Embryonic Stem Cell Culture and Differentiation
Embryonic stem cells, or ES cells, have a number of unique properties that distinguish them from regular adult cells. Therefore, scientists have devised special culturing techniques to maintain or take advantage of these properties. When cultured properly, ES cells can divide indefinitely to make more of themselves. At the same time, by altering the culturing conditions, ES cells can be directed to differentiate into almost any cell type found in our body; this ability of ES cells is called "pluripotency."
In this video, you are going to learn how to culture and passage ES cells so that they maintain their unique properties, how to induce differentiation in ES cells to form specific cell types, and the ways in which ES cell culturing and differentiation techniques are being used and refined by researchers today.
Before going into details of the procedures for ES cell maintenance, it is important to first understand what prevents or drives ES cell differentiation. In order for ES cells to retain their unique properties of pluripotency and self-renewal, they must be cultured with specific factors in their growth medium that suppress spontaneous differentiation.
This is most commonly done by culturing ES cells on a layer of "feeders," usually mouse embryonic fibroblasts, or MEFs. Feeder cells "feed" the ES cells certain factors, such as activin A, that help to maintain ES cells in their undifferentiated state.
Recently, scientists have also developed feeder-free culturing methods in which ES cells are grown in media with the necessary factors added in a defined recipe. This approach reduces variability and removes the non-human component from ES cell cultures, which is a prerequisite for clinical applications.
Another characteristic of ES cells is that they naturally grow in clusters or colonies, and tend to have low survival rate when dissociated into single cells, especially human ES cells. As a result, passaging human ES cells generally requires retaining them in intact clumps through mechanical "picking."
When ES cells are grown in non-adherent conditions, they form 3D aggregates known as embryoid bodies, or EBs, in which the ES cells will differentiate into all different cell types. By varying the differentiation conditions, such as the addition of specific growth factors to the medium, scientists are developing methods to direct ES cell differentiation into specific cell types.
Now that you understand the principles behind ES cell maintenance and differentiation, let's look at how to culture and passage ES cells on MEF feeders.
MEFs are obtained from early stage mouse embryos, and are then inactivated by chemicals or radiation to prevent them from further dividing. Inactivated MEFs should be plated onto gelatinized tissue culture dishes at least one day before starting to culture ES cells. When feeders are fully settled, ES cells are thawed and seeded onto the plates.
Over the next few days, ES cells will grow into large colonies. Before the colonies begin to touch and fuse, the ES culture should be passaged. One should observe the ES cells' morphology to see if they are showing signs of differentiation. Undifferentiated ES colonies generally look well-defined and homogeneous, while differentiated cells would look like dull, irregular-shaped "cobblestones."
If colonies show 70% or more of differentiation, or if they are sparse, mechanically cut out the undifferentiated colonies and transfer them to a new feeder plate. Otherwise, simply remove the differentiated areas or colonies and proceed with passaging.
Once the differentiated colonies have been cleaned up, proteolytic enzymes, like collagenase, are added to lift the cells from the plate with incubation at 37°C for the appropriate amount of time. Fresh ES media is then added to stop the enzymatic reactions. The colonies are mechanically dislodged from the plate and transferred to a centrifuge tube, where they are broken up into desired sizes with gentle pipetting. The ES cells are collected by centrifugation, resuspended in ES media, and plated onto the prepared feeder plates.
After learning how to passage ES cells, let's look at one of the more common techniques used to differentiate ES cells into embryoid bodies-the hanging drop method.
To begin, ES cells are detached with the help of proteolytic enzymes like collagenase, and diluted to the desired concentration in media containing lineage-specific differentiation factors. The ES cell suspension is then deposited in drops onto the lid of a bacterial petri dish. The lid is quickly inverted and placed on the dish, so the media drops will hang upside down and allow the embryoid bodies to develop.
After about two days, the drops with EBs can be collected and plated onto non-adherent plates for further culturing. At the appropriate time point for the desired cell lineage, embryoid bodies are plated back onto gelatinized adherent tissue culture dishes for further differentiation.
Now that we've covered the basic techniques of culturing and differentiating ES cells, let's look at how they are tailored for specific experimental needs.
As mentioned earlier, ES cells can be directed to differentiate into specific cell types under different culturing conditions. In this experiment, scientists produced motor neurons from human ES cells. This was done by first adding factors to differentiating embryoid bodies that direct them towards the neural lineage, followed by chemicals and plating conditions that turn these cells specifically into motor neurons. Using similar approaches, researchers have been successful in differentiating ES cells into rhythmically beating heart muscle cells.
Because differentiating ES cells mimic events that occur as embryos develop naturally, we can also use them to investigate the biology of early embryonic development. One of the phenomena being studied is X-chromosome inactivation, or XCI, the crucial process whereby one of the two X-chromosomes is silenced in each cell of a female mammal. By visualizing the distribution of specific RNAs and proteins in developing EBs, scientists have gained valuable insights into the biology of XCI.
Finally, to increase the consistency and efficiency of ES cell experimentation, scientists are continually trying to devise better techniques to culture ES cells. One approach is to grow ES cells in a single layer, called non-colony type monolayer culturing, or NCM.
Remember that human ES cells tend to be unhappy when they are not growing as 3D aggregates? Scientists have shown that chemicals known as ROCK inhibitors increase the survival of dissociated ES cells, which allow them to be passaged as single-cell suspensions, and to be plated at high density. The advantage of the NCM technique is that every cell will be more equally exposed to growth factors and nutrients in the medium, reducing heterogeneity within the culture while making it easier to grow ES cells in large numbers.
You've just watched JoVE's video on how to culture and differentiate embryonic stem cells. You should now know what factors are important for maintaining ES cells in their undifferentiated state, and how we can take advantage of ES cell pluripotency to generate specific cell types in a dish. Going forward, scientists will be working to develop more efficient and well-defined culturing and differentiation conditions to produce specialized cell types that can be used in regenerative medicine applications. Thanks for watching!
Culturing embryonic stem (ES) cells requires conditions that maintain these cells in an undifferentiated state to preserve their capacity for self-ren…
Embryonic stem cells, or ES cells, have a number of unique properties that distinguish them from regular adult cells. Therefore, scientists have devised special culturing techniques to maintain or take advantage of these properties. When cultured properly, ES cells can divide indefinitely to make more of themselves. At the same time, by altering the culturing conditions, ES cells can be directed to differentiate into almost any cell type found in our body; this ability of ES cells is called "pluripotency."
In this video, you are going to learn how to culture and passage ES cells so that they maintain their unique properties, how to induce differentiation in ES cells to form specific cell types, and the ways in which ES cell culturing and differentiation techniques are being used and refined by researchers today.
Before going into details of the procedures for ES cell maintenance, it is important to first understand what prevents or drives ES cell differentiation. In order for ES cells to retain their unique properties of pluripotency and self-renewal, they must be cultured with specific factors in their growth medium that suppress spontaneous differentiation.
This is most commonly done by culturing ES cells on a layer of "feeders," usually mouse embryonic fibroblasts, or MEFs. Feeder cells "feed" the ES cells certain factors, such as activin A, that help to maintain ES cells in their undifferentiated state.
Recently, scientists have also developed feeder-free culturing methods in which ES cells are grown in media with the necessary factors added in a defined recipe. This approach reduces variability and removes the non-human component from ES cell cultures, which is a prerequisite for clinical applications.
Another characteristic of ES cells is that they naturally grow in clusters or colonies, and tend to have low survival rate when dissociated into single cells, especially human ES cells. As a result, passaging human ES cells generally requires retaining them in intact clumps through mechanical "picking."
When ES cells are grown in non-adherent conditions, they form 3D aggregates known as embryoid bodies, or EBs, in which the ES cells will differentiate into all different cell types. By varying the differentiation conditions, such as the addition of specific growth factors to the medium, scientists are developing methods to direct ES cell differentiation into specific cell types.
Now that you understand the principles behind ES cell maintenance and differentiation, let's look at how to culture and passage ES cells on MEF feeders.
MEFs are obtained from early stage mouse embryos, and are then inactivated by chemicals or radiation to prevent them from further dividing. Inactivated MEFs should be plated onto gelatinized tissue culture dishes at least one day before starting to culture ES cells. When feeders are fully settled, ES cells are thawed and seeded onto the plates.
Over the next few days, ES cells will grow into large colonies. Before the colonies begin to touch and fuse, the ES culture should be passaged. One should observe the ES cells' morphology to see if they are showing signs of differentiation. Undifferentiated ES colonies generally look well-defined and homogeneous, while differentiated cells would look like dull, irregular-shaped "cobblestones."
If colonies show 70% or more of differentiation, or if they are sparse, mechanically cut out the undifferentiated colonies and transfer them to a new feeder plate. Otherwise, simply remove the differentiated areas or colonies and proceed with passaging.
Once the differentiated colonies have been cleaned up, proteolytic enzymes, like collagenase, are added to lift the cells from the plate with incubation at 37°C for the appropriate amount of time. Fresh ES media is then added to stop the enzymatic reactions. The colonies are mechanically dislodged from the plate and transferred to a centrifuge tube, where they are broken up into desired sizes with gentle pipetting. The ES cells are collected by centrifugation, resuspended in ES media, and plated onto the prepared feeder plates.
After learning how to passage ES cells, let's look at one of the more common techniques used to differentiate ES cells into embryoid bodies-the hanging drop method.
To begin, ES cells are detached with the help of proteolytic enzymes like collagenase, and diluted to the desired concentration in media containing lineage-specific differentiation factors. The ES cell suspension is then deposited in drops onto the lid of a bacterial petri dish. The lid is quickly inverted and placed on the dish, so the media drops will hang upside down and allow the embryoid bodies to develop.
After about two days, the drops with EBs can be collected and plated onto non-adherent plates for further culturing. At the appropriate time point for the desired cell lineage, embryoid bodies are plated back onto gelatinized adherent tissue culture dishes for further differentiation.
Now that we've covered the basic techniques of culturing and differentiating ES cells, let's look at how they are tailored for specific experimental needs.
As mentioned earlier, ES cells can be directed to differentiate into specific cell types under different culturing conditions. In this experiment, scientists produced motor neurons from human ES cells. This was done by first adding factors to differentiating embryoid bodies that direct them towards the neural lineage, followed by chemicals and plating conditions that turn these cells specifically into motor neurons. Using similar approaches, researchers have been successful in differentiating ES cells into rhythmically beating heart muscle cells.
Because differentiating ES cells mimic events that occur as embryos develop naturally, we can also use them to investigate the biology of early embryonic development. One of the phenomena being studied is X-chromosome inactivation, or XCI, the crucial process whereby one of the two X-chromosomes is silenced in each cell of a female mammal. By visualizing the distribution of specific RNAs and proteins in developing EBs, scientists have gained valuable insights into the biology of XCI.
Finally, to increase the consistency and efficiency of ES cell experimentation, scientists are continually trying to devise better techniques to culture ES cells. One approach is to grow ES cells in a single layer, called non-colony type monolayer culturing, or NCM.
Remember that human ES cells tend to be unhappy when they are not growing as 3D aggregates? Scientists have shown that chemicals known as ROCK inhibitors increase the survival of dissociated ES cells, which allow them to be passaged as single-cell suspensions, and to be plated at high density. The advantage of the NCM technique is that every cell will be more equally exposed to growth factors and nutrients in the medium, reducing heterogeneity within the culture while making it easier to grow ES cells in large numbers.
You've just watched JoVE's video on how to culture and differentiate embryonic stem cells. You should now know what factors are important for maintaining ES cells in their undifferentiated state, and how we can take advantage of ES cell pluripotency to generate specific cell types in a dish. Going forward, scientists will be working to develop more efficient and well-defined culturing and differentiation conditions to produce specialized cell types that can be used in regenerative medicine applications. Thanks for watching!
Embryonic stem cells, or ES cells, have a number of unique properties that distinguish them from regular adult cells. Therefore, scientists have devised special culturing techniques to maintain or take advantage of these properties. When cultured properly, ES cells can divide indefinitely to make more of themselves. At the same time, by altering the culturing conditions, ES cells can be directed to differentiate into almost any cell type found in our body; this ability of ES cells is called "pluripotency."
In this video, you are going to learn how to culture and passage ES cells so that they maintain their unique properties, how to induce differentiation in ES cells to form specific cell types, and the ways in which ES cell culturing and differentiation techniques are being used and refined by researchers today.
Before going into details of the procedures for ES cell maintenance, it is important to first understand what prevents or drives ES cell differentiation. In order for ES cells to retain their unique properties of pluripotency and self-renewal, they must be cultured with specific factors in their growth medium that suppress spontaneous differentiation.
This is most commonly done by culturing ES cells on a layer of "feeders," usually mouse embryonic fibroblasts, or MEFs. Feeder cells "feed" the ES cells certain factors, such as activin A, that help to maintain ES cells in their undifferentiated state.
Recently, scientists have also developed feeder-free culturing methods in which ES cells are grown in media with the necessary factors added in a defined recipe. This approach reduces variability and removes the non-human component from ES cell cultures, which is a prerequisite for clinical applications.
Another characteristic of ES cells is that they naturally grow in clusters or colonies, and tend to have low survival rate when dissociated into single cells, especially human ES cells. As a result, passaging human ES cells generally requires retaining them in intact clumps through mechanical "picking."
When ES cells are grown in non-adherent conditions, they form 3D aggregates known as embryoid bodies, or EBs, in which the ES cells will differentiate into all different cell types. By varying the differentiation conditions, such as the addition of specific growth factors to the medium, scientists are developing methods to direct ES cell differentiation into specific cell types.
Now that you understand the principles behind ES cell maintenance and differentiation, let's look at how to culture and passage ES cells on MEF feeders.
MEFs are obtained from early stage mouse embryos, and are then inactivated by chemicals or radiation to prevent them from further dividing. Inactivated MEFs should be plated onto gelatinized tissue culture dishes at least one day before starting to culture ES cells. When feeders are fully settled, ES cells are thawed and seeded onto the plates.
Over the next few days, ES cells will grow into large colonies. Before the colonies begin to touch and fuse, the ES culture should be passaged. One should observe the ES cells' morphology to see if they are showing signs of differentiation. Undifferentiated ES colonies generally look well-defined and homogeneous, while differentiated cells would look like dull, irregular-shaped "cobblestones."
If colonies show 70% or more of differentiation, or if they are sparse, mechanically cut out the undifferentiated colonies and transfer them to a new feeder plate. Otherwise, simply remove the differentiated areas or colonies and proceed with passaging.
Once the differentiated colonies have been cleaned up, proteolytic enzymes, like collagenase, are added to lift the cells from the plate with incubation at 37°C for the appropriate amount of time. Fresh ES media is then added to stop the enzymatic reactions. The colonies are mechanically dislodged from the plate and transferred to a centrifuge tube, where they are broken up into desired sizes with gentle pipetting. The ES cells are collected by centrifugation, resuspended in ES media, and plated onto the prepared feeder plates.
After learning how to passage ES cells, let's look at one of the more common techniques used to differentiate ES cells into embryoid bodies-the hanging drop method.
To begin, ES cells are detached with the help of proteolytic enzymes like collagenase, and diluted to the desired concentration in media containing lineage-specific differentiation factors. The ES cell suspension is then deposited in drops onto the lid of a bacterial petri dish. The lid is quickly inverted and placed on the dish, so the media drops will hang upside down and allow the embryoid bodies to develop.
After about two days, the drops with EBs can be collected and plated onto non-adherent plates for further culturing. At the appropriate time point for the desired cell lineage, embryoid bodies are plated back onto gelatinized adherent tissue culture dishes for further differentiation.
Now that we've covered the basic techniques of culturing and differentiating ES cells, let's look at how they are tailored for specific experimental needs.
As mentioned earlier, ES cells can be directed to differentiate into specific cell types under different culturing conditions. In this experiment, scientists produced motor neurons from human ES cells. This was done by first adding factors to differentiating embryoid bodies that direct them towards the neural lineage, followed by chemicals and plating conditions that turn these cells specifically into motor neurons. Using similar approaches, researchers have been successful in differentiating ES cells into rhythmically beating heart muscle cells.
Because differentiating ES cells mimic events that occur as embryos develop naturally, we can also use them to investigate the biology of early embryonic development. One of the phenomena being studied is X-chromosome inactivation, or XCI, the crucial process whereby one of the two X-chromosomes is silenced in each cell of a female mammal. By visualizing the distribution of specific RNAs and proteins in developing EBs, scientists have gained valuable insights into the biology of XCI.
Finally, to increase the consistency and efficiency of ES cell experimentation, scientists are continually trying to devise better techniques to culture ES cells. One approach is to grow ES cells in a single layer, called non-colony type monolayer culturing, or NCM.
Remember that human ES cells tend to be unhappy when they are not growing as 3D aggregates? Scientists have shown that chemicals known as ROCK inhibitors increase the survival of dissociated ES cells, which allow them to be passaged as single-cell suspensions, and to be plated at high density. The advantage of the NCM technique is that every cell will be more equally exposed to growth factors and nutrients in the medium, reducing heterogeneity within the culture while making it easier to grow ES cells in large numbers.
You've just watched JoVE's video on how to culture and differentiate embryonic stem cells. You should now know what factors are important for maintaining ES cells in their undifferentiated state, and how we can take advantage of ES cell pluripotency to generate specific cell types in a dish. Going forward, scientists will be working to develop more efficient and well-defined culturing and differentiation conditions to produce specialized cell types that can be used in regenerative medicine applications. Thanks for watching!
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Q1: What factors maintain embryonic stem cells in an undifferentiated state?
Embryonic stem cells require specific factors in their growth medium to suppress spontaneous differentiation and retain pluripotency. Feeder cells, typically mouse embryonic fibroblasts (MEFs), provide factors like activin A that maintain ES cells undifferentiated. Alternatively, feeder-free methods use defined media recipes with necessary factors added directly, reducing variability and enabling clinical applications.
Q2: Why do human embryonic stem cells require mechanical picking during passaging?
Human ES cells naturally grow in clusters and have low survival rates when dissociated into single cells. Mechanical picking preserves intact cell clumps, maintaining viability during passaging. This approach contrasts with feeder-free methods using ROCK inhibitors, which allow single-cell suspension passaging at high density while improving cell survival and reducing culture heterogeneity.
Q3: What is the hanging drop method used for in embryonic stem cell differentiation?
The hanging drop method directs ES cell differentiation into specific cell types by forming embryoid bodies (EBs). ES cells are suspended in media containing lineage-specific differentiation factors and deposited as drops on inverted petri dish lids. After two days, EBs are collected and plated onto non-adherent plates, then transferred to adherent dishes at appropriate timepoints for further differentiation.
Q4: How can embryonic stem cells be directed to differentiate into specific cell types?
Scientists direct ES cell differentiation by varying culturing conditions and adding specific growth factors to the medium. For example, motor neurons are produced by first adding factors that direct embryoid bodies toward the neural lineage, followed by chemicals and plating conditions that specify motor neuron identity. Similar approaches have successfully generated rhythmically beating heart muscle cells.
Q5: What are embryoid bodies and how do they form?
Embryoid bodies (EBs) are three-dimensional aggregates that form when ES cells grow in non-adherent conditions. Within EBs, ES cells differentiate into multiple cell types, mimicking early embryonic development. By controlling differentiation conditions and growth factors, researchers can direct EB development toward specific cell lineages for regenerative medicine applications.
Q6: What is pluripotency and why is it important for embryonic stem cell research?
Pluripotency is the ability of ES cells to differentiate into almost any cell type found in the body. This unique property allows researchers to generate specialized cells for studying embryonic development and developing treatments for degenerative diseases. Maintaining pluripotency requires culturing ES cells with specific factors that suppress spontaneous differentiation.
Q7: How do scientists study early embryonic development using differentiated embryonic stem cells?
Because differentiating ES cells mimic events occurring during natural embryonic development, researchers use them to investigate developmental biology. For instance, scientists study X-chromosome inactivation (XCI) by visualizing specific RNAs and proteins in developing embryoid bodies. This approach provides valuable insights into crucial developmental processes without requiring intact embryos.
Chapters in this video
0:00
Overview
1:07
Principles of ES Cell Culturing and Differentiation
3:13
Growing and Passaging ES Cells on MEFs
5:26
ES Differentiation by the Hanging Drop Method
6:32
Applications
9:08
Summary