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Generation of induced pluripotent stem cells from human cord blood cells with only two factors: Oct4 and Sox2

Nature Protocols volume 5, pages 811–820 (2010)Cite this article

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Abstract

Induced pluripotent stem cells (iPSC) provide an invaluable resource for regenerative medicine as they allow the generation of patient-specific progenitors with potential value for cell therapy. However, in many instances, an off-the-shelf approach is desirable, such as for cell therapy of acute conditions or when the patient's somatic cells are altered as a consequence of a chronic disease or aging. Cord blood (CB) stem cells appear ideally suited for this purpose as they are young cells expected to carry minimal somatic mutations and possess the immunological immaturity of newborn cells; additionally, several hundred thousand immunotyped CB units are readily available through a worldwide network of CB banks. Here we present a detailed protocol for the derivation of CB stem cells and how they can be reprogrammed to pluripotency by retroviral transduction with only two factors (OCT4 and SOX2) in 2 weeks and without the need for additional chemical compounds.

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Figure 1: Quantitative RT-PCR analysis for pluripotent markers and the two transcription factors c-MYC and KLF4.
Figure 2: Umbilical cord blood collection and separation.
Figure 3: Phoenix Amphotropic 293 cells post transfection.
Figure 4: Morphology of cord blood (CB) CD133+ cells after infection.
Figure 5: Induced pluripotent stem cells (iPSC) from cord blood CD133+ cells.
Figure 6: Characterization of CD133+ and cord blood-induced pluripotent stem (CBiPS) cells.

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Acknowledgements

We thank Dr. M. Torrabadella, Director of the Banc de Sang i Teixits, Vall d'Hebron for providing cord blood units. We are grateful to Y. Muñoz Santos for expert assistance with cell culture techniques. N.M. was partially supported by the Juan de la Cierva program. This work was partially supported by grants from MICINN, CIBER, the Fondo de Investigaciones Sanitarias (RETIC-RD06/0010/0016), TERCEL, the G. Harold and Leila Y. Mathers Charitable Foundation and Fundación Cellex.

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Author notes

  1. Alessandra Giorgetti and Nuria Montserrat: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Regenerative Medicine in Barcelona, Dr. Aiguader, Barcelona, Spain

    Alessandra Giorgetti, Nuria Montserrat, Ignacio Rodriguez-Piza, Anna Veiga & Juan Carlos Izpisúa Belmonte

  2. Banc de Sang i Teixits, Vall d'Hebron, Paseo Vall d'Hebron, Barcelona, Spain

    Carmen Azqueta

  3. Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA

    Juan Carlos Izpisúa Belmonte

  4. Networking Center of Biomedical Research in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain

    Juan Carlos Izpisúa Belmonte

Authors
  1. Alessandra Giorgetti
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  2. Nuria Montserrat
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  3. Ignacio Rodriguez-Piza
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  4. Carmen Azqueta
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  5. Anna Veiga
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  6. Juan Carlos Izpisúa Belmonte
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Contributions

A.G.: protocol design, isolation of CB CD133+ cells, generation of iPS cell lines and preparation of manuscript; N.M.: protocol design, isolation of CB CD133+ cells, generation of iPS cell lines and preparation of manuscript; I.R.-P.: characterization of iPS cell lines; C.A.: characterization, selection and provision of CB units; A.V.: preparation of manuscript; and J.C.I.B.: preparation of manuscript.

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Correspondence to Juan Carlos Izpisúa Belmonte.

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Giorgetti, A., Montserrat, N., Rodriguez-Piza, I. et al. Generation of induced pluripotent stem cells from human cord blood cells with only two factors: Oct4 and Sox2. Nat Protoc 5, 811–820 (2010). https://doi.org/10.1038/nprot.2010.16

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