A chemical platform for improved induction of human iPSCs - PubMed (original) (raw)

A chemical platform for improved induction of human iPSCs

Tongxiang Lin et al. Nat Methods. 2009 Nov.

Abstract

The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we describe a chemical approach that dramatically improves (200-fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, nonviral methods for reprogramming human somatic cells.

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Figures

Figure 1

Figure 1. Compound treatment for seven days is sufficient to induce pluripotent stem cells from human fibroblasts transduced with the four reprogramming factors

(a) Timeline for human iPSC induction using combined SB431542 and PD0325901 treatment along with 4TFs. Treatment began with cell re-seeding at day 7 after 4TF transduction and was maintained for 7 days. (b) Staining for ALP+ colonies that emerged in the untreated (left) or 2 compound-treated (right) cultures within seven days. (c) RT-PCR showing elevated endogenous mRNA expression of pluripotency markers OCT4 and NANOG in 2 compound-treated cultures. (d) Tra-1-81 staining at day 14 without (left) or with (right) 2 compound treatment. (e) The numbers of NANOG+ colonies at day 14 under different treatment conditions are plotted. (f) Typical staining for hESC-specific markers (NANOG and SSEA4) exhibited by D14 iPSCs. Scale bars, 50 µm in (d & f)

Figure 2

Figure 2. Prolonged compound treatment and cell passaging dramatically increased the number of reprogrammed colonies

(a) Timeline of human iPSC induction using SB431542, PD0325901 and thiazovivin. (b) Day 30 iPSCs expressed pluripotency markers NANOG, SSEA4 and TRA-1-81. Scale bars, 50 µm (c) ALP staining of day 30 cultures with (upper panels) or without (lower panels) 3 compound treatment. Boxed areas in the left panels are enlarged in the right panels. Scale bars, 200 µm (d) Number of NANOG+ colonies on day 30 under different treatment conditions, without splitting. (e) Number of NANOG+ colonies on day 30 from 3 compound-treated cultures trypsinized as indicated. (f) RT-PCR on iPSC colonies obtained with 3 compound treatment showing reactivated expression of endogenous pluripotency markers. HDF: Human Dermal Fibroblast.

Figure 3

Figure 3. In vitro and in vivo differentiation of iPSCs generated with 3 compound treatment

(a) Micrographs show embryoid bodies (EB) generated from iPSCs and in vitro differentiation into ectodermal (βIII tubulin), mesodermal (brachyury) and endodermal (PDX1) cell types. Scale bars, EB: 100 µm; others 10 µm (b) RT-PCR showing expression of representative lineage markers and the absence of OCT4 mRNA expression in differentiating cells. U- undifferentiated, D- differentiated. (c) Teratomas generated in nude mice from iPSCs (3 independent colonies tested) consist of tissues from all three germ layers. Left panel: 1- muscle, 2- neural epithelium; middle panel: 1- skin, 2- gut epithelium; right panel: 1- bone, 2- cartilage. Scale bars, 20 µm.

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