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Doi, D. Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson’s disease. Stem Cells 30, — Grigoriadis, A. Directed differentiation of hematopoietic precursors and functional osteoclasts from human ES and iPS cells. Blood , — Kunisada, Y. Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells.
Stem Cell Res 8, — Nakatsuji, N. HLA-haplotype banking and iPS cells. Nat Biotechnol 26, — Saha, K. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions. Ido, H. The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin gamma chains in integrin binding by laminins.
J Biol Chem , — Suemori, H. Efficient establishment of human embryonic stem cell lines and long-term maintenance with stable karyotype by enzymatic bulk passage. Biochem Biophys Res Commun , — Watanabe, K. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25, — Generation of germ-line competent induced pluripotent stem cells. Nature , — Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.
Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Kajiwara, M. Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells. Download references. Knut Woltjen for valuable help in preparing the manuscript CiRA. James A. Institute for Innovation, Ajinomoto CO.
You can also search for this author in PubMed Google Scholar. Conceived and designed the experiments: M. Nakagawa, S. Performed the experiments: M. Nishizawa, Y. Analyzed the data: M. Wrote the paper: M. The authors declare no competing financial interests. Reprints and Permissions. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells.
Sci Rep 4 , Download citation. Received : 09 October Accepted : 06 December Published : 08 January Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. By submitting a comment you agree to abide by our Terms and Community Guidelines.
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Skip to main content Thank you for visiting nature. Download PDF. Subjects Embryonic stem cells Induced pluripotent stem cells. Abstract In order to apply human embryonic stem cells hESCs and induced pluripotent stem cells hiPSCs to regenerative medicine, the cells should be produced under restricted conditions conforming to GMP guidelines. Introduction Human embryonic stem cells hESCs and induced pluripotent stem cells hiPSCs hold promise as tools for regenerative medicine. Figure 1. Full size image.
Figure 2. Establishment of iPS cell clones under the feeder-free Ff and xeno-free Xf culture system. Figure 3. Establishment of Ff-hiPSCs from fibroblasts and blood cells Human primary fibroblasts were derived from biopsied skin tissue samples. Directed differentiation into dopaminergic neurons Dopaminergic neurons were induced from Ff-hiPSCs under xeno-free conditions.
Directed differentiation into blood cells Ff-hiPSCs were differentiated into blood cells as described previously 30 with some key modifications. Directed differentiation into insulin-producing cells Ff-hiPSCs were differentiated into insulin-producing cells as described previously References Schwartz, S. View author publications.
Ethics declarations Competing interests The authors declare no competing financial interests. Electronic supplementary material. Supplementary Information Fano resonance in anodic aluminum oxide based photonic crystals. About this article Cite this article Nakagawa, M. Copy to clipboard. Comments By submitting a comment you agree to abide by our Terms and Community Guidelines.
Publish with us For authors Submit manuscript. Search Search articles by subject, keyword or author. Show results from All journals This journal. Close banner Close. Second, it does not require viral packaging. Third, no repeated treatments with reprogramming factors are needed.
A single transfection of episomal vectors is sufficient for the derivation of human iPSCs. In this report, we have made significant improvement of the episomal reprogramming method. Using chemically defined media, we have established a small molecule-aided feeder-free reprogramming condition for the efficient derivation of footprint-free human iPSCs from skin fibroblasts, adipose tissue derived cells and cord blood cells.
This method can be readily adapted to the derivation of clinical-grade human iPSCs. Of particular interest, iPSC derivation with this method appeared to progress through a distinct intermediate stage. It will be interesting to find out how this small-molecule aided episomal reprogramming method compares to other reprogramming methods in terms of the quality of iPSCs they generate.
Thus, small molecules that could either accelerate reprogramming process, or reduce episomal vector loss and transgene silencing during the first two weeks post-transfection, were expected to improve episomal reprogramming. Human leukemia inhibitory factor hLIF , though did not significantly improve episomal reprogramming efficiency, increased the proliferation of reprogramming intermediates and the reprogramming pace.
The increase in the episomal reprogramming efficiency correlated with the duration of small molecule treatment Fig. Early treatment, i. A Effects of PD P, 0. Transfected human foreskin fibroblasts were plated to MEF feeder cells. Different combinations of small molecules and hLIF were added throughout reprogramming starting on day 4 post-transfection. B Temporal requirement of small molecule treatment for improved episomal reprogramming. C Extensive differentiation of the newly derived iPSCs p3 in the presence of small molecules.
The addition of bFGF in the culture medium had no effect. Black arrow: undifferentiated iPSC colonies; white arrows: differentiated colonies. On the contrary, these molecules were able to expand mouse ESC-like human iPSCs from reprogramming cultures that were not previously exposed to these small molecules . Surprisingly, the human episomal iPSCs obtained in the continuous presence of small molecules expanded well under conditions for human ESCs without small molecules , but underwent extensive differentiation when cultured in the same condition used for their derivation, i.
The seemingly conflicting results could be explained by the presence of activities that mitigated the effectiveness of small molecules in the MEF feeder cells and human ESC medium used for reprogramming e.
Since the MEF feeder cells and human ESC medium used for reprogramming likely contains small molecule-mitigating activities, it is possible to further improve episomal reprogramming by using matrix and defined culture media. Such defined reprogramming conditions will not only reduce the quality variations associated with feeder cells and KnockOut serum replacement in the human ESC medium, but also can be readily adapted to the production of clinical-grade human iPSCs.
To find defined media that could support episomal reprogramming, we initially tested the N2B27 medium since it has a simple formulation and was able to support the proliferation of human ESCs when supplemented with cytokines . As shown in Figure 2A , the N2B27 medium supplemented with small molecules gave rise to nearly 6-fold higher number of colonies stained positive for alkaline phosphatase a human pluripotent stem cell marker test 2 vs.
They could be expanded for more than 10 passages in the N2B27 medium supplemented with small molecules. Interestingly, the piPSCs contained abundant episomal vectors and maintained high-level transgene expression even after multiple passages in the N2B27 medium supplemented with small molecules Fig.
S2B , suggesting a likely mechanism that these small molecules improved episomal reprogramming was through the retention of episomal vectors and transgene expression. A Effects of MEF feeder cells, matrigel and culture media on episomal reprogramming. Transfected human foreskin fibroblasts were plated to MEF feeder cell-seeded or matrigel-coated cm dishes, and subjected to different reprogramming culture conditions.
Total: both endogenous and transgene expression. D Temporal requirement of small molecule treatment for feeder-free episomal reprogramming test 4 Fig. Transfected human foreskin fibroblasts were plated to matrigel-coated cm dishes. Alkaline phosphatase positive iPSC colonies were counted on day 22 post-transfection. To this end, we divided the reprogramming process into three stages: transfection of human somatic cells with episomal reprogramming vectors stage 1 , reprogramming using N2B27 medium supplemented with small molecules stage 2 , and expansion with mTeSR1 medium stage 3 Fig.
When the N2B27 medium supplemented with small molecules was used at stage 2 to support reprogramming, only rare conversion of piPSCs to human ESC-like iPSCs could be observed, suggesting that the transgene expression during expansion in mTeSR1 was insufficient to reactivate the expression of the endogenous pluripotent genes in most piPSCs. Thus we examined whether it was possible to improve episomal reprogramming by adding additional cytokines in the N2B27 medium supplemented with small molecules stage 2.
Importantly, removal of MEF feeder cells by replacing with matrigel yielded even higher reprogramming efficiency test 4 Fig.
Time-course experiments showed a requirement for an optimal time window of small molecule treatment Fig. Thus, using small molecules and defined media test 4 Fig. With the newly developed feeder-free reprogramming condition, we have successfully derived human ESC-like iPSCs from neonatal and adult skin fibroblasts.
When injected into immunocompromised mice, they formed teratomas consisting of derivatives of all three germ layers, demonstrating the pluripotency of these iPSCs Fig. Open circles indicate unmethylated, and filled circles indicate methylated CpG dinucleotides.
Teratomas were obtained from all iPSC clones. Left panel: neural tissue ectoderm ; middle panel: cartilage mesoderm ; right panel: gut epithelium endoderm. Using human fibroblasts, we have successfully established a feeder-free small molecule-aided episomal reprogramming method. Though the reprogramming efficiency was high enough to enable routine iPSC derivation from human adult fibroblasts, we sought to further improve the efficiency by modifying the episomal vectors.
Our previous work demonstrated that the balance between the expression of different transgenes had great impact on the reprogramming efficiency .
Since the transgene expression from different episomal vectors differs, we tested the two episomal vector combinations that were previously shown to be functional  Fig. S1A and S1B : a two-vector combination 7F-1, which was used for the studies above, and a three-vector combination 7F As shown in Figure 4A , the three-vector combination 7F-2 consistently yielded more iPSC colonies than the two-vector combination 7F Thus with LMYC , the small molecule-aided episomal reprogramming method is robust for human skin fibroblasts.
A Effects of different episomal vector combinations and transformation-deficient LMYC on the reprogramming of human fibroblasts. Transfected human foreskin fibroblasts HFFs were plated to matrigel-coated cm dishes. B Effects of different transgene combinations on the reprogramming of human adult adipose tissue-derived stem cells AdSCs. Transfected adipose tissue-derived stem cells were plated to matrigel-coated cm dishes.
Since skin fibroblast might contain higher number of somatic mutations, they might not be an ideal cell type for the production of clinical-grade human iPSCs. We tested episomal reprogramming of additional cell types that are readily available from human donors, specifically, adipose tissue derived cells and cord blood cells.
As shown in Figure 4B and 4C , the small molecule-aided episomal reprogramming method could be easily adapted to these two cell types. For adipose tissue-derived cells, the three-vector combination 7F-2 only yielded very few iPSCs, although many piPSC colonies were present.
Cord blood cells, compared to other somatic cell types, have unique advantages as donor cells for the production of clinical-grade human iPSCs, since these cells potentially contain less somatic mutations, and are readily available.
In this report, using a combined genetic and chemical approach, we have successfully established a robust feeder-free episomal reprogramming method using defined media. Though developed with fibroblasts, this method was readily adapted to adipose tissue-derived cells, cord blood cells, and potentially other cell types easily accessible from human donors. Additional features can be introduced into episomal vectors to further improve reprogramming efficiency. For example, the current episomal vectors have elements necessary for bacterial propagation, which contain many CpG islands known to contribute to transgene silencing .
Nevertheless, the current method is robust enough for the routine derivation of footprint-free iPSCs from different somatic cell types and from many human donor samples. Selection of the right episomal vector combination and transgene combination appeared to be important for achieving optimal reprogramming efficiency for different somatic cell types Fig.
Since only a single transfection is needed for episomal reprogramming, this method could be readily applied to many cell types including blood cells such as cord blood cells, although peripheral blood cells might represent an ideal cell source for large-scale studies as established procedures are readily available for blood handling e. It is interesting to see how well this method can be applied to peripheral blood cells, particularly cells without genomic rearrangements.
The current episomal reprogramming method used matrigel as the matrix to support reprogramming and iPSC expansion due to its affordability.