Introduction

Umbilical cord blood transplantation (UCBT) has become an established therapy for patients without matched related or unrelated donors, leading to cures of previously incurable disease. Owing to a limited number of stem cells available in a typical UCB unit and delayed engraftment when compared to marrow or mobilized peripheral blood, the prime focus for this therapy continues to be the pediatric patient. Nevertheless this form of transplant is emerging as a viable alternative in adults, provided there are sufficient cells in the UCB unit and the patient is a small to average-sized adult. Although UCBT permits the use of mismatched donors and are available more quickly than matched unrelated donor grafts,¹ the main problems after the infusion of an UCB unit are delayed engraftment of neutrophils and platelets and aberrant immune reconstitution – both leading to a higher mortality. In 2004, two large retrospective UCBT studies on adults transfused with a median of 2.3 10⁷ total nucleated cells (TNC)/kg showed no difference in leukemia-free survival between the human leukocyte antigen (HLA)-mismatched cord blood group and the HLA-matched marrow groups using allele matching at HLA-DRB1 and serologic matching at HLA-A and -B.^{2, 3} Based on these results, UCBT could become the treatment of choice if a HLA class I and class II (eight out of eight) matched unrelated adult donor is not available.

Unfortunately 90% of adults referred for a UCBT to our institution are ineligible to receive an UCB graft based on their weight^{4, 5} at the recommended cell dose and with no more than two HLA mismatches.⁶ Thus efforts to increase the number of hematopoietic stem cells (HSC) infused are paramount. Ongoing efforts to achieve cell dose guidelines include improvements in the harvesting of cord blood units,⁷ modifications of post-thaw procedures to decrease the loss of TNC,⁸ and simultaneous infusion of two cord units. One of the earliest considerations that was investigated that could speed engraftment and improve immune reconstitution was to expand the primitive UCB progenitors ex vivo and transplant with or without the unmanipulated portion of UCB. Initial efforts were largely unsuccessful because mature rather than immature cells were expanded.⁹ However, with increased knowledge of the hematopoietic niche and new methods to promote progenitor cell expansion without differentiation, UCBT in adults may become more widely available. This review will focus on the current problems associated with UCB expansion: more rapid reconstitution of hematopoiesis and immune reconstitution.

Early clinical experience of HSC ex vivo expansion

Techniques of ex vivo expansion have been developed over the past decade in in vitro systems and murine models using unfractionated marrow and CD34+ selected marrow and peripheral blood cells. Caldwell et al.¹⁰ and Schwartz et al.¹¹ demonstrated that slow perfusion and local oxygenation of flat hematopoietic cultures would allow stem cell survival with 10–20-fold progenitor cell expansion with sufficient cells in one or two closed culture vessels. Haylock et al.¹² showed that, if sufficient quantities of interleukin-1b (IL-1), IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage-CSF (GM-CSF), and stem cell factor (SCF) were added to suspension cultures of CD34+-enriched peripheral blood progenitor cells, progenitor cell expansions of 50-fold or more could be achieved. Murine stem cell transplants demonstrated that SCF, Flt-3 ligand (FL) and thrombopoietin (TPO) are crucial cytokines^{13, 14, 15} in combination with fibronectin contact¹⁶ for promoting stem cell expansion. Our group successfully ex vivo expanded a small aliquot of marrow (median volume 36.7 ml) in the Aastrom/Replicell stromal-based closed system in serum-containing medium using GM-CSF-IL3 fusion protein, flt-3 ligand and erythropoietin then successfully infused this as the sole autologous stem cell source in 19 patients with breast cancer.¹⁷ Long-term hematopoietic reconstitution was achieved with marrow volumes as low as 13 ml, without an increase in infections or late graft failure to 8 years so far.

At first liquid culture systems were used to expand UCB based on animal models showing that non-contact and stroma-free cultures can maintain long-term engrafting cells, defined by their capacity to engraft secondary or tertiary murine hosts.¹⁸ The process began with the isolation of CD34+ or CD133+ progenitor cells and incubation in serum or alternative media with additional growth factors. McNiece et al.¹⁹ described two 7-day expansions in Teflon-coated bag culture systems in the presence of SCF, G-CSF and megakaryocyte growth and differentiation factor (MGDF) which increased median TNC, GM-CFC and highly proliferative potential-CFCs and yielded more than a 20-fold increase in CD34+ cells. DiGiusto et al.²⁰ expanded CD34+/Thy-1+/Lin- UCB cells in another stroma-free system with IL-3, IL-6 and SCF and found no measurable loss of long-term culture or in vivo engrafting potential in severe combined immunodeficient (SCID) mice.

Early attempts to move these impressive preclinical results to patients failed to show improvements in the delayed engraftment typically seen in these patients. Shpall et al.²¹ expanded CD34+ cells from a portion of a UCB unit for 10 days with SCF, G-CSF and MGDF. The median time to engraftment of neutrophils was 28 days and platelets were 106 days, and at a follow-up at 30 months and 37% were alive. Acute grade III/IV graft-versus-host disease (GVHD) was seen in 40% of patients, similar to unmodified grafts. Our group also expanded unfractionated UCB cells in the Aastrom/Replicell system as described above.^{22, 23} We reported a 2.4-fold increase in TNC expansion, but a loss of 36% of CD34+ cells and a similar loss of LTC-IC of 40%. Engraftment was not enhanced in our patients nor in another trial where expanded cells were infused also on day 12.²⁴ Unlike our earlier experience with bone marrow cells, these cultures did not develop a potentially critical stromal cell layer, likely due to a lack of significant number of mesenchymal stem cells (MSC) in UCB. The concern was that without the stabilizing influence of a supporting stromal layer, growth factors would cause the expanded cells to differentiate at the expense of self-renewal.

Current needs for successful HSC expansion

Stem cell assays to evaluate UCB expansion

With the clinical failure of ex vivo expansion when the expanded product was used as the sole stem cell source²⁵ and the lack of improvement when the manipulated and unmanipulated products were both infused,^{21, 24} the current preclinical models and predictive assays for engraftment of all cell lineages deserve further development. Long-term engraftment is dependent upon the number and engraftment ability of the HSC transplanted; therefore, the ideal assay for this purpose must be simple, quantitative and reliably demonstrate engraftment capacity. These assays have been recently reviewed²⁶ and to date no such in vitro assays have been developed to measure long-term engraftment of HSC (Table 1).

Table 1 - Stem cell assays.

Full table

Assays to test products of UCB ex vivo expansion may improve on current techniques or incorporate new technologies. Lodging assays test HSC homing and niche-retaining capacity, correlating well with HSC long-term engraftment ability in murine models.²⁷ Hypothesis-generating results from gene expression profiling data may lead to surface or DNA markers that can quickly be assessed after expansion, for example, telomere length measurements by flow cytometry of fluorescein isothiocyanate-labeled telomere-specific peptide nucleic acid probes (flow fluorescent in situ hybridization)²⁸ rather than time-consuming xenogeneic engraftment models.

Assessment of engraftment

The final common denominator to assess the success of ex vivo expansion is engraftment. Current methods to ex vivo expand umbilical cord blood cells may introduce defects that lead to engraftment failures as has been reported using expanded²⁵ or transduced²⁹ autologous CD34+ cells. Guenechea et al.³⁰ transplanted fresh and ex vivo-expanded CD34+ cord blood cells into irradiated non-obese diabetic (NOD)/SCID mice and while they found no differences in engraftment at 4 months, engraftment at 3 weeks was impaired when the expanded cells were used, suggesting that expanded cells might do not facilitate engraftment. Larger animal models also indicate that ex vivo expansion introduces cellular defects that affect engraftment. McNiece et al.³¹ expanded UCB CD34+ cells with SCF, MGDF, and G-CSF for 2 weeks and transplanted them into preimmune (day 60 gestation) fetal sheep with long-term engraftment evaluated in secondary and tertiary recipients. Expanded cells were capable of faster engraftment at the expense of long-term hematopoietic reconstitution. Additional preclinical studies suggest that ex vivo expansion interferes with engraftment by introducing defects that promote apoptosis,^{32, 33, 34} disrupt marrow homing,^{35, 36, 37} and initiate cell cycling^{38, 39} (Table 2).

Table 2 - Cellular defects acquired during ex vivo expansion.

Full table

Recent attempts to optimize HSC culture conditions

In order to carefully choose the ex vivo conditions that will most likely expand the number of long-term reconstituting cell (LTRC), studies will need to carefully evaluate the characteristics of the expanded cells and their interaction with both the soluble and insoluble components of ex vivo culture.

Cells

Isolating cells to expand based on only their surface protein expression is likely to include undifferentiated and mature cells. The surface phenotype can change depending on the activation status of the precursor cells and does not provide information on the functional ability of the cells in vivo. Early data suggested that spleen repopulating cells (SRC) were CD34+CD38- in contrast to CFC and LTC-IC which were also found in the CD34+CD38+ fraction.^{13, 40, 41} Although CD34 expression has been the most commonly selected surface marker for ex vivo expansion,^{15, 42} CD34 is often expressed on more differentiated cells and large animal models suggest that CD34+ cells are not the cells primarily involved in marrow reconstitution,^{43, 44} and following ex vivo culture, dissociation between CD34+CD38- cell expansion and SCID-repopulating capacity has been observed.⁴⁵ Preliminary data suggests that as UCB units are made up of progenitor cells that possess both a CD34+ and a CD34- phenotype,^{46, 47} isolating a more primitive marker CD133+ may identify cells that are less mature than those that express CD34.^{48, 49} Murine models using this cell type showed excellent engraftment.⁵⁰ Functional assays, such as aldehyde dehydrogenase activity,⁵¹ may be a more accurate method to isolate the most effective UCB progenitors.

Cytokines

The proper mixture of growth factors and cytokines used in ex vivo culture conditions has not yet been determined. Different cytokine components can affect speed of recovery of white cells and platelets as well as affecting long-term donor engraftment.⁵²

Most ex vivo conditions involve SCF, FL and TPO. SCF has been shown to improve homing capacity of UCB cells in preclinical models.⁵³ FL leads to short-term expansion^{54, 55} and helps regulate⁵⁶ the expression of very late antigen (VLA)-4 and VLA-5, adhesion molecules which play a part in proliferation and differentiation either directly or through the modulation of cytokine-induced signals.^{57, 58} In a study to evaluate cytokine combinations that lead to expansion without change in repopulating potential, Levac et al.⁵⁹ cultured CD34+CD38-Lin- cord blood cells in serum-free media with SCF and FL and found that TPO may be able to replace IL-3, IL-6 and G-CSF without changing the number of SRC. In addition the combination of FL and TPO may prevent apoptosis⁶⁰ and support the self-renewal of primitive stem cells by preventing telomere degradation.⁶¹

Stroma

Schofield's⁶² 'niche hypothesis' suggested that true HSC are in essence fixed tissue cells, existing in association with one or more other supporting cells. It is these microenvironmental cells that were postulated to form the niche that enable HSC to indefinitely self-renew, while effectively inhibiting differentiation and maturation. In vitro and in vivo studies have shown that stromal cells provide a rich environment of signals (cytokines, extracellular matrix proteins and adhesion molecules) that control proliferation, survival and differentiation of hematopoietic progenitor and stem cells.⁶³ It is currently not known whether non-contact conditions are sufficient for ex vivo expansion or whether stromal binding is required.⁶⁴

MSCs have been used in preclinical models as a method to maintain stem cells in an immature state. MSCs give rise to osteoblasts, chondrocytes, adipocytes and myelosupportive stroma.⁶⁵ MSC express adhesive ligands and soluble factors critical for hematopoiesis, and have been demonstrated to support hematopoiesis in vitro in co-culture with unmanipulated cord cells.⁶⁶ Third-party MSC can be used to alleviate donor deviation in dual-cord transplantation and to facilitate engraftment of multidonor UCBT as shown in NOD/SCID mice.^{67, 68, 69} It is unclear whether the immunomodulatory properties of MSC^{70, 71} when co-cultured with unmanipulated cord cells yielding a 16–37-fold increase in CD34+ cells⁷² will prove useful in the setting of cord blood transplant or adoptive immunotherapy.

The future of HSC ex vivo expansion: understanding molecular pathways involved in HSC proliferation and maintenance

As current ex vivo systems have not been proven to enhance LTRC, it will be necessary to further study and manipulate molecular pathways that expand the stem cell niche in culture without impinging on recipient engraftment. Such pathways involve cell signaling with Notch, Wnt/-catenin, the receptor tyrosine kinase Tie2 and bone morphogenetic proteins (BMP) in combination with transcription factors BMi-1 and homeobox gene HoxB4 (Figure 1). Cytoplasmic mediators of these pathways such as phosphatase and tensin homolog (PTEN) have become areas of intense investigation as PTEN may be the most important switch between the leukemic and normal stem cell,⁷³ an exciting arena for translational research as the mTor inhibitor Rapamycin (Sirolimus) has been shown to at least partially make up for the loss of PTEN function.⁷⁴

Figure 1.

Quiescent HSCs (G0) are located in the endosteal osteoblastic niche in which a special type of osteoblastic cell (SNO cell) serves as a key component. Adhesive molecules glue HSCs to their osteoblastic niche and provide quiescent signals to maintain HSCs in an undifferentiated state. N-cadherin is expressed on both the SNO cell and HSC and form an adhesive junction. The interaction of the integrin (e.g. VLA-4 and -5) with VCAM and fibronectin is also involved in the HSC-niche adhesion. The SNO cell provides inhibitory signals, such as BMP, Jagged and Ang-1, to restrict HSCs from activation and protect HSCs from differentiation and apoptosis. PTEN plays a role in inhibition of HSC cell cycle entry by repressing growth factor induced phosphoinositol-3-kinase/AKT activation and enhances HSC-niche adhesion by stabilizing the N-cadherin/-catenin-mediated adhesive junction. HSC cell cycle entry is promoted by growth factor stimulation and can be independently induced by decreasing PTEN activity, allowing HSCs to detach from niche cells owing to destabilization of N-cadherin/-catenin adhesive junctional complex. Wnt signal induced -catenin activation and nuclear transfer is also critical for HSC activation and proliferation. Noggin, a BMP antagonist, expressed by the HSC and surrounding cells during the process of HSC activation may also be involved in HSC activation by blocking BMP signal activity. Violet hues promote stem cell differentiation whereas blue hues maintain stem cell immaturity. HSCs, hematopoietic stem cells; SNO cell, spindle shaped N-cadherin+CD45- osteoblastic type cells; VLA-4 and -5, very late antigen integrins -4 and -5; VCAM, vascular cell adhesion molecule-1; PTEN, phosphotase and tensin homolog; BMP, bone morphogenetic protein; Ang-1, angiopoietin-1; Wnt, blah; HOXB4, homeobox B4; mTor, B lymphoma Mo-MLV insertion region; Bmi-1, B lymphoma Mo-MLV insertion region 1.

Full figure and legend (190K)

Notch

Notch signaling is involved in cellular differentiation, proliferation, apoptosis, adhesion and epithelial-to-mesenchymal transition. There are five Notch ligands in mammals, Jagged-1/2 and Delta-1/3/4. Binding of a Notch ligand to a Notch receptor results in cleavage and release of the intracellular domain of the Notch receptor by a membrane-associated protease complex. The intracellular domain then translocates to the nucleus to join with CBF1/RBP-J⁷⁵ and mastermind-like (MAML)⁷⁶ among others. The assembled nuclear complex regulates transcription of several Notch effector genes, including homolog of Drosophila Hairy and Enhancer of Split.⁷⁷

Early studies suggested that Jagged-1 expanded early progenitor cells in ex vivo culture.^{78, 79} Engineered and immobilized Delta-1 expanded human CD34+CD38- cord blood progenitors cells in serum-free media with fibronectin and growth factors. These culture conditions inhibited myeloid differentiation and induced a 100-fold increase in the number of CD34+ cells compared with control cultures. Common lymphoid precursors (CD34+CD7+CD45RA+) and CD3+ T/natural killer cell precursors also were expanded.⁸⁰ The number of Sca-1+c-kit+ precursors with short-term lymphoid and myeloid repopulating ability increased.⁸¹ The reconstitution of NOD/SCID mice was improved for cells expanded at low concentrations of the Notch ligand unlike cells expanded with higher ligand concentrations which appeared to cause apoptosis.⁸² Thus the optimal concentration of Notch ligands in ex vivo culture has yet to be established and the differing effects of Notch ligands has not been fully studied; for instance Delta-1, unlike Jagged-1, inhibits B-cell differentiation⁸³ and decreases CFU-GM, CFU-G and CFU-M.⁸⁴

Wnt signaling

Wnt-mediated signaling involves the binding of Wnt proteins to their receptor–coreceptor complexes, frizzled–LRP, which leads to the accumulation of -catenin and its translocation to the nucleus. In the nucleus, -catenin forms a bipartite transcription-factor complex with T-cell factor to activate target genes, such as cyclin D1 and c-Myc. The Wnt-signaling pathway is crucial for the development of many organ systems.

Early studies demonstrated that activation of Wnt signaling induced both mouse fetal liver HSC (AA4+cKIT+SCA1+) and human bone-marrow (Lin)- CD34+ proliferation when HSC were co-cultured on Wnt transduced stromal feeder layers.^{85, 86} Although stromal cells transduced with WNT5A were unable to induce the proliferation of cord-blood CD34+ in in vitro culture, intraperitoneal injection of WNT5A-conditioned medium into mice engrafted with human CD34+ cells led to increased multilineage reconstitution by more than threefold compared with controls.⁸⁷ Direct evidence of the involvement of Wnt signaling in HSC self-renewal was demonstrated by Murdoch et al.⁸⁷ and Reya et al.⁸⁸ when transduction with a constitutively active form of -catenin or purified recombinant WNT3A promoted the proliferation of HSC in vitro, maintained the immature phenotype of HSC in long-term cultures, and enhanced long-term hematopoietic engraftment ability. Interestingly, Wnt-mediated maintenance of HSC immaturity in vitro requires Notch signaling, although HSC survival and entry into the cell cycle is Notch independent.⁸⁹

Tie2/Ang-1

The interaction of receptor tyrosine kinase Tie2⁹⁰ with its ligand angiopoietin-1 (Ang-1) may maintain progenitors grown ex vivo in an immature state. Long-term HSC that express the receptor tyrosine kinase Tie2 are quiescent and adhere to osteoblasts in the marrow. Ang-1 expressed by the osteoblastic niche cell activates Tie2-mediated signals in HSC, which enhance the ability of HSC by inducing HSC adhesion to the bone marrow niche, restricting HSC in a quiescent state, and protecting HSC from apoptosis. Enhancing the Tie2 signaling activity by overexpression of Ang-1 or injecting the mice with recombinant Ang1 results in protection of the HSC compartment from myelosuppressive stress. Furthermore the interaction of Tie2 with Ang-1 induced cobblestone formation of HSC in vitro and maintained in vivo long-term repopulating activity of HSC. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSC in a quiescent state in the BM niche.⁹¹

BMP

BMP are members of the TGF- superfamily that have long been established to function in the development and regulation of a wide range of biological systems. BMP also play key roles in regulating fate choices during stem cell differentiation. BMP plays a role in maintaining murine embryonic stem cell self-renewal,⁹² although it promotes differentiation of human embryonic stem cells.⁹³ BMP4 functions downstream of the sonic hedgehog signal and plays a role in HSC maintenance. High concentrations of BMP4 can repress UCB CD34-CD38- HSC differentiation and maintain stem cell engraftment ability in a cell-free culture system. BMP may also indirectly control HSC by regulating the size of the HSC niche.

Zhang et al.⁹⁴ showed that BMP receptor activation of spindle shaped N-cadherin+CD45- osteoblastic type (SNO) cells maintains the stem cell niche. In a murine model, Zhang et al. showed that the SNO cells on the bone surface of cancellous/trabecular bone connect to HSCs via N-cadherin and -catenin and help control the size of the stem cell niche via BMP signaling.

Bmi-1

B lymphoma Mo-MLV insertion region 1 (Bmi-1), a proto-oncogene and member of the Polycomb group repressive complex 1, is expressed in mouse and human HSC and functions by chromatin-structure modulation to repress downstream genes p16Ink4a, a modulator of the retinoblastoma pathway, and p19Arf (p14Arf in humans), a modulator of the p53 pathway – both are cyclin-dependent kinase inhibitors which function to inhibit cell division. The importance of Bmi-1 in HSC self-renewal has been demonstrated in mouse models of both genetic gene deletion and transduced overexpression.⁹⁵ Bmi-1 null mice show normal development of embryonic hematopoiesis but develop hypocellular bone marrows after birth and die within 2 months because of marrow HSC exhaustion. Forced expression of Bmi-1 enhances symmetrical cell division of HSC and results in HSC expansion⁹⁶ by p16Ink4a and p19Arf expression inhibition in a telomere-independent manner.⁹⁷ As a central player in HSC self-renewal, Bmi-1 could be a target for therapeutic manipulation of HSC.

HOXB4

HOX genes encode a large family of transcription factors that have a highly conserved DNA-binding motif known as the homeodomain. In mammals, there are four main families of HOX factors that are arranged in a co-linear manner at different loci (groups A, B, C and D). HOXB4 may be one of the most important regulators of HSC self-renewal. It is expressed in the stem cell fraction of the bone marrow and subsequently downregulated during differentiation in humans^{98, 99} and mice.¹⁰⁰ HOXB4-deficient mice have HSC with a reduced proliferative capacity but normal differentiation and lineage pathways.^{101, 102} Ectopic expression of HOXB4 can mediate a significant expansion of HSC of mice and humans in vitro and in vivo.¹⁰³ Forced expression of HOXB4 also rapidly triggers an increase of human UCB HSC detected by both in vitro and in vivo assays¹⁰⁴ and the effect of HOXB4 on HSC expansion can be achieved by direct delivery of the recombinant HOXB4 protein¹⁰⁵ thus providing an opportunity to develop effective HSC expansion for future stem cell therapies.

Recent preclinical/clinical HSC expansion investigations

Transcription inhibition

A number of novel methods are in development to improve upon ex vivo culture systems in order to expand UCB progenitors without introducing defects that will affect engraftment (Table 3). Because growth factor infused ex vivo conditions promote differentiation, agents to maintain an immature state are ideal. Preclinical data suggested that repressing transcription by epigenetic modification via methylation of cytosine within cytidine-phosphate-guanosine dinucleotides¹⁰⁶ or by decreased acetylation of histones¹⁰⁷ may slow the process of differentiation. The methyltransferase inhibitor 5-aza-2'-deoxycytidine (5azaD) and histone deacetylase inhibitor trichostatin A (TSA) promote demethylation and histone acetylation, thereby sustaining self-renewal gene transcription and expanding HSC in vitro.^{108, 109} Murine data suggest that the treatment of CD34+ selected UCB cells with 5azaD and TSA in the presence of SCF, IL-3, MGDF and Flt-3 ligand may result in a 9.6-fold expansion of SRC.^{110, 111}

Table 3 - Novel expansion technologies.

Full table

Copper chelation

The copper chelation system utilizing tetraethylenepentamine^{112, 113, 114} may also maintain a primitive phenotype, murine models have shown improved engraftment, and early clinical trials of this system are underway at MD Anderson Cancer Center.¹¹⁵ All-trans retinoic acid (ATRA) has been implicated in the regulation of hematopoiesis^{116, 117, 118} and CD34+CD38-Lin- cells from UCB cultured in a non-contact system with AFT024 stromal cells, FL and TPO, with or without ATRA showed increased LTC-IC, ML-IC and SRC expansion via a stromal cell non-contact dependent mechanism.¹¹⁹

Wnt pathway activation

Agents that can activate the Wnt pathway such as the glycogen synthase kinase-3 inhibitor 6-bromoindurubin-3'-oxime may ultimately enhance HSC repopulation.^{120, 121}

Immunodeficiencies after UCBT and ex vivo expansion

The reported worldwide experience of UCBT is complicated by a 40% rate of infectious deaths.^{2, 3, 122, 123} Nearly 5000 UCB transplants have been performed with approximately 20% of all pediatric transplants now using this stem cell source.¹²⁴ UCBT in adults who meet cell dose minimums can be effective; however, infections are a major cause of death owing to delayed hematopoietic and immune reconstitution.

The delayed immune reconstitution in adult recipients compared to pediatric recipients is likely due both to the immaturity of cells in UCB and to the decline in lymphopoiesis with age.¹²⁵ Development of T and B cells in thymus and bone marrow, respectively, declines in adults, and thus, after infusion with comparable numbers lymphoid progenitors/kg body weight of recipient, adults will require a longer time to reconstitute a normal immune system from HSC.

The immaturity of cells in UCB is exemplified by V CDR3 spectratype analysis of T cells which show that this T-cell repertoire is polyclonal and fully constituted, but naïve.¹²⁵ The naivety of this repertoire likely contributes to the delay in immune reconstitution. Other kinds of immaturity in neonatal host defense that likely contribute to the delay in immune reconstitution¹²⁶ include significantly reduced mRNA expression of GM-CSF, G-CSF, IL-3, TGF-1 and MIP-1 in mononuclear cells (MNCs).^{127, 128, 129} Further, decreased Th1 type cytotoxic cellular responses may result from impaired synthesis of IL-2 and IFN-,^{130, 131} and to decreased capacity of dendritic cells to produce IL-12.¹³² Microarray analysis of UCB and adult CD4T cells showed profoundly different groups of genes were transcribed after activation, indicating that the T-cell response from UCB T cells are considerably different than T cells from adults.¹³³ The decreased effectiveness of UCB CD4+ T cells, while delaying immune reconstitution of transplant recipients, likely contributes to the decrease in GVHD seen after UCB transplants.¹³⁴

Ex vivo expansion of HSC may introduce additional immune dysregulation after UCBT. Although ex vivo expansion does not seem to harm T-cell development, it decreases dendritic cell functions. Rosenzwajg et al.¹³⁵ reported that day 14 expanded CD34+ cells have a similar capacity to generate lymphocytes as unexpanded CD34+ cells, and the TCR-V repertoire was not skewed. However, the investigators found a 13-fold decreased capacity of expanded cells to generate dendritic cells compared to unexpanded CD34+ cells.

Selective expansion strategies to control GVHD and improve thymopoiesis

Controlling the development of GVHD and quickly resolving this immune-mediated process in favor of a graft-versus-disease effect is essential. One strategy to control the severity of GVHD is to capture the specific suppressive activity of T regulatory (Treg) cells, a subset of CD4+CD25+ cells that express the Foxp3 transcription factor, against the allogeneic reactive T cells.^{136, 137} In UCB, Treg cells are present at 2–5% of UCB MNCs¹³⁸ and are predominantly CD45RA+.¹³⁹ When activated, Treg cells display antigen specific suppressive activity which could be exploited to target alloreactive T cells, mitigating GVHD without affecting disease relapse.

In addition to controlling alloreactive T cells, it is also critical to improve recipient thymic function post transplant. Clinical studies have demonstrated that decreased thymopoiesis is associated with aging, preparative regimen intensity and GVHD.^{140, 141} In mice, expression of Foxn1 on epithelial stem cells promotes maturation and differentiation of both cortical and medullary thymic epithelial cells (TEC).¹⁴² We have demonstrated that reduced expression of Foxn1 correlates with a reduced number of thymocytes with age.¹⁴³ Thus, it may be possible to expand UCB mesenchymal cells, engineer these cells to express the FOXN1 gene, and then test their potential to generate functional cortical and medullary TECs and restore thymic function in patients receiving a HSC transplant. Maximizing thymic function should be combined with a strategy to promote T lineage commitment from UCB HSC CD34+CD38- cells to maximize T-cell reconstitution and engraftment. The CD34+CD45RA+CD7+ cells in UCB have T, B and some GM precursor activities.^{144, 145} Commitment of these cells to the T lineage is initiated by Notch signaling¹⁴⁶ and in vitro can be induced by culturing with a stromal cell line that overexpresses the Notch ligand Delta-like 1.^{147, 148} Future studies should focus on the generation and identification of T lineage committed cells from UCB that have thymic homing potential. The combination of the T lineage committed cells with UCB would improve T-cell reconstitution and engraftment. Careful models will be required to balance enhanced immune reconstitution¹⁴⁹ with GVHD, graft rejection and relapse.

Current/planned HSC expansion clinical trials

ViaCell (www.viacellinc.com) has initiated a clinical trial using their proprietary ex vivo expansion methods that concentrates on the removal of committed cells to improve expansion. In a phase I study, ViaCell attempted to remove differentiated cells from an umbilical cord blood unit before expansion. Their phase I trial has been completed and results are expected shortly.

Shpall et al.¹¹⁵ at MD Anderson are currently performing a randomized controlled trial in which patients receive either two unmanipulated cords or one unmanipulated and one from which all cells are expanded. On day-14 the CliniMACS system removes and freezes the T-cell rich CD133- fraction whereas the CD133+ fraction is cultured ex vivo for 14 days containing SCF, G-CSF and TPO. All cells are infused on day zero. The future of ex vivo expansion may indeed be optimized using this dual cord approach as this meets the TNCs per kilogram guidelines without changing engraftment capacity, homing tendencies or apoptotic pathways.¹⁵⁰

Gamida Cell is in the process of organizing a second clinical trial based on the process of copper chelation as described above which should get underway in 2007.

Another advantage for using a dual cord approach is that one could be infused without modification and the other cord could be used as a means for adoptive immunotherapy. A recent development involves the genetic manipulation of the naïve T cells in a cord graft into CD19-specific effector cells, followed by expansion of these cells ex vivo, with subsequent reinfusion into patients with CD19+ tumors with the hopes of eliminating any residual disease cells. This technique has been validated by time-lapse microscopy in NOD/SCID mice.¹⁵¹ Another adoptive immunotherapy technique designed to decrease the infectious complications would be to expand UCB T-cells using anti-CD3/anti-CD28-coated beads with subsequent expansion for reinfusion post transplant.¹⁵² Both of these techniques will require validation in phase I trials.

Conclusions

The ultimate clinical value of infusing expanded UCB is yet to be realized. With a better understanding of the molecular mechanisms of HSC homing, expansion and presentation, a second generation of clinical trials are being initiated and more are sure to follow based on our recent enhanced understanding of hematopoiesis. This is occurring at the same time that other clinical trials exploring the use of dual UCB units as the transplant source. Although dual cord transplants may ultimately supplant our need for HSC expansion, it remains to be determined if this approach will minimize relapse and optimize immune reconstitution as compared to that seen after a living related or unrelated donor transplant. Until this is clarified, expansion studies should continue.

References

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Acknowledgements

This work was supported in part by a grant from the Illinois Regenerative Medicine Institute.