 Differentiated cells and maintenance of tissues

Stem cell differentiation is the generation of specialized cells from stem cells:

Scientists and doctors are excited about the potential of stem cells to revolutionize medicine. The
promise of stem cells lies in their ability to change from an unspecialized cell into a specialized cell
such as a nerve, muscle or bone cell. Currently, doctors do not have access to a renewable source of
tissue that can be used in transplantations to restore damaged or destroyed tissue. Stem cells could be
used as a tissue source for transplantation therapy. For example, insulin-secreting pancreas cells
created from stem cells have the potential to treat Diabetes. These stem cell-derived pancreas cells
could replace dysfunctional cells in the pancreas, allowing a patient to regulate their blood sugar levels.
Another use of stem cells is to make specific cell types for studying human disease in the laboratory.
For example, in Parkinson’s disease, neurons of the brain die resulting in motor and brain defects. But
the reason why these neurons die is unknown. Scientists cannot easily access neurons from
Parkinson’s patients, so they are trying to make neurons directly from stem cells, in the laboratory.
Scientists are now using these neurons made from the stem cells of Parkinson’s patients to research
the causes of this disease. In order to use stem cells to treat disease or as research tools to study
disease, scientists must be able to differentiate stem cells into fully functioning specialized cells.
Differentiation is the process where an unspecialized cell acquires cellular traits that allow it to perform
specialized functions. Scientists are currently working on methods to effectively differentiate stem cells
into functional specialized cells.

How do stem cells differentiate during embryonic development? The best approach to stem cell
differentiation is to apply what developmental biologists have learned about embryonic development.
During development, cells of the embryo make a number of cell fate decisions that result in decreasing
potency eventually producing specialized cells (The two main stem cell types are embryonic stem cells
(ES) cells and adult stem cells (i.e., somatic stem cells)). Cells of the embryo make cell fate decisions
based on a number of chemical and physical signals that are produced within the embryo including;
secreted chemicals from neighbouring cells, direct communication with neighbouring cells by cell-cell
contact or mechanical strain caused by movements of the embryo as it develops. All of these
processes activate signaling that ultimately leads to changes in gene activity, which determines cellular
differentiation. Stem cell biologists try to push stem cells to differentiate into specialized cell types by
recreating an embryonic environment in a petri dish.

How do scientists differentiate stem cells in a dish? To recreate embryonic development in a dish, stem
cells are exposed to chemical or physical signals that will cause cellular differentiation including:

• Growth Factors Signalling molecules that cause cellular differentiation can be added to stem cell
culture medium to transform stem cells into specialized cell types.

• Cell Culture Substrate: Stem cells can be cultured on top of extracellular matrix proteins (proteins
secreted by cells into the extracellular space) to cause differentiation.

• Co-culture Environments: Cells produce signalling molecules that neighbouring cells can respond to.
Stem cells can be grown together with other cells that produce growth factors that will cause
differentiation.
• Three-dimensional Culture: Stem cells can be cultured in three-dimensional aggregates (spherical
clumps of cells) called embryoid bodies to create mechanical stimulation and cell contact. Embryoid
bodies recreate an early developmental process called gastrulation, a process that establishes the
three primary germ layers.

• Signal Inhibition: In addition to providing activating signals to cause cellular differentiation, signals that
cause the differentiation of stem cells to cell types other than the cell type of interest can be inhibited.
This can be accomplished by using inhibitory growth factors.

The production of pancreas cells from pluripotent stem cells is an example of stem cell differentiation
using a number of the tools described above. To create pancreas cells, stem cells are grown as
embryoid bodies in combination with a number of pancreas-inducing growth factors. After a few days of
three-dimensional culture, the cells are separated into single cells and then grown in a two-dimensional
environment with another set of growth factors that will drive differentiation to mature pancreas cells.
How do scientists determine if stem cells have differentiated into a specialized cell type? Scientific
Proof of Differentiated Cells Before a scientist can claim that a stem cell has differentiated into a
specialized cell, a number of criteria must be met to prove that a stem cell has turned into a functional
specialized cell:

• Appearance: Does the differentiated cell LOOK like the target cell type? The unique combination of
genes that are on and off determines the characteristics of a unique cell type. Scientists can use a
number of laboratory techniques to determine which genes are turned on or off in a cell. For example,
does the neuron that has been created from a stem cell have the same sets of gene turned on and off
as a neuron from the brain? Cells are also unique in shape depending on the function they perform
within the body. Neurons have long, thin projections that are used to transmit signals throughout the
body. Scientists use imaging techniques to determine whether a neuron made from a stem cell has the
same type of long, thin projections as a neuron from the brain.

• Behaviour: Does the differentiated cell BEHAVE like the target cell type? Specialized cells perform
specialized functions within the body. For example, heart muscle cells contract and relax to pump blood
throughout the body. In the laboratory, a number of experiments can be performed to test whether a
differentiated cell can perform the function of a specialized cell. Scientists can see heart cells created
from stem cells beating (contacting and relaxing) in the dish using a microscope (in fact, you can see
heart cells beating on YouTube). In the body, heart muscle cells respond to electrical signals for the
cue to contract. In the laboratory, the ability of heart cells to respond to electrical signals can be tested.

• Safety: Are the differentiated cells SAFE for use in transplantation therapy? If specialized cells
generated from stem cells are to be used to treat human disease, they must be fully differentiated, that
is, they must have lost their ability to generate any other cell type. When undifferentiated stem cells are
transplanted into a mouse, the cells will form a tumour known as a teratoma. Teratomas are a mass
made up of various cell types. Following differentiation of stem cells, scientists must be sure that the
stem cells have lost their ability to form teratomas before they are transplanted into patients.

Storage by freezing
Long term freezing of cells can be carried out to, preserve cells, avoid senescence, reduce the risk of
contamination, and minimize effects of genetic drift. Cryoprotective agents are used for the freezing
purpose. Without cryoprotective agent freezing would be lethal to the cells in most cases. Commonly, a
cryoprotective substance like dimethylsulfoxide is used in conjunction with complete medium for the
preservation of cells at -70°C or lower. DMSO can reduce the freezing point and allows a reduced
cooling rate. Gradual freezing decreased the risk of cell damage and ice crystal formation.

Resuscitation of Frozen Cell Lines

When cryopreserved cells are needed for study, it is vital to thaw cells correctly in order to maintain the
viability of the culture and enable the culture to recover quickly. Some cryoprotectants, such as DMSO,
are toxic above 4°C therefore it is essential that cultures are thawed quickly and diluted in culture
medium to reduce the toxic effects.

CAUTION
Protective clothing, particularly insulated gloves and goggles, should be worn when removing frozen
vials or ampules from the liquid nitrogen freezer. The room having the liquid nitrogen freezer should be
well-ventilated. Make sure that liquid nitrogen does not spill on the skin.

Additional Materials

70% (v/v) ethanol

Complete medium /20% FBS (e.g. supplemented DMEM-20).
NOTE: All incubations should be performed in a humidified 37°C, and 5% CO2 incubator unless
otherwise specified.