Genome-wide analysis of mRNA and microRNA confirms efficient pluripotent reprogramming of mGriPSCs and hGriPSCs.

<p>mGriPSC-EBs and mESC-EBs have comparable microarray expression profiles <b>(A)</b>. All ESC or EB samples show minimal steroidogenic mRNA compared to adult ovary samples <b>(B).</b> Principal component analysis (PCA) indicates attached and unattached mGriPSC-EBs are more disparate relative to the analogous mESC-EBs <b>(C)</b>. microRNA heatmaps support efficient stem cell reprogramming of mGriPSCs <b>(D)</b>.</p

mGriPSCs are pluripotent.

<p>Using G4-mESCs as a standard <b>(A-D, I)</b>, mGriPSCs are alkaline phosphatase reactive <b>(E)</b> and express stem cell antigens Oct4 <b>(F)</b>, SSEA1 <b>(G)</b> and Nanog <b>(H, I)</b>. mGriPSCS express additional stem cell markers by RT-PCR <b>(I)</b> and are karyotypically normal <b>(J)</b>. mGriPSC express endogenous copies of the introduced reprogramming genes and retroviral [trans] copies are present to varying degrees <b>(K).</b> mGriPSCs form EBs <b>(L)</b> that differentiate into three germ layers—ectodermal neurofilament <b>(M)</b>, and mesodermal SMA <b>(N)</b>, and endoderm alpha-fetoprotein (<b>O,</b> AFP). Teratomas <b>(P)</b> also show differentiation into three germ layers <b>(Q-S)</b>. Scale bars: <b>A-H</b> 20 μm; <b>L</b> 200 μm; <b>M-O, Q-S</b> 10 μm.</p

An autologous iPSC model in regenerative medicine.

<p>Terminally-differentiated cells from various adult organs may generate iPSC lines for homotypic differentiation into desired tissues.</p

Methylation analysis of GC-derived iPS cells shows stem cell reprogramming with retention of target tissue methylation.

<p>The stem cell gene Oct4 in mGriPSC is less methylated than it is in GCs (mGriPSC vs. GC, P<0.001) and is therefore more similar to Oct4 in G4 ESCs <b>(A)</b>. Methylation patterns of ovarian and germ cell genes Foxl2 and Figla are comparable in both mGriPSC and GCs (<b>B</b>, Foxl2, P = 0.84; Figla, P = 0.606).</p

mGriPSC-EBs differentiate to express ovarian and primordial oocyte markers.

<p>Differentiating mGriPSC-EBs express ovarian markers AMHR <b>(A)</b>, FSHR <b>(B)</b>, Cyp19a <b>(C)</b>, Inha <b>(D)</b>, and ER <b>(E)</b> (phase contrast; <b>F)</b> at higher frequencies than G4- and mFiPSC-EB <b>(G)</b>. Expression of germ cell markers Mvh <b>(H)</b>, Dazl <b>(I)</b>, Gdf9 <b>(J)</b>, Boule <b>(K)</b>, and Zp1 <b>(L)</b> (phase contrast; <b>M</b>) are also increased in mGriPSC-EBs <b>(N).</b> Two-tailed t-test, *<i>P</i> < 0.05. Scale bars: <b>A-F</b> 10 μm; <b>H-M</b> 20 μm.</p

mGriPSC-EBs preferentially synthesize estradiol.

<p>Mouse GCs <b>(A)</b> infected with pluripotency genes generate iPSC colonies. Arrow indicates primary granulosa cells. mGriPSC-EBs, when allowed to attach, synthesize estradiol <b>(B)</b> at concentrations 5 to 12 times those of G4-EBs and mFiPSC-EBs. Two-tailed t-test, *<i>P</i><0.001, different from other two cell lines at same time point. G4-EBs primarily secreted progesterone <b>(C)</b> while mFiPSC-EBs produced negligible amounts of either. Two-tailed t-test, *<i>P</i><0.005, different from other two cell lines at same time point. Scale bar: <b>A</b> 100 μm.</p