L14 Flashcards
What are the 2 properties/pathways of a stem cell
- Self renewal
2. Differentiate into a specialized cell type
What can hESC differentiate into
Any cell types in the vbody
Where was the initial source of hESCs?
Leftover embryos from IVF
What controls pluripotency of SCs and factors that stimulate differentiation of SCs into discrete cell types? How do you drive a cell that has potential to become anything to drive it down a particular developmental path?
- GFs, hormones, small peptides
- Activate stage-specific and tissue-specific TFs, which then direct differentiation of pluripotent SCs into differentiated cell type
Where do adult-derived stem cells (ASCs) reside?
- In differentiated tissue of the body and can be isolated
- Brain, intestine, hair, skin, pancreas, bone marrow, fat, mammary glands, teeth, muscle, and blood and almost every adult tissue.
Describes the test for pluripotency? How to know whether what u have is a SC?
- Give rise to the 3 primary germ layers: ectoderm (skin cells), mesoderm (RBCs), and endoderm (lung cell). E.g. if had some cells and thought were SCs, implant into mouse and see what grew. Looked for embryo body or teratoma (little tumors that grow where SCs injected). See if all 3 cell types are there, if so, evidence of ESCs.
- In vivo: Inject ESCs into developing blastocyst; resulting embryo can be examined to determine whether the injected ESCs contributed to development of all 3 primary germ layers.
- start out with 2 black mice. fur colour serves as marker for DNA. Mate the 2 black mice; from the blastocyst remove the ESCs. Grow the ESCs in culture. Then get 2 white mice and mate them. Then inject the grow ESCs removed from the blastocyst derived from the black mice, into the blastocyst of the white mice. Then implant that blasocyst into surrogate mother. If there is successful integration of ESC that came from black mice, then embryonic donor cells will contribute to coat color. Examine interal organs to see donor contribution to tissues derived from diff germ layers.
Describe the stages of fetal development
- Egg is fertilized by a sperm cell = zygote.
- zygote divides n becomes collection of cells = blastocyst
- blastocyst implants in the wall of uterus and develops into PLACENTA and EMBRYO
- An embryo represents the early stage of human development, roughly corresponding to 5th-10th weeks of pregnancy
- embryonic period is the stage in which most organs are formed. heart and main blood vessels are formed, and heart starts beating by 6th week.
- primitive brain and spinal cord (neural tube) also begin to form.
- Fetal period is time of growth of the organs formed during embryonic period
What are the methods of animal cloning? Describe each.
- Embryo splitting
- Egg fertilized in vitro and allowed to develop to 8-16 cell stage
- Cells then separated and each grown to form genetically identical embryos
- Embryos then transplanted into surrogate moms
- Extension of how identical twins or triplets r produced naturally - Cell fusion
- Used to produce large #’s of genetically identical embryos
- Uses unfertilized eggs (nucleus removed). A fertilized embryo is split into single cells. Embryo cells and enucleated eggs are fused, each then containing an identical nucleus. Eggs are implanted in surrogate mothers. Genetically identical calves are born - Nuclear transfer
- Donor eggs and door nuclei are fused
How is somatic cell nuclear transfer different from cell fusion with embryonic nucleus?
In embryonic nucleus, we were taking taking the nucleus of a single cell from the embryo and fusing it with an enucleated egg. But in SCNT, we’re taking a somatic cell, smtg that is past the embryonic stage and has been differentiated and shouldn’t have hte potency SC has.
Describe SCNT
Take nucleus of somatic cell (2N) e.g. adult fibroblast, transplant into an oocyte that has been enucleated. The cell can then divide and be grown in culture and allow to develop into blastocyst.
Describe reproductive cloning using Dolly as an example
- Remove udder cell from white-face sheep; remove DNA from unfertilized egg. Fuse cells
- Get early embryo with donor DNA
- Cloned embryo
- Remove some cells from embryo and plate (ESCs). Implant cloned embryo in surrogate.
- Clone of white face sheep
- Remove skin cell from patient. Remove DNA from unfertilized egg. Fuse cells
- Get early embryo with donor DNA
- Cloned embryo
- Remove some cells from embryo and culture (ESCs). Implant cloned embryo in surrogate.
- Infant clone of patient
What are the 2 types of human cloning? Pos and neg arguments for human cloning
- Two types
Therapeutic cloning
Reproductive cloning - Positive arguments
- Infertile couples or couples suffering from genetic disease on one side of the family could choose to make a clone of one of the parents to raise a biologically related child
- Cloning cells in vitro could provide tissue to treat serious diseases - Negative arguments
- Many- related to ideas of identity, human rights, ethics
What is the difference btwn DNA/chromatin in differentiated cell vs. DNA/chromatin in SC?
A simplified way of thinking about DNA/chromatin in SC vs. differentiated cell is compared euchromatin and heterochromatin. The DNA in SC/young cells/tumor cells likely resemble euchromatin, which is accessible and plastic. Whereas DNA in differentiated cell/old cells/normal cells likely resemble heterochromatin.
There are many genes that drive the developmental processes, but once organs form these genes are no longer needed so they’re shut down (as seen in heterochromatin)
What happens during nuclear transfer?
TFs present in the egg occupy their DNA targets and initiate program of embryonic gene expression
How does regulation of gene expression during development lead to differentiation of specific cell types? Provid example
Overexpression of TF MyoD can elicit the entire program of muscle differentiation when introduced into non-muscle cell types
Describe nuclear reprogramming of somatic cells
Take a differentiated, adult cell and alter its patterns of gene expression in order to reprogram the cell to an early stage in its differentiation pathway—an undifferentiated pluripotent state.
Describe the process and goal of Yamanaka’s exp
Goal: nuclear reprogramming of somatic cell: Taking a differentiated adult cell and alter its pattern of gene expression in order to reprogram the cell to an early stage in its differentiation pathway- an undifferentiated pluripotent state
- Based on the literature they derived a list of 24 candidate genes that were active in ES cells
- When all 24 were introduced by viral vectors they could reprogram fibroblasts
- Started eliminating single genes to see which were absolutely required for this process
- Identified 4 (all transcriptional regulators) that were essential for reprogramming
What are the diff models oc cellular reprogramming.
- Differentiated cell to IPSC
- slow reprogramming, low efficiency - adult stem/progenitor cell to IPSC
- fast reprogramming, high efficiency
Describe the steps in the process of generating an iPS derived mouse; as well as the proof of principle that these are true pluripotent cells
- Took skin fibroblasts from transgenic mouse (transgenic mouse with antibiotic gene in its genome driven by SC promoter; only expressed if these cells are in fact reprogrammed into SC)
- Grew in dish and transfect with retroviruses carrying yamanaka factors
- Antibiotic selection
- Growth in culture
- iPS cell line
- Normal blastocyst
- Normal mouse
- Chimeric mouse
- Normal mouse
- iPS-derived mouse
, took fibroblasts from mouse, grew in dish, transfected w/retroviruses carrying yamanka factors. In this pics starting out with transgenic mouse with antibiotic gene in its genome driven by SC promoter; only expressed if these cells are in fact reprogrammed into SC. If expressing antibiotic resistance gene must be SC that came from the original skin cell and u can inject into blastocyst. Starting out mouse strain with one coat colour and putting into blastocyst of another colour so can track them. Get chimeric mouse, then after breeding u can have mouse entirely derived from cells that were originally reprogrammed. Proof of principle that completely pluripotent even tho originally come from differentiated adult cell
What are the diff modes of delivery of TFs to cells
- Integrating
- retrovirus
- lentivirus - Non integrating
a) DNA
- plasmids
- adenovirus
- mini circle/episomal
b) Non DNA
- proteins
- miRs
- mRNAs
Why are RNA-induced pluripotent stem cells so much faster to make?
If put in TF mRNA, it’ll make protein, protein will go to endogenous locus and find its own promoter and turn on the endogenous so don’t need the one you transfected in for very long cause pos feedback loop.
Potential Applications of Stem Cells
- Patients with leukemia undergo chemotherapy/radiation treatment to destroy defective WBCs. Using stem cells to make WBCs = effective way to treat leukemia
- SCs from umbilical cord blood used to provide RBCs for sickle-cell patients
- Most effective and safest SC treatments in the future likely involve differentiating SCs in vitro into desired cell and tissue types and then introducing those cells into a patient as appropriate instead of directly injecting stem cells into a patient
What is transcription factor induced transdifferentiation
Transdifferentiation is going from a differentiated adult cell type to another adult cell type. Diff than iPSC reprogramming cause bypass ESC stage
