Week 6

Question 1

Bipotential Gonad (+ Paramesonephric Duct)

Answer 1

1. Y-Chromosome Present (SRY/TDF) –> Testis (androgen): male genitalia
2. Y-Chromosome Absent –> Ovary (female genitalia)
   Gametes fuse at fertilization –> diploid zygote

Question 2

Fertilization

Answer 2

Gametes fuse into diploid zygote
> DNA insert via HR into ICM of blastocyst from ES cell line –> select –> reimplant into foster mother
> Egg –> rotational cleavage –> 4-cell –> morula –> tight junction in compacted morula –> grow in mature blastocyst with 64-200 cell stage and ICM –> cross with heterozygote to identify AOI

Question 3

Embryo Development and Cell Differentiation

Answer 3

1. Function: Treat Disease, prevent cancer, and replace bad cells
2. 64-200 cell stage blastocyst: (in to out)
   > ICM: ESC here –> all human cells
   > Blastococel: cavity filled with fluid around ICM
   > Trophectoderm: derives trophoblast (placental embryo); surrounds everything else

Question 4

Stem Cells (Overview)

Answer 4

1. Unspecialized –> indefinite renewal

2. Can be induced –> specialized/differentiate

Question 5

ESC

Answer 5

From ICM of embryo –> specialized

Question 6

ASC

Answer 6

1. Somatic tissue only (undifferentiated in other tissues self-renew into cells specialized in one type of cell it originates from, usually)
2. Difficult to obtain (neuronal), but blood SC can be obtained from BM cells

Question 7

BSC

Answer 7

1. From multipotent hemapoietic SC progenitor –> lymphoid/myeloid progenitor –> thymus T-cells/eosinophils
2. X-irradiation kills BC + newly injected BM colonize donor hema-tissues and generate new BC –> survival
3. BM-based monoclonal Ab –> identify cell-surface protein (CLUSTER OF DIFFERENTIATION) for precursors –> Fluorescence-Activated Cell Sorter (FACS)
4. Different growth factors and TF’s to stimulate colonies –> increase survival and differentiation

Question 8

Repair/Replacement of Human Tissues

Answer 8

Problems: 1. Difficulty Directing ESC to only 1 pathway; 2. Immune Rejection
> Sol (SCNT/iPS): patient cell and unfertilized donor egg
> Use growth factors to control differentiation path
> Moral embryo treatment: end human suffering, but MEANS is disputed since ESC therapy uses donated embryo and it never enters the uterus (human?)

Question 9

Reprogram Mature Cells to Pluripotent SC

iPS

Answer 9

iPS: introduce few genes –> PSC
> Expresses Oct4, Sox2, KLF2, and C-Myc (4 TF)
> Skin fibroblast –> retroviral transduction –> selected iPS in normal blastocyst –> crossbreed normal and chimeric mice –> iPS-desired mouse

Question 10

Reprogramming of Somatic

Answer 10

Patient-specific SC’s that bypass both ethical and practical concerns (SCNT, hES)
> C-Myc overexpression = oncogenic (20% CA)
> C-Myc underexpression = inefficiency and chimeric don’t immediately get CA

Question 11

Solutions to Abnormal C-Myc Expression

Answer 11

1. Insert retroviral LTR –> active promoter/enhancer controls gene –> HIGH proto-oncogene GE/ LOW TSG
2. Adenoviral Reprogram: lost after several cell division/not present in genome
3. Recombinant proteins –> disease-specific iPS –> study disease mech, screen drugs and new therapies
   > hES, iPS-myogenic progenitors restore dystrophin to increase contractility
   > Derive phenotypes (DMD) with hiPSC model: different types of mutations identified (reduced fusion competence, etc) –> myogenic specification and myoblast purification form rescued multi-nucleated myotubes –> correct defect