Disease Model iPS Cell Lines User Manual
Contents
1. 1 Remove the vial from liquid nitrogen and thaw quickly in 37 C 2 Remove the vial from the water bath as soon as the cells are half thawed and sterilize by spraying with 70 ethanol 3 Transfer the cells with 10 ml of HFF medium to a 15 cm conical tube and pellet the cells by centrifugation at 200 g for 5 min 4 Discard the supernatant and resuspend the cells with 10 ml fresh HFF medium and plate the cells at seed density of 1 x 104 cells cm 5 Incubate at 37 C with 5 COs until the cells reach 80 90 confluency 6 Change medium twice a week or when pH decreases C Growth Conditions for Human Disease Specific iPS cells 1 Required media and reagents Reagent SC100M 1 SC150M 1 ZRD Y 01 05 25 Invitrogen Millipore 888 266 5066 Toll Free 650 968 2200 outside US Information Human ESC iPSC Growth Medium for feeder dependent conditions CRYO GOLD Human ESC iPSC Cryopreservation Medium Y27632 ROCK Inhibitor Knockout DMEM F12 Accutase Page 7 System Biosciences SBI User Manual 2 Thawing human iPS cells To insure the highest level of viability be sure to warm medium to 37 C before using it on the cells Due to the low survival rate of cryopreserved human iPS cells the recovery is expected to take at least one week Remove the vial from liquid nitrogen and thaw quickly in a 37 C water bath Remove the vial from the water bath as soon as the cells are half thawed and s2. SBI System Biosciences Disease Specific iPS Cells Cat SC60XA Series User Manual Store in Gas Phase of Liquid Nitrogen A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement ver 1 031011 contained in this user manual Disease Specific 3 3 iPS Cell Lines Cat SC60xA B xxx Contents l Human Disease Specific iPS CellS ceeeeeeeeeeeneeeeeeees 2 A DOSCFIPUOM cese cccecesheecexaneccesttbastins deat dedasd net einari aeia 2 B Human Wild type iPSC cell line Cat SC600A B WT 2 C Type Diabetes iPS cell line Cat SC602A B DT 3 D Metachromatic Leukodystrophy MLD iPS cell line Cat SC601A B MLD E Amyotrophic Lateral Sclerosis iPS cell line Cat SC603A B ALS F Muscular Dystrophy MD iPS cell line Cat SC604A B MD 3 G Glioblastoma Multiforme GBM iPS cell line Cat SC605A B GBM H Parkinson s Disease PD iPS cell lines SC607A B PD amp SC608A B PD 3 Il Culture and Growth Conditions for Disease Specific iPSC and Feeder Cells 4 A Culture Conditions for MEF feeder cells 0 cccseeee 4 B Culture Conditions for HFF cells 0 cccecceeeeeeeeeeeeeeeees 6 C Growth Conditions for Human Disease Specific iPS cells 7 IIl Validation Data ccccccccececceeeeseceeeeseeeeceeeseeeeeseaesteeeseaeeas 9 IV References ceeece cece eeeneeeeeeeceeeeeeeaee
3. cells The magic brew Nature 448 260 262 Takahashi K and Yamanaka S 2006 Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors Cell 126 663 676 Takahashi K et al 2007 Induction of pluripotent stem cells from adult human fibroblasts by defined factors Cell 131 861 72 Park IH et al 2008 Reprogramming of human somatic cells to pluripotency with defined factors Nature 451 141 6 Baker Monya 2007 Adult cells reprogrammed to pluripotency without tumors Nature Reports Stem Cells 2007 12 11 Nakagawa M et al 2008 Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts Nature Biotechnology 26 101 106 Okita K et al 2007 Generation of germline competent induced pluripotent stem cells Nature 448 313 7 Yu J et al 2007 Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells Science 318 1917 1920 V Technical Support For more information about SBI products or to download manuals in PDF format please visit our website http www systembio com For additional information or technical assistance please call or email us at tech systembio com 650 968 2200 VI Licensing and Warranty Statement Limited Use License Use of the Disease Specific iPS cells e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components
4. followed by fluorescent labeled Alexa Fluor secondary antibodies Invitrogen Phase contrast bottom left shows the morphology of the MLD iPS cell line Positive AP staining is shown on the bottom right Cat SAB KIT 1 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual ALS iPS cell line Amyotrophic lateraksclerosis A TRA 60 1 Figure 4 Stem cell markers for Nanog top left SSEA3 top right and TRA 1 60 bottom right were determined by immunocytochemistry using antibodies for Nanog SSEA3 and TRA 1 60 followed by fluorescent labeled Alexa Fluor secondary antibodies Invitrogen Phase contrast middle left shows the morphology of the ALS iPS cell line Positive AP staining is shown on the middle right Cat SAB KIT 1 Page 12 ver 2 093013 www systembio com Disease Specific iPS Cell Lines Cat SC60xA B xxx Muscular Dystrophy iPS cell line TRA 60 1 Figure 5 Stem cell markers for Nanog top left SSEA3 top right Oct4 bottom left and TRA 1 60 bottom right were determined by immunocytochemistry using antibodies for Nanog SSEA3 Oct4 and TRA 1 60 followed by fluorescent labeled Alexa Fluor secondary antibodies Invitrogen Phase contrast middle left shows the morphology of the MD iPS cell line Positive AP staining is shown on the middle right Cat SAB KIT 1 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manua
5. of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions e The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use e The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI e This Product should be used in accordance with the NIH guidelines developed for stem cell research SBI has pending patent applications related to the Product For information concerning licenses for commercial use contact SBI Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by Page 16 ver 2 093013 www systembio com Disease Specific iPS Cell Lines Cat SC60xA B xxx patents or subject to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Limited Warranty SBI warrants
6. that the Product meets the specifications described in this manual If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBs liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2013 System Biosciences SBI 888 266 5066 Toll Free 650 968 2200 outside US Page 17
7. LD Human ESC iPSC Cryopreservation Medium Aliquot it at 1 ml per vial Put the vials in a cell freezing container and store the vials at 80 C overnight 10 Transfer the vials to liquid nitrogen for long term storage Validation Data 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual Figure 1 Stem cell markers for SSEA3 middle left and Nanog middle right Oct4 bottom left and TRA 1 60 bottom right were determined by immunocytochemistry using antibodies for SSEA3 Nanog Oct4 and TRA 1 60 followed by fluorescent labeled Alexa Fluor secondary antibodies Invitrogen Phase contrast top left shows the morphology of the Wild Type iPS cell line Positive AP staining is shown on the top right Cat SAB KIT 1 Type Diabetes iPS cell line Figure 2 Stem cell markers for Nanog top left and SSEA4 top right were determined by immunocytochemistry using antibodies for SSEA4 and Nanog followed by fluorescent labeled Alexa Fluor secondary antibodies Invitrogen Phase contrast bottom left shows the morphology of the Type Diabetes iPS cell line Positive AP staining is shown on the bottom right Cat SAB KIT 1 Page 10 ver 2 093013 www systembio com Disease Specific iPS Cell Lines Cat SC60xA B xxx MLD iPS cell line Figure 3 Stem cell markers for Nanog top left and SSEA4 top right were determined by immunocytochemistry using antibodies for SSEA4 and Nanog
8. ce and incubate for 1 min at room temperature 5 Add 5 ml MEF medium and break up the cells to a single cell suspension by pipetting up and down Count the number of cells 6 Seed the cells on gelatin coated dishes 2 x 10 cells per 100 mm dish or 3 x 10 cells per well of a 6 well plate 7 Cells should be ready to use by the next day B Culture Conditions for HFF cells HFF cells for feeder cells can be obtained from SBI Cat PC502B HFF The HFF cells provided in the iPSC kits are not suitable for use as feeder cells 1 Required media and reagents Reagent Information HFF Medium DMEM containing 10 FBS 2mM glutamine 0 1 M nonessential amino acids and 50 U and 50 ug ml penicillin and streptomycin 2x Cold 20 DMSO and 80 FBS Freezing Media 2 Gelatin treatment of plates for HFF feeder cells optional 1 Add enough sterile autoclaved 0 1 gelatin to cover the bottom of the wells Approximate amounts 10cm plate 5 ml 6 well 1 5 ml well 24 well 0 5 ml well 96 ell 200 ul well 2 Incubate the gelatin coated dishes for at least 15 min at 37 C 3 Aspirate excess gelatin solution before using 3 Thawing HFF cells To insure the highest level of viability be sure to warm medium to 37 C before using it on the cells Cells should be plated at a minimum cell density of 1 x 10 cells cm Page 6 ver 2 093013 www systembio com Disease Specific iPS Cell Lines Cat SC60xA B xxx
9. e cell pellet in 10 ml MEF medium 6 Count the number of cells plate cells at 0 5x10 cells cm and incubate at 37 C with 5 COs 5 Freezing MEF cells 1 Follow steps 1 4 from the Passaging MEF cells protocol above N Discard the supernatant and resuspend the pellet in MEF medium Add approximately 1 ml for each T75 flask 3 Count the number of cells and dilute the cell suspension to 1 x 10 cells ml 4 Add an equal volume of cold 2X Freezing Media to the cell suspension 5 Aliquot 1 ml of suspension into each cryovial 5 x 10 cells vial 6 Place the vials in a cell freezing container and keep itat 80 C overnight 7 Transfer the vials to a liquid nitrogen take for long term storage 6 Mitomycin C treatment of MEF Mitomycin C acts to halt the division of MEF cells so that they can be used to condition the medium for human iPS cells MEF cells should be at confluence when treated with mitomycin C 1 Add 6 ml of fresh MEF medium contain 50 ul of mitomycin C solution 1 mg ml to one T75 flask of confluent MEF cells and swirl it briefly The final concentration of mitomycin C is 8 ug ml 2 Incubate at 37 C for at least 3 hrs 3 Aspirate the mitomycin C containing medium off the cells and wash the cells twice with 10 ml PBS 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual 4 Aspirate PBS and add 1 ml of 0 25 trypsin EDTA swirl to cover the entire surfa
10. eeaaeeeeeeetaeeeeaaeeseneeee 16 V Technical SUPPOSt ceccceceeeceeeeeeeeeeeeeeeeeseeeeseaeeteeeeennees 16 Vi Licensing and Warranty Statement cceceeeeeeeeeees 16 888 266 5066 Toll Free 650 968 2200 outside US Page 1 3 System Biosciences SBI User Manual List of Components Each disease specific iPS cell line set comes as one vial with 2 x 10 cells The product is shipped on dry ice and should be immediately stored in the gas phase of liquid nitrogen In general iPS cells are challenging to culture and should only be operated by researchers experienced in the intricacies of human embryonic stem hES cell culture The methods for culture are nearly identical to hES cell culture although more care and increased maintenance will be required Disease specific iPS cells must be grown on feeder cells for culture I Human Disease Specific iPS Cells A Description SBI and DV Biologics have partnered to develop novel human iPS cell lines from patient derived sources Utilizing iPS cell lines from these disorders represents an opportunity to recapitulate both normal and pathological tissue formation in vitro for the ideal drug development screening cell line to facilitate new therapeutic discovery and disease modeling Human disease specific induced pluripotent stem cells iPSCs cat 4 SC60xA B xxx were generated by transducing source cells with retroviruses individually encoding the four human transcription fact
11. in Early in the course of the disease the most prevalent symptoms are movement related including tremors shaking rigidity slowness of movement and difficulty with walking As Parkinson s disease progresses cognitive and behavioral problems may develop and dementia commonly occurs in the advanced stages of the disease The human patient dermal fibroblasts are derived from cultured skin explants from a single donor The patient with late onset Parkinson s disease was 81 year old Hispanic Caucasian male who was diagnosed with 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual Parkinson s disease at age 79 The patient with early onset Parkinson s disease was 41 year old Hispanic Caucasian male ll Culture and Growth Conditions for Disease Specific iPSC and Feeder Cells A Culture conditions for MEF feeder cells 1 Required media and reagents Reagent Information MEF Medium DMEM containing 10 FBS 2 mM glutamine 1x10 M nonessential amino acids and 50 U and 50 ug ml penicillin and streptomycin 2x Cold 20 DMSO and 80 FBS Freezing Media Mitomycin C 1 mg ml solution 2 Gelatin treatment of plates for MEF feeder cells 1 Add enough sterile autoclaved 0 1 gelatin to cover the bottom of the wells Approximate amounts 10cm plate 5 ml 6 well 1 5 ml well 24 well 0 5 ml well 96 well 200 ul well 2 Incubate the gelatin coated dishes for at least 15 min a
12. l Glioblastoma multiforme iPS cell line TRA 60 1 Figure 6 Stem cell markers for Nanog top left SSEA3 top right and TRA 1 60 bottom right were determined by immunocytochemistry using antibodies for Nanog SSEA3 and TRA 1 60 followed by fluorescent labeled Alexa Fluor secondary antibodies Invitrogen Phase contrast middle left shows the morphology of the ALS iPS cell line Positive AP staining is shown on the middle right Cat SAB KIT 1 Late Onset Parkinson s Disease iPS cell line TRA 1 60 Figure 7 Stem cell markers for Nanog top left SSEA3 top right TRA 1 60 bottom left were determined by immunocytochemistry using antibodies for Nanog SSEA3 and TRA 1 60 followed by fluorescent labeled Alexa Fluor secondary antibodies Invitrogen Positive AP staining is shown on the bottom right Cat SAB KIT 1 Page 14 ver 2 093013 www systembio com Disease Specific iPS Cell Lines Cat SC60xA B xxx Early Onset Parkinson s Disease iPS cell line TRA 1 60 Figure 8 Stem cell markers for Nanog top left Oct4 top right TRA 1 60 bottom left were determined by immunocytochemistry using antibodies for Nanog SSEA3 and TRA 1 60 followed by fluorescent labeled Alexa Fluor secondary antibodies Invitrogen Positive AP staining is shown on the bottom right Cat SAB KIT 1 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual IV References Rossant J 2007 Stem
13. nic insulin therapy is required for survival The type diabetes human iPS cells were derived from human patient fibroblasts from cultured skin explants from a single donor The patient was a 29 year old female of Hispanic Caucasian descent who was diagnosed with Type Diabetes at age 12 D Metachromatic Leukodystrophy MLD iPS cell line Cat SC601A B MLD Metachromatic leukodystrophy MLD is a group of disorders marked by storage buildup in the white matter of the central nervous system in the peripheral nerves and to some extent in the kidneys Similar to Krabb disease MLD affects the myelin that covers and protects the nerves This autosomal recessive disorder is caused by a deficiency of the enzyme arylsulfatase A Both males and females are affected by this disorder and death generally occurs within 6 to 14 years after onset of symptoms MLD has a juvenile onset that is marked by proximal and distal weakness hypotonia decreased or absent tendon reflexes and mild distal sensory loss The MLD iPS cell line were drived from human patient fibroblasts cultured from skin explants from a single donor They can be used as a model to study other polyneuropathies as well E Amyotrophic Lateral Sclerosis iPS cell line Cat SC603A B ALS ALS is the most common form of motor neuron disease Sporadic forms of unknown cause account for about 90 95 percent of ALS cases The human patient dermal fibroblasts are derived from cultured skin explant
14. ors Oct4 Sox2 KIf4 and c Myc that have been shown to induce the reprogramming of somatic cells to a pluripotent state The cells were derived using morphological selection criteria and without the use of fluorescent markers or drug selection When cultured under standard human ES cell culture conditions the morphology of SBI human disease specific iPSCs is identical to that of human ES cells The cells also express the pluripotency markers Nanog SSEA3 TRA 1 60 etc and demonstrate a strong endogenous AP activity Human iPS cells must be grown on a feeder cell layer Appropriate feeder cells for human iPS cells are mouse embryonic fibroblasts or human foreskin fibroblasts B Human Wild type iPSC cell line Cat SC600A B WT The SC600A B WT human iPS cell line is derived from cultured skin explants from dermal fibroblasts from a single donor The donor is a 64 year old Caucasian male with no known disease The human wild type iPS cell Page 2 ver 2 093013 www systembio com Disease Specific iPS Cell Lines Cat SC60xA B xxx line was generated and cultured in the same conditions as the diseased iPS cell lines It is an ideal control cell line for the disease and patient specific iPS cell lines C Type I Diabetes iPS cell line Cat SC602A B DTIl Type Diabetes is an autoimmune condition where the pancreatic beta cells are destroyed Patients display diabetes related autoantibodies The cause of type diabetes is polygenic and chro
15. s from a single human donor The patient was a 61 year old female of Latino Caucasian descent who was diagnosed with agressive and sporatic ALS F Muscular Dystrophy MD iPS cell line Cat SC604A B MD Muscular dystrophies are a group of genetic conditions characterized by progressive muscle weakness and wasting atrophy The Duchenne and Becker types of muscular dystrophy primarily affect the skeletal muscles which are used for movement and the muscles of the heart Mutations in the Dystrophin X linked gene cause Duchenne and Becker muscular dystrophy The human patient dermal fibroblasts are derived from cultured skin explants from a single donor The patient was one year old male of Latino Caucasian descent who had a deletion of Dystrohpin gene at exons 3 6 G Glioblastoma Multiforme GBM iPS cell line Cat SC605A B GBM Glioblastoma multiforme GBM is the most common and most aggressive malignant primary brain tumor in humans GBM is the most invasive type of glial tumor that grows rapidly and spreads to nearby tissues The human patient dermal fibroblasts are derived from cultured skin explants from a single donor The patient was 41 year old male of Latino Caucasian descent who was diagnosed by both MRI and CT SCAN at age 40 H Parkinson s Disease PD iPS cell lines SC607A B PD amp SC608A B PD Parkinson s disease PD is the result from the progressive death of dopamine generating cells in the substantia nigra in the midbra
16. t 37 C 3 Aspirate excess gelatin solution before use 3 Thawing MEF cells To insure the highest level of viability be sure to warm medium to 37 C before using it on the cells Cells should be plated at a minimum cell density of 1 x 104 cells cm 1 Remove the vial from liquid nitrogen and thaw quickly in a 37 C water bath 2 Remove the vial from the water bath as soon as the cells are half thawed and sterilize the tube by spraying with 70 ethanol 3 Transfer the cells with 10 ml of MEF medium to a 15 cm conical tube and pellet the cells by centrifugation at 200 g for 5 min Page 4 ver 2 093013 www systembio com Disease Specific iPS Cell Lines Cat SC60xA B xxx 4 Discard the supernatant and resuspend the cells with 10 ml fresh MEF medium and plate the cells at seed density of 10 cells cm 5 Incubate at 37 C with 5 COs until the cells reach 80 90 confluency 6 Change medium twice a week or when pH decreases 4 Passaging MEF cells Cells should be split when they reach confluency We recommend splitting the cells based on 0 5x10 cells cm a Discard the medium and wash the cells twice with PBS 2 Aspirate PBS and add 1 ml per T75 flask of 0 25 trypsin EDTA and incubate for 1 min 3 Add 5 ml of MEF medium and break up the cell clumps by gently pipetting up and down several times 4 Transfer cells into a conical tube and centrifuge at 200 g for 5 min 5 Discard the supernatant and resuspend th
17. terilize by spraying with 70 ethanol Transfer the cells with 10 ml of Human ESC APSC Growth Medium to a 15 ml conical tube and pellet the cells by centrifugation at 200g for 5 min While centrifuging remove MEF HFF medium from the feeder cell plates and wash the wells twice with Knockout DMEM F12 Then add 1 ml of Human ESC iPSC Growth Medium with 10 uM ROCK inhibitor Y 27632 Discard the supernatant from the human iPS cells and resuspend the cells with 1 ml fresh human ES medium containing 10 uM ROCK inhibitor Y 27632 Plate the cells on MEF or HFF feeder cells After 24 hours remove the media and replace with Human ESC iPSC Growth Medium without ROCK inhibitor Incubate at 37 C with 5 CO until the cells reach 80 confluency The Human ESC iPSC Growth Medium must be changed every day and human iPS cells subcultured every 5 7 days 3 Maintenance and Passage of human iPS cells In order to maintain pluripotency it is important not to keep human iPS cells in culture for long periods of time Aspirate the medium and wash the cells twice with 1 ml of PBS Remove PBS completely and add 0 5 ml of 1x Accutase and incubate for 2 min at room temperature Tap the bottom of the plate to dislodge the cells from the bottom of the plate Then aspirate the accutase Add 1 ml of DMEM F12 to the plate and carefully wash the feeder cells and aspirate the medium Repeat Add 1 ml of Human ESC iPSC Growth Medium containing 10 UM ROCK inhibi
18. tor to the plate and suspend the cell colonies by pipetting up and down It is important not to break up the colonies into single cells Remove a plate of MEF or HFF feeder cells from the incubator Aspirate the MEF medium Wash once with KO DMEM F12 medium Distribute 0 2 0 3 ml of the human iPS cell suspension to each well of a 6 well plate Add Human ESC iPSC Growth Medium with ROCK inhibitor to a final volume of 2 ml per well Right after plating the iPS cells gently swirl the plate back and forth and side to side and incubate at 37 C After 24 hours remove the media and replace with Human ESC iPSC Growth Medium without ROCK inhibitor The Human ESC iPSC Growth Medium must be changed every day and human iPS cells subcultured every 5 7 days Track the passage number of the iPS cells Page 8 ver 2 093013 www systembio com Disease Specific iPS Cell Lines Cat SC60xA B xxx 4 Freezing human iPS Cells Grow cells to the exponential phase in a 6 well plate Aspirate the medium and wash the cells twice with 2 ml PBS Add 0 5 ml of 1x accutase and incubate 2 min at 37 C Aspirate the accutase Add 1 ml of Knockout DMEM F 12 to the plate and carefully wash the feeder cells Add 1 ml of Human ESC iPSC Growth Medium to the plate Scrape the colonies off using a cell scraper Transfer the cell suspension to a 15 ml conical tube and spin the cells at 200 g for 5 min Discard the supernatant and resuspend the cells with 1 ml CRYO GO
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