CSB349H1 Lecture 14: Section 2- L14b
14 May 2018
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Lecture 14(b): Heterokaryons & Nuclear Transfer
Somatic (Fibroblast) Cells + Embryonic Stem Cells:
• Generated a cell fusion via chemical treatment with: Fibroblast Cell (DJ1) and ESCs
o Hybrid cells were then analyzed and found to behave like ESCs:
§ Expressed pluripotent transcription factors (i.e. OCT4)
§ Capable of differentiating into multiple cell type in vivo and in vitro
ð This does not conclude that nuclear reprogramming was seen in DJ1 cells:
o Expression analyzed can be due to only ESCs nuclei and not DJ1 nuclei
§ To verify these findings the following experiment was completed:
Experiment:
• Generated a construct: GFP marker was fused with a pluripotent gene promotor
a) Construct + ESCs:
§ GFP signal was expressed – expected because ESCs express the pluripotent gene
b) Construct + DJ1:
§ GFP signal was not expressed – expected because differentiated cell do not express
the following pluripotent gene
• Construct + DJ1 was then fused with an ESCs (no GFP marker):
o Hybrid cell expressed GFP – therefore indicating the ESCs nuclei was capable of
reprogramming the DJ1 nuclei which led to observe the stem-cell specific expression
Reprogramming Without Cell Fusion:
• Pre-neuronal cells (adult neuronal stem cells) have the fate of differentiating into neurons
o Is it possible to reprogram the fate of pre-neuronal stem cells into a different fate?
§ Phenotypic plasticity exists within these cells if placed in the right
environment; i.e. proper environmental cues (growth factors, hormones…)
ð Concern: during experiments cell fusion may occur, therefore must distinctly differentiate cells
and whether we are looking at a heterokaryon (2 distinct nuclei)
Experiment:
• Pre-neuronal stem cells were fused with a LacZ marker (LacZ expression = blue cells)
o Pre-neuronal stem cells were injected into blastocysts which contain inner cell mass
• Pluripotent embryonic stem cells (inner cell mass) will differentiate into different cell fates to
generate all three germ layers
o Pre-neuronal stem cells were capable of being reprogrammed and were found in
different tissue-types in the regenerated embryo
ð Indicates that integrating the
pre-neuronal stem cells in the inner
Document Summary
Somatic (fibroblast) cells + embryonic stem cells: generated a cell fusion via chemical treatment with: fibroblast cell (dj1) and escs, hybrid cells were then analyzed and found to behave like escs: Capable of differentiating into multiple cell type in vivo and in vitro. This does not conclude that nuclear reprogramming was seen in dj1 cells: expression analyzed can be due to only escs nuclei and not dj1 nuclei. To verify these findings the following experiment was completed: Experiment: generated a construct: gfp marker was fused with a pluripotent gene promotor, construct + escs: Gfp signal was expressed expected because escs express the pluripotent gene: construct + dj1: Phenotypic plasticity exists within these cells if placed in the right environment; i. e. proper environmental cues (growth factors, hormones ) Concern: during experiments cell fusion may occur, therefore must distinctly differentiate cells and whether we are looking at a heterokaryon (2 distinct nuclei)
