**4. Assessing the quality and potency of UCB cells**

UCB dosages are often 5–10% of those obtained for BM and mPB and as many as 10–20% of UCB HSCTs can result in graft failure [65, 66]. The total nucleated cell (TNC) and CD34+ cell counts have most often been used in selecting UCB units for transplantation as a particular threshold dosage of these cells in a graft correlates with better engraftment and better clinical outcomes [3]. Poorer outcomes of HLA-mismatched UCB HSCTs are reported with dosages of CD34+ cells and TNCs of less than 1.7 × 105 cells/kg and 2.5 × 107 cells/kg recipient body weight, respectively, and much improved outcomes with median TNCs of 10 × 107 cells/kg [66–69]. Although double cord blood transplants increase cell numbers in the graft, the time for engraftment does not increase in comparison with single UCB HSCTs that are appropriately dosed [70].

Because the potency of the unmanipulated UCB unit following cryopreservation has been cited as the most important parameter in predicting engraftment [71], Kurtzberg and colleagues have developed an Apgar Score for enhancing the quality and hence the potency of UCB HSPCs at the time of collection and banking [72]. For Caucasoid babies, they predict the best quality UCBs are likely to be obtained if birth weights are >3500 g, deliveries are between 34 and 38 weeks of gestation, and the UCB units are processed within 10 h of collection. This is because both CD34+ cells and progenitor cell number decrease at and after 40 weeks gestation, even though TNCs increase, and also due to loss in cell viabilities with delays in processing and cryopreservation postcollection [72, 73]. It is of note that in the USA, African-American UCB units contain 30% less TNC after processing and therefore, may not meet the criteria for TNC numbers that have been set for Caucasoid donors in some banks [72]. Although there are moves by some cord blood banks to bank UCB units with >1.75 × 109 TNCs, Kurtzberg's studies provide a note of caution and suggest that this would limit banking of UCB units collected to 5% of UCBs collected, adding to cost, but more importantly, potentially compromising the potency of UCBs for transplantation since as many as 25% of such units would be predicted to have insufficient progenitors for successful engraftment.

Measuring the HSC content of UCB presents some difficulty as the gold standard is in vivo transplantation over an individual's lifespan or alternatively in surrogate nonhuman primate models, which are likely to be closer to the human situation than in vivo immune-deficient murine models [3, 16, 74]. However, this may be particularly important in the genome-editing context. Viability of banked UCB units is generally assessed using in vitro colony forming unit (CFU) content. Although this is not a measure of the repopulating HSCs, CFU content correlates with neutrophil engraftment and posttransplant survival and can be performed in 2–3 weeks [75, 76]. In human UCB, aldehyde dehydrogenase (ALDH)bright cells as assessed by flow cytometry of viable CD45+CD34+ or CD45+CD133+ cells have been strongly correlated with CFU content and may represent a more rapid surrogate potency assay for predicting at least early neutrophil engraftment [76] provided that the thaw-wash protocol is followed after thawing cryopreserved UCB units. The coexpression of MA6 on CD34+ cells has been reported recently as predictor of platelet recovery [77].

More complex phenotyping of HSCs is not generally carried out in the clinical setting. However, combinations of biomarkers have been used to identify and segregate human HSCs from their immediate progeny and to define the HSPC lineage hierarchy of unmanipulated UCB units in the research setting [15]. The classical hematological hierarchy comprises rare, durable long-term repopulating HSC, which give rise to short-term repopulating HSC and multipotent progenitors (MPP) and subsequently to oligopotent, and finally, unipotent progenitors that differentiate into more than 10 hematopoietic lineages. Based on this hierarchical lineage tree, experimental studies have demonstrated that UCB HSCs can be enriched in the Lineage (Lin)–, CD133+, CD34+ or CD34–, CD90+, CD38lo/–, CD45RA–, and CD49f+ subsets [19–27, 47, 78–86]. The CD90– fraction that contains multipotent progenitors (MPPs) also contains HSCs and, when measured by 20-week repopulation in NSG mice, 1 in 20 of the CD90+ and 1 in 100 of the CD90– UCB cells were defined as HSCs [15]. Both the CD90+ and CD90– subsets could be serially transplanted at least for a further 14–16 weeks [15]. Notably, the CD90+ (50–70%) and CD90– (10–20%) cells that expressed CD49f (CD49+/high), and those CD90– cells that become CD90+ after in vitro culture on OP9 stroma retained their 20-week long-term repopulating ability in NSG mice [15]. When the CD90+ UCB cells were further segregated on the basis of CD49f expression, only the CD90+ CD49f+/high cells could be serially transplanted [15]. Notta et al. [15] defined the MPPs that were CD90− CD49f− as transiently engrafting cells (2–4 weeks in NSG mice) or short-term repopulating HSCs and concluded that most HSCs reside in the CD90+ fraction, but 1 in 5.5 UCB HSCs lacked CD90 expression, and approximately 10% of the UCB CD90+CD49f+ cells fraction are HSCs [15]. More recent experiments from Notta et al. [86] have added further to our understanding of the human hematopoietic lineage and the developmental changes it undergoes from fetal liver to UCB to bone marrow hematopoiesis. Importantly, these investigators developed an in vitro single-cell assay that exclusively assesses myeloid, including erythroid and megakaryocytic, lineage potential of individual CD34+ cells by combining MS-5 stromal cultures with LDL and eight cytokines, stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin 6, interleukin 3, interleukin 11, granulocyte macrophage colony stimulating factor and erythropoietin (SCF, TPO, Flt3L, IL6, IL3, IL11, GM-CSF and Epo respectively) and found that 72% of the enriched UCB CD49f+ HSCs gave rise to such clones [86]. Of these, approximately half formed high proliferative potential (HPP)-CFU [86]. This level of clonogenic potential was not observed in semisolid methocel cultures used to assay CFU content.


**Table 4.** Biomarkers that segregate human UCB HSPC subsets.

murine models [3, 16, 74]. However, this may be particularly important in the genome-editing context. Viability of banked UCB units is generally assessed using in vitro colony forming unit (CFU) content. Although this is not a measure of the repopulating HSCs, CFU content correlates with neutrophil engraftment and posttransplant survival and can be performed in 2–3 weeks [75, 76]. In human UCB, aldehyde dehydrogenase (ALDH)bright cells as assessed by flow cytometry of viable CD45+CD34+ or CD45+CD133+ cells have been strongly correlated with CFU content and may represent a more rapid surrogate potency assay for predicting at least early neutrophil engraftment [76] provided that the thaw-wash protocol is followed after thawing cryopreserved UCB units. The coexpression of MA6 on CD34+ cells has been reported

More complex phenotyping of HSCs is not generally carried out in the clinical setting. However, combinations of biomarkers have been used to identify and segregate human HSCs from their immediate progeny and to define the HSPC lineage hierarchy of unmanipulated UCB units in the research setting [15]. The classical hematological hierarchy comprises rare, durable long-term repopulating HSC, which give rise to short-term repopulating HSC and multipotent progenitors (MPP) and subsequently to oligopotent, and finally, unipotent progenitors that differentiate into more than 10 hematopoietic lineages. Based on this hierarchical lineage tree, experimental studies have demonstrated that UCB HSCs can be enriched in the Lineage (Lin)–, CD133+, CD34+ or CD34–, CD90+, CD38lo/–, CD45RA–, and CD49f+ subsets [19–27, 47, 78–86]. The CD90– fraction that contains multipotent progenitors (MPPs) also contains HSCs and, when measured by 20-week repopulation in NSG mice, 1 in 20 of the CD90+ and 1 in 100 of the CD90– UCB cells were defined as HSCs [15]. Both the CD90+ and CD90– subsets could be serially transplanted at least for a further 14–16 weeks [15]. Notably, the CD90+ (50–70%) and CD90– (10–20%) cells that expressed CD49f (CD49+/high), and those CD90– cells that become CD90+ after in vitro culture on OP9 stroma retained their 20-week long-term repopulating ability in NSG mice [15]. When the CD90+ UCB cells were further

CD49f+/high cells could be serially

as transiently

CD49f−

recently as predictor of platelet recovery [77].

146 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

segregated on the basis of CD49f expression, only the CD90+

transplanted [15]. Notta et al. [15] defined the MPPs that were CD90−

observed in semisolid methocel cultures used to assay CFU content.

engrafting cells (2–4 weeks in NSG mice) or short-term repopulating HSCs and concluded that most HSCs reside in the CD90+ fraction, but 1 in 5.5 UCB HSCs lacked CD90 expression, and approximately 10% of the UCB CD90+CD49f+ cells fraction are HSCs [15]. More recent experiments from Notta et al. [86] have added further to our understanding of the human hematopoietic lineage and the developmental changes it undergoes from fetal liver to UCB to bone marrow hematopoiesis. Importantly, these investigators developed an in vitro single-cell assay that exclusively assesses myeloid, including erythroid and megakaryocytic, lineage potential of individual CD34+ cells by combining MS-5 stromal cultures with LDL and eight cytokines, stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, interleukin 6, interleukin 3, interleukin 11, granulocyte macrophage colony stimulating factor and erythropoietin (SCF, TPO, Flt3L, IL6, IL3, IL11, GM-CSF and Epo respectively) and found that 72% of the enriched UCB CD49f+ HSCs gave rise to such clones [86]. Of these, approximately half formed high proliferative potential (HPP)-CFU [86]. This level of clonogenic potential was not Oligopotent or bipotent myeloid progenitors, as the offspring of HSCs, have previously been categorized into common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and megakaryocyte-erythroid progenitors (MEP) on the basis of differential expression on CD34+CD38+ cells of CD123 [15, 86] or CD135 [87, 88] and CD45RA [84]. Using additional biomarkers to those described above or in Ref. [15], particularly CD110 and CD71, but not lineage markers (used in [15]) as shown in **Table 4** [86], has allowed progeny of HSCs to be segregated further (e.g., into 3 MPP, 3 MEP, 3 CMP, and 2 GMP subsets), with the erythroid/ megakaryocytes now being shown to originate from the HSC compartment predominantly and with megakaryocytes emerging from the multipotent HSC and MPP compartments, rather than the CMP subset. This again redefines the lineage relationships in UCB and has implications for expanding distinct HSPC subsets ex vivo.
