Newsletter - September 2014

CORD:USE
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How Long Can Cord Blood Be Stored?

Hal E. Broxmeyer, PhD

Hal E. Broxmeyer, PhD, Indiana University School of Medicine

Cord blood transplantation is a clinically effective form of treatment for many patients with cancer and blood diseases who need a stem cell transplant (1-3). To date over 30,000 patients have been treated with cord blood transplants (3).

The precious ingredients in cord blood are the blood-forming (hematopoietic) stem cells and progenitor cells that can replicate and diversify to replace a patient's entire immune system. These cells are rare, comprising less than one percent of the cells in cord blood, but nonetheless a typical cord blood collection contains millions of blood-forming stem and progenitor cells.

Cord blood transplants can come from either public banks or from private/family banks in which the cord blood stem cells are stored in a cryopreserved frozen state. The key to cord blood banking is to properly cryopreserve the stem cells so that when they are thawed for therapy they are still alive and maintain the functional capacity of the cells to repopulate the blood cells in a patient's body.

A question that often comes up about stored cord blood, is how long can the stem and progenitor cells be maintained in frozen form and still be viable when they are thawed?

In theory, if the cord blood stem and progenitor cells were properly cryopreserved, it should be possible to keep them in a frozen state for many decades, if not longer, with subsequent retrieval of viable stem and progenitor cells.

Practically, this depends on the quality of the cryopreservation procedure. It depends on whether the storage facility assured that the cryogenic nitrogen tanks were maintained at a constant very low temperature. Finally it depends on the competence of the laboratory staff who thaw the cells to revive them.

There have been a number of studies on retrieval and viability testing of cord blood after many years of storage in liquid nitrogen-containing tanks. Our own studies, first reported in the late 1980's when we suggested that cord blood stem cells could serve as a substitute for bone marrow transplants, noted highly efficient cell recovery after a few months of storage in cryopreserved form (4).

This led us to establish the first proof-of-principle cord blood bank in my laboratory (1-3). We supplied the frozen cells for the first five cord blood transplants ever performed, which were all between HLA-identical siblings. We also banked cells for two of the next five cord blood transplants performed.

Over the decades since, we have demonstrated efficient cell recovery at 5 years (5), 10 years (6), 15 years (7), and most recently 23.5 years (8) after the cells were frozen in cryopreserved form. The accuracy of these tests rests on the fact that we have had continuous custody of these cells, and we have performed their post-thaw analysis with the same tests as their pre-freeze measurements.

Coming up in another 2-3 years we will perform a 30 year assessment of our oldest cord blood specimens. Until then, the longest time that cord blood has been frozen and subsequently thawed with efficient recovery of stem and progenitor cells is 23.5 years in a laboratory setting. The longest storage interval of frozen cells that were given to a patient as a cord blood transplant is at least 14 years (pers. comm., Dr. P. Rubinstein).

Based on the studies in our laboratory, it is likely that cord blood can be stored frozen for decades and still be a potent source of cells for transplantation.

It may take some time before clinical studies demonstrate the viability of stem cells from long-term storage that we have established in the laboratory. Clinical proof would require treating patients with cord blood units that had been in storage for decades. But public cord blood banks tend not to use older stored cord blood collections if they have newer ones. Hence clinical proof of long-term storage success may have to come from private/family cord blood banks, when their clients eventually use the cord blood as therapy for the baby it came from or for a related family member.

Dr. Broxmeyer is a Distinguished Professor, Mary Margaret Walther Professor Emeritus, and Professor of Microbiology and Immunology at the Indiana University School of Medicine (IUSM), Indianapolis, Indiana, and is Co-Leader of the National Cancer Institute Designated Indiana University Simon Cancer Center on Hematopoiesis, Malignant Hematology and Immunology. He is the Founder of the CORD:USE Family Cord Blood Bank and is on the Medical Scientific Advisory Board of the CORD:USE Public Cord Blood Bank. He is a member of the National Marrow Donor Program (NMDP) Cord Blood Advisory Group.

References

  1. Gluckman, E., Broxmeyer, H.E., Auerbach, A.D., Friedman, H., Douglas, G.W., Devergie, A., Esperou, H., Thierry, D., Socie, G., Lehn, P., Cooper, S., English, D., Kurtzberg, J., Bard, J. and Boyse, E.A. 1989. Hematopoietic reconstitution in a patient with Fanconi anemia by means of umbilical-cord blood from an HLA-identical sibling. New Engl. J. Medicine 321:1174-1178. PubMed: PMID2571931
  2. Broxmeyer, H.E. and Smith, F.O. 2009. Cord Blood Hematopoietic Cell Transplantation. In: Thomas' Hematopoietic Cell Transplantation 4th Edition. Eds: Appelbaum, F.R., Forman, S.J., Negrin, R.S., and Blume, K.G. Wiley-Blackwell, West Sussex, United Kingdom, Section 4, Chapter 39, pp. 559-576.
  3. Ballen, K.K., Gluckman, E., and Broxmeyer, H.E. 2013. Umbilical Cord Blood Transplantation - the first 25 years and beyond. Blood. 122:491-498. PubMed: PMC3952633
  4. Broxmeyer, H.E., Douglas, G.W., Hangoc, G., Cooper, S., Bard, J., English, D., Arny, M., Thomas, L., and Boyse, E.A. 1989. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc. Natl. Acad. Sci. USA. 86:3828-3832. PubMed: PMC287234
  5. Broxmeyer, H.E., Hangoc, G., Cooper, S., Ribeiro, R.C., Graves, V., Yoder, M., Wagner, J., Vadhan-Raj, S., Benninger, L., Rubinstein, P. and Broun, E.R. 1992. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation of adults. Proc. Natl. Acad. Sci. USA 89:4109-4113. PubMed: PMC525642
  6. Broxmeyer, H.E. and Cooper, S. 1997. High efficiency recovery of immature hematopoietic progenitor cells with extensive proliferative capacity from human cord blood cryopreserved for ten years. Clin. and Exp. Immunol. 107:45-53. PubMed: PMID9020936
  7. Broxmeyer, H.E., Srour, E.F., Hangoc, G., Cooper, S., Anderson, J.A., and Bodine, D. 2003. High efficiency recovery of hematopoietic progenitor cells with extensive proliferative and ex-vivo expansion activity and of hematopoietic stem cells with NOD/SCID mouse repopulation ability from human cord blood stored frozen for 15 years. Proc Natl Acad Sci USA. 100:645-650. PubMed: PMC141050
  8. Broxmeyer, H.E., Lee, M-R, Hangoc, G., Cooper, S., Prasain, N., Kim, Y-J, Mallett, C., Ye, Z., Witting, S., Cornetta, K., Cheng, L., and Yoder, M.C. 2011. Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood. Blood. 117:4773-4777. PubMed: PMC3100689
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Adriana's cord blood therapy for cerebral palsy

Irene Martini, PhD

Irene Martini, PhD, Director SmartBank, Italy

Adriana was born in Italy in 2009 after an in vitro fertilization and her parents were finally achieving the dream of their lives. Everything appeared to be fine with the pregnancy until the last moment when the mother started to feel contractions and ran to the clinic. She had forgotten to bring the kit for storing cord blood stem cells, but luckily Francesco from SmartBank reached them at the clinic right on time.

The clinical status of Adriana before birth kept getting worse and worse. She was suffering and the gynecologist was busy with another delivery. It took a while before a C-section was performed. Later, Adriana was diagnosed with a cerebral lesion caused by hypoxia at birth. Hypoxia is a medical term for when a region of the body does not get adequate oxygen supply.

Adriana's symptoms of cerebral palsy were not evident at the beginning and it took a while before her parents decided to seek therapy. They began seeing neurologists in Italy. Again and again they asked the neurologists if the stem cells in Adriana's cord blood might have therapeutic potential for her brain lesion, but all of the neurologists dismissed the cord blood stem cells as useless. Finally, three years after Adriana's birth, her parents called back SmartBank.

As the scientific director at SmartBank, I immediately suggested that the parents should get in touch with Duke Medical Center, where a clinical trial was enrolling children from all over the world for therapy of brain injury with their own cord blood stem cells.

Adriana's stem cells were identified in the SmartBank storage facility at BioVault Ltd in the UK. Then BioVault sent the cord blood unit to Duke Medical center for further testing according to the UK and US regulations. Adriana had enough stem cells for at least two therapies.

Adriana and her family traveled to the United States for the cord blood therapy. She was infused the first time in May 2013 and the second time in December 2013. Prior to this therapy, Adriana was not able to speak. She also suffered from muscle contractions called dystonia, such that she was not able to walk or even to sit without a support.

One evening before Adriana left for the second infusion in North Carolina, I received a phone call. A little voice was whispering at the other end, saying that she was happy to go and see Dr. Kurtzberg once again after 6 months. Adriana could talk!! This was one of the few moments in my life when I felt that so many efforts and battles to advocate for stem cell therapy were rewarded.

Adriana had a repeat MRI one year after the first infusion and her cerebral lesion had disappeared. She still needs to train herself like a little soldier every day in order to recover competely from dystonia, and she requires an exoskeleton in order to walk. However she can now speak fluently.

SmartBank s.r.l., Italy Irene Martini, PhD, is the co-founder and Scientific Director of SmartBank s.r.l., a family cord blood bank based in Rome, Italy, which uses laboratory storage facilities at BioVault in the UK. She has more than fifteen years experience as a researcher, and has written several scientific publications as well as patent documents. As part of her work with SmartBank, Dr. Martini has appeared frequently on national television and radio to explain the benefits of cord blood storage. She also runs monthly courses for prospective parents on the potential benefits of storage, including the latest research developments, as well as courses for gynaecologists and mid-wives. SmartBank advocates storage of cord blood both for the family and for the public via donation. Their lab, BioVault, is the only bank in the UK which offers both kinds of storage. She has three children herself, and was able to save cord blood for the youngest.

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Inducing Tolerance for Hand Transplants with Stem Cells

Curtis L. Cetrulo, Jr., MD

Curtis L. Cetrulo, Jr., MD,Head of the Hand Transplantation Service at Massachusetts General Hospital

Massachusetts General Hospital (MGH) investigators have made an important step towards greater availability of hand transplants, face transplants and other transplants involving multiple types of tissue, and stem cells play an important role. Hand, face and soft tissue transplants are collectively known as vascularized composite allografts, or VCAs. A VCA transplant is one that involves transplantation of multiple tissue types: muscle, bone, nerves and skin. They are most frequently used to replace amputated hands and arms, and to repair severe facial injuries.

In recent publication in the American Journal of Transplantation (1), our team describes how a procedure developed at the MGH to induce immune tolerance to organ transplants has also succeeded in an animal model of VCAs. Transplantation of donor hematopoietic stem cells - either several months before or simultaneous with the limb transplant - allowed the animals to accept VCAs from immunologically-mismatched donors.

Although VCA transplants offer recipients significantly improved quality of life, they come with a price. Currently, patients who undergo a VCA transplant must take immune suppressing drugs for the rest of their lives to prevent graft rejection. These drugs leave patients more vulnerable to infection and have numerous long-term side effects. If the immune system of the transplant recipient could be induced to accept the donor tissue, it would greatly reduce the risks of the surgery.

A personal view of the new procedure is offered by Curtis L. Cetrulo, Jr., MD, senior author of the current study: "The need for lifelong immunosuppression to prevent graft rejection is the most important challenge in this type of procedure, since most potential VCA recipients are young and would face increased risks of infection, diabetes or kidney problems, and even some types of cancer over many years."

"Bringing immunologic tolerance to hand and face transplantation would result in a paradigm shift in the way we will be able to treat the horrific injuries our service members are sustaining in the current military conflicts in Iraq and Afghanistan, as well for the types of blast-injury extremity loss seen in the Boston Marathon bombing. Tolerance would give us a unique tool - a real game changer - with which to help these patients. Importantly, a number of different stem cell sources - both adult and perinatal - may provide the final pieces of this puzzle."

The MGH is a world leader in the development of tolerance-inducing protocols. Several decades of research led by David H. Sachs, MD, founder and scientific director of the MGH Transplantation Biology Research Center (TBRC), led to a protocol in which transplant patients receive both the needed organ and bone marrow from a living donor, producing a state called mixed chimerism, in which the patient's immune system contains both donor and recipient elements.

A number of patients have received kidney transplants using versions of the protocol pioneered at MGH, and were subsequently able to discontinue immunosuppressive drugs. Most of these patients have been able to remain off immunosuppressive medications long term, some for more than a decade.

The current VCA study was designed to test whether a similar protocol could induce tolerance to VCAs from immunologically mismatched donors in an animal model. An additional challenge is posed by the fact that skin, an essential part of a VCA, carries what could be considered its own immune system, making its acceptance by a recipient's immune system particularly problematic. In several previous attempts to induce VCA tolerance, bone and muscle tissue were accepted but the skin was rejected and eventually separated from the underlying tissue.

Building on previous TBRC animal studies, the researchers tested whether combining bone marrow transplantation with VCA could induce chimerism and tolerance. In the first phase of the study, four recipient animals received bone marrow transplants from immunologically mismatched donors in advance, allowing time to confirm that chimerism had been established before the VCA procedure - involving transplantation of components of a hind limb from the same donor - was carried out three to five months later. Even though the recipients received no immunosuppression after the transplant procedure, all animals accepted the transplant with no sign of rejection.

Since pretransplant induction of chimerism would not be practical for hand or face transplants from deceased donors, the researchers next tested VCA surgery conducted simultaneously with the bone marrow transplantation to induce tolerance in two recipient animals. The immune systems of the animals were conditioned prior to the procedure. Chimerism was successfully induced in both recipients, and overall results were the same as in the other group - immune tolerance of all components of the VCA with no evidence of rejection throughout the follow-up period, which for one recipient was more than 480 days.

Since the availability of human cadaver tissues cannot be precisely predicted, the MGH team is exploring two approaches to the issue of timing the procedures. In one, the patient begins immune conditioning as soon as a donor is identified and the patient receives a simultaneous transplant of VCA and bone marrow from the donor. In the second, the patient receives the VCA first with conventional immunosuppression, and then months later is conditioned to receive the bone marrow transplant. This second protocol is currently in clinical trials for organ transplants. However in the case of VCAs from deceased donors, any donor bone marrow would have to be collected at the same time that the transplant tissues are collected.

In addition, our group is investigating the use of other stem cell types that may make the procedure more effective. "Perinatal stem cells of various phenotypes - for example, mesenchymal stem cells or MSCs - may provide an effective adjunct to our protocol for inducing chimerism and make the approach both safer and more efficient." An advantage of MSC from perinatal tissues is that they can be stored in cryogenic banks and are always available at the time of need.

Curtis L. Cetrulo, Jr., MD, is the head of the Hand Transplantation Service in the MGH Division of Plastic and Reconstructive Surgery. He is affiliated with Harvard Medical School. Dr. Cetrulo received his MD from Tufts Medical School and served residencies at both Lahey Clinic and Long Island Plastic Surgical Group, followed by a Fellowship at California Pacific Medical Center. He is the lead author of the recent MGH study on induced immunologic tolerance for vascularized composite allografts (VCAs) from immunologically mismatched donors.

Reference

  1. DA Leonard, JM. Kurtz, C Mallard, A Albritton, R Duran-Struuck, EA Farkash, R Crepeau, A Matar, BM Horner, MA Randolph, DH Sachs, CA Huang, & CL Cetrulo Jr.
    Vascularized Composite Allograft Tolerance Across MHC Barriers in a Large Animal Model
    American Journal of Transplantation 2014; 14(2):343-355. DOI:10.1111/ajt.12560