Introduction

Umbilical cord blood (CB) transplants have been performed successfully since 1989 and provide an alternative graft source in many patients for whom a matched donor is not available [1, 2]. CB grafts offer many advantages, including a less stringent tissue matching requirement, a low rate of graft-versus-host disease (GVHD), and ready availability. The main disadvantage of CB transplants is the protracted time to full blood and immune cell reconstitution, which leaves the recipient vulnerable to infection and bleeding. It is believed that this is a consequence of the relatively low absolute numbers of hematopoietic stem progenitor cells (HSPCs) infused [3]; however, this issue can be overcome by increasing the infused cell dose present in a single CB unit by instead transplanting two CB units. Although this has been relatively successful in reducing the time taken to achieve blood cell reconstitution following double-unit CB transplantation, the cost of transplanting two CB units is prohibitive for many institutions [4].

An alternative strategy is the ex vivo expansion of one CB unit to generate sufficient HSPCs [5, 6], with a retrospective review showing no difference in mortality between one and two CB unit recipients [7]. Initial attempts have included the use of a cocktail containing human recombinant cytokines, such as thrombopoietin (TPO), fetal liver tyrosine 3 ligand (Flt3L), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), interleukin 3 (IL-3), megakaryocyte growth and development factor (MGDF), erythropoietin (EPO), and interleukin 6 (IL-6) to expand CB-derived HSPCs in culture [8], with additional small molecules and feeder cell layers included at a later stage. Several phase I/II clinical trials have been conducted [9] with varying degrees of success (Table 1). However, the latest iterations of CB expansion, including copper chelation tetraethylenepentamine (TEPA) [10], Notch ligand [11], StemRegenin-1 (SR-1) [12], UM171, a pyrimido indole derivative that likely promotes hematopoietic stem cell (HSC) self-renewal in vitro [13], and nicotinamide (NAM; NiCord) [14] have shown promising results and are currently being evaluated in phase I/ II clinical trials (Table 1) [15,16,17].

Table 1. Clinical trial studies using expanded CB cells
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To date, none of these approaches have been thoroughly evaluated in a randomized trial comparing double CB transplantation versus one expanded and one unexpanded CB unit or the transplantation of a single expanded CB unit alone. A phase I/II clinical trial has been conducted for the copper chelator TEPA (StemEx), which attenuates the differentiation of ex vivo cultured HSPCs resulting in the preferential expansion of early progenitors and hematopoietic engraftment; however, the protocol has not yet been optimized for clinical implementation [10]. Here, we discuss recent progress and encouraging results from the transplantation of expanded CB grafts using small molecules, including SR-1, NiCord, and UM171, which have all shown early clinical success.

Expansion Mechanisms

To date, the discovery of ex vivo HSPC expansion using small molecules termed next-generation molecules (NGMs), including notch ligand, TEPA, SR-1, NAM, and UM171, has been either serendipitous or the result of extensive chemical screenings rather than the product of knowledge regarding the processes that govern the decision between maintaining multipotency and differentiation in hematopoietic progenitor cells (HPC). Several studies have reported that epigenetic modification by chromatin-modifying agents (CMAs) promotes HSC expansion, likely by promoting symmetrical HSC self-renewal divisions [18]. Recently, it has been proposed that relatively mature HPC can be reverted to more primitive HSCs via mitochondrial network reprogramming by epigenetic modifiers, although this requires further validation [19]. Thus, for this review article HSPC is considered as a population of cells consisting of both primitive multipotent HSCs that possess self-renewing ability and relatively mature HPCs that lack self-renewal capacity. The holy grail of CB expansion strategies is to achieve symmetric HSC division in culture as the primary mechanism of expansion (Fig. 1) since asymmetric division will only maintain a multipotent HSC pool under the best-case scenario without rendering true expansion. In contrast, symmetric differentiating division gives rise to two daughter cells that lack HSC properties as they commit to become progenitors and lose their self-renewal ability. As shown in Fig. 1, although asymmetric HSC division only maintains HSC numbers, the newly generated relatively mature progenitors HPCs can serve to repopulate blood cells to shorten the neutropenic period immediately after transplant. Thus the primary goal of CB expansion is twofold, namely to reduce the time taken to achieve blood cell reconstitution following transplantation, and ensuring sustained long-term trilineage hematopoiesis. Preclinical models indicate that the recovery of blood cells immediately after transplantation is repopulated primarily by short term HPCs while sustenance of trilineage hematopoiesis is the contribution of long-term multipotent HSCs that have the ability to self-renew. While long-term HSCs drive sustained lifelong hematopoiesis, these cells are relatively quiescent, hence resulting in a prolonged period of leukopenia. The period immediately after transplantation is primarily repopulated by relatively mature progenitors (HPC). Ex vivo expansion strategies resulting in expansion of HSPCs would, in theory, provide progenitors the capability of giving rise to blood cells immediately after transplantation as well as provide long-term blood cell production for the host. Various strategies have been employed to either augment HSPC numbers or increase the ability of infused HPC progenitors to repopulate the bone marrow (BM) niche via manipulation (Table 1). The foremost ex vivo CB expansion strategies include: (1) HSPC expansion with cytokines including SCF, TPO, and Flt-3 L in liquid culture; (2) mesenchymal stromal cell (MSC) co-culture; (3) chemical molecules such as TEPA (copper chelator), NAM (sirtuin 1 inhibitor), SR1 (aryl hydrocarbon receptor antagonist), immobilized delta-1 (Notch ligand), and bioreactors that allow continuous perfusion culture [9]; and (4) improving BM homing following intravenous graft infusion, intra-bone (i.b.) CB graft infusion, or CB co-infusion with mesenchymal stromal cells [20].

Fig. 1
figure 1

Fate of hematopoietic stem cell (HSC) division: HSC expansion, HSC maintenance, and HSC extinction

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Enhanced CB graft homing following transplantation has been attempted more recently using pharmacological strategies such as CD26/Dipeptidylpeptidase-4 (DPP-4) inhibition with sitagliptin [21]. In a phase I clinical trial, DPP-4 inhibition showed only modest benefits in terms of the speed of hematopoietic engraftment (day 21, comparable with the controls at day 19) [21]. CD34 + cells possess complement C3a receptors that allow calcium influx into the cells to modulate SDF-1-dependent chemotaxis. This system has been exploited by using complement C3a priming to expand HSPCs [22], with the transplantation of C3a-primed CB alongside a second unmanipulated CB unit resulting in a median time to neutrophil engraftment of seven days (historical control of D12 at the same institution) [23]. It has also been shown that enhancing prostaglandin E2 (PGE2) synthesis in a zebrafish model increases the number of HSPCs [24], while the transplantation of CB cells briefly exposed to PGE2 in a clinical trial reduced the time to neutrophil engraftment by 3.5 days in one of the cohorts [25]; a phase II randomized trial of PGE2-treated CB cell transplantation is also underway. The homing of transplanted HSCs requires interactions between cell surface molecules, including E and P-selectins. Binding to selectins requires glycoprotein fucosylation to form cell surface glycan determinants such as sialyl Lewis X (sLex) [26]. CD34 + CB cells have been shown to display significantly lower alpha 1–3 fucosylation levels [27], which may explain their reduced ability to populate the BM niche. Several strategies have been used to correct this deficit, including exposing CD34 + CB cells to human fucosyl-transferase VI (FTVI), FTVII, or its substrate GDP-fucose to increase fucosylation levels, with preclinical studies demonstrating improved BM homing following CD34 + cell fucosylation [26,27,28]. An ongoing clinical trial at the M.D. Anderson Cancer Center is exploring this strategy further [Clinical trial.gov NCT01471067] (Table 1).

Another common strategy for improving ex vivo HSPC expansion has been the use of different combinations of cytokines and small molecules (Table 2) [29]. For instance, a preclinical study found that the use of CMA, the hypomethylating drug 5-aza-2’ deoxycytidine (5azaD), and then trichostatin a (TSA) in fetal bovine serum (FBS)-containing culture for nine days in the presence of a cytokine cocktail (SCF, TPO, FL, and IL-3), increased the number of severe combined immune deficiency (SCID) repopulating cells (SRC) by 9.6-fold (Table 2) [30]. Various other studies have found that SR1 with cytokines (SCF, TPO, FL, IL-6) increases the number of SRCs by 17-fold after 21 days of culture [31], UNC0638 with cytokines (SCF, TPO, FL, IL-6) yields a 2-fold increase in 14 days [32], NAM with a cytokine cocktail (SCF, TPO, FL, IL-6, and 10% FBS) increases the number of SRCs by 9-fold in 21 days [33], and the small molecule UM171 with a cytokine cocktail (SCF, TPO, FL) yields a 13-fold increase in 12 days [13]. In our laboratory, we are currently investigating the potential role of stress signals (i.e., pro-inflammatory pathways) in promoting HSC expansion [unpublished data]. The ex vivo recapitulation of physiological events that culminate in the generation of multipotent HSPCs may provide reproducible expansion strategies that can optimize CB grafts, ultimately allowing successful and timely blood cell reconstitution following transplantation. The reliable amplification of HSPC precursors may have significant economic impacts and provide potent CB grafts for an increasing number of patients in need.

Table 2 Preclinical studies of experimental strategies to expand CB cells
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Epigenetic Modification As an Initiation Event for Promoting HSC Expansion

The process via which HSCs either maintain a relatively primitive multipotent state or adopt a more differentiated state is thought to be regulated by epigenetic modifications. Therefore, several groups have attempted to use CMAs to induce cultured HSCs to retain a multipotent state and generate two daughter HSCs rather than one primitive multipotent HSC and one relatively committed HPC [18]. By doing so, a small number of HSCs could be amplified by self-renewal divisions to facilitate successful transplantation capable of sustaining long-term hematopoiesis. The main problem with these approaches is that they generate numerous relatively mature HPCs rather than primitive HSCs and may not yield true functional, transplantable HSC expansion in culture. Besides, the sequence in which the agents are added in culture and intrinsic cellular determinants may significantly affect the outcome. Since the initial chromatin state is not known when the modification process starts, cell membrane surface markers are used to identify potential HSCs (i.e., CD34, CD90), and the quality of the starting cell pool is only inferred to be equal. Moreover, it is unclear how many replication cycles HSPCs can undergo before triggering senescence and apoptosis (Hayflick’s limit) or whether HSPCs can bypass these crucial cellular transformation checkpoints.

To date, our group and others have performed preclinical studies on several CMAs, including valproic acid (VPA) and 5azaD/TSA [18, 19, 34,35,36,37,38] (Table 2), intending to induce epigenetic reprogramming to either allow symmetric HSC division or allow committed HPCs to return to HSC status. The ability to promote symmetrical HSC division may potentiate the effects of NGMs and improve their in vivo hematopoietic engraftment ability. For instance, it has been shown that CB grafts expanded with NGMs (NAM, UM171, etc.) become detectable as the engrafted CB unit in approximately 40% of recipients when an unmanipulated CB graft is transplanted simultaneously [12, 14]. Since double-unit CB transplantation was performed, it remains unclear whether these molecules promote symmetrical or asymmetrical HSC division; however, a more recent clinical trial transplanted a UM171-expanded CB graft as a single-unit [39], which may indirectly demonstrate that self-renewing HSC divisions occur in culture.

To determine the mechanisms via which CMA-mediated epigenetic modification promotes the expansion of transplantable HSCs in culture [18], we performed global microarray and pathway analyses. Using expanded enriched CD34 + cells from culture, we demonstrated the expansion and maintenance of transplantable HSCs when cultured with 5azaD/TSA and VPA, respectively, compared to culture lacking these NGMs (control), which lacked the ability to repopulate human CB cells following transplantation in a xenogeneic host. Intriguingly, the sequential addition of a hypomethylating drug (5-azaD) followed by a histone deacetylase (HDAC) inhibitor (TSA) transiently activated inflammatory molecules such as S100A8 and leukotriene B4 (LTB4); however, it remains unclear whether this transient activation promotes the symmetric self-renewing division of transplantable HSCs in culture [unpublished observations, [40]. Mitochondrial activity may also be a crucial determinant of HSC fate [41], since low mitochondrial mass, reactive oxygen species (ROS), and functionally immature mitochondria are all hallmarks of self-renewing HSCs [42]. Upon cellular commitment, HSCs switch to mitochondrial oxidative phosphorylation (OXPHOS) to increase the generation of energy required for differentiation [19]. Indeed, it has been postulated that mitochondrial activity is a critical driver of HSC fate [42] and that interactions with the BM milieu may trigger mitochondrial programs linked to the ultimate destiny of HSCs, such as quiescence, self-renewal, and differentiation [42].

Environmental cues are also thought to play a crucial role in controlling HSPC division, with previous studies indicating that interactions between cell-intrinsic factors are likely influenced by epigenetic regulators and environmental factors such as growth factors and cytokines [37]. For instance, Araki et al. demonstrated that environmental cues could augment epigenetic influences on HSCs in culture and that intricate control over the rate of cell division may be a critical determinant of the maintenance of HSC functional potency during ex vivo expansion. The same study also found that the combination of 5azaD/TSA with an optimal cytokine cocktail in culture increases the expression of the adhesion molecule CD62L and diminishes CD26 expression on HSPCs, likely improving BM homing and ultimately the hematopoietic engraftment of the expanded cells in a xenotransplant model. CD62L also has a crucial role in targeting cells to inflamed tissues and may upregulate inflammatory molecules upon HSPC expansion. Previous work in our laboratory revealed that the demethylation of CpG sites in the promoters of individual inflammatory/stress response genes corresponds to increases in the expression of genes encoding a calcium-binding protein (S100A8) and cytochrome P450 (Cyp11A1) in 5azaD/TSA-expanded culture [18]. S100A8 is a known agonist of toll-like receptor 4 (TLR-4), which plays a role in the maintenance and proliferation of endothelial progenitors [43]. In addition, the transcript levels of inflammatory cytokines such as IL-8, TNFα, and Alox 5, which are involved in the production of the inflammatory molecule leukotriene, were also found to be higher in 5azaD/TSA-expanded cells by quantitative polymerase chain reaction (qPCR) [18]. Consistently, the levels of the inflammatory mediators/cytokines leukotriene B4, TNFα, and IL-8 were found to be significantly higher in the conditioned medium of 5azaD/TSA-expanded cells by ELISA [unpublished data, [40]. Notably, CMA-expanded human CB cells displayed a reduced allostimulatory capacity, as determined by mixed lymphocyte culture (MLC), likely due to the reversible inhibition of STAT3-dependent differentiation in these cells [44]. Automated process development for ex vivo expansion of growth factor-mobilized peripheral blood (mPB)-derived autologous CD34 + cells has also been reported [45]. Previous studies from our laboratory have also shown that human BM [35], CB [30, 37, 38], and mPB [34] grafts promote transplantable HSC expansion. Interestingly, CB-derived CD34 + cells exposed to CMA in culture displayed the highest expansion potential, followed by mPB and BM [35]. Although the underlying mechanisms remain unclear, the apparent correlation between expansion potential and cell ontogeny is intriguing.

It would be interesting to determine whether CMA pretreatment followed by the sequential addition of NGMs (SR-1, UM171, or NAM) in culture promotes symmetrical HSC division, thus improving HSC expansion in culture. Although improved blood cell reconstitution kinetics have begun to emerge following expanded single-unit CB transplantation (i.e., UM171, NiCord), only long-term follow-ups in these patients will allow us to ascertain the benefits of ex vivo expansion reliably. Once established, double-unit CB transplantation may be deemed unnecessary, and many patients who do not currently qualify for HSCT will likely be eligible to receive an expanded CB unit.

Combining Multiple Agents Activating Sequential Pathways

The transcriptional analysis of VPA-expanded CD34 + CB cells has revealed the activation of pathways that were not expected to be involved in cell fate choices; for instance, inflammatory mediators are upregulated as a consequence of CMA treatment [18, 40], unpublished observations]. A cascade of events may, therefore, occur after epigenetic activation, indicating that a sequence of events is needed to promote symmetrical HSC division; however, the cascade of events is currently unknown. Our laboratory is currently studying the possible involvement of stress signaling pathway components [unpublished observations] and attempting to position each cascade event in hierarchical order. Determining the sequence of events may allow them to be recapitulated by sequentially activating each pathway to generate the HSPC graft, while the addition of HSPC amplification agents may further improve the yield and engraftment potential of the expanded cells.

Our group has also proposed, and is currently investigating, a ‘Composite Graft’ approach, that involves high numbers of both short-term (ST) - and long-term (LT)-HSCs expanded in culture with VPA and 5azaD/TSA, respectively (Fig. 2). The rationale behind this approach is to bridge the neutropenic period following CB transplantation and ensure that blood cell production is sustained in the long-term [manuscript under preparation]. Therefore, the integration of the composite CB graft strategy with the NGM-expanded CB graft could serve as a powerful strategy for circumventing cell dose limitations. VPA-expanded grafts do not expand LT-HSCs in culture instead maintains its number, as evidenced by lack of a serial transplantability [18]; however, VPA-expanded CB grafts are primarily enriched in ST-HSCs which could serve as a bridge graft when the hematopoietic function needs to be supplemented transiently prior to the endogenous recovery of blood cells from residual HSPCs (i.e., after myeloablative treatments, graft failure, or ionizing radiation exposure). In contrast, using serum-free culture conditions, Chaurasia et al. suggested that VPA results in expansion of HSCs [36]. Whether VPA is capable of promoting symmetric HSC divisions in culture, or rather reverts more committed HPC to HSC to render HSC expansion, has yet to be fully proven. Although the combination of CMA and NGMs has yet to be studied in CD34 + CB cell culture, it would be interesting to determine whether the symmetrical HSC division promoted by CMA can be improved further by the subsequent addition of another HSC division agonist, such as SR-1 or UM171. Similar to CMAs, the presence of prostaglandin E synthase 3 (PTGES3; an inflammatory mediator) in an inactive cytoplasmic complex with aryl hydrocarbon receptor (AhR) can upregulate inflammatory pathway molecules during HSPC expansion [46]. Further studies of the AhR pathway in conjunction with CMAs may elucidate the role of inflammatory molecules in promoting symmetrical and asymmetrical HSC division.

Fig. 2
figure 2

Composite graft. Expansion of short-term HSCs by VPA and long-term HSCs by 5azaD/TSA

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Recent Progress in Ex Vivo CB Expansion

In recent years, significant advancements have been achieved using ex vivo expanded CB grafts [6]. Here, we explore the three main HSPC-promoting agents that have demonstrated benefits in clinical trials.

  1. 1)

    The AhR antagonist SR-1 results in CB expansion

AhR is a ligand-activated basic helix-loop-helix transcription factor that is involved in the biological responses to aromatic (aryl) hydrocarbons and upregulates enzymes (cytochrome P450) involved in xenobiotic detoxification. Non-ligand-bound AhR is retained in the cytoplasm as an inactive protein complex along with a dimer of HSP90, PTGES3, XAP2, AIP, and ARA9 [46], which prevent its inappropriate translocation to the nucleus. A recent phase I/II trial at the University of Minnesota revealed that SR-1 could increase the number of CB CD34 + cells by 330-fold after 15 days in culture, resulting in successful engraftment in 17/17 patients after a median of 15 days for neutrophils and 49 days for platelets [12]. Although these results are encouraging, further follow-up studies are needed to determine the relapse-free survival and long-term utilization of expanded CB grafts, given that early expansion may lead to later exhaustion (12 months follow-up) and graft failure.

A novel product using SR-1, known as MGTA-456 (Magenta Therapeutics, Cambridge, MA), has been developed by combining CD34 + cells expanded by culture with an AhR antagonist (AhRa) for 15 days and the CD34 negative fraction from the same CB unit. An industry-sponsored clinical trial revealed that MGTA-465 promoted faster hematopoietic engraftment than historical controls in 36 patients with hematological malignancies and five patients with non-malignant diseases, with a median follow-up of 2.5 years (range: 0.1-5 years) and 75 days (80–203 days), respectively [47]. The effects of SR-1 in symmetric and asymmetric HSC division or earlier versus later expansion have not yet been evaluated; however, such studies would help to elucidate whether expanded CB cells can sustain long-term hematopoiesis or provide the milieu for the hematopoietic engraftment of a second unmanipulated CB unit.

  1. 2)

    The epigenetic modifier NAM results in CB graft expansion (NiCord)

NAM is a vitamin B3 complex co-factor, also known as niacinamide that acts as a precursor of NAM adenine dinucleotide (NAD+) and potently inhibits enzymes that require NAD for their activities, such as mono-ADP-ribosyltransferases, poly-ADP-ribose polymerases, CD38, and cyclic ADP ribose/NADase. NAM is also a potent inhibitor of the sirtuin family of histone/protein deacetylases and the NAD-dependent class III histone deacetylase (HDAC). SIRT1 catalyzes the deacetylation of acetyl-lysine residues in a reaction that cleaves NAD + and generates NAM, which inhibits SIRT1 in a feedback loop mechanism that blocks NAD + hydrolysis. The genetic deletion of SIRT1 in knock-out mice has been shown to increase HSC proliferation, while the ex vivo treatment of CB CD34 + cells with NAM delays differentiation and enhances migration, homing, and engraftment [48]. The same group also showed that another specific SIRT1 inhibitor mediated the same effects [33]. Based on these preclinical successes, a phase I clinical trial was initiated to test whether a CB-derived cell product consisting of HSPCs expanded with NAM (NiCord) for 21 days and a non-cultured T cell fraction could accelerate hematopoietic recovery and provide long-term hematopoietic engraftment [14]. In another clinical trial, 11 adult patients with hematologic malignancies were recruited from two participating institutions and received a pre-transplant myeloablative-conditioning regimen followed by CB transplantation with NiCord and a second unmanipulated CB unit [14]. NiCord infusion resulted in no detectable adverse events, while complete or partial neutrophil recovery and NiCord-derived T cell engraftment was observed in eight patients and the graft remained stable in all transplanted patients (median follow-up, 21 months). Another clinical trial of 21 patients found that two achieved long-term engraftment with the unmanipulated CB unit while 19 achieved engraftment from the NAM-expanded CB unit [14], in marked contrast to similar previous strategies [11]. Furthermore, patients transplanted with NiCord have been shown to achieve earlier median neutrophil recovery (13 versus 25 days, P < 0.001) than historical controls [14], with one-year overall and progression-free survival rates of 82 and 73%, respectively. In conclusion, rapid blood cell recovery following the NAM-expanded CB transplantation approach is associated with early clinical benefits [48]. Based on these successes, an international multicenter phase I/II clinical trial has been launched utilizing a single NAM-expanded NiCord CB unit [NCT01816230].

  1. 3)

    CB expansion using UM171

A landmark paper demonstrated that the next-generation pyrimidoindole derivative, UM171, which acts independently of AhR and targets cells with a more limited regenerative potential, attenuates cell differentiation and promotes the ex vivo expansion of LT-HSCs [13]. This molecule appears to inhibit transcripts involved in erythroid and megakaryocytic differentiation while most commonly upregulating cell surface molecules. Ex vivo treatment of very small embryonic-like stem cells with UM171 resulted in expansion without compromising their hematopoietic- and endothelial lineage differentiating capacity [49]. An open-label, non-randomized phase I-II clinical trial was conducted in Montreal (Quebec, Canada) to evaluate the use of CB cells expanded with UM171 in ex vivo culture in patients without an available suitable matched donor graft [NCT02668315]. A total of 17 adult patients (out of 25 targeted in the trial NCT02668315) were treated with a myeloablative conditioning regimen prior to transplantation, and CD34 + cells were supplemented with UM171-containing media in a closed culture system until day 0 when the cells were washed and infused. A 36-fold net increase in the absolute number of CD34 + cells was observed alongside relatively rapid neutrophil engraftment (day 10). The clinical trial study (NCT02668315) that was presented as an abstract was recently published showing results from UM171-expanded single CB transplantation trial, demonstrating the feasibility and safety of expanded single-unit CB transplantation [39]. Notably, the patients who received the UM171-expanded CB graft were free of fever much earlier than the historical controls post-transplant (day 7 vs. day 15 p < 0.001), while the length of overall hospitalization was reduced by 11 days and all patients who received UM171-expanded CB grafts achieved full donor chimerism. Since this was a rather small-scale study, its findings should be interpreted with caution and require validation by randomized studies with other CB expansion strategies. Notably, other ex vivo expansion strategies utilizing SR-1 or NAM successfully achieved CD34 + cell expansion, yet the survival benefit observed for UM171-expanded grafts was not apparent in other trials using expanded CB grafts. However, these were small-scale studies and retrospective analyses compared to historical controls, only future randomized trials comparing multiple ex vivo expansion strategies will facilitate the identification of the most potent CB expansion strategy.

Conclusions

In general, mPB and BM grafts provide an optimal number of CD34 + cells as a hematopoietic graft source; however, around one-third of patients are unable to find an appropriately matched allogeneic graft from an adult source. The ex vivo manipulation of human CB cells under appropriate conditions has the potential to expand a limited number of HSPCs in a single-unit of CB to improve the kinetics of hematopoietic engraftment. The epigenetic manipulation of CB HSPCs in culture has resulted in successful preclinical stem cell transplantation and is being actively pursued in clinical trials with some success; however, significant obstacles remain before CB transplantation can be considered a viable alternative source of HSPC grafts [50]. Adding a secondary agent in culture may allow the amplification of functional HSPCs present in a single CB unit; therefore, the identification of non-redundant agonistic pathways may yield a compound effect and thus increase the efficacy of HSPC expansion ex vivo [51].

Given that several NGMs are reportedly effective in promoting HSPC expansion in culture, carefully selected combinatorial approaches may further improve the expansion of HSPCs. Indeed, the identification and validation of a given sequence of compounds that result in enhanced functional HSPC expansion would be an additional advantage in the field of stem cell expansion. To determine the safety of these procedures, long-term follow-ups are needed to demonstrate whether expanded CB cells continue to repopulate the marrow without untoward side effects successfully. An attractive alternative strategy could be to split the CB graft into several components that could be independently manipulated, given that long-term, sustained repopulation appears to be the concerted action of LT-HSCs rather than ST-HSCs. Emerging cell-based therapies under development also include induced pluripotent stem cell (iPSC)-derived tissue-restricted differentiated cells (i.e., HSCs), which also require ex vivo expansion. Besides, genetic modification via the viral delivery of a therapeutic gene to HSPCs for gene therapy or gene editing purposes [52, 53] will likely require brief ex vivo manipulation without triggering HSPC differentiation. The preclinical CB expansion strategies that are currently under investigation may allow the identification of novel sequences of events that are essential for the self-renewing division of HSCs in culture, with the aid of chemical agents that specifically activate symmetric or asymmetric HSC division. Agents that specifically target LT- or ST-HSCs may help to develop tailored HSPC grafts that either facilitate sustained blood cell reconstitution or temporarily improve blood cell counts as a bridge following anti-cancer radiation therapy or chemotherapy. In summary, the use of expanded CB grafts is on the cusp of becoming a clinical reality, and the combination of current ex vivo CB expansion strategies may ultimately result in a better understanding of how to stimulate self-renewing HSC division to engineer an optimal graft for transplantation. Consequently, chemical agonists (alone or in combination) that promote HSC self-renewal could be developed as in vivo drugs to rescue or boost failing HSPC grafts post-transplant and to treat acquired BM failure syndromes.