Study Guide 17 - Embryonic Stem Cells

Distinguish between totipotent, pluripotent and multipotent stem cells.

Totipotent cells - can develop into ANY cell in the body, including extra-embryonic membranes (such as the trophectoderm or chorion). Totipotent cells are also called blastomeres, and are *capable of forming an entire organism capable of reproduction.* Totipotent cells can be derived from a days 3-8 blastocyst. Pluripotent stem cells - can develop into derivates from all 3 germ layers (the ectoderm, mesoderm, and endoderm) and germ cells (eggs/sperm). Pluripotent stem cells come only from the inner cell mass of an embryo where all 3 germ layers are observed. Multipotent aka precursor aka progenitor stem cells - can become a specialized cell, but generally only in a tissue or organ in which it resides. *Multipotent stem cells are considered as adult stem cells* Examples include hematopoietic stem cells that can differentiate into RBCs or WBCs.

Discuss the challenges facing regenerative medicine.

Challenges to regenerative medicine include: 1) being able to generate sufficient amounts of tissue for patients 2) Differentiate tissues into the desired cell type(s) reliably 3) Be free of animal products 4) Survive in recipient after transplant, integrate into surrounding tissues, and function appropriate for the rest of their life 5) Avoid harming the recipient, find potential side effects

Define chimera (i.e., a chimeric organism).

Chimera - an individual organism derived from two or more genetic sources.

Distinguish between embryonic and adult stem cells.

Embryonic cells are pluripotent stem cells (if an inner cell mass is apparent in a day 5 embryo) that can develop into derivatives of all 3 germ layers. In contrast, adult stem cells are multipotent stem cells that can only differentiate into several different types of related cells , usually in the same tissue.

Distinguish between the endoderm, mesoderm, ectoderm and germ cells. Give examples of cells arising from each of the germinal layers.

Endoderm - gives rise to digestive tract (liver, gallbladder, and pancreas), respiratory system (lungs and related tubing) Mesoderm - skeletal muscle, skeleton, circulatory system, kidneys Ectoderm - epidermis (outer layer of skin, hair, nails, tooth enamel) and neural tissue

Describe the tests used to identify embryonic stem cells. Explain each of the tests for pluripotency. Include a description of embroid bodies, teratomas, and tetraploid complementation. Distinguish between spontaneous and directed differentiation. Explain the difference between finding for the presence of Oct-4 and PAX6 in cell lines.

General steps outlining how to test embryonic (pluripotent) stem cells: 1) Grow and sub-culture cells for many months, then can look for surface proteins or markers only found in undifferentiated cells. *For example, the Oct-4 transcription factor is only present in pluripotent cells) 2) Can examine the chromosomes for embryonic stem cells, aka their karyotype, to search for any damage or change in chromosome number 3) Determine if cells can be grown and sub-cultured after freezing, thawing, and replating 4) Demonstrate differentiation potential. The differentiation potential of embryonic stem cells can be tested in two different ways: a) *Spontaneous differentiation* - involves the development of *embroid bodies* which are 3D aggregates that result from cells clumping together. Inefficient for producing cultures of a specific cell type. b) *Directed differentiation* - used to generate a large number of specific cell types indicated by a change in the surface of the culture dish, its chemical composition, or modification of cells by inserting specific genes. Undifferentiated cells (such as those in embroid bodies) can be guided down a certain developmental pathway and become a specific cell type. Can determine whether embroid bodies have in fact differentiated into a specific cel type by testing for the presence of markers that are only found in differentiated cells, such as *PAX6.*

Describe potential uses/goals of stem cell research.

Stem cell research can be used to conduct further research into stem cell differentiation, the study and development of treatments to birth defects, the use of certain cells for treatments or toxicity testing for new medications, and research into transplantation of tissues developed from ES cells (embryonic stem cells). Stem cell research can be used in *regenerative medicine* which is the study of methods to regrow, repair, or replace damaged, diseased cells, organs, or tissues.

Explain why termatoma formation is the "Gold Standard" of pluripotency, while tetraploid complementation is the "Most rigorous" test of pluripotency.

Teratoma formation is considered the gold standard of pluripotency, since it can very clearly show the differentiation potential of embryonic stem cells. Involves injection of pluripotent stem cells into an immunosuppressed organism (such as a mouse), and their differentiation into many different tissue types (that are all derivates of the three main germ layers) that forms into a teratoma, a benign tumor showing differentiated or partly differentiated cell types (teeth, hair, muscle, bone, etc.). A successful teratoma formation will demonstrate a mixed population of all three embryonic germ layers (endoderm, mesoderm, and ectoderm), which is why it's considered as the gold standard. Tetraploid complementation is considered as the most rigorous test of pluripotency due to how thorough its procedure is. Tetraploid complementation involves combining two different blastomeres, resulting in a 4n zygote; tetraploidy is fatal for humans so its inner cell mass will not develop, however tetraploidy will have no effect on the trophectoderm and resulting placenta. Embryonic stem cells are then injected into the 4n blastocyst, and eventually develops into an inner cell mass; if the test is successful, the inner cell mass will result in the development of a fetus which may result in the live birth of an infant that may grow to become a fertile adult.

Explain how embryonic stem cells are created.

1) Create embryo, through IVF 2) Remover inner cell mass from the blastocyst 3) Culture cells from the inner cell mass Controversial because harvesting embryonic stem cells destroys the embryo.

Describe three defining characteristics of stem cells.

A stem cell is defined by the following 3 characteristics: 1) Self renewing - capable of dividing and renewing themselves for long periods 2) Unspecialized - do not have any tissue-specific structures that allow them to perform specialized functions 3) Can give rise to specialized cells - differentiation of a stem cell causes changes to how its DNA is expressed, and these changes stay and are passed down through cell division.