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Lecture 5

MCD BIO 90 Lecture 5: Cell Programming

Molecular, Cell, and Developmental Biology
Course Code
Mitra Hooshmand

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This involves manipulating the gene expression of a adult fully-differentiated cell and turning it into a
pluripotent cell.
The easiest adult cell to use is the skin cell, as it is the least invasive procedure.
From skin to Neuron cell
Direct Reprogramming:
From skin cell to pluripotent stem cell to neuron
Treatment with Pluripotent Cell Extract
Yamanaka Technique
Methods 1 to 6, all of which are inducing pluripotent cells (iPSCs)
Indirect Reprogramming:
Process by which an unfertilized oocyte becomes activated and begins to divide.
Does not require sperm
Mimics the calcium-dependent processes of post-fertilization using specific factors to take oocyte
out of MII arrest.
Because the hardening of the zona pellucida begins the process of fertilization (after the sperm cell
releases certain chemicals), by releasing the chemicals, we could recreate the hardening and enable this
process. The egg will begin dividing, but due to the lack of half of the genetic material, they are limited
in their use. It gives us a half haploid population of embryonic cells.
Somatic Cell Nuclear Transfer
This is known as "therapeutic cloning". It involves using the machinery of an oocyte to reset the identity
of a somatic cell. It was first discovered in amphibians by John Gurdon 1958.
To study diseases: you can replicate a disease in a dish and study it (disease modeling), and see
which cures work.
Cell-based therapies with Population of pluripotent cells: if no genetic problem, it can be used to
make other needed cells.
It has many applications:
Adult fibroblast is taken from skin,
The nucleus of the oocyte taken out
Cell Programming
Friday, January 26, 2018
1:06 PM
Stem Cells Page 1

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The nucleus of the oocyte taken out
The nucleus of the fibroblast put into the oocyte.
You give it an electric shock to fertilize it.
Division begins and the organism begins to develop.
Teratoma: proves it can become 3 germ layers.
Chimera: cell put into embryo of a mouse, creating a hybrid.
Epigenetics: still that of an oocyte
Test for pluripotency
Sterile: It can't produce sex cells.
Low efficiency: it is very difficult to remove the nucleus of the oocyte without disrupting any other
part of the cell.
Hwang Controversy: good egg cells from female graduate students and fabricated data.
Problems to cloning:
In 2005, North Korean Scientist Hwang went to jail for publishing a paper with fabricated date in
Science about a study where he did SCNT human cells. In the investigation, they found that Hwang got
the oocytes from his graduate students. This controversy lies in the fact that the donation operation is
risky, even of death, and very invasive. The reputation of SCNT was forever harmed.
Cell Fusion
A differentiated somatic cell is reprogrammed by fusion with a pluripotent stem cell to express stem cell
genes and functions. It occurs through a complicated fusion of membranes: intercellular membranes
fusion between transport vesicles and the plasma membrane. It involves many proteins like SNARE and
Rab proteins.
This involves the fusion of nucleus of two cells, which results in 4 copies of DNA. It involves one
immature cell and one fully differentiated cell. The membranes have to break down and the nucleus
have to fuse by first fusing the membrane of the nucleus, and then the genetic material fuses. People
still don’t know why this happens.
This is not technically cell programming, as this is a mistake. Scientist have to prove the lack of cell
fusion in many experiments involving cell programming.
Neural stem cells attach to B-Galactosidase, a reporter gene (in nucleus) with a blue marker. We know
it’s a neural stem cell because the cell is blue. If we put this neural stem cell with a myoblast (muscle
cell) / embryoid body (three-dimensional aggregates of pluripotent stem cells) in a dish, it can become
muscle cells expressing B-galactosidase.
Neural stem cell expressing GFP (green marker) and puromycin resistance protein was put in a dish with
an ES cell expressing hygromycin-resistance protein. The neural stem cell would die if exposed to
hygromycin and the ES cell would die if exposed to puromycin. When puromycin was added, the result
was a hybrid cell expressing GPF, puromycin-resistance protein and hygromycin-resistance protein.
Bone marrow cell expressing GFP and puromycin-resistance protein was put in the same cell culture as
ES cell. Scientist put a selection on puromycin, and the result was a hybrid cell.
Stem Cells Page 2
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