Lecture 17- Stem cells and regenerative medicine

What are the challenges of embryonic stem cells for transplantation therapies?

-Purity/production problems -Cancer problems -Immunology problems -Ethics problems.

Spontaneous embryonic stem cell differentiation in vitro

By taking away certain growth factors you stop embryonic stem cell expansion. Instead the embryonic stem cells can form a mixture of differentiated cells into the 3 different germ layers.

Therapeutic cloning

Cells are genetically identical to the patient so won't get rejection issue!

Teratomas from embryonic stem cells

ES cells give rise to disorganised growths called teratomas. = A non-malignant tumor in an animal consisting of tissues from the three embryonic germ layers. Usually found in ovary and testis. -Produced experimentally by the growth and differentiation of injected pluripotent embryonic stem cells.

Ethics of embryonic stem cell:

Ethical concerns --> You would have to destroy the blastocyst. The blastocyst is believed in some religions to be a possible prospective human being so you shouldn't destroy it.

Directed ES differentiated in vitro:

Goal: to determine how to consistently make pure populations of therapeutically relevant cell types (e.g. neurons, pancreatic beta cells) Need to understand the molecules that mediate fate decisions in ES culture.

Another advantage of adult stem cells:

Importantly, adult stem cells have already been proven to be therapeutically effective: -Skin grafts for burn victims -Bone marrow transplants

What are the markers present in undifferentiated cells?

Oct4, Sox2, Nanog. (These markers are only found in pluripotent stem cells).

What is REPRODUCTIVE cloning?

Practically the same thing as therapeutic cloning but instead of taking the inner cell mass from the blastocyst, you take the embryo and implant it, so the baby is genetically identical to the starting woman. (The method about therapeutic clonign is ethically debated as some people believe it could lead to creating genetically identical babies).

Challenges of IPS

Reduce risk of cancer Remove the requirement for viral integration Demonstrate long-term stability of re-programmed cells Improvement of technical efficiency.

Niches of stem cells

Stem cells are in a niche environment. They are effected by their environment around it. -some things surrounding the stem cells that might effect what they do: Other stem cells, growth factors, cellular matrix components, gap junctions, basement membranes, adhesion molecules. Other cells in that region = niche cells. They can send signals to the stem cells that affect it. -If you remove the stem cell from this niche it will most likely differentiate. Also if the basal lamina changes, thus changing the niche, can cause the stem cells to proliferat uncontroledly and can give rise to cancer.

Stem cell niches

Stem cells are thought to be maintained (and perhaps defined) by the environment produced by surrounding differentiated cells. The differentiated cells may: > secrete specific factors into the surrounding matrix > communicate with the stem cells via gap junctions Changes within the niche may induce a stem cell to die, divide or differentiate.

Stem cell division

Symmetric Cell Division: Stem cell + Stem Cell Differentiated cell + Differentiated cell Assymetric Cell Division: Stem Cell + Differentiated Cell.

State of an embryo at 5-7 days

The blastocyst = PLURIPOTENT. In the blastocyst are pluriptent stem cels can give rise to all 3 germ layers. When you are older, i.e. a newborn or an adult, you come to have some multipotent stem cells - they give rise to more specialised tissues. They have lost some of the ability of the pluripotent stem cells.

Stem cells

They vary in their multi potential capacity-- i.e. they vary in the number of different progeny they can generate.

Tissue stem cells: What they can do

Tissue stem cells can often make several kinds of specialized cell, but they are more limited than embryonic stem cells. Tissue stem cells can ONLY make the kinds of cell found in the tissue they belong to. So, blood stem cells can only make the different kinds of cell found in the blood. Brain stem cells can only make different types of brain cell. Muscle stem cells can only make muscle cells. And so forth. Scientists say that tissue stem cells are multipotent because they can make multiple types of specialized cell, but NOT all the kinds of cell in your body.

How are embryonic stem cells identified?- their characteristics:

What are their characteristics? - Immortality -Clonality -Undifferentiated -Wide development potential. There is an unlimited supply --> this enables them to carry out tests as there are so many!

What is THERAPEUTIC cloning?

You take a donor cells and isolate it. You take an unfertilised egg with the nucleus removed. You combine them ie by shocking the cells. You develop a somatic oocyte. You isolate the cells of the inner cell mass of the blastocsyt which can give rise to the stem cells. These stem cells are PLURIPOTENT gives rise to all 3 germ lines. They are identical to the adult cell nucleus. Ethical problem = blastocyst removal. Second problem = where do you get the egg from? Ie discarded eggs from IVF clinics.

Directed embryonic stem cell differentiation in vitro

You want to direct the differentiation in vitro. Requires signals ie growth factors, etc. to make sure it goes down ONE LINEAGE rather than giving rise to cells from all 3 of the lineage lines. For diabetes want for the beta cells for the islets to replace.

Tissue stem cells: what can they do?

i.e. blood stem cells found in bone marrow --> can give rise to only specialised types of blood cells: red blood cells, white blood cells, platelets. -They can differentiate to give rise to the specialized blood cells, but they cant cross boundaries.

What does the endoderm give rise to?

pancreas and liver.

Understand how stem cells can be used in a research laboratory to study disease

review images.

What is an embryonic stem cell and where does it come from?

You separate the cells from the inner cell mass. You culture then in the lab and maintain them as an embryonic stem cell lines.

What does the mesoderm give rise to?

-Blood, endothelial, kidney, heart, bone, muscle.

Adult stem cells

An example of adult stem cells to treat a patient = bone marrow.

What are the 2 characteristic of embryonic stem cells?

ES cell lines are derived from individual ES cells The daughter cells are alike and grow indefinitely.

Ethical debate

-Are humans playing god? -Will embryo farms be around in the future? -Will healthcare costs skyrocket? -Is it morally right? -Are embryos alive? -Will cloning factories produce human organs?

How are embryonic stem cells identified?

-Grow indefinitely in culture in the primitive embryonic state > unlimited supply permits meaningful experiments >give rise to clinically relevant numbers -Maintain normal karyotype and expression of telomerase. - Presence of markers that are only found in undifferentiated cells > e.g. Oct4, Sox2, Nanog -Able to differentiate into a wide range of cell types in vitro and in vivo.

What does the ectoderm give rise to?

-Nerve, skin, adrenal

Potential advantages of using adult stem cells for cell therapies:

-No legal or ethical concerns. -Using a patient's own cells circumvents the problem of immunological rejection. -Injection of normal somatic stem cells has not lead to tumour formation. Also, by learning to manipulate endogenous adult stem cells in the body, we may learn how to repair ourselves (avoiding the problems of cell transplantation).

What are ADULT stem cells?

-Rare, undifferentiated cells found among differentiated cells in a tissue or organ. - Capable of differentiating to produce the major specialised cell types of the tissue. - Multipotent, not pluripotent. -Primary role: to maintain and repair the tissue in which they are FOUND.

Stem cell division symmetry

-Symmetric division = the stem cells divides and gives rise to either two stem cells, or to differentiated cells. The prodgeny are the same! Either stem cells OR differentiated cells. -Asymmetric division = gives rise to one stem cell and one differentiated cell.

Early embryonic development

-The cells of the inner cell mass undergo GASTRULATION --> becomes specialised either >ectoderm - the external layer; skin, neutrons, pituitary glands, ears, eyes. >mesoderm - the middle layer; bone marrow, skeletal/ smooth/ cardiac muscle, heart and blood vessels and the kidney tubules. >endoderm - the internal layer; pancreas, liver, thyroid, lung, bladder, urethra.

Induced pluripotent stem cells (IPS)

-they are PLURIPOTENT. You take a somatic stem cell from the body, add certain genes to the cell to genetically reprogramming it, giving rise to a pluripotent stem cell. It acts like an embryonic stem cell as it can differentiate into all types of cells.

Benefits of therapeutic cloning

1) Ideal tissue for regenerative medicine because genetically identical to patient - circumvents problem of tissue rejection 2) Use to study diseases • Create lung cells growing in culture having cystic fibrosis mutation • Screen for effective treatments.

Challenges to therapeutic cloning

1) Percentage of successful clones is low - long time-frame to generate. 2) Creating potential embryo (not fertilized) for research/treatment 3) Practical - source of oocytes? Should women donate for such research/treatments? 4) Slippery slope to reproductive cloning.

What are the defining characteristics of stem cells?

1. Are primitive, or UNDIFFERENTIATED <-- they have the capacity to give rise to a number of different cells. 2. They can DIVIDE INDEFINITELY (stem cell line). 3. Are SELF- RENEWING. 4. Give rise to PROGENY that DIFFERENTIATE INTO SPECIALISED CELLS.

Define 1. Pluripotent 2. Multipotent 3. Oligopotent 4. Unipoten 5. Totipotent

1. PLURIPOTENT = can differentiate into cells derived from any of the three germ layers, but not the placenta. 2. MULTIPOTENT= can give rise to several specialised cells or tissues of an organism (often tissue-specific). 3. OLIGOPOTENT = can generate a few cell types within a particular tissue . 4. UNIPOTENT = can produce only one cell type, but still capable of self-renewal. 5. TOTIPOTENT = can give rise to all three of the germ layers of the embryo AND the placenta!

Advantages of IPS

= No need for embryos Circumvents immunlogical rejection Reprogramming needs to be safe and stable!

Progeny produced from pluripotent human embryonic stem cells

= can give rise to a large range of progeny. i.e. the 3 different germ layers give rise to different things.

An example

=Hemapoetic stem cells. -they can renew. -as you go down the tree, they become more restricted in what they can give rise to. Ie by the end you have different cells giving rise to T cells, B cells, mast calls, granulocytes etc.

The immune rejection issue in embryonic stem cell based therapy

Another problme is the problem of immunology- you need to match the stem cells to the recipient of a transplant/ donor etc. --> a possible solution = therapeutic cloning. Potential Solutions: - large banks of ES cells - manipulation of histocompatibility genes in ES cells - replacement of hematopoietic tissue of patient with ES-derived cells prior to graft - immunosuppressive drugs or antibodies - therapeutic cloning