Mid-term review. Recitation5 03/31/2014. Stem Cell Biology and Function W4193 1

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1 Mid-term review Recitation5 03/31/2014 Stem Cell Biology and Function W4193 1

2 What to expect in the midterm2 It will be all so far but mainly whatever was NOT covered by the previous mid-term- not a huge amount of material. I haven't figured out Qs yet but I am looking into whether it is practical to include something about understanding a paper. -Professor Kalderon Stem Cell Biology and Function W4193 2

3 Mid-term1 review Position, neighboring signals in development Mouse ESC features and application Mouse and human SCNT generation Ipsc generation, features and deficits Reprogramming efficiency Human v.s. mouse ESC, ipsc, mepisc Stem Cell Biology and Function W4193 3

4 What s not been covered in Midterm1 ESC, Ipsc, SCNT-techniques and comparison read Note documents and check what s not covered in midterm 1 Differentiation into different cell types T6 Clinical application and trials T7 T9: 3 phases in clinical trials Direct conversion strategy and methods T8 Beta cell (grade assignment) Guest speaker (insulin-producing cells) HSC-related topics Stem Cell Biology and Function W4193 4

5 Differentiation: how to create certain cell types copying normal development in vitro has been (and will be) the successful guiding principle Step 1. Specify germ layer (specific signals) 2. Separate each germ layer further by certain signals 3. Greater refinements by manipulating signaling pathways How to manipulate signaling pathway Forced expression of specific genes Transient induced expression External signals: proteins or small molecules other approaches Stem Cell Biology and Function W4193 5

6 Murry & Keller, 2008

7 Critical points Cell population, identities, mixture, support cells Mouse v.s. human Embryonic v.s. adult cell types for cell therapy Exogenous gene expression: virus Proteins v.s. small chemicals (screening strategy) Co-culture? In vitro tissue engineering Stem Cell Biology and Function W4193 7

8 Direct conversion: some Qs regarding beta-cell reprogramming Works in humans? How to deliver factors if in human? Different source tissues? Did not work in vitro, why? Specific lineage methods Stem Cell Biology and Function W4193 8

9 HSC: key definition and methods Irradiation? Rescued by transplantation of? Results? Immunological positive & negative? Why? Hematopoietic failure Cell mixture from blood marrow Restore bloods cells GVT, GVHD Mixed cells: lineage. HOW? Purified cells: identity. HOW? Stem Cell Biology and Function W4193 9

10 Colony forming unit-spleen (CFU-S) assay Cells from marrow (or spleen or blood) are prepared as a single suspension and are injected by tail vein into mice subjected to lethal doses of irradiation (how much, why?) Recognizable chromosomal abnormality; Function intact Random and different At varying time intervals are killed, their spleen removed and fixed, and nodules on the spleen counted. The nodule of CFU-S represents the derivatives of a single cell implanted on the spleen (why?) All cells of a colony have same chromosomal abnormalities Stem Cell Biology and Function W

11 What to expect in CFU-S? Chromosomal abnormality (how to observe) Common precursor cells in a colony CFU-S can generate colonies: ERYTHROID, GRANULOCYTIC, MACROPHAGE AND MEGAKARYOCYTIC CELLS Time-dependent progressive determination CFU-S are considered to be derived from shortterm repopulating HSCs, which represent the early progeny of stem cells (why?) Stem Cell Biology and Function W

12 clonal cell tracking CFU-S + chromosomal abnormality CFU-S + fluorescence labeling (how to achieve this) Cre-Dox, GFP dilution Stem Cell Biology and Function W

13 Cell identity in the transplanted population (purification) Key tech(2) MAb FACS Key assay Reconstitute all blood cells Stem Cell Biology and Function W

14 Mab development Against WHAT? Bone marrow cells Surface molecules Stem Cell Biology and Function W

15 Limitation of FACS Surface marker only Purity of cells with same markers How many channels Stem Cell Biology and Function W

16 Key assay for HSC After purification functional tests Fully reconstitute all blood cells Duration: transient, short, long (each represents a group of cells) LT-HSC Stem Cell Biology and Function W

17 Sum up: how to get a standard HSC lineage diagram for mice CFU-S (in vivo) Purification (in vitro) Reconstitute Stem Cell Biology and Function W