#mini-brain

Building organs for repair or replacement. Image by Barbara Gilhooly

Regenerative Medicine.  What does that phrase mean?  We know that regeneration seems like a good thing…. invoking regenerative power, or regenerating tissue, that is, a renewal of something lost.  In high school biology we learned that salamanders (including newts and axolotls)  could lose a tail or a leg and it will grow back.  We are all pretty certain we don’t have the same ability. 

Reproduction of the Gaudi salamander Parc Guell de Barcelona

Even while scientists are trying to find why we can’t regrow lost limbs in the hope that we can learn how to actually make it happen, the phrase regenerative medicine has taken on a larger meaning.  In fact humans do have the ability to regenerate lost tissue but that ability is very limited. In disease our organs lose function often because they lose the cells that comprise them.  But organs also have special cells, cells of renewal, known as stem cells.   When disease kills cells in some organs the body tries to compensate by inducing these resident stem cells to repopulate the organ and reestablish function.  It is a very inefficient process for us humans.  Regenerative medicine then is the attempt to understand how to make the process of renewal of organ function more efficient.  

 Newt!  Image by Holger Krisp, Ulm, Germany

Stages of salamander limb regeneration.  At the 3 o’clock position is a picture of a normal limb before amputation.  Proceeding clockwise are pictures of the limb growing back, taking place over 30 days involving wound healing and dedifferentiation (return to embryonic state), then redevelopment of a normal limb.  Many genes are involved many of which are known and understood (genes identified by new gene expression technologies shown in the center). Image from Biology Open, Oct. 15, 2012, Monaghan et al. (doi: 10.1242/bio.20121594)

We know how to take any adult cell and by manipulating a few genes turn back the genetic clock of the cell so that it thinks its fate is not yet determined, that it can become (differentiate into) any type of cell and participate in the function of any organ. In other words become a stem cell. The use of these cells in bioengineering new tissue is a bold new science that aims for nothing less than the replacement of diseased organs with a patient’s own cells.

We have learned a great deal about how to do this in the more than 15 years since Japanese scientists first described how to turn an adult cell into a stem cell.  Biologists have many ways to study this process and its ramifications but one method particularly useful to regenerative medicine is with structures grown in a dish called organoids.  This is important as the cells used to create stem cells are initially obtained from adults, no embryos involved.  The cells can be manipulated in the lab without creating moral dilemmas and without having to conduct the research on human patients.  By manipulating the stem cells while they are growing they can create structures remarkably like the organs we would like to induce to repair. Organoids are small, three dimensional structures that recapitulate the structure and function of organs.  They are simple, relatively inexpensive test beds for development, physiology and drug testing that allow the development of approaches in advance of animal modeling and allow investigators to cycle through many more iterations of approaches than would be practical with intact animals or with humans.

A striking example of this is the eye. By placing stem cells in a dish and providing them with environmental and genetic cues the cells develop a structure that recapitulates the embryonic eye cup and begin to turn into retinal cells and self assemble in the dish into morphologically recognizable retina. The structures develop photoreceptors that can respond to light with electrical signals showing they are electrophysiologically functional.  Currently human trials are starting to determine if they can cure or ameliorate serious retinal diseases.

Eye structure developed from stem cells in a dish!  Closely resembles a normal embryonic eye.  © 2011 Yoshiki Sasai

When eyes grow in a dish from stem cells the retina organizes (top picture) with similar anatomy and gene expression as normal retinal development (bottom picture, red is Pax6).  Images from Manuela Völkner, et al., Stem Cell Reports. 2016 Apr 12; 6(4): 525–538; 10.1016/j.stemcr.2016.03.001

Similar remarkable results have been obtained for the intestine, prostate and the kidney.  But most remarkable of all is the ability to create little mini-brains in a dish that reveal many normal morphological and patterning similarities to developing brain.  These organoids allow formal investigations of physiology, function, toxicity, and even to test therapeutic approaches to diseases.

Dish with cerebral organoids comprising neural cells differentiated from stem cells, i.e. mini-brains!  Insert: microscopic view of histological section through a cerebral organoid showing gene expression, cortical patterning, and ventricle-like vesicles all reminiscent of normal embryonic brain development. © 2017 Magdalena Renner, Madeline A Lancaster, Shan Bian,Heejin Choi,Taeyun Ku,Angela Peer, 1 Kwanghun Chung,  and  Juergen A Knoblich; EMBO J. 2017 May 15; 36(10): 1316–1329.

As if this weren’t enough very recently scientists have managed to combine two types of stem cells (derived from the two types of cells in very early embryos) in a dish with a matrix that supports 3-D growth and to develop a normal looking and behaving mouse embryo!  This embryo, at what is known as the blastocyst stage, expresses normal genes in the normal place and at the correct time when compared with natural mouse embryos.  This is stunning confirmation of the remarkable ability of stem cells to self organize and direct normal development. This was most recently followed by demonstration that by adding another type of stem cell into the mix synthetic embryos could go through gastrulation, a critical early step of embryonic development, showing the astounding ability of these cells to communicate with each other and self organize into a developing embryo.

Synthetic mouse embryo with embryonic (pink) and extraembryonic (blue) cells. These structures are remarkably like the normal mouse embryo at the time of implantation.  They proceed to mature with normal patterning steps and gene expression in time and place. Zernicka-Goetz lab, University of Cambridge

The ability to create models of the brain and intact embryos in a dish from individual cells and potentially from humans, as in the case of mini-brains should provoke profound ethical concerns.  The dilemmas presented by these capabilities are obvious but have attracted relatively little attention.  Given what is at stake this is remarkable for it would be far better to thoroughly consider ramifications of this technology now.

On the other hand, as scientists learn more about these amazing abilities the cells and the conditions under which they are grown will provide an incredible future for the field of regenerative medicine. We will have in our grasp the tools needed to repair damaged organs with the patient’s own cells!

New Post has been published on http://www.newsnish.com/technology/science/an-accessible-approach-to-making-a-mini-brain/

An accessible approach to making a Mini-Brain

If you need a working miniature brain — say for drug testing, to test neural tissue transplants, or to experiment with how stem cells work — a new paper describes how to build one with what the Brown University authors say is relative ease and low expense. The little balls of brain aren’t performing any cogitation, but they produce electrical signals and form their own neural connections — synapses — making them readily producible testbeds for neuroscience research, the authors said.

“We think of this as a way to have a better in vitro [lab] model that can maybe reduce animal use,” said graduate student Molly Boutin, co-lead author of the new paper in the journal Tissue Engineering: Part C. “A lot of the work that’s done right now is in two-dimensional culture, but this is an alternative that is much more relevant to the in vivo [living] scenario.”

Just a small sample of living tissue from a single rodent can make thousands of mini-brains, the researchers said. The recipe involves isolating and concentrating the desired cells with some centrifuge steps and using that refined sample to seed the cell culture in medium in an agarose spherical mold.

The mini-brains, about a third of a millimeter in diameter, are not the first or the most sophisticated working cell cultures of a central nervous system, the researchers acknowledged, but they require fewer steps to make and they use more readily available materials.

“The materials are easy to get and the mini-brains are simple to make,” said co-lead author Yu-Ting Dingle, who earned her Ph.D. at Brown in May 2015. She compared them to retail 3-D printers which have proliferated in recent years, bringing that once-rare technology to more of a mass market. “We could allow all kinds of labs to do this research.”

The spheres of brain tissue begin to form within a day after the cultures are seeded and have formed complex 3-D neural networks within two to three weeks, the paper shows.

25-cent mini-brains

There are fixed costs, of course, but an approximate cost for each new mini-brain is on the order of $0.25, said study senior author Diane Hoffman-Kim, associate professor of molecular pharmacology, physiology and biotechnology and associate professor of engineering at Brown.

“We knew it was a relatively high-throughput system, but even we were surprised at the low cost per mini-brain when we computed it,” Hoffman-Kim said.

Hoffman-Kim’s lab collaborated with fellow biologists and bioengineers at Brown — faculty colleagues Julie Kauer, Jeffrey Morgan, and Eric Darling are all co-authors — to build the mini-brains. She wanted to develop a testbed for her lab’s basic biomedical research. She was interested, for example, in developing a model to test aspects of neural cell transplantation, as has been proposed to treat Parkinson’s disease. Boutin was interested in building working 3-D cell cultures to study how adult neural stem cells develop.

Morgan’s Providence startup company, MicroTissues Inc., makes the 3-D tissue engineering molds used in the study.

The method they developed yields mini-brains with several important properties:

Diverse cell types: The cultures contain both inhibitory and excitatory neurons and several varieties of essential neural support cells called glia. Electrically active: the neurons fire and spike and form synaptic connections, producing complex networks. 3-D: Cells connect and communicate within a realistic geometry, rather than merely across a flat plane as in a 2-D culture. Natural density: Experiments showed that the mini-brains have a density of a few hundred thousand cells per cubic millimeter, which is similar to a natural rodent brain.

Physical structure: Cells in the mini-brain produce their own extracellular matrix, producing a tissue with the same mechanical properties (squishiness) as natural tissue. The cultures also don’t rely on foreign materials such as scaffolds of collagen.

Longevity: In testing, cultured tissues live for at least a month.

Hoffman-Kim, who is affiliated with the Brown Institute for Brain Science and the Center for Biomedical Engineering, said she hopes the mini-brains might proliferate to many different labs, including those of researchers who have questions about neural tissue but not necessarily the degree of neuroscience and cell culture equipment required of other methods.

“If you are that person in that lab, we think you shouldn’t have to equip yourself with a microelectronics facility, and you shouldn’t have to do embryonic dissections in order to generate an in vitro model of the brain,” Hoffman-Kim said.

The paper’s other authors are Anda Chirila, Liane Livi, Nicholas Labriola, and Lorin Jakubek.