L4: stem cells and reproductive ethics

What are stem cells?

Unspecialised cells which can reproduce indefinitely or differentiate into one or more specialised cell types (potency)

What are the types of stem cells?

Embryonic and adult

What can stem cells generate?

Tissues, organs or organisms

What are the 4 stem cell potencies?

Totipotent, pluripotent, multipotent, unipotent

Totipotent Stem Cells

Generate all the tissues of the embryo and extra -embryonic tissues, such as the placenta

E.g. zygote/fertilised ovum

Pluripotent stem cells

Generate cells all 3 germ layers but not the extra - embryonic tissues

E.g. embryonic stem cells

Multipotent Stem cells

Able to differentiate into multiple lineages but not to all germ layers

E.g. haemopoietic stem cells, mesenchymal stem cells

Unipotent stem cells

Able to differentiate along only one lineage

E.g. most adult stem cells in differentiated undamaged tissues

What are the 3 germ layers?

• Ectoderm outside layer (forming exoskeleton)

• Mesoderm middle layer (develops into organs)

• Endoderm Bottom layer (forms inner lining of organs)

What tissues can stem cells be isolated from and how?

• Adult tissue: biopsy & bone marrow

• Umbilical cord blood: at birth

• Foetal tissues & organs: after pregnancy termination

• IVF embryos

Autologous stem cells meaning

Take from an individual and return to the same person

Allogeneic stem cells meaning

Take from an individual & return to a different person

What happens in somatic cell nuclear transplantation?

The nucleus is removed from an egg cell and a somatic cell. The nucleus from the somatic cell is implanted or transferred into the egg cell. The egg cell forms a morula(ball of cells)

What happens in somatic cell nuclear transplantation if the donor nucleus maintains full genetic potential?

The recipient cell develops into any tissue/ organ of the organism

What happened in John Gurdon’s nuclear transplantation in xenopus laevis?

• Frog egg cell was enucleated

• The nucleus was transplanted to both a less differentiated frog embryo cell and a fully differentiated cell

• The cells with donor nucleus activated to begin development

What happened in nuclear transplantation of Xenopus Laevis when the nuclei came from a less differentiated cell?

Most recipient eggs developed into tadpoles

What happened in nuclear transplantation when nuclei came from fully differentiated intestinal cells?

Fewer than 2% of the eggs developed into normal tadpoles as most embryos stopped developing at an earlier stage

What decreases as the donor cell becomes more differentiated?

Its efficiency

What happens to the nucleus as animal cells differentiate?

It changes , potential is restricted more and more as embryonic development & cell differentiation progress

Describe the mammalian reproductive cloning of Dolly the sheep

In 1997 at Roslin institute, a lamb was cloned from adult sheep by somatic cell nuclear transplantation of a differentiated cell. From 700 embryos, one successfully completed normal development (Dolly) where her confirmed chromosomal DNA was identical to the donor nucleus.

Range of mammals subsequently were cloned (mice, rats, cows, horses, pigs dogs & monkey)

What are the challenges of mammalian reproductive cloning?

• Small percentage of cloned embryos develop normally to birth

• Cloned animals don't always look or behave identically

• At age 6, Dolly developed a lung condition associated with older sheep & was euthanised

• Dolly's cells were not as healthy as normal sheep due to incomplete reprogramming

• Cloned nice prone to obesity, pneumonia, liver failure & premature death

What organism is reproductive cloning banned and what is a reason for this?

Humans because there is a good chance the clone would have serious health conditions.

What are the stages of human reproductive cloning?

• Nucleus from patient cell is transferred into a denucleated oocyte

• The cell forms a morula and blastocyst

• The blastocyst is implanted into the uterus

• Foetus is developed and clone is born

How long could human reproductive cloning take?

14 days Max

What is there interest in for cloning?

The ability to generate stem cells for therapeutic cloning

Features of human embryonic stem cells

• Derived from inner cell mass of blastocyst

• Self renew and expand indefinitely in culture

• Pluripotent - can derive cells from all 3 dermal layers

What are the uses of therapeutic cloning?

• The application of SCNT to produce patient- specific cell lines isolated from an embryo

• Designed to replace injured/ diseased tissue

• Not intended for in utero transfer

What is a challenge of therapeutic cloning?

It is very difficult

What are therapeutic uses of stem cells?

• Repair

• Replace

• Restore

• Regenerate

• Replacement of cells or tissues after injury or disease with stem cell derived tissue

The applications of embryonic stem cells

• Basic research

• Drug testing: disease & patient - specific cells

• Testing Toxicology of new drugs - normal, human cell supply

• Drug discovery

• Therapy

What was wrong with Hwang Woo Suk cloning paper?

• The data was fabricated

• Unethically sourced oocytes

What are the ethical objections of human embryonic stem cells?

• Derived from embryo at very early stage - when is it considered a life?

• Range of policies across the world for their use

• religious beliefs

What is a major risk of SCNT.?

Teratoma

Teratoma

Often benign tumour containing tissues of more than one germ layer arising from totipotent cells and often found in the testes or ovaries. It can grow teeth & hair

When might a teratoma arise?

If pluripotent cells were left in therapies of differentiated cells.

What are the concerns about the clinical use of human embryonic stem cells?

• Mainly mixed differentiation still, methods have to be refined

• Potential for teratoma development from non- differentiated ones

• Small experimental scull

• Off the shelf therapy requires a huge bank to tissue match or immune rejection issues occur

• Animal products used in culture pose infection & immune risks

What are murine embryonic stem cells used to make?

Transgenic mice

What are Hematopoietic stem cells used for?

Bone marrow transplants

Which patients need blood system replaced?

• Leukaemia

• Sickle cell anaemia and thalassaemia

• Immune deficiencies

• MS replacement auto reactive immune system

• Other cancer recovery of blood system after extreme chemo

Adult vs embryonic stem cells in regenerative medicine

• Origin - adult = autologous or allogeneic embryonic = allogeneic

• Expansion - adult =poor embryonic = limitless

• Differentiation - adult = limited lineage embryonic = all cells of body

• Ethics - adult = few issues embryonic = pro life concerns

What cells can somatic cells be reprogrammed into?

Iadult cells can be de-differentiated into Induced pluripotent stem cells

• Re-programme differentiated cells to act like embryonic stem cells

• Essential to Introduce 4 stem cell master regulatory genes (Myc, Oct3/4, Sox2, Klf4)

• Improved efficiency by adding Nanog and LIN28

What are the major potential uses for human induced pluripotent stem cells?

• Cell therapy: patients own cells reprogrammed into iPS cells to replace non functioning tissues such as pancreatic insulin producing cells.

• Research & drug discovery: reprogrammed to form iPS cells to provide a cellular model of disease to understand disease and develop treatments

What is the therapeutic potential of induced pluripotent stem cells?

Can reverse type one diabetes: patients adipose tissue can be reprogrammed

What is potency?

The ability of a cell to differentiate

What do mature cells lose?

Their potency

What happens in non-reproductive therapeutic cloning?

• Nucleus from patient cell transferred into denucleated oocyte

• Cell forms morula & blastocyst

• Totipotent cells in culture form pluripotent ES cells

• Differentiation to cell of interest transplanted back to patient.

What are hematopoïetic stem cells the precursor for?

Blood cells and can be used for bone marrow transplant

Advantages and disadvantages of induced pluripotent stem cells

• Advantages; can make person - specific cell lines, no embryos damaged providing an ethical alternative, can make lines from people with genetic disease and study images.

• Problems: genetically modified, potential encogenesis or damage to host genome, differentiation and threat of teratoma.

Key questions of stem cell regenerative therapy

1. Is cell replacement a practical solution?

2. Can cells be delivered effectively?

3. Is it a mechanism of repair? - direct or indirect effect? Host or donor cell recovery?