What are they thinking!?

Birgitt: Thank you, Alex, for coming to California last week for the Festival of Genomics. People at the conference were very inspired by your story and that you put yourself out there despite having Parkinson's disease.

Alex: It was good being in San Francisco. Thanks again to our sponsors Thermo Fisher and the organizers of the meeting to make this happen. What are you up to?

Birgitt: As I pointed out at the meeting, I am planning to write about your genetic make-up and potential factors that might predispose you to Parkinson's disease. But today I will share with you how to make neurons from your skin-derived stem cells.

Alex: Would it be possible to transplant these neurons back into my brain and replace the neurons that are gone?

Birgitt: In principle, yes, that is an approach many research laboratories are working on. But to make cells safe for human use, they have to be handled under special good manufacturing practices, which means for example that cells cannot come in contact with any animal products. Your cells have been grown for research purposes only.

Alex: Got it, makes sense.

Timeline of neuronal differentiation

Birgitt: Here is a timeline that shows the neuronal differentiation from pluripotent stem cells over an intermediate stage of neural stem or progenitor cells, which take about 10-12 days and then 20-30 days for maturation into neurons.

Alex: I am quite amazed that this is possible and the potential for drug discovery these cell cultures have. I really hope this will lead to faster discoveries and validation of novel compounds in the future.

Birgitt: That is exactly the concept and idea behind developing these models. Here you can see how your neurons look after about 30 days differentiation. The blue dots are cell bodies and in green you see the processes of the neurons.

Differentiated neurons after 30 day in culture

Alex: Incredible and really beautiful. Are these really my neurons? I wonder what they are thinking about. 

Birgitt: I am sure they want  cure for Parkinson's disease.

Alex: And so do I!

Alex at Festival of Genomics in California

Alex: The Festival of Genomics in San Mateo/San Francisco is getting closer (http://www.festivalofgenomicscalifornia.com/). I am really looking forward to visit San Francisco and be on stage with you on November 5th, 2015 at 3p.m. at the San Mateo Event Center, CA discussing what really matters to conquer Parkinson's disease.

Birgitt: I am excited as well. I am thankful to be invited by the organizers and that you received sponsorship from Thermo Fisher Scientific for your trip (https://www.thermofisher.com/us/en/home.html).

Alex: BIG THANKS from me as well! The support is very much appreciated.

Birgitt: We will meet you at the conference. We will talk about Parkinson's disease from Alex's perspective as a patient and my view as a scientist and how we can work together to make change happen for people with this disease.

Alex:  Let's hope El Niño hasn't started yet. See you in a couple of weeks. BTW: what is the attire for the meeting? California casual?

More Than Meets the Eye

In the last post we discussed the potential for stem cells to be pluripotent, and which steps need to be taken to test for their pluripotency. In this post, we will explain to Alex how his pluripotent stem cells are transformed into neural stem cells.

Alex: We should talk more often. It is very interesting to learn about the science and potential implications for new therapies. I am excited to learn about the transformation of my stem cells into nerve cells. Tell me, what are neural stem cells?

Birgitt: Neural stem cells are the precursor cells of the brain. They can differentiate into multiple cell types such as nerve cells and cells that support neurons which are called glia cells. They are the stem cells of the central nervous system. Even in the adult brain, we can find neural stem cells. Recent studies have shown that physical exercise helps with the regeneration of neural stem cells. So, keep moving!

Alex: I am running and keep moving, that's my motto. It helps a lot with my Parkinson's. What is the concept behind the transformation into neural stem cells?

Birgitt: To understand cell differentiation one must understand the concept of the three germ layer. A germ layer is a group of cells that interacts with one another to create organs and tissues. Humans have three germ layers: the inner layer (endoderm), the middle layer (mesoderm), and the outer layer (ectoderm).  Each one of these layers is responsible for creating different tissues of the human body.  Nerve cells are derived from the outer layer. In the culture of your induced pluripotent stem cells, we promote the preferential growth of the  outer layer cells and suppress the mesoderm and endoderm layers using specific small molecules and proteins in the cell culture media.

Alex: Fascinating, there is indeed more than meets the eye in this process.

Birgitt: Well, the process to transform stem cell into neurons or neural precursor cells has been a tedious and lengthy procedure in the past. However, newer protocols allow us to circumvent several difficulties, and has made the transformation much faster. One procedure in particular has made it possible for pluripotent stem cells to be turned into neural stem cells in only seven to ten days. You can see your stem cells at day 2 and day 6 during the neural induction in the next two images.

Alex's stem cells at day 2 of neural induction

Alex's stem cells at day 6 of neural induction

Alex: Interesting. On the next two images below, my cells look completely different and I can see individual cells, almost like my skin cells.

Birgitt: Exactly, after seven days and one enzymatic treatment to divide the cells, the cells grow in a single layer. They look similar to skin cells, but have a rounder body and a few small processes. The images below are in two different magnifications, the first one is an overview and then a close-up. Looking good!

Alex's neural stem cells have formed

Alex's neural stem cells at higher magnification

Alex: And after that? These are not the dopaminergic neurons that are dying in my brain. I assume there is another step or steps?

Birgitt: Right on, the neural stem cells can be further differentiated into different types of neurons or glia through several different processes. Many research labs develop protocols to make all kinds of different cells of the brain, such as cortical neurons, inhibitory GABAergic neurons, dopaminergic neurons, Purkinje cells, and motor neurons as well as glia cells like astrocytes or oligodendrocytes. The methods for differentiating all these types of neurons can require extended periods of time (up to several months).

Alex: How long does it take to make dopaminergic neurons?
  
Birgitt: It takes about 4-8 weeks to have fully mature dopaminergic neurons. A long way to go and it needs passion, endurance, and commitment, just as running a marathon. Will talk about them in the next blogs.
  
Alex: Thanks so much, as always, fascinating and it gives me hope that this research will -some day, hopefully soon- change the lives of people with Parkinson's disease. 

Movie Monday Episode 5 "Alex Strikes Back"

In this week's video Alex discusses his future plans for raising awareness for Parkinson's. 

Music: "Broken" by Alialujah Choir

Movie Monday Episode 3 "Revenge of Alex"

In this week's video Alex discusses how he finds motivation to keep advocating for research for Parkinson's Disease. 

Music: "People Like You" by Weinland

Movie Monday Episode 4 "A New Hope"

In this week's video Alex explains how he raises awareness for Parkinson's.

Music: "Solo Acoustic" by Jason Shaw

Movie Monday Episode 2 "Stem Cell Wars"

In this week's video Alex discusses how Parkinson's has affected his professional and personal life.

Music: "Sunken Eyes" by Weinland

New! Movie Mondays with Alex: Episode 1 "Alex's Menace"

We've gotten a lot of questions about Alex and how Parkinson's Disease has affected his life. To answer some of these questions we will be releasing weekly clips from an interview we conducted with Alex in 2013. The interview answers many of the same questions our fans have asked and will hopefully be insightful, informative, and interesting. In this first clip Alex discusses when he discovered his first symptoms of Parkinson's Disease  Enjoy.

Music: "Cold Weather" by Alialujah Choir

Yes, they can – testing Alex’s cells for pluripotency

In the last blog we gave an overview on how to characterize iPS cells. Today, we dive in and explain how we test the cells for pluripotency, the potential to turn into different cell types of the human body.

 

Alex: Hi, it's been a while, but it's summer and I heard Lauren has accepted a new position!

Birgitt: Yes, we'll miss Lauren and we wish her the best of luck for her future.

Alex: So what's next?

Birgitt: I'll explain how we test your cells to see if they have the potential to show "yes we can" "Change"  into other cells. To test proteins and markers in the cell you need to visualize them. This can be done with a method called immunostaining. Immunostaining is an antibody-based method that detects specific proteins on the surface or inside of the cells. The picture below is an example of how we can immunostain the cells. We use an antibody specific for a certain marker on the cell, called epitope. Once this antibody binds to the epitope we then incubate with a fluorescently labeled antibody that binds to the first antibody. We can then visualize this fluorescence with a fluorescent microscope and take the colorful images you will see in the next few blogs. 

More info about immunostaining here

Alex: Hmm sounds a lot like 2008. I posted some pretty fluorescence pictures on my Facebook page that you shared with me in the past. I am looking forward to understanding more about these immunostaining techniques.

Birgitt: We used three different antibodies to detect whether or not your stem cells are pluripotent. The three markers we used were OCT4, SOX2, and SSEA4, these are proteins that are expressed in pluripotent cells and therefore called pluripotency markers. These are abbreviations for protein names octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), and stage-specific embryonic 4 (SSEA4). Since these protein names are so complicated we usually only use the abbreviations.

See the results in the three images below.  The blue color is the nuclear stain or counterstain which shows where the nucleus of the cell is located. The green fluorescence corresponds one of the pluripotency markers.

iPS colony stained with SSEA-4 antibody

iPS colony stained with OCT4 antibody

iPS colony stained with SOX2 antibody

Alex: I hope we can talk about this more next time and I am also very excited to come out to San Francisco for the Festival of Genomics on November 5^th, 2015 to chat with you on stage about my Parkinson’s disease, stem cells,  and more (http://www.festivalofgenomicscalifornia.com/).

Birgitt: I am thrilled to have the opportunity to talk with you on stage, can't wait! And maybe the topic of pluripotency will be reelected in the next post.

About our New Co-editors

Bastian Schuele, senior at Menlo-Atherton Highschool, Gautam Bordia, student at UC Santa Barbara and Raghav Bordia, student at the University of Washington, are interns at the Parkinson's Institute. They all share an interest in neurobiology, stem cells, and genetics. They assist Birgitt Schuele with the skin cell blog, brain archive, and subject data entry.

Gautam Bordia, Bastian Schuele, Raghav Bordia

Alex's stem cells pass the practice test

In the last blog we talked about how we bank your cells. Today, we will give you an overview of how to make sure that the cell we have are actually pluripotent stem cells, a process we call characterization.

Lauren: We use four methods to characterize your cells. In the image below you can see a summary of these methods. Two methods test the pluripotency of the newly derived cells and the other two methods make sure that the DNA was not damaged or changed during the reprogramming process.

Summary of characterization process

Alex: I know we have talked about pluripotency before, but can you remind me what that is?

Lauren: Pluripotency refers to the ability of the cells to give rise to all of the cell types that make up the human body. The first way we test pluripotency is by staining the cells for certain markers. All cell types have markers on the surface of their cells and within the cells. These markers are specific to each type of cell so that we can distinguish different cells from one another. We use multiple markers that are specific for pluripotency to be sure that the cells are indeed pluripotent.

Alex: Well that seems fairly straightforward. How else do you test for pluripotency?

Lauren: Another way that we test for pluripotency is to spontaneously differentiate these stem cells. The spontaneous differentiation begins by forming embryoid bodies. Embryoid bodies are 3-D cell aggregates as shown in the picture below. The cells are no longer adherent to the bottom of the dish but are floating in the media. Embryoid bodies are meant to mimic the early development of an embryo. This stage primes the stem cells and gets them ready to differentiate into all cell types. We use specific cell markers to confirm that the stem cells can differentiate into various tissue types.

Example of embryoid bodies

Alex: I did not realize that so much work went into these processes. What else needs to be done in order to call my newly made stem cells "induced pluripotent stem cells"?

Lauren: The next two methods of characterization ensure that the DNA within the cells is not damaged and that the virus has not "sneaked into" the DNA by the reprogramming process. The first method is called a karyotype which shows the number and shape of chromosomes (packaged DNA) of the cell. For humans, a normal karyotype consists of 22 pairs of chromosomes and the two sex chromosomes X and/or Y. The image below is an example of how a normal karyotype looks like.

Example of a normal karyotype, 46 XY

Alex: I hope my karyotype comes out normal. What is the next step?

Lauren: To make sure that the genetic material of these reprogrammed cells has not been modified, we measure the virus in the cells which we used to deliver the reprogramming factors.  After 10 to 12 passages, we should not detect the virus anymore. We can detect the virus by an amplification method that will be explain in more detail later.

Alex: Thank you for the overview. I am looking forward to learning more about each of these methods and seeing if my cells pass these tests. It is a bit nerve racking almost like passing a practice driving test.

Lauren: Indeed, I have quite nervous about some of the test results of your cells. In the next few blogs we will go into more detail about each of these methods and show the results in you cells. The first process we will discuss is staining for the pluripotency cell markers.