Frontiers in Cell Biology Technology Cloning an Organism

Frontiers in Cell Biology Technology: Cloning an Organism, Stem Cells and Organ Regeneration

Gurden 1962 – Nuclear transfer Nucleus of intestinal cell replaced nucleus of egg cell; Normal tadpole produced Note: This experiment supports the hypothesis that transcription factors are present in the cytosol of the egg cell which activate the genes in the transplanted nucleus to stimulate development.

Fig. 20 -18 CLONING AN ORGANISM TECHNIQUE Issues: Technique is successful less than 1% of the time. Clones typically have shortened lives (DNA teleomeres are shorter than normal for age – match adult lengths) Mammary cell donor Egg cell donor Link to cloning 101 Dolly video 2 1 Egg cell from ovary 3 Cells fused Cultured mammary cells 3 4 Grown in Nucleus removed Nucleus from mammary cell culture Early embryo Is cloning of a sheep ethical? Is cloning a person ethical? 5 Implanted in uterus of a third sheep Surrogate mother Benefits: Could make many identical copies of an organism that has been genetically engineered with useful properties such as high production of a particular protein. Click and clone Mouse expts Utah 6 Embryonic development RESULTS Lamb (“Dolly”) genetically identical to mammary cell donor Dolly 1996

Fig. 20 -23 GENETICALLY MODIFIED GOAT THAT PRODUCES A HUMAN BLOOD PROTEIN FOR CLOTTING IN ITS MILK

Fig. 20 -19 CLONED CATS

Jan. 2018 First Primates cloned

Stem Cells

What is a stem cell? A single cell that can REPLICATE ITSELF AN UNLIMITED NUMBER OF TIMES OR. . . Intro to stem cells Utah DIFFERENTIATE INTO MANY CELL TYPES

Increasing Differentiation of Cells Least S T E M C E L L S Most EMBRYONIC STEM CELLS TOTIPOTENT PLURIPOTENT MOST USEFUL POTENTIAL TO BECOME COMPLETE ORGANISM POTENTIAL TO BECOME ANY CELL TYPE MULTIPOTENT UNIPOTENT (FULLY DIFFERIENTIAGED) Link to stem cells

Note that Pluripotent cells cannot form a placenta, but they can form every other tissue hence practically they are of tremendous value. Multipotent and Unipotent (Adult stem cells) could be potentially useful to regenerate a particular tissue but in practice are very difficult to isolate because they are present in such small amounts.

Embryonic Stem Cells Common Source: Discarded embryos from in vitro-fertilization (5 -6 day old blastocysts). RELATIVELY EASY TO ISOLATE FROM EMBRYO (In contrast, Adult stem cells are present in small amounts and scattered throughout our bodies) PRACTICAL PROBLEM – LIMITED SUPPLY OF EMBRYO’S AVAILABLE

Embryonic Stem Cells EASY TO MAINTAIN IN CULTURE – ESSENTIALLY IMMORTAL (no obvious limit to how many times can divide – telomerase actively renews teleomeres) Freeman Stem Cells POTENTIAL TO BECOME ANY TYPE OF CELL AND REPAIR DAMAGED OR DISEASED TISSUE

Fig. 20 -20 Embryonic Stem Cells have the potential to become any type of cell Stem cells could be converted into different cell types to replace damaged cells without Cultured risk of immune system cells rejection. Embryonic stem cells Adult stem cells Early human embryo at blastocyst stage (mammalian equivalent of blastula) From bone marrow in this example Cells generating all embryonic cell types Cells generating some cell types Different culture conditions Different types of differentiated cells Liver cells Nerve cells Blood cells Link to Utah Stem Cells animation

Bone Marrow transplant can cure Leukemia and Multiple Myeloma Bone Marrow Cancers Bone Marrow produces red blood cells, white blood cells and platelets. Autologous Source of stem cells can be either from patient or close relative donor. Transplant to bone marrow Use chemotherapy or radiation therapy to kill all cells in bone marrow, then replace cells with stem cells. animation Video story

2012 Nobel Prizes in Medicine and Physiology Gurdon expts 1962 Yamanaka (2007) • Specialization of Cells is Reversible • Inactivated nucleus in frog egg cell and replaced nucleus with mature intestinal cell • Modified egg cell developed into normal tadpole • Induced Pluripotency • Mature cells can be reprogrammed back to stem cell state by introducing genes for 4 transcription factors Link to IPS video

Out of 24 possible proteins expressed in only stem cells – only 4 are needed to transform differentiated cells • 24 known proteins specifically expressed in stem cells not expressed in differentiated cells • Add all 24 genes for all 24 proteins using virus; differentiated cells become stem cells • Reduce genes to find minimum # required: Only 4 required. • These proteins are transcription factors associated with epigenetic reprograming. • Later: Methylation patterns in i. PS cells not fully identical to embryonic stem cells. Link to IPS video Link to IPS 2

2007 2010

Benefits and Risks of Stem Cell Applications Benefits • Stem cells can be used in basic research of cell function and disease • Stem cells could be converted into other types of cells damaged by diseases, e. g. nerve or brain cells and used to restore normal function without risk of immune system rejection Risks • Mice injected with differentiated cells produced from stem cells all developed tumors (problem- conversion of stem cells to differentiated cells is not 100% efficient) • Moral/ethical objections – humans changing nature/playing God and objections to embryos as source of stem cells (concern from aborted fetus? )

2017

Growing Replacement Organs by Regeneration • Human ear: The synthetic scaffold of an ear sits bathed in cartilage-producing cells, part of an effort to grow new ears for wounded soldiers. Ear link video Link to Tissue Regeneration

(2010)

Growing a Replacement Bladder Replacement Heart 3 -D printing from stem cells Link to Video Beating heart Link to Rice • The replacements which have used only 1 or 2 types of cells thus far have been grown from differentiated cells from patients specific tissues. • Complex organs, heart, liver, pancreas, nerves will probably need stem cells.

Update April 2013 • Published in Nature Medicine: Researchers in Pittsburgh have succeeded in growing a replacement kidney for rats. The regenerated kidneys have 10 -15% of normal function in vitro and 5% when transplanted into the rat. • Techniques need improvement before human implantation but a significant forward step.

Update Fall 2016 • Researchers at Advanced Cell Technology reported the ability to remove a single viable stem cell from a developing embryo without destroying the viability of the embryo for normal development. (Fall 2015) • Nov. 2016, U of Michigan researchers were able to take grow mice stem cells on a mini-lung scaffold and then transplant the cells into mice lung tissue. Over time the stem cells differentiating into different types of lung cells because of the signals they received from surrounding cell tissue.

• Utah – applications of stem cells link Link to 3 -D bioprinting

Link to Making New Hearts • Link to Tissue Regeneration

• Link to Utah Stem Cells animation • Intro to stem cells Utah

Link to cloning 101 Link to stem cells

Link to Jaenisch interview Link to animation Link to Nature IPS video

The developmental potential and epigenetic states of cells at different stages of development. Hochedlinger K , and Plath K Development 2009; 136: 509523

2013 Human stem cell created by somatic nuclear transfer Nuclear transfer video link

Differentiated (unipotent) cells – are specialized cells that carry out very specific tasks; for example the primary job of red blood cells is transport oxygen to tissues

Freeman Stem Cells

Multipotent have the potential to become cells of a particular tissue. For example, a hematopoietic stem cells have the potential to develop into any type of blood cell (but not for example, an eye cell or a liver cell) “Adult” stem cells = multipotent stem cells