Potential clinical applications using stem cells derived from human umbilical cord blood

RBMOnline - Vol 13. No 4. 2006 562-572 Reproductive BioMedicine Online; www.rbmonline.com/Article/ www.rbmonline.com/Article/2372 on web 18 July 2006

Article Potential clinical applications using stem cells derived from human umbilical cord blood Dr Ghen is one of the pioneers of stem cell research and transplantation and has been using human cord blood for more than 4 years. As the Chief Medical Officer for Eden, Dr Ghen also heads the team as the principal investigator, who will have primary clinical responsibility for Eden’s undertakings. He has already successfully made transplants to dozens of individuals suffering from varying disorders without serious adverse events associated with transplantation. Dr Ghen holds a Doctorate of Osteopathy from the Philadelphia College of Osteopathic Medicine, a Master’s degree in biomechanical trauma, a PhD in anti-ageing and psychoneuroimmunology, and is a certified nutrition specialist.

Dr MJ Ghen MJ Ghen, R Roshan, RO Roshan, DJ Blyweiss, N Corso, B Khalili, WT Zenga Eden Laboratories Ltd, Frederick House, Frederick Street, PO Box SS-19392, Nassau, The Bahamas Correspondence: e-mail: [email protected]

Abstract There is an abundance of clinical applications using human umbilical cord blood (HUCB) as a source for stem cell populations. Other than haematopoietic progenitors, there are mesenchymal, endothelial stem cells and neuronal precursors, in varying quantities, that are found in human umbilical cord blood. These may be useful in diseases such as immune deficiency and autoimmune disorders. Considering issues of safety, availability, transplant methodology, rejection and side effects, it is contended that a therapeutic stem cell transplant, utilizing stem cells from HUCB, provides a reliable repository of early precursor cells that can be useful in a great number of diverse conditions. Drawbacks of relatively smaller quantities of mononucleated cells in one unit of cord blood can be mitigated by in-vitro expansion procedures, improved in-vivo signalling, and augmentation of the cellular milieu, while simultaneously choosing the appropriate transplantation site and technique for introduction of the stem cell graft. Keywords: human umbilical cord blood, stem cells

Introduction

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There are many potential clinical applications for human umbilical cord blood (HUCB) derived stem cells. HUCB has several distinct advantages associated with its use. It is readily available with new cord blood banks opening throughout the United States and the world on a regular basis. Although still classified as a medical waste by-product, as a rich source of stem cells, it is a precious commodity for the growing medical cellular industry (Rogers et al., 2004; Moise Jr, 2005; Sanberg et al.,, 2005; Watt et al., 2005). The cost of testing per unit is also relatively low. Testing per unit typically includes studies for HIV, hepatitis A, B and C, cytomegalovirus, toxoplasmosis, blood typing, Rh factor and other tests that may be required for endemic diseases (McCullough et al., 2005; Tamburini et al., 2005). The above studies are typically performed on the mother; those studies that are typically performed on the cord blood are blood typing and Rh along with aerobic and anaerobic cultures and fungal cultures (Ademkoun et al., 1997; Jimenez et al., 2005). The procedure for extraction of cord blood has been demonstrated to be extremely safe for both mother and

baby (Elchalal et al., 2000; Gurcheva et al., 2001; Bornstein et al., 2005). Ethical issues surrounding HUCB have been considered by both political and religious organizations, and the use of human cord blood is considered to be a moral and ethical solution, with its useful collection of stem cells. In addition, human cord blood offers a wealth of growth factors that accompany the stem cells and are present in all units of cord blood (Ruggieri et al., 1994; Bracci-Laudiero et al., 2003; Fan et al., 2005; Tsao et al., 2005). Of course, the earlier the delivery the greater likelihood of having earlier precursors, and therefore more multipotent stem cells present with their associated growth factors (Shields et al., 1998). Transdifferentiation, as suggested with the use of human cord blood, may be secondary to non-haematopoietic early precursors in the cord blood and not to the plasticity of the mononuclear haematopoietic CD34+ cells, as was previously thought (Ramos, 2002). Using HUCB has distinct advantages over adult stem cells (Summers et al., 2004). Earlier cell cycle phase is seen with

Article - Uses for stem cells derived from umbilical cord blood - MJ Ghen et al. human cord blood. One of the challenges associated with adult stem cells is their decreased capability for differentiation (stemness) because they are often harvested in a late cell cycle phase (Kirchstein et al., 2001). In addition, long standing environmental abuse, often associated with adult stem cells limits their use. Lifetime exposure to pesticides, insecticides, preservatives, heavy metals, allergens and volatile organic compounds may contribute to loss of effectiveness in adult stem cells.

marrow or human stem cell transplantation is directly related to the conditioning regimen used, or is an extension of the immunoablation that occurs. It is not, in most instances, a process caused by the stem cell graft. Transplant conditioning regimens generally cause a wide range of toxicities that differ based on the regimen used. They range from minor complications such as nausea, vomiting and skin erythema to haemorrhagic cystitis and veno-occlusive disease leading to a hypercoagulable state (Kasper et al., 2004).

Human cord blood components contain haematopoietic progenitors, mesenchymal progenitors, endothelial cell progenitors and non-haematopoietic stem cells (Erices et al., 2000; Goodwin et al., 2001; Song et al., 2002). Some of these cells can be utilized in disorders that are classified as immune deficiencies. HUCB transplants can function as both reparative at the cellular or organ level, while subsequently improving and restoring function (Abbatista et al., 2004; Humes et al., 2004; Ma et al., 2005). Expectations for DNA repair, such as histone methylation and chromatin acetylation, have clinical implications for patients with neoplastic disorders (Ryan et al., 1986; Worm et al., 2002; Szyf, 2003, 2005; Szyf et al., 2004; Madhusudan et al., 2005).

Infections resulting from total ablation of the immune system are also a serious complication post-transplantation. Most patients, soon after transplantation, are severely neutropenic and are at risk of developing bacterial and fungal infections. The patient will continue to be at risk of developing such infections until 3 months post-transplant (Kasper et al., 2004).

At present, bone marrow and or human cord blood transplants are accepted practice for haematological malignancies. Typically, they are performed after myeloablative techniques have been employed (Gluckman, 1995, 1996, 2000; Michallet et al.,, 2001; Barker et al., 2004; Koh et al., 2004). Haematopoietic transplants can improve chemotherapy-induced anaemias and bone marrow arrest, as well as stimulate the white blood cell repertoire (Bishop, 1997). Tumour reduction strategies, coupled with enhancement of white blood cell lineages, also improve long-term survival in patients with solid tumours (Ende et al., 2006). In addition, reduction in common adverse side effects such as mucositis, with its subsequent nutritional deficiencies, also improves with the combination of stem cell transplantation and aggressive nutritional repletion (Anderson et al., 1998; Peterson, 2006; Ucuncu et al., 2006). In addition, autoimmune disorders are amenable to HUCB transplantation. These include patients with amyotrophic lateral sclerosis (ALS), multiple sclerosis, rheumatoid arthritis, systemic lupus erythematous and diabetes mellitus (Ende et al., 2000, 2004; Alaez et al., 2005). Miscellaneous applications include cerebrovascular accidents, as demonstrated by the work of Dr Paul Sandberg at University of South Florida (Newman et al., 2005). There are also some early studies utilizing HUCB for cardiac, urological and certain infectious diseases (Ringe et al., 2002; Abbattsista et al., 2004; Humes et al., 2004; Ma et al., 2005). Drawbacks of human cord blood are the relatively low total nucleated cell counts and mononucleated cell counts. In addition, finding an appropriate human leukocyte antigen (HLA) match is difficult, particularly in minority groups (Beatty et al., 1995). Although reconstitution of the entire immune system requires large quantities of cells (1.2−4.9 × 107 per kg of nucleated cells) (Sanz et al., 2001), with transplantations not requiring total immune reconstitution, the requirement would be for much smaller quantities of these cells. Typically, 85 ml of human cord blood contains between 4 and 36 × 106 CD34+ cells (Kogler et al., 1998). Most of the toxicity experienced by patients undergoing bone

The recent understanding that total myeloablation is not critical to the achievement of engraftment has resulted in a switch to the less intensive and safer non-myeloablative regimens. Such regimens may include the minimum to achieve engraftment, such as fludarabine plus 200 cGY total body irradiation, or those of higher intensity such as fludarabine and melphalan (Kasper et al., 2004). By utilizing the same agents as used in the myeloablative model, although at a reduced dose, the potential for similar toxicity still remains. Graft-versus-host disease (GVHD), a result of allogeneic T cells transferred with the donor stem cell inoculum, is often encountered in allogeneic transplantations utilizing the conventional transplant methods. Reduction or a total cessation of GVHD would be a priority in any new transplant preparative regimen and procedure. In general, a cord blood unit that is at least a 4/6 HLA match with sufficient cells has a similar outcome in engraftment compared with matched bone marrow grafts (Juliet et al., 2001; Rocha et al., 2001). In this article, 27 ALS patients and two with solid tumours previously transplanted with HUCB, were reviewed. Different degrees of success, depending on the stage of the disease, were observed. The early data not only suggest safety but efficacy as well in several of these difficult (and in ALS, fatal) disorders (Tables 1 and 2). It has been shown that multiple units of mismatched umbilical cord blood can safely be transplanted into patients without the typical side effects associated with myeloablative and immunosuppressive techniques (Tables 1 and 3). Recently, an article demonstrated that the number of disparities in an unrelated cord blood used in transplants was not directly associated with an increase in graft-versus-host disease, especially grades 2−4 (Rocha et al., 2004; Kleen et al., 2005). Graft T cell reduction techniques were used so that aggressive leukocyte subsets would not be present, therefore reducing the probability for graft-versus-host disease. In order to take advantage of all the potential applications that HUCB offers, it is necessary to develop and establish the most efficacious transplantation methods while preserving and improving safety margins. Transplant procedures have varied little in the past 20 years. Reduced intensity stem cell transplants (RIST) still utilize total

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Article - Uses for stem cells derived from umbilical cord blood - MJ Ghen et al.

Table 1. Parameters measuring safety. Statistical analysis included Pearson correlation and tests for two paired samples for mean comparison. Statistical significance was set at α = 0.05. Safety parameters such as systolic, diastolic, temperature, pulse and respiration were two-tailed. All other tests were one-tailed. ALT = alanine transaminase; AST = aspartate transaminase; BUN = blood urea nitrogen. Measures Vital signs (n = 29) Systolic (mm HG) Diastolic (mm HG) Temperature (°F) Pulse (per min) Respiration (per min) Haemoglobin (g/dl; n = 19) Haematocrit (%; n = 19) Total bilirubin (mg/dl; n = 14) Hepatic function (n = 16) ALT (IU/l) AST (IU/l) Renal function (n = 18) BUN (mg/dl) Creatinine (mg/dl) Skin rash (n = 29) Haematuria (n = 29) Proteinuria (n = 29)

t

P-value

127.1 (19.6) 82.9 (12.8) 97.2 (1.1) 74.1 (10.1) 16.0 (1.1) 14.4 (2.3) 42.9 (5.3) 0.6 (0.3)

2.04 2.04 2.04 2.04 2.04 1.73 1.73 1.77

0.007 0.02 0.0006 0.01 <0.0009 0.001 0.006 0.04

50.1 (38.9) 34.9 (22.8)

48.3 (24.4) 30.6 (12.0)

1.76 1.75

0.04 0.02

14.2 (3.6) 0.8 (0.2) Negative Negative Negative

14.7 (3.2) 0.9 (0.7) Negative 27% (Transient <12 h) Negative

1.73 1.74

0.02 0.04

Mean (SD) Pretransplantation

Post-transplantation

120.3 (17.7) 79.9 (11.4) 96.7 (1.0) 73.1 (12.1) 15.4 (1.4) 14.1 (2.5) 43.2 (4.5) 0.6 (0.2)

Table 2. Karnofsky performance scale (KPS) and forced vital capacity (FVC%) values. Measures

Mean (SD) Pretransplant value

Post-transplant values

t

P-value

KPS (n = 29) FVC% (n = 7)

51.0 (12.6) 42.9 (30.6)

54.8 (10.2) 48 (21.9)

1.70 1.94

0.03 0.02

Table 3. Cord blood characteristics (n = 21).

TNCa MNCa Viabilityb No. of cord blood units transfused HLA type

Mean

SD

1.08 × 1010 6.28 × 109 92.9 19.8 Mismatch

1.40 × 109 8.36 × 108 1.5 1.1

TNC = total nucleated cells; MNC = mononucleated cells; HLA = human leukocyte antigen. a Total number of cells transfused per patient. b Prior to cryopreservation.

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Article - Uses for stem cells derived from umbilical cord blood - MJ Ghen et al. body irradiation and chemotherapeutic agents. A critical review of transplant variables could potentially lead to an improved methodology that will advance engraftment outcomes and safety.

Location of the transplant centre may play an important part in the outcome. Patients should be hospitalized prior to, during and for an initial period post-transplant. Reverse isolation procedures must be strictly adhered to.

Transplantation and clinical observations

Classic screening procedures of both maternal and infant cord blood maintain safety margins. All procedures as related to extraction, differentiation, expansion and production of stem cells used as a donor, should be performed in a laboratory with conditions that can safely produce a pharmaceutical grade product.

The team identified many factors that when addressed simultaneously improved engraftment. The key is to ensure that the transplant in general is without side effects and eventually elicits a positive change in the patients’ morbidity and mortality, while always respecting risk versus benefit ratios. The main issue one must be concerned with when considering transplantation using HUCB, is the overall safety of the procedure in question. The typical conventional preparation, which includes total body irradiation and myeloablation with chemotherapy, has been shown to warrant serious concern (Couriel et al., 2006; Paveletic et al., 2006; Shulman et al., 2006). New techniques, such as reduced intensity stem cell transplants (RIST) using lower doses of total body irradiation and conditioning regimens and considered less toxic, have been more helpful (Del Toro et al., 2004). However, graftversus-host disorder is promoted by conditioning regimens that ultimately damage host tissues, which includes the intestinal mucosa (Couriel et al., 2006; Kandabashi et al., 2006; Paveletic et al., 2006; Shulman et al., 2006). Up-regulation of adhesion molecules and major histocompatibility complexes enhances the recognition of the host major and minor antigens by donor T cells of the graft (Eyrich et al., 2005). During phase II, when the donor T cells enter the host system and traverse the host’s vascular system, alloantigens prime the cells in the vascular network to secrete interleukin-2 and interferon-γ (Iwasaki, 2004). These type 1 cytokines are mediators of acute graft-versus-host disease. Subsequent cytotoxic lymphocytes (CTL) cause the majority of damage that is seen with acute graft versus host disorder. Of interest, even when there is not full reconstitution of the immune system and there is only microchimerism (as defined by <1% in the peripheral blood), the cytotoxic lymphocytes are maintained in a state of hypo responsiveness (Ichinohe et al., 2005). The case for immunosuppression also needs to be studied carefully. Traditionally, long-term immunosuppressive agents are utilized during and for a period after a transplant. The presence of these agents, of course, lends itself to a more compromised host with a subsequent increase in infectious diseases (Goebel et al., 2005; Narimatsu et al., 2005) and an increased nidus for neoplastic genesis (Swinnen et al., 1990; McCann, 2003; Durando et al., 2005). It has been found that short-term immunosuppression (from day −1 to day +30) is sufficient for obtaining acceptable levels of chimeras. This procedure reduces unnecessary risk to the patient. Careful choice of the appropriate candidate for cellular transplant is important. Strict attention must be paid to both inclusion and exclusion criteria. Patients with compromised cardiac, renal and or hepatic dysfunction would make poor candidates for cellular transplant. In addition, it has been shown that candidates with poor nutritional status should also be considered as inappropriate for transplant. However, in the latter, aggressive repletion prior to day 0 may allow some of these candidates to undergo successful transplant.

Engraftment improving procedures must also be examined critically. Since the present technique does not include myeloablation, procedures are instituted to improve engraftment and outcome. It has been demonstrated that by increasing the number of cells in the graft, while preparing the graft with certain factors, improves the outcome. These factors may include neurotrophic substances such as brain-derived neurotrophic factor, heat shock protein 90, neurotrophin, nerve growth factor, vascular endothelial growth factor, epidermal growth factor and insulin-like growth factor (Kofidis et al., 2004; Namura et al., 2005). Even signalling chemical messengers, such as stromal cell-derived factor-1 (SDF 1) released as a consequence of total body irradiation or chemotherapeutic agents, can also be used to improve engraftment (Glimm et al., 2002). It is advocated that tandem and serial grafts, utilizing the same cell culture batch, can provide the additional necessary number of stem cells for appropriate engraftment (Slatter et al., 2005). Age and microenvironment considerations are also subject to examinations. The matrix upon which cells reside, influences the cellular response and defines a direct effect on the stem cell differentiation (Calvi et al., 2003; Wynn et al., 2004; Zhu et al., 2004; Adams et al., 2005; Heissig et al.,, 2005; Stier et al., 2005; Stein et al., 2005). Therefore, meticulous attention to matrix health pre-, peri- and post-operatively can make the difference for cellular homing after differentiation. As well, improved signalling mechanisms support engraftment. Findings show chemokines, inflammation and hypoxia play a pivotal role in stem cell signalling (Imitola et al., 2004; Mueller et al.,, 2005; Fryer et al., 2006). Timing in the use of these parameters in the patient undergoing transplant is extremely important. For example, it is believed that in certain instances it may be appropriate to allow innate inflammatory mechanisms to go unchecked, while prior to transplant and at some time post-transplantation, using agents that are anti-inflammatory. A recent study has examined the use of hyperbaric oxygen in the release of peripheral stem cells. It maintains that there is an added utility in using hyperbaric oxygen at some time posttransplant to improve release of the implanted stem cells (Thom et al., 2006). Aggressive attention to cellular mitochondrial function issues is equally important. Agents that either decrease cellular toxicity allowing for improved mitochondrial function or agents that stimulate mitochondrial function are supportive to the outcome (Wallace, 1999; Rousset et al., 2004; Beal, 2005; Liu et al., 2005; Linford et al., 2006). The synthetic or natural compounds that are used improve the intracellular milieu promoting optimal cellular function. Molecular cross-talk is enhanced by hormonal considerations and the appropriate hormones should be simultaneously replete in these individuals, especially

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Article - Uses for stem cells derived from umbilical cord blood - MJ Ghen et al. if deficiencies have been identified. There are even times when a blockade of certain hormones may be advantageous. For example, discussion recently has ensued considering sex hormone blockade to be used in the prevention of thymus gland atrophy (Marchetti et al., 1989; Bodey et al., 1997). Minimizing physical barriers to the engrafting material, allowing it to become more available, is also important. For example, using mannitol will decrease the blood−brain barrier’s capacity for preventing the excursion of the stem cells into the brain (Borlongan et al., 2004). In addition, new research demonstrating that dendritic cells require serotonin for presentation to T cells becomes increasingly important, considering the large population of patients that currently ingest selective serotonin re-uptake inhibitors on a daily basis (O’Connell et al., 2005). Medications that the patients presently take, or are prescribed as part of the procedure, must be carefully evaluated in light of the physiological responses that could alter either the stem cell engraftment or the subsequent cellular differentiation. Narrow-based transplants should be replaced with broaderbased stem cell transplantation. Clearly, studies support the fact that transplants performed with CD34 cells alone often are accompanied by a high rate of infections due to the delay of CD4 and CD8 subsets returning to adequate levels for up to 12 months (Peggs, 2004). In addition, mixed stem cell lineages may be required for transplant in the absence of the ability to use embryonic or embryonic like precursors. In many diseases, the presentation has several distinct aberrations. ALS has components of both autoimmune dysfunction as well as neurological considerations being an anterior horn cell disease (Smith et al., 1992). Therefore, the use of haematopoietic stem cell progenitors along with neuronal progenitors may be a far better graft than either one alone. When appropriate, increasing cellular elements will aid in engraftment. T regulatory cells best characterized in mice, comprise only 5−10% of the T cell population and do not fit into either T helper 1 (TH1) or TH2 morphology (Godfrey et al., 2005). This important group of cells that express CD25, act as a vital immunosuppressive agent via IL-10 and transforming growth factor β (TGFβ) (Kriegel et al., 2006; Taylor et al., 2006). Therefore, in the appropriate disorders, the use of an agent or agents that will increase T regulatory cells would be important for short-term immunosuppression and long-term success. It would be best to utilize an agent that has the greatest margin of safety. New research in the animal model has demonstrated that high-dose 1,25 di-hydroxy vitamin D3 is an appropriate choice (Wood, 2003).

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Maintaining the stem cell niche and providing a healthy thymic microenvironment will also improve the success for active chimerism. Control over sympathetic nervous system signalling will also eventually improve stem cell release into the periphery (Katayama et al., 2006). Sympathomimetic agents may be considered in the future as part of the armamentarium that transplant physicians will use. Environmental inputs such as diet, air and water quality, patient toxic load and psychosocial issues may become important factors in whether or not the patient’s ultimate prognosis is favourable. Increasing insulin resistance and metabolic syndrome noted in large populations within Western cultures, have increased

release of chemicals that alter the normal cellular chemistry (Einstein et al., 2005; Maeda et al., 2005). In addition to sodium and potassium altered distributions, plasminogenactivating inhibitor is also increased (Smith, 2003). Therefore, increased inflammation, which could misdirect donor grafts are a potential for outcome failure. Toxic loads with heavy metals, such as mercury, aluminium, platinum, cadmium and lead, interfere with normal cell function (Choi et al., 1978; Hamada et al., 1998; Suarez-Fernandez et al., 1999; Lacson et al., 2002; El-Demerdash et al., 2004; Jurasovic et al. 2004; Ali et al., 2006; Nersesyan et al., 2006). Insecticides, herbicides, pesticides and simple foods, such as trans fatty acids, not only inhibit normal functioning cellular mechanisms but also cellular membrane morphology, with subsequent negative physiological changes (Ostlund-Lindquist et al., 1985; Amr et al., 1993; Daniel et al., 1995). Genetic factors will also play a role in successful transplantation. This role may be overstated, and can be compensated by improved biochemical balancing through either medication or amino acid and nutritional precursors. A recent article looked at a large pooled cohort of mono and dizygotic twins and the incidence of development of cancer. Familial factors accounted for 27−42% of the incidence of colorectal, breast and prostate cancer in the twins studied; however, non-shared environmental inputs as listed above were responsible for 58−73% (Lichtenstein et al., 2000). This again underscores the scope of environmental factors that play a role in stem cell transplantation. The site used for transplantation, whether peripheral, intra -bone marrow, or intrathecal, may affect the short-term as well as long-term engraftment. The contention is that in cases requiring immune system repletion, the intra-bone marrow injection technique may offer distinct advantages over others. In cases of neurological disorders or those demonstrating central nervous system pathology, both peripheral infusion technique and direct intrathecal injection, combined with agents that reduce the blood−brain barrier interference, may improve outcome. Integrity of the MALT (mucus-associated lymph tissue) and subsequently the gut-associated lymphoid tissue is an important component in the patients continued health status post transplantation. Portal circulation between the gut and liver typically detoxifies nutrients, toxins and allergens to a weight of 1538 lb or 700 kg per year (Vasquez, 2005). With more than 500 species of bacteria and yeast residing in the gut and the already known importance of the lymphoid tissue present in the Peyer’s patches, attention to optimizing mucosal integrity is imperative (Moise Jr, 2005). The patient’s overall immunological response, including negative responses such as graft-versus-host disease, as described above, are often mediated in the gut. Agents often employed in improving this segment of the immune system include, vitamin A, L-glutamine and Saccharomyces boulardii (increasing immunoglobulin A production) (van der Hulst et al., 1996; Elitsur et al., 1997; Calder and Yagoob, 1999; Johansson-Lindbom et al., 2000; Quadro et al., 2000; Rodrigues et al., 2000; Ziegler, 2002). Lastly, particular attention to thymic function at the appropriate time can make the difference in a successful formation of chimeras (Shizuru et al., 2000; Talvernsaari et

Article - Uses for stem cells derived from umbilical cord blood - MJ Ghen et al. al., 2002). Typical atrophy of the thymus, as expected with age, has to be combated with agents that can demonstrate hypertrophic effects and subsequent functional improvement. For example, studies have suggested that human growth hormone can enlarge an already atrophied thymus (Savino et al., 1995, 2002, 2003). Pre- and post-ultrasonography of the thymus may demonstrate which agent or combination of agents is most effective. Experience with transplantation, and the results derived, has led the authors to the conclusion that there are safer alternatives than the standard myeloablative procedures. A retrospective review of 29 patients who received 20 units of HLA mismatched human umbilical cord blood has been undertaken. All patients had signed an informed consent for the treatment. In all cases ABO/RH was respected in the donor units. Twenty-seven patients had ALS with a median onset of 2 years prior to diagnosis. Ten patients (37%) had bulbar onset and 17 (63%) had limb onset. Two patients had cancer, one with metastatic breast cancer and the other with metastatic colon cancer. Demographics of the files reviewed revealed eight women (28%) and 21 men (72%), with a median age of 49 years (Table 4). All patients received aggressive preoperative nutritional support, which was maintained posttransplant with oral repletion. Prior to the infusion of the cord blood, patients were given oral medications to reduce any potential anaphylactic or allergic reactions. Pre-operative history and physical, laboratory evaluations, pulmonary function tests and muscle testing were included and obtained prior to the procedure. Post-transplant testing prescriptions for pulmonary and muscle testing were given to all patients but due to the widespread geographical diversity amongst the patients, collection of data was often impaired. The total number of units were transfused peripherally on three separate occasions, i.e. 3 days in a row. Close monitoring for transfusion reactions and possible acute graft versus host disorder were also monitored carefully. During the procedure, vital signs were monitored every 15 min. Table 3 shows the mean values of the cord blood transfused. Most patients received 20 units of mismatched cord blood. A decrease in the mixed lymphocyte culture reaction was obtained by a method as described by Ende et al. (2000). Total nucleated cell (TNC) counts were averaged at 1.08 × 1010 and mononucleated cells at 6.28 × 109, these counts reflect the total number of cells transfused per patient. No direct CD34+ counts were performed; however, by extrapolation (0.38 ± 0.2% of TNC) (Kogler et al., 1998), the estimated average given to each patient was 4.1 × 107. Data obtained from this retrospective review of mismatched human umbilical cord blood transplantation clearly suggest that safety can be maintained in such procedures (Table 1). Observations of the parameters measuring safety, such as vital signs, liver function (alanine transaminase P = 0.04 and aspartate transaminase P = 0.02), renal function (blood urea nitrogen P = 0.02 and creatinine P = 0.04), urinalysis and complete blood count, support this claim. Safety was further demonstrated by the absence of graft versus host disease post-transplant supported by the absence of skin rash and the fall in liver enzymes. Haematuria, the most common side effect observed in eight (28%) patients, was transient (12 h)

and was resolved by the administration of intravenous fluids. There was no evidence of other transfusion reactions such as fever and proteinuria. No serious adverse event was reported. Additionally, pulmonary, hepatic and infectious complications often observed after conventional haematopoietic transplantation were not seen (Lambertenghi Deliliers et al., 2000; Parimon et al., 2005; van Tol et al., 2005). There was an overall improvement in pulmonary function as measured by FVC (forced vital capacity) (P = 0.02) and over all wellbeing as measured by Karnofsky performance scale (P = 0.03) as indicated in Table 2. Karnofsky scores were reported on an average 6 months post-treatment date. To find out if this overall improvement was consistent in both bulbar and limb onset ALS, the Karnofsky performance scale was analysed in these two groups of patients, as shown in Tables 5 and 6 respectively, and Figure 1. The bulbar group had a substantial improvement over the pretransplant values (P = 0.02) (Figure 2a). On the other hand, there was no improvement in the Karnofsky mean value of the limb onset group and in fact there is a slight decrease in Karnofsky mean values (Figure 2b). Even though there was a slight decrease in the limb onset group, this compares favourably to the natural progression of the disease. It is believed that this treatment has a significant impact on the disease progression (P = 0.02) in this group. Unfortunately, due to geographical limitations, serial transplantation could not be performed in these patients. It is contended that due to the nature of ALS, subsequent transplantation is extremely important. It is also believed that the earlier the intervention into neurodegenerative disorders, the more likely is the presence of either inflammation or other signalling mechanisms to enhance a positive outcome. These signalling mechanisms would improve the stem cells homing to the areas requiring repair and regeneration. The introduction of improved culture techniques and/or cytokines and chemokines may eventually reduce the requirement for large quantities of cord blood units (Edwards, 2004). Extraction of the necessary cell lines with appropriate expansion of these lines should provide the necessary quantity of appropriate stem cells for adequate transplantation. Maintaining some of the graft stem cells in cryopreservation will allow for future expansion and transplants as required.

Table 4. Patient characteristics. Characteristic

Patients (n = 29)

Median age at the time of treatment (years) Gender (%) Male Female Median time with disease (years) ALS bulbar onset n = 10 (%) ALS limb onset n = 17 (%)

46

ALS = amyotrophic lateral sclerosis.

72 28 2 37 63

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Article - Uses for stem cells derived from umbilical cord blood - MJ Ghen et al.

Table 5. Bulbar onset (n = 10), Karnofsky performance scale.

Table 6. Limb onset (n = 17), Karnofsky performance scale.

Patient ID

Pretransplant values

Posttransplant values

Patient ID

Pretransplant values

Posttransplant values

1 3 5 8 12 13 17 19 23 24

60 30 60 40 50 50 30 60 60 60

70 50 60 50 50 50 40 60 60 70

Mean SD

50.0 12.5

56.0 9.7

2 4 6 7 9 10 11 14 15 16 20 21 22 25 26 28 29

50 30 50 40 50 40 50 50 50 70 60 70 40 60 60 30 70

40 50 50 50 50 50 50 50 50 70 60 70 40 50 60 40 60

Mean SD

55.5 12.9

54.5 10.4

t = 1.83; P = 0.02.

t = 1.75; P = 0.02.

Figure 1. Combined bulbar and limb onset amyotrophic lateral sclerosis (ALS); Karnofsky performance scale.

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a

b

Figure 2. (a) Bulbar onset of ALS (n = 10); Karnofsky performance scale. (b) Limb onset of ALS (n = 17); Karnofsky performance scale.

Conclusions

Acknowledgements

Umbilical cord blood transplantation (UCBT) is an expanding field for both paediatric and adult patients. Rapid availability, low risk of infectious disease transmission, lower risk of graft-versus-host disease, lack of risk for the donor and immune tolerance allowing successful transplantation despite HLA disparity, makes UCB an attractive alternative source of haematopoietic stem cells for transplantation. UCB will address needs in both transplantation and regenerative medicine fields. Observations suggest that UCB from unrelated donors is a feasible alternative source of stem cells for transplantation, resulting in durable although delayed haematopoietic reconstitution. It has distinct advantages over adult stem cells as previously discussed, as they must be HLA matched prior to transplantation.

The authors thank Ralph Dittman MD for his support and proofreading of the manuscript. They also wish to thank Johnny Thatil for his assistance with the research.

Experience has demonstrated that the use of HUCB in ALS, and some solid tumour cancers, is safe and is often efficacious. An obvious difference was seen in the ALS subgroups with patients with bulbar onset receiving a greater positive effect. Improvements in Karnofsky scale varied widely from patient to patient. These changes included improved muscle strength, pulmonary function and improved activity of daily living. Although the findings reported are only based on approximately 6 months follow-up, it suggests that HUCB transplant holds a significant potential for a disease that up to the present time has had no effective treatment. For unknown reasons, some patients achieved remarkable improvements in several parameters, including an improved ability to smile, speak, move previously paralysed limbs and the ability to selftoilet. One patient (ID3) sustained all of these improvements for greater than 18 months following the transplant. The greater the improvement in Karnofsky scores, the longer the received benefits are likely to be sustained. At 1 year posttransplant, 18 (62%) of the patients reviewed were still alive. The rest were lost to follow-up. Several factors have been identified which influence transplantation and optimize the chance of early and long-lasting engraftment. These factors minimize the risk of GVHD and transplant related deaths. A clinical trial is now warranted to fully explore the efficacy of umbilical cord blood transplantation using these methods.

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Paper based on contribution presented at the PGDIS Annual Meeting ‘Nuclear transfer and reprogramming’ in Belize, Central America, February 2–5, 2006. Received 7 April 2006; refereed 23 May 2006; accepted 23 June 2006.