**5. Improving UCB HSPC grafts with cytokine and small molecule treatments ex vivo**

Different approaches to improve the efficacy of UCB units clinically have been and are being taken. Whether UCB is used in the related, unrelated, or autologous HSCT settings described above or to generate blood cells ex vivo, enhancing UCB HSC self-renewal or graft content, improving delayed hematological reconstitution, and improving UCB homing to and engraftment in patient bone marrow niches are important issues to address and some of the approaches are described below.

#### **5.1. Biologics to enhance UCB homing to and engraftment in the bone marrow niche**

UCB HSCs demonstrate a defect in homing to the bone marrow [87, 89–97]. This is related to the expression of homing receptors and adhesion molecules on UCB HSC and their progeny. Fucosylated selectins are required for HSCs to roll on bone marrow sinusoidal endothelium [87, 89–91], before being attracted into the bone marrow niche via specific chemokines. CXCL12 is a key chemotactic factor controlling HSC homing to and more specifically engraftment and retention in the bone marrow niche [92–94]. Its cognate receptor, CXCR4, is expressed on HSC and their progeny including pre-B and T lymphoid cells [92–96]. Coreceptors or other factors that regulate CXCR4 signaling in response to CXCL12 on HSCs include CD26 (DPPIV), endolyn, JAM-A, VCAM-1, thrombin, fibrinogen, hyaluronic acid, and C3a [17, 95–100].

Clinical trials have been based on double or single UCB HSCTs and are exemplified in the following findings:


The latter two approaches [83, 102–107] require relatively short exposures of UCB cells to fucosyl transferases or dmPGE2 prior to transplant. dmPGE2 was first identified as a potential agent for expanding HSC in a high throughput screen in zebrafish [105]. The initial clinical study on nine patients receiving nonmyeloablative conditioning and double UCB HSCTs (with 1 unit primed with dmPGE2) did not demonstrate improved engraftment [104]. In a subsequent similar clinical trial, but for which the dmPGE2 treatment of the UCB cells was first optimized, improved time to neutrophil recovery (median 17.5 days) was observed when compared with historical controls (21 days) and with the dmPGE2 primed UCB unit engrafting in over 80% of the patients. This has preceded to a Phase II clinical trial [106], and recent studies suggest that dmPGE2 may modulate Wnt signaling in UCB T cells and enhance immune reconstitution posttransplant [103]. The effects of dmPGE2 in patients undergoing myeloablative conditioning are unknown. Two Phase II clinical trials are being conducted to examine the effects of CD34+ cell fucosylated on engraftment. Double UCB HSCTs in which patients receive myeloablative conditioning for high-risk hematological malignancies and where 1 UCB CD34+ cell graft is fucosylated for 30 min prior to HSCT demonstrate a median time to neutrophil and platelet engraftment of 17 and 35 days, respectively, compared with 26 and 45 days for historical controls [87, 107]. However, the fucosylated UCB unit contributed to engraftment in approximately half of the patients. Biologics that specifically modulate homing and engraftment activities of UCB grafts thus warrant further investigation and optimization.

#### **5.2. Ex vivo expansion of UCB units prior to infusion**

It has been predicted that a fourfold expansion of HSCs in UCB would allow the majority of banked UCB units to be used for single UCB HSCT. Currently, the principal practice is to transplant an unmanipulated UCB unit with an ex vivo expanded UCB unit, with the former generally engrafting longer term and the latter contributing to early neutrophil engraftment. The preferred aim is to move to a single UCB unit where a portion of the UCB unit is expanded and transplanted with the unexpanded portion or to expand a single UCB unit ensuring that the manipulated cells can enhance short-term engraftment without compromising long-term engraftment. Approaches have used cytokines with or without small molecules or MSCs to expand UCB units.

#### *5.2.1. Classical cytokine expansion*

**5.1. Biologics to enhance UCB homing to and engraftment in the bone marrow niche**

148 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

UCB HSCs demonstrate a defect in homing to the bone marrow [87, 89–97]. This is related to the expression of homing receptors and adhesion molecules on UCB HSC and their progeny. Fucosylated selectins are required for HSCs to roll on bone marrow sinusoidal endothelium [87, 89–91], before being attracted into the bone marrow niche via specific chemokines. CXCL12 is a key chemotactic factor controlling HSC homing to and more specifically engraftment and retention in the bone marrow niche [92–94]. Its cognate receptor, CXCR4, is expressed on HSC and their progeny including pre-B and T lymphoid cells [92–96]. Coreceptors or other factors that regulate CXCR4 signaling in response to CXCL12 on HSCs include CD26 (DPPIV), endolyn, JAM-A, VCAM-1, thrombin, fibrinogen, hyaluronic acid, and C3a [17, 95–100].

Clinical trials have been based on double or single UCB HSCTs and are exemplified in the

**i.** Systemic infusion of stigaliptin, a CD26 inhibitor, aimed at enhancing the HSC

**ii.** Priming of UCB grafts with C3a. This complement pathway protein is produced by

**iii.** Ex vivo treatment of one of the two UCB units using prostaglandin E2 (dmPGE2)

**iv.** Ex vivo treatment of one of the two UCB units using fucosyl transferases, fucT-VI, or

of homing receptors, e.g., the selectins (ASC101) [75, 87, 107].

aimed at enhancing HSC homing by upregulating CXCR4 and HSC survival (FT1050;

fucT-VII, aimed at enhancing HSC entry to the bone marrow niche by fucosylation

bone marrow MSCs and interacts with the C3aR on UCB HSPCs to enhance CXCL12 mediated migration [100, 101]. A Phase I clinical trial involving C3a priming of a one of the two UCB units in the nonmyeloablative conditioning double cord blood setting [101] did not, however, demonstrate preferable neutrophil recovery in the manipulated UCB unit in most cases, although the CD3 content of the graft correlated with engraftment. Lund et al. [102] have reviewed this trial recently and concluded that the study has added value for designing further clinical trials using manipulated and

for this treatment to be more effective [99].

unmanipulated double UCB units.

ProHema) [102–106].

homing/engraftment response to the key chemokine CXCL12 by inhibiting CXCL12 degradation. The mechanism of CD26 action has been studied and found not only to degrade CXCL12 but also to truncate a number of other growth factors such as GM-CSF, IL3, M-CSF, EPO, Flt3L, and SCF. Truncation alters their growth factor activity. For example, truncated EPO blocks the activity of the full-length EPO molecule, while truncated GM-CSF binds to its receptor with higher affinity [96, 97]. Two multicentre Phase II trials have commenced where Stigaliptin is given orally for 3 days in the myeloablative conditioning, single UCB HSCT setting. Although initial outcomes indicated that oral treatment with Stigaliptin showed a median time to neutrophil engraftment of 21 days, if the UCB unit were red-cell depleted, neutrophil engraftment correlated with DPPIV suppression [98]. This approach does not require ex vivo manipulation of the UCB graft, but dosage of Stigaliptin needs further investigation

following findings:

Current expansion protocols for UCB HSPCs are still in development and evolving continually with improvements in understanding the bone marrow niches and advances in cell and molecular technologies. However, they have evolved from many studies conducted over the past four decades or more commencing with studies in the 1960s and 1970s on in vitro cultures of murine hematopoietic stem/progenitor cells [108, 109] and the identification of monoclonal antibodies [110] to define specific cell surface biomarkers on HSPC subsets. Notably, some of the initial basic cytokine cocktails, such as SCF, TPO, and Flt3L, are still used and are supplemented with new cytokines or factors or better characterized supportive cells and factors either in static or perfusion bioreactor culture conditions in the presence of extracellular matrix molecules that amplify their efficacy ([18, 80, 111–117] and references therein). Notably, removal of inhibitory factors is also beneficial in promoting human UCB HSPC expansion using a fed-batch approach [111, 112]. Our own unpublished studies also indicate that expansion of HSPCs is a multistage process in which exposure to different cytokine combinations over time influences HSC self-renewal and differentiation ex vivo. In the initial clinical trials using cytokine-based expansion, in which part of the graft was unmanipulated and CD34+ cells from part of the same UCB unit were expanded in a limited number of cytokines (e.g., SCF, granulocyte-colony stimulating factor (G-CSF) and megarkaryocyte growth and differentiation factor (MGDF) for 10 days or in a perfused bioreactor with Flt3L, Epo, and GM-CSF-IL3 fusion protein for 12 days) and then both manipulated and unmanipulated cells transplanted, no improvements in neutrophil or platelet recovery were observed [66]. These studies, however, provided the impetus for the identification of new factors for HSPC expansion and for the design of further clinical trials.

#### *5.2.2. Further cytokine and small molecule addition to expand UCB HSPCs*

With an improving knowledge of stem cell niches and microenvironments [118, 119] and technological advances, numerous factors have been identified that regulate HSPC proliferation and differentiation and some may potentially also control HSC self-renewal. Here, we will restrict our discussion to UCB expansion ex vivo and, as appropriate, discuss clinical applications, while other approaches to generate and assay (in xenograft in vivo models, e.g., in zebrafish) patient-specific HSPCs derived from ES or iPS cells have recently been reviewed and will not be discussed further [118].

Additional factors or small molecules that enhance UCB HSPC proliferation or function include the Notch Delta-like ligand 1 (DLL1), StemRegenin1 (SR 1), the copper chelator tetraethylenepentamide (TEPA), GSK3β inhibitors of WNT signaling, p18—a specific inhibitor of cyclin-dependent kinase (CDK), pyrimidoindole derivatives such as UM171, specific miRNAs, and epigenetic modifiers such as histone deacetylase (HDAC) inhibitors and nicotinamide, as well as additional growth factors such as the designer cytokine hyper-IL-6, oncostatin M, IL-11, angiopoietin-like 5, and other angiopoietin-like molecules and IGFBP2 [120–139]. Coculture of UCB cells with mesenchymal stromal cells has also been examined [140].

Human UCB CD34+CD38– HSPCs cultured in SCF, Flt3L, TPO, IL6, and IL3 and with Fc immobilized DLL1 Notch ligand over several weeks in vitro demonstrate a tenfold increase in CD34+ cells with enhanced repopulating ability in immunodeficient mice [124, 125]. Similar to Notch signaling, another key developmental signaling pathway, Wnt, also serves as a potential target for maintaining the HSPC multipotency during ex vivo expansion. Increased early engraftment and chimaerism levels in immunodeficient mice were observed when UCB CD34+ cells were expanded in cytokines supplemented with an inhibitor of glycogen synthase kinase-3β (GSK3β), BIO [141]. Another GSK3β inhibitor CHIR99021 also appears to maintain HSC functionality, at least when tested on murine HSPCs [142]. A higher level of chimaerism was observed 4 months postsecondary HSC transplantation for UCB cells expanded with CHIR99021 and the mTOR inhibitor rapamycin for a week [142]. Thus activating Wnt/β-catenin signaling and inhibiting mTOR may serve to increase UCB HSC numbers in future studies. Wnt and TGFβ pathways also have opposing roles in regulating the balance between HSC selfrenewal, quiescence, and differentiation [143], and these are controlled by miRNAs. By regulating the balance of these two signaling pathways, the miR-99a/100∼125b tricistronic miRNAs are reported to promote human HSCs expansion and to favor megakaryocytic differentiation [143]. Additionally, miR-126 regulates HSC proliferation and differentiation by targeting PI3K/AKT/mTOR signaling [131]. Regulating these miRNAs may serve as a potential strategy to modulate HSPCs by targeting multiple functional, but opposing, signaling pathways.

removal of inhibitory factors is also beneficial in promoting human UCB HSPC expansion using a fed-batch approach [111, 112]. Our own unpublished studies also indicate that expansion of HSPCs is a multistage process in which exposure to different cytokine combinations over time influences HSC self-renewal and differentiation ex vivo. In the initial clinical trials using cytokine-based expansion, in which part of the graft was unmanipulated and CD34+ cells from part of the same UCB unit were expanded in a limited number of cytokines (e.g., SCF, granulocyte-colony stimulating factor (G-CSF) and megarkaryocyte growth and differentiation factor (MGDF) for 10 days or in a perfused bioreactor with Flt3L, Epo, and GM-CSF-IL3 fusion protein for 12 days) and then both manipulated and unmanipulated cells transplanted, no improvements in neutrophil or platelet recovery were observed [66]. These studies, however, provided the impetus for the identification of new factors for HSPC expan-

With an improving knowledge of stem cell niches and microenvironments [118, 119] and technological advances, numerous factors have been identified that regulate HSPC proliferation and differentiation and some may potentially also control HSC self-renewal. Here, we will restrict our discussion to UCB expansion ex vivo and, as appropriate, discuss clinical applications, while other approaches to generate and assay (in xenograft in vivo models, e.g., in zebrafish) patient-specific HSPCs derived from ES or iPS cells have recently been reviewed

Additional factors or small molecules that enhance UCB HSPC proliferation or function include the Notch Delta-like ligand 1 (DLL1), StemRegenin1 (SR 1), the copper chelator tetraethylenepentamide (TEPA), GSK3β inhibitors of WNT signaling, p18—a specific inhibitor of cyclin-dependent kinase (CDK), pyrimidoindole derivatives such as UM171, specific miRNAs, and epigenetic modifiers such as histone deacetylase (HDAC) inhibitors and nicotinamide, as well as additional growth factors such as the designer cytokine hyper-IL-6, oncostatin M, IL-11, angiopoietin-like 5, and other angiopoietin-like molecules and IGFBP2 [120–139]. Coculture of UCB cells with mesenchymal stromal cells has also been examined

Human UCB CD34+CD38– HSPCs cultured in SCF, Flt3L, TPO, IL6, and IL3 and with Fc immobilized DLL1 Notch ligand over several weeks in vitro demonstrate a tenfold increase in CD34+ cells with enhanced repopulating ability in immunodeficient mice [124, 125]. Similar to Notch signaling, another key developmental signaling pathway, Wnt, also serves as a potential target for maintaining the HSPC multipotency during ex vivo expansion. Increased early engraftment and chimaerism levels in immunodeficient mice were observed when UCB CD34+ cells were expanded in cytokines supplemented with an inhibitor of glycogen synthase kinase-3β (GSK3β), BIO [141]. Another GSK3β inhibitor CHIR99021 also appears to maintain HSC functionality, at least when tested on murine HSPCs [142]. A higher level of chimaerism was observed 4 months postsecondary HSC transplantation for UCB cells expanded with CHIR99021 and the mTOR inhibitor rapamycin for a week [142]. Thus activating Wnt/β-catenin signaling and inhibiting mTOR may serve to increase UCB HSC numbers in future studies.

sion and for the design of further clinical trials.

150 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

and will not be discussed further [118].

[140].

*5.2.2. Further cytokine and small molecule addition to expand UCB HSPCs*

p18 [144] as a specific inhibitor of cyclin-dependent kinase (CDK) is a potential direct target of cell cycle regulation. Two small-molecule compounds P18IN003 and P18IN011 were identified in this study as being able to enhance the proliferation of mouse HSC cells and increase the reconstitution to bone marrow by at least threefold. This may have a potential for human UCB HSC expansion.

Fares et al. screened 5289 small molecules for expansion of mPB CD34+CD45RA– cells, and this and subsequent synthesis of derivatives of one compound UM729 led to the identification of UM171, a pyrimidoindole derivative, which expanded human UCB CD34+ cells over 100 fold with limited differentiation in 12-day fed-batch cultures supplemented with three essential cytokines, SCF, TPO, and Flt3L [122, 145]. Cells expanded with UM171 show improved hematological reconstitution in NSG mice for at least 18 weeks postsecondary transplantation (13-fold higher than DMSO control). By comparing the RNAseq profiling of cells treated with DMSO or UM171 at different concentrations, UM171 suppressed erythroid/ megakaryocyte transcripts. Of note, TMEM183A and PROCR (CD201) encoding genes were found to be significantly upregulated after UM171 treatment. Both TMEM183A and PROCR are cell surface molecules, with the latter being expressed highly on murine HSCs [146]. A direct comparison [122] has been made between the effects of UM171 and the purine derivative SR 1, which was also identified by a high throughput screen on mPB CD34+ cells in the presence of SCF, TPO, Flt3L, and IL6 and which functions as an aryl hydrocarbon receptor antagonist. SR 1 was reported to expand UCB CD34+ and immunodeficient mouse in vivo repopulating cells by 670- and 17-fold, respectively, over 3 weeks of culture [123]. Fares et al. [122] have suggested, however, that SR 1 expands less durable engrafting cells than UM171.

Other small molecules used for HSPC expansion include nicotinamide and TEPA. The vitamin, nicotinamide, generates oxidized nicotinamide adenine dinucleotide (NAD) that regulates the function of the sirtuins (SIRTs). As well as generating oxidized NAD, nicotinamide acts as a specific inhibitor of SIRT-1, and when it is added to human UCB cultures containing SCF, TPO, Flt3L, and IL6 for 3 weeks, then expansion of in vivo (in NOD/SCID mice) repopulating HSCs occurs [129, 132]. When UCB CD133+ cells were cultured in TEPA with SCF, TPO, Flt3L, and IL6 for 3 weeks, an 89-fold increase in CD34+ cells was observed, together with increases in in vivo NOD/SCID repopulating cells [132].

Poycomb group (PcG) genes, identified as global epigenetic transcriptional repressors, have been demonstrated to work through negatively regulating Hox genes [147]. Genes in the Hox families appear to be highly expressed in murine long-term repopulating HSCs [148]. This supports the idea that HSPC expansion can be regulated epigenetically. Histone deacetylases (HDACs) are classed as important epigenetic modifiers of the eraser type. There are 11 HDAC family members that function as zinc dependent deacetylases of histone and nonhistone proteins. These are divided into four classes. Class I comprises HDAC1-3 and HDAC8, class II consists of HDAC4-7 and HDAC9-10, class III are the sirtuins SIRT1-7 that require NAD as a cosubstrate for their activity, and class IV comprises HDAC11 [149]. Elizalde et al. [150] demonstrated HDAC3 as a potential target for regulating CD34+ cells. Hoffman and colleagues subsequently tested eight HDAC inhibitors, which inhibit class I and II HDACs and found that VPA had a robust influence in promoting the expansion of human UCB CD34+ C90+ CD184+ CD49f+ CD45A− HSCs, which expressed key biomarkers such as CD90, CD49f, and CXCR4 and were ALDHhigh [121]. To evaluate the repopulating activity of expanded cells, sublethally irradiated NSG mice were injected with cells expanded in cytokines (SCF, IL-3, FtL-3, and TPO) and HDAC inhibitors for 7 days. Assessment of the human CD45+ cell chimaerism 13–14 weeks posttransplant demonstrated higher levels of chimaerism in grafts expanded with VPA plus cytokines (32.2±11.3%) than grafts with cytokines alone (13.2±6.4%). The former cells showed secondary transplantation activity (measured at 15–16 weeks posttransplant). Additionally, when compared with the uncultured cells, VPA with cytokines generated 36-fold more SCID-repopulating cells (SRCs) ex vivo. Using limiting dilution analyses, VPA-expanded grafts were also found to contain significantly more SRCs (1 in 31) than control primary grafts (1 in 1115) or cytokine alone expanded grafts (1 in 9223). However, although three of eight HDAC inhibitors were more effective in improving HSC expansion, further studies are required to more accurately define the mode of action of these HDAC inhibitors in HSC expansion. As indicated above, it has been suggested that HDAC3 serves as a target for regulating HSC expansion [150], but not all HDAC inhibitors, and particularly not VPA, target HDAC3 only. Further investigations are warranted to determine the mechanism of action by which HDAC inhibitors provide improved UCB HSC expansion.

#### *5.2.3. Clinical trials of cytokine and small molecule expanded UCB HSPCs*

In these clinical trials, a second, unmanipulated UCB unit with adequate cell numbers and/or the unmanipulated CD34– or CD133– fraction of the expanded UCB unit are generally cotransplanted to ensure the presence of durable long-term engrafting HSCs. Outcomes have generally reported improved times to early neutrophil engraftment.

Clinical trials which have been or are being progressed include the following:

**i.** Notch ligand (DLL1) enhanced expansion of HSC/HPC ex vivo of one of the two UCB units prior to transplant. Delaney et al. [151] expanded CD34+ UCB with SCF, Flt3L, TPO, IL6, IL3, and DLL1 and noted an improved median neutrophil engraftment of 16 days in a double UCB transplant setting. T cells were not expanded, whereas myeloid cells (CD33+, CD14+) from the expanded UCB unit predominated. Over 3 weeks, TNC expansion averaged 562-fold and CD34+ cell expansion averaged 164 fold. Longer-term engraftment (of <1 year) was observed for two of the nine patients evaluated, but in one patient, this was not maintained and in the second, the patient died from sepsis at 6 months posttransplant [102].

**ii.** Ex vivo expansion of one of the two UCB units prior to transplant using bone marrowderived MSCs from a third party haploidentical family member or purified bone marrow Stro3+ MPCs protected under patent to Mesoblast. For the Mesoblast clinical trial [140], where Stro3+ MSCs were cocultured with UCB CD34+ cells for 2–3 weeks in the presence of SCF, G-CSF, FL, TPO, median TNC, and CD34+ cell expansions were c.12- and 30-fold, respectively. Expansion of the HSPC generated more myeloid cells, while NK cells were preserved. Neutrophil engraftment was enhanced (median 15d) in patients receiving HSCT with myeloablation, and the MSCs were equally efficient if used as an off-the-shelf product or sourced as a haploidentical product. Chimaerism from both expanded and unmanipulated UCB units was 46% on days 21–30; this reduced to 13% at 6 months, and by 1 year, the unmanipulated UCB unit had engrafted. The expanded cells, therefore, contributed to early neutrophil recovery, and the unmanipulated UCB unit to longer-term repopulation.

(HDACs) are classed as important epigenetic modifiers of the eraser type. There are 11 HDAC family members that function as zinc dependent deacetylases of histone and nonhistone proteins. These are divided into four classes. Class I comprises HDAC1-3 and HDAC8, class II consists of HDAC4-7 and HDAC9-10, class III are the sirtuins SIRT1-7 that require NAD as a cosubstrate for their activity, and class IV comprises HDAC11 [149]. Elizalde et al. [150] demonstrated HDAC3 as a potential target for regulating CD34+ cells. Hoffman and colleagues subsequently tested eight HDAC inhibitors, which inhibit class I and II HDACs and found that VPA had a robust influence in promoting the expansion of human UCB

CD49f, and CXCR4 and were ALDHhigh [121]. To evaluate the repopulating activity of expanded cells, sublethally irradiated NSG mice were injected with cells expanded in cytokines (SCF, IL-3, FtL-3, and TPO) and HDAC inhibitors for 7 days. Assessment of the human CD45+ cell chimaerism 13–14 weeks posttransplant demonstrated higher levels of chimaerism in grafts expanded with VPA plus cytokines (32.2±11.3%) than grafts with cytokines alone (13.2±6.4%). The former cells showed secondary transplantation activity (measured at 15–16 weeks posttransplant). Additionally, when compared with the uncultured cells, VPA with cytokines generated 36-fold more SCID-repopulating cells (SRCs) ex vivo. Using limiting dilution analyses, VPA-expanded grafts were also found to contain significantly more SRCs (1 in 31) than control primary grafts (1 in 1115) or cytokine alone expanded grafts (1 in 9223). However, although three of eight HDAC inhibitors were more effective in improving HSC expansion, further studies are required to more accurately define the mode of action of these HDAC inhibitors in HSC expansion. As indicated above, it has been suggested that HDAC3 serves as a target for regulating HSC expansion [150], but not all HDAC inhibitors, and particularly not VPA, target HDAC3 only. Further investigations are warranted to determine the mechanism

of action by which HDAC inhibitors provide improved UCB HSC expansion.

Clinical trials which have been or are being progressed include the following:

In these clinical trials, a second, unmanipulated UCB unit with adequate cell numbers and/or the unmanipulated CD34– or CD133– fraction of the expanded UCB unit are generally cotransplanted to ensure the presence of durable long-term engrafting HSCs. Outcomes have

**i.** Notch ligand (DLL1) enhanced expansion of HSC/HPC ex vivo of one of the two UCB

units prior to transplant. Delaney et al. [151] expanded CD34+ UCB with SCF, Flt3L, TPO, IL6, IL3, and DLL1 and noted an improved median neutrophil engraftment of 16 days in a double UCB transplant setting. T cells were not expanded, whereas myeloid cells (CD33+, CD14+) from the expanded UCB unit predominated. Over 3 weeks, TNC expansion averaged 562-fold and CD34+ cell expansion averaged 164 fold. Longer-term engraftment (of <1 year) was observed for two of the nine patients evaluated, but in one patient, this was not maintained and in the second, the patient

*5.2.3. Clinical trials of cytokine and small molecule expanded UCB HSPCs*

generally reported improved times to early neutrophil engraftment.

died from sepsis at 6 months posttransplant [102].

HSCs, which expressed key biomarkers such as CD90,

CD34+

C90+

CD184+

CD49f+

CD45A−

152 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine


expansion of UCB CD34+ cells with SR 1 and cytokines. The median time to neutrophil recovery for 17 patients has been reported as being 15 days. Further studies are planned based on infusion of the expanded unit only. Recent in vitro studies [153] indicate that SR 1 promotes the production of megakaryocyte precursors from CD34+ cells with 90% reaching the proplatelet stage with TPO addition, thereby potentially contributing to the ex vivo production of platelets from normal cells for transfusion.

**vi.** A Phase I/II clinical trial (NCT02668315) involving UM171-expanded UCB HSCs produced in a fed-batch culture system is recruiting patients with hematological malignancies from January 2016.

With most of these clinical studies, the common denominator is that it is possible to reduce the time to neutrophil engraftment to 15–17 days and possibly down to 10–11 days. Thus, improved early neutrophil engraftment is possible with current expansion protocols and the key question going forward relates to whether it is possible to maintain or promote long-term hematopoietic engraftment, particularly where genome editing is applied and where there is a need to expand blood cells ex vivo for difficult-to-transfuse patients.
