Scientists Grow Small Chunks of Brain Tissue From Induced Pluripotent Stem Cells

Induced pluripotent stem cells are made from adult cells by means of genetic engineering techniques that introduce into the cells a combination for four different genes that drive the cells to de-differentiate into a cell that has many of the characteristics of embryonic stem cells without the destruction of embryos.

A new study from the laboratory of Juergen Knoblich at the Institute of Molecular Biotechnology in Vienna has mixed induced pluripotent stem cells (iPSCs) to form structures of the human brain. He largely left the cells alone to allow them to form the brain tissue, but he also placed them in a spinning bioreactor that constantly circulates the culture medium and provides nutrients and oxygen to the cells. One other growth factor he supplied to the cells was retinoic acid, which is made by the meninges that surround our brains. All of this and the cells not only divided, differentiated and assembled, but they formed brain structures that had all the connections of a normal brain. These brain-like chunks of tissue are called “mini-brains” and the recent edition of the journal Nature reports their creation.

“It’s a seminal study to making a brain in a dish,” says Clive Svendsen, a neurobiologist at the University of California, Los Angeles. Svendsen was not involved in this study, but wishes he was. Of this study, Svendsen exclaimed, “That’s phenomenal” A fully formed artificial brain is still years and years away, but the pea-sized neural clumps developed in Knoblich’s laboratory could prove useful for researching human neurological diseases.

Researchers have previously used pluripotent human stem cells to grow structures that resemble the developing eye (Eiraku, M. et al. Nature 472, 51–56 (2011), and even tissue layers similar to the cerebral cortex of the brain (Eiraku, M. et al. Cell Stem Cell 3, 519–532 (2008). However, this latest advance has seen bigger and more complex neural-tissue clumps by first growing the stem cells on a synthetic gel that resembled natural connective tissues found in the brain and elsewhere in the body. After growing them on the synthetic gel, Knoblich and his colleagues transferred the cells to a spinning bioreactor that infuses the cells with nutrients and oxygen.

“The big surprise was that it worked,” said Knoblich. The clump formed structures that resembled the brains of fetuses in the ninth week of development.

Under a microscope, the blobs contained discrete brain regions that seemed to interact with one another. However, the overall arrangement of the different proto-brain areas varied randomly across tissue samples. These structures were not recognizable physiological structures.

A cross-section of a brain-like clump of neural cells derived from human stem cells.
A cross-section of a brain-like clump of neural cells derived from human stem cells.

“The entire structure is not like one brain,” says Knoblich, who added that normal brain maturation in an intact embryo is probably guided by growth signals from other parts of the body. The tissue balls also lacked blood vessels, which could be one reason that their size was limited to 3–4 millimeters in diameter, even after growing for 10 months or more.

Despite these limitations, Knoblich and his collaborators used this system to model key aspects of microcephaly, which is a condition that causes extremely stunted brain growth and cognitive impairment. Microcephaly and other neurodevelopmental disorders are difficult to replicate in rodents because the brains of rodents develop differently than those of humans.

Knoblich and others found that tissue chunks cultured from stem cells derived from the skin of a single human with microcephaly did not grow as large as clumps grown from stem cells derived from a healthy person. When they traced this effect, they discovered that it was due to the premature differentiation of neural stem cells inside the microcephalic tissue chunks, which depleted the population of progenitor cells that fuels normal brain growth.

The findings largely confirm prevailing theories about microcephaly, says Arnold Kriegstein, a developmental neurobiologist at the University of California, San Francisco. But, he adds, the study also demonstrates the potential for using human-stem-cell-derived tissues to model other disorders, if cell growth can be controlled more reliably.

“This whole approach is really in its early stages,” says Kriegstein. “The jury may still be out in terms of how robust this is.”

Microparticles and Local Control of Stem Cells

Using stem cells to grow three-dimensional structures, such as organs or damaged body parts, requires that scientists have the ability to control the growth and behavior of those cells. Also, adapting such a technology to an off-the-shelf kind of process so that it does not cost an arm and a leg is also important.

A research project by scientists from Atlanta, Georgia has used gelatin-based microparticles to deliver growth factors to specific areas of aggregates of stem cells that are differentiating. This localized delivery of growth factors provides spatial control of cell differentiation, which enables the creation of complex, three-dimensional tissues. The local delivery of growth factors also decreases the amount of growth factor used and, consequently, the cost of the procedure.

This particular microparticle technique was used on mouse embryonic stem cells and it proved to provide better control over the kinetics of cell differentiation since it delivered that promote cell differentiation or inhibit it.

Todd McDevitt, associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, said, “By trapping these growth factors within microparticle materials first, we are concentrating the signal they provide to the stem cells. We can then put the microparticle materials physically inside the multicellular aggregate system that we use for differentiation for the stem cells. We have good evidence that this technique can work, and that we can use it to provide advantages in several areas.”

The differentiation of stem cells is largely controlled by external cues, including protein growth factors that direct cell proliferation, and differentiation that are available in the three-dimensional environment in which the cells live. In most experiments, stem cells are grown in liquid culture and growth factor is equally accessible to the growth factors. This makes the cultures quite homogeneous. But delivering the growth factors via microparticles gives better control of the spatial and temporal presentation of these growth factors to the stem cells. This gives scientists the means to make heterogeneous structures from stem cell cultures.

When embryonic stem cells grow in culture, they tend to clump together. When the growth medium is withdrawn or if growth factors that induce differentiation are added, the cells form an “embryoid body” that is stuff with cells differentiating into all kinds of cell types. When McDevitt and his co-workers added microparticles with the growth factors BMP4 (bone morphogen protein 4) or Noggin (which inhibits BMP4 signaling), they centrifuged the cells and found that the microparticles found their way into the interior of the embryoid bodies.

When they examined the embryoid bodies, with confocal microscopy they found that BMP4 directed the cells to make mesodermal and endodermal derived cell types. However, because the microparticles were in direct contact with the cells, they needed 12 times less growth factor than was required by solution-based techniques.

“One of the major , in a practical sense, is that we are using much less growth factor,” said McDevitt. “From a bioprocessing standpoint, a lot of the cost involved in making stem cell products is related to the cost of the molecules that must be added to make the stem cells differentiate.”

Beyond more focuses signaling, the microparticles also provided localized control that was not available through other techniques. It allowed researchers to create spatial differences in the aggregates and this is an important possible first step toward forming more complex structures with different tissue types such as vascularization and stromal cells.

“To build tissues, we need to be able to take stem cells and use them to make many cell types which are grouped together in particular spatial patterns,” explained Andres M. Bratt-Leal, the paper’s first author and a former graduate student in McDevitt’s lab. “This spatial patterning is what gives the ability to perform higher order functions.”

Once the stem cell aggregates were made and treated with growth factor-endowed microparticles, McDevitt and his colleagues saw spheres of cells with differentiating cells.

“We can see the microparticles had effects on one population that were different from the population that didn’t have the particles,” said McDevitt. “This may allow us to emulate aspects of how development occurs. We can ask questions about how tissues are naturally patterned. With this material incorporation we have the ability to better control the environment in which these cells develop.”

The microparticles could provide better control over the kinetics of cell differentiation; slowing it down with molecules that antagonize differentiation or speed up with other molecules that promote stem cell differentiation.

Despite the fact that McDevitt and his colleagues used mouse embryonic stem cells in this paper, he and his co-workers are already testing this technology on human embryonic stem cells, and the results have been comparable.

“Our findings will provide a significant new tool for tissue engineering, bioprocessing of stem cells and for better studying early development processes such as axis formation in embryos,” said Bratt-Leal. “During development, particular tissues are formed by gradients of signaling molecules. We can now better mimic these signal gradients using our system.”

Clinical-Scale NK Cells for Cancer Therapy Made from Pluripotent Stem Cells

Dan Kaufman’s laboratory has done it again. The Kaufman laboratory at the University of Minnesota in collaboration with scientists from MD Anderson Cancer Center in Houston, Texas have designed a protocol to make natural killer cells from embryonic stem and induced pluripotent stem cells.

Natural killer cells provide a very important contribution to the innate immune response. These cells produce molecules called cytokines and they also kill virally infected cells and malignant cells. NK cells are unique among the cells of the immune system in that they have the ability to recognize foreign, infected or stressed cells in the absence of antibodies and Major Histocompatibility Complex proteins (the cell surface proteins that act as bar codes used by the immune system uses to determine if a cell is yours or not yours). Therefore, NKs typically work faster than the rest of the immune system.

Natural killer cells or NK cells have been used to treat patients with refractory cancers. Unfortunately, a major problem with using NK cells is growing a sufficient quantity of cells for therapy. Using pluripotent stem cells to make NK cells is an intriguing possibility, but the protocols for differentiating NK cells from embryonic stem cells (ESCs) is tedious and inefficient. However, the Kaufman laboratory has provided a much more efficient and straight-forward way to derive NK cells, thus allowing for the production of clinical scale quantities of NK cells.

The Kaufman lab protocol involves first deriving embryoid bodies from ESCs or induced pluripotent stem cells (iPSCs), which are made from adult cells through genetic engineering techniques that causes the cells to de-differentiate into ESC-like cells known as iPSCs. Embryoid bodies are three-dimensional aggregates of pluripotent stem cells that assume a kind of spherical shape and have a variety of cells differentiating into a wide range of cell types. Embryoid bodies can contain beating heart muscle, neural-type cells, blood progenitors cells, and even muscle or bone cells in their interiors in a haphazard arrangement. Forming embryoid bodies or EBs from cultured ESCs or iPSCs is rather easy, but controlling the differentiation of the cells in the EBs is quite another matter.

embryoid bodies
embryoid bodies

Kaufman and others discovered that if the EBs were incubated with artificial antigen-presenting cells that expressed a surface-bound version of the protein IL21 (interleukin 21) plus a cocktail of cytokines, these pluripotent stem cells could efficiently form NK cells.

Functional assays of the NK cells differentiated from ESCs and iPSCs easily showed that the NK cells for functional in every way and expressed all the cell surface molecules characteristic of NK cells. Furthermore, all ESC and iPSC lines examined were able to make NK cells, but the efficiency with which they made them different rather widely.

In conclusion, Kaufman and others state in their paper, “our ability to now produce large numbers of cytotoxic NK cells means that prospect hESC- and iPSC-derived hematopoietic products for diverse clinical therapies can be realized in the not-too-distant future.” For some cancer patients, that day cannot come soon enough.