Stem Cells to Treat Spinal Cord Injury

Journal Club: Intravenous multipotent adult progenitor cell treatment decreases inflammation leading to functional recovery following spinal cord injury

Written by DePaul, et al. in the journal Scientific Reports. The complete article is here.

Background: Stem cells are unique in that they can divide indefinitely and can turn into multiple different types of cells (see image below).  For example, during human development, embryonic stem cells have the ability to turn into all of the different cell types throughout the body – such as liver cells, lung cells, and skin cells. Embryonic stem cells are called pluripotent stem cells for this reason.  Stem cells have also been found in adults, but they are usually able to turn into only one or several types of cells (called multipotent stem cells).  Scientists have thought that this ability for stem cells to continually grow and turn into different cell types could be harnessed to treat a number of diseases that involve cell death, including spinal cord injury.

Stem cells - wikipedia

By OpenStax College [CC BY 3.0], via Wikimedia Commons


The authors of this paper are interested spinal cord injury (SCI).  When the spinal cord (the bundle of nerves that goes down your back) is injured when something disrupts the vertebrae (like it being hit during a car accident) this can tear or push on the spinal cord.  This causes damage to the nerves and prevents electrical signals between the body and the brain from being transmitted properly, often resulting in paralysis. The authors were interested in studying whether stem cells could help reverse the damage that’s caused by this type of injury.

Results: In this publication, the authors isolate stem cells called multipotent adult progenitor cells (MAPCs) from the bone marrow. To break this down, “multipotent” means that the stem cells can turn into several different cell types. “Progenitor” cells are actually more specified than stem cells – stem cells can divide forever, but progenitor cells can only divide a certain number of times. “Adult” means that these cells are taken from the adult bone marrow.

Because you can’t just inject cells into humans with spinal cord injury (that would have to be a clinical trial WAY down the road once there is evidence that it works in animals), the authors use a “model” of spinal cord injury in rats. Technically – they crush the rat’s spine (eek).  In case you’re interested, here’s the device they use to do this. The rat then has decreased mobility and inability to urinate on it’s own – like what might happen in the case of a paralyzed person with a spinal cord injury.

The researchers injected multipotent adult progenitor cells into the rat after injury to see if they reverse these mobility and urination effects.  Interestingly, when MAPCs are injected into a rat vein the day of injury, nothing happened. However, if the MAPCs are injected 1 day after injury, the rats recover some mobility and the ability to urinate on their own compared to rats without treatment.

One might assume that the MAPCs do this by going to the injured area of the spinal cord and re-growing nerve cells.  However, the authors found that this wasn’t the case.  In fact, the MAPCs moved to the outside edges of the injury and even more to the spleen (see image below).  The spleen is where many of the body’s immune cells are stored. In spinal cord injury, the immune system is a double-edged sword.  The immune system cleans up the damage from the injury itself but also attacks the injury and makes more damage.  There is evidence from this paper that the MAPCs in the spleen decrease the damaging effect on the spinal cord injury from the immune cells in the spleen.

figure 6 SCI

Figure 6 from the paper. The green dots are labeled multipotent adult progenitor cells (MAPCs). They are labeled green so that you can see where they are located in the rat’s body.

Conclusion: This paper presents a promising result that provides hope for this type of therapy in spinal cord injury patients.

Journal Club – A mutation in mice to study ALS

Amyotrophic Lateral Sclerosis (ALS) is a debilitating neurodegenerative disease that affects neurons in the brain, brain stem, and spinal cord. When these neurons die, it affects the connections that they have to muscles throughout the body resulting in muscle weakness that affects speaking, swallowing and breathing leading to paralysis and eventual dead.  The cause of ALS isn’t known in 90-95% of cases.  However, recently scientists have identified a mutation in a gene nondescriptly called “chromosome 9 open reading frame 72,” which is abbreviated to C9ORF72.  This mutation results in six nucleotides, GGGGCC, being repeating up to 1000 times within this gene in ALS patients.  Even though this is the most common mutation found in ALS patients, it’s still unclear exactly how these repeats affect neurons or the progression of the disease. The two hypotheses that are most studied are that the mutated C9ORF72 makes mutated RNA transcripts (RNA is the molecule that usually helps DNA be translated into proteins) or makes unusual proteins called dipeptide repeat proteins (DRPs), each of which can aggregate (clump) together and disrupt the normal activity of the neurons leading to neurodegeneration.

neuron_als_c9ORF72

The pink dots show clumped up RNA from the many many repeats in the C9ORF72 gene. Each panel shows these clumps in different areas of the mouse brain. Taken from the article Peters et al. 2015 Neuron

In two recent publications in the journal Neuron, researchers have used mice as a model system to look at how these large GGGGCC repeats in C9ORF72 affect the mouse nervous system.  Why use a mouse? First, scientists know how to experimentally change the mouse genome in order to add hundreds or thousands of repeats to a single gene, like C9ORF72.  Second, mouse and human genes are about 85% identical, so if scientists can understand how a gene like the mutated C9ORF72 affects neurons in mice, it may also help scientists understand how it works in humans. Third, by creating a mouse “model” of ALS, scientists can use this model to better understand ALS and to test potential future therapies (it’s worth noting that only one drug currently exists for ALS and it typically extends life only by a few months).

In each article, the mouse model was slightly different – one had a mutated C9ORF72 with 500 GGGGCC repeats and the other varied between 100-1000 repeats. However, both sets of researchers found the same results.  The mice had aggregated RNA transcripts and DRPs in their neurons just like what are found in human patients with ALS, but none of the mice had behavioral changes or neurodegeneration that are seen in human ALS patients.  So why didn’t the clumped up RNA and proteins cause neurodegeneration in mice like they do in humans?  There are lots of potential reasons – including the fact that even though mice and humans are similar, there are still lots of differences and mice may respond to these aggregated RNA and proteins differently than humans.  However, the authors of these papers suggest that other environmental and/or genetic factors along with the aggregates caused by the C9ORF72 mutation must be involved in developing the neurodegeneration.  It may also mean that getting rid of these aggregates before neurodegeneration occurs may prevent development of ALS.  Now that these mice are available to study, they should help in identifying the other factors involved in ALS development along with developing possible treatments for this debilitating disease.

Want to read the articles?  Unfortunately, they are behind a paywall, but you can see the abstracts here:

O’Rourke et al. (2015) C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD. Neuron. Volume 88, Issue 5, p892–901, 2 December 2015 Article

Peters et al. (2015) Human C9OFR72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins but Not Neurodegeneration in BAC Transgenic Mice. Neuron. Volume 88, Issue 5, p902–909, 2 December 2015 Article

Read the Article from PN News that I contributed to here

The Cancer Genome Atlas Project (TCGA): Understanding Glioblastoma

TCGAIn 2003, Cold Spring Harbor Laboratory (CSHL) and researchers around the world celebrated the 50th Anniversary of the discovery of the structure of DNA by Jim Watson and Francis Crick.  I was a graduate student in the Watson School of Biological Science at CSHL, named after James Watson who was the chancellor of the CSHL, and in 2003, I participated in (and planned!) some of the 50th anniversary events. Coinciding with this celebration was a meeting about DNA that brought world-renowned scientists and Nobel Prize winners from around the world to CSHL to celebrate how much had been accomplished in 50 years (including sequencing the human genome) and to look to the future for what could be done next. That meeting was the first time I had heard about the Cancer Genome Atlas Project. At this point, the TCGA (as the project was affectionately called) was just a pipe dream – a proposal by the National Cancer Institute and the National Human Genome Research Institute (two institutes in the National Institutes of Health – the NIH).  The idea was to use DNA sequencing and other techniques to understand different types of cancer at the genome level. The goal was to see what changes are happening in these cancer cells that might be exploited to detect or treat these cancers.  I remember that there was a heated debate about whether or not this idea would work. I was actually firmly against it, but now with the luxury of hindsight, the scientific advances of the TCGA seem to be clearly worth the time and cost.

The first part of the TCGA started in 2006 as a pilot project to study glioblastoma multiforme, lung, and ovarian cancer. In 2009, the project was expanded, and in the end, the TCGA consortium studied over 33 cancer types (including 10 rare cancers).  All of the data that was made publically available so that any results could be used by any scientist to better understand these diseases. To accomplish this goal, the TCGA created a network of institutions to provide the tissue for over 11,000 tumor and normal samples (from biobanks including the one that I currently manage).  These samples were analyzed using techniques like Next Generation Sequencing and researchers used heavy-duty computing power to put all of the data together. So what did they find? This data has contributed to hundreds of publications, but the one I’m going to talk about today is the results from the analysis of the glioblastoma multiforme tumors.

Title: Comprehensive genomic characterization defines human glioblastoma genes and core pathways published in Nature in October 2008.

Authors: The Cancer Genome Atlas Network

gbmBackground: Glioblastoma is a fast-growing, high grade, malignant brain tumor​ that is the most common brain tumor found in adults.  The most common treatments are surgery​, radiation therapy​, and/or chemotherapy (temozolomide​). Researchers are also testing new treatments such as NovoTFF, but these have not yet been approved for regular use. However, even with these treatments the median survival for someone diagnosed with glioblastoma is only ~15 months.  At the time that this study was published, little was known about the genetic cause of glioblastoma – a small handful of mutations were known, but nothing comprehensive. Because of the poor prognosis and lack of understanding of this disease, the TGCA targeting it for a full molecular analysis.

Methods: The TCGA requested tissue samples from glioblastoma patients from biobanks around the country. They received 206 samples that were of good enough quality to use for these experiments.  143 of these also had matching blood samples.  Because the DNA changes in the tumor only happen in the tumor, the blood is a good source of normal, unchanged DNA to compare the tumor DNA to. To these samples, the study sites did a number of different analyses:

  • They looked at the number of copies of each piece of DNA. This is called DNA copy number, and copy number is often changed in tumor cells (see more about what changes in the number of chromosomes can do here)
  • They looked at gene expression.  The genes are what makes proteins, which do all of the stuff in your body.  If you have a mutation in a gene, it could change the protein so that it contributes to the development of cancer.
  • They also looked at DNA methylation.  Methylation is a mark that can be added to the DNA telling the cell to turn off that part of DNA.  If there is methylation on gene that normally stops a cell from growing like crazy, that methylation would turn that gene off and the cell could grow out of control.
  • In a subset of samples, they performed next generation sequencing to know the full sequence of the tumor genomes.

Results and Discussion: From all of this data, the researchers found  quite a bit.

  • Copy number results: There were many differences in copy number including deletions of genes important for slowing growth and duplications of genes the told the cell to grow more.
  • Gene expression results: Genes that are responsible for cell growth, like the gene EGFR, were expressed more in glioblastoma tumor cells.  This has proven to be an interesting result because there are drugs that inhibit EGFR.  These drugs are currently being tested in the clinic to see if this EGFR drug is a good treatment for patients with a glioblastoma that expresses a lot of EGFR.
  • Methylation results: They found a gene called MGMT that is responsible for fixing mutated DNA was highly methylated.  This mutation was actually beneficial to patients because it made them more sensitive to the most common chemotherapy, temozolomide.  However, this result also suggests that losing MGMT methylation may cause treatment resistance.
  • Sequencing results: From all of the sequencing they created over 97 million base pairs of data! They found mutations in over 200 human genes. From statistical analysis, seven genes had significant mutations including a gene called p53, which usually prevents damaged cells from growing, but when mutated the cell can more easily grow out of control
glioblastoma_pathways

This is the summary figure from this paper that shows the three main pathways changed in glioblastoma and the evidence they found to support these genes’ involvement. Each colored circle or rectangle represents a different gene. Blue means that the gene is deleted and red means that there is more of that gene in glioblastoma tumors.

Bringing all of this data together, scientists found three main pathways that lead to cancer in glioblastoma (see the image above for these pathways).  These pathways provide targets for treatment by targeting drugs to specific genes in these pathways. Scientists also identified a new glioblastoma subtype that has improved survival​. This is great for patients who find out that they have this subtype!  Changes in the methylation also show how patients could acquire resistance to chemotherapy. Although chemotherapy resistance is bad for the patient, understanding how it happens allows scientists to develop drugs to overcome the resistance based on these specific pathways.

Although this is where the story ended for this article, the TCGA data has been used for many more studies about glioblastoma.  For example, in 2010, TCGA data was used to identify four different subtypes of glioblastoma: Proneural, Neural, Classical, and Mesenchymal that have helped to tailor the type of treatments use for each group. For example proneural glioblastoma does not benefit from aggressive treatment, whereas other subtypes do. Other researchers are using the information about glioblastoma mutations to develop new treatments for the disease

To learn more about the Cancer Genome Atlas Project, check out this article “The Cancer Genome Atlas: an immeasurable source of knowledge” in the journal or watch this video about the clinical implications of the TCGA finding about glioblastoma

Journal Club: The Microbiome Autism Connection

As I’ve mentioned in other posts, scientists have to read about and understand the current scientific literature. Lots of the time this is done alone, at your desk in the office or the lab, for hours and hours so that you can really understand whatever topic it is that you’re studying. But one of my favorite ways to share scientific papers is through a weekly meeting with the whole lab called a Journal Club. Although my husband laughs about this kind of nerdy science “club” (akin to his amusement about scientific societies), it’s a great way to discuss a particular topic and dive deep into a discussion about how the researchers got their results and came to their conclusions. This is the first of many Journal Clubs where we will do an abbreviated version of what we would discuss in a typical journal club in the lab. 

Paper TItle:  Reduced Incidence of Prevotella and Other Fermenters in Intestinal Microflora of Autistic Children

Authors: Dae-Wook Kang , Jin Gyoon Park ,Zehra Esra Ilhan, Garrick Wallstrom, Joshua LaBaer, James B. Adams, Rosa Krajmalnik-Brown
Full disclosure, Dr. LaBaer is the Director of the center I previously worked in at the Biodesign Institute at ASU. Drs. Park and Wallstrom worked in offices down the hall from me and Dr. Krajmalnik-Brown was in another center at the Biodesign Institute.

Journal: PLOS One (PLOS stands for “Public Library of Science”). In case you want to read the whole article, it can be downloaded (for free) here

Background/Introduction: Before this paper was published, scientists knew that many children with autism also had gastrointestinal (GI) issues suggesting that there may be a connection between the two. There have been some studies looking at antibiotic treatment (which could change the gut microbiome) before 3 years of age and how this might be connected with autism.  There have also been studies connecting the gut microbiome and the brain. So there was evidence that the gut microbiome and autism may be related in some way.

When this paper was written, scientists also knew about the microbiome and how changes in the bacteria (all 1,000,000,000,000,000 of them) that are in the gut are found in patients with many different diseases – from C. diff infection to obesity to depression.

Goal of this paper: Look closely at the changes in the gut microbiome of children with autism to better understand how these two might be related.

Methods/what did they do?: Bacteria in fecal samples is considered representative of bacteria in the gut microbiome. Therefore, the researchers collected fecal samples from children both with (20 children) and without (20 children) autism.  The samples from patients without autism were used as a “control” to compare to the autistic samples. The researchers also asked the children (or their parents) questions to help determine the level of GI issues, the severity of their autism, and their environmental factors like their diet. The researchers isolated bacterial DNA from each of these fecal samples and then sequenced the DNA to determine what types of bacteria are in the gut.

autism microbiome diversity

A figure from the paper comparing the phylogenetic diversity (PD) of the bacteria in autistic versus non-autistic children. you can see that the red boxes (for autistic children) are lower than the blue boxes (for non-autistic children) indicating a lower microbiome diversity.

Results: Through sequence analysis and other statistical methods, the authors found that children who did not have autism have a more diverse microbiome compared to autistic children.  If there is higher diversity, it means that the gut contains more different types of bacteria, and lower diversity means a smaller variety of bacteria in the gut. They also found that in the autistic patients with a greater diversity in their microbiome, their autism was generally less severe. They also did not find any correlation between age, gender or diet with these microbiome changes.

The scientists also looked at what specific genus and species of bacteria were more represented in non-autistic versus autistic children. Specifically the bacteria from genus Veillonellacaea, Provetella, and Coprococcus  are less abundant in autistic children.

Discussion/Significance: What does this all mean?  The researchers did find a correlation between decreased gut microbiome diversity and autism. It should be clarified that just because GI problems are often found in autistic children and the severity of the GI issues correlates with the severity of autism, this does not necessary mean that GI issues cause autism or vice versa.  That still needs to be determined. Also because the diversity of bacteria in autistic children is low, it is not clear if this is a cause of autism or an effect of a child having autism.  However, this paper does provide a “stepping stone” to better understand what is happening in the gut of autistic children and may help define a target for diagnosing autism (by looking at the decreased diversity in the gut as a diagnostic test) or treatment (perhaps through fecal transplant).

What has been done since? This paper was published in 2013.  So what has changed since the paper was published?  Do we know whether or not changes in the gut microbiome cause autism or not?  Unfortunately, this is still unclear.  However, if these microbiome changes are a cause of the neurological changes in autism, then one would want to do a clinical trial to test what happens to autism symptoms when the microbiome has been altered.  This could be done in a number ways including diet modulations, prebiotics, probiotics, synbiotics, postbiotics, antibiotics, fecal transplantation, and activated charcoal.  Researchers have started this process by holding a meeting that included patients and their families to figure out how this type of trial could be designed (for more details, check out this journal article).

For more information about the microbiome/autism connection, check out Autism Speaks. To read more Journal Clubs, visit the archives here.