Induced pluripotent stem cell lines derived from human somatic cells

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells.

These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

Introduction

In vitro reprogramming of somatic cells to an undifferentiated pluripotent state by viral transfer of defined factors such as SOX2, OCT4, NANOG and LIN28 or SOX2, OCT4, c-Myc, and KLF4 [1], [2] has opened the way for the generation of patient-specific human iPSCs using multiple cell types [3], [4]. This premise has been further advanced by derivation of iPSCs via transient expression of genes or by using protein transduction of appropriate transcription factors [5], [6]. To date, the majority of iPSC research in humans has focused on fibroblasts as a cell source.

While fibroblasts offer certain advantages as a starting material due to their commercial availability and ease of gene delivery, they are suboptimal for large-scale clinical derivation of iPSC lines due to the need for invasive skin biopsies and the difficulty of establishing stable lines from primary tissue. Non-mobilized peripheral blood is perhaps the ideal cell source for reprogramming due to the ease of obtaining patient samples [7]. Additionally, large numbers of frozen blood samples, from living and deceased donors, are stored in biorepositories worldwide [8].

Investigators have recently reported successful reprogramming of primary CD34⁺ hematopoietic progenitor cells from both mobilized and non-mobilized blood donors [3], [9]. These findings represent an important advance in iPSC research, however, non-mobilized adult peripheral blood contains approximately 100–1000 CD34⁺ cells/ml [10], [11] making these rare progenitors a challenging cell source for iPSC line derivation from small blood volumes.

As an alternative, more abundant and tractable blood cell source we report the derivation of iPSCs from T lymphocytes obtained from the equivalent of 1 ml whole blood. These T-cell derived iPSCs (“TiPS”) share essential characteristics with hESCs as well as fibroblast-derived iPSC lines. Additionally, they retain their characteristic T-cell receptor (TCR) gene rearrangements, a property which could be exploited, for example, as a genetic tracking marker or in re-differentiation experiments to study human T-cell development.

Results and Discussion

T-cells are well suited as a starting material for reprogramming due to their abundance in whole blood (∼6.5×10⁵–3.1×10⁶/ml in healthy adults) [12] and ease of culture using well-established protocols [13], [14]. To facilitate T-cell proliferation and efficient retroviral transduction, peripheral blood mononuclear cells (PBMCs) were isolated from a leukapheresis or a standard venipuncture (Vacutainer© CPT tube) and cultured in serum-free media with IL-2 and anti-CD3 antibody (Figure 1). This led to preferential expansion of mature CD3+ T-cells consisting of an average day 3 CD3⁺ purity of 90% +/− 7% (Figure 2A).

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