Adding Cyclosporin to Bone Marrow Might Increase Stem Cell Numbers, Quality, and Engraftment Efficiency


In the bone marrow, we have an army of blood cell-making stem cells called hematopoietic stem cells (HSCs) that make all the blood cells that course through our blood vessels. These cells divide throughout our lifetimes, and they replacement themselves while they generate all the red and white cells found in our blood.

hematopoietic-stem-jpg

HSCs are also the cells that are harvested during bone marrow aspirations and biopsies. Transplantation of HSCs can save the lives of patients with blood cancers or other types of blood-or bone marrow-based diseased.

Harvesting and transplanting HSCs is, therefore, a very important clinical strategy for treating many different types of blood disorders and diseases. However, this crucial strategy is limited by the relative rarity of HSCs in isolated bone marrow. Additionally, the number and function of HSCs deteriorate both during their collection from the bone marrow (BM) and during their manipulation outside the body. Fortunately, the development of culture conditions that best mimic the environment these cells experience in bone marrow (the so-called “HSC niche environment”) may help to minimize this loss.

Scanning electron microscopy of stem cells (yellow / green) in a scaffold structure (blue) serving as a basis for the artificial bone marrow.
Scanning electron microscopy of stem cells (yellow / green) in a scaffold structure (blue) serving as a basis for the artificial bone marrow.

One of the most important variables for HSC viability is oxygen concentration, since various studies have shown that the oxygen concentrations found in ambient air seems to be damaging to HSCs, which normally are found in rather oxygen-poor reaches in bone marrow. Researchers from the laboratory of Hal Broxmeyer at the Indiana University School of Medicine have discovered that HSCs suffer from ‘‘extra-physiologic oxygen shock/stress (EPHOSS)” if they are harvested under ambient oxygen conditions. On top of that, treatment of the collected HSCs with the immunosuppressant drug cyclosporin A (CSA) can inhibit this stress, enhance the yield of collected HSCs, and increase their transplantation efficiency.

When Broxmeyer and his colleagues compared mouse BM that had been harvested under normal oxygen concentrations (21% O2) and low-oxygen concentrations (3% O2), they observed that the hypoxic (low-oxygen) treatment caused a 5-fold increase in the number of Long Term (LT) self-renewing HSCs, and a decrease in harmful reactive oxygen species (ROS) and mitochondrial activity. Broxmeyer and others also confirmed the positive effect of hypoxia on HSC collection from human cord blood. When mouse BM collected under different conditions were assayed by competitive transplantation, the “hypoxic HSCs” engrafted more efficiently in recipient mice. This increased engraftment was not due to enhanced homing or reduced cell death. Instead it seems that the stress response to non-physiological oxygen concentrations (EPHOSS) has a rapid and significant damaging effect in HSCs.

Broxmeyer decided to take this study one step further. In mitochondria (the powerhouse of the cell), increased expression of the mitochondrial permeability transition pore (MPTP) seems to be one of the key mechanism by which oxidative stress affects HSCs.

mitochondrial permeability transition pore
mitochondrial permeability transition pore

Induction of the MPTP leads to mitochondrial swelling and uncoupled energy production (which leads to the generation of reactive oxygen species, otherwise known as “free radicals). This leads to cell death apoptosis and necrosis, and intermittent MPTP activation may also decrease stem cell function in general without killing the cells. Broxmeyer and his coworkers came upon a rather ingenious idea to use the drug cyclosporin A (CSA) to antagonize MPTP induction, since CSA inhibits the associated CypD (cyclophilin) protein. When HSCs were collected under high-oxygen conditions in the presence of CSA, there was a 4-fold increase in the recovery of LT-HSCs and enhanced engraftment levels compared to HSCs harvested in high-oxygen conditions without CSA. This link was further strengthened by examining the HSCs of mice with a deletion of the CypD gene. In these mice, HSCs collected under high-oxygen conditions showed increased LT-HSC recovery and decreased LT-HSC ROS levels compared to wild-type mice.

Cyclophilin
Cyclophilin

How, harvesting and processing HSCs from bone marrow in a low-oxygen environment within a transplant clinic is generally not possible. However, given the observed advantages, the application of CSA may represent an easy and attractive alternative. The authors of this paper (which was published in the journal Cell) note that CSA is already used in the clinic as an immunosuppressant. Therefore, this technique could potentially be rapidly adapted into bone marrow harvesting techniques.

An additional thought is that studies that use other types of stem cells for transplantation might also need to consider the effects of EPHOSS and oxygen concentration while preparing their cells in other model systems.

See “Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock” from Cell by Stuart P. Atkinson

Gestational Diabetes Affects the Quality of Umbilical Cord Mesenchymal Stromal Cells


The laboratories of Drs. Jene Choi and Chong Jai Kim from the University of Ulsan College of Medicine in Seoul, South Korea have collaboratively shown that the therapeutic quality of umbilical cord mesenchymal stem cells is profoundly affected by gestational diabetes. Their work was published in a recent issue of the journal Stem Cells and Development and has profound implications for regenerative medicine.

Choi and Kim and their coworkers collected umbilical cords from mothers who had been given birth by Cesarian section and had also been diagnosed with gestational diabetes and mothers who had also just given birth by Cesarian section and showed normal blood sugar control. These umbilical cord tissues were processed and the mesenchymal stem cells from the cord tissue were isolated and cultured. These cells were grown and then subjected to a rather extensive battery of tests. These tests were a reflection of the ability of these to perform in regenerative treatments.

First umbilical cord mesenchymal stem cells (UCMSCs) from mothers with gestational diabetes (GD) did not grow as well as UCMSCs from mother who did not have GD.  As you can see in the graphs below, these are not small growth differences.  The UCMSCs from non-GD mothers (on the left) grow substantially better than those from GD mothers.  This result is also consistent for different cell lines.  This also means that transplanted cells would not grow very well if they were used for therapeutic purposes.

Umbilical cord mesenchymal stromal cells (UC-MSCs) derived from gestational diabetes mellitus (GDM) patients exhibit retarded growth proliferation. The growth of 7.5×103 UC-MSCs isolated from patients with normal pregnancies (A) and GDM (B) was monitored over a period of 12 days. GDM-UC-MSCs consistently showed decreased proliferation compared with normal pregnant women (N-UC-MSCs). Points represent the mean values from three independent experiments; bars denote standard deviation (SD).
Umbilical cord mesenchymal stromal cells (UC-MSCs) derived from gestational diabetes mellitus (GDM) patients exhibit retarded growth proliferation. The growth of 7.5×103 UC-MSCs isolated from patients with normal pregnancies (A) and GDM (B) was monitored over a period of 12 days. GDM-UC-MSCs consistently showed decreased proliferation compared with normal pregnant women (N-UC-MSCs). Points represent the mean values from three independent experiments; bars denote standard deviation (SD).

Secondly, UCMSCs from GD mothers showed a greater tendency to undergo premature senescence.  When MSCs are grown in culture, they usually grow rather well for several days and then the cells go to sleep and they stop growing.  This is called culture senescence and it is due to intrinsic properties of the cells.  When the cells go into senescence tends to be a cell line-specific property, but one thing is certain; the sooner cells become senescent, the few cells they will generate in culture.  The UCMSCs from GD mothers go into senescence early and easily and this is one of the reasons they grow so poorly relative to normal cells – because they are running to their beds to take a nap (so to speak).  Such cells are usually not good candidates for regenerative medicine.

Third, UCMSCs from GD mothers show poor lineage-specific differentiation.  MSCs have the ability to differentiate into fat cells, bone cells, and cartilage cells if particular well-established protocols are used.  However, UCMSCs from GD mothers showed inefficient differentiation and that is one of the things that MSCs must do if they are to repair bone or cartilage problems or if they are to help make smooth muscle for new blood vessels formation. 

Stem cell differentiation potentials are largely different between normal and GDM-affected UC-MSCs. Three different cell lines of normal and GDM-affected pregnancies were cultured in a control medium or induction medium for 5 days. Upregulation of the expression of the adipogenic-specific gene PPARγ (A) and the osteogenic genes alkaline phosphatase (ALP) (B), osteocalcin (OC) (C), and collagen type 1 alpha 1 (Col1α1) (D) was evaluated by real-time RT-PCR and normalized to GAPDH. All assays were performed in triplicate; bars denote SD (*P<0.05).
Stem cell differentiation potentials are largely different between normal and GDM-affected UC-MSCs. Three different cell lines of normal and GDM-affected pregnancies were cultured in a control medium or induction medium for 5 days. Upregulation of the expression of the adipogenic-specific gene PPARγ (A) and the osteogenic genes alkaline phosphatase (ALP) (B), osteocalcin (OC) (C), and collagen type 1 alpha 1 (Col1α1) (D) was evaluated by real-time RT-PCR and normalized to GAPDH. All assays were performed in triplicate; bars denote SD (*P<0.05).

The figure above shows the disparity between these established UCMSC cell lines.  The dark, solid bars indicate non-induced cells that were grown in normal culture media, and the striped bars are cells grown in media that designed to induce the differentiation of these cells into either bone, fat, or cartilage cells.  The cell lines with “N” in their name are from non-GD mothers and those with “D” in their designations are from GD mothers.  These assays are for genes known to be strongly induced when cells begin to differentiate into fat (PPARgamma), bone (ALP or osteocalcin or collagen 1 alpha 1).  As you can clearly see, the Ns outdo the Ds every time.

Finally, when the mitochondria, the compartments in cells that generate energy, from these two cell populations were examined it was exceedingly clear that UCMSCs from GD mothers had mitochondria that were abnormal and did not make every very well.  Mitochondria from UCMSCs taken from GD mothers showed decreased expression of the energy-making components.  Thus the energy-making pathways in these cell compartments were sub-par from a structural perspective.  Functional assays for mitochondria showed that mitochondria from UCMSCs from GD mothers consistently underperformed those from UCMSCs taken from non-GD mothers.  Also, when markers of mitochondrial dysfunction were measured (reactive oxygen species and indicators of mitochondrial damage from reactive oxygen species), such markers were consistently higher in mitochondria from UCMSCs from GD mothers relative to those from non-GD mothers.  This shows that the energy-making or powerhouses of the cells are dysfunctional in UCMSCs from GD mothers.  Without the ability to properly make energy from food molecules, the cells have a diminished capacity to heal damaged tissues and organs.

Several studies have established a positive link between mitochondrial dysfunction and accelerated aging.  Therefore, these cells, because they have more extensive indications of mitochondrial damage, may show profound accumulation of mitochondrial damage and accelerated aging.

In summary, this study shows that integral biological properties of human UC-MSCs differ according to obstetrical conditions.  These data also stress the importance of maternal–fetal conditions in biological studies of hUC-MSCs and the development of future therapeutic strategies using hUC-MSCs.

Loss of Antioxidant Protein Prevents Muscle Regeneration During Aging


Nrf2 is a protein that regulates the response of cells to oxidative damage, This protein normally sits in the cytoplasm of cells where it is routinely degraded by other proteins. However, once cells are exposed to oxidative damage by ultraviolet light, reactive oxygen species, various chemicals, or other conditions that damage cellular structures, the degradation of Nrf2 slows way down and this protein moves into the nucleus where it binds DNA and stimulates the expression of a host of genes that encode proteins with anti-oxidant activity. Thus Nrf2 is one of the primary cellular defenses against the toxic effects of oxidative stress.

Nrf2 pathway

Researchers at the University of Utah School of Medicine have made mice that lack functional Nrf2 and they found that the stem cells of these mice were seriously impaired.

Raj Soorappan and his colleagues have discovered that the muscles of these Nrf2-deficient mice do not regenerate as they get older.

Soorappan explained: “Physical activity is the key to everything.” He continued: “After this study we believe that moderate exercise could be one of the key ways to induce stem cells to regenerate especially during aging.”

Sarcopenia or the age-related loss of muscle mass, begins in most people around the age of 30. To delay this inevitable slide, muscle=producing stem cells help regenerate muscle lost by means of aging and the production of antioxidant molecules help protect stem cells populations so that they can maintain muscle mass.

However, as we age, the production of reactive oxygen species (ROS) overwhelms our endogenous antioxidant systems, and our stem cell populations take a hit. This compromises our ability to regenerate muscle and other tissues as well.

As previously mentioned, Nrf2 regulates the production of these antioxidant molecules. Soorappan used mice that were 23 months old or older (these are rodent senior citizens to be sure). One group of old mice made normal levels of Nrf2, but the other group had no functional Nrf2 protein. Soorappan and his colleagues put these mice through endurance training to determine the effects of ROS on these animals. Interestingly, the Nrf2-deficient mice showed an inability to mobilize their muscle stem cells (satellite cells) to regenerate their muscles. The Nrf2-containing mice, however, were able to properly regenerate their muscles.

“We now know that the antioxidant protein Nrf2 guards the muscle regeneration process in elderly mice and loss of Nrf2, when combined with endurance exercise stress, can cause severe muscle stem cell impairment,” said Mudhusudhanan Narasimhan, the primary author of this research and a research associate with Soorappan.

Soorappan thinks that by understanding the precise role of Nrf2 in muscle regeneration, he an his co-workers will be able to design more informed therapies of muscle loss in aging animals and humans.

Next on Soorappan’s agenda is to examine the effects of exercise on Nrf2 and whether or not an active lifestyle affects the function of Nrf2 and the efficiency of the anti-oxidant pathway it mediates.

The take-home message for now seems to be: “If you don’t use your muscles, you will lose them. At the same time, overdoing endurance training may detract from muscle regeneration,” said Soorappan.

Inhibition of a Heart-Specific Enzyme After a Heart Attack Decreases Heart Damage and Prevents Remodeling


Cardiac Troponin I-interacting Kinase or TNNI3K is an enzyme that was initially identified in fetal and adult heart tissue, but was undetectable in other tissues. The function of this enzyme remains unknown, but Chinese scientists showed that overexpression of TNNI3K in cultured heart muscle cells causes them to blow up and get large (hypertrophy). Earlier this year, a research team from Peking Union Medical College showed that overexpression of TNNI3K in mice caused enlargement of the heart (Tang H., et al., J Mol Cell Cardiol 54 (2013): 101-111). These results suggested that TNNI3K is a potential therapeutic target for heart attack patients.

To that end, Ronald Vagnozzi and his colleagues in the laboratory of Thomas Force at Temple University School of Medicine and their collaborators designed small molecules that can inhibit TNNI3K activity, and these small molecules decrease cardiac remodeling after a heart attack in rodents. Large animal trials are planned next.

In the first experiments of this paper, Vagnozzi and others showed that the levels of TNNI3K in the heart increase after a heart attack. Measurements of TNNI3K protein levels failed to detect it in all tissue other than the heart. Furthermore, it was present throughout the heart, and mainly in heart muscle and not in blood vessels, fibroblasts, and other types of non-muscle heart tissues.

Next, Vagnozzi and others measured TNNI3K protein levels in heart transplant patients. The heart tissues of these patients, who had badly dysfunctional hearts showed higher than usual levels of TNNI3K protein. Thus, TNNI3K is associated with heart tissue and is up-regulated in response to heart dysfunction.

The next experiment examined the effects of overexpressing the human TNNI3K gene in mice. While the overexpression of TNNI3K did not affect heart function of structure under normal circumstances, under pathological conditions, however, this is not he case. If mice that overexpressed TNNI3K where given heart attacks and then “reperfused,” means that the blood vessel that was tied off to cause the heart attack was opened and blood flowed back into the infarcted area. In these cases, mice that overexpressed TNNI3K had a larger area of cell death in their hearts than their counterparts that did not overexpress TNNI3K. The reason for this increased cell death had to do with the compartment in the cell that generated most of the energy – the mitochondrion. TNNI3K causes the mitochondria in heart muscle cells to go haywire and kick out all kinds of reactive oxygen-containing molecules that damage cells.

Cell damage as a result of reactive oxygen-containing molecules (known as reactive oxygen species or ROS) activates a pathway in heart cells called the “p38” pathway, which leads to programmed cell death.

p38 signaling

Once Vagnozzi and his colleagues nailed down the function of TNNI3K in heart muscle cells after a heart attack, they deleted the gene that encodes TNNI3K and gave those TNNI3K-deficient mice heart attacks. Interestingly enough, after a heart attack, TNNI3K-deficient mice showed much small dead areas than normal mice. Also, the levels of the other mediators of TNNI3K-induced cell death (e.g., oxygen-containing molecules, p38, ect.) were quite low. This confirms the earlier observations that TNNI3K mediates the death of heart muscle cells after a heart attack, and inhibiting TNNI3K activity decreases the deleterious effects of a heart attack.

And now for the pièce de résistance – Vagnozzi and his crew synthesized small molecules that inhibited TNNI3K in the test tube. Then they gave mice heart attacks and injected these molecules into the bellies of the mice. Not only were the infarcts, or areas of dead heart muscle cells small in the mice injected with these TNNI3K inhibitors, but the heart of these same mice did not undergo remodeling and did not enlarge, showed reduced scarring, and better ventricular function. This is a proof-of-principle that inhibiting TNNI3K can reduce the pathological effects of a heart attack.

This strategy must be tested in large animals before it can move to human trials, but the strategy seems sound at this point, and it may revolutionize the treatment of heart attack patients.