LIVER BUD PRODUCTION FROM HUMAN PLURIPOTENT STEM CELLS
By Lauren Smart
Technology Summary
- Liver buds (LBs) are produced in a U-bottom-shaped microwell plate, which is made of a fine film, and placed on a glass slide to be observed
- There are approximately twenty thousand microwells per dish, each with a very high aspect ratio (the ratio of width to height of the microwell, specifically 500μm diameter and 400μm depth)
- The wells are arranged triangularly and closely together (30μm apart)
- The LB culture is produced by mixing three cells, all of which are derived from human induced pluripotent stem cells (hiPSCs):
- Hepatic endoderm cells (HECs)
- Human umbilical cord vein endothelial cells (HUVECs)
- Bone marrow mesenchymal stem cells (BMSCs)
Background:
https://www.cell.com/cell-reports/pdf/S2211-1247(17)31625-X.pdf
https://www.yokohama-cu.ac.jp/en/community/medcenter/
From Cells to Liver Buds
- hiPSCs must be removed from the subject, induced, and coaxed into their “final form” either as HECs, HUVECs, or BMSCs
- The three cells are self-organizing, such that they will form three dimensional LBs by repeating interactions to form the organ during the development
- These interactions occur between the HUVECs and BMSCs
https://www.sciencedirect.com/science/article/pii/S1934590915001150
- Research occurred at the Yokohama City University in Japan
- The paper was published by a Japanese research team in 2013
- Organoid technology has been around for the past decade
- The term organoid refers to a three dimensional cell culture that has some key functions of the full organ
https://www.cincinnatichildrens.org/-/media/cincinnati%20childrens/home/research/divisions/c/custom/organoid-cycle-300x300.jpg?hash=A7155CEB2FD139E6699509141B541FC217E518CB&la=en
Liver Bud Culture
- The optimal culture was determined to be after 8 days, where the expression signature is the most similar to a human adult liver tissue cell
- An expression signature is the result of a biological process or condition, where one or multiple genes has a unique pattern of gene expression
- 100 MILLION LBs are produced in a single production batch
https://www.cell.com/cell-reports/pdf/S2211-1247(17)31625-X.pdf
Benefits
1. hiPSCs come directly from the patient, so the developed liver cells are a genetic match, eliminating tissue matching and rejection problems in transplants
2. Self-organization approach can be applicable to other organs such as lungs, pancreas, and kidney
3. Stem cells used are not harvested from embryos
4. Creates hope for the possibility to generate virtually any organ for a patient from their own cells
More Genetics
http://dev.biologists.org/content/develop/142/18/3113/F2.large.jpg?download=true
Disadvantages
1. Use of postnatal tissue cells (HUVECs and BMSCs) could help streamline the process, and would could be easier to obtain
2. It will take ten years before the possibility of human clinical trials in adults and in full transplants
3. hiPSCs can be traditionally difficult to obtain
4. Risk of inserting mutation, possibly cancer, when developing hiPSCs
https://i2.wp.com/www.cocoona.in/wp-content/uploads/2016/09/skin_biopsy.gif?resize=242%2C257&ssl=1
Genetics
- Modification to histone proteins and DNA methylation
- Histone: protein around which DNA is wrapped in the nucleus
- DNA methylation: process of adding methyl groups to a molecule of DNA
- Changes gene expression in the chromatin structure similar to the embryonic stage
- When combining the cells together, they begin to form LBs similar to liver production in a developing embryo
- The role of the nucleus/DNA/genes is relatively unknown in self-organizing three dimensional structures
Social Controversies
- The genetics of production lie in the development of hiPSCs
- Pluripotent stem cells are referred to as master cells
- Can possibly produce any cell required by the body to repair its organs
- The process involves taking an ordinary cell, usually a skin cell, and reprogramming it by introducing several genes that convert it into a pluripotent cell
- Hence it is an induced pluripotent cell
Effectiveness
https://www.rndsystems.com/resources/articles/differentiation-potential-induced-pluripotent-stem-cells
http://placentadonor.org/how-to-donate/
1. Use of postnatal tissue cells (HUVECs and BMSCs)
2. Significant animal and human testing will be required before it can become a reliable treatment, often requiring induced liver failure in animals
3. Demand for human donors rather than lab grown organs, one may be considered socially superior to the other
4. Cost of developing hiPSCs, LB cultures, or fully functional organs
5. Playing God by creating new organs
https://undsci.berkeley.edu/article/_0_0/sciencetoolkit_06
Induced Pluripotency
Ethical Implications
https://www.cell.com/cell-reports/pdf/S2211-1247(17)31625-X.pdf
Works Cited
Anwar, et al. “Pluripotent Stem Cells to Hepatocytes, the Journey so Far (Review).” Molecular Medicine Reports,
Spandidos Publications, 1 Apr. 2017, www.spandidos-publications.com/10.3892/br.2017.867.
“Boston Children's Hospital.” Boston Childrens Hospital,
stemcell.childrenshospital.org/about-stem-cells/pluripotent-stem-cells-101/.
“Differences between in Vitro, in Vivo, and in Silico Studies.” Marshall
Protocol Knowledge Base, mpkb.org/home/patients/assessing_literature/in_vitro_studies.
“Gene Signature.” Wikipedia, Wikimedia Foundation, 14 May 2018,
en.wikipedia.org/wiki/Gene_signature.
“Miniature Human Liver Grown in Mice.” Nature News, Nature Publishing
Group, www.nature.com/news/miniature-human-liver-grown-in-mice-1.13324.
“New Liver Made from Stem Cells.” NewsComAu, 3 July 2013,
www.news.com.au/lifestyle/health/japans-yokohama-city-university-grows-new-human-liver-made-from-stem-cells-inside-mouse/news-story/6eabcd8c7aca9a49cb4e937df35332ff.
“Organoid Research.” Stemcell Technologies, www.stemcell.com/
technical-resources/area-of-interest/organoid-research.html.
“Scientists Have Created Bioengineered Mini Livers from Stem Cells.”
Daily Accord, 25 Sept. 2017, dailyaccord.com/scientists-have-created-bioengineered-mini-livers-from-stem-cells/.
Takebe, Takanori, et al. “Vascularized and Functional Human Liver from
an IPSC-Derived Organ Bud Transplant.” Nature News, Nature Publishing Group, 3 July 2013, www.nature.com/articles/nature12271.
Takebe, Takanori et al. “Massive and Reproducible Production of Liver
Buds Entirely from Human Pluripotent Stem Cell.” Cell Reports , Volume 21 , Issue 10 , 2661 - 2670. www.cell.com/cell-reports/pdf/S2211-1247(17)31625-X.pdf
Technology Networks. “Reprogramming and IPSC Formation - Then and
Now.” Neuroscience from Technology Networks, Technology Networks, 30 Jan. 2018, www.technologynetworks.com/cell-science/articles/reprogramming-and-ipsc-formation-then-and-now-296977.
“Transduction (Genetics).” Wikipedia, Wikimedia Foundation, 1 June
2018, en.wikipedia.org/wiki/Transduction_(genetics).
Image Sources
www.yokohama-cu.ac.jp/en/community/medcenter/
www.cincinnatichildrens.org/-/media/cincinnati%20childrens/home/research/divisions/c/custom/organoid-cycle-300x300.jpg?hash=A7155CEB2FD139E6699509141B541FC217E518CB&la=en
www.cell.com/cell-reports/pdf/S2211-1247(17)31625-X.pdf
www.pinterest.com/pin/400609329326487267/
www.sciencedirect.com/science/article/pii/S1934590915001150
www.sandiegouniontribune.com/business/biotech/sdut-liver-buds-pluripotent-2013jul03-story.html
www.alpfmedical.info/endothelial-cells/chronic-window-preparations.html
www.mdpi.com/1422-0067/19/4/936
www.uu.nl/sites/default/files/styles/image_385x257/public/images/uls_platform_for_organoid_technology.jpg?itok=EikZksFn×tamp=1517998482
en.wikipedia.org/wiki/Histone
en.wikipedia.org/wiki/DNA_methylation
dev.biologists.org/content/develop/142/18/3113/F2.large.jpg?download=true
i2.wp.com/www.cocoona.in/wp-content/uploads/2016/09/skin_biopsy.gif?resize=242%2C257&ssl=1
placentadonor.org/how-to-donate/
undsci.berkeley.edu/article/_0_0/sciencetoolkit_06
www.animal-ethics.org/animal-experimentation-introduction/
https://www.researchgate.net/figure/Model-for-the-generation-of-iPS-cells-by-the-retroviral-transduction-of-four-genes-It-is_fig1_23424081
1. There would need to be consent on behalf of parents to use postnatal tissue cells
2. Ethical concerns regarding chemically induced liver failure in animals as a part of test...the age old question: is medical testing on animals ethical?
3. Patient’s right to choose a cultured organ transplant versus a human donor organ
4. Technology could be misused by those who can afford it as an excuse to mistreat their liver (alcohol)
5. Process of hiPSCs is debatably more ethical than embryonic stem cells from eggs/embryos
- In order to induce pluripotency, specifically as a stem cell, four proteins were discovered to be necessary for reprogramming:
- OSKM: OCT4, SOX2, KLF4, and cMYC are the four proteins initially used for inducing pluripotency
- Inserted into adult human dermal fibroblasts (cells traditionally used for induced pluripotency) via RETROVIRAL TRANSDUCTION
- A retrovirus is a group of RNA viruses that target the host cell and insert the DNA copy of their complete set of DNA in order to be reproduced
- Virus uses reverse transcriptase enzyme to produce its complementary DNA strand
- Risk of inserting mutation or causing cancer, as seen in animals
- DNA strand is added to the host’s genome via an integrase enzyme
- Host cell adopts the DNA strand as its own and transcribes and translates the DNA until the cell is completely infected
- Grafted livers had full functional capability of the adult organ
- LB tissue performs liver-specific functions without full liver replacement
- Transplant trials were successful at minimum dosages, particularly in pediatric patients
- Requires further production scaling to produce successful results in metabolic disorders for animal testing
- The livers implanted in mice with induced liver failure prolonged their survival
- There is no current method or standard for the use of LBs in humans
https://en.wikipedia.org/wiki/DNA_methylation
http://www.animal-ethics.org/animal-experimentation-introduction/
https://en.wikipedia.org/wiki/Histone
Liver Bud Grafts
Liver Grafts in Crainial Window
https://www.cell.com/cell-reports/pdf/S2211-1247(17)31625-X.pdf
- Some livers were implanted into the cranial windows of mice, which were replaced with a glass cover, to observe the connection of the LBs to the circulatory system
https://www.alpfmedical.info/endothelial-cells/chronic-window-preparations.html
Potential Uses & Applications
http://www.sandiegouniontribune.com/business/biotech/sdut-liver-buds-pluripotent-2013jul03-story.html
"Blood vessels in liver buds, in green, connect with mouse blood vessels (blue), within 48 hours after implantation."
http://www.mdpi.com/1422-0067/19/4/936
https://www.uu.nl/sites/default/files/styles/image_385x257/public/images/uls_platform_for_organoid_technology.jpg?itok=EikZksFn×tamp=1517998482
1) Drug Testing: there are endless possibilities when it comes to drug testing and the developed LBs
- Effectiveness of medications
- Possible side effects for medications on liver
2) Regenerative Applications: transplants of liver cells cells have already produced clinical success
3) Organ Transplant: future uses could include generating fully functional organs for transplantation
- The immature cells were then developed into a miniature adult human liver after being grafted into a mouse
- Grafted livers were observed to engage in liver-specific functions, including protein production and drug metabolism