Feeding Alex's cells with "Orangina"

Alex’s cells are taking off, they divide and multiply in the culture dish.

Skin biopsy with outgrowth of keratinocytes and fibroblasts

Alex: Where do my cells live now?

Outside of a tissue culture incubator

Inside of a tissue culture incubator

Birgitt: Your cells are kept in an incubator at 37C body temperature -like you see on these pictures- and a mixture of gas with 5% CO₂. These incubators are located in our tissue culture laboratory. Only trained staff have access to these rooms, which regularly get inspected to ensure proper safety conditions.

Alex:What do you feed my cells? I love pizza and chocolate but that's not what you feed them, right?

Birgitt: Your cells are fed with a cocktail of sugar, protein, and amino acids. It looks like Orangina. Feeding means, you remove the media with a glass pipette under vacuum and then add an ounce or so –depending on the size of the dish- of media with a new sterile pipette to the cells. We change the media every 2-3 days. We also supplement the media with antibiotics to prevent contamination of the cultures with bacteria or fungi.

Alex: I am just a bit competitive. Are my cells competitive too?

Birgitt: Definitely, your cells are growing great. On Day 8, we noticed the first fibroblasts growing out of the biopsy. See the labeled example above to illustrate the outgrowth of keratinocytes and fibroblasts from a skin biopsy.

Below you can see three pictures comparing the growth of your own cells over five days. Fibroblast cells typically double every 24 to 36 hours. The culture dish has some white markings which allow us now to take images from the same area and to follow the dividing cells. You can see how the space between the markings is filling up with cells. Soon this culture dish will be too small and we will have to transfer the cells to a larger dish. In technical jargon we call it “splitting the cells”.

Alex: Thanks so much. I am really amazed that these are my cells.

Glossary

Terms are arranged in alphabetical order

2008: 2008 was the year Barack Obama won the presidential elections. Some of his campaign slogans during his election were "change" and "yes we can".

Alive...and in perfect hibernation: A quote from Star Wars: Episode V - The Empire Strikes Back (1980) when Han Solo was frozen in carbonite: Darth Vader "Well, Calrissian? Did he survive?" Lando Calrissian "Yes, he's alive...and in perfect hibernation." http://www.imdb.com/character/ch0000031/quotes
Star Wars Blog

Antibody: Any of a large number of proteins of high molecular weight that are produced normally by specialized B cells after stimulation by an antigen. They act specifically against the antigen in an immune response, that are produced abnormally by some cancer cells, and that typically consist of four subunits. These include two heavy chains and two light chains called immunoglobulin.
http://www.merriam-webster.com/dictionary/antibody 
 
Biological safety cabinet: A laminar flow safety cabinet designed to provide three basic types of protection; Personnel protection from harmful agents inside the cabinet,product protection to avoid contamination of the work, experiment, or process and environmental protection from contaminants contained within the cabinet. http://www.bakerco.com/introduction-biological-safety-cabinets
Watch a video about Sterile Tissue Culture Techniques
Coffee blog

Contact inhibition: Biological process that prevents further division of proliferating cells. (Illustrated Dictionary of Podiatry and Foot Science by Jean Mooney. 2009. Elsevier Limited 24 Feb. 2015)

Growing Out of Clothes Blog

Cryopreservation: The process of cooling and storing cells, tissues, or organs at very low temperatures to maintain their viability. For example, the technology of cooling and storing cells at a temperature below the freezing point ('196' C) permits high rates of survivability of the cells upon thawing. http://www.medicinenet.com/script/main/art.asp?articlekey=7252

Differentiated cell: A cell that has undergone the process of becoming progressively more specialized; developmental process through which cells mature.

Rejuvenation Blog

Embryoid bodies: Spheroid colonies seen in culture produced by the growth of embryonic stem cells in suspension. Embryoid bodies are of mixed cell types, and the distribution and timing of the appearance of specific cell types corresponds to that observed within the embryo. http://www.isscr.org/visitor-types/public/stem-cell-glossary#embryoid

Epitope: The surface portion of an antigen capable of eliciting an immune response and of combining with the antibody produced to counter that response. http://medical-dictionary.thefreedictionary.com/epitope

Feeder cell:Inactivated (non-dividing by radiation or chemically) mouse embryonic fibroblasts (MEFs) provide structural support and release of growth factors for optimal growth of stem cells.

Good, Bad, Ugly Blog

Fibroblasts: Skin fibroblasts are a critical cell type of the skin; they produce and organize the extracellular matrix of the skin and communicate with each other and other cell types. Skin fibroblasts play a crucial role in regulating skin physiology (Sorrell, J.M., and Caplan, A.I. (2004)). Fibroblast heterogeneity: more than skin deep. Journal of cell science 117, 667-675). The fibroblasts have characteristic morphological features of elongated, spindle-like cell bodies, round to oval cell nuclei, and the fibroblasts grow aligned and in bundles when confluent (Vangipuram, M., Ting, D., Kim, S., Diaz, R., and Schule, B. (2013). Skin punch biopsy explant culture for derivation of primary human fibroblasts. Journal of visualized experiments : JoVE, e3779).

Start A Cell Culture Blog

The Force: is a metaphysical, spiritual, binding, omnipotent and ubiquitous power that holds enormous importance for both the Jedi and Sith monastic orders. "Well, the Force is what gives a Jedi his power. It's an energy field created by all living things. It surrounds us and penetrates us; it binds the galaxy together. " Obi-Wan Kenobi. http://starwars.wikia.com/wiki/The_Force
Star Wars Blog

The Good, the Bad and the Ugly: Italian Western film directed by Sergio Leone, starring Clint Eastwood, Lee Van Cleef, and Eli Wallach in the title roles respectively.The screenplay was written by Age & Scarpelli, Luciano Vincenzoni and Leone. http://en.wikipedia.org/wiki/The_Good,_the_Bad_and_the_Ugly

Good, Bad, Ugly Blog

Han Solo is a character in the Star Wars movies. Han Solo is portrayed by George Lucas as "a loner who realizes the importance of being part of a group and helping for the common good". He and his co-pilot, Chewbacca, become involved in the Rebel Alliance which opposes the Galactic Empire. During the course of the Star Wars story, he becomes a chief figure in the Alliance and succeeding galactic governments. http://en.wikipedia.org/wiki/Han_Solo
Star Wars Blog

Immunostaining: the staining of a specific substance by using an antibody against it which is complexed with a staining medium (as horseradish peroxidase) http://www.merriam-webster.com/medical/immunostaining 

Human embryonic stem (hES) cells: Embryonic stem cells are derived from embryos that develop from eggs that have been fertilized and then donated for research purposes with informed consent of the donors.("Definition of Differentiation". MedicineNet. MedicineNet, 28 Aug. 2013.

Induced pluripotent stem (iPS) cells: Embryonic stem cell-like cells that were reprogrammed from adult differentiated cells, e.g. skin, blood. The iPS cells have the potential to differentiate into any cell type of the human body. ("What Are Induced Pluripotent Stem Cells? [Stem Cell Information]. National Institute of Health)

Growing Out of Clothes Blog

Karyotype: To arrange and classify the chromosome complement of an organism based on the number, size, shape, and other characteristics of the chromosomes. http://www.biology-online.org/dictionary/Karyotype

Keratinocytes: Keratinocytes are specialized cells that  are predominantly found in the outermost layer of the skin, called epidermis. The primary function of keratinocytes is the formation of a barrier against environmental damage (McGrath JA, Eady RAJ, Pope FM. (2004). "Anatomy and Organization of Human Skin". In Burns T, Breathnach S, Cox N, Griffiths C. Rook's Textbook of Dermatology (7th ed.). Blackwell Publishing. doi:10.1002/9780470750520.ch3. ISBN 978-0-632-06429-8).
Start A Cell Culture Blog

Liquid nitrogen: Liquefied form of nitrogen at an extremely low temperature. Nitrogen boils at −195.79 C (77 K; −320 F) and can cause rapid freezing on contact with tissue. It is used in cryotherapy for treating potentially malignant skin lesions or for the cryopreservation of biological samples such as cells or tissues. http://en.wikipedia.org/wiki/Liquid_nitrogen

Star Wars Blog

Neubauer chamber: The counting chamber or also called hemocytometer is a device used to count cells. It consists of a thick glass microscope slide with a rectangular indentation that creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. http://insilico.ehu.es/counting_chamber/neubauer_improved.php

Good, Bad, Ugly Blog

Nuclear or cellular reprogramming: Technology to turn adult differentiated cells into stem cell-like cells using a factors that modulate the expression in the cell nucleus.

Growing Out of Clothes Blog

Orangina: Orangina is a carbonated citrus beverage made from orange, lemon, mandarin, and grapefruit juices and containing orange pulp. The concept of Orangina originated at a trade fair in France and was first marketed in Algeria by the French Algerian Léon Beton. It is a popular beverage in Europe and to a much lesser extent in North America. http://en.wikipedia.org/wiki/Orangina
Orangina Blog

Philz Coffee: A local California coffee house whose mission is "to better people's day. http://www.philzcoffee.com/

Coffee blog

Pluripotent: Not fixed as to developmental potentialities; capable of differentiating into one of many cell types http://www.merriam-webster.com/medical/pluripotent

Rejuvenation: The process of making young again; illustrated in “Fountain of Youth” by Lucas Cranach the Elder (1472-1553)
Rejuvenation Blog

Skin Biopsy: A procedure in which a sample of skin tissue is removed, processed, and examined ("Skin Biopsy.". WebMD.). A skin biopsy can be performed to diagnose unusual skin growth, such as a mole. It is a typically done under local anesthesia with a small pen-like instrument.
Start A Cell Culture Blog

Split (aka Passaging, Subculture): A procedure that enables for further propagation of cell line through the removal of the medium and transfer of cells from a previous culture into fresh growth medium. "Guidelines for Maintaining Cultured Cells". Life Technologies
Growing Out of Clothes Blog

Starbucks: Coffee chain whose motto is "Our mission to inspire and nurture the human spirit – one person, one cup, and one neighborhood at a time." http://www.starbucks.com/

Coffee blog

Substantia Nigra: An area in the midbrain which normally includes cells which contain melanin, a byproduct of dopamine synthesis. Parkinson’s disease occurs due to the lack of a dopamine transmitter, causing the degeneration of melanin-containing cells in the Nigra. In deeply affected parkinsonian brains, the band of melanin is absent from the Substantia Nigra. http://www.neuroanatomy.wisc.edu/virtualbrain/BrainStem/20Substantia.html

Trypsin: An enzyme found in the digestive system that hydrolyses proteins. For cell culture, trypsin is used to detach adherent cells from the culture dish during cell harvesting. "Trypsin-EDTA (0.25%)", Stem Cell Technologies.
Growing Out of Clothes Blog

Starting a skin cell culture

Two days ago, Alex donated a small piece of his skin. We are now growing and expanding skin cells from his biopsy and will turn them into stem cells.

Alex: What’s the brown area in the upper left corner of the picture?

Birgitt: This is a piece of your skin how it looks under a microscope. When I took the biopsy two days ago, I used a small instrument that looks like a pen to cut your skin, about the size of a small pea. Later in the lab, I cut the piece into 15 little pieces, which I then transferred to a tissue culture vessel.
We actually have a video showing this process in more detail
http://www.youtube.com/watch?v=pNNL5St3Yhs.

Alex:  Interesting, what else do I see on the picture? There is a rim around the skin tissue with little dots. What are those dots?

Birgitt:  Those dots are cells growing out of the skin biopsy and attach to the bottom of the culture vessel. These cells are called keratinocytes. Soon, we will observe another cell type also known as fibroblasts. These fibroblasts are the cells that we will turn into stem cells.

Alex:  Wow, those are my cells, how exciting! How many skin cells are you growing from me?

Birgitt: In four to six weeks, we will have grown about 20 Million of your skin cells. The journey of your skin cells has begun.

Introduction to Skin Cell Blog

We will tell you the story of how skin cells are turned into stem cells from a patient with young onset sporadic Parkinson’s disease. His name is Alex. Alex has volunteered to have a skin biopsy taken. As we progress, we will explain to him how his cells are becoming a model for Parkinson’s disease, to better understand the causes of neurodegeneration.

We will take you on the “journey” from a skin biopsy, how it grows, how it gets coaxed into becoming stem cells. We will also find out how the skin-derived stem cells are characterized and how the stem cells will become dopamine producing neurons.

What is Parkinson's disease? Parkinson’s disease is a multifactorial disease. Genetic and environmental causes have been reported and many pathways in the neurons can be compromised. There is no one type of Parkinson’s disease. Therefore, it is critical to understand the cause in every Parkinson’s patient when we want to find better therapies and potentially a cure.

Patient-derived stem cell models are powerful tools to understand disease and to test drugs. These cells models have a human genetic background, mimic the neuronal cell type that is most susceptible in Parkinson’s disease. These cells are derived from patients with Parkinson's disease.

We are very excited about this project. Alex’s motto is ‘keep moving’. Please keep moving with us on this journey. Stay tuned.

Lauren, Jessica, and Birgitt

About Lauren and Birgitt

Lauren Pijanowski got her Master’s in Biotechnology  and undergraduate degree at San Jose State University (SJSU) in Forensic Science. As a graduate student she joined the CIRM sponsored Stem Cell Internship in Laboratory based Learning program which prepared her with a broad understanding of stem cell biology through lectures, seminars, and in-depth laboratory work.
She also attended an intensive human stem cell workshop at Stanford University prior to beginning her internship at the Parkinson’s Institute. The goal of her work is to take skin cells, restore them to the stem cell state and use these stem cells to study Parkinson’s disease and the mechanisms of this disease with a more personalized approach to research.

Birgitt Schuele is an Associate Professor and Director of Gene Discovery and Stem Cell Modeling at the Parkinson’s Institute in Sunnyvale, California. She heads the neurogenetics program, which includes clinical genetic studies of families and sporadic cases with parkinsonism.

The Schuele team applies novel stem cell technologies to generate stem cells from patients with Parkinson’s disease. These skin-derived human stem cells build the foundation for further studies in the culture dish. The stem cells can be differentiated into neurons, specifically neurons that produce and release dopamine resembling the neurons that die in the brain of Parkinson’s patients. This unique human cellular model allows replicating conditions as in the human brain. These neurons are also electrically active and can ”fire” action potentials.

Parkinson’s research has been hampered for a long time because no pre-clinical model could faithfully replicate human disease. Also autopsy tissues from brain donors only have limited use as the disease process has already destroyed most of the dopamine neurons in the brain.

With these novel stem cell-derived neuronal model systems, living human dopamine neurons can be manipulated and studied for changes between patients and controls. This modeling approach has great promise for advancing science and discovery by having the tools to study early changes in the disease process as well as understanding environmental exposure which are the basis for drug screening.

For more information about The Schuele Team at The Parkinson's Institute, visit www.thepi.org

About Alex

Alex was diagnosed with Parkinson’s disease five years ago at age 36. He is a lawyer, ultra athlete, film producer, writer and father of three boys. It all started with a little shaking of his right hand. Parkinson’s disease has changed Alex’s life. He was on an uphill path in his successful career as a lawyer with an attitude that life has many more opportunities and challenges to come.

Parkinson’s disease is now his biggest challenge but it has also given him a new direction. He is raising awareness and funds for Parkinson’s disease that gives many of his fellow sufferers hope and inspiration to keep moving http://www.alexflynn.co.uk .

We don’t know why Alex got the disease so early and we currently don’t have a therapeutic strategy that allows us to treat the cause of his disease and not just his symptoms. The journey of Alex’s skin cells is a personalized approach to find the cause for Parkinson's disease.

Parkinson’s disease can have many different faces: some forms are slowly progressing and the disease has only a minor impact on daily activities for decades, other forms are more aggressive and within a few years patients are bedridden. Patients also respond very differently to the therapies and the type of drugs and the dosage needs to be carefully adjusted because of the changes in effectiveness of the medication over time.

We think that there is a significant need to develop approaches to realize a better understanding of what the underlying causes are in respect of each individual patient –a personalized approach.

Recent advancements in disease modeling allow us to now create patient derived dopamine producing neurons from skin cells; similar to the ones dying in a patient’s brain.

We will take you on a journey to develop a model of Parkinson's disease in a dish from a little piece of skin. Alex has donated a skin biopsy for our stem cell program. We have grown and have banked about 20 Million cells of his skin. Follow us and learn more about the disease and the novel individualized research that lays the foundation for causal treatments.