Stem Cells Derived from Cord Blood

14 Stem Cells Derived from Cord Blood Julie G. Allickson

INTRODUCTION Cord blood was first seen as biological waste product post childbirth. One of the first publications reported on the colony-forming capacity of cord blood summarizing its cloning efficiency to be similar to bone marrow was reported in 1980 (Di Landro et al., 1980). In 1988, the first cord blood transplant took place in France for Fanconi’s anemia with the donor being an identical human leukocyte antigen (HLA)-matched sibling (Gluckman et al., 1989). The transplant was successful without graft versus host disease (GVHD) (Gluckman et al., 2005) and the patient is reported to be alive and well 18 years after the transplant (Kurtzberg, personal communication). In 1989, Broxmeyer reported on the colony-forming potential of umbilical cord blood assessing its growth for colony-forming granulocyte-macrophage (CFU-GM), burst-forming erythroid (BFU-E), and colony-forming capacity for multipotent progenitors (CFU-GEMM) the most immature assessed. In 1990, it was reported that three patients had been transplanted for Fanconi’s anemia and it was suggested that cord blood transplantation maybe applicable to other diseases with a possibility of also transplanting adults. The cord blood cellular product viewed as a source of hematopoietic progenitors cells coupled to the immaturity of the immune system at birth is one of the advantages of using these cells for transplantation (Gluckman et al., 1990). In 1990, Thierry et al. reported difficulty in processing the procured cord blood in regards to cell recovery. It was also discovered that the total stem cell content correlated significantly with the time of delivery; the earlier in gestation the cord blood was collected the higher the number of stem cells retrieved (Thierry et al., 1990). In 1991 was the first report of a Public Cord Blood Bank for unrelated cord blood transplants (Rubinstein et al., 1993). One of the first reports in 1992 on the characterization of cord blood by flow cytometry was reported by Dr. Gluckman’s Laboratory to demonstrate that the content of the cord blood graft represented both suppressive and naive cells. Naive cells were noted by the T-cell content and its ability to produce receptors for interleukin (IL)-2 and HLA-DR6 (Rabian-Herzog et al., 1992). In 1994, researchers investigated the incidence of maternal cell contamination in the cord blood. It was demonstrated that only rarely are they discovered at birth and at an extremely low percentage as displayed in the lymphocyte population which was less than 1% (Socie et al., 1994). In 2000, Rocha reported a lower risk of acute and chronic GVHD in cord blood as compared to bone marrow in HLA-matched identical sibling transplants (Rocha et al., 2000). He was able to demonstrate the colony-forming capacity of cord blood which would remain viable 3 days after procurement if stored at 4°C or at room temperature, but not at 37°C (Broxmeyer et al., 1989). In this chapter, cord blood procurement, processing, and storage are briefly reviewed. The pluripotent capabilities of the umbilical cord blood stem cells have recently demonstrated differentiation potential in all

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three germ cell layers referring to the ectoderm, the endoderm, and the mesoderm. Researchers have studied the potential of differentiating in vivo and in vitro to not only characterize the cell, but to test its proliferative and clonogenic capacities. The most recent investigations will be discussed and summarized relating to the current efforts in the field of umbilical cord blood transplantation as it relates to regenerative medicine.

PROCUREMENT AND PROCESSING OF CORD BLOOD Cord blood procurement is generally performed by a health care professional or trained staff of the cord blood bank where the product will be processed and stored. The postnatal collection occurs after the cord blood vein is disinfected similar to a process used for whole blood collection utilizing a combination of iodine and alcohol through a series of steps. The procurement most commonly is harvested in a bag with citrate phosphate dextrose (CPD), acid citrate dextrose (ACD), or heparin as the anticoagulant, but may also be collected via a syringe with anticoagulant. Procurement of cord blood may be initiated while the placenta is in utero or ex utero. Most publications to date have shown statistically similar results by comparing methodology (Lasky et al., 2002; Pafumi et al., 2002; Solves et al., 2006), but some studies demonstrated the in utero collection yielded a higher recovery of hematopoietic cell content (Solves et al., 2003a, b; 2005). The collection usually takes place in a closed system mimicking the collection procedure used for whole blood. One other method published that yielded a higher cell recovery used a saline wash after the routine collection to procure residual cells residing in the vein after collection (Elchalal et al., 2000) although feasibility at the bedside may be difficult. Processing cord blood to enrich for hematopoietic progenitor cells most frequently depends on hydroxyethyl starch (HES), which was the method first published by Dr. Rubinstein and others (Rubinstein et al., 1995; Alonso et al., 2001; Liu et al., 2003) demonstrating great success. Cord blood banks reached a consensus that HES sedimentation is a reliable method which can easily be adapted to process large quantities of cord blood products. This method incorporates HES at a 1:5 ratio with the cord blood and allows it to sediment after a centrifugation step. The enriched hematopoietic progenitor cell product is expressed from the concentrated cord blood product. This fraction is then further volume reduced prior to cryopreservation with dimethylsulfoxide (DMSO). Alonso et al. in 2001 reported on a modified method according to Rubinstein et al. (1995). The modified method includes an inverted positioning of the cord blood product in a refrigerated centrifuge during the HES incubation and to reduce red blood cells they are drained from the bottom of the bag. Both methods yield a high recovery of nucleated and hematopoietic progenitor cells. Rubinstein et al. reported a minimum of 91% leukocyte and progenitor cell recovery and Alonso et al. reported an 87% recovery for total nucleated cells and 97% recovery for CD34 positive cells. Other methods used for manual processing include density gradient separations (using Percoll™ or Ficoll™) (Sato et al., 1995; Rogers et al., 2001) and gelatin (Oldak et al., 2000). Automated devices that have been evaluated for cell processing include the Optipress II (Armitage et al., 1999; U-pratya et al., 2003), the Biosafe, and Sepax (Tiumina et al., 2005) and the AutoXpress™ Platform (AXP™) by Thermogenesis (Dobrila et al., 2006) which have demonstrated a high cell recovery post processing. In 1995, a Request for Proposal was solicited by the National Heart, Lung, and Blood Institute entitled, “Transplant Centers for Clinical Research on Transplantation of Umbilical Cord Stem and Progenitor Cells” (Fraser et al., 1998). The study was designed to determine if cord blood transplantation is a viable option for bone marrow transplant. The study would also help to build standard operating protocols for cord blood banking and focused on building an ethnically diverse unrelated donor pool to supply nationalities under-represented (http://spitfire.emmes.com/study/cord/sop.htm). The study was initiated in 1996 and conducted with the United States Food and Drug Administration (FDA) under an Investigational New Drug (IND). The study end point was survival at 180 days with other end points including engraftment, GVHD, relapse, and long-term survival (Cairo et al., 2005). In summary, the report in 2005 states that cord blood transplant should continue

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along with research focusing on cord blood transplantation. The study consisted of approximately 11,000 donors (64% of the total donors collected) stored for potential future use; with 79% meeting donor eligibility criteria stated for the study. The study defined protocols for collection and processing, characterized a unique cell population in the cord blood which includes CD34 positive/CD38 negative a highly proliferative cell population capable of forming early uncommitted blast-like colonies in culture and CD34 positive/CD61 positive demonstrated a correlation to rapid platelet recovery (Cairo et al., 2005). A population of cells CD34 positive/ CD90 positive demonstrated significant correlation with colony-forming capacity (Cairo et al., 2005). They also examined factors of the collection such as sex of the donor, ethnicity, type of delivery, and gestational age which illustrated a significant effect on the progenitor cell content and the lymphocyte subsets (Cornetta et al., 2005).

CORD BLOOD STORAGE Cord blood products when stored long term will either be in liquid nitrogen vapor phase or stored directly in the liquid nitrogen. Concerns in the past with liquid nitrogen vapor storage erupted from temperature changes occurring at the top of the tank when it was opened, but liquid nitrogen storage tank models are available that can retain a temperature of less than
190°C on a consistent basis at the top and the bottom of the tank which allows consistent storage in the vapor phase. Since 1995, when a reported case of hepatitis B transmission occurred (Tedder et al., 1995) in the liquid storage of a nitrogen tank many moved to an overwrap bag system to add a second layer of protection and/or storage in the vapor phase of liquid nitrogen. Since cord blood banking allows an indefinite time period for storage, studies have evaluated the stability of these products for transplantation post cryopreservation. The most common functional viability assay to evaluate clonogenic potential is the colony-forming assay which frequently assesses these four parameters: colony-forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM), colony-forming unit-granulocyte, macrophage (CFU-GM), burst-forming unit-erythrocyte (BFU-E), and colony-forming unit-erythrocyte (CFU-E). Current studies performed have assessed cryopreserved products that have been stored for 10–15 years (Broxmeyer et al., 1997, 2003; Kobylka et al., 1998; Mugishima et al., 1999). Eight samples were assessed after 15 years of storage in liquid nitrogen. At post-thaw the recovery of mononuclear cells averaged 80% proliferative capability and demonstrated cytotoxic response potential against foreign HLA antigens (Kobylka et al., 1998). Proliferative capacities were demonstrated by assessing colony-forming units and replating CFU-GEMM as described by Broxmeyer as a test of “self-renewal” for hematopoietic progenitor cells (Broxmeyer et al., 2003). An assay testing repopulation and engraftment capability in a non-obese diabetic/ severe combined immune deficiency (NOD/SCID) mouse was tested and exhibited similar results compared to using fresh cord blood CD34 positive cells (Broxmeyer et al., 2003). They were also able to demonstrate that an average of 83% of the total nucleated cells was recovered from the products after 15 years in storage. HEMATOPOIETIC AND TISSUE REGENERATION Cord blood cells are now considered a standard product for hematopoietic reconstitution and a potential product for regenerative medicine. Hematopoietic cell transplantation is now a standard of care worldwide for a long list of different diseases which includes but is not limited to leukemia, myelodysplastic syndrome, myeloproliferative and lymphoproliferative disorders, phagocyte disorders, inherited metabolic disorders, inherited immune disorders, inherited platelet disorders, and other malignancies. Transplantation of umbilical cord blood attributes includes low immunogenicity as illustrated by reduced acute GVHD (Rocha et al., 2000) with graft versus leukemia effect remaining intact (Howrey et al., 2000), ease of collection as described earlier, generally a biohazardous discard product with no alternative use, lower risk of infectious disease transmission, potential for ex vivo expansion and the possibility of use in gene therapy.

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Cord blood in the past was viewed as a product for transplantation only in children due to the number of cells that could be harvested from a single cord blood collection, but recently adult cord blood transplantation has been successfully studied along with double cord blood unit transplantation. Umbilical cord blood cells have been demonstrated by Laughlin and colleagues to provide long-term hematopoietic reconstitution in adults over 40 kg in weight, but this cellular product also demonstrated significant delay in the time to hematopoietic engraftment by delayed neutrophil, red blood cell, and platelet recovery similar to the delay seen in children (Laughlin et al., 2001, 2004). A recent study examined high-risk malignancy patients where the median day to engraftment was day 23 when they infused two cord blood units. Twenty-four percent of patients demonstrated engraftment from both units with 76% demonstrating unit dominance on day 100. They were able to demonstrate the safety of transplantation of two partially HLA-matched cord blood products and demonstrated the possibility of adequate cell dose for hematopoietic reconstitution from two cord blood units in adults (Barker et al., 2005).

PLURIPOTENT CELLS FROM UMBILICAL CORD BLOOD CELLS Umbilical cord blood stem cells are not only considered for hematopoietic stem cell reconstitution, but also for other uses demonstrated by its pluripotent stem cell capabilities. The source of umbilical cord cells is almost endless as globally the birth rate is approximately 130 million which would allow for a source of cells easily retrievable. These cells are able to differentiate and expand without a feeder layer and are generally a more ethically accepted cell source. Advantages of this cell source include its naive nature and relatively unshortened telomere length (McGuckin et al., 2005). Kogler and colleagues have identified a cell population in the cord blood which is CD45 negative that they refer to as unrestricted somatic stem cells (USSC). They have demonstrated the potential for this cell population to differentiate into osteoblasts, adipocytes, chondroblasts, hematopoietic, and neural cells in vitro and bone, cartilage, heart, and liver cells in vivo. They were also able to show a time frame greater than 40 population doubling without recombinant cytokines and a longer telomere length as compared to mesenchymal stem cells from bone marrow (Kogler et al., 2004). NEUROLOGICAL REGENERATION Stroke There is an enormous potential for cord blood stem cells to assist in the repair and regeneration of cells and tissues that are afflicted by neurological diseases. Currently there is a wide range of neurological disorders in which scientists are studying the effects of cord blood as a treatment modality in small animal models and there is also a significant amount of work being done on the characterization of these cells. Umbilical cord blood stem cells are one current source of adult stem cells involved in this research today. Many researchers have demonstrated cells co-transplanted with other cells such as sertoli cells (Sanberg et al., 2002) or growth factors (cytokines and chemokines) (Newman et al., 2005) to produce a synergistic response toward therapeutic benefit. Some of the neurological diseases that have been proposed to be treated with stem cells derived for cord blood include stroke, Alzhemier’s disease, Parkinson’s disease, Huntington’s disease, spinal cord injury, central nervous system (CNS) injuries, amyotrophic lateral sclerosis (ALS), cerebral palsy, and generalized brain injuries. In review of the literature for treatment of non-hematopoietic disorders with cord blood cells, stroke is one of the more widely studied disorders. Stroke is also one of the leading causes of death, later-life dementia, and adult disability world-wide today ranking third as the cause of death in the United States behind heart disease and cancer. Researchers in the field of neurological disorders are proposing that adult stem cells may be able to differentiate into neurological tissue or cells and assist with repair at the site by promoting neogenesis specifically by the release of factors that will stimulate the growth of cells already at the site (Borlongan et al., 2004).

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In a review of the historical literature on umbilical cord blood cells in the treatment of neurological disorders, one of the first reports was issued by researchers in 2000 (Erices et al., 2000) where they reported the production of an adherent cell population with potential to differentiate into osteoclasts and mesenchymal-like phenotyped cells. About the same time, Ende and colleagues were studying the effects of cord blood in SOD1 mice used as an animal model for the disease of ALS. They were able to demonstrate that large doses of human umbilical cord blood mononuclear cells were able to prolong the lifespan of SOD1 mice (Ende et al., 2000). In 2001, Sanchez-Ramos reported on the presence of molecular markers on umbilical cord blood cells that are generally associated with neurons and glia cells (Sanchez-Ramos et al., 2001). Ha and colleagues also reported on the current status of neural markers (neurofilament (NF), microtubule associated protein (MAP2), glial fibrillary acidic protein(GFAP)) demonstrated in cultured human umbilical cord blood cells along with the classic neural morphology (Ha et al., 2001). Both groups suggested these cells may be used in the future for therapeutic applications related to neurological disorders. Cord blood cells may be a viable option compared to the use of neural progenitors due to the ease of collection. Researchers compared stromal cells in bone marrow to cells with multilineage potential found in cord blood and were able to demonstrate these cells could differentiate into neural cells as identified by immunofluorescent labeling and Western blot analysis, but lacked some of the neural markers seen in bone marrow cells which alluded to a more immature cell population in cord blood (Goodwin et al., 2001). Researchers were able to demonstrate a neural stem-like cell population from human umbilical cord blood after cell isolation and fractionation that yielded a high-potency cell population expressing the surface marker nestin. When fractionated cells were placed in culture with growth factors or rat brain the researchers were able to demonstrate the three main neural phenotypes representing neurons, astroglia, and oligodendroglia at 30%, 40%, and 11% of the population respectively (Buzanska et al., 2002). Chen and colleagues studied the effects of human umbilical cord blood infused intravenously after stroke in a rat model. The rats were subjected to middle cerebral artery occlusion prior to cell infusion. Cord blood cells significantly improved function as demonstrated by two behavioral tests where as 7 days after occlusion improvement in only one of the behavioral tests was observed. The investigators were able to determine that the cord blood cells could enter brain tissue, survive, and improve neurological recovery in this model which demonstrates the potential of using these cells in the future for therapeutic applications related to stroke (Chen et al., 2001). Zigova and colleagues infused human umbilical cord blood cells into a developing rat brain to evaluate cell survival and phenotypic properties of the cells after infusion. They cultured the cells in retinoic acid (RA) and nerve growth factor (NGF) prior to infusion and then cell suspensions were injected into the anterior part of the subventricular zone. When the brain tissue was assessed for neural markers the cells were found to be positive for the following neural markers; GFAP and beta-III-tubulin. They determined 1 month post infusion into a rat brain that approximately 20% of the cells infused into the brain survived (Zigova et al., 2002). Other researchers measured the effects of human umbilical cord blood cells infused into a rat after traumatic brain injury and the cell migrated to the site of injury in the brain and expressed neural markers. The rat model demonstrated the potential of cord blood cells in treatment of traumatic brain injury. The cells not only expressed neural markers within the brain, but also integrated into vascular tissue surrounding the injured brain tissue (Lu et al., 2002). Taguchi in 2004 was able to demonstrate neurogenesis and angiogenesis in a mouse model after the infusion of CD34 positive cells selected from human umbilical cord blood cells. The infusion was given to immunocompromised mice 48 h after injury. They were able to demonstrate endogenous neurogeneration accelerated by homing neural progenitors to the site of injury. They proposed that the CD34 positive cord blood cells promote neovascularization either directly or indirectly providing the environment for neovascularization (Taguchi et al., 2004). Borlongan and colleagues investigated why human umbilical cord blood transplants in a rat stroke model exhibited neuroprotection. They infused cord blood with mannitol to

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permeabilize the blood brain barrier and this reduced the cerebral infarcts and improved behavioral function in the animals that received the cord blood cells. Other animals that received the cord blood without the mannitol did not have an effect on the cerebral infarct or behavioral tests. The researchers concluded that the neuroprotection was transferred via secreted chemical factors rather than cells homing to the site of injury (Borlongan et al., 2004). These research groups both discuss the potential of indirect neovascularization that occurs in an in vivo stroke model. Vendrame and colleagues examined the behavioral recovery and stroke infarct volume before and after infusion of human umbilical cord blood cells assessing cell dose in a rat model. Four weeks after the cord blood infusion, there was a significant recovery in behavioral performance when a minimum of 1 million cells were infused. When doses of cord blood cells increased, they were able to demonstrate behavioral improvement, and neuronal sparing correlating directly to the number of cells infused. The researchers discussed the large cell dose required for potential human infusion and possibly using ex vivo expansion in this situation (Vendrame et al., 2004). This same group also reported on studies in a rat model of stroke that human umbilical cord blood cells may be effective by decreasing pro-inflammatory cytokines to result in enhanced neuroprotection. Testing results demonstrated a decrease in mRNA and protein expression of pro-inflammatory cytokines and a decrease in nuclear factor kappa B DNA binding activity in the brain of stroke animals treated with cord blood cells (Vendrame et al., 2005). Newman and colleagues investigated the migration of human cord blood cells to ischemic tissue extracts which correlated with an increase in certain cytokines and chemokines. They were also able to illustrate that the time frame for treatment may be extended out from a suggested 3 h to 24–72 h after a stroke when using approved anticoagulant therapy and cord blood cell infusion (Newman et al., 2005). In summary the research on human umbilical cord blood cells in relationship to stroke has demonstrated that neuronal cell markers are present on cells and that some of these cells actually demonstrate a more primitive status than the cells found in bone marrow. The cells when infused not only could enter the brain but also vascular tissue. Neuroprotection has a strong correlation to chemical factors produced at the site of injury. Decreasing pro-inflammatory cytokines appears to be a major factor and it may be possible to extend the treatment after cord blood infusion greater than 3 h as previously considered. Huntington’s disease, Alzheimer’s disease, Parkinson’s disease and ALS In the examination of neurodegenerative diseases one publication was found in the literature related to Huntington’s disease reported by Ende and colleagues where they treated mouse models for the disease with mega-doses of human umbilical cord cells. They infused approximately 70–100 million cells to treat a mouse to increase their lifespan from 88 days to 97.8–103.4 days with the largest dose of cells (Ende et al., 2001). Ende and colleagues also examined Alzheimer’s and Parkinson’s disease in a small animal model with human cord blood cells. Their reports included considerable life extension in the mouse model for Alzheimer’s disease after umbilical cord blood mononuclear infusion. A high dose of 110 million cells per mouse infused compared to control animals demonstrated a longer lifespan (Ende et al., 2001). In the mouse model for Parkinson’s disease three groups were studied: infused with congenic marrow mononuclear cells with one out of ten alive, infused with cord blood mononuclear cells with four out of twelve alive, and a control group with one out of ten alive. The experiments were terminated at day 200 and results demonstrated a delay in the onset of symptoms and prolonged lifespan in the group infused with umbilical cord blood mononuclear cells (Ende and Chen, 2002). Researchers examined ALS which is characterized by motor neuronal degeneration. An ALS mouse model (G93A) was used to study the infusion of human umbilical cord blood mononuclear cells into systemic circulation. The researchers demonstrated that the infusion delayed disease progression 2–3 weeks and increased the lifespan of the mice. The infused cells migrated to the parenchyma of the brain and spinal cord

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where neural markers were expressed on these cells including nestin, beta-III-tubulin, and GFAP (Garbuzova et al., 2003). Other researchers utilizing a mouse model for ALS, the SOD1 mice, infused a mega-dose of umbilical cord blood mononuclear cells after irradiation. They demonstrated that doubling the mega-dose of cells further increased the lifespan of the mice. To produce a mega-dose for infusion donors were pooled and no negative effects were observed (Chen and Ende, 2000). Willing and colleagues examined different sites of infusion in the mouse model for ALS. They demonstrated through behavioral tests that intraspinal infusion did not demonstrate improvements but intravenous did demonstrate improvements not only by behavioral tests, but also demonstrated by life-span (Willing et al., 2002). Spinal Cord Injury and CNS injuries Researchers recently examined the functional effects from an umbilical cord blood infusion in a rat model for spinal cord injury. Three groups were assessed for treatment by infusion of umbilical cord blood cells, umbilical cord blood cells with brain-derived neurotrophic factor, and a control group with media alone injected directly into the spinal cord. Groups that included the infusion of cord blood demonstrated improvement weekly over the control group in the locomotor rating scale. They also demonstrated that the transplanted cells differentiated into various neural cells (Kuh et al., 2005). Other researchers examined the effects of cord blood cell infusion in a sex-mismatched mouse model which demonstrated cells were generated in the CNS but concerns arose surrounding the available cell dose in a product and HLA disparity of the cells (Korbling et al., 2005). Researchers in 2005 published a case study on a spinal cord-injured patient transplanted with umbilical cord blood cells. The cells were HLA-matched and transplanted directly into the spinal cord. The case study demonstrated an improvement in the sensory perception and movement in the patient’s hips and thighs. An MRI and CT scan also demonstrated regeneration of the spinal cord at the site of injury (Kang et al., 2005). These are potentially exciting applications that will need further investigation for the site of infusion and possible assessment of the minimal cell dose required prior to scale up studies in larger animal models. Neural Cell Surface Markers It is known that researchers are able to differentiate adult multipotent cells into neurons, astrocytes, and oligodendrocytes in the CNS, but the mechanisms involved in the differentiation are a critical component of current research. More recently human umbilical cord blood cells specifically have been assessed for potential to produce neural progenitors some of the markers used to identify the cell population are discussed. Buzanska and colleagues selected CD34 negative umbilical cord blood cells after density gradient and expanded the cells in media to support the growth of neurogenic cells. Post culture of the cells expressed nestin, which is a primitive marker for neural cells and in culture with selected growth factors 30% of the cell population was neuronal, 40% astrocytic, and 11% were oligodendrocytes (Buzanska et al., 2002). Although nestin is a well-known neural progenitor cell marker it is also associated with other cell types such as pancreas, kidney, hair follicle cells, and blood vessels in the skin (Amoh et al., 2005). Nestin is also a filament protein that has been shown to play a role in cytoskeleton regulation (Chen et al., 2006). Jang and colleagues isolated CD133 via magnetic cell sorting by bead sorting and fluorescence-activated cell sorter (FACS). After selection, umbilical cord blood cells were cultured in RA and cells expressed neuronal and glial phenotypes. Post culture, the cells demonstrated transcription factors important for early neurogenesis including Otx2, Pax6, Wnt1, Olig2, Hash1, and NeuroD1 (Jang et al., 2004). Other researchers also isolated CD133 positive progenitor cells from human umbilical cord blood and cultured the selected cells in media containing Flt3-ligand (FL), thrombopoietin (TPO), and stem cell factor (SCF). The cells post culture expressed pluripotent markers including Sox-1, Sox-2, FGF-4, Rex-1, and Oct-4. After cell culture with RA the cells demonstrated a neural morphology coupled to the expression of beta-III-tubulin (Baal et al., 2004).

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McGuckin and colleagues performed a negative hematopoietic lineage depletion where they were able to recover 0.1% of the starting cell population and adherent cells were capable of demonstrating neuroglial progenitor cell morphology. Neuroglial progenitor cell markers were identified by gene expression analysis including, beta-III-tubulin (McGuckin et al., 2004). In summary the markers associated with identification of the neural cell lineage in umbilical cord blood cells include nestin, CD133, GFAP, NF, and MAP2. Nestin is a marker for primitive neural tissue and CD133 is a known cell surface marker associated with the production of neural and glial cells. Glial fibrillary acidic protein more commonly known as GFAP marks the astrocytes which is a type of glial cell. Neurofilament known as NF is an important structural component of the neuron and MAP2 is a protein found in the dendritic branching of the neuron. Cardiac Function Treatment Cardiac disease is the number one killer of men and women worldwide. Approximately 70 million Americans have some form of the disease and this is one of the reasons many investigators are studying how to treat the disease with cellular therapy. Researchers as early as 2001 discussed potential therapeutic applications, such as genetic modulation, cell transplantation, and tissue engineering as a novel approach to myocardial regeneration and tissue repair after myocardial infarction (Etzion et al., 2001). Current animal studies and human clinical trials are evaluating infusion of cells directly into the damaged myocardium or infusion of cells via intravenously to repair damaged and infarcted tissue. These cells may have the potential in the future to replace whole organ transplants with cell transplants derived from umbilical cord blood cells. Myocardial Infarction Regenerative medicine after a myocardial infarction may include the replacement of the damaged cells by either an intravenous infusion or infusion at the site of ischemia. Recently several articles have been published on the phenotypic properties of umbilical cord blood stem cells used in cardiac repair. The most common phenotypic marker published in the literature is CD34 which is a cell surface glycoprotein, generally marking the hematopoietic progenitor cell. In 2004, Botta and colleagues discussed the production of hematopoietic and endothelial cells from the hemangioblast. They were investigating a progenitor cell in cord blood with a phenotype of CD34 positive/KDR positive. KDR is an endothelial growth factor receptor. They assessed the potential of these cells in a NOD–SCID mouse model and were able to demonstrate beneficial effects of cord blood cells CD34 positive/KDR positive illustrating improvement in cardiac hemodynamics by resistance to apoptosis and their angiogenic action (Botta et al., 2004). Looking at a different disease process Cogle et al. (2004) also demonstrated the functional hemangioblast potential of CD34 positive human umbilical cord blood cells. They assessed an NOD–SCID mouse model for retinal ischemia which resulted in human retinal neovascularization in the mouse model. Hirata and colleagues in 2005 studied the effects of human umbilical cord blood CD34 positive cells in a rat model for myocardial infarction produced by ligation of the left coronary artery. The CD34 positive cells survived and improved cardiac function (Hirata et al., 2005). Two groups examined the effects of CD133 positive cells which has been identified as a neural and hematopoietic cell marker and recently was published as a marker for embryonic stem cell-derived progenitors (Kania et al., 2005). Leor and colleagues discussed the possibility of human umbilical cord blood stem cells for use in repair of infarcted myocardium. They infused approximately 1.2–2 million cells intravenously 7 days after coronary artery ligation in a rat model and were able to demonstrate that the cell infusion produced functional recovery by preventing scar thinning and left ventricular systolic dilation (Leor et al., 2006). Wu and colleagues expanded CD133 positive cells from human umbilical cord blood stem cells to produce endothelial progenitor cells (EPC) (Wu et al., 2004).

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Ott and colleagues suggested that cell therapy for myocardial infarction may be limited by the number of cells available. They expanded CD34 positive cord blood and cultured the cells in endothelial medium where cells were expanded up to 46 population doublings. These cells were able to form vascular structures and improve left ventricular function after experimental myocardial infarction in an athymic nude mouse model (Ott et al., 2005). Delorme and colleagues demonstrated that EPC in umbilical cord blood are CD146 positive cells, which is an adhesion marker on endothelial cells. These cells were selected from the non-adherent cell population in umbilical cord blood cells. They were able to demonstrate proliferation in long-term culture while maintaining the same phenotypic properties and the colonization of a matrigel plug in immunodeficient NOD–SCID mice. This study suggests that the CD146 positive cells contain a subpopulation of circulating EPC that may be used in pro-angiogenic therapy (Delorme et al., 2005). Other scientists investigated the use of mononuclear cells from human umbilical cord blood cells for treatment in acute myocardial infarction. They infused 1 million cells in a rat model that underwent left anterior descending coronary artery ligation and the cells were injected directly into the infarct border. The results of the experiments demonstrated a reduction in the infarction size in the rat model. Left ventricular functional measurements and ejection fractions were greater in the cord blood infusion group (Henning et al., 2004). Kim and colleagues discussed the USSC potential to differentiate into myogenic cells and induce angiogenesis. A porcine model demonstrated regional and global function of the heart after a myocardial infarction. These cells have been proposed to be used for cellular cardiomyoplasty due to efficacy and safety of the cells (Kim et al., 2005). Ishikawa and colleagues tested the potential of human umbilical cord blood stem cells to give rise to cardiomyocytes in vivo. They infused cord blood lineage negative cells which generated cardiomyocytes following transplantation into immune deficient mice (Ishikawa et al., 2006). Chen and colleagues assess the potential use of human umbilical cord blood cells with gene therapy to enhance angiogenesis via a mouse model after acute myocardial infarction. The goal of the study was to improve myocardial infarction by new vessel formation. A mouse model for acute myocardial infarction was infused intramyocardially with purified CD34 positive cells. The mouse model demonstrated a reduction in the infarct size with increased capillary density which resulted in a reversal of cardiac dysfunction (Chen et al., 2005). Ma and colleagues isolated human umbilical cord blood CD34 positive cells to inject them into the tail vein of an NOD–SCID mouse model with ligation of the left anterior coronary artery. Post infusion they analyzed capillaries for chimerism, but only occasionally human and mouse endothelial cells were discovered with most new vessels displaying mouse cells only. Post analysis, it was determined that up to 70% of the cord bloodderived cells in the heart were CD45 positive. The cells did not appear to differentiate, but did demonstrate migration to the infarcted tissue selectively where they engrafted to assist in neogenesis (Ma et al., 2005). Umbilical cord blood cells appear to be an attractive target for cell therapy after myocardial infarction due to the low immunogenicity of the cells and the ease of collection and storage of the cryopreserved product which render it easily accessible. It has no ethical concerns as embryonic stem cells and is currently used as an alternative for bone marrow in hematopoietic reconstitution in standard treatment protocols. All the recent data are encouraging for the use of human umbilical cord blood stem cells to assist in the reversal of cardiac dysfunction in the above described applications. Stem cell expansion maybe a major limiting factor if the cell dose used in the animal model needs to be translated to humans. The cord blood cell infusion may in the future eliminate the need to procure tissue or blood vessels from the patient for cardiac reconstruction. Clinical Trials for Cardiac Disorders Several phase I clinical trials are active involving progenitor cells derived from bone marrow for the treatment of myocardial infarction. The trials include the use of mesenchymal stem cells infused intravenously and autologous bone marrow mononuclear cells infused directly into the coronary artery. One other study

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involves the assessment of the safety of the autologous skeletal myoblasts cells via catheter delivery. Clinical studies with bone marrow cells in the past have shown improvement in function and decreased infarct size although the treatment is still an area of debate, but none the less is able to proceed with clinical trials to determine the process is safe. Currently bone marrow stem cells appear to be the cell of choice for treatment of myocardial infarction. The optimal cell type may allow for the promotion of angiogenesis and myogenesis, but further studies are required to determine the best cell type. Treatment of Diabetes Pancreatic Cells, Insulin-Producing Cells, Treatment of Type I/Type II Diabetes and Diabetic Neuropathy Recently a lot of work has been done in the area of regenerative medicine for Type I diabetes including the use of hepatocytes, bone marrow, intestinal epithelial cells, and pancreatic stem cells. Yoshida and colleagues demonstrated the production of insulin-producing cells from human umbilical cord blood via a mononuclear cell preparation that was infused into an NOD–SCID mouse. They were able to demonstrate cord bloodderived cells resulted, in insulin-producing cells at a rate of 0.65%  0.64% in xenogeneic hosts by fusion dependent and independent functions (Yoshida et al., 2005). Ende and colleagues assessed the use of human umbilical cord blood mononuclear cells in pre-diabetic stage NOD mice with autoimmune Type I diabetes. The outcome of the experiments demonstrated significantly lower glucose levels and increased their lifespan. The mice that received the highest dose had the most significant response with the highest dose at 200 million cells. The researchers were able to demonstrate that cord blood mononuclear cells infused at the pre-diabetic stage in the NOD mouse model without any immunosuppression is able to lower glucose levels and increase lifespan (Ende et al., 2004a). They also examined the effects of human umbilical cord blood cells for the treatment of Type II diabetes. They assessed blood glucose levels, survival, and renal pathology. In the obese mice with Type II diabetes infused with umbilical cord blood improvement was seen not only in blood glucose levels and survival rate, but also normalization of glomerular hypertrophy and tubular dilation (Ende et al., 2004b). Pessina and colleagues discuss a panel of markers required for human umbilical cord blood cells to form multipotent progenitor cells of the pancreas. The markers included nestin, which is generally viewed as a neuronal or pancreatic progenitor cell marker; other markers listed include cytokeratin (CK)-8 and CK-18. Transcription factors associated with islet-derived progenitors are Isl-1, Pdx-1, Pax-4, and Ngn-3. They were able to demonstrate that human umbilical cord blood cells contain a population of phenotyped cells similar to endocrine cell precursors forming beta cells (Pessina et al., 2004). Naruse and colleagues have assessed the use of EPCs from human umbilical cord blood cells for use in the reversal of diabetic neuropathy. Cord blood mononuclear cells were cultured and EPCs were isolated and expanded. The EPCs were injected intramuscular into the hindlimb skeletal muscles of streptozotocininduced diabetic nude rat model. The study results demonstrated an increased number of microvessels in hindlimb skeletal muscles in the diabetic rats compared to the controls (Naruse et al., 2005). Clinical Trials for Type I Diabetes Currently there is one active clinical trial assessing autologous cord blood infusion for Type I diabetes in an attempt to regenerate pancreatic islet insulin-producing beta cells and therefore improving glucose control. The researchers will track migration of the infused stem cells and study changes in metabolism and immune function leading to islet regeneration. The study is a phase I/phase II clinical trial so that it will evaluate safety and efficacy.

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Hepatocyte-Like Cells Currently research has focused on the search for alternatives, such as liver progenitors, fetal hepatoblasts, embryonic, bone marrow, or umbilical cord blood stem cells to replace hepatocytes in a disease state. Several investigators have examined the specific potential of human umbilical cord blood cells assessing the in vitro and in vivo potential to form hepatic cells. This is a relatively new area of experimentation as a significant amount of the work completed appeared in the literature over the last year. Most of the researchers assessed the potential of in vivo generation of hepatic cells after injury in a mouse model and a few others examined the potential of in vitro differentiation to the hepatic cell lineage. In the assessment of in vitro differentiation Kang and colleagues assessed the capability of human umbilical cord blood cells to differentiate into hepatocyte-like cells. Cord blood mononuclear cells were collected and cultured with hepatocyte growth factor (HGF), fibroblast growth factor 4 (FGF4), both, and no growth factor. The authors were able to demonstrate that HGF- and FGF4-induced cord blood mononuclear cells were capable of differentiation into hepatocyte-like cells (Kang et al., 2005). Other investigators reported on a cell population; cord-blood-derived embryonic-like stem cells (CBE) positive for TRA-1-60, TRA-1-81, SSEA-4, SSEA-3, and Oct-4, which are also embryonic stem cell markers. CBE were also cultured with hepatocyte growth medium and post culture the cells expressed characteristic hepatic markers, CK-18, alpha-fetoprotein, and albumin (McGuckin et al., 2005). Researchers investigated the potential of human cord blood to be used as cell therapy for an injured liver in vitro and in vivo. Cord blood cells post culture expressed albumin and hepatocyte lineage markers. When investigating liver-injured severe combined immunodeficient mice infused with human umbilical cord blood cells, they were able to demonstrate the development of functional hepatocytes in the liver (Kakinuma et al., 2003). Researchers suggest that these cells may have potential for treatment of hepatic diseases. Other researchers examined the potential of CD34 selected cells from human umbilical cord blood cells for production of hepatocytes in vitro. They also assessed NOD/SCID mice for the in vivo studies where it was exposed to liver injury by a Fas ligand-carried adenoviral vector. As demonstrated by RT-PCR the cord cells were able to differentiate into hepatocyte-like cells in the mouse liver and it was demonstrated that liver injury was essential during this process. There were no differences between the use of CD34 positive and CD34 negative cells (Nonome et al., 2005). Kashofer and colleagues also evaluated hepatic in vivo differentiation from human cord blood mononuclear cells selected for CD34 positive cells or lineage negative cells. The cells were infused after liver damage in NOD/SCID mice. To identify the infused cells they transduced, the stem cell population, with lentivirus construct expressing enhanced green fluorescent protein (eGFP) and fluorescent in situ hybridization (FISH) analysis performed as the cells were sex mismatched. The results of the study revealed that very little human chromosomes were present in the hepatocyte-like cells and they may have fused with host hepatocytes (Kashofer et al., 2005). Other researchers examined the potential of inducing hepatic differentiation in human umbilical cord blood cells. They assessed for newly formed hepatocyte-like cells in the liver of NOD–SCID mice after transplantation of human cord blood or murine bone marrow. Liver injury was induced by carbon tetrachloride and they detected clusters of hepatocyte-like cells derived from cord blood cells. FISH demonstrated mostly host-derived hepatocyte-like cells with murine bone marrow infusion. They demonstrated that human cord blood in an NOD–SCID mouse model has contrasting differentiation potential from murine bone marrow cells (Sharma et al., 2005). Investigators assessed the efficacy of human umbilical cord blood cells to decrease histologic damage and the mortality rate of animals previously damaged by allyl alcohol. NOD/SCID mice were treated with allyl alcohol with and without intraperitoneal infusion of human cord blood cells. The cord blood cells infused were able to transdifferentiate into hepatocytes and demonstrate a significant decrease in mortality rate in the mouse model. Researchers believe that endogenous regeneration occurs for early stage of damage (Di Campli et al.,

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2005). Investigators studied human umbilical cord blood cells that were CD34 positive or CD45 positive and they were transplanted into NOD/SCID/beta-II-microglobulin null mice. The livers were examined for evidence of human hepatocyte engraftment. Analysis of the mouse bone marrow revealed that 21.0–45.9% of the cells were human and FISH analysis excluded spontaneous cell fusion for the generation of human hepatocytes. The researchers demonstrated that human cord blood cells can give rise to hepatocytes in an xenogeneic transplantation model (Ishikawa et al., 2003). Researchers investigated the potential of human cord blood to be used as cell therapy for an injured liver in vitro and in vivo. Cord blood cells post culture expressed albumin and hepatocyte lineage markers. When investigating liver-injured severe combined immunodeficient mice infused with human umbilical cord blood cells they were able to develop into functional hepatocytes in the liver (Kakinuma et al., 2003). All researchers were able to demonstrate the in vitro repopulating capability of hepatic cells derived from human umbilical cord blood cells with the appropriate growth factors. Others working with the mouse model were able to demonstrate the in vivo differentiation potential of cord blood cells when hepatic injury occurs due to carbon tetrachloride or allyl alcohol. McGuin and colleagues were able to demonstrate CBE that may have the potential in the future as a source of transplantable hepatic progenitor cells. Endothelial Progenitors Angiogenic therapy by using EPC is currently a topic of debate. These cells have been used to treat of ischemic diseases for revascularization. They may also be used in diagnosis to assess the disease state in the patient; cord blood is fairly new in this arena. These cells have not only been assessed phenotypically by markers, but they also need to be evaluated for the proliferative and clonogenic potential. Ingram and colleagues described a group of EPCs derived from replating colonies by a single cell method in culture from umbilical cord blood cells. This culture gave rise to a new cell population capable of at least 100 population doublings and was able to retain high levels of telomerase activity (Ingram et al., 2004). Murga and colleagues isolated CD34 negative cells including endothelial precursor cells from human umbilical cord blood cells. The CD34 negative cell population with angiogenic factors produced cells that express the endothelial cell markers: vascular endothelial-cadherin, vascular endothelial growth factor receptor1 (VEGFR-1) and VEGFR-2, Tie-1 and Tie-2, von Willebrand factor, and CD31 and can be expanded in vitro for over 20 passages. Researchers were able to demonstrate endothelial precursors in the CD34 negative cell population of cord blood (Murga et al., 2004). Salven and colleagues were able to demonstrate that human CD34 positive and CD133 positive cells expressing VEGFR-3 constitute a phenotypically and functionally distinct population of endothelial stem and precursor cells that may play a role in angiogenesis (Salven et al., 2003). Other researchers were able to identify a cell population of circulating endothelial precursors expressing VEGFR2, CD34, and CD133 from human cord blood which may have a role in neogenesis (Peichev et al., 2000). Shin and researchers examined the cytokines and culture conditions required for large amounts of endothelial cells that may be required for vasculogenesis. The CD34 positive cells from human cord blood were selected and cultured in various cytokine cocktails. The quantity of cells adherent and non-adherent was the greatest with use of SCF, FL, and TPO cytokines. When growth factors were added: VEGF, IL-1 beta, FGFbasic (FGF-b); endothelial cells were identified by morphology and endothelial-specific markers (Shin et al., 2005). Researchers are investigating autologous patches engineered from human umbilical cord-derived fibroblasts and EPCs as a ready-to-use cell source for pediatric cardiovascular tissue engineering. EPCs were isolated from umbilical cord blood by density gradient centrifugation and myofibroblasts were harvested from umbilical cord tissue. Cells were differentiated and expanded in vitro. The investigators believe that these cells may be used for autologous replacement materials for congenital cardiac interventions (Schmidt et al., 2005). The possibility exists in the future to be able to use the differentiated cells produced from umbilical

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cord blood for vascular cell therapy, but more in vivo studies are required to assess the homing capabilities of the cells. Chondrocytes One article was published on the differentiation of cord blood cells into chondrocytes. Researchers investigated human umbilical cord blood cells lineage negative, CD45 negative, CD34 negative for the potential of chondrocyte differentiation. They cultured the cells with mouse embryonic limb bud cells which demonstrated that cord blood cells have the potential to differentiate into chondrocytes (Jay et al., 2004). Ex vivo Expansion A limiting factor for the use of cord blood cells is the low cell dose harvested at procurement. In clinical transplantation the low dose is often associated with delayed engraftment of neutrophils and platelets. Cell expansion is being investigated, but concerns lie in the low quantity of primitive cells observed in the expanded cell population. Different methods currently in use for ex vivo expansion include CD34 or CD133 positive selection. These selected cells are cultured to proliferate primitive cells, they can also be co-cultured with mesenchymal stem cells with growth factors or cells can be cultured in a bioreactor with continuous perfusion (Robinson et al., 2005). Robinson and colleagues compared two cord blood expansion methods. They studied the effects of CD133 positive cells expanded in culture and cord blood unmanipulated co-cultured with bone marrow mesenchymal stem cells both supplemented with growth factors. They were able to conclude through analysis of the total nucleated count, CD133 positive and CD34 positive cells that the cord blood co-cultured with mesenchymal stem cells performed better than the CD133 selected cells (Robinson et al., 2006). Tetraethylenepentamine (TEPA) enables preferential expansion of early hematopoietic progenitor cells in human umbilical cord blood-derived CD34 positive cell cultures as reported by Peled and colleagues in 2004. The copper chelation appears to modulate the balance between self-renewal and differentiation of hematopoietic progenitor cells (Peled et al., 2004). CD133 selected cells were cultured and expanded. The authors reported CD34 cells increased by 89-fold, CD34 positive/CD38 negative increased by 30-fold and colony-forming unit cells by 172-fold over the number of cells seeded (Peled et al., 2004). Subsequently they were transplanted into NOD/SCID mice which demonstrated the CD133 expanded cells faired better compared to the unexpanded for engraftment in terms of CD45 positive and CD45, CD34 positive cells (Peled et al., 2004). Peled and colleagues have demonstrated the enhancement effect of TEPA when they examined human umbilical cord blood selected for CD133 positive cells, cultured in a closed system with cytokines (SCF, TPO, IL-6, and FL). The cell yield of CD34 positive population made a 89-fold and a 172-fold increase in colony-forming units. Infusion into an irradiated non-obese diabetic (NOD/SCID) mice demonstrated superiority with the expanded product (Peled et al., 2005). McNiece and colleagues investigated the potential of human cord blood mononuclear cells in co-culture with mesenchymal stem cells. The expansion demonstrated 10- to 20-fold increase in total nucleated cells, 7to 18-fold increase in committed progenitors, 2- to 5-fold expansion of primitive progenitors and 16- to 37fold increase in CD34 positive cells which may allow for significant expansion without the use of pre-cell selection (McNiece et al., 2004). Delany and colleagues elaborate on the effect of Notch ligand density on induction of Notch signaling and the effect on expansion of human CD34 positive, CD38 negative cord blood progenitors. Lower densities of Delta1 (ext-IgG) enhanced production of CD34 positive cells while higher densities induced apoptosis of these cells. The density of Notch ligands may be an important factor in expansion of cord blood cells (Delaney et al., 2005). Jang and colleagues also examined expansion of cord blood cells in co-culture with mesenchymal stem cells lacking cytokines which demonstrated CFU-GM, CFU-GEMM, BFU-E, and CFU-E increased to 3.46-, 9.85-, 3.64-, and 2.03-folds, respectively (Jang et al., 2006). It appears

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that cord blood expansion has made progress in increasing not only the CD34 positive and the CD34 positive/CD38 negative hematopoietic progenitor cell population, but also capable of expanding the colonyforming unit capacity of the cell product. Aldehyde Dehydrogenase Expressing Cells Jones and colleagues described aldehyde dehydrogenase as an enzyme observed in high amounts in primitive cells initially discovered in bone marrow (Jones et al., 1995). Storms and colleagues discussed the stem cell and progenitor potential of umbilical cord blood with aldehyde dehydrogenase positive cells. They were able to demonstrate progenitors were highly enriched within the aldehyde dehydrogenase bright and CD34 positive population, but when compared to the aldehyde dehydrogenase negative and CD34 positive population few primitive progenitors were identified. They suggest the use of aldehyde dehydrogenase to discriminate between stem cell and progenitor cell populations in umbilical cord blood (Storms et al., 2005).

CONCLUSION For many years bone marrow and mobilized peripheral blood were the leaders in reconstitution for hematopoietic disorders, but now umbilical cord blood is gaining speed and is viewed as an alternative to bone marrow for transplantation. Mesenchymal stem cells were first described in bone marrow and now there are several reports on the unrestricted pluripotent cells identified in cord blood. Several advantages of these cells may assist in its leadership in regenerative medicine as one cell source lacking ethical concerns. Advantages are vast reaching including its ease of procurement, its naive immune status, its low contamination potential of infectious disease, its relatively unshortened telomere length and its homing capabilities that have been demonstrated in small animal models and in humans for hematopoietic reconstitution. With approximately 130 million births a year worldwide this is a largely under-utilized precious source of stem cells. An abundance of work has been done in the area of cord blood transplantation since the first reported case in 1988 which includes transplantation of umbilical cord blood stem cells for children to treat leukemia, lymphoma, and certain cancers including genetic disorders that affect the blood and immune system. Cord blood cells have also been used in adult transplants and double cord transplants which have been able to treat patients over 40 kg in weight. Due to the limiting nature of the number of cells in a cord blood product a significant amount of work has been done on the expansion of these cells focusing on the retention of the primitive stem cells required for engraftment. Amazing strides have been made to demonstrate that cord blood does in fact contain pluripotential cells that have proven differentiation to the lineages within all three germ cell layers. Publications on differentiation capability included neural related cells, cardiac cells, pancreatic progenitor cells, hepatocyte-like cells, endothelial cells, and chondrocytes. Ex vivo expansion is an active area of research due to the quantity of cells available at time of procurement. Researchers are examining different cells to expand and different culture conditions including a study assessing the bioreactor for continuous perfusion culture. The current challenges in umbilical cord blood stem cells include the quantity of cells in the procured product to be used for children and adults. Also tied into the quantity is the fact that a number of studies performed in small animal models required a significant amount of cells to demonstrate effective treatment; to be able to translate this dose to large animal models or human clinical trials may require an optimal technique for the expansion of these critical cells. FUTURE DEVELOPMENTS In evaluation of the pluripotent cells from umbilical cord blood investigators will be able to demonstrate safety in the infusion of the cells. Umbilical cord blood has already had years of safety data with routine transplant for

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leukemia and lymphoma. Unlike embryonic stem cells cord blood may have an abbreviated path to clinical trials as demonstrated with bone marrow mesenchymal stem cells in limited cases. More studies will be assessing the efficacy of cord blood transplants in adults. Basic research will continue to thrive in an effort to fuel significant changes in this area. Pre-clinical trials and phase I clinical trials will continue to move transplantation into an era where it will immensely expand the number of diseases it will have potential to treat or cure.

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