Takahashi and Yamanaka 2006 Fig 1 Takahashi and

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 (i. PS 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 i. PS cells: OCT 4, SOX 2, KLF 4, c-Myc (Takahashi and Yamanka, 2006)

Reprogramming Factors OCT 4: (TF) strongly expressed in ES cells, Oct 4 null embryos have ICM degeneration and fail to produce ES cells. Reduction of Oct 4 in ES cell lines leads to trophoblast differentiation. Oct 4 is strongly expressed in PGCs and is required for their survival. SOX 2: (TF) Co-factor for OCT 4. Sox 2 null embryos die and ES cells can not be recovered. ES cells with reduced Sox 2 activity become trophoblast. Overexpression of Oct 4 rescues Sox 2 null defects. SOX 2 is upstream of Oct 4 and turns it on. KLF 4: (TF) Has genetic redundancy with KLF 2, -4, and -5 and deletion of all has ES cell defects similar to described above. Represses p 53, which represses Nanog. 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. 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. LIN 28: (RNA binding protein) Inhibits the mi. RNA Let 7 which inhibits endogenous c-MYC.

A few different i. PS cell cocktails used successfully… Takahashi and Yamanaka, 2006 Mouse OCT 4 SOX 2 KLF 4 c-Myc Takahashi et al. , 2007 Human OCT 4 SOX 2 KLF 4 c-Myc Nakagawa et al. , 2008 Mouse OCT 4 SOX 2 KLF 4 Huangfu et al. , 2008 Human OCT 4 SOX 2 Valproic Acid (inhibitor of HDAC) Kim et al. , 2009 Human Neural Stem Cells OCT 4 Liu et al. , 2012 Human Amniotic Fluid OCT 4 Yu et al. , 2007 Human OCT 4 SOX 2 NANOG LIN 28 (RNA binding protein) Hong et al. , 2009 Mouse and Human OCT 4 SOX 2 KLF 4 p 53 knockout Extrinsic signals are also provided in the form of feeder cells, serum, or LIF and BMP.

OCT 4, SOX 2, and NANOG Target Genes in Human ES Cells Ch. IP-Chip experiments were used to identify target genes of key TF’s in pluripotent cells – they have overlapping targets. Venn diagram representing the overlap of OCT 4, SOX 2, and NANOG promoter bound regions. Boyer et al. , 2005

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

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

Amabile and Meissner, 2009

Rolletschek and Wobus, 2009

Are i. PS 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 i. PS Cells – One Example