Establishment of retinal progenitor cell clones by transfection with Pax6 gene of mouse induced pluripotent stem (iPS) cells
Highlights
► Mouse iPS cells were transfected with Pax6 gene, followed by limiting dilution culture. ► Almost half of the cells were CD73+, a marker of photoreceptor precursors. ► Western blotting confirmed cytoplasmic expression of rhodopsin protein. ► KCl stimulation increased free Ca influx into the cells on Ca imaging. ► Thus, they became retinal progenitors including photoreceptor precursors.
Introduction
Retinal progenitors are multipotent and are able to generate different cell types, such as six major neuronal cell types and one glial cell type [20], [32]. Several transcription factors, including Paired box6 (Pax6) [6], [19], [22], Retinal homeobox (Rax) [21], Visual system homeobox2 (Vsx2) [3], SIX homeobox6 (Six6) [17], and SRY-box2 (Sox2) [31] regulate their subsequent maturation.
Pax6 gene contains a paired domain and a paired-type homeodomain [6], [7], [19], [20]. Pax6 gene is expressed during early neuroretina development [7], [27], and is required for multipotency of retinal progenitors [24]. We thus consider that Pax6 transfection may induce retinal progenitors not only from ES cells as has been reported [13] but also from iPS cells.
Induced pluripotent stem (iPS) cells are artificially derived from a somatic cell, by inducing transfected expression of the four genes [23]. iPS cells are similar to ES cells and may become an unlimited source of cells for transplantation without harmful immune reaction and ethical concerns. They differentiate preferentially into multiple neural cell types [28].
We reported establishment of retinal progenitors from mouse ES cells [13]. The cell clones have an ability to differentiate into at least three cell types of retinal neurons, retinal ganglion cells, bipolar cells and photoreceptors. However, their differentiation beyond retinal ganglion cells, actually bipolar cells and photoreceptors was less efficient. Here, we established several retinal progenitors from mouse iPS cells and report characterization of them.
Section snippets
Mouse iPS cells
We used a mouse iPS cell line, iPS-MEF-Ng-20D-17 [23]. The culture (growth) medium consisted of DMEM, supplemented with 2 mM glutamine, 0.1 mM beta-mercaptoethanol, 1 mM pyruvate, and 15% FCS. Undifferentiated iPS cells were maintained in the presence of LIF (1000 U/ml, Millipore, Tokyo, Japan) [13], [23]. We found that our undifferentiated iPS cell preparation expressed nanog promoter-driven GFP, suggesting its undifferentiated status (Fig. 1D) [23].
Pax6 cDNA expression vectors
Mouse Pax6 cDNA was donated by Dr. Barbara
Transfection of mouse iPS cells and cloning of them with limiting dilution culture
We reported that transfection with Pax6 gene of mouse ES cells induced their differentiation and brought about emergence of retinal progenitors [13].
Here, we conducted transfection of mouse iPS cells with the same Pax6 expression vectors. After selection and limiting dilution cultures (Fig. 1A), we picked up the cloned cells expressing Nestin mRNA and Musashi1 mRNA by RT-PCR (Fig. 1A) [12], [16]. Then we conducted second screening where cell clones expressing Chx10 mRNA and Six3 mRNA
Discussion
Previously, we reported that transfection of Pax6 gene induced mouse ES cells to differentiate into corneal epithelial cells as well as retinal progenitors [13]. Thus, Pax6 gene induced ES cell differentiation in two cell lineages, neural cells and epithelial cells. It is possible that Pax6 gene transfection by itself brings about differentiation of ES cells and iPS cells into the stage of “theoretical neuro-epithelial lineage”, which represents a stage capable of differentiating into neural
Acknowledgment
This study was supported in part by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science.
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