Dual Antibody Treatment Knocks Down HIV Viral Loads in HIV-Infected Patients

According to two early-stage clinical trials reported in the journals Nature and Nature Medicine, a combination of two different antibodies against the Human Immunodeficiency Virus (HIV) have successfully diminished the virus levels in patients not taking anti-HIV medications.  Infusions of these antibodies kept virus levels low in the blood of a percentage of these patients for two-three months, after which, blood quantities of HIV in these patients rebounded to their previous levels.

HIV is a member of a special class of viruses called “retroviruses.”  Retroviruses receive their name from their ability to take an RNA copy of their genome and make a DNA copy of it.  This process, called “reverse transcription,” is backwards from the way cells normally do things.  In cells, genetic information is stored by DNA (deoxyribonucleic acid), and enzymes called RNA polymerases make an RNA copy of stretches of DNA.  The process by which RNA copies are made from DNA is called “transcription.”  In retroviruses, an enzyme called “reverse transcriptase” makes a double-stranded DNA copy of a single-stranded RNA genome.  This transcriptional process reverses the direction of transcription; hence the name “retroviruses.”

Below is a picture of a HIV particle.  The virus is surrounded by an envelope that is derived from the membranes of the host cell from which it budded.  Inserted into those membranes are viral proteins that are decorated by sugars.  These sugar-coated proteins are called “glycoproteins.”  The HIV surface glycoproteins are called gp120, which means that the glycoprotein weighs 120 kilodaltons (a Dalton is the weight of a hydrogen atom and a kilodalton is the weight of 1,000 hydrogen atoms), and this viral glycoprotein is anchored to the viral membrane by means of another protein called gp41.

HIV capsid


These two glycoproteins are the first thing that the acquired immune response responds to.  Unfortunately, the viral reverse transcriptase is extremely error-prone and makes lots of mistakes as it copies its viral RNA genome into DNA copies.  This means that when HIV infects cells, it produces progeny virus particles that vary tremendously.  When these variant viruses particles are put under intense selective pressure, as in the case when the immune system makes antibodies against it or when the viruses is brushed back by means of highly active antiretroviral therapy (HAART), the variant viruses that survive the best (and show the highest levels of resistance to the treatment of immune system-based attack) will emerge as the dominant strain of virus in the infected person and as the treatment strategies change, so will the virus in adaptation to the latest attempts to eradicate it.  It is an insidiously efficient mode of evading antiretroviral drugs and the immune system.  HIV also infects and destroys T4 lymphocytes (T-helper lymphocytes), which are essential for activating B lymphocytes.  Thus, HIV not only evades the immune response, but it permanently hamstrings it as well.

When a patient makes antibodies against the HIV surface glycoproteins, the virus simply changes the amino acid sequence of the protein and those neutralizing antibodies no longer prevent the virus from infecting cells.  Therefore, scientists have worked very hard to make custom antibodies that neutralize the virus in such a way that would make it difficult for the virus to adapt.

Michel Nussenzweig, an immunologist at the Rockefeller University who led these studies and his colleagues, have made custom (“monoclonal” antibodies against specific pieces of gp120.  Specifically, their custom antibodies target the base of the so-called “V3 loop” and the surrounding sugars.  Past work shows these antibodies (3BNC117 and 10-1074) that target this one spot reduces virus levels in human patients infected with HIV (Caskey, M. and others, Nature 522, 487–491 (2015); Lynch, R. M. and others, Sci. Transl. Med. 7, 319ra206 (2015); and Caskey, M. and others, Nat. Med. 23, 185–191 (2017).  Infusions of the antibody called 3BNC117 reduced virus levels of about 28 days in HIV-infected patients and 10-1074 reduced virus levels for one week in HIV-infected patients after which antibody-resistant viruses began to emerge.  Nussenzweig and his group then had the novel idea of treatment patients with both antibodies and not just one of them.  This way, the virus would have more hurdles to jump in order to adapt to this treatment.

In one of the clinical trials, 15 HIV-infected patients who had stopped taking all antiretroviral medications received three infusions of both monoclonal antibodies.  Of these 15 patients, nine of them showed a significant reduction in viral load for 15 – 30 weeks, after which, virus levels in the blood rebounded.

In the second clinical trial, seven HIV-infected patients who had never taken antiretroviral drugs were given infusions of the two monoclonal antibodies.  Initially, patients received infusions of 30 mg kg−1 of each of the antibodies and these infusions were very well tolerated.  In those four patients whose HIV viral profile showed sensitivity to the dual antibody treatment in laboratory tests, this therapy reduced HIV-1 viral load by two factors of ten.  This viral load reduction continued for three months following the first of up to three infusions. Additionally, and this is potentially the most exciting part of this study, none of these individuals developed resistance to both antibodies.  According to The Scientist, the viral loads of these patients rebounded; it is possible that these patients stopped receiving the antibody treatments (I only have access to the abstract of the Nature Medicine article).  However, the fact that the sensitive viruses stayed susceptible to the dual antibody treatment is quite encouraging.

Antibody treatments are diabolically expensive because making hybridoma cell lines that create the monoclonal antibodies is ridiculously labor- and resource-intensive.  For this reason, Nussenzweig said, “Ultimately, this may not be good for everybody, and it’s expensive.”

When HIV-infected patients are placed on HAART, they must take those drugs daily, for the rest of their lives.  These drugs have some potentially serious side effects, but they do tend to reduce progression of HIC infection to full-blown AIDS and reduce HIV-induced mortality (Kitahata, MM and others (2009), New England Journal of Medicine. 360 (18): 1815–1826; Lundgren, JD and others (2015), New England Journal of Medicine. 373 (9): 795–807; Danel, Christine and others (2015), The New England Journal of Medicine. 373 (9): 808–822).  Neutralizing antibodies against the virus may provide longer-lasting treatments, but, to date, HIV quickly adapts to experimental antibody treatments (for example, see Trkola, A. and others, Nature Medicine 11, 615–622 (2005).


“Antibodies can be used as a safe, new treatment to allow people to go ‘off’ of drugs for several months,” Nancy Haigwood, an AIDS researcher at Oregon Health & Science University who was not part of these studies.

Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases, said that this work represents an “early step” toward antibody-based HIV interventions.

Both these studies are small, and only included a few patients.  What we can glean from there, therefore, is limited.  However, there are some definite bright spots from them.  First of all, the dual antibody treatment was well tolerated in these patients.  This suggests that antibody treatments might be rather safe.  Again, larger studies are needed, but this is a potential bright spot of this study.  Secondly, the dual antibody treatment did significantly reduce viral loads for some time in a percentage of both groups of patients.  Thus, if the right antibody combination is found, it could potentially do some real damage to HIV levels in patients with susceptible viral populations.  Third, the antibody-based treatment strategy might actually work in some cases.

The caveats are many.  One is the cost.  Such mass treatments of HIV patients around the world will prove horribly expensive and third-world countries with the largest HIV load will simply not be able to sustain such costs.  Secondly, how long can such treatments give relief to patients when the virus simply adapts after a few weeks or months.  Therefore, feasibility of antibody-based treatments are a second problem.  Once potential use for antibody-based treatments that occurs to me are for those patients whose toxicity from HAART is just too onerous and the antibody-based treatments can give them a drug holiday to heal.  Such patients would need to be pre-screened to ensure that their virus population is susceptible to the antibody treatment before starting it.  Such HAART holidays may also cause a shift in their viral population to strains that are more susceptible to the HAART regimen they are taking.  Therefore, such drug holidays/dual antibody treatments might (a big might) have knock-off benefits.

In any case, this early, preliminary data is hopeful, but it is just the beginning if antibody-based treatments are to make it to the clinic.


Stem Cells from Baby Teeth Regenerate Dental Pulp after Implantation into Injured Teeth

Going to the dentist is usually not anyone’s idea of fun.  In particular, root canals are no fun.  However, if you have an abscessed tooth that hurts like the dickens, then a root canal may be your best bet for resuming normalcy.

In younger patients, the innermost part of the tooth, the pulp, may die off for a variety of reasons.  This phenomenon, known as “pulp necrosis” arrests root development and may result in tooth loss.  Injury to the pulp as a result of trauma, inflammation, tooth decay, or infection can result in the irreversible loss of teeth.


Regenerating dental pulp has proven to be a bear.  Getting the mass of blood vessels and nerves to regrow is not straight forward. However, teeth, fortunately, are blessed with a host of stem cell populations.  This includes the pulp, which contains “human deciduous pulp stem cells” or hDPSCs.  These cells can be extracted from baby teeth.  Can they be tamed to regenerate the pulp?

A new paper from Science Translational Medicine by Kun Xuan and others have used hDPSCs to regenerate the pulp in two different animals.  However, this Chinese team did not stop there, since they turned around and tried hDPSCs in human patients.

In their animal model, implantation of hDPSCs into damaged teeth regenerated dental pulp with blood vessels and nerves.  However, it also with a layer that deposited dentine. In short, the regenerated pulp saved the damaged teeth.

On the strength of these results, Xuan and others enrolled 40 patients with pulp necrosis after traumatic dental injuries in a randomized, controlled clinical trial. In this trial, Xuan and his colleagues randomly assigned 30 patients to the hDPSC group and 10 to the group that received a traditional dental treatment called apexification.   Apexification materials like calcium hydroxide and mineral trioxide aggregates to form a calcified barrier in the lower parts of the tooth root to seal it and prevent it from further degradation. 


They lost four patients, whose teeth experienced new trauma and were lost.  In the 26 patients they examined after hDPSC implantation and 10 patients (10 teeth) after apexification treatment, Xuan and others found that hDPSC implantation, but not apexification treatment, regenerated the pulp tissue complete with blood vessels and sensory nerves at 12 months after treatment. hDPSC implantation also led to regeneration of sensory nerves in the pulp.

They further followed 20 of the hDPSC-implanted patients for 24 months to determine safety risks.  In these observations Xuan and others did not observe any adverse events.

This is a small study, but it is a very encouraging study.  It suggests that hDPSCs can regenerate whole dental pulp and may potentially revolutionize the treatment of tooth injuries due to trauma.  Larger studies are needed and all of this must be verified before commercialization of this treatment is possible, but it seems like a great start.

Building a Better CAR-T cell to Attack Solid Tumors

Chimeric Antigen Receptor T-cells or CAR T-cells are genetically engineered white blood cells that have been taken from a patient’s own blood, genetically engineered to express a receptor that can tightly bind to cancer cells, expanded in culture, and then re-administered into the patient’s blood.  These CAR T-cells then act like guided anti-cancer missiles that find, attack, and kill cancer cells.  The results of CAR T-cell treatments have been astoundingly successful, and the FDA has just approved several such treatments for specific cancers.  On May 1, the Food and Drug Administration (FDA) approved the CAR T-cell therapy tisagenlecleucel (Kymriah) for adults with certain types of non-Hodgkin lymphoma.  Last year, FDA approved another CAR T-cell therapy, axicabtagene ciloleucel (Yescarta), for the treatment of diffuse large B-cell lymphoma (DLBCL).


CAR-T cell therapies are effective against blood cancers like lymphoma and leukemia, but when it comes to solid tumors, this treatment has been less effective.  Can CAR T-cell treatments be improved to attack solid tumors?  Research teams at Memorial Sloan-Kettering Cancer Center (MSKCC) and Eureka Therapeutics have bet that they can.  In a proof-of-concept study, MSKCC and Eureka Therapeutics scientists designed tumor-specific T-cells that express “checkpoint inhibitor” antibodies that protect the T-cell and allow it to evade the immunosuppressive tumor microenvironment found within a solid tumor.  The results of this study were published in the journal Nature Biotechnology.

Checkpoint inhibitors are monoclonal antibodies bind to cell surface proteins on white blood cells that are used by cancer cells to disengage the white cells from the tumor.  These checkpoint inhibitors, such as PD-1, when bound by cancer cells, cause the white blood cells to “forget” that they ever encountered the tumor.  This effectively permits the tumor to hide from the immune system.  Checkpoint inhibitors have successfully treated some solid tumors. In this experiment, the engineered CAR-T cells produced a single-variable fragment (scFv) PD-1 blocking antibody that is similar to already commercially available checkpoint inhibitor drugs.  This checkpoint inhibitor antibody innocuously binds to PD-1 and prevents the cancer cell engaging it.  This pulls back the tumor’s invisibility cloak and the CAR T cell recruits other neighboring immune cells to gang up on the tumor and kill it.

By using a mouse model, this study examined two different types of the anti-PD-1-expressing CAR-T cells; one of which targeted B-cell cancers and another that targeted solid tumors from in the ovary and the pancreas. ovarian and pancreatic cancer.  These groups discovered that their anti-PD-1-expressing CAR-T cells stayed near the tumor site longer and, once inside the tumor, they recruited other neighboring tumor-fighting cells to wake up and sock it to the tumor.

This very exciting finding may be foundational for future targeted therapies. “We can build CAR-T cells to secrete a variety of different molecules, tailored to the needs of the patient,” he says. “It’s not just limited to this one drug,” said Renier Brentjens, Director of the Cellular Therapeutics Center at MSK and one of the pioneers of CAR therapy.


Cynata’s Cymerus MSC Treatment Improves Cardiac Function Recovery in Preclinical Heart Attack Study

Cynata Therapeutics Limited, an Australian stem cell and regenerative medicine company, has announced the results of a preclinical trial in which it tests its Cymerus mesenchymal stem cell treatment in a preclinical heart attack model.

Cynata uses induced pluripotent stem cell technology to make large numbers of mesenchymal stem cells for therapeutic treatments.  To review, induced pluripotent stem cells (iPSCs) are made from body cells by means of genetic engineering and cell culture techniques.  Briefly, four different genes – OCT4, KLF4, SOX2, and c-MYC – are transfected into bodily cells.  Transient expression of these genes in regular body cells drives a small proportion of them (0.1%-0.5%) to undergo dedifferentiation into developmentally immature cells that resemble embryonic stem cells.  These embryonic stem cell-like cells, or iPSCs, will outgrow the other cells and can be successfully cultured in cell culture systems.  Once purified, cloned, and established as a viable cell line, the iPSC cell line can be grown to large numbers and differentiated into specific cell types.

Cynata has differentiated iPSCs derived from cells provided a healthy donor to make a recently identified precursor cell, known as a mesenchymoangioblast (MCA).  MCAs were first identified by Professor Igor Slukvin and his coworker from the University of Wisconsin-Madison. MCAs are early clonal mesoendodermal precursor cells that are the common precursor for both mesenchymal stem cells (MSCs) and endothelial cells, which are the main component of blood vessels. MCAs can also differentiate into pericytes and smooth muscle cells.

By making MCAs from iPSCs, Cynata seeks to overcome some of the largest hurdles for adult stem cell-based treatments, those being the dependence on donors for a steady supply of stem cells, the extensive variability of donated adult stem cells, the contamination of isolated adult stem cells with other cell types that do not have established therapeutic activities, and, probably most importantly, the limited scalability of stem cells from donors. Thus, in principle, Cynata’s Cymerus technology incorporates iPSC-derived MCA, which are used to make MCA-derived MSCs. This platform can potentially address the shortcomings of adult stem cell-based treatments, since iPSCs can proliferate indefinitely, and MCAs themselves can expand into extremely large quantities of MSCs. To quote the Cyanata web site; “Cynata should be able to manufacture all of the MCAs that it will ever need from a single Master Cell Bank of iPSCs – derived from a single donor.”

This most recent experiment was conducted by James Chong, Associate Professor at the Westmead Institute for Medical Research in Sydney, Australia, and colleagues. Chong and his coworkers induced heart attacks in three groups of rats (15 groups per group) and treated them four days later.  All the laboratory animals were assessed for 28 days after the heart attack (I wish they had observed them for longer periods of time). The rat groups consisted of three treatment groups: Group 1 received infusions of Cymerus MSCs, which were derived from iPSC-derived MCAs; Group 2 received MSCs from bone marrow; Group 3 was a placebo group that received infusions of buffer. All post-heart attack assessments were performed in a blinded manner, which simply means that the staff conducting the assessments had no idea which treatment the animal they tested had received.

The results of this study were rather encouraging.  The first category examined was “fractional shortening,” which is an estimate of the ability of the heart to contract effectively. Improvements in fractional shortening is indicative of recovery of the pumping function of the heart after a heart attack. Treatment with Cynata’s Cymerus MSCs resulted in an improvement in fractional shortening 28 days after the heart attack. Statistical comparisons showed that the Cymerus MSC-induced improvements were better than those observed in the placebo group (p=0.013) and the BM-MSC group (p=0.003).

The next assessment examined the effects of these treatments on left ventricular end-systolic diameter (LVESD). If, after a heart attack, the LVESD values are higher, then the heart is not contracting well.  However, reduced LVESD values are associated with improved cardiac function and correlated quite well with a reduced risk of further cardiac events. In this study, LVESD was lower in the Cymerus MSC group compared to those rats that had been treated with the placebo (p=0.054) and bone marrow-derived MSCs (p<0.001).

Finally, Chong and his gang examined the scar size as a proportion of the left ventricle size. For all three groups, there were no statistically significant differences between groups when it came to scar size as a proportion of the size of the left ventricle. It is possible that 28 weeks is far too short a time for the stem cells to produce any reduction in the size of the cardiac scar.  Therefore, further assessments of scar are ongoing.

To summarize, the Cymerus MSC treatments improved recovery of cardiac function post heart attack and reduced left ventricular end-systolic diameter (LVESD) compared to either placebo or bone marrow-derived MSCs, and further demonstrates the broad applicability of the Cymerus cell-manufacturing platform for clinical treatments.

In response to these positive results, Cynata’s Vice President of Product Development, Dr. Kilian Kelly said: “These very encouraging results add to a growing body of evidence showing that Cymerus MSCs may have an important role to play in the treatment of a wide range of diseases. There is still a huge unmet medical need associated with heart attacks, which cause over 8,000 deaths and more than 50,000 hospitalizations each year in Australia alone. We are optimistic about the potential benefits that Cymerus MSCs could bring to patients who experience these life-changing events.”

Steminent Biotherapeutics Inc Receives FDA Approval to Test Fat-Based Stem Cell Product to Treat Spinocerebellar Ataxia

Steminent Biotherapeutics Inc. is a biotechnology company based in Taipei, Taiwan, with subsidiary offices in San Diego and Shanghai. It is developing stem cell-based treatments for neurological conditions for which there are presently no treatment options.

The main product developed by Steminent is “Stemchymal.” Stemchymal consists of fat-based stem cells isolated from healthy donors. The cells are isolated from the fat (collected by means of liposuction), isolated, & standardized according to Good Manufacturing Practices that allows them to be administered to human patients. These fat-based mesenchymal stem cells contain a cornucopia of growth factors, cytokines, and other molecules that promote healing. They are also safe. Steminent scientists have shown that Stemchymal cells can be grown in culture for extended periods of time without becoming genetically altered. Stemchymal cells also neither form tumors in laboratory animals, nor elicit inflammatory reactions. Therefore, tissue matching is not required before administering them. Finally, Phase I clinical trials in human patients established the safety of Stemchymal when administered to people.

In Taiwan, Stemchymal has been approved for three different clinical trials. At the Taipei Veterans General Hospital, physicians are testing Stemchymal to treat osteoarthritis of the knee, spinocerebellar ataxia (a neurodegenerative condition), and vascular conditions. A recently approved application also allows testing Stemchymal to treat patients with diabetes mellitus.

In the United States, the Food and Drug Administration has “raised no objections” Steminent’s Investigational New Drug (“IND”) application that proposes to test Stemchymal as a treatment for polyglutamine spinocerebellar ataxia (“PolyQ SCA”).

Spinocerebellar Ataxias refer to a cluster of devastating, inherited neurodegenerative diseases that are relatively rare (between 2-7 per 100,000). These diseases are characterized by degeneration of the cerebellum, a part of the brain that regulates movement, and, sometimes, the spinal cord. Spinocerebellar ataxias (SCAs) are classified according to the altered genes that cause the disease. The symptoms of SCAs tend to include an uncoordinated gait, poor hand-eye coordination, and abnormal speech (dysarthria). There are no treatments for SCAs, and supportive measures are usually used.

Some SCAs are caused by the expansion of a portion of genes that encode stretches of the amino acid glutamine. Glutamine stretches seem to act as a flexible region that allows different portions of the protein to interact with each other.  When these glutamine stretches expand, the protein does not fold properly and aggregates, forming insoluble, toxic globules in the cell that cause cell death. Other mechanisms may be at work as well, such as mRNA toxicity, loss of protein function, or some other, as yet, uncharacterized mechanisms. There are more than 30 subtypes of SCA, and the following types of SCAs include poly-glutamine expansions: SCA1, SCA2, SCA3, SCA6, SCA7 and SCA17. The amino acid glutamine is encoded by the codons “CAG” and “CAA” stretches of these codons can cause DNA polymerase to slip, which causes the insertion of extra codons and expansion of the polyglutamine stretches.

The age of onset associated with PolyQ SCA disease patients can range from 20-50 years old. Not only are SCAs life-threatening diseases, but the extended physical handicaps imposed on the patient place a heavy burden on the patient’s family and healthcare providers.

As stated, there are no cures for SCAs, but Steminent has conducted a Phase I/II trial with SCA patients and showed that Stemchymal is safe for SCA patients. There were “no biological-related adverse effects observed in the 12-month follow-up. Additionally, patients seemed to improve while on Stemchymal. These functional improvements were maintained for up to 6 months.

In December 2015, the FDA designated Stemchymal as an Orphan Drug for the treatment of PolyQ SCAs. The Orphan Drug Designation grants “orphan status” to treatments of rare indications that affect fewer than 200,000 people in the U.S. This Orphan Drug Designation allows Steminent a seven-year window during they will enjoy “market exclusivity upon approval of Stemchymal® and other development incentives including tax credits for clinical research costs and Prescription Drug User Fee Act (PDUFA) fee exemption.”

Managing Director of Steminent USA, Dr. Jennifer Ho, said, “Our Phase II Stemchymal® SCA program includes double blinded, randomized, and placebo-controlled trials to evaluate Stemchymal® SCA for safety and evidence of efficacy for treating PolyQ SCA in three countries. The first of these Phase II trials is currently enrolling patients in Taipei, and now with FDA consent, we are very pleased to initiate this US orphan designated drug trial. ReproCELL, our Japan partner, has also submitted its CTN to the PMDA to assess Stemchymal® SCA in treating PolyQ SCA in Japan.”

Dr. Susan Perlman, Clinical Director, UCLA Ataxia Center, Professor of Neurology, UCLA, and Medical Director; National Ataxia Foundation, said of Steminent’s clincal trial, “as there are currently no approved treatments for this progressive, irreversible disease, we are encouraged by the possibility that Stemchymal® cell therapy may demonstrate safety and therapeutic benefit in these patients.” According to Dr. Perlman, “It is estimated that about 15,000 people in the USA suffer from PolyQ SCA disease.”

With FDA approval in hand, Steminent will prepare the US trial sites and commence patient enrollment.

Aegle Therapeutics is Awarded IND to Test Extracellular Vesicles from Stem Cells in Burn Patients

Aegle Therapeutics is a Miami, FL-based biotechnology company that has taken a completely novel approach to regenerative medicine.  Aegle Therapeutics has developed new techniques to isolated extracellular vesicles made by cultured stem cells.  Specifically, Aegle Therapeutics isolate extracellular vesicles from bone marrow-based mesenchymal stem cells for therapeutic purposes.

Scientists at Aegle Therapeutics have shown that standard protocols used to isolated extracellular vesicles tend to badly damage them.  If these damaged extracellular vesicles are administered to injured animals, they tend to induce inflammation and fail to promote healing.  Aegle has demonstrated this very fact by administering extracellular vesicles (EVs) isolated by standard protocols to pigs suffering from skin injuries.  These damaged EVs did not promote healing and made the injuries worse.  However, if similarly, injured pigs were administered undamaged, whole EVs isolated with Aegle Therapeutics’ proprietary protocols, they not only accelerated healing, but they significantly decreased scarring and promoted the formation of blood vessels and hair follicles and nerve regeneration.

Aegle’s isolation process is also easily scalable and low-cost.

Aegle Therapeutics has used their whole EV preparations to treat severe dermatological disorders, with a specific focus on the treatment of burns. In May 2018, Aegle announced that the US Food and Drug Administration (FDA) approved an Investigational New Drug (IND) application. This IND will examine the use of whole EVs from mesenchymal stem cells to treat severe second degree burn patients. This is an open label dose escalation study, which means that the patients and their physicians will know that they are being treated with the experimental product, but randomly-assigned groups of patients will receive gradually increasing doses of the EVs. This clinical trial will test both safety and efficacy (a Phase 1/2a clinical trial) of Aegle Therapeutics’ lead product, AGLE-102. This clinical trial will enroll burn patients at several U.S. sites.

According to the founder and Chief Science Officer of Aegle Therapeutics, Evangelos Badiavas, M.D., Ph.D., “We are excited to be moving our EV therapy into the clinic to treat burns, an indication with a substantial unmet medical need. We believe this product has the potential for functional regeneration and organization of complex tissue structures that can enhance healing, reduce scarring, minimize contraction and improve overall cosmesis. Currently, patients with burns suffer scarring, disfigurement, loss of mobility and chronic pain. There’s a real need for better therapies.”

According to Shelly Hartman, Chief Executive Officer of Aegle, “This achievement is an important step as the company launches a Series A capital raise in 2018 to fund its clinical development.”

Aegle Therapeutics is also developing another product, AGLE-103 for the treatment of a genetic skin condition called epidermolysis bullosa (EB), which causes the skin to be fragile and blister.

Rexgenero Treats Its First Patient In Phase III Clinical Trial of Bone Marrow-Based Critical Limb Ischemia Treatment

According to their website, Rexgenero is a “regenerative medicine company and developer of advanced cell-based therapeutics for the treatment of serious diseases that are poorly treated with existing therapies.”   The head office of Rexgenero is in London, but their Research and Development laboratories are in Seville, Spain.  They also have a United Kingdom office in Brighton, which is a town with which I have some familiarity, since I lived there at one time in my life.

Rexgenero has focused on developing regenerative products from bone marrow cells.  In particular, they have used bone marrow-derived mononuclear cells (BM-MNCs).  BM-MNCs are extracted from the patient’s bone marrow after a bone marrow aspiration.  To isolate the mononuclear fraction, the white blood cells fraction from bone marrow is usually isolated by centrifugation with a material called Ficoll.  The pelleted material is the mononuclear fraction and contains a potent mixture of lymphocytes, monocytes, mesenchymal stem cells, hematopoietic progenitor cells, and other less well-characterized cell populations.


Another component of bone marrow mononuclear cells is endothelial progenitor cells (EPCs), which give rise to the walls of blood vessels.  When locally infused locally at the site of diseased vascular tissue, a well-processed mononuclear fraction can potentially form new blood vessels and restore tissue that was formerly starved for oxygen (see Haider, KH, Aziz S, and Al-Reshidi MA, Regenerative Medicine, 2017 12(8):969-982 for a review).

A bone marrow-based product developed by Rexgenero, called REX-001, has been used in human clinical trials to revascularize patients with a rather nasty condition called Critical Limb Ischemia (CLI).  CLI occurs when the blood vessels that feed and nourish an arm or leg are severely damaged or obstructed.  This results markedly reduces blood flow to the limb and the oxygen-deprived tissues can begin to die off, which causes excruciating pain, and disfiguring skin ulcers or sores.

CLI skin ulcer

These ulcers can become infected, gangrenous, and may require debridement or, in the most severe cases, limb amputation (yikes!).

Can REX-001 administration treat patients with CLI?  In an earlier Phase II clinical trial, which was completed in 2016, REX-001 was administered to over 100 patients in three clinical trials.  The results were rather encouraging. Patients who had severe ischemic pain at rest without skin ulcers (Rutherford scale in category 4), or resting pain with skin ulcers (category 5) were treated with REX-001 for 12 months.  At the end of 12 months, most patients (the website does not say what percentage of patients) in both patient populations were devoid of CLI and showed significant improvement in their clinical condition, as assessed by changes in Rutherford category.  Patients showed complete ulcer healing and alleviation of rest pain.  Angiographic imaging of treat patients definitively showed that REX-001-treated patients had extensive new networks that blood vessels that had sprouted.  It is reasonable, in my view to suspect that these new blood vessel networks in the limb were responsible for the corresponding improvement in patient’s clinical condition.

REX-001-induced new vasculature
Angiograms showing blood vessels prior to treatment (left) and after 6 months following treatment with REX-001 (right)

Rexgenero has recently announced the treatment of its first patient in a Phase III clinical trial that will evaluate REX-001, in patients with CLI and Diabetes Mellitus (DM).  In these two placebo-controlled, double-blind, adaptive Phase III trials, patients with CLI and DM with severe ischemic limb pain will be treated with REX-001 or a placebo to assess the efficacy and safety of REX-001 in the relief of CLI-associated resting limb pain.  In a second leg to this Phase III study, CLI patients with ischemic limb pain and skin ulcers (Rutherford stage 5) will be treated with REX-001 or a placebo to ascertain the efficacy and safety of REX-001 in the healing of CLI-induced skin ulcers. The relief of pain and the complete healing of ulcers are the primary endpoints of this clinical trial and amputation-free survival is the secondary endpoint in both legs of the study.

The European Medicines Agency (EMA) has fully endorsed the design of both legs of the trial, and this includes the target patient population and primary endpoints.  Clinical improvement in CLI patients increases the probability of successful treatment. Rexgenero also plans to enroll a total of 138 patients at approximately 35 clinical sites in Europe with first interim results expected in circa 18 months’ time and full data in 2020.

Joe Dupere, Rexgenero’s CEO, said “We are extremely pleased to announce the first patient infusion in our Phase III program with REX-001, which if successful could significantly improve the treatment of patients with CLI.”  He continued: “The program has been designed following advice from the EMA and in close collaboration with our Scientific Advisory Board. CLI is a medical condition with a clear need for new improved treatment options. Our goal is to bring innovative cell, gene and tissue therapies to the market addressing high unmet needs which cannot be treated with available therapies. We believe that REX-001 has the potential to be one of the first effective cell therapy products available for patients with CLI.”

This is definitely one clinical trial to keep an eye on.  If bone-marrow-based cell preparations can help patients sprout new blood vessel networks, there might be applications for other medical conditions as well, including cardiac ischemia, intestinal blood vessel blockage, and liver disease too.

Artificial blood vessels made by University of Minnesota Scientists

In patients who must receive dialysis to accommodate failing kidneys, ports are placed in their blood vessels, and a vein and an artery are tied together.  The name for the connection of an artery and a vein is a Cimino-Brescia fistula. Such fistulas are necessary for dialysis, and they are usually made in the arm. Since blood, like other fluids takes the path of least resistance, such fistulas generate high volume flow rates. Blood flow will prefer the fistula over capillary beds, which are high resistance flow areas. Also, native blood vessels are usually used to generate these fistulas because they are less likely to narrow and fail. Unfortunately, these surgical connections tend to fail. Worse still, they cannot be used in some patients because of the bad shape of their vascular system. Therefore, the answer in those cases is a graft. That seems onerous and likely to fail too.  Is there a better way?

Zeeshan H. Syedain and his coworkers from the laboratory of Robert Tranquillo at the University of Minnesota have used tissue engineering approach to generate vascular grafts from fibrin scaffolds and skin-based human fibroblasts.  In short, Tranquillo and his colleagues have made “off-the-shelf” blood vessels that were grown in the laboratory and do not have any living cells. Such lab-grown vessels might serve as blood vessel replacements for hard-up dialysis patients and others.  Tranquillo and his group published their findings in the journal Science Translational Medicine.

To make blood vessel substitutes, Tranquillo and others embedded human skin cells in a gel-like material made of cow fibrin. This concoction was grown in a bioreactor for seven weeks, after which, the cells were washed away. This left vessel-like tubes made of collagen and other proteins secreted by the cells.

Synthetic blood vessels
Researchers at the University of Minnesota have created a new lab-grown blood vessel replacement that is the first-of-its-kind nonsynthetic, decellularized graft that becomes repopulated with cells by the recipient’s own cells when implanted. Image courtesy of University of Minnesota.

Tranquillo said of this study, “We harnessed the body’s normal wound-healing system in this process by starting with skin cells in a fibrin gel, which is Nature’s starting point for healing.” He continued, “Washing away the cells in the final step reduces the chance of rejection. This also means the vessels can be stored and implanted when they are needed because they are no longer a living material.”

The vessel-like tubes looked like blood vessels, and they lacked any human cells.  Therefore, the immune system should not reject them if they were implanted into a human body.  However, can they function as blood vessels? To address this concern, Tranquillo and others implanted their laboratory-produced tubes into adult baboons. Six months after transplantation, the engrafted vessels looked like blood vessels and healthy cells from the recipient had grown into them and seemed to adapt to them without any ill effects. These laboratory-made vessels could withstand 30 times the average human blood pressure without bursting.  Additionally, there was no indication of an immune response and the grafts even self-healed when punctured with a needle.

Tranquillo and the team are in the process of FDA approval to test their synthetic blood vessels in clinical trials. In particular, Tranquillo and his team would like to test them in children with pediatric heart defects.

Infusion of high-dose umbilical cord blood cells normalized brain connectivity and improves motor function in children with cerebral palsy

Cerebral palsy is a congenital disorder that adversely affects movement, muscle tone and posture. Because those who suffer from congenital cerebral palsy are bone with it, there is often little that can be done to predict or prevent it. Cerebral palsy or CP is usually due to abnormal brain development prior to birth, but it can also result from in utero strokes, or oxygen deprivation during development or delivery. CP causes exaggerated reflexes, floppy or rigid limbs, and involuntary motions and there is a generalized weakness of skeletal muscles. CP affects 2-3 per 1,000 live births and the investment required by schools to accommodate CP children is substantial.  Furthermore, the personal investment of the heroic parents of CP children is substantial and, at times, exhausting.

Fortunately, animal models of CP have shown that the infusion of stem cells into the brains of young AP animals improves motor (movement-based) function.  In particular, human umbilical cord blood cells seem to facilitate repair of neural networks in the brain and improve movement. One study (Pediatric Research 2006; 59(2): 244-249) by Carola Meier and others from Ruhr-University in Bochum, Germany used an oxygen-deprivation model of CP in rats to show that treatment of these animals with human Umbilical Cord Blood Cells (hUBCs) substantially alleviated spastic paresis as assessed by walking track analysis. Also, examination of brain slices established that administered hUCBs incorporated themselves around the brain lesion (a phenomenon called “homing”) in large numbers. This study showed that the administration of hUCB stem cells after perinatal brain damage to could significantly reduce potential motor deficits.  A second paper (Developmental Neuroscience 2015;37(4-5):349-62) by Drobyshevsky and others from North Shore University Health System in Evanston, IL and collaborators from Duke University used a CP rabbit model to assess the efficacy of hUBCs to treat CP. In this experiment, Drobyshevsky and others induced oxygen deprivation when the rabbits were at 70% of their in utero lives. Then a group of the newborn rabbits were treated with hUCBs while others were not. The hUBC-treated animals showed significant improvements in posture, righting reflex, locomotion, tone, and dystonia (involuntary muscle contractions that cause repetitive or twisting movements). Unfortunately, the swimming test however showed that joint function was not restored by the hUBC treatment, but these other functions were. Tracking studies of the infused hUBC cells did not indicate that the cells penetrated into the brain with any efficiency, and Drobyshevsky and others suggested that the cells exerted their beneficial effects by means of “paracrine signaling,” which is to say that the cells secreted molecules that induced healing by activating native cells rather than differentiating into new neurons that created neural networks.


On the strength of these animal experiments, Jessica Sun from Duke University Medical Center and her colleagues and collaborators from multiple institutions extended these studies into human CP patients.  This, I’m sure, was a very dicey experiment to run because the subjects were children.  Getting approval for clinical experiments on children is very difficult and time-consuming.  Sun and her colleagues had shown that her hUBC infusion protocol was safe in a previous publication (Transfusion 2010; 50: 1980-1987) in which Sun and others reported treating 184 children with a single infusion of their own umbilical cord blood. The paper reported that the adverse effects of this treatment were rare and minimal. Because this was a Phase I study, it was only designed to assess the safety of the hUBC infusions and not their efficacy.


In a second publication, Sun and others have reported the results of their Phase II study in which they treated 63 CP children with various doses of hUBCs. This was a rigorous double-blind, placebo-controlled, crossover study in which Sun and her colleagues gave 10-50 million hUBCs per kilogram body weight to CP children between the ages 1 to 6 years.  These children received either their own umbilical cord blood or a placebo at the start of the experiment, followed by an alternate infusion 1 year later. After 1 year, those children who had received their own UBCs at the beginning of the trial, received the placebo, and those who had received the placebo at the start of the trial received their own UBCs. The children were assessed by means of specific motor function tests and their brains were imaged by means of magnetic resonance imaging brain connectivity studies. These assessments were done at the start of the trial, and then 1, and 2 years after the treatment.  To assess their motor skills, children were tested with a clinical tool called the Gross Motor Function Measure-66 tool.  This clinical tool evaluates changes in motor function in CP children.  Children are asked to perform a range of everyday activities from lying and rolling to walking, running, and jumping.  The children are given a composite score for all 66 tasks they are asked to do and this score reflects the depth of their motor skill. Changes in the Gross Motor Function Measure-66 (GMFM-66) indicates an improvement or decrease in motor function.  The primary endpoint was change in motor function 1 year after baseline infusion.

Two years after the initial treatments, the children were given further evaluations. Of the 63 CP children, 32 received their own umbilical cord blood and 31 received the placebo at the start of the experiments. One year after the trial began, Sun and her team detected no average change in GMFM-66) scores between the placebo and treated groups.  However, two years after the start of the trials, those CP children who had received higher doses of their own umbilical cord blood (20 million cells or more) showed significantly greater increases in their GMFM-66 scores.  In fact, their GMFM-66 scores were above what CP children at this specific age usually score. Another test that was administered was the Peabody Developmental Motor Scales test, which consists of six subtests that measure abilities in early motor development and assesses gross and fine motor skills in children from birth through five years of age. Gross Motor Quotient scores from the Peabody Developmental Motor Scales tests also revealed that children who had received the higher dose UBC treatments showed normalized scores, which indicates that the motor development of these children had become more normal rather than delayed.

Finally, the MRIs revealed normalized brain connectivity in the CP children who had received the higher doses of their own umbilical cord blood cells.

While this study is still preliminary, it suggests that appropriately doses of a child’s own umbilical cord blood stem cells improves brain connectivity and gross motor function in young children with CP.


Using Light to Guide T Lymphocytes to Attack Tumors

Solid tumors have a whole bag of tricks to avoid the immune system. Fortunately, new therapies aim at these strategies to sensitize the patient’s immune system to the tumor. A new study from the University of Rochester Medical Center laboratory has discovered a simple, practical way that uses light and optics to turn killer immune cells to the tumors.

The lead author of this study, Minsoo Kim, Ph.D., works as a professor of Microbiology and Immunology, and is also an investigator at the Wilmot Cancer Institute. This work from Kim’s laboratory were published in the online journal Nature Communications. Kim described the method devised in his laboratory as similar to “sending light on a spy mission to track down cancer cells.”

A new therapy for treating hard-to-crack cancers is called immunotherapy. Immunotherapy does not utilize radiation or chemotherapy, but instructs the patient’s T lymphocytes to attack the cancerous cells. For example, CAR T-cell therapy removes the patient’s T cells, grows them in the laboratory, genetically engineers them to recognize, attack and kill the cancer, and then reintroduces these cells back into the patient. This is one type of immunotherapy. While this ingenuous technique shows remarkable promise, the immune system can overreact or under-react sometimes. Also, slippery cancers can find ways to hide from marauding T cells. Likewise, aggressive tumors often have mechanisms by which they suppress the immune system and surround themselves with a kind of “no-go” zone that prevents any immune cells from coming near the tumor. These immunosuppressive microenvironments that surround the malignant tumor keeps T cells out.

While it is true that T cells can be engineered to be more efficient killers, unleashing such supercharged T cells into the body can produce a tempest of toxicities. Is there a safer way?

Kim and his colleagues tried to find a kinder, gentler way to crack the tumor. They used a two-prong approach. First, Kim and others discovered that light-sensitive molecules could effectively guide T cells toward tumors. Kim and his coworkers even discovered that a molecule from algae called “channelrhodopsin” (CatCh) that is light-sensitive, could be introduced into immune cells by genetically engineering them with viruses. This technology is so novel that the university’s technology transfer office has filed for patent protection on the invention. Secondly, Kim collaborated with University of Rochester optics and photonics experts to design a Light Emitting Diode (LED) chip that could be implanted and shine light on the tumor.

Next, the Kim group fitted their mice with a small battery pack that sent a wireless signal to the implanted LED chip. When the ears of the mice were implanted with aggressive melanoma cells taken from a patient, the chip remotely shines light on the implanted tumor and surrounding areas. The light-guided T cells ran headlong to the tumor, ignoring the no-go zone where they killed the implanted tumor.

Even more interestingly, the LED chip with the battery pack were used in many control mice and no toxic side effects were observed. In the tumor-implanted mice, the light-guided T cells completely destroyed the implanted melanoma was destroyed without dangerous side effects.

In the future, Kim wants to determine if the wireless LED signal can deliver light to tumors deep within the body instead only on the surface. Also, can light shined into deep areas of the body still guide the T cells to the tumor to attack the tumor.

Kim cautiously emphasized that while his discovery is exciting, it is only meant to be combined with immunotherapy to make it safer, more effective, and traceable. Perhaps with additional improvements, Kim’s optical method might allow doctors to see, in real-time, if cancer therapies are reaching their target. Currently when patients receive immunotherapy, they must wait for several weeks and then have imaging scans to determine if the treatment worked.

“The beauty of our approach is that it’s highly flexible, non-toxic, and focused on activating T cells to do their jobs,” Kim said.

Making Blood Cells in Culture – Done


One of the “Holy Grails” of stem cell biology has been growing blood cells in culture for use in clinical settings. Such a feat would provide large quantities of blood cells for post-surgical patients, or those with leukemia or other blood-based illness. The clinical applications are manifold and extensive.

Unfortunately, growing blood-making stem cells in the laboratory has proven to be a difficult task for even the most inventive and skilled stem cell laboratories. Nevertheless, several laboratories have been able to recapitulate the differentiation of pluripotent stem cells into cells that have the capacity to form T-cells and myeloid (non-lymphoid) cells (see Kennedy, M. et al. Cell Rep. 2, 1722–1735 (2012); Ditadi, A. et al. Nat. Cell Biol. 17, 580–591 (2015); and Elcheva, I. et al. Nat. Commun. 5, 4372 (2014)). Unfortunately, these experiments generated cells that were not able to engraft in the bone marrow of irradiated mice. Such an experiment is essential because radiation destroys the bone marrow of the mouse, and if a cultured cell is indeed and blood-cell-forming stem cell, then placing it into the bone marrow of irradiated mice should result in a functional restoration of the bone marrow. This, however, was not the case, which shows that whatever these pluripotent stem cells in these experiments differentiated into, they were not blood-cell-forming hematopoietic stem cells (HSCs).

Now, after a hiatus of almost 20 years, two different research groups have use two very different approaches to transform mature cells into primitive HSCs that are self-propagating and also form the cellular components of blood.

The first of these research teams was led by George Daley of Boston Children’s Hospital in Massachusetts. Daley’s group used induced pluripotent stem cell technology to reprogram adult human cells into cells that function as HSCs, even though they are not precisely like those found in the bone marrow in people. The second research team was led by Shahin Rafii of the Weill Cornell Medical College in New York City. Rafii and his coworkers used direct programming to differentiate mature cells from mice into fully functional HSCs.


The Daley group isolated skin-based fibroblasts from adult donors and then reprogrammed then through a combination of genetic engineering and cell culture techniques. This technology is similar to that designed by Shinya Yamanaka and his colleagues at Kyoto University, for which Yamanaka won the Nobel Prize in Medicine in 2012.   Once these reprogrammed cells formed induced pluripotent stem cells (iPSCs), Daley and his group did something very creative. They inserted the genes that encode seven different transcription factors into the genomes of their iPSCs. Transcription factors are proteins that activate gene expression. Transcription factors do so either by binding specific sequences of DNA, or by tightly binding to other proteins involved in gene expression and activating them. The genes for these seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) are known to be sufficient to convert hemogenic endothelium (the cells from which HSCs develop) into HSCs.

After engineering their iPSCs with these seven genes, Daley’s group did yet another highly creative thing. Daley and his colleagues injected their modified human cells into developing mice. This provided the cells with the proper environment to differentiate into HSCs. Twelve weeks after injecting them into mouse embryos, the engineered iPSCs had differentiated into progenitor cells that could produce the full range of blood cells found in human blood. This included immune cells, platelets, and other types of red and white blood cells. These progenitor cells are, according to Daley, “tantalizingly close” to naturally occurring HSCs.

Rafii and his team made their mouse HSCs from mature mouse cells without going through an embryonic intermediate. The Rafii grouop isolated endothelial cells that line blood vessels from adult mice and genetically engineered them to overexpress four different genes (Fosb, Gfi1, Runx1, and Spi1). Upon culturing their genetically engineered cells in a culture system that mimicked blood vessels, these cells, over time, differentiated into HSCs.

For the next test, Rafii and others injected their cultures-derived HSCs into irradiated mice. These mice survived and showed a completely recapitulated bone marrow that produced immune cells, and all types of red and white blood cells, and lived than 1.5 years in the lab.

Rafii told Nature’s Amy Maxmen that his approach is like “a direct airplane flight, and Daley’s procedure to a flight that takes a detour to the Moon before reaching its final destination.” Rafii noted that how the cells are made matters when it comes to using them in the clinic. Every time genes are transformed into cultured cells, a significant percentage of the cells fail to incorporate one or all of these genes. Such cells must be removed from those cells that were successfully transformed. Genetically engineered cells also run the risk of having experienced mutations as a side effect of the genetic manipulation. If implanted into people, such cells might cause problems.

Daley, however, and other stem cell researchers remain sanguine about the possibility of making such cells in safer, more efficient and even cheaper ways that can be brought to the clinic. For example, Jeanne Loring from the Scripps Research Institute in La Jolla, CA has suggested that using techniques that cause transient rather than permanent expression of introduced genes might very well make such cells inherently safer. Loring also noted that the iPSCs had by Daley’s group are initially made from skin-based fibroblasts, which are easy to acquire and isolate, whereas Rafii’s method begins with endothelial cells, which are more difficult to gather and to keep alive in the lab.

“For many years, people have figured out parts of this recipe, but they’ve never quite gotten there,” says Mick Bhatia, a stem-cell researcher at McMaster University in Hamilton, Canada, who was not involved with either study. “This is the first time researchers have checked all the boxes and made blood stem cells.” Bhatia added: “A lot of people have become jaded, saying that these cells don’t exist in nature and you can’t just push them into becoming anything else. . . I hoped the critics were wrong, and now I know they were.”

So making blood cells and HSCs in the laboratory is possible.  Bring this into the clinic is going to be even tougher.

Repeated Administrations of Stem Cells are More Effective than Single Administration

After a heart attack, the heart can undergo several structural and functional changes. Even after oxygen delivery to the heart muscle has been restored, a temporary loss of contractile function can persist for several hours or even days. This phenomenon is called myocardial stunning and it can also occur in people who have undergone cardiovascular procedures or central nervous system trauma. Myocardial stunning seems to result from the release of toxic molecules by the dying heart muscle cells, and an imbalance in ions required for heart muscle contraction, such as calcium ions.

If the heart muscle remains deprived of oxygen for some time, then the heart muscle adapts to low-oxygen conditions and “hibernates.” Hibernating myocardium contracts very little, and has “battened down the hatches,” metabolically speaking, in order to survive. However, hibernating myocardium takes even more heart muscle cells off-line and further reduces the performance of the heart.

Reduced heart performance leads to the production of molecules by the blood-starved kidneys that enlarge the heart and further compromise its efficiency. This leads to congestive heart failure and death. The term for such post-heart attack heart disease is ischemic cardiomyopathy” and it refers to a heart after a heart attack that is deprived of oxygen in many places and has heart walls filled with dead tissue that struggles to properly supply to body with blood, and often deteriorates as a result of this struggle.

Several different cell types have been used in many different studies to treat ischemic (oxygen-deprived) heart disease. The results, though positive in many cases, only show modest improvements in most cases. Furthermore, clinical studies and pro-clinical studies in laboratory animals tend to produce inconsistent results in which some patients or animals significantly improve while others either fail to improve at all. However, all of these studies have one feature in common: the subject is treated with only one infusion of stem cells. What if only one treatment is not enough to properly heal the damaged heart after a heart attack?

Workers from Roberto Bolli’s laboratory at the University of Louisville, Kentucky have treated laboratory rodents with heart attacks with three doses of cardiac progenitor cells. These treatments were given 35 days apart and were compared with single treatments and placebo treatments.

In their paper, which was published in Circulation Research (DOI:10.1161/CIRCRESAHA.116.308937), Bolli and his group gave heart attack to 85 Fischer 344 rats, but 13 died one week after the procedure. The remaining 72 rats were split into three groups: vehicle only, single-treatment, and triple-treatment. The vehicle-only rats received injections of saline into their heart muscle 30 days after the heart attack, and then again 35 days later and then again 35 days after that. The single-treatment rats received an injection of 12 million cardiac progenitor cells into their heart muscle 30 days after their heart attacks, but then received injections of saline 35 days later and 35 days after that. The triple-treatment rats received an injection of 12 million cardiac progenitor cells into their heart muscle 30 days after their heart attack, and then another injection 35 days later and a third inject 35 days after that. Of the 72 rats that survived the laboratory-induced heart attacks, 9 died as a result of injections into the heart. Thus, 63 rats completed the protocol.

Cardiac progenitor cells (CPCs) are resident stem cells in the heart that can be isolated with small heart tissue biopsies (see Tang XL, et al., Circ Res 2016;118:1091-1105). They can differentiate into heart muscle, blood vessels, or other heart-specific cell types (See Parmacek and Epstein, Cell 2005;120;295-298). Bolli and his co-workers have shown that the infusion of CPCs into the hearts of laboratory rodents after a heart attack can improve heart function, but the CPCs do not engraft into the heart at a terribly high rate. Furthermore, the functional improvements in the heart of these rodent elicited by CPC implantation are long-term (see Circ Res 2016;118:1091-1105).

When heart function of the laboratory rats were assessed prior to the procedure, no significant differences were observed between any of the rats in the three groups. However, after the procedure, the hearts of those rats that were injected with saline continued to deteriorate throughout the duration of the experiment. This deterioration was functional in nature and structural.

Animals that had received only one injection of 12 million CPCs into the left ventricles of their hearts showed significant improvements over the saline-injected animals. These animals showed less dilation of the heart (lower end-systolic volume), their hearts pumped more blood per beat (stroke volume), better thickness in the damaged heart wall, and improved ejection fraction (average percentage of blood pumped from the left ventricle during each beat). These hearts also had smaller heart scars, great elasticity, and greater amounts of viable muscle.

While all that sounds great, the triply-injected hearts that received three injections of CPCs, showed even more robust and significant improvements in heart structure and function. Whatever the single injection of CPCs did, the triple injections did even better. However, it must be noted that even with three injections of CPCs, the level of engraftment of the injected cells remained poor.

To summarize these results observed in this paper, the 3 doses of CPCs 35 days apart resulted in increases in local and global heart function that were, roughly, triple that produced by a single dose. The multiple CPC administrations were associated with more viable tissue, less scar tissue, less collagen, and greater heart muscle cell density in the infarcted region. Still, the level of engraftment and differentiation of the injected cells accounted for <1% of total heart muscle cells.

Bolli and his coworkers believe that their work suggests that all clinical and preclinical trials should at least try multiple stem cell treatments in order to maximize the clinical benefit of the injected stem cells. Furthermore, Bolli and others suggest that the use of single injection protocols are the reason so many stem cell-based clinical trials have resulted in inconsistent and inconclusive results.

This study is a large preclinical trial that used large numbers of animals. The data are solid and the results are believable. My problem with the clinical implications of this study are as follows: A heart attack causes a good deal of inflammation in the heart that culminates in wound healing. Within 24 hours of the heart attack, white blood cells infiltrate the damaged area of the heart. Protein-degrading enzymes from scavenger neutrophils (a type of white blood cell) degrade dead tissue. The damaged cells degenerate, and collagen-making fibroblasts divide and lay down scar tissue. The initially-deposited collagen deposited in the wall of the heart is weak, mushy, and vulnerable to re-injury. Unfortunately, this is precisely the period of time (10-14 days after the heart attack) that patients feel better and want to increase their activity levels. However, this greater activity level can stress the heart and cause rupture of the heart wall. After 6 weeks, the dead (necrotic) area is completely replaced by scar tissue, which is strong but incapable of contracting or relaxing.

In light of this timeline, multiple injections of stem cells into the heart after a heart attack might very increase the risk of rupture of the heart wall. This is particularly the case if implanted cells secrete tissue proteases that degrade surrounding tissue. Thus, timing and dose will be extremely important in such multiple treatments. Too many cells can rupture the heart, treatments too early when the healing heart walls are sift and weak will prove inimical to the heart and treatments given too late might very well be too late for the cells to do any good. Therefore, while this paper seems to move the ball down the field of regenerative medicine, it creates a fair number of questions that will need to be answered before such a strategy can come to the clinic.

Minneapolis Heart Institute Foundation Tests Stem Cell Combination in Heart Attack Patients

The Minneapolis Heart Institute Foundation has announced a new clinical trial that will examine the ability of a stem cell combination to treat patients with ischemic heart failure.

In patients who have suffered from former heart attacks, clogged coronary blood vessels and heart muscle that hibernates can result in a heart that no longer works well enough to support the life of the patient. The lack of blood flow to vital parts of the heart and an increasing work load can result is so-called “Ischemic heart failure.” Such heart failure after a previous heart attack is one of the leading cause of death and morbidity in the world. According to the World Health Organization, ischemic heart disease affects more than 12% of the world’s population.

Stem cell therapy has been tested as a potential treatment for ischemic heart disease. Despite flashes of remarkable success, the overall efficacy of these treatments has been relatively modest. Most clinical trials have used the patient’s own bone marrow cells. In this case, the cell population is very mixed and it might not even be stem cell populations in the bone marrow that are eliciting recovery. Also, the quality of each patient’s bone marrow is probably quite varied, which makes standardizing such experiments remarkably difficult. Other clinical trials have used bone marrow derived mesenchymal cells [MSCs]. Several clinical trials with MSCs have seen some improvement in patients. MSCs seem to induce the formation of new blood vessels and also seem to induce endogenous stem cell populations in the heart to come to life and fix the heart. Other trials have used cardiac stem cells (CSCs) that were derived from biopsies of the heart. Even though fewer clinical trials have tested the efficacy of CSCs in human patients, the trials that have been conducted suggest that these cells can truly regenerate damaged heart tissue.

The Minneapolis Heart Institute Foundation® (MHIF) has announced a new clinical trial which will examine the combination of MSCs with CSCs to treatment patients with ischemic heart failure. This clinical trial, the CONCERT study, will be led by Principal Investigator Jay Traverse, MD. The CONCERT study will implant MSC’s and CSC’s in order to determine if the combination would be more successful than using either alone based on pre-clinical studies in swine demonstrating an enhanced synergistic effect of the combination.

CONCERT is sponsored by the National Institutes of Health and the Cardiovascular Cell Therapy Research Network (CCTRN), of which MHIF is a charter member. This will be a phase II clinical trial, which means that the focus of this leg of the study is to assess the relative safety of CSCs and MSCs, delivered either alone, or in combination, in comparison to placebo, and to measure the efficacy of the stem cell cocktail as well. To that end, researchers will measure and note any change or improvement in left ventricular (LV) function by cardiac MRI as well as changes in various clinical outcomes (survival, 6-minute walking, blood pressure, etc.), and quality of life.

This phase II study is a randomized, blinded, placebo-controlled study that will enroll 160 subjects at seven different CCTRN sites throughout the U.S. All recruited subjects will have ischemic cardiomyopathy and an ejection fraction 5%). This is significant, because some work in animals suggests that CSCs can make new heart muscle tissue that can shrink the heart scar. The first 16 patients were recently enrolled in a FDA-required safety run-in phase, but the remaining patients will be enrolled in the fall after a three-month safety analysis is performed. Incidentally, this is the first cardiac stem cell trial to perform MRIs on patients with defibrillators and pacemakers

“This combination of cells represents the most potent cell therapy product ever delivered to patients,” said Dr. Traverse. “Confirming that both types of stem cells together work better than either individual cell type could lead to improved patient outcomes and better quality of life for ischemic heart failure patients.”

Dosing Recent Heart Attack Patients with G-CSF Doesn’t Seem To Work

Granulocyte-Colony Stimulating Factor (G-CSF)is a small protein that stimulates the bone marrow to produce more of a particular class of white blood cells called granulocytes and release them into the bloodstream. A commercially available version of G-CSF called Filgrastim (Neupogen) is used to boost the immune system of cancer patients whose immune systems have taken a beating from chemotherapy.

Because several clinical trials have shown that implanting bone marrow mononuclear fractions into the hearts of heart attack patients can improve the heart health of some heart attack patients, clinicians have supposed that injecting heart attack patients with drugs like filgrastim, which moves many bone marrow-derived cells into the bloodstream might also provide some relief for heart attack patients.

Nice idea, but it does not seem to work. Two clinical trials, STEMMI and REVIVAL-2, have given G-CSF to heart attack patients at different times after their heart attacks. Unfortunately both studies have failed to show a difference from the placebo.

In the REVIVAL-2 study, 114 patients were enrolled, and 56 received 10 micrograms per kilogram body weight G-CSF for five days, and the remaining patients received a placebo treatment.  G-CSF and the placebo were administered to patients five days after the hearts were successfully reperfused by percutaneous coronary intervention (this is a fancy way of saying stenting).  This study was double-blinded, placebo-controlled and well designed.  Unfortunately, when patients were studied seven years after treatment, there were no statistically significant differences between the treatment and the placebo groups when it came to the number of deaths, heart attacks, and strokes.  Thus, the authors conclude that G-CSF administration did not improve clinical outcomes for patients who had a heart attack (see Birgit Steppich, et al, Atherosclerosis and Ischemic Disease 115.4, 2016).

A second clinical trial, the STEMMI trial, was a prospective trial in which G-CSF treatment was begun 10-65 hours after reperfusion.  Here again, there were no structural differences between the placebo group and the G-CSF-treated group six months after treatment and a five-year follow-up analysis of 74 patients revealed no differences in the occurrence of major cardiovascular incidents between the two treatment groups (R.S. Ripa, and others, Circulation 2006; 113: 1983-1992).

The STEM-AMI clinical trial also showed no differences in clinical outcomes after G-CSF treatment as compared to placebo in 60 patients after three years (F. Achilli, and others, Heart 2014, 100: 574-581).

Why does this technique fail?  It is possible that the white blood cells that are mobilized by G-CSF are low-quality and do not express particular genes.  A study in rats has shown that G-CSF infusion increases the number of progenitor cells in the bloodstream, but fails to increase the number of progenitor cells in the heart after a heart attack (D. Sato, and others, Experimental Clinical Cardiology, 2012; 17:83-88).  In order for cells to home to the infarcted heart, they must express particular proteins on their surfaces.  For example, the cell surface protein CXCR4 is known to play an integral role in progenitor cell homing, along with several other proteins (see Taghavi and George, American Journal of Translational Research 2013; 5:404-411; Shah and Shalia, Stem Cells International 2011;2011:536758; Zaruba and Franz, Expert Opinion in Biological Therapy 2010; 10:321-335).  Indeed, Stein and others have shown that progenitor cells mobilized with G-CSF in human patients lack CXCR4 and other cell adhesion proteins thought to play a role in homing to the infarcted heart (Thromb Haemost 2010;103:638-643).

Therefore, even though all of these studies have not uncovered a risk in G-CSF treatment, the consensus of the data seems to be there no clinical benefit is conferred by treating heart attack patients with G-CSF.

Hair Follicles Can Direct Wound-Based Cells to Induce Scar-Free Healing

News from the University of Pennsylvania reports a new method that involves the use of fat to help heal skin without the formation of scar tissue.  This work comes from the Perelman School of Medicine at the University of Pennsylvania, and it is the result of a large-scale, multi-year study that collaborated with the Plikus Laboratory for Developmental and Regenerative Biology at the University of California, Irvine.  Their findings were published online in the journal Science on January 5th, 2017.

A fancy name for fat is “adipose tissue.”  Adipose tissue is actually a rather complicated pastiche of different cell types.  Specialized cells in adipose tissue that stores fat are called “adipocytes,” but they are more colloquially called fat cells.  Fat cells are normally found in the skin, but when wounds in the skin heal and form, those underlying population of fat cells are lost.  In skin tissue that is undergoing the process of healing, the most common cell types are known as “myofibroblasts.”  Myofibroblasts are large cells with ruffled membranes, that are kind of a cross between smooth muscle cells and fibroblasts.  They have the ability to contract like smooth muscle cells when exposed to molecules that induce smooth muscle to contract, such as angiotensin II or epinephrine.  Fibroblasts, which are numerous throughout the skin and other organs, can readily differentiate into myofibroblasts, as can stellate cells found in liver or the pancreas, some smooth muscle cells, progenitor cells in stromal tissue, epithelial cells, or circulating progenitor cells (see B. Hinz, et al, The myofibroblast: one function, multiple origins, Am J Pathol. 2007 Jun;170(6):1807-16).  Once it forms, scar tissue also does not properly form any hair follicles and this can give it a rather odd appearance relative to the rest of the skin. The Perelman researchers designed a new strategy to limit scar formation during healing by converting wound-based myofibroblasts into fat cells, which prevents the formation of scarring.

“Essentially, we can manipulate wound healing so that it leads to skin regeneration rather than scarring,” said George Cotsarelis, MD, the chair of the Department of Dermatology and the Milton Bixler Hartzell Professor of Dermatology at Penn, and the principal investigator of this project. “The secret is to regenerate hair follicles first. After that, the fat will regenerate in response to the signals from those follicles.”

Cotsarelis and his colleagues showed that the formation of fat in the skin and hair follicles are separate developmental events, but they are, nevertheless, linked.  Hair follicles form first, and the factors required to induce hair follicle formation that are produced by the regenerating hair follicle can also convert surrounding myofibroblasts into fat cells instead of a scar.  This underlying fat does not form without the formation of these new hair follicles.  These new fat cells are indistinguishable from pre-existing skin-based fat cells that give the healed wound a natural look instead of leaving a scar.  Cotsarelis and his gang discovered that a factor secreted by hair follicles called Bone Morphogenetic Protein (BMP) instructs the myofibroblasts to become fat.  This single finding represents a tectonic shift on our understanding of myofibroblasts.

“Typically, myofibroblasts were thought to be incapable of becoming a different type of cell,” Cotsarelis said. “But our work shows we have the ability to influence these cells, and that they can be efficiently and stably converted into adipocytes.” This was shown in both the mouse and in human keloid cells grown in culture.

“The findings show we have a window of opportunity after wounding to influence the tissue to regenerate rather than scar,” said the study’s lead author Maksim Plikus, PhD, an assistant professor of Developmental and Cell Biology at the University of California, Irvine. Plikus began this research as a postdoctoral fellow in the Cotsarelis Laboratory at Penn, and the two institutions have continued to collaborate.

These new findings might very well revolutionize dematological wound treatments.  These data might be useful for developing therapies that drive myofibroblasts to differentiate into adipocytes that can help wounds heal without scarring.

As Cotsarelis put it: “It’s highly desirable from a clinical standpoint, but right now it’s an unmet need.”

However, wound treatments are not the only use for this work.  Fat cell loss is a common complication of other clinical conditions.  HIV treatments, cancer, scleroderma, are just a few of the diseases that can cause wasting and drastic weight loss.  Also, because fat cells are also lost naturally because of the aging process, especially in the face, which leads to permanent, deep wrinkles, something that available anti-aging treatments cannot satisfactorily address.

“Our findings can potentially move us toward a new strategy to regenerate adipocytes in wrinkled skin, which could lead us to brand new anti-aging treatments,” Cotsarelis said.

The Cotsarelis Lab is now examining how hair follicle regeneration can promote skin regeneration.  The Plikus Laboratory would like to know more about the role of BMP in wound healing and are conducting further studies with using human cells and human scar tissue.

Stem Cell-Based Skin Graft for Severe Burns

Severe wounds are typically treated with full thickness skin grafts. Some new work by researchers from Michigan Tech and the First Affiliated Hospital of Sun Yat Sen University in Guangzhou, China might provide a way to use a patient’s own stem cells to make split thickness skin grafts (STSG). If this technique pans out, it would eliminate the needs for donors and could work well for large or complicated injury sites.

This work made new engineered tissues were able to capitalize on the body’s natural healing power. Dr. Feng Zhao at Michigan Tech and her Chinese colleagues used specially engineered skin that was “prevascularized, which is to say that Zhao and other designed it so that it could grow its own veins, capillaries and lymphatic channels.

This innovation is a very important one because on of the main reasons grafted tissues or implanted fabricated tissues fail to integrate into the recipient’s body is that the grafted tissue lacks proper vascular support. This leads to a condition called graft ischemia. Therefore, getting the skin to form its own vasculature is vital for the success of STSG.

STSG is a rather versatile procedure that can be used under unfavorable conditions, as in the case of patients who have a wound that has been infected, or in cases where the graft site possess less vasculature, where the chances of a full thickness skin graft successfully integrating would be rather low. Unfortunately, STSGs are more fragile than full thickness skin grafts and can contract significantly during the healing process.

In order to solve the problem of graft contraction and poor vascularization, Zhao and others grew sheets of human mesenchymal stem cells (MSCs) and mixed in with those MSCs, human umbilical cord vascular endothelial cells or HUVECs. HUVECs readily form blood vessels when induced, and growing mesenchymal stem cells tend to synthesize the right cocktail of factors to induce HUVECs to form blood vessels. Therefore this type of skin is truly poised to form its own vasculature and is rightly designated as “prevascularized” tissue.

Zhao and others tested their MSC/HUVEC sheets on the tails of mice that had lost some of their skin because of burns. The prevascularized MSC/HUVEC sheets significantly outperformed MSC-only sheets when it came to repairing the skin of these laboratory mice.

When implanted, the MSC/HUVEC sheets produced less contracted and puckered skin, lower amounts of inflammation, a thinner outer skin (epidermal) thickness along with more robust blood microcirculation in the skin tissue. And if that wasn’t enough, the MSC/HUVEC sheets also preserved skin-specific features like hair follicles and oil glands.

The success of the mixed MSC/HUVEC cell sheets was almost certainly due to the elevated levels of growth factors and small, signaling proteins called cytokines in the prevascularized stem cell sheets that stimulated significant healing in surrounding tissue. The greatest challenge regarding this method is that both STSG and the stem cell sheets are fragile and difficult to harvest.

An important next step in this research is to improve the mechanical properties of the cell sheets and devise new techniques to harvest these cells more easily.

According to Dr. Zhao: “The engineered stem cell sheet will overcome the limitation of current treatments for extensive and severe wounds, such as for acute burn injuries, and significantly improve the quality of life for patients suffering from burns.”

This paper can be found here: Lei Chen et al., “Pre-vascularization Enhances Therapeutic Effects of Human Mesenchymal Stem Cell Sheets in Full Thickness Skin Wound Re-pair,” Theranostics, October 2016 DOI: 10.7150/ thno.17031.

Activation of the Proteasome Enhances Stem Cell Function and Lifespan

As we age, the capacity of our stem cells to heal and replace damaged cells and tissues decline. This age-associated decrease in adult stem cell function seems to be a major contributor to the physiological decline during aging. A new paper, by Efstathios Gonos and his colleagues at the National Hellenic Research Foundation in Athens, Greece gives one possible technique that might improve the function of stem cells in an aging body.

Cells contain a multiprotein complex called the “proteasome” that degrades unneeded or defective proteins. The proteasome controls protein half-lives, function, and the protein composition of the cell. Functional failure of the proteasome has been linked to various biological phenomena including senescence and aging. The role of the proteasome in stem cells aging, however has received little attention to date.

Proteasome figure

Gonos and his coworkers used mesenchymal stem cells from umbilical cord Wharton’s Jelly and human fat. Because they were able to compare the proteasome activity in very young and aged stem cells, Gonos and others discovered a significant age-related decline in proteasome content and activity between these two types of stem cells. The proteasome from Warton’s Jelly mesenchymal stem cells were consistently more active and displayed more normal function and activity than those from human fat.  In fact, not only were the protease activities of the proteasomes from the aging stem cells decreased, but they also displayed structural alterations.

These differences in proteasomal activity were not only reproducible, but when the proteasome of young stem cells were compromised, the “stemness,” or capacity of the stem cells to act as undifferentiated cells, was negatively affected.

Even more surprisingly, once after mesenchymal stem cells from human donors lost their ability to proliferate and act as stem cells (their stemness, that is) their decline could be counteracted by artificially activating their proteasomes. Activating the proteasome seems to help the cell “clean house,” get rid of junk proteins, and rejuvenate themselves.


Gonos and his team found that the stem cell-specific protein, Oct4, binds to the promoter region of the genes that encode the β2 and β5 proteasome subunits. Oct4 might very well regulate the expression of these proteasome-specific genes.

From this paper, it seems that a better understanding the mechanisms regulating protein turnover in stem cells might bring forth a way to stem cell-based interventions that can improve health during old age and lifespan.

This paper was published in Free Radical Biology and Medicine, Volume 103, February 2017, Pages 226–235.

Better Ways to Make Dopamine-Producing Neurons From Stem Cells

Producing dopamine-making neurons from stem cells for transplantation into Parkinson’s disease patients remains challenging. Differentiating stem cells into dopaminergic neurons is not as efficient a process as we would like it to be. While several laboratories have managed to make pretty good batches of dopaminergic neurons, reliably producing large and pure batches of dopamine-making neurons from pluripotent stem cells is still somewhat problematic. Secondly, transplanting dopamine-making neurons into either the midbrain or the striatum of the brain represents another patch of problems because the production of too much dopamine can cause unwanted, uncontrollable movements. Preclinical assessments of stem cell-derived dopamine neurons in laboratory animals have produced positive, but highly varied results, even though the transplanted cells are very similar at the time of transplantation.

“This has been frustrating and puzzling, and has significantly delayed the establishment of clinical cell production protocols,” said Malin Parmar, who led the study at Lund University.

To address this issue, Parmar and his colleagues used modern global gene expression studies to gain a better understand the molecular changes that drive the differentiation of stem cells into dopamine-making neurons. Parmar conducted these experiments in collaboration with a team of scientists at Karolinska Institute. In their paper, which appeared in the journal Cell Stem Cell, Parmar and his colleagues used single-cell RNA seq to construct the neuronal development of dopaminergic neurons.


These neurons are characterized by the expression of a gene called LMX1a. However, it turns out that LMX1a-expressing neurons includes not only midbrain dopaminergic neurons (see below at the substantia nigra), but also subthalamic nuclear neurons.


These findings reveal that markers used to identify midbrain dopaminergic neurons do not specifically isolate midbrain dopaminergic neurons, but isolate a mixture of cells. Is there a way to separate these two populations?


Indeed, there is. Parmar and his colleagues in the laboratory of Thomas Perlmann showed that although dopaminergic neurons from the midbrain and subthalamic nuclear neurons are related, they do express a distinct profile of genes that are specific to the two cell types. The authors argue that the application of these distinct marker genes can help optimize those protocols that differentiate dopaminergic neurons from pluripotent stem cells.

See Nigel Kee and others, “Single-Cell Analysis Reveals a Close Relationship between Differentiating Dopamine and Subthalamic Nucleus Neuronal Lineages,” Cell Stem Cell, 2016; DOI: 10.1016/j.stem.2016.10.003.

Hitting Acute Myeloid Leukemia Where It Hurts

Research teams from Massachusetts General Hospital and the Harvard Stem Cell Institute have teamed up to devise a new strategy for treating acute myeloid leukemia (AML). This new strategy is an outgrowth of new findings by these research groups that have identified an enzyme that plays a central role in the onset of AML.

During blood cell development in the bone marrow, hematopoietic stem cells divide to produce daughters cells, one of which remains a stem cells and the other of which becomes a progenitor cell. The progenitor cells can either differentiate toward the lymphoid lineage, in which it will become either a B-lymphocyte, T-lymphocyte, or a Natural Killer cell, or a myeloid precursor that can give rise to neutrophils, megakarocytes (that produce platelets), monocytes, eosinophils, or red blood cells. However, the means by which myeloid cells are formed in the bone marrow of AML patients is abnormal, and the myeloid precursor cells do not differentiate into a specific white blood cells, but, instead, remain immature and proliferate and crowd out and suppress the development of normal blood cells.

David Scadden, MD, director of the MGH Center for Regenerative Medicine (MGH-CRM), co-director of the Harvard Stem Cell Institute (HSCI), and senior author of this Cell paper, had this to say about AML: “AML is a devastating form of cancer; the five-year survival rate is only 30 percent, and it is even worse for the older patients who have a higher risk of developing the disease.” Dr. Scadden continued: “New therapies for AML are extremely limited – we are still using the protocols developed back in the 1970s – so we desperately need to find new treatments.”

What genetic changes cause these developmental abnormalities that lead to AML? As it turns out, a wide range of genetic changes occur in AML (see Medinger M, Lengerke C, Passweg J. Cancer Genomics Proteomics. 2016 09-10;13(5):317-29; and Prada-Arismendy J, Arroyave JC, Röthlisberger S. Blood Rev. 2016 Sep 2. pii: S0268-960X(16)30060-1). In this paper, however, the authors proposed that the effects on differentiation had to transition through a few shared events. Using a method created by lead author David Sykes of the MGH-CRM and HSCI, the team discovered that a single dysfunctional point in the developmental pathway common to most forms of AML that could be a treatment target.

Previous studies had demonstrated that expression of the HoxA9 transcription factor, a transcription factor that must be inactivated during normal myeloid cell differentiation, is actually quite active in the myeloid precursors of 70 percent of patients with AML.  Unfortunately, no inhibitors of HoxA9 have been identified to date.  Therefore, Scadden and others used a different, albeit freaking ingenious, approach to screen small molecules that were potential Hox9A inhibitors based not on their interaction with a particular molecular target but on whether they could overcome the differentiation blockade characteristic of AML cells.

First, they induced HoxA9 overexpression in cultured mouse myeloid cells to design a cellular model of AML.  They also genetically engineered these cultured cells to glow green once they differentiated into the mature white blood cell types.  These groups screened more than 330,000 small molecules to find which would produce the green signal in the cultured cells.  The green glow indicated that the HoxA9-induced differentiation blockade had been effectively overcome. Only these 330,000 compounds, only 12 induced terminal differentiation of these cells, but 11 of then acted by suppressing a metabolic enzyme called DHODH.  DHODH, or dihydroorotate dehydrogenase, is a biosynthetic an enzyme that is a member of the pyrimidine biosynthesis pathway, which catalyzes the oxidation of dihydroorotate to orotate.


This is a shocking discovery because the DHODH enzyme is not known to play any significant role in myeloid differentiation.  Corroboratory experiments demonstrated that inhibition of DHODH effectively induced differentiation in both mouse and human AML cells.

The next obvious step would be to use known inhibitors of DHODH in mice with AML.  They were able to identify a dosing schedule that reduced levels of leukemic cells and prolonged survival that caused none of the adverse effects of normal chemotherapy.  Even though six weeks of treatment with DHODH inhibitors did not prevent eventual relapse, treatment for up to 10 weeks seemed to have led to long-term remission of AML.  This remission included reduction of the leukemia stem cells that can lead to relapse.  Similar results were observed in mice into which human leukemia cells had been implanted.

“Drug companies tend to be skeptical of the kind of functional screening we used to identify DHODH as a target, because it can be complicated and imprecise. We think that with modern tools, we may be able to improve target identification, so applying this approach to a broader range of cancers may be justified,” says Scadden, who is chair and professor of Stem Cell and Regenerative Biology and Jordan Professor of Medicine at Harvard University. Additional investigation of the mechanism underlying DHODH inhibition should allow development of protocols for human clinical trials.

Scadden noted that this manuscript describes six years of work and, also, is a true reflection of the collaborative nature of science in pursuit of clinically relevant therapies.