Intravenous Bone Marrow For Stroke: Clinical Trial


Akihiko Taguchi from the Institute of Biomedical Research and Innovation in Kobe, Japan, in collaboration with a whole host of colleagues from various places treated stroke with their own bone marrow. This is a Phase 1/2 clinical trial but it is a very small trial that was neither blinded not placebo-controlled. Therefore, while this trial is useful, the results are of limited value.

In this clinical trial, 12 stroke patients were divided into two groups, one of which received 25 milliliters and the other of which received 50 milliliters of bone marrow cells 7-10 days after their strokes. The bone marrow cells were administered intravenously. To isolate bone marrow cells, the so-called “mononuclear fraction” was isolated from whole bone marrow samples that came from bone marrow aspirations. Patients were evaluated by means of brain imaging to measure blood flow in their brains, and a series of neurological tests. The National Institute of Health Stroke Scale or NIHSS scores were used to grade the neurological capabilities of each patient. Patients were examined 1 month and then 6 months after treatment.

All treated patients were compared with the records of other stroke patients in the past who were not treated with bone marrow cells. These comparisons showed that the bone marrow-treated patients showed a trend towards improved neurological outcomes. Statistically, the bone marrow-treated patients had significantly better blood flow and oxygen consumption in their brains 6 months after treatment compared to the historic controls. Also, the NIHSS scores of the bone marrow-treated patients were also significantly better than those of the historic controls. Patients who received the higher doses of bone marrow cells did better than those who received the lower doses.

There were also no apparent adverse effects to administering the bone marrow cells. One patient experienced pneumonia and sepsis 3 months after cell therapy, but data monitoring largely eliminated the cell therapy as being a contributing factor to this issue. Another patient experienced a seconded stroke that was detected the day after the cell therapy. Because the patient had shown signs of a stroke the day before treatment, the association between the cell therapy and the recurrent stroke is rather unclear. None of the other patients showed any worsening of their present strokes, seizures, or other complications.

All in aloe, it seems as though this procedure is safe, and there is a trend towards increased metabolic and neurological recovery. However, this is a very small study and these trends may not hold in a larger study. Secondly, these patients must be followed for an extended period of time in order to determine if these improvements are durable or transient. Finally, these improvements must be compared with a placebo if there are going to convince the FDA.

Bone marrow cells contain a variety of stem cells and other types of cells that may release cocktails of healing molecules that help cells survive, make new blood vessels, and tamp down inflammation. Additionally, bone marrow cells might stimulate resident populations of stem cells to proliferate and make new neurons and glial cells. Until these positive results can be reproduced in larger, better controlled studies, these results will remain interesting and hopeful, but ultimately inconclusive.

These results were published in Stem Cells and Development 2015 DOI: 10.1089/scd.2015.0160.

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.

Print

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.

A Patient’s Own Bone Marrow Stem Cells Defeat Drug-Resistant Tuberculosis


People infected with multidrug-resistant forms of tuberculosis could, potentially, be treated with stem cells from their own bone marrow. Even though this treatment is in the early stage of its development, the results of an early-stage trial of the technique show immense promise.

British and Swedish scientists have tested this procedure, which could introduce a new treatment strategy for the estimated 450,000 people worldwide who have multi drug-resistant (MDR) or extensively drug-resistant (XDR) TB.

This study, which was published in the medical journal, The Lancet, showed that over half of 30 drug-resistant TB patients treated with a transfusion of their own bone marrow stem cells were cured of the disease after six months.

“The results … show that the current challenges and difficulties of treating MDR-TB are not insurmountable, and they bring a unique opportunity with a fresh solution to treat hundreds of thousands of people who die unnecessarily,” said TB expert Alimuddin Zumla at University College London, who co-led the study.

TB initially infects the lungs but can rapidly spread from one person to another through coughing and sneezing. Despite its modern-day resurgence, TB is often regarded as a disease of the past. However, recently, drug-resistant strains of Mycobacterium tuberculosis, the microorganism that causes TB, have spread globally, rendering standard anti-TB drug treatments obsolete.

The World Health Organisation (WHO) estimates that in Eastern Europe, Asia and South Africa 450,000 people have MDR-TB, and close to half of these cases will fail to respond to existing treatments.

Mycobacterium tuberculosis, otherwise known as the “tubercle bacillus, trigger a characteristic inflammatory response (granulomatous response) in the surrounding lung tissue that elicits tissue damage (caseation necrosis).

Bone-marrow stem cells are known to migrate to areas of lung injury and inflammation. Upon arrival, they initiate the repair of damaged tissues. Since bone marrow stem cells also they also modify the body’s immune response, they can augment the clearance of tubercle bacilli from the body. Therefore, Zumla and his colleague, Markus Maeurer from Stockholm’s Karolinska University Hospital, wanted to test bone marrow stem cell infusions in patients with MDR-TB.

In a phase 1 trial, 30 patients with either MDR or XDR TB aged between 21 and 65 who were receiving standard TB antibiotic treatment were also given an infusion of around 10 million of their own bone marrow-derived stem cells.

The cells were obtained from the patient’s own bone marrow by means of a bone marrow aspiration, and then grown into large numbers in the laboratory before being re-transfused into the same patient.

During six months of follow-up, Zumla and his team found that the infusion treatment was generally safe and well tolerated, and no serious side effects were observed. The most common non-serious side effects were high cholesterol levels, nausea, low white blood cell counts and diarrhea.

Although a phase 1 trial is primarily designed only to test a treatment’s safety, the scientists said further analyses of the results showed that 16 patients treated with stem cells were deemed cured at 18 months compared with only five of 30 TB patients not treated with their own stem cells.

Maeurer stressed that further trials with more patients and longer follow-up were needed to better establish how safe and effective the stem cell treatment was.

But if future tests were successful, he said, this could become a viable extra new treatment for patients with MDR-TB who do not respond to conventional drug treatment or for those patients with severe lung damage.

Priming Cocktail for Cardiac Stem Cell Grafts


Approximately 700,000 Americans suffer a heart attack every year and stem cells have the potential to heal the damage wrought by a heart attack. Stem cells therapy has tried to take stem cells cultured in the laboratory and apply them to damaged tissues.

In the case of the heart, transplanted stem cells do not always integrate into the heart tissue. In the words of Jeffrey Spees, Associate Professor of Medicine at the University of Vermont, “many grafts simply didn’t take. The cells would stick or would die.”

To solve this problem, Spees and his colleagues examined ways to increase the efficiency of stem cell engraftment. In his experiments, Spees and others used mesenchymal stem cells from bone marrow. Mesenchymal stem cells are also called stromal cells because they help compose the spider web-like filigree within the bone marrow known as “stroma.” Even though the stroma does not make blood cells, it supports the hematopoietic stem cells that do make all blood cells.  Here is a picture of bone marrow stroma to give you an idea of what it looks like:

Immunohistochemistry-Paraffin: Bone marrow stromal cell antigen 1 Antibody [NBP2-14363] Staining of human smooth muscle shows moderate cytoplasmic positivity in smooth muscle cells.
Immunohistochemistry-Paraffin: Bone marrow stromal cell antigen 1 Antibody [NBP2-14363] Staining of human smooth muscle shows moderate cytoplasmic positivity in smooth muscle cells.
Stromal cells are known to secrete a host of molecules that protect injured tissue, promote tissue repair, and support the growth and proliferation of stem cells.

Spees suspected that some of the molecules made by bone marrow stromal cells could enhance the engraftment of stem cells patches in the heart. To test this idea, Spees and others isolated proteins from the culture medium of bone marrow stem cells grown in the laboratory and tested their ability to improve the survival and tissue integration of stem cell patches in the heart.

Spees tenacity paid off when he and his team discovered that a protein called “Connective tissue growth factor” or CTGF plus the hormone insulin were in the culture medium of these stem cells. Furthermore, when this culture medium was injected into the heart prior to treating them with stem cells, the stem cell patches engrafted at a higher rate.

“We broke the record for engraftment,” said Spees. Spees and his co-workers called their culture medium from the bone marrow stem cells “Cell-Kro.” Cell-Kro significantly increases cell adhesion, proliferation, survival, and migration.

Spees is convinced that the presence of CTGF and insulin in Cell-Kro have something to do with its ability to enhance stem cell engraftment. “Both CTGF and insulin are protective,” said Spees. “Together they have a synergistic effect.”

Spees is continuing to examine Cell-Kro in rats, but he wants to take his work into human trials next. His goal is to use cardiac stem cells (CSCs) from humans, which already have a documented ability to heal the heart after a heart attack. See here, here, and here.

“There are about 650,000 bypass surgeries annually,” said Spees. “These patients could have cells harvested at their first surgery and banked for future application. If they return for another procedure, they could then receive a graft of their own cardiac progenitor cells, primed in Cell-Kro, and potentially re-build part of their injured heart.”

Stem Cell Treatments to Improve Blood Flow in Angina Patients


Angina pectoris is defined as chest pain or discomfort that results from poor blood flow through the blood vessels in the heart and is usually activated by activity or stress.

In Los Angeles, California, physicians have initiated a double-blind, multicenter Phase III clinical trial that uses a patient’s own blood-derived stem cells to restore circulation to the heart of angina patients.

This procedure utilizes state-of-the-art imaging technology to map the heart and generate a three-dimensional image of the heart. These sophisticated images will guide the physicians as they inject stem cells into targeted sites in the heart.

This is a double-blinded study, which means that neither the patients nor the researcher will know who is receiving stem-cell injections and who is receiving the placebo.

The institution at which this study is being conducted, University of Los Angeles (UCLA), is attempting to establish evidence for a stem cell treatment that might be approved by the US Food and Drug Administration for patients with refractory angina. The subjects in this study had received the standard types of care but did not receive relief. Therefore by enrolling in this trial, these patients had nothing to lose.

Dr. Ali Nasir, assistant professor of cardiology at the David Geffen School of Medicine and co-principal investigator of this study, said: “We’re hoping to offer patients who have no other options a treatment that will alleviate their severe chest pain and improve their quality of life.”

Before injecting the stem cells or the placebo, the team examined the three-dimensional image of the heart and ascertained the health of the heart muscle and voltage it generated. Damaged areas of the heart fail to produce adequate quantities of voltage and show low levels of energy.

Jonathan Tobis, clinical professor of cardiology and director of interventional cardiology research at Geffen School of Medicine, said: “We are able to tell by the voltage levels and motion which area of the [heart] muscle is scarred or abnormal and not getting enough blood and oxygen. We then targeted the injections to the areas just adjacent to the scarred and abnormal heart muscle to try to restore some of the blood flow.”

What did they inject? The UCLA team extracted bone marrow from the pelvic bones and isolated CD34+ cells. CD34 refers to a cell surface protein that is found on bone marrow stem cells and mediates the adhesion of bone marrow stem cells to the bone marrow matrix. It is found on the surfaces of hematopoietic stem cells, placental cells, a subset of mesenchymal stem cells, endothelial progenitor cells, and endothelial cells of blood vessels. These are not the only cells that express this cell surface protein, but it does list the important cells for our purposes. Once the CD34+ cells were isolated, the were injected into the heart through a catheter that was inserted into a vein in the groin.

CD34

The team hopes that these cells (a mixture of mesenchymal stem cells, hematopoietic stem cells, and endothelial progenitor cells) will stimulate the growth of new blood vessels (angiogenesis) in the heart, and improve blood flow and oxygen delivery to the heart muscle.

“We will be tracking patients to see how they’re doing,” said William Suh MD, assistant clinical professor of medicine in the division of cardiology at Geffen School of Medicine.

The goal of this study is to enroll 444 patients nation-wide, of which 222 will receive the stem cell treatment, 111 will receive the placebo, and 111 who will be given standard heart care.

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.”