A high-efficiency system for the generation and study of human induced pluripotent stem cells

Abstract

Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28. Little is known, however, about the mechanisms by which reprogramming occurs, which is in part limited by the low efficiency of conversion. To this end, we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem cells (hiPSCs). hiPSCs generated with this system were molecularly and functionally similar to human embryonic stem cells (hESCs), demonstrated by gene expression profiles, DNA methylation status, and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts, we found that 10 days was sufficient for the reprogramming of keratinocytes. Using our inducible system, we developed a strategy to induce hiPSC formation at high frequency. Upon addition of doxycycline to hiPSC-derived differentiated cells, we obtained "secondary" hiPSCs at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells at high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming.

Figure 1. Generation of hiPS cells using inducible lentiviruses

A. Experimental scheme for the generation of hiPS cells. Primary human fibroblasts and keratinocytes were infected with separate lentiviruses (LV) containing a constitutively active rtTA and doxycycline-inducible reprogramming factors. After infection, cells were seeded to feeders and doxycycline (dox) was applied for 30 days. hiPS clones were picked based on hESC-like morphology and doxycycline-independent growth. B. Morphology and marker expression in hiPS colonies. Doxycycline-independent fibroblast- and keratinocyte-derived hiPS cells express OCT4 protein. C. Microarray analysis of gene expression in hiPS cells. Genes with greater than twofold expression level between HUES8 hES cells and BJ fibroblasts were analyzed. Shown are BJ fibroblasts, HUES8 hES cells, and BJ fibroblast-derived hiPS clones. D. In vitro differentiation of fibroblast-derived hiPS cells into lineages from all three germ layers. Immunostaining for i) Tuj1 (neuronal), ii) cardiac troponin T (cTnT; cardiac muscle), and iii) alpha-fetoprotein (AFP; epithelial, early endodermal). E. In vitro differentiation of keratinocyte-derived hiPS cells into lineages from all three germ layers. Immunostaining for i) Tuj1, ii) skeletal muscle (MF20), and iii) alpha-fetoprotein. F. Hematoxylin and eosin stain of teratomas generated from fibroblast-derived hiPS cells. Differentiated structures from all three germ layers were present. i) Pigmented epithelium (ectoderm), ii) cartilage (mesoderm), iii) gut-like epithelium (endoderm), and iv) muscle (mesoderm).

Figure 2. Generation of secondary hiPS cells

A. Experimental scheme depicting the generation of secondary hiPS cells. hiPS cells were differentiated in vitro as embryoid bodies for 7 days, then plated to adherent conditions. Fibroblast-like colonies were picked and expanded for at least three passages prior to undergoing re-induction by doxycycline. B. Alkaline phosphatase staining of reprogrammable cells grown in the absence or presence of doxycycline. Doxycycline was withdrawn at day 21, and colonies were stained and counted at day 30. C. Morphology and expression of OCT4 and Tra-1–81 in doxycycline-independent secondary hiPS cells. D. Microarray analysis of gene expression between BJ fibroblasts, HUES8 hES cells, primary fibroblast-derived hiPS cells, and a resulting secondary hiPS clone. Shown are genes with >2-fold expression value between BJ fibroblasts and HUES8 hES cells. E. In vitro differentiation of secondary hiPS cells into lineages from all three germ layers. Immunostaining for i) Tuj1, ii) cardiac troponin T, and iii) alpha-fetoprotein. F. Temporal requirement of factor expression in hiPS-derived fibroblast-like cells. 10⁴ cells were plated per time-point and doxycycline was withdrawn daily from days 4 through 19. The number of hES-like colonies that expressed Tra-1–81 were counted at day 25.