Conceived and designed the experiments: HH NK. Performed the experiments: HH. Analyzed the data: HH MF NK. Contributed reagents/materials/analysis tools: MF. Wrote the paper: HH NK.
The authors have declared that no competing interests exist.
Human induced pluripotent stem cells (iPSCs) can be divided into a leukemia inhibitory factor (LIF)-dependent naïve type and a basic fibroblast growth factor (bFGF)-dependent primed type. Although the former are more undifferentiated than the latter, they require signal transduction inhibitors and sustained expression of the transgenes used for iPSC production. We used a transcriptionally enhanced version of OCT4 to establish LIF-dependent human iPSCs without the use of inhibitors and sustained transgene expression. These cells belong to the primed type of pluripotent stem cell, similar to bFGF-dependent iPSCs. Thus, the particular cytokine required for iPSC production does not necessarily define stem cell phenotypes as previously thought. It is likely that the bFGF and LIF signaling pathways converge on unidentified OCT4 target genes. These findings suggest that our LIF-dependent human iPSCs could provide a novel model to investigate the role of cytokine signaling in cellular reprogramming
Mouse pluripotent stem cells can be divided into naïve and primed pluripotent cells, depending on their level of pluripotency, patterns of gene expression, and the cytokines required to maintain an undifferentiated state (self-renewal)
Unlike their mouse counterparts, human ESCs and iPSCs appear to be primed pluripotent stem cells
(A) Immunofluorescence staining of iPSCs prepared with LIF or bFGF on day 8. Antibodies against NANOG and TRA1-60 were used, and DNA was counterstained with Hoechst 33342. Bar, 100 µm in (A) and (C). (B) Formation of iPSC colonies that were double-positive for NANOG and TRA1-60 with LIF, bFGF, or no cytokines. Cells (1.7×104) were seeded in each well, and iPSC colonies were counted on day 10. Mean + standard deviation (SD) was obtained from three independent experiments. (C) Immunofluorescence staining of iPSCs prepared with LIF or bFGF on day 8. Antibodies against SSEA4 and NANOG were used. (D) Formation of iPSC colonies that were double-positive for SSEA4 and NANOG with bFGF, LIF, or no cytokines. Cells were seeded and iPSC colonies counted as described in (B).
We recently established a novel strategy to significantly improve the efficiency of making mouse and human iPSCs by using a fusion protein, called M3O, between OCT4 and the powerful transactivation domain of the myogenic master transcription factor MYOD
(A) Phase contrast images of L-iPSC colonies obtained on day 90. Bar, 100 µm. (B) Immunofluorescence staining of an L-iPSC colony with antibodies against NANOG and SSEA4 on day 28. Bar, 100 µm. (C) Quantitative RT-PCR analysis of the
Adult human dermal fibroblasts were transduced with retroviruses expressing M3O-SKM or OSKM. Fibroblasts were subcultured onto feeder cells three days later, when addition of bFGF or LIF was also started. Control culture was prepared without bFGF and LIF. ESC-like colonies were defined as iPSC colonies when they were double-positive for the pluripotency markers NANOG and TRA1-60 (
(A) Immunofluorescence staining of L-iPSCs with antibodies against OCT4, NANOG, TRA1-60 and TRA1-81 on day 120. Alkaline phosphatase staining is also shown. Bar, 100 µm. (B) Quantitative RT-PCR analysis of genes that are typically expressed in pluripotent stem cells. Expression levels of each gene were normalized to GAPDH. The normalized value for ESCs was defined as 1.0. Mean + SD obtained from three independent experiments are shown. (C) Karyotype analysis of an L-iPSC on day 120. (D) Haematoxylin and eosin staining of histological sections of teratomas derived from L-iPSCs. Bar, 200 µm.
(A) Quantitative RT-PCR analysis of genes that are typically expressed in mouse naïve (
We picked up 20–48 iPSC colonies from each culture condition between day 10 and 12 when colonies were available. These clones were then expanded on feeder cells in culture with the same cytokine to compare their efficiency of establishing pluripotent cell lines in the absence of signaling inhibitors. More than 90% of iPSC clones prepared with M3O-SKM in the presence of bFGF (F-iPSCs) remained undifferentiated for 120 days (25–30 passages) as monitored by the expression of TRA1-60 and NANOG. In the absence of bFGF and LIF, however, all iPSC colonies prepared with M3O-SKM morphologically differentiated by day 15 when the transgenes were suppressed although they remained undifferentiated if bFGF was added from day 15 onwards. In addition, most of the iPSC colonies prepared with M3O-SKM in the presence of LIF (L-iPSCs) differentiated around the same time. However, a few L-iPSC clones retained morphologically undifferentiated colonies scattered among numerous differentiated colonies beyond day 15. These apparently undifferentiated colonies were also unstable, with central undifferentiated parts being surrounded by differentiated derivatives (
(A) Immunofluorescence staining and alkaline phosphatase staining of LF-iPSCs on day 140. Bar, 100 µm. (B) Haematoxylin and eosin staining of histological sections of teratomas derived from LF-iPSCs. Bar, 200 µm.
L-iPSCs established with this procedure were characterized with several approaches. First, suppression of the M3O-SKM transgenes was verified by comparing the levels of total mRNA (derived from endogenous genes and transgenes) encoding
(A) Scatter plots comparing levels of whole transcripts obtained from two WA09 ESC cultures, two dermal fibroblast cultures, two lines each for L-iPSCs, F-iPSCs and LF-iPSCs. Two independently cultured cells were used for WA09 cells. All these iPSCs were established from fibroblasts obtained from a single adult female. Two different cultures of fibroblasts obtained from the same individual were used. (B) Cluster analysis of the genome-wide gene expression patterns of cells analyzed in (A). (C) Scatter plots comparing levels of whole transcripts between L-iPSCs and other cell types used in this study. Average values obtained from two cultures or two lines for each cell type were used.
To rule out the possibility that autocrine bFGF was compensating for the lack of exogenous bFGF in L-iPSC culture, we blocked the FGF receptor with SU5402, an inhibitor of the FGF receptor tyrosine kinase
To understand whether L-iPSCs belonged to the naïve or primed type, we compared them with F-iPSCs, which are primed pluripotent stem cells. First, L-iPSC colonies were flat and morphologically indistinguishable from F-iPSC colonies. Second, L-iPSCs did not form colonies after single-cell dissociation with trypsin during subculture, similar to F-iPSCs. Third, F-iPSCs and L-iPSCs expressed
The similarity between F-iPSCs and L-iPSCs was further tested by examining if LIF in the culture medium of L-iPSCs could be replaced with bFGF on day 90 without disrupting self-renewal. After culture for an additional 50 days, the colonies, now called LF-iPSCs, maintained an ESC-like morphology and expressed OCT4, NANOG, TRA1-60, TRA1-81 and alkaline phosphatase (
Finally, genome-wide transcription patterns were compared among ESCs, F-iPSCs, L-iPSCs, LF-iPSCs and parent fibroblasts with a microarray analysis. Overall gene expression patterns were quite similar among ESCs, two F-iPSC clones, two L-iPSC clones and two LF-iPSC clones (
Our study established L-iPSCs in the absence of sustained overexpression of transgenes and signaling inhibitors, unlike previously reported methods of making LIF-dependent human iPSCs. This was possible because of the usage of the transcriptionally augmented M3O. Reflecting differences in methods, L-iPSCs were also phenotypically different from previously reported LIF-dependent iPSCs and more closely related to standard bFGF-dependent iPSCs. This result has three important implications for our understanding of the regulation of pluripotency and self-renewal in iPSCs. First, dependency on LIF or bFGF is not the primary determinant of naïve or primed pluripotency. The activity of transgenes and modulations to cytokine signaling systems appear to be more critical in determining the type of pluripotent stem cell. Second, human and mouse cells are fundamentally different in terms of their response to LIF because this cytokine induces different types of pluripotent stem cells: primed-type human iPSCs and naïve-type mouse iPSCs
All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Minnesota (1002A78174).
Human M3O,
Following fixation with 4% formaldehyde in phosphate-buffered saline for 10 min, iPSCs were treated with 0.5% Triton X-100 for 3 min to permeabilize the plasma membrane. Primary and secondary antibodies (
Total RNA was prepared from each iPSC colony with a PureLink RNA Micro Kit (Life Technologies). cDNA was synthesized using a SuperScript III First-Strand Synthesis System for RT-CR (Life Technologies) and applied for qRT-PCR with a GoTaq qPCR Master mix (Promega) on a Realplex 2S system (Eppendorf). Primers are listed in
RNA was prepared from WA09 ESCs, the dermal fibroblasts from which all iPSCs in the microarray study were derived, two F-iPSC clones, two L-iPSC clones and two LF-iPSC clones on day 100–140 with the PureLink RNA total RNA purification system (Life Technologies). RNA quality was evaluated with a NanoDrop 8000 and an Agilent Bioanalyzer 2100. Biotin-labeled cRNA was synthesized with a TotalPrep-96 RNA Amplification Kit (Life Technologies/Applied Biosystems) following the manufacturer’s instructions. cRNA was hybridized onto a HumanHT-12 v.4.0 Expression Beadchip (Illumina) following the Direct Hybridization Gene Expression protocol. The Beadchips were then scanned using an Illumina’s iScan instrument. Data were created using Illumina’s GenomeStudio software and processed with Spotfire DecisionSite for Functional Genomics software. DNA microarray data have been deposited in the NCBI GEO database under the accession number GSE37077.
Colcemid-arrested iPSCs were treated with 0.75 M KCl hypotonic solution, and fixed with methanol and acetic acid at a 3∶1 ratio. Cell spreads were stained with Wright-Giemsa on glass slides for G band analyses with the Applied Spectral Imaging software.
NOD/SCID mice were injected with 1–2×106 iPSCs into the hind limb muscle. Seven to eight weeks later, teratomas were isolated, fixed with 10% formalin and embedded with paraffin. Histological sections (5 µm thick) were prepared and stained with haematoxylin and eosin.
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We thank Michael Franklin for critical reading of the manuscript; Wei-Shou Hu, Dong Seong Cho, Shikha Sharma, and Andrew Yongky for DNA microarray analyses; Kristin Voltzke for feeder cells; Toshio Kitamura for the pMXs-IP retroviral vector; and the BioMedical Genomics Center at the University of Minnesota for the microarray experiments. We also thank the University of Minnesota Comparative Pathology Shared Resource and the Cytogenetics Core Laboratory for histological preparation of teratomas and karyotyping, respectively.