medium in the absence of inhibitoryfactors (i.e., leukemia inhibitory factor or ADF) will re-main in a quiescent inactive state. Thus, they do notdemonstrate cell proliferation, cell differentiation, or celldegeneration. This result suggests that the adultPPELSCs are not preprogrammed to form all somatic cellsof the body like embryonic stem cells, but rather must waitfor regulatory signals to dictate growth and/or subsequentdifferentiation.

A second difference between embryonic stem cells andadult PPELSCs is their activities in vivo after injection ortransplantation of undifferentiated cells. Implanted-transplanted embryonic stem cells form teratomas (Thom-son et al., 1998; Amit et al., 2000; Pera et al., 2000;Reubinoff et al., 2000; Lin et al., 2003; Watkitani et al.,2003). In contrast, undifferentiated adult pluripotentstem cells incorporate into all tissues in need of repair. Wehave noted this activity with respect to skeletal musclerepair, vascular repair, bone repair, cartilage repair, bonemarrow incorporation, and myocardial repair (Young etal., 2004; this study) (Fig. 7B–G). This suggests that theadult PPELSCs are acting as a true precursor stem cell,allowing the body to dictate what cell type the stem cellswill become in order to repair the appropriate damagedtissues.

197ADULT PLURIPOTENT EPIBLASTIC-LIKE STEM CELLS

Comparison of PPELSCs to MAPCsRecently, Verfaillie and colleagues reported the isola-

tion of precursor cells, designated multipotent adult pro-genitor cells (MAPCs), with attributes similar to embry-onic stem cells (Jiang et al., 2002a, 2002b; Reyes et al.,2002; Schwartz et al., 2002). These precursor cells were

located within adult mammalian bone marrow, brain, andmuscle. MAPCs, expanded from an initial population of 10cells, demonstrated a CD13�, Flk1dim, c-kit–, CD44–,CD45–, major histocompatibility complex (MHC) class I–,and MHC class II– cell surface profile. These cells dis-played capabilities for extended self-renewal through 120

Figure 8.

198 YOUNG ET AL.

population doublings and were induced to differentiateinto cells from all three primary germ layer lineages, i.e.,neuroectoderm (neurons and glial-like cells), mesoderm(endothelium), and endoderm (hepatocyte-like cells).

Young and colleagues (Young, 2004; Young and Black,2004; Young et al., 2004; this study) isolated an undiffer-entiated cell from skeletal muscle and dermis of adultmammals, including newborn to geriatric humans, anddesignated it as PPELSC. Sorted human PPELSCs dis-play a CD10�, CD66e�, CD1a–, CD2–, CD3–, CD4–, CD5–,CD7–, CD8–, CD9–, CD11b–, CD11c–, CD13–, CD14–,CD15–, CD16–, CD18–, CD19–, CD20–, CD22–, CD23–,CD24–, CD25–, CD31–, CD33–, CD34–, CD36–, CD38–,CD41–, CD42b–, CD45–, CD49d–, CD55–, CD56–, CD57–,CD59–, CD61–, CD62E–, CD65–, CD68–, CD69–, CD71–,CD79–, CD83–, CD90–, CD95–, CD105–, CD117–, CD123–,CD135–, CD166–, Glycophorin-A–, MHC-I–, HLA-DRII–,FMC-7–, Annexin-V–, and LIN– cell surface profile (Youngand Black, 2004). Scl-40� was derived from a single cell byrepetitive single-cell clonogenic analysis followed bytransfection with LacZ to provide a genomic marker (thisstudy). The PPELSCs exhibit capabilities for extendedself-renewal, i.e., over 400 population doublings for sortedhuman cells and Rat-A2B2 and a minimum of 287 popu-lation doublings for clone Scl-40�, without loss of pluripo-tentiality (Young, 2004; Young et al., 2004; this study).Both the parental clone, Rat-A2B2, and its transfectedprogeny, Scl-40�, are telomerase positive (Young et al.,2004; this study). The PPELSCs were induced to differen-tiate into cells from all three primary germ layer lineages,i.e., 7 or more ectodermal cell types (neuronal progenitorcells, neurons, ganglia, astrocytes, oligodendrocytes, ra-dial glial cells, keratinocytes) (Tables 1 and 2; Figs. 2A–I,

6E–G), 20 or more mesodermal cell types (skeletal muscle,cardiac muscle, smooth muscle, white fat, brown fat, hy-aline cartilage, elastic cartilage, growth plate cartilage,articular cartilage, fibrocartilage, cortical bone, trabecularbone, loose fibrous connective tissues, tendon, ligament,scar-connective tissue, dermal connective tissues, endo-thelial cells, erythrocytes, monocyte/macrophages, T-cells,B-cells, neutrophils) (Tables 1 and 2; Figs. 3A–U, 6H–J,and 7), and 11 or more endodermal cell types (endodermalprogenitor cells, gastrointestinal epithelial cells, PanPCs,insulin-secreting �-cells, glucagon-secreting �-cells, soma-tostatin-secreting �-cells, pancreatic ductal cells, liver ovalcells, liver hepatocytes, liver biliary cells, and liver cana-licular cells) (Tables 1–3; Figs. 4A–R, 6K–M, and 8)(Young et al., 2004).

While published reports of MAPCs (Verfaillie and col-leagues) and PPELSCs (Young and colleagues) suggestthat they have similar activities, we would propose thatthey are different subsets of adult stem cells. This pro-posed difference is based on a comparison of their cellsurface antigenic profiles. MAPCs of Verfaillie and col-leagues display a CD10not determined (nd), CD13�, Flk1dim,c-kit–, CD44–, CD45–, CD66end, MHC class I–, and MHCclass II– cell surface profile. In contrast, PPELSCs ofYoung and colleagues display a CD10�, CD13–, Flk1nd,c-kit–, CD44nd, CD45–, CD66e�, MHC class I–, and MHCclass II– cell surface profile. Currently the two adult-derived precursor cell populations differ with respect tothe presence or absence of the cell surface antigen CD13,e.g., aminopeptidase. Further studies are necessary tocharacterize the similarities and differences betweenthese two populations of adult-derived stem cells.

TABLE 3. Glucose challenge: nanograms of secreted insulin per well

TM only 24 hr 5 mM—24 hr 5 mM—1 hr 25 mM—1 hr

Native islets 0 0 2215 282 658 36 308 51Islet-like structure 0 0 482 81 325 35 136 26

Fig. 8. Expression of insulin, glucagon, and somatostatin in Scl-40�(A–C); Scl-40� induced to form EndoSCs (D–F); and EndoSCs inducedto form PanPCs (G–I). Twenty-four hours after plating respective celltypes, i.e., Scl-40�, EndoSCs, and PanPCs, the cultures were switchedto islet-inductive medium (Bonner-Weir et al., 2000), containing serumwith endodermal inductive activity. Cultures were incubated for twoweeks and then processed for enzyme-linked immunoculture assay(ELICA) using primary antibodies to insulin, glucagon, and somatostatin.Visualization of bound antibody occurred with DAB. Original magnifica-tions, �100 (A–F), �400 (G), �300 (H), �200 (I). A–C: Scl-40� expandedin medium containing PDGF-like (proliferative) and ADF-like (inhibitory)activities. A: Minimal intracellular staining for insulin. B: Minimal intra-cellular staining for glucagon. C: Minimal intracellular staining for soma-tostatin. D–F: EndoSCs were generated from the Scl-40� by directedlineage induction. Scl-40� was expanded in serum-free medium con-taining PDGF-like (proliferative) and ADF-like (inhibitory) activities. Twenty-four hours after initial plating Scl-40� was switch to serum-free mediumcontaining endodermal inductive activity (EIM) for two passages. By the endof the second passage in EIM the cells increased to a uniform size andshape and assumed contact inhibition, forming a single confluent layer ofEndoSCs. D: Diffuse distribution of individual cells stained intracellularly forinsulin. E: Diffuse distribution of individual cells stained intracellularly forglucagon. F: Diffuse distribution of individual cells stained intracellularly for

somatostatin. G–I: PanPCs were generated from EndoSCs by directedlineage induction. EndoSCs were expanded in EIM. Twenty-four hours afterreplating, EndoSCs were switched to PanPC-induction medium. A mini-mum of two passages were required for the induction process. G: Three-dimensional nodular ILSs and surrounding mononucleated cells showingmoderate to heavy intracellular staining for insulin. H: Three-dimensionalnodular ILSs with a few centrally located cells showing heavy intracellularstaining for glucagon. I: Three-dimensional nodular ILSs and some sur-rounding mononucleated cells showing moderate to heavy intracellularstaining for somatostatin. J–M: Three-dimensional ILSs (J and K) inducedfrom Scl-40� and native rat pancreatic islets (L and M). Cultures werephotographed with phase-contrast microscopy, original magnification,�100. J and K: Three-dimensional ILSs (3D-ILSs) were induced from Scl-40� clone by sequential directed lineage induction, i.e., Scl-40� to En-doSCs to PanPCs to ILSs. For an abbreviated induction protocol, seeabove. The induced transition was monitored by changes in phenotypiclineage expression markers (Table 1). Cultures were photographed withphase-contrast microscopy, original magnification, �100. J: Induced single3D-ILS. K: Induced group of 3D-ILSs. L and M: Pancreatic islets from 9- to10-week-old male Wistar-Furth rats (�220 g) were isolated as described.Cultures were incubated for 24 hr and photographed with phase-contrastmicroscopy, original magnification, �100. L: Native Wistar-Furth pancreaticislet. M: Native Wistar-Furth islet grouping.

199ADULT PLURIPOTENT EPIBLASTIC-LIKE STEM CELLS

Adult Pluripotent Stem Cells vs. EmbryonicStem Cells

Young et al. (2004) proposed that it would be advanta-geous to use adult-derived pluripotent stem cells for genetherapy and tissue engineering rather than using embry-onic stem cells. The PPELSCs can be isolated as a smallbiopsy of skeletal muscle or dermis from newborn to geri-atric individuals. In addition, these stem cells are telom-erase positive, indicating that vast quantities of cells canbe produced from a few harvested cells. This suggests thatpatients awaiting transplantation therapies could becometheir own stem cell donors. The use of autologous stemcells could eliminate the need for immunosuppressanttherapy with its associated morbidity and mortality basedon donor/host human leukocyte antigen (HLA) mis-matches. This is especially important where an identicalHLA match is essential for survival. PPELSCs remainquiescent in serum-free defined medium in the absence ofinhibitory agents to induction or differentiation. Indeed,the addition of exogenous or endogenous inductive agentsis a crucial step for lineage commitment and differentia-tion of these adult stem cells. Further, PPELSCs could beinduced to form cells from all three primary germ layerlineages, i.e., ectoderm, mesoderm, and endoderm. Onceinduced to commit to a particular tissue lineage, theseadult stem cells assume the normal biological clock of50–70 population doublings before programmed cell se-nescence and cell death occurs. The above attributes sug-gest that adult PPELSCs, unlike embryonic stem cells, areunder very strict regulatory control. Adult-derived undif-ferentiated PPELSCs do not lose their inherent stem cellcharacteristics when transfected with a genomic se-quence. When undifferentiated stem cells were deliveredin vivo after injury, they were recruited and retainedwithin tissues undergoing repair. And PPELSCs could beinduced to form a biologically functional three-dimen-sional tissue construct. Thus, reports by Young and col-leagues (Young, 2004; Young and Black, 2004; Young etal., 2004; this study) support the proposal that undiffer-entiated PPELSCs derived from adult skeletal muscleand/or dermis can serve as a source of donor cells for genetherapy and/or tissue engineering.

CONCLUSIONThis is the first report of a clonal population of adult

pluripotent stem cells generated from a single postnatalcell by repetitive single-cell clonogenic analysis andthereby forming a pure population of undifferentiatedadult stem cells. The clone expressed stem cell character-istics parallel to embryonic stem cells with respect to size,ratio of nucleus to cytoplasm, expression of embryonicmarkers in the undifferentiated state (i.e., SSEA-4, CEA-CAM, and Oct-4), telomerase activity, extensive capabili-ties for self-renewal, and pluripotentiality, i.e., the abilityto form cells from all three primary germ layer lineages.The clone differed from embryonic stem cells with respectto not having the capacity to spontaneously differentiatein culture in the absence of inhibitory agents and induc-tive factors. The clone was stably transfected by a genomicsequence. Transfection did not alter the expressed stemcell characteristics of the undifferentiated clone. The clonewas recruited and retained within damaged myocardialtissues undergoing repair. The clone differed from embry-onic stem cells with respect to not forming teratomaswhen implanted as undifferentiated cells in vivo. The

clone can generate biologically functional tissue by di-rected lineage induction. This study thus supports theproposal of Young et al. that undifferentiated PPELSCsderived from adult skeletal muscle and/or dermis com-prise a potential source of donor cells for gene therapyand/or tissue engineering.

ACKNOWLEDGMENTSI thank Paul A. Lucas for the generous exchange of

ideas and reagents. I thank my collaborators, co-authors,and technical assistants for their insight and work ethicand John Knight for photographic assistance. SS10 andSS12 were the generous gifts of T. Ryusaki, MorphoGenPharmaceuticals, Inc., San Diego, CA. The following anti-bodies were obtained from the Developmental Studies Hy-bridoma Bank developed under the auspices of theNICHD and maintained by the University of Iowa, De-partment of Biological Sciences, Iowa City, IA 52242:MC480, MC631, and MC813-70 developed by D. Solter;FORSE-1 developed by P. Patterson; RAT-401 and Ripdeveloped by S. Hockfield; RT-97 developed by J. Wood;8A2 developed by V. Lemmon; SV2 developed by K.M.Buckley; VM-1 developed by V.B. Morhenn; 151-Ig devel-oped by A. Hubbard; 40E-C developed by A. Alvarez-Buylla; F5D developed by W.E. Wright; MF-20 andALD-58 developed by D.A. Fischman; A4.74 developed byH.M. Blau; CIIC1 developed by R. Holmdahl and K. Ru-bin; D1-9 developed by X.-J. Ye and K. Terato; 9/30/8A4and 12/21/1C6 developed by B. Caterson; 12C5 developedby R.A. Asher; WV1D1(9C5) and MP111B101 developedby M. Solursh and A. Frazen; and HA4c19 developed by A.Hubbard. We thank Robert L. Price, PhD, director of In-strumentation Resource Facility at the University ofSouth Carolina School of Medicine, for the use of hisfacility for the confocal microscopy images. This researchwas supported by grants from Rubye Ryle Smith Charita-ble Trust (H.E.Y.), Lucille M. and Henry O. Young EstateTrust (H.E.Y.), MedCen Community Health Foundation(H.E.Y., T.A.S., J.H., F.P.B., A.C.B.), MorphoGen Pharma-ceuticals, Inc. (H.E.Y.), NIH grants K25-HL67097 andHL072096 (M.J.Y.), NASA Cooperative Agreement NCC5-575 (M.J.Y.), and the University of South Carolina Re-search and Productive Scholarship Program (M.J.Y.).

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