Special Brain Cell Helps New Neurons Survive


A specialized type of brain cell that down-regulates stem cell activity seems to encourage the survival of stem cell progeny, according to new research from the laboratory of Hongjun Song, professor of neurology and director of Johns Hopkins Medicine’s Institute for Cell Engineering’s Stem Cell Program.

Uncovering the precise mechanism by which these cells regulate the life and death of neurons is a central to understanding neurodegenerative diseases, aging, and Alzheimer’s disease, since the activity of these cells is linked to these conditions.

“We’ve identified a critical mechanism for keeping newborn neurons alive,” said Song.  “Not only can this help us understand the underlying causes of some diseases, it may also be a step toward overcoming barriers to therapeutic cell transplantation.”

Song collaborated with Guo-li Ming and the members of his research group. Ming is a professor of neurology at the Institute for Cell Engineering.  Song’s team first reported last year that special brain cells called “parvalbumin-expressing interneurons” signal to nearby stem cells not to divide.  They means by which the parvalbumin-expressing interneurons (PEIs) signal to nearby stem cells is by releasing a neurotransmitter called “gamma-aminobutyric acid” (GABA).  In this present study, Ming and Song examined how GABA from surrounding PEIs affects nearby neurons produced by stem cells.

arvalbumin-expressing interneurons
parvalbumin-expressing interneurons

Many of these newborn neurons naturally die soon after they are born.  According to Song, if the new cells survive, these neurons will migrate to a permanent home in the brain and forge connections called synapses with other cells.

To determine whether GABA is a factor in the survival of newborn neurons and their behavior, Song’s team tagged neurons in mouse brains with a fluorescent protein and watched their response to GABA.

“We didn’t expect these immature neurons to form synapses, so we were surprised to see that they had built synapses from surrounding interneurons and that GABA was getting to them that way,” Song said.

In an earlier study, this research team had found that GABA was getting to the synapse-less stem cells by a less direct route – it was drifting across the spaces between cells.

To confirm the finding, the team engineered the interneurons to be stimulated or suppressed by light.  When stimulated by light, the cells activated nearby neurons.  Then they used this light stimulation procedure in live mice, they found that when the specialized neurons were stimulated and gave off more GABA, the newborn neurons survived in greater numbers than otherwise.  This was the opposite of the response of the neural stem cells, which become dormant when given GABA.

Song interpreted these data in the following manner: “This appears to be a very efficient system for tuning the brain’s response to its environment.  When you have a high level of brain activity, you need more newborn neurons, and when you don’t have high activity, you don’t need newborn neurons, but you need to prepare yourself by keeping the stem cells active.  It’s all regulated by the same signal.”

According to Song, the PEIs behave abnormally in neurodegenerative diseases such as Alzheimer’s disease and mental illnesses such as schizophrenia.

“Now we want to see what the role of these interneurons is in the newborn neurons’ next steps” migrating to the right place and integrating into the existing circuitry.  That may be the key to their role in disease,” said Song.  His team is also interested in using the GABA signal to keep transplanted cells alive without affecting other brain processes as a side effect.

See Song J, Sun J, Moss J, Wen Z, Sun GJ, Hsu D, Zhong C, Davoudi H, Christian KM, Toni N, Ming GL, Song H. Parvalbumin interneurons mediate neuronal circuitry-neurogenesis coupling in the adult hippocampus. Nat Neurosci. 2013 Dec;16(12):1728-30. doi: 10.1038/nn.3572. Epub 2013 Nov 10.

Porous Material Helps Deliver Molecules to Stem Cell-Derived Cells


A Swedish group has successfully tested a new porous material that allows for the efficient delivery of key molecules to transplanted cells that have been derived from stem cells. Such a material can dramatically improve the way stem cell-based treatments for neurodegenerative diseases.

This research project included a collaboration between Danish, Swedish and Japanese laboratories, and it tested a new type of porous material that efficiently delivers key molecules to transplanted cells derived from stem cells in an animal model.

Mesoporous silica loaded with differentiation factors induce motor neuron differentiation in vitro. (A): Top: Scanning and transmission (inset) electron micrographs of Meso. Scale bars = 200 nm (main panel) and 50 nm (inset). Bottom: CNTF with the Cintrofin motif shown in magenta and GDNF with the Gliafin motif shown in magenta. Amino acid residues are numbered according to UniProtKB entry nos. P26441 (Cintrofin) and Q07731 (Gliafin). (B): Differentiating motor neurons (MNs) extended numerous bTUB-labeled neurites (red) on poly-D-lysine (PDL)/laminin-coated coverslips after direct administration of CNTF and GDNF or treatment with MesoMim. Neurite formation was absent from MN precursors exposed to unloaded Meso. Scale bar = 75 μm. (C): Quantitative analysis of neurite length from MNs on PDL/laminin-coated coverslips after direct administration of CNTF and GDNF, treatment with MesoMim, or treatment with unloaded Meso. Results from 7–10 experiments are expressed as mean ± SEM, and the MesoMim group is set at 100%. Direct and MesoMim administration of the factors induced a significantly greater extent of neurite outgrowth compared with the unloaded Meso group; ***, p  .05). (D): HB9-GFP+ MNs expressed the MN markers ChAT and Isl1 in a 3-day differentiation assay after treatment with CNTF and GDNF or MesoMim but not in the absence of factors. Scale bar = 25 μm. (E, F): Almost all GFP+ cells expressed Isl1 (E) and ChAT (F) after treatment with CNTF and GDNF or MesoMim. Abbreviations: bTUB, β-tubulin; ChAT, choline acetyltransferase; CNTF, ciliary neurotrophic factor; D, day; GDNF, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; Isl1, Islet 1; Meso, mesoporous silica; MesoMim, mesoporous silica loaded with peptide mimetics; Rel., relative.
Mesoporous silica loaded with differentiation factors induce motor neuron differentiation in vitro. (A): Top: Scanning and transmission (inset) electron micrographs of Meso. Scale bars = 200 nm (main panel) and 50 nm (inset). Bottom: CNTF with the Cintrofin motif shown in magenta and GDNF with the Gliafin motif shown in magenta. Amino acid residues are numbered according to UniProtKB entry nos. P26441 (Cintrofin) and Q07731 (Gliafin). (B): Differentiating motor neurons (MNs) extended numerous bTUB-labeled neurites (red) on poly-D-lysine (PDL)/laminin-coated coverslips after direct administration of CNTF and GDNF or treatment with MesoMim. Neurite formation was absent from MN precursors exposed to unloaded Meso. Scale bar = 75 μm. (C): Quantitative analysis of neurite length from MNs on PDL/laminin-coated coverslips after direct administration of CNTF and GDNF, treatment with MesoMim, or treatment with unloaded Meso. Results from 7–10 experiments are expressed as mean ± SEM, and the MesoMim group is set at 100%. Direct and MesoMim administration of the factors induced a significantly greater extent of neurite outgrowth compared with the unloaded Meso group; ***, p < .001. No statistically significant differences were observed between groups with direct or MesoMim administration of the factors (p > .05). (D): HB9-GFP+ MNs expressed the MN markers ChAT and Isl1 in a 3-day differentiation assay after treatment with CNTF and GDNF or MesoMim but not in the absence of factors. Scale bar = 25 μm. (E, F): Almost all GFP+ cells expressed Isl1 (E) and ChAT (F) after treatment with CNTF and GDNF or MesoMim. Abbreviations: bTUB, β-tubulin; ChAT, choline acetyltransferase; CNTF, ciliary neurotrophic factor; D, day; GDNF, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; Isl1, Islet 1; Meso, mesoporous silica; MesoMim, mesoporous silica loaded with peptide mimetics; Rel., relative.

This potentially versatile and widely applicable strategy for the efficient differentiation and functional integration of stem cell derivatives upon transplantation, and it can serve as a foundation for improving stem cell-based neurodegenerative protocols, for example, Parkinson’s disease.

Alfonso Garcia-Bennett of Stockholm University, one of the lead authors of this study, said: “We are working to provide standard and reproducible methods for the differentiation and implementation of stem cell therapies using this type of approach, which coupled material science with regenerative medicine.”

Garcia-Bennett continued: “We demonstrated that delivering key molecules for the differentiation of stem cells in vivo with these particles enabled not only robust functional differentiation of motor neurons from transplanted embryonic stem cells but also improved their long-term survival.”

This research group is already working together with two companies to speed up the commercialization of a standard differentiation kit that will allow other scientists and clinicians to reproduce their work in their own laboratories.

“Noncontroversial” Embryonic Stem Cells?


An article from Bioscience Technology, a working scientist’s rag, has argued that everyone can have their lifetime supply of embryonic stem cells. Below is a summary of the article, after which I will comment on it.

Susan Fisher is the director of the UCSF Human Embryonic Stem Cell program. Last week, her lab reported that they have efficiently created embryonic stem cell lines from the cells removed from early embryos for Preimplantation Genetic Diagnosis (PGD) clinics. PGD takes a single cell from an early embryo that was created by means of in vitro fertilization, and subjects that single cell to genetic analyses to determine if the embryo carries a genetic disease. Because early human embryos have the ability to “regulate,” the removal of a single simply spurs the cells of the embryo to undergo extra cell divisions. The embryos subjected to PGD are then either destroyed, if they harbor a genetic disease, or implanted into the mother’s womb and gestated.

However, these cells removed from embryos could also be used to make an embryonic stem cell culture, since they could be seeded in culture to make an embryonic stem (ES) cell line. Therefore, in theory, cells could now be routinely removed from in vitro fertilization (IVF) clinic embryos, to provide them with a lifetime supply of their own embryonic stem cells. Because these cells were made without destroying embryos, they would be uncontroversial.

“Back in the mid-2000’s, when California was trying to decide whether to fund ES cell research, thousands of interested people would come out to hear us speak about topics like this,” says Fisher, interviewed after her report to the New York Stem Cell Foundation conference last week. “It is possible this particular, refined approach will generate that kind of interest now.”

ES cells have the greatest potency of any human stem cells and they can potentially form every cell type in the adult human body. Because such cells were recently harvested, they would not possess any of the mutations that ES cultures can acquire when they are grown for long periods of time in culture.

Traditionally, ES cell lines have been derived from stored, spare embryos from IVF clinics that were donated by other patients. Therefore, they are not immunologically identical to patients who potentially need them. Patients who receive non-matching tissues must take harsh immunosuppressive drugs for years to avoid rejecting the cells, and even then, over time the immune eventually wins the fight in some cases.

In recent years, scientists have turned to induced Pluripotential Stem Cells (IPSCs). IPSCs are made by genetically engineering adult cells to express four genes that de-differentiate the cells so that they are embryonic-like cells. IPSCs have been a boon to research, since scientists hace used them to make “disease in a dish” models on which to try drugs. But IPSCs are often riddled with mutations, as they come from adults. They have not yet hit the clinic as a result (although trials are upcoming).

However, Fisher, following on the heels of very preliminary work published in the journal Nature by the biotechnology company ACT, has refined the ability to create possibly uncontroversial stem cells—that are immunological matches to patients. By removing one cell from a very young human embryo, Fisher thinks that scientist might be able to produce a veritably unlimited supply of ES cells that are immunologically identical to the embyros from which they came. And as the embryos aren’t destroyed, but implanted into the mothers’ uteruses, the derivation of these tailor-made ES cells should be uncontroversial. “We will see how this is received,” Fisher says.

The process, she reported, is robust, if still not easy to pull off. This procedure, however, is labor-intensive and required a great deal of skill to pull off. In Fisher’s lab at UCSF, they derived ten human ES cell lines from four eight-cell embryos and one 12-cell embryo from a single couple.

When compared to standard ES cells, the UCSF lines were healthy and “formed derivatives of the three germ layers” like standard ES cells. Furthermore, these cells could form trophoblasts (placental cells), and Fisher’s team used them to create the first human trophoblast stem cell line. This is something that standard ES cells cannot do and this could make the UCSF cells useful in the clinic for diseases affecting the placenta.

Will patients begin turning to such cells? A few companies in the mid-2000s started offering designer ES cells like these, but that practice ended due to lack of interest or understanding, Fisher says. Additionally, some technical problems—later fully rectified—associated with the earlier Nature ACT paper may have cast a pall on enthusiasm for the approach, others in the field note.

“It remains to be seen if a place will be found for both iPS and ES cells,” Fisher concludes.

Now follows my comments:

Human embryos are very young human beings.  They do not have the right to vote, own property, or get a driver’s license, but they at least have the right not to be harmed.  By withdrawing cells from the embryo, you are potentially harming it.  “But wait,” proponents will tell you, “there are hundreds or even thousands of children who have been born who grew from embryos that were subjected to PGD and their rates of birth defects are no higher than everyone else’s.”  So their rates of birth defects are lower, but have we followed them for the rest of their lives to establish that removing a blastomere during early development does no harm?

“Oh come on,” you say.  But there are studies in mice that show that removing blastomere from early embryos does not cause higher rates of birth defects, but it does cause higher rates of neurological defects that manifest later in life.  Yu and others found that “mice generated after blastomere biopsy showed weight increase and some memory decline compared with the control group. Further protein expression profiles in adult brains were analyzed by a proteomics approach. A total of 36 proteins were identified with significant differences between the biopsied and control groups, and the alterations in expression of most of these proteins have been associated with neurodegenerative diseases. Furthermore hypomyelination of the nerve fibers was observed in the brains of mice in the biopsied group. This study suggested that the nervous system may be sensitive to blastomere biopsy procedures and indicated an increased relative risk of neurodegenerative disorders in the offspring generated following blastomere biopsy.”  In another paper, Yang and others showed that “blastomere biopsy, increases the rate of embryo death at 4.5-7.5 dpc, but does not affect the development of surviving 7.5 dpc embryos.”  In human embryos, time-lapse photography of biopsied embryos by Kirkegaard K, Hindkjaer JJ and Ingerslev HJ showed that “blastomere biopsy prolongs the biopsied cell-stage, possibly caused by a delayed compaction and alters the mechanism of hatching.”  Finally, Sugawara and others showed that “The data demonstrate that blastomere biopsy deregulates steroid metabolism during pregnancy. This may have profound effects on several aspects of fetal development, of which low birth weight is only one. If a similar phenomenon occurs in humans, it may explain low birth weights associated with PGD/ART and provide a plausible target for improving PGD outcomes.”

There is reason to believe that this procedure potentially hurts the embryo.  Also, not all blastomeres in the early embryo are equally competent to make ES lines (see Lorthongpanich et al., Reproduction. 2008 Jun;135(6):805-1).  Therefore, if more than one blastomere must be taken from the embryo, the risks to it definitely increases (see Groossens et al., Hum. Reprod. (2008) 23 (3): 481-492).  The embryo has a basic right not to be harmed, but PGD potentially harms it without its consent.  This is barbaric.  With any other procedure we would say so, but this seems to be alright because we are dealing with embryos and they are too small and young.  This is ageism and size discrimination.  These are not “uncontroversial stem cells.”  They are anything but.