LIF Increases Muscle Satellite Expansion in Culture and Transplantation Efficiency

Transplantation of satellite stem cells, which are found in skeletal muscles, might potentially treat degenerative muscle diseases such as Duchenne muscular dystrophy. However, muscle satellite cells have an unfortunate tendency to lose their ability to be transplanted then they are grown in culture.

In order to generate enough cells for transplantation, the cells are isolated from the body and then they must be grown in culture. However, in order to properly grow in culture, the cells must be prevented from differentiating because fully differentiated cells stop growing and die soon after transplantation. Several growth factors, cytokines, and chemicals have been used in muscle satellite cell culture systems. Unfortunately, the optimal culture conditions required to maintain the undifferentiated state, inhibit differentiation, and enhance eventual transplantation efficiency have not yet been established satisfactorily.

Because it is impossible to extract enough satellite cells for therapeutic purposed from biopsies, these cells must be expanded in culture. However this very act of culturing satellite cells renders them inefficient for clinical purposes. How can we break away from this clinical catch-22?

Shin’ichi Takeda from the National Center of Neurology and Psychiatry and his colleagues have used growth factors to maintain muscle satellite cell efficiency during cell culture. In particular, Takeda and others used a growth factor called leukemia inhibitory factor (LIF). LIF effectively maintains the undifferentiated state of the satellite cells and enhances their expansion and transplantation efficiency. LIF is also thought to be involved in muscle regeneration.

This is the first study on the effect of LIF on the transplantation efficiency of primary satellite cells,” said Shin’ichi Takeda of the National Center of Neurology and Psychiatry. “This research enables us to get one step closer to the optimal culture conditions for muscle stem cells.”

The precise mechanisms by which LIF enhances transplantation efficiency remain unknown. Present work is trying to determine the downstream targets of LIF. Identifying the precise mechanisms by which LIF enhances satellite cell transplantation efficiency would help to clarify the functional importance of LIF in muscle regeneration, and, even more importantly, further its potential application in cell transplantation therapy.

The reference for this paper is: N. Ito et al., “Enhancement of Satellite Cell Transplantation Efficiency by Leukemia Inhibitory Factor,” Journal of Neuromuscular Diseases, 2016; 3 (2): 201. DOI: 10.3233/JND-160156.

Umbilical Cord Blood Mesenchymal Stem Cells do Not Cause Tumors in Rigorous Tests

Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) have the ability to self-renew and also can differentiate into a wide range of cell types. However, many clinicians and scientists fear that even these very useful cells might cause tumors.

To that end, Moon and colleagues from the Korean Institute of Toxicology have rigorously tested the tendency for hUBC-MSCs to cause tumors. They used a large battery of tests in living organisms and in culture. hUCB-MSCs were compared to MRC-5 and HeLa cells. MRC-5 cells are known to have no ability to cause tumors and HeLa cells have a robust ability to form tumors, and therefore, constitute negative and positive controls,

To evaluate the ability of cells to cause tumors, Moon and others examined the tendency of these cells to grow without being attached to a substratum. This is a hallmark of tumor cells and is called “anchorage-independent growth” (AIG). To assess AIG, the cells were grown in soft agar, which is a standard assay for AIG. hUCB-MSCs and MRC-5 cells formed few colonies in soft agar, but HeLa cells formed a greater number of larger colonies. This indicated that hUCB-MSCs and MRC-5 cells do not show AIG, a common trait of tumorigenic cells.

The next assay implanted these cells into live laboratory animals. hUCB-MSCs were implanted as a underneath the skin of BALB/c-nu mice (nasty creatures – they bite). All the mice implanted with hUCB-MSCs and NRC-5 cells showed any sign of tumors. Both gross and microscopic examination failed reveal any tumors. However, all mice transplanted with HeLa cells developed tumors that were clearly derived from the implanted cells.

These experiments, though somewhat mundane, rigorously demonstrate that hUCB-MSCs do not exhibit tumorigenic potential. This provides further evidence of these cells clinical applications.

The paper appeared in Toxicol Res. 2016 Jul;32(3):251-8. doi: 10.5487/TR.2016.32.3.251.

A New Tool for Gene Editing In Stem Cells Can Drive Changes in Cell Fate Without Causing Mutations

Recently, a new tool is now available to control gene expression in order to understand gene function and manipulate cell fate. This new tool is called CRIPSR/Cas9, which is a gene-editing tool that employs a genetic system that naturally occurs in bacteria, who use it as a protection against viruses. CRISPR/Cas9 allows scientists to precisely add, remove or replace specific sequences of DNA. It is the most efficient, inexpensive and easiest gene editing tool available to date.

Several laboratories have tried to use CRISPR/Cas9 to activate genes in cells, but such an effort has not always succeeded. However a research team at Hokkaido University’s Institute of Genetic Medicine has developed a powerful new method that uses CRISPR/Cas9 to do exactly that.

In cells, genes have an expression switch called “promoters.” Genes are switched off, or silenced, when their promoters are methylated, which means that islands of C-G bases have a methyl group (a –CH3 group) attached to the cytosine base. The Hokkaido University team wanted to turn an inactivated gene on. The ingeniously combined a DNA repair mechanism, called MMEJ (microhomology-mediated end-joining), with CRISPR/Cas9 to do this. They excised a methylated promoter using CRISPR/Cas9 and then used MMEJ to insert an unmethylated promoter. Thus, they replaced the off-switch signal with an on-switch signal.

DNA Methylation

The gene that was activated was the neural cell gene OLIG2 and the embryonic stem cell gene NANOG in order to test the efficiency of this technology in cultured cells. Within five days, they found evidence that the genes were robustly expressed. When they activated the OLIG2 gene in cultured human stem cells, the cells differentiated to neurons in seven days with high-efficiency.

Toru Kondo and his colleagues also discovered that their editing tool could be used to activate other silenced promoters. They also found that their system didn’t cause unwanted mutations in other non-target genes in the cells. According to Kondo, this gene editing tool has wide potential to manipulate gene expression, create genetic circuits, or to engineer cell fates.

See Shota Katayama et al., “A Powerful CRISPR/Cas9-Based Method for Targeted Transcriptional Activation,” Angewandte Chemie International Edition, 2016; 55(22): 6452 DOI: 10.1002/anie.201601708.

Using Small Molecules to Covert Donated Stomach Cells into Stem Cells that Can Cure Liver Disease

A collaboration between Yunfang Wang from the Tissue Engineering Lab at the Beijing Institute of Transfusion Medicine in Beijing, China and Xuetao Pei, from the Stem Cell and Regenerative Medicine Lab, also at the Beijing Institute of Transfusion Medicine has produced a remarkable result.

Wang and Pei and their colleagues have used small molecules to convert human gastric epithelial cells into endodermal progenitors.  Endodermal stem/progenitor cells have diverse potential applications in research and regenerative medicine.  They can differentiate into just about any cell type in the gastrointestinal tract, including liver cells, and pancreatic cells.  Thus these cells may have a variety of uses in research, and perhaps even in the clinic.

In this experiment, gastric epithelial cells were isolated from human donors and cultured in the presence of a carpet of tissue-specific mesenchymal cells as support cells to help the gastric epithelial cells survive in primary culture.  These cells were then treated with a cocktail of small molecules that drove the cells to dedifferentiate into induced endodermal progenitor cells (hiEndoPCs). These cells can grow in culture, expand rapidly and express a variety of stem cell markers.

In culture, the hiEndoPCs were able to differentiate into liver cells (hepatocytes, pancreatic endocrine cells, and intestinal epithelial cells.  The differentiation protocols used in this paper all involved the use of small molecules – no genetic engineering of the cells was necessary.

Small molceules that convert gastric epithelial cells into hiEndoPCs

Then liver cells made from hiEndoPCs were transplanted into mice that had messed up livers (Fah -/- Rag2-/-).  Fah-mutant mice lack the enzyme fumarylacetoacetate hydrolase, and without this enzyme, the liver is incapable to processing toxic ammonium, which builds up in the liver and kills it.  Rag2 mutations prevent the mouse from rejecting implanted livers cells.  In these experiments, transplanted hepatocytes rescued liver failure in these mice.  hiEndoPCs had the added advantage of no causing teratomas, like embryonic stem cells.

Human gastric epithelial cells are easily isolated from human donors without causing harm to the donor.  They can be isolated from patients of a variety of ages and this strategy can easily convert those cells into a cell population that is readily expandable and can be personalized into cells from treating patients with abnormal livers, pancreases, or intestinal tracts.  This fascinating proof-of-principle paper brings such personalized medicine one step closer.

The article was published in Cell Stem Cells 2016, 

Genetic Switch to Making More Blood-Making Stem Cells Found

A coalition of stem cell scientists, co-led in Canada by Dr. John Dick, Senior Scientist, Princess Margaret Cancer Centre, University Health Network (UHN) and Professor, Department of Molecular Genetics, University of Toronto, and in the Netherlands by Dr. Gerald de Haan, Scientific Co-Director, European Institute for the Biology of Ageing, University Medical Centre Groningen, the Netherlands, have uncovered a genetic switch that can potentially increase the supply of stem cells for cancer patients who need transplantation therapy to fight their disease.

Their findings were published in the journal Cell Stem Cell and constitute proof-of-concept experiments that may provide a viable new approach to making more stem cells from umbilical cord blood.

“Stem cells are rare in cord blood and often there are not enough present in a typical collection to be useful for human transplantation. The goal is to find ways to make more of them and enable more patients to make use of blood stem cell therapy,” says Dr. Dick. “Our discovery shows a method that could be harnessed over the long-term into a clinical therapy and we could take advantage of cord blood being collected in various public banks that are now growing across the country.”

Currently, all patients who require stem cell transplants must be matched to an adult donor. The donor and the recipient must share a common set of cell surface proteins called “human leukocyte antigens” HLAs. HLAs are found on the surfaces of all nucleated cells in our bodies and these proteins are encoded by a cluster of genes called the “Major Histocompatibility Complex,” (MHC) which is found on chromosome six.

Map of MHC

There are two main types of MHC genes: Class I and Class II.

MHC Functions

Class I MHC contains three genes (HLA-A, B, and C). The three proteins encoded by these genes, HLA-A, -B, & -C, are found on the surfaces of almost all cells in our bodies. The exceptions are red blood cells and platelets, which do not have nuclei. Class II MHC genes consist of HLA-DR, DQ, and DP, and the proteins encoded by these genes are exclusive found on the surfaces of immune cells called “antigen-presenting cells” (includes macrophages, dendritic cells and B cells). Antigen-presenting cells recognize foreign substances in our bodies, grab them and, if you will, hold them up for everyone to see. The cells that usually respond to antigen presentation are immune cells called “T-cells.” T-cells are equipped with an antigen receptor that only binds antigens when those antigens are complexed with HLA proteins.

If you are given cells from another person who is genetically distinct from you, the HLA proteins on the surfaces of those cells are recognized by antigen-presenting cells as foreign substances. The antigen-presenting cells will them present pieces of the foreign HLA proteins on their surfaces, and T-cells will be sensitized to those proteins. These T-cells will them attack and destroy any cells in your body that have those foreign HLA proteins. This is the basis of transplant rejection and is the main reason transplant patients must continue to take drugs that prevent their T-cells from recognizing foreign HLA proteins as foreign.

When it comes to bone marrow transplantations, patients can almost never find a donor whose HLA surface proteins match perfectly. However, if the HLA proteins of the donor are too different from those of the recipient, then the cells from the bone marrow transplant attack the recipient’s cells and destroy them. This is called “Graft versus Host Disease” (GVHD). The inability of leukemia and lymphoma and other patients to receive bone marrow transplants is the unavailability of matching bone marrow. Globally, many thousands of patients are unable to get stem cell transplants needed to combat blood cancers such as leukemia because there is no donor match.

“About 40,000 people receive stem cell transplants each year, but that represents only about one-third of the patients who require this therapy,” says Dr. Dick. “That’s why there is a big push in research to explore cord blood as a source because it is readily available and increases the opportunity to find tissue matches. The key is to expand stem cells from cord blood to make many more samples available to meet this need. And we’re making progress.”

Umbilical cord blood, however, is different from adult bone marrow. The cells in umbilical cord blood are more immature and not nearly as likely to generate GVHD. Therefore, less perfect HLA matches can be used to treat patients in need of a bone marrow transplant. Unfortunately, umbilical cord blood has the drawback of have far fewer stem cells than adult bone marrow. If the number of blood-making (hematopoietic) stem cells in umbilical cord blood can be increased, then umbilical cord blood would become even more useful from a clinical perspective.

There has been a good deal of research into expanding the number of stem cells present in cord blood, the Dick/de Haan teams took a different approach. When a stem cell divides it produces a large number of “progenitor cells” that retain key properties of being able to develop into every one of the 10 mature blood cell types. These progenitor cells, however, have lost the critical ability to self-renew.

Dick and his colleagues analyzed mouse and human models of blood development, and they discovered that a microRNA called miR-125a is a genetic switch that is on in stem cells and controls self-renewal, but gets turned off in the progenitor cells.

“Our work shows that if we artificially throw the switch on in those downstream cells, we can endow them with stemness and they basically become stem cells and can be maintained over the long-term,” says Dr. Dick.

In their paper, Dick and de Haan showed that forced expression of miR-125 increases the number of hematopoietic stem cells in a living animal. Also, miR-125 induces stem cell potential in murine and human progenitor cells, and represses, among others, targets of the MAP kinase signaling pathway, which is important in differentiation of cells away from the stem cell fate. Furthermore, since miR-125 function and targets are conserved in human and mouse, what works in mice might very well work in human patients.

graphical abstract CSC_v9

This is proof-of-concept paper – no human trials have been conducted to date, but these data may be the beginnings of making more stem cells from banked cord blood to cure a variety of blood-based conditions.

Here’s to hoping.

How Stem Cells Exit The Bloodstream

New research from a laboratory at North Carolina State University has changed our understanding of how therapeutic stem cells exit the bloodstream.  Understanding this new process, which has been given the name “angiopellosis” may not only increase our understanding of how intravenous stem cells home to their target tissues, but also how metastatic cancer cells invade new sites.

When white blood cells are summoned to a site of infection, they exit the bloodstream by means of a rather well understood process called “Leukocyte extravasation” or “diapedesis.”.

Leukocyte extravasation mostly occurs in post-capillary venules, where hemodynamic shear force are low.  This process is characterized by 4 steps: 1)  “chemoattraction;” 2)  “rolling adhesion;” 3) “tight adhesion;” and 4)  “(endothelial) transmigration.”. If any of these steps are inhibited, diapedesis does not occur.

White blood cells or leukocytes phagocytose or gobble up foreign particles, produce antibodies, secrete inflammatory response triggers (histamine and heparin), and neutralize histamine.  In general, leukocytes defend an organism and protect it from disease by promoting or inhibiting inflammatory responses. Leukocytes do most of their specific functions in tissues and they use the blood as a transport medium to reach the tissues of the body.


Below is a brief summary of each of the four steps involved in leukocyte extravasation:

1) Chemoattraction

Upon recognition of and activation by pathogenic organisms, resident macrophages in the affected tissue release small signaling proteins called “cytokines” such as IL-1, TNFα and chemokines (small molecules that induce cell migration). IL-1, TNFα and other blood-based  molecules induce the endothelial cells that line blood vessels near the site of infection to express cellular adhesion molecules, including selectins.  Circulating leukocytes are localized to the site of injury or infection as a result of secreted chemokines.

2) Rolling adhesion

Sugar residues on they surfaces of circulating leukocytes bind to these selectin molecules on the inner wall of the blood vessels.  This interaction, however, is relatively modest in its binding strength.  The sugar-selectin interaction causes the leukocytes to roll along the inner surface of the vessel wall as transient bounds are constantly broken and reformed between selectins and cell-bound sugars.

The carbohydrate binding partner for P-selectin, P-selectin glycoprotein ligand-1 (PSGL-1), is an expressed by different types of leukocytes. The binding of PSGL-1 on the leukocyte to P-selectin on the endothelial cell allows for the leukocyte to roll along the endothelial surface. This interaction can be fine-tuned by the different ways that sugars are attached to PSGL-1.   These different forms of PSGL-1 that have distinct patterns of sugar attachment have unique affinities for different selectins.  This gives different leukocytes varying abilities to migrate to distinct specific sites within the body.

3) Tight adhesion

The chemokines released by macrophages activate the rolling leukocytes and induce them to synthesize surface integrin molecules.   Integrin molecules create high-affinity associations between cells and bind tightly to complementary receptors expressed on endothelial cells. This immobilized the leukocytes, despite the shear forces of the ongoing blood flow.

4) Transmigration

The internal cytoskeleton of the leukocytes are reorganizes that the leukocytes spread out over the endothelial cells. In this form, leukocytes extend pseudopodia and pass through gaps between endothelial cells.  This migratory step requires the expression of PECAM proteins on both the surface  of the leukocytes and the endothelial cells.  PECAM interaction effectively pulls the cell through the endothelium. Once through the endothelium, the leukocyte must penetrate the underlying basement membrane.  The mechanism by which the leukocytes does this remains a source of some dispute.  Once in the interstitial fluid, leukocytes migrate along a gradient of attractant molecules towards the site of injury or infection.

When stem cells are administered intravenously, they too Havre a similar ability to leave the bloodstream, but the means by which they do so was poorly understood.

Ke Cheng and colleagues examined zebrafish and used genetically engineered fish whose blood vessels glowed a fluorescent color.  Next, these fish were injected with leukocytes, and stem cells from rats, humans, and dogs that had been labeled with a red fluorescent protein.  These cells were followed by means of time-lapsed, three-dimensional light sheet microscopic imaging.  This technology allowed Cheng and others to view the stem cells as they left the blood vessels.

As predicted, the leukocytes exited the bloodstream by means of leukocytes extravasation.  The stem cells, however, were actively expelled from the blood vessels by the endothelial cells.  The endothelial cells membranes moved around the stem cells, surrounded them, moved them through the endothelial cells and then extruded them on the opposite side of the blood vessel.  This is a very different process than diapedesis in which the leukocyte is the active participant.  In the case of the stem cells, the endothelial cells are the active participants and the stem cells passively exit the bloodstream.  Cheng and company called this process angiopellosis.

Other differences between angiopellosis and diapedesis involved the time of the process.  Diapedesis can occur rather quickly whereas angiopellosis takes hours.  During diapedesis, one cell moves at a time, but during angiopellosis, several cells are moved at a time.

How effective of a method is this to leave the bloodstream?  If cancer cells used angiopellosis to facilitate metastasis, cent we inhibit it?

Further work should answer these important questions.  This work was published in the journal Stem Cells, 2016; DOI:10.1002/stem.2451.

Antiaging Glycoprotein Quadruples Viability of Stem Cells in Retina

When pluripotent stem cells are differentiated into photoreceptor cells, and then implanted into the retina at the back of the eye of a laboratory animal, they do not always survive.  However, pre-treatment of those cells with an antiaging glycoprotein (AAGP), made by ProtoKinetix, causes those transplanted cells to be 300 times more viable than cells not treated with this protein according to a study recently accepted for publication.

AAGP was invented by Dr. Geraldine-Castelot-Deliencourt and developed in partnership with the Institute for Scientific Application (INSA) of France. For her work in this area Dr. Castelot-Deliencourt was honored with France’s highest award for scientific accomplishment, the Francinov Award, in 2006.

ProtoKinetix, Incorporated said that a paper submitted by Kevin Gregory-Evans on the company’s AAGP was accepted for publication by the Journal of Tissue Engineering and Regenerative Medicine for publication.

AAGP significantly improves the viable yield of stem cells transplanted in retinal tissue, according to experiments conducted at the University of British Columbia in the laboratory of Dr. Kevin Gregory-Evans.

AAGP seems to protect cells from inflammation-induced cell death. This is based on experiments in which cultured cells that were treated with AAGP were significantly more resistant to hydrogen peroxide, ultraviolet A (wavelengths of 320-400 nanometers), and ultraviolet C (shorter than 290 nm). In addition, when exposed to an inflammatory mediator, interleukin β (ILβ), AAGP exposure reduced COX-2 expression three-fold. COX-2 is an enzyme that is induced by the various stimuli that stimulate Inflammation. It is, therefore, an excellent read-out of the degree to which inflammation has been induced. The fact that AAGP prevented the induction of COX-2 shows that this protein can inhibit the induction of inflammation. These data suggest that AAGP™ may not just be usable in cell and organ storage but also in pharmacological treatments.