Services

The Human Pluripotent Stem Cell Core is committed to enabling and accelerating collaborative studies in stem cell biology by providing a nourish and cost effective environment with the final goal of advancing stem cell research. We offer expertise in generating patient-specific induced pluripotent stem cells (iPSCs) for disease modeling and prospective therapies, as well as differentiating them into specialized cellular lineages. We also provide full genome editing services using TALENs and CRISPR technology, along with gene regulation based on ZFP/TALENs/CRISPRs. Our expertise is available to facilitate the development of joint proposals, provide technical support and consultation for researchers seeking to work with human pluripotent stem cells.

The Human Pluripotent Stem Cell Core offers the following services:

  • Generation of hiPS cells using integration-free methods
    • Reprograming of fibroblast, blood cells (B and T cells or erythroblast) and other custom cell types using Episomal, Sendai virus and Repro-RNA methods.
  • Characterization of IPS cells
    • Immunocytochemistry staining for pluripotency markers (Oct 4, Nanog, TRA-160, SSEA3, SSEA40), quantitative analysis of trilineage differentiation potential by qRT-PCR, EB formation, karyotyping and teratoma formation
  • Differentiation of hES and hiPS cells into specialized cell types
    • Differentiation of stem cells into neural progenitor cells, mature neurons, cardiomyocytes, hepatocytes, mesenchymal stem cells, osteoblasts, adipocytes and endothelial cells.
  • Maintenance, expansion and banking of hES and hiPS cells.

Genome editing service is offered for any mammalian cell type (hESC, hIPS and cancer cell lines).

TALENs and CRISPR/Cas9 based technologies are offered to engineer cell lines for research use.  We perform cell targeting experiments, selection and genotyping of resistant clones, expansion and freeze of positive cells clones.

Genome editing of mammalian cells

  • Construction and testing TALEN/CRISPR vectors.
  • TALEN/CRISPR-mediated gene knock-out.
  • TALEN/CRISPR-mediated gene knock-in.
  • CRISPR-mediated point mutation introduction and/or repair to create isogenic cell lines.
  • Custom targeting vectors to be used in combination with TALEN or CRISPRs for tagging endogenous genes with fluorescent proteins.  

Gene regulation services

ZFP, TALENs CRISPR/Cas9 technologies can be used to alter gene regulation when coupled with activators, repressors or chromatin remodeling enzymes. Full service includes design, construction and testing gene regulation in any mammalian cell type.

  • Preparation of cell culture dishes I (e.g. matrigel preparation, coating and storage)
  • Preparation of cell culture dishes II (mitomycin C inactivation and plating of MEF cells)
  • iPS media preparation (mTeSR, TeSR 8 and DMEM/F12)
  • Culture and passage of mouse iPS on MEF 
  • Culture and passage of human iPS cells on matrigel and MEF 
  • Identifying differentiated and undifferentiated human iPS colonies  
  • Methods of removal differentiated colonies from iPS cultures  
  • Passaging, thawing and cryo-preservation of mouse and human iPS cells 
  • Transitioning of human iPS cells on MEF to feeder free system
Cost: $1500  

The following services are offered by the Core. Please contact the  to set up a strategy meeting before initiating any project.

Important:

  • All UPenn users requesting services and/or cell lines involving human cells must acknowledge that they have obtained all necessary permission from UPenn HUMAN STEM CELL RESEARCH ADVISORY COMMITTEE and have obtained all applicable IRB approval for research relating to human ESC/iPSC research.
  • All materials (cells, tissues) delivered to the iPSC Core are not known or suspected to contain any infectious or etiologic agent.
  • The investigator(s) requesting Services shall acknowledge the UPenn iPSC Core Facility in any publication resulting from services/cell lines provided by the Core.

Fibroblast isolation and expansion from skin biopsy

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 

iPSC derivation from fibroblasts using lentiviral vector or Sendai virus

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 blood using lentiviral vector or Sendai virus

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 other somatic cell sources

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

iPSC derivation hands-on training option

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.

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.

The Core has several established human iPS cell lines for purchase for research purposes by non-profit institutions. Please contact the director for available cell lines. For purchases from researchers outside of UPenn, an MTA between UPenn and your institution is required.
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.
Please contact the  if you require custom services not listed above. We will do our best to accommodate you.