Generation of Induced Pluripotent Stem Cells from the Prairie Vole

The vast majority of animals mate more or less promiscuously. A few mammals, including humans, utilize more restrained mating strategies that entail a longer term affiliation with a single mating partner. Such pair bonding mating strategies have been resistant to genetic analysis because of a lack of suitable model organisms. Prairie voles are small mouse-like rodents that form enduring pair bonds in the wild as well as in the laboratory, and consequently they have been used widely to study social bonding behavior. The lack of targeted genetic approaches in this species however has restricted the study of the molecular and neural circuit basis of pair bonds. As a first step in rendering the prairie vole amenable to reverse genetics, we have generated induced pluripotent stem cell (IPSC) lines from prairie vole fibroblasts using retroviral transduction of reprogramming factors. These IPSC lines display the cellular and molecular hallmarks of IPSC cells from other organisms, including mice and humans. Moreover, the prairie vole IPSC lines have pluripotent differentiation potential since they can give rise to all three germ layers in tissue culture and in vivo. These IPSC lines can now be used to develop conditions that facilitate homologous recombination and eventually the generation of prairie voles bearing targeted genetic modifications to study the molecular and neural basis of pair bond formation

Generation of induced pluripotent stem cells from the prairie vole.

The vast majority of animals mate more or less promiscuously. A few mammals, including humans, utilize more restrained mating strategies that entail a longer term affiliation with a single mating partner. Such pair bonding mating strategies have been resistant to genetic analysis because of a lack of suitable model organisms. Prairie voles are small mouse-like rodents that form enduring pair bonds in the wild as well as in the laboratory, and consequently they have been used widely to study social bonding behavior. The lack of targeted genetic approaches in this species however has restricted the study of the molecular and neural circuit basis of pair bonds. As a first step in rendering the prairie vole amenable to reverse genetics, we have generated induced pluripotent stem cell (IPSC) lines from prairie vole fibroblasts using retroviral transduction of reprogramming factors. These IPSC lines display the cellular and molecular hallmarks of IPSC cells from other organisms, including mice and humans. Moreover, the prairie vole IPSC lines have pluripotent differentiation potential since they can give rise to all three germ layers in tissue culture and in vivo. These IPSC lines can now be used to develop conditions that facilitate homologous recombination and eventually the generation of prairie voles bearing targeted genetic modifications to study the molecular and neural basis of pair bond formation

PVi lines are pluripotent in tissue culture.

<p>PVi lines generate embryoid bodies (EBs) that contain cell types representing all 3 somatic germ layers as revealed by RT-PCR for molecular markers of ectoderm, endoderm, and mesoderm. This analysis also shows that embryoid bodies from many PVi lines contain <i>Vasa</i>-expressing cells, thereby suggesting the presence of germ cells.</p

PVi lines are pluripotent in vitro and in vivo.

<p>(<i>A–B</i>) Differentiation of PVi lines in tissue culture yields embryoid bodies. (<i>C</i>) Teratoma obtained following subcutaneous implantation of PVi cells (PVi3) into a NOD/SCID mouse. (<i>D–I</i>) Hematoxylin and eosin stained tissue from teratomas obtained from PVi3 (D, F, H) and PVi6 (E, G, I) shows cellular differentiation into mesodermal, endodermal, and ectodermal lineages. Scale bars equal 100 µm (A, B, D–I).</p

Induction and characterization of PVi lines.

<p>(<i>A</i>) Protocol for reprogramming PVEFs. All brightfield and immunolabeling images (B, C, E–J) depict colonies of a single representative PVi line (line 6). (<i>B–C</i>) Colony morphology of a PVi line. Colonies display morphology similar to that of mouse ES cells, including distinct raised colonies (B), with tightly-packed cells and well-defined, phase-bright margins (C). (<i>D</i>) RT-PCR for endogenous reprogramming factors in PVi lines. RT-PCR for pv-<i>Nat1</i> was performed as a positive control for an endogenous, ubiquitously expressed gene that should be expressed in all cells irrespective of their reprogramming state. Lanes 1–11 show PCR products from PVi lines 1–11, respectively. Lane 12 (“F”) shows PCR products from PVEFs. (<i>E</i>) PVi colony exhibiting alkaline phosphatase (Alk. phosphatase) activity. (<i>F</i>) Live immunofluorescent labeling of a PVi colony for SSEA-1. (<i>G–J</i>) Immunofluorescent labeling of PVi colonies for Nanog, Oct3/4, Klf4, and Sox2. Scale bars equal 500 µm (<i>B</i>), 100 µm (<i>C, E</i>), and 50 µm (<i>F–J</i>).</p