Final review. Recitation8 05/08/2014. Stem Cell Biology and Function W4193 1

Transcription

1 Final review Recitation8 05/08/2014 Stem Cell Biology and Function W4193 1

2 Neuron Fibroblast ESC Same Different Stem Cell Biology and Function W4193 2

3 A Cell s ID? Category of parameters Protein mrna DNA Gene Expression Morphology Genome Cell mechanics Cell stiffness Size Shape Non-coding RNA Karyotype DNA modification Stem Cell Biology and Function W4193 3

4 Gene Expression Measurement Location Dead or alive In vivo (wholeanimal) Mass extraction In vitro (cellculture) Fix and in situ Live imaging Protein Western blot Immunostaining GFP-labeled Mass spectrometry Cell number Large group Small group Single cell Data size High throughput Few target of interest RNA Northern blot Q-PCR in situ hybridization Live-labeling of RNA Microarray RNAseq Stem Cell Biology and Function W4193 4

5 Genome characterization Karyotype number and appearance of chromosomes in the nucleus of a eukaryotic cell after staining (stable genome) Chromatin modification Stem Cell Biology and Function W4193 5

6 chromatin immune precipitation (ChIP) for histone modification Or? Stem Cell Biology and Function W4193 6

7 DNA-Me bisulphite sequencing different positions within the sequenced region; black-me Stem Cell Biology and Function W4193 7

8 Injection of sperm RNAs from traumatized males into fertilized wild-type oocytes reproduced the behavioral and metabolic alterations in the resulting offspring. Stem Cell Biology and Function W4193 8

9 How to alter DNA sequence in a cell? Forward genetics Natural mutation rate of a target gene Induced mutation UV, carcinogen Reverse genetics Gene of interest (complete genome seq) Over-expression Knockout Site-mutagenesis Stem Cell Biology and Function W4193 9

10 Demo: express protein A in cell C Background Protein A not expressed in this type of cells C Gene A in its genome, but not transcribed (WHY?) How do you get protein A expressed? Endogenous Force the original promoter of A to be active (Possible ways?) Exogenous Express another copy of gene A with an active promoter Stem Cell Biology and Function W

11 cdna of A restriction endonuclease Circular DNA is more stable Extra nuclear plasmids in bacteria Eukaryotic cells? Virus Retro (replicating) lenti Calcium phosphate of plasmids (non-integrate?) Stem Cell Biology and Function W

12 cdna + promoter + termination replication defective particles w/o particle proteins helper RNA Cell A packaging produces a high virus titer Way to express concentration of particle packaging Likely more than 10 7 particles per ml of culture supernatant in about 5-10ml Measurement: functional titer p24 assay cell infection efficiency is very efficient (particle not replicate) entering RNA genomes are converted into DNA and integrated into the genome efficiently followed by gene expression. Stem Cell Biology and Function W

13 How to select efficient infection and expression of gene A if the rate is quite low? Selection (antibiotic resistance/sensitivity) Reporter Multiple genes expression in Yamanaka s paper? Stem Cell Biology and Function W

14 Integration of gene A to ONE given site? Homologous recombination where a designed variant segment of DNA flanked by normal DNA is introduced into cells and exchanged precisely for a segment of genomic DNA bounded by the same flanking DNA 10-6) CRISPR Efficient DNA editing Introduce gene A to mice Stem Cell Biology and Function W

15 mesc AB-AB One copy (?) Neomycin analogue G418 (+) Tk expression killed by ganc(anti-viral drug) (-) Stem Cell Biology and Function W

16 Ab-Ab Brown dominant(b) Knockout recessive(a) Gene A and B are linked Ab-Ab Ab-Ab ab-ab Ab-Ab Gonad expression Ab-Ab Ab-aB Ab-aB Genotyping Ab-AB Ab-aB Ab-Ab ab-ab Genotyping Ab-aB Stem Cell Biology and Function W

17 Medium matters WHY? WHAT FACTORS may be important? Pay attention to the recipe of ESC and ipsc in the paper Stem Cell Biology and Function W

18 Stem cell standard ID? Once the authors convert/induce/generate some cells (name: X) different from the original cells (MEF or Lymphocytes), how do they know these cells X are stem cells? Stem Cell Biology and Function W

19 A Cell s ID? Category of parameters Protein mrna DNA Gene Expression Morphology Genome Cell mechanics Development Cell stiffness Size Shape Non-coding RNA Karyotype DNA modification Stem Cell Biology and Function W

20 Stem cell ISO In vivo injection teratomas chimeric embryo In vitro embryoid body formation Stem Cell Biology and Function W

21 Revisit Zhou & Melton 2008 Direction conversion Cell Type A Characterize cell type Marker Expression profile Morphology Etc. Cell Type B induced conversion of cell Type A Exogenous DNA delivery Transfection marker Adenoviral expression GFP Stem Cell Biology and Function W

22 Stem Cell Biology and Function W

23 Cell Type A Cell Type B Characterize cell typeb Morphology Ultrastructure Function Insulin (early selection marker) Animal physiology The exact same cell changes identity? Lineage tracking Cpa1CreERT2 CELL-SPECIFIC MARKER (amy)? Position Stem Cell Biology and Function W

24 Reporter Stem Cell Biology and Function W

25 Stem Cell Biology and Function W

26 βgal GFP Insulin intersection Exocrine cells w/o transfection After 2 month, GFP diminished Innate Insulin+ beta cells Innate beta cells w/ transfection Exocrine cells w/ transfection Other pancreatic cells w/ transfection Induced beta cells from exocrine cells producing insulin Stem Cell Biology and Function W

27 Other direct conversion-1 Lena, Yamanaka Screen for critical factors (HOW?) Fibroblasts into Functional Cardiomyocytes 2. Characterize GFP+ cells 3. Candidate factor combination 4. Origin of new cell-type-population based marker Stem Cell Biology and Function W

28 Other direct conversion-2 direct conversion of fibroblasts to neurons 2010 TauEGFP: knock-in label of neurons (no neurons in the MEF culture) Tuj1: induced neurons mesodermal cell type is being converted to an ectodermal sub-type Wernig, 2010 Stem Cell Biology and Function W

29 Other direct conversion-3 Guo Chen (2013) Glia-specific transfection In vivo retroviral delivery for in vivo injection because, unlike lentiviruses or adenoassociated viruses, retroviruses only infect dividing cells such as progenitor cells or reactive glial cells, and do not infect nondividing cells such as neurons Stem Cell Biology and Function W

30 Stem Cell Therapies Bone marrow stem cells hematopoietic stem cells blood cancer Human spinal cord stem cells surgical transplantation of stem cells into the cervical region trial testing the use of human spinal cord stem cells to treat Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig s Disease Human mesenchymal stem cells protecting pancreatic beta islet cells in adults and children with newly diagnosed type 1 diabetes Adult Eye Stem Cells for Eye Disease or Damage hesc hesc-derived retinal cells to treat patients with an eye disease called Stargardt's Macular Dystrophy (SMD). hesc-derived retinal cells to treat patients with age-related macular degeneration.(transplantation/engraftment) hipsc Japan age-related macular degeneration Stem Cell Biology and Function W

31 ipsc/direct conversion therapy pros and cons Safety Virus Teratoma Quality Homogeneity Mixture 3D In vivo Engraftment In vivo functionality (VGEF) Stem Cell Biology and Function W

32 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 W

33 Murry & Keller, 2008

34 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 W

35 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 W

36 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

37 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

38 clonal cell tracking CFU-S + chromosomal abnormality CFU-S + fluorescence labeling (how to achieve this) CreER GFP/LacZ Stem Cell Biology and Function W

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

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

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

42 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

43 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

44 Key concepts in cancer development Clonal evolution CSC Stem Cell Biology and Function W

45 CE v.s. CSC Hetero/homogenous Genetic/epigenetic Cancer-initiating potential Lineage tracing Driver mutation Etc CE CSC Stem Cell Biology and Function W

46 How can you identify which cells can suffice to transmit cancers? But not necessary will Conditions Environment Niche Tracing -MarkerS -Dye-labeling -Epi/Genetic Single cell Mixture Certain mixture Contribution from different cells Define sufficient Infectious v.s. Metastatic Stem Cell Biology and Function W

47 Transplantation-xenograft (across species) Across species Mice v.s. human Stem cell niche Immuno-deficient mice Cancer niche Stress-induced new mutation Stem Cell Biology and Function W

48 Why use immunodeficient mice? Any problem with this model? Stem Cell Biology and Function W

49 Critical role of immuno-protection in cancer How do our immune system kill a foreign/cancer cell? Innate: NK, macrophage, cytokine Adapted: Antigen presented, T/B cell Immunodeficient v.s. immunocompetent Stem Cell Biology and Function W

50 Adult stem cells Features Self-renewal Differentiation Compare embryonic stem cell ipsc Importance Renew your body periodically (Why this is necessary Examples Hair, gut, ovary, skin (epithelial cells) Neural Bone marrow Stem Cell Biology and Function W

51 How to identify a stem cell marker candidates: which molecules to look at? Normal development signaling pathways Lineage tracing (hematopoietic) Characterize candidate cell types Gut: why wnt targets? Because Wnt signals constitute the principal driving force behind the (developmental) biology of the crypt5, we hypothesized that some Wnt target genes may be specifically expressed in the stem cells. Wnt targets in colorectal cancer Stem Cell Biology and Function W

52 How to detect/identify/prove the existence of new stem cell 1. Wnt targets new candidate marker for gut stem cells Lgr5 1) Expression in crypt (in situ) and cancer 2) Wnt target: inhibit wnt block expression 3) Unique expression pattern in crypt different other known markers (what s known about GSC) What cells does each marker represent? Cell cycle marker Stem Cell Biology and Function W

53 How to detect/identify/prove the existence of new stem cell 2. A new marker (in situ mrna) Lgr5 Promoter + reporter (knock-in) = null Lgr5 In situ v.s. reporter Stem Cell Biology and Function W

54 How to detect/identify/prove the existence of new stem cell 3. Lgr5 express in the crypt base stem cell or not? ONLY in Lgr5 expressing cells get EGFP ONLY after adding TXF get LacZ in cells that have expressed Lgr5 (why?) Stem Cell Biology and Function W4193

55 How to detect/identify/prove the existence of new stem cell 4. CreERT2+ reporter lineage tracing Other reporters? Stem Cell Biology and Function W

56 Lineage tracing CreER2 (promoter specific) + Reporter Chromosomal labeling by irradiation Dye injection Brainbow in stem cell study Stem Cell Biology and Function W

57 Brainbow2.1 (inversion and excision) Stem Cell Biology and Function W

58 Crypt Homeostasis to renew or to differentiate Hans Clevers 2010 Stem Cell Biology and Function W

59 A big picture Start from scratch 1. How do you distinguish one cell from other cells? 2. How do you identify an adult stem cell from a group of cells? 3. How do you convert one type of cells to another (stem cell or differentiated cell)? 4. How do you get a particular type of cells from an ESC or ipsc? 5. Think again about the concept of niche, how would you define a niche of a cell? What external factors do you think are critical for the normal growth and functions of a cell? 6. Stem Cell Biology and Function W

60 Good luck! Stem Cell Biology and Function W