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Umbilical Cord Blood
A Scientific Review
The field of regenerative biomedicine is a promising but still poorly understood one. In the past several years, there have been many significant discoveries in developmental biology and stem cell biology. But, understanding the main issues presented by regenerative medicine goes far beyond grasping the intricacies of stem cell differentiation, isolation and lineage diversity. Clarifying the mechanisms behind stem cell mobilization, recruitment and integration into various tissue types, is also of key importance. How do we adequately mobilize stem and progenitor cells? And what challenges might scientists have to overcome in order to provide a suitable niche environment for stem cell homing and engraftment? These and other such questions are currently being explored in the laboratory today, and umbilical cord blood transplantation (UCBT) provides a key exploratory model.
The History of Umbilical Cord Blood Transplantation
Fifty years ago, the seminal work in UCBT was begun when the Gengozian and Makinodan experiment showed that transplanted bone marrow (BM) could reconstitute the hematopoietic system of irradiated mice. This discovery led to further research in the area, and 16 years later, the first account of UCBT in humans occurred with the publication of a clinical intervention which involved using transfused cord blood to treat a teenage boy suffering from acute lymphoblastic leukemia (1). However, it took another twenty years for allogeneic UCBT to be successfully achieved in a child with Fanconi’s anemia (2). This accomplishment was welcomed by the biomedical community because until then, attention had been focused primarily on bone marrow transplantation. And, although transplanting bone marrow between relatives is feasible, it is a finite and costly source of hematopoietic stem cells. This 1989 discovery led by Broxmeyer also found that umbilical cord blood (UCB) contained progenitors that responded to bone marrow stimulating factors in a manner conducive to transplantation. And so began the quest to store and use UCB which until then had been discarded as waste.
Why Use Hematopoietic Stem Cells from UCB?
Hematopoietic stem cells (HSCs) derived from UCB provide several advantages: 1) they are relatively easy to obtain, 2) there is a reduced possibility of transmitting infections, 3) there is a reduced risk for mothers and donors, 4) there is a lower risk of graft versus host disease (GVHD), and 5) the criteria for HLA matching for donor-recipient selection is less stringent.
Myeloablative Interventions
In myeloablative preparative regimens, patients either receive total body irradiation or busulfan-based myeloablative treatment before being transfused with UCB. HSCs derived from UCB have been shown to be sufficient in number to achieve engraftment in adult patients, without the attendant complication of GVHD. But, although UCBT is now an accepted alternative source for hematopoietic stem cells for transplantation in children, there is reason for caution when it comes to UCB transplantation in adults. Myeloablative preparatory treatments are known to be highly toxic to all patients, and are especially so in immuno-compromised adults. When this therapeutic regimen is combined with the many known engraftment delays of all cell lineages, and the enrollment of seriously ill patients with advanced disease states, the possibility of death or long term disability is a significant concern. More recently though, there are reports of successful allogeneic transplantation with UCB. This, however, has been done by following a less intense myeloablative treatment regimen. Based on these results, it has been suggested that a lower intensity preparative regimen might allow for more efficient engraftment of UCB stem cells and decreased toxicity in adult patients.
Values were taken from different studies testing for each variable in CML patients (24,25,26).
Non-Myeloablative Interventions
The rationale behind non-myeloablative preparatory regimens is based on the fact that GVHD plays a significant role in the therapeutic success of allogeneic transplantation. Non-myeloablative stem cell transplantation of UCB allows immunotherapy to be administered to older and sicker patients, who might be excluded from a more toxic curative approach. Patients without appropriate donors, who might ordinarily be disallowed from treatment, can also be included here. But, unlike myeloablative treatment regimens, there is an increased risk of GVHD. However, two patients with relapsed lymphoma, who had no partially matched family members, matched siblings, or unrelated donors, were reported to have undergone successful non-myeloablative therapy, followed by matched unrelated donor UCB infusion. Both patients were found to have 100% engraftment after 90 days, and remained in remission for between 6 to 12 months (3,4). The positive outcome here, points to the feasibility of using mismatched unrelated UCB stem cells, even in non-myeloablative therapeutic treatments.
Strategies for Dealing with Adult Transplantation Problems
Currently, there are three strategies for dealing with adult transplantation problems. These include: 1) sequential/multiple UCB units, 2) expansion, and 3) combinations with haploid identical cells (5,6). Transplanting more than one unit of partially matched UCB has also been shown to be successful, since this causes an increase in cell count, as well as better engraftment with little immune rejection and mortality (7). In addition, researchers have observed that one of the UCB transplants is dominant over the other. However, current debate still rages over whether a graft vs graft response keeps one unit long term or whether the inferior UCB somehow helps the dominant UCB engraft.
Combination Interventions
Combination treatments of UCB with haploid identical CD34+ cells, (probably from mobilized peripheral blood {MPB}), helps with delayed neutrophil engraftment. Fernandez et al found that the transplantation of UCB with MPB CD34+ cells actually decreases neutrophil engraftment time from approximately 1 month to about 10 days (8). Combining UCB with Mesenchymal Stem Cells (MSCs), the cells that create the niche environment for HSCs, is thought to be useful in repairing damaged niche areas in UCB transplants. MSC co-transplantation has also shown to engraft both units of UCB-HSCs when given with more than one UCB unit (6).
Ethical Concerns
The use of stem cells from UCB differs from the use of other stem cell types in that there is a general lack of ethical guidance. Because UCB is taken from the placenta and umbilical cord, there is a reduced chance of endangering the safety of the newborn or mother (although there is a slight possibility of extracting too much blood – which may harm the newborn and/or mother) (9). But, the biggest ethical consideration today involves issues of informed consent. Due to the fact that maternal HSCs may contaminate cord blood; to whom the cord blood actually belongs (mother or infant), is still unclear (10). And, it is also being debated at present whether or not cord blood should be used for research before the blood donor is known.
The Future of UCB Transplantation in Adults – Applications in Stroke, Leukemia, Breast Cancer and Traumatic Brain Injury
Human umbilical cord blood contains hematopoietic stem cells and mesenchymal stem cells, both of which are regarded as valuable sources for cell transplantation and therapy. Under proneurogenic conditions, mesenchymal cells rapidly assume the morphology of multipolar neurons and express neural markers (Tuj1, TrkA, GFAP and CNPases). The neurogenic potential of umbilical cord blood derived cells may facilitate stem cell therapeutic approaches to neurodegenerative disease (11). Human umbilical cord blood cells were cultured with brain-derived neurotrophic factor (BDNF) and after ten days, the differentiated cells expressed glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein, a prerequisite for formation of neurons and glial cells (12). The neural stem cell like sub-population from human UCB cells can be selected and expanded in vitro. These cells and resulting clones express nestin, a neurofilament protein and specific marker of multipotent neural stem cells. With selected growth factors, the progeny can be oriented towards neurons, astroglia or oligodendroglia. The cells show high commitment (about 30-40%) to neuronal (30%) and astrocytic (40%) fate and about 11% of the total population of cells give rise to oligodendrocytes (13).
Umbilical cord blood, due to its primitive nature and its unproblematic collection, appears to be a promising candidate for multipotent stem cell harvest. Umbilical cord blood negative lineage stem cells could be expanded to produce slow-dividing adherent cells with neuroglial progenitor cell morphology over 8 weeks. Gene expression analysis showed up regulation of primitive neuroglial progenitor cell markers, including GFAP, nestin, musashi-1, and necdin (14). The DNA micro-array data of mononuclear cells from human UCB showed a down regulation of several genes associated with blood cell lines. One month after these cells were transplanted into 1 day old rat brains, approximately 20% of the cells survived and some of the cells differentiated into cells with distinct glial or neuronal phenotypes (15). CD45-, a rare population from human cord blood can be expanded without losing pluripotency and can differentiate into osteoblasts, chondroblasts, adipocytes, hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein and various neurotransmitter phenotypes. Implantation of these cells into the intact rat brain showed that they persisted for up to 3 months and showed migratory activity and a typical neuron-like morphology (16).
Human umbilical cord stem cells administered intravenously into the stroke affected rat’s femoral (leg) vein or directly into the striatum (brain) found similar behavioral recovery, suggesting that therapy with umbilical cord stem cells may be effective for brain injuries and neurodegenerative disorders for long-term functional improvement in stroke animal models (17). Systemic administration of human cord blood-derived CD34+ cells to immuno compromised mice subjected to stroke 48 hours earlier induces neovascularization in the ischemic zone and provides a favorable environment for neuronal regeneration. Endogenous neurogenesis is accelerated as a result of enhanced migration of neuronal progenitor cells to the damaged area, followed by their maturation and functional recovery of ischemic brain (18,19).
Twenty-four hour post traumatic rats administered with human umbilical cord blood, showed significant reduction in motor and neurological deficits. The cells preferentially entered the brain, migrating into the injured area expressing markers for neurons (NeuN and MAP-2) and astrocytes (GFAP) (20). It is possible that these stem cells might be useful in the treatment of central nervous system injury.
A significant increase was also observed in the survival of IL-2 activated UCB cells or Peripheral Blood Cells (PBCs) on day 3 and day 5 after tumor transplantation in mice. Similar anti-tumor cytotoxicity of UCB cells and PBCs was also observed against MDA-231 human breast cancer grown in severe combined immunodeficient mice (21).
Dendritic cells (DCs) are important accessory cells that are capable of initiating an immune response. Functional DCs were generated form mononuclear cells isolated from human umbilical cord blood cells and peripheral blood cells, using a defined medium (Prime Complete Growth Medium {PCGM}). These DCs increased the allogeneic mixed lymphocyte reaction, confirming their immune accessory functions compared to a control mixed lymphocyte reaction (MLR) without DCs. The DCs generated using PCGM medium also significantly enhanced the hematopoietic colony (CFU-C) forming ability. Addition of 5% DCs derived from cord blood loaded with tumor antigen also significantly increased peripheral and cord blood-derived antigen-specific cytotoxic T lymphocyte (CTL)-mediated killing of human leukemic cells (K562) and breast cancer cells indicating that a DC of UCB is a source of accessory cells for augmenting CTL-mediated cytotoxicity with potential use in cellular therapy for human leukemia and breast cancer (22).
Cord blood, a potent source of hematopoietic stem cells, has been shown to successfully reconstitute hematopoiesis following allogeneic transplantation in a variety of disorders. A major drawback of cord blood has been the risk of transfusion reactions in ABO blood group incompatibility and drastic reduction in the stem cell pool if the cord blood is manipulated to remove red cells prior to cryopreservation or after thawing. Pahwa et al describes an erythrocyte depletion method employing 3% gelatin-induced erythrocyte sedimentation for the selective removal of red cells from cord blood to enrich progenitor cells and cells secreting hematopoietic cytokines: interleukin 3, granulocyte/macrophage colony-stimulating factor, and interleukin 6. A major source for cytokines was umbilical cord T cells, illustrating the successful use of this method for transplanting cord cells from patients with malignant/nonmalignant disease (23).
Conclusion
The use of less toxic non-myeloablative regimens in adult patients is an unequivocal advance in the field of UCB transplantation. But, there are many unanswered questions worthy of consideration.
1) What is the incidence of GVHD in non-myeloablative versus myeloablative interventions?
2) Is patient recovery affected by the choice of treatment regimen? And if so, what is the difference in recovery time?
3) Is there an optimal UCB dose for adult patients?
These and other such considerations will undoubtedly be addressed by the biomedical research community. However, because of the success of UCB transplantation in recent years, several UCB banks have now been established for UCB transplants. It is clear that although there still are many issues to consider (both biomedical and ethical), UCB is a good alternative to bone marrow and peripheral blood for the reconstitution of the hematopoietic system in adults, when a suitable bone marrow match is unavailable.
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Acknowledgements
This review was prepared by the following graduate students in the Stem Cell Biology Class, Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey: Monique H. Johnson, Katherine Liu, Christopher Komurek, George Lambrinos, Raghavendra A Shamanna (in alphabetical order).
Teaching Assistant: Edward Garay
The review was edited by two stem cell biologists.
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