Biphasic Electrical Stimulation Increases Stem Cell Survival


One of the challenges of stem cell-based therapies is cell survival. Once stem cells are implanted into a foreign site, many of them tend to pack up and die before they can do any good. For this reason, many scientists have examined strategies to improve stem cell survival.

A new technique that improves stem cells survival have been discovered by Yubo Fan and his colleagues at Beihang University School of Biological Science and Medical Engineering. This non-chemical technique, biphasic electrical stimulation (BES) might become important for spinal cord injury patients in the near future.

The BES incubation system. (a) Schematic diagram of a longitudinal section of the incubation chamber including: the upper and lower electric conductive glass plates (FTO glass), a closed silicone gasket, the incubation chamber, and a pair of electrode wires; (b) Schematic diagram of a longitudinal section of the entire BES incubation system including the incubation chamber, the fluid inflow-outflow system, the air filter system, a pair of electrode wires, and a fixed cover and base. Conditions of BES: the NPCs were exposed to 12 h of BES at 25mV/mm and 50mV/mm electric field strengths with a pulse-burst pattern and 8ms pulses (20% duty cycle). Cells that were not exposed to BES served as controls. (A color version of this figure is available in the online journal)
The BES incubation system. (a) Schematic diagram of a longitudinal
section of the incubation chamber including: the upper and lower electric  conductive glass plates (FTO glass), a closed silicone gasket, the incubation
chamber, and a pair of electrode wires; (b) Schematic diagram of a longitudinal
section of the entire BES incubation system including the incubation chamber,
the fluid inflow-outflow system, the air filter system, a pair of electrode wires, and
a fixed cover and base. Conditions of BES: the NPCs were exposed to 12 h of
BES at 25mV/mm and 50mV/mm electric field strengths with a pulse-burst
pattern and 8ms pulses (20% duty cycle). Cells that were not exposed to BES
served as controls. 

Spinal cord injury affects approximately 250,000 Americans, with 52% being paraplegic and 47% quadriplegic. There are 11,000 new spinal cord injuries each year and 82% are male.

Stem cell transplantions into the spinal cord to regenerate severed neurons and associated cells provides a potentially powerful treatment. However, once stem cells are implanted into the injured spinal cord, many of them die. Cell death is probably a consequence of several factors such as a local immune response, hypoxia (lack of oxygen), and probably most importantly, limited quantities of growth factors.

Fan said of his work, “We’ve shown for the very first time that BES may provide insight into preventing growth factor deprivation-triggered apoptosis in olfactory bulb precursor cells. These findings suggest that BES may thus be used as a strategy to improve cell survival and prevent cell apoptosis (programmed cell death) in stem cell-based transplantation therapies.”

The olfactory bulb is in green in this mouse brain.
The olfactory bulb is in green in this mouse brain.

Since electrical stimulation dramatically accelerates the speed of axonal regeneration and target innervation and positively modulates the functional recovery of injured nerves, Fan decided to test BES. His results showed that BES upregulated all the sorts of responses in stem cells that you would normally see with growth factors. Thus BES can increase stem cell survival without exogenous chemicals or genetic engineering.

Fan and his team examined the effects of BES on olfactory bulb neural precursor cells and they found that 12 hours of BES exposure protected cells from dying after growth factor deprivation. How did BES do this? Fan and other showed that BES stimulated a growth factor pathway called the PI3K/Akt signaling cascade. BES also increase the output of brain-derived neurotrophic factor.

“What was especially surprising and exciting,” said Fan, “was that a non-chemical procedure can prevent apoptosis in stem cell therapy for spinal cord patients.” Fan continued: “How BES precisely regulates the survival of exogenous stem cells is still unknown but will be an extremely novel area of research on spinal cord injury in the future.”

BES alters the ultrastructure of NPCs. The ultrastructural morphological changes of cells were investigated by TEM. In the control group (unstimulated), cells had a necrotic appearance: most cells lost the normal cellular structure with a consequent release of cell contents. In the 25mV/mm and 50mV/mm BES groups, the NPCs showed an apoptotic morphology with nuclear fragmentation and condensation
BES alters the ultrastructure of NPCs. The ultrastructural morphological changes of cells were investigated by TEM. In the control group (unstimulated), cells had a necrotic appearance: most cells lost the normal cellular structure with a consequent release of cell contents. In the 25mV/mm and 50mV/mm BES groups, the NPCs showed an apoptotic morphology with nuclear fragmentation and condensation

BES can improve the survival of neural precursor cells and will provide the survival of neural precursor cells and will provide the basis or future studies that could lead to novel therapies for patients with spinal cord injury.

Nanometer Scaffolds Regulate Neural Stem Cells


In the laboratory, stem cells can grow in liquid culture quite well in many cases, but this type of culture system, though convenient and rather inexpensive, does not recapitulate the milieu in which stem cells normally grow inside our bodies. Inside our bodies, stem cells stick to all kinds of surfaces and interact with and move over a host of complex molecules. Many of the molecules that stem cells contact have profound influences over their behaviors. Therefore, reconstituting or approximating these environments in the laboratory is important even though it is very difficult.

Fortunately nanotechnology is providing ways to build surfaces that approximate the kinds of surfaces stem cells encounter in our bodies. While this field is still in its infancy, stem cell-based nanotechnology may provide strategies to synthesize biologically relevant surfaces for stem cell growth, differentiation, and culture.

One recent contribution to this approach comes from Jihui Zhou and his team from the Fifth Hospital Affiliated to Qiqihar Medical University. Zhou and his co-workers prepared randomly oriented collagen nanofiber scaffolds by spinning them with an electronic device. Collagen is a long, fibrous protein that is found in tendons, ligaments, skin, basement membranes (the substratum upon which sheets of cells sit), bones, and is also abundant in cornea, blood vessels, cartilage, intervertebral disc, muscles, and the digestive tract. Collagen is extremely abundant in the human body; some 30% of all the proteins in our bodies are collagen. It is the main component in connective tissues.

There are many different types of collagen. Some types of collagen form fibers, while others for sheets. There are twenty-eight different types of collagen. Mutations in the genes that encode collagens cause several well-known genetic diseases. For example, mutations in collagen I cause osteogenesis imperfecta, the disease made famous by the Bruce Willis/Samuel T. Jackson movie, “Unbreakable.” Mutations in Collagen IV cause Alport syndrome, and mutations in either collagen III or V cause Ehlers-Danlos Syndrome.

Wen cells make fibrous collagen, they weave three collagen polypeptides together to form a triple helix protein that is also heavily crosslinked. This gives collagen its tremendous tensile strength.

Collagen fibers
Collagen fibers

In this experiment, electronic spinning technology made the collagen fibers and these fibers had a high swelling ratio when placed in water, high pore size, and very good mechanical properties.

Zhou grew neural stem cells from spinal cord on these nanofiber scaffolds and the proliferation of the neural stem cells was enhanced as was cell survival. Those genes that increase cell proliferation (cyclin D1 and cyclin-dependent kinase 2) were increased, as was those genes that prevent cells from dying (Bcl-2). Likewise, the expression of genes that cause cells to die (caspase-3 and Bax) decreased.

Thus novel nanofiber scaffolds could promote the proliferation of spinal cord-derived neural stem cells and inhibit programmed cell death without inducing differentiation of the stem cells. These scaffolds do this by inducing the expression of proliferation- and survival-promoting genes.

Making Preneurons from White Blood Cells for ALS Patients


ALS or amyotrophic lateral sclerosis is a disease that results in he death of motor neurons. Motor neurons enable skeletal muscles to contract, which drives movement. The death of motor neurons robs the patient of the ability to move and ALS patients suffer a relentless, progressive, and sad decline that culminates in death from asphyxiation. Treatments are largely palliative, but stem cells treatments might delay the onset of the disease, or even regenerate the dead neurons.

To this end a Mexican group from Monterrey has used a protocol to isolate white blood cells from the circulating blood of ALS patients, and differentiate a specific population of stem cells from peripheral blood into preneurons. Although these cells were not used to treat the patients in this study, such cells do show neuroprotective features and using them in a clinical study does seem to be the next step.

In this study, CD133 cells were isolated from peripheral blood and subjected to a special culture system called a neuroinduction system. After 2-48 hours in this system, the cells showed many features that were similar to those of neurons. The cells express a cadre of neural genes (beta-tubulin III, Oligo 2, Islet-2, Nkx6.1, and Hb9). Some of the ells also grew extensions that resemble the axons of true neurons.

Interestingly, the conversion of the CD133 cells into preneurons showed similar efficiency regardless of the age, sex, or health of the individual. Even those patients with more advanced ALS had CD133 cells that differentiated into preneurons with efficiencies equal to those of their healthier counterparts. While each patient showed variation with regards to the efficiency at which their CD133 cells differentiated into preneurons, these variations could not be correlated with the age, health or sex of the patient.

The fact that these preneurons expressed Oligo2, suggests that they could differentiate into motor neurons. Therefore, even though this study was small (13 patients), it certainly shows that cells that might provide treatment possibilities for ALS patients can be made from the patient’s own blood cells.

See Maria Teresa Gonzalez-Garza et al., Differentiation of CD133+ Stem Cells from Amyotrophic Lateral Sclerosis Patients into Preneuron Cells. Stem Cells Translational Medicine 2013;2:129-35.

When To Use Umbilical Cord Blood Stem Cells


Umbilical cord blood stem cells (UCB-SCs) have been used in a variety of clinical trials and treatments. Their use in treatment bone marrow-based conditions is very well-known, but they have also been used in other experimental treatments as well.

Treatments with UCB-SCs suffer from inconsistent results that stem from a variable number of viable cells in UCB-SC samples. Establishing high numbers of viable cells in UCB-SC samples is not easy, and there is a great interest in being able to grow UCB-SCs in culture and expand them. However, even though UCB-SCs can be grown in culture, the effects of culturing UCB-SCs is presently unclear.

To address this question in a rigorous fashion, Miguel Alaminos at the University of Granada and his colleagues grew UCB-SCs in culture and analyzed cell viability and gene expression at every passage.

What they discovered was astounding. When UCB-SCs were passaged two or three times, the cells showed signs of cells death, and gene expression studies revealed that many of the cells expressed genes associated with programmed cell death. Cells passaged eight, nine, or ten times also showed extensive cell death. However, cells passaged five or six times showed the highest viability.

This suggests that different studied have used cells that were grown for different periods of time and probably had different viabilities. This explains why UCB-SCs have performed so variably in experiments and clinical trials. This suggests that therapies that utilize UCB-SCs should use them after they are passaged for the fifth or sixth time in order to ensue the highest levels of viability.