The Therapeutic Potential of Fat-Based Stem Cells Decreases With Age


Fat is a rich source of stem cells for regenerative medicine.  Treating someone with their own stem cells from their own fat certainly sounds like an attractive option.  However, a new study shows that demonstrates that the therapeutic value of fat-based stem cells declines when those cells come from older patients.

“This could restrict the effectiveness of autologous cell therapy using fat, or adipose-derived mesenchymal stromal cells (ADSCs), and require that we test cell material before use and develop ways to pretreat ADSCs from aged patients to enhance their therapeutic potential,” said Anastasia Efimenko, M.D., Ph.D.  Dr Efimenko and Nina Dzhoyashvili, M.D., were first authors of the study, which was led by Yelena Parfyonova, M.D., D.Sc., at Lomonosov Moscow State University, Moscow.

Heart disease remains the most common cause of death in most countries.  Mesenchymal stromal cells (MSCs) collected from either bone marrow or fat are considered one of the most promising therapeutic agents for regenerating damaged tissue because of their ability to proliferate in culture and differentiate into different cell types.  Even more importantly they also have the ability to stimulate the growth of new blood vessels (angiogenesis).

In particular, fat is considered an ideal source for MSCs because it is largely dispensable and the stem cells are easily accessible in large amounts with a minimally invasive procedure.  ADSCs have been used in several clinical trials looking at cell therapy for heart conditions, but most of the studies used stem cells from relatively healthy young donors rather than sick, older ones, which are the typical patients who suffer from heart disease.

“We knew that aging and disease itself may negatively affect MSC activities,” Dr. Dzhoyashvili said. “So the aim of our study was to investigate how patient age affects the properties of ADSCs, with special emphasis on their ability to stimulate angiogenesis.”

The Russian team analyzed age-associated changes in ADSCs collected from patients of different age groups, including some patients who suffered from coronary artery disease and some without.  The results showed that ADSCs from the older patients in both groups showed some of the characteristics of aging, including shorter telomeres (the caps on the ends of chromosomes that protect them from deterioration), which confirms that ADSCs do age.

“We showed that ADSCs from older patients both with and without coronary artery disease produced significantly less amounts of angiogenesis-stimulating factors compared with the younger patients in the study and their angiogenic capabilities lessened,” Dr. Efimenko concluded. “The results provide new insight into molecular mechanisms underlying the age-related decline of stem cells’ therapeutic potential.”

“These findings are significant because the successful development of cell therapies depends on a thorough understanding of how age may affect the regenerative potential of autologous cells,” said Anthony Atala, M.D., director of the Wake Forest Institute for Regenerative Medicine, and editor of STEM CELLS Translational Medicine, where this research was published.

Brown Fat Stem Cells for Treating Diabetes and Obesity


Mammals have two main types of fat: brown fat and white fat.  Brown fat is especially abundant in newborns and in mammals undergoing hibernation.  The primary function of brown fat is to produce body heat so that the animal does not shiver.  In contrast to white fat cells, which contain a single lipid droplet, brown fat cells contain numerous smaller droplets and a higher number of mitochondria, and it is these mitochondria and their high iron content that makes this fat tissue brown.  Brown fat also contains more small blood vessels than white fat, since it has a greater need for oxygen than most tissues.

Recently, researchers at the University of Utah School of Medicine have identified stem cells from brown fat.  The principal researcher of this project, Amit Patel, associate professor of medicine, refuted an old dogma – that adult humans do not possess brown fat.  Children have large amounts of brown fat that is highly metabolically active.  Children can eat a great deal and not gain any weight, relative to adults.  Adults, on the other hand, have an abundance of white fat, and accumulation of white fat leads to weight gain and cardiovascular disease (something not seen in brown fat).  As people age, the amount of white fat increases and the amount of brown fat decreases, which contributes to the onset of diabetes and high cholesterol.

As Patel put it, “If you have more brown fat, you weigh less, you’re metabolically efficient, and you have fewer instances of diabetes and high cholesterol.  The unique identification of human brown fat stem cells in the chest of patients aged 28-34 years is profound.  We were able to isolate the human stem cells, culture and grow them, and implant them into a pre-human model which has demonstrated positive effects on glucose levels.”

In vitro differentiation of brown adipose derived stem cells (BADSCs). (A) Gene expression profile comparing undifferentiated BADSCs to undifferentiated white adipose derived stem cells derived from subcutaneous adipose tissue. Genes in red are associated with brown fat phenotype. (B) Gene expression profile comparing undifferentiated brown adipose derived stem cells to differentiated brown adipocytes. Biological replicates performed in triplicate from a single clone were used for gene expression profile. (C) Transmission electron microscopy of 21 day brown adipocyte differentiation induced with fibronectin type III domain containing 5 (FNDC5) demonstrate multiocular intracytoplasmic lipid vacuoles and mitochondria (arrows). (D) Alizarian red staining of brown adipose derived stem cells induced to undergo osteogenesis. (E) Alcian blue staining of brown adipose derived stem cells directionally differentiated into chondrocytes. (F) Fatty acid binding protein 4 (FABP4) immunocytochemistry of brown adipose derived stem cells induced to undergo white adipogenesis. (G) Undifferentiated BADSCs. (H) Western blot 21 days post FNDC5 induction. Lane 1 brown adipose derived stem cells directionally differentiated into brown adipocytes. Lane 2 non- FNDC5 cells.
In vitro differentiation of brown adipose derived stem cells (BADSCs). (A) Gene expression
profile comparing undifferentiated BADSCs to undifferentiated white adipose derived stem cells
derived from subcutaneous adipose tissue. Genes in red are associated with brown fat phenotype. (B)
Gene expression profile comparing undifferentiated brown adipose derived stem cells to differentiated
brown adipocytes. Biological replicates performed in triplicate from a single clone were used for gene
expression profile. (C) Transmission electron microscopy of 21 day brown adipocyte differentiation
induced with fibronectin type III domain containing 5 (FNDC5) demonstrate multiocular
intracytoplasmic lipid vacuoles and mitochondria (arrows). (D) Alizarian red staining of brown
adipose derived stem cells induced to undergo osteogenesis. (E) Alcian blue staining of brown adipose
derived stem cells directionally differentiated into chondrocytes. (F) Fatty acid binding protein 4
(FABP4) immunocytochemistry of brown adipose derived stem cells induced to undergo white
adipogenesis. (G) Undifferentiated BADSCs. (H) Western blot 21 days post FNDC5 induction. Lane 1
brown adipose derived stem cells directionally differentiated into brown adipocytes. Lane 2 non-
FNDC5 cells.

This new discovery of finding brown fat stem cells may help in identifying potential drugs that may increase the body’s own ability to make brown fat or find novel ways to directly implant brown fat stem cells into patients.

Induced Pluripotent Stem Cells Recapitulate ALS in Culture and Suggest New Treatment


Induced pluripotent stem cells are made from the adult cells of an individual by means of genetic engineering techniques. After introducing four different genes into adult cells, some of the cells de-differentiate to form cells that grow indefinitely in culture and have most of the characteristics of embryonic stem cells. However, if iPSCs are made from a patient who suffers from a genetic disease, then those stem cells will have the same mutation as the patient, and any derivatives of those iPSCs will show the same behaviors and pathologies of the tissues from the patient. This strategy is called the “disease in a dish” model and it is being increasingly used to make seminal discoveries about diseases and treatment strategies.

Scientists from Cedars-Sinai Regenerative Medicine Institute have used iPSC technology to study Lou Gehrig’s disease, and their research has provided a new approach to treat this horrific, debilitating disease.

Because I have previously written about Lou Gehrig’s disease or Amyotrophic Lateral Sclerosis (ALS), I will not describe it further.

Cedar Sinai scientists isolated skin scrapings from each patient and used the skin fibroblasts from each sample to make iPSCs. According to Dhruv Sareen, the director of the iPSC facility and faculty research scientist with the Department of Biomedical Sciences and the first author on this article, skins cells of patients who have ALS were converted into motor neurons that retained the genetic defects of the disease, thanks to iPSC technology. Then they focused on gene called C9ORF72, which was found to be the most common cause of familial ALS and frontotemporal lobar disease, and is even responsible for some cases of Alzheimer’s and Parkinson’s disease.

Mutations in a gene that has the very non-descriptive name “chromosome 9 open reading frame 72” or C9ORF72 for short seems to play a central role in the onset of Lou Gehrig’s disease. Mutations in C9orf72 have been linked with familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). FTD is a brain disorder that typically leads to dementia and sometimes occurs in tandem with ALS.

Mutations in C9ORF72 result from the expansion of a hexanucleotide repeat GGGGCC. When the C9ORF72 gene is replicated, the enzyme that replicates DNA (DNA polymerase) has a tendency to slip when comes to this stretch of nucleotides and this polymerase slip causes the hexanucleotide GGGGCC sequence to wax and wane (expand and shrink). Normally, there are up to 30 repeats of this GGGCC sequence, but in people with mutations in C9ORF72, this GGGGCC repeat can occur many hundreds of times. Massive expansions of the GGGGCC repeat interferes with normal expression of the protein made by C9ORF72. The presence of messenger RNAs (mRNAs) with multiple copies of GGGGCC in the nucleus and cytoplasm is toxic to the cell, since it gums up protein synthesis, RNA processing and other RNA-dependent functions. Also the lack of half of the C9ORF72 protein contributes to the symptoms of this conditions.

Robert Baloh, director of Cedars-Sinai’s Neuromuscular Division and the lead researcher of this research project, said, “We think this buildup of thousands of copies of the repeated sequence GGGGCC in the nucleus of patient’s cells may become toxic by altering the normal behavior of other genes in the motor neurons. Because our studies supported the toxic RNA mechanism theory, we used to small segments of genetic material called antisense oligonucleotides – ASOs – to block the buildup and degrade the toxic RNA. One ASO knocked down overall C9ORF72 levels. The other knocked down the toxic RNA coming from the gene without suppressing overall gene expression levels. The absence of potentially toxic RNA, and no evidence of detrimental effect on the motor neurons, provides a strong basis for using this strategy to treat patients suffering from these diseases.”

Baloh continued: “In a sense, this represents the full spectrum of what we are trying to accomplish with patient-based stem cell modeling. It gives researchers the opportunity to conduct extensive studies of a disease’s genetic and molecular makeup and develop potential treatments in the laboratory before translating them into patient trials.”

Researchers from another institution recently began a phase one clinical trial that used a similar ASO strategy to treat ALS caused by a different mutation. No safety issues were reported in this clinical trial.

Clive Svendsen, director of the Regenerative Medicine Institute and one of the authors, has investigated ALS for more than a decade, said, “ALS may be the cruelest, most severe neurological disease, but I believe the stem cell approach used in this collaborative effort holds the key to unlocking the mysteries of the and other devastating disorders. Within the Regenerative Medicine Institute, we are exploring several other stem cell-based strategies in search of treatments and cures.”

ALS affects 30,000-50,000 people in the US alone, but unlike other neurodegenerative diseases, it is almost always fatal within three to five years.

Long-Lasting Blood Vessels Regenerated from Reprogrammed Human Cells


Researchers from Massachusetts General Hospital (MGH) in Boston, MA have used human induced pluripotent stem cells to make vascular precursor cells to produce functional blood vessels that lasted as long as nine months.

Rakesh Jain, director of the Steele Laboratory for Tumor Biology at MGH and his team derived human induced pluripotent stem cells (iPSCs) from adult cells of two different groups of patients. One group of individuals were healthy and the second group had type 1 diabetes. Remember that iPSCs are derived from adult cells through the process of genetic engineering. By introducing specific genes into these adult cells, the adult cells are de-differentiated into an embryonic-like state. The embryonic-like cells can be cultured and grown into a cell line that can be differentiated into various cell types in the laboratory. These differentiated cells types can then be transplanted into laboratory animals for regenerative purposes.

“The discovery of ways to bring mature cells back to a ‘stem-like’ state that ca differentiate into many different types of tissue has brought enormous potential to the field of cell-based regenerative medicine, but the challenge of deriving functional cells from these iPSCs still remains,” said Rakesh. “our team has developed an efficient method to generate vascular precursor cells from human iPSCs and used them to create networks of engineered blood vessels in living mice.”

The ability to regenerate or repair blood vessels could be a coup for regenerative medicine. Cardiovascular disease, for example, continues to be the number one cause of death in the United States and other conditions caused by blood vessel damage (e.g., the vascular complications of diabetes) continue to cause a great deal of morbidity and mortality each year. Also, providing a blood supply to newly generated tissue remains one of the greatest barriers to building solid organs through tissue engineering.

Some studies have used iPSCs to build endothelial cells (the cells that line blood vessels), and connective tissue cells that provide structural support. These cells, unfortunately, tend to not produce long-lasting vessels once they are introduced into laboratory animals. A collaborator with Jain, Dai Fukumura, stated, “The biggest challenge we faced during the early phase of this project was establishing a reliable protocol to generate endothelial cell lines that produced great quantities or precursor cells that could generate durable blood vessels.”

Jain’s group adapted a protocol that was originally designed to derived endothelial cells from human embryonic stem cells. They isolated cells based on the presence of more than one cell surface protein that marked out vascular potential. Then they expanded this population of cells using a culture system developed with embryonic stem cells that had been differentiated into endothelial cells. Further experiments confirmed that only those iPSCs that expressed all three cell surface proteins on their surfaces had the potential to form blood vessels.

To test the capacity of those cells to generate blood vessels, they implanted them onto the surface of the brain of mice in combination with mesenchymal precursors that generate smooth muscles that surround blood vessels.

Within two weeks after transplantation, the implanted cells had formed entire networks of blood vessels with blood flowing through them that has also fused with the already existing blood vessels. These engineered blood vessels continued to function for as long as 280 days in the living animal. Implantation under the skin, however, was a different story. It took 5 times the number of cells to get them to form blood vessels and they were short-lived. This is similar to the results observed in other studies.

Because type 1 diabetes can ravage blood vessels, Jain’s team made iPSCs from patients with type 1 diabetes to determine if iPSCs from such patients would generate functional blood vessels. Similarly to the cells generated from healthy individuals, vascular precursors generated from type 1 diabetics were able to form long-lasting blood vessels. However, these same cell lines showed variability in their ability to form vascular precursors. The reason for this is uncertain.

Rekha Samuel, one of the lead authors of this paper, said “The potential applications of iPSC-generated blood vessels are broad – from repairing damaged vessels supplying the heart or brain to preventing the need to amputate limbs because of the vascular complications of diabetes. But first we need to deal with such challenges as the variability of iPSC lines and the long-term safety issues involved in the use of these cells, which are being addressed by researchers around the world. We need better ways of engineering the specific type of endothelial cell needed for specific organs and functions.”