A group of Japanese researchers is at the forefront of the quest to grow complete organs in the lab.
In the latest of a series of remarkable studies, researchers from the RIKEN Center for Developmental Biology in Kobe, Japan report that embryonic stem cells grown under special conditions can spontaneously organize themselves into a partial pituitary gland that is fully functional when transplanted into mice.
|Embryonic stem cells self-organize themselves into functional pituitary gland. Image: Yoshiki Sasai, RIKEN Center for Developmental Biology. |
Over the past four years, Yoshiki Sasai and his colleagues of RIKEN's Organogenesis and Neurogenesis Group have developed a novel cell culture technique for growing embryonic stem (ES) cells in floating three-dimensional aggregates. In their new study, published recently in Nature, they used it to grow mouse ES cells and then altered the culture conditions to mimic the chemical environment that gives rise to the adenohypophysis, or anterior pituitary.
Development of the adenophyophysis requires interactions between two different types of tissue – the oral ectoderm, which goes on to form the skin inside the mouth, and the hypothalamic neuroectoderm, which generates a small structure called the hypothalamus. These interactions cause a region of the oral ectoderm to invaginate, or fold in on itself, and then detach itself to form a small sac called Rathke's pouch.
Cells within Rathke's pouch then continue to differentiate further, generating the six different types of hormone-producing cell found in the mature adenohypophysis. Each one synthesizes a different hormone, under the control of the hypothalamus; the hormones are secreted into the blood stream and perform a wide range of critical functions, including the regulation of growth and blood pressure, metabolism and sex organ function.
ES cells recapitulated this process when grown in 3D cultures and treated with the appropriate signalling molecules. First, they differentiated into oral ectoderm and hypothalamic neuroectoderm cells, and separated themselves into two separate but adjacent layers. Cells at the interface between the two layers then spontaneously formed oval-shaped structures resembling Rathke's pouch. When treated with additional signalling molecules, cells inside the pouch then differentiated into four different types, each of which synthesized and secreted a different hormone.
"ES cells produced both oral ectoderm and hypothalamic tissues in 3D culture," says Sasai. "When both are induced together, they spontaneously form layers back to back by self-organization. When they are in contact, signals from hypothalamic tissues act on oral ectoderm to induce Rathke's pouch. It takes about two weeks to develop Rathke's pouch and a total of three weeks for hormone-producing pituitary."
The researchers then transplanted into the kidneys of mice whose pituitary glands had been surgically removed. Normally, the hormone deficiency would cause the animals to die about two months after surgery. But the lab-grown pituitaries rescued the animals, by restoring the hormones to their normal levels.
In 2008, Sasai's group showed that ES cells grown in 3D cultures can recapitulate the earliest stages of neural development to self-organize into functional brain tissue, which integrated into existing neural circuits when transplanted into the brains of newborn mice. And earlier this year, they reported ES cells can also generate embryonic eyes with retinas.
Growing complete, fully functional organs for transplantation is the holy grail of regenerative medicine, one which is being pursued by many groups of researchers around the world. This usually involves using a synthetic scaffold that guides the formation of the tissue. Researchers from University College London and the Karolinska Institute in Stockholm, recently grew a synthetic windpipe using such a scaffold, and then successfully transplanted it into a patient whose own cancerous windpipe had been removed.
Sasai's group is at the forefront of these efforts. Their work shows that ES cells can spontaneously form complex three-dimensional structures when grown under the right conditions, in the absence of a scaffold. With each new study, they demonstrate the generation of increasingly complex structures, and the pituitary gland is the most complex one yet.
The work builds on decades of research into the molecular mechanisms by which embryonic tissues are specified to differentiate into different tissue types, and then into specific types of cells. But brain development is an incredibly complex process, involving much more than just chemical signals. Huge numbers of immature neurons jostle within the developing neural tissue, undergoing changes in shape and mass migrations that cause the embryonic brain to fold in on itself multiple times in different ways at different locations.
These processes are tightly co-ordinated, and give rise to the characteristic folded structure of the cerebral cortex. Yet, we still have very little understanding of the mechanical forces involved and, indeed, Sasai and his colleagues do not know exactly how ES cells self-organize themselves into such complex 3D structures.
Growing a complete, functioning brain is unfeasible, but there is real potential in growing functional neural tissue containing specific types of cells, for transplantation into the human brain. This is one avenue of research that the team are investigating. Last year, they showed that ES cells can be coaxed to differentiate into functional cerebellar Purkinje cells, which integrate themselves into the brain when transplanted into mouse foetuses.
"We are now attempting to generate Purkinje cells from human ES and induced pluripotent stem cells," says Sasai. This would be useful in establishing animal models for the spino-cerebellar ataxias, to investigate their pathogenesis and to explore the possibility of gene therapy for these diseases."
The 3D culture technique could also be used to grow midbrain tissue containing neurons that synthesize the neurotransmitter dopamine, which could eventually prove to be very useful in the development of therapies for Parkinson's Disease.
"It is a gradual process," says Sasai, "but regenerative medicine is proceeding into this direction. The self-organizing technology of 3D ES cell culture should be applicable to many other types of organs, and we are now using computer-based modelling and simulations to facilitate the design of more complex structures."
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2. Eiraku, M., et al (2011). Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature, 427, 51-56. doi: 10.1038/nature09941
3. Eiraku, M., et al (2008). Self-Organized Formation of Polarized Cortical Tissues from ESCs and Its Active Manipulation by Extrinsic Signals. Cell Stem Cell, 3: 519-532. DOI: 10.1016/j.stem.2008.09.002
4. Mugurama, K., et al (2010). Ontogeny-recapitulating generation and tissue integration of ES cell–derived Purkinje cells. Nat. Neurosci, 13: 1171–1180. DOI: 10.1038/nn.2638
Souce: The Guardian