iPSC Core Services

This service is normally requested in order to establish fibroblasts for iPSC reprogramming.

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

This service is normally requested in order to isolate PBMCs from blood draws for iPSC reprogramming. 

Freshly isolated cells can be reprogrammed immediately or can be revived at a later time for iPSC reprogramming.  This service takes 1 day to complete and includes the following:

• Isolation of PBMCs from blood using ficoll gradient separation
• Cryopreservation of several vials of PBMCs at >4million cells per vial.
• Freezing of cell pellets for future genomic DNA isolation

 Note:  This service does not include storage of cryopreserved vials in the Core facility

Prerequisites:

• All required IRB approval must be in place. 
• A Service Agreement must be completed for non-Penn users
• 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 passage (preferably <5) and are proliferating as non-dividing cells are refractory to reprogramming.

This service takes 4-6 months to complete and includes the following:

• Expansion, mycoplasma testing, cryopreservation of fibroblast culture
• Transduction of fibroblasts with Sendai viral reprogramming vectors
• iPSC colony picking
• Expansion of iPSC clones in feeder-free conditions
• Mycoplasma testing of expanded clones
• FACS analysis of 2 pluripotency markers
• qPCR-based analysis for common karyotypic abnormalities in iPSCs
• PCR assay for loss of Sendai viral reprogramming vectors
• Cryopreservation of iPSC lines (4 vials/clone)
• Will deliver 3 clones at passage ≥10

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).

Prerequisites:

• All required IRB approval must be in place. Please submit IRB approval document here:
• A Service Agreement must be completed for non-Penn users
• Users should request the PBMC isolation from blood service or submit previously isolated PBMCs.
• If submitting PBMCs, evidence showing their mycoplasma free status must be shown before they are accepted.

This service takes 4-6 months to complete and includes the following:

• Culture of PBMCs for enrichment of erythroid progenitors
• Transduction of cultured cells with Sendai viral reprogramming vectors
• iPSC colony picking
• Expansion of iPSC clones in feeder-free conditions
• Mycoplasma testing of expanded clones
• FACS analysis of 2 pluripotency markers
• qPCR-based analysis for common karyotypic abnormalities in iPSCs
• PCR assay for loss of Sendai viral reprogramming vectors
• Cryopreservation of iPSC lines (4 vials/clone)
• Will deliver 3 clones at passage ≥10

Prerequisites:

• All required IRB approval must be in place. 
• A Service Agreement must be completed for non-Penn users.
• When submitting LCL cells, evidence of their mycoplasma free status must be shown before they are accepted.

This service takes 4-6 months to complete and includes the following:

• Culture of LCL cells for expansion
• Neucleofection of cultured cells with Episomal reprogramming vectors
• iPSC colony picking
• Expansion of iPSC clones in feeder-free conditions
• Mycoplasma testing of expanded clones
• FACS analysis of 2 pluripotency markers
• qPCR-based analysis for common karyotypic abnormalities in iPSCs
• PCR assay for no genomic insertion of episomal reprogramming vectors
• Cryopreservation of iPSC lines (4 vials/clone)
• Will deliver 3 clones at passage ≥10

Prerequisites:

• Cell line must be generated by the Core

This service takes 1 week to complete and includes the following:

• Isolation of total RNA from iPSC line
• qRT-PCR analysis of 5 stem cell marker genes
• Gene expression of parental cells and a previously established iPSC line included as controls
• Will deliver analysis report

Prerequisites:

• Cell line must be generated by the Core

This service will take 3-4 weeks to complete and includes the following:

• Expansion and plating of iPSC line for tri-lineage differentiation
• Directed differentiation of iPSCs to mesoderm, endoderm and ectoderm lineages separately
• Flow cytometry analysis of 2 marker proteins per cell lineage
• Will deliver analysis report

Prerequisites:

• iPS Cell line must be generated by the Core
• When submitting a non-iPSC line (i.e. parental cell line for iPSC derivation), user must submit a frozen cell pellet

This service will take 2-3 weeks to complete and includes the following:

• Expansion of iPSC line
• Freezing down of a cell pellet
• Submission of frozen cell pellet to a commercial vendor for STR analysis
• Delivery of STR analysis report

Prerequisites:

• Cell line must be generated by the Core

 This service will take 3-4 weeks to complete and includes the following:

• Expansion of the iPSC line
• Submission of live culture to a commercial vendor for G-banding of metaphase cells
• Delivery of karyotyping report to the user

This service provides thawing, expansion and banking of iPSC lines derived by the Core or external laboratories under feeder-dependent or feeder-free conditions.

Prerequisites:

• Designation of iPSC line to be banked
• iPSC lines new to the facility will need to produce evidence of being mycoplasma-free

This service takes 2-5 weeks to complete and includes the following:

• Thaw cells on mouse feeders or under feeder-free conditions
• Transition cells from feeder-dependent culture to feeder-free conditions if requested

• Expansion of cells
• Cryopreservation of expanded cells into 20 vials per cell line
• Mycoplasma testing
• Transfer of all frozen vials

This service provides thawing and expansion of iPSC line(s) with approved MTAs for one passage (~2 weeks) and delivers high quality undifferentiated cells (1 plate of 6-well plate) on mouse feeders or coated wells.

The lines need to be mycoplasma free.

The service provides passaging feeder-free iPSC cells after thaw or transition from feeders every 5-7 days with routine maintenance.

This service will check whether the iPSCs reprogrammed by Sendai virus method still contain the virus.

• Total RNA will be purified from the cells.
• cDNA will be generated by reverse transcription with random primer.
• Four sets of Sendai virus, reprogramming factor specific primer-probes together with a housekeeping set will be used in the qPCR.
• Sendai virus loss will be decided when all 4 sets are undetectable with robust amplification of housekeeping gene.

This service provides genomic DNA extraction and PCR detection for mycoplasma from cell pellets.

This service establishes genome modified hESCs/hiPSCs using CRISPR/Cas9 technology

Prerequisites:

• Selection of a hESC or hiPSC line to be gene edited
• A Service Agreement must be executed for non-Penn users
• MTA must be executed if transferring a cell line to the Core
• All cell lines not generated by the Core must be shown evidence of being mycoplasma- free

This service takes 6-9 months to complete and includes the following:

• Consultation with the user to determine targeting strategy
• Design and construction of CRISPR gRNA(s) for targeting
• Validation of gRNA(s) in the targeting cell line
• Targeting of the cell line using validated gRNA(s)
• Screening of targeted clones
• Establishment of at least 2 correctly targeted clones
• Mycoplasma testing of the clones
• qPCR-based analysis for common karyotypic abnormalities of the clones
• Delivery of cryopreserved cells and written report

This service establishes genome modified hESCs/hiPSCs using CRISPR/Cas9 technology

Prerequisites:

• Selection of a hESC or hiPSC line to be gene edited
• A Service Agreement must be executed for non-Penn users
• MTA must be executed if transferring a cell line to the Core
• All cell lines not generated by the Core must be shown evidence of being mycoplasma- free

This service takes 6-9 months to complete and includes the following:

• Consultation with the user to determine targeting strategy
• Design and construction of CRISPR gRNA(s) for targeting
• Validation of gRNA(s) in the targeting cell line
• Targeting of the cell line using validated gRNA(s) and HDR DNA donor template
• Screening of targeted clones
• Establishment of at least 2 correctly targeted clones
• Mycoplasma testing of the clones
• qPCR-based analysis for common karyotypic abnormalities of the clones
• Delivery of cryopreserved cells and written report

This service establishes genome modified hESCs/hiPSCs using CRISPR/Cas9 technology

Prerequisites:

• Selection of a hESC or hiPSC line to be gene edited
• A Service Agreement must be executed for non-Penn users
• MTA must be executed if transferring a cell line to the Core
• All cell lines not generated by the Core must be shown evidence of being mycoplasma-free

This service takes 6-9 months to complete and includes the following:

• Consultation with the user to determine targeting strategy
• Design and construction of CRISPR gRNA(s) for targeting
• Validation of gRNA(s) in the targeting cell line
• Design and synthesis of the ssODN donor template
• Targeting of the cell line using validated gRNA(s) and ssODN donor template
• Screening of targeted clones
• Establishment of at least 2 correctly targeted clones
• Mycoplasma testing of the clones
• qPCR-based analysis for common karyotypic abnormalities of the clones
• Delivery of cryopreserved cells and written report

This Service provides the design and the construction (cloning) of a plasmid template used for targeting iPSCs using homology directed repair (e.g. template for generating a gene reporter)

Prerequisites:

• Selection of a hESC or hiPSC line to be gene edited
• A Service Agreement must be executed for non-Penn users
• MTA must be executed if transferring a cell line to the Core
• All cell lines not generated by the Core must be shown evidence of being mycoplasma- free

This service takes 3-5 weeks to complete and includes the following:

• Consultation with the user to determine targeting strategy
• Design and construction of the donor vector
• Delivery of the donor vector as a DNA maxiprep

This service provides iPSC-derived cardiomyocytes in monolayer cultures.  iPSCs are differentiated using cytokines and small molecules in defined media.  Cultures are then metabolically selected to enrich for cardiomyocytes and re-plated. These cells resemble immature cardiomyocytes and but also display a number of properties of adult cardiomyocytes. 

Prerequisites:

• Designation of iPSC line to be differentiated
• New iPSC lines will need to show evidence of being mycoplasma-free or this will be performed by the Core at extra charge (see Mycoplasma Testing service)

This service takes 5-8 weeks to complete and includes the following:

• Thaw cells and expand for differentiation
• Up to two rounds of pilot studies to determine optimal seeding density and conditions for differentiation 
• Directed differentiation in monolayer cultures
• Transfer of live monolayer cultures of beating cells in two 10-cm plates
• Approximate yield: 12-20 million cells

Additional Notes:

• Additional plates can be purchased at a reduced price.
• If differentiation fails in both pilot studies, PI will be notified immediately and the project will be discontinued.  Service fee still applies.
• More pilot studies can be performed using a different CM differentiation protocol after submitting a new request.

This service provides iPSC-derived pulmonary alveolar type 2 cells (iAT2s) derived from the BU-SPC2-ST-B2 line (Boston University).  Cells were differentiated and maintained as alveolospheres embedded in 3D Matrigel as previously described (Jabob et al., 2019) and passaged every two weeks.  The iAT2s express the tdTomato fluorescent marker protein from the endogenous locus of surfactant protein-C (SFTPC).  tdTomato expression is assayed by flow cytometry as each passage and should be between 50-90%.

This service takes 2-4 weeks to complete and will be provided in single cell suspensions in growth media or plated in 2D format.  One unit = 10 millions cells. 

Additional Notes:

• If cell expansion fails or tdTomato expression is suboptimal, the PI will be notified immediately.
• Additional units of cells can be purchased, but at least 2 week advance notice is required.

This service provides HLCs in monolayer cultures.   iPSCs undergo stage-specific directed differentiation to establish definitive endoderm followed by, hepatic progenitors, immature hepatocytes, and hepatocyte-like cells (HLCs).  These cells resemble fetal hepatocytes but do display a number of properties of mature hepatocytes.

Prerequisites: 

• Designation of iPSC line to be differentiated
• New iPSC lines will need to show evidence of being mycoplasma-free

This service will take 5-8 weeks to complete and includes the following:

• Thaw cells and expand for differentiation
• Pilot study to determine optimal seeding density for differentiation (if working with new line)
• Directed differentiation in monolayer cultures
• Flow cytometry analysis of definitive endoderm markers (Sox17/CXCR4) at day 5 and flow cytometry analysis of albumin on day 20
• Transfer of live, monolayer culture supplied in either four 6-well plates or four 12-well plates (approx. 25,000 cells/cm²)

Additional Notes:

• If differentiation fails, PI will be notified immediately
• Additional plates can be generated at a reduced price

This service provides iPSC-derived endothelial cell in monolayer cultures.  iPSCs undergo directed differentiation using cytokines and small molecules to endothelial progenitors.   Cultures are then purified with endothelial specific cell surface markers to enrich for purity and further passaged.  These cells express CD31(PECAM) & CD144 (VE-cadherin) surface markers, uptake Ac-LDL, and form vessels when cultured with stromal cells in 3D cultures.

Prerequisites:

• Designation of iPS line to be differentiated
• New iPSC lines will need to show evidence of being mycoplasma-free

This service takes 4-6 weeks to complete and includes the following:

• Thaw cells and expand for differentiation
• Directed differentiation in monolayer cultures
• Magnetic activated cell sorting against endothelial markers
• Passaged in endothelial growth media for cell expansion
• Flow cytometry analysis of endothelial markers CD31/CD144
• Passage 3 live culture supplied in at least 2 6-well plates
• Approximate yield: 6 million cells
• Guaranteed purity > 90% CD31⁺/CD144⁺

Additional Notes:

• If differentiation fails, PI will be notified immediately
• Additional plates can be purchased at a reduced price

Prerequisites:

• Students must have at least one year of hands-on experience in mammalian cell culture

This is a hands-on training course in basic techniques in hESC/iPSC culture including cell thawing, expansion, identification by morphology, manual and enzymatic passaging, freezing, and feeder-free culture.  This course will take 2 weeks to complete (1-2 hours per day)

The Core has a number of wild-type control iPSC lines available to users.   Please inquire with the Core director at wenliyan@pennmedicine.upenn.edu.

The iPSC Core offers a number of quality-tested reagents and for use in stem cell culture. For pricing and a full list of reagents, please visit the iPS Core iLab website.