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Cell Reprogramming and Differentiation Platform

The Cell Reprogramming and Differentiation Core Facility has started in June 2010 as a part of the Inbiomed Foundation.

The general aim of the facility is to facilitate and to optimize the derivation of human pluripotent cells from donors with different pathologies, using integrative and non-integrative approaches.

In our facility we provide service to researchers from Inbiomed and collaborating institutions as well as to external users.

Induced Pluripotent Stem Cells (iPSCs) are terminally differentiated somatic cells that have been reprogrammed into an Embryonic Stem Cell (ESC)-like state. The discovery of iPSCs has rised great expectation, given the potencial of these cells for modeling and treating certain diseases, and has potentiated the interest for understanding the mechanisms involved in nuclear reprogramming.

The most direct way to induce pluripotency is through the transduction of cells with transcription factors able to reprogram the cell nucleus.In 2006, Takashi and Yamanaka first discovered that four factors, Oct4, Sox2, Klf4 and c-Myc (OSKM), were sufficient to induce pluripotency in mouse fibroblasts.

Infection with integrative vectors

We have four retroviral (pMXs) or bicistronic lentiviral vectors encoding each of the factors from the Yamanaka Cocktail (OSKM). The latest vectors co-express a fluorescent reporter under regulation of the hPGK promoter. The combination of fluorescent proteins allows a real time follow-up of the expression of each factor by fluorescent microscopy.

Schematic representation of vexGFP-OCT4, mCitrine-SOX2, mCherry-KLF4 and mCerulean-cMYC lentiviral vectors. LTR, Long Terminal Repeat

Infection with Sendai virus (SeV) usando el Cyto Tune™-iPS Reprogramming Kit

The most promising non-integrative method is based on Sendai virus (SeV)-based vectors. The system provided in the Cyto Tune –iPS Reprogramming System is based on replication-incompetent SeV for the effective expression of the reprogramming factors in somatic cells. The kit provides non-integrative viruses that remain in the cytoplasm of the cells.

Schematic representation of the life cycles of the non-integrative SeV (left) and integrative viral vectors (right). Modified from CytoTuneTM-iPS Reprogramming Kit user´s guide

Methodology

Human somatic cells will be infected with viral concentrates (lentivirus, retrovirus or Sendai virus), containing the four factors OSKM during 16 hours. One to seven days post-infection, the cells will be seeded on a feeder layer of irradiated human fibroblasts and incubated in hESC-specific medium that will be replaced every 48 hours. At day 15-25 post-infection, the colonies with a morphology similar to ESC cells will be manually picked and expanded. Following this, we will analyze the expression of pluripotency markers and the differentiation potential.

Schematic representation of the methodology and times for generating iPSCs

The researcher placing the request must be aware that once the iPSCs have been derived and their pluripotent state has been tested, they must be banked according to Spanish law (14/2007 for Biomedical Research). Inbiomed counts on Inbiobank, a biobank belonging to the net of the National Cell Lines Bank in Spain (BNLC), through which the requests for iPSCs banking may be placed.

Summary of services:

Module 1: fibroblasts isolation from skin biopsy.

This service takes about 1 month to be completed.

  • Fibroblasts isolation and expansion will be performed according to the SOPs established in Inbiobank.
  • Only freshly collected skin biopsies will be accepted.
  • The researcher must provide the required tests for viral pathogens.

Module 2: fibroblasts reprogramming using integrative or non-integrative methods.

This service can take up to 6 months to be completed, it includes:

  • Fibroblasts expansion, mycoplasma test, low-passage samples freezing and plating for viral infection.
  • Infection 35-mm tissue culture dish (75-100 x103/ well) with the viral vectors. When integrative vectors are used, viral supernatants and concentrates will be produced at the Viral Vectors Core Facility from Inbiomed.
  • 5-7 days post-infection, transgene expression will be checked by fluorescence microscopy or qRT-PCR, and 50 x103 infected cells are seeded on two 10-cm plates containing a feeder layer of irradiated human fibroblasts (one plate without compound, one plate with VPA).
  • Picking and expansion of colonies (30-40 days post-infection).
  • Mycoplasma test.
  • Freezing (up to 10 vials per line).

If the researcher chooses to supply the fibroblasts, the culture should be at a low passage (ideally < 5) and the cells must be proliferating. If the cells show abnormal morphology or are not proliferating, it will be evaluated with the researcher whether it is convenient to obtain new samples or attempt to transduce.

In some samples, iPSCs are not obtained after the first round of reprogramming. If this is the case, we will try different strategies (small compounds, changing infection, or plating conditions, etc.). If the second round of reprogramming is not successful at day 40 post-infection, we will not repeat the reprogramming. Once tested the expression of the transgenes, failure in reprogramming is due to intrinsic properties of the cells. Cell reprogramming is a stochastic process and occurs at a very low frequency (0.001-0.1%), depending on cell type, donor’s age, passage number, sample collection method, etc.

Depending on the number of colonies obtained from the sample, we will pick manually 3-10 colonies. However, the researcher must take into account that some samples may produce less than 3 colonies. Transgenes silencing will be checked by qRT-PCR and fluorescence microscopy. iPSCs will be sent to the researcher either as a live culture or frozen in vials. In this case, 5 frozen vials for each line will be sent.

Module 3: basic characterization: pluripotency and in vitro differentiation markers.

This module takes about one month to be completed. It includes:

  • Alkaline Phosphatase assay.
  • qRT-PCR for two pluripotency markers.
  • EB formation, qRT-PCR for 2 mesoderm markers, 2 endoderm markers and 2 ectoderm markers.

Module 4: teratoma formation.

This service takes 3 months to be completed and will be carried out at The Animal Models Core Facility from Inbiomed, it includes:

  • iPSCs expansion.
  • iPSCs collection and transportation to The Animal Models Core Facility.
  • Subcutaneous injection of cells (1 NOD/SCID mouse per iPS cell line).
  • Histology.
  • Analysis.

Module 5: karyotyping.

This service takes about 1 month to be completed and will be carried out at Genetics Lab from the Policlínica Gipuzkoa, it includes:

  • iPS colonies expansion in T25 flasks.
  • Mycoplama test.
  • Short time treatment with colchicine for detection of cells in metaphase and transportation to the Policlínica Gipuzkoa.

Module 6: DNA fingerprinting.

This service takes about 1 month to be completed. It includes:

  • Genomic DNA extraction from the starting sample.
  • Expansion of iPSCs in feeder-free conditions and genomic DNA extraction.
  • Short Tandem Repeat Polymorphisms (STRPs) analysis.

Inversión cofinanciada por el Ministerio de Ciencia e Innovación

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