Elsevier

Neuroscience Letters

Volume 509, Issue 2, 16 February 2012, Pages 116-120
Neuroscience Letters

Establishment of retinal progenitor cell clones by transfection with Pax6 gene of mouse induced pluripotent stem (iPS) cells

https://doi.org/10.1016/j.neulet.2011.12.055Get rights and content

Abstract

We previously reported that transfection of Pax6 gene which regulated early events in eye development into mouse ES cells brought about their differentiation into retinal progenitors. Here, we attempted to establish cloned retinal progenitors which had ability to further differentiate into photoreceptor like cells by transfecting mouse induced pluripotent stem (iPS) cells with Pax6 gene. Undifferentiated iPS cells were transfected with Pax6 cDNA, followed by selection with G418. After limiting dilution culture, we selected cloned Pax6-transfected cells, which simultaneously expressed mRNAs of Nestin, Musashi1, Six3 and Chx10 for further characterization. We obtained totally 8 clonally expanding Pax6-transfected cells. They started to express mRNAs of Brn3b, Cone-rod homeobox (Crx), pkc, CD73, rhodopsin and the γ-subunit of rod cGMP phosphodiesterase (PDEγ). Flow cytometric analysis revealed that almost half of the cells were CD73+, a marker of photoreceptor precursors. Western blotting confirmed cytoplasmic protein expression of rhodopsin. High KCl stimulation increased free Ca influx into the cells on Ca2+ imaging. iPS cells transfected with Pax6 gene, followed by subsequent limiting dilution culture became retinal progenitors including photoreceptor like cells. The cloned cell lines may be useful for analyzing differentiation requirement of retinal progenitors.

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.

References (37)

  • M. Burmeister et al.

    Ocular retardation mouse caused by CHX10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation

    Nat. Genet.

    (1996)
  • W.A. Catterall

    Structure and regulation of voltage-gated Ca2+ channels

    Annu. Rev. Cell Dev. Biol.

    (2000)
  • S.M. Chambers et al.

    Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling

    Nat. Biotechnol.

    (2009)
  • R.L. Chow et al.

    Early eye development in vertebrates

    Annu. Rev. Cell Dev. Biol.

    (2001)
  • N. Davis-Silberman et al.

    Genetic dissection of Pax6 dosage requirements in the developing mouse eye

    Hum. Mol. Genet.

    (2005)
  • U. Demkow et al.

    Kinetics of calcium ion concentration accompanying transduction of signals into neutrophils from diabetic patients and its modification by insulin

    J. Physiol. Pharmacol.

    (2009)
  • J.D. Dignam et al.

    Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei

    Nucleic Acids Res.

    (1983)
  • M. Ide et al.

    Characterization of intracellular free Ca2+ movements in neural progenitor cells derived from ES cells transfected with MASH1 transcription factor gene

    Inflamm. Regen.

    (2005)
  • Cited by (18)

    • Bioactive functional scaffolds for stem cells delivery in wound healing and skin regeneration

      2022, Reactive and Functional Polymers
      Citation Excerpt :

      They used physiological electric fields for this work [136]. The iPSCs may differentiate into many types of progenitor cells, which are necessary to treat disease or for tissue engineering applications [137]. Some of these cells are cardiomyocytes [116], oligodendrocytes [138], hepatocyte-like cells [139], fibroblast-like cells [120], neurons [140], and pancreatic hormone-positive cells [141].

    • Induced pluripotent stem cells for modeling open-angle glaucoma

      2022, Novel Concepts in iPSC Disease Modeling
    • Cellular and molecular mechanisms of the restoration of human APP transgenic mouse cognitive dysfunction after transplant of human iPS cell-derived neural cells

      2015, Experimental Neurology
      Citation Excerpt :

      Complementary DNA (cDNA) was synthesized with High-Capacity cDNA Reverse Transcription Kits (Applied Bioscience, Waltham, MA). Total RNA extraction, cDNA synthesis, and PCR amplification methods were previously described (Suzuki et al., 2012). We found that the neural cells expressed mRNA of several neural markers, including nestin, neurofilament M (NFM), βIII-tubulin, ChAT, and α7nAchR.

    View all citing articles on Scopus
    View full text