The Founder Cell Identity Does Not Affect iPS Cell Differentiation to Hematopoietic Stem Cell Fate

Induced pluripotent stem cells (iPSCs) have many of the characteristics of embryonic stem cells, but are made from mature cells by means of a process called cell reprogramming. To reprogram cells, particular genes are delivered into mature cells, which are then cultured until they h:ave the growth properties of pluripotent cells. Further tests are required to demonstrate that the growing cells actually are iPSCs, but once they pass these tests, these cells can be grown in culture indefinitely and, ideally, differentiated into just about any cell type in our bodies (caveat: some iPSC lines can only differentiate into particular cell lineages). Theoretically, any cell type can be reprogrammed into iPSCs, but work from many laboratories has demonstrated that the identity of the founder cell influences the type of cell into which it can be reprogrammed.

Founder cells can be easily acquired from a donor and come in one of four types: fibroblasts (in skin), keratinocytes (also from skin), peripheral and umbilical cord blood, and dental pulp cells (from baby teeth). A variety of laboratories from around the world have made iPSC lines from a gaggle of different founder cells. Because of the significant influence of founder cells for iPSC characteristics, the use of iPSCs for regenerative medicine and other medical applications requires that the desired iPSC line should be selected based on the founder cell type and the characteristics of the iPSC line.

However, the founder cell identity is not the only factor that affects the characteristics of derived iPSC lines. The methods by which the founder cells are reprogrammed can also profoundly contribute to the differentiation efficiency of iPSC lines. According to Yoshinori Yoshida, Associate Professor at the Center for iPSC Research and Application (CiRA) at Kyoto University, the most commonly used methods of cell reprogramming utilize retroviruses, episomal/plasmids, and Sendai viruses to move genes into cells.

The cells found in blood represent a diverse group of cells that includes red blood cells that carry oxygen, platelets that heal wounds, and white blood cells that fight off infection. All the cells in blood are made by bone marrow-specific stem cells called “hematopoietic stem cells.” The production of clinical grade blood has remained a kind of “holy grail” for cellular reprogramming studies. Some scientists have argued that in order to make good-quality hematopoietic cells, the best founder cells are hematopoietic cells. Is this true? Yoshida and his colleagues examined a very large number of iPSC lines that were made from different founder cells and with differing reprogramming methods.  The results of these experiments were published in the journal Cell Stem Cell (doi:10.1016/j.stem.2016.06.019).

Remarkably, Yoshida and his crew discovered that neither of these factors has a significant effect. What did have a significant effect were the expression of certain genes and the position of particular DNA methylations. These two factors were better indicators of the efficiency at which an iPSC line could differentiate into the hematopoietic stem cells.

“We found the IGF2 (Insulin-like Growth Factor-2) gene marks the beginning of reprogramming to hematopoietic cells”, said Dr. Masatoshi Nishizawa, a hematologist who works in Yoshida’s lab and is the first author of this new study. Higher expression of the IGF2 gene is indicative of iPSCs initiating differentiation into hematopoietic cells. Even though IGF2 itself is not directly related to hematopoiesis, its uptake corresponded to an increase in the expression of those genes involved in directing differentiation into hematopoietic stem cells.

Although IGF2 marked the beginnings of differentiation to hematopoietic lineage, the completion of differentiation was marked by the methylation profiles of the iPS cell DNA. “DNA methylation has an effect on a cell staying pluripotent or differentiating,” explained Yoshida. Completion of the final stages of differentiation was highly correlated with less aberrant methylation during the reprogramming process. Blood founder cells showed a much lesser tendency to display aberrant DNA methylation patterns than did other iPSC lines made from other founder cells. This probably explains why past experiments seemed to indicate that the founder cell contributes to the effectiveness of differentiating iPS cells to the hematopoietic stem cell lineage.

These findings reveal molecular factors that can be used to evaluate the differentiation potential of different iPSC lines, which should, hopefully, expedite the progression of iPSCs to clinical use. Nishizawa expects this work to provide the basis for evaluating iPSC lines for the preparation of other cell types. “I think each cell type will have its own special patterns,” he said.

Gamida Cell Announces First Patient with Sickle Cell Disease Transplanted in Phase 1/2 Study of CordIn™ as the Sole Graft Source

An Israeli regenerative therapy company called Gamida Cell specializes in cellular and immune therapies to treat cancer and rare (“orphan”) genetic diseases. Gamida Cell’s main product is called NiCord, which provides patients who need new blood-making stem cells in their bone marrow an alternative to a bone marrow transplant. NiCord is umbilical cord blood that has been expanded in culture. In clinical trials to date, NiCord has rapidly engrafted into patients and the clinical outcomes of NiCord transplantation seem to be comparable to transplantation of peripheral blood.

Gamida Cell’s two products, NiCord and CordIn, as well as some other products under development utilize the company’s proprietary NAM platform technology to expand umbilical cord cells. The NAM platform technology has the remarkable capacity to preserve and enhance the functionality of hematopoietic stem cells from umbilical cord blood. CordIn is an experimental therapy for those rare non-malignant diseases in which bone marrow transplantation is the only currently available cure.

Gamida Cell has recently announced that the first patient with sickle cell disease (SCD) has been transplanted with their CordIn product.  Mark Walters, MD, Director of the Blood and Marrow Transplantation (BMT) Program is the Principal Investigator of this clinical trial. The patient received their transplant at UCSF Benioff Children’s Hospital Oakland.

CordIn, as previously mentioned, is an experimental therapy for rare non-malignant diseases, including hemoglobinopathies such as Sickel Cell Disease and thalassemia, bone marrow failure syndromes such as aplastic anemia, genetic metabolic diseases and refractory autoimmune diseases. CordIn potentially addresses a presently unmet medical need.

“The successful enrollment and transplantation of our first SCD patient with CordIn as a single graft marks an important milestone in our clinical development program. We are eager to demonstrate the potential of CordIn as a transplantation solution to cure SCD and to broaden accessibility to patients with rare genetic diseases in need of bone marrow transplantation,” said Gamida Cell CEO Dr. Yael Margolin. “In the first Phase 1/2 study with SCD, the expanded graft was transplanted along with a non-manipulated umbilical cord blood unit in a double graft configuration. In the second phase 1/2 study we updated the protocol from our first Phase 1/2 study so that patients would be transplanted with CordIn as a standalone graft, which is expanded from one full umbilical cord blood unit and enriched with stem cells using the company’s proprietary NAM technology.”

Somewhere in the vicinity of 100,000 patients in the U.S suffer from SCD; and around 200,000 patients suffer from thalassemia, globally. The financial burden of treating these patients over their lifetime is estimated at $8-9M. Bone marrow transplantation is the only clinically established cure for SCD, but only a few hundred SCD patients have actually received a bone marrow transplant in the last ten years, since most patients were not successful in finding a suitable match. Unrelated cord blood could be available for most of the patients eligible for transplantation, but, unfortunately, the rate of successful engraftment of un-expanded cord blood in these patients is low. Therefore, cord blood is usually not considered for SCD patients. Without a transplant, these patients suffer from very high morbidity and low quality of life.

Eight patients with SCD were transplanted in the first Phase 1/2 study performed in a double graft configuration. This study is still ongoing. Preliminary data from the first study will be summarized and published later this year. A Phase 1/2 of CordIn for the treatment of patients with aplastic anemia will commence later this year.

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.

Musashi-2 Protein Increases Number Hematopoietic Stem Cells in Umbilical Cord Blood

Umbilical cord blood infusions save the lives of many children and adults each year. Umbilical cord blood contains hematopoietic stem cells (HSCs) that can replace those lost to anticancer treatments, chemicals, or bone marrow collapse. However, despite their advantages for transplantation, the clinical use of umbilical cord blood is limited by the fact that HSCs in cord blood are found only in small numbers.

Small molecules that enhance hematopoietic stem and progenitor cell (HSPC) expansion in culture have been identified (see Boitano, A. E. et al. Science 329, 1345–1348 (2010), and Fares, I. et al. Science 345, 1509–1512 (2014). Unfortunately, the mechanisms of action or the nature of the pathways they impinge on are poorly understood.

Now a research team from McMaster University’s Stem Cell and Cancer Research Institute have discovered a key protein in the HSC/HSPC regenerative signaling pathway.

Kristin J. Hope and her team have elucidated the role of a protein called Musashi-2 in the function and development of HSCs.

Dr. Hope says that this discovery could help the tens of thousands of patients who suffer from blood-based disorders, including leukemia, lymphoma, aplastic anemia, sickle-cell disease, and more.

“We’ve really shone a light on the way these stem cells work,” she said. “We now understand how they operate at a completely new level, and that provides us with a serious advantage in determining how to maximize these stem cells in therapeutics. With this newfound ability to control over the regeneration of these cells, more people will be able to get the treatment they need.”

Only about five percent of all umbilical cord blood samples contain enough HSCs for a transplant, which is unfortunate because umbilical cord blood is less likely to be rejected by the immune system, because of the immaturity of the cells, and is also rather abundant.

Growing HSCs in culture is a possibility, but this remains a somewhat poorly understood and ill-defined procedure.

Musashi-2 is an RNA-binding protein in cells and was actually named for the Japanese samurai who fought using two swords.

In collaboration with researchers in Dr. Gene Yeo’s lab at the University of California San Diego, Dr. Hope’s lab has found that the Musashi-2 protein plays a pivotal role in controlling stem cell production in human cord blood HSCs. When Musashi-2 levels in HSCs are the knocked down, the cod blood HSCs were no longer able to regenerate the blood system. Conversely, when the levels of Musashi-2 were increased, the number of HSCs in the cord blood sample increased significantly.

The Hope’s group new discovery has identified a new way to tightly control on the development of HSCs. Essentially, Hope and her colleagues have discovered a new way to make more cord blood stem cells in a dish.

In the past, attempts to control HSC function and development has been approached at the level of transcriptional factors. The Hope lab’s approach of directing stem cell function through manipulation of an RNA-binding protein is somewhat novel, and represents a paradigm shift in the way we think about stem cell biology.

“This discovery really highlights the underappreciated role that RNA-binding protein-mediated control has on the properties of stemness in the blood system,” explained Dr. Hope.

This paradigm shift provides new targets for pharmaceuticals that may be able to expand these cells in a safe and targeted manner.

These findings represent an important step forward in surmounting the obstacles associated with stem cell transplants. According to Dr. Hope, the ability to increase the number of available cord blood stem cells has the potential to “mitigate a lot of the problems that arise post-transplantation.” Elaborating further, Dr. Hope explained that stem cells from cord blood are a “safer and more efficient transplant product,” and detailed how their use could reduce the number of patient follow-up visits and treatments required post-transplantation. Streamlining the transplantation process could help to alleviate the stress on the healthcare system and open up space for more transplant patients.

SEPCELL Trial Tests Fat-Derived Stem Cells as a Treatment for Sepsis

The Belgium-based biotechnology company, TiGenix, has launched a clinical trial entitled SEPCELL that uses fat-derived stem cells (called Cx611) to treat severe sepsis secondary to acquired pneumonia (also known as sCAP). SEPCELL is a randomized, double-blind, placebo-controlled, Phase 1b/2a study of sCAP patients who require mechanical ventilation and/or vasopressors.

SEPCELL will, hopefully, enroll 180 patients and will be conducted at approximately 50 centers throughout Europe. Subjects who participate in this trial will be randomly assigned to receive either an investigational product or placebo on days 1 and 3. All patients will be treated with standard care, which usually includes broad-spectrum antibiotics and anti-inflammatory drugs.

The primary endpoint of this clinical trial will examine the number, frequency, and type of adverse reactions during the 90-day period of the trial. The secondary endpoints of the SEPCELL trial include reduction in the duration of mechanical ventilation and/or vasopressors, overall survival, clinical cure of sCAP, and other infection-related endpoints. SEPCELL will also assess the safety and efficacy of the expanded allogeneic adipose stem cells (eASCs) that will be intravenously delivered to some of the patients in this study.

The SEPCELL trial will be managed by TFS International, a company based in Lund, Sweden. TFS has extensive experience in running sepsis trials and hospital-based trials.

Sepsis is a potentially life-threatening complication of infection that occurs when inflammatory molecules (cytokines and chemokines) released into the bloodstream to fight the infection trigger systemic inflammation.  This body-wide inflammation has the ability to trigger a cascade of detrimental changes that damage multiple organ systems and cause them to fail. If sepsis progresses to “septic shock,” blood pressure drops dramatically, which may lead to death. Patients with “severe sepsis” require close monitoring and treatment in a hospital intensive care unit. Drug therapy is likely to include broad-spectrum antibiotics, corticosteroids, vasopressor drugs to increase blood pressure, as well as oxygen and large amounts of intravenous fluids. Supportive therapy may be needed to stabilize breathing and heart function and to replace kidney function. Patients with severe sepsis have a low survival rate so there is a critical need to improve the effectiveness of current therapy. Only a small number of new molecular entities are currently in development for severe sepsis.

Severe sepsis and septic shock significantly affect public health and these event also are leading causes of mortality in intensive care units.

Severe sepsis and septic shock have an incidence of about 3 cases per 1,000, but due to the aging of the population and an increase in drug resistant bacteria.

Cx611 is an intravenously-administered concoction that consists of allogeneic eASCs. These cells are largely mesenchymal stem cells that secrete an impressive array of molecules that suppress the type of immune responses that damage organs during events like septic shock.  eASCs have a higher proliferation rate in culture and faster attachment than bone marrow-based mesenchymal stem cells in cell culture.  ASCs are also less prone to senescence and differentiation.  Their differentiation capacity decreases with expansion time without losing immunomodulatory properties.  These eASCs also have superior inflammation targeting capacities than bone marrow-based mesenchymal stem cells, and are safe, since they do not express ligands for receptors on Natural Killer cells that, and therefore, are unlikely to elicit an immune rejection.

In May 2015, TiGenix completed a Phase 1 sepsis challenge that demonstrated that Cx611 is safe and well tolerated. That trial began in December 2014, and was a placebo-controlled dose-ranging study (3 doses of eASC’s) in which 32 healthy male volunteers were randomized to receive Cx611 or placebo in a ratio of 3:1. Primary endpoints were vital signs and symptoms, laboratory measures and functional assays of innate immunity. All 32 volunteer subjects were recruited and dosed by March 2015. By May, 2015, the phase I trial data essentially demonstrated the safety and tolerability of Cx611.  On the strength of that phase I trial, TiGenix designed a Phase 1b/2a trial in severe sepsis secondary to sCAP in which they expecet to enroll 180 subjects across Europe.

SEPCELL was funded by a €5.4 million grant ($6.14 million) from the European Union.

Hematopoietic Stem Cells Use a Simple Heirarchy

New papers in Science magazine and the journal Cell have addressed a long-standing question of how the descendants of hematopoietic stem cells in bone marrow make the various types of blood cells that course through our blood vessels and occupy our lymph nodes and lymphatic vessels.

Hematopoietic stem cells (HSCs) are partly dormant cells that self-renew and produce so-called “multipotent progenitors” or MPPs that have reduced ability to self-renew, but can differentiate into different blood cell lineages.

The classical model of how they do this goes like this: the MPPs lose their multipotency in a step-wise fashion, producing first, common myeloid progenitors (CMPs) that can form all the red and white blood cells except lymphocytes, or common lymphoid progenitors (CLPs) that can form lymphocytes (see the figure below as a reference). Once these MPPs form CMPs, for example, the CMP then forms either an MEP that can form either platelets or red blood cells, or a GMP. which can form either granulocytes or macrophages. The possibilities of the types of cells the CMP can form in whittled down in a step-by-step manner, until there is only one choice left. With each differentiation step, the cell loses its capacity to divide, until it becomes terminally differentiated and becomes platelet-forming megakarocyte, red blood cell, neutrophil, macrophage, dendritic cells, and so on.


These papers challenge this model by arguing that the CMP does not exist. Let me say that again – the CMP, a cell that has been identified several times in mouse and human bone marrow isolates, does not exist. When CMPs were identified from mouse and human none marrow extracts, they were isolated by means of flow cytometry, which is a very powerful technique, but relies on the assumption that the cell type you want to isolate is represented by the cell surface protein you have chosen to use for its isolation. Once the presumptive CMP was isolated, it could recapitulate the myeloid lineage when implanted into the bone marrow of laboratory animals and it could also produce all the myeloid cells in cell culture. Sounds convincing doesn’t it?

In a paper in Science magazine, Faiyaz Notta and colleagues from the University of Toronto beg to differ. By using a battery of antibodies to particular cell surface molecules, Notta and others identified 11 different cell types from umbilical cord blood, bone marrow, and human fetal liver that isolates that would have traditionally been called the CMP. It turns out that the original CMP isolate was a highly heterogeneous mixture of different cell types that were all descended from the HSC, but had different developmental potencies.

Notta and others used single-cell culture assays to determine what kinds of cells these different cell types would make. Almost 3000 single-cell cultures later, it was clear that the majority of the cultured cells were unipotent (could differentiate into only one cell type) rather than multipotent. In fact, the cell that makes platelets, the megakarocyte, seems to derive directly from the MPP, which jives with the identification of megakarocyte progenitors within the HSC compartment of bone marrow that make platelets “speedy quick” in response to stress (see R. Yamamoto et al., Cell 154, 1112 (2013); S. Haas, Cell Stem Cell 17, 422 (2015)).

Another paper in the journal Cell by Paul and others from the Weizmann Institute of Science, Rehovot, Israel examined over 2700 mouse CMPs and subjected these cells to gene expression analyses (so-called single-cell transriptome analysis). If the CMP is truly multipotent, then you would expect it to express genes associated with lots of different lineages, but that is not what Paul and others found. Instead, their examination of 3461 genes revealed 19 different progenitor subpopulations, and each of these was primed toward one of the seven myeloid cell fates. Once again, the presumptive CMPs looked very unipotent at the level of gene expression.

One particular subpopulation of cells had all the trappings of becoming a red blood cell and there was no indication that these cells expressed any of the megakarocyte-specific genes you would expect to find if MEPS truly existed. Once again, it looks as though unipotency is the main rule once the MPP commits to a particular cell lineage.

Thus, it looks as though either the CMP is a very short-lived state or that it does not exist in mouse and human bone marrow. Paul and others did show that cells that could differentiate into more than one cell type can appear when regulation is perturbed, which suggests that under pathological conditions, this system has a degree of plasticity that allows the body to compensate for losses of particular cell lineages.

A model of the changes in human My-Er-Mk differentiation that occur across developmental time points. Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.
A model of the changes in human My-Er-Mk differentiation that occur across developmental time points.
Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.

Fetal HSCs, however, are a bird of a different feather, since they divide quickly and reside in fetal liver.  Also, these HSCs seem to produce CMPs, which is more in line with the classical model.  Does the environmental difference or fetal liver and bone marrow make the difference?  In adult bone marrow, some HSCs nestle next to blood vessels where they encounter cells that hang around blood vessels known as “pericytes.”  These pericytes sport a host of cell surface molecules that affect the proliferative status of HSCs (e.g., nestin, NG2).  What about fetal liver?  That’s not so clear – until now.

In the same issue of Science magazine, Khan and others from the Albert Einstein College of Medicine in the Bronx, New York, report that fetal liver also has pericytes that express the same cell surface molecules as the ones in bone marrow, and the removal of these cells reduces the numbers of and proliferative status of fetal liver HSCs.

Now we have a conundrum, because the same cells in bone marrow do not drive HSC proliferation, but instead drive HSC quiescence.  What gives? Khan and others showed that the fetal liver pericytes are part of an expanding and constantly remodeling blood system in the liver and this growing, dynamic environment fosters a proliferative behavior in the fetal HSCs.

When umbilical inlet is closed at birth, the liver pericytes stop expressing Nestin and NG2, which drives the HSCs from the fetal liver to the other place were such molecules are found in abundance – the bone marrow.

These models give us a better view of the inner workings of HSC differentiation.  Since HSC transplantation is one of the mainstays of leukemia and lymphoma treatment, understanding HSC biology more perfectly will certainly yield clinical pay dirt in the future.


Fat-Based Stem Cell Product HemaXellerate Will be Tested in Clinical Trials for Aplastic Anemia

A regenerative medicine company called Regen BioPharma, Inc., has announced that it received a communication from the U.S. Food and Drug Administration that grants it permission to initiate clinical trials under its Investigational New Drug (IND) #15376.

Granting of the IND gives the green light to Regen BioPharma to begin testing their product HemaXellerate in clinical trials with human patients. HemaXellerate is a personalized stem cell treatment for patients whose bone marrow no longer works (aplastic anemia). It uses fat-based stem cells from a patient’s own belly fat to treat bone marrow that has been damaged. HemaXellerate uses the patient’s own fat-based stem cells as a source of endothelial (blood vessel) cells to heal damaged bone marrow.

Aplastic anemia occurs when the bone marrow stops producing sufficient numbers of blood cells. It is a potentially fatal disease of the bone marrow that leads to bleeding, infection and fever. Patients with severe or even very severe aplastic anemia have a mortality rate of greater than 70%. Current treatments for aplastic anemia include blood transfusions, immunosuppression and stem cell transplantation.

This Phase I clinical trial will treat patients who have been diagnosed with refractory aplastic anemia, which includes those patients with aplastic anemia who were unsuccessfully treated with first-line immunosuppressive therapy. Patients treated with HemaXellerate with be followed for safety parameters and signals of treatment efficacy. Since this will be an unblinded trial, all data will be available as the study progresses.

“Current drug-based approaches for healing bone marrow dysfunction involve flooding the body with growth factors, which is extremely expensive and causes unintended consequences because of lack of selectivity,” said Harry Lander, Ph.D., President and Chief Scientific Officer of Regen Biopharma. “By utilizing a cell-based approach that both modulates the immune system and stimulates production of blood cells, we aim to offer alternatives to the current approaches to treating patients with aplastic anemia. This product will complement our immune-modulatory pipeline that includes a potential novel checkpoint inhibitor.”

If HemaXellerate passes this clinical trial, Regen Biopharma would like to position HemaXellerate as a treatment for bone marrow dysfunction on par with other members of the hematopoietic growth factor market that includes drugs such as Neupogen®, Neulasta®, Leukine® and Revolade®.

“The FDA clearance marks a substantial step for Regen, in that we are now a clinical-stage company. We are grateful to our collaborators and scientific advisory board members who have worked tirelessly in bringing our product to the point where the FDA has permitted treatment of patients,” said David Koos, Ph.D., Chairman and Chief Executive Officer of Regen BioPharma. “We believe the success of today will not only allow for the rapid execution of HemaXellerate’s development plan, but will also allow for more rapid translation of the company’s other immune modulatory products to the clinic.”

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.

Skin Cells Converted into Blood Cells By Direct Reprogramming

Making tissue-specific progenitor cells that possess the ability to survive, but have not passed through the pluripotency state is a highly desirable goal of regenerative medicine. The technique known as “direct reprogramming” uses various genetic tricks to transdifferentiate mature, adult cells into different cell types that can be used for regenerative treatments.

Juan Carlos Izpisua Belmonte and his colleagues from the Salk Institute for Biological Studies in La Jolla, California and his collaborators from Spain have used direct reprogramming to convert human skin cells into a type of white blood cells.

These experiments began with harvesting skin fibroblasts from human volunteers that were then forced to overexpress a gene called “Sox2.” The Sox2 gene is heavily expressed in mice whose bone marrow stem cells are being reconstituted with an infusion of new stem cells. Thus this gene might play a central role is the differentiation of bone marrow stem cells.

Sox2 overexpression in human skin fibroblasts cause the cells express a cell surface protein called CD34. Now this might seem so boring and unimportant, but it is actually really important because CD34 is expressed of the surfaces of hematopoietic stem cells. Hematopoietic stem cells make all the different types of white and red blood cells in our bodies. Therefore, the expression of these protein is not small potatoes.

In addition to the expression of CD34, other genes found in hematopoietic stem cells were also induced, but not strongly. Thus overexpression of SOX2 seems to induce an incipient hematopoietic stem cell‐like status on these fibroblasts. However, could these cells be pushed further?

Gene profiling of hematopoietic stem cells from Umbilical Cord Blood identified a small regulatory RNA known as miR-125b as a factor that pushes SOX2-generated CD34+ cells towards an immature hematopoietic stem cell-like progenitor cell that can be grafted into a laboratory animal.

When SOX2 and miR-125b were overexpressed in combination, the cells transdifferentiated into monocytic lineage progenitor cells.

What are monocytes? They are a type of white blood cells and are, in fact, the largest of all white blood cells. Monocytes compose 2% to 10% of all white blood cells in the human body. They play multiple roles in immune function, including phagocytosis (gobbling up bacteria and other stuff), antigen presentation (identifying and altering other cells to the presence of foreign substances), and cytokine production (small proteins that regulate the immune response).

Monocytes express a molecule on their cell surfaces called CD14, and when human fibroblasts overexpressed Sox2 and miR-125b, they became CD14-expressing cells that looked and acted like monocytes. These cells were able to gobble up bacteria and other foreign material, and when transplanted into a laboratory animal, these directly reprogrammed cells generated cells that established the monocytic/macrophage lineage.

Cancer patients, and other patients with bone marrow diseases can have trouble making sufficient white blood cells. A technique like this can generate transplantable monocytes (at least in laboratory animals) without many of the drawbacks associated with reprogramming human cells into hematopoietic stem cells that possess true clinical potential. Also because this technique skips the pluipotency stage, it is potentially safer.

A Genetic Recipe To Convert Stem Cells into Blood

University of Wisconsin at Madison Stem Cell researchers led by Igor Slukvin discovered two genetic programs that can convert pluripotent stem cells into the wide array of white and red blood cells found in human blood (pluripotent means “capable of developing into more than one organ or tissue and not fixed as to potential development).

This research has ferreted out the actual pathway used by the developing human body to make blood-based cells at the early stages of development.

During embryonic development, blood formation, which includes the formation of blood cells and blood vessels from the same progenitor cell; a cell called a hemangioblast. This begins in week three of development in the extraembryonic mesoderm or the primary embryonic umbilical sac, which is also known as the yolk sac. Also, the connecting stalk and chorion contain blood islands as well. These blood islands are rich in particular growth factors such as vascular endothelial growth factor (VEGF) and placental growth factor (PIGF). The blood islands form clusters with two cell populations; peripheral cells (angioblasts) that form the endothelial cells that form vessels. These networks of vessels extend and fuse together to form a robust a network. The cores of the blood islands (hemocytoblasts) form blood cells. Initially all vessels (arteries and veins) look the same. Blood formation occurs later in week 5, and occurs throughout the embryonic mesenchyme (connective tissue), and then moves to the liver, and then the spleen, and then bone marrow.

Embryonic red blood cells
Embryonic red blood cells

Hematopoietic stem cells (HSCs), the stem cells that form the blood cells, form from the wall of the aorta, which is the major blood vessel in the embryo. In the aortic wall, cells called hemogenic endothelial cells bud off progenitor cells that become HSCs.

A course of transcription factors have now been identified by Slukvin and his team as the triggers that switch these cells into HSCs. Two groups of transcriptional regulators can induce distinct developmental programs from pluripotent stem cells. The first developmental program, directed by the transcription factors ETV2 and ​GATA2, the pan-myeloid pathway, switches cells into the myeloid lineage (the myeloid lineage includes red blood cells, platelets, neutrophils, macrophages, basophils and eosinophils). The second developmental pathway, directed by the transcription factors GATA2 and ​TAL1, directs cells into the erythro-megakaryocytic pathway. In either cases, these transcription factors directly convert human pluripotent stem cells into an endothelium, which subsequently transform into blood cells with pan-myeloid or erythro-megakaryocytic potential.


In Slukvin’s laboratory, treatment of either ETV2 and ​GATA2 or GATA2 and ​TAL1 induced cells to make the complete range of human blood cells. Slukvin said of these experiments, “This is the first demonstration of the production of different kinds of cells from human pluripotent stem cells using transcription factors.” Transcription factors bind to DNA at specific sites and regulate gene expression.

Slukvin continued: “By overexpressing just two transcription factors, we can, in the laboratory dish, reproduce the sequence of events we see in the embryo.”

Slukvin and his co-workers showed that his technique produced blood cells by the millions. For every million stem cells, it was possible to produce 30 million blood cells.

Slukvin and his colleagues did not use viruses to genetically modify these stem cells. Instead they used modified RNA to induce overexpression of these transcription factors. Such a technique avoids genetic modification of cells and is inherently safer.

“You can do it without a virus, and genome integrity is not affected,” said Slukvin.  This technique might also work to differentiate pluripotent stem cells into other cell types, such as pancreatic beta cells, brain-specific cells, or liver cells.

Despite these successes, Slukvin says that the “Holy grail” of hematopoietic research is to differentiate pluripotent stem cells into HSCs.  Since HSC transplants are used to treat multiple myeloma and other types of blood-based cancers as well, making HSCs in the laboratory remains a significant goal and challenge as well.

“We still don’t know how to do that,” said Slukin, “but our new approach to making blood cells will give us an opportunity to model their development in a dish and identify novel hematopoietic stem cell factors.”

Correcting Mutations Associated with a Blood Disorder

The protein hemoglobin carries oxygen from our lungs to our tissues. Mutations in the genes that encode the protein chains that form hemoglobin can cause inherited blood disorders like sickle-cell anemia, or the so-called Thalassemias. Thalassemias come from the Greek word from sea (θάλασσα or thalassa), because these blood disorders are found in Mediterranean populations. Thalassemias are found in these populations because they convey some resistance to malaria, which was endemic to that area. People with thalassemias tend to have fatigue, weakness, a pale appearance, yellow discoloration of skin (jaundice), facial bone deformities, slow growth, abdominal swelling, or dark urine, although some people have no symptoms.

Now this common genetic blood disorder has been genetically corrected in cultured induced pluripotent stem cells by using cutting-edge genome-editing techniques.

β-Thalassaemia shows reduced levels of hemoglobin, and these reduced levels are due to mutations in the gene that encodes the β-globin protein. Hemoglobin consists of four protein chains, two of which are alpha-globin proteins, and the other two of which are beta-globin proteins. Mutations in the beta-globin gene reduces the levels of functional beta-globin protein and this reduces the levels of functional hemoglobin.

Yuet Kan and his colleagues at the University of California, San Francisco, made induced pluripotent stem cells from skin fibroblasts from a person who suffered from β-thalassemia. Kan and his colleagues then used the CRISPR–Cas9 gene-editing technique to correct the mutation responsible for β-thalassemia. The CRISPR–Cas9 gene-editing technique allows for precise and accurate correction of the mutation without affecting other genes.

After the genetic editing, the iPSCs were differentiated into the precursors of red blood cells in culture and demonstrated that the modified cells showed higher expression of hemoglobin than unmodified cells.

Hopefully transplantation of such corrected cells back into the original patient could one day provide a cure for β-thalassaemia, according to the authors.

Expanding Functional Cord Blood Stem Cells for Transplantation

Patients who suffer from blood-based diseases such as leukemia, lymphoma, and other blood-related diseases sometimes require bone marrow transplants in order to live. The paucity of available bone marrow necessitates the use of umbilical cord blood for these patients, but cord blood suffers from one flaw and that is small volumes of blood and low numbers of stem cells. Scientists have tried to grow cord blood stem cells in culture in order to beef up the numbers of stem cells, but cord blood stem cells sometimes lose their ability to repopulate the bone marrow while in culture.

To solve this problem, researchers at the Icahn School of Medicine at Mount Sinai have designed a new technique to expand the number of cord blood stem cells without causing any loss of potency.

“Cord blood stem cells have always posed limitations for adult patients because of the small number of stem cells present in a single collection,” said Partita Chaurasia of the Tisch Cancer Institute at Mount Sinai. “These limitations have resulted in a high rate of graft failure and delayed engraftment in adult patients.”.

Chaurasia and coworkers used a technique called “epigenetic reprogramming” to reshape the structure of the genome of the stem cells. They used a combination of a drug called valproic acid and histone deacetylase inhibitors (HDACIs). The valproic acid-treated cells produced greater numbers of marrow repopulating stem cells in culture. These expanded cord blood stem cells were also able to reconstitute the bone marrow of immune-deficient mice, and when the reconstituted bone marrow of that mouse could be used to reconstitute the bone marrow of another immune-deficient mouse. Bone marrow from this second mouse could also reconstitute the bone marrow of a third immune deficient mouse.

These results have extremely important implications for patients who are in the midst of a battle with blood cancers, and might mean the difference between a successful cord blood transplant and one that fails.

Mouse Blood Cells Reprogrammed into Blood Cell Stem Cells

Boston Children’s Hospital researchers have directly reprogrammed mature blood cells from mice into blood-forming hematopoietic stem cells by using a cocktail of eight different transcription factors.

These reprogrammed cells have been called induced hematopoietic stem stem cells or iHSCs. These cells have all the hallmarks of mature mouse HSCs and they have the capacity to self-renew and differentiate into all the blood cells that circulate throughout the body.

These findings are highly significant from a clinical perspective because they indicate that it might be entirely possible to directly reprogram a patient’s existing, mature blood cells into a hematopoietic stem cell for transplantation purposes. Such a procedure, known as hematopoietic stem cells transplantation or HSCT, is a common treatment for patients whose bone marrow has suffered irreparable damage due to environmental causes (heavy metals, chloramphenicol, etc) or disease (cancer). The problem with HSCT is finding a proper match for the patient and then procuring clinically significant quantities of high-quality bone marrow for HSCT.

Derrick J. Rossi, an investigator in the Program in Cellular and Molecular Medicine at Boston Children’s Hospital and Assistant Professor in the Department of Stem Cell & Regenerative Biology, explained: “HSCs comprise only about one in every 20,000 cells in the bone marrow. If we could generate autologous (a patient’s own) HSCs from other cells, it could be transformative for transplant medicine and for our ability to model diseases of blood development.”

Rossi and his collaborators have screened genes that are expressed in 40 different types of blood progenitor cells in mice. This screen identified 36 different genes that control the expression of the other genes. These 36 genes encode so-called “transcription factors,” which are proteins that bind to DNA and turn gene express on or shut it off.

Blood cell production tends to go from the stem cells to progeny cells called progenitor cells that can still divide to some limited extent, and to effector cells that are completely mature and, in most cases, do not divide (the exception is lymphocytes, which expand when activated by specific foreign substances called antigens).

Further work by Rossi and others identified six transcription factors (Hlf, Runx1t1, Pbx1, Lmo2, Zfp37, and Prdm5) of these 36 genes, plus two others that were not part of their original screen (N-Myc and Meis1) that could robustly reprogram myeloid progenitor cells or pro/pre B lymphocytes into iHSCs.


To put these genes into these blood cells, Rossi and others uses souped-up viruses that introduced all either genes under the control of sequences that only allowed expression of these eight genes in the presence of the antibiotic doxycycline. Once these transfected cells were transplanted into mice, the recipient mice were treated with doxycycline, and the implanted cells formed HSCs.

When this experiment utilized mice that were unable to make their own blood cells, because their bone marrow had been wiped out, the implanted iHSCs reconstituted the bone marrow and blood cells of the recipient mice.

To further show that this reconstituted bone marrow was normal, high-quality bone marrow, Rossi and others used these recipient mice as bone marrow donors for sibling mice whose bone marrow had been wiped out. This experiment showed that the mice that had received the iHSCs had bone marrow that completely reconstituted the bone marrow of their siblings. This established that the iHSCs could completely reestablish the bone marrow of another mouse.

Thus Rossi and others had established that iHSCs could in fact created HSCs from progenitor cells, but could they do the same thing with mature blood cells that were not progenitor cells? Make that another yes. When Rossi and others transfected their eight-gene cocktail into mature myeloid cells, these mature cells also made high-quality iHSCs.

Rossi noted that no one has been able to culture HSCs in the laboratory for long periods of time. A few laboratories have managed expand HSCs in culture, but only on a limited basis for short periods of time (see Aggarwal R1, Lu J, Pompili VJ, Das H. Curr Mol Med. 2012 Jan;12(1):34-49).  In these experiments, Rossi used his laboratory mice as living culture systems to expand his HSCs, which overcomes the problems associated with growing these fussy stem cells in culture.

Gene expression studies of his iHSCs established that, from a gene expression perspective, the iHSCs were remarkably similar to HSCs isolated from adult mice.

This is certainly an exciting advance in regenerative medicine, but it is far from being translated into the clinic.  Can human blood progenitor cells also be directly reprogrammed using the same cocktail?  Can mature myeloid cells be successfully reprogrammed?  Will some non-blood cell be a better starting cell for iHSC production in humans?  As you can see there are many questions that have to be satisfactorily answered before this procedure can come to the clinic.

Nevertheless, Rossi and his team has succeeded where others have failed and the results are remarkable.  HSCs can be generated and transplanted with the use of only a few genes.  This is certainly the start of what will hopefully be a fruitful regenerative clinical strategy.

On a personal note, my mother passed about almost a decade ago after a long battle with myelodysplastic syndrome, which is a pre-leukemic condition in which the bone marrow fails to make mature red blood cells.  Instead the bone marrow fills up with immature red blood cells and the patient has to survive on blood transfusions.

The reason for this condition almost certainly stems from defective HSCs that do not make normal progeny.  Therefore the possibility of using a patient’s own cells to make new HSCs that can repopulate the bone marrow is a joyful discovery for me to read about, even though it is some ways from the clinic at this point.

Human Stem Cell Gene Therapy Appears Safe and Effective

Two recent studies in the journal Science have reported the outcome of virally-mediated gene correction in hematopoietic stem cells (HSCs) to treat human patients. These studies may usher in a new era of safe and effective gene therapy. These exciting new clinical findings both come from the laboratory of Luigi Naldini at the San Raffaele Scientific Institute, Milan, Italy. The first experiment examined the treatment of metachromatic leukodystrophy (MLD), which is caused by mutations in the arylsulfatase A (ARSA) gene, and the second, investigated treatments for Wiskott-Aldrich syndrome (WAS), which is caused by mutations in the gene that encodes WASP.

MLD is one of several diseases that affects the lysosome; a structure in cells that acts as the garbage disposal of the cell. So called “lysosomal storage diseases” result from the inability of cells to degrade molecules that come to the lysosome for degradation. Without the ability to degrade these molecules, they build up to toxic levels and produce progressive motor and cognitive impairment and death within a few years of the onset of symptoms.

To treat MLD, workers in Naldini’s laboratory isolated blood-making stem cells from the bone marrow of three pre-symptomatic MLD patients (MLD01, 02 and 03). These stem cells were infected with genetically engineered viruses that encoded the human ARSA gene. After expanding these stem cells in culture, they were re-introduced into the MLD patients after those same patients had their resident bone marrow wiped out. The expression of the ARSA gene in the reconstituted bone marrow was greater than 10 fold the levels measured in healthy controls and there were no signs of blood cancers or other maladies. One month after the transplant, the implanted cells showed very high-level and stable engraftment. Between 45%-80% of cells isolated and grown from bone marrow samples harbored the fixed ARSA gene. AS expected, the levels of the ARSA protein rose to above-normal levels in therapeutically relevant blood cells and above normal levels of ARSA protein were isolated from hematopoietic cells after one month and cerebrospinal fluid (CSF) one to two years after transfusion. This is remarkable when you consider that one year before, no ARSA was seen. This shows that the implanted cells and their progeny properly homed to the right places in the body. The patient evaluations at time points beyond the expected age of disease onset was even more exciting, since these treat patients showed normal, continuous motor and cognitive development compared to their siblings who had MLD, but were untreated. The sibling of the patient designated “MLD01” was wheelchair-bound and unable to support their head and trunk at 39 months, but excitingly, after treatment, patient MLD01 was able to stand, walk and run at 39 months of age and showed signs of continuous motor and cognitive development. Lastly, and perhaps most importantly, there was no evidence of implanted cells becoming cancerous, even though they underwent self-renewal, like all good stem cells. This is the first report of an MLD patient at 39 months displaying such positive clinical features.

The second study treated WAS, which is an inherited disease that affects the immune system and leads to infections, abnormal platelets, scaly skin (eczema), blood tumors, and autoimmunity. In this second study, blood-making stem cells were collected from three patients infected with genetically engineered viruses that expressed the WASP gene. These stem cells were then reinfused intravenously (~11 million cells ) three days after collection. Blood tests and bone marrow biopsies showed evidence of robust engraftment of gene-corrected cells in bone marrow and peripheral blood up to 30 months later. WASP expression increased with time in most blood cells. Although serious adverse infectious events occurred in two patients, overall clinical improvement resulted in reduced disease severities in all patients. None of the three patients demonstrated signs of blood cancers and the platelet counts rose, but, unfortunately, not to normal levels. Again, no evidence for adverse effects were observed.

Simply put, these authors have presented a strategy for ex vivo gene correction in HSCs for inherited disorders which works and appears safe in comparison to previous strategies. Long-term analyses will undoubtedly need to be intensely scrutinized, but this research surely represents a huge step forward in the safe treatment of these and similar genetic disorders.