Blocking Differentiation is Enough to Turn Mature Cells into Stem Cells

Hiroshi Kawamoto led a collaboration between the RIKEN Center for Integrative Medical Science and other institutions in Japan and Europe that examined the possibility that adult cells can be maintained in a stem cell-like state where they can proliferate without undergoing differentiation. They discovered that in immune cells, blocking the activity of one transcription factor can maintain the cells in a stem cell-like state where they continue to proliferate and still have the capacity to differentiate into different mature cell types.

Kawamoto and his team genetically engineered hematopoietic progenitor cells from mice to overexpress the Id3 protein. Id3, or inhibitor of DNA binding 3, is an inhibitory protein that forms nonfunctional complexes with other transcription factors. In particular, Id3 inhibits so-called “E-proteins,” (such as TCF3) which drive the progenitor cells to differentiate into immune cells.

Overexpression of Id3, in addition to soaking the cells in a cocktail of cytokines, cause the cells to continue to divide as stem cells. However, when the cytokines were withdrawn, the cells differentiated into various types of immune cells.

Next, Kawamoto and his collaborators infused these engineered hematopoietic progenitors into mice that had been depleted of white blood cells. They discovered that their Id3-overexpressing cells could expand and replenish the white blood cell population of these.

In a follow-up experiment, Kawamoto and his crew recapitulated this experiment using human umbilical cord blood hematopoietic progenitors. Just like their mouse counterparts, these umbilical cord cells could be maintained in culture, and then, upon change of culture conditions, could differentiate into blood cells.

Because these cells can be kept in an undifferentiated state and can extensively proliferate, this culture system provides a model for studying the genetic and epigenetic basis of stem cell self-renewal. And it might also allow scientists to inexpensively grow large quantities of immune cells for regenerative medicine or immune therapies.

This work was published in Stem Cell Reports, October 2015 DOI: 10.1016/j.stemcr.2015.09.012.

Clincal Trial Validates Stem Cell-Based Treatments of Sickle Cell Disease in Adults

Santosh Saraf and his colleagues at the University of Illinois have used a low-dose irradiation/alemtuzumab plus stem cell transplant procedure to cure patients of sickle-cell disease. 12 adult patients have been cured of sickle-cell disease by means of a stem cell transplantation from a healthy, tissue-matched donor.

This new procedure obviates the need for chemotherapy to prepare the patient to receive transplanted cells and offers the possibility of curing tens of thousands of adults from sickle-cell disease.

Sickle cell disease is an inherited disease that primarily affects African-Americans born in the United States. The genetic lesion occurs in the beta-globin gene that causes hemoglobin molecules to assemble into filaments under low-oxygen conditions. These hemoglobin filaments deform red blood cells and cause them to plug small capillaries in tissues, causing severe pain, strokes and even death.

Fortunately, a bone marrow transplant from a healthy donor can cure sickle-cell disease, but few adults undergo such a procedure because the chemotherapeutic agents that are given to destroy the patient’s bone marrow leaves from susceptible to diseases, unable to make their own blood cells, and very weak and sick.

Fortunately, a gentler procedure that only partially ablate the patient’s bone marrow was developed at the National Institutes of Health ()NIH) in Bethesda, Maryland. Transplant physicians there have treated 30 patients, with an 87% success rate.

In the Phase I/II clinical trial at the University of Illinois, 92% of the patients treated with this gentler procedure that was developed at the NIH.

Approximately 90% of the 450 patients who received stem cells transplants for sickle-cell disease have been children. However, chemotherapy has been considered too risky for adult patients who are often weakened far more than children by it.

Adult sickle-cell patients live an average of 50 years with a combinations of blood transfusions and pain medicines to manage the pain crisis. However, their quality of life can be quite low. Now, with this chemotherapy-free procedure, adults with sickle-cell disease can be cured of their disease within one month of their transplant. They can even go back to work or school and operate in a pain-free fashion.

In the new procedure, patients receive immunosuppressive drugs just before the transplant, with a very low dose of whole body radiation. Alemtuzumab (Campath, Lemtrada) is a monoclonal antibody that binds to the CD52 glycoprotein on the surfaces of lymphocytes and elicits their destruction, but not the hematopoietic stem cells that gives rise to them.  Next, donor cells from a healthy a tissue-matched sibling or donor are transfused into the patient. Stem cells from the donor home to the bone marrow and produce healthy, new blood cells in large quantities. Patients must continue to take immunosuppressive drugs for at least a year.

In the University of Illinois trial, 13 patients between the ages of 17-40 were given transplants from the blood of a healthy, tissue-matched sibling. Donors must be tested for human leukocyte antigen (HLA) markers on the surfaces of cells. Ten different HLA markers must match between the donor and the recipient for the transplant to have the best chance of evading rejection. Physicians have transplanted two patients with good HLA matches, to their donor, but had a different blood type than the donor. In many cases, the sickle cells cannot be found in the blood after the transplant.

In all 13 patients, the transplanted cells successfully engrafted into the bone marrow of the patients, but one patient failed to follow the post-transplant therapy regimen and reverted to the original sickle-cell condition.

One year after the transplantation, the 12 successfully transplanted patients had normal hemoglobin concentrations in their blood and better cardiopulmonary function. They also reported significantly less pain and improved health and vitality,

For of the patients were able to stop post-transplantation immunotherapy, without transplant rejection or other complications.

“Adults with sickle-cell disease can be cured with chemotherapy – the main barrier that has stood in the way for so long,” said Damiano Rondelli, Professor of Medicine and Director of the Stem Cell Transplantation Program at the University of Illinois. “Our data provide more support that this therapy is safe and effective and prevents patients from living shortened lives, condemned to pain and progressive complications.”

These data were published in the journal Biology of Blood and Marrow Transplantation, 2015; DOI 10.1016/j.bbmt.2015.08.036.

Stem Cell-like Megakaryocyte Progenitors Replenish Platelets After Inflammatory Episodes

A paper that appeared in the journal Cell Stem Cell from the laboratory of Marieke A.G. Esters, from the Heidelberg Institute for Stem Cell Technology and Experimental Medicine in Heidelberg, Germany has answered a long-standing question about how our bodies regenerate platelets using so many of them.

When we suffer damage to our bodies from surgery, accidents, infections, or other physical insults, we tend to use a lot of platelets. Platelets are small cells in the blood that help the blood clot once we cut ourselves. Platelets, however, take some time to form. How then do we rapidly regenerate the platelet pool during such stressful conditions?

Esters and her team have shown that the bone marrow contains stem-like cell called a “single-lineage megakaryocyte-committed progenitor” or SL-MkPs. Platelets bud from a large cell called a “megakaryocyte,” and megakaryocytes form from the hematopoietic stem cells that reside in the bone marrow. Hematopoietic stem cells make all the blood cell that course through our blood vessels and continue to replace those cells throughout our lifetime. Hematopoietic stem cells personify what it means to be a multipotent stem cell.

Haas et al, graphical abstract 5.5x5.5

This newly-identified stem cell population, the SL-MkP actually shares many features with multipotent hematopoietic stem cells and provides a stem cell population that is lineage-restricted (that means they can only form one type of cell) for emergency purposes.

Normally, SL-MkPs are maintained in an inactive, almost sleep-like state. In this state, SL-MkPs do not contribute very much to making platelets in the blood. There is some gene expression in this sleepy state, but protein synthesis is turned way down.

In response to acute inflammation, SL-MkPs wake up and become activated. Upon activation, these cells ramp up protein synthesis and mature into full-blown SL-MkPs that efficiently replenishment of platelets that are lost during high levels of inflammation. Thus, there is an emergency system that accommodates platelet depletion during acute inflammation and replenishes the platelet pool.

Stem Cell Researchers Develop New Method to Treat Sickle Cell Disease

Stem cells researchers from the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at the University of California, Los Angeles (UCLA) have shown that a new stem cell gene therapy protocol can potentially lead to a one-time, lasting treatment for sickle-cell disease, which remains the nation’s most common inherited blood disorder.

This study was led by Dr. Donald Kohn and was published March 2 in the journal Blood. This paper details a method that repairs a mistake in the beta-globin that causes sickle-cell disease and, for the first time, shows that such a gene therapy technique can lead to the production of normal red blood cells.

People with sickle-cell disease are born with a mutation in their beta-globin gene.


Beta-globin is one of the protein chains that compose the protein hemoglobin. Hemoglobin is the protein in red blood cells that ferries oxygen from the lungs to the tissues and then returns to the lungs to load up with oxygen again and then goes back to the tissues. Red blood cells, which are made in the bone marrow, are packed from stem to stern with hemoglobin molecules, and normally are round, and slightly dished and flexible enough to squeeze through small capillary beds in tissues. The mutation in the beta-globin gene that causes sickle-cell disease, however, causes hemoglobin to form long, stiff rods of protein rather than tight, compactly packed clusters of hemoglobin. These protein rods deform the red blood cells and make them stiff, sickle-shaped, and unable to pass through tissue capillary beds.


These abnormally shaped red blood cells not only move poorly through blood vessels, but they also do not sufficiently carry oxygen to vital organs.

Sickle_cell 2

The stem cell gene therapy method described by Kohn and his colleagues corrects the mutation in the beta-globin gene in the bone marrow-based stem cells so that they produce normal, circular-shaped blood cells. The technique uses specially engineered enzymes, called zinc-finger nucleases, to eliminate the mutation and replace it with a corrected version that repairs the beta-globin mutation. Kohn’s research showed that this method has the potential to treat sickle-cell the disease if the gene therapy achieves higher levels of correction.

“This is a very exciting result,” said Dr. Kohn, professor of pediatrics and microbiology, immunology and molecular genetics. “It suggests the future direction for treating genetic diseases will be by correcting the specific mutation in a patient’s genetic code. Since sickle-cell disease was the first human genetic disease where we understood the fundamental gene defect, and since everyone with sickle-cell has the exact same mutation in the beta-globin gene, it is a great target for this gene correction method.”

Forcing Sugars on the Surfaces of Cord Blood Cell Increases Their Engraftment

When a child or adult needs new bone marrow, a bone marrow transplant from a donor is usually the only way to save their life. Without properly functioning bone marrow, the patient’s blood cells will die off, and there will be too few red blood cells to ferry oxygen to tissues or white blood cells to fight off infections.

An alternative to bone marrow from a bone marrow donor if umbilical cord blood. Umbilical cord blood does not require the rigorous tissue matching that bone marrow requires because the blood making stem cells from cord blood are immature and not as likely to cause tissue rejection reactions.. However, umbilical cord blood cells suffer from two drawbacks: low numbers of stem cells in cord blood and poor engraftment efficiencies.

Fortunately, some progress has been made at expanding blood-making stem cells from umbilical cord blood, and it is likely that such technologies might be ready for common use in the future. As to the poor engraftment efficiencies, a new paper in the journal Blood from the laboratory of Elizabeth J. Shpall at the University of Texas MD Anderson Cancer Center, in Houston, Texas reports a new way to increase cord blood stem cells engraftment efficiencies.

As previously discusses, delayed engraftment is one of the major limitations of cord blood transplantation (CBT). Delayed engraftment seems to be due to the diminished ability of the cord blood stem cells to home to the bone marrow. How are cells channeled to the bone marrow? A protein receptor called P- and E-selectins is expressed on the surfaces of bone marrow blood vessels. Cells that can bind these selectin receptors will pass from the circulation to the bone marrow. Thus binding selectin receptors is kind of like having the “password” for the bone marrow.

What does it take to bind the selectin proteins? Selectins bind to specific sugars that have been attached to proteins. These sugars are called “fucose” sugars. As it turns out, cord blood stem cells do not express robust levels of these fucosylated proteins. Could increasing the levels of fucosylated proteins on the surfaces of cord blood stem cells increase their engraftment? Shpall and her colleagues tested this hypothesis in patients with blood-based cancers.

Patients with blood cancers had their cancer-producing bone marrow stem cells destroyed with drugs and radiation. Then these same patients had their bone marrows refurbished with two units of umbilical cord blood. However, these cells in these cord blood units were treated with the enzyme fucosyltransferase-VI and guanosine diphosphate fucose for 30 minutes before transplantation. This treatment should have increased the content of fucosylated proteins on the surfaces of cells in the hope of enhancing their interaction with Selectin receptors on the surfaces of bone marrow capillaries.

The results of 22 patients enrolled in the trial were then compared with those for 31 historical controls who had undergone double unmanipulated CBT. There was a clear decrease in the length of time it took for cells to engraft into the bone marrow.  For example, the median time to neutrophil (a type of white blood cell) engraftment was 17 days (range 12-34) compared to 26 days (range, 11-48) for controls (P=0.0023). Platelet (a cell used in blood clotting) engraftment was also improved: median 35 days (range, 18-100) compared to 45 days (range, 27-120) for controls (P=0.0520).  These are significant differences.

These findings support show that treating cord blood cells with a rather inexpensive cocktail of enzymes for a short period of time before transplantation is a clinically feasible means to improve engraftment efficiency of CBT.  This is a small study.  Therefore, these data, though very hopeful, must be confirmed with larger studies.

Americord Registry Funds Research in the Use of Stem Cells for Cancer Patients

Headquartered in New York City, the Americord Registry is one of the leaders in umbilical cord blood, cord tissue and placenta tissue banking. Americord collects, processes, and stores newborn stem cells from umbilical cord blood for future medical or therapeutic use. These uses include the treatment of many blood diseases, including sickle-cell anemia and leukemia.

The Americord Registry has announced that it will fund a research project by the Masonic Cancer Center at the University of Minnesota. This research will examine the potential use of donor stem cells in patients who have been previously treated for three different cancers of the blood or bone marrow; lymphoma, myeloma, or chronic lymphocytic leukemia.

Masonic Cancer Center researchers would like to use donor stem cells to further treat patients who have previously received chemotherapy. Two chemotherapeutic agents, cyclophosphamide and busulfan, for example, arrests the growth of cancer cells, and additionally, prevents the patient’s immune system from rejecting implanted stem cells from a donor. Donated stem cells, for bone marrow or umbilical cord blood, will not share the same array of cell surface proteins as the patient, and might be rejected by the patient’s immune system. However, cancer patients who have been treated with chemotherapeutic agents might be able to tolerate implanted cells, since the anti-cancer drugs might also dull the immune system to the implanted stem cells. These donated stem cells may replace the patient’s immune cells and help destroy any remaining cancer cells.

Americord has a Corporate Giving Program that was established to support research into the therapeutic uses of stem cells from umbilical cord blood, cord tissue, and placenta tissue. The funding for this research comes from Americord’s Corporate Giving Program.

“Americord is committed to supporting the advancement of stem cell treatments and technologies,” said Americord CEO Martin Smithmyer. “We are excited about the research being done at the Masonic Cancer Center and the potential it has for future treatment options.”

The study at the Masonic Cancer Center began in February 2008 and is scheduled to be completed by January 2015. It is registered with in accordance with best practices and requirements of the U.S. Food and Drug Administration.

Directly Reprogramming Skin Cells into White Blood Cells

Scientists from the Salk Institute have, for the first time, directly converted human skin cells into transplantable white blood cells, which are the soldiers of the immune system that fight infections and invaders. This work could prompt the creation of new therapies that introduce new white blood cells into the body that can attack diseased or cancerous cells or augment immune responses for other conditions.

This work, which shows that only a small amount of genetic manipulation could prompt this direct conversion, was published in the journal Stem Cells.

“The process is quick and safe in mice,” says senior author Juan Carlos Izpisua Belmonte, who holds the Salk’s Roger Guillemin Chair. “It circumvents long-standing obstacles that have plagued the reprogramming of human cells for therapeutic and regenerative purposes.”

The problems that Izpisua Belmonte mentions, includes the long time (at least two months) numbingly tedious cell culture work it takes to produce, characterize and differentiate induced pluripotent stem (iPS) cells. Blood cells derived from iPSCs also have other obstacles: they engraft into organs or bone marrow poorly and can cause tumors.

The new method designed by Izpisua Belmonte and his team, however, only takes two weeks, does not produce tumors, and engrafts well.

“We tell skin cells to forget what they are and become what we tell them to be—in this case, white blood cells,” says one of the first authors and Salk researcher Ignacio Sancho-Martinez. “Only two biological molecules are needed to induce such cellular memory loss and to direct a new cell fate.”

This faster reprogramming technique developed by Belmonte’s team utilized a form of reprogramming that does not go through a pluripotency stage. Such techniques are called indirect lineage conversion or direct reprogramming. Belmonte’s group has demonstrated that such approaches can reprogram cells to form the cells that line blood vessels. Thus instead of de-differentiating cells into an embryonic stem cell-type stage, these cells are rewound just enough to instruct them to form the more than 200 cell types that constitute the human body.

Direct reprogramming used in this study uses a molecule called SOX2 to move the cells into a more plastic state. Then, the cells are transfected with a genetic factor called miRNA125b that drives the cells to become white blood cells. Belmonte and his group are presently conducting toxicology studies and cell transplantation proof-of-concept studies in advance of potential preclinical and clinical studies.

“It is fair to say that the promise of stem cell transplantation is now closer to realization,” Sancho-Martinez says.

Study co-authors include investigators from the Center of Regenerative Medicine in Barcelona, Spain, and the Centro de Investigacion Biomedica en Red de Enfermedades Raras in Madrid, Spain.