Molecular Genetics

Does the reversibility of epigenetic information enable reprogramming?

Imprinting and “nuclear cloning”

Reproductive cloning

“should not work”

Dolly (1996-2003)

Successful cloning was

obtained for cattle, mice,

goats, pigs and cats.

Hochedlinger and Jaenisch (2003)

ICM Embryonic

Stem (ES)

cells

Copycat

Reproductive vs. therapeutic

cloning

Somatic Cell

Nuclear Transfer

(SCNT)

Few facts about reproductive cloning

Reproductive cloning is inefficient

• 1-5 live birth out of 100 cloned embryos

Inefficiency is due to the inability of the recipient oocyte to reprogram

the somatic epigenome of incoming nucleus. Imprinted genes, in

particular, are the hardest to reprogram correctly.

Many cloned embryos die during development and show abnormal

placentation. Viable clones show “large offspring syndrome”

(deregulation of IGF2 ?) and die of respiratory or cardiovascular failure.

Live cloned animals represent the few winners of the “epigenetic

lottery”. Nonetheless, they can show a host of abnormalities.

Reproductive cloning is banned for humans and fraught with

multiple ethical issues, including ones revolving around oocyte

donations

Therapeutic cloning is a HUGE breakthrough

➢ Therapeutic cloning: reprogramming of differentiated somatic cells back

to a pluripotent embryonic stem cell state

➢ Opens the possibility of generating patient-matched ES cells

➢ Resolves thorny ethical issues surround ESC research

➢ Opens up possibilities of “regenerative medicine”

Embryonic stem cells: • derived from Inner Cell

Mass of blastocysts

• capable of self renewal

• pluripotent = can

differentiate into all three

germ layers (no Xtra

embryonic development)

https://www.jove.com/video/52010/high-efficiency-differentiation-human-pluripotent-stem-cells-to

http://www.nature.com/nature/journal/v510/n7504/full/nature13233.html

Combining Gene Therapy with Somatic Cell Nuclear Transfer technology

The end of genetic diseases?

Rideout et al., (2002) Cell, 109:17

How does the oocyte mediate genome- wide reprogramming?

“The oocyte doesn’t accomplish magic – it’s a biochemical reaction” Rudolf Jaenisch

Oct4, Sox2 and NANOG are

critical pluripotency transcription

factors (TFs). - Have thousands of targets in the

genome (promoters and

enhancers), including other

important transcription factors.

- Can BOTH repress cell

differentiation genes and

enhance proliferation and self-

renewal genes

c-Myc and Klf4 are also TFs and

oncogenes associated with

cellular proliferation

Behold the power of “Master” TFs: OCT4, SOX2, NANOG

Jaenisch and Young Cell 2008

Yamanaka and Blau (2010) Nature

Turning the clock back on differentiated cells: TF-mediated reprogramming

• Expression of four “Yamanaka factors” is sufficient to revert

differentiated adult cells to induced pluripotent stem cells (iPS)

• Are iPS cells indistinguishable from ESCs??

Are iPS cells really equivalent to ES cells?

Most stringent test for pluripotency is formation of a whole organism

• iPSCs can support formation of chimeras

Wernig et al., 2007 Nature

• iPSCs can support complete embryo development

Wernig et al., 2007 Nature

Have iPS cells really undergone epigenetic reprogramming?

• Generating iPS cells involves re-wiring the transcriptional circuitry

and epigenetic memory of donor cells

• Low efficiency process: 0.05-0.5%.

• Rate-limiting step involves reprogramming the somatic epigenome

• Efficiency can be improved up to 10-20x by : ➢ drugs which favor reversal of epigenetic memory (5-aza-C, TSA, Vitamin C)

➢kicking cell proliferation in high gear (p53 knockouts, inducer of metabolism)

Cellular reprogramming and iPS cells open the door to corrective regenerative medicine

Power and Rasko (2011) Annals of Internal Medicine

Even better than iPS cells? induced neuronal cells (iN)

Transcription factor-mediated

Reprogramming

Vierbuchen and Wernig

Molecular Cell Sept 2012

Cellular Alchemy: the science of reprogramming cells

Oct4

Sox2 Other master TFs

Normal development:

cells differentiate,

reach terminal fate and

maintain fate

Reprogramming “back

in time” to an

undifferentiated

pluripotent state

Direct fate conversion

through cellular

reprogramming at the

adult stage

Reprogramming / trans-differentiation + gene therapy = regenerative medicine

Next frontiers:

Growing organs?

Rat pancreas grown in mouse

genetically deficient in making

pancreas (and vice-versa)

Cell (2010)

Nature (2016)

Next: human cells in larger animal models (pig, sheep, cattle)

http://www.sciencemag.org/news/2017/01/human-organs-grown-

pigs-not-so-fast

Only few human cells survived after birth

Next step: - Finding more robust human cell types to inject

- Avoiding pig immune system (modify it)

Caveat: the pig genomes carries active

Porcine Endogenous RetroViruses (PERVs)

that could infect the developing human

organs

https://www.statnews.com/2017/10/20/human-pig-chimera/

Science, September 2017