Most of the prior hair stem cell functional studies wereperformed using rodent tissues. In this study, we havedeveloped a new method to isolate human adult stemcells by culturing cells from human hair follicles in ahuman embryonic stem cell (hESC) culture condition. Wehave shown that the isolated cells are capable of differ-entiating into neurons, smooth muscle cells, and melano-cytes in specific induction medium. The differentiatedcells not only acquire lineage-specific markers but alsodemonstrate appropriate functions in
ex vivo
conditions.These cells appear to be located in the bulge area ofhuman hair follicles.
Materials and Methods
Cell Culture
Human hair follicles were isolated as described previous-ly.
23–25
Human scalp tissues (0.5
2 cm
2
or less) from13 patients (50 to 65 years old) and 3 autopsies wereobtainedthroughtheCooperativeHumanTissueNetworkwith approval from the Institutional Review Board of theUniversity of Pennsylvania. Tissues were rinsed, trimmedto remove excess adipose tissues, cut into small pieces,and subjected to enzymatic dissociation in 12.5 mg/mldispase (Invitrogen, Carlsbad, CA) in Dulbecco’s modi-fied Eagle’s medium (DMEM) for 24 hours at 4°C. Aftertreatment, the epidermis was peeled off from the dermis,and hair follicles were plucked from the dermis. Hairfollicles were rinsed thoroughly with phosphate-bufferedsaline to prevent contaminating epidermal or dermal cellsand examined under an inverted microscope. To confirmthe integrity of plucked hair follicles, we fixed some hairfollicles in 10% buffered formalin, embedded them inparaffin, and sectioned and stained them with hematox-ylin and eosin (H&E). To obtain viable single cells fromfollicular epithelium, hair follicles were treated twice with0.25% trypsin/ethylenediamine tetraacetic acid (Invitro-gen) for 30 minutes at 37°C. The cell suspension wasfiltered through a 40-
m cell strainer (BD Falcon, Bed-ford, MA), and cell numbers were counted. Single cellswere cultured in noncoated flasks in media designatedfor hESCs. Human ESC medium consisted of 80% knock-out DMEM/F-12 medium (Invitrogen), 20% knockout se-rum replacer (Invitrogen), 200 mmol/L
L
-glutamine (In-vitrogen), 0.1 mmol/L
-mercaptoethanol (Sigma, St.Louis, MO), 1% nonessential amino acids (Invitrogen),and 4 ng/ml basic fibroblast growth factor (bFGF) (Re-search Diagnostics, Concord, MA).
26,27
This mediumwas conditioned by using it for 48 hours as growth me-dium for mouse embryonic fibroblasts (MEFs) as de-scribed previously.
28
Human ESC medium conditionedby MEFs was mixed with fresh hESC medium at a 3:1ratio, sterilized by filtration, and supplemented with addi-tional bFGF at 4 ng/ml before use.For self-renewal, dissociated individual cells from hairspheres were serially diluted in hESC medium in un-coated 96-well plates. Each well was assessed micro-scopically for the presence of a single cell. The wellscontaining no cells or more than one cell were excluded.The human ESC line H9 was obtained from the WiCellResearch Institute, Inc., (Madison, WI). Cells were cul-tured on mitotically inactivated MEF feeder layers asdescribed previously.
26,27
Embryoid bodies were derivedfrom hESCs as described previously.
28
Differentiation Assays
Hair spheres and attached cells were enzymatically dis-sociated into single cells before plating onto tissue cul-ture-grade plastic coated with 10 ng/ml fibronectin(melanogenic differentiation) or 0.1% Matrigel (BDBiosciences, San Jose, CA; neuronal and smooth muscledifferentiation) in differentiation medium. Melanogenicdifferentiation medium exclusively differentiated hESCsinto melanocytic lineages.
29
It contains dexamethasone(0.05
mol/L; Sigma), insulin-transferrin-selenium (1
;Sigma), linoleic acid-bovine serum albumin (1 mg/ml;Sigma), low-glucose DMEM (30%; Invitrogen), MCDB201 (20%; Sigma),
L
-ascorbic acid (10
4
mol/L; Sigma),conditioned media of mouse L-Wnt3a cells (AmericanType Culture Collection, Manassas, VA) (50%), stem cellfactor (100 ng/ml; R&D Systems, Minneapolis, MN), en-dothelin-3 (100 nmol/L; American Peptide, Sunnyvale,CA), cholera toxin (20 pmol/L; Sigma), the phorbol ester12-
O
-tetradecanoylphorbol-13-acetate (50 nmol/L; Sigma),and bFGF (4 ng/ml; R&D Systems). For smooth muscledifferentiation, dissociated cells were cultured in a mediumcontaining 90% knockout DMEM/F-12 medium, 1% nones-sential amino acids solution, 10% fetal calf serum, and 10ng/ml transforming growth factor-
1 (R&D Systems). Neu-ron differentiation medium and steps were used as previ-ously described.
20
Immunocytochemistry and Immunohistochemistry
Cells were fixed with 4% paraformaldehyde and stainedwith primary antibodies specific for Oct4 (mouse mono-clonal, 1:200; Santa Cruz Biotechnology Inc., Santa Cruz,CA), Nanog (goat polyclonal, 1:100; R&D Systems), mi-crophthalmia-associated transcription factor (MITF)(mouse monoclonal, 1:200; NeoMarkers), HMB45/SILV(mousemonoclonal,1:500;DakoCytomation,Carpinteria,CA), microtubule-associated protein-2 (MAP2, mousemonoclonal, 1:1000; Sigma), chromogranin (rabbit poly-clonal, 1:100; Zymed), Neurofilament-M (NFM) (mousemonoclonal,1:100;a giftfromDr.Virginia Lee),glutamate(rabbit polyclonal, 1:1000; Sigma), pan-cytokeratin(mouse monoclonal, 1:500; Sigma), and smooth muscleactin (SMA) (mouse monoclonal, 1:100; Chemicon, Te-mecula, CA). Monoclonal antibodies against the surfacemarkers of hESCs—stage-specific embryonic antigenstage-specific embryonic antigen (SSEA)-3 and SSEA-4—were obtained from The Wistar Institute (where theywere originally developed). Isotype-matched mouse an-tibodies or normal rabbit IgG was used as control. Afterwashings, primary antibody binding was detected viacorresponding goat anti-mouse or anti-rabbit Alexa Fluor488-conjugated,ordonkeyanti-goatAlexaFluor568sec-
1880 Yu et al
AJP June 2006, Vol. 168, No. 6
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