Masayo Takahashi

A more efficient method to generate integration-free human iPS cells

We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match ∼20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.

Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.

We previously reported the differentiation of mouse embryonic stem (ES) cells into retinal progenitors. However, these progenitors rarely differentiate into photoreceptors unless they are cultured with embryonic retinal tissues. Here we show the in vitro generation of putative rod and cone photoreceptors from mouse, monkey and human ES cells by stepwise treatments under defined culture conditions, in the absence of retinal tissues. With mouse ES cells, Crx+ photoreceptor precursors were induced from Rx+ retinal progenitors by treatment with a Notch signal inhibitor. Further application of fibroblast growth factors, Shh, taurine and retinoic acid yielded a greater number of rhodopsin+ rod photoreceptors, in addition to default cone production. With monkey and human ES cells, feeder- and serum-free suspension culture combined with Wnt and Nodal inhibitors induced differentiation of Rx+ or Mitf+ retinal progenitors, which produced retinal pigment epithelial cells. Subsequent treatment with retinoic acid and taurine induced photoreceptor differentiation. These findings may facilitate the development of human ES cell–based transplantation therapies for retinal diseases.

Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity

We previously identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells, including midbrain tyrosine hydroxylase-positive (TH+) dopaminergic neurons, from mouse embryonic stem cells. We report here that SDIA induces efficient neural differentiation also in primate embryonic stem cells. Induced neurons contain TH+ neurons at a frequency of 35% and produce a significant amount of dopamine. Interestingly, differentiation of TH+ neurons from undifferentiated embryonic cells occurs much faster in vitro (10 days) than it does in the embryo (≈5 weeks). In addition, 8% of the colonies contain large patches of Pax6+-pigmented epithelium of the retina. The SDIA method provides an unlimited source of primate cells for the study of pathogenesis, drug development, and transplantation in degenerative diseases such as Parkinsons disease and retinitis pigmentosa.

GENERATION OF RETINAL CELLS FROM MOUSE AND HUMAN INDUCED PLURIPOTENT STEM CELLS

We previously reported a technique for generating retinal pigment epithelia (RPE) and putative photoreceptors from embryonic stem (ES) cells. Here we tested whether our procedure can promote retinal differentiation of mouse and human induced pluripotent stem cells (iPSCs). Treating iPSCs with Wnt and Nodal antagonists in suspension culture induced expression of markers of retinal progenitor cells and generated RPE cells. Subsequently, treatment with retinoic acid and taurine generated cells positive for photoreceptor markers in all but one human cell lines. We propose that iPSCs can be induced to differentiate into retinal cells which have a possibility to be used as patient-specific donor cells for transplantation therapies.

Characterization of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cell Sheets Aiming for Clinical Application

Summary Age-related macular degeneration (AMD) causes severe visual impairment due in part to age-dependent impairment of retinal pigment epithelium (RPE). It has been suggested that autologous human induced pluripotent stem cells (hiPSCs) may represent a useful cell source for the generation of graft RPE. We generated hiPSC-derived RPE (hiPSC-RPE) cell sheets optimized to meet clinical use requirements, including quality, quantity, consistency, and safety. These cell sheets are generated as a monolayer of cells without any artificial scaffolds, express typical RPE markers, form tight junctions that exhibit polarized secretion of growth factors, and show phagocytotic ability and gene-expression patterns similar to those of native RPE. Additionally, upon transplantation, autologous nonhuman primate iPSC-RPE cell sheets showed no immune rejection or tumor formation. These results suggest that autologous hiPSC-RPE cell sheets may serve as a useful form of graft for use in tissue replacement therapy for AMD.

Widespread Integration and Survival of Adult-Derived Neural Progenitor Cells in the Developing Optic Retina

Adult rat hippocampus-derived neural progenitor cells (AHPC) show considerable adaptability following grafting to several brain regions. To evaluate the plasticity of AHPCs within the optic retina, retrovirally engineered AHPCs were grafted into the vitreous cavity of the adult and newborn rat eye. Within the adult eye, AHPCs formed a uniform nondisruptive lamina in intimate contact with the inner limiting membrane. Within 4 weeks of grafting to the developing eye, the AHPCs were well integrated into the retina and adopted the morphologies and positions of Müller, amacrine, bipolar, horizontal, photoreceptor, and astroglial cells. Although the cells expressed neuronal or glial markers, none acquired end-stage markers unique to retinal neurons. This suggests that the adult-derived stem cells can adapt to a wide variety of heterologous environments and express some but not all features of retinal cells when exposed to the cues present late in retinal development.

In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction

The use of stem-cell therapy to treat retinal degeneration holds great promise. However, definitive methods of retinal differentiation that do not depend on recombinant proteins produced in animal or Escherichia coli cells have not been devised. Here, we report a defined culture method using low-molecular-mass compounds that induce differentiation of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into retinal progenitors, retinal pigment epithelium cells and photoreceptors. The casein kinase I inhibitor CKI-7, the ALK4 inhibitor SB-431542 and the Rho-associated kinase inhibitor Y-27632 in serum-free and feeder-free floating aggregate culture induce retinal progenitors positive for RX, MITF, PAX6 and CHX10. The treatment induces hexagonal pigmented cells that express RPE65 and CRALBP, form ZO1-positive tight junctions and exhibit phagocytic functions. Subsequent treatment with retinoic acid and taurine induces photoreceptors that express recoverin, rhodopsin and genes involved in phototransduction. Both three-factor (OCT3/4, SOX2 and KLF4) and four-factor (OCT3/4, SOX2, KLF4 and MYC) human iPS cells could be successfully differentiated into retinal cells by small-molecule induction. This method provides a solution to the problem of cross-species antigenic contamination in cell-replacement therapy, and is also useful for in vitro modeling of development, disease and drug screening.

Wnt Signaling Promotes Regeneration in the Retina of Adult Mammals

Regeneration in the mammalian CNS is severely limited. Unlike in the chick, current models hold that retinal neurons are never regenerated. Previously we demonstrated that, in the adult mammalian retina, Müller glia dedifferentiate and produce retinal cells, including photoreceptors, after acute neurotoxic injury in vivo. However, the number of newly generated retinal neurons is very limited. Here we demonstrate that Wnt (wingless-type MMTV integration site family)/β-catenin signaling promotes proliferation of Müller glia-derived retinal progenitors and neural regeneration after damage or during degeneration. Wnt3a treatment increases proliferation of dedifferentiated Müller glia >20-fold in the photoreceptor-damaged retina. Supplementation with retinoic acid or valproic acid induces differentiation of these cells primarily into Crx (cone rod homeobox)-positive and rhodopsin-positive photoreceptors. Notably, injury induces nuclear accumulation of β-catenin, cyclin D1 upregulation, and Wnt/β-catenin reporter activity. Activation of Wnt signaling by glycogen synthase kinase-3β inhibitors promotes retinal regeneration, and, conversely, inhibition of the signaling attenuates regeneration. This Wnt3a-mediated regeneration of retinal cells also occurs in rd mice, a model of retinal degeneration. These results provide evidence that Wnt/β-catenin signaling contributes to CNS regeneration in the adult mammal.

Stepwise differentiation of pluripotent stem cells into retinal cells

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos. They can maintain an undifferentiated state indefinitely and can differentiate into derivatives of all three germ layers, namely ectoderm, endoderm and mesoderm. Although much progress has been made in the propagation and differentiation of ES cells, induction of photoreceptors has generally required coculture with or transplantation into developing retinal tissue. Here, we describe a protocol for generating retinal cells from ES cells by stepwise treatment with defined factors. This method preferentially induces photoreceptor and retinal pigment epithelium (RPE) cells from mouse and human ES cells. In our protocol, differentiation of RPE and photoreceptors from mouse ES cells requires 28 d and the differentiation of human ES cells into mature RPE and photoreceptors requires 120 and 150 d, respectively. This differentiation system and the resulting pluripotent stem cell-derived retinal cells will facilitate the development of transplantation therapies for retinal diseases, drug testing and in vitro disease modeling. It will also improve our understanding of the development of the central nervous system, especially the eye.

Autologous Induced Stem-Cell–Derived Retinal Cells for Macular Degeneration

We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age‐related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age‐related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC‐derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN‐CTR] number, UMIN000011929.)