Stem Cell Through Present and Future - medIND

Special Article

Stem Cell Through Present and Future Vijay K. Sharma1, Utpal K. Singh3, Rajniti Prasad2 and Sophie Fletcher1 1

Dewsbury District Hospital, Dewbury, UK, 2Institute of Medical Sciences, BHU, Varanasi, 3Nalanda Medical College, Patna, India

ABSTRACT Stem cell transplantation (SCT) has the potential to transform the lives of children with a wide variety of genetic diseases, ranging from inherent defects of hemopoietic cell production or function through to metabolic diseases mostly affecting solid organs. For these children life expectancy or quality of life would otherwise be very poor.1 It ranks as one of the most remarkable therapeutic advances of the past 40 years. Despite rapid technological improvements, however, there are still many short term risks and potential long term toxicities. Consequently, the rapid emergence of alternative therapies (including new drugs, enzyme and gene therapies), necessitate constant re-evaluation of the risk/benefit ratio for each disease and hence the appropriateness of SCT. This review describes the major aspects of the transplant process, indications for transplantation, outcome statistics, and areas where alternative therapies are becoming available. SCT remains a highly experimental therapy. Due to the relatively short history of the discipline no data exists on truly long term follow up. This is important as some organs benefit relatively poorly or problems may emerge which were never apparent as part of the untreated disease. The speed of technological change makes randomised trials on these diseases, which are individually quite rare, almost impossible to perform. [Indian J Pediatr 2009; 76 (1) : 51-56] E-Mail: [email protected] Key words : Stem cell transplantation; Genetic diseases; Inborn; Severe combined immunodeficiency; Metabolism; Inborn errors; Chimaerism

What are stem cells? Stem cells are unique, they have three basic characteristics; Differentiation; they are still at an early stage of development and retain the potential to turn into many different types of cell; they are also capable of self-renewal and proliferation. Theoretically, it should be possible to use stem cells to generate healthy tissue to replace that either damaged by trauma, or compromised by disease. Some conditions which scientists believe may eventually be treated by stem cell therapy are given in table 1. Progress in the field of bone marrow stem cell plasticity must be made on multiple fronts simultaneously. To discern the mechanisms underlying plasticity, we must optimise and standardise the experimental approaches used so that the data obtained are as reproducible and definitive as possible. By assessing the potential clinical benefits of stem cell administration in different disease models, we will gain insight not only into their therapeutic potential but also into the mechanisms by which plasticity occurs. Indications of HSCT

Abbreviations GVHD: Graft Versus Host Disease; MSD: Matched Sibling Donor; MUD: Matched Unrelated Donor; PCR: Polymerase Chain Reaction; SCID: Severe Combined Immunodeficiency; SCT: Stem Cell Transplantation; TCD: T-Cell Depletion; RIC: Reduced Intensity Conditioning

• By directly replacing diseased marrow or blood cells operating within the blood system; Thalassemia major, sickle cell anaemia, severe combined immunodeficiency disorder and bone marrow failure syndromes. • By replacing phagocytic cells of the monocyte/ macrophage lineage which operate in solid organs; Osteoclasts in osteopetrosis, macrophages and histiocytes in haemophagocytic conditions. • By acting as a source of indwelling enzyme therapy in metabolic diseases. Haemopoetic stem cell transplantation is already established in a variety of indications see table 2.

SCT acts in one of three ways in treating genetic diseases: Correspondence and Reprint requests : Dr Utpal Kant Singh 8, Rajender Nagar, Patna - 800016, India Ph: +91-9431012727 [Received November 12, 2007; Accepted March 25, 2008]

Indian Journal of Pediatrics, Volume 76—January, 2009

TYPES OF STEM CELLS There are different types of stem cell. The most useful 51

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V.K. Sharma et al TABLE 1. Indications of Hematopoietic Stem Cell Transplantation Being Used for “Repair” Indication

Current status

Post myocardial infarction-repair of heart muscle

Early evidence for autologous stem cell transplantation.2Cells would be inserted via an angioplasty catheter. Experimental stage. Stem cells have been used to produce comparable hepatocytes. Has not been peer reviewed.3 Experimental stage. Early research has shown potential in delaying onset of disease, growing new nerve endings and slower disease progression. More work needs to be done.4 Experimental stage. Early animal studies suggest that some diseases can be cured by autologous or allogeneic transplantations.5 Autologous stem cell transplanatation shows promise for the future. Autologous stem cell transplantation has been used in over 40 children.

Production of liver cells- hepatocytes. Motor Neurone Disease

Autoimmune Disease and Arthritis Cartilage growth for transplant in osteoporosis Juvenile Idiopathic arthritis. Shows promise. More work needed.6 Muscular dystrophy

Experimental stage. Shows promise.7

T ABLE 2. Current Indications of Hempoietic Stem Cell Transplantation Autologous Transplantation Indication Current Status Malignant Multiple myeloma Non-Hodgkin lymphoma Neuroblastoma Hodgkin disease Non-malignant

In use In use In use In use Non-malignant

Autoimmune Diseases

Experimental

Allogeneic Transplantation Current status

Indication Malignant Chronic myeloid leukaemia Multiple myeloma Acute myeloblastic leukaemia Acute lymphoblastic leukaemia Haemoglobinopathies Thalassaemia major

Single largest indication for SCT among genetic diseases. In use. Concerns about the risk of developing solid tumours. In use In use rarely. Only severe SS anaemia in under 16 year olds with severe sickle complications.

Fanconi anaemia Severe aplastic anaemia Sickle cell anaemia Immunodeficiency Disorders Autoimmune disorders Wiskott-Aldrich syndrome Severe combined immunodeficiency (SCID) Lysosomal storage disorders

Type 1 Gauchers disease

Chronic Granulomatous Disease Inborn errors of metabolism

stem cells come from the tissue of embryos. These cells are totipotent, they are capable of giving rise to all the types of differentiated cell that are found in an organism. A single totipotent cell could, by division, reproduce a whole organism. Pluripotent cells on the other hand have the potential to give rise to virtually any foetal or adult cell e.g. bone marrow cells. However, alone they are unable to develop into a foetus or adult because they lack the potential to produce extraembryonic tissue, such as the placenta.

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In use Experimental In use 8 In use

Experimental stage In use. Best results in those transplanted before 5 years. In use with impressive results. Replacing bone marrow provides lifelong enzyme therapy. Donor cells penetrate the blood-brain barrier and differentiate into microglia, carrying enzyme into the CNS effectively. This mechanism cannot be used in x-linked adrenoleukodystrophy. Only those who develop complications or patients with type 3 disease stand to benefit from transplantation. In use In use

SOURCES OF CELLS Sources of stem cells and the age of the recipient and donor have an impact on the success of the stem cell transplant. Cells for clinical use may come from self (autologous) or from others (allogeneic), such as a related family member or an adult unrelated donor or an anonymously donated umbilical cord blood sample.9 Umbilical cord blood banks have been established which are either anonymous and publicly funded or Indian Journal of Pediatrics, Volume 76—January, 2009

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Stem Cell Transplantation: Present and Future TABLE 3. Sources of Stem Cells Source of Cells

Advantages

Disadvantages

Numbers Required

Umbilical

Ease of collection Availability No risk to donor Decreased risk of adverse side effects Low risk of acute and chronic graft versus host disease Adequate stem cell content Adequate progenitor cell content Fewer mature auto-reactive T cells. Good stem cell content

Low stem cell content Slow engraftment Higher primary graft failure

1 x 107 cells/kg

Bone Marrow

Peripheral Blood

High progenitor cell content Low risk of tumour cell contamination Fastest engraftment time Patient recovers more quickly

High risk of tumour cell contamination Close HLA matching required Average engraftment time Close HLA matching required

> 2x10 6 cells/kg

Highest risk of acute graft versus host disease

TABLE 4. Conditioning Therapy Myeloablative

Non-myeloablative

Aims

Eradicates the patient’s marrow. Creates space for the incoming donor cells to engraft. Suppresses rejection reactions Total-body irradiation (TBI) and cyclophosphamide or busulfan

Regime

Advantages

Kills residual cancer cells

Disadvantages

Aggressive. TBI can cause growth and endocrine problems, impaired neuropsychological function, organ damage and malignancy.10,13-15

run as a private business whereby individuals direct that umbilical cord blood from their children be cryopreserved for their subsequent use at a later time. A third situation has already developed whereby when a child has a genetic disease that is best treated with SCT, parents have undergone collection of oocytes, fertilization of the oocytes in the laboratory, typing of some of the early cells in the blastocysts, and selection of those embryos developing with an identical tissue type and without the underlying genetic disease. Then at the time of birth, umbilical cord blood is collected from the fully matched and otherwise genetically normal child and used for transplantation purposes in the child who needs new haematopoietic stem cells for disease correction.10 A Recent study suggests that stem cells taken from the fluid that fills the womb in pregnancy could be used to create new cells. US researchers have successfully extracted the amniotic cells from samples, and then grew them in a laboratory. In-utero transplantation has a niche role in immunodeficiency diseases.11 CD34 is a cell surface marker on haematopoietic Indian Journal of Pediatrics, Volume 76—January , 2009

Immunosuppression but not myeloablation. Less ablative drugs and more intense immunosuppression Rely on a graft-versus-tumour effect to kill tumour cells with donor T lymphocytes. Involves drugs such as fludarabine, melphalan, CAMPATH-1H anti-T-cell antibody and cuclosporin/mycophenolate mofetil Decreased acute and chronic toxicities. Can be used in patients with co-morbidities.

stem cells. There is a good correlation between the CD34 count in the peripheral blood and the number of cells harvested. It is currently being used to determine the appropriate time for collection of the stem cells. Conditioning Therapy Successful SCT relies on the use of pre-transplant “conditioning therapy” to both eradicate the patient’s marrow (“myeloablation”: creating space for the incoming donor cells to engraft) and to suppress rejection reactions. In malignant disease the most popular conditioning regime has been a combination of cyclophosphamide and total body irradiation (TBI). However, there is now an increasing trend to use “reduced intensity conditioning” (RIC) therapy, with less ablative drugs coupled to more intense immunosuppression. RIC transplants have transformed the management of some conditions and are now being much more widely used, especially in immune deficiencies. 12 It is possible that such transplants may completely supplant conventional SCT for genetic diseases, so reducing the risks and widening the potential indications for SCT. Busulfan can cause veno-occlusive disease of the liver, fits and infertility. 53

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V.K. Sharma et al TREATMENT RESULTS

Chimaerism

followed by spontaneous improvement of chimaerism to 100% donor. This implies a competitive advantage for the normal cells and may be an important clue as to whether gene therapy can be successful in these conditions in the future.

Donor/recipient chimaerism can now be monitored accurately and quickly in most patients by PCR methods. One of the most fascinating aspects of SCT is the level of donor chimaerism which can cure a given disease. 29 Partial chimaerism will cure most blood diseases, although the level required varies depending on the disease treated. For example in Fanconi anaemia and thalassaemia major; it is common to see mixed chimaerism in the early months after the transplant

Variable number tandem repeats (VNTR) and short tandem repeats (STR) along with cytogenetics and fluorescence in situ hybridisation (FISH) are techniques used for analysing chiamerism. Two-colour FISH analysis of sex chromosomes is a valuable tool for chimaerism monitoring. It can be used in sexmismatched allogeneic stem cell transplants to detect host and donor cells and to examine the chimaerism.30

Complications of SCT

Condition

Survival

Complications

Thalassaemia major

>90% event free in young children, for whom iron overload and HLA sensitisation have not become established.16-18

Graft failure, toxicity, iron overload. Recurrent venesection used to reduce iron overload. Non-sibling donation should only be used in exceptional circumstances.

Sickle cell anaemia

75-84% event-free at 6-11 years post-SCT 18 inexplicably several children who have rejected grafts have subsequently developed increased foetal haemoglobin levels which have rendered them symptom free. 77% three year survival with sustained engraftment for HLA matched donors for all patients transplanted from 1968 to 1999. 54% three year survival with sustained engraftment for mismatched transplants19 In some groups survival now exceeds 90% especially if transplantation occurs before 6 months of age. Haploidentical transplantation shows 78% long term survival.20 Best results if transplanted before five years of age. 22

Severe combined immunodeficiency disorder (SCID)

Wiskott-Aldrich Syndrome

Chronic Granulomatous Disease

Restricted to patients with significant complications.

Fanconi Anemia

Acceptable transplant results have only been achieved by marked reductions in doses of conditioning therapy.

Osteopetrosis (caused by defective formation or function of osteoclasts)18 Hemophagocytic disorders (mechanisms which control histiocytes are defective)19 Metabolic Diseases

SCT results in gradual replacement of cells and normalises bone density within 12 months of transplant.

Hurler syndrome

Sanfilippo syndrome

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Poorest results are seen in patients with absent T and B cells but reasonable natural killer cell function. Some subtypes have better cure rates than others, which will in turn allow better stratification of therapy.21

Selection of appropriate patients is difficult as early SCT is preferable but the risk of death with SCT means that conservative treatments are often tried first leaving SCT until an age when the results are less desirable. Prophylactic approaches are improving but many patients will develop serious infective complications. Transplantation must only be performed by expert physicians. Development of solid tumours. High risk of developing squamous carcinoma of the mouth or pharynx23

Transplantation even with donor cells at levels as low as 10-20% can re-impose immune control and hence cure the disease. Success determined by degree of tissue damage present at time of transplantation. Metachromatic leukodystrophy in infancy and type 2 Gauchers disease tend to do the least well. IQ can be stabilised and breathing difficulties, hepatosplenomegaly, facial appearance, corneal clouding and cardiac degeneration can be greatly ameliorated. Does not respond at all.24

Dysostosis multiplex remains a major factor. In order to preserve maximum mobility, many orthopaedic operations are required.


Stem Cell Transplantation: Present and Future In many matched transplants for immunodeficiency, little or no conditioning therapy has been used since graft rejection is rare due to lack of T-cell function. As a consequence, many patients engraft exclusively with donor T-cells but these coexist with progenitor cells, myeloid cells, and B-cells largely of recipient origin. Defective B-cell function means that patients often need intravenous immunoglobulin infusions for many years after transplantation. In metabolic diseases, controversy remains as to (a) whether homozygous donors with normal enzyme levels produce superior outcome over carrier siblings/other relatives (who usually have 50% normal enzyme levels), and (b) whether mixed chimaerism is important since this lowers the proportion of donor cells and so reduces the amount of enzyme available. New Directions Despite major advances in the applicability and safety of

SCT, substantial risks remain. It is therefore critical to restrict SCT to patients with life threatening disease or complications in whom alternative therapies are not available e.g., the use of enzyme therapies for patients with less aggressive forms of MPS and Gaucher’s disease.24 There have been advances in HLA typing which can now be performed using DNA-based methods as opposed to the serology HLA typing which was previously employed. Polymerase chain reaction (PCR) amplification of specific HLA genes from genomic DNA is used to accurately HLA type patients receiving allogeneic stem cell transplantations. Sequence-specific oligonucleotide probe hybridisation (SSOPH) has allowed identification of an HLA allele. This is an essential component of allogeneic stem cell transplantation and is critical for selecting an appropriate unrelated donor. Accurate matching by molecular methods is associated with reduced post-transplant complications such as graft rejection, and acute and chronic graft-versus-host

TABLE 5. Complications of SCT Complications Acute Mucositis Nausea and vomiting Infection-one of the greatest challenges of SCT25 Bacterial infection-common due to co-existent neutropenia and presence of indwelling central venous catheters Fungal-Candida, Aspergillus (due to inhalation of airborne spores) Viral-RSV, Influenza, parainfluenza Reactivation of viruses resident in the patient or donor e.g., CMV, adenovirus, post-transplant lymphoproliferative disease (PTLD) due to EBV

Primary graft rejection Acute Graft vs Host disease (GVHD). Affects the skin, gut or liver, manifesting as rashes, diarrhea and liver dysfunction in varying degree from mild and transient to therapy resistant and life threatening.28 Veno-occlusive Disease Transplantation related lung injury

Prevention

Nursing in isolation with positive pressure ventilation and HEPA filtration. Prophylactic therapy-aciclovir to prevent Herpes infections, fluconazole and itraconazole to prevent fungal infections. Ciprofloxacin to reduce gram negative bacterial infections and cotrimoxazole to combat Pneumocystis Viral reactivation can be monitored accurately in blood by PCR analyses, allowing careful pre-emptive use of toxic antiviral drugs. Ganciclovir or foscarnet-reduces frequency of CMV Anti-B-cell antibody Rituximab to prevent causes of PTLD Technological advances include specific selection of antiviral T cells but carry a low risk of producing GVHD.26 New antifungal drugs e.g., caspofungin, voriconazole and mobilisation of granulocytes from volunteer donors by G-CSF and dexamethasone. 27 Future strategies may rely on immunisation of donors against common viruses. Immunosuppressive drugs e.g. cyclosporin A, mycophenolate mofetil and tacrolimus are given to most patients for 3-12 months after SCT and then gradually withdrawn this is to prevent the allogeneic recognition of patient tissue by donor T cells Drugs that interrupt the coagulation cascade

Late Osteoporosis, Avascular necrosis Restrictive and chronic obstructive lung disease Hypothyroidism Impaired growth and development Infertility Cataracts, retinopathy, infectious retinitis, and hemorrhage. Secondary graft rejection Chronic graft versus host disease (GVHD) Resembles a severe autoimmune disease

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V.K. Sharma et al disease (GVHD). It is thought that also matching for natural killer cell immunoglobulin receptors (KIR) which recognise HLA-class I molecules may have an important role in haplo-identical or mismatched haemopoietic stem cell transplantation. Recent observations published in the New England Journal suggest that natural killer cells and CD4+, CD25+ T cells in the stem cell graft can prevent GVHD without preventing the graft antitumour activity in patients. This is promising and could reduce the morbidity and mortality due to acute GVHD. The study found that if patients were treated with non myeloablative conditioning therapy and administration of antithymocyte globulin prior to the transplantation the incidence of acute graft versus host disease was reduced.31 Graft cell dose is an important determinant of haematopoietic recovery and overall outcome following umbilical cord blood (UCB) transplantation. Previously the limited cell dose of single UCB units has been a major barrier to its more widespread use. However, it has been suggested that transplantation with two unrelated UCB units is feasible, safe and effective and can overcome the limitation of cell dose of single UCB units.32 REFERENCES 1. Steward CG. Stem cell transplantation for non-malignant disorders. Baillieres Best Pract Res Clin Haematol 2000; 13: 343–363. 2. Wollert KC, Drexler H. Clinical applications of stem cells for the heart. Circ Res 2005; 96: 151–163. 3. Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000; 6: 1229-1234. 4. Xu Leyan, Yan Jun, Chen David et al. Human Neural Stem Cell Grafts Ameliorate Motor Neuron Disease in SOD-1 Transgenic Rats. Transplantation 2006; 82 : 865-875. 5. de Kleer I, Vastert B, Klein M et al. Autologous stem cell transplantation for autoimmunity induces immunologic selftolerance by reprogramming autoreactive T cells and restoring the CD4+CD25+ immune regulatory network. Blood 2006 15; 107 : 1696-702. 6. Wulffraat NM, de Kleer IM, Prakken B et al. Refractory juvenile idiopathic arthritis: using autologous stem cell transplantation as a treatment strategy. Expert Rev Mol Med 2006; 8 : 1-11 7. Sampaolesi M, Blot S, D’Antona G Mesoangioblast et al. Stem cells ameliorate muscle function in dystrophic dogs. Nature 2006; 444 : 574-579. 8. Ravindranath Y, Chang M, Steuber CP et al. Pediatric Oncology Group (POG) studies of acute myeloid leukemia (AML): a review of four consecutive childhood AML trials conducted between 1981 and 2000. Leukemia 2005; 19 : 2101 –2116. 9. Thomas ED. Bone marrow transplantation: a review. Semin Hematol 1999; 36: 95-103. 10. Verlinsky Y, Rechitsky S, Schoolcraft W, Strom C, Kuliev A. Preimplantation diagnosis for Fanconi anemia combined with HLA matching. JAMA 2001; 285: 3130-3133. 11. Flake AW, Zanjani, eds. In utero hematopoietic stem cell transplantation: ontogenic opportunities and biologic barriers.

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Blood 1999; 94 : 2179-2191. 12. MacKintosh FR, Adlish J, Hall SW et al. Suppression of normal human hematopoiesis by cytomegalovirus in vitro. Exp Hematol 1993; 21: 243. 13. Young NS. Acquired aplastic anemia. Ann Intern Med 2002; 136 : 534-546. 14. Lum LG. The kinetics of immune reconstitution after human marrow transplantation. Blood 1987; 69 : 369-380. 15. Storek J, Ferrara S, Ku N, Giorgi JV, Champlin RE, Saxon A. B cell reconstitution after human bone marrow transplantation: recapitulation of ontogeny? Bone Marrow Transplant 1993; 12: 387-398. 16. Lucarelli G, Andreani M, Angelucci E. The cure of thalassemia by bone marrow transplantation. Blood Rev 2002; 16: 81–85. 17. Sevilla J, Fernandez-Plaza S, Diaz MA et al. Hematopoietic transplantation for bone marrow failure syndromes and thalassemia. Bone Marrow Transplant 2005; 35 : S17–S21. 18. Vassiliou G, Amrolia P, Roberts IA. Allogeneic transplantation for haemoglobinopathies. Best Pract Res Clin Haematol 2001; 14: 807-822. 19. Antoine C, Muller S, Cant A et al. Long-term survival and transplantation of haemopoietic stem cells for immunodeficiencies: report of the European experience 1968–99. Lancet 2003; 361 : 553-560. 20. Buckley RH, Schiff SE, Schiff RI et al. Hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency. N Engl J Med 1999; 340: 508-5016. 21. Buckley RH. Treatment options for genetically determined immunodeficiency. Lancet 2003; 361: 541-542. 22. Ozsahin H, Le Deist F, Benkerrou M et al. Bone marrow transplantation in 26 patients with Wiskott-Aldrich syndrome from a single center. J Pediatr 1996; 129 : 238-244. 23. Rosenberg PS, Socie G, Alter BP et al. Risk of head and neck squamous cell cancer and death in patients with Fanconi anemia who did and did not receive transplants. Blood 2005; 105 : 67–73. 24. Vellodi A. Lysosomal storage disorders. Br J Haematol 2005; 128: 413-431. 25. Foot ABM, Potter MN, Donaldson C et al. Immune reconstitution after BMT in children. Bone Marrow Transplant 1993; 11 : 7-13. 26. Kook H, Goldman F, Padley D et al. Reconstruction of the immune system after unrelated or partially matched T-celldepleted bone marrow transplantation in children: immunophenotypic analysis and factors affecting the speed of recovery. Blood 1996; 88 : 1089-1097. 27. Kook H, Goldman F, Giller R et al. Reconstruction of the immune system after unrelated or partially matched T-cell-depleted bone marrow transplantation in children: functional analyses of lymphocytes and correlation with immunophenotypic recovery following transplantation. Clin Diagn Lab Immunol 1997; 4 : 96103. 28. Hochberg EP, Chillemi AC, Wu CJ et al. Quantitation of T-cell neogenesis in vivo after allogeneic bone marrow transplantation in adults. Blood 2001; 98: 1116-1121. 29. Hayashi S, Peranteau WH, Shaaban AF, Flake AW. Complete allogeneic hematopoietic chimerism achieved by a combined strategy of in utero hematopoietic stem cell transplantation and postnatal donor lymphocyte infusion. Blood 2002; 100: 804 – 812. 30. Turkiewicz D et al. Monitoring of hematopoietic chimerism after sex-mismatched allogeneic stem cell transplantation (alloSCT) by dual-color FISH analysis of X and Y chromosomes. Leuk Res 2003; 27: 993-998 . 31. Robert Lowsky, MD et al. Protective Conditioning for Acute Graft-versus-Host Disease NEJM 2005; 353; 1321-1331. 32. Navneet S. Majhail et al. Double umbilical cord blood transplantation. Current Opinion in Immunology 2006; 18: 571-575.
