1 Lecture 2: Nuclear Reprogramming

Nuclear Reprogramming 2 Switch of gene expression from one cell type to another Switch from a differentiated, specialized cell type to a developmental more primitive and pluripotent state No modification of the genome Alteration of the epigenome (DNA methylation, histone modification)

3 How to reprogram towards pluripotency 1.Somatic cell nuclear transfer 1.Somatic cell fusion with pluripotent cells 2.Transduction of pluripotent genes into somatic cells a.k.a. Direct reprogramming Yamanaka and Blau, Nature, 465(7299):704{12, Jun 2010.

4 History of nuclear reprogramming Yamanaka and Blau, Nature, 465(7299):704{12, Jun nuclear transfer (blue), cell fusion (pink) and transcription-factor transduction (green)

5 How to reprogram towards pluripotency (reminder) 1.Somatic cell nuclear transfer 1.Somatic cell fusion with pluripotent cells 2.Transduction of pluripotent genes into somatic cells Yamanaka and Blau, Nature, 465(7299):704{12, Jun 2010.

Somatic-derived stem cells via nuclear transfer Create ES cells that match the donor’s genetic makeup for therapeutic purposes. Currently, no human ES stem cell lines have been derived from this method (only 3N so far). ES cells derived from patients can be directed to differentiate into specific lineages (e.g. dopaminergic neurons) to study a particular disease (e.g. Parkinson’s disease). This method may be used for cell-based therapies that would circumvent immune rejection. Not extensively used at present, because: 1) iPS strategies are more feasible, 2) stress to the egg causes a reduced efficiency for ES cell generation, 3)ethics. 6 ES cell Neuron Progenitor Enucleated oocyte Fibroblasts from patients Nucleus directed differentiation

7 1.Somatic cell nuclear transfer 1.Somatic cell fusion with pluripotent cells 2.Transduction of pluripotent genes into somatic cells Yamanaka and Blau, Nature, 465(7299):704{12, Jun How to reprogram towards pluripotency (reminder)

Cell Fusion-Mediated Nuclear Reprogramming 8 Several nuclei are forced to share one common cytoplasm (viral, chemical or electric cell fusion technologies) If fused cells proliferate they will become hybrids and on division the nuclei fuse to become 4n or greater Ratio of different nuclei and culture medium conditions favors towards the desired cell type. Direct and fast method (1-2 days)

9 How to reprogram towards pluripotency 1.Somatic cell nuclear transfer 1.Somatic cell fusion with pluripotent cells 2.Transduction of pluripotent genes into somatic cells Yamanaka and Blau, Nature, 465(7299):704{12, Jun 2010.

Induced pluripotent stem cells (iPS cells) 10

Induced pluripotent stem cells (iPS cells) A type of pluripotent stem cell artificially derived from an adult somatic cell by "forcing" expression of specific genes. Forced expression in somatic cells is realized by: – Viral transduction – Proteins – Plasmids – mRNA 11 Skin cell iPSC Reprogramming Strategy With few reprogramming factors OCT3/4, SOX2, KLF4, cMYC OCT3/4, SOX2, LIN28, NANOG Yamanaka factors Thomson factors

iPS cells – using retrovirus/lentivirus 12 Advantages: - easy to use - reproducible - good efficiency - controlled expression (inducible) Disadvantages: - increased risk of insertional mutagenesis - possibility of transgene reactivation - incomplete silencing - clone to clone variation

iPS cells – using Proteins 13 Advantages: - no genomic modification - non-DNA approach Disadvantages: - very slow process - very inefficient process (0.006%, Zhou et al. 2009) - requires the addition of other molecules (VPA) Reprogramming factors are fused to cell-penetrating peptide (CCP) Proteins can be recombinant (produced in bacteria) or in mammalian cells (HEK293) Proteins need to be active and functional in order to work

iPS cells – using Plasmids 14 Advantages: - no genomic modification Disadvantages: - very inefficient process (0.006%, Zhou et al. 2009) - repeated transfection

iPS cells – mRNA 15 Advantages: - no genomic modification - highly efficient approach - faster kinetics - factors titratable - transient nature of mRNA - Biosafety Disadvantages: - repeated transfection

Day 1Day 3Day 5 Day 7Day 9Day 10 Nuclear reprogramming with mRNA

17 it allows researchers to obtain pluripotent stem cells, which are important in research and potentially have therapeutic uses, without the controversial use of embryos. Reprogramming adult cells to obtain iPS cells may pose significant risks that could limit their use in humans. If viruses are used to alter the cells’ genome, the expression of cancer-causing genes or oncogenes may potentially be triggered after these cells are introduced into animals. iPS cells - a recent advance

RNA iPSC Embryonic stem cell iPS cells versus ES cells iPS cells are believed to be similar to ES cells with respect to: A) stem cell gene and protein expression B) ability to differentiate into all lineages in vitro C) forming viable chimeras after injection into blastocysts or tumors when transplanted into adult tissues D) potential to form an entire organism, such as a mouse

iPSC reprogramming factors 19 Retroviruses (viruses that contain RNA, and convert RNA into DNA) that infect fibroblast cells are commonly used. Virus encodes four transcription factors: Oct4, Sox2, Klf-4 and c-Myc. C-Myc is a tumor-inducing gene (oncogene). Oct4 and Sox2 are necessary to induce pluripotency of fibroblasts. Transcription factors increase the efficiency of iPS production. Currently, reprogramming is inefficient and slow. Transcription factors modify gene expression in infected cells. Factors turn OFF genes that are part of the differentiated phenotype. Factors turn ON genes that both maintain pluripotency and the ability to self-renew.

iPSC reprogramming factors – OCT3/4 20 transcription factor (one slide of what a transcription factor is!!!) key reprogramming factor for derivation of iPS cells master regulator of pluripotency specifically expressed in ES cells and the early embryo knock-down of OCT3/4 in ES cells leads to differentiation to date specific function of OCT3/4 during reprogramming is not known

iPS reprogramming factors – SOX2 21 another key factor for nuclear reprogramming expressed in ES cells, early embryos, germ cells and neural stem cells May act as an OCT3/4 cofactor and even regulate expression of OCT3/4 itself SOX2 forms heterodimers with OCT3/4 to synergistically control ES cell-specific gene expression

Reprogramming different cell types 22 Sun et al., 2010

Epigenetic modifications during reprogramming 23

What exactly happens during reprogramming? 24 Suggested model #1 Suggested model #2 Suggested model #3

Transcriptional Regulatory Circuitry 25 Suggested model #1Suggested model #2

Nuclear Reprogramming Concept Mapping Terms Add the key terms/concepts from today’s lecture to your previous concept map. You should include (but are not limited to) the following terms/concepts: Induced pluripotent stem cell Nuclear transfer Transcription factor Direct reprogramming Reprogramming factor Epigenome Transgene Transcriptional Regulatory Circuitry Yamanaka factors Due by xxx 26