![]() ![]() hPGCLCs are aggregated with mouse embryonic ovarian somatic cells to form xenogeneic reconstituted ovaries, which are cultured under an air–liquid interface condition for ~4 months for hPGCLCs to differentiate into oogonia and immediate precursory states for oocytes. iMeLCs, or, alternatively, hPSCs cultured with divergent signaling inhibitors, are induced into hPGC-like cells (hPGCLCs) in floating aggregates by cytokines including bone morphogenic protein 4. hiPSCs are induced into incipient mesoderm-like cells (iMeLCs) using activin A and a WNT pathway agonist. Here, we describe a protocol to differentiate human induced pluripotent stem cells (hiPSCs) into oogonia in vitro. hPGCs undergo genome-wide epigenetic reprogramming and differentiate into oogonia or gonocytes, precursors for oocytes or spermatogonia, respectively. (I) Amplitude, maximal upstroke velocity (Vmax upstroke), and maximal decay velocity (Vmax decay) of calcium transients.The human germ-cell lineage originates as human primordial germ cells (hPGCs). (H) Representative tracings of rhythmic spontaneous and caffeine-induced calcium transients. Bottom: action potential duration at 90 and 50% repolarization (APD90 and APD50). (G) Top: maximal diastolic potential (MDP). Data correspond to clone UC C0406-iPS-C4P17 (also in G through I) similar results were observed with UC C0406-iPS-C1P17. (F) Representative spontaneous action potential tracing recorded by whole-cell patch clamp technique. ASGPR stands for asialoglycoprotein receptor. Glycogen accumulation was detected with periodic acid-Schiff staining. (D and E) Phase contrast and immunofluorescence photographs of hepatocytes and cardiomyocytes produced from representative UiPSC clones scale bars, 50 μm (phase contrast of cardiomyocytes), 200 μm (phase contrast of hepatocytes) and 50 μm (all immunofluorescence photographs). Bottom: confocal immunofluorescence of neurons and astrocytes (glial fibrillary acidic protein ) produced from the same UiPSC clone scale bars, 50 μm. Middle: confocal immunofluorescence microscopy for the indicated markers of neural rosettes produced from the same UiPSC clone scale bars, 50 μm. (C) Top, from left to right: phase contrast photographs of EBs growing in suspension, neural rosettes, and neurons produced from a representative UiPSC clone scale bars, 50 μm. AFP stands for α-fetoprotein scale bars, 50 μm. (B) Confocal immunofluorescence microscopy for markers of the 3 germ layers in differentiating embryoid bodies (EBs) of a representative UiPSC clone. (A) Teratomas comprising derivatives of the 3 germ layers. Multidifferentiation potential of urinary induced pluripotent stem cells (UiPSCs). (J) Bisulfite sequencing analysis for the Oct4 and Nanog proximal promoters in 2 representative UiPSC clones from the same donor. (I) Normal karyotype of representative male and female UiPSC clones. (H) Semiquantitative PCR showing integration of the exogenous transgenes in the genome of the indicated UiPSC clones, urine donor cells, water, and H9 ESCs were included as controls. (G) Scatter plot of DNA microarrays data for 2 representative UiPSC clones and H9 ESCs. (F) qPCR showing silencing of the exogenous transgenes in the indicated UiPSC clones values are referred to transduced cells extracted at day 6. Values are referred to donor urine cells H9 ESCs were used as control. hTERT indicates human telomerase reverse transcriptase. (E) Quantitative real-time PCR (qPCR) for endogenous ESC transcription factors in the indicated UiPSCs. (D) Confocal immunofluorescence microscopy for the indicated human embryonic stem cell (ESC) markers of a representative UiPSC clone scale bars, 100 μm. Bottom: representative phase contrast photographs of passage (P) 1 UiPSCs grown on feeders (alkaline phosphatase staining is also included) or Matrigel. (C) Top: representative phase contrast photographs of emerging urinary induced pluripotent stem cells (UiPSCs) colonies at different time points. Cells infected with the exogenous factors are also included note the early morphologic changes (clustering) indicative of reprogramming. (B) Phase contrast and immunofluorescence photographs of urine cells at day 3 after infection with control green fluorescent protein (GFP) retrovirus. SKOM refers to the four exogenous factors Sox2, Klf4, Oct4, and c-Myc. (A) Schematic representation of iPSC generation from urine cells (UC). Generation of induced pluripotent stem cells (iPSCs) from urine cells.
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