Hard Deck 2 Flashcards
Pulse-chase experiment: • To measure turnover of cells • T1/2
- Labelled BrUridine deoxythymidine Short time (PULSE)
- Unlabelled Thymidine (Chase)
- label 4 cells, how long for it to get to 1/2 tabled cells
- measure radioactivity at zero then a X minutes
Intestinal Stem Cells Niche
- Wnt - promotes survival and proliferation of stem cells in intestine
- cells undergoing continuous dividing replace microvilli and are in the crypt
- progenitor cells form transient amplifying zone. They move forward till reach epithelium
- progenitor not cabable of unlimited self renual (unlike stem cells)
- BMP - promotes differentiation, maturation in the direction of willis
Fluorescence activated Cell sorting (FACS)
- CFP and GFP expressing cells -stem cells have specific surface markers
- cell goes through resiviour one at a time. Excited by laser and florescent light detected by photomultiplier tube
- certain changes (that’s related to surface markers) and separated into fractions
Bones
- osteoblasts -form bone cells
- osteoclasts -degrade bone cells (arise from hemapoetic stem cells)
- osteoblasts arise from mesenchymal stem cells
Formation of ICM Cells
Trophectoderm undergoes asymmetrical division perpendicular to apicobasal axis (instead on the normal symmetrical division to expands the trophectoderm) This creates an ICM (inner cell mass)
Reprogramming of somatic cells induced pluripotent stem (iPS) cell
- EScells can be differentiated in somatic cells then reprogrammed to induced pluripotent sen cell by TF such as Sox2 and Oct4
- Regenerative Medicine: Disease treatment Organ repair
iPS derived HSC correct sickle cell anemia
1) harvest tail tip fibroblasts
2) infect with Oct 4, Sox2
3) correct sickle cell mutation in iPS cells by specific gene targeting
4) differentiate into embryo bodies
5) transplant corrected hematopoietic progenitors back into irradiated mice
Natural Stem cell therapy: Trafficking cells from Fetus across placenta Stem Cells
- embryo to maternal circulation to moms heart which was experimentally damaged
- generated diverse cardiac lineages
Mesenchymal Stem Cells differentiation
if cells sit on colagen coat thats similar elasticity to brain then MSC differentiated into cells with neural markers but not mucle or bone markers. gel stiffer, cells hsve muscle markers.
Regeneration in Planaria
- In planarian flatworms, regeneration is a kind of stem-cell mediated event, and occurs in a way that suggest a morphogen gradient is at work in the regeneration that is observed.
- At the cut surface clonogenic neoblasts (pluripotent stem cell) direct development of the missing structures—heads make tails, and tails make heads.
- Colognenic neoblasts migrate to the site of wound and regenerate the tissues.
- One cologenic neoblast is sufficient to regenerate into head or tail region.
Polarity in Planaria regeneration: DV and Ant-Post
DV Axis: •BMP defines the dorsal region in flat worms.
•Noggin in the Ventral Anterior Posterior:
•Anterior posterior polarity is regulated by Wnt and β-catenins.
•β-catenin is activated by Wnt in the posterior regions (generating tail).
•Repressors of Wnt prevent β- catenin production in the anterior– facing regenerating neoblast cells.
•If Wnt pathway is blocked in the posterior blastema then the result is a worm with heads at both the ends.
Two possible mechanisms for neoblast specification during regeneration
1) •pluripotent neoblast cell gets activated by injury
•becomes multipotent post mitotic blastemacell
•geys wound specific signal and differentiates
2)
•pluripotent neoblast cell gets regiional signal before injury •becomes multipotent regional lineage restrictedproginators •gets wond specific signal
•becomes unipotent post mitotic blastema precureser
•differetiates
Epimorphic regeneration of salmander limbs
- Relatively undifferentiated cells arise from originally differentiated tissue, which then proliferates and re-differentiates into new limb parts.
- Bone, dermis, cartilage and muscles contribute to the regeneration blastema.
Upon amputation of salamander limb
- plasma clot forms at the site of injury.
- epidermis from the stump migrate and form wound epidermis.
- Nerves innervating the limb degenererate for a short distance proximal to the plane of amputation.
- Extracellular matrices are degenerated by protease and single cells that undergo dedifferentiation are formed.
- These cells redifferentiate to form new structures of the limb.
- Wound epidermis thickens to form AER equivalent structure called apical ectodermal cap (AEC).
- cut wait 72 days whole limb regrown
- AEC much bigger than AER
- accumulation of blaster under AEC
Blastema is similar to progress zone of developing limb
- The anterior-posterior axis is established by sonic hedgehog activation and HoxD gene activation, just as in limb buds
- Proximal-distal axis is established by interactions between HoxA gene activti’on and retinoic acid (RA)
- The most distal cells are fated to become autopod structures
- A transplanted limb blastema can substitute for an AER on a developing limb
- The full ‘nested’ Hox gene pacern is restored during regeneration, and is influenced by RA which PROXIMALIZES blastema cells
