The Mechanism Behind Blood Stem Cell Longevity


The blood stem cells that live in bone marrow divide and send their progeny down various pathways that ultimately produce red cells, white cells and platelets. These “daughter” cells must be produced at a rate of about one million cells per second in order to constantly replenish the body’s blood supply.

A nagging question is how these stem cells to persist for decades even though their progeny last for days, weeks or months before they need to be replaced. A study from the University of Pennsylvania has uncovered one of the mechanisms, and these cellular mechanisms allow these stem cells to keep dividing in perpetuity.

Dennis Discher and his colleagues in the Department of Chemical and Biomolecular Engineering in the School of Engineering and Applied Science found that a form of a protein called “myosin,” the motor protein that allow muscles to contract, helps bone marrow stem cells divide asymmetrically. This asymmetric cell division helps one cell remains a stem cell while the other cell becomes a daughter cell. Discher’s findings might provide new insights into blood cancers, such as leukemia, and eventually lead to ways of growing transfusable blood cells in a laboratory.

The participants in this study were members of the Discher laboratory, which include lead author Jae-Won Shin, Amnon Buxboim, Kyle R. Spinler, Joe Swift, Dave P. Dingal, Irena L. Ivanovska and Florian Rehfeldt. Discher collaborated with researchers at the Univ. de Strasbourg, Lawrence Berkeley National Laboratory and Univ. of California, San Francisco. This paper was published in Cell Stem Cell.

“Your blood cells are constantly getting worn out and replaced,” Discher said. “We want to understand how the stem cells responsible for making these cells can last for decades without being exhausted.”

Presently, scientists understand the near immortality of hematopoietic stem cells (HSCs) as a result of their asymmetric cell division, although how this asymmetric cell division enables stem cell longevity was unknown. To ferret out this mechanism, Discher and his coworkers analyzed all of the genes expressed in the stem cells and compared them with the genes expression in their more rapidly dividing progeny. Those proteins that only went to one side of the dividing cell might play a role in partitioning other key factors responsible for keeping one of the cells a stem cell and the other a progeny cell.

One of the proteins that showed a distinct expression pattern was the motor protein myosin II, which has two forms, myosin A and myosin B. Myosin II is the protein that enables the body’s muscles to contract, but in nonmuscle cells also it used during cell division. During the last phase of cell division, known as cytokinesis, myosin II helps cleave and close off the cell membranes as the cell splits apart.

“We found that the stem cell has both types of myosin,” Shin said, “whereas the final red and white blood cells only had the A form. We inferred that the B form was key to splitting the stem cells in an asymmetric way that kept the B form only in the stem cell.”

With these myosins as their top candidate, Discher and others labeled key proteins in dividing stem cells with different colors and put them under the microscope.

“We could see that the myosin IIB goes to one side of the dividing cell, which causes it to cleave differently,” Discher said. ”It’s like a tug of war, and the side with the B pulls harder and stays a stem cell.”

The researchers then performed in vivo tests using mice that had human stem cells injected into their bone marrow. By genetically inhibiting myosin IIB production, Shin and others saw the stem cells and their early progeny proliferating while the amount of downstream blood cells dropped.

“Because the stem cells were not able to divide asymmetrically, they just kept making more of themselves in the marrow at the expense of the differentiated cells,” Discher said.

HSC cell division mechanism

Discher and his team then used a drug that temporarily blocked both myosin A and myosin B. They observed that myosin inhibition increased the prevalence of non-dividing stem cells, blocking the more rapid division of progeny.

Discher believes that these findings could eventually help regrow blood stem cells after chemotherapy treatments for blood cancers or even grow blood products in the lab. Finding a drug that can temporarily shut down only the B form of myosin, while leaving the A form alone, would allow the stem cells to divide symmetrically and make more of themselves without preventing their progeny from dividing themselves.

“Nonetheless, the currently available drug that blocks both the A and B forms of myosin II could be useful in the clinic,” Shin said, “because donor bone marrow cultures can now easily be enriched for blood stem cells, and those are the cells of interest in transplants. Understanding the forces that stem cells use to divide can thus be exploited to better control these important cells.”

Producing blood cells from stem cells could yield a purer, safer cell therapy


The journal Stem Cells Translational Medicine has published a new protocol for reprogramming induced pluripotent stem cells (iPSCs) into mature blood cells. This protocol uses only a small amount of the patient’s own blood and a readily available cell type. This novel method skips the generally accepted process of mixing iPSCs with either mouse or human stromal cells. Therefore, is ensures that no outside viruses or exogenous DNA contaminates the reprogrammed cells. Such a protocol could lead to a purer, safer therapeutic grade of stem cells for use in regenerative medicine.

The potential for the field of regenerative medicine has been greatly advanced by the discovery of iPSCs. These cells allow for the production of patient-specific iPSCs from the individual for potential autologous treatment, or treatment that uses the patient’s own cells. Such a strategy avoids the possibility of rejection and numerous other harmful side effects.

CD34+ cells are found in bone marrow and are involved with the production of new red and white blood cells. However, collecting enough CD34+ cells from a patient to produce enough blood for therapeutic purposes usually requires a large volume of blood from the patient. However, a new study outlined But scientists found a way around this, as outlined by Yuet Wai Kan, M.D., FRS, and Lin Ye, Ph.D. from the Department of Medicine and Institute for Human Genetic, University of California-San Francisco has devised a way around this impasse.

“We used Sendai viral vectors to generate iPSCs efficiently from adult mobilized CD34+ and peripheral blood mononuclear cells (MNCs),” Dr. Kan explained. “Sendai virus is an RNA virus that carries no risk of altering the host genome, so is considered an efficient solution for generating safe iPSC.”

“Just 2 milliliters of blood yielded iPS cells from which hematopoietic stem and progenitor cells could be generated. These cells could contain up to 40 percent CD34+ cells, of which approximately 25 percent were the type of precursors that could be differentiated into mature blood cells. These interesting findings reveal a protocol for the generation iPSCs using a readily available cell type,” Dr. Ye added. “We also found that MNCs can be efficiently reprogrammed into iPSCs as readily as CD34+ cells. Furthermore, these MNCs derived iPSCs can be terminally differentiated into mature blood cells.”

“This method, which uses only a small blood sample, may represent an option for generating iPSCs that maintains their genomic integrity,” said Anthony Atala, MD, Editor of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine. “The fact that these cells were differentiated into mature blood cells suggests their use in blood diseases.”

Using Sleeping Stem Cells to Treat Aggressive Leukemias


British scientists have discovered that aggressive forms of leukemia (blood cancers) do not displace normal stem cells from the bone marrow, but instead, put them to sleep. If the normal stem cells are asleep, it implies that they can be awakened. This offers a new treatment strategy for acute myeloid leukemia or AML.

This work comes from researchers at Queen Mary, University of London with the support of Cancer Research UK’s London Research Institute.

In the United Kingdom, approximately 2,500 people are diagnosed with AML each year. The disease strikes young and old patients and the majority of patients die from AML.

In healthy patients, the bone marrow contains hematopoietic stem cells (HSCs) that divide to form either a common myeloid precursor (CMP) or a common lymphoid precursor (CLP) that differentiate into various kinds of white blood cells or red blood cells or lymphocytes. Individuals afflicted with AML, however, have bone marrow invaded by leukemic myeloid blood cells. Since red blood cells are derived from the myeloid lineage, AML causes red blood cell deficiencies (anemia), and the patient becomes tired, and is at risk for excessive bleeding. AML patients are also more vulnerable to infection those white blood cells that fight infections are not properly formed.

HSC differentiation2

David Taussig from the Barts Center Institute at Queen Mary, University of London said that the widely accepted explanation for these symptoms is that the cancerous stem cells displace or destroy the normal HSCs.

However, Taussig and his colleagues have found in bone marrow samples from mice and humans with AML contain plenty of normal HSCs. Thus, AML is not destroying or displacing the HSCs. Instead, the cancerous stem cells appear to be turning them off so that they cannot form HSCs. If Taussig and his coworkers and collaborators had determine how these leukemic myeloid blood cells are shutting off the normal HSCs, they might be able to design treatments to turn them back on.

Such a treatment strategy would increase the survival of AML patients. Only 40% of younger patients are cured of AML, and the cure rate for older patients in much lower. Current treatments that include chemotherapy and bone marrow transplants are not terribly successful with older patients.

Taussig’s group examined the levels of HSCs in the bone marrow of mice that had been transplanted with human leukemic myeloid cells from AML patients. They discovered that the numbers of HSCs stayed the same, but these same HSCs failed to transition through the developmental stages that result in the formation of new blood cells. When Taussig and his group examined bone marrow from 16 human AML patients, they discovered a very similar result.

Even though AML treatment has come a long way in the last ten years, there is still an urgent need for more effective treatments to improve long-term survival. This present study greatly advances our understanding of what’s going on in the bone marrow of AML patients. The future challenge is to turn this knowledge into treatments.

Under normal circumstances, stress on the body will boost HSC activity. For example, when the patient hemorrhages, the HSCs kick into action to produce more red blood cells that were lost during the bleed. However, the cancer cells in the bone marrow are somehow over-riding this compensatory mechanism and the next phase of this research will determine exactly how they do it.

Stem Cells to Make Red Blood Cells and Platelets in Culture


A collaborative study between Boston University School of Public Health and researchers at Boston Medical Center has used induced pluripotent stem cells to make unlimited numbers of human red blood cells and platelets in culture.

This finding could potentially reduce the need for blood donations to treat patients who require blood transfusions. Such research could also help researchers examine fresh and new therapeutic targets in order to treat blood diseases such a sickle-cell anemia.

The lead scientist on this project was George Murphy, assistant professor of medicine at Boston University School of Medicine and co-director of the Center for Regenerative Medicine at Boston University. Murphy’s main collaborator was David Sherr, professor of environmental health at Boston University School of Medicine and the Boston University School of Public Health.

Induced pluripotent stem cells or iPSCs are made from adult cells by applying genetic engineering technology to the adult cells that introduces genes into them. The introduction of four specific genes de-differentiates the adult cells into pluripotent stem cells that can, potentially, differentiate into any adult cell type. This makes iPSCs powerful tools for research and potential therapeutic agents for regenerative medicine.

In this study, Murphy and others used iPSCs from the CreM iPS Cell Bank and exposed them to a battery of different growth factors in order to push them to differentiate into different adult cell types. They were looking for the precise cocktail to differentiate iPSCs into red blood cells, since they wanted to further study red blood cell development in detail.

One group of compounds given to the set of iPSCs were molecules that activate “aryl hydrocarbon” receptors. Aryl hydrocarbon receptors (AHRs) play important roles in the expansion of hematopoietic stem cells, which make blood cells, since antagonism of AHRs promotes expansion of hematopoietic stem cells (see AE Boitano et al.,Science 10 September 2010: Vol. 329 no. 5997 pp. 1345-1348). In this case, however, Murphy and his colleagues observed a dramatic increase in the production of functional red blood cells and platelets in a short period of time. THis suggests that the ARH is important for normal blood cell development.

Aryl Hydrocarbon Receptor
Aryl Hydrocarbon Receptor

“This finding has enabled us to overcome a major hurdle in terms of being able to produce enough of these cells to have a potential therapeutic impact both in the lab and, down the line, in patients,” said Murphy. “Additionally, our work suggests that AHR has a very important biological function in how blood cells form in the body.”

“Patient-specific red blood cells and platelets derived from iPSC cells, which would solve problems related to immunogenicity and contamination, could potentially be used therapeutically and decrease the anticipated shortage and the need for blood donation,” added Murphy.

iPS-derived cells have tremendous potential as model systems in which scientists can test and develop new treatments for disease, given that such diseases can be constructed in the laboratory. These iPSC-derived red blood cells could be used by malaria researchers, and IPSC-derived platelets could be used to explore cardiovascular disease and treatments for blood clotting disorders.

Because my mother died from myelodysplasia, this finding has some personal interest to me. Mom had a difficult blood type to match, since she had the Bombay blood type (H). Finding blood for her was a major tour de force, and as she received blood that was less and less well matched to her body, she suffered the ravages of poorly matched blood. A treatment of red blood cells made from IPSCs derived from her own cells might have extended her life and even improved her quality of life in her later years.

I look forward to this research eventually culminating in clinical trials.

Discarded White Blood Cells Induce Relocation of Blood Stem Cells


Researchers at the Fundación Centro Nacional de Investigaciones Cardiovasculares or CNIC in Madrid, Spain have discovered that the clearance of the white blood cells called neutrophils induces the release of blood cell making stem cells into the bloodstream.

Our blood consists of a liquid component known as plasma and cells collectively known as “formed elements.” Formed elements include red blood cells and a whole encore of white blood cells. Red blood cells contain hemoglobin that ferry oxygen from the lungs to the tissues. White blood cells come in two flavors: granulocytes, which contain granules, and agranulocytes, which are devoid of granules.

Granulocytes are a subgroup of white blood cells characterized by the presence of cytoplasmic granules. Granulocytes are formed in the bone marrow and can be classified as basophils, eosinophils, or neutrophils. These particular cell types are named according to their distinct staining characteristics using hematoxylin and eosin (H&E) histological preparations. Granules in basophils stain dark blue, eosinophilic components stain bright red, and neutrophilic components stain a neutral pink.

Granulocytes

The most abundant white blood cells is known as a neutrophil. Neutrophils comprise 50-70% of all white blood cells and are a critical component of the immune system. When immature, neutrophils have a distinct band-shaped nucleus that changes into a segmented nucleus following maturation. Neutrophils are normally in circulating blood, but they migrate to sites of infection via chemotaxis under the direction of molecules such as Leukotriene B4. The main function of neutrophils is to destroy microorganisms and foreign particles by phagocytosis.

Granulocytes-blood smear

Because neutrophils are packed with granules that are toxic to microorganisms and our own cells, damaged neutrophils can spill a plethora of pernicious chemicals into our bodies. To prevent neutrophils from aging and becoming a problem, they live hard and die young. in the vicinity of 1011 neutrophils are eliminated every day. They are rapidly replaced, however, and the means of replacement includes stem cell mobilization from the bone marrow to the bloodstream.

Workers in the laboratory of Andrés Hidalgo have discovered what happens to the discarded neutrophils. Earlier work in mice showed that injections of dead or dying neutrophils increase the number of circulating blood cell-making stem cells. Therefore, something about dead neutrophils causes the hematopoietic stem cells to move from the bone marrow to the bloodstream. By following marked, dying neutrophils, Hidalgo and his coworkers showed that the neutrophils went to the bone marrow to die. While in the bone marrow, the dying neutrophils were phagocytosed (gobbled up) by special cells called macrophages.

Once these bone marrow-located macrophages phagocytose aged neutrophils, they begin to signal to hematopoietic stem cells in the bone marrow, and these signals drive them to move from the bone marrow to the bloodstream to replenish the neutrophil population.

Hidalgo admits that even though his research has produced some unique answers to age-old questions, it also poses almost as many questions as it answers. For example, Hidalgo and his colleagues showed that neutrophils follow a circadian or day/night rhythm and this has implications for diseases. For instance, the vast majority of heart attacks are in the morning. Does this have something to do with neutrophil aging cycles?

“Our study shows that stem cells are affected by day/night cycles thanks to this cell recycling . It is possible that the malign stem cells that cause cancer use this mechanism to relocate, for example, during metastasis,” said Hidalgo.

Daily changes in neutrophil function could be part of the reason that acute cardiovascular and inflammatory events such as heart attack, sepsis or stroke tend to occur during particular times of the day.

“Given that this new discovery describes fundamental processes in the body that were unknown before, it will now be possible to interpret the alterations to certain physiological patterns that occur in many diseases,” Hidalgo said.

See Cell 2013; 153(5): 1025 DOI:10.1016/j.cell.2013.04.040.

The Nooks and Crannies in Bone Marrow that Nurture Stem Cells


Stems cells in our bodies often require a specific environment to maximize their survival and efficiency. These specialized locations that nurture stem cells is called a stem cell niche. Finding the right niche for a stem cell population can go a long way toward growing more stem cells in culture and increasing their potency.

To that end, a recent discovery has identified the distinct niches that exist in bone marrow for hematopoietic stem cells (HSCs), which form the blood cells in our bodies.

A research team from Washington university School of Medicine in St. Louis has shown that stem niches in bone marrow can be targeted, which may potentially improve bone marrow transplants and cancer chemotherapy. Drugs that support particular niches could encourage stem cells to establish themselves in the bone marrow, which would greatly increase the success rate of bone marrow transplants. Alternatively, tumor cells are known to hide in stem cell niches, and if drugs could disrupt such niches, then the tumor cells would be driven from the niches and become more susceptible to chemotherapeutic agents.

Daniel Link, the Alan A. and Edith L. Wolff Professor of Medicine at Washington University, said, “Our results offer hope for targeting these niches to treat specific cancers or to impress the success of stem cell transplants. Already, we and others are leading clinical trials to evaluate whether it is possible to disrupt these niches in patients with leukemia or multiple myeloma.”

Working in mice, Link and his colleagues deleted a gene called CXCL12, only in “candidate niche stromal cell populations.” CXCL12 which encodes a receptor protein known to be crucial for maintaining HSC function, including retaining HSCs in the bone marrow, controlling  HSCs activity, and repopulating the bone marrow with HSCs after injury.

CXCL12 crystal structure
CXCL12 crystal structure

CXCL12 signaling pathways

In bone marrow, HSCs are surrounded by a whole host of cells, and it is difficult to precisely identify which type of cells serve as the niche cells. These bone marrow cells are known collectively as “stroma,” but there are several different types of cells in stroma. Cells that have been implicated in the HSC niche include endosteal osteoblasts (osteoblasts are bone-making cells and the endosteum in the layer of connective tissue that lines the inner cavity of the bone), perivascular stromal cells (cells that hang out around blood vessels), CXCL12-abundant reticular cells, leptin-receptor-positive stromal cells, and nestin–positive mesenchymal progenitors. Basically, there are a lot of cells in the stroma and figuring out which one is the HSC niche is a big deal.

bone marrow stromal cells

When HSCs divide, they form two cells, one of which replaces the HSC that just divided and a new cells called a hematopoietic progenitor cell (HPC), which can divide and differentiate into either a lymphoid progenitor or a myeloid progenitor. The lymphoid progenitor differentiates into either a B or T lymphocyte and the myeloid progenitor differentiates into a red blood cell, or other types of white blood cells (neutrophil, basophil, macrophage, platelet or eosinophil). As the cells become more differentiated, they lose their capacity to divide.

HSC differentiation

Deleting CXCL12 from mineralizing osteoblasts (bone making cells) did nothing to the HSCs or those cells that form lymphocytes (lymphoid progenitors). Deletion of Cxcl12 from osterix-expressing stromal cells, which include CXCL12-abundant reticular cells and osteoblasts, causes mobilization of hematopoietic progenitor cells (HPCs) from the bone marrow into the bloodstream, and loss of B-lymphoid progenitors, but HSC function is normal. Cxcl12 deletion from blood vessel cells causes a modest loss of long-term repopulating activity. Deletion of Cxcl12 from nestin-negative mesenchymal progenitors causes a marked loss of HSCs, long-term repopulating activity, and lymphoid progenitors. All of these data suggest that osterix-expressing stromal cells comprise a distinct niche that supports B-lymphoid progenitors and retains HPCs in the bone marrow. Also, the expression of CXCL12 from stromal cells in the perivascular region, including endothelial cells and mesenchymal progenitors, supports HSCs.

Link summarized his results this way: “What we found was rather surprising. There’s not just one niche for developing blood cells in the bone marrow. There’s a distinct niche for stem cells, which have the ability to become any blood cell in the body, and a separate niche for infection-fighting cells that are destined to become T cells and B cells.”

These data provide the foundation for future investigations whether disrupting these niches can improve the effectiveness of cancer chemotherapy.

In a phase 2 study at Washington University, led by oncologist Geoffrey Uy, assistant professor of medicine, Link and his team are evaluating whether the drug G-CSF (granulocyte colony stimulating growth factor) can alter the stem cell niche in patients with acute lymphoblastic leukemia and whose disease is resistant to chemotherapy or has recurred. The FDA approved this drug more than 20 years ago to stimulate the production of white blood cells in patients undergoing chemotherapy, who have often weakened immune systems and are prone to infections.

Uy and his colleagues want to evaluate G-CSF if it is given prior to chemotherapy. Patients enrolled at the Siteman Cancer Center will receive G-CSF for five days before starting chemotherapy, and the investigators will determine whether it can disrupt the protective environment of the bone marrow and make cancer cells more sensitive to chemotherapy.

This trial is ongoing, and the results are not yet in, but Link’s work has received a welcome corroboration of his work. A companion paper was published in the same issue of Nature by Sean Morrison, the director of the Children’s Medical Center Research Institute at the University of Texas Southwestern Medical Center in Dallas. Morrison and his team used similar methods as Link and his colleagues and came to very similar conclusions.

Link said, “There’s a lot of interest right now in trying to understand these niches. Both of these studies add new information that will be important as we move forward. Next, we hope to understand how stem cells niches can be manipulated to help patients undergoing stem cell transplants.”