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Lecture 9

November 19 Lecture 9 -Nov 15, 19) - LIFESCI 2A03.docx

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Life Sciences
Joe Kim

LECTURE 9 LIFESCI 2A03 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 1 LECTURE 9 LIFESCI 2A03  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 2 LECTURE 9 LIFESCI 2A03  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 3 LECTURE 9 LIFESCI 2A03 o Turn on promoter by adding 4 factors – haven’t indicated transdifferenetiation of reprogramming has occurred yet  The addition of three factors, Mef2C, Mesp1 and Tbx5 were necessary for induction of GFP and cardio
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