Because BM transplantation has a longer history than UCB transplantation, fundamental questions remain about UCB transplantation. For example, “what is special in UCB and what advantages does UCB (HSCs/HPCs) have?” Clinical trials have basically shown that UCB transplantation causes less GVHD and graft failure [6–9, 32, 33]. Recipients seem to be less sensitive to HLA-mismatching when UCB is used as a source of HSCs/HPCs.

One clinically interesting fact is that UCB myeloid progenitor cells are relatively chemoresistant [34]. This is very important because blood cell transplantation is often combined with intensive chemotherapies to reconstruct host immune systems. Use of chemoresistant UCB cells with more intensive chemotherapies may avoid the relapse of diseases that are sometimes observed after the treatment of hematopoietic malignancy.

If UCB cells have better potential than BM cells to be engrafted into recipients, then what is the underlying mechanism(s)? The immunological potential of T cells is thought to be one of the key determinants of successful long-term engraftment of transplanted HSCs. A study by Harris et al., which was the first to systematically measure a significant number of samples, showed that UCB cells were unresponsive compared to peripheral blood cells, although IL-2 stimulation could still induce NK cell-mediated lysis of target cells [35]. A slightly lower level of T cell receptor expression was also reported. The authors found that UCB contained CD3⁻ cells, which were not present in peripheral blood [35]. In addition, although the percentage of B cells was comparable between peripheral blood and UCB, approximately 50% of B cells in UCB were CD5⁺, a marker of immature B cells [35]. This finding led the authors to suggest that UCB lymphocytes are functionally immature. UCB cells are less likely to cause GVHD than BM or peripheral blood cells. Interestingly, UCB also showed less cytotoxicity against NK cell-resistant tumor cells. This finding requires careful investigation, as less antitumor activity in reconstituted hematopoietic cells could increase the risk of relapse of hematopoietic malignancies. That is, functional immaturity of UCB lymphocytes may be a “double-edged sword.” UCB transplantation apparently yields better responses in recipients, such as decreased GVHD; however, the long-term clinical outcomes of using UCB may not be significantly better than those achieved with other blood sources.

Although T-cell depletion does not seem to be necessary for UCB transplantation [36], subpopulations of patients undergoing UCB transplantation still experience problems such as GVHD and a relatively poor survival rate, possibly due to the higher percentage of HSCs or immaturity of hematopoietic cells in UCB [8, 10]. A small dose of transplanted cells is also thought to be associated with graft rejection. Progenitor cell content may be particularly important for successful engraftment [37]. Potential solutions are discussed in Section 4.

A comparison of UCB, BM, and peripheral blood cells has shown that UCB cells engraft better than cells from the latter two origins when transplanted into nonobese/severe combined immunodeficient (NOD/SCID) mice. In vitro and in vivo studies conducted by Kim et al. revealed that UCB-derived CD34⁺ cells not only have better colony-forming ability than BM-derived CD34⁺ cells, but also show significantly better engraftment in NOD/SCID mice [38]. One very interesting finding of this study is that the level of chimerism is better in mice transplanted with total UCB cells than partially purified UCB cells (CD34⁺ cell fraction) [38]. This finding implies that the CD34⁻ fraction may contain some cells that support the engraftment of transplanted cells. If this finding is applicable to humans, again, enrichment of cells based on cell surface CD34 may not be necessary for transplantation. Interestingly, UCB is thought to contain a significantly higher number of multipotent and CD34⁺CD38⁻ cells than BM. Hoffman's group developed an in vivo competition assay to analyze the BM repopulating potential of UCB cells. They established mice implanted with human fetal bones [39]. After irradiation, HLA-mismatched UCB or adult BM cells were injected individually or in combination. UCB and adult BM CD34⁺ cells showed similar multilineage hematopoiesis when equal numbers of UCB or BM cells were injected individually. However, UCB outcompeted BM cells in repopulating BM when both were simultaneously injected [39]. This result suggests UCB HSCs/HPCs have superior capacity to engraft and repopulate. Thus, studies in animal model systems support the idea that UCB is superior source of hematopoietic cells than BM.

Aside from increasing the dose of cells, accelerating the homing of HSCs/HPCs to hematopoietic tissues could considerably improve engraftment. Although the exact molecular mechanisms underlying HSC/HPC homing remain unclear, a chemokine, stroma-derived factor-1α (SDF-1α), has been shown to play an important role in BM engraftment of HSCs [40]. In vitro experiments showed that this factor induced chemotaxis of UCB progenitors as well as BM cells [41]. In spite of this in vitro chemotactic activity, clinical trials have shown that neutrophil recovery is slower after UCB transplantation than after BM transplantation [32, 36], a finding that might reflect slower migration of UCB cells than BM cells toward the BM after transplantation. The functional immaturity of UCB cells may make it more difficult for these cells to rapidly home to the correct location (i.e., BM). This might explain why UCB transplantation requires a longer time to show beneficial effects. Interestingly, the cell surface dipeptidylpeptidase, CD26, is suggested to negatively regulate in vivo homing and engraftment of HSCs [42]. This protease can cleave the N-terminal dipeptide from several cytokines, including a chemokine SDF-1 (CXCL12) [43]. This proteolysis dampens the biological activity of SDF-1 in chemotaxis [44]. Moreover, a recent study by the same group demonstrated that the percentage of CD26⁺ cells is higher in immature, primitive cells (CD34⁺CD38⁻) than in mature cells [45]. These findings suggest that inhibition of CD26 may be a promising strategy for improving clinical outcomes. Enhancing HSC/HPC homing may reduce the dose of cells required for transplantation.

Infection is the major complication following transplantation. In one study, all patients experienced at least one infection after transplantation [46]. One characteristic of UCB transplantation is the slow neutrophil recovery that occurs relative to BM transplantation [36]. Although increasing the dose of transfused cells certainly shortens the neutrophil recovery time [8], this may not be a realistic solution because of the low availability of UCB cells. Unfortunately, treatment of patients with granulocyte-colony stimulating factor or granulocyte macrophage-colony stimulating factor is not effective in improving neutrophil recovery [32]. It is not yet clear whether slower neutrophil recovery increases morbidity and mortality by infection. Nevertheless, cotransplantation of neutrophils or their precursors might improve clinical outcomes. Indeed, in one study, such cotransplantation accelerated neutrophil recovery and reduced bacterial and fungal infections [47]. Because the number of neutrophils is limited in blood samples, ex vivo expansion of neutrophils is necessary. Few studies have tackled this topic. The most recent, conducted by Timmins and colleagues, has yielded very encouraging results with regard to clinical application of neutrophil coinfusion. The authors successfully expanded functional neutrophils in a large-scale bioreactor at a volume up to 10 L [48]. Expanded neutrophils showed respiratory burst activity to generate reactive oxygen species and killed the fungus, Candida albicans. The system that the authors used is capable of processing culture volumes up to 500 L [48].

Most recently, a new strategy has been introduced to combat infections and leukemia relapse, major factors causing death of transplant recipients. Michlethwaite and colleagues have generated cytotoxic T lymphocytes (CTLs) harboring both antiviral and antileukemic activities [49]. A retrovirus-coded single-chain anti-CD19 fragment was introduced into T cells prepared from peripheral blood and UCB. The resulting CTLs showed specific and strong cytolytic activity against B-cell acute lymphoblastic leukemia as well as against cell lines, demonstrating its clinical potential in combating leukemia relapse and viral infections concomitant with blood transplantation. Although the introduction of a retrovirus-encoded chimeric gene may bring some concerns, this strategy is very straightforward, and the outcome of the study suggests that this approach holds promise in reducing infections and leukemia relapse-related mortality in patients in the future.

The limited number of HSCs/HPCs is the major obstacle in blood transplantation. Until the number of transplantable cells can be increased to amounts necessary for adult patients, the application of UCB should be limited to pediatric patients. Efficient methods are needed to obtain a larger quantity of primitive cells from UCB. For this reason, developing simple and realistic protocols for industrial-scale expansion of primitive cells in UCB is essential.

Usually, the use of animal products such as fetal bovine serum is inevitable for the propagation of SCs. Since the HSC field is well established, a serum-free culture system is available to expand HSCs/HPCs, though room for improvement still exists. A combination of several cytokines is typically used for ex vivo culture of HSCs/HPCs. Although the exact combination differs among laboratories, it usually consists of several cytokines such as Flt3/Flk2 ligand [50, 51], stem cell factor (SCF) [52, 53], interleukin-3 (IL-3) [54–56], IL-6 [57], and thrombopoietin [58–60]. Among these cytokines, SCF probably has the longest history as a supplement. Flt3/Flk2 ligand may be more effective than SCF [61]. Flt3 ligand knockout mice exhibit serious problems in the early development of HPCs [62], suggesting that including Flt3/Flk2 ligand in the media is essential. Interestingly, gp130 signaling (through IL-6) was reported to synergistically enhance the effect of SCF [63]. The same trend was also reported for Flt3 ligand [64]. Thus, gp130 signaling appears to be supportive. It should be noted that IL-6 alone was not shown to be effective [54, 63, 64]. The effect of IL-3 may need to be carefully evaluated, because the addition of IL-3 was reported to suppress the number of colony-forming cells [65]. It also reduced the reconstituting activity of HSCs [65]. Other cytokines, such as IL-7 and IL-11, are also suggested to promote proliferation of HSCs/HPCs. IL-7 is likely more effective in promoting T-cell expansion [66]. Thus, it might not be suitable for expanding primitive HSC/HPC populations. An unbalanced increase in T-cell populations in ex vivo culture may increase the risk of graft rejection. IL-11 might not be essential, since the absence of IL-11 did not result in a dramatic difference after 10 weeks of culture [67]. Although some suppliers can offer bulk recombinant cytokines in these days, the use of cytokines still increases the cost of expansion. Using a combination of several cytokines also makes it difficult to know the exact molecular mechanism facilitating the expansion of HSCs/HPCs. Further evidence suggests that a feeder layer-based coculture system may also be good for ex vivo culture. For example, porcine microvascular endothelial cells (PMVECs) have been reported to be effective for ex vivo expansion of UCB cells [68]. Among several studies investigating different types of feeder layer-based culture systems, that by Rosler et al. showed that the PMVEC coculture system was 5-fold and 241-fold more effective than a stroma-free culture system (supplemented with cytokines) for expansion of CD34⁺ and CD34⁺CD38⁻ cells, respectively (Figure 2 in [39]). However, coculture systems are probably not suitable for large-scale production of cells. New culture methods or supplements should provide more advantages, while remaining simple. Two recent lines of studies, discussed below, may bring important breakthroughs in the ex vivo expansion of HSCs/HPCs.

Notch signaling has been proposed as a target to promote efficient expansion of HSCs. The human homolog of Drosophila Notch was originally discovered as a gene highly expressed in CD34⁺ hematopoietic cells [69]. Incubation of immobilized Notch ligand dramatically increases (by approximately 100-fold) the number of CD34⁺ cells and enhances repopulating ability in NOD/SCID mice [70]. Although Notch signaling was shown to initiate lymphoid differentiation and enhance self-renewal, a repopulation assay using NOD/SCID mice clearly demonstrated that Notch ligand-incubated cells have far greater ability to reconstitute host marrow. Notch ligand-treated cells injected into mice were also capable of thymic engraftment, which is typically unsuccessful [70]. Recently, the same group has provided encouraging data, including preliminary results of a phase 1 trial showing that CD34⁺ UCB progenitors expanded with Notch ligand engraft better than control cells [71]. One of the important observations in this study is that transplantation of Notch ligand-expanded cells substantially shortens neutrophil recovery time [71], a feature thought to predict successful engraftment. Because clinical trials are ongoing, Notch ligand-mediated ex vivo expansion may alter clinical regimens soon. One potential pitfall relates to the fact that the successful results of this study were obtained using an immobilized Delta1 fusion protein. It should be noted that Delta1-mediated Notch activation can induce apoptosis of cells under certain conditions (such as when there is a high density of ligand) [72]. The development of a more effective Delta1 ligand with reduced cytotoxicity would be desirable for Notch ligand-mediated large-scale expansion.

Biological compounds such as growth factors/cytokines may not be the only compounds that can be used to promote expansion. A recent study has shown that a chemical compound could also be used for this purpose [73]. Chemical compounds would be superior to biological compounds because of lower production costs and greater ease of quality control. Microscope-based high-throughput screening has identified aryl hydrocarbon receptor antagonists as potential drug candidates for promoting ex vivo expansion of HSCs [73]. Currently, these compounds require a cytokine cocktail. However, further development of this finding may eventually lead to chemically defined culture media for clinical-scale production of HSCs. Table 1 shows currently available supplements (including those for feeder cell-based cultures) for ex vivo expansion of HSCs/HPCs and shortly summarizes the advantages and disadvantages of each.

Conventional culture flasks and gas-permeable blood bags [31] are the most widely used systems for the expansion of hematopoietic cells. The first automated bioreactor culture system for BM HPCs was described in 1993 [74]. This first system used a continuous perfusion device, which was connected to a syringe pump to allow fresh cell culture medium to be supplied. This system, with supplements, allowed for up to a 30-fold expansion of granulocyte-macrophage progenitor cells; however, it was less effective for other progenitors [74]. Advantages/disadvantages and other factors (e.g., oxygen, pH, shear stress) are well summarized in Nielsen's review [75]. Recently, a rotating wall vessel was used for ex vivo expansion of UCB cells [76]. Although the total cell number was increased by 435 times, the expansion of CD34⁺ cells was approximately 30-fold [76]. Since a simple Teflon cell culture bag yields an average expansion of 113-fold in CD34⁺ cells [31], further improvements should be explored for more efficient large-scale culture.