LUMC hiPSC core facility: generation of high-quality hiPSC for LUMC researchers and external parties.
The LUMC iPSC core facility offers the generation of high quality hiPSCs from different tissue sources using state of the art reprogramming techniques, and full characterization of the generated lines**. This service is available to LUMC researchers and external parties, although the generation of hiPSCs for LUMC researchers is prioritized. Generation of hiPSCs for external, not-profit as well as commercial parties, is scheduled according to free capacity of the LUMC hiPSC core facility. For LUMC researchers the reprogramming standard services are free of costs (for exceptions see list below), whereas external users will be charged a service fee (see below).
The LUMC iPSC core facility service includes:
- Isolation of somatic cells from tissue samples (skin, milk teeth (dental pulp), urine or peripheral blood) for reprogramming. Isolated cells will be tested for mycoplasma, and cryopreserved at low passage. Skin biopsies (4 mm) are the most convenient source of cells to be reprogrammed since they can be stored for up to two weeks in PBS at 4°C prior to isolation of fibroblasts ( Dambrotet al . ). In the Netherlands skin biopsies are not admitted for children unless being part of a necessary surgery. In those cases milk teeth or urine represent an alternative non-invasive tissue source. Nevertheless both tissue types have to be processed within hours after collection and therefore require detailed planning in advance. Erythroblasts isolated from peripheral blood (10 ml) represent an alternative tissue source for reprogramming.
- Reprogramming of somatic cells with three alternative vector systems. The customer can choose between an integrating, excisable polycistronic lentiviral vector (figure of lentiviral vector ), non-integrating episomal plasmids (figure of plasmids ) or non-integrating Sendai virus (SeV) based vectors (figure of SeV vector ) or RNA (Schematic of VEE-RF RNA replicons). The use of vector systems may be restricted by the somatic cell type used for reprogramming. Please contact the hiPSC core facility for details.
- Reprogramming with polycistronic lentivirus: The LUMC core facility has optimized reprogramming with this system resulting in a maximum of 1 or 2 proviral integrations per hiPSC clone made from skin fibroblasts. Transgenes can be excised by the use of FLP enzyme. Generation of hiPSCs with polycistronic lentivirus does not include screening of proviral integrations (sites or numbers) or excision of transgenes on a standard basis. Using the polycistronic lentivirus for reprogramming includes the possibility to negotiate a license for commercial use with the original provider.
- For reprogramming with episomal vectors rare cases of integration of the (partial) vector into the host genome have been reported. The LUMC hiPSC core facility will not screen hiPSC cloons for the absence of (partial) integrations. Using the episomal vectors for reprogramming includes the possibility to negotiate a license for commercial use with the original provider.
- Reprogramming with SeV: The LUMC hiPSC core facility has a limited capacity for reprogramming with the SeV-based vector system. Please contact the facility for availability. In the Netherlands hiPSCs generated with the SeV system used by the LUMC iPSC core facility have to be maintained at ML-2 level at all times unless absence of SeV has been confirmed by RT-PCR and immunofluorescent staining. The LUMC hiPSC core facility will test presence of SeV of the 3 clones which are characterized as described below at a passage number indicated to the end user. Customers will be responsible to test for absence of SeV if lines are used at lower passage or other clones are used. Protocols for SeV detection can be provided by the LUMC hiPSC core facility.Using SeV for reprogramming excludes commercial use of the resulting hiPSC lines.
- For reprogramming with synthetic RNA additional reagent costs apply also for LUMC researchers (see list below).
- Generation of 6 hiPSC clones per patient (somatic tissue sample). hiPSC lines are generated on feeder cells (mouse embryonic fibroblasts), and subsequently maintained under defined conditions (vitronectin/ TESRE8). In rare cases cells cannot be reprogrammed for unknown reasons (e.g. due to a (un)known mutation). In case that reprogramming fails in the 1st attempt we will repeat the reprogramming. If the 2nd round is not successful no more attempts will be made. For paying customers the fee is reduced by 50% when two rounds of reprogramming were unsuccessful.
- Expansion and freezing of hiPSC clones cultured on vitronectin/ TESR E8: 3 clones will be frozen at passage ~3, whereas the remaining 3 clones will be frozen at passages ~3 and 5. Three clones are further characterized (see below)
- Longterm banking (backup) of hiPSCs in liquid nitrogen. Note : hiPSC lines made for LUMC researchers are property of the LUMC but will not be distributed to third parties without permission of the original customer. Lines generated for paying customers will be owned exclusively by the purchaser
- Analysis of expression of pluripotency markers in three undifferentiated hiPSC clones (ICC: SSEA-4, NANOG, OCT3/4). The customer will receive representative images for each clone (Image pluripotency markers ).
- Analysis of differentiation potential of three hiPSC clones by spontaneous differentiation into derivatives of the three germ layers (ICC: βIII-TUBULIN (ectoderm); α-Fetoprotein (endoderm); PECAM-1 (mesoderm). The customer will receive representative images for each clone (Image three germlayer differentiation ).
- Further in depth characterization of hiPSCs (optional) by
- Karyotyping (COBRA-FISH)
- Teratoma assay (3 mice/ clone)
- Note:Planning and execution of these services (partially) involves third parties.
- Provision of hiPSC control lines. The facility has generated a variety of hiPSC control lines (female or male; different ages; several tissues of origin, different reprogramming techniques). These control lines are freely available (excluding shipment)#.
- Supply of defined differentiation protocols developed in the departments of Anatomy & Embryology and Molecular Cell Biology (e.g. for derivation of cardiomyocytes, endothelial cells, pericytes, neural progenitors, blood cells etc.). Note: Further involvement of the LUMC core facility after the generation of hiPSC lines (e.g. for directed differentiation, in vitro modeling etc.) can be discussed.
- Teaching courses: 1-2 times per year, 4 days, 8-10 participants. "Hands on" course on TESR1 and TESR E8 culture of undifferentiated hiPSCs (thawing, passaging, freezing) and selection of differentiated cells etc. Dates of courses will be announced on the LUMC website.
The LUMC hiPSC core facility is a non-profit facility and the fee will only cover the costs resulting from hiPSC generation#. Note: Prices are exclusive shipment fees.
|LUMC researchers||External non-profit|
|Karyotyping||€750/ clone||€1000/ clone|
|Teratoma assay||€1000/ clone||€3500/ clone|
|PluriTest||€550/ clone||€800/ sample|
|Control hiPSCs||free||shipment fees|
* limited capacity/ year
All prices include BTW.
# Prices for external for-profit on request.
** Nota Bene
The technology used for reprogramming is state of the art, and generally reliable and robust. Notwithstanding the consistency of the procedures, no guarantee can be made on the quality and quantity of the reprogrammed cell lines generated by the LUMC hiPSC core facility. However all reasonable steps will be taken to provide that customers with the cell lines and services that are requested. With the order it is assumed that the customers will attribute the contribution of the LUMC iPS core facility in the manner that is customary in scientific outings such as publications and presentations.