Lecture 22

Question 1

what are stem cells and teratomas?

Answer 1

stem cells have the ability to divide for indefinite periods in culture and five rise to specialized cells. they are ‘master cells’ in the body that can grow into various types of cells.
capable of forming teratomas: tumor with tissue of organ components resembling normal derivatives of more than one germ layer

Question 2

define undifferentiated and unipotent/multipotent cells

Answer 2

undifferentiated cells: unlimited or prolonged self renewal, can give rise to differentiated cells
unipotent/multipotent: specific type of stem cells that produce one or more of the 4 classes of mature differentiated cells

Question 3

three categories of stem cells

Answer 3

embryonic stem cells (ESC): come from early embryo (blastocyst) and are pluripotent. can develop into a new individual, all three germ layers

adult stem cells (Adult SCs): derived from early adult differentiated tissues and are multipotent, can form a certain number of tissues within the lineage

induced pluripotent stem cells (iPSC): derived from any cells that are reprogrammed back to pluripotent state (blastocyst) with Yamanaka factors. can form any cell type, all 3 germ layers but not extra embryonic tissue (placenta)

Question 4

describe steps of ES cell derivation

Answer 4

derived from the inner cell mass of the blastocyst (embryo proper). Use irradiated feeder cell layer and growth the isolate inner cell mass on top to establish embryonic stem cell cultures. very sensitive process, requires frequent media changes and perfect temp, etc

Question 5

describe how to characterize pluripotent stem cells

Answer 5

pluripotent stem cell marker proteins are detected using specific antibodies. some common proteins are:
NANOG, nuclear protein harder to detect bc must kill cell
OCT4, nuclear protein harder to detect bc must kill cell
SSEA1 (3 and 4), surface proteins easier to detect on live cells
hTERT, other telomerase proteins

Question 6

describe ES cell morphology and specific culture requirements

Answer 6

morphology changes, depends on culture conditions (media, feeders) and species of origin
specific culture medium and additives are required for stem cell maintenance: LIF (leukemia inhibitory factor) prevents differentiation, B-mercaptoethanol is antioxidant (ES cells are very low oxygen naturally), FGF-2 and Forskolin to maintain pluripotent

Question 7

examples of somatic stem cells

Answer 7

hematopoietic stem cell (HSCs): come from fetal (liver and umbilical cord blood) or adult (bone marrow and peripheral blood). Can make a huge range of mature cells depending on transcription factors (eg cytokines). Have surface markers to use as tools to isolate HSCs using FACS sorting. HSC represent the most important cells for transplantation and cancer research!

Mesenchymal stem cells (MSCs): bone marrow derived, multipotent and can form mesoderm derived cells limited to one germ layer. using the right conditions is important for directing differentiation

Question 8

describe iPS cells and applications

Answer 8

iPS are pluripotent cells that can be generated directly form adult cells. derived from skin or blood cells, reprogrammed into embryonic pluripotent state by expression of Yamanaka factors: c-Myc, Klf4, Oct4, Sox2.

can develop any type of human cell type for therapeutic purposes. Regenerative Medicine Paradigm: donor tissue is differentiated and reprogrammed, then multiplied in culture, combined with scaffolds and other agents, then administered and integrated into tissue (replacement) or stimulates existing repair mechanisms (trophic support)