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Flashcards in Stem Cells and Cloning Deck (18)
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1
Q

What are the properties of embryonic stem cells

A

When fertilised embryo reach the blastocyst stage it forms an inner cell mass, and have unique properties

Immortality
Unspecialized
Developmental Potential (POTENCY)

2
Q

Discuss ES cell division

A

Symmetric Cell Division - mitosis, undergone by differentiated cells as well as stem cells

Asymmetric cell division - divides into 2 cells; 1 = self-renewing cell, 2 = differentiated progenitor cell

They can differentiate into different types of progenitors (developmental potential/potency)

3
Q

Discuss derivation of human stem cells

A

ESC’s are derived at a developmental stage before implantation
Fertilisation occurs in the oviduct, and a series of cleavage stages occur as it travels down

Each cleavage-stage embryo (blastomere) is undifferentiated and has potential to give rise to any cell of the body

1st differentiation event at ~5 days
Outer layer of cells committed to becoming part of the placenta (trophectoderm) separates from inner cell mass (ICM)

ICM cells have potential to generate any cell type of body
Cultured ICM cells continue to proliferate & replicate indefinitely, maintaining developmental potential to form any cell type of the body
Removal and culturing allows them to replicate indefinitely
These pluripotent, ICM-derived cells are embryonic stem (ES) cells

4
Q

Discuss how stem cells can differentiate

A

ESC’s remain undifferentiated (unspecialized) in culture

If cells are allowed to clump together to form embryoid bodies, they begin to differentiate spontaneously
They can form muscle cells, nerve cells, and many other cell types - uncontrolled

Directed Differentiation
Change the chemical composition of the culture medium
Alter the surface of the culture dish
Modify the cells by inserting specific genes

5
Q

Discuss directed differentiation

A

Directed Differentiation
Change the chemical composition of the culture medium
Alter the surface of the culture dish
Modify the cells by inserting specific genes

6
Q

Discuss sources of embryos for hESC’s

A

Two groups obtained the ES cells from different sources:
ICM from IVF embryos-
Primordial germ cells from Aborted Fetuses

Both sets of hESC lines were capable of differentiating into cell types of all the major tissues representing the three primary germ layers (mesoderm, ectoderm, and endoderm)

Questions of ethics arise because embryos are destroyed as the cells are extracted, such as: When does human life begin? What is the moral status of the human embryo?

7
Q

What are sources of stem cells in adult tissues and what are their limitations

A

These can be found in the brain, muscle, liver, retina, hair follicle, adipose tissue

Critical for maintenance of tissues
Responsive to injury and trauma
Illuminate our understanding of cancer

Adult stem cells are not as potent as ESC
Relatively rare un-differentiated cells found in many organs & differentiated tissues

Limited capacity for both self-renewal (in the laboratory) & differentiation
Differentiation capacity usually limited to cell types in the organ of origin

8
Q

Discuss developmental potential of stem cells

A

Totipotent - fertilisation, as a zygote , can form embryonic and extra-embryonic tissues e.g. placenta

Pluripotent - once implanted forms a blastocyst with the inner cell mass and 3 germ layers

Multipotent - after differentiation, includes adults SC’s

Oligopotent - blood cells, can only differentiate into a very specific lineage

Unipotent - can self-renew but only differentiates into one cell type

9
Q

Discuss nuclear reprogramming

A

As cells become more specialized they undergo epigenetic changes (DNA methylation and Histone modification)

This results in a specific pattern of gene expression in a differentiated cell that gives it the characteristics of that particular cell type

But is it possible to redirect the gene expression to that of a different cell type
Reprogramming backwards
Induced pluripotent cells

10
Q

What are the types of stem cells

A

Embryonic
Adult
iPS
Nuclear transfer embryonic stem cells - derived form therapeutic cloning

11
Q

What is the difference between fertilisation and cloning

A

Fertilised embryo - combined genetic material of parents

Clonal embryo - genetically identical to donor

12
Q

How does cloning occur

A

Donor (what you want to clone) and recipient

Enucleate the recipient oocyte
DNA stain localises the genetic material, which is then removed

Prepare the donor nucleus from the donor cell and transfer into the donor egg

Apply electricity which starts cell division

The epigenetic marks needs to be removed from the donor DNA - needs to undergo nuclear reprogramming
This can occur when it is under the right conditions - in oocytes

13
Q

What are the problems with cloning

A

Dolly 1/276 attempts - Success rate 1-3% V IVF success rate 20-50%

30% clones born alive have “large-offspring syndrome”
Likely due to incomplete reprogramming

Serious inexplicable respiratory or circulatory problems, which causes them to die soon after birth
Very few clones actually survive to adulthood

14
Q

Discuss reproductive V therapeutic cloning

A

Reproductive cloning - produces a clonal embryo which is implanted in a womb with intent to create a fully formed living offspring - a clone
Embryo implanted

Therapeutic cloning - produces a clonal embryo, but instead of being implanted in a womb and brought to term it is used to generate stem cells
Embryo is cultured and grown into ES cell lines, can be used to be transplanted back to the patient

15
Q

What is therapeutic cloning

A

Purpose
Find cures and therapies for diseases
Awaken the natural capacity for self-repair that resides in our genes

Potential Results
Patients will receive own stem cells to treat disease
No need for donor match if using ASC/iPSC
Like transplantation, but without rejection

16
Q

Discuss somatic gene therapy

A

Cloning of somatic nucleus from patient, derivation of ES cell

Correction of genetic defect by gene targeting

In vitro differentiation of “repaired” ES cells

Transplantation of differentiated cells into patient resulting in cure

17
Q

Compare embryonic V adult stem cells

A

Source - embryo, bone marrow, brain…
Availability - ESC’s are unlimited in availability
Longevity - ESC’s can be kept indefinitely, ASC’s have limited longevity
Potency - ASC’s can give rise to cells you want, but some are harder to get e.g. brain… ESC’s can form any cell
Safety - ASC’s has been researched much longer
Immune sensitivity - ASC’s can be derived from the patient themselves

18
Q

How do you create iPS and what are their uses

A

It was found introduction of 4 key genes - Oct 3/4, SOX2, c-Myc, KLF4 allows for reprogramming
These show similar gene expression levels to ESC’s

Usage
Treating sickle cell anaemia in a mouse model by extracting differentiated adult skin cells, and inducing pluripotency, correcting the mutation and directing differentiation into blood cells thus curing the mouse