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Stem cell medicine: Umbilical cord blood

and its stem cell potential

Suzanne M. Watt

a,b,

*

, Marcela Contreras

a

aNational Blood Service, Oxford and Colindale, UK b

Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, UK

KEYWORDS Stem cells; Cord blood; Potential; Plasticity; Transplantation; Banking; Expansion; Myeloablation; Non-myeloablation; Definition

Summary The ultimate aim of stem cell research is to improve patient outcomes and quality of life, and/or to effect a cure for a variety of inherited or acquired diseases. Improved treatments rely on developments in stem cell therapies and the discovery of new therapeutic drugs that regulate stem cell functions. These complement each other for the repair, regeneration and replacement of damaged or defective tissues. Stem cells may be sourced or derived from blood and tissues postnatally (‘adult’ stem cells), from the fetus (fetal stem cells) or from the blastocyst in the developing embryo prior to implantation (embryonic stem cells), each forming a unique component of the revolution in stem cell research and therapies. This review will concentrate on recent developments in the use of haemopoietic stem cells from umbilical cord blood for the transplantation of patients with haematological disorders. It will conclude with a summary of the potential of other umbilical cord blood e derived stem cells for tissue repair or regeneration.

Ó 2005 Elsevier Ltd. All rights reserved.

Successful haemopoietic stem cell (HSC) thera-pies have been carried out for 30e40 years.1These provide curative therapies for patients with

hae-matological and non-haehae-matological disorders. Currently accepted indications are the acute and chronic myeloid and lymphocytic leukaemias, myelodysplastic syndrome, myeloma, Hodgkin’s and non-Hodgkin’s lymphoma, solid tumours (e.g. neuroblastoma, retinoblastoma), osteopetrosis, liposarcoma, bone marrow failure syndromes, haemoglobinopathies, severe combined immuno-deficiencies, inborn errors of metabolism and * Corresponding author. Stem Cell Laboratory, National Blood

Service e Oxford Centre, The John Radcliffe Hospital, Head-ington, Oxford OX3 9DU, UK. Tel.: C44 1865 447919; fax: C44 1865 764367.

E-mail address:suzanne.watt@nbs.nhs.uk(S.M. Watt).

1744-165X/$ - see front matterÓ 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.siny.2005.02.001

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autoimmune diseases.2 HSC transplantation permits the dose escalation of chemo- or radio-therapy in an effort to eradicate the underlying disease and effect a cure. Patients with lymphoma and myeloma often undergo autologous HSC transplantation as part of dose escalation therapy, whereas for patients with some leukaemias and a range of inherited and metabolic disorders, allogeneic HSC transplantation offers the best chance of cure.1,2

HSCs for transplantation may be sourced from umbilical cord blood (UCB) or bone marrow, or from mobilized peripheral blood (MPB). The choice of allogeneic donors is often restricted to one unique family (related) member or to an unrelated UCB or adult volunteer HSC donor, with the degree of human leukocyte antigen (HLA) matching being critical for rapid engraftment without excessive graft-versus-host disease (GvHD).3 Better engraftment outcomes are re-corded for HLA-matched, rather than mis-matched, patients.3 The National Blood Service, processes and stores cellular products for approx-imately 40% of HSC transplants in England, about 78% of these being autografts and about 22% allografts. In the latter case, 30% of the donors are unrelated to the patients receiving trans-plants. The National Blood Service also maintains the NHS Cord Blood Bank, which currently has over 7000 UCB units banked, HLA-typed, microbiologi-cally screened and ready for transplantation. The National Blood Service also manages and co-ordinates the British Bone Marrow Registry, which searches for and provides donors nationally and internationally.4

UCB HSCs have a particular advantage over other HSC sources because:

 they may be the only source of allogeneic HSCs available to patients with rare HLA types and hence to ethnic minorities, to siblings suffering from diagnosed haematological disor-ders and for urgent unrelated donor trans-plants3e11;

 they can be easily collected from autologous, or related and unrelated donors, tested, HLA-typed and banked for immediate use4,7;

 allogeneic HSC transplant recipients receiving HLA-mismatched allografts tolerate a higher level of HLA locus disparities in UCB than in bone marrow or MPB stem cell, grafts.5,12 In addition, the lessons learnt from research into the use of UCB for new stem cell therapies for cardiovascular, skeletal, bone, cartilage and other disorders13,14 will provide useful information for

related therapies using other adult, fetal and embryonic stem cells.

Haemopoietic stem cells: their

definition and distribution

The haemopoietic compartment comprises a hier-archy of cells with different potentials. At its apex are the HSCs, which are a rare heterogeneous population of immature haemopoietic precursor cells, occurring at a frequency of approximately 1 in 104e1 in 105cells postnatally. Their genetic and phenotypic heterogeneity resides in their different proliferative and differentiative abilities, and ac-tivation or cell cycle states. They are multipotent, with the ability to commit to one of 10 or 11 functional haemopoietic lineages. These HSCs, through their multipotent and long-term repopu-lating ability, are able to populate the whole haemopoietic system at least over an individual’s lifespan.13,14 They differ functionally from short-term repopulating ability cells, which promote multipotent haemopoietic reconstitution over a more limited time. HSCs have the potential to ‘self-renew’ to maintain their ‘stemness’ charac-teristics during cell division, while having a high potential for proliferation. UCB has a higher con-tent of primitive haemopoietic precursor cells than MPB and bone marrow, but fewer than earlier trimester fetal bone marrow, blood and liver.15 UCB and MPB haemopoietic precursors are slowly cycling, whereas those in fetal and adult bone marrow have a higher proportion in G2, M and S

phases.16

Human haemopoietic cells emerge from a com-mon haemopoietic and endothelial stem cell, the haemangioblast, which forms during the process of mesodermal commitment.17 They appear first in the extra-embryonic yolk sac blood islands at week 3 of gestation, and subsequently and indepen-dently 1 week later in the embryo proper from the ventral floor of the dorsal aorta and vitelline artery.17 Human HSCs migrate via the circulation to colonize the fetal liver, thymus, gastrointestinal tract and bone marrow, but may also develop in situ from haemogenic endothelium in the fetal tissues and possibly even in adult bone marrow.17 Circulating HSCs are more highly concentrated in fetal blood than in UCB at term,5,13e16 with the haemopoietic progenitor content of UCB being higher for higher birthweight children and for mothers with fewer previous live births, but de-creasing with inde-creasing gestational age at term.18 In the adult human, the bone marrow remains the major site of HSC production.13,14

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Surrogate assays in vivo as indicators

of human HSC function

The gold standard for defining the human HSC is its ability to reconstitute all haemopoietic lineages during a lifetime.13,14A number of model systems, either in vitro or xenogeneic animal models in vivo, are used as surrogates to estimate either directly or indirectly (by assuming that the ratio of the cells being assayed to the HSC content in the sample remains constant) long-term repopulating ability cell numbers in the human graft. The best available surrogate animal models include the transplantation of human HSCs into non-human primates,19fetal sheep in utero20and immunode-ficient mice.5

Due to costs, time constraints and ethical con-siderations, the murine model is more often used than the other model systems. In efforts to promote and improve human HSC engraftment, variations include engineering mice to provide a ‘humanized’ microenvironment by the transplantation of human tissues (e.g. fetal bone, liver, thymus), the co-transplantation of human interleukin (IL)-3-engi-neered stroma or mesenchymal stem cells (MSCs) with the human HSCs, the injection of human haemopoietic cytokines and the intrafemoral in-jection of the human HSCs.5,21 The number of human cells able to repopulate immunodeficient mice is higher in UCB than in MPB or adult bone marrow (approximately 1 in 106compared to 1 in 3e6 ! 106nucleated cells).5However, variations in HSC numbers are genetically defined, and this will ultimately determine the number of HSCs within an individual graft.22Significant improvements to the immunodeficient mouse assays based on the intra-hepatic injection of human CD34C

UCB cells into newborn Ragÿ/ÿgcÿ/ÿdouble knockout mice pro-vide models with a functioning human adaptive immune system,23,24which are likely to be of value

for further assessing HSC transplantation and newer stem cell and related therapies.

The in utero sheep xenograft model was initially developed using human fetal liver cells, which were injected into the fetal sheep just before or around day 65 of gestation, at the time of haemopoietic cell expansion and prior to the de-velopment of immunocompetence in the rec-ipent.5Human cells can be detected in the bone marrow, blood and other tissues of the trans-planted sheep for up to 4 years post-transplanta-tion. Variations of this model include the use of T-depleted or CD34/CD133-enriched human hae-mopoietic progenitor cells, and/or of human cyto-kines, and the co-transplantation of human bone

marrow stromal or mesenchymal cells with HSCs in order to improve human HSC engraftment.5,20

An alternative model in vivo is the non-human primate.19 Here, their genetic similarity and ex-tended lifespan make this the most appropriate model for in vivo human haemopoiesis. Compar-isons of the transplantation of similarly marked non-human primate cells into NOD/SCID mice and non-human primates have suggested that the former measure shorter-term engrafting cells.19,25 It is, however, difficult to compare these models as xenogeneic versus autologous transplants were carried out, and the cells used for transplantation were derived from non-human primate genetically marked and cultured, cytokine-primed adult bone marrow CD34C

cells.

Surrogate assays in vitro

Clonogenic assays in vitro are also used as surro-gates for determining the viability of haemopoietic precursors and predicting HSC numbers in the graft prior to and after cryopreservation. The most common in vitro assays are the clonogenic colony-forming unit granulocyteemacrophage (CFUeGM) and granulocyteeerythroidemacrophageemega-karyocyte assays.16Overall, UCB CFUs form larger colonies and have a higher secondary replating ability, and hence a higher proliferative ability, than those found in adult bone marrow or MPB stem cells, but less than in week 16e20 fetal bone marrow.16Other assays in vitro that identify more immature precursors include the long-term culture-initiating cell (LTC-IC), cobblestone area-forming cell (CAFC) and high proliferative potential colony-forming cell assays. These much more specialized assays are less often used in the clinical transplant setting than is the CFU assay.5,13,14The assumption is that the ratio of these in vitro precursors to HSCs remains constant in unmanipulated UCB, bone marrow and MPB stem cells and hence provides an indirect measure of HSC content.

Molecular phenotypes of HSCs: the

stem cell signature

A specific phenotype for the HSC has been difficult to define easily. A combination of markers (in-cluding their relative density) has been used to identify the human HSC, or at least a primitive haemopoietic precursor subset. This phenotype can, however, change depending on the activation

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status of the precursor cell population and on the source of cells, and does not provide information on the functional ability of these cells in vivo. Historically, the best defined surface marker is CD34,13,14,26 which is expressed on primitive and more mature haemopoietic precursors, as well as endothelial and stromal cells. In their steady state, human HSCs and their immediate progeny are often CD133C

and CD38ÿ, and lack many haemopoietic lineage-defined surface molecules, yet CD133 again is not a specific marker of HSCs.26e28They are present in CD34hicell subsets, although more primitive or less activated progen-itors may lack external CD34.27,28CD34hicells may be CD90lo, CD117lo, CD164C, CXCR-4C,

P-glyco-proteinC

or rhodamine-123lo and VEGFR-2C

.5,6,13,14,25e30The more primitive haemopoietic progenitors in UCB are HLA-DRC

, whereas those in adult bone marrow demonstrate low or negligible levels of HLA-DR.5 In the HSC transplant setting, CD34 is the marker often used to enumerate HSC content, yet by itself, it measures both immature and more mature haemopoietic precursors, and like the majority of markers analysed it is not specific for HSCs. More recently, HSCs have been shown to express aldehyde dehydrogenase, allow-ing their further enrichment.31

Hopes of defining ‘stemness’ characteristics or the stem cell signature of HSCs have been raised with the completion of the Human Genome Pro-ject; with new protocols for their enrichment and tracking; with the development of knockout and knock-down, database mining, gene array and proteomics technologies; and with newer animal models of disease. The salient points from gene and protein profiling studies and reviews5are that:

 stemness genes may be shared between HSCs and other stem cells;

 a significant proportion of genes identified in HSCs have unknown functions;

 most of the shared stemness genes are not exclusively expressed in stem cells;

 stemness genes include cell cycle regulators, transcription factors, signalling molecules, extra-cellular matrix receptors and stress-resistance and chromatin-remodeling molecules;

 the gene profiles of circulating HSCs may differ from those of HSCs resident in the bone marrow.

Thus, even with these more sophisticated tech-niques, it is likely to be the combination of genetic or cellular phenotypes that allows the HSC to be defined.

UCB banking and transplantation of

HSCs: history, current practice,

controversies and innovations

History and current practices

In 1988, Gluckman and colleagues demonstrated that the UCB contained sufficient HSCs for success-ful haematological reconstitution following trans-plantation into an HLA-identical child suffering from Fanconi’s anaemia.32The transplanted child remains alive and well 16 years later. UCB is now an established source of HSCs for transplantation, particularly for children with a variety of malig-nant and non-maligmalig-nant disorders.5,33e35

UCB has the advantage over other HSC sources in that it can be relatively easily collected from related and unrelated donors, tested and HLA-typed, and then banked for immediate use, allow-ing shorter times to transplantation.4,36Units can be entered onto worldwide registries (e.g. Bone Marrow Donors Worldwide and Netcord) and easily resourced.36Maternity units with a high number of deliveries from ethnic minority mothers can be targeted, thus trying to compensate for the mainly white Caucasian population represented in inter-national bone marrow registries. These UCB banks have a particular importance for patients with rare HLA types and hence for ethnic minorities, for the donors of siblings suffering from diagnosed hae-matological disorders and for urgent unrelated donor transplants.4,5,8 Although there are poten-tially over 9 million registered bone marrow donors worldwide, the proportion of matched unrelated donors that cannot be found in these registries may be, for some racial groups, of the order of 20e 50%.5,8,9 Such patients therefore have the oppor-tunity for UCB transplants when an autologous, related or unrelated bone marrow donor cannot be sourced.

It has been stated by some that UCB can also be stored in order to provide autologous stem cells for the transplantation of individuals who might de-velop a variety of clinical disorders that will require transplantation in the future. There is no evidence that this approach will work, and it is not recommended by the Royal College of Obstetri-cians and Gynaecologists. Collection and cryostor-age costs over the longer term need to justify this approach until the newer cellular therapies have been fully developed.5 Accessing banked cord blood units takes on an average about 2 weeks, whereas the work-up for an unrelated bone mar-row donor from a registry may require up to 4 months.36Other advantages include:

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 a lack of risk to and attrition of donors compared with bone marrow donors (the annual donor loss in bone marrow registries being around 7%);

 a better tolerance of 1e2 HLA mismatches compared with other sources of allogeneic HSCs, such as bone marrow and MPB;

 fewer risks of viral infection;

 a lower incidence of severe GvHD.4e6

In public cord blood banks, over 175 000 banked UCB units are available for transplantation, and more than 5000e6000 transplants are estimated to have been carried out with UCB worldwide.8,9It is predicted that, worldwide, in the region of 2000 UCB transplants were conducted during 2004.8,9

Current UCB banking practices have recently been fully described.4,5,7 These cover donor re-cruitment, consent, medical evaluation and histo-ries, screening for infectious diseases, the logistics of in utero and ex utero collections, processing, cryopreservation, quality assurance and accredita-tion schemes. In the UK, the NHS Cord Blood Bank has achieved both Medicines and Health Products Regulatory Agency and Federation for the Accred-itation of Cellular Therapies Netcord (the second worldwide) accreditation. This bank has issued 83 UCB units for transplantation, approximately three-quarters being for children (median age 8.7 years). Median nucleated cell dosages for transplantation were of the order of 5 ! 107cells/kg recipient body weight. With approximately 25% of transplants being HLA-matched (6 out of 6 HLA-A, -B, -DR loci), 50% with 5 out of 6 HLA matches and the remaining 25% having 2 or 3 HLA mismatches, the median time to neutrophil (O500/ml) and platelet (O20 000/ml) engraftment were 24 and 34 days, respectively.

Indications for transplantation have included acute lymphocytic leukaemia, acute and chronic myeloid leukaemia, non-Hodgkin’s lymphoma, myelodysplastic syndrome, Fanconi’s anaemia, se-vere aplastic anaemia and paroxysmal nocturnal haemoglobinuria. Items of note for UCB banking for transplantation and for research, as managed by the National Blood Service in England, are summarised in Fig. 1. Thus, the NHS Cord Blood Bank provides UCB units within the recommended threshold cell dosages for patients undergoing myeloablative therapies (R1.7 ! 105 CD34C cells and R2 !

107nucleated cells/kg recipient body weight, with fewer than 3 out of 6 HLA locus disparities).5,6,37

Controversies and innovations

The major disadvantages in the use of UCB have been in (1) the availability of single donations

from a specific donor (i.e. no possibility of going back to the donor for a repeat stem cell donation or for the donation of lymphocytes), and (2) a limit to the number of HSCs within the UCB unit. In addition, there is often delayed haemato-logical and immune reconstitution,5,6 and in adults UCB has often been used as a last resort for transplants with advanced disease, generally undergoing myeloablative therapies. Despite the preferred use of UCB for transplantation in chil-dren rather than adults, there is now a renewed interest in the use of UCB for the transplantation of adults and for older patients undergoing non-myeloablative therapies; in methods to increase or have accessible higher HSC numbers for trans-plantation; and in addressing the issue of delayed haematological reconstitution.5,6 These are sum-marised below.

UCB transplantation in adults

Data relating to UCB transplantation in adults are much more limited than in children, the issue being the concern over the reduced cell numbers in the graft, with the consequent lower cell dose transplanted per kilogram of recipient body weight. Thus, allogeneic UCB transplants have often been used as a last resort in high-risk adults and when matched unrelated bone marrow donors are unavailable.5,6 Despite this, there have been

some good outcomes, and there has recently been renewed interest in this area, with the develop-ment of new approaches to the use and manipu-lation of UCB for transplanting adults with haematological disorders.

Chao et al.6have recently reviewed transplant outcomes for 339 adult patients receiving UCB grafts from eight published clinical studies. Indi-cations for transplantation were haematological malignancies (90%) and bone marrow failure or myelodysplasia (10%), with patients aged between 15 and 60 years, and with body weights ranging from 43 to 115 kg. Patients received a range of standard myeloablative therapies prior to the re-ceipt of a median of 1.5e2.51 ! 107nucleated

cells or, where measured, a median of 0.79e 1.37 ! 105CD34C

cells/kg recipient body weight. One or two or more HLA locus disparities (mea-sured serologically for HLA-A and -B, and molecu-larly for HLA-DR) occurred in 30 and 65% of the transplant recipients, respectively. These clinical trials may be summarised as follows:

 The median times to engraftment (i.e. the time to reach 500 neutrophils/ml and O20 000 plate-lets/ml) were 1 month and 2e4 months, re-spectively.

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 Comparisons with bone marrow and MPB stem cell transplants are difficult due to the limited quantity of UCB HSCs transplanted into some patients, differences in the quality or immatu-rity of the transplanted HSCs, poor patient prognosis and the prior failure of other treat-ment regimes in the UCB transplanted patients.  Survival rates of 50% were similar to those

expected for this group of patients.

 Severe GvHD was less frequent with UCB trans-plants than with those involving allogeneic bone marrow or MPB HSCs.

 There were better outcomes in younger patients and patients receiving adequate cell numbers.  The speed of engraftment correlated with the

number of CD34 cells infused.

Several other studies comparing the outcomes for UCB and unrelated or related bone marrow transplants in adults with leukaemia have recently

been published.10,11 The bone marrow recipients were not matched equally with those receiving UCB transplants, who were generally at higher risk, received lower cell doses, had more HLA loci disparities and were younger.

Laughlin et al.3 compared 150 UCB with 450 bone marrow transplant recipients. In the former, 77% had two HLA antigen mismatches, whereas in the latter 82% were HLA-matched, the remainder (18%) possessing one HLA antigen mismatch. The median UCB nucleated cell counts were 2.2 ! 107cells/kg recipient body weight. Acute GvHD was less severe in the HLA-mismatched UCB than mismatched bone marrow transplant patients, and the time to engraftment was slower with both mismatched UCB and bone marrow than with matched bone marrow transplants. No differences were seen in the overall mortality nor in the recurrence of leukaemia for mismatched UCB or bone marrow, and the presence of one or two HLA

Figure 1. NHS Cord Blood Bank: umbilical cord blood for banking and research

(HLA, human leukocyte antigen; HSC, haemopoietic stem cells; UCB, umbilical cord

blood).

 Altruistic donors are recruited in the prenatal period through the distribution of information to the mother, midwives and physicians at prenatal visits to clinics in London and Oxfordshire

 Because of improved outcomes from related, as opposed to unrelated, HSC transplants, a directed UCB donation programme also exists. Kits are provided by the National Blood Service to obstetricians in various hospitals in England for the collection of UCB from the healthy sibling donors of patients with disorders requiring allogeneic HSC transplantation

 Consent is obtained from mothers donating UCB either before delivery or within 24 h of UCB collection

 With local ethical committee approvals, unrelated UCB units for banking and transplantation are currently collected at three London hospitals and, for research, at the John Radcliffe Hospital in Oxford. The former units are obtained from populations with a high proportion of ethnic minorities  Collections are currently ex utero, although in utero collections may yield increased volumes of UCB  UCB volumes R 40 ml are processed within 24 h of collection, usually by volume reduction, with the removal of red cells (80% reduction) and plasma, using the Biosafe Sepax closed processing system, prior to the addition of DMSO and cryostorage in liquid nitrogen

 UCB units for banking are tested for sterility, for hepatitis viruses (HCV, HBV), HIV, human T-lymphotrophic virus, cytomegalovirus and syphilis, and for rhesus and ABO blood groups. Blood films are made for haemoglobinopathy screening and for the assessment of other haematological abnormalities

 UCB units are typed by DNA molecular techniques to medium resolution for HLA class I and II alleles including HLA-A, -B, -C and -DRBI loci, and registered with Netcord, the British Bone Marrow Registry and Bone Marrow Donors Worldwide, ready for searching. Additional high-resolution HLA typing is performed when a unit is selected by the transplant unit

 The identity of the selected unit is confirmed by short tandem repeat analysis from the bleedline of the frozen unit

 Nucleated and viable CD34Ccell counts are carried out prior to cryopreservation and upon issue for

transplantation. In the latter case, the UCB unit is also assessed for its colony-forming unit granulocyteemacrophage content

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mismatches in the UCB grafts did not alter patient outcome.

Rocha et al.10compared 682 adults with acute leukaemia. Of these, 85% received bone marrow transplants and 15% were transplanted with UCB between 1998 and 2002. The UCB recipients were younger, lighter in body weight and had more advanced disease. Comparisons were, however, made between matched bone marrow and mis-matched (94%) UCB transplants. The median nucleated cell numbers infused averaged 2.3 ! 107nucleated cells/kg recipient body weight. Acute GvHD was less severe in the UCB transplant recipients. There was no significant difference in transplant-related mortality, relapse rate or survival between the groups, but time to engraftment was delayed for UCB transplants.

A Japanese report has demonstrated a better outcome for UCB transplantations performed after 1998 when compared with unrelated bone marrow donor transplants, with respect to acute GvHD, disease-free survival and transplant-related mortality.38

Conclusions drawn from these studies were that HLA-mismatched UCB is acceptable for HSC trans-plantation in adults lacking an HLA-matched donor, particularly when R2 ! 107nucleated cells/kg recipient body weight are infused and with fewer than 3 of 6 HLA mismatches.

Transplantation of multiple UCB units

Approaches to the increasing numbers of HSCs for UCB transplantation have included the use of multiple UCB units. This procedure was first carried out in 1972, when 8 UCB units were trans-planted into a patient with acute lymphoblastic leukaemia.39At least 1 UCB unit engrafted in the short term, but this was not sustained. Subse-quently, between 2 and 12 UCB units were co-transplanted into 7 patients with either hae-matological disorders or solid tumours in a number of case studies.5 Coinciding with these studies were the analyses in murine and sheep models of multiple UCB transplants. Results demonstrated improved rates of survival in mice receiving more than one UCB unit for transplantation and the predominance of a single UCB unit in fetal sheep over the longer term.5

A recently published phase I/II clinical trial by Barker et al.40is the most comprehensive study to date considering the transplantation of multiple UCB units into patients with haematological disorders. This study of 23 high-risk patients, between the ages of 13 and 53 years and receiving myeloablative therapies, revealed that when 2 partially HLA-matched UCB units were transplanted together,

both donor UCB cell types engrafted in the short term in 24% of cases, with one donor UCB predom-inating over the longer term in all recipients by 100 days post-transplantation. There was no recorded increase in severe GvHD, but a higher engraftment rate associated with a low transplant-related mor-tality was observed. There was no correlation of graft success with neither nucleated or CD34 cell content nor with the HLA locus disparity of the original grafted units. The longer-term engrafting unit contained a higher number of CD3C

cells, suggesting that a graft-versus-graft reaction may be responsible for the preferential long-term en-graftment of a single unit, or that cells within the non-sustained UCB unit may have promoted en-graftment of the sustained unit.

These studies provide support for more detailed experimental analyses in improved animal models in order to understand the mechanisms, and for larger clinical trials using double UCB units for improving the outcome of transplantation, partic-ularly in adults.

Non-myeloablative transplants

Myeloablative regimens have traditionally been used to reduce the burden of malignant cells prior to HSC transplantation and to prevent graft re-jection following allogeneic transplantation. Re-duced-intensity conditioning regimes for patients undergoing HSC transplantation include the re-ceipt of mega- or increased doses of HSCs, to-gether with specific immunosuppressive drugs/ reagents that ameliorate GvHD and graft rejec-tion.5,6These have the advantage of (1) reduced transplant-related morbidity and mortality, (2) the potential benefits of versus-tumour or graft-versus-leukaemia effects of the transplanted im-mune cells, and (3) improved and more robust haematological reconstitution. Potential concerns when using mismatched UCB for such transplants include low transplanted cell numbers, increased risks of GvHD and graft rejection, increased dis-ease relapse or progression, and the non-achieve-ment of chimaerism.

Chao et al.6have recently reviewed UCB trans-plants using non-myeloablative therapies, includ-ing the largest study of 43 high-risk patients with haematological malignancies based at the Univer-sity of Minnesota, USA. Diseases that may respond to UCB HSC transplantation combined with re-duced-intensity conditioning include chronic lymphocytic leukaemia, low-grade non-Hodgkin’s lymphoma and multiple myeloma, particularly for older, unwell patients who cannot tolerate myeloablative therapies and who do not have an appropriately matched donor. Although there is

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a recent report of UCB transplantation using re-duced conditioning regimes in a 6-month-old infant with congenital neutropenia,41 Chao et al.6 have recommended that non-myeloablative regimes be used in clinical trials for selected older adult patients without a matched sibling donor in the first instance, while issues related to cell dosages, conditioning regimes and the development of re-sistance to infection are addressed. Those involved in these trials therefore do not currently recom-mend the use of non-myeloablative regimens in younger patients receiving UCB transplants for treatments for malignant conditions.

Improvements in haematological reconstitution by transplantation of UCB with other sources of CD34C

cells, ex vivo expanded cells and MSCs. Other innovative approaches to improving the out-comes of UCB transplantation by improving the haematological and immune reconstitution of UCB transplant recipients have been directed towards the supplementation or co-transplantation of the UCB graft with:

 low numbers of haploidentical CD34C

cells from sources such as MPB;

 ex vivo expanded UCB haemopoietic cells or possibly the selected use of T-cell subsets that might enhance HSC engraftment or ameliorate viral infections;

 MSCs.

The rationale for the co-transplantation of low numbers of haploidentical CD34C

MPB stem cells relates generally to the shorter time taken for these cells to achieve neutrophil engraftment (10e 20 days) compared with UCB (approximately 1 month). 6,42For MPB stem cells, there is a recog-nized correlation between the time to neutrophil engraftment and the content of haemopoietic progenitor (CFU-GM and CD34C

) cells in the graft, although, with dosages above 5 ! 106CD34C

cells infused/kg recipient body weight, the time to haemopoietic reconstitution was not acceler-ated.43In a study involving 11 adult UCB transplant recipients with haematological disorders and sup-ported with small numbers of haploidentical MPB CD34C

cells, the median time to neutrophil en-graftment (mediated by the haploidentical CD34C

graft) was 10 days, whereas the UCB HSCs provided the cell source for longer-term engraftment.42

In adult bone marrow, HSCs reside in microen-vironmental niches, adjacent to the trabecular bone.44,45 These niches are composed of MSC-derived stromal niche osteoblasts that in-teract directly with HSCs via such molecules as

N-cadherin.44,45 These stromal niche osteoblasts cells express growth regulators that control intrinsic genetic programmes within the HSC, allowing it to remain quiescent, survive, self-renew or undergo lineage commitment, differentiation or pro-grammed cell death. MSC co-transplantation aims to supplement the supply of stromal niche osteo-blasts cells damaged by transplant conditioning regimes or to promote HSC engraftment by regu-lating their expression of homing receptors or by suppressing alloreactive T-cell responses.46e48 MSCs may be derived from bone marrow, UCB, fetal blood or other tissues, but their quantity and quality may vary depending on the source of these cells.49It is of note that:

 recent animal studies suggest that MSC co-transplantation with dual UCB units may pro-mote the engraftment of HSCs from both units;  the treatment of 8 paediatric leukaemic pa-tients by the co-transplantation of haploident-ical MSCs with unrelated UCB demonstrated more rapid median times to neutrophil engraft-ment (19 days) than expected.47,48,50

Further experimental animal studies and clini-cal trials are required to adequately evaluate the potential clinical benefits of this approach.

Finally, for a number of years, attempts have been made to increase UCB HSC numbers without the loss of their ‘stemness’ characteristics, or to promote HSC differentiation and use these differ-entiated cells for haematological support during the transplant period. Protocols to expand UCB HSCs ex vivo have been hindered by the lack of simple procedures to identify HSCs phenotypically or in surrogate functional assays (see above). Expansion protocols ex vivo have, until recently, been directed towards the use of available cytokine cocktails that promote HSC survival and prolifera-tion. The read-out assays have included CD133 and CD34 phenotypes, LTC-IC and CAFC assays in vitro, and functional engraftment assays in vivo, partic-ularly in murine and fetal sheep models.5,13,14The

cytokines most commonly used for the experimen-tal expansion of HSC or their progeny from UCB have included FLT3 ligand, Thrombopoietin (TPO), Stem Cell Factor (SCF), IL-II, and hyper-IL-6.5,6,13,14,51,52 These demonstrate large expansions of the CD133 or CD34 cells, lesser expansions of LTC-IC or CAFC cell subsets and either negligible or up to 4e5-fold expansions of cells that repopulate immunodefi-cient mice.51e53It is not always clear whether these expansion protocols extinguish or do not promote HSC self-renewal, or whether there is an effect on homing receptors that affects HSC engraftment.

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With the recent advances in identifying ‘stem-ness’ genes in HSCs and with improved understand-ings of the mechanisms whereby stromal niche osteoblasts cells in bone marrow maintain the stem cell features of HSCs, new approaches to HSC expansion protocols are being devised. One aim is to use drug discovery programmes to produce soluble transcription factors or transcription factor peptide mimetics to promote the HSC cell cycling. Transcription factors of interest include NF-Y, which regulates such genes as telomerase, LEF-1 (in the Wnt pathway) and homeobox (HOX) genes, as well as the Wnt/LEF-1, Bmi-1 and HOXB4 mole-cules.54e56Soluble Tat-HOXB4, when added to HSCs for short periods of time has been shown to increase murine HSC numbers 20e100-fold.57It is essential that such careful preclinical studies are conducted with human UCB cells prior to the use of these new compounds in clinical trials to ensure that such methods do not promote cellular abnormalities or diminish to any great extent the HSC lifespan.

Clinical trials conducted using expanded UCB ex vivo to support the haematological reconstitution of an unmanipulated UCB graft have recently been reviewed.5,6There was no significant acceleration in the time to neutrophil or platelet engraftment. These studies were, however, hampered by the unavailability of clinical grade cytokines (SCF, Flt3, TPO only were used) and the clinical trial design, with expanded cells being infused 10e12 days after transplantation of the unmanipulated UCB; this would therefore have been unlikely to have contributed to improvements in the time taken to haematological engraftment.

Genetic selection of UCB units

Preimplantation diagnosis during in vitro fertiliza-tion procedures has allowed the ‘selecfertiliza-tion’ of HLA-matched donors of UCB units for the transplanta-tion of siblings suffering from life-threatening haematological and related disorders, particularly genetically inherited conditions and situations where an appropriate donor cannot be found. This approach has been practised in the UK under Human Fertilisation and Embryology Authority regulations, but has raised many ethical issues and remains controversial.58

Non-haemopoietic stem cells in

UCB: new opportunities for

cellular therapies

It is of particular interest that a variety of non-haemopoietic stem cells, in addition to HSCs, has been identified in haemopoietic tissues. This has

come about over the past 5 years in relation to the development of newer concepts of ‘adult’ stem cell plasticity and potentiality.13,14,59 Cell types identified in UCB include pluripotent stem cells,60 endothelial precursors and MSCs.61e63 Although the pluripotent stem cell subset is rare and its value in the clinical context requires definition, endothelial precursors are routinely found in UCB. Such precursors have a particular importance for improving the revascularization of organs or tis-sues affected by ischaemic diseases (e.g. wound healing, cardiovascular diseases, etc.), and for tissue engineering.61,64,65 MSCs are reported to occur in 30e60% of low-volume UCB units, with a frequency of up to 2.3 MSC clones/108 mono-nuclear cells. Although MSCs are known to rou-tinely generate bone, cartilage and fat, there is a preference for the generation of osteoblasts and chondrocytes from UCB.63,64Thus, such a source of UCB stem cells are likely to have a particular relevance to the treatment of neonates with skeletal malformations or diseases.

Other cell types that are reported to be gener-ated from MSCs in UCB include muscle, liver and neural cells.66e73These are much more controver-sial, as although such cells express markers that are present on muscle and neural cells, it is unclear whether these cells will act as functional muscle, liver and neural cells in vivo in the human or whether they mediate such effects by the production of growth factors that have an indirect effect on cell and tissue repair.

Practice points

 UCB sourced at birth is a viable and acceptable alternative to bone marrow and mobilised peripheral blood for alloge-neic HSC transplantation of children, when an HLA-matched donor is unavailable.  Family-directed UCB donations are

accept-able for transplantation of siblings with life-threatening diseases.

 UCB HSC transplants in adults are the subject of clinical trials and are acceptable in the absence of an appropriate HLA matched donor and when critical thresholds of nucleated and CD34+cells are available for transplantation.

 With 4 of 6 HLA matches and with sufficient cells, outcomes are similar following UCB transplantation to those of matched or one antigen mismatched unrelated bone mar-row donor grafts.

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Acknowledgements

The authors wish to thank Ms Maggie Maguire for assistance with the manuscript, Ms Sue Armitage for information related to the NHS Cord Blood Bank and Dr Cristina Navarrete. The research of SMW is supported by the National Blood Service, Leukae-mia Research Fund, British Heart Foundation and the Wellcome Trust, and benefits from R&D fund-ing received from the NHS R&D Directorate.

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 UCB HSC transplants are associated with less severe acute graft-versus-host disease, but will delayed haematological reconsti-tution.

 UCB HSC transplants are particularly bene-ficial for ethnic minorities where bone marrow donors are not available and for urgent HSC transplants.

 Transplant-related mortality and limited HSC dosages remain a major obstacle to UCB transplantation in adults.

Research directions

 Strategies being evaluated in clinical trials in order to overcome the limitations of UCB transplantation include:

multiple UCB transplants,

transplantation of UCB units with small numbers of haploidentical CD34 cells, transplantation of UCB units with MSCs, transplantation of UCB units with ex vivo expanded UCB units.

 UCB contains non-haemopoietic stem/pro-genitor cells, such as vascular endothelial cell precursors, mesenchymal stem cells, osteoblast and chondrocyte precursors etc. These may have a future role in:

tissue revascularisation,

repair of bone and joint diseases, wound healing,

cardiac repair, and

possibly in diseases requiring repair of muscle, neural, liver cells etc.

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References

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