LIFESCI 2A03 Lecture Notes - Lecture 9: Hematopoietic Stem Cell, Cd90, Transdifferentiation

58 views8 pages
LECTURE 9 LIFESCI 2A03
1
Transdifferentiation
November 15, 2013
Turritopsis nutricul jellyfish-like hyrazoan; only animal known to be potentially immortal
o Adult can revert back to a polyp stage (similar to larval stage) and develop anew repair damaged tissues, regenerate
lost parts, reorganize cells and go backward to embryonic stage (polyp), then can develop back into adult
Suggests that differentiated cells can dedifferentiate and form new cell types can adult stem cell lines switch to another?
Three possible processes
o Reprogramming restore pluripotency in a differentiated cell state; eg/ ASC to iPSC; can potentially assume any cell
type
o Dedifferentiation induce the differentiated cell to proliferate, a differentiated cell goes back to a less differentiated
state, divides and then dedifferentiates to the same cell type
Not full restoration of pluripotency; go to less differentiated stage
Eg/ blood cell haemopoetic SC different blood cell
o Transdifferentiation switch to another cell type lineage
Examples of transdifferentiation (1998-2004) reports were made of various differentiated tissues containing cells derived
from bone marrow grafts
1. Radiation to destroy bone marrow (mice); ultimately lethal if animals cannot replace blood cells and immune cells
2. Inject donor bone marrow (with the hematopoietic stem cells (HSCs)) provides source for new blood cells and
immune cells
3. Labeled donor mice cells (eg/ with green fluorescent protein) revealed that a small number of cells of donor origin
appeared throughout the body of the recipient animal, including the brain, skeletal and cardiac muscle and epithelium
tissues such as liver and kidney
Labeled HSC become not only blood cells, but other cells as well
Philippe Collas (Nucleotech) published results that seem to show that some cells can be transformed into other types of cells
o Paper showed in vitro reprogramming of fibroblasts through the uptake of regulatory factors that caused the
fibroblast cells to express molecules characteristic of immune cells while down-regulating fibroblast genes
o Fluorescently labeled cells, introduce into different systems, identify whether cells maintain same cell type or others
o Further investigation did not support these conclusions (the reports could not be replicated)
o Explanation for initial results
Donor cells had fused with host cells (genetic marker of the donor was still displayed in the fused cells)
Donor cells were embedded in the host tissues, but the cells had not transdifferentiated not actually
assuming function and morphology of cells around them; misplaced haemopoetic cells
o Given these problems, the consensus was that transdifferentiation of grafted HSCs did not occur
Overview of reprogramming, transdifferenetiation and dedifferentiation
o Addition of collection of transcription factors (TF) to fibroblast goes all the way back to pluripotent state
o Reprogramming reprogramming of progenitor cell (not entirely differentiated) to pluripotency
o Dedifferentiation less differentiated; redifferentiates into same cell lineage
o Transdifferentiation one differentiated cell type to another
Importance of these studies transdifferenetiation is much easier than going back to pluripotent state to differentiate into
different cell types
Paper 1 Ieda, M et al. 2010. Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors
Report a combination of three developmental transcription factors (i.e. Gata4, Mef2c, Tbx5) rapidly and efficiently
reprogrammed postnatal cardiac or dermal fibroblasts directly into differentiated cardiomyocyte-like cells
Unlock document

This preview shows pages 1-3 of the document.
Unlock all 8 pages and 3 million more documents.

Already have an account? Log in
LECTURE 9 LIFESCI 2A03
2
Skin cell to heart muscle cell? Fibroblast to cardiac myocyte?
o Cardiac fibroblast provide support structure and secrete signals
o Cardiomyocytes are specialized muscle cells of the heart
Cardiac Myocytes specifically for muscle movement in heart; extensive actin cytoskeletal filament and
organization of actin into striated sarcomere structures allowing for muscular movement
o Derived from different progenitor cells
Screening for Cardiomyocyte-Inducing Factors need to assay to identify cardiomyocytes
o A myosin heavy chain genes is expressed only in cardiac myocytes
o MHC-GFP (MHC promoter + GFP) is expressed only in the cardiac myocytes in the mouse
o Figure Left = bright field; right = UV light
Isolation of cardiac fibroblast cells
o In vitro reprogramming and differentiation
o Left Petri Dish cardiac fibroblast cells are growing; genome manipulated to contain MHC-GFP; fibroblast not
expressing GFP no fluorescence
Vimentin and Thy1 both proteins always expressed in fibroblast cells (positive); GFP not expressed
(negative); Troponin T not expressed (negative)
o Right Petri Dish presence of TF; MHC promoter is on, turning on transcription of GFP
If no longer fibroblast and now myocytes, then they will fluoresce green
o FAC = fluorescence-activated cell sorter; detect and separate fluorescing cells
o Pink Cells
GFP negative not cardiomyocytes
Negative for Troponin T (cardiac muscle protein or marker)
Positive for fibroblast proteins (vimentin and Thy1)
o Graph
X-axis MHC-GFP
Control no GFP or Thy1 protein
TF added some cells express new marketer (Thy1 fibroblast cell, confirm normal functioning fibroblast)),
not expressing GFP
Unlock document

This preview shows pages 1-3 of the document.
Unlock all 8 pages and 3 million more documents.

Already have an account? Log in
LECTURE 9 LIFESCI 2A03
3
14 candidate genes/factors
o Effect on GFP+ cell induction with 14 factors or the removal of individual factors from the pool of 14 factors (n=3)
o Removal of Baf60c, Hand2, Hopx, Hrt2, or Putx2c did not decrease the percent of GFP+ cells and were excluded for
further analyses
o Data presented as means +/- SD. *p<0.01; **p<0.05 vs. relevant control, here relative to “14 factors”
o Observed
Induce expression of HHC GFP by adding all 14 factors
Control fibroblast expressing no GFP
Add all 14 factors GFP expression
14 factors, subtract 1 causes increase or decrease in expression of GFP
* Indicates significant difference in GFP expressed when protein removed shows that gene is related to
myocytes/GFP expression
Effect on GFP+ cell induction with 5 factors or the removal of individual factors from the pool of 5 factors
o Look for factor that, when removed causes a reduction in GFP expression
o * indicates significant difference from control
Mef2C, Mesp1 and Tbx5 were necessary for induction of GFP (shown by loss of GFP expression when removed)
o Included Gata4 based on other experiments, but removed Myocd (showed an increase)
o Fibroblast cells exposed to 4 factors look to see if there is a change in the behaviour of the cells and to isolate the
cells
o Cell sorting experiment
o X-axis myosin heavy chain fused to GFP
o PI = pidium iodide; # of nuclei (counted by ability to stain)
o All 4 factors added see a shift in cells
CONTROL left 100%; right 0%
FACTORS 75% on left, 21% on right (have not activated muscle GFP activation?? Increased level of GFP
expression??)
Not every cell underwent what appears to be transdifferenetiation (~20% appears to have)
o Dots = single cell able to sort
Unlock document

This preview shows pages 1-3 of the document.
Unlock all 8 pages and 3 million more documents.

Already have an account? Log in

Document Summary

Not full restoration of pluripotency; go to less differentiated stage. Eg/ blood cell haemopoetic sc different blood cell: transdifferentiation switch to another cell type lineage. Examples of transdifferentiation (1998-2004) reports were made of various differentiated tissues containing cells derived from bone marrow grafts: radiation to destroy bone marrow (mice); ultimately lethal if animals cannot replace blood cells and immune cells. Labeled hsc become not only blood cells, but other cells as well. Donor cells had fused with host cells (genetic marker of the donor was still displayed in the fused cells) Importance of these studies transdifferenetiation is much easier than going back to pluripotent state to differentiate into different cell types. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Report a combination of three developmental transcription factors (i. e. gata4, mef2c, tbx5) rapidly and efficiently reprogrammed postnatal cardiac or dermal fibroblasts directly into differentiated cardiomyocyte-like cells. Fibroblast to cardiac myocyte: cardiac fibroblast provide support structure and secrete signals.

Get access

Grade+
$10 USD/m
Billed $120 USD annually
Homework Help
Class Notes
Textbook Notes
40 Verified Answers
Study Guides
Booster Classes
Class+
$8 USD/m
Billed $96 USD annually
Homework Help
Class Notes
Textbook Notes
30 Verified Answers
Study Guides
Booster Classes