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Pax6- and Six3-mediated induction of lens cell fate in mouse and human ES cells.

Anchan RM, Lachke SA, Gerami-Naini B, Lindsey J, Ng N, Naber C, Nickerson M, Cavallesco R, Rowan S, Eaton JL, Xi Q, Maas RL - PLoS ONE (2014)

Bottom Line: Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture.In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms.These findings should facilitate investigations of lens development.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology and Reproductive Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America.

ABSTRACT
Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.

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Derivation of a Pax6-GFP reporter mES cell line.(A–H) Stem cell identity of new ES cell line confirmed by immunofluorscence for undifferentiated ES cell markers Oct4, SSEA-1, Nanog, and by histochemical staining for alkaline phosphatase (Alk Phos) activity. (I) Oct4 and Nanog expression confirmed in two Pax6-GFP mES cell clones by RT-PCR. (J–L) Differentiation of mES cells into mesodermal, neuroectodermal and endodermal derivatives confirmed by immunofluorscence for smooth muscle actin (SMA), neurofilament (NF), and alpha-fetoprotein (AFP), respectively. (M–T) Pax6-GFP reporter expression in mES cells detected following transfection with Pax6 (M–O) or Six3 (Q–T) expression vectors 3 days post-transfection. M,O,Q,S, phase contrast; N,P,R,T GFP detection. Scale bars: A-D, H 40 µm; E-G, J-L 10 µm; M–T 20 µm.
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pone-0115106-g001: Derivation of a Pax6-GFP reporter mES cell line.(A–H) Stem cell identity of new ES cell line confirmed by immunofluorscence for undifferentiated ES cell markers Oct4, SSEA-1, Nanog, and by histochemical staining for alkaline phosphatase (Alk Phos) activity. (I) Oct4 and Nanog expression confirmed in two Pax6-GFP mES cell clones by RT-PCR. (J–L) Differentiation of mES cells into mesodermal, neuroectodermal and endodermal derivatives confirmed by immunofluorscence for smooth muscle actin (SMA), neurofilament (NF), and alpha-fetoprotein (AFP), respectively. (M–T) Pax6-GFP reporter expression in mES cells detected following transfection with Pax6 (M–O) or Six3 (Q–T) expression vectors 3 days post-transfection. M,O,Q,S, phase contrast; N,P,R,T GFP detection. Scale bars: A-D, H 40 µm; E-G, J-L 10 µm; M–T 20 µm.

Mentions: A novel mES cell line, designated Pax6-GFP mES (FVB/N mice) was derived from the previously described transgenic mouse line Pax6-GFP Cre, (P0-3.9-GFPCre mice on an FVB background) which expresses GFP reporter under the control of the Pax6 P0 3.9 ectoderm enhancer (Pax6 EE-GFP) [30], [31] (S1A Figure). All animal studies were conducted in accordance with protocols defined in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Animal Care and Use Committee of Harvard Medical School (Boston, MA). Blastocysts from these mice were isolated using standard protocols and the inner cell mass was manually separated and cultured with mES cell media on mitotically inactivated mouse embryonic fibroblasts (MEFs). Based on morphologic appearance, presumptive Pax6-GFP mES colonies were picked, clonally expanded and characterized by immunohistochemistry, RT-PCR analysis, and differentiation assays, to confirm their stem cell identity (Fig. 1A–L).


Pax6- and Six3-mediated induction of lens cell fate in mouse and human ES cells.

Anchan RM, Lachke SA, Gerami-Naini B, Lindsey J, Ng N, Naber C, Nickerson M, Cavallesco R, Rowan S, Eaton JL, Xi Q, Maas RL - PLoS ONE (2014)

Derivation of a Pax6-GFP reporter mES cell line.(A–H) Stem cell identity of new ES cell line confirmed by immunofluorscence for undifferentiated ES cell markers Oct4, SSEA-1, Nanog, and by histochemical staining for alkaline phosphatase (Alk Phos) activity. (I) Oct4 and Nanog expression confirmed in two Pax6-GFP mES cell clones by RT-PCR. (J–L) Differentiation of mES cells into mesodermal, neuroectodermal and endodermal derivatives confirmed by immunofluorscence for smooth muscle actin (SMA), neurofilament (NF), and alpha-fetoprotein (AFP), respectively. (M–T) Pax6-GFP reporter expression in mES cells detected following transfection with Pax6 (M–O) or Six3 (Q–T) expression vectors 3 days post-transfection. M,O,Q,S, phase contrast; N,P,R,T GFP detection. Scale bars: A-D, H 40 µm; E-G, J-L 10 µm; M–T 20 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4269389&req=5

pone-0115106-g001: Derivation of a Pax6-GFP reporter mES cell line.(A–H) Stem cell identity of new ES cell line confirmed by immunofluorscence for undifferentiated ES cell markers Oct4, SSEA-1, Nanog, and by histochemical staining for alkaline phosphatase (Alk Phos) activity. (I) Oct4 and Nanog expression confirmed in two Pax6-GFP mES cell clones by RT-PCR. (J–L) Differentiation of mES cells into mesodermal, neuroectodermal and endodermal derivatives confirmed by immunofluorscence for smooth muscle actin (SMA), neurofilament (NF), and alpha-fetoprotein (AFP), respectively. (M–T) Pax6-GFP reporter expression in mES cells detected following transfection with Pax6 (M–O) or Six3 (Q–T) expression vectors 3 days post-transfection. M,O,Q,S, phase contrast; N,P,R,T GFP detection. Scale bars: A-D, H 40 µm; E-G, J-L 10 µm; M–T 20 µm.
Mentions: A novel mES cell line, designated Pax6-GFP mES (FVB/N mice) was derived from the previously described transgenic mouse line Pax6-GFP Cre, (P0-3.9-GFPCre mice on an FVB background) which expresses GFP reporter under the control of the Pax6 P0 3.9 ectoderm enhancer (Pax6 EE-GFP) [30], [31] (S1A Figure). All animal studies were conducted in accordance with protocols defined in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Animal Care and Use Committee of Harvard Medical School (Boston, MA). Blastocysts from these mice were isolated using standard protocols and the inner cell mass was manually separated and cultured with mES cell media on mitotically inactivated mouse embryonic fibroblasts (MEFs). Based on morphologic appearance, presumptive Pax6-GFP mES colonies were picked, clonally expanded and characterized by immunohistochemistry, RT-PCR analysis, and differentiation assays, to confirm their stem cell identity (Fig. 1A–L).

Bottom Line: Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture.In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms.These findings should facilitate investigations of lens development.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology and Reproductive Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America.

ABSTRACT
Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.

Show MeSH
Related in: MedlinePlus