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Induced Pluripotent Stem Cell Core Facility (Penn)

Directors: Morrisey, Edward, Ph.D.; Yang, Wenli, Ph.D.

Summary:

The Penn Institute for Regenerative Medicine (IRM), which has been at the forefront of stem cell research and translational medicine, established the iPSC Core in 2009 to promote this powerful technology on campus and surrounding institutions. The goals of the Core are:

• to facilitate derivation of induced pluripotent stem (iPS) cells from somatic cells;
• to provide expertise and training to researchers in embryonic stem (ES)/iPS cell culture;
• and to serve as a resource for sharing iPS cell lines and iPSC technology within the UPenn and the broader scientific community.

Affiliations:

People:

Resources:

Protocols

Reagents

  • Induced pluripotent stem cell lines ( Induced pluripotent stem cell line )

    "The Core has several established mouse and human iPSC lines for purchase for research purposes by non-profit institutions. For outside purchases, an MTA between UPenn and your institution is required."

Services

  • Basic hESC/iPSC culture training course ( Training service )

    "Basic techniques in hESC/iPSC culture including cell thawing, expansion, identification by morphology, manual and enzymatic passaging, freezing, and feeder-free culture will be covered. This training course will take 2 weeks to complete. Previous experience in mammalian cell culture is a prerequisite."

  • Characterization and validation services ( Material analysis service )

    "Newly derived iPS cell lines can be transferred to the investigator without further characterization and validation or they can be further expanded and characterized at the Core. The Core will ask the PI to make a decision for characterization and validation services before freezing down all newly derived iPSC cultures.

    New iPSC line characterization
    This service takes about 1 month to complete and the following assays are performed:
    • Immunocytochemistry for 4 pluripotency markers
    • qRT-PCR analysis for Oct4, Sox2, Nanog, Rex1, and Dmnt3b
    • Embryoid body formation and qRT-PCR analysis for 2 differentiation markers of each of the three cell lineages

    Karyotyping
    iPS cell culture will be expanded and live culture will be sent to Cell Line Genetics for G-band karyotyping.

    DNA finger printing
    Feeder-free iPS cell culture will be expanded and cells will be harvested. Genomic DNA will be isolated from iPS cells and the corresponding parental cell line. DNA will be sent to Cell Line Genetics for Short Tandem Repeat (STR) analysis for originating vs. resulting cell verification."

  • Custom services ( Support service )

    "Please contact the Core director if you require custom services not listed above. We will do our best to accommodate you."

  • Fibroblast isolation and expansion from skin biopsy ( Material processing service )

    "This service takes 3-4 weeks to complete and includes the following:

    • Fibroblast isolation from freshly collected skin biopsies
    • Establishment of fibroblast culture
    • Mycoplasma testing
    • Cryopreservation of several vials of early passage cells
    • Freezing of cell pellets for future RNA and DNA isolation

    Live cultures will then be used for iPSC reprogramming"

  • Genome editing using CRISPR technology ( Material processing service )

    Genome editing using CRISPR technology in hESCs and hiPSCs (reporter lines, KO, and KI)

  • iPSC derivation from blood using lentiviral vector or Sendai virus ( Material production service )

    "A minimum of 8 mls of blood is required for this service. Blood should be collected in CPT tube (BD biosciences) or other heparinized or EDTA tubes and delivered fresh to the Core. Please contact the Core director if you are submitting peripheral blood mononuclear cells (PBMCs). This service takes 3-6 months to complete and includes the following:

    • Isolation and cryopreservation of PBMCs from blood
    • Expansion of erythroblasts from PBMCs and mycoplasma testing
    • Transduction of erythroblasts with lentiviral vector or Sendai viral vectors
    • iPSC colony picking
    • Expansion of 4 iPSC clones
    • Mycoplasma testing
    • FACS analysis of 2 pluripotency markers (TRA-1-60 and SSEA4)
    • Cryopreservation of iPSC lines (4-6 vials/clone)
    • Will deliver 4 clones (frozen or live culture) at passage >5

    Notes for iPSC reprogramming services: Inherent to iPSC reprogramming, a certain percentage of cells will fail to produce iPS cells that is likely cell specific and not due to technical reasons. If we do not obtain stable colonies after the first round of reprogramming, we will repeat the process. However, if the second round fails, we will not repeat again and will only charge 50% of the service fee."

  • iPSC derivation from fibroblasts using lentiviral vector or Sendai virus ( Material production service )

    "The Core accepts either live or frozen cell stocks. Submitted cells MUST be mycoplasma free before they are accepted. Cells should also be at low passages (preferably <10) and are proliferating as non-dividing cells are refractory to reprogramming. This service takes 3-6 months to complete and includes the following:

    • Expansion, mycoplasma testing, cryopreservation of fibroblast culture
    • Transduction of fibroblasts with lentiviral vector or Sendai viral vectors
    • iPSC colony picking
    • Expansion of 4 iPSC clones
    • Mycoplasma testing
    • FACS analysis of 2 pluripotency markers (TRA-1-60 and SSEA4)
    • Cryopreservation of iPSC lines (4-6 vials/clone)
    • Will deliver 4 clones (frozen or live culture) at passage >5"

  • iPSC derivation from other somatic cell sources ( Material production service )

    "We will work with you to reprogram iPS cells from other cell sources. Please contact the director for details."

    Somatic cell sources other than blood or fibroblasts.

  • iPSC derivation hands-on training option ( Training service )

    "For researchers who would like to learn to reprogram iPSC cells on their own, the Core accepts a limited number of researchers with advanced cell culture experience to derive iPS cells in our facility. With direction from the Core staff, the researcher will be responsible for all work involving cell expansion, quality control and reprogramming and abide by all rules of the Core. All reagents and supplies are provided by the Core. This training period is limited to 4 months, and up to 4 parental cell lines may be used for reprogramming during this time period."

  • Primary cell expansion and quality control ( Material processing service )

    "The Core will accept primary cells (fibroblasts, PBMCs) for iPSC derivation. Cells should be at low passage and proliferating as non-dividing cells are not likely to generate iPSCs. We will assess cell growth and determine optimal media conditions; mycoplasma testing will also be performed. Parental cell lines will also be cryopreserved to serve as back-up stock. This service will take about 2-3 weeks to complete."

  • Supply of stem cell culture reagents ( Support service )

    Discounted supply of quality tested stem cell culture reagents

  • Technical consulting for induced pluripotent stem cell research ( Support service )

    "The Core offers technical consulting for iPSC related research projects. Each session typically lasts 1-2 hours. Please contact the Director for more information."


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Last updated: 2016-06-14T15:50:28.348-04:00

Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016