Curing HIV Disease With Stem Cell Therapy

Science Highlights by Ann A. Kiessling, PhD

What is HIV disease?

Human Immunodeficiency Virus (HIV) infects specific types of cells in the immune system. Like most viruses, in order for HIV to infect a cell, the virus must bind to a specific protein, termed a receptor, on the cell’s surface. There are many different types of cells in our immune system, and each plays a specific role in fighting infections, both bacterial and viral. Our bodies produce billions of new immune cells every day from stem cell reservoirs in bone marrow.

Patient Specific Stem Cells

HIV has a complex life cycle that includes becoming part of the genetic information of the host cell so the cell is infected for life. Infection can be dormant, with no new virus produced, or active, with new virus produced continuously

HIV infects immune cells that have a protein termed CD4 on their surface. Some HIV-infected CD4 cells die, but others remain in the body, prepared to fight another infection at a later date. When the HIV infected person encounters a new infection, such as the flu, or an infected injury, the HIV-infected CD4 cell responds like a reliable member of the immune system. It becomes activated, multiplies, and as a side effect, produces new HIV particles before it dies. The new HIV particles then infect new CD4 cells, setting up a repeat of the cycle. Because billions of new immune cells are made every day, it generally takes several years for an HIV infected individual to lose enough CD4 cells to have a negative impact on his/her ability to fight other infections. Once the number of CD4 cells is depleted to the point that the individual can no longer effectively fight new infections, their HIV disease has advanced to a new condition termed Acquired Immunodeficiency Syndrome (AIDS).


Virus receptor: the protein on the surface of a cell that allows the virus to bind to, and then enter, the cell to infect it


Is there a cure for HIV infection?

No. It is currently treated with drugs that block specific steps in the life cycle of HIV infection in the CD4 cells, but because some CD4 cells live for decades, and are not killed by the HIV drugs, the potential for them to activate, multiply, and give rise to new virus particles persists for decades. The long life of immune cells is important for disease memory, i.e. it is the reason adults don’t get childhood diseases, such as chicken pox, and the reason that vaccination is effective against diseases, such as polio, for many decades.


Immune system: the collection of cells that respond to and eliminate infection and foreign cell invaders


Can stem cells cure HIV disease?

Over 50 years ago, treatments for some diseases of the immune system were developed, and are the original stem cell therapies. The treatments involve destroying all the diseased immune cells, such as leukemias, with radiation treatment and cancer drugs. (6,7,8). Once the diseased immune system is destroyed, it is replaced by transplanting new immune cells from the bone marrow of a healthy donor.


Bone marrow transplant: the transfer of healthy bone marrow stem cells from a donor to a recipient whose own immune system has been destroyed


This has now become a routine treatment for many cancers and diseases of the blood(1). Early in the HIV pandemic, it was recognized that bone marrow transplants might cure HIV disease. But obstacles have stood in the way of this therapeutic approach:

First, all of the HIV-infected CD4 cells in the recipient must be destroyed before the transplant. If not, the donor bone marrow cells will become infected with HIV, and the transplant will have been for naught. Since not all CD4 cells everywhere in the body are destroyed by the radiation and drugs, infection of transplanted bone marrow was observed (2). Since bone marrow is limited in supply, the medical community was reluctant to “waste” valuable bone marrow to infection by HIV.

Second, the transplanted bone marrow must be a perfect match to the recipient’s cells, or the new immune system will attack them as “foreign,” leading to a life threatening condition known as “graft versus host disease” (see: Patient Specific Stem Cells). Since few matches are perfect, bone marrow recipients are usually treated with immune suppressing drugs. Since immune suppression of HIV infected persons leads to AIDS, this possibility further limited enthusiasm for bone marrow transplant treatment for HIV disease, and restricted it to those individuals who also developed a cancer for which bone marrow transplant was needed.

Importantly, proof-of-concept for the efficacy of bone marrow transplant for HIV disease was reported in 2009 in the New England Journal of Medicine(3). A team of German physicians treating an HIV-infected man with a cancer, lymphoma, by bone marrow transplant, was able to use a bone marrow match from an individual who was naturally resistant to HIV infection. Unlike earlier reports, the new bone marrow cells did not become infected with HIV.

What is natural resistance to HIV infection?

Studies of persons routinely exposed to HIV, but who did not become infected, revealed that in addition to cells having the CD4 protein, efficient infection also needs one of two additional receptor proteins, termed CXCR4 and CCR5. CXCR4 is a protein expressed on the surface of many cells, not just CD4 cells, but CCR5 is less commonly expressed. Individuals genetically lacking CCR5 appear normal and demonstrate remarkable resistance to HIV infection. The bone marrow donor for the German patient was genetically lacking the CCR5 protein.

How can stem cells provide therapy for HIV disease?

The proof-of-concept report from Germany supports the value of bone marrow transplant for HIV disease. New developments in stem cell science open new avenues to solve the main barriers to this therapeutic approach.

First, the possibility of deriving patient-specific stem cells (see: Patient-specific stem cells) will eliminate wasting valuable bone marrow.

Second, the laboratory methods for developing bone marrow stem cells from patient-specific stem cells have greatly advanced in the past two years (4), thus eliminating the need for a good tissue match from a bone marrow bank.

Third, the laboratory methods for silencing genes in stem cells has also greatly advanced in the past two years(5).

Taken together, it is now possible to derive patient-specific stem cells from HIV-infected individuals, differentiate them into bone marrow stem cells, and knock-out the CCR5 protein, rendering them resistant to HIV infection. This source of cells would then be available for transplant into the HIV infected individual, who may or may not have to prepare by going through radiation and drug treatment for complete ablation of all HIV-infected cells. Because the new cells will not be susceptible to HIV infection, it may be possible that over time, they would simply replace the individual’s HIV infected cells.

What is the timeline to develop patient-specific, CCR5 negative, bone marrow stem cells for HIV treatment?

The science of patient-specific stem cells is moving rapidly. By mid-2011, the best sources could be at hand. Within the same time frame, the most efficient laboratory methods for developing stem cells into bone marrow stem cells will also be identified. Hence, 2012 is a realistic time frame for the development of reliable methods to derive patient-specific bone marrow stem cells.

Laboratory methods to knock-out the CCR5 protein may also take 2 to 3 years. Several approaches are currently under study(5).

Once the CCR5 negative, patient-specific bone marrow stem cells are at hand, possibly by 2013, they must be studied for safety and efficacy. This may be the longest phase of the work since it will be necessary to prove long-term survival and lack of negative side effects in an animal model. A conservative estimate for this phase is 3 to 5 years.

Hence, if funding is available, it will be known within 5 to 8 years if patient-specific, CCR5-negative, bone marrow stem cells are a useful tool in the fight against HIV disease.

Will the cost be too high?

Until the efficiencies with which patient-specific, CCR5-negative, bone marrow stem cells can be derived are known, it will not be possible to predict overall costs per treatment.

However, given the current cost of $25,000 to $50,000 per year per patient for monitoring and treating HIV disease in the U.S., it is highly likely that stem cell therapy may be substantially less costly.

Bedford Research scientists will begin the patient-specific Testis Stem Cell Project in 2010, as soon as funding is available.

References:

  1. Kiessling AA and Anderson SC 2007 Human Embryonic Stem Cells, Jones and Bartlett plublishers
  2. Krishnan A,Zaia J, and Forman SJ 2003. Should HIV-positive patients with lymphoma be offered stem cell transplants? Bone Marrow Transplantation 32: 741-748
  3. Hutter G, Nowak D, Mosner M, Ganepola S, Mubig A, Allers K, Schneider T, Hofmann J, Kucherer C, Blau O, Blau I, Hofmann W, Thiel E 2009. New England Journal of Medicine 360: 693-698.
  4. Goodrich A, Ersek A, Varain N, Groza D, Cenariu M, Thain D, Almeida-Porada G, Porada C, Zanjani E 2010. In vivo generation of b-cell-like cells from CD34+ cells differentiated from human embryonic stem cells. Experimental Hematology 38: 516-525.
  5. Shimizu S, Hong P, Arumugam B, Pokomo Ll, Boyer J, Koizumi N, Kittipongdaja P, Chen A, Bristol G, Ballic Z, Zack J, Yang O, Chen I, Lee B, An D 2010. A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model. Blood 115: 1534-1544.

Day Two of the Int’l Conference: Reports from the Front Lines of Stem Cell Therapy Around the World

Day Two: April 23, 2010

stem cell conference international

The conference speakers, organizers and some attendees.

Our second day of the International Conference of Stem Cells and Regenerative Medicine for Neurodegenerative Diseasesbegan with a talk by Dr. Wise Young, of Rutgers University (USA), entitled, “Lithium Effects on Blood and Brain Stem Cells,” in it he summarized the clinical trial design for using umbilical cord blood stem cell therapies for spinal cord injury in China and the US.

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Day One: Int’l Conference of Stem Cells and Regenerative Medicine for Neurodegenerative Diseases

April 22, 2010

Dr. Kiessling and Dr. Shyr

Dr. Kiessling and Dr. Shyr

The first day of the International Conference of Stem Cells and Regenerative Medicine for Neurodegenerative Diseases began with a full auditorium in the beautiful and immaculate Tzu Chi Hospital conference center.

The conference was opened by Dr. Ann Kiessling and Dr. Ming-Hwang Shyr (Superintendent of Tzu Chi General Hospital) together they emphasized the importance of international cooperation in stem cell research. Dr. Kiessling said, “International scientific collaboration is absolutely fundamental to moving stem cell treatments forward as fast as possible.”

Dr. Kiessling’s talk, What is a Pluripotent Cell? And Is Pluripotency Important to Neuronal Differentiation? highlighted her recent research into the importance of circadian rhythms in stem cell biology. Discovering the impact of light and dark cycles for developing cells may be key to our understanding of developing stem cell lines.

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Early Human Embryos Make “Mistakes” as a Matter of Survival, Could Be Key to Understanding Stem Cell

FOR IMMEDIATE RELEASE

EARLY HUMAN EMBRYOS MAKE “MISTAKES” AS A MATTER OF SURVIVAL, COULD BE KEY TO UNDERSTANDING STEM CELL DEVELOPMENT

Early human embryos may be naturally prone to making mistakes in chromosome allocation to new cells, according to a report by Bedford Stem Cell Research Foundation scientists. Their new findings indicate rapid increases in total genetic information may be more important to embryo survival than accurate allocation of genetic information to each new cell.

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Patient-Specific, Pluripotent Stem Cells – Testis is a New Source

Patient Specific Stem Cells

The challenge is not only producing pluripotent stem cells, but patient-specific pluripotent stem cells

Pluripotent stem cell: cell with unlimited potential to multiply and differentiate into all the tissues in the body.

Science Highlights by: Ann A. Kiessling, PhD

Improving treatments for damaged organs and tissues is the promise of human pluripotent stem cells. The power of pluripotent stem cells to alleviate damage to organs, a form of regenerative medicine, has been amply demonstrated in many animal and laboratory model systems (see: State of the Stevm Cell). In some studies, the pluripotent stem cells need to differentiate into the type of cell needed for normal function prior to transplantation, whereas in other studies, the presence of the transplanted stem cells themselves appears to alleviate damage and help restore organ function (1). It is not hype to assert that pluripotent stem cells are the foundation upon which regenerative medicine will grow.

Several problems are currently hampering advances in stem cell therapy, one of which is the lack of readily available sources of patient-specific, pluripotent stem cells. Bedford Research scientists have focused on deriving pluripotent stem cells from unfertilized eggs, termed parthenote stem cells, for the past decade (2,3). Parthenote stem cells are patient-specific cells for the woman whose eggs were activated for their derivation, a highly promising source, but current protocols are too inefficient for routine therapeutic use. Bedford Research scientists are currently developing milestones during egg activation to improve the efficiency of parthenote stem cell derivation (4,5).

Recent reports of the derivation of patient-specific, pluripotent stem cells from testis biopsies indicate a readily available source of stem cells for men (6,7,8). Bedford Research scientists will begin testing the efficiency of pluripotent stem cell derivation from testis biopsies in 2010, as soon as funding is available. Once the stem cell lines are derived, they need to be tested for pluripotency, stability and safety. They will be compared with all other known sources of human pluripotent stem cells.

BACKGROUND

Pluripotent stem cells derived from eggs: embryonic stem cells derived from fertilized eggs (usually left-over embryos), and parthenote stem cells derived from artificially activated, unfertilized human eggs (9), are undeniably the most robust and stable human stem cells currently known. They are the “gold standard” against which all other pluripotent stem cells are judged. They continue to divide and expand for years in culture, in sharp contrast to stem cells derived from tissues, such as cord blood and adult bone marrow.

But the clinical value of egg-derived stem cells as therapeutic agents is debated.

First Clinical Trial with Human Embryonic Stem Cells — Spinal Cord Injury

On the one hand, trillions of identical embryonic stem cells can be grown under the careful laboratory conditions specified by the Food and Drug Administration for cell therapies. This characteristic is termed “stable cell line,” meaning they maintain a constant, correct number of chromosomes after each cell division, and they remain pluripotent, capable of differentiating into any cell desired.

These stable, reproducible, reliable characteristics hold the promise of the development of off-the-shelf reagents to treat diseases. Indeed, the first embryonic stem cell therapy clinical trial approved in 2008 by the U.S. Food and Drug Administration (FDA) will use early stage nerve cells derived from human embryonic stem cells to treat acute spinal cord injury. The cells were developed in Geron corporation laboratories from one of the first lines of embryonic stem cells derived in 1998. According to the study protocol, the cells will be injected into the cord just below the injury site. The clinical study protocol was under review by the FDA for 3 years before its approval. The FDA review included detailed information about the cell culture procedures, precisely how the cells will be delivered into the spinal cord, extensive animal studies of safety, and approval of the devise developed for cell delivery to ensure all participating neurosurgeons treat the acute spinal cord injury patients uniformly. The approved study protocol is designed to assess safety of the cell delivery treatment. One concern is that the cells placed at the site of the spinal cord injury may not be 100% “early stage nerve cells” designed to replace the protective coating around the injured spinal cord nerve pathways, but may also include undetected embryonic stem cells that could develop into a tumor at the injury site. Many trials with rats and mice have indicated this will not happen, but it will not be known for certain until the human clinical safety trial is conducted.

Histocompatibility: the genetic match of cellular proteins between the patient and the stem cells.

On the other hand, the early stage nerve cells differentiated from the embryonic stem cell line may not be compatible with the patient’s immune system. Just like blood type, every person has a unique repertoire of proteins on the surface of every cell. These proteins help the body’s immune system distinguish between “self” and “foreign”, such as bacterial or viral invaders. This important protection causes the immune system to attack cells from other people as foreign, along with bacteria and viruses. This is the reason people needing kidney or liver transplants must wait for an organ that “matches” most of their proteins.

Host versus graft disease: the attack of the stem cells (the “graft”) by the patient’s immune system (the “host”)

The term for this is “histocompatibility,” meaning the two tissues can get along with each other. If the histocompatibility match is not good, a condition termed “host versus graft disease” results and the patients will need to take drugs that suppress their immune systems for the rest of their lives.

Because there is only one line of embryonic stem cells being used for the first spinal cord clinical trial, the histocompatibility of the cells with each and every person entering the trial is unknown. There is evidence that embryonic stem cells may have fewer histocompatibility problems than adult organs such as a kidney, possibly due to their embryonic nature. But it will not be known until the trial is conducted if the spinal cord victims participating in the trial need to take immunosuppressive drugs for life, although it seems likely that at least some patients will. An immunosuppressive drug regimen has a number of side effects, including increased susceptibility to infections with both bacteria and viruses. One caveat to this is the possibility that the positive effect of the stem cells may not be needed forever, but only during the period of healing of the spinal cord injury. If this is the case, immunosuppressive therapy may only be needed for a few months or years. These considerations will not be understood until the trial is conducted.

Proposed Clinical Trial with Umbilical Cord Blood Cells — Spinal Cord Injury

Another clinical trial approach being developed takes advantage of a growing body of information from other countries, such as China, that umbilical cord blood cells delivered to the site of the spinal cord injury has beneficial effect in reducing the severity of the injury. In this instance, the cord blood stem cells are not treated in the laboratory to become immature nerve cells before being injected into the spinal cord at the injury site. This clinical trial design has the advantage that cord blood stem cell banks already exist, and the histocompatibility type of each cord blood sample is known, so a match may be found for the spinal cord victim. The additional advantage is research that has demonstrated umbilical cord blood cells do not develop into tumors. Moreover, some of the studies using umbilical cord blood cells have treated chronic spinal cord injury, injuries more than one year old. The surgical approach has been to open the spinal cord at the site of the injury, remove the cellular debris that has accumulated because of the large scar that forms at the injury site, and instill umbilical cord blood cells into the scar cavity. Although controversial, reports of improved function have appeared.

Sources of Patient-specific pluripotent stem cells

There is no way to know at this time which is more important, being able to coat the damaged spinal cord with new protective nerve cells, or supporting the re-growth of the spinal cord by inhibiting the damage and the scar tissue that forms. What is clear, however, is that if the stem cells being used for therapy were derived from the patient’s own body, the problem of histocompatibility would not exist.

The question is, how to create patient-specific stem cells?

Bone marrow pluripotent stem cells
If bone marrow stem cells prove therapeutically useful for acute injuries, such as heart attack, stroke, spinal cord injury and severe burns, they could be harvested from the patient at the time of the injury. Recent studies are promising (10,11), but require the isolation and expansion of a specific sub-population of cells that requires several days to weeks to accomplish, rather than cardiac injection of an entire sample of bone marrow cells.

For chronic conditions, such as diabetes, Parkinson’s disease, chronic spinal cord injury, deafness, congestive heart failure, kidney failure, Huntington’s disease and Lou Gherig’s disease, there is time to derive stable, pluripotent stem cells from the patient’s own tissues.

Induced pluripotent stem cells from tissue biopsies
The recent reports of developing pluripotent stem cells, (“induced pluripotent” stem cells) from biopsies of patient’s skin or liver are exciting and may prove broadly applicable to people of all ages. At this time the cell manipulations necessary to achieve pluripotency render the cells not suitable for therapeutic use, but many laboratories are working to circumvent this problem (1).

Egg-derived (parthenote) pluripotent stem cells 
As described above, for younger women still producing eggs every month (before menopause), stem cells could be derived from artificially activated eggs, termed parthenotes, for their own use. The eggs could be collected by procedures that are routine for women undergoing assisted reproductive therapies for infertility. More than 80,000 women undergo hormone stimulation and egg collection every year in the U.S., so the risks and side effects are well documented. Hormone administration ensures the collection of 10 to 20 eggs, rather than the one or two normally produced by a women’s ovaries each month. Studies in animal models have demonstrated that parthenote stem cells are histocompatible when transplanted back into the egg donor (12). For this to become clinically useful, however, the efficiency of deriving parthenote stem cell lines must reach at least 10% of eggs to ensure the derivation of one stem cell line for each cycle of egg collection.

Research into this possible source of stem cells for therapy cannot be conducted with federal dollars in the U.S., according to the new National Institutes of Health guidelines for stem cell research. The rationale for the moratorium on government funding is thought to relate to the “Dickey” amendment which is a rider attached to the annual congressional budget for the National Institutes of Health that specifically prohibits federal funding for research on fertilized or artificially activated human eggs. This restriction is surprising, because parthenotes are not capable of development into offspring, and as such would seem to be less controversial for research than left-over embryos from fertility treatments. The restriction may relate to an over-arching concern about the ethics of asking women to donate eggs for research purposes. In my view, although the concern is real, and egg donation cycles must be carefully conducted to ensure safety to the egg donor (see: BSCRF egg donor program), the federal moratorium is an unnecessary restriction of research funding for women interested in supporting stem cell research.

Concerns over egg donation for stem cell research were fueled by the research scandal of a few years ago that fraudulently reported the derivation of another form of patient-specific stem cells, “nuclear transplant” stem cells. These stem cells were touted by scientists for several years as being the best source of patient-specific stem cells (13). A South Korean research team reported deriving several lines of patient-specific stem cells by this method, which was greeted by major enthusiasm all over the world, only to be quickly exposed by South Korean scientists as a fraudulent report. To date, no such nuclear transplant stem cells have been reported for human eggs, although this is a relatively common procedure in lower animals.

New: testis-derived pluripotent stem cells
The recent reports of deriving pluripotent stem cells from testis biopsies is an exciting new development in the field of patient-specific stem cells for men. No genetic manipulations of the cells are necessary, the efficiency appears to be high, and it may only take a few weeks to grow sufficient cells in the laboratory for therapeutic use.

First reported by a German research team, it was a surprise to many scientists that pluripotent stem cells existed in the testis. For several decades, it has been known that sperm are abundantly produced (billions per week) in the adult male testis for life, and that the sperm arise from “sperm stem cells” termed spermatogonia. There are several stages to sperm cell maturation, analogous to the several stages in blood cell maturation, and ultimately each sperm precursor cell gives rise to four adult sperm.

The new reports indicate that in addition to the spermatogonia, there is a less committed stem cell within the testis, a pluripotent stem cell, that may be called upon to divide only in extreme circumstances. It is the pluripotent testis stem cell that has been shown to be as versatile as embryonic stem cells, potentially capable of developing into all tissues of the body. How stable it is in culture long term will take more time to learn, but results to date indicate this is a highly promising source of patient-specific stem cells that does not require genetic manipulation and can be derived from men of all ages in a relatively short time.

Bedford Research scientists will begin the Testis Stem Cell Project in 2010, as soon as funding is available.

 

References:

  1. Watt FM and Driskell RR 2010 The therapeutic potential of stem cells (pdf). Philosophical Transactions of the Royal Society, Biological Sciences, 365: 155-163
  2. Kiessling AA 2005 Eggs Alone (pdf). Nature 434:145
  3. Polak de Fried E, Ross P, Zang G, Divita A, Cunniff K, Denaday F, Salamone D, Kiessling AA, Cibelli J 2007 Human parthenogenetic blastocysts derived from noninseminated cryopreserved human oocytes (pdf). Fertility and Sterility 89: 943-947.
  4. Kiessling AA, Bletsa R, Desmarais B, Mara C, Kallianidis K, Loutradis D 2009 Evidence that human blastomere cleavage is under unique cell cycle control. Journal of Assisted Reproduction and Genetics 26: 187-195.
  5. Kiessling AA, Bletsa R, Desmarais B, Mara C, Kallianidis K, Loutradis D 2010 Genome-wide microarray evidence that 8-Cell human blastomeres over-express cell cycle drivers and under-express checkpoints. In Press, J of Assisted Reproduction and Genetics
  6. Conrad s, Renninger M, Hennenlotter J, Wiesner T, Just L, Bonin M, Aicher W, Buhring HJ, Mattheus U, Mack A, Wagner HJ, Minger S, Matzkies M, Reppel M, Hescheler J, Sievert KD, Stenl A, Skutella T 2008 Generation of pluripotent stem cells from adult human testis. Nature 456: 344-9.
  7. Kossack N, Meneses J, Shefi S, Nguyen HN, Chavez S, Nicholas C, Gromoll J, Turek PJ, Reijo-Pera RA 2009 Isolation and characterization of pluripotent human spermatogonial stem cell-derived cells. Stem Cells 27: 138-149.
  8. Golestaneh N, Kokkinaki M, Pant D, Jiang J, DeStefano D, Fernandez-Bueno C, Rone JD, Haddad BR, Gallicano GI, Dym M 2009 Pluripotent stem cells derived from adult human testes. Stem Cells and Development 18:1115-1126.
  9. Revazova ES, Turovets NA, Kochetkova OD, Kindarova LB, Kuzmichev LN, Janus JD, Pryzhkova MV 2007 Patient-specific stem cell lines derived from human parthenogenetic blastocysts. Cloning Stem Cells 9: 432-449.
  10. Tang XL, Rokosh DG, Guo Y, Bolli R 2010 Cardiac progenitor cells and bone-marrow derived very small embryonic-like stem cells for cardiac repair after myocardial infarction. Circulation Journal 74: 390-404.
  11. Zhu WZ, Hauch K, Xu C, Laflamme M 2009 Human embryonic stem cells and cardiac repair. Transplantation Reviews 23: 53-68.
  12. Kim K, Lerou P, Yabuuchi A, Lengerke C, Ng K, West J, Kirby A, Daly MJ, Daley GQ.
    2007 Histocompatible embryonic stem cells by parthenogenesis. Science: 315:482-6.
  13. The research involved the collection of over 2,000 human eggs from South Korean women, removing all the chromosomes from the eggs, and transplanting into the eggs a cell nucleus containing all the chromosomes of the patient in need of stem cells. The reconstructed entity was activated artificially, by methods similar to parthenote egg activation, and stem cells would be derived from the resulting egg divisions. The South Korean research team was skilled in the relevant animal work, so it remains a mystery why they were not successful with 2,000 eggs.

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The State of the Stem Cell

Science Highlights by Ann A. Kiessling, PhD

Too many choices?
state of the union

The big question facing scientists is: what type of pluripotent stem cell will ultimately prove to be the most therapeutically valuable?

As we enter a new decade of this new millennium, stem cell science is in a state of confusion. The power of pluripotent stem cells to alleviate damage to organs has been amply demonstrated in many model systems (1). It is not hype to assert that pluripotent stem cells are the foundation upon which regenerative medicine will grow. The over arching problem now, however, is lack of consensus about which stem cells to use and how to use them.

Simply put, the big question facing scientists is: what type of pluripotent stem cell will ultimately prove to be the most therapeutically valuable? This report on the state of the stem cell outlines the choices, the concerns, and the unknowns, for each candidate therapeutic stem cell.

Egg-derived stem cells

Embryonic stem cells derived from discarded human embryos, and parthenote stem cells derived from artificially activated, unfertilized human eggs, are undeniably the most robust and stable cells currently known. Once we learned the laboratory conditions necessary to support these remarkable cells, they have proven to multiply endlessly, maintain the integrity of their chromosomes, and retain through many, many cell divisions, the capacity to develop into all the tissues of the body. This is not surprising given that the purpose of the fertilized egg is to give rise to cells that can multiply rapidly enough to signal the mother to conserve the lining of her uterus in order to nourish the embryo, and ultimately all the tissues and organs that comprise a fetus (2,3).

Somewhat surprisingly, an unfertilized, artificially activated egg (a parthenote) also has the capacity to give rise to cells that can multiply rapidly and give rise to all the same types of cells, but they do not organize themselves into a fetus. Sperm are needed for the higher order organization required to form functioning organs and a viable fetus. Many studies in animal models have demonstrated the value of injecting embryonic or parthenote stem cells into animals with diseased hearts or kidneys or injuries, such as spinal cord injury (1). In some instances it appears the embryonic stem cells differentiate into the cell type that needs to be replaced, in other instances, the mere presence of the stem cells themselves appears to support repair and regeneration of the injured or diseased cells and tissue.

Why are egg-derived stem cells not yet available for treatments of human diseases? The answer is partly medical and partly social.

Medically, the same tissue match needed for a blood transfusion or a kidney or liver transplant applies to stem cell therapies. Tissues are comprised of individual cells, bonded together in specific ways. The surface of cells is a protein matrix, unique to each person. To protect us from bacterial and virus infections, immune cells that circulate through our bodies by the trillions each hour are looking for foreign proteins. Such proteins are ruthlessly attacked and destroyed. The same thing happens to foreign kidneys and stem cells. So, in the absence of patient-specific stem cells, each individual would have to be matched to a specific type of stem cell. Theoretically, it is possible to create embryonic and parthenote stem cell banks that would contain lines of stem cells to match most people, analogous to present day blood banks. This could alleviate the need for patient-specific stem cells for chronic diseases, but the need to conduct a tissue match rules out the use of banked stem cells for emergency treatments. Moreover, since perfect matches are rare, individuals undergoing therapy with banked stem cells would need suppression of their immune system to keep the engrafted stem cells alive. This problem would be overcome by having everyone tissue-matched early in life, analogous to being blood-typed, or by having patient-specific stem cells banked for everyone — both enormous, but not impossible, undertakings.

The possibility of creating embryonic and parthenote stem cell banks for therapeutic use has been derailed more by social considerations than by science. Concerns about the destruction of discarded embryos, or women donating eggs for scientific and therapeutic uses instead of procreation, have occupied thousands of hours of air time and created new careers for religious and medical ethicists.

Thus, many scientists and clinicians initially focused on developing clinical approaches with embryonic or parthenote stem cells have changed their research focus and sought more pragmatic sources of stem cells.

Other sources of stem cells

Stem cell therapy is, in fact, not new. There are stem cells in our bone marrow that give rise to all the types of cells in our blood stream, both red cells and white cells. The bone marrow stem cells produces billions and billions of blood cells daily, and can entirely re-populate the bone marrow of a patient undergoing bone marrow transplantation to treat various cancers and anemias (3). Bone marrow stem cells have been studied for many years, but to date, laboratory conditions have not been found that support their cell division in the same robust way embryonic and parthenote stem cells multiply. As a consequence, there are currently not enough bone marrow stem cells available for all the folks who need them for proven therapeutic uses, leaving very few for experimental stem cell therapies. Moreover, most studies reveal they lack the versatility of egg-derived stem cells. They can become all the cells in blood, but do not become all the cell types in the body. Nonetheless, some scientists and clinicians have abandoned their work with stem cells derived from eggs, and turned to trying to adapt bone marrow stem cells to laboratory conditions that will support their endless multiplication and subsequent development into all the tissues in the body. Once they succeed, bone marrow stem cells will still need to be tissue-matched to the patient.

Another source of stem cells already in clinical practice for stem cell therapy is umbilical cord blood. These cells can be recovered from the umbilical cord and placenta of every baby that is delivered. They, too, have been studied for many years (3) and, like bone marrow stem cells, laboratory conditions have not been found that support their cell multiplication to the numbers needed for therapies for adults. At this time, umbilical cord blood treatments are limited to treatment of children because there are not enough cells to treat adults. Tissue matching is also needed. Nonetheless, some scientists and clinicians have focused their research efforts on stem cells derived from umbilical cord blood.

Many other sources of stem cells have also been reported, such as stem cells from fat pads, placentas, amniotic fluid, roots of teeth and hair follicles, and are currently being characterized.

Induced pluripotent stem cells

Discovered by Shinya Yamanaka, MD, PhD at Japan’s Kyoto University in 2007, these new stem cells give rise to a totally new category of pluripotent stem cell.

“Yamanaka screened 24 candidate proteins before finding four that were able to reprogram adult cells, reverting them to their embryonic state. He and others then showed that these factors are also effective in human cells. Developmental biologist James Thomson, of the University of Wisconsin was the first to identify a slightly different group of factors that do the same.”
– Ian Wilmut, Time, April ’08

In the midst of this research melee, a Japanese team reported that ordinary cells, fibroblasts, cultured from small skin biopsies, could be manipulated in the laboratory to behave similarly to egg-derived stem cells (1). This was accomplished by changing the expression of a specific, small set of genes in the cells (link to iPS cartoon). After a few weeks, the new type of cell, termed “induced pluripotent stem cell” seemed to multiply as rapidly as egg-derived stem cells, and retain the capacity to differentiate into all cell types. The news rocked the stem cell scientific community. This could be the sought after source of patient-specific stem cells for therapies that would not require a tissue match or suppressing the immune system. The problem is that the manipulation to gene expression takes several weeks and resulted in some induced pluripotent stem cells that behaved like cancer cells. The formation of cancerous tumors if used therapeutically remains the major unknown for these iPS cells at this time.

Which stem cell will be the best for therapy?

The result of the many sources of stem cells is the current chaos.

The result of the many sources of stem cells is the current chaos. There are only a few thousand stem cell scientists in the world, and to develop therapies it is essential to pick a cell type and stick with it for the several years it will take to test safety and efficacy and qualify for treatments in humans.

The big question facing scientists: what type of pluripotent cell will ultimately prove to be the most therapeutically valuable? Value will be measured by alleviation of disease, and the absence of side effects, such as the growth of tumors.

Stem cells from testis

And in the midst of it all, a German research team quietly reported the derivation of stem cells from adult human testis. Like bone marrow, it is clear there is a large reservoir of stem cells in the testis because men produce billions of sperm each day throughout their lives. But most studies indicated the testis stem cells were restricted to giving rise solely to sperm. Now, however, two other research teams have derived stem cells from testis that so far behave like egg-derived stem cells. No manipulation of gene expression is needed and the cells multiply stably for many generations. Could this be the sought after patient-specific stem cell for therapeutic purposes? As of this writing, it is not clear how many stem cell scientists have diverted their research efforts to focus on testis-derived stem cells.

Bedford Stem Cell Research Foundation goals

The frustration at the lack of progress in patient-specific stem cell therapies is high at BRF. Patient-specific stem cells will alleviate one of the barriers to moving therapies forward because they will be tissue matched, avoiding the need for immune suppression. Although depending on patient-specific stem cells for therapeutic purposes is deemed by many to be impractical because of the effort needed to establish each line, characterize it for safety, and prove efficacy, the relevant parameters have not been established. BRF scientists are working toward this goal.

Patient-specific parthenote stem cells

BRF scientists are halfway through the analysis of expression of all the genes turned on and turned off in the cells of 8-Cell human embryos. This stage of development was chosen for study because the cells are totipotent (can give rise to all the cells in the body plus the placenta), and it is the stage at which the parthenote stem cells frequently arrest in culture. The goal is to increase the efficiency of deriving parthenote stem cells to at least 20% of artificially activated eggs, ensuring the derivation of a stable line of stem cells every time a woman undergoes an egg collection.

To date, several previously unknown and surprising characteristics of totipotent cells have been discovered and reported by BRF scientists, including possible control of cell division by an internal circadian clock. The work is ongoing.

Patient-specific testis stem cells

In parallel with this ongoing gene expression analyses, BRF scientists are working to establish the efficiency with which stem cells can be derived from testis. Reports from some laboratories are as low as 5%, from others as high as 80%. The success rates from mouse testis in the BRF lab are currently 75%.

Testis biopsies are a routine procedure for infertility treatment, and there is a large body of medical evidence that there are few negative side effects from the procedure. The goal will be to determine the cost and speed of deriving patient-specific testis stem cells from normal men and from men with specific diseases such as spinal cord injury, diabetes, Parkinson’s disease, ALS, heart failure and HIV infection.

The future

Although it appears costly at first, the rising cost of health care suggests patient-specific stem cell therapies for chronic, expensive diseases may ultimately lessen treatment costs. Parthenote stem cell research cannot be federally funded, even  now. The moratorium on parthenote stem cell research was not lifted by President Obama’s executive order. The testis stem cell work might be federally funded, but such funding would create a need for BRF to put in place the costly and elaborate accounting practices needed during the Bush administration to separate federally funded projects from privately funded projects. To avoid this, federal funds are not being currently sought for this work.

Once derived, the stem cell lines require six to twelve months to characterize for safety and potential to differentiate into the cell types needed for therapy. Given the ongoing work throughout the world to develop therapeutic approaches for stem cell delivery, the near future may see the dawn of a new era in regenerative medicine.

(1) ILAR Journal (51) Jan, 2010 Regenerative Medicine: From Mice to Men. dels.nas.edu/ilar_n/ilarjournal/51_1/html/
(2) What is an Embryo? Connecticut Law Review, 2004.
(3) Human Embryonic Stem Cells, 2007, Jones and Bartlett.

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Can stem cells cure HIV?

Ann A. Kiessling, PhD

 

A promising new case report of stem cell therapy for leukemia re-opens the possibility of curing HIV infection with stem cell transplantation

Revisiting HIV and Stem Cells

In February, 2009, a medical team in Germany (N Engl J Med 360(7):692; February 12, 2009) reported that their treatment for leukemia in an HIV-infected man may have cured his HIV disease. He was reported to be free of HIV 20 months after transplantation with bone marrow stem cells from a donor who was naturally resistant to HIV. This report has special meaning to Bedford Foundation scientists because of their core research area: patient-specific stem cells.

hiv stem cell
HIV may be cured by a bone marrow transplant similar to common treatments for leukemia.

A brief review:
HIV infects cells of the immune system, some of which reside in reservoirs, such as lymph nodes. Eradicating all HIV infected cells using methods developed for other diseased immune cells, e.g. cancers, is a highly promising treatment strategy.

There have, however, been two principle problems with this strategy:(1) bone marrow cells for transplantation must closely match the cells of the recipient to avoid the serious complication of “graft-versus-host-disease,” in which the transplanted cells fight the body, and (2) the transplanted bone marrow cells become infected with HIV, which not only fails to cure the patient’s HIV disease, but is a wasted use of valuable bone marrow cells needed to treat cancers in non-HIV infected individuals.

Solving Problem 1: Stem Cell Research

The first problem would be solved by generating stem cells directly from the HIV-infected patient. Such patient-specific cells would be a perfect match, thus
avoiding the risk of graft-versus-host-disease.

What is a Parthenote Stem Cell? (1 min)
Unlike most embryonic stem cell research, the Foundation’s research with Parthenotes does not require the use of fertilized human eggs.

Deriving patient-specific stem cells has been the focus of Bedford Research Foundation scientists for several years. Bedford Research is raising funds to continue the work of their scientists to derive stem cells from unfertilized eggs. Termed “parthenote stem cells” this research was begun in 2000, paused in 2004 to conduct more basic research to improve the efficiency of stem cell derivation, and the hope is to resume the work 2010. This work, which would provide patient-specific stem cells for younger women, cannot be federally funded.

In 2008, BRF scientists began studies to also derive patient-specific stem cells for men: from the testis. Work with mouse models has been very promising, and Bedford Research is now raising funds to develop methods to
derive stem cells from human testis, including men with HIV disease.

CCR5

In Humans, the CCR5 gene location is on the short (p) arm at position 21 on chromosome 3. Certain populations have inherited the Delta 32 mutation resulting in the Genetic deletion of the CCR5 gene. Homozygous carriers of this mutation are resistant to HIV-1 infection.
http://en.wikipedia.org/wiki/CCR5

Solving Problem 2: Resistance to HIV infection

HIV must bind to specific receptors, e.g. CCR5, on the surface of cells in order to infect them. People born with genetic defects in the CCR5 gene are resistant to infection by HIV. The German medical team used bone marrow stem cells lacking CCR5, and thus they were resistant to infection after transplantation. Methods for eliminating CCR5 expression are also under advanced stages of study. Work to date is very promising.

Research needed

Many research hurdles remain, but they can be surmounted. With proper funding, patient-specific stem cells lacking CCR5 could be developed by Bedford Research scientists in the near future. These cells would form the basis for an entirely new, potentially curative therapy for HIV disease. Personnel and supplies to move this project forward as fast as possible will cost $345,000 per year. Every donation counts.

Dr. Ann A. Kiessling honored with the Jacob Heskel Gabbay Award for Biotechnology and Medicine

Bedford Research Foundation

 

Dr. Ann A. Kiessling
Photo by Blake Gardner (hi-res jpg)

This is the twelfth year this award has been presented and the first year it has been awarded to a female scientist. This year, the award recognizes significant contributions to the field of assisted human reproduction. This international honor reflects Dr. Kiessling’s leading position in stem cell research and reproductive technologies.

Dr. Kiessling’s ground breaking Special Program of Assisted Reproduction (SPAR) allows HIV positive men to safely father children. The program’s success has been due to a revolutionary methodology developed to test semen for HIV, and a patented process for safely transporting specimens throughout the world by mail.

As of October, 2009, 101 healthy babies have been born from SPAR – a shining example of this pioneering science in action. This methodology helped solve a problem for families previously overlooked by the reproductive medicine community.

Dr. Kiessling’s work has also helped bridged a gap between assisted reproduction and basic research in stem cells. Using the micro-array technologies of previous Gabbay Awardees, Patrick O. Brown and Stephen P. A. Fodor, she is revealing the cellular machinery that gives rise to human embryonic stem cells. Understanding of this cell cycle regulation is urgently needed. It will revolutionize outcomes in assisted reproduction, the derivation of stem cells and our understanding of cancer.

Dr. Kiessling will present a talk entitled, “Retroviruses, Reproduction and Regenerative Medicine” on Monday, November 16 at 3:45 PM on Brandeis University campus in Gerstenzang Hall #123, admission is free. The award ceremony will be at 6 PM. tickets required.

 

BSCRF awards Representative Daniel E. Bosley with a Certificate of Appreciation

For Immediate Release:

Daniel Bosley and Ann KiesslingThe Bedford Stem Cell Research Foundation awards Representative Daniel E. Bosley (1st Berkshire) with a Certificate of Appreciation at the Activated Egg Symposium 2009.

The Foundation honored Representative Daniel E. Bosley in recognition of his dedication and tireless efforts on behalf of Stem Cell Science and Regenerative Medicine in Massachusetts.

“Representative Bosley was instrumental to the passage of the Massachusetts Stem Cell Bill in 2005. And we thank him for taking a leadership position in educating his fellow legislators about the importance of stem cell science,” said Dr. Ann Kiessling, director of the Bedford Stem Research Foundation as she presented the award.

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Stem Cell scientists from major universities meet for a small, focused conference in Weston, Massach

Discussions will include estimates of time to develop new stem cell based therapies for degenerative diseases, including diabetes, Parkinson’s disease, spinal cord diseases, AIDS and heart failure.

At this crucial time, when the federal government is not funding any new human embryonic stem cell grants while federal guidelines are being organized, the Bedford Stem Cell Research Foundation’s Seventh Annual Activated Egg Symposium is bringing together an elite group of speakers from Connecticut, Massachusetts and California to share their latest research with other scientists, mostly funded by state stem cell research initiatives.

This year’s keynote, Renee A. Reijo Pera, director of Stanford’s Center for Human Embryonic Stem Cell Research and Education was interviewed last year in the New York Times for her controversial research using embryonic stem cells to create sperm and eggs. She will be joined by internationally known leaders including, Dr. Gary Stein, director of Massachusetts’ new International Stem Cell RegistryDr. Haifan Lin, director of the Yale Stem Cell CenterDr. Laura Grabel, Wesleyan University, Dr. Ann Kiessling, director of the Bedford Stem Cell Research Foundation, Dr. Ren-He Xu, director of the University of Connecticut-Wesleyan Stem Cell CoreDr. Jesse Mager, University of Massachusetts, Amherst, and I. Glenn Cohen, Harvard Law School’s new Petri Flom Center for Health Law Policy, Biotechnology and Bioethics. With attendance limited to 100, the event provides a unique environment for investigators from academia, industry and infertility clinics to meet, discuss their most recent science and form new collaborations.

Special guests this year also include, Senator Jack Hart and Representative Daniel E. Bosley, legislators behind Massachusetts’s stem cell bill passed in 2005.

Bedford Stem Cell Research Foundation is Massachusetts’ only independent, non-profit, – stem cell laboratory, currently located in Somerville, MA.