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.”

Using Bone Marrow Stem Cells to Reprogram Neurons and Regenerate the Retina

Spanish researchers from the Center for Genomic Regulation (CGR) have regenerated the retina in mice by reprogramming neurons with bone marrow stem cells.

Cell reprogramming normally uses genetic engineering techniques that introduces genes into cells that push them into another cell fate without taking them through an embryonic-like state. One strategy for reprogramming cells fuses those cells with other cells that express genes that drive the fused cell into a different cell fate.

Pia Cosma and her team have used cell fusion to reprogram retinal neurons in mice. The mechanism consisted of introducing bone marrow stem cells into the damaged retina. The transplanted stem cells fused with existing retinal neurons, which conveyed to these retinal neurons the ability to regenerate the retina.

“For the first time we have managed to regenerate the retina and reprogram its neurons through in vivo cell fusion. We have identified a signaling pathway that, once activated, allows the neurons to be reprogrammed through their fusion with bone marrow cells,” said Pia Cosma, who is the head of the Reprogramming and Regeneration group at the CGR and ICREA (Institució Catalana de Recerca i Estudis Avançats) research professor.

Daniela Sanges, first author or the work and postdoctoral researcher in Pia Cosma’s laboratory, said, “This discovery is important not only because of the possible medical applications for retinal regeneration but also for the possible regeneration of other nervous tissues.”

The study demonstrates that the regeneration of nervous tissue by means of cell fusion is possible in mammals and describes this new technique as a potential mechanism for the regeneration of more complex nervous tissue.

This research is in the very early stages but already there are laboratories interested in being able to continue the work and take it to a more applied level.

Daniela Sanges, Neus Romo, Giacoma Simonte, Umberto Di Vicino, Ariadna Diaz Tahoces, Eduardo Fernández, Maria Pia Cosma. Wnt/β-Catenin Signaling Triggers Neuron Reprogramming and Regeneration in the Mouse Retina . Cell Reports – 25 July 2013 (Vol. 4, Issue 2, pp. 271-286)

Tests to Improve Stem Cell Safety

Stem cell scientists from the Commonwealth Scientific and Industrial Research Organisation or CSIRO (the Australian version of the NIH) have developed a test to identify unsafe pluripotent stem cells that can potentially cause tumors. This test is one of the first tests specifically designed for human induced pluripotent stem cells or iPSCs.

The development of this test marks a significant breakthrough in improving the quality of iPSCs and identifying unwanted stem cells that can form tumors. The test also directly assesses the stability of iPSCs when they are grown in the lab.

Andrew Laslett and his team have spent the last five years working on this research project and perfecting their test.

Laslett explained: “The test we have developed allows us to easily identify unsafe iPSC cells. Ensuring the safety of these cell lines is paramount and we hope this test will become a routine screen as part of developing safe and effective iPS-based cell therapies.”

Laslett’s research focused on comparing different types of iPS cells with human embryonic stem cells. Induced pluripotent stem cells are, at this time, the most commonly used type of pluripotent stem cell in research.

Laslett’s method has established that iPSCs made in certain ways are inherently less stable and riskier than those made by alternative means. For example, the classical way of making iPSCs, with genetically engineered retroviruses that insert their genes into the chromosomes of the cells they infect, can cause insertional mutations and are inherently more likely to cause tumors. In comparison, iPSCs made with viruses that do not integrate into the host cell’s DNA (that is, with genetically engineered adenoviruses), or made with plasmid DNA, mRNA or modified proteins, do not form tumors.

Laslett hopes the study and the new test method will help to raise the awareness and the importance of stem cell safety. He also predicts that tests like his will promote a kind of quality control over the production of iPSC lines.

“It is widely accepted that iPS cells made using viruses should not be used for human treatment, but they can also be used in research to understand diseases and identify new drugs. Having the assurance of safe and stable cells in all situations should be a priority,” said Laslett.

This test utilizes laser technology that activates fluorescent dyes attached to antibodies that are bound to specific cell surface proteins.  If the cell has the cell surface protein bound by the antibody, the cell and its surface proteins fluoresce, and it is sent into the positive test tube.  If it does not fluoresce, it is sent to the negative test tube.  This technique is called fluorescence activated cell sorting or FACS.  In order to identify proteins found the surfaces of iPSCs, Laslett’s team used dye-conjugated antibodies that bound to surface proteins TG30 (CD9) and GCTM-2.  The presence of these specific cell-surface proteins provides a means to separate cells into safe and unsafe cell lines.  Very early-stage differentiated stem cells that expressed TG30 (CD9) and GCTM-2 on their cell surfaces tend to dedifferentiate into pluripotent cells after differentiation and cause tumors, whereas those very early-stage differentiation stem cell lines that do not express TG30 (CD9) and GCTM-2 on their cell surfaces do not cause tumors.  After separation of the stem cell lines by FACS, the iPSC lines were further monitored as they grew in culture.  Unsafe iPS cell lines that form tumors usual clump together to make recognizable clusters of cells.  However, the safe iPS cell lines do no such thing. This test can also be applied to somatic cell nuclear transfer human embryonic stem cells.

Professor Martin Pera, the Program Leader of Stem Cells, Australia said, “Although cell transplantation therapies based on iPS cells are being fast tracked for testing in humans, there is still much debate in the scientific community over the potential hazards of this new technology.”

Engineered Mesenchymal Stem Cells Make Blood Vessels that Help Heal Ailing Hearts

Another term for a heart attack is a myocardial infarction (MI). A heart attack or an MI occurs when the blood supply to the heart that flows through coronary blood vessels is interrupted. The interruption of blood flow deprives the heart of nourishment and oxygen, and the downstream blood vessels and heart muscle die as a result. The decrease in blood vessel density after a MI can increase cell death, which increases the amount of cell death and the size of the heart scar. Therefore, growing more blood vessels in the heart after a heart attack, which is known as therapeutic angiogenesis, is a potentially strategy in treating an MI (see Ziebart T, et al., (2008) Circ Res 103: 1327–1334)..

To this end, a few clinical trials have attempted to used stem cells that can make blood vessels to reverse heart damage caused by an MI (see Ripa RS, et al. (2007) Circulation 116: I24–I30 and Schachinger V, et al. (2006) N Engl J Med 355: 1210–21).

Among those therapeutic agents for heart attack patients, mesenchymal stem cells (MSCs) are considered excellent candidates. MSCs have the ability to differentiate into smooth muscle, or blood vessels, which means that they can help revascularize the heart after a MI. The problem with MSCs is their tendency to die off rapidly after transplantation into the heart after a heart attack (see Ziegelhoeffer T, et al. (2004) Circ Res 94: 230–38 & O’Neill TT, et al., Circ Res 97: 1027–35; & Perry TE, et al. (2009) Cardiovasc Res 84: 317–25).

To fix this problem, MSCs can be either preconditioned before implantation (see previous posts) or genetically engineered to withstand the hostile conditions inside the heart after a heart attack.

Previously, Muhammad Ashraf and Yigang Wang from the University of Cincinnati genetically engineered MSCs to express a surface protein called CXCR4.  CXC4R is the receptor for a chemokine known as CXCL12/SDF-1.  SDF-1 is a rather potent stem cell recruitment molecule.

When transplanted into the hearts of rodents that had just experienced a heart attack, MSCs that expressed CXCR4 showed increased mobilization and engraftment into the damaged areas of the heart. Also, the pumping abilities of the heart regions into which the MSC-CXCR4s were infused increased, and the MSC-CXCR4 cells cranked up their secretion of blood vessel-inducing growth factors (vascular endothelial growth factor-A or VEGF-A), This led to increased formation of new blood vessels and a decrease in the early signs of left ventricular remodeling (see Zhang D, et al. (2010) Am J Physiol Heart Circ Physiol 299: H1339– H1347; Huang W, et al. (2010) J Mol Cell Cardiol 48: 702–712; &.Zhang D, et al. (2008) J Mol Cell Cardiol 44: 281–292). While these papers show truly stunning results, it was still, even after all this work, unclear if the MSCs were actually differentiating into blood vessel cells and making blood vessels.

To nail this down, Wang and his group used a clever little technique. They engineered MSCs to express CXCR4 and the viral TK gene. TK stands for “thymidine kinase,” which is an enzyme involved in nucleotide synthesis from a virus. The TK enzyme is not found in human cells, and is therefore a target for antiviral drugs. If treated with antiviral drugs that target the TK enzyme, only cells with the TK gene will be killed.

When Wang and his group used their CXCR4-engineered MSCs to treat the heart of mice that had recently suffered a heart attack, they found that their hearts improved and that these same heart were covered with new blood vessels. However, when this experiment was repeated with CXCR4-MSCs that also had the TK gene, Wang his co-workers fed the mice a drug called ganciclovir, which kills only those cells that possess the TK gene. In these mice, their heart failed to improve and also were completely devoid of the new blood vessels.

This paper nicely shows that without viable MSCs, no new blood vessels were made. This strongly suggests that the engineered MSCs are differentiating into blood vessel cells and making new blood vessels, which helps the heart recover from the heart attack and shrinks the size of the dead area of the heart.

What are the implications for human clinical trial\? This is difficult to say. Before clinical trials with genetically engineered cells are approved those cells will need to go through piles of safety tests before they can be used in clinical trials. Once that hurdle is passed, then they can be used in human clinical trials, and they will certainly prove efficacious for human patients.

Direct Conversion of Skin Cells into Neural Precursor Cells

Cell reprogramming involves the use of genetic engineering techniques to push cells into a new cell type WITHOUT passing those cells through the embryonic stage. Several different studies have shown that transferring particular genes into specific cell types or removing distinct genes from them can drive them to become other cell types. There are several published examples of transdifferentiation:
1) In 1989, Weintraub and colleagues overexpressed a gene called MyoD in cultured fibroblasts to convert them into muscle cells. Unfortunately, this conversion was incomplete and required continuous expression of MyoD (Weintraub H et al., Proc. Natl. Acad. Sci. USA 1989;86:5434-8).
2) Tachibana and colleagues overexpressed a gene called MITF to transdifferentiate fibroblasts into pigment-synthesizing melanocytes (Tachibana et al., Nature Genetics 1996;14:50-4).
3) Xie and others overexpressed genes that encode two transcription factors (C/EBP and PU.1) in B cells, T cells, and fibroblasts into transdifferentiated them into cells that looked like macrophages (Xie et al., Cell 2004;117:663-76).
4) Deletion of a gene called Pax5 can transdifferentiate antibody-secreting B lymphocytes into common lymphoid progenitors, macrophages and antigen-presenting T cells (Cobaleda C, Jochum W, and Busslinger M. Nature 2007;449:473-7).
5) Doug Melton’s laboratory at Harvard University transferred a specific combination of three transcription factor genes (Ngn3, which is also known as Neurog3, Pdx1 and Mafa), into pancreatic exocrine cells (those cells that produce and secrete digestive enzymes).  This reprogrammed the cells into insulin-secreting beta cells (Qiao Zhou et a., In vivo reprogramming of adult pancreatic exocrine cells to β-cells. Nature 2008;455, 627-632).
6) Deletion of a gene that encodes a transcription factor called Foxl2 converts granulosa and thecal cells (found in the ovary) into Sertoli and Leydig cells, which are found in the testes (Uhlenhaut et al., Cell 2009;139:1130-42).
7) Thomas Vierbuchen and colleagues in the laboratory of Marius Wernig at Sanford University School of Medicine used a combination of three genes (Asc1, Brn2 and Myt1l) to convert fibroblasts into functional neurons (Vierbuchen et al., Nature 2010;463:1035-42).
8) In 2010, Ieda and co-workers in the laboratory of Deepak Srivastava have used ectopic expression of three genes (GATA4, MEF2C, and TBX5) to directly convert heart-based fibroblasts into heart muscle cells. These reprogrammed cells did not require expression of the introduced transgenes (Ieda et al., Cell 2010;142:375-86).

A recent study has extended these results even further. In an earlier study, Marius Wernig’s lab at Stanford University School of Medicine showed that skin fibroblasts can be transdifferentiated into functional neurons. Wernig’s lab has followed up in a paper that was published online on Jan. 30, 2012 in the Proceedings of the National Academy of Sciences.

In this study, Wernig’s lab used mouse skin cells and directly transdifferentiated them into the three main parts of the nervous system. These transdifferentiation experiments show that pluripotency (a term that describes the ability of stem cells to become nearly any cell in the body) is NOT necessary for a cell to transform from one cell type to another. Together, these results raise the possibility that embryonic stem cell research and induced pluripotency could be superseded by a more direct way of generating specific types of cells for therapy or research.

In the new study, Wernig and his colleagues converted fibroblasts in to neural precursor cells (NPCs). NPCs have the capacity to differentiate into neurons, but they can also become the two other main cell types in the nervous system: astrocytes and oligodendrocytes. In addition to their greater versatility, newly derived NPCs offer another advantage over neurons because they can be cultivated to large numbers in the laboratory — a feature critical for their long-term usefulness in transplantation or drug screening..

The switch from skin cells to NPCs occurred with high efficiency and only took about three weeks after the addition of just three transcription factors. Wernig’s research group used a different combination of three transcription factors than those used to generate mature neurons (Brn2, Sox2 and FoxG1) than was used to generate mature neurons. This combination of transcription factors drove the fibroblasts to transdifferentiate into “tripotent” NPCs that have the ability to form neurons and astrocytes but also into oligodendrocyte. The finding implies that it may one day be possible to generate a variety of neural-system cells for transplantation that would perfectly match a human patient.

The lab’s previous success with transdifferentiation experiments led Wernig to wonder if his lab could convert skin-based fibroblasts into the more-versatile NPCs. To do so, Wernig’s research group infected embryonic mouse skin cells — a commonly used laboratory cell line — with a virus that encoded 11 transcription factors known to be expressed at high levels in NPCs. Just over three weeks later, about 10 percent of the cells began to look and act like NPCs.

They then winnowed down the original panel of 11 transcription factors to just three that still converted fibroblasts to NPCs. Three of these genes (Brn2, Sox2 and FoxG1; in contrast, the conversion of skin cells directly to functional neurons requires the transcription factors Brn2, Ascl1 and Myt1l.) drove fibroblasts to differentiate into NPCs that were “tripotential” – that is, the NPCs could differentiate into not just neurons and astrocytes, but also oligodendrocytes, which make myelin that insulates nerve fibers and allows them to effectively transmit nerve impulses. Wernig’s lab workers dubbed the newly converted population “induced neural precursor cells,” or iNPCs.

In vitro experiments showed that the astrocytes, neurons and oligodendrocytes made from iNPCs expressed the same genes and morphologically resembled that they resembled astrocytes, neurons and oligodendrocytes found in living organisms. However, Wernig’s lab wanted to know how iNPCs would react when transplanted into an animal. Therefore, they injected them into the brains of newborn laboratory mice that were bred to lack the ability to myelinate neurons. After 10 weeks, they found that the injected cells had differentiated into oligodendroytes and had begun to coat the animals’ neurons with myelin.

Marius Wernig, MD, assistant professor of pathology and a member of Stanford’s Institute for Stem Cell Biology and Regenerative Medicine, said: “We are thrilled about the prospects for potential medical use of these cells. We’ve shown the cells can integrate into a mouse brain and produce a missing protein important for the conduction of electrical signal by the neurons. This is important because the mouse model we used mimics that of a human genetic brain disease. However, more work needs to be done to generate similar cells from human skin cells and assess their safety and efficacy.”

Pediatric cardiologist Deepak Srivastava, MD, who was not involved in these studies noted, “Dr. Wernig’s demonstration that fibroblasts can be converted into functional nerve cells opens the door to consider new ways to regenerate damaged neurons using cells surrounding the area of injury. It also suggests that we may be able to transdifferentiate cells into other cell types.” Srivastava is the director of cardiovascular research at the Gladstone Institutes at the University of California-San Francisco. In 2010, Srivastava’s lab transdifferentiated mouse heart fibroblasts into beating heart muscle cells.

The first author of this article, Ernesto Lujan, added: “Direct conversion has a number of advantages. It occurs with relatively high efficiency and it generates a fairly homogenous population of cells. In contrast, cells derived from iPS cells must be carefully screened to eliminate any remaining pluripotent cells or cells that can differentiate into different lineages.” Pluripotent cells can cause cancers when transplanted into animals or humans.

“Not only do these cells appear functional in the laboratory, they also seem to be able to integrate appropriately in an in vivo animal model,” said Lujan.

Wernig’s group is now working to replicate the work with skin-based fibroblasts from adult mice and humans, but Lujan emphasized that more research is needed before any human transplantation experiments could be conducted. Until that time, the ability to quickly and efficiently generate NPCs that can be grown in the laboratory to mass quantities and maintained over time will be valuable in disease and drug-targeting studies.

“In addition to direct therapeutic application, these cells may be very useful to study human diseases in a laboratory dish or even following transplantation into a developing rodent brain,” said Wernig.