Stem Cell-Tweaking Drug Might Treat Osteoporosis


A research group from the Florida campus of The Scripps Research Institute (TSRI) has identified a new therapeutic approach that could promote the development of new bone-forming cells in patients suffering from bone loss.

The study was published in the journal Nature Communications, and it focused on a protein called PPARγ, which is a master regulator of fat, and the impact of this molecule on the fate of mesenchymal stem cells derived from bone marrow. Since these mesenchymal stem cells can differentiate into several different cell types, including fat, connective tissues, bone and cartilage. Consequently mesenchymal stem cells have a number of potentially important therapeutic applications.

A partial loss of PPARγ in a genetically modified mouse model led to increased bone formation. Could the use of drugs to inhibit PPARγ and potentially mimic that effect? This group combined a variety of structural biology approaches and then tried to design drugs that could fit PPARγ. This type of strategy is called “rational design,” and this yielded a new compound that could repress the biological activity of PPARγ.

The new drug, SR2595 (SR=Scripps Research), when applied to mesenchymal stem cells, significantly increased bone cell or osteoblast formation, a cell type known to form bone.

“These findings demonstrate for the first time a new therapeutic application for drugs targeting PPARy, which has been the focus of efforts to develop insulin sensitizers to treat type 2 diabetes,” said Patrick Griffin, chair of the Department of Molecular Therapeutics and director of the Translational Research Institute at Scripps Florida. “We have already demonstrated SR2595 has suitable properties for testing in mice; the next step is to perform an in-depth analysis of the drug’s efficacy in animal models of bone loss, aging, obesity and diabetes.”

In addition to identifying a new, potential therapeutic use for bone loss, this study may have even broader implications.

“Because PPARG is so closely related to several proteins with known roles in disease, we can potentially apply these structural insights to design new compounds for a variety of therapeutic applications,” said David P. Marciano, first author of the study, a recent graduate of TSRI’s PhD program and former member of the Griffin lab. “In addition, we now better understand how natural molecules in our bodies regulate metabolic and bone homeostasis, and how unwanted changes can underlie the pathogenesis of a disease.” Marciano will focus on this subject in his postdoctoral work in the Department of Genetics at Stanford University.

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.

Recreating an Inherited Heart Condition in the Laboratory


Patients that suffer from an inherited condition known arrhythmogenic right ventricular dysplasia/cardiomyopathy or ARVD/C usually have no idea that they had a heart problem until they are in their 20s. The lack of symptoms at a younger age makes it virtually impossible for researchers to study this condition or to know how it develops. Fortunately, by making induced pluripotent stem cells (iPSCs) from patients with ARVD/C, researchers now have way to solve this very problem.

Small skin biopsies of ARVD/C patients can yield enough cellular material to make iPSCs. These iPSCs can be differentiated into heart muscle cells that are immature. These may not be terribly useful, since the goal is to model a disease that manifest itself during adulthood.

Researchers at Sanford-Burnham Medical Research Institute and Johns Hopkins University have created the first maturation-based “disease in a dish” model for ARVD/C. They created this model system by using iPSCs technology and employing a new method to mimic maturity that makes the metabolism of the hearts muscle cells more like those adult hearts. Thus, this model is likely more relevant to human ARVD/C than other models and therefore better suited for studying the disease and testing new treatments.

Huei-Sheng Vincent Chen, associate professor at Sanford-Burnham and the senior author of this study said, “It’s tough to demonstrate that a disease-in-a-dish model is clinically relevant for an adult-onset disease. But we made a key finding here – we can recapitulate the defects in this disease only when we induce adult-like metabolism. This is an important breakthrough considering that ARVD/C symptoms usually don’t arise until young adulthood. Yet the stem cells we’re working with are embryonic in nature.”

Daniel Judge, associate professor of medicine at Johns Hopkins University School of Medicine, said, “There is currently no treatment to prevent progression of ARVD/C, as rare disorder that preferentially affects athletes. With this new model, we hope we are not on a path to develop better therapies for this life-threatening disease.”

To make this model system, Cheng and his collaborators took skin samples from ARVD/C patients and converted various skin-based cells into iPSCs. After iPSC lines had been established, they differentiated them into heart muscle cells that had the characteristics of embryonic heart muscle cells. Unfortunately, these heart muscle cells showed no signs of ARVD/C, even when grown for over a year in culture.

What was the key? Metabolism. The young heart muscle cells primarily burn sugar for energy, but more mature heart muscle cells burn fat. Therefore, Chen’s group used a cocktail of molecules to get the heart muscle cells to preferentially burn fat.

As it turns out, metabolic malfunction is at the heart of ARVD/C. Chen and his group managed to track down the one piece that would get heart muscle cells made from patient-specific iPSCs to behave like sick ARVD/C heart muscle cells. The answer was the over-stimulation of a protein called PPAR-gamma.

PPARgamma

PPAR-gamma plays an absolutely central role in type II diabetes. It regulates fatty acid storage and sugar metabolism. When PPAR-gamma activates genes, those genes stimulate lipid uptake and the production of fat in fat cells. If mice are made that do not have functional versions of PPAR-gamma, these mice fail to make fat, even when fed a high-fat diet.

PPAR stands for “peroxisome proliferator-activated receptor,” which is a subfamily of nuclear receptors. PPAR proteins bind DNA in combination with retinoid X receptors (RXRs), and these two proteins pair up to regulate transcription of various genes. There are three subtypes of PPARs: PPAR-alpha, PPAR-delta, and PPAR-gamma.

ppar activity

The fact that PPAR-gamma plays such a central role in the pathology of ARVD/C suggests a link between those mechanisms that cause type II diabetes and ARVD/C. According to Chen and Judge, ARVD/C heart muscle cells undergo exaggerated fat production, which leads to cell death. Because PPAR-gamma is a target for a group of drugs known as “glitazones,” perhaps these drugs can play a role in treating ARVD/C.

Glitazones