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Human Eyelid Adipose-Derived Stem Cells (hEA) hEA-00 bcellbio Inc. • TEL +82-2-974-2472 • Fax +82-2-974-2473 • www. bcellbio.co.kr • email@example.com bcellbio Inc. 420 Science Hall I, Seoul Women‟s University 623 Hwarangro, Nowon-gu, Seoul, 139-774, Korea Tel. +82-2-974-2472 Fax. +82-2-974-2473 Human Eyelid Adipose-Derived Stem Cells (hEA) Instructions for Use Safety Statements THESE PRODUCTS ARE FOR RESEARCH USE ONLY. Not approved for application to human, or for use in vitro diagnostic or clinical procedures. Cryopreservation Medium Cells were cryopreserved in „Cryoscarless-DMSO free‟ (BioVerde, Japan) which is FBS and DMSO–free medium. Unpacking and Storage Instructions Transfer cryovials from the dry ice packing into liquid nitrogen storage. Otherwise, immediately thaw and use for the culture of cells. If no dry ice remains, please contact Customer Service. Cell Thawing of Cells for the Initiation of Culture Process 1. Thaw quickly the cryovial in a 37℃ water bath. 2. Wipe cryovial with 70% ethanol and transfer to a sterile field. 3. (e.g., RPMI1640 containing 5.5 mM glucose and 10% FBS). * FBS could be substituted with human serum. 4. Centrifuge at 1,500 rpm for 5 min at room temperature (RT, 18-25℃). 5. Discard the suspension solution and resuspend the cell pellet in 5 ml of temperature-equilibrated medium and centrifuge at 1,500 rpm for 5 min at RT. 6. Repeat step 5. 7. Resuspend the pellet in culture medium (e.g., RPMI1640 containing 5.5 mM glucose and 10% FBS) and assess the cell viability using trypan blue staining. 8. Cultivate cells in the culture medium (e.g., RPMI1640 containing 5.5 mM glucose and 10% FBS) at 37℃, 5% CO2. 2 * The recommended seeding density for hEA is 1,500-2,000 cells per cm . Subculturing 1. Discard all medium from the culture flasks. 2. Wash the adhering cell layer twice with a basic medium (e.g. RPMI1640). 3. Add sufficient amount of 37℃ trypsin/EDTA solution to the adhering cell layer. 4. Incubate the culture flasks on a 37℃ warming tray for 1 to 2 min. 5. When cells are detached, add an equal volume of the culture medium. 6. Transfer the cell suspension into conical tubes. 7. Centrifuge at 1,500 rpm for 5 min at RT. 8. Discard the supernatant and suspend the cells with the above basic medium. 9. Centrifuge at 1,500 rpm for 5 min at RT. 10. Repeat steps 8 and 9. 11. Count the number of cells using a haemocytometer. 2 12. Seed cells into a new flask at a density of 1,500-2,000 cells per cm . 13. Cultivate cells in flasks at 37℃, 5% CO2. * hEA culture medium should be changed at 3-4 days interval after plating. * Typical period from seeding to subculture is 6-7 days. Ordering Information Product Cat. No Cell No. 6 hAM-00 1 x 10 / cryovial 6 hEA-00 1 x 10 / cryovial Human Adult Stem Cells 6 hAA-00 1 x 10 / cryovial 6 hTA-00 1 x 10 / cryovial Related Products Cryopreserved Cells Product Cat. No Cell No. 6 Human Amniotic Membrane- hAM-ISC-00 1 x 10 / cryovial 7 derived Insulin-Secreting Cells hAM-ISC-01 1 x 10 / cryovial 6 Human Eyelid Adipose-derived hEA-ISC-00 1 x 10 / cryovial 7 Insulin Secreting Cells hEA-ISC-01 1 x 10 / cryovial Insulin-Secreting Cells Differentiation Media Kit Description Cat. No. Unit IS1-00 1 Kit Stock Solution IS1-01 1 mL (10X) IS1-02 2 mL IS1-03 3 mL IS1-10 1 Kit IS1-11 1 mL IS1-12 2 mL IS1-13 3 mL IS1-20 1 Kit Stock Solution IS1-21 5 mL (50X) IS1-22 10 mL IS1-23 15 mL IS1-30 1 Kit IS1-31 30 mL IS1-32 60 mL IS1-33 90 mL Quality Control The hEA were prepared from abdominal adipose tissues donated from a participant under “informed consent”. Cell viability and morphology were assessed after recovery from cryopreservation. Data and further information are available upon request. firstname.lastname@example.org Product Warranty bcellbio Inc. warrants cell products only if the recommended protocols are followed. Cryopreserved hAA is assured to be viable and cell numbers at recovery to be accurate, when handled exactly according to our instructions for thawing and subculturing. Ordering Order on-line at http://www.bcellbio.co.kr/eng/eng_infomail.php Email us at email@example.com or please call us. INTRODUCTION Adult stem cells isolated from the human eyelid adipose tissue (hEA) exhibit neuron-like bipolar morphology (Fig. 1A) until their senescence at p14 or later. Throughout the culture, they undergo an average of 36.3 16 doublings and produce 6.9 x 10 cells (Fig. 1B). hEA both at p2 and p14 express stem cell-specific genes of OCT4, REX1, and SCF, endoderm-specific genes of BMP4, AFP, GATA4, and HNF4A, and mesoderm/endoderm specific genes of vimentin (VIM) and CK18. However, they do not express mesoderm- specific brachyury (T) and endoderm-specific BMP2 genes (Fig. 1C). Immunocytochemical analyses show that hEA at p4 express stem cell-specific proteins of TRA 1-60, SSEA-3, SSEA-4, and CD90, and other proteins, but not proteins of CD31 and HLA-DR (Fig. 1D). When they are cultured in appropriate differentiation media, they show adipogenic, osteogenic, or chondrogenic differentiation potential (Fig. 2). Flow cytometric analyses reveal that hEA are positive for the antigens of SSEA-4, HLA-ABC, CD44, CD49d, CD59, CD90, and CD105, weakly positive for TRA 2-54 and CD34, and negative for HLA-G, HLA-DR, CD31, CD45, CD106, and CD117 antigens (Fig. 3). hEA also express many neural cell-related mRNAs and proteins such as GABA, GalC, serotonin, b-tubulin III, and GFAP, most of which are typically present in human neural crest cells and in neuroblastoma cells (Fig. 4). When hEA are induced to differentiate into insulin-secreting cells by cultivating in the IS1 medium (bcellbio, Korea), the differentiated cells secrete insulin and c-peptide into the medium in a high glucose-dependent manner (Fig. 5A). They, particularly in colony-forming areas, are intensively stained with dithizone (DTZ), a zinc-chelating agent known to stain pancreatic β-cells (Fig. 5B). Stem Cell-Related Characters of hEA A B Growth Curve of hEA C D Fig. 1. Stem cell-related characters of hEA. A, Morphology of hEA at p2 and p14; B, Cumulative doubling number of the four hEA lines; C, Stem cell-related gene expression of hEA at p2 and p14; D, Immunocytochemistry analysis. Scale bar = 200 μm. Fig. 2. Multi-differentiation potential of hEA. A, Oil red O-stained adipogenic cells; B, von Kossa-stained osteogenic cells; C, Alcian blue-stained chondrogenic cells. Fig. 3. Flow cytometric analyses of hEA. The hEA at passage 3 are strongly positive for SSEA-4, HLA-ABC, CD44, CD49d, CD59, CD90, and CD105; weakly positive for TRA 2-54 and CD34; and negative for HLA-DR, CD31, CD45, CD106, and CD117 antigens. Abbreviations: APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin. Fig. 4. Neural properties of hEA. A, expression of neuron-related genes by hEA at p2, p8, and p14. B, immunocytochemistry of hEA at p4, p8, and p12. Scale bar = 500 μm. Abbreviations: AA, human abdominal adipose-derived stem cells at p3; NB, neuroblastoma cells; p, passage. A B Fig. 5. Insulinogenic differentiation of hEA. A, ELISA analyses of insulin and c-peptide secreted by differentiated hEA in response to low (5.5 mM) or high (25 mM) glucose challenge following culture in DMEM or RPMI1640 medium containing IS1 Kit or not (Control). The amount of insulin and c-peptide were assessed by using human-specific insuln ELISA kit (Mercodia, Sweden) and human-specific c- peptide ELISA kit (Alpco, USA), respectively. Cell donors were female Asian within 20-40s of age. B, Dithizone staining of hEA at passage 3 before (a) and after differentiation into insulin-secreting cells (b). Dithizone is a zinc-chelating agent known to identify insulin-producing β-cells.
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