Takahashi and Yamanaka, 2006 Fig 1

Takahashi and Yamanaka, 2006 Fig 1

Takahashi and Yamanaka, 2006 Fig 2

Takahashi and Yamanaka, 2006 Fig 3

Takahashi and Yamanaka, 2006 Fig 4

Takahashi and Yamanaka, 2006 Fig 5

Takahashi and Yamanaka, 2006 Fig 5

Takahashi and Yamanaka, 2006 Fig 6

Induced Pluripotent Stem Cells (iPS cells) Fully differentiated adult cells can be reprogrammed to become ES cell-like by addition of a few important transcription factors. List of ES cell associated genes ~20 genes strongly enriched in mouse ES cells over non-pluripotent cell types Reprogramming Factors 4 factors needed to make iPS cells: OCT4, SOX2, KLF4, c-Myc (Takahashi and Yamanka, 2006)

OCT4: (TF) strongly expressed in ES cells, Oct4 null embryos have ICM degeneration and fail to produce ES cells. Reduction of Oct4 in ES cell lines leads to trophoblast differentiation. Oct4 is strongly expressed in PGCs and is required for their survival. SOX2: (TF) Co-factor for OCT4. Sox2 null embryos die and ES cells can not be recovered. ES cells with reduced Sox2 activity become trophoblast. Overexpression of Oct4 rescues Sox2 null defects. SOX2 is upstream of Oct4 and turns it on. NANOG: (TF) Nanog null embryos have a very early defect in which all cell differentiate as extraembryonic tissues. Nanog knockdown in ES cells makes them prone to differentiation likely because it functions to suppresses differentiation. KLF4: (TF) Has genetic redundancy with KLF2, -4, and -5 and deletion of all has ES cell defects similar to described above. Represses p53, which represses Nanog. c-MYC: (TF) Promotes proliferation and survival and thus increases the efficiency and time it takes to induce pluripotency, but also promotes tumorigenesis. Interacts with HAT. LIN28: (RNA binding protein) Inhibits the miRNA Let7 which inhibits endogenous c-MYC. Reprogramming Factors

A few different iPS cell cocktails used successfully… Takahashi and Yamanaka, 2006 Mouse OCT4 SOX2 KLF4 c-Myc Nakagawa et al., 2008 Mouse OCT4 SOX2 KLF4 Takahashi et al., 2007 Human OCT4 SOX2 KLF4 c-Myc Yu et al., 2007 Human OCT4 SOX2 NANOG LIN28 (RNA binding protein) Huangfu et al., 2008 Human OCT4 SOX2 Valproic Acid (inhibitor of HDAC) Extrinsic signals are also provided in the form of feeder cells, serum, or LIF and BMP. Hong et al., 2009 Mouse and Human OCT4 SOX2 KLF4 p53 knockout Liu et al., 2012 Human Amniotic Fluid OCT4 Kim et al., 2009 Human Neural Stem Cells OCT4

OCT4, SOX2, and NANOG Target Genes in Human ES Cells Boyer et al., 2005 Venn diagram representing the overlap of OCT4, SOX2, and NANOG promoter bound regions. ChIP-Chip experiments were used to identify target genes of key TF’s in pluripotent cells – they have overlapping targets.

Boyer et al., 2005 Core Transcriptional Regulatory Network in Human ES 3 TF’s regulate many global processes to maintain a state of pluripotentiality.

Progress and Problems with iPS Cells 1. Viral integration of reprogramming factors Why is this a problem? What can we do about this? 2. Identification of iPS cells Why is this difficult? What can we do to make identification easier? 4. Efficient and specific differentiation of iPS cells Why is this so difficult? What can we do to figure this out? 3. Tumorigenesis Why does this happen? What can we do to stop this?

Amabile and Meissner, 2009

Rolletschek and Wobus, 2009

Are iPS Cells the same as ES Cells? How can we even tell if they are the same? How can we identify them at all?

Specific Differentiation of iPS Cells – One Example