Among various biotechnology inventions of the last few years with potential to revolutionize medicine, nothing excites us more than growing of three- dimensional human organoids on matrigel. Therefore, we plan to devote a number of posts on this topic to keep our readers aware of the practices, potentials and challenges.
If you have not heard of organoids at all, the following few news articles will help you get started. In future posts, we plan to cover the primary literature in depth, but let us start with the popular media.
There are many diseases that attack specific organs, landing patients on a transplant list. Unfortunately, our bodies have markers that identify an organ as self, which makes it difficult to find an organ match. Many individuals die waiting for an organ transplant because a match can’t be found.
Research on stem cellsa type of cell that is able to transform into nearly any cell typehas raised hopes of treating organ failure. Researchers envision using these cells to grow fully functional organs.
A functional organ is similar to a machine. Organs contain many interacting parts that must be positioned in a specific configuration to work properly. Getting all the right cell types in the appropriate locations is a real challenge. Recently, a team of scientists has met that challenge by using stem cells to grow a tissue, termed an organoid, that resembles a developing kidney.
Scientists at The Ohio State University have developed a nearly complete human brain in a dish that equals the brain maturity of a 5-week-old fetus.
The brain organoid, engineered from adult human skin cells, is the most complete human brain model yet developed, said Rene Anand, professor of biological chemistry and pharmacology at Ohio State.
The lab-grown brain, about the size of a pencil eraser, has an identifiable structure and contains 99 percent of the genes present in the human fetal brain. Such a system will enable ethical and more rapid and accurate testing of experimental drugs before the clinical trial stage and advance studies of genetic and environmental causes of central nervous system disorders.
An investigation on zika has shown that it modifies the function of the molecule and causes a TLR3 cell ‘suicide’ in the brain. The experiment conducted in laboratory brains, seeks to reduce the aggressiveness of infection, resulting in microcephaly fetuses, using an inhibitor. Early results indicate that cells infected by the virus decreased by 16% in five days.
What is an organoid?
Organoids are tiny organs grown in the lab on petri-dish.
How are they grown?
Usually the researcher starts with pluripotent stem cells and applies a series of enzymes over several days. The experiment is done in a special material (e.g. Matrigel) that allows the growth to take place in three-dimension. Over time, the stem cell divides and develops specific organs depending on the chosen enzymes.
For example, here is the ‘recipe’ to grow human lungs, based on the 2015 paper
- “In vitro generation of human pluripotent stem cell derived lung organoids”. Let us quickly explain what is going on and then you can read the actual methods.
There are three steps explained in highly simplified form.
(i) The researchers start with pluripotent stem cells (PSC) and apply Activin A for 3 consecutive days. This process turns PSCs into definitive endoderm.
(ii) The researchers apply Noggin and SB431542 for another 4 days to turn definitive endoderm into anterior foregut.
(iii) The researchers apply Noggin, SB431542 and FGF4 for another 4 days to turn anterior foregut into lung organoid. This part of the experiment is done in Matrigel so that the tissue can maintain its three-dimensional pattern.
Differentiation of PSCs into definitive endoderm
Differentiation into definitive endoderm was carried out as previously described (D’Amour et al., 2005; Spence et al., 2011). Briefly, a 4-day Activin A (R&D; systems, Minneapolis, MN) differentiation protocol was used. Cells were treated with Activin A (100 ng ml?1) for 3 consecutive days in RPMI 1640 media (Life Technologies, Grand Island, NY) with increasing concentrations of 0%, 0.2% and 2% HyClone defined fetal bovine serum (dFBS, Thermo Scientific, West Palm Beach, FL).
Differentiation of definitive endoderm into anterior foregut
After differentiation into definitive endoderm, foregut endoderm was differentiated, essentially as described (Green et al., 2011). Briefly, cells were incubated in foregut media: Advanced DMEM/F12 plus N-2 and B27 supplement, 10 mM Hepes, 1 L-Glutamine (200 mM), 1 Penicillin-streptomycin (5000 U/ml, all from Life Technologies) with 200 ng/ml Noggin (NOG, R&D; Systems) and 10 M SB431542 (SB, Stemgent, Cambridge, MA) for 4 days. For long term maintenance, cultures were maintain in basal foregut media without NOG and SB, or in the presence of growth factors including 50, 500 ng/ml FGF2 (R&D; systems), 10 M Sant-2 (Stemgent), 10 M SU5402 (SU, Stemgent), 100 ng/ml SHH (R&D; systems), and SAG (Enzo Life Sciences, Farmingdale, NY) for 8 days.
Directed differentiation into anterior foregut spheroids and lung organoids
After differentiation into definitive endoderm, cells were incubated in foregut media with NOG, SB, 500 ng/ml FGF4 (R&D; Systems), and 2 M CHIR99021 (Chiron, Stemgent) for 46 days. After 4 days with treatment of growth factors, three-dimensional floating spheroids were present in the culture. Three- dimensional spheroids were transferred into Matrigel to support 3D growth as previously described (McCracken et al., 2011). Briefly, spheroids were embedded in a droplet of Matrigel (BD Bioscience #356237) in one well of a 24 well plate, and incubated at room temperature for 10 min. After the Matrigel solidified, foregut media with 1% Fetal bovine serum (FBS, CAT#: 16000044, Life Technologies) or other growth factors and small molecules were overlaid and replaced every 4 days. Organoids were transferred into new Matrigel droplets every 1015 days.
To procedure is similar for other organs except for the series of enzymes being selected. If the steps appear too mysterious, do not worry. We will cover the scientific details in a future post.
What are the medical potentials of the organoid technology?
One can take tissue from any person, turn it back into pluripotent stem cells (iPSC) and then grow that person’s organs from the iPSC. That means it is possible to check whether a drug will work or have bad effect on one person’s brain or kidney before applying the drug on the person himself. That marks the arrival of truly personalized medicine and that too without the person !
To understand how it all started, you need to go back to stem cell pioneer Shinya Yamanaka.
Shinya Yamanaka discovered more than 40 years later, in 2006, how intact mature cells in mice could be reprogrammed to become immature stem cells. Surprisingly, by introducing only a few genes, he could reprogram mature cells to become pluripotent stem cells, i.e. immature cells that are able to develop into all types of cells in the body.
Continue reading here.